Kajana Satkunendrarajah

The effects of intrathecal injection of a hyaluronan-based hydrogel on inflammation, scarring and neurobehavioural outcomes in a rat model of severe spinal cord injury associated with arachnoiditis

Traumatic spinal cord injury (SCI) comprises a heterogeneous condition caused by a complex array of mechanical forces that damage the spinal cord - making each case somewhat unique. In addition to parenchymal injury, a subset of patients experience severe inflammation in the subarachnoid space or arachnoiditis, which can lead to the development of fluid-filled cavities/syringes, a condition called post-traumatic syringomyelia (PTS). Currently, there are no therapeutic means to address this devastating complication in patients and furthermore once PTS is diagnosed, treatment is often prone to failure. We hypothesized that reducing subarachnoid inflammation using a novel bioengineered strategy would improve outcome in a rodent model of PTS. A hydrogel of hyaluronan and methyl cellulose (HAMC) was injected into the subarachnoid space 24 h post PTS injury in rats. Intrathecal injection of HAMC reduced the extent of fibrosis and inflammation in the subarachnoid space. Furthermore, HAMC promoted improved neurobehavioural recovery, enhanced axonal conduction and reduced the extent of the lesion as assessed by MRI and histomorphometric assessment. These findings were additionally associated with a reduction in the post-traumatic parenchymal fibrous scar formation as evidenced by reduced CSPG deposition and reduced IL-1α cytokine levels. Our data suggest that HAMC is capable of modulating inflammation and scarring events, leading to improved functional recovery following severe SCI associated with arachnoiditis.

Delayed Post-Injury Administration of Riluzole Is Neuroprotective in a Preclinical Rodent Model of Cervical Spinal Cord Injury

Riluzole, a sodium/glutamate antagonist has shown promise as a neuroprotective agent. It is licensed for amyotrophic lateral sclerosis and is in clinical trial development for spinal cord injury (SCI). This study investigated the therapeutic time-window and pharmacokinetics of riluzole in a rodent model of cervical SCI. Rats were treated with riluzole (8 mg/kg) at 1 hour (P1) and 3 hours (P3) after injury or with vehicle. Afterward, P1 and P3 groups received riluzole (6 (mg/kg) every 12 hours for 7 days. Both P1 and P3 animals had significant improvements in locomotor recovery as measured by open field locomotion (BBB score, BBB subscore). Von Frey stimuli did not reveal an increase in at level or below level mechanical allodynia. Sensory-evoked potential recordings and quantification of axonal cytoskeleton demonstrated a riluzole-mediated improvement in axonal integrity and function. Histopathological and retrograde tracing studies demonstrated that delayed administration leads to tissue preservation and reduces apoptosis and inflammation. High performance liquid chromatography (HPLC) was undertaken to examine the pharmacokinetics of riluzole. Riluzole penetrates the spinal cord in 15 min, and SCI slowed elimination of riluzole from the spinal cord, resulting in a longer half-life and higher drug concentration in spinal cord and plasma. Initiation of riluzole treatment 1 and 3 hours post-SCI led to functional, histological, and molecular benefits. While extrapolation of post-injury time windows from rat to man is challenging, evidence from SCI-related biomarker studies would suggest that the post-injury time window is likely to be at least 12 hours in man.

A self-assembling peptide reduces glial scarring, attenuates post-traumatic inflammation and promotes neurological recovery following spinal cord injury

The pathophysiology of spinal cord injury (SCI) involves post-traumatic inflammation and glial scarring which interfere with repair and recovery. Self-assembling peptides (SAPs) are molecules designed for tissue engineering. Here, we tested the performance of K2(QL)6K2 (QL6), a SAP that attenuates inflammation and glial scarring, and facilitates functional recovery. We injected QL6 into the spinal cord tissue of rats 24 h after clip compression SCI. QL6 led to a significant reduction in post-traumatic apoptosis, inflammation and astrogliosis. It also resulted in significant tissue preservation as determined by quantitative histomorphometry. Furthermore, QL6 promoted axonal preservation/regeneration, demonstrated by BDA anterograde and Fluorogold retrograde tracing. In vitro experiments found that a QL6 scaffold enhanced neuronal differentiation and suppressed astrocytic development. The electrophysiology confirmed that QL6 led to significant functional improvement of axons, including increased conduction velocity, reduced refractoriness and enhanced high-frequency conduction. These neuroanatomical and electrophysiological improvements were associated with significant neurobehavioral recovery as assessed by the Basso-Beattie-Bresnahan technique. As the first detailed examination of the pathophysiological properties of QL6 in SCI, this work reveals the therapeutic potential of SAPs, and may suggest an approach for the reconstruction of the injured spinal cord.

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.

Immunoglobulin G (IgG) attenuates neuroinflammation and improves neurobehavioral recovery after cervical spinal cord injury

BackgroundEvidence suggests that the inflammatory events in the acute phase of spinal cord injury (SCI) exacerbate the initial trauma to the cord leading to poor functional recovery. As a result, minimizing the detrimental aspects of the inflammatory response after SCI is a promising treatment strategy. In this regard, immunoglobulin G (IgG) from pooled human serum is a promising treatment candidate. Due to its putative, though poorly characterized immuno-modulatory effects, IgG has been used clinically to treat neuroinflammatory disorders such as Guillain-Barré syndrome, but its effects in neurotrauma remain largely unexplored.MethodsThis study examines the potential neuroprotective effects of IgG in a well-characterized cervical model of SCI. Female Wistar rats were subject to moderate-severe clip compression injury at the C7-T1 level. IgG (0.4 g/kg) or saline was injected intravenously to randomly selected animals at 15 min post SCI. At several time points post SCI, biochemical assays, histology and immunohistochemistry analyses, and neurobehavioral assessments were used to examine the neuroprotective effects of IgG at the molecular, cellular, and neurobehavioral levels.ResultsWe found that intravenous treatment of IgG following acute clip-compression SCI at C7-T1 significantly reduced two important inflammatory cytokines: interleukin (IL)-1β and IL-6. This early reduction in pro-inflammatory signaling was associated with significant reductions in neutrophils in the spinal cord and reductions in the expression of myeloperoxidase and matrix metalloproteinase-9 in the injured spinal cord at 24 h after SCI. These beneficial effects of IgG were associated with enhanced tissue preservation, improved neurobehavioral recovery as measured by the BBB and inclined plane tests, and enhanced electrophysiological evidence of central axonal conduction as determined by motor-evoked potentials.ConclusionThe findings from this study indicate that IgG is a novel immuno-modulatory therapy which shows promise as a potential treatment for SCI.

Evaluating the role of IL-11, a novel cytokine in the IL-6 family, in a mouse model of spinal cord injury

BackgroundSpinal cord injury (SCI) is a devastating condition with substantial functional and social morbidity. Previous research has established that the neuroinflammatory response plays a significant role in cord damage post-SCI. However, global immunosuppressive therapies have demonstrated mixed results. As a result, more specific therapies modulating inflammation after injury are needed. In this regard, research into cytokine signaling has demonstrated that cytokines of the gp130 family including IL-6 and leukemia inhibitory factor (LIF) play key roles in mediating damage to the spinal cord. Since members of the gp130 family all share a common signal transduction pathway via the JAK/STAT system, we performed the first study of a relatively new member of the gp130 family, IL-11, in SCI.MethodsA validated clip-compression mouse model of SCI was used to assess for temporal changes in expression of IL-11 and its receptor, IL-11Rα, post-SCI. To elucidate the role of IL-II in the pathophysiology of SCI, we compared differences in locomotor recovery (Basso Mouse Score; CatWalk), electrophysiological spinal cord signaling, histopathology, and the acute inflammatory neutrophil response in IL-11Rα knockouts with littermate wild-type C57BL/6 mice.ResultsWe found an increase in gene expression of IL-11 in the spinal cord to a peak at twenty-four hours post-SCI with increases in IL-11Rα gene expression, peaking at seven days post-SCI. In spite of clear changes in the temporal expression of both IL-11 and its receptor, we found that there were no significant differences in motor function, electrophysiological signaling, histopathology, or neutrophil infiltration into the spinal cord between wild-type and knockout mice.ConclusionsThis is the first study to address IL-11 in SCI. This study provides evidence that IL-11 signaling may not play as significant a role in SCI as other gp130 cytokines, which will ideally guide future therapy design and the signaling pathways those therapies target.

Development and Characterization of a Novel Rat Model of Cervical Spondylotic Myelopathy: The Impact of Chronic Cord Compression on Clinical, Neuroanatomical, and Neurophysiological Outcomes

Cervical spondylotic myelopathy (CSM) is the most common cause of spinal cord impairment worldwide and is a risk factor for traumatic central cord syndrome. Despite advances in surgery, there are no effective neuroprotective treatments for CSM, which reflects a limited understanding of its pathophysiology. In order to develop therapeutic strategies, we have developed a novel rat model of chronic progressive cervical spinal cord compression that mimics CSM. A titanium-screw-based chronic compression device (CCD) was designed to achieve progressive cord compression at the C6 level. The CCD was fixed to the C2 and T2 spinous processes and a threaded screw was turned to induce compression. Sprague-Dawley rats (n=75) were divided into three groups: (1) sham (no compression, n=6), (2) mild compression (1.4 mm stenosis, n=27), and (3) severe compression (2.6 mm stenosis, n=42). Compression was evaluated using micro-computed tomography (micro-CT). The area of spared white matter, extent of cord flattening ratio, and loss of neurons were assessed. Functional deficits were characterized using sensory-evoked potential (SEP) recordings, and with neurobehavioral tests: the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, inclined plane, paw grip strength, and assessment of mechanical and thermal allodynia. Micro-CT confirmed progressive canal stenosis. The loss of intact white matter and cord flattening were significantly greater in rats with severe cord compression, and the number of neurons was reduced at the epicenter of cord compression. With chronic cord compression there was a significant decline in locomotor function, forelimb function, trunk stability/coordination, an increase in mechanical allodynia, and impaired axonal conduction. The CCD model results in chronic and precise cervical cord compression. The compression is associated with mechanical allodynia and measurable neurobehavioral, neurophysiological, and neuropathological deficits. We anticipate that the CCD model will enable the investigation of translationally-relevant therapeutic strategies for CSM.

Neural precursor cell transplantation enhances functional recovery and reduces astrogliosis in bilateral compressive/contusive cervical spinal cord injury.

Spinal cord injury has a significant societal and personal impact. Although the majority of injuries involve the cervical spinal cord, few studies of cell transplantation have used clinically relevant models of cervical spinal cord injury, limiting translation into clinical trials. Given this knowledge gap, we sought to examine the effects of neural stem/precursor cell (NPC) transplants in a rodent model of bilateral cervical contusion‐compression spinal cord injury. Bilateral C6‐level clip contusion‐compression injuries were performed in rats, which were then blindly randomized at 2 weeks after injury into groups receiving adult brain‐derived NPCs, vehicle, or sham operation. Long‐term survival of NPCs was evident at 10 weeks after transplant. Cell grafts were localized rostrocaudally surrounding the lesion, throughout white and gray matter. Graft‐derived cells were found within regions of gliotic scar and motor tracts and deposited myelin around endogenous axons. The majority of NPCs developed an oligodendroglial phenotype with greater neuronal profiles in rostral grafts. Following NPC transplantation, white matter was significantly increased compared with control. Astrogliosis and glial scar deposition, measured by GFAP‐positive and chondroitin sulfate proteoglycan‐positive volume, was significantly reduced. Forelimb grip strength, fine motor control during locomotion, and axonal conduction (by in vivo electrophysiology) was greater in cell‐treated animals compared with vehicle controls. Transplantation of NPCs in the bilaterally injured cervical spinal cord results in significantly improved spinal cord tissue and forelimb function, warranting further study in preclinical cervical models to improve this treatment paradigm for clinical translation.

Delayed Administration of a Bio-Engineered Zinc-Finger VEGF-A Gene Therapy Is Neuroprotective and Attenuates Allodynia Following Traumatic Spinal Cord Injury

Following spinal cord injury (SCI) there are drastic changes that occur in the spinal microvasculature, including ischemia, hemorrhage, endothelial cell death and blood-spinal cord barrier disruption. Vascular endothelial growth factor-A (VEGF-A) is a pleiotropic factor recognized for its pro-angiogenic properties; however, VEGF has recently been shown to provide neuroprotection. We hypothesized that delivery of AdV-ZFP-VEGF – an adenovirally delivered bio-engineered zinc-finger transcription factor that promotes endogenous VEGF-A expression – would result in angiogenesis, neuroprotection and functional recovery following SCI. This novel VEGF gene therapy induces the endogenous production of multiple VEGF-A isoforms; a critical factor for proper vascular development and repair. Briefly, female Wistar rats – under cyclosporin immunosuppression – received a 35 g clip-compression injury and were administered AdV-ZFP-VEGF or AdV-eGFP at 24 hours post-SCI. qRT-PCR and Western Blot analysis of VEGF-A mRNA and protein, showed significant increases in VEGF-A expression in AdV-ZFP-VEGF treated animals (p<0.001 and p<0.05, respectively). Analysis of NF200, TUNEL, and RECA-1 indicated that AdV-ZFP-VEGF increased axonal preservation (p<0.05), reduced cell death (p<0.01), and increased blood vessels (p<0.01), respectively. Moreover, AdV-ZFP-VEGF resulted in a 10% increase in blood vessel proliferation (p<0.001). Catwalk™ analysis showed AdV-ZFP-VEGF treatment dramatically improves hindlimb weight support (p<0.05) and increases hindlimb swing speed (p<0.02) when compared to control animals. Finally, AdV-ZFP-VEGF administration provided a significant reduction in allodynia (p<0.01). Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and functional benefits.

Riluzole promotes motor and respiratory recovery associated with enhanced neuronal survival and function following high cervical spinal hemisection.

Cervical spinal cord injury (SCI) can result in devastating functional deficits that involve the respiratory and hand function. The mammalian spinal cord has limited ability to regenerate and restore meaningful functional recovery following SCI. Riluzole, 2-amino-6-trifluoromethoxybenzothiazole, an anti-glutamatergic drug has been shown to reduce excitotoxicity and confer neuroprotection at the site of injury following experimental SCI. Based on promising preclinical studies, riluzole is currently under Phase III clinical trial for the treatment of SCI (ClinicalTrials.gov: NCT01597518). Riluzoles anti-glutamatergic role has the potential to regulate neuronal function and provide neuroprotection and influence glutamatergic connections distal to the initial injury leading to enhanced functional recovery following SCI. In order to investigate this novel role of riluzole we used a high cervical hemisection model of SCI, which interrupts all descending input to motoneurons innervating the ipsilateral forelimb and diaphragm muscles. Following C2 spinal cord hemisection, animals were placed into one of two groups: one group received riluzole (8 mg/kg) 1 h after injury and every 12 h thereafter for 7 days at 6 mg/kg, while the second group of injured rats received vehicle solution for the same duration of time. A third group of sham injured rats underwent a C2 laminectomy without hemisection and served as uninjured control rats. Interestingly, this study reports a significant loss of motoneurons within the cervical spinal cord caudal to C2 hemisection injury. Disruption of descending input led to a decrease in glutamatergic synapses and motoneurons caudal to the injury while riluzole treatment significantly limited this decline. Functionally, Hoffmann reflex recordings revealed an increase in the excitability of the remaining ipsilateral cervical motoneurons and significant improvements in skilled and unskilled forelimb function and respiratory motor function in the riluzole-treated animals. In conclusion, using a C2 hemisection injury model, this study provides novel evidence of motoneuron loss caudal to the injury and supports riluzoles capacity to promote neuronal preservation and function of neural network caudal to the SCI resulting in early and sustained functional improvements.