Slow progression of sciatic nerve degeneration and regeneration after loose ligation through microglial activation and decreased KCC2 levels in the mouse spinal cord ventral horn.

Peripheral nerve injury affects motor functions. To reveal the mechanisms underlying motor dysfunction and recovery after nerve compression, which have not been precisely examined, we investigated the temporal relationship among changes in motor function, nerve histopathology, and marker molecule expression in the spinal cord after loose ligation of the mouse sciatic nerve. After ligation, sciatic motor function suddenly declined, and axons gradually degenerated. During degeneration, galanin was localized in motor neuron cell bodies. Then, in the ventral horn, microglia were activated, and expression of choline acetyltransferase (ChAT), a synthetic enzyme of acetylcholine, and potassium chloride co-transporter 2 (KCC2), which shifts the action of γ-amino butyric acid (GABA) and glycine to inhibitory, decreased. Motor function recovery was insufficient although axonal regeneration was complete. ChAT levels gradually recovered during axonal regeneration. When regeneration was nearly complete, microglial activation declined, and KCC2 expression started to increase. The KCC2 level sufficiently recovered when axonal regeneration was complete, suggesting that the excitatory action of GABA/glycine may participate in axonal regeneration. Furthermore, these changes proceeded slower than those after severance, suggesting that loose ligation, compression, may mediate slower progression of degeneration and regeneration than severance, and these changes may cause the motor dysfunction and its recovery.

Astrocytes lure CXCR2-expressing CD4+ T cells to gray matter via TAK1-mediated chemokine production in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system driven by peripheral immune cell infiltration and glial activation. The pathological hallmark of MS is demyelination, and mounting evidence suggests neuronal damage in gray matter is a major contributor to disease irreversibility. While T cells are found in both gray and white matter of MS tissue, they are typically confined to the white matter of the most commonly used mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Here, we used a modified EAE mouse model (Type-B EAE) that displays severe neuronal damage to investigate the interplay between peripheral immune cells and glial cells in the event of neuronal damage. We show that CD4+ T cells migrate to the spinal cord gray matter, preferentially to ventral horns. Compared to CD4+ T cells in white matter, gray matter-infiltrated CD4+ T cells were mostly immobilized and interacted with neurons, which are behaviors associated with detrimental effects to normal neuronal function. T cell-specific deletion of CXCR2 significantly decreased CD4+ T cell infiltration into gray matter in Type-B EAE mice. Further, astrocyte-targeted deletion of TAK1 inhibited production of CXCR2 ligands such as CXCL1 in gray matter, successfully prevented T cell migration into spinal cord gray matter, and averted neuronal damage and motor dysfunction in Type-B EAE mice. This study identifies astrocyte chemokine production as a requisite for the invasion of CD4+T cell into the gray matter to induce neuronal damage.

Tissue engineered axon-based "living scaffolds" promote survival of spinal cord motor neurons following peripheral nerve repair.

Peripheral nerve injury (PNI) impacts millions annually, often leaving debilitated patients with minimal repair options to improve functional recovery. Our group has previously developed tissue engineered nerve grafts (TENGs) featuring long, aligned axonal tracts from dorsal root ganglia (DRG) neurons that are fabricated in custom bioreactors using the process of axon "stretch-growth." We have shown that TENGs effectively serve as "living scaffolds" to promote regeneration across segmental nerve defects by exploiting the newfound mechanism of axon-facilitated axon regeneration, or "AFAR," by simultaneously providing haptic and neurotrophic support. To extend this work, the current study investigated the efficacy of living versus nonliving regenerative scaffolds in preserving host sensory and motor neuronal health following nerve repair. Rats were assigned across five groups: naïve, or repair using autograft, nerve guidance tube (NGT) with collagen, NGT + non-aligned DRG populations in collagen, or TENGs. We found that TENG repairs yielded equivalent regenerative capacity as autograft repairs based on preserved health of host spinal cord motor neurons and acute axonal regeneration, whereas NGT repairs or DRG neurons within an NGT exhibited reduced motor neuron preservation and diminished regenerative capacity. These acute regenerative benefits ultimately resulted in enhanced levels of functional recovery in animals receiving TENGs, at levels matching those attained by autografts. Our findings indicate that TENGs may preserve host spinal cord motor neuron health and regenerative capacity without sacrificing an otherwise uninjured nerve (as in the case of the autograft) and therefore represent a promising alternative strategy for neurosurgical repair following PNI.

Upregulation of proteoglycans in the perilesion perimeter in ventral horns after spinal cord injury.

Spinal cord injury (SCI) results in pronounced focal tissue damage with subsequent formation of a glial scar that blocks axon regeneration and regrowth. Cellular changes and the composition of the extracellular matrix in regions distal from the injured area remain poorly characterized. In the present study, in the spinal cord distal to the damaged area (perilesion perimeter) there were minimal gross histological changes, but there were pronounced alterations in the extracellular proteoglycans even at 30 days after SCI. These abnormalities coincided with the appearance of reactive astrocytes and a reduction in main astrocytic glutamate transporter 1. Proteoglycan levels exhibited different kinetics and changes after SCI in areas near neuronal cell bodies and in areas distal from them. The results of the study suggest that SCI induces widespread changes in the spinal cord that may be responsible for neuronal dysfunction far from the damaged area and further aggravation of the SCI.

Predeterminative role of Onuf's nucleus ischemia on mesenteric artery vasospasm in spinal subarachnoid hemorrhage: A preliminary experimental study.

BACKGROUND: Although posttraumatic mesenteric artery ischemia is attributed to various etiologies, sacral parasympathetic network/mesenteric artery relations have not been studied so far. The primary objective of this study is to elucidate whether there is a relationship between Onuf's nucleus ischemia and mesenteric artery vasospasm following subarachnoid hemorrhage (SAH). METHODS: This study was conducted on 22 rabbits. The animals were grouped as follows: 5 of animals control, 5 SHAM which saline was given, and 12 animals study group that was homologous blood injected into the spinal subarachnoid space at the Li level. Neurodegeneration in Onuf's nucleus, axonal degeneration of S2 roots, and mesenteric arteries vasospasm indexes (VSI; Wall surface/Lumen surface), brachias of mesentery arteries in various tissues and ischemic mucosal changes of intestines of all animals were determined histopathologically. Important degenerative changes were detected in axons in S2 roots and Onuf's nucleus in severe mesenteric artery vasospasm observed. RESULTS: The mean degenerated neuron density of Onuf's nucleus (n/mm3), degenerated axon density in S2 roots (n/mm2), and VSI values of mesenteric arteries of control, SHAM, and study groups were estimated as 5.00 ± 1.58, 4.00 ± 1.58, 1.76 ± 0.13; 18.29 ± 4.31, 11.00 ± 2.24, 2.23 ± 0.20; and 135.21 ± 30.75, 117.33 ± 22.11, 2.81 ± 0.44, respectively. Statistical analyses between the VSI values, mucosal ischemic changes degenerated neurons in Onuf's nucleus, and axons in S2 levels were meaningful (p < 0.005). CONCLUSION: We interestingly noticed that Onuf's nucleus-S2 roots complex degeneration plays an important role in mesenteric artery vasospasm and the development of intestinal ischemic mucosal changes following SAH which has not been extensively mentioned in the literature.

Neural crest stem cells protect spinal cord neurons from excitotoxic damage and inhibit glial activation by secretion of brain-derived neurotrophic factor.

The acute phase of spinal cord injury is characterized by excitotoxic and inflammatory events that mediate extensive neuronal loss in the gray matter. Neural crest stem cells (NCSCs) can exert neuroprotective and anti-inflammatory effects that may be mediated by soluble factors. We therefore hypothesize that transplantation of NCSCs to acutely injured spinal cord slice cultures (SCSCs) can prevent neuronal loss after excitotoxic injury. NCSCs were applied onto SCSCs previously subjected to N-methyl-D-aspartate (NMDA)-induced injury. Immunohistochemistry and TUNEL staining were used to quantitatively study cell populations and apoptosis. Concentrations of neurotrophic factors were measured by ELISA. Migration and differentiation properties of NCSCs on SCSCs, laminin, or hyaluronic acid hydrogel were separately studied. NCSCs counteracted the loss of NeuN-positive neurons that was otherwise observed after NMDA-induced excitotoxicity, partly by inhibiting neuronal apoptosis. They also reduced activation of both microglial cells and astrocytes. The concentration of brain-derived neurotrophic factor (BDNF) was increased in supernatants from SCSCs cultured with NCSCs compared to SCSCs alone and BDNF alone mimicked the effects of NCSC application on SCSCs. NCSCs migrated superficially across the surface of SCSCs and showed no signs of neuronal or glial differentiation but preserved their expression of SOX2 and Krox20. In conclusion, NCSCs exert neuroprotective, anti-apoptotic and glia-inhibitory effects on excitotoxically injured spinal cord tissue, some of these effects mediated by secretion of BDNF. However, the investigated NCSCs seem not to undergo neuronal or glial differentiation in the short term since markers indicative of an undifferentiated state were expressed during the entire observation period.

Dorsal and ventral horn atrophy is associated with clinical outcome after spinal cord injury.

OBJECTIVE: To investigate whether gray matter pathology above the level of injury, alongside white matter changes, also contributes to sensorimotor impairments after spinal cord injury. METHODS: A 3T MRI protocol was acquired in 17 tetraplegic patients and 21 controls. A sagittal T2-weighted sequence was used to characterize lesion severity. At the C2-3 level, a high-resolution T2*-weighted sequence was used to assess cross-sectional areas of gray and white matter, including their subcompartments; a diffusion-weighted sequence was used to compute voxel-based diffusion indices. Regression models determined associations between lesion severity and tissue-specific neurodegeneration and associations between the latter with neurophysiologic and clinical outcome. RESULTS: Neurodegeneration was evident within the dorsal and ventral horns and white matter above the level of injury. Tract-specific neurodegeneration was associated with prolonged conduction of appropriate electrophysiologic recordings. Dorsal horn atrophy was associated with sensory outcome, while ventral horn atrophy was associated with motor outcome. White matter integrity of dorsal columns and corticospinal tracts was associated with daily-life independence. CONCLUSION: Our results suggest that, next to anterograde and retrograde degeneration of white matter tracts, neuronal circuits within the spinal cord far above the level of injury undergo transsynaptic neurodegeneration, resulting in specific gray matter changes. Such improved understanding of tissue-specific cord pathology offers potential biomarkers with more efficient targeting and monitoring of neuroregenerative (i.e., white matter) and neuroprotective (i.e., gray matter) agents.

Neurodegeneration in the Spinal Ventral Horn Prior to Motor Impairment in Cervical Spondylotic Myelopathy.

Remote gray matter pathology has been suggested rostral to the compression site in cervical spondylotic myelopathy (CSM). We therefore assessed neurodegeneration in the gray matter ventral and dorsal horns. Twenty patients with CSM and 18 healthy subjects underwent a high-resolution structural and diffusion magnetic resonance imaging protocol at vertebra C2/C3. Patients received comprehensive clinical assessments. T2*-weighted data provided cross-sectional area measurements of gray matter ventral and dorsal horns to identify atrophy. At the identical location, mean diffusivity (MD) and fractional anisotropy (FA) determined the microstructural integrity. Finally, the relationships between neurodegeneration occurring in the gray and white matter and clinical impairment were investigated. Patients suffered from mild-to-moderate CSM with mainly sensory impairment. In the ventral horns, cross-sectional area was not reduced (p = 0.863) but MD was increased (p = 0.045). The magnitude of MD changes within the ventral horn was associated with white matter diffusivity changes (MD: p = 0.013; FA: p = 0.028) within the lateral corticospinal tract. In contrast, dorsal horn cross-sectional area was reduced by 16.0% (p < 0.001) without alterations in diffusivity indices, compared with controls. No associations between the magnitude of ventral and dorsal horn neurodegeneration and clinical impairment were evident. Focal cord gray matter pathology is evident remote to the compression site in vivo in CSM patients. Microstructural changes in the ventral horns (i.e., motoneurons) related to corticospinal tract integrity in the absence of atrophy and marked motor impairment. Dorsal horn atrophy corresponded to main clinical representation of sensory impairment. Thus, neuroimaging biomarkers of cord gray matter integrity reveal focal neurodegeneration prior to marked clinical impairment and thus could serve as predictors of ensuing impairment in CSM patients.

Increased Expression of 15-Hydroxyprostaglandin Dehydrogenase in Spinal Astrocytes During Disease Progression in a Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease caused by selective loss of motor neurons. Both ALS model mice and patients with sporadic ALS have increased levels of prostaglandin E2 (PGE2). Furthermore, the protein levels of microsomal PGE synthase-1 and cyclooxygenase-2, which catalyze PGE2 biosynthesis, are significantly increased in the spinal cord of ALS model mice. However, it is unclear whether PGE2 metabolism in the spinal cord is altered. In the present study, we investigated the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin metabolism, in ALS model mice at three different disease stages. Western blotting revealed that the 15-PGDH level was significantly increased in the lumbar spinal cord at the symptomatic stage and end stage. Immunohistochemical staining demonstrated that 15-PGDH immunoreactivity was localized in glial fibrillary acidic protein (GFAP)-positive astrocytes at the end stage. In contrast, 15-PGDH immunoreactivity was not identified in NeuN-positive large cells showing the typical morphology of motor neurons in the anterior horn. Unlike 15-PGDH, the level of PGE2 in the spinal cord was increased only at the end stage. These results suggest that the significant increase of PGE2 at the end stage of ALS in this mouse model is attributable to an imbalance of the synthetic pathway and 15-PGDH-dependent scavenging system for PGE2, and that this drives the pathogenetic mechanism responsible for transition from the symptomatic stage.

Spinal Cord Ventral Horns and Lymphoid Organ Involvement in Powassan Virus Infection in a Mouse Model.

Powassan virus (POWV) belongs to the family Flaviviridae and is a member of the tick-borne encephalitis serogroup. Transmission of POWV from infected ticks to humans has been documented in the USA, Canada, and Russia, causing fatal encephalitis in 10% of human cases and significant neurological sequelae in survivors. We used C57BL/6 mice to investigate POWV infection and pathogenesis. After footpad inoculation, infected animals exhibited rapid disease progression and 100% mortality. Immunohistochemistry and immunofluorescence revealed a very strong neuronal tropism of POWV infection. The central nervous system infection appeared as a meningoencephalitis with perivascular mononuclear infiltration and microglial activation in the brain, and a poliomyelitis-like syndrome with high level of POWV antigen at the ventral horn of the spinal cord. Pathological studies also revealed substantial infection of splenic macrophages by POWV, which suggests that the spleen plays a more important role in pathogenesis than previously realized. This report provides a detailed description of the neuroanatomical distribution of the lesions produced by POWV infection in C57BL/6 mice.

Astrocytes and Microglia-Mediated Immune Response in Maladaptive Plasticity is Differently Modulated by NGF in the Ventral Horn of the Spinal Cord Following Peripheral Nerve Injury.

Reactive astrocytes and activated microglia are the key players in several pathophysiologic modifications of the central nervous system. We used the spared nerve injury (SNI) of the sciatic nerve to induce glial maladaptive response in the ventral horn of lumbar spinal cord and examine its role in the remodeling of the tripartite synapse plasticity. Imaging the ventral horn revealed that SNI was associated with both an early microglial and astrocytic activation, assessed, respectively, by analysis of Iba1 and GFAP expression. Microglia, in particular, localized peculiarly surrounding the motor neurons somata. Perineuronal astrocytes, which play a key role in maintaining the homeostasis of neuronal circuitry, underwent a substantial phenotypic change following peripheral axotomy, producing reactive gliosis. The gliosis was associated with the reduction of glial aminoacid transporters (GLT1 and GlyT1) and increase of neuronal glutamate transporter EAAC1. Although the expression of GABAergic neuronal marker GAD65/67 showed no change, glutamate increase, as demonstrated by HPLC analysis, shifted the excitatory/inhibitory balance as showed by the net increase of the glutamate/GABA ratio. Moreover, endogenous NGF levels were altered in SNI animals and not restored by the intrathecal NGF administration. This treatment reverted phenotypic changes associated with reactive astrocytosis, but failed to modify microglia activation. These findings on one hand confirm the correlation between gliopathy and maladaptive plasticity of the spinal synaptic circuitry, on the other hand add new data concerning the complex peculiar behavior of different glial cells in neuronal degenerative processes, defining a special role of microglia in sustaining the inflammatory response.

Autologous adipose-derived stem cells attenuate muscular atrophy and protect spinal cord ventral horn motor neurons in an animal model of burn injury.

BACKGROUND AIMS: Burn injuries might increase muscle mass loss, but the mechanisms are still unclear. In this study, we demonstrated that burn injury induced spinal cord ventral horn motor neuron (VHMN) apoptosis and subsequently caused muscle atrophy and revealed the potential protection of autologous adipose-derived stem cells (ASCs) transplantation on spinal cord VHMNs and muscle against burn injury. METHODS: Third-degree hind-paw burns were established by contact with a 75°C metal surface for 10 seconds. Adipose tissues were harvested from the groin fat pad, expanded in culture and labeled with chloromethyl-benzamido/1,1'-dioctadecyl-3,3,3',3'- tetramethyl indocarbocyanine perchlorate. The ASCs were transplanted into the injured hind paw at 4 weeks after burn injury. The lumbar spinal cord, sciatic nerve, gastrocnemius muscle and hind-paw skin were processed for immunofluorescent staining at 4 weeks after transplantation, including terminal deoxynucleotidyl transferase (TUNEL) assay, caspase-3, caspase-9, CD 90 and S100, and the gastrocnemius muscle was evaluated through the use of hematoxylin and eosin staining. RESULTS: Caspase-3-positive, caspase-9-positive and TUNEL-positive cells were significantly increased in the corresponding dermatome spinal cord VHMNs after burn injury. Moreover, the decrease of Schwann cells in sciatic nerve and the increase of denervation atrophy in gastrocnemius muscle were observed. Furthermore, ASCs transplantation significantly attenuated apoptotic death of VHMNs and the area of muscle denervation atrophy in the gastrocnemius muscle fibers. CONCLUSIONS: The animal model of third-degree burns in the hind paw showed significant apoptosis in the corresponding spinal cord VHMNs, which suggests that neuroprotection might be the potentially therapeutic target in burn-induced muscle atrophy. ASCs have potential neuroprotection against burn injuries through its anti-apoptotic effects.

Third-degree hindpaw burn injury induced apoptosis of lumbar spinal cord ventral horn motor neurons and sciatic nerve and muscle atrophy in rats.

BACKGROUND: Severe burns result in hypercatabolic state and concomitant muscle atrophy that persists for several months, thereby limiting patient recovery. However, the effects of burns on the corresponding spinal dermatome remain unknown. This study aimed to investigate whether burns induce apoptosis of spinal cord ventral horn motor neurons (VHMNs) and consequently cause skeletal muscle wasting. METHODS: Third-degree hindpaw burn injury with 1% total body surface area (TBSA) rats were euthanized 4 and 8 weeks after burn injury. The apoptosis profiles in the ventral horns of the lumbar spinal cords, sciatic nerves, and gastrocnemius muscles were examined. The Schwann cells in the sciatic nerve were marked with S100. The gastrocnemius muscles were harvested to measure the denervation atrophy. RESULT: The VHMNs apoptosis in the spinal cord was observed after inducing third-degree burns in the hindpaw. The S100 and TUNEL double-positive cells in the sciatic nerve increased significantly after the burn injury. Gastrocnemius muscle apoptosis and denervation atrophy area increased significantly after the burn injury. CONCLUSION: Local hindpaw burn induces apoptosis in VHMNs and Schwann cells in sciatic nerve, which causes corresponding gastrocnemius muscle denervation atrophy. Our results provided an animal model to evaluate burn-induced muscle wasting, and elucidate the underlying mechanisms.