Melissa J. Walker

Systemic bisperoxovanadium activates Akt/mTOR, reduces autophagy, and enhances recovery following cervical spinal cord injury.

Secondary damage following primary spinal cord injury extends pathology beyond the site of initial trauma, and effective management is imperative for maximizing anatomical and functional recovery. Bisperoxovanadium compounds have proven neuroprotective effects in several central nervous system injury/disease models, however, no mechanism has been linked to such neuroprotection from bisperoxovanadium treatment following spinal trauma. The goal of this study was to assess acute bisperoxovanadium treatment effects on neuroprotection and functional recovery following cervical unilateral contusive spinal cord injury, and investigate a potential mechanism of the compounds action. Two experimental groups of rats were established to 1) assess twice-daily 7 day treatment of the compound, potassium bisperoxo (picolinato) vanadium, on long-term recovery of skilled forelimb activity using a novel food manipulation test, and neuroprotection 6 weeks following injury and 2) elucidate an acute mechanistic link for the action of the drug post-injury. Immunofluorescence and Western blotting were performed to assess cellular signaling 1 day following SCI, and histochemistry and forelimb functional analysis were utilized to assess neuroprotection and recovery 6 weeks after injury. Bisperoxovanadium promoted significant neuroprotection through reduced motorneuron death, increased tissue sparing, and minimized cavity formation in rats. Enhanced forelimb functional ability during a treat-eating assessment was also observed. Additionally, bisperoxovanadium significantly enhanced downstream Akt and mammalian target of rapamycin signaling and reduced autophagic activity, suggesting inhibition of the phosphatase and tensin homologue deleted on chromosome ten as a potential mechanism of bisperoxovanadium action following traumatic spinal cord injury. Overall, this study demonstrates the efficacy of a clinically applicable pharmacological therapy for rapid initiation of neuroprotection post-spinal cord injury, and sheds light on the signaling involved in its action.

Treadmill training induced lumbar motoneuron dendritic plasticity and behavior recovery in adult rats after a thoracic contusive spinal cord injury.

Spinal cord injury (SCI) is devastating, causing sensorimotor impairments and paralysis. Persisting functional limitations on physical activity negatively affect overall health in individuals with SCI. Physical training may improve motor function by affecting cellular and molecular responses of motor pathways in the central nervous system (CNS) after SCI. Although motoneurons form the final common path for motor output from the CNS, little is known concerning the effect of exercise training on spared motoneurons below the level of injury. Here we examined the effect of treadmill training on morphological, trophic, and synaptic changes in the lumbar motoneuron pool and on behavior recovery after a moderate contusive SCI inflicted at the 9th thoracic vertebral level (T9) using an Infinite Horizon (IH, 200 kDyne) impactor. We found that treadmill training significantly improved locomotor function, assessed by Basso-Beattie-Bresnahan (BBB) locomotor rating scale, and reduced foot drops, assessed by grid walking performance, as compared with non-training. Additionally, treadmill training significantly increased the total neurite length per lumbar motoneuron innervating the soleus and tibialis anterior muscles of the hindlimbs as compared to non-training. Moreover, treadmill training significantly increased the expression of a neurotrophin brain-derived neurotrophic factor (BDNF) in the lumbar motoneurons as compared to non-training. Finally, treadmill training significantly increased synaptic density, identified by synaptophysin immunoreactivity, in the lumbar motoneuron pool as compared to non-training. However, the density of serotonergic terminals in the same regions did not show a significant difference between treadmill training and non-training. Thus, our study provides a biological basis for exercise training as an effective medical practice to improve recovery after SCI. Such an effect may be mediated by synaptic plasticity, and neurotrophic modification in the spared lumbar motoneuron pool caudal to a thoracic contusive SCI.

A controlled spinal cord contusion for the rhesus macaque monkey

Most in vivo spinal cord injury (SCI) experimental models use rodents. Due to the anatomical and functional differences between rodents and humans, reliable large animal models, such as non-human primates, of SCI are critically needed to facilitate translation of laboratory discoveries to clinical applications. Here we report the establishment of a controlled spinal contusion model that produces severity-dependent functional and histological deficits in non-human primates. Six adult male rhesus macaque monkeys underwent mild to moderate contusive SCI using 1.0 and 1.5mm tissue displacement injuries at T9 or sham laminectomy (n=2/group). Multiple assessments including motor-evoked potential (MEP), somatosensory-evoked potential (SSEP), MR imaging, and monkey hindlimb score (MHS) were performed. Monkeys were sacrificed at 6 months post-injury, and the lesion area was examined for cavitation, axons, myelin, and astrocytic responses. The MHS demonstrated that both the 1.0 and 1.5mm displacement injuries created discriminative neurological deficits which were severity-dependent. The MEP response rate was depressed after a 1.0mm injury and was abolished after a 1.5mm injury. The SSEP response rate was slightly decreased following both the 1.0 and 1.5mm SCI. MRI imaging demonstrated an increase in T2 signal at the lesion site at 3 and 6months, and diffusion tensor imaging (DTI) tractography showed interrupted fiber tracts at the lesion site at 4h and at 6 months post-SCI. Histologically, severity-dependent spinal cord atrophy, axonal degeneration, and myelin loss were found after both injury severities. Notably, strong astrocytic gliosis was not observed at the lesion penumbra in the monkey. In summary, we describe the development of a clinically-relevant contusive SCI model that produces severity-dependent anatomical and functional deficits in non-human primates. Such a model may advance the translation of novel SCI repair strategies to the clinic.

Controlled cervical laceration injury in mice.

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of ±0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

Anatomical and functional effects of lateral cervical hemicontusion in adult rats.

PURPOSE Cervical injuries are the most common form of spinal cord injury (SCI), and are often complicated by pathological secondary damage. Therefore, cervical SCI is of great clinical importance for understanding pathology and potential therapies. Here we utilize a weight drop cervical hemi-contusion injury model using a NYU/MASCIS impactor that produced graded anatomical and functional deficits. METHODS Three groups of rats were established: 1) Sham (laminectomy only) (n = 6), 12.5 mm weight drop (n = 10), and 25 mm weight drop (n = 10) SCI groups. Forelimb functional assessments of grooming ability, cereal manipulation, and forepaw adhesive removal were performed weekly after injury. Using transcranial magnetic motor evoked potentials (tcMMEPs), supraspinal motor stimulations were recorded in both forelimbs and hindlimbs at 5 and 28d post-injury. Lesion volume and myelinated tissue area were assessed through histological analysis. RESULTS A 12.5 mm weight drop height produced considerable tissue damage compared to Sham animals, while a 25 mm drop induced even greater damage than the 12.5 mm drop (p < 0.05). Forelimb functional assessments showed that increased injury severity and tissue damage was correlated to the degree of forelimb functional deficits. Interestingly, the hindlimbs showed little to no motor function loss. Upon tcMMEP stimulation, surprisingly little motor signal was recorded in the hindlimbs despite outward evidence of hindlimb motor recovery. CONCLUSIONS Our findings highlight a correlation between anatomical damage and functional outcome in a graded cervical hemi-contusion model, and support a loss of descending motor control from supraspinal inputs and intraspinal plasticity that promote spontaneous hindlimb functional recovery in this model.

Transplantation of Pro-Oligodendroblasts, Preconditioned by LPS-Stimulated Microglia, Promotes Recovery After Acute Contusive Spinal Cord Injury.

Spinal cord injury (SCI) is a significant clinical challenge, and to date no effective treatment is available. Oligodendrocyte progenitor cell (OPC) transplantation has been a promising strategy for SCI repair. However, the poor posttransplantation survival and deficiency in differentiation into myelinating oligodendrocytes (OLs) are two major challenges that limit the use of OPCs as donor cells. Here we report the generation of an OL lineage population [i.e., pro-oligodendroblasts (proOLs)] that is relatively more mature than OPCs for transplantation after SCI. We found that proOLs responded to lipopolysaccharide (LPS)-stimulated microglia conditioned medium (L+M) by preserving toll-like receptor 4 (TLR4) expression, improving cell viability, and enhancing the expression of a myelinating OL marker myelin basic protein (MBP), compared to other OL lineage cells exposed to either LPS-stimulated (L+M) or nonstimulated microglia conditioned medium (L-M). When L+M-stimulated proOLs were intrathecally delivered through a lumbar puncture after a T10 thoracic contusive SCI, they promoted behavioral recovery, as assessed by the Basso–Beattie–Bresnahan (BBB) locomotor rating scale, stride length, and slips on the grid tests. Histologically, transplantation of L+M proOLs caused a considerable increase in intralesional axon numbers and myelination, and less accumulation of invading macrophages when compared with the vehicle control or OPC transplantation. Thus, transplantation of proOLs, preconditioned by L+M, may offer a better therapeutic potential for SCI than OPCs since the former may have initiated the differentiation process toward OLs prior to transplantation.

A Novel Vertebral Stabilization Method for Producing Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Remodeling of lumbar motor circuitry remote to a thoracic spinal cord injury promotes locomotor recovery

Retrogradely-transported neurotrophin signaling plays an important role in regulating neural circuit specificity. Here we investigated whether targeted delivery of neurotrophin-3 (NT-3) to lumbar motoneurons (MNs) caudal to a thoracic (T10) contusive spinal cord injury (SCI) could modulate dendritic patterning and synapse formation of the lumbar MNs. In vitro, Adeno-associated virus serotype two overexpressing NT-3 (AAV-NT-3) induced NT-3 expression and neurite outgrowth in cultured spinal cord neurons. In vivo, targeted delivery of AAV-NT-3 into transiently demyelinated adult mouse sciatic nerves led to the retrograde transportation of NT-3 to the lumbar MNs, significantly attenuating SCI-induced lumbar MN dendritic atrophy. NT-3 enhanced sprouting and synaptic formation of descending serotonergic, dopaminergic, and propriospinal axons on lumbar MNs, parallel to improved behavioral recovery. Thus, retrogradely transported NT-3 stimulated remodeling of lumbar neural circuitry and synaptic connectivity remote to a thoracic SCI, supporting a role for retrograde transport of NT-3 as a potential therapeutic strategy for SCI.