Spinal cord injury, acute pain, and morphine tolerance are important issues in the clinical practice. A primary injury to the spinal cord causes both morphological and biochemical changes with initiation of the devastating secondary pathophysiological pathways that ultimately destroy CNS cells and cause degeneration of nerve fibers. Tissue injury is associated with sensitization of nociceptors and subsequent changes in the excitability of central neurons, known as central sensitization. Nociceptor sensitization and central sensitization are believed to underlie the development of primary and secondary hyperalgesia, respectively. The most efficacious drugs used to relieve pain are the opioid analgesics. Chronic administration leads to the development of tolerance. Tolerance is manifested as a decreased potency of the drug, so that progressively larger doses must be administered to achieve a given level of analgesia. The processes underlying opioid tolerance still need to be elucidated. Recently, it is found calpain-Cdk5 (cyclin-dependent kinase-5)-p35 pathway modulation implicated in neuroprotection, acute nociceptive response, and morphine analgesia. In this thesis, we evaluate calpain inhibitor-MDL28170 and Cdk5 inhibitor-roscovitine against rat spinal cord hemisection, formalin-induced acute nociceptive responses, and chronic morphine tolerance. We found calpain-Cdk5-p35 pathway inhibition could protect spinal cord hemisection and subsequent neurodegeneration, inhibit formalin-induced flinch response involving DARPP-32 (dopamine and c-AMP regulated phosphoprotein, MW=32 kDa) phosphorylation, and reverse right shifted morphine dose-response curve with upregulated ED50 (50% of effective dose) reduction. Taken together, calpain-Cdk5-p35 pathway inhibition is useful in the management of spinal cord injury, acute inflammatory pain, and attenuate morphine tolerance development with further clinical application.

Abstract The first part of this research was focused on the relationship between injury severity and cell death mechanisms after spinal cord ischemia-reperfusion injury. The major blood supply to the thoracolumbar spinal cord comes from the segmental arteries originating from the thoracoabdominal aorta. Paraplegia cause by spinal cord ischemia is a devastating complication of thoracoabdominal aortic surgery. Previous studies indicated that ischemia-reperfusion injury of the central nervous system causes two distinct types of cell death, necrosis and apoptosis. It was also implicated that the intensity of injury can somehow affect the cell death mechanisms. In the first series of our experiments, by occluding the descending thoracic aorta with or without simultaneously inducing hypovolemic hypotension in rats, we established a model of experimental spinal cord ischemia-reperfusion (SCIR) in which the injury severity can be controlled. Recordings of carotid blood pressure (CBP) and spinal cord blood flow (SCBF) showed that aortic occlusion induced dramatic CBP elevation but SCBF drop in both the normotensive (NT) and hypotensive (HT) groups. However, the HT group demonstrated significantly lower SCBF during aortic occlusion, and much slower elevation of SCBF after reperfusion, and extremely poor neurological performance. Spinal cord lesions were characterized by infarction associated with extensive necrotic cell death, but little apoptosis and caspase-3 activity. In contrast, in the NT group, SCIR resulted in minor tissue destruction associated with persistently abundant apoptosis, augmented caspase-3 activity, and favorable functional outcome. The relative sparing of motoneurons in the ventral horns from apoptosis might have accounted for the minor functional impairment in the NT group. The severity of ischemia-reperfusion (I/R) injury was found to have substantial impact on the histopathological changes and cell death mechanisms, which correlated with neurological performance. These findings implicate that injury severity and duration after injury are two critical factors to be considered in therapeutic intervention. Based on the knowledge that bPrevious studies have implicated both excitotoxicity and apoptosis are involved in the pathogenesis of SCIR injury, we proposedtested the possibility that the N-methyl-D-aspartate (NMDA) receptor antagonist (dizocilpine maleate: (MK801) and the protein synthesis inhibitor (cycloheximide) would produce a synergic effect in the treatment of SCIR injury. In the second series of experiments, I/R iSpinal cord ischemia-reperfusion injury was induced by a thoracic aortic occlusion and blood volume reduction, followed by reperfusion and volume restoration. ischemia-reperfusion Rats were treated with vehicle, MK801, cycloheximide, or combination of MK801 and cycloheximide in combination. The MK801 and combined therapy group got a better recovery of hHind limb motor function recovery was better in the MK801 and combined-therapy groups than in the control and cycloheximide groups. On the 7th day after ischemia-reperfusion injury, all three treated groups showed significantly higher neuronal survival rates (NSR) than that of the control group. Among the three treated groups, the combined-treatment group showed the highest NSR. In addition, the Ttherapeutic effect of the combined-treatment group (27.4% increase of NSR) iwas better than the anticipated by the addition of MK801 and cycloheximide based on NSR data group. The number of apoptotic cells of was significantly reduced in the cycloheximide group and the combined-treatment group, as compared to that of the control group. It was unchanged in the MK-801 group. These results suggest that combined treatments directed at blocking both NMDA receptor-mediated excitotoxic necrosis and caspase-mediated apoptosis might have synergic therapeutic potential in reducing SCIR injury. Mitogen-activated protein kinases (MAPKs) including c-Jun N-terminal kinases (JNK), p38, and extracellular signal-regulated kinases (ERK), play important roles in the transduction of stressful signals and the integration of cellular responses. Although it has been generally held that the JNK and p38 pathways are related to cell death and degeneration, while the ERK pathway, cell proliferation and survival, controversy still exists. The roles of the ERK pathway in I/R injury of the CNS, in particular, remain to be clarified, because contradictory data have been reported by different investigators. Given this controversy, in the third series of experiments, we examined in injured spinal cords the temporal and spatial profiles of ERK1/2 activation following SCIR, and the effects of inhibiting the kinase that phosphorylates ERK1/2, MEK. The results showed that I/R injury induced an immediate phosphorylation of ERK1/2 in the spinal cord, which was alleviated by a MEK inhibitor, U0126. The control group was characterized by poorer neurological outcome, more severe tissue destruction, pronounced apoptosis, and lower neuronal survival. In contrast, the U0126-treated group demonstrated more apparent improvement of hind limb motor function, less tissue destruction, lack of apoptosis, and higher neuronal survival. In addition, administration of U0126 also significantly increased the activation of nuclear factor-£eB (NF-£eB) and the expression of cellular inhibitor of apoptosis protein 2 (c-IAP2). These findings implicate that the mechanisms underlying the neuroprotection afforded by ERK1/2 inhibition may be through the NF-£eB-c-IAP2 axis. The activation of the MEK-ERK signaling pathway appeared to be harmful in SCIR injury. Strategies aimed at blocking this pathway may bear potential therapeutic benefits in the treatment of SCIR injury. The second part of the research was focused on the pathophysiology of surgery-associated paraspinal muscle injury and measures to protect surgically violated paraspinal muscles. The wide dissection and forceful retraction of paraspinal muscles which are often required for posterior spinal sugery may severely jeopardize the muscles structurally and functionally. Immediate posteoperative pathological changes in the surgically violated paraspinal muscles may cause severe pain and a delay of patient ambulation. Long-term sequelae of surgical injury of paraspinal muscles include chronic back pain and impaired back muscle strength. Ironically, being a common complication of posterior spinal surgery, paraspinal muscle injury is so often neglected. Limited previous data indicate that the underlying pathophysiology of muscle damage involve both mechanical and ischemic mechanisms. We hypothesized that surgical dissection and retraction may produce oxidative stress within the paraspinal muscles. Meanwhile, we also proposed that the oxidative stress may trigger certain protective mechanisms within the insulted muscles. The first part of our study was a human study conducted to assess the significance of oxidative stress, and the relationship between it and the stress response mediated by heat shock protein 70 (HSP70) induction within paraspinal muscles under intraoperative retraction. A group of patients with lumbar spondylolisthesis treated with posterolateral lumbar spinal fusion, pedicle fixation and laminectomy were enrolled. Multifidus muscle specimens were harvested intraoperatively before, at designated time points during, and after surgical retraction. Muscle samples were analyzed for HSP70 and malondialdehyde (MDA) levels. Both HSP70 expression and MDA production within multifidus muscle cells were increased significantly by retraction. HSP70 expression then dropped after a peak at 1.5 hr of retraction, whereas MDA levels remained elevated even after release of retractors for reperfusion of the muscles. Histopathological and immunohistochemical evidence indicated that the decline of HSP70 synthesis within muscle cells after prolonged retraction was the result of severe muscle damage. These results highlighted the noxious impact of intraoperative retraction on human paraspinal muscles, and the significance of oxidative stress at the cellular and molecular levels. It is also implicated that intraoperative maneuvers aimed at reducing the oxidative stress within the paraspinal muscles may help attenuating surgery-associated paraspinal muscle damage. Given the findings of the first part of our study, and the knowledge that inflammation is a major postoperative pathological finding in surgically injured paraspinal muscles, we proceeded to examine the roles of two important inflammatory mediators, cyclooxygenase (COX)-2 and nuclear factor (NF)-£eB, in the pathogenesis of retraction-associated paraspinal muscle injury. A rat model of paraspinal muscle dissection and retraction that mimicks the conditions in human posterior spinal surgery was established. In the control group, paraspinal muscles were dissected from the spine through a dorsal incision, and then laterally retracted. Paraspinal muscle specimens were harvested before, and at designated time points during and after persistent retraction. The time course of NF-£eB activation as well as the expression of COX-2 were examined. Severity of inflammation was evaluated based on histopathology and myeloperoxidase (MPO) activity. NF-£eB activation was inhibited by the administration of pyrrolidine dithiolcarbamate (PDTC) in the PDTC-treated group. In the control group, retraction induced an early increase of NF-£eB/DNA binding activity in paraspinal muscle cells, which persited throughout the whole course of retraction. COX-2 expression was not detectable until 1 day after surgery, and reached a peak at 3 days. The time course of COX-2 expression correlated with that of inflammatory pathology and MPO activity. Extensive muscle fiber loss and collagen fiber replacement were observed at 7 days after surgery. Pretreatment with PDTC inhibited intraoperative NF-£eB activation and greatly downregulated postoperative COX-2 expression and inflammation in the muscles. Fibrosis following inflammation was also significantly abolished by PDTC administration. These findings indicate that NF-£eB-regulated COX-2 expression and inflammation play an important role in the pathogenesis of surgery-associated paraspinal muscle injury. Therapeutic strategies involving NF-£eB inhibition may be applicable to the prevention of such injury.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Neurosciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Current treatments for spinal cord injury (SCI) are extremely limited due to the fact that the central nervous system lacks the intrinsic ability to regenerate, and constitutes a poor environment for regenerative axon growth. Nerve tissue engineering is an emerging field with the aim of repairing or creating new nerve tissues to promote functional recovery by using artificial tissue repair scaffolds. The design of a stable and consistent animal model of SCI is essential to study the effectiveness of scaffolds in promoting nervous system repair. In this study, a partial transection animal model was created with a three dimensional lesion at T8-T9 that disrupts axonal pathways unilaterally in the dorsal columns of the rat spinal cord. Alginate hydrogel scaffolds incorporating living Schwann cells were fabricated to evaluate the abilities of those scaffolds to foster axonal regeneration. The surgical technique was improved to provide better outcomes related to bleeding during surgery, weight control, neurological function and surgery duration. The survival rate of animals during the surgical procedure and post-surgery period was ultimately increased to 100%. Histology and immunohistochemistry results indicated that implanted alginate scaffolds may induce larger cavities and extenuate harmful inflammation responses, but that effect was ameliorated by inclusion of Schwann cells in the scaffold. However, neither plain alginate scaffolds nor scaffolds containing living Schwann cells were able to improve regeneration of identified axon tracts in the spinal dorsal columns. This research also employed a synchrotron based x-ray phase contrast imaging technique coupled with computed-tomography to visualize the low optical density structural features of scaffolds and spinal cord tissues in formaldehyde fixed specimens. The imaging results suggest that this is a promising method for analyzing the structure of tissue repair scaffolds within the spinal cord. This degree of structural characterization, potentially applicable to living tissue, is not afforded by other conventional image analysis techniques.

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Medical Sciences

Spinal cord injury is a devastating source of suffering in the spectrum of human pathophysiology; advancement for clinical therapy in this area has been stagnant in comparison to modern medical development. Current treatments are palliative, and functional recovery is minimal. During the first two weeks after injury, dense glial scar forms that is impenetrable by regenerating axons. Intervention is imperative to minimize scar formation and provide a supportive environment for axonal regeneration. Oligodendrocytes are critical to maintain the health of growing axons during development and after injury. Obtaining these cells through differentiation of neural progenitor cells (NPCs) is a viable option, but current clinical trials involving stem cells are plagued by poor cell survival and undirected differentiation. Research indicates that local extracellular matrix (ECM) is vital to progenitor differentiation and tissue regeneration. During development, spinal cord ECM is comprised of high concentrations of laminin and hyaluronic acid (HA), which provide essential cues to direct NPC migration and differentiation. The purpose of this research is to create a biomaterial optimized to direct NPC differentiation to oligodendrocytes. Natural biomaterials were optimized from distinct combinations of collagen I, HA, and laminin I to model the native ECM signals found during oligodendrocyte maturation. Four material combinations (collagen, collagen-HA-laminin, collagen-HA, and collagen-laminin) were fabricated into injectable hydrogels to mimic the range of compressive and shear mechanical properties present in neonatal central nervous system (CNS) tissue. Differentiation was assessed by culturing rodent fetal NPCs in these materials without specific soluble factors to direct cellular behavior. The three-component hydrogel performed optimally and achieved a 66% oligodendrocyte differentiation rate compared to approximately 15% in the collagen alone hydrogel. An in vivo study was then conducted using a rat contusion model of spinal cord injury with intervention using the injectable, three-component hydrogel seeded with rat NPCs. Functional recovery was assessed using six behavioral tests. Significant recovery was observed using two behavioral tests six weeks post-treatment. Lesion size was measured and correlated well with behavioral outcomes. The data obtained in this research indicate that a multi-component hydrogel mimicking native, developmental CNS tissue may address problems associated with current clinical practice. / text

Abstract Purpose: To characterize physical activity of people using manual wheelchairs with spinal cord injury in Manitoba. Methods: An observational study of manual wheelchairs users with spinal cord injury. Participants completed surveys related to self-efficacy for exercise, physical activity participation, and shoulder pain. Accelerometers were worn for 7 days on the wrist and trunk (GT3X, 100 Hz, 5 s epochs) and completed an activity log concurrently. Individual specific thresholds were determined for moderate intensity during a pace graded wheeling trial. Physical activity and sedentary time were characterized using various derived variables. Results: Twenty five participants (12 tetra:13 para, 21M:4F) demonstrated excellent accelerometer adherence achieving an average of 6.2 days worn for over 13 hours per day. A total of 74.6 min (all activity) and 115 min (contiguous bouts of activity) were achieved over time worn (6.2 days), corresponding to 11.8 and 18.5 min/day respectively. The participants substantially exceeded the published SCI guidelines (40 min/week, P<0.01) but were under the able bodied threshold of 150 min/week (P<0.01). No relationships were observed between surveys and objectively measured PA. Characterization of PA bouts revealed few participants (n=7) exhibiting single bout durations greater than 10 minutes, with an average contiguous bout duration of 30 s. A new functional classification scheme revealed positive correlations to PA variables and wheeling performance. Sedentary times ranged from 6.25 to 8.4 hours per day depending upon accelerometer placement. Conclusion: Arm based accelerometry can be used to determine PA and sedentary characteristics of manual wheelchair users with individual specific moderate intensity thresholds. Participants exceeded the SCI specific activity guidelines in terms of time per week, and failed to reach bout durations of 20 min. This study supports the use of able-bodied PA guidelines as a target. A new functional classification scheme was derived based upon wheeling dependent muscle innervation that had enhanced prediction of PA relative to standard anatomical classification / October 2015

Individuals with spinal cord injury (SCI) demonstrate reduced limb blood flow and muscle spasticity. It is plausible that the accumulation of metabolites, resulting from reduced perfusion, could exacerbate spasticity via activation of fusimotor neurons by Group III and IV afferents. PURPOSE: To determine the association between peripheral blood flow and muscle spasticity in persons with SCI. METHODS: A total of 16 individuals with SCI were classified into high (N=6), low (N=5), and no (N=5) spasticity groups according to their spasticity levels indicated by the modified Ashworth scale scores. Blood flow was measured in femoral and brachial arteries using duplex Doppler ultrasound and was normalized to limb lean mass obtained with dual energy X-ray absorptiometry. RESULTS: There were no significant group differences in age (30.5±4.15, 38.48±4.61, 32.6±4.89 years), time post SCI (8.5±4.2, 12.6±4.74, 6.8±1.66 years), American SCI Association motor scores (39.2±7.78, 59±12.34, 53.4±1.08), or sensory scores (96±22.1, 144.4±13.97, 130±13.8). Femoral artery blood flow, adjusted for limb lean mass, was significantly different (p=0.002) across the three leg spasticity groups (high 76.03±6.44, low 95.12±15.49, no 142.53±10.86 ml/min/kg).Total leg muscle spasticity scores were significantly and negatively correlated with femoral artery blood flow (r=-0.60, p=0.014). There was no significant difference in brachial artery blood flow between the three groups, indicating that the reduction in blood flow was confined to injured limbs and not due to systemic cardiovascular disorder. CONCLUSION: Among SCI patients, whole-leg blood flow is progressively lower in individuals with greater spasticity scores. These results suggest that a reduction in lower limb perfusion, among other factors, plays a significant role in the pathogenesis leading to muscle spasticity after SCI. / text