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1.
J Neurotrauma ; 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33397170

RESUMO

After a severe, high-level spinal cord injury (SCI), plasticity to intraspinal circuits below injury results in heightened spinal sympathetic reflex activity and detrimentally impacts peripheral organ systems. Such sympathetic hyperreflexia is immediately apparent as an episode of autonomic dysreflexia (AD), a life-threatening condition characterized by sudden hypertension and reflexive bradycardia following below-level sensory inputs; for example, pressure sores or impacted fecal matter. Over time, plasticity within the spinal sympathetic reflex (SSR) circuit contributes to the progressive intensification of AD events, as the frequency and severity of AD events increase greatly beginning ∼2 weeks post-injury (wpi). The neuroimmune system has been implicated in driving sympathetic hyperreflexia, as inhibition of the cytokine soluble tumor necrosis factor-alpha (sTNFα) using the biological mimetic XPro1595 beginning within days post-SCI has been shown to attenuate the development of AD. Here, we sought to further understand the effective therapeutic time window of XPro1595 to diminish sympathetic hyperreflexia, as indicated by AD. We delayed the commencement of continuous intrathecal administration of XPro1595 until 2 weeks after a complete, thoracic level 3 injury in adult rats. We examined the severity of colorectal distension-induced AD biweekly. We found that initiation of sTNFα inhibition at 2 wpi does not attenuate the severity or intensification of sympathetic hyperreflexia compared with saline-treated controls. Coupled with previous data from our group, these findings suggest that central sTNFα signaling must be targeted prior to 2 weeks post-SCI in order to decrease sympathetic hyperreflexia.

2.
Nat Commun ; 11(1): 6131, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33257677

RESUMO

After a dorsal root crush injury, centrally-projecting sensory axons fail to regenerate across the dorsal root entry zone (DREZ) to extend into the spinal cord. We find that chemogenetic activation of adult dorsal root ganglion (DRG) neurons improves axon growth on an in vitro model of the inhibitory environment after injury. Moreover, repeated bouts of daily chemogenetic activation of adult DRG neurons for 12 weeks post-crush in vivo enhances axon regeneration across a chondroitinase-digested DREZ into spinal gray matter, where the regenerating axons form functional synapses and mediate behavioral recovery in a sensorimotor task. Neuronal activation-mediated axon extension is dependent upon changes in the status of tubulin post-translational modifications indicative of highly dynamic microtubules (as opposed to stable microtubules) within the distal axon, illuminating a novel mechanism underlying stimulation-mediated axon growth. We have identified an effective combinatory strategy to promote functionally-relevant axon regeneration of adult neurons into the CNS after injury.

3.
J Neurotrauma ; 2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33297828

RESUMO

Traumatic spinal cord injury (SCI) often causes micturition dysfunction. We recently discovered a low level of spinally-derived dopamine (DA) that regulates recovered bladder and sphincter reflexes in SCI female rats. Considering substantial sexual dimorphic features in the lower urinary tract, it is unknown if the DA-ergic mechanisms act in the male. Histological analysis showed a similar distribution of tyrosine hydroxylase (TH)+ neurons in the lower cord of male rats and the number increased following thoracic SCI. Subsequently, focal electrical stimulation in slices obtained from L6/S1 spinal segments of SCI rats elicited detectable DA release with fast scan cyclic voltammetry. Using bladder cystometrogram and external urethral sphincter (EUS) electromyography in SCI male rats, intravenous (i.v.) administration of SCH 23390, a D1-like receptor (DR1) antagonist, induced significantly increased tonic EUS activity and a trend of increased residual volume, whereas activation of these receptors with SKF 38393 did not influence the reflex. Meanwhile, blocking spinal D2-like receptors (DR2) with remoxipride had no effect but stimulating these receptors with quinpirole elicited EUS bursting to increase voiding volume. Furthermore, intrathecal (i.t.) delivery of SCH 23390 and quinpirole resulted in similar responses to those with i.v. delivery, respectively, which indicates the central action regardless of delivery route. In addition, metabolic cage assays showed that quinpirole increased the voiding frequency and total voiding volume in spontaneous micturition. Collectively, spinal DA-ergic machinery regulates recovered micturition reflex following SCI in male rats; spinal DR1 tonically suppress tonic EUS activity to enable voiding and activation of DR2 facilitates voiding.

4.
Front Cell Neurosci ; 14: 187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32792908

RESUMO

Following an injury to the central nervous system (CNS), spontaneous plasticity is observed throughout the neuraxis and affects multiple key circuits. Much of this spontaneous plasticity can elicit beneficial and deleterious functional outcomes, depending on the context of plasticity and circuit affected. Injury-induced activation of the neuroimmune system has been proposed to be a major factor in driving this plasticity, as neuroimmune and inflammatory factors have been shown to influence cellular, synaptic, structural, and anatomical plasticity. Here, we will review the mechanisms through which the neuroimmune system mediates plasticity after CNS injury. Understanding the role of specific neuroimmune factors in driving adaptive and maladaptive plasticity may offer valuable therapeutic insight into how to promote adaptive plasticity and/or diminish maladaptive plasticity, respectively.

5.
J Neurosci ; 40(6): 1248-1264, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31896670

RESUMO

Cardiovascular dysfunction often occurs after high-level spinal cord injury. Disrupting supraspinal vasomotor pathways affects basal hemodynamics and contributes to the development of autonomic dysreflexia (AD). Transplantation of early-stage neurons to the injured cord may reconstruct the descending projections to enhance cardiovascular performance. To determine the specific role of reestablishing serotonergic regulation of hemodynamics, we implanted serotonergic (5-HT+) neuron-enriched embryonic raphe nucleus-derived neural stem cells/progenitors (RN-NSCs) into a complete spinal cord transection lesion site in adult female rats. Grafting embryonic spinal cord-derived NSCs or injury alone served as 2 controls. Ten weeks after injury/grafting, histological analysis revealed well-survived grafts and partial integration with host tissues in the lesion site. Numerous graft-derived serotonergic axons topographically projected to the caudal autonomic regions. Neuronal tracing showed that host supraspinal vasomotor pathways regenerated into the graft, and 5-HT+ neurons within graft and host brainstem neurons were transsynaptically labeled by injecting pseudorabies virus (PRV-614) into the kidney, indicating reconnected serotonergic circuits regulating autonomic activity. Using an implanted telemeter to record cardiovascular parameters, grafting RN-NSCs restored resting mean arterial pressure to normal levels and remarkably alleviated naturally occurring and colorectal distension-induced AD. Subsequent pharmacological blockade of 5-HT2A receptors with ketanserin in RN-NSC-grafted rats reduced resting mean arterial pressure and increased heart rate in all but 2 controls. Furthermore, spinal cord retransection below RN-NSC grafts partially eliminated the recovery in AD. Collectively, these data indicate that RN-NSCs grafted into a spinal cord injury site relay supraspinal control of serotonergic regulation for sympathetic activity to improve cardiovascular function.SIGNIFICANCE STATEMENT Disruption of supraspinal vasomotor pathways results in cardiovascular dysfunction following high-level spinal cord injury. To reestablish the descending regulation of autonomic function, we transplanted serotonergic neuron enriched embryonic raphe nucleus-derived neural stem cells/progenitors into the lesion site of completely transected rat spinal cord. Consequently, grafted raphe nucleus-derived neural stem cells/progenitors acted as a neuronal relay to reconnect supraspinal center and spinal sympathetic neurons below the injury. The reconstituted serotonergic regulation of sympathetic activity led to the improvement of hemodynamic parameters and mitigated autonomic dysreflexia. Based on morphological and physiological results, this study validates the effectiveness of transplanting early-stage serotonergic neurons into the spinal cord for cardiovascular functional recovery after spinal cord injury.


Assuntos
Disreflexia Autonômica/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Hemodinâmica/fisiologia , Células-Tronco Neurais/transplante , Neurônios Serotoninérgicos/transplante , Animais , Células-Tronco Embrionárias/transplante , Feminino , Núcleos da Rafe/citologia , Ratos , Ratos Endogâmicos F344 , Traumatismos da Medula Espinal/fisiopatologia , Transplante de Células-Tronco/métodos , Sistema Nervoso Simpático/fisiopatologia
6.
J Neurosci ; 40(2): 478-492, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31754014

RESUMO

Spinal cord injury (SCI) disrupts critical physiological systems, including the cardiovascular and immune system. Plasticity of spinal circuits below the injury results in abnormal, heightened sympathetic responses, such as extreme, sudden hypertension that hallmarks life-threatening autonomic dysreflexia. Moreover, such sympathetic hyperreflexia detrimentally impacts other effector organs, including the spleen, resulting in spinal cord injury-induced immunodeficiency. Consequently, infection is a leading cause of mortality after SCI. Unfortunately, there are no current treatments that prophylactically limit sympathetic hyperreflexia to prevent subsequent effector organ dysfunction. The cytokine soluble tumor necrosis factor α (sTNFα) is upregulated in the CNS within minutes after SCI and remains elevated. Here, we report that commencing intrathecal administration of XPro1595, an inhibitor of sTNFα, at a clinically feasible, postinjury time point (i.e., 3 d after complete SCI) sufficiently diminishes maladaptive plasticity within the spinal sympathetic reflex circuit. This results in less severe autonomic dysreflexia, a real-time gauge of sympathetic hyperreflexia, for months postinjury. Remarkably, delayed delivery of the sTNFα inhibitor prevents sympathetic hyperreflexia-associated splenic atrophy and loss of leukocytes to dramatically improve the endogenous ability of chronic SCI rats to fight off pneumonia, a common cause of hospitalization after injury. The improved immune function with XPro1595 correlates with less noradrenergic fiber sprouting and normalized norepinephrine levels in the spleen, indicating that heightened, central sTNFα signaling drives peripheral, norepinephrine-mediated organ dysfunction, a novel mechanism of action. Thus, our preclinical study supports intrathecally targeting sTNFα as a viable strategy to broadly attenuate sympathetic dysregulation, thereby improving cardiovascular regulation and immunity long after SCI.SIGNIFICANCE STATEMENT Spinal cord injury (SCI) significantly disrupts immunity, thus increasing susceptibility to infection, a leading cause of morbidity in those living with SCI. Here, we report that commencing intrathecal administration of an inhibitor of the proinflammatory cytokine soluble tumor necrosis factor α days after an injury sufficiently diminishes autonomic dysreflexia, a real time gauge of sympathetic hyperreflexia, to prevent associated splenic atrophy. This dramatically improves the endogenous ability of chronically injured rats to fight off pneumonia, a common cause of hospitalization. This preclinical study could have a significant impact for broadly improving quality of life of SCI individuals.


Assuntos
Disreflexia Autonômica/etiologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Feminino , Injeções Espinhais , Pneumonia Bacteriana/etiologia , Pneumonia Bacteriana/prevenção & controle , Ratos , Ratos Wistar , Baço/efeitos dos fármacos , Baço/imunologia , Fator de Necrose Tumoral alfa/farmacologia
7.
J Neurosci ; 39(11): 2011-2024, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30647150

RESUMO

Fidgetin is a microtubule-severing protein that pares back the labile domains of microtubules in the axon. Experimental depletion of fidgetin results in elongation of the labile domains of microtubules and faster axonal growth. To test whether fidgetin knockdown assists axonal regeneration, we plated dissociated adult rat DRGs transduced using AAV5-shRNA-fidgetin on a laminin substrate with spots of aggrecan, a growth-inhibitory chondroitin sulfate proteoglycan. This cell culture assay mimics the glial scar formed after CNS injury. Aggrecan is more concentrated at the edge of the spot, such that axons growing from within the spot toward the edge encounter a concentration gradient that causes growth cones to become dystrophic and axons to retract or curve back on themselves. Fidgetin knockdown resulted in faster-growing axons on both laminin and aggrecan and enhanced crossing of axons from laminin onto aggrecan. Strikingly, axons from within the spot grew more avidly against the inhibitory aggrecan concentration gradient to cross onto laminin, without retracting or curving back. We also tested whether depleting fidgetin improves axonal regeneration in vivo after a dorsal root crush in adult female rats. Whereas control DRG neurons failed to extend axons across the dorsal root entry zone after injury, DRG neurons in which fidgetin was knocked down displayed enhanced regeneration of axons across the dorsal root entry zone into the spinal cord. Collectively, these results establish fidgetin as a novel therapeutic target to augment nerve regeneration and provide a workflow template by which microtubule-related targets can be compared in the future.SIGNIFICANCE STATEMENT Here we establish a workflow template from cell culture to animals in which microtubule-based treatments can be tested and compared with one another for their effectiveness in augmenting regeneration of injured axons relevant to spinal cord injury. The present work uses a viral transduction approach to knock down fidgetin from rat neurons, which coaxes nerve regeneration by elevating microtubule mass in their axons. Unlike previous strategies using microtubule-stabilizing drugs, fidgetin knockdown adds microtubule mass that is labile (rather than stable), thereby better recapitulating the growth status of a developing axon.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/fisiologia , Axônios/fisiologia , Gânglios Espinais/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Regeneração Nervosa/fisiologia , Proteínas Nucleares/fisiologia , ATPases Associadas a Diversas Atividades Celulares/genética , Agrecanas/fisiologia , Animais , Feminino , Técnicas de Silenciamento de Genes , Masculino , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Neuroglia/fisiologia , Proteínas Nucleares/genética , Ratos Sprague-Dawley
8.
J Neurotrauma ; 36(9): 1478-1486, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30362884

RESUMO

Selection of a proper spinal cord injury (SCI) rat model to study therapeutic effects of cell transplantation is imperative for research in cardiovascular functional recovery, due to the local harsh milieu inhibiting cell growth. We recently found that a crushed spinal cord lesion can minimize fibrotic scarring and grafted cell death compared with open-dura injuries. To determine if this SCI model is applicable for studying cardiovascular recovery, we examined hemodynamic consequences following crushed SCI and tested cardiovascular responses to serotonin (5-HT) or dopamine (DA) receptor agonists. Using a radio-telemetric system, multiple cardiovascular parameters were recorded prior to, 2, and 4 weeks after SCI, including resting mean arterial pressure (MAP) and heart rate (HR), as well as spontaneous or colorectal distension (CRD)-induced autonomic dysreflexia (AD). The results showed that this injury caused tachycardia at rest as well as the occurrence of spontaneous or artificially induced dysreflexic events. Four weeks post-injury, specific activation of 5-HT2A receptors by subcutaneous (s.c.) or intrathecal (i.t.) delivery of Dimethoxy-4-iodoamphetamine (DOI) remarkably increased resting MAP levels in a dose-dependent fashion. During CRD-induced autonomic dysreflexia, systemic administration of DOI alleviated the severity of bradycardia responsive to episodic hypertension. In contrast, selective stimulation of 5-HT1A receptors with 8-OH-DPAT or non-selective activation of DA receptors with apomorphine did not affect cardiovascular performance. Thus, crush injuries induce cardiovascular abnormalities in rats that are sensitive to 5-HT2A receptor stimulation, indicating a reliable SCI model to study how cell-based approaches impact the severity of autonomic dysreflexia and identify a possible target for pharmacological interventions.


Assuntos
Disreflexia Autonômica/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Hemodinâmica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Disreflexia Autonômica/etiologia , Modelos Animais de Doenças , Feminino , Compressão Nervosa , Ratos , Ratos Endogâmicos F344 , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Traumatismos da Medula Espinal/complicações
9.
Neurourol Urodyn ; 37(8): 2487-2494, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29999191

RESUMO

AIMS: This study was designed to determine specific cell groups of the raphe nuclei (RN) that give rise to supraspinal serotonergic projections regulating the bladder reflex. METHODS: Anesthetized rats underwent surgery to open the abdomen and expose the bladder. A total of 6 µL transsynaptic neuronal tracer pseudorabies virus (PRV-152), encoding for green fluorescent protein (GFP), was injected into the bladder detrusor. After 72 or 96 h, animals were perfused and the brain was dissected for processing transverse and sagittal sections. Subsequently, fluorescent immunohistochemistry for GFP and Serotonin (5-hydroxytryptamine [5-HT]) was performed in the brain sections. Under the microscope, each RN subset was characterized individually from caudal to rostral according to the atlas. GFP+ or GFP/5-HT double labeled neurons in each subset were quantified for statistical analysis. RESULTS: At 72-h post-infection, very few GFP+ or GFP/5-HT double-labeled neurons appeared in the brainstem and beyond. In contrast, many labeled neurons were found at these levels after 96 h. Quantitative analysis showed that the majority of infected 5-HT+ neurons were located in the pallidus, obscurus, and magnus nuclei. Conversely, very few infected neurons were found in other raphe subsets, that is the pontis, median, dorsal, or linear nuclei. Overall, the raphe magnus had the highest number of GFP-labeled and GFP/5-HT double-labeled cells. CONCLUSIONS: The caudal subsets of RN, especially the raphe magnus, are the main sources of serotonergic input to the lower spinal cord controlling bladder activity.


Assuntos
Reflexo/fisiologia , Neurônios Serotoninérgicos/fisiologia , Medula Espinal/fisiologia , Sinapses/fisiologia , Bexiga Urinária/inervação , Bexiga Urinária/fisiologia , Anestesia , Animais , Tronco Encefálico/fisiologia , Feminino , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Núcleos da Rafe/fisiologia , Ratos , Ratos Wistar , Serotonina/metabolismo
10.
J Neurosci ; 38(17): 4146-4162, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29610439

RESUMO

Cardiovascular disease and susceptibility to infection are leading causes of morbidity and mortality for individuals with spinal cord injury (SCI). A major contributor to these is autonomic dysreflexia (AD), an amplified reaction of the autonomic nervous system (hallmarked by severe hypertension) in response to sensory stimuli below the injury. Maladaptive plasticity of the spinal sympathetic reflex circuit below the SCI results in AD intensification over time. Mechanisms underlying this maladaptive plasticity are poorly understood, restricting the identification of treatments. Thus, no preventative treatments are currently available. Neuroinflammation has been implicated in other pathologies associated with hyperexcitable neural circuits. Specifically, the soluble form of TNFα (sTNFα) is known to play a role in neuroplasticity. We hypothesize that persistent expression of sTNFα in spinal cord underlies AD exacerbation. To test this, we intrathecally administered XPro1595, a biologic that renders sTNFα nonfunctional, after complete, high-level SCI in female rats. This dramatically attenuated the intensification of colorectal distension-induced and naturally occurring AD events. This improvement is mediated via decreased sprouting of nociceptive primary afferents and activation of the spinal sympathetic reflex circuit. We also examined peripheral vascular function using ex vivo pressurized arterial preparations and immune function via flow cytometric analysis of splenocytes. Diminishing AD via pharmacological inhibition of sTNFα mitigated ensuing vascular hypersensitivity and immune dysfunction. This is the first demonstration that neuroinflammation-induced sTNFα is critical for altering the spinal sympathetic reflex circuit, elucidating a novel mechanism for AD. Importantly, we identify the first potential pharmacological, prophylactic treatment for this life-threatening syndrome.SIGNIFICANCE STATEMENT Autonomic dysreflexia (AD), a disorder that develops after spinal cord injury (SCI) and is hallmarked by sudden, extreme hypertension, contributes to cardiovascular disease and susceptibility to infection, respectively, two leading causes of mortality and morbidity in SCI patients. We demonstrate that neuroinflammation-induced expression of soluble TNFα plays a critical role in AD, elucidating a novel underlying mechanism. We found that intrathecal administration after SCI of a biologic that inhibits soluble TNFα signaling dramatically attenuates AD and significantly reduces AD-associated peripheral vascular and immune dysfunction. We identified mechanisms behind diminished plasticity of neuronal populations within the spinal sympathetic reflex circuit. This study is the first to pinpoint a potential pharmacological, prophylactic strategy to attenuate AD and ensuing cardiovascular and immune dysfunction.


Assuntos
Disreflexia Autonômica/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Animais , Disreflexia Autonômica/fisiopatologia , Células Cultivadas , Feminino , Artérias Mesentéricas/fisiopatologia , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Baço/imunologia , Baço/fisiopatologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia
11.
J Neurosci Methods ; 293: 144-150, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28947264

RESUMO

BACKGROUND: Cellular transplantation to repair a complete spinal cord injury (SCI) is tremendously challenging due to the adverse local milieu for graft survival and growth. Results from cell transplantation studies yield great variability, which may possibly be due to the surgical techniques employed to induce an SCI. In order to delineate the influence of surgery on such inconsistency, we compared lesion morphology and graft survival as well as integration from different lesion methodologies of SCI. NEW METHOD: Surgical techniques, including a traditional approach cut+microaspiration, and two new approaches, cut alone as well as crush, were employed to produce a complete SCI, respectively. Approximately half of the rats in each group received injury only, whereas the other half received grafts of fetal brainstem cells into the lesion gap. RESULTS: Eight weeks after injury with or without graft, histological analysis showed that the cut+microaspiration surgery resulted in larger lesion cavities and severe fibrotic scars surrounding the cavity, and cellular transplants rarely formed a tissue bridge to penetrate the barrier. In contrast, the majority of cases treated with cut alone or crush exhibited smaller cavities and less scarring; the grafts expanded and blended extensively with the host tissue, which often built continuous tissue bridging the rostral and caudal cords. COMPARISON WITH EXISTING METHODS: Scarring and cavitation were significantly reduced when microaspiration was avoided in SCI surgery, facilitating graft/host tissue fusion for signal transmission. CONCLUSION: The result suggests that microaspiration frequently causes severe scars and cavities, thus impeding graft survival and integration.


Assuntos
Sobrevivência de Enxerto , Procedimentos Neurocirúrgicos , Traumatismos da Medula Espinal/cirurgia , Regeneração da Medula Espinal , Animais , Tronco Encefálico/embriologia , Tronco Encefálico/transplante , Sobrevivência Celular/fisiologia , Cicatriz/etiologia , Cicatriz/patologia , Cicatriz/fisiopatologia , Modelos Animais de Doenças , Células-Tronco Embrionárias/transplante , Feminino , Sobrevivência de Enxerto/fisiologia , Microcirurgia , Células-Tronco Neurais/transplante , Ratos Endogâmicos F344 , Ratos Transgênicos , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Medula Espinal/cirurgia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Regeneração da Medula Espinal/fisiologia , Sucção
12.
Mol Ther ; 25(12): 2715-2726, 2017 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-28967557

RESUMO

After spinal cord injury (SCI), severed axons in the adult mammalian CNS are unable to mount a robust regenerative response. In addition, the glial scar at the lesion site further restricts the regenerative potential of axons. We hypothesized that a combinatorial approach coincidentally targeting these obstacles would promote axonal regeneration. We combined (1) transplantation of a growth-permissive peripheral nerve graft (PNG) into an incomplete, cervical lesion cavity; (2) transduction of neurons rostral to the SCI site to express constitutively active Rheb (caRheb; a Ras homolog enriched in brain), a GTPase that directly activates the growth-promoting pathway mammalian target of rapamycin (mTOR) via AAV-caRheb injection; and (3) digestion of growth-inhibitory chondroitin sulfate proteoglycans within the glial scar at the distal PNG interface using the bacterial enzyme chondroitinase ABC (ChABC). We found that expressing caRheb in neurons post-SCI results in modestly yet significantly more axons regenerating across a ChABC-treated distal graft interface into caudal spinal cord than either treatment alone. Excitingly, we found that caRheb+ChABC treatment significantly potentiates the formation of synapses in the host spinal cord and improves the animals' ability to use the affected forelimb. Thus, this combination strategy enhances functional axonal regeneration following a cervical SCI.


Assuntos
Axônios/efeitos dos fármacos , Axônios/fisiologia , Condroitina ABC Liase/farmacologia , Expressão Gênica , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Traumatismos da Medula Espinal/genética , Adenoviridae/genética , Animais , Comportamento Animal , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Genes Reporter , Genes fos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Atividade Motora , Neurônios/metabolismo , Ratos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Transdução Genética
13.
Biomaterials ; 112: 62-71, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27744221

RESUMO

Many mechanisms contribute to the secondary injury cascades following traumatic spinal cord injury (SCI). However, most current treatment strategies only target one or a few elements in the injury cascades, and have been largely unsuccessful in clinical trials. Minocycline hydrochloride (MH) is a clinically available antibiotic and anti-inflammatory drug that has been shown to target a broad range of secondary injury mechanisms via its anti-inflammatory, anti-oxidant, and anti-apoptotic properties. However, MH is only neuroprotective at high concentrations. The inability to translate the high doses of MH used in experimental animals to tolerable doses in human patients limits its clinical efficacy. In addition, the duration of MH treatment is limited because long-term systemic administration of high doses of MH has been shown to cause liver toxicity and even death. We have developed a drug delivery system in the form of hydrogel loaded with polysaccharide-MH complexes self-assembled by metal ions for controlled release of MH. This drug delivery system can be injected into the intrathecal space for local delivery of MH with sufficient dose and duration, without causing any additional tissue damage. We show that local delivery of MH at a dose that is lower than the standard human dose (3 mg/kg) was more effective in reducing secondary injury and promoting locomotor functional recovery than systemic injection of MH with the highest dose and duration reported in experimental animal SCI (90-135 mg/kg).


Assuntos
Implantes de Medicamento/administração & dosagem , Nanopartículas Metálicas/química , Minociclina/administração & dosagem , Regeneração Nervosa/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologia , Animais , Cristalização/métodos , Implantes de Medicamento/química , Feminino , Hidrogéis/química , Íons , Nanopartículas Metálicas/administração & dosagem , Minociclina/química , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Nanocápsulas/ultraestrutura , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/química , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/patologia , Resultado do Tratamento
14.
Front Mol Neurosci ; 9: 49, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27458339

RESUMO

While the peripheral branch of dorsal root ganglion neurons (DRG) can successfully regenerate after injury, lesioned central branch axons fail to regrow across the dorsal root entry zone (DREZ), the interface between the dorsal root and the spinal cord. This lack of regeneration is due to the limited regenerative capacity of adult sensory axons and the growth-inhibitory environment at the DREZ, which is similar to that found in the glial scar after a central nervous system (CNS) injury. We hypothesized that transduction of adult DRG neurons using adeno-associated virus (AAV) to express a constitutively-active form of the GTPase Rheb (caRheb) will increase their intrinsic growth potential after a dorsal root crush. Additionally, we posited that if we combined that approach with digestion of upregulated chondroitin sulfate proteoglycans (CSPG) at the DREZ with chondroitinase ABC (ChABC), we would promote regeneration of sensory axons across the DREZ into the spinal cord. We first assessed if this strategy promotes neuritic growth in an in vitro model of the glial scar containing CSPG. ChABC allowed for some regeneration across the once potently inhibitory substrate. Combining ChABC treatment with expression of caRheb in DRG significantly improved this growth. We then determined if this combination strategy also enhanced regeneration through the DREZ after dorsal root crush in adult rats in vivo. After unilaterally crushing C4-T1 dorsal roots, we injected AAV5-caRheb or AAV5-GFP into the ipsilateral C5-C8 DRGs. ChABC or PBS was injected into the ipsilateral dorsal horn at C5-C8 to digest CSPG, for a total of four animal groups (caRheb + ChABC, caRheb + PBS, GFP + ChABC, GFP + PBS). Regeneration was rarely observed in PBS-treated animals, whereas short-distance regrowth across the DREZ was observed in ChABC-treated animals. No difference in axon number or length between the ChABC groups was observed, which may be related to intraganglionic inflammation induced by the injection. ChABC-mediated regeneration is functional, as stimulation of ipsilateral median and ulnar nerves induced neuronal c-Fos expression in deafferented dorsal horn in both ChABC groups. Interestingly, caRheb + ChABC animals had significantly more c-Fos(+) nuclei indicating that caRheb expression in DRGs promoted functional synaptogenesis of their axons that regenerated beyond a ChABC-treated DREZ.

15.
Neural Regen Res ; 11(2): 189-94, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27073353

RESUMO

Both sensorimotor and autonomic dysfunctions often occur after spinal cord injury (SCI). Particularly, a high thoracic or cervical SCI interrupts supraspinal vasomotor pathways and results in disordered hemodynamics due to deregulated sympathetic outflow. As a result of the reduced sympathetic activity, patients with SCI may experience hypotension, cardiac dysrhythmias, and hypothermia post-injury. In the chronic phase, changes within the CNS and blood vessels lead to orthostatic hypotension and life-threatening autonomic dysreflexia (AD). AD is characterized by an episodic, massive sympathetic discharge that causes severe hypertension associated with bradycardia. The syndrome is often triggered by unpleasant visceral or sensory stimuli below the injury level. Currently the only treatments are palliative - once a stimulus elicits AD, pharmacological vasodilators are administered to help reduce the spike in arterial blood pressure. However, a more effective means would be to mitigate AD development by attenuating contributing mechanisms, such as the reorganization of intraspinal circuits below the level of injury. A better understanding of the neuropathophysiology underlying cardiovascular dysfunction after SCI is essential to better develop novel therapeutic approaches to restore hemodynamic performance.

17.
Exp Neurol ; 285(Pt B): 136-146, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26655672

RESUMO

Dopamine (DA) neurons in the mammalian central nervous system are thought to be restricted to the brain. DA-mediated regulation of urinary activity is considered to occur through an interaction between midbrain DA neurons and the pontine micturition center. Here we show that DA is produced in the rat spinal cord and modulates the bladder reflex. We observed numerous tyrosine hydroxylase (TH)+ neurons in the autonomic nuclei and superficial dorsal horn in L6-S3 spinal segments. These neurons are dopamine-ß-hydroxylase (DBH)- and some contain detectable dopamine decarboxylase (DDC), suggesting their capacity to produce DA. Interestingly, following a complete thoracic spinal cord injury (SCI) to interrupt supraspinal projections, more TH+ neurons emerged in the lumbosacral spinal cord, coincident with a sustained, low level of DA expression there and a partially recovered micturition reflex. Non-selective blockade of spinal DA receptors reduced bladder activity whereas activation of spinal D2-like receptors increased bladder activity and facilitated voiding. Additionally, depletion of lumbosacral TH+ neurons with 6-hydroxydopamine (6-OHDA) decreased bladder non-voiding contractions and voiding efficiency. Furthermore, injecting the transsynaptic neuronal tracer pseudorabies virus (PRV) into the bladder detrusor labeled TH+ cells in the lumbosacral cord, confirming their involvement in spinal micturition reflex circuits. These results illustrate that DA is synthesized in the rat spinal cord; plasticity of lumbosacral TH+ neurons following SCI may contribute to DA expression and modulate the spinal bladder reflex. Thus, spinally-derived DA and receptors could be a novel therapeutic target to improve micturition recovery after SCI.


Assuntos
Dopamina/metabolismo , Reflexo/fisiologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Animais , Animais Recém-Nascidos , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Dopamina/análogos & derivados , Dopaminérgicos/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Gânglios Parassimpáticos/patologia , Gânglios Simpáticos/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/metabolismo , Neurônios/metabolismo , Oxidopamina/toxicidade , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Ratos Wistar , Traumatismos da Medula Espinal/induzido quimicamente , Estilbamidinas/farmacocinética , Tiocarbamatos/metabolismo , Transdução Genética , Tirosina 3-Mono-Oxigenase/metabolismo , Bexiga Urinária/inervação , Bexiga Urinária/fisiopatologia
18.
J Neurosci ; 35(31): 11068-80, 2015 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-26245968

RESUMO

UNLABELLED: After a spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to: (1) the presence of inhibitory molecules, e.g., chondroitin sulfate proteoglycans (CSPG), in the glial scar at the lesion; and (2) the diminished growth capacity of adult neurons. We sought to determine whether expressing a constitutively active form of the GTPase Rheb (caRheb) in adult neurons after a complete SCI in rats improves intrinsic growth potential to result in axon regeneration out of a growth-supportive peripheral nerve grafted (PNG) into the SCI cavity. We also hypothesized that treating the glial scar with chondroitinase ABC (ChABC), which digests CSPG, would further allow caRheb-transduced neurons to extend axons across the distal graft interface. We found that targeting this pathway at a clinically relevant post-SCI time point improves both sprouting and regeneration of axons. CaRheb increased the number of axons, but not the number of neurons, that projected into the PNG, indicative of augmented sprouting. We also saw that caRheb enhanced sprouting far rostral to the injury. CaRheb not only increased growth rostral and into the graft, it also resulted in significantly more regrowth of axons across a ChABC-treated scar into caudal spinal cord. CaRheb(+) neurons had higher levels of growth-associated-43, suggestive of a newly identified mechanism for mTOR-mediated enhancement of regeneration. Thus, we demonstrate for the first time that simultaneously addressing intrinsic and scar-associated, extrinsic impediments to regeneration results in significant regrowth beyond an extremely challenging, complete SCI site. SIGNIFICANCE STATEMENT: After spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to the diminished growth capacity of adult neurons and the presence of inhibitory molecules in the scar at the lesion. We sought to simultaneously counter both of these obstacles to achieve more robust regeneration after complete SCI. We transduced neurons postinjury to express a constitutively active Rheb to enhance their intrinsic growth potential, transplanted a growth supporting peripheral nerve graft into the lesion cavity, and enzymatically modulated the inhibitory glial scar distal to the graft. We demonstrate, for the first time, that simultaneously addressing neuron-related, intrinsic deficits in axon regrowth and extrinsic, scar-associated impediments to regeneration results in significant regeneration after SCI.


Assuntos
Condroitina ABC Liase/uso terapêutico , Cicatriz/terapia , Proteínas Monoméricas de Ligação ao GTP/genética , Regeneração Nervosa/genética , Neuropeptídeos/genética , Traumatismos da Medula Espinal/terapia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Condroitina ABC Liase/farmacologia , Cicatriz/tratamento farmacológico , Cicatriz/genética , Modelos Animais de Doenças , Feminino , Regeneração Nervosa/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/genética , Resultado do Tratamento
19.
Exp Neurol ; 263: 172-6, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25447935

RESUMO

While it is well established that the axons of adult neurons have a lower capacity for regrowth, some regeneration of certain CNS populations after spinal cord injury (SCI) is possible if their axons are provided with a permissive substrate, such as an injured peripheral nerve. While some axons readily regenerate into a peripheral nerve graft (PNG), these axons almost always stall at the distal interface and fail to reinnervate spinal cord tissue. Treatment of the glial scar at the distal graft interface with chondroitinase ABC (ChABC) can improve regeneration, but most regenerated axons need further stimulation to extend beyond the interface. Previous studies demonstrate that pharmacologically inhibiting kinesin-5, a motor protein best known for its essential role in mitosis but also expressed in neurons, with the pharmacological agent monastrol increases axon growth on inhibitory substrates in vitro. We sought to determine if monastrol treatment after an SCI improves functional axon regeneration. Animals received complete thoracic level 7 (T7) transections and PNGs and were treated intrathecally with ChABC and either monastrol or DMSO vehicle. We found that combining ChABC with monastrol significantly enhanced axon regeneration. However, there were no further improvements in function or enhanced c-Fos induction upon stimulation of spinal cord rostral to the transection. This indicates that monastrol improves ChABC-mediated axon regeneration but that further treatments are needed to enhance the integration of these regrown axons.


Assuntos
Axônios/efeitos dos fármacos , Cinesina/antagonistas & inibidores , Regeneração Nervosa/efeitos dos fármacos , Pirimidinas/farmacologia , Traumatismos da Medula Espinal/patologia , Tionas/farmacologia , Animais , Condroitina ABC Liase/farmacologia , Modelos Animais de Doenças , Feminino , Ratos , Ratos Sprague-Dawley
20.
J Neurosci ; 33(43): 17138-49, 2013 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-24155317

RESUMO

High-level spinal cord injury can lead to cardiovascular dysfunction, including disordered hemodynamics at rest and autonomic dysreflexia during noxious stimulation. To restore supraspinal control of sympathetic preganglionic neurons (SPNs), we grafted embryonic brainstem-derived neural stem cells (BS-NSCs) or spinal cord-derived neural stem cells (SC-NSCs) expressing green fluorescent protein into the T4 complete transection site of adult rats. Animals with injury alone served as controls. Implanting of BS-NSCs but not SC-NSCs resulted in recovery of basal cardiovascular parameters, whereas both cell grafts alleviated autonomic dysreflexia. Subsequent spinal cord retransection above the graft abolished the recovery of basal hemodynamics and reflexic response. BS-NSC graft-derived catecholaminergic and serotonergic neurons showed remarkable long-distance axon growth and topographical innervation of caudal SPNs. Anterograde tracing indicated growth of medullar axons into stem cell grafts and formation of synapses. Thus, grafted embryonic brainstem-derived neurons can act as functional relays to restore supraspinal regulation of denervated SPNs, thereby contributing to cardiovascular functional improvement.


Assuntos
Células-Tronco Embrionárias/transplante , Coração/inervação , Hemodinâmica , Regeneração Nervosa , Células-Tronco Neurais/transplante , Traumatismos da Medula Espinal/cirurgia , Fibras Adrenérgicas/fisiologia , Neurônios Adrenérgicos/fisiologia , Animais , Disreflexia Autonômica/cirurgia , Fibras Autônomas Pré-Ganglionares/fisiologia , Axônios/fisiologia , Tronco Encefálico/citologia , Processos de Crescimento Celular , Feminino , Coração/fisiopatologia , Ratos , Ratos Endogâmicos F344 , Reflexo , Neurônios Serotoninérgicos/fisiologia , Medula Espinal/citologia , Medula Espinal/fisiopatologia , Transplante de Células-Tronco , Sinapses/fisiologia
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