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

• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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., 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.
• 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.
• 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.