Blocking connexin43 expression reduces inflammation and improves functional recovery after spinal cord injury

After traumatic CNS injury, a cascade of secondary events expands the initial lesion. The gap-junction protein connexin43 (Cx43), which is transiently up-regulated, has been implicated in the spread of 'bystander' damage. We have used an antisense oligodeoxynucleotide (asODN) to suppress Cx43 up-regulation in two rat models of spinal cord injury. Within 24 h of compression injury, rats treated with Cx43-asODN scored higher than sense-ODN and vehicle-treated controls on behavioural tests of locomotion. Their spinal cords showed less swelling and tissue disruption, less up-regulation of astrocytic GFAP, and less extravasation of fluorescently-labelled bovine serum albumin and neutrophils. The locomotor improvement was sustained over at least 4 weeks. Following partial spinal cord transection, Cx43-asODN treatment reduced GFAP immunoreactivity, neutrophil recruitment, and the activity of OX42(+) microglia in and around the lesion site. Cx43 has many potential roles in the pathophysiology of CNS injury and may be a valuable target for therapeutic intervention.     

Neutralization of the chemokine CXCL10 reduces apoptosis and increases axon sprouting after spinal cord injury

Spinal cord injury (SCI) is followed by a secondary degenerative process that includes cell death. We have previously demonstrated that the chemokine CXCL10 is up-regulated following SCI and plays a critical role in T-lymphocyte recruitment to sites of injury and inhibition of angiogenesis; antibody-mediated functional blockade of CXCL10 reduced inflammation while enhancing angiogenesis. We hypothesized, based on these findings, that the injury environment established by anti-CXCL10 antibody treatment would support greater survival of neurons and enhance axon sprouting compared with the untreated, injured spinal cord. Here, we document gene array and histopathological data to support our hypothesis. Gene array analysis of treated and untreated tissue from spinal cord-injured animals revealed eight apoptosis-related genes with significant expression changes at 3 days postinjury. In support of these data, quantification of TUNEL-positive cells at 3 days postinjury indicated a 75% reduction in the number of dying cells in treated animals compared with untreated animals. Gene array analysis of treated and untreated tissue also revealed six central nervous system growth-related genes with significant expression changes in the brainstem at 14 days postinjury. In support of these data, quantification of anterograde-labeled corticospinal tract fibers indicated a 60-70% increase in axon sprouting caudal to the injury site in treated animals compared with untreated animals. These findings indicate that anti-CXCL10 antibody treatment provides an environment that reduces apoptosis and increases axon sprouting following injury to the adult spinal cord.     

Senegenin inhibits neuronal apoptosis after spinal cord contusion injury

Senegenin has been shown to inhibit neuronal apoptosis, thereby exerting a neuroprotective effect. In the present study, we established a rat model of spinal cord contusion injury using the modiifed Allen’s method. Three hours after injury, senegenin (30 mg/g) was injected into the tail vein for 3 consecutive days. Senegenin reduced the size of syringomyelic cavities, and it substantially reduced the number of apop-totic cells in the spinal cord. At the site of injury, Bax and Caspase-3 mRNA and protein levels were decreased by senegenin, while Bcl-2 mRNA and protein levels were increased. Nerve ifber density was increased in the spinal cord proximal to the brain, and hindlimb motor function and electrophysiological properties of rat hindlimb were improved. Taken together, our results suggest that senegenin exerts a neuroprotective effect by suppressing neuronal apoptosis at the site of spinal cord injury.     

VX-765 reduces neuroinflammation after spinal cord injury in mice

Inflammation is a major cause of neuronal injury after spinal cord injury. We hypothesized that inhibiting caspase-1 activation may reduce neuroinflammation after spinal cord injury, thus producing a protective effect in the injured spinal cord. A mouse model of T9 contusive spinal cord injury was established using an Infinite Horizon Impactor, and VX-765, a selective inhibitor of caspase-1, was administered for 7 successive days after spinal cord injury. The results showed that: (1) VX-765 inhibited spinal cord injury-induced caspase-1 activation and interleukin-1β and interleukin-18 secretion. (2) After spinal cord injury, an increase in M1 cells mainly came from local microglia rather than infiltrating macrophages. (3) Pro-inflammatory Th1Th17 cells were predominant in the Th subsets. VX-765 suppressed total macrophage infiltration, M1 macrophages/microglia, Th1 and Th1Th17 subset differentiation, and cytotoxic T cells activation; increased M2 microglia; and promoted Th2 and Treg differentiation. (4) VX-765 reduced the fibrotic area, promoted white matter myelination, alleviated motor neuron injury, and improved functional recovery. These findings suggest that VX-765 can reduce neuroinflammation and improve nerve function recovery after spinal cord injury by inhibiting caspase-1/interleukin-1β/interleukin-18. This may be a potential strategy for treating spinal cord injury. This study was approved by the Animal Care Ethics Committee of Bengbu Medical College (approval No. 2017-037) on February 23, 2017.

Chinese Library Classification No. R453; R392.3; R744     

Elamipretide reduces pyroptosis and improves functional recovery after spinal cord injury

Aims

Elamipretide (EPT), a novel mitochondria-targeted peptide, has been shown to be protective in a range of diseases. However, the effect of EPT in spinal cord injury (SCI) has yet to be elucidated. We aimed to investigate whether EPT would inhibit pyroptosis and protect against SCI.

Methods

After establishing the SCI model, we determined the biochemical and morphological changes associated with pyroptosis, including neuronal cell death, proinflammatory cytokine expression, and signal pathway levels. Furthermore, mitochondrial function was assessed with flow cytometry, quantitative real-time polymerase chain reaction, and western blot.

Results

Here, we demonstrate that EPT improved locomotor functional recovery following SCI as well as reduced neuronal loss. Moreover, EPT inhibited nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome activation and pyroptosis occurrence and decreased pro-inflammatory cytokines levels following SCI. Furthermore, EPT alleviated mitochondrial dysfunction and reduced mitochondrial reactive oxygen species level.

Conclusion

EPT treatment may protect against SCI via inhibition of pyroptosis.     

Differential effects of neurotrophins on neuronal survival and axonal regeneration after spinal cord injury in adult rats

Spinal cord injury (SCI) induces retrograde cell death in descending pathways, which can be prevented by long-term intrathecal infusion of neurotrophins (Novikova et al. [2000] Eur J Neurosci 12:776-780). The present study investigates whether the same treatment also leads to improved regeneration of the injured tracts. After cervical SCI in adult rats, a peripheral nerve graft was attached to the rostral wall of the lesion cavity. The animals were treated by local application into the cavity of Gelfoam soaked in (1) phosphate buffered saline (untreated controls) or (2) a mixture of the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (local treatment), or by intrathecal infusion of BDNF + NT-3 for (3) 2 weeks (short-term treatment) or (4) 5-8 weeks (long-term treatment). Despite a very strong survival effect, long-term treatment failed to stimulate ingrowth of descending tracts into the nerve graft. In comparison with untreated controls, the latter treatment also caused 35% reduction in axonal sprouting of descending pathways rostral to the lesion site and 72% reduction in the number of spinal cord neurons extending axons into the nerve graft. Local and short-term treatments neither prevented retrograde cell death nor enhanced regeneration of descending tracts, but induced robust regeneration of spinal cord neurons into the nerve graft. These results indicate that the signal pathways promoting neuronal survival and axonal regeneration, respectively, in descending tracts after SCI respond differently to neurotrophic stimuli and that efficient rescue of axotomized tract neurons is not a sufficient prerequisite for regeneration.     

Gliopathy ensures persistent inflammation and chronic pain after spinal cord injury

Research focused on improving recovery of function, including the reduction of central neuropathic pain (CNP) after spinal cord injury (SCI) is essential. After SCI, regional neuropathic pain syndromes above, at and below the level or spinal injury develop and are thought to have different mechanisms, but may share common dysfunctional glial mechanisms. Detloff et al., [Detloff, M.R., Fisher, L.C., McGaughy, V., Longbrake, E.E., Popovich, P.G., Basso, D.M., Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats. Exp. Neurol. (2008), doi: 10.1016/j.expneurol.2008.04.009.] describe events in the lumbar region of the spinal cord after a midthoracic SCI injury, the so called “below-level” pain and compares the findings to peripheral nerve lesion findings. This commentary briefly reviews glial contributions and intracellular signaling mechanisms, both neuronal and glial, that provide the substrate for CNP after SCI, including the persistent glial production of factors that can maintain sensitization of dorsal horn neurons in segments remote from the spinal injury; ie. dorsal horn hyperexcitability to formerly non noxious stimuli that become noxious after SCI resulting in allodynia. The term “gliopathy” is proposed to describe the dysfunctional and maladaptive response of glial cells, specifically astrocytes and microglia, to neural injury that is initiated by the sudden injury induced increase in extracellular concentrations of glutamate and concomitant production of several proinflammatory molecules. It is important to understand the roles that different glia play in “gliopathy”, a condition that appears to persist after SCI. Furthermore, targeted treatment of gliopathy will attenuate mechanical allodynia in both central and peripheral neuropathic pain syndromes.     

Modulation of spinal neuronal excitability by spinal direct currents and locomotion after spinal cord injury

ObjectiveSpinal neuronal function is impaired after a severe spinal cord injury (SCI) and can be assessed by the analysis of spinal reflex (SR) behavior. We applied transcutaneous spinal direct current stimulation (tsDCS) and locomotor activity, to determine whether the excitability of spinal neuronal circuitries underlying locomotion can be modulated after motor complete SCI.MethodSRs were evoked by non-noxious electrical stimulation of the tibial nerve. SR behavior was assessed before, immediately after, and 20 min after four different interventions (anodal, cathodal, sham tsDCS, or locomotion) in subjects with motor complete SCI and healthy subjects.ResultsSR amplitudes in SCI subjects were increased after anodal tsDCS by 84% (p < 0.05). Cathodal, sham tsDCS and locomotion had no influence on SR amplitudes. In addition, reflex threshold was lower after anodal tsDCS and locomotion in SCI subjects (p < 0.05).ConclusionAnodal tsDCS is able to modulate spinal neuronal circuitries after SCI.SignificanceThis novel, noninvasive approach might be used as a tool to excite spinal neuronal circuitries. If applied repetitively within a training approach, anodal tsDCS might prevent adverse alterations in spinal reflex function in severely affected SCI subjects, i.e., a manifestation of a spinal neuronal dysfunction taking part below the level of a spinal lesion.     

FAS deficiency reduces apoptosis, spares axons and improves function after spinal cord injury

After spinal cord injury (SCI), apoptosis of neurons and oligodendrocytes is associated with axonal degeneration and loss of neurological function. Recent data have suggested a potential role for FAS death receptor-mediated apoptosis in the pathophysiology of SCI. In this study, we examined the effect of FAS deficiency on SCI in vitro and in vivo. FAS(Lpr/lpr) mutant mice and wildtype background-matched mice were subjected to a T5-6 clip compression SCI, and complementary studies were done in an organotypic slice culture model of SCI. Post-traumatic apoptosis in the spinal cord, which was seen in neurons and oligodendrocytes, was decreased in the FAS-deficient mice both in vivo and in vitro particularly in oligodendrocytes. FAS deficiency was also associated with improved locomotor recovery, axonal sparing and preservation of oligodendrocytes and myelin. However, FAS deficiency did not result in a significant increase in surviving neurons in the spinal cord at 6 weeks after injury, likely reflecting the importance of other cell death mechanisms for neurons. We conclude that inhibition of the FAS pathway may be a clinically attractive neuroprotective strategy directed towards oligodendroglial and axonal preservation in the treatment of SCI and neurotrauma.     

TGF-alpha increases astrocyte invasion and promotes axonal growth into the lesion following spinal cord injury in mice

Astrocytes respond to environmental cues and play a multifaceted role in the response to trauma in the central nervous system. As the most prevalent contributors to the glial scar, astrocytes are targeted as barriers to regeneration. However, there is also strong evidence that astrocytes are vital for neuroprotection and metabolic support after injury. In addition, consistent with their role during development, astrocytes may be capable of supporting the growth of injured axons. Therefore, we hypothesized that with appropriate stimulation, the reparative functions of endogenous astrocytes could be harnessed to promote axon growth and recovery after spinal cord injury. Transforming growth factor-α (TGF-α) is a mitogenic growth factor that is active on astrocytes and is poised to contribute to such a strategy. Recombinant TGF-α was administered intrathecally to adult C57BL/6 mice for two weeks following a moderate mid-thoracic spinal cord contusion. By three weeks post-injury, TGF-α infusion had not affected locomotor recovery, but promoted extensive axon growth and altered the composition of the lesion site. The center of the lesion in the treated mice contained greater numbers of new cells and increased astrocyte invasion. Despite the expression of inhibitory proteoglycans, there was a marked increase in axons expressing neurofilament and GAP-43 immunoreactivity, and the new axons were closely associated with increased laminin expression within and beyond the astrocyte matrix. The results demonstrate that astrocytes are dynamic players in the response to spinal cord injury, and the growth-supportive role of these cells can be enhanced by TGF-α infusion.     

Leukemia inhibitory factor promotes oligodendrocyte survival after spinal cord injury

Injury to the mammalian spinal cord is accompanied by a delayed, secondary wave of oligodendrocyte apoptosis that arises several days after the initial injury. A strong candidate to support oligodendrocyte survival after spinal cord injury is the pleiotropic cytokine, leukemia inhibitory factor (LIF). In vitro, LIF potentiates the differentiation and survival of oligodendrocyte precursors. LIF can also prevent oligodendrocyte apoptosis in response to either growth factor removal or cytotoxic challenge. More recently, in vivo studies have demonstrated that LIF is effective in preventing oligodendrocyte death in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). We therefore asked whether systemic delivery of LIF could ameliorate oligodendrocyte death in a mouse model of spinal cord injury. We have found that daily administration of LIF (25 microg/kg/day) promotes oligodendrocyte survival after spinal cord injury. Interestingly however, this effect does not appear to be mediated by a direct action of LIF on the oligodendrocyte but rather via an ancillary cell type, which results in augmented expression of another trophic factor capable of supporting oligodendrocyte survival, insulin-like growth factor 1 (IGF-1).     

PAF antagonist treatment reduces pro-inflammatory cytokine mRNA after spinal cord injury

Platelet-activating factor (PAF) is a pro-inflammatory molecule which contributes to secondary damage after spinal cord injury (SCI).To test if PAF contributes to cytokine induction following SCI, female Long-Evans rats were pretreated with the PAF antagonist WEB 2170 prior to receiving a contusion injury at spinal cord level T10 using the NYU impactor. RNase protection assay (RPA) analysis revealed that IL-1alpha mRNA peaked at I h post-injury while IL-1beta and IL-6 mRNA levels were higher and peaked at 6 h.TNF-alpha mRNA was almost undetectable. All mRNA levels approached baseline by 24 h. Treatment with WEB 2170 (1 mg/kg, i.p.) 15 min prior to injury significantly decreased mRNA levels for all three cytokines at 6 h post-injury, but not at I h post-injury. These results demonstrate a role for PAF in proinflammatory cytokine induction after SCI.     

AIDA reduces glutamate release and attenuates mechanical allodynia after spinal cord injury

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations, resulting in glutamate receptor-mediated excitotoxicity and central sensitization. To test contributions of group I metabotropic glutamate receptors (mGluRs) in SCI induced release of glutamate and in behavioral outcomes of central sensitization following injury, we administered 1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.1 nmol intraspinally), a potent group I mGluR antagonist, to rats immediately after spinal cord contusion injury. EAAs were collected by microdialysis and quantified using HPLC. AIDA significantly decreased extracellular glutamate but not aspartate concentrations and significantly attenuated the development of mechanical but not thermal allodynia. These results suggest mGluRs play an important role in injury-induced EAA release and in central sensitization following SCI.     

Seven-year survival following spinal cord injury

This retrospective study of 5131 persons who sustained a spinal cord injury between 1973 and 1980 sought to determine the overall seven-year survival rate and the effect of several prognostic factors on survival. All study subjects had been treated at one of seven federally designated Model Regional Spinal Cord Injury Care Systems and each had survived at least 24 hours after injury. The cumulative seven-year survival was 86.7%. Advancing age at time of injury and being rendered a neurologically complete quadriplegic were significant prognostic factors. The cumulative seven-year survival among neurologically complete quadriplegics who were at least 50 years of age when injured, was only 22.7%. Spinal cord injury mortality rates ranged from 3.26 to 20.78 times higher than corresponding rates for nonspinal injured persons. Although mortality rates for spinal cord injury patients have declined dramatically since World War II, life expectancies for these patients are still substantially below normal.     

Neuronal release and successful astrocyte uptake of aminoacidergic neurotransmitters after spinal cord injury in lampreys

In contrast to mammals, the spinal cord of lampreys spontaneously recovers from a complete spinal cord injury (SCI). Understanding the differences between lampreys and mammals in their response to SCI could provide valuable information to propose new therapies. Unique properties of the astrocytes of lampreys probably contribute to the success of spinal cord regeneration. The main aim of our study was to investigate, in the sea lamprey, the release of aminoacidergic neurotransmitters and the subsequent astrocyte uptake of these neurotransmitters during the first week following a complete SCI by detecting glutamate, GABA, glycine, Hu and cytokeratin immunoreactivities. This is the first time that aminoacidergic neurotransmitter release from neurons and the subsequent astrocytic response after SCI are analysed by immunocytochemistry in any vertebrate. Spinal injury caused the immediate loss of glutamate, GABA and glycine immunoreactivities in neurons close to the lesion site (except for the cerebrospinal fluid‐contacting GABA cells). Only after SCI, astrocytes showed glutamate, GABA and glycine immunoreactivity. Treatment with an inhibitor of glutamate transporters (DL‐TBOA) showed that neuronal glutamate was actively transported into astrocytes after SCI. Moreover, after SCI, a massive accumulation of inhibitory neurotransmitters around some reticulospinal axons was observed. Presence of GABA accumulation significantly correlated with a higher survival ability of these neurons. Our data show that, in contrast to mammals, astrocytes of lampreys have a high capacity to actively uptake glutamate after SCI. GABA may play a protective role that could explain the higher regenerative and survival ability of specific descending neurons of lampreys. GLIA 2014;62:1254–1269     

Reducing inflammation decreases secondary degeneration and functional deficit after spinal cord injury

Injury to the spinal cord is followed by degeneration, which leads to progressive tissue loss and usually cystic cavitation. Cellular and humoral immune responses have been implicated as mediators of secondary degeneration, and the expression of leukocyte chemoattractants has been shown to precede immune cell influx. However, the relationship between the increased expression of chemoattractants, the invasion of lymphocytes, and overall lesion evolution is poorly understood. Here, we show that the T-lymphocyte chemoattractant CXCL10 is upregulated after dorsal hemisection injury to the adult mammalian spinal cord of C57/BL6 mice, and that antibody neutralization of CXCL10 beginning 1 day prior to injury dramatically reduces the T-lymphocyte invasion that normally occurs after trauma. Notably, this treatment resulted in a significant reduction of secondary tissue loss and functional deficit. We conclude that CXCL10 plays a critical role in recruitment of T lymphocytes to sites of spinal cord injury, and that a reduction of T-lymphocyte recruitment significantly enhances tissue preservation and functional outcome.     

Clomethiazole (ZENDRA, CMZ) improves hind limb motor function and reduces neuronal damage after severe spinal cord injury in rat

The neuroprotective role of the expression of heat shock protein (HSP) and immediate early gene remains unclear. Using immunoelectron microscopy, we examined the ultrastructural integrity of the neurons with expression of c-Fos, c-Jun and HSP70 in gerbils after transient cerebral ischemia and repefusion. Induction of c-Fos and c-Jun was observed in the CA3 region resistant to ischemia, while HSP70 was expressed not only in the CA3 but also in the vulnerable CA1 region. With immunoelectron microscopy, the expression of c-Fos/c-Jun and HSP70 was observed in the neurons which retained neuronal integrity except for mitochondrial swelling and polyribosomal disaggregation. In contrast, the CA1 neurons without immunoreaction for HSP70 showed cytoplasmic vacuoles and parallel stacking of rough endoplasmic reticulum, the features associated with the process of delayed neuronal death. These findings suggested that c-Fos and c-Jun were induced selectively in reversibly damaged neurons, whereas HSP70 was up-regulated even in neurons with irreversible damage, but was more preferentially and intensely expressed in neurons with reversible damage. Received: 23 January 1998 / Revised, accepted: 12 June 1998     

Bone morphogenetic proteins mediate cellular response and, together with Noggin, regulate astrocyte differentiation after spinal cord injury

Bone morphogenetic proteins (BMPs) play a critical role in regulating cell fate determination during central nervous system (CNS) development. In light of recent findings that BMP-2/4/7 expressions are upregulated after spinal cord injury, we hypothesized that the BMP signaling pathway is important in regulating cellular composition in the injured spinal cord. We found that BMP expressions were upregulated in neural stem cells (NSCs), neurons, oligodendrocytes and microglia/macrophages. Increased expression levels of pSmad1/5/8 (downstream molecules of BMP) were detected in neurons, NSCs, astrocytes, oligodendrocytes and oligodendroglial progenitor cells (OPCs). Active astrocytes which form the astroglial scar were probably derived from NSCs, OPCs and resident astrocytes. Since quiescent NSCs in the normal adult spinal cord will proliferate and differentiate actively into neural cells after traumatic injury, we proposed that BMPs can regulate cellular components by controlling NSC differentiation. Neurosphere culture from adult mouse spinal cord showed that BMP-4 promoted astrocyte differentiation from NSCs while suppressing production of neurons and oligodendrocytes. Conversely, inhibition of BMP-4 by Noggin notably decreased the ratio of astrocyte to neuron numbers. However, intrathecal administration of Noggin in the injured spinal cord failed to attenuate glial fibrillar acidic protein (GFAP) expression even though it effectively reduced pSmad expression. Noggin treatment did not block phosphorylation of Stat3 and the induction of GFAP in the injured spinal cord, suggesting that in addition to the BMP/Smad pathway, the JAK/STAT pathway may also be involved in the regulation of GFAP expression after spinal cord injury.