Although neural plasticity has traditionally been studied within the brain, evidence indicates that the spinal cord is quite plastic as well. Spinal neurons can even support a simple form of instrumental learning (Grau et al., 1998), as indicated by spinally transected rats’ ability to exhibit an increase in hind limb flexion duration when limb extension is associated with shock (controllable shock). If limb extension is not associated with shock (uncontrollable shock), a learning deficit develops. Recent research indicates that other forms of plasticity, such as long-term potentiation and central sensitization, do not depend on neural activity alone, but also on glial cells. I examined whether glial cells are also necessary in spinal instrumental learning and the learning deficit. Therefore, two glial inhibitors were selected: minocycline and fluorocitrate. To examine the role of glial cells in spinal instrumental learning, rats received intrathecal minocycline, fluorocitrate, or saline prior to testing with 30-minutes of controllable leg-shock. Results indicate that both drugs dose-dependently reduced acquisition, with higher doses resulting in shorter response durations. Once the response was acquired, fluorocitrate did not alter response maintenance. This suggests that glial cells are involved in the acquisition, but not the maintenance, of spinal learning. To examine the role of glial cells in the spinal learning deficit rats were given intrathecal minocycline, fluorocitrate, or saline prior to testing with 6-minutes of uncontrollable tail shock or no shock. Twenty-four hours later all rats were tested with 30-minutes of controllable leg-shock. Results indicated the learning deficit induced by uncontrollable shock was prevented by prior administration of fluorocitrate. Minocycline did not prevent the deficit; moreover, it appears that even in the absence of shock, minocycline caused a learning deficit. Overall, this data indicate that glial cells are necessary for the acquisition of spinal instrumental learning and the learning deficit. Furthermore, it provides further evidence for the role of glial cells in plasticity.

Previous work has shown that the spinal cord is capable of supporting a simple form of instrumental learning. Subjects that receive controllable shock to an extended hind limb will increase the duration of limb flexion over time in order to reduce net shock exposure. Exposure to as little as 6 minutes of uncontrollable stimulation prior to instrumental testing can elicit a long-lasting learning deficit. Prior work has suggested that this deficit may reflect an overexcitation of spinal neurons akin to central sensitization, and that learning is inhibited by the saturation of plasticity. The experiments in this dissertation were designed to test the role of the cytokine tumor necrosis factor alpha (TNFa) in the induction and expression of the deficit. It is believed that the inflammatory properties of TNFa may mediate the excitatory processes that lead to maladaptive spinal functioning. Experiments 1 and 2 tested the necessity of endogenous TNFa in the deficit produced by uncontrollable shock. These experiments showed that the inhibition of endogenous TNFa blocks both the induction and expression of the shock-induced deficit, suggesting a necessary role for TNFa in mediating the inhibition of spinal learning. Conversely, Experiment 3 was designed to test the sufficiency for TNFa in producing a learning deficit. I found that treatment with exogenous TNFa undermined spinal learning in a dose-dependent fashion, whether given immediately, or 24 hours prior to testing. Experiment 4 demonstrated that the long-term TNFa-induced deficit is mediated by TNFa receptor activity, as a TNF inhibitor given prior to testing blocked the expression of this deficit. As TNFa has been shown to be predominantly of glial origin, I next assessed the role that glia play in the TNFa-induced deficit. Experiment 5 showed that inhibiting glial metabolism prior to TNFa treatment blocked the capacity for TNFa to produce a long-term deficit. Experiment 6 assessed the potential for TNFa inhibition to block the deficit induced by lipopolysaccharide (LPS), an agent known to induce TNFa. TNFa has also been shown to drive neural excitation by increasing the trafficking of calciumpermeable AMPA receptors to the active zone of the post-synaptic bouton. Experiment 7 showed that selectively antagonizing these receptors prior to testing blocked the TNFa- induced deficit, suggesting a possible post-synaptic mechanism by which TNFa exerts its effects. Finally, histological evidence was sought to reinforce the previous behavioral findings. Experiment 8 used quantitative RT-PCR to assess the differential expression of TNFa mRNA in uncontrollably shocked subjects as compared to those receiving controllable shock and no shock. To determine concentrations of TNFa protein, an ELISA was run in Experiment 9 comparing uncontrollably shocked subjects to unshocked controls.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成4年7月20日 新田弘幸氏の博士論文として提出された

The purpose of this study is to quantify the dynamic mechanical properties of human cervical ligaments following whiplash. Cervical ligaments function to provide spinal stability, propioception, and protection during traumatic events to the spine. The function of cervical ligaments is largely dependant on their dynamic biomechanical properties, which include force and energy resistance, elongation capability, and stiffness. Whiplash has been shown to injure human cervical spine ligaments, and ligamental injury has been shown to alter their dynamic properties, with potential clinical consequences such as joint degeneration and pain. In this study we quantified the dynamic properties of human lower cervical ligaments following whiplash and compared their properties to those of intact ligaments. Whiplash simulation was performed using biofidelic whole cervical spine with muscle force replication (WCS-MFR) models. Next, ligaments were elongated to failure at a fast elongation rate and peak force, peak elongation, peak energy, and stiffness values were calculated from non-linear force-elongation curves. Peak force was highest in the ligamentum flavum (LF) and lowest in the intraspinous and supraspinous ligaments (ISL+SSL). Elongation was smallest in middle-third disc (MTD) and greatest in ISL+SSL. Highest peak energy was found in capsular ligament (CL) and lowest in MTD. LF was the stiffest ligament and ISL+SSL least stiff. These findings were similar to those found in intact ligaments. When directly comparing ligaments following whiplash to intact ligaments in a prior study it was found that the anterior longitudinal ligament (ALL) and CL had altered dynamic properties that were statistically significant, suggesting that whiplash may alter the dynamic properties of cervical ligaments. These findings may contribute to the understanding of whiplash injuries and the development of mathematical models simulating spinal injury.

Thesis (Ph. D.)--Case Western Reserve University, 2009. / [School of Medicine] Department of Neurosciences. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 123-155).

Thesis (Ph. D.)--Drexel University, 2004. / Includes abstract and vita. Includes bibliographical references (leaves 168-182).

Neuromyelitis optica (NMO) is a central nervous system inflammatory demyelinating disorders (CNS IDD) characterized by acute myelitis (AM) and optic neuritis (ON), especially clinically severe longitudinally extensive transverse myelitis (LETM) and simultaneous bilateral ON. Patients with recurrent AM especially LETM without ON, and patients with recurrent ON without AM may have disorders belonging to the spectrum of NMO, neuromyelitis optica spectrum disorders (NMOSD). NMO is likely autoimmune in nature as a significant proportion of patients are seropositive for aquaporin-4 (AQP4) autoantibodies. I studied the clinical features of local Chinese NMOSD patients and their AQP4 autoantibodies seropositivity rates of by indirect immunofluorescence using tissue slides containing primate cerebellum (tissued-based immunofluorescence assay) in patients with 1) NMO, 2) classical multiple sclerosis (CMS), 3) acute disseminated encephalomyelitis (ADEM), 4) single attack or relapsing AM, 5) single attack or relapsing ON, and 6) other neurological disorders. The results showed that NMOSD are severe CNS IDD affecting patients with a wide range of onset ages. Chinese NMOSD patients predominantly have relapsing NMO and relapsing LETM with severe attack of LETM and/or ON. The six-year mortality rate of patients with NMO or relapsing myelitis with LETM was about 12%. Two-thirds of patients have poor neurological outcome at a mean duration of 6.0 years. The results confirmed that AQP4 autoantibodies are specific for NMOSD, and detection of AQP4 autoantibodies is clinically useful for early diagnosis of NMOSD and distinction from CMS. I proceeded to study a cell-based immunofluorescence assay using transfected human embryonic kidney cells overexpressing human AQP4 on cell membrane and found that cell-based assay has higher sensitivity than tissue-based assay in detection of AQP4 autoantibodies in NMO (78% versus 61%). As our NMOSD patients frequently presented clinically with severe brainstem symptoms and signs and lesions in brainstem and other brain regions on magnetic resonance imaging (MRI), I studied the clinical and neuroradiological characteristics of Chinese NMOSD patients with brain involvement. I found that 59% of NMOSD patients have clinical and/or radiological evidence of brain involvement. Importantly, brainstem is the most frequently affected brain region and 24% of NMOSD patients had clinical manifestation of brainstem encephalitis. I also studied the pathogenicity of AQP4 autoantibodies in the absence of complement activation by passive transfer of IgG isolated from sera of NMOSD patients into mice pretreated with complete Freund’s adjuvant (CFA, containing heat-killed mycobacterium tuberculosis) and pertussis toxin (PTx). I observed that pretreatment with CFA and PTx led to breach of BBB in mouse, and IgG isolated from sera of NMOSD patients seropositive for AQP4 autoantibodies led to asymptomatic loss of AQP4 in gray and white matter in mouse spinal cord without inflammatory cell infiltration, demyelination or astrocytic loss in the absence of complement activation (human IgG cannot activate mouse complements). My findings support that 1) AQP4 autoantibodies binding to astrocytic AQP4 per se can cause downregulation of AQP4 in the absence of complement activation, and 2) complement activation with resultant complement activation products play key roles in the inflammation, demyelination and astrocyte cytotoxicity in NMO. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Spinal cord injury (SCI) results in extensive demyelination, leading to deleterious axon degeneration and inability of functional recovery. Remyelination has become a part of the fundamental strategy for SCI repair. Endogenous neural progenitor cells (NPCs) respond to SCI producing progenies and provide a possible source of regenerated oligodedrocytes for remyelination. During development of the central nervous system, glycogen synthase kinase-3 isoform beta (GSK-3β) is involved in multiple pathways that regulate oligodendrocyte differentiation and myelination, and thus may also play an important part in remyelination after SCI. This study aims to investigate (1) the role of GSK-3β in the differentiation of adult spinal cord derived-neural progenitor cells (ASC-NPCs); (2) whether AR-A014418 as a GSK-3β inhibitor, can promote oligodendroglial differentiation of ASC-NPCs; (3) the effect of LiCl, another GSK-3β inhibitor, on functional recovery after SCI; (4) the effects of LiCl on the myelin and axonal preservation after SCI. Neurosphere culture from adult mouse spinal cord was performed to test the effect of GSK-3β inhibitors, LiCl and AR-A014418, on differentiation of ASC-NPCs. Phenotyping of differentiated ASC-NPCs by immunocytochemistry (ICC) was performed to identify oligodendroglia progenitor cells (OPCs) at different stages. It was shown that LiCl (1 mM) and AR-A014418 (5 μM) promoted differentiation of OPCs as labeled by oligodendrocyte lineage-specific markers: PDGFR-α, NG2 and O4, while AR-A014418 was more potent in the OPC differentiation. Moreover, preliminary data from western blot confirmed that ARA014418 (5 μM) treatment increased the expression level of pGSK (inactive form of GSK-3) in differentiated ASC-NPCs. This suggests a possible strategy to modulate endogenous NPC response to SCI: to induce the preferential differentiation of NPCs into oligodendrocyte lineage by inhibiting GSK-3β activity and thus leading to enhanced remyelination by the differentiated oligodendrocytes. Basso Mouse Scale (BMS) open field test was used to evaluate the locomotive function of the spinal cord injured mice. The result showed that LiCl (4 mM, 200 μl) administration delivered locally at the lesion site by osmotic pump for 2 weeks improved functional recovery after SCI. Furthermore, immunohistochemistry (IHC) analyses revealed that LiCl treatment inhibited GSK-3β activity in the 〖Olig2〗^+ OPCs/oligodendrocytes, confirming LiCl as a GSK-3β inhibitor in vivo. Moreover, LiCl treatment better preserved myelin and axons detected by myelin basic protein (MBP) immunostaining and neurofilment-200 (NF-200) immunostaining respectively in the injured spinal cords. All together, the data from our in vitro and in vivo experiments suggested that LiCl treatment after spinal cord injury is beneficial for functional recovery by preventing the loss of myelin and axons after SCI and this effect is mediated via GSK-3β inhibition This study provided evidence for the involvement of GSK-3β in the regulation of OPC differentiation and the subsequent remyelination in the injured adult spinal cord. We propose GSK-3β as an important therapeutic target for SCI repair, LiCl as a potential candidate for SCI clinical treatment and the possibility to manipulate endogenous NPCs after SCI to enhance oligodendrocyte differentiation, remyelination, and ultimately better functional recovery.. / published_or_final_version / Anatomy / Master / Master of Philosophy

Many experimental therapies have been used in the search for effective approaches to improve recovery after spinal cord injury (SCI). One of the most promising approaches is the augmentation of spontaneously occurring plasticity in uninjured neural pathways. Acute intermittent hypoxia (AIH-brief exposures to reduced O2 levels alternating with normal O2 levels) elicits plasticity in respiratory and non-respiratory spinal systems in experimental animals. AIH treatment has also been shown to improve walking abilities in persons with chronic incomplete SCI. In this thesis, I first examined the effect of AIH treatment, alone or in combination with motor training, on functional recovery in a rat model of incomplete cervical SCI. Second, I examined the effect of AIH on the expression of plasticity- and hypoxia-related proteins in the spinal cords of SCI rats. In a randomized, blinded, normoxia-controlled study, rats were trained to cross a horizontal ladder and footslip errors were measured before surgery for SCI, 4 wks post-surgery, each day of daily AIH treatment, and 1, 2, 4 and 8 weeks after treatment. dAIH treatment consisted of 10 episodes of AIH: (5 min 11% O2: 5 min 21% O2) for 7 days beginning at 4 wks post-SCI. AIH-treated rats made fewer footslips on the ladder task compared to normoxia-treated control rats after 4 days of treatment and this improvement was sustained for 8 wks post-treatment. Importantly, daily ladder training was required for AIH treatment to facilitate recovery. AIH treatment + motor training also increased the expression of Hypoxia-inducible factor-1α (HIF-1α), Vascular endothelial growth factor (VEGF), Brain-derived neurotrophic factor (BDNF), tyrosine kinase B receptors (trkB) and phospho-trkB in spinal motor neurons in SCI rats compared to normoxia-treated SCI rats. In particular these hypoxia- and plasticity-related proteins were differentially expressed both temporally and spatially in the spinal cord during AIH treatment. These findings demonstrate that AIH + motor training can augment neural plasticity and improve motor recovery in an animal model of SCI. Taken together with the promising findings from human SCI studies, the results of this thesis suggest that AIH has potential as an effective therapy to restore motor function after nervous system injury.