Riluzole attenuates neuropathic pain and enhances functional recovery in a rodent model of cervical spondylotic myelopathy.

Cervical spondylotic myelopathy (CSM) is the commonest cause of spinal cord impairment worldwide and despite surgical treatment, it is commonly associated with chronic neuropathic pain and neurological impairment. Based on data suggesting a key role of sodium and glutamate mediated cellular injury in models of spinal cord compression, we examined whether riluzole, a sodium channel/glutamate blocker, could improve neurobehavioral outcomes in a rat model of CSM. To produce chronic progressive compression of the cervical spinal cord, we used an established model of graded mechanical cord compromise developed in our laboratory. The chronic (8weeks) mechanical compression of the cervical spinal cord resulted in persistent mechanical allodynia and thermal hyperalgesia at 8weeks. Moreover, we found increased expression of phosphorylated NR1 and NR2B in the dorsal horns as well as astrogliosis and increased microglia expression in the dorsal horns after mechanical compression. Following daily systemic administration for 7weeks after the induction of compression, riluzole (8mg/kg) significantly attenuated forelimb and hindlimb mechanical allodynia and alleviated thermal hyperalgesia in the tail. Importantly, riluzole led to a decrease in swing phase duration, an increase in hind leg swing speed and an increase paw intensity in gait analysis. Riluzole also decreased the number of phosphorylated NR1 and phosphorylated NR2B positive cells in the dorsal horns and the microglia activation in the dorsal horns. Together, our results indicate that systemic riluzole administration during chronic cervical spinal cord compression is effective at protecting spinal cord tissue, preserving neurobehavioral function and alleviating neuropathic pain, possibly by decreasing NMDA receptor phosphorylation in astrocytes and by eliminating microglia activation. As such, riluzole represents a promising clinical treatment for CSM.

A novel experimental model of cervical spondylotic myelopathy (CSM) to facilitate translational research.

Cervical spondylotic myelopathy (CSM) is the most common form of spinal cord impairment in adults. However critical gaps in our knowledge of the pathobiology of this disease have limited therapeutic advances. To facilitate progress in the field of regenerative medicine for CSM, we have developed a unique, clinically relevant model of CSM in rats. To model CSM, a piece of synthetic aromatic polyether, to promote local calcification, was implanted microsurgically under the C6 lamina in rats. We included a sham group in which the material was removed 30s after the implantation. MRI confirmed postero-anterior cervical spinal cord compression at the C6 level. Rats modeling CSM demonstrated insidious development of a broad-based, ataxic, spastic gait, forelimb weakness and sensory changes. No neurological deficits were noted in the sham group during the course of the study. Spasticity of the lower extremities was confirmed by a significantly greater H/M ratio in CSM rats in H reflex recordings compared to sham. Rats in the compression group experienced significant gray and white matter loss, astrogliosis, anterior horn cell loss and degeneration of the corticospinal tract. Moreover, chronic progressive posterior compression of the cervical spinal cord resulted in compromise of the spinal cord microvasculature, blood-spinal cord barrier disruption, inflammation and activation of apoptotic signaling pathways in neurons and oligodendrocytes. Finally, CSM rats were successfully subjected to decompressive surgery as confirmed by MRI. In summary, this novel rat CSM model reproduces the chronic and progressive nature of human CSM, produces neurological deficits and neuropathological features accurately mimicking the human condition, is MRI compatible and importantly, allows for surgical decompression.

Fas/FasL-mediated apoptosis and inflammation are key features of acute human spinal cord injury: implications for translational, clinical application.

The Fas/FasL system plays an important role in apoptosis, the inflammatory response and gliosis in a variety of neurologic disorders. A better understanding of these mechanisms could lead to effective therapeutic strategies following spinal cord injury (SCI). We explored these mechanisms by examining molecular changes in postmortem human spinal cord tissue from cases with acute and chronic SCI. Complementary studies were conducted using the in vivo Fejota™ clip compression model of SCI in Fas-deficient B6.MRL-Fas-lpr (lpr) and wild-type (Wt) mice to test Fas-mediated apoptosis, inflammation, gliosis and axonal degeneration by immunohistochemistry, Western blotting, gelatin zymography and ELISA with Mouse 32-plex cytokine/chemokine panel bead immunoassay. We report novel evidence that shows that Fas-mediated apoptosis of neurons and oligodendrocytes occurred in the injury epicenter in all cases of acute and subacute SCI and not in chronic SCI or in control cases. We also found significantly reduced apoptosis, expression of GFAP, NF-κB, p-IKappaB and iba1, increased number of CD4 positive T cells and MMP2 expression and reduced neurological dysfunction in lpr mice when compared with Wt mice after SCI. We found dramatically reduced inflammation and cytokines and chemokine expression in B6.MRL-Fas-lpr mice compared to Wt mice after SCI. In conclusion, we report multiple lines of evidence that Fas/FasL activation plays a pivotal role in mediating apoptosis, the inflammatory response and neurodegeneration after SCI, providing a compelling rationale for therapeutically targeting Fas in human SCI.

Human neuropathological and animal model evidence supporting a role for Fas-mediated apoptosis and inflammation in cervical spondylotic myelopathy.

Although cervical spondylotic myelopathy is a common cause of chronic spinal cord dysfunction in humans, little is known about the molecular mechanisms underlying the progressive neural degeneration characterized by this condition. Based on animal models of cervical spondylotic myelopathy and traumatic spinal cord injury, we hypothesized that Fas-mediated apoptosis and inflammation may play an important role in the pathobiology of human cervical spondylotic myelopathy. We further hypothesized that neutralization of the Fas ligand using a function-blocking antibody would reduce cell death, attenuate inflammation, promote axonal repair and enhance functional neurological outcomes in animal models of cervical spondylotic myelopathy. We examined molecular changes in post-mortem human spinal cord tissue from eight patients with cervical spondylotic myelopathy and four control cases. Complementary studies were conducted using a mouse model of cervical spondylotic myelopathy (twy/twy mice that develop spontaneous cord compression at C2-C3). We observed Fas-mediated apoptosis of neurons and oligodendrocytes and an increase in inflammatory cells in the compressed spinal cords of patients with cervical spondylotic myelopathy. Furthermore, neutralization of Fas ligand with a function-blocking antibody in twy/twy mice reduced neural inflammation at the lesion mediated by macrophages and activated microglia, glial scar formation and caspase-9 activation. It was also associated with increased expression of Bcl-2 and promoted dramatic functional neurological recovery. Our data demonstrate, for the first time in humans, the potential contribution of Fas-mediated cell death and inflammation to the pathobiology of cervical spondylotic myelopathy. Complementary data in a murine model of cervical spondylotic myelopathy further suggest that targeting the Fas death receptor pathway is a viable neuroprotective strategy to attenuate neural degeneration and optimize neurological recovery in cervical spondylotic myelopathy. Our findings highlight the possibility of medical treatments for cervical spondylotic myelopathy that are complementary to surgical decompression.

Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injury.

Activation of the Fas receptor has been recently linked to apoptotic cell death after spinal cord injury (SCI). Although it is generally considered that Fas activation mediates apoptosis predominantly through the extrinsic pathway, we hypothesized that intrinsic mitochondrial signaling could be involved in the underlying mechanism of Fas-induced apoptosis after SCI. In the present study, we utilized the Fejota clip compression model of SCI at T5-6 in C57BL/6 Fas-deficient (lpr) and wild-type mice. Complementary studies were conducted using an in vitro model of trauma or a Fas-activating antibody to induce apoptosis in primary neuronal-glial mixed spinal cord cultures. After in vivo SCI, lpr mice, in comparison with wild-type mice, exhibited reduced numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells at the lesion, reduced expression of truncation of Bid (tBid), apoptosis-inducing factor, activated caspase-9 and activated caspase-3, and increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. After in vitro neurotrauma or the induction of Fas signaling by the Jo2 activating antibody, lpr spinal cord cultures showed an increased proportion of cells retaining mitochondrial membrane integrity and a reduction of tBid expression, caspase-9 and caspase-3 activation, and TUNEL-positive cells as compared to wild-type spinal cord cultures. The neutralization of Fas ligand (FasL) protected against traumatically induced or Fas-mediated caspase-3 activation and the loss of mitochondrial membrane potential and tBid expression in wild-type spinal cord cultures. However, in lpr spinal cord cultures, FasL neutralization had no protective effects. In summary, these data provide direct evidence for the induction of intrinsic mitochondrial signaling pathways following Fas activation after SCI.

Molecular mechanisms of spinal cord dysfunction and cell death in the spinal hyperostotic mouse: implications for the pathophysiology of human cervical spondylotic myelopathy.

Cervical spondylotic myelopathy (CSM) is the most common cause of spinal cord dysfunction in adults in Western society. Paradoxically, relatively little is known about the pathobiological mechanisms associated with the progressive loss of neural tissue in the spinal cord of CSM patients. In this report we have utilized the twy/twy mutant mouse, which develops ossification of the ligamentum flavum at C2-C3 and exhibits progressive paralysis. This animal model represents an excellent in vivo model of CSM. This study reports novel evidence, which demonstrates that chronic extrinsic cervical spinal cord compression leads to Fas-mediated apoptosis of neurons and oligodendrocytes which is associated with activation of caspase-8, -9 and -3 and progressive neurological deficits. While surgical decompression will remain the mainstay of management of CSM, molecular therapies, which target Fas-mediated apoptosis could show promise as a complementary approach to maximize neurological recovery in this common spinal cord condition.