RESUMEN
Our earlier work generated a powerful platform technology of polymeric scaffolding of stem cells to investigate and treat the injured or diseased central nervous system. However, the reciprocal sequelae between biophysical properties of the polymer and responses of the stem cell have not been examined in situ in lesioned spinal cords. We postulated that implantable synthetic scaffolds, acting through physical features, might affect donor cell behavior and host tissue remodeling. To test this hypothesis, poly(d,l-lactic-co-glycolic acid) (PLGA) in either low/soft or high/hard rigidity was fabricated for carrying adult human bone marrow mesenchymal stromal stem cells (hMSCs). The construct was transplanted into the epicenter of a rat model of acute T9-10 segmental hemisection to evaluate the effect of PLGA rigidity on the therapeutic potential and fate of hMSCs for neural repair. Compared to controls, only treatment with soft PLGA-scaffolded hMSCs significantly improved sensorimotor function via activation of recovery neurobiology mechanisms. The main benefits included inhibiting neuroinflammation and enhancing tissue protection. Also detected in the treated lesion region were expressions of neurotrophic and anti-inflammatory factors together with proliferation of endogenous neural stem cells, impacts likely derived from hMSCs' functional multipotency maintained by soft PLGA-scaffolding. Conversely, hard rigidity PLGA activated mechanotransduction and mesoderm lineage differentiation of hMSCs that ectopically produced bone, cartilage and muscle markers in neural parenchyma. The findings collectively suggested that the physical texture of polymeric scaffolds should be tailored for sustaining the stemness of hMSCs to constructively interact with the spinal cord for functional restoration.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas/métodos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Traumatismos de la Médula Espinal , Andamios del Tejido/química , Animales , Femenino , Humanos , Ratas , Ratas Sprague-DawleyRESUMEN
Human mesenchymal stromal stem cells (hMSCs) hold regenerative medicine potential due to their availability, in vitro expansion readiness, and autologous feasibility. For neural repair, hMSCs show translational value in research on stroke, spinal cord injury (SCI), and traumatic brain injury. It is pivotal to establish multimodal in vitro systems to investigate molecular mechanisms underlying neural actions of hMSCs. Here, we describe a platform protocol on how to set up organotypic co-cultures of hMSCs (alone or polymer-scaffolded) with explanted adult rat dorsal root ganglia (DRGs) to determine neural injury and recovery events for designing implants to counteract neurotrauma sequelae. We emphasize in vitro hMSC propagation, polymer scaffolding, hMSC stemness maintenance, hMSC-DRG interaction profiling, and analytical formulas of neuroinflammation, trophic factor expression, DRG neurite outgrowth and tropic tracking, and in vivo verification of tailored implants in rodent models of SCI. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Técnicas de Cocultivo/métodos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Traumatismos del Sistema Nervioso/terapia , Animales , Ganglios Espinales/citología , Ganglios Espinales/lesiones , Humanos , RatasRESUMEN
BACKGROUND: Adipose tissue-derived mesenchymal stem cells (AT-MSCs) offer potential as a therapeutic option for chronic discogenic low back pain (LBP) because of their immunomodulatory functions and capacity for cartilage differentiation. The goal of this study was to assess the safety and tolerability of a single intradiscal implantation of combined AT-MSCs and hyaluronic acid (HA) derivative in patients with chronic discogenic LBP. METHODS: We performed a single-arm phase I clinical trial with a 12-month follow-up and enrolled 10 eligible chronic LBP patients. Chronic LBP had lasted for more than 3 months with a minimum intensity of 4/10 on a visual analogue scale (VAS) and disability level ≥ 30% on the Oswestry Disability Index (ODI). The 10 patients underwent a single intradiscal injection of combined HA derivative and AT-MSCs at a dose of 2 × 107 cells/disc (n = 5) or 4 × 107 cells/disc (n = 5). Safety and treatment outcomes were evaluated by assessing VAS, ODI, Short Form-36 (SF-36), and imaging (lumbar spine X-ray imaging and MRI) at regular intervals over 1 year. RESULTS: No patients were lost at any point during the 1-year clinical study. We observed no procedure or stem cell-related adverse events or serious adverse events during the 1-year follow-up period. VAS, ODI, and SF-36 scores significantly improved in both groups receiving both low (cases 2, 4, and 5) and high (cases 7, 8, and 9) cell doses, and did not differ significantly between the two groups. Among six patients who achieved significant improvement in VAS, ODI, and SF-36, three patients (cases 4, 8, and 9) were determined to have increased water content based on an increased apparent diffusion coefficient on diffusion MRI. CONCLUSIONS: Combined implantation of AT-MSCs and HA derivative in chronic discogenic LBP is safe and tolerable. However, the efficacy of combined AT-MSCs and HA should be investigated in a randomized controlled trial in a larger population. TRIAL REGISTRATION: ClinicalTrials.gov NCT02338271 . Registered 7 January 2015.
Asunto(s)
Ácido Hialurónico/uso terapéutico , Degeneración del Disco Intervertebral/terapia , Dolor de la Región Lumbar/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Tejido Adiposo/fisiología , Adulto , Diferenciación Celular , Enfermedad Crónica , Femenino , Estudios de Seguimiento , Humanos , Inyecciones Intralesiones , Disco Intervertebral/efectos de los fármacos , Disco Intervertebral/patología , Disco Intervertebral/fisiopatología , Degeneración del Disco Intervertebral/patología , Degeneración del Disco Intervertebral/fisiopatología , Dolor de la Región Lumbar/patología , Dolor de la Región Lumbar/fisiopatología , Masculino , Células Madre Mesenquimatosas/fisiología , Persona de Mediana Edad , Dimensión del Dolor , Seguridad del Paciente , Trasplante Autólogo , Resultado del Tratamiento , Agua/metabolismoRESUMEN
Mesenchymal stromal stem cells (MSCs) isolated from adult tissues offer tangible potential for regenerative medicine, given their feasibility for autologous transplantation. MSC research shows encouraging results in experimental stroke, amyotrophic lateral sclerosis, and neurotrauma models. However, further translational progress has been hampered by poor MSC graft survival, jeopardizing cellular and molecular bases for neural repair in vivo. We have devised an adult human bone marrow MSC (hMSC) delivery formula by investigating molecular events involving hMSCs incorporated in a uniquely designed poly(lactic-co-glycolic) acid scaffold, a clinically safe polymer, following inflammatory exposures in a dorsal root ganglion organotypic coculture system. Also, in rat T9-T10 hemisection spinal cord injury (SCI), we demonstrated that the tailored scaffolding maintained hMSC stemness, engraftment, and led to robust motosensory improvement, neuropathic pain and tissue damage mitigation, and myelin preservation. The scaffolded nontransdifferentiated hMSCs exerted multimodal effects of neurotrophism, angiogenesis, neurogenesis, antiautoimmunity, and antiinflammation. Hindlimb locomotion was restored by reestablished integrity of submidbrain circuits of serotonergic reticulospinal innervation at lumbar levels, the propriospinal projection network, neuromuscular junction, and central pattern generator, providing a platform for investigating molecular events underlying the repair impact of nondifferentiated hMSCs. Our approach enabled investigation of recovery neurobiology components for injured adult mammalian spinal cord that are different from those involved in normal neural function. The uncovered neural circuits and their molecular and cellular targets offer a biological underpinning for development of clinical rehabilitation therapies to treat disabilities and complications of SCI.
Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Traumatismos de la Médula Espinal/terapia , Adulto , Animales , Técnicas de Cocultivo , Femenino , Ganglios Espinales , Miembro Posterior , Humanos , Ácido Láctico , Lipopolisacáridos , Locomoción , Masculino , Células Madre Mesenquimatosas , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas Sprague-Dawley , Recuperación de la Función , Traumatismos de la Médula Espinal/fisiopatología , Andamios del TejidoRESUMEN
Diverse mechanisms including activation of NMDA receptors, microglial activation, reactive astrogliosis, loss of descending inhibition, and spasticity are responsible for â¼40% of cases of intractable neuropathic pain after spinal cord injury (SCI). Because conventional treatments blocking individual mechanisms elicit only short-term effectiveness, a multimodal approach with simultaneous actions against major pain-related pathways may have value for clinical management of chronic pain. We hypothesize that [-]-huperzine A (HUP-A), an alkaloid isolated from the club moss Huperzia serrata, that is a potent reversible inhibitor of acetylcholinesterase and NMDA receptors, could mitigate pain without invoking drug tolerance or dependence by stimulating cholinergic interneurons to impede pain signaling, inhibiting inflammation via microglial cholinergic activation, and blocking NMDA-mediated central hypersensitization. We tested our hypothesis by administering HUP-A i.p. or intrathecally to female Sprague-Dawley rats (200-235 g body weight) after moderate static compression (35 g for 5 min) of T10 spinal cord. Compared with controls, HUP-A treatment demonstrates significant analgesic effects in both regimens. SCI rats manifested no drug tolerance following repeated bolus i.p. or chronic intrathecal HUP-A dosing. The pain-ameliorating effect of HUP-A is cholinergic dependent. Relative to vehicle treatment, HUP-A administration also reduced neural inflammation, retained higher numbers of calcium-impermeable GluR2-containing AMPA receptors, and prevented Homer1a up-regulation in dorsal horn sensory neurons. Therefore, HUP-A may provide safe and effective management for chronic postneurotrauma pain by reestablishing homeostasis of sensory circuits.
Asunto(s)
Alcaloides/uso terapéutico , Dolor/tratamiento farmacológico , Sesquiterpenos/uso terapéutico , Compresión de la Médula Espinal/complicaciones , Animales , Conducta Animal , Femenino , Dolor/etiología , Ratas , Ratas Sprague-Dawley , Compresión de la Médula Espinal/fisiopatologíaRESUMEN
We sought to determine whether neural stem cells (NSCs) can be isolated from the amniotic fluid in the setting of neural tube defects (NTDs), as a prerequisite for eventual autologous perinatal therapies. Pregnant Sprague-Dawley dams (n=62) were divided into experimental (n=42) and control (n=20) groups, depending on prenatal exposure to retinoic acid for the induction of fetal NTDs. Animals were killed before term for analysis (n=685 fetuses). Amniotic fluid samples from both groups underwent epigenetic selection for NSCs, followed by exposure to neural differentiation media. Representative cell samples underwent multiple morphological and phenotypical analyses at different time points. No control fetus (n=267) had any structural abnormality, whereas at least one type of NTD developed in 52% (217/418) of the experimental fetuses (namely, isolated spina bifida, n=144; isolated exencephaly, n=24; or a combination of the two, n=49). Only amniotic samples from fetuses with a NTD yielded cells with typical neural progenitor morphology and robust expression of both Nestin and Sox-2, primary markers of NSCs. These cells responded to differentiation media by displaying typical morphological changes, along with expression of beta-tubulin III, glial fibrillary acidic protein, and/or O4, markers for immature neurons, astrocytes, and oligodendrocytes, respectively. This was concurrent with downregulation of Nestin and Sox-2. We conclude that the amniotic fluid can harbor disease-specific stem cells, for example, NSCs in the setting of experimental NTDs. The amniotic fluid may be a practical source of autologous NSCs applicable to novel forms of therapies for spina bifida.
Asunto(s)
Líquido Amniótico , Células-Madre Neurales , Disrafia Espinal , Líquido Amniótico/citología , Líquido Amniótico/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Diferenciación Celular , Modelos Animales de Enfermedad , Femenino , Feto/citología , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/trasplante , Embarazo , Ratas , Ratas Sprague-Dawley , Disrafia Espinal/embriología , Disrafia Espinal/patología , Disrafia Espinal/terapia , Trasplante Autólogo , Trasplante HomólogoRESUMEN
Genetic modification of stem cells could be applied to initiate/enhance their secretion of therapeutic molecules, alter their biological properties, or label them for in vivo tracking. We recently developed a negatively charged gene carrier ("anioplex") based on pullulan-spermine, a conjugate prepared from a natural polysaccharide and polyamine. In rat mesenchymal stem cells (MSCs), anioplex-derived reporter gene activity was comparable to or exceeded that obtained using a commercial cationic lipid reagent. Transfection in the growth medium with 15% serum and antibiotics was approximately sevenfold more effective than in serum-free conditions. Cytotoxicity was essentially indiscernible after 24 h of anioplex transfection with 20 µg/mL DNA, in contrast to cationic lipid transfection that resulted in 40%-60% death of target MSCs. Anioplex-derived reporter gene activity persisted throughout the entire 3-week study, with post-transfection MSCs appearing to maintain osteogenic, adipogenic, and chondrogenic multipotency. In particular, chondrogenic pellet formation of differentiating human MSCs was significantly inhibited after lipofection but not after aniofection, which further indicates the biological inertness of pullulan-spermine/DNA anioplexes. Collectively, these data introduce a straightforward technology for genetic engineering of adult stem/progenitor cells under physiological niche-like conditions. Moreover, reporter gene activity was observed in rat spinal cords after minimally invasive intrathecal implantation, suggesting effective engraftment of donor MSCs. It is therefore plausible that anioplex-transfected MSCs or other stem/progenitor cells with autologous potential could be applied to disorders such as neurotrauma or neuropathic pain that involve the spinal cord and brain.
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ADN/metabolismo , Ingeniería Genética/métodos , Glucanos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Suero/metabolismo , Espermina/metabolismo , Tejido Adiposo/citología , Animales , Transporte Biológico , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Muerte Celular , Diferenciación Celular , Supervivencia Celular , Humanos , Espacio Intracelular/metabolismo , Células Madre Mesenquimatosas/citología , Ratas , Ratas Wistar , Médula Espinal/patología , Células del Estroma/citología , Células del Estroma/metabolismo , Células del Estroma/trasplante , TransfecciónRESUMEN
Recently, the binding sequence Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) was found adjacent to the RGD sequence in osteopontin, suggesting involvement in osteo-immune cross-talk. The aim of this study was to investigate bioactive functions of a synthetic SVVYGLR peptide in osteoprogenitor cells and osteoclasts, and to examine potential applications in bone regeneration. The SVVYGLR peptide significantly enhanced the adhesion and proliferation of several human mesenchymal cells including bone marrow-derived mesenchymal stem cells, and alphavbeta3 integrin was involved in cell attachment to the peptide. Additionally, the peptide reduced the number of TRAP-positive multinucleated cells during osteoclastogenesis of RAW264.7 cells and normal murine pre-osteoclasts, and also suppressed NFAT activity and expression of osteoclastogenesis-related mRNAs. When standardized bone defects in rat calvariae were filled with a collagen sponge containing the peptide or PBS (control), the number of TRAP-positive osteoclasts in the grafted sites after 3 weeks was significantly lower in the peptide group. By the 5th week, significantly enhanced resorption of the grafted collagen sponge and new bone formation was observed within and surrounding the sponge in the peptide group. These data suggest that SVVYGLR is an effective bioactive peptide for bone tissue regeneration that promotes attachment and proliferation of osteogenic cells while also suppressing osteoclastogenesis.
Asunto(s)
Regeneración Ósea/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Péptidos/química , Péptidos/farmacología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Calcificación Fisiológica/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Integrina alfaVbeta3 , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Datos de Secuencia Molecular , Osteoclastos/citología , Osteogénesis/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Cráneo/efectos de los fármacos , Cráneo/patología , Células Madre/citologíaRESUMEN
BACKGROUND: Neuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury. RESULTS: After unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states. CONCLUSION: Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Dolor/genética , Dolor/fisiopatología , Nervio Ciático/metabolismo , Nervio Ciático/fisiopatología , Canales de Sodio/genética , Regulación hacia Arriba/genética , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Masculino , Canal de Sodio Activado por Voltaje NAV1.8 , Síndromes de Compresión Nerviosa/inducido químicamente , Síndromes de Compresión Nerviosa/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Poliadenilación/efectos de los fármacos , Transporte de ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Nervio Ciático/efectos de los fármacos , Canales de Sodio/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
Nonviral gene transfer to neurons remains unreliable due to a lack of effective and nontoxic vectors. Here, we achieved effective neuronal gene delivery through salt-free complexation of plasmid DNA and pullulan-spermine, a conjugate prepared from a naturally derived polysaccharide and polyamine. Specifically, at low spermine nitrogen:DNA phosphate (N:P) ratios, complexes formed with zeta-potential and diameter of approximately-40mV and 350nm, respectively. Higher N:P ratios increased the zeta-potential to approximately +10mV. All complexes were stable for at least 1 week and protected DNA from degradation. In vitro transfection of rat sensory neurons occurred at all N:P ratios, but uniquely, efficiency was highest for anionic complexes (anioplexes). Subsequent analyses revealed the inhibition of reporter gene expression by asialofetuin (1mg/ml) and methyl-beta-cyclodextrin (5mm), indicating utilization of glycoprotein-specific interactions and lipid rafts for uptake and intracellular trafficking. In marked contrast to a commercial cationic lipid reagent, anioplexes did not exhibit measurable cytotoxicity at up to 20microg/ml DNA. Additionally, transfection efficiency was maintained in the presence of serum and antibiotics. Based on these favorable properties, we successfully established two transfection methods for cultured adult sensory neurons and tissue explants. Collectively, these data suggest that negatively charged pullulan-spermine/DNA anioplexes could represent an effective gene delivery technology, particularly for neurons.
Asunto(s)
ADN/química , Técnicas de Transferencia de Gen , Glucanos/química , Neuronas/metabolismo , Espermina/química , Animales , Muerte Celular/efectos de los fármacos , ADN/toxicidad , Endocitosis/efectos de los fármacos , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Glucanos/toxicidad , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Ratas , Receptores de Superficie Celular/metabolismo , Suero , Espermina/toxicidad , TransfecciónRESUMEN
Bone marrow- and adipose tissue-derived stromal cells (BMSCs and ASCs, respectively) exhibit a similar capacity for osteogenic differentiation in vitro, but it is unclear whether they share a common differentiation process, because they originate from different tissues. The aim of this study was to explore BMSC and ASC osteogenic differentiation by focusing on the expression of extracellular matrix-related genes (ECMGs), which play a crucial role in osteogenesis and bone tissue regeneration in vivo. We characterized the gene expression profiles of BMSCs and ASCs using a custom complementary deoxyribonucleic acid microarray containing 55 ECMGs. Undifferentiated BMSCs and ASCs actively expressed a wide range of ECMGs. Once BMSCs and ASCs were placed in an osteogenic differentiation medium, 24 and 17 ECMGs, respectively, underwent considerable downregulation over the course of the culture period. The remaining genes were maintained at a similar expression level to corresponding uninduced cell cultures. Although the suppression phenomenon was consistent irrespective of stromal cell origin, collagen (COL)2A1, COL6A1, COL9A1, parathyroid hormone receptor, integrin (INT)-beta3, and TenascinX genes were only downregulated in osteogenic BMSCs, whereas COL1A2, COL3A1, COL4A1, COL5A2, COL15A1, osteopontin, osteonectin, and INT-beta1 genes were only downregulated in osteogenic ASCs. During this time period, cell viability was sustained, suggesting that the observed downregulation did not occur by selection and elimination of unfit cells from the whole cell population. These data suggest that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern, reflecting their multipotency toward a configuration specifically meeting the requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues.
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Adipocitos/citología , Adipocitos/fisiología , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Familia de Multigenes/fisiología , Osteogénesis/fisiología , Diferenciación Celular , Células Cultivadas , Humanos , Células del Estroma/citología , Células del Estroma/fisiologíaRESUMEN
Selective modulation of sensory neuron gene expression could have numerous applications for the peripheral nervous system. Here, we report that subcutaneous peripheral injection of plasmid DNA complexed with a non-viral cationized gelatin (CG) vector led to transgene expression in rat lumbar dorsal root ganglia (DRGs). CG/DNA polyplexes appeared to undergo rapid retrograde transport through sciatic and spinal nerves, with reporter gene messenger RNA (mRNA) expression detectable in L4 and L5 DRGs within 60 hours. Maximum transgene expression was observed for polyplexes formed at 7.5:1 CG-to-DNA weight ratio under salt-free conditions, which generated 615 +/- 112 nm nanoparticles with zeta-potential of 9.4 +/- 0.19 mV. Six days after injection of the CG/DNA polypex, reporter gene protein immunofluorescence was observed in 1,164 +/- 176 DRG neurons, representing an estimated transfection rate of 47% of targeted neurons. Reporter gene expression was not detected in heart, lung, or liver tissues, suggesting a lack of systemic uptake. Measurements of tactile sensitivity indicate that CG/DNA injection did not cause behavioral toxicity. The injection platform was further used for plasmid-driven short hairpin RNA-mediated suppression of glyceraldehyde-3-phosphate dehydrogenase. This non-invasive gene delivery system could be used for the mechanistic study and targeted molecular evaluation of peripheral nervous system pathologies such as neuropathic pain.