Mario Romero-Ortega

Interests

Research

Our research aims to understand the cellular and molecular mechanisms involved in axon regeneration, and to develop tools and applications of clinical neuromodulation of the PNS.Specific areas include, peripheral nerve gap repair, regenerative peripheral neural interfaces, and bioelectronics medicines. A long-term goal of our research is to develop devices that allow the bilateral neural interfacing in peripheral nerves with the capability for specific recording from motor axons and stimulation of modality-specific sensory neurons with high selectivity and stability. In the Bioelectronics field, our aim is to develop devices that allow the neuromodulation of small peripheral nerves for the selective control of visceral targets for applications that include Stress Urinary Incontinence (SUI) and Hypertension.

Teaching

My teaching combines fundamental basic science and biomedical engineering and is focused on translational research and clinical solutions, which allow our students to have an early exposure to pressing clinical problems, and to work side-by-side with medical collaborators in the clinic and surgery rooms. My goal is to prepare them for a successful independent career by focusing on significant challenges that make an impact, training them to excel in experimental and device designs, and develop rigorous analytical and critical thinking.

Courses

Scholarly Contributions

Journals/Publications

• Lloyd, D. A., Alejandra Gonzalez-Gonzalez, M., & Romero-Ortega, M. I. (2024). AxoDetect: an automated nerve image segmentation and quantification workflow for computational nerve modeling. Journal of Neural Engineering, 21(Issue 2). doi:10.1088/1741-2552/ad31c3
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  Objective. Bioelectronic treatments targeting near-organ innervation have unprecedented clinical applications. Particularly in the spleen, the inhibition of the cholinergic inflammatory response by near-organ nerve stimulation has potential to replace pharmacological treatments in chronic and autoimmune diseases. A caveat is that the optimization of therapeutic stimulation parameters relies on in vivo experimentation, which becomes challenging due to the small nerve diameters (2 μ m), complex anatomy, and mixed axon type composition of the autonomic nerves. Effective development of in silico models requires tools which allow for fast and efficient quantification of axonal composition of specific nerves. Current approaches to generate such information rely on manual image segmentation and quantification. Approach. We developed a combined image-segmentation and model-generation software called AxoDetect: a target- and format-agnostic computer vision algorithm which can segment myelin, endo/epineurium, and both myelinated and unmyelinated fibers from a nerve image without training. Main results. AxoDetect is over 10 times faster on average when compared with current automatic methods while maintaining flexibility through the use of tunable pixel threshold filters to detect different types of tissue. When compared to a distribution-based and a manually segmented model of the splenic nerve terminal branch 1, the model generated with AxoDetect had comparable threshold prediction and was able to accurately detect an increase in activation threshold caused by the addition of surrounding fat tissue to the modeled nerve. Significance. AxoDetect contributes to the acceleration of neuromodulation treatment development through faster model design and iteration without requiring training. Furthermore, the computer vision approach and tunable nature of the filters in our method allow for its use in a variety of histological applications. Our approach will impact not only the study of nerves but also the design of implantable neural interfaces to enhance bioelectronic therapeutic options.
• Rahman, F., Yousuf, Z., Castelan, F., Martinez-Gomez, M., Akay, M., Zimmern, P., Romero-Ortega, M., & Akay, Y. (2024). Neuromodulation Improves Stress Urinary Incontinence-like Deficits in Female Rabbits. IEEE Open Journal of Engineering in Medicine and Biology, 6(Issue). doi:10.1109/ojemb.2024.3408454
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  Objective: Stress urinary incontinence (SUI) affects a third of the female population and is characterized by involuntary urine leakage during abdominal efforts such as sneezing, laughing, or coughing. Acute neuromodulation of the bulbospongiosus nerve (BsN) was shown to increase bladder efficiency in aged and multiparous rabbits. This study investigates the efficacy of sub-chronic BsN neuromodulation in alleviating SUI-like deficits in mature multiparous rabbits, characterized by increased urine leakage and reduced leak point pressure. Results: Using the voiding spot assay, we observed a 40% reduction in urine leakage events after 30 days of BsN stimulation, which correlated with a 60% increase in daily micturition volume, a 10- fold increase in voided volume, and improvements in voiding efficiency and leak point pressure compared to negative control animals. Conclusion: In multiparous rabbits, BsN neuromodulation improves important SUI-like metrics including bladder capacity and urethral closure, supporting the use of this bioelectronic modality as treatment for SUI.
• Romero, K., Gonzalez-Gonzalez, M., Lloyd, D., Nguyen, K., Eli, N., Akay, Y., Vongpatanasin, W., Smith, S., Akay, M., & Romero-Ortega, M. (2024). Sub-Chronic Peroneal Nerve Stimulation Lowers Ambulatory Blood Pressure in Spontaneously Hypertensive Rats. IEEE Open Journal of Engineering in Medicine and Biology. doi:10.1109/ojemb.2024.3477411
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  Objective: Acute electrical stimulation of the common peroneal nerve (cPNS) has been shown to cause an immediate reduction in systolic blood pressure (SBP) in spontaneous hypertense rats (SHR), but the effect of this treatment in sub-chronic ambulatory SBP is unknown. Here we developed an implantable wireless WNClip neural stimulator to test the efficacy of 5-week cPNS as a treatment for hypertension. Results: Daily cPNS 2Hz monophasic stimulation at threshold for 8 minutes every day for five weeks, reduced SBP in WKY animals by -4 mm Hg, and in SHR animals by -21 mmHg in week 5(p
• Hernandez-Reynoso, A. G., Rahman, F. S., Hedden, B., Castelán, F., Martínez-Gómez, M., Zimmern, P., & Romero-Ortega, M. I. (2023). Secondary urethral sphincter function of the rabbit pelvic and perineal muscles. Frontiers in Neuroscience, 17(Issue). doi:10.3389/fnins.2023.1111884
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  Perineal and pelvic floor muscles play an important role in continence by providing mechanical support to pelvic organs. It is also known that the pubococcygeus muscle (PcM) contracts in the storage phase and is inactive during voiding, while the bulbospongiosus muscle (BsM) is active during the voiding phase. Recent evidence suggested an additional role of these muscles in supporting urethral closure in rabbits. However, the individual role of perineal and pelvic muscles as urethral sphincters is not well-defined. Here we evaluated the individual, sequential and synergistic roles of the PcM and BsM in assisting urethral closure and defined the optimal electrical stimulation parameters that can effectively contract these muscles and increase the urethral pressure (Pura) in young nulliparous animals (n = 11). Unilateral stimulation of either the BsM or PcM at 40 Hz induced modest increases in average Pura (0.23 ± 0.10 and 0.07 ± 0.04 mmHg, respectively). Investigation on the changes in Pura evoked by stimulation frequencies between 5 and 60 Hz show that sequential contralateral PcM-BsM activation at 40 Hz induced a 2-fold average Pura increase (0.23 ± 0.07 mmHg) compared to that evoked by PcM stimulation. Simultaneous activation of PcM and BsM at 40 Hz also showed an increased average Pura (0.26 ± 0.04 mmHg), with a 2-fold increase in average Pura observed during the unilateral sequential PcM-BsM stimulation at 40 Hz (0.69 ± 0.2 mmHg). Finally, stimulation at 40 Hz of the bulbospongiosus nerve (BsN) induced an approximate 4-fold increase in average Pura (0.87 ± 0.44 mmHg; p < 0.04) compared to that elicited by BsM stimulation, confirming that direct nerve stimulation is more effective. Together, this study shows that in the female rabbit, both perineal and pelvic muscles support of the urethral function during continence, and that unilateral stimulation of the BsN at 40–60 Hz is sufficient to achieve maximal secondary sphincter activity. The results also support the potential clinical value of neuromodulation of pelvic and perineal nerves as bioelectronic therapy for stress urinary incontinence.
• Tasmim, S., Yousuf, Z., Rahman, F. S., Seelig, E., Clevenger, A. J., VandenHeuvel, S. N., Ambulo, C. P., Raghavan, S., Zimmern, P. E., Romero-Ortega, M. I., & Ware, T. H. (2023). Liquid crystal elastomer based dynamic device for urethral support: Potential treatment for stress urinary incontinence. Biomaterials, 292(Issue). doi:10.1016/j.biomaterials.2022.121912
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  Stress urinary incontinence (SUI) is characterized by the involuntary loss of urine due to increased intra-abdominal pressure during coughing, sneezing, or exercising. SUI affects 20–40% of the female population and is exacerbated by aging. Severe SUI is commonly treated with surgical implantation of an autologous or a synthetic sling underneath the urethra for support. These slings, however, are static, and their tension cannot be non-invasively adjusted, if needed, after implantation. This study reports the fabrication of a novel device based on liquid crystal elastomers (LCEs) capable of changing shape in response to temperature increase induced by transcutaneous IR light. The shape change of the LCE-based device was characterized in a scar tissue phantom model. An in vitro urinary tract model was designed to study the efficacy of the LCE-based device to support continence and adjust sling tension with IR illumination. Finally, the device was acutely implanted and tested for induced tension changes in female multiparous New Zealand white rabbits. The LCE device achieved 5.6% ± 1.1% actuation when embedded in an agar gel with an elastic modulus of 100 kPa. The corresponding device temperature was 44.9 °C ± 0.4 °C, and the surrounding agar temperature stayed at 42.1 °C ± 0.4 °C. Leaking time in the in vitro urinary tract model significantly decreased (p < 0.0001) when an LCE-based cuff was sutured around the model urethra from 5.2min ± 1min to 2min ±0.5min when the cuff was illuminated with IR light. Normalized leak point force (LPF) increased significantly (p = 0.01) with the implantation of an LCE-CB cuff around the bladder neck of multiparous rabbits. It decreased significantly (p = 0.023) when the device was actuated via IR light illumination. These results demonstrate that LCE material could be used to fabricate a dynamic device for treating SUI in women.
• Conde, S. V., Sacramento, J. F., Melo, B. F., Fonseca-Pinto, R., Romero-Ortega, M. I., & Guarino, M. P. (2022). Blood Pressure Regulation by the Carotid Sinus Nerve: Clinical Implications for Carotid Body Neuromodulation. Frontiers in Neuroscience, 15(Issue). doi:10.3389/fnins.2021.725751
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  Chronic carotid sinus nerve (CSN) electrical modulation through kilohertz frequency alternating current improves metabolic control in rat models of type 2 diabetes, underpinning the potential of bioelectronic modulation of the CSN as a therapeutic modality for metabolic diseases in humans. The CSN carries sensory information from the carotid bodies, peripheral chemoreceptor organs that respond to changes in blood biochemical modifications such as hypoxia, hypercapnia, acidosis, and hyperinsulinemia. In addition, the CSN also delivers information from carotid sinus baroreceptors—mechanoreceptor sensory neurons directly involved in the control of blood pressure—to the central nervous system. The interaction between these powerful reflex systems—chemoreflex and baroreflex—whose sensory receptors are in anatomical proximity, may be regarded as a drawback to the development of selective bioelectronic tools to modulate the CSN. Herein we aimed to disclose CSN influence on cardiovascular regulation, particularly under hypoxic conditions, and we tested the hypothesis that neuromodulation of the CSN, either by electrical stimuli or surgical means, does not significantly impact blood pressure. Experiments were performed in Wistar rats aged 10–12 weeks. No significant effects of acute hypoxia were observed in systolic or diastolic blood pressure or heart rate although there was a significant activation of the cardiac sympathetic nervous system. We conclude that chemoreceptor activation by hypoxia leads to an expected increase in sympathetic activity accompanied by compensatory regional mechanisms that assure blood flow to regional beds and maintenance of hemodynamic homeostasis. Upon surgical denervation or electrical block of the CSN, the increase in cardiac sympathetic nervous system activity in response to hypoxia was lost, and there were no significant changes in blood pressure in comparison to control animals. We conclude that the responses to hypoxia and vasomotor control short-term regulation of blood pressure are dissociated in terms of hypoxic response but integrated to generate an effector response to a given change in arterial pressure.
• Gonzalez-Gonzalez, M. A., Romero, K., Beitter, J., Lloyd, D., Lam, D. V., Hernandez-Reynoso, A. G., Kanneganti, A., Kim, H. K., Bjune, C. K., Smith, S., Vongpatanasin, W., & Romero-Ortega, M. I. (2022). Renal Nerve Activity and Arterial Depressor Responses Induced by Neuromodulation of the Deep Peroneal Nerve in Spontaneously Hypertensive Rats. Frontiers in Neuroscience, 16(Issue). doi:10.3389/fnins.2022.726467
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  Hypertension is a main cause of death in the United States with more than 103 million adults affected. While pharmacological treatments are effective, blood pressure (BP) remains uncontrolled in 50–60% of resistant hypertensive subjects. Using a custom-wired miniature electrode, we previously reported that deep peroneal nerve stimulation (DPNS) elicited acute cardiovascular depressor responses in anesthetized spontaneously hypertensive rats (SHRs). Here, we further study this effect by implementing a wireless system and exploring different stimulation parameters to achieve a maximum depressor response. Our results indicate that DPNS consistently induces a reduction in BP and suggests that renal sympathetic nerve activity (RSNA) is altered by this bioelectronic treatment. To test the acute effect of DPNS in awake animals, we developed a novel miniaturized wireless microchannel electrode (w-μCE), with a Z-shaped microchannel through which the target nerves slide and lock into the recording/stimulation chamber. Animals implanted with w-μCE and BP telemetry systems for 3 weeks showed an average BP of 150 ± 14 mmHg, which was reduced significantly by an active DPNS session to 135 ± 8 mmHg (p < 0.04), but not in sham-treated animals. The depressor response in animals with an active w-μCE was progressively returned to baseline levels 14 min later (164 ± 26 mmHg). This depressor response was confirmed in restrained fully awake animals that received DPNS for 10 days, where tail-cuff BP measurements showed that systolic BP in SHR lowered 10% at 1 h and 16% 2 h after the DPNS when compared to the post-implantation baseline. Together, these results support the use of DPN neuromodulation as a possible strategy to lower BP in drug-resistant hypertension.
• Liu, X., Wang, K., González-González, M. A., Romero-Ortega, M., & Wallace, G. G. (2022). Sensing and Stimulating Electrodes for Electroceuticals. Frontiers in Sensors, 3(Issue). doi:10.3389/fsens.2022.873862
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  Electroceuticals is an emerging field that combines the technology in conductive materials with their ability to interface with biological systems. The development of highly conductive electrodes to monitor human health in real-time while simultaneously delivering stimulation promises to revolutionize medical science. Aspects to consider during development include the desired shape, electrode material properties, number of active sites, carriers used, and methods of deployment and activation. Novel organic-conductor based electrode compositions offer properties unattainable with conventional metal electrodes. Emerging innovative deployment strategies communicate directly with target tissues while minimizing damage to the surrounding biological environment. Here we highlight the recent reported technology on platinized graphene fibers (sutrode), a high performance electrode, capable of recording electrophysiological signals from small autonomic nerves, which could bring us closer to the ultimate goal: modulating the activity of individual organs with high selectivity and precision for a therapeutic medical outcome. An in-depth understanding of electrode materials and methods of fabrication and deployment can provide unprecedented opportunities for electroceutical research.
• Gonzalez-Gonzalez, M. A., Bendale, G. S., Wang, K., Wallace, G. G., & Romero-Ortega, M. (2021). Platinized graphene fiber electrodes uncover direct spleen-vagus communication. Communications Biology, 4(Issue 1). doi:10.1038/s42003-021-02628-7
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  Neural interfacing nerve fascicles along the splenic neurovascular plexus (SNVP) is needed to better understand the spleen physiology, and for selective neuromodulation of this major organ. However, their small size and anatomical location have proven to be a significant challenge. Here, we use a reduced liquid crystalline graphene oxide (rGO) fiber coated with platinum (Pt) as a super-flexible suture-like electrode to interface multiple SNVP. The Pt-rGO fibers work as a handover knot electrodes over the small SNVP, allowing sensitive recording from four splenic nerve terminal branches (SN 1–4), to uncover differential activity and axon composition among them. Here, the asymmetric defasciculation of the SN branches is revealed by electron microscopy, and the functional compartmentalization in spleen innervation is evidenced in response to hypoxia and pharmacological modulation of mean arterial pressure. We demonstrate that electrical stimulation of cervical and sub-diaphragmatic vagus nerve (VN), evokes activity in a subset of SN terminal branches, providing evidence for a direct VN control over the spleen. This notion is supported by adenoviral tract-tracing of SN branches, revealing an unconventional direct brain-spleen projection. High-performance Pt-rGO fiber electrodes, may be used for the fine neural modulation of other small neurovascular plexus at the point of entry of major organs as a bioelectronic medical alternative.
• Hernández-Reynoso, A. G., Corona‐Quintanilla, D. L., López-García, K., Horbovetz, A. A., Castelán, F., Zimmern, P. E., Martı́nez–Gómez, M., & Romero-Ortega, M. I. (2021). Targeted neuromodulation of pelvic floor nerves in aging and multiparous rabbits improves continence. Scientific Reports, 11(1). doi:10.1038/s41598-021-90088-8
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  Abstract Pelvic floor muscle stretch injury during pregnancy and birth is associated with the incidence of stress urinary incontinence (SUI), a condition that affects 30–60% of the female population and is characterized by involuntary urine leakage during physical activity, further exacerbated by aging. Aging and multiparous rabbits suffer pelvic nerve and muscle damage, resulting in alterations in pelvic floor muscular contraction and low urethral pressure, resembling SUI. However, the extent of nerve injury is not fully understood. Here, we used electron microscopy analysis of pelvic and perineal nerves in multiparous rabbits to describe the extent of stretch nerve injury based on axon count, axon size, myelin-to-axon ratio, and elliptical ratio. Compared to young nulliparous controls, mid-age multiparous animals showed an increase in the density of unmyelinated axons and in myelin thickness in both nerves, albeit more significant in the bulbospongiosus nerve. This revealed a partial but sustained damage to these nerves, and the presence of some regenerated axons. Additionally, we tested whether electrical stimulation of the bulbospongiosus nerve would induce muscle contraction and urethral closure. Using a miniature wireless stimulator implanted on this perineal nerve in young nulliparous and middle age multiparous female rabbits, we confirmed that these partially damaged nerves can be acutely depolarized, either at low (2–5 Hz) or medium (10–20 Hz) frequencies, to induce a proportional increase in urethral pressure. Evaluation of micturition volume in the mid-age multiparous animals after perineal nerve stimulation, effectively reversed a baseline deficit, increasing it 2-fold ( p = 0.02). These results support the notion that selective neuromodulation of pelvic floor muscles might serve as a potential treatment for SUI.
• Loper, H., Leinen, M., Bassoff, L., Sample, J., Romero-Ortega, M., Gustafson, K. J., Taylor, D. M., & Schiefer, M. A. (2021). Both high fat and high carbohydrate diets impair vagus nerve signaling of satiety. Scientific Reports, 11(Issue 1). doi:10.1038/s41598-021-89465-0
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  Obesity remains prevalent in the US. One potential treatment is vagus nerve stimulation (VNS), which activates the sensory afferents innervating the stomach that convey stomach volume and establish satiety. However, current VNS approaches and stimulus optimization could benefit from additional understanding of the underlying neural response to stomach distension. In this study, obesity-prone Sprague Dawley rats consumed a standard, high-carbohydrate, or high-fat diet for several months, leading to diet-induced obesity in the latter two groups. Under anesthesia, the neural activity in the vagus nerve was recorded with a penetrating microelectrode array while the stomach was distended with an implanted balloon. Vagal tone during distension was compared to baseline tone prior to distension. Responses were strongly correlated with stomach distension, but the sensitivity to distension was significantly lower in animals that had been fed the nonstandard diets. The results indicate that both high fat and high carbohydrate diets impair vagus activity.
• Corona-Quintanilla, D. L., López-Juárez, R., Pacheco, P., Romero-Ortega, M. I., Castelán, F., & Martínez-Gómez, M. (2020). Bladder and urethral dysfunction in multiparous and mature rabbits correlates with abnormal activity of pubococcygeus and bulbospongiosus muscles. Neurourology and Urodynamics, 39(Issue 1). doi:10.1002/nau.24176
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  Aims: To evaluate the combined effect of age and multiparity on the micturition reflex, including pelvic floor muscle activation. Methods: Young and mature nulliparous rabbits were compared to young and mature multiparas (n = 6 per group). Cystometrograms and urethral pressure (UP) were performed while simultaneously recording the electromyogram (EMG) activity of the pubococcygeus and bulbospongiosus muscles to establish their functional correlation to urological function. Results: Multiparity and age significantly influence the bladder and UP affecting the voiding efficiency and intercontraction interval. Such interaction also reduced the UP threshold, timing, and duration. Other bladder and urethral variables were predominantly affected only by age. Urodynamic alterations correlated with abnormal patterns or absent EMG activity of the pubococcygeus and bulbospongiosus muscles. Conclusions: The present findings strongly suggest that multiparity and age affects specific pelvic floor muscle reflex activation during micturition, and may contribute to alterations in bladder and urethral function. This data broadens our understanding of the critical role of the appropriate activity of the individual pelvic floor muscles in micturition.
• Ghazavi, A., González-González, M. A., Romero-Ortega, M. I., & Cogan, S. F. (2020). Intraneural ultramicroelectrode arrays for function-specific interfacing to the vagus nerve. Biosensors and Bioelectronics, 170(Issue). doi:10.1016/j.bios.2020.112608
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  Selective interfacing to small multifunctional nerves such as the vagus nerve (VN) which is the main multimodal autonomic nerve that provides a major communication pathway from vital peripheral organs to the brain, can have significant potential in treating and diagnosing diseases as well as enhancing our understanding of peripheral nerve circuits. Here we describe the fabrication of a 16-channel intraneural electrode array with ultramicro-dimensioned electrodes to achieve improved functionally selective recording. We demonstrate that the amorphous silicon carbide ultramicroelectrode arrays (a-SiC UMEAs) provide selectivity in the detection of neural activity in the cVN related to changes in systemic oxygenation and blood pressure. We will also demonstrate spatially selective recording of micro-compound action potentials (μCAPs) by electrical stimulation of the subdiaphragmatic branches of the VN. Distinct neural activity was recorded on electrodes separated by less than about 100 μm. This is the first time that this level of spatially selectivity recording has been demonstrated in the cVN with an intraneural multielectrode array.
• Bucksot, J. E., Wells, A. J., Rahebi, K. C., Sivaji, V., Romero-Ortega, M., Kilgard, M. P., Rennaker, R. L., & Hays, S. A. (2019). Flat electrode contacts for vagus nerve stimulation. PLoS ONE, 14(Issue 11). doi:10.1371/journal.pone.0215191
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  The majority of available systems for vagus nerve stimulation use helical stimulation electrodes, which cover the majority of the circumference of the nerve and produce largely uniform current density within the nerve. Flat stimulation electrodes that contact only one side of the nerve may provide advantages, including ease of fabrication. However, it is possible that the flat configuration will yield inefficient fiber recruitment due to a less uniform current distribution within the nerve. Here we tested the hypothesis that flat electrodes will require higher current amplitude to activate all large-diameter fibers throughout the whole crosssection of a nerve than circumferential designs. Computational modeling and in vivo experiments were performed to evaluate fiber recruitment in different nerves and different species using a variety of electrode designs. Initial results demonstrated similar fiber recruitment in the rat vagus and sciatic nerves with a standard circumferential cuff electrode and a cuff electrode modified to approximate a flat configuration. Follow up experiments comparing true flat electrodes to circumferential electrodes on the rabbit sciatic nerve confirmed that fiber recruitment was equivalent between the two designs. These findings demonstrate that flat electrodes represent a viable design for nerve stimulation that may provide advantages over the current circumferential designs for applications in which the goal is uniform activation of all fascicles within the nerve.
• Hernandez-Reynoso, A. G., Nandam, S., O'Brien, J. M., Kanneganti, A., Cogan, S. F., Freeman, D. K., & Romero-Ortega, M. I. (2019). Miniature electroparticle-cuff for wireless peripheral neuromodulation. Journal of Neural Engineering, 16(Issue 4). doi:10.1088/1741-2552/ab1c36
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  Objective. Recent developments in peripheral nerve electrodes allow the efficient and selective neuromodulation of somatic and autonomic nerves, which has proven beneficial in specific bioelectronic medical applications. However, current most clinical devices are wired and powered by implantable batteries which suffer from several limitations. We recently developed a sub-millimeter inductively powered neural stimulator (electroparticle; EP), and in this study, we report the integration of the EP onto commercial cuff electrodes (EP-C) allowing the wireless activation of peripheral nerves. Approach. The current output of this device was defined at different magnetic field strenghts, and with respect to external antenna distance and activation angles. In acute in vivo testing, stimulation of the rat sciatic nerve (ScN) with the EP-C was able to evoke motor responses quantified by 3D tracking of the hind limb movement. Motor recruitment curves were obtained in response to variations in magnetic field strength (0-92.91 A m-1), stimulation frequencies (2-7 Hz), and pulse widths (50-200 μs). Main results. The results show constant output voltage throughout 50 400 stimulating cycles on a benchtop setting, and successful ScN motor activation with a 4 cm distance between external antenna and receiver. We achieved optimal motor recruitment indicated by maximizing range of hindlimb movement (6.01 ± 2.92 mm) with a magnetic field of 40.02 ± 2.85 A m-1 and 150 μs pulse width. Stimulating pulse width or frequency did not significantly influence motor recruitment. Significance. We confirmed that continuous stimulation for 14 min using monophasic pulses did not deleteriously affect the evoked motor responses when compared to wired charge-balanced biphasic electrical stimulation. We observed, however, a 36%-44% decrease in the evoked limb movement in both groups over time due to muscle fatigue. This study shows that the EP-C device can be used effectively for peripheral nerve neuromodulation.
• Meyers, E. C., Kasliwal, N., Solorzano, B. R., Lai, E., Bendale, G., Berry, A., Ganzer, P. D., Romero-Ortega, M., Rennaker, R. L., Kilgard, M. P., & Hays, S. A. (2019). Enhancing plasticity in central networks improves motor and sensory recovery after nerve damage. Nature Communications, 10(Issue 1). doi:10.1038/s41467-019-13695-0
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  Nerve damage can cause chronic, debilitating problems including loss of motor control and paresthesia, and generates maladaptive neuroplasticity as central networks attempt to compensate for the loss of peripheral connectivity. However, it remains unclear if this is a critical feature responsible for the expression of symptoms. Here, we use brief bursts of closed-loop vagus nerve stimulation (CL-VNS) delivered during rehabilitation to reverse the aberrant central plasticity resulting from forelimb nerve transection. CL-VNS therapy drives extensive synaptic reorganization in central networks paralleled by improved sensorimotor recovery without any observable changes in the nerve or muscle. Depleting cortical acetylcholine blocks the plasticity-enhancing effects of CL-VNS and consequently eliminates recovery, indicating a critical role for brain circuits in recovery. These findings demonstrate that manipulations to enhance central plasticity can improve sensorimotor recovery and define CL-VNS as a readily translatable therapy to restore function after nerve damage.
• Stiller, A. M., González-González, M. A., Boothby, J. M., Sherman, S. E., Benavides, J., Romero-Ortega, M., Pancrazio, J. J., & Black, B. J. (2019). Mechanical considerations for design and implementation of peripheral intraneural devices. Journal of Neural Engineering, 16(Issue 6). doi:10.1088/1741-2552/ab4114
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  Objective. Neural interfaces designed to stimulate or record electrical activity from peripheral nerves have applications ranging from the electrical modulation of nerve activity as a therapeutic option (e.g. epilepsy and depression) to the design of prosthetics. Currently, most peripheral nerve interfaces are either cuff-style devices that wrap around the target nerve or intraneural devices that are implanted within the nerve. While the latter option offers higher specificity and signal-to-noise ratio, penetrating devices can cause significant damage to the nerve due to the high degree of mechanical mismatch. Because of this, there is interest in developing penetrating devices fabricated from soft or softening materials (materials having a low elastic modulus). However, there is currently a lack of understanding regarding implantation forces required for successful insertion, which is a constraint for soft device design. Softer devices require robust designs to achieve a critical buckling force that is larger than forces experienced during device insertion. Approach. This study comprehensively assesses insertion force under different implantation conditions, with three variations for implantation speed, angle, and device tip angle, during insertion of silicon shanks in rat sciatic nerve. Additionally, we report compression moduli for rat sciatic nerve at different compression rates to inform computational modeling. Main results. We found that insertion speed and angle had significant effects on peak insertion force. We observed lower insertion forces (10-60 mN) when the device was implanted at higher angles relative to perpendicular insertion (80-125 mN). We also demonstrate the use of a nerve-stabilizing device to keep the nerve immobile during implantation. Additionally, we found that compression moduli were significantly different in small and large strain regions of the stress-strain curve with values between 1500-4500 Pa depending on compression rate. Significance. This study provides information imperative to the design and successful implementation of soft penetrating peripheral nerve interfaces.
• Wang, K., Frewin, C. L., Esrafilzadeh, D., Yu, C., Wang, C., Pancrazio, J. J., Romero-Ortega, M., Jalili, R., & Wallace, G. (2019). High-Performance Graphene-Fiber-Based Neural Recording Microelectrodes. Advanced Materials, 31(Issue 15). doi:10.1002/adma.201805867
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  Fabrication of flexible and free-standing graphene-fiber- (GF-) based microelectrode arrays with a thin platinum coating, acting as a current collector, results in a structure with low impedance, high surface area, and excellent electrochemical properties. This modification results in a strong synergistic effect between these two constituents leading to a robust and superior hybrid material with better performance than either graphene electrodes or Pt electrodes. The low impedance and porous structure of the GF results in an unrivalled charge injection capacity of 10.34 mC cm −2 with the ability to record and detect neuronal activity. Furthermore, the thin Pt layer transfers the collected signals along the microelectrode efficiently. In vivo studies show that microelectrodes implanted in the rat cerebral cortex can detect neuronal activity with remarkably high signal-to-noise ratio (SNR) of 9.2 dB in an area as small as an individual neuron.
• Alsmadi, N. Z., Bendale, G. S., Kanneganti, A., Shihabeddin, T., Nguyen, A. H., Hor, E., Dash, S., Johnston, B., Granja-Vazquez, R., & Romero-Ortega, M. I. (2018). Glial-derived growth factor and pleiotrophin synergistically promote axonal regeneration in critical nerve injuries. Acta Biomaterialia, 78(Issue). doi:10.1016/j.actbio.2018.07.048
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  The repair of nerve gap injuries longer than 3 cm is limited by the need to sacrifice donor tissue and the morbidity associated with the autograft gold standard, while decellularized grafts and biodegradable conduits are effective only in short nerve defects. The advantage of isogenic nerve implants seems to be the release of various growth factors by the denervated Schwann cells. We evaluated the effect of vascular endothelial growth factor, neurotrophins, and pleiotrophin (PTN) supplementation of multi-luminal conduits, in the repair of 3 and 4 cm nerve gaps in the rabbit peroneal nerve. In vitro screening revealed a synergistic regenerative effect of PTN with glial-derived neurotrophic factor (GDNF) in promoting sensory axon density, and motor axonal growth from spinal cord explants. In vivo, pleiotrophins were able to support nerve regrowth across a 3 cm gap. In the 4 cm lesions, PTN-GDNF had a modest effect in the number of axons distal to the implant, while increasing the mean axon diameter (1 ± 0.4; p ≤ 0.001) over PTN or GDNF alone (0.80 ± 0.2, 0.84 ± 0.5; respectively). Some regenerated axons reinnervated muscle targets as indicated by neuromuscular junction staining. However, many were wrapped in Remak bundles, suggesting a delay in axonal sorting, explaining the limited electrophysiological function of the reinnervated muscle, and the modest recovery in toe spreading in the PTN-GDNF repaired animals. These results support the use of synergistic neurotrophic/pleiotrophic growth factors in long gap repair and underscore the need for re-myelination strategies distal to the injury site. Statement of Significance: Nerve injuries due to trauma or tumor resection often result in long gaps that are challenging to repair. The best clinical option demands the use of autologous grafts that are associated with serious side effects. Bioengineered nerves are considered a good alternative, particularly if supplemented with growth factors, but current options do not match the regenerative capacity of autografts. This study revealed the synergistic effect of neurotrophins and pleiotrophins designed to achieve a broad cellular regenerative effect, and that GDNF-PTN are able to mediated axonal growth and partial functional recovery in a 4 cm nerve gap injury, albeit delays in remyelination. This report underscores the need for defining an optimal growth factor support for biosynthetic nerve implants.
• Black, B. J., Atmaramani, R., Kumaraju, R., Plagens, S., Romero-Ortega, M., Dussor, G., Price, T. J., Campbell, Z. T., & Pancrazio, J. J. (2018). Adult mouse sensory neurons on microelectrode arrays exhibit increased spontaneous and stimulus-evoked activity in the presence of interleukin-6. Journal of Neurophysiology, 120(Issue 3). doi:10.1152/jn.00158.2018
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  Following inflammation or in-jury, sensory neurons located in the dorsal root ganglia (DRG) may exhibit increased spontaneous and/or stimulus-evoked activity, contributing to chronic pain. Current treatment options for peripherally mediated chronic pain are highly limited, driving the development of cell-or tissue-based phenotypic (function-based) screening assays for peripheral analgesic and mechanistic lead discovery. Extant assays are often limited by throughput, content, use of tumorigenic cell lines, or tissue sources from immature developmental stages (i.e., embryonic or postnatal). Here, we describe a protocol for culturing adult mouse DRG neurons on substrate-integrated multiwell microelectrode arrays (MEAs). This approach enables multiplexed measurements of spontaneous as well as stimulus-evoked extracellular action potentials from large populations of cells. The DRG cultures exhibit stable spontaneous activity from 9 to 21 days in vitro. Activity is readily evoked by known chemical and physical agonists of sensory neuron activity such as capsaicin, bradykinin, PGE2, heat, and electrical field stimulation. Most importantly, we demonstrate that both spontaneous and stimulus-evoked activity may be potentiated by incubation with the inflammatory cytokine interleukin-6 (IL-6). Acute responsiveness to IL-6 is inhibited by treatment with a MAPK-interacting kinase 1/2 inhibitor, cercosporamide. In total, these findings suggest that adult mouse DRG neurons on multiwell MEAs are applicable to ongoing efforts to discover peripheral analgesic and their mechanisms of action. NEW & NOTEWORTHY This work describes methodologies for culturing spontaneously active adult mouse dorsal root ganglia (DRG) sensory neurons on microelectrode arrays. We characterize spontaneous and stimulus-evoked adult DRG activity over durations consistent with pharmacological interventions. Furthermore, persistent hyperexcitability could be induced by incubation with inflammatory cytokine IL-6 and attenuated with cercosporamide, an inhibitor of the IL-6 sensitization pathway. This constitutes a more physiologically relevant, moderate-throughput in vitro model for peripheral analgesic screening as well as mechanistic lead discovery.
• Castelán, F., López-García, K., Moreno-Pérez, S., Zempoalteca, R., Corona-Quintanilla, D. L., Romero-Ortega, M. I., Jiménez-Estrada, I., & Martínez-Gómez, M. (2018). Multiparity affects conduction properties of pelvic floor nerves in rabbits. Brain and Behavior, 8(Issue 10). doi:10.1002/brb3.1105
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  Introduction: Women often develop pelvic floor dysfunction due to damage to the pelvic musculature during childbirth; however, the effect on pelvic floor nerves function is less understood. This study used adult rabbits to evaluate the electrophysiological and histological characteristics of the bulbospongiosus (Bsn) and pubococcygeus nerves (Pcn) in multiparity. Methods: Compound nerve action potentials (CNAP) were compared between age-matched nulliparous and multiparous animals and associated to the histological characteristics of myelinated axons from the Bsn and Pcn nerves. The extensor digitorum longus nerve (EDLn) was used as negative control. Data were analyzed with unpaired two-tailed Student's t test or Mann–Whitney U test to determine significant differences between groups. Results: The onset and peak latencies, duration, and conduction velocity of the motor fibers in these pelvic nerves were not significantly different between nulliparous and multiparous animals. However, the peak-to-peak amplitude and area of the CNAP in both Bsn and Pcn were reduced in multiparous rabbits. Histology showed a higher percentage of axons with myelin disorganization caused by multiparity in these pelvic nerves. Together, the data indicate a reduction in the number of functional pelvic axons due to multiparity. As expected, no effect of parity was observed in the EDLn controls. Conclusions: Present findings demonstrated that multiparity affects myelination and consequently conduction properties in the small pelvic floor nerves.
• González-González, M. A., Kanneganti, A., Joshi‐Imre, A., Hernández-Reynoso, A. G., Bendale, G., Modi, R., Ecker, M., Khurram, S. A., Cogan, S. F., Voit, W., & Romero-Ortega, M. I. (2018). Thin Film Multi-Electrode Softening Cuffs for Selective Neuromodulation. Scientific Reports, 8(1). doi:10.1038/s41598-018-34566-6
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  Abstract Silicone nerve cuff electrodes are commonly implanted on relatively large and accessible somatic nerves as peripheral neural interfaces. While these cuff electrodes are soft (1–50 MPa), their self-closing mechanism requires of thick walls (200–600 µm), which in turn contribute to fibrotic tissue growth around and inside the device, compromising the neural interface. We report the use of thiol-ene/acrylate shape memory polymer (SMP) for the fabrication of thin film multi-electrode softening cuffs (MSC). We fabricated multi-size MSC with eight titanium nitride (TiN) electrodes ranging from 1.35 to 13.95 × 10 −4 cm 2 (1–3 kΩ) and eight smaller gold (Au) electrodes (3.3 × 10 −5 cm 2 ; 750 kΩ), that soften at physiological conditions to a modulus of 550 MPa. While the SMP material is not as soft as silicone, the flexural forces of the SMP cuff are about 70–700 times lower in the MSC devices due to the 30 μm thick film compared to the 600 μm thick walls of the silicone cuffs. We demonstrated the efficacy of the MSC to record neural signals from rat sciatic and pelvic nerves (1000 µm and 200 µm diameter, respectively), and the selective fascicular stimulation by current steering. When implanted side-by-side and histologically compared 30 days thereafter, the MSC devices showed significantly less inflammation, indicated by a 70–80% reduction in ED1 positive macrophages, and 54–56% less fibrotic vimentin immunoreactivity. Together, the data supports the use of MSC as compliant and adaptable technology for the interfacing of somatic and autonomic peripheral nerves.
• Shimizu, E. N., Seifert, J. L., Johnson, K. J., & Romero-Ortega, M. I. (2018). Prophylactic Riluzole Attenuates Oxidative Stress Damage in Spinal Cord Distraction. Journal of Neurotrauma, 35(Issue 12). doi:10.1089/neu.2017.5494
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  Spinal cord injury (SCI) without radiographical abnormalities (SCIWORA) presents a significant challenge because of the loss of function despite an apparent normal anatomy. The cause of dysfunction is not understood, and specific treatment options are lacking. Some scoliosis corrective surgeries result in SCIWORA, where stretching of the spinal cord can lead to vascular compromise and hypoxia. The iatrogenic nature of this injury allows for the implantation of neuroprotective strategies that are designed to prevent damage. We utilized a model of atraumatic SCI to evaluate the efficacy of the sodium-channel blocker, riluzole, as a prophylactic neuroprotectant. As expected, the stretch injury caused a significant reduction in intraparenchymal oxygen in distraction (-53.09 ± 22.23%) and riluzole pre-treated distraction animals (-43.04 ± 22.86%). However, in contrast to the oxidative stress and metabolic impairments observed in vehicle-treated distraction animals, in which protein carbonylation increased significantly (5.88 ± 1.3 nmol/mL), riluzole kept these levels within the normal range (1.8 ± 1.0 nmol/mL). This neurprotection also prevented ventral motor neuron hypoplasia and pyknosis, characteristic features of this atraumatic SCI model, and maintained normal gait function (e.g., stride length and stance time). This study provides evidence for the use of prophylactic neuroprotective strategies in which thoracic or spine surgeries present the risk of causing atraumatic SCI.
• Anand, S., Desai, V., Alsmadi, N., Kanneganti, A., Nguyen, D. H., Tran, M., Patil, L., Vasudevan, S., Xu, C., Hong, Y., Cheng, J., Keefer, E., & Romero-Ortega, M. I. (2017). Asymmetric Sensory-Motor Regeneration of Transected Peripheral Nerves Using Molecular Guidance Cues. Scientific Reports, 7(Issue 1). doi:10.1038/s41598-017-14331-x
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  Neural interfaces are designed to decode motor intent and evoke sensory precepts in amputees. In peripheral nerves, recording movement intent is challenging because motor axons are only a small fraction compared to sensory fibers and are heterogeneously mixed particularly at proximal levels. We previously reported that pain and myelinated axons regenerating through a Y-shaped nerve guide with sealed ends, can be modulated by luminar release of nerve growth factor (NGF) and neurotrophin-3 (NT-3), respectively. Here, we evaluate the differential potency of NGF, glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), pleiotrophin (PTN), and NT-3 in asymmetrically guiding the regeneration of sensory and motor neurons. We report that, in the absence of distal target organs, molecular guidance cues can mediate the growth of electrically conductive fascicles with normal microanatomy. Compared to Y-tube compartments with bovine serum albumin (BSA), GDNF and NGF increased the motor and sensory axon content, respectively. In addition, the sensory to motor ratio was significantly increased by PTN (12.7:1) when compared to a BDNF + GDNF choice. The differential content of motor and sensory axons modulated by selective guidance cues may provide a strategy to better define axon types in peripheral nerve interfaces.
• Bell, J. E., Seifert, J. L., Shimizu, E. N., Sucato, D. J., & Romero-Ortega, M. I. (2017). Atraumatic Spine Distraction Induces Metabolic Distress in Spinal Motor Neurons. Journal of Neurotrauma, 34(Issue 12). doi:10.1089/neu.2016.4779
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  Corrective forces during spine deformity surgery, including distraction, impart significant stresses to the spinal cord that may result in permanent injury. Intraoperative neuromonitoring is commonly used by surgeons to recognize possible damage to the spinal cord in cases of evident traumatic or vascular damage to the spinal cord. However, mild insult to the spinal cord that does not result in obvious trauma or electrophysiological changes present a major clinical challenge as the mechanisms of this type of spinal cord injury (SCI) remain largely unknown, and thus preventive strategies are lacking. We used a sustained bidirectional spinal distraction animal model to determine the role of stretch-induced hypoxia in mild SCI. Direct measurement of intraparenchymal oxygen revealed an immediate decrease in partial pressure (47.08 ± 5.79% pO2) distal to the injury site following a 5-mm distraction. This hypoxic insult induced mitochondrial dysfunction as evidenced by an acute increase (216%) in protein oxidation 30 min post-injury, as well as a 37% decrease in perikaryal size and a 42% decrease in nuclear area (pyknosis) in ventral motor neurons at the injury site. These results indicate that hypoxic events during mild spine distraction may lead to cellular metabolic impairments and permanent functional deficits. The development of strategies targeting the prevention of hypoxic injury during spine distraction may be useful in protecting the cellular metabolic damage that may occur during spine surgery in the absence of overt mechanical or vascular SCI.
• Freeman, D. K., O'Brien, J. M., Kumar, P., Daniels, B., Irion, R. A., Shraytah, L., Ingersoll, B. K., Magyar, A. P., Czarnecki, A., Wheeler, J., Coppeta, J. R., Abban, M. P., Gatzke, R., Fried, S. I., Lee, S. W., Duwel, A. E., Bernstein, J. J., Widge, A. S., Hernandez-Reynoso, A., , Kanneganti, A., et al. (2017). A sub-millimeter, inductively powered neural stimulator. Frontiers in Neuroscience, 11(Issue). doi:10.3389/fnins.2017.00659
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  Wireless neural stimulators are being developed to address problems associated with traditional lead-based implants. However, designing wireless stimulators on the sub-millimeter scale (< 1 mm3) is challenging. As device size shrinks, it becomes difficult to deliver sufficient wireless power to operate the device. Here, we present a sub-millimeter, inductively powered neural stimulator consisting only of a coil to receive power, a capacitor to tune the resonant frequency of the receiver, and a diode to rectify the radio-frequency signal to produce neural excitation. By replacing any complex receiver circuitry with a simple rectifier, we have reduced the required voltage levels that are needed to operate the device from 0.5 to 1 V (e.g., for CMOS) to ~0.25-0.5 V. This reduced voltage allows the use of smaller receive antennas for power, resulting in a device volume of 0.3-0.5 mm3. The device was encapsulated in epoxy, and successfully passed accelerated lifetime tests in 80°C saline for 2 weeks. We demonstrate a basic proof-of-concept using stimulation with tens of microamps of current delivered to the sciatic nerve in rat to produce a motor response.
• Meyers, E. C., Granja, R., Solorzano, B. R., Romero-Ortega, M., Kilgard, M. P., Rennaker, R. L., & Hays, S. (2017). Median and ulnar nerve injuries reduce volitional forelimb strength in rats. Muscle and Nerve, 56(Issue 6). doi:10.1002/mus.25590
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  Introduction: Peripheral nerve injuries (PNI) are among the leading causes of physical disability in the United States. The majority of injuries occur in the upper extremities, and functional recovery is often limited. Robust animal models are critical first steps for developing effective therapies to restore function after PNI. Methods: We developed an automated behavioral assay that provides quantitative measurements of volitional forelimb strength in rats. Multiple forelimb PNI models involving the median and ulnar nerves were used to assess forelimb function for up to 13 weeks postinjury. Results: Despite multiple weeks of task-oriented training following injury, rats exhibit significant reductions in multiple quantitative parameters of forelimb function, including maximal pull force and speed of force generation. Discussion: This study demonstrates that the isometric pull task is an effective method of evaluating forelimb function following PNI and may aid in development of therapeutic interventions to restore function. Muscle Nerve 56: 1149–1154, 2017.
• Black, B., Granja-Vazquez, R., Johnston, B. R., Jones, E., & Romero-Ortega, M. (2016). Anthropogenic radio-frequency electromagnetic fields elicit neuropathic pain in an amputation model. PLoS ONE, 11(Issue 1). doi:10.1371/journal.pone.0144268
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  Anecdotal and clinical reports have suggested that radio-frequency electromagnetic fields (RF EMFs) may serve as a trigger for neuropathic pain. However, these reports have been widely disregarded, as the epidemiological effects of electromagnetic fields have not been systematically proven, and are highly controversial. Here, we demonstrate that anthropogenic RF EMFs elicit post-neurotomy pain in a tibial neuroma transposition model. Behavioral assays indicate a persistent and significant pain response to RF EMFs when compared to SHAM surgery groups. Laser thermometry revealed a transient skin temperature increase during stimulation. Furthermore, immunofluorescence revealed an increased expression of temperature sensitive cation channels (TRPV4) in the neuroma bulb, suggesting that RF EMF-induced pain may be due to cytokine-mediated channel dysregulation and hypersensitization, leading to thermal allodynia. Additional behavioral assays were performed using an infrared heating lamp in place of the RF stimulus. While thermally-induced pain responses were observed, the response frequency and progression did not recapitulate the RF EMF effects. In vitro calcium imaging experiments demonstrated that our RF EMF stimulus is sufficient to directly contribute to the depolarization of dissociated sensory neurons. Furthermore, the perfusion of inflammatory cytokine TNF-α resulted in a significantly higher percentage of active sensory neurons during RF EMF stimulation. These results substantiate patient reports of RF EMF-pain, in the case of peripheral nerve injury, while confirming the public and scientific consensus that anthropogenic RF EMFs engender no adverse sensory effects in the general population.
• Farajidavar, A., Seifert, J. L., Delgado, M. R., Sparagana, S., Romero-Ortega, M. I., & Chiao, J. C. (2016). Electromagnetic interference in intraoperative monitoring of motor evoked potentials and a wireless solution. Medical Engineering and Physics, 38(Issue 2). doi:10.1016/j.medengphy.2015.11.007
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  Intraoperative neurophysiological monitoring (IONM) is utilized to minimize neurological morbidity during spine surgery. Transcranial motor evoked potentials (TcMEPs) are principal IONM signals in which the motor cortex of the subject is stimulated with electrical pulses and the evoked potentials are recorded from the muscles of interest. Currently available monitoring systems require the connection of 40-60 lengthy lead wires to the patient. These wires contribute to a crowded and cluttered surgical environment, and limit the maneuverability of the surgical team. In this work, it was demonstrated that the cumbersome wired system is vulnerable to electromagnetic interference (EMI) produced by operating room (OR) equipment. It was hypothesized that eliminating the lengthy recording wires can remove the EMI induced in the IONM signals. Hence, a wireless system to acquire TcMEPs was developed and validated through bench-top and animal experiments. Side-by-side TcMEPs acquisition from the wired and wireless systems in animal experiments under controlled conditions (absence of EMI from OR equipment) showed comparable magnitudes and waveforms, thus demonstrating the fidelity in the signal acquisition of the wireless solution. The robustness of the wireless system to minimize EMI was compared with a wired-system under identical conditions. Unlike the wired-system, the wireless system was not influenced by the electromagnetic waves from the C-Arm X-ray machine and temperature management system in the OR.
• Lozano, R., Gilmore, K. J., Thompson, B. C., Stewart, E. M., Waters, A. M., Romero-Ortega, M., & Wallace, G. G. (2016). Electrical stimulation enhances the acetylcholine receptors available for neuromuscular junction formation. Acta Biomaterialia, 45(Issue). doi:10.1016/j.actbio.2016.08.006
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  Neuromuscular junctions (NMJ) are specialized synapses that link motor neurons with muscle fibers. These sites are fundamental to human muscle activity, controlling swallowing and breathing amongst many other vital functions. Study of this synapse formation is an essential area in neuroscience; the understanding of how neurons interact and control their targets during development and regeneration are fundamental questions. Existing data reveals that during initial stages of development neurons target and form synapses driven by biophysical and biochemical cues, and during later stages they require electrical activity to develop their functional interactions. The aim of this study was to investigate the effect of exogenous electrical stimulation (ES) electrodes directly in contact with cells, on the number and size of acetylcholine receptor (AChR) clusters available for NMJ formation. We used a novel in vitro model that utilizes a flexible electrical stimulation system and allows the systematic testing of several stimulation parameters simultaneously as well as the use of alternative electrode materials such as conductive polymers to deliver the stimulation. Functionality of NMJs under our co-culture conditions was demonstrated by monitoring changes in the responses of primary myoblasts to chemical stimulants that specifically target neuronal signaling. Our results suggest that biphasic electrical stimulation at 250 Hz, 100 μs pulse width and current density of 1 mA/cm2 for 8 h, applied via either gold-coated mylar or the conductive polymer PPy, significantly increased the number and size of AChRs clusters available for NMJ formation. This study supports the beneficial use of direct electrical stimulation as a strategic therapy for neuromuscular disorders. Statement of Significance The beneficial effects of electrical stimulation (ES) on human cells in vitro and in vivo have long been known. Although the effects of stimulation are clear and the therapeutic benefits are known, no uniform parameters exist with regard to the duration, frequency and amplitude of the ES. To this end, we are answering several important questions on the parameters for ES of nerve and muscle monocultures and co-cultures by probing the effects on the enhancement of acetylcholine receptors (AChR) clustering available for neuromuscular junction formation using a conductive platform. This work opens the possibility to combine electrical stimulus delivered via conductive polymer substrates, from which biomolecules could also be delivered, providing opportunities to further enhance the therapeutic effect.
• Lozano, R., Stevens, L., Thompson, B. C., Gilmore, K. J., Gorkin, R., Stewart, E. M., Panhuis, M. I., Romero-Ortega, M., & Wallace, G. G. (2016). Brain on a bench top: Cortical neurons within a 3D printed structure. Materials Today, 19(Issue 2). doi:10.1016/j.mattod.2016.01.018
• Alsmadi, N. Z., Patil, L. S., Hor, E. M., Lofti, P., Razal, J. M., Chuong, C. J., Wallace, G. G., & Romero-Ortega, M. I. (2015). Coiled polymeric growth factor gradients for multi-luminal neural chemotaxis. Brain Research, 1619(Issue). doi:10.1016/j.brainres.2015.01.055
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  In the injured adult nervous system, re-establishment of growth-promoting molecular gradients is known to entice and guide nerve repair. However, incorporation of three-dimensional chemotactic gradients in nerve repair scaffolds, particularly in those with multi-luminal architectures, remains extremely challenging. We developed a method that establishes highly tunable three-dimensional molecular gradients in multi-luminal nerve guides by anchoring growth-factor releasing coiled polymeric fibers onto the walls of collagen-filled hydrogel microchannels. Differential pitch in the coiling of neurotrophin-eluting fibers generated sustained chemotactic gradients that appropriately induced the differentiation of Pheochromocytoma (PC12) cells into neural-like cells along an increasing concentration of nerve growth factor (NGF). Computer modeling estimated the stability of the molecular gradient within the luminal collagen, which we confirmed by observing the significant effects of neurotrophin gradients on axonal growth from dorsal root ganglia (DRG). Neurons growing in microchannels exposed to a NGF gradient showed a 60% increase in axonal length compared to those treated with a linear growth factor concentration. In addition, a two-fold increment in the linearity of axonal growth within the microchannels was observed and confirmed by a significant reduction in the turning angle ratios of individual axons. These data demonstrate the ability of growth factor-loaded polymeric coiled fibers to establish three-dimensional chemotactic gradients to promote and direct nerve regeneration in the nervous system and provides a unique platform for molecularly guided tissue repair.
• Gu, L., Uhelski, M. L., Anand, S., Romero-Ortega, M., Kim, Y. T., Fuchs, P. N., & Mohanty, S. K. (2015). Pain inhibition by optogenetic activation of specific anterior cingulate cortical neurons. PLoS ONE, 10(Issue 2). doi:10.1371/journal.pone.0117746
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  Cumulative evidence from both humans and animals suggests that the anterior cingulate cortex (ACC) is important for pain-related perception, and thus a likely target for pain relief therapy. However, use of existing electrode based ACC stimulation has not significantly reduced pain, at least in part due to the lack of specificity and likely co-activation of both excitatory and inhibitory neurons. Herein, we report a dramatic reduction of pain behavior in transgenic mice by optogenetic stimulation of the inhibitory neural circuitry of the ACC expressing channelrhodopsin-2. Electrophysiological measurements confirmed that stimulation of ACC inhibitory neurons is associated with decreased neural activity in the ACC. Further, a distinct optogenetic stimulation intensity and frequency-dependent inhibition of spiking activity in the ACC was observed. Moreover, we confirmed specific electrophysiological responses from different neuronal units in the thalamus, in response to particular types of painful stimuli (i,e., formalin injection, pinch), which we found to be modulated by optogenetic control of the ACC inhibitory neurons. These results underscore the inhibition of the ACC as a clinical alternative in inhibiting chronic pain, and leads to a better understanding of the pain processing circuitry of the cingulate cortex.
• Kim, Y. t., Kanneganti, A., Nothnagle, C., Landrith, R., Mizuno, M., Wijesundara, M. B., Smith, S., & Romero-Ortega, M. I. (2015). Microchannel Electrode Stimulation of Deep Peroneal Nerve Fascicles Induced Mean Arterial Depressor Response in Hypertensive Rats. Bioelectronic Medicine, 2(Issue 1). doi:10.15424/bioelectronmed.2015.00001
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  Hypertension (HTN) affects over 1 billion people in the world, and while most are treated effectively with pharmacological regimens, 10–30% of them do not show a beneficial response. Electrical stimulation of the renal sympathetic, vagus and carotid sinus nerves has shown depressor effects and has been proposed as an alternative treatment for resistant hypertension (R-HTN). However, these nerves are heterogeneous in afferent/efferent composition, and their stimulation often results in unwanted side effects. We evaluated the possibility of eliciting a depressor response from stimulation of single fascicle of the somatic deep peroneal nerve (fDPN). A microchannel electrode array (µCEA) was used to stimulate the fDPN at low frequency, which induced a significant (p ≤ 0.03) transient reduction in mean arterial pressure (MAP) with no significant effects on heart rate. The depressor response was prolonged for several hours by extending the fDPN stimulation to 5 min, which induced significant reduction (17–25%) in MAP for up to 4 h. Immunofluorescence evaluation of the axonal marker, myelin, and active macrophages in the fDPN revealed no indication of nerve damage or overt inflammation in response to the procedure. This study provides evidence supporting the use of µCEA interfacing of small somatic nerve fascicles associated with cardiovascular relevant acupoints to induce significant reductions in MAP and opens the possibility of neuromodulation of small fascicles as an alternative strategy to treat R-HTN with minimal side effects. Further, the µCEA multielectrode array offers an effective tool for neuromodulation of small nerve fascicles, enabling a number of possible future medical bioelectronic applications.
• Logan, S., Romero, M., Nguyen, D., & Benson, M. (2015). Corrigendum to "Ephrin-B2 expression in the proprioceptive sensory system" [Neurosci. Lett. 545 (2013) 69-74]. Neuroscience Letters, 587(Issue). doi:10.1016/j.neulet.2014.12.032
• Lozano, R., Stevens, L., Thompson, B. C., Gilmore, K. J., Gorkin, R., Stewart, E. M., Panhuis, M. i., Romero-Ortega, M. I., & Wallace, G. G. (2015). 3D printing of layered brain-like structures using peptide modified gellan gum substrates. Biomaterials, 67. doi:10.1016/j.biomaterials.2015.07.022
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  The brain is an enormously complex organ structured into various regions of layered tissue. Researchers have attempted to study the brain by modeling the architecture using two dimensional (2D) in vitro cell culturing methods. While those platforms attempt to mimic the in vivo environment, they do not truly resemble the three dimensional (3D) microstructure of neuronal tissues. Development of an accurate in vitro model of the brain remains a significant obstacle to our understanding of the functioning of the brain at the tissue or organ level. To address these obstacles, we demonstrate a new method to bioprint 3D brain-like structures consisting of discrete layers of primary neural cells encapsulated in hydrogels. Brain-like structures were constructed using a bio-ink consisting of a novel peptide-modified biopolymer, gellan gum-RGD (RGD-GG), combined with primary cortical neurons. The ink was optimized for a modified reactive printing process and developed for use in traditional cell culturing facilities without the need for extensive bioprinting equipment. Furthermore the peptide modification of the gellan gum hydrogel was found to have a profound positive effect on primary cell proliferation and network formation. The neural cell viability combined with the support of neural network formation demonstrated the cell supportive nature of the matrix. The facile ability to form discrete cell-containing layers validates the application of this novel printing technique to form complex, layered and viable 3D cell structures. These brain-like structures offer the opportunity to reproduce more accurate 3D in vitro microstructures with applications ranging from cell behavior studies to improving our understanding of brain injuries and neurodegenerative diseases.
• Billo, R. E., Wilson, P. A., Priest, J. W., Romero-Ortega, M., Brunskill, S. R., & Keens, D. (2014). Slump molding of microchannel arrays in soda-lime glass for bioanalytical device development. Journal of Micro and Nano-Manufacturing, 2(Issue 4). doi:10.1115/1.4028487
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  A slump molding process was developed to place microchannel geometries in a sodalime glass substrate for a lab-on-chip bioanalytical device. The process was developed to overcome the biological and chemical reactivity associated with current polymer lab-ona- chip substrates, and as an alternative to using more expensive glass material. A high speed micro mill and UV laser micromachining center were used to fabricate the negative geometries in the graphite mold material that was used. The slumping process of the soda-lime glass was done using a glass kiln. Microchannel dimensions were in the mesa scale range of 50 μm width × 10 μm depth. The heating schedule for slump molding of the soda-lime glass to take its final shape to these dimensions was determined and documented. The functionality of the slumping process and resultant soda-lime glass device was validated through murine nerve tissue experiments conducted through the bioanalytical device that was developed. The research represented a novel use of slump molding, a process traditionally known for producing artistic works for: (a) embossing engineered microchannels and (b) reliably processing a soda-lime glass substrate, a material known to be difficult to work with due to its poor physical properties.
• Jia, X., Romero-Ortega, M. I., & Teng, Y. D. (2014). Peripheral nerve regeneration: mechanism, cell biology, and therapies. BioMed Research International, 2014(Issue). doi:10.1155/2014/145304
• Bouché, E., Romero-Ortega, M. I., Henkemeyer, M., Catchpole, T., Leemhuis, J., Frotscher, M., May, P., Herz, J., & Bock, H. H. (2013). Reelin induces EphB activation. Cell Research, 23(Issue 4). doi:10.1038/cr.2013.7
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  The integration of newborn neurons into functional neuronal networks requires migration of cells to their final position in the developing brain, the growth and arborization of neuronal processes and the formation of synaptic contacts with other neurons. A central player among the signals that coordinate this complex sequence of differentiation events is the secreted glycoprotein Reelin, which also modulates synaptic plasticity, learning and memory formation in the adult brain. Binding of Reelin to ApoER2 and VLDL receptor, two members of the LDL receptor family, initiates a signaling cascade involving tyrosine phosphorylation of the intracellular cytoplasmic adaptor protein Disabled-1, which targets the neuronal cytoskeleton and ultimately controls the positioning of neurons throughout the developing brain. However, it is possible that Reelin signals interact with other receptor-mediated signaling cascades to regulate different aspects of brain development and plasticity. EphB tyrosine kinases regulate cell adhesion and repulsion-dependent processes via bidirectional signaling through ephrin B transmembrane proteins. Here, we demonstrate that Reelin binds to the extracellular domains of EphB transmembrane proteins, inducing receptor clustering and activation of EphB forward signaling in neurons, independently of the 'classical' Reelin receptors, ApoER2 and VLDLR. Accordingly, mice lacking EphB1 and EphB2 display a positioning defect of CA3 hippocampal pyramidal neurons, similar to that in Reelin-deficient mice, and this cell migration defect depends on the kinase activity of EphB proteins. Together, our data provide biochemical and functional evidence for signal integration between Reelin and EphB forward signaling. © 2013 IBCB, SIBS, CAS All rights reserved.
• Logan, S., Romero, M., Nguyen, D., & Benson, M. (2013). Ephrin-B2 expression in the proprioceptive sensory system. Neuroscience Letters, 545(Issue). doi:10.1016/j.neulet.2013.04.025
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  Recent studies have shown that ephrin-B2 on sensory afferent fibers from the dorsal root ganglia (DRG) controls transmission of pain sensation to the spinal cord. We examined ephrin-B2 expression in mouse DRG and spinal cord using an ephrin-B2/ß-galactosidase chimeric allele. We found that ephrin-B2 is expressed exclusively in proprioceptive neurons and fibers in neonates, while expression in lamina III and IV of the adult spinal cord was observed in addition to that in the deeper laminae. We confirmed that ephrin-B2 protein causes co-clustering of EphB2 and glutamate receptors in spinal cord neurons. Our data are consistent with a role for ephrin-B2 in transmission of positional information to the CNS, and thus suggest a role in synaptic plasticity of spinal cord locomotor circuits that are known to be sensitive to proprioceptive sensory input after spinal cord injury. © 2013 The Authors.
• Kim, Y. T., & Romero-Ortega, M. I. (2012). Material considerations for peripheral nerve interfacing. MRS Bulletin, 37(Issue 6). doi:10.1557/mrs.2012.99
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  Multi-fingered prosthetic limbs are now capable of performing complex movements, closely simulating those of the lost human arm/hand in amputees. Conveying natural control and perception of such sophisticated robotic limbs requires direct interfacing with the nervous system of the users. Both the brain and transected peripheral nerves have been proposed as appropriate locations for such neural interfaces. In the peripheral nerves, several electrode designs have been used successfully to record motor signals, and through stimulation, used to convey sensation in long-term amputee human volunteers. Here we review the advantages and limitations of peripheral nerve interfaces (PNIs) in relation to the desired characteristics for safety and performance. We compare current PNI electrodes and materials designed to increase recording sensitivity and to achieve selective stimulation of different nerves such as those carrying mechanical sensation and limb position information. Emphasis is placed on novel electrode materials, including biological coatings, conductive polymers, and nanostructured modifications. Successful development of reliable PNIs will not only enable natural control and feel of future robotic prosthetics, but will likely be a centerpiece technology in the development of multiple bionic organs. © 2012 Materials Research Society.
• Seifert, J., Desai, V., Watson, R., Musa, T., Kim, Y., Keefer, E., & Romero, M. (2012). Normal molecular repair mechanisms in regenerative peripheral nerve interfaces allow recording of early spike activity despite immature myelination. IEEE Transactions on Neural Systems and Rehabilitation Engineering, 20(2). doi:10.1109/tnsre.2011.2179811
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  Clinical use of neurally controlled prosthetics has advanced in recent years, but limitations still remain, including lacking fine motor control and sensory feedback. Indwelling multi-electrode arrays, cuff electrodes, and regenerative sieve electrodes have been reported to serve as peripheral neural interfaces, though long-term stability of the nerve-electrode interface has remained a formidable challenge. We recently developed a regenerative multi-electrode interface (REMI) that is able to record neural activity as early as seven days post-implantation. While this activity might represent normal neural depolarization during axonal regrowth, it can also be the result of altered nerve regeneration around the REMI. This study evaluated high-throughput expression levels of 84 genes involved in nerve injury and repair, and the histological changes that occur in parallel to this early neural activity. Animals exhibiting spike activity increased from 29% to 57% from 7 to 14 days following REMI implantation with a corresponding increase in firing rate of 113%. Two weeks after implantation, numbers of neurofilament-positive axons in the control and REMI implanted nerves were comparable, and in both cases the number of myelinated axons was low. During this time, expression levels of genes related to nerve injury and repair were similar in regenerated nerves, both in the presence or absence of the electrode array. Together, these results indicate that the early neural activity is intrinsic to the regenerating axons, and not induced by the REMI neurointerface. © 2011 IEEE.
• Dawood, A., Lotfi, P., Dash, S., Kona, S., Nguyen, K. T., & Romero-Ortega, M. I. (2011). VEGF Release in Multiluminal Hydrogels Directs Angiogenesis from Adult Vasculature In Vitro. Cardiovascular Engineering and Technology, 2(3). doi:10.1007/s13239-011-0048-4
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  Large bioengineered organs such as the heart and kidney need immediate perfusion by the host vascular network to avoid ischemia, support implant survival, and prevent implant failure. Vascularization of new bioengineered tissues can be stimulated by porous scaffold design and vascular growth factors. However, comprehensive vascularization of thick tissues in vitro remains a formidable challenge. We developed a simple and reproducible vascularization method that integrates a transparent biodegradable multiluminal scaffold for guided endothelial migration stimulated by intraluminal controlled release of Vascular Endothelial Growth Factor (VEGF). Two- and three-dimensional in vitro vasculogenesis induced by human umbilical vein endothelial cells (HUVEC)/fibroblast co-cultures formed discontinuous vessel-like structures. In sharp contrast, hydrogel microchannels with luminal collagen matrix, enticed the angiogenic growth from postnatal and adult aortic explants into the agarose microchannels, forming continuous vascular tubes identified by CD31 expression, in which the number of cells can be controlled by intraluminal VEGF concentration. This study suggests that multiluminal scaffolds can be used to promote angiogenesis from mature blood vessels and form vascular networks within thick bioengineered scaffolds. This method might offer a viable alternative in the prevascularization of bio-artificial organs. © 2011 Biomedical Engineering Society.
• Farajidavar, A., Seifert, J., Bell, J., Seo, Y., Delgado, M., Sparagana, S., Romero, M., & Chiao, J. (2011). A wireless system for monitoring transcranial motor evoked potentials. Annals of Biomedical Engineering, 39(1). doi:10.1007/s10439-010-0152-x
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  Intraoperative neurophysiological monitoring (IONM) is commonly used as an attempt to minimize neurological morbidity from operative manipulations. The goal of IONM is to identify changes in the central and peripheral nervous system function prior to irreversible damage. Intraoperative monitoring also has been effective in localizing anatomical structures, including peripheral nerves and sensorimotor cortex, which helps guide the surgeon during dissection. As part of IONM, transcranial motor evoked potentials (TcMEPs), and somatosensory evoked potentials (SSEPs) are routinely monitored. However, current wired systems are cumbersome as the wires contribute to the crowded conditions in the operating room and in doing so not only it limits the maneuverability of the surgeon and assistants, but also places certain demand in the total anesthesia required during surgery, due to setup preoperative time needed for proper electrode placement, due to the number and length of the wires, and critical identification of the lead wires needed for stimulation and recording. To address these limitations, we have developed a wireless TcMEP IONM system as a first step toward a multimodality IONM system. Bench-top and animal experiments in rodents demonstrated that the wireless method reproduced with high fidelity, and even increased the frequency bandwidth of the TcMEP signals, compared to wired systems. This wireless system will reduce the preoperative time required for IONM setup, add convenience for surgical staff, and reduce wire-related risks for patients during the operation. © 2010 Biomedical Engineering Society.
• Lotfi, P., Garde, K., Chouhan, A. K., Bengali, E., & Romero-Ortega, M. I. (2011). Modality-specific axonal regeneration: Toward selective regenerative neural interfaces. Frontiers in Neuroengineering. doi:10.3389/fneng.2011.00011
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  Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control of robotic prosthetic devices. However, sensory and motor axons at the neural interface are of mixed sub-modality types, which difficult the specific recording from motor axons and the eliciting of precise sensory modalities through selective stimulation. Here we evaluated the possibility of using type specific neurotrophins to preferentially entice the regeneration of defined axonal populations from transected peripheral nerves into separate compartments. Segregation of mixed sensory fibers from dorsal root ganglion neurons was evaluated in vitro by compartmentalized diffusion delivery of nerve growth factor (NGF) and neurotrophin-3 (NT-3), to preferentially entice the growth of TrkA+ nociceptive and TrkC+ proprioceptive subsets of sensory neurons, respectively. The average axon length in the NGF channel increased 2.5-fold compared to that in saline or NT-3, whereas the number of branches increased threefold in the NT-3 channels. These results were confirmed using a 3D "Y"-shaped in vitro assay showing that the arm containing NGF was able to entice a fivefold increase in axonal length of unbranched fibers. To address if such segregation can be enticed in vivo, a "Y"-shaped tubing was used to allow regeneration of the transected adult rat sciatic nerve into separate compartments filled with either NFG or NT-3. A significant increase in the number of CGRP+ pain fibers were attracted toward the sural nerve, while N-52+ large-diameter axons were observed in the tibial and NT-3 compartments. This study demonstrates the guided enrichment of sensory axons in specific regenerative chambers, and supports the notion that neurotrophic factors can be used to segregate sensory and perhaps motor axons in separate peripheral interfaces. © 2011Daly,Galán,Peters and Staudacher.
• Seifert, J., Bell, J., Elmer, B., Sucato, D., & Romero, M. (2011). Characterization of a novel bidirectional distraction spinal cord injury animal model. Journal of Neuroscience Methods, 197(1). doi:10.1016/j.jneumeth.2011.02.003
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  Scoliosis corrective surgery requires the application of significant multidirectional stress forces, including distraction, for correction of the curved spine deformity and the application of fixation rods. If excessive, spine distraction may result in the development of new neurological deficits, some as severe as permanent paralysis. Current animal models of spinal cord injury, however, are limited to contusion, transection, or unidirectional distraction injuries, which fail to replicate the multidirectional forces that occur during spine corrective surgery. To address such limitation, we designed a novel device that relies on intervertebral grip fixation and linear actuators to induce controllable bidirectional distraction injuries to the spine. The device was tested in three (i.e., 3, 5, and 7. mm) distention paradigms of the rat T9-T11 vertebra, and the resulting injuries were evaluated through electrophysiological, behavioral, and histological analysis. As expected, 3. mm bilateral spine distractions showed no neurological deficit. In contrast, those with 5 and 7. mm showed partial and complete paralysis, respectively. The relationship between the severity of the spine distraction and injury to the spinal cord tissue was determined using glial fibrillary acidic protein immunocytochemistry for visualization of reactive astrocytes and labeling of ED1-positive activated macrophages/microglia. Our results demonstrate that this device can produce bidirectional spine distraction injuries with high precision and control and, thus, may be valuable in contributing to the testing of neuroprotective strategies aimed at preventing unintended new neurological damage during corrective spine surgery. © 2011 Elsevier B.V.
• Tansey, K. E., Seifert, J. L., Botterman, B., Delgado, M. R., & Romero, M. I. (2011). Peripheral nerve repair through multi-luminal biosynthetic implants. Annals of Biomedical Engineering, 39(Issue 6). doi:10.1007/s10439-011-0277-6
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  Peripheral nerve damage is routinely repaired by autogenic nerve grafting, often leading to less than optimal functional recovery at the expense of healthy donor nerves. Alternative repair strategies use tubular scaffolds to guide the regeneration of damaged nerves, but despite the progress made on improved structural materials for the nerve tubes, functional recovery remains incomplete. We developed a biosynthetic nerve implant (BNI) consisting of a hydrogel-based transparent multichannel scaffold with luminar collagen matrix as a 3-D substrate for nerve repair. Using a rat sciatic nerve injury model we showed axonal regeneration through the BNI to be histologically comparable to the autologous nerve repair. At 10 weeks post-injury, nerve defects repaired with collagen-filled, single lumen tubes formed single nerve cables, while animals that received the multi-luminal BNIs showed multiple nerve cables and the formation of a perineurial-like layer within the available microchannels. Total numbers of myelinated and unmyelinated axons in the BNI were increased 3-fold and 30%, respectively, compared to collagen tubes. The recovery of reflexive movement confirmed the functional regeneration of both motor and sensory neurons. This study supports the use of multi-luminal BNIs as a viable alternative to autografts in the repair of nerve gap injuries. © 2011 Biomedical Engineering Society.
• Fenghua, T., Delgado, M., Dhamne, S., Khan, B., Alexandrakis, G., Romero, M., Smith, L., Reid, D., Clegg, N., & Liu, H. (2010). Quantification of functional near infrared spectroscopy to assess cortical reorganization in children with cerebral palsy. Optics Express, 18(25). doi:10.1364/oe.18.025973
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  Cerebral palsy (CP) is the most common motor disorder in children. Currently available neuroimaging techniques require complete body confinement and steadiness and thus are extremely difficult for pediatric patients. Here, we report the use and quantification of functional near infrared spectroscopy (fNIRS) to investigate the functional reorganization of the sensorimotor cortex in children with hemiparetic CP. Ten of sixteen children with congenital hemiparesis were measured during finger tapping tasks and compared with eight of sixteen age-matched healthy children, with an overall measurement success rate of 60%. Spatiotemporal analysis was introduced to quantify the motor activation and brain laterality. Such a quantitative approach reveals a consistent, contralateral motor activation in healthy children at 7 years of age or older. In sharp contrast, children with congenital hemiparesis exhibit all three of contralateral, bilateral and ipsilateral motor activations, depending on specific ages of the pediatric subjects. This study clearly demonstrates the feasibility of fNIRS to be utilized for investigating cortical reorganization in children with CP or other cortical disorders. © 2010 Optical Society of America.
• Khan, B., Tian, F., Behbehani, K., Romero, M., Delgado, M., Clegg, N., Smith, L., Reid, D., Liu, H., & Alexandrakis, G. (2010). Identification of abnormal motor cortex activation patterns in children with cerebral palsy by functional near-infrared spectroscopy. J Biomed Opt., 15(3). doi:10.1117/1.3432746
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  We demonstrate the utility of functional near-infrared spectroscopy (fNIRS) as a tool for physicians to study cortical plasticity in children with cerebral palsy (CP). Motor cortex activation patterns were studied in five healthy children and five children with CP (8.4±2.3 years old in both groups) performing a finger-tapping protocol. Spatial (distance from center and area difference) and temporal (duration and time-to-peak) image metrics are proposed as potential biomarkers for differentiating abnormal cortical activation in children with CP from healthy pediatric controls. In addition, a similarity image-analysis concept is presented that unveils areas that have similar activation patterns as that of the maximum activation area, but are not discernible by visual inspection of standard activation images. Metrics derived from the images presenting areas of similarity are shown to be sensitive identifiers of abnormal activation patterns in children with CP. Importantly, the proposed similarity concept and related metrics may be applicable to other studies for the identification of cortical activation patterns by fNIRS. © 2010 Society of Photo-Optical Instrumentation Engineers.
• Garde, K., Keefer, E., Botterman, B., Galvan, P., & Romero-Ortega, M. (2009). Early interfaced neural activity from chronic amputated nerves. Frontiers in Neuroengineering, 2(MAY). doi:10.3389/neuro.16.005.2009
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  Direct interfacing of transected peripheral nerves with advanced robotic prosthetic devices has been proposed as a strategy for achieving natural motor control and sensory perception of such bionic substitutes, thus fully functionally replacing missing limbs in amputees. Multi-electrode arrays placed in the brain and peripheral nerves have been used successfully to convey neural control of prosthetic devices to the user. However, reactive gliosis, micro hemorrhages, axonopathy and excessive inflammation currently limit their long-term use. Here we demonstrate that enticement of peripheral nerve regeneration through a non-obstructive multi-electrode array, after either acute or chronic nerve amputation, offers a viable alternative to obtain early neural recordings and to enhance long-term interfacing of nerve activity. Non-restrictive electrode arrays placed in the path of regenerating nerve fibers allowed the recording of action potentials as early as 8 days post-implantation with high signal-to-noise ratio, as long as 3 months in some animals, and with minimal inflammation at the nerve tissue-metal electrode interface. Our findings suggest that regenerative multi-electrode arrays of open design allow early and stable interfacing of neural activity from amputated peripheral nerves and might contribute towards conveying full neural control and sensory feedback to users of robotic prosthetic devices. © 2009 Garde, Keefer, Botterman, Galvan and Romero.
• Cho, S. H., Lu, H. M., Cauller, L., Romero-Ortega, M. I., Lee, J. B., & Hughes, G. A. (2008). Biocompatible SU-8-based microprobes for recording neural spike signals from regenerated peripheral nerve fibers. IEEE Sensors Journal, 8(Issue 11). doi:10.1109/jsen.2008.2006261
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  A biocompatible neural microprobe constructed using well-established SU-8 microfabrication techniques is described that was designed to record fiber spike signals from regenerated axons within peripheral nerves. These microprobes features bipolar longitudinal gold electrodes recessed below the surface within "grooves" designed to guide the growth of regenerating axons along the length of the grooves and limit the number of fibers that come in contact with the longitudinal electrodes. In addition, screening microprobe toxicity using cultures of human skin fibroblasts, the biocompatibility of these SU-8 microprobes for neural interface applications, in particular, was specifically verified using primary cultures of two sensitive cell types found in peripheral nerves: purified Schwann cells and explanted dorsal root ganglion (DRG) neurons and their fibers. The SU-8 microprobes were surgically implanted into transected rat Sciatic nerves within a unique peripheral nerve regeneration tube. Long-term fiber spike signals were recorded with these SU-8 microprobes in 13 chronically implanted rats for periods from 4 to 51 weeks without any signs of tissue damage or inflammatory reaction. © 2008 IEEE.
• Keefer, E., Botterman, B., Romero, M., Rossi, A., & Gross, G. (2008). Carbon nanotube coating improves neuronal recordings. Nature Nanotechnology, 3(7). doi:10.1038/nnano.2008.174
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  Implanting electrical devices in the nervous system to treat neural diseases is becoming very common. The success of these brain-machine interfaces depends on the electrodes that come into contact with the neural tissue. Here we show that conventional tungsten and stainless steel wire electrodes can be coated with carbon nanotubes using electrochemical techniques under ambient conditions. The carbon nanotube coating enhanced both recording and electrical stimulation of neurons in culture, rats and monkeys by decreasing the electrode impedance and increasing charge transfer. Carbon nanotube-coated electrodes are expected to improve current electrophysiological techniques and to facilitate the development of long-lasting brain-machine interface devices. © 2008 Macmillan Publishers Limited. All rights reserved.
• Galvan-Garcia, P., Keefer, E., Yang, F., Zhang, M., Fang, S., Zakhidov, A., Baughman, R., & Romero, M. (2007). Robust cell migration and neuronal growth on pristine carbon nanotube sheets and yarns. Journal of Biomaterials Science, Polymer Edition, 18(10). doi:10.1163/156856207782177891
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  Carbon nanotubes (CNTs) have unique chemical and physical properties anticipated to enable broad novel biomedical applications. Yet the question concerning their biocompatibility remains controversial. We recently reported a method for rapidly preparing strong, highly electrically conducting sheets and yarns from multi-walled CNTs. The present studies demonstrate that highly oriented 50-nm-thick semi-transparent CNT sheets and yarns, produced with a minimal residual content of catalytic transition materials, support the long-term growth of a variety of cell types ranging from skin fibroblasts and Schwann cells, to postnatal cortical and cerebellar neurons. We show that CNT sheets stimulate fibroblast cell migration compared to plastic and glass culture substrates; entice neuronal growth to the level of those achieved on polyornithine-coated glass and can be used for directed cellular growth. These findings have positive implications for the use of CNTs in applications such as tissue engineering, wound healing, neural interfaces and biosensors. © 2007 VSP.
• Romero, M., Lin, L., Lush, M., Lei, L., Parada, L., & Zhu, Y. (2007). Deletion of Nf1 in neurons induces increased axon collateral branching after dorsal root injury. Journal of Neuroscience, 27(8). doi:10.1523/jneurosci.4363-06.2007
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  Ras-mediated signaling pathways participate in multiple aspects of neural development and function. For example, Ras signaling lies downstream of neurotrophic factors and Trk family receptor tyrosine kinases to regulate neuronal survival and morphological differentiation, including axon extension and target innervation. Neurofibromin, the protein encoded by the tumor suppressor gene Nf1, is a negative regulator of Ras [Ras-GAP (GTPase-activating protein)], and we previously demonstrated that Nf1 null embryonic sensory and sympathetic neurons can survive and differentiate independent of neurotrophin support. In this report, we demonstrate that Nf1 loss in adult sensory neurons enhances their intrinsic capacity for neurite outgrowth and collateral branching in vitro and in vivo after dorsal root injury. In contrast to the permanent sensory deficits observed in control mice after dorsal rhizotomy, neuron-specific Nf1 mutant mice spontaneously recover proprioceptive function. This phenomenon appears to be mediated both by a cell-autonomous capacity of spared Nf1-/- DRG neurons for increased axonal sprouting, and by non-cell-autonomous contribution from Nf1-/- neurons in the denervated spinal cord. Copyright © 2007 Society for Neuroscience.
• Benson, M., Romero, M., Lush, M., Lu, Q., Henkemeyer, M., & Parada, L. (2005). Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proceedings of the National Academy of Sciences of the United States of America, 102(30). doi:10.1073/pnas.0504021102
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  The inability of CNS axons to regenerate after traumatic spinal cord injury is due, in part, to the inhibitory effects of myelin. The three major previously identified constituents of this activity (Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein) were isolated based on their potent inhibition of axon outgrowth in vitro. All three myelin components transduce their inhibitory signals through the same Nogo receptor/p75 neurotrophin receptor/LINGO-1 (NgR1/p75/LINGO-1) complex. In this study, we considered that molecules known to act as repellants in vertebrate embryonic axonal pathfinding may also inhibit regeneration. In mice, ephrin-B3 functions during development as a midline repellant for axons of the corticospinal tract. We therefore investigated whether this repellant was expressed in the adult spinal cord and retained inhibitory activity. We demonstrate that ephrin-B3 is expressed in postnatal myelinating oligodendrocytes and, by using primary CNS neurons, show that ephrin-B3 accounts for an inhibitory activity equivalent to that of the other three myelin-based inhibitors, acting through p75, combined. Our data describe a known vertebrate axon guidance molecule as a myelin-based inhibitor of neurite outgrowth. © 2005 by The National Academy of Sciences of the USA.
• Lei, L., Laub, F., Lush, M., Romero, M., Zhou, J., Luikart, B., Klesse, L., Ramirez, F., & Parada, L. (2005). The zinc finger transcription factor Klf7 is required for TrkA gene expression and development of nociceptive sensory neurons. Genes and Development, 19(11). doi:10.1101/gad.1227705
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  TrkA, the high affinity receptor for nerve growth factor (NGF), is essential for the development of nociceptive sensory and sympathetic neurons. The zinc finger transcription factor Klf7 interacts with an important cis element of the TrkA minimal enhancer and is coexpressed with TrkA in these neurons. We show that Klf7 binds to the endogenous TrkA minimal enhancer and can activate transcription from the TrkA minimal enhancer in a sequence-dependent manner. In Klf7-/- newborn mice, we find a significant reduction in sensory neurons due to increased apoptosis. The neuronal loss is restricted to nociceptive neurons that normally depend on TrkA for neurotrophic support, while other populations of somatosensory neurons appear normal. The reduction of TrkA expression in sensory neurons is a direct effect of Klf7 gene ablation, rather than a secondary effect of cell death. As a result, Klf7-/- mice have deficient response to noxious stimuli. Finally, removal of one TrkA allele exacerbates the loss of TrkA(+) neurons in Klf7-/- mice. Thus, Klf7 specifically regulates TrkA gene expression and is required for the development of a subset of nociceptive sensory neurons. © 2005 by Cold Spring Harbor Laboratory Press.
• Aimone, J. B., Leasure, J. L., Perreau, V. M., Thallmair, M., Anderson, A. J., Arvanian, V. L., Bareyre, F. M., Bunge, M. B., Cotman, C. W., Craveiro, L., Crown, E. D., Edgerton, V. R., Engesser-Cesar, C., Gage, F. H., Garcia, A. J., Garraway, S. M., Horner, P. J., Howley, S., Ichiyama, R. M., , Mendell, L. M., et al. (2004). Spatial and temporal gene expression profiling of the contused rat spinal cord. Experimental Neurology, 189(Issue 2). doi:10.1016/j.expneurol.2004.05.042
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  Microarray technology was used to examine gene expression changes following contusive injury of the adult rat spinal cord. To obtain a global understanding of the changes triggered by the injury, differential gene expression was examined spatially, using tissue samples from the epicenter of injury as well as 1 cm rostral and 1 cm caudal to the epicenter, and temporally, at 3 h, 24 h, 7 days, and 35 days post-injury. To filter out gene expression changes that were due to the laminectomy, samples of contused tissue were compared to laminectomy-only controls. We took advantage of four different, complementary methods of data analysis to detect differentially expressed genes. We have identified functional groups of genes that are differentially regulated in our model, including those associated with apoptosis, cell cycle, inflammation, and cholesterol metabolism. Our analysis has led to the identification of novel potential therapeutic targets within each group of genes that is discussed. © 2004 Elsevier Inc. All rights reserved.
• Phelps, C., Romero, M., & Hurley, D. (2003). Prolactin replacement must be continuous and initiated prior to 21 d of age to maintain hypothalamic dopaminergic neurons in hypopituitary mice. Endocrine, 20(1-2). doi:10.1385/endo:20:1-2:139
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  The prolactin (PRL) deficit in mice homozygous for the spontaneous Ames dwarf (df) mutation coincides with a marked reduction in the number of PRL-regulating tuberoinfundibular dopaminergic (TIDA) neurons. The TIDA deficit develops after 14-21 d postnatally and may be prevented by PRL replacement initiated at 12, but not at 60, d of age. The present study was designed to define further the developmental period during which PRL can prevent the deficit in the number of TIDA neurons in df/df mice, as well as to evaluate whether exposure to PRL neonatally affects the response to PRL by TIDA neurons in later development. To address the first aim, litters of df/df and normal (DF/df) mice were treated daily with ovine PRL (50 μg intraperitoneally), starting at 12, 21, or 30 d of age. To address the second aim, DF/df and df/df animals treated with PRL for 30 d starting at 12 d of age were subjected to PRL withdrawal (15 d of saline vehicle treatment), followed by PRL retreatment. All brains were evaluated using both catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry. Total numbers of TH-immunostained cells were counted in area A12 (TIDA neurons) and in A13 (medial zona incerta). Qualitatively, catecholamine fluorescence in A12 perikarya and terminals in df/df mice was enhanced by PRL treatment initiated at 12 or 21, but not at 30, d of age. TH immunostaining intensity was enhanced in all df/df PRL-treated groups, compared with saline treatment. However, total numbers of TH-positive TIDA neurons were reduced significantly in df/df mice treated with PRL beginning at 21 or 30 d, as well as with saline at 12 d, compared with similarly treated DF/df groups and with df/df animals treated with PRL beginning at 12 d (p < 0.01 for all comparisons). Among dwarf mice treated with PRL beginning at 12 d, followed by PRL withdrawal, the numbers of TH-positive TIDA neurons were greater than those of saline-treated dwarfs, but less than those in DF/df mice (p < 0.05 for both comparisons). In dwarfs retreated with PRL after withdrawal, the TIDA population was also smaller than that in normal animals (p < 0.05), although it was larger than in vehicle-treated dwarfs of the same age (p < 0.05). No effect of PRL treatment on TIDA cell numbers in normal mice, or of treatment or mouse phenotype on the number of TH-positive cells in zona incerta, occurred in either experiment. These results indicate that the effect of PRL on preventing the reduction in the TIDA population in df/df mice is limited to a developmental period prior to 21 d postnatally. In addition, this study provides evidence that continuous PRL feedback is required to maintain normal numbers of TIDA neurons. These findings extend the evidence for a critical role of PRL feedback in the differentiation and preservation of phenotype in TIDA neurons.
• Ma, L., Harada, T., Harada, C., Romero, M., Hebert, J., McConnell, S., & Parada, L. (2002). Neurotrophin-3 is required for appropriate establishment of thalamocortical connections. Neuron, 36(4). doi:10.1016/s0896-6273(02)01021-8
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  In the vertebrate brain, the thalamus serves as a relay and integration station for diverse neuronal information en route from the periphery to the cortex. Formation of the thalamocortical tract occurs during pre- and postnatal development, with distinct thalamic nuclei projecting to specific cortical regions. The molecular forces that underlie the invasion by axons into specific cortical layers followed by activity-dependent maturation of synapses are poorly understood. We show that genetic ablation of neurotrophin-3 (NT-3) in the mouse neocortex results in reduction of a set of anatomically distinct axonal bundles projecting from thalamus through cortical white matter. These bundles include thalamocortical axons that normally establish connections with retrosplenial and visual cortex, sites of early postnatal NT-3 expression. These results implicate neurotrophins in the critical stage of precise thalamocortical connections.
• Romero, M., Rangappa, N., Garry, M., & Smith, G. (2001). Functional regeneration of chronically injured sensory afferents into adult spinal cord after neurotrophin gene therapy. Journal of Neuroscience, 21(21). doi:10.1523/jneurosci.21-21-08408.2001
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  Lesioned axons within the dorsal roots fail to regenerate through the peripheral nerve transition zone and into the spinal cord. This regenerative failure leads to a persistent loss of sensory function. To induce axonal growth across this barrier, we used recombinant adenovirus to express fibroblast growth factor-2 (FGF2), nerve growth factor (NGF), L1 cell adhesion molecule (L1), or β-galactosidase (LacZ) within the endogenous glia of the dorsal spinal cord 16 d after injury. Expression of either FGF2 or NGF, but not L1 or LacZ, induced robust axonal regeneration into normal as well as ectopic locations within the dorsal spinal cord. This regeneration led to near-normal recovery of thermal sensory function. Functional recovery and the majority of regenerating axons within the dorsal horn disappeared with recutting of the sensory roots. Injections of adenovirus encoding NGF, but not FGF2, also resulted in extensive sprouting of noninjured sensory axons, which we previously demonstrated could cause hyperalgesia and chronic pain. Thus, neurotrophic factor gene therapy administered as late as 16 d after injury may serve as a useful treatment to elicit recovery after dorsal root avulsion; however, the choice of neurotrophin is important to induce selective regeneration of damaged axons.
• Yokoyama, N., Romero, M., Cowan, C., Galvan, P., Helmbacher, F., Charnay, P., Parada, L., & Henkemeyer, M. (2001). Forward signaling mediated by ephrin-B3 prevents contralateral corticospinal axons from recrossing the spinal cord midline. Neuron, 29(1). doi:10.1016/s0896-6273(01)00182-9
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  To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling resulted in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract was defective as axons failed to respect the midline boundary of the spinal cord and bilaterally innervated both contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-B3 protein lacking its cytoplasmic domain did not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal corticospinal neurons as their fibers pathfind down the contralateral spinal cord. Our data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons.
• Zhu, Y., Romero, M., Ghosh, P., Ye, Z., Charnay, P., Rushing, E., Marth, J., & Parada, L. (2001). Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Genes and Development, 15(7). doi:10.1101/gad.862101
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  Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.
• Romero, M. I., Rangappa, N., Li, L., Lightfoot, E., Garry, M. G., & Smith, G. M. (2000). Erratum: Extensive sprouting of sensory afferents and hyperalgesia induced by conditional expression of nerve growth factor in the adult spinal cord (Journal of Neuroscience (June 15, 2000) (4435-4445)). Journal of Neuroscience, 20(Issue 22).
• Romero, M., Rangappa, N., Li, L., Lightfoot, E., Garry, M., & Smith, G. (2000). Extensive sprouting of sensory afferents and hyperalgesia induced by conditional expression of nerve growth factor in the adult spinal cord. Journal of Neuroscience, 20(12). doi:10.1523/jneurosci.20-12-04435.2000
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  Genetic transfer of growth-promoting molecules was proposed as a potential strategy to modify the nonpermissive nature of the adult CNS to induce axonal regeneration. To evaluate whether overexpression of neurotrophins or cellular adhesion molecules would effect axonal plasticity, adenoviruses encoding fibroblast growth factor-2 (FGF-2/Adts), nerve growth factor (NGF/Adts), neurotrophin-3, and the cell adhesion molecules N-cadherin and L1 were injected into the dorsal horn of the adult spinal cord. Transgene expression was primarily localized to astrocytes in the dorsal horn and motor neurons within the ventral horn. Overexpression of these factors, with the exception of NGF/Adts, failed to increase axonal sprouting. Eight days after NGF/Adts injections, axonal sprouting within the dorsal horn was apparent, and after 4 weeks, extensive spouting was observed throughout the entire dorsal horn, extending into the ventral horn and the white matter of the lateral funiculus. These axons were identified primarily as a subpopulation of nociceptive fibers expressing calcitonin gene-related peptide and substance-P. Behavioral analysis revealed thermal hyperalgesia and perturbation of accurate paw placement on gridwalking tasks for both FGF-2- and NGF-treated animals. These results indicate that the administration of growth-promoting molecules can induce robust axonal plasticity of normal adult primary sensory neurons into areas of transgene expression, causing significant alterations in behavioral responses. This observation also indicates that gene transfer protocols that aim to reconstruct diseased or injured pathways should also be designed to prevent the sprouting of the normal circuitry from adjacent unaffected neurons.
• Romero, M., Romero, M., & Smith, G. (1999). Visualization of axonally transported horseradish peroxidase using enhanced immunocytochemical detection: A direct comparison with the tetramethylbenzidine method. J Histochem Cytochem, 47(2). doi:10.1177/002215549904700216
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  Visualization of the neuronal tract tracer horseradish peroxidase (HRP) is commonly achieved through the histochemical detection of its enzymatic activity using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen. However, the TMB product is unstable and is incompatible with tissue processing methods that render the enzyme inactive, or when a combination of HRP tract tracing with neuronal phenotype identification is required. In this study we evaluated the applicability of the immunocytochemical detection method for horseradish peroxidase (HRP) visualization using an enhanced detection method based on the Elite ABC peroxidase amplification protocol. The results provide evidence for the immunocytochemical visualization of both anterograde and transganglionic HRP transport in the rat spinal cord. This immunocytochemical method not only showed similar sensitivity to the TMB protocol in detecting HRP-labeled motor neuron perikarya but provided enhanced resolution in the identification of individual neuronal fibers compared to the TMB method. Immunodetection of the HRP tracer also allowed its co-localization with specific neuronal markers using double immunofluorescence techniques. These results offer the first demonstration that sensitive identification of axonally transported HRP can be achieved by immunocytochemistry and provides further support for its use in HRP tract tracing studies.
• Smith, G., & Romero, M. (1999). Adenoviral-mediated gene transfer to enhance neuronal survival, growth, and regeneration. Journal of Neuroscience Research, 55(2). doi:10.1002/(sici)1097-4547(19990115)55:2<147::aid-jnr2>3.0.co;2-8
• Romero, M., & Smith, G. (1998). Adenoviral gene transfer into the normal and injured spinal cord: Enhanced transgene stability by combined administration of temperature-sensitive virus and transient immune blockade. Gene Therapy, 5(12). doi:10.1038/sj.gt.3300774
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  This study characterized gene transfer into both normal and injured adult rat dorsal spinal cord using first (E1-/E3-) or second (E1-/E2A125/E3-, temperature sensitive; ts) generation of replication-defective adenoviral (Ad) vectors. A novel immunosuppressive regimen aimed at blocking CD4/CD45 lymphocytic receptors was tested for improving transgene persistence. In addition, the effect of gene transfer on nociception was also evaluated. Seven days after treatment, numerous LacZ-positive cells were observed after transfection with either viral vector. By 21 days after transfection, β-galactosidase staining was reduced and suggestive of ongoing cytopathology in both Ad-treated groups, despite the fact that the immunogenicity of LacZ/Ad(ts) appeared less when compared with that elicited by the LacZ/Ad vector. In contrast, immunosuppressed animals showed a significant (P ≤ 0.05) increase in the number of LacZ-positive cells not displaying cytopathology. In these animals, a concomitant reduction in numbers of macrophages/microglia and CD4 and CD8 lymphocytes was observed. Only animals that received LacZ/Ad(ts) and immunosuppression showed transgene expression after 60 days. Similar results were observed in animals in which the L4-L5 dorsal roots were lesioned before transfection. Gene transfer into the dorsal spinal cord did not affect nociception, independent of the adenovirus vector. These results indicate that immune blockade of the CD4/CD45 lymphocytic receptors enhanced transgene stability in adult animals with normal or injured spinal cords and that persistent transgene expression in the spinal cord does not interfere with normal neural function.
• Romero, M., & Phelps, C. (1997). Identification of growth hormone-releasing hormone and somatostatin neurons projecting to the median eminence in normal and growth hormone-deficient ames dwarf mice. Neuroendocrinology, 65(2). doi:10.1159/000127170
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  In the spontaneous mutant Ames dwarf mouse, GH deficiency coincides with a dramatic increase in the expression of both mRNA and peptide for stimulatory GHRH and reduced expression of GH-inhibitory somatostatin (SRIH) mRNA and peptide. However, both GHRH and SRIH are markedly reduced in the dwarf median eminence (ME), suggesting that ME innervation by GHRH and SRIH neurons may be aberrant in the absence of GH. In order to test this hypothesis, the number of GHRH and SRIH ME-projecting neurons was evaluated in normal and dwarf mice using a combination of retrograde tract-tracing and neuron phenotype identification by immunocytochemistry (ICC). Adult animals were injected intraperitoneally with the fluorescent tract-tracer fluorogold (FG), which, in the brain, is taken up only by axons terminating in areas deprived of the blood-brain barrier, such as the ME. Visualization of FG was achieved by either UV illumination or ICC, and was combined as appropriate with fluorescence or bright-field ICC for GHRH or SRIH. Cells immunoreactive for GHRH or SRIH and labeled with FG were quantified at each 180-μm rostral-to-caudal level through the hypothalamus. As reported previously, the total number of hypophysiotropic GHRH neurons was markedly increased in dwarf compared with that in normal mice. However, a similar percentage of ME-innervating GHRH cells was estimated in dwarf (73 ± 4%) and normal (76 ± 3%) animals. Such a percentage in dwarfs thus represents a larger population of ME-projecting GHRH cells (749 ± 53) than in normal mice (128 ± 15). Increased numbers of FG-labeled GHRH neurons in dwarfs were located at the middle and posterior levels of the arcuate nucleus (2.08, 2.26 and 2.44 mm posterior to bregma). The percentage of FG-labeled SRIH neurons was also similar for dwarf (83 ± 2%) and normal (87 ± 2%) mice. Because the total SRIH-immunoreactive neuronal population in dwarfs is significantly reduced compared to that in normal animals, the similar FG-labeled percentage reflects a reduced number of SRIH cells projecting to ME in dwarf (1,376 ± 104) compared with normal (3,192 ± 267) mice. Fewer FG-labeled SRIH cells were found in dwarfs at every anterior-to-posterior level of the periventricular nucleus (p < 0.01 for comparisons at 0.28, 0.46, 0.64, and 1.0, and p < 0.05 for comparison at 1.18 mm posterior to the bregma). The present study indicates that the reduction in GHRH and SRIH immunoreactivity in the dwarf ME may result from different phenomena for each neuronal population. The reduction in GHRH immunostaining in the ME, despite a marked increase in the total ME-projecting GHRH neurons, may be interpreted as increased GHRH release, with consequent depletion of the ME stores. In contrast, the deficit in ME SRIH may be proportional to the deficit in the number of detectable SRIH periventricular nucleus neurons. © 1997 S. Karger AG, Basel.
• Phelps, C., Saleh, N., & Romero, M. (1996). Hypophysiotropic somatostatin expression during postnatal development in growth hormone-deficient ames dwarf mice: Peptide immunocytochemistry. Neuroendocrinology, 64(5). doi:10.1159/000127140
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  Based on previous findings that the inhibitory hypophysiotropic factor somatostatin (somatotropin-release-inhibiting hormone, SRIH) is markedly reduced in growth hormone (GH)-deficient transgenic or spontaneous Snell dwarf mice, the present study was undertaken to determine whether hypophysiotropic SRIH expression was reduced in a type of dwarf mouse (Ames, df/df) in which SRIH had not been assessed, and whether the supposed reduction was present throughout life or was the result ofregression after initial normal differentiation. Brain sections from normal (DF/?) and df/df mice were immunostained for SRIH using both standard and ‘Elite’ avidin-biotin complex reagents (Vectastain kits, Vector Laboratories, Inc., Burlingame, Calif., USA). Selected adult mice were treated with intracerebroventricular colchicine to maximize SRIH retention in perikarya. The developmental pattern of hypophysiotropic SRIH was assessed in brains of DF/? and df/df mice at 1, 3, 7,14, 21, 60, and 90 days (d) postnatally. SRIH-immunoreactive neurons in the anterior periventricular nucleus (PeN) were quantified at each age. Although the use of Elite reagents or Elite and colchicine pretreatment increased (p < 0.001) thenumber of immunoreactive cells that were detectable in adult (60- to 90-day-old) df/df mice, the number of PeN SRIH neurons was reduced to 28% (p < 0.01) in untreated, and to 47% (p < 0.01) in colchicine-treated, df/df compared with DF/?, mice. In other CNS areas, SRIH immunostaining was comparable for df/df and DF/? mice, including neuron numbers in the medial basal hypothalamus of untreated mice. In postnatal development, SRIH was detectable in median eminence (ME) terminals at birth in some mice of both phenotypes, and at 3 d in all DF/? mice; ME SRIH was detectable in all mice by 7 d. In PeN, SRIH cells were first detectable consistently in normals at 3 d, and in dwarfs at 7 d. In DF/? mice, numbers of immunoreactive SRIH perikarya increased from 3 to 21 d, then plateaued. In dwarfs, SRIH cell numbers increased through 14 d. Numbers of SRIH perikarya were lower in df/df than in DF/? at 7, 14, 21, 60, and 90 d (all p < 0.05 or less). Thus, in Ames dwarf mice, as in other GH-deficient models, SRIH is markedly reduced in hypophysiotropic, ME-projecting neurons. The developmental pattern of hypophysiotropic SRIH in Ames dwarf mice is different from that of hypophysiotropic dopaminergic (DA) neurons in these animals, which are also prolactin (PRL)-deficient. Although DA levels and cell numbers are reduced markedly in adult df/df mice, both parameters have been found to be comparable tothose of DF/? mice for the first 2-3 weeks postnatally. The consistent PeN SRIH deficit in dwarfs may reflect the importanceof GH feedback earlier in development, because GH production in normal mice begins before birth, whereas PRL is not detectable until 7 d postnatally. The findings indicate that absent GHproduction has a marked negative effect on differentiation andlevels of peptide expression in hypophysiotropic SRIH neurons.© 1996 S.Karger AG, Basel.
• Phelps, C., Romero, M., & Hurley, D. (1995). Role of prolactin in developmental differentiation of hypophysiotropic tuberoinfundibular dopaminergic neurons. Recent Progress in Hormone Research, 50(1). doi:10.1016/b978-0-12-571150-0.50039-1
• Romero, M., & Phelps, C. (1995). Prolactin replacement in adult dwarf mice does not reverse the deficit in tuberoinfundibular dopaminergic neuron number. Endocrinology, 136(8). doi:10.1210/en.136.8.3238
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  The lack of PRL synthesis in Ames dwarf mice coincides with a marked reduction in dopamine (DA) and in numbers of PRL-inhibiting tuberoinfundibular dopaminergic neurons in the hypothalamus (catecholaminergic area A12), as assessed by tyrosine hydroxylase (TH) immunoreactivity. This DA/TH deficit develops postnatally and can be prevented by PRL replacement initiated at 12 days of age. The present study tested whether a similar PRL treatment in adult dwarfs would reverse the A12 deficit, indicating that these neurons are quiescent due to absent PRL feedback stimulation, or would not reverse the deficit, suggesting that A12 neurons are either absent or refractory to PRL effect. At 60 days of age, Ames dwarf (df/df) mice received renal pituitary allografts from normal (DF/df) donors as a source of mouse PRL. Separate groups of dwarfs were treated sequentially with ovine PRL (50 μg/day, ip; 30 days/and vehicle (15 days) to assess whether the putative restorative effect of PRL regressed after hormone withdrawal. Brains were evaluated using DA histofluorescence and TH immunocytochemistry. Total numbers of TH-immunostained cells in A12 and medial zona incerta (area A13) regions were counted, and the intensity of TH immunostaining was assessed by computerized image analysis. The total A12 TH-positive cell number was reduced (P < 0.01) in all PRL-treated dwarfs (1826 ± 58) compared with that in normal mice (3340 ± 180), and was not different from that in untreated dwarfs (1953 ± 304) regardless of the PRL regimen. However, A12 perikarya in all PRL-treated dwarfs showed qualitatively increased histofluorescence and quantitatively increased TH immunostaining (P < 0.01) intensity compared with that in untreated dwarfs, an effect that regressed after ovine PRL withdrawal. Neither cell number nor staining intensity differed by gender. There were no significant differences in A13 cell numbers or staining intensity according to phenotype or PRL treatment. The present results indicate that the tuberoinfundibular dopaminergic neuronal population in adult Ames dwarf mice is permanently reduced, although extant A12 cells in dwarfs are responsive to either homologous or heterologous PRL feedback. Together with the previously reported effect of PRL treatment in neonatal dwarfs, the reduction appears to be the result of absent PRL stimulation during development.
• Phelps, C., Vaccarella, M., Romero, M., & Hurley, D. (1994). Postnatal reduction in number of hypothalamic tuberoinfundibular dopaminergic neurons in prolactin-deficient dwarf mice. Neuroendocrinology, 59(3). doi:10.1159/000126658
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  Mice homozygous for the recessive ‘Ames’ dwarf mutation have undetectable serum or pituitary prolactin (PRL). Accompanying this pituitary deficiency is a marked reduction of dopamine (DA) and its rate-limiting synthetic enzyme tyrosine hydroxylase (TH) in PRL-regulating tuberoinfundibular hypothalamic neurons. In order to determine whether this deficit in adult Ames dwarf mice is congenital or arises postnatally, brains of dwarf (df/df) and normal (DF/?) littermate mice were assessed for TH immunoreactivity from 7 days through 2 months of age. Numbers of TH-positive neurons were counted in three hypothalamic DA areas: tuberoinfundibular A12, medial zona incerta A13, and anterior periventricular A14. There was an increase in the number of TH-positive neurons between 7 and 21 days of age in A12 and A14, but not in A13, for both DF/? and df/df mice. Between 21 days and 2 months of age, cell numbers were the same in all three areas in DF/? mice and in A13 and A14 in df/df mice. However, A12 TH-positive cell number in dwarfs decreased significantly (p < 0.01) between 21 days and 2 months, and was markedly lower (p < 0.001) in df/df than in DF/? mice at 2 months of age. The results emphasize the specificity of the dopaminergic neuron reduction in the Ames dwarf, which is restricted to the PRL-regulating tuberoinfundibular region. The data also indicate that the dwarf DA/TH deficit has an onset in late postnatal development, suggesting a response to absence of target PRL, rather than a primary hypothalamic effect of the dwarf mutation. © 1994 S. Karger AG, Basel.
• Romero, M., & Phelps, C. (1993). Prolactin replacement during development prevents the dopaminergic deficit in hypothalamic arcuate nucleus in prolactin-deficient ames dwarf mice. Endocrinology, 133(4).
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  PRL-deficient dwarf mice exhibit marked reduction in dopamine (DA) and in tyrosine hydroxylase (TH) immunoreactivity in the PRL-regulating neurons of the hypothalamic arcuate nucleus (catecholaminergic area A12). Recent studies in this laboratory have revealed that this condition develops postnatally, in that A12 DA fails to increase and the number of TH-positive cells decreases after 21 days of age. The present study was designed to test whether PRL replacement during the early postnatal period would increase DA and TH expression in dwarfs. Ames dwarf (df/df) and normal sibling (DF/?) mice were treated with daily injections of ovine PRL (50 μg, ip) or vehicle for 30 days starting on postnatal day 12. Brains were evaluated by catecholamine histofluorescence and TH immunocytochemistry at the end of the treatment period. TH-positive cells were counted in A12 and medial zona incerta (area A13) and also differentially within A12, in dorsal and ventral regions, and at anterior, middle, and posterior levels. Histofluorescence and TH-positive cell number (P < 0.01) in vehicle-treated dwarfs were greatly reduced compared with those in DF/? mice in A12, but not in A13. However, A12 fluorescence in PRL-treated dwarfs was comparable to that in DF/? mice. TH cell counts in A12 of PRL-treated dwarfs were significantly higher (P < 0.01) than those in vehicle-treated dwarfs and not different from those in either group of DF/? mice. Within A12, both dorsal and ventral TH cell numbers were reduced in vehicle-treated dwarfs (P < 0.01); the reduction was greater in the ventral subpopulation (P < 0.01). TH cell counts were lower in middle and posterior (P < 0.05), but not anterior, areas of A12 in vehicle-treated df/df mice compared with those in DF/? mice. TH cell numbers in all A12 regions in PRL-treated dwarfs were not different from those in DF/? mice. Thus, PRL replacement initiated before 2 weeks of age in dwarfs is effective in supporting DA and TH expression in both A12 neurons and median eminence external zone at normal levels, providing direct evidence that the DA/TH deficit in dwarfs is secondary to endogenous PRL deficiency.
• Domino, G., Romero, M., Blair, G., Villalobos, S., Suriano, T., & Bridges, A. (1985). Cross-Cultural Aspects of Sleep in the Elderly. Research on Aging, 7(Issue 2). doi:10.1177/0164027585007002003
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  A sleep questionnaire (SQ) was administered to 850 adults, 50 years and older, drawn from urban areas in 7 countries (Australia, Greece, Italy, Mexico, Spain, Venezuela, United States). All were functioning adults in relatively good physical and mental health. The samples were equated on sex, age, and occupational level. Of the 8 scales on the SQ, 7 showed statistically significant differences between countries, and 3 between males and females. At the same time, substantial similarities were found and are discussed from a normative point of view. © 1985, Sage Publications. All rights reserved.

Proceedings Publications

• Gonzalez-Gonzalez, M. A., Ghazavi, A., Cogan, S., & Romero-Ortega, M. (2023). Intraneural SiC multi electrodes to detect multimodal sensory signals. In 11th International IEEE/EMBS Conference on Neural Engineering, NER 2023, 2023-.
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  Limb prosthesis technology now incorporates actuation and touch sensing information, however evoking such specific types of sensation to amputee users remains a challenge, in part due to the low spatial resolution of microelectrodes in peripheral neural interfaces relatively large active areas (i.e.., 2000 um2) at the tip and shafts of 100-200 um2 OD. Here we implemented a sixteen-channels ultra-small multi electrode arrays (UMEA) with 10 um OD shafts and 200 um2 surface area using amorphous silicon carbide (a-SiC) for high precision intraneural detection of cutaneous sensory signals. This technology allowed to discriminate between slow and rapid adapting mechanoceptors associated to light and deep pressure in response to touch stimulation using von Frey filaments. We observed specific electrophysiological profiles evoked from five glabrous dermatomes, which inform neural subtypes and spatial arrangement in the nerve. The results demonstrate that ultra-small UMEA can be used reliably to record specific sensory modalities from the peripheral nerves, which could offer a viable alternative to inform closed loop systems to decode movement intent and to evoke specific sensations through microstimulation.
• Hussein, K. R., & Romero-Ortega, M. I. (2023). Sensory and Motor Intent Signals Recorded by Regenerative Multielectrode Arrays. In 11th International IEEE/EMBS Conference on Neural Engineering, NER 2023, 2023-.
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  Advanced robotic limbs have human-like degrees-of-freedom and are sensorized. However, offering amputees naturalistic control and sensation/perception requires interfacing to the nervous system of the user with selectivity and sensitivity. Current peripheral neural interfaces include targeted muscle re-innervation, and myoelectric control and peripheral nerve electrodes, including cuff or FINE electrodes, indwelling tf-LIFE, TIME or regenerative sieve or Regenerative Multielectrode Interface (REMI) electrodes. However, identification of the nature of the electrical activity recorded by these interfaces remains challenging. Here, we report waveform characterization of neurophysiological activity recorded from a motor nerve (tibial), from a sensory nerve (sural) recorded using a dual REMI interface. We analyzed neural spike data recorded in animals 1-2 months after regeneration to their natural muscle and skin targets, that correlated with bipedal locomotion or evoked by von Frey activation of skin touch sensors. Motor waveforms exhibited biphasic and triphasic shapes, while sensory activity was monophasic. These results provide insight as to the shape of action potentials in the REMI and contribute to the characterization of motor and sensory neural activity.
• Lloyd, D. A., Akay, Y. M., Akay, M., & Romero-Ortega, M. (2023). Investigating Neuronal Feature Extraction Using Deep Learning Techniques: A Comparative Study. In 2023 IEEE EMBS Special Topic Conference on Data Science and Engineering in Healthcare, Medicine and Biology, IEEECONF 2023.
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  High quality feature sets are vital to the development of neuroprosthetic and bioelectronic therapies. For neural signals, the primary features are 'spikes' extracted from neuronal signals. Current spike detection is a subjective process with no certain ground truth. Leveraging synthetic neuronal data, we can train a deep learning model to provide continuous pointwise spike probability and leverage this to improve feature detection quality. Our results indicate that high-fidelity, variable-length spikes can be extracted using continuous probabilityClinical Relevance: This work improves feature quality and diversity over current static thresholding techniques. Furthermore, focusing on small-amplitude neuronal signals gives vastly superior insight into autonomic regulatory encoding. This is particularly important for the development of bioelectronics pain treatment and synthetic organ development, as the smaller-amplitude longer-time features they need are frequently missed by current approaches.
• Wang, Y., Zimmern, P., & Romero-Ortega, M. I. (2023). Direct Bladder Contraction by Single and Multi-Electrode Stimulation. In 11th International IEEE/EMBS Conference on Neural Engineering, NER 2023, 2023-.
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  Detrusor sphincter dyssynergia (DSD) is characterized by the uncoordinated activity between contraction of the detrusor and relaxation of the external urinary sphincter, affecting 70%-84% of individuals with spinal cord injury (SCI). Current treatments include catheterization, medication, sphincterotomy, and neuromodulation. Here we divided the bladder in quarters and used point (0.78 mm2) electrical stimulation at 0.5, 1.0 and 2 mA to map those that evoked maximal contraction (9.88 mm2), as determined by vascular fiduciary markers before and after stimulation. A multi-electrode array was then used to reduce the electrodes size (2000μm2) which evoked a more efficient bladder contraction (16.51 mm2) and at a lower threshold (0.15 mA). The relative change of bladder size evoked by single electrodes was -7.3 %, whereas that evoked by the multichannel electrode was 12.9% (p = 0.007). This result defines the optimal targets sites and parameters that can be used to improve the neuromodulation treatment of DSD.
• Al-Amin, S. T., Varghese, R., Lloyd, D., Gonzalez-Gonzalez, M. A., Romero-Ortega, M. I., & Ordonez, C. (2022). Discovering Similar Spike Patterns in High Dimensional Biomedical Signals. In 2022 IEEE International Conference on Big Data, Big Data 2022.
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  We discuss our progress towards solving a challenging biomedical problem: identifying similar patterns among multiple physiological nerve signals hidden in high throughput data, collected from micro electrical sensors implanted in several animal organs. The problem is difficult because patterns come as spikes within millisecond time-windows, data sets have high dimensionality and there is background electrical noise. A previous analytic system discovers patterns combining PCA dimensionality reduction and K-means clustering, which is slow and misses important patterns hidden by noise. Moreover, it requires reading the data set several times and it requires multiple languages and tools. With such limitations in mind, we present an improved, integrated system that effectively allows the discovery of more accurate patterns, with automated algorithm parameter tuning, by learning model parameters incrementally exploiting summarization. Our integrated solution combines signal filtering, variable construction (feature engineering) and multidimensional data summarization, for a tighter and more effective integration of PCA and K-means clustering. We present preliminary experiments on signals collected from key nerves in a rat. We show our method discovers more patterns in larger time-windows, with better noise filtering, taking less time. In the future, we plan to link signal patterns to specific physiological functions, paving the way for innovative medical treatment via nerve stimulation.
• Tong, Y., Murbach, J. M., Subramanian, V., Chhatre, S., Delgado, F., Martin, D. C., Otto, K. J., Romero-Ortega, M., & Johnson, B. N. (2018, NA). A Hybrid 3D Printing and Robotic-assisted Embedding Approach for Design and Fabrication of Nerve Cuffs with Integrated Locking Mechanisms. In NA, 3, 2365-2372..
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  The ability to interface electronic materials with the peripheral nervous system is required for stimulation and monitoring of neural signals. Thus, the design and engineering of robust neural interfaces that maintain material-tissue contact in the presence of material or tissue micromotion offer the potential to conduct novel measurements and develop future therapies that require chronic interface with the peripheral nervous system. However, such remains an open challenge given the constraints of existing materials sets and manufacturing approaches for design and fabrication of neural interfaces. Here, we investigated the potential to leverage a rapid prototyping approach for the design and fabrication of nerve cuffs that contain supporting features to mechanically stabilize the interaction between cuff electrodes and peripheral nerve. A hybrid 3D printing and robotic-embedding (i.e., pick-and-place) system was used to design and fabricate silicone nerve cuffs (800 m diameter) containing conforming platinum (Pt) electrodes. We demonstrate that the electrical impedance of the cuff electrodes can be reduced by deposition of the conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) on cuff electrodes via a post-processing electropolymerization technique. The computer-aided design and manufacturing approach was also used to design and integrate supporting features to the cuff that mechanically stabilize the interface between the cuff electrodes and the peripheral nerve. Both 'self-locking' and suture-assisted locking mechanisms are demonstrated based on the principle of making geometric alterations to the cuff opening via 3D printing. Ultimately, this work shows 3D printing offers considerable opportunity to integrate supporting features, and potentially even novel electronic materials, into nerve cuffs that can support the design and engineering of next generation neural interfaces.
• Bredeson, S., Kanneganti, A., Deku, F., Cogan, S., Romero-Ortega, M., & Troyk, P. (2015). Chronic in-vivo testing of a 16-channel implantable wireless neural stimulator. In 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBC 2015, 2015-.
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  Here, we report on chronic in-vivo testing of a 16-channel wireless floating microelectrode array (WFMA) in a rat sciatic nerve model. Muscle threshold currents, charge injection levels, and charge density were monitored for electrodes of two WFMA devices implanted into animal subjects over a five month period. This type of wireless stimulation device could eliminate problems associated with percutaneous connectors for a variety of neural prostheses and other medical devices.
• Romero-Ortega, M., Kanneganti, A., Bendale, G., Seifert, J., Bredeson, S., Troyk, P., Deku, F., & Cogan, S. (2015). Chronic and low charge injection wireless intraneural stimulation in vivo. In 37th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBC 2015, 2015-.
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  Functional stability and in-vivo reliability are significant factors determining the longevity of a neural interface. In this ongoing study, we test the performance of a wireless floating microelectrode array (WFMA) over a period of 143 days. The topography of the microelectrodes has allowed for selective stimulation of different fascicles of the rat sciatic nerve. We confirmed that motor evoked thresholds remain stable over time and that the nerve stimulation charges were within tissue safety limits. Importantly, motor evoked responses were elicited at threshold currents in fully awake animals without causing pain or discomfort. These data validate the use of the WFMA system for intraneural interfacing of peripheral nerves for neuroprosthetic and bioelectronics medical applications.
• Troyk, P., Bredeson, S., Cogan, S., Romero-Ortega, M., Suh, S., Hu, Z., Kanneganti, A., Granja-Vazquez, R., Seifert, J., & Bak, M. (2015). In-vivo tests of a 16-channel implantable wireless neural stimulator. In 7th International IEEE/EMBS Conference on Neural Engineering, NER 2015, 2015-.
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  Wireless stimulation of neural tissue could enable many emerging neural prosthesis designs, and eliminate problems associated with percutaneous wires and connectors. Our laboratory has developed a 16-channel wireless floating microelectrode array (WFMA) for chronic implantation. Here, we report on its first use within in-vivo experiments, using a rat sciatic nerve model. Stimulus currents and associated muscular movements were determined for electrodes of two WFMA devices implanted into four animal subjects.
• Billo, R. E., Wilson, P. A., Priest, J. W., Romero-Ortega, M., Brunskill, S., & Keens, D. (2014). Slump molding inexpensive soda-lime glass to produce microchannel arrays. In 24th International Conference on Flexible Automation and Intelligent Manufacturing, FAIM 2014.
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  A slump molding process was developed to form microchannel geometries on the surface of soda-lime glass substrates. The process helped to overcome biological and chemical reactivity associated with current polymer lab-on-a-chip substrates, and permitted the use of low-cost soda-lime glass. A high speed micro mill and UV laser micromachining center produced negative geometries in the graphite mold material used as mold. An electric programmable kiln processed the soda-lime glass through a schedule of firing and cooling. Microchannel dimensions measured 50 μm width x 10 μm depth. The heating and cooling schedule required the soda-lime glass to slump into its final shape to these dimensions was determined and documented. Murine nerve tissue experiments validated the functionality of the slumping process and resultant soda-lime glass device.
• Desai, V. H., Anand, S., Tran, M., Kanneganti, A., Vasudevan, S., Seifert, J. L., Cheng, J., Keefer, E. W., & Romero-Ortega, M. I. (2014). Chronic sensory-motor activity in behaving animals using regenerative multi-electrode interfaces. In 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBC 2014.
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  Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control and feel of robotic prosthetic limbs. We have developed a Regenerative Multi-electrode Interface (REMI) that guides re-growing axons through an electrode array deployed in the lumen of a nerve guide. While acute studies have shown the use of the REMI in the rat sciatic nerve, the quality of chronic signal recording has not been reported. Here we show that implantation of this interface in the sciatic nerve is stable with high quality recordings up to 120 days and failures mainly attributable to abiotic factors related to pedestal detachment and wire breakage. We further tested the interfacing of REMI with fascicles of the sciatic nerve that primarily innervate muscles (tibial) and skin (sural). When implanted into the tibial nerve, bursting activity was observed synchronous to stepping. However, implantation of REMI into the sural nerve failed due to its small size. While fascicles smaller than 300 μm are a challenge for regenerative interfacing, we show that a modified REMI can be used in an insertion mode to record sensory signals from skin. In summary, the REMI represents an effective tool for recording firing patterns of specific axon types during voluntary movement, which may be used to improve the motor control and sensory feedback in closed loop control systems for robotic prosthesis.
• Kim, Y. T., Kanneganti, A., Fatemi, S., Nothnagle, C., Wijesundara, M., & Romero-Ortega, M. I. (2014). A novel Microchannel Electrode Array: Towards bioelectronic medical interfacing of small peripheral nerves. In 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBC 2014.
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  Bioelectronic medicine is an emerging field that relies on electrical signals to modulate complex neuronal circuits, particularly in the peripheral nervous system, as an alternative to drug-enabled therapeutics. Small autonomic nerves are one of the targets in this field, however, interfacing peripheral nerves smaller than 300 □m remains a challenge. Here we report the development of a Microchannel Electrode Array (□CEA) capable of interfacing nerve fascicles as small as 50-300□m. The current μCEA records and stimulates from 28 channels and is designed for easy implantation and removal, bearing promise to enable neural interfacing in BM.
• Farajidavar, A., McCorkle, P., Wiggins, T., Rao, S., Hagains, C., Peng, Y., Seifert, J., Romero, M., O'Grady, G., Cheng, L., Sparagana, S., Delgado, M., Tang, S., Abell, T., & Chiao, J. (2011). A miniature power-efficient bidirectional telemetric platform for in-vivo acquisition of electrophysiological signals. In 2011 IEEE MTT-S International Microwave Symposium, IMS 2011.
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  The need for in vivo wireless acquisition of biological signals is emerging in various medical fields. Electrophysiological applications including recording myoelectric signals in-vivo gastric electrical activity (GEA) to study gastric dysmotility, electrocorticography (ECoG) to study pain, and transcranical motor evoked potentials (TcMEP) for intraoperative neurophysiological monitoring of spinal cord integrity require physically miniaturized devices with low power consumption and capability of implantation. These systems should provide reliable communication in real time with sufficient data rates. We have developed three telemetric systems for GEA, ECoG and TcMEP applications based on a common transceiver platform but with different design considerations. Each has been successfully validated in appropriate animal models, to demonstrate the feasibility of wireless acquisition of key electrophysiological signals. © 2011 IEEE.
• Khan, B., Wildey, C., Francis, R., Tian, F., Romero, M., Delgado, M., Clegg, N., Smith, L., Liu, H., MacFarlane, D., & Alexandrakis, G. (2011). Functional near infrared brain imaging with a brush-fiber optode to improve optical contact on subjects with dense hair. In Photonic Therapeutics and Diagnostics VII, 7883.
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  A major obstacle in functional near infrared spectroscopy (fNIRS) and imaging is poor optical contact of the optodes with the scalp due to obstruction by hair. To overcome this problem a novel brush-fiber extension was made as an attachment to existing commercial flat-face fiber bundle optodes. The goal was that the brush-fiber extension would thread through hair and improve optical contact on subjects with dense hair. Simulations and experiments were performed to assess the magnitude of these improvements. Firstly, a Monte Carlo (MC) simulation of photon propagation through the scalp with hair and hair follicles was performed showing a drop in light transmission for different hair densities in the range of 3 to 9 dB. Similar levels of signal loss at individual optodes were determined experimentally in preliminary measurements on a human subject, when comparisons were made for finger tapping activation measurements with and without the brush-fiber extensions. The effects of such losses on reconstructed images were also simulated by using neutral density filters during tissue phantom measurements. As brush-fibers attenuated the signal by approximately 2.5 dB due to imperfect optical contact with the flat-face optode tips we explored the idea of using larger diameter brush-fibers to overcome that loss. © 2011 Copyright SPIE - The International Society for Optical Engineering.
• Lotfi, P., & Romero-Ortega, M. I. (2011). Control of neural interfacing in peripheral nerves through regenerative molecular guidance. In 33rd Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS 2011.
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  Regenerative peripheral nerve interfaces have been proposed as viable alternatives for the natural control of robotic prosthetic devices. However, random axonal pathfinding during peripheral nerve regeneration leads to mixed populations of sensory and motor neurons at the electrode interfaces preventing the precise identification of the modality nature of the recorded action potentials; motor or specific sensory sub-modalities. This study present evidence that supports the notion that type-specific neurotrophins can be used to preferentially entice and segregate the growth of defined axonal populations from transected peripheral nerves. Segregation of mixed sensory fibers from dorsal root ganglion neurons was evaluated in vitro by compartmentalized diffusion delivery of nerve growth factor (NGF) and neurotrophin-3 (NT-3), to preferentially entice the growth of trkA nociceptive and trkC proprioceptive subsets of sensory neurons, respectively. © 2011 IEEE.
• Romero-Ortega, M. I., & Ezaki, M. (2009). Nerve pathology in unregulated limb growth. In JBJS, 91, 53-57..
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  The causes of unregulated limb growth remain poorly understood. However, the converging fields of nerve-tumor biology and limb regeneration have provided evidence that points toward the participation of nerve pathology in some limb abnormalities. Copyright © 2009 by The Journal of Bone and Joint Surgery, Incorporated.
• Romero-Ortega, M. I., Butt, A. R., & Iqbal, S. M. (2009). Carbon nanotube coated high-throughput neurointerfaces in assistive environments. In 2nd International Conference on PErvasive Technologies Related to Assistive Environments, PETRA 2009.
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  Loosing motor activity due to impaired or damaged nerves or muscles affects millions of people world-wide. The resulting lack of mobility and/or impaired communication bears enormous personal, economical and social costs. While several assistive technologies exist, they rely on device surrogates to compensate for the lack of movement and thus provide limited utility and unnatural interface with the user. The ability of interfacing populations of neurons with super high-density multielectrode arrays (SD-MEA) can provide the sensing from and control of bionics devices by thought. Here we propose a neurointerfacing approach using SD-MEAs coated with carbon nanotubes and high-speed computing to overcome latency and long-term electrical viability bottlenecks that are essential in assistive environments. The proposed approach provides ability for fast integration of recording/stimulation from thousands of individually addressable electrodes, while coordinating a real-time computing approach to register, recognize, analyze and respond appropriately to the biological signals from the motor neurons and sensory signals from the robotic prosthesis. Copyright 2009 ACM.
• Kmecko, T., Hughes, G., Cauller, L., Lee, J. B., & Romero-Ortega, M. (2006). Nanocomposites for neural interfaces. In 2006 MRS Spring Meeting, 926.
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  We have fabricated micro-probes consisting of gold microelectrode sites (500 μm long and 12 μm wide) modified with conductive polymers and carbon nanotubes to achieve intimate contact with the nervous system. The fabrication process includes photolithography, electroplating and micromachining techniques. In order to obtain a high quality neural contact, we have investigated the preparation and characterization of neural interface materials. Electrochemical polymerization using potentiostatic and galvanostatic methods was used to optimize the surface of the metal electrode sites. Scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used to study the surface morphology, electrochemical properties, and stability of electrodeposited polymers. Cytotoxicity tests using fibroblasts and Schwann cells were performed to evaluate the biocompatibility of the micro-probes and neural interface materials. Normal rat kidney (NRK) cells and dorsal root ganglia (DRG) in vitro preparation was used to evaluate neuronal cell adhesion to the electrode. Polypyrrole (PPy), poly(3,4- ethylendioxythiophene) (PEDOT) were deposited onto microelectrode sites from aqueous solution with various thicknesses, dopants and electrochemical growth through self-assembly strategy to improve adhesion of PPy and PEDOT films to the electrode. The phenomenon of autoadsorption of thiolates on gold was used to anchor either monomer or CNT functionalization/dispersive agent to the electrode surface. Our results demonstrate that we can control the morphology, size and electrical properties of PPy and PEDOT by changing the polymerization conditions and adding dopant structures, such as chloride and CNTs. It was observed that the addition of carbon nanotubes favors the formation of nodules and increases the surface roughness. Also, electrochemical impedance spectroscopy revealed that conductive polymer composites lower the impedance of gold microelectrodes by three orders of magnitude. © 2006 Materials Research Society.
• Lu, H., Cho, S. H., Lee, J. B., Romero-Ortega, M., Cauller, L., & Hughes, G. (2006). SU8-based micro neural probe for enhanced chronic in-vivo recording of spike signals from regenerated axons. In 2006 5th IEEE Conference on Sensors.
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  In this paper, a fully biocompatible and flexible micro neural probe based on SU-8 to record neural spike signals from regenerated axons from amputated peripheral nerves is presented. The channel structures were designed at the tip of the probe to capture a single axon and guide its growth to ensure good quality contact between the single axon and the gold micro electrode. The gold electrodes placed at the bottom of the channel structures were exposed to allow contact with axons. The test results of in-vitro cytotoxicity tests showed that the SU-8 microprobe is biocompatible. Dorsal root ganglion (DRG) was used to evaluate neuronal cell adhesion to the microelectrodes. The probe was assembled into a fully biodegradable biosynthetic nerve regeneration guide (BNRG) filled with nerve growth factor (NGF) that promotes regeneration of specific sensory or motor axons. Neural spike signals from regenerated axons from amputated peripheral nerves were chronically recorded (> 6 months) without any sign of tissue damage. © 2006 IEEE.