Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 May 24

Primary spinal cord injury (SCI) results from direct mechanical damage to the spinal cord. The resulting pathochemical and pathophysiological events, including oxidative stress and inflammation, lead to secondary injury. The ability to decrease secondary injury may lead to improved recovery. Increasing glutathione production after SCI leads to decreased secondary injury. Glutamine is an important precursor to glutathione following trauma. Skeletal muscle phenotype is strongly influenced by neuromuscular activity. SCI causes myosin heavy chain (MyHC) profiles to shift towards faster isoforms in slow muscles and slower isoforms in fast muscles. The hypothesis was that glutamine, as a precursor of glutathione, administration to SCI rats would lead to better functional recovery and a more preserved MyHC phenotype in locomotory muscles. <p> Rats were assigned to one of four groups; healthy, laminectomy only, untreated SCI, and SCI treated with an intraperitoneal injection of 1mmol/kg glutamine every 12 hours for one week after injury. SCIs were performed at T6 with a modified aneurism clip. Functional recovery was measured weekly using the Basso-Beattie-Bresnahan scale and the angle board method. Six weeks later, all rats were killed, and their extensor digitorum longus and soleus muscles excised and weighed. MyHC composition of the muscles was determined using SDS-PAGE.<p>The hypothesis that glutamine treatment following SCI would lead to better functional recovery and a more preserved MyHC profile was validated. Glutamine treated rats received significantly higher BBB scores (p<0.01) and angle board scores (p<0.001) than untreated SCI rats. Glutamine treatment also reduces muscle atrophy in the soleus muscle, but not the extensor digitorum longus (EDL). In untreated rats the soleus muscle accounted for significantly (p<0.001) less of the percentage of total body weight than the soleus muscle from glutamine treated rats. Finally, SCI rats with preserved functional abilities displayed a significantly better preserved MyHC profile compared to untreated SCI rats. In the soleus healthy rats contain 94% type 1 myosin, treated rats maintained 68% which was significantly (p<0.001) greater than 28% maintained by untreated rats. In the EDL healthy rats contain 55% type 2b myosin, treated rats maintained 32% which was greater than 26% type 2b myosin maintained by untreated rats.

Neurotrophin-3 (NT-3) and anti-NogoA have shown promise in regenerative strategies after spinal cord injury; however, conventional methods for localized release to the injured spinal cord are either prone to infection or not suitable for sustained release. To address these issues, we have designed a composite drug delivery system that is comprised of poly(lactic-co-glycolic acid) (PLGA) nanoparticles dispersed in an injectable hydrogel of hyaluronan and methyl cellulose (HAMC). Achieving sustained and bioactive protein release from PLGA particles is a known challenge; consequently, we studied the effects of processing parameters and excipient selection on protein release, stability, and bioactivity. We found that embedding PLGA nanoparticles in HAMC results in more linear drug release due to the formation of a diffusion-limiting layer of methyl cellulose on the particle surface. Co-encapsulated MgCO3 was able to significantly improve NT-3 bioactivity, while trehalose + hyaluronan was able to improve anti-NogoA bioactivity and release.

Neurotrophin-3 (NT-3) and anti-NogoA have shown promise in regenerative strategies after spinal cord injury; however, conventional methods for localized release to the injured spinal cord are either prone to infection or not suitable for sustained release. To address these issues, we have designed a composite drug delivery system that is comprised of poly(lactic-co-glycolic acid) (PLGA) nanoparticles dispersed in an injectable hydrogel of hyaluronan and methyl cellulose (HAMC). Achieving sustained and bioactive protein release from PLGA particles is a known challenge; consequently, we studied the effects of processing parameters and excipient selection on protein release, stability, and bioactivity. We found that embedding PLGA nanoparticles in HAMC results in more linear drug release due to the formation of a diffusion-limiting layer of methyl cellulose on the particle surface. Co-encapsulated MgCO3 was able to significantly improve NT-3 bioactivity, while trehalose + hyaluronan was able to improve anti-NogoA bioactivity and release.

Spinal cord injury occurs at a rate of 11.5 - 53.4 per million in developed countries with great emotional and financial consequences. The damage caused by the initial injury is followed by secondary damage, a complex cascade of mechanisms including ischemia, oxidative stress, inflammation and apoptosis. Although nothing can be done to reverse the initial damage to the spinal cord once it occurs, the secondary damage can be targeted by therapeutics to improve recovery. Following injury, concentrations of the potent antioxidant glutathione (GSH) are decreased in the spinal cord which potentiates mechanisms of secondary damage. In an attempt to maintain the GSH concentrations, the non-essential amino acid glutamine was tested as it was shown to increase GSH concentrations both in vivo and in vitro. Glutamine is being used extensively in clinical research in an expansive number of physiological and pathological conditions including brain trauma. To examine the therapeutic potential of glutamine after spinal cord trauma, two compression injury models, the modified aneurysm clip and the modified forceps, were used to induce an injury in male Wistar rats. We have demonstrated the ability of glutamine treatment (1 mmol/kg), given 1 hour after a 30 g aneurysm clip injury to increase GSH not only in whole blood samples but within the spinal tissue at the site of injury. Increasing GSH in this way also resulted in improved locomotor scores and maintenance of white matter tissue at the injury epicenter. Experiments using the forceps model were then performed to determine if the potency of glutamine treatment would be carried over to a different model and at a variety of severities. Glutamine, again, demonstrated the ability to improve maintenance of whole blood GSH, locomotor scores and tissue histology. In our experiments, glutamine has proven to be a potent therapeutic for spinal cord injury with an effect that is matched by few compounds currently being studied and well exceeding the standard therapeutic, methylprednisolone. Given the breadth of knowledge regarding the effects of glutamine clinically in numerous paradigms and the potency of the therapeutic effect seen in these studies, we believe that glutamine is fit for clinical trial and has a high potential for success.

Primary spinal cord injury (SCI) results from direct mechanical damage to the spinal cord. The resulting pathochemical and pathophysiological events, including oxidative stress and inflammation, lead to secondary injury. The ability to decrease secondary injury may lead to improved recovery. Increasing glutathione production after SCI leads to decreased secondary injury. Glutamine is an important precursor to glutathione following trauma. Skeletal muscle phenotype is strongly influenced by neuromuscular activity. SCI causes myosin heavy chain (MyHC) profiles to shift towards faster isoforms in slow muscles and slower isoforms in fast muscles. The hypothesis was that glutamine, as a precursor of glutathione, administration to SCI rats would lead to better functional recovery and a more preserved MyHC phenotype in locomotory muscles. <p> Rats were assigned to one of four groups; healthy, laminectomy only, untreated SCI, and SCI treated with an intraperitoneal injection of 1mmol/kg glutamine every 12 hours for one week after injury. SCIs were performed at T6 with a modified aneurism clip. Functional recovery was measured weekly using the Basso-Beattie-Bresnahan scale and the angle board method. Six weeks later, all rats were killed, and their extensor digitorum longus and soleus muscles excised and weighed. MyHC composition of the muscles was determined using SDS-PAGE.<p>The hypothesis that glutamine treatment following SCI would lead to better functional recovery and a more preserved MyHC profile was validated. Glutamine treated rats received significantly higher BBB scores (p<0.01) and angle board scores (p<0.001) than untreated SCI rats. Glutamine treatment also reduces muscle atrophy in the soleus muscle, but not the extensor digitorum longus (EDL). In untreated rats the soleus muscle accounted for significantly (p<0.001) less of the percentage of total body weight than the soleus muscle from glutamine treated rats. Finally, SCI rats with preserved functional abilities displayed a significantly better preserved MyHC profile compared to untreated SCI rats. In the soleus healthy rats contain 94% type 1 myosin, treated rats maintained 68% which was significantly (p<0.001) greater than 28% maintained by untreated rats. In the EDL healthy rats contain 55% type 2b myosin, treated rats maintained 32% which was greater than 26% type 2b myosin maintained by untreated rats.

Background: Persons who sustain a spinal cord injury (SCI) experience a dramatic loss of muscle and bone, and a dramatic increase in adipose tissue. It has been suggested that the muscle atrophy, obesity, and sublesional osteoporosis (SLOP) that occurs after SCI is due in part to the loss of voluntary control of the skeletal muscles in the lower extremities, impaired energy metabolism below the level of the lesion, and cessation of sufficient mechanical strain on bone. The prevalence of obesity and SLOP after SCI leads to increased cardiovascular disease and fracture risk, respectively. Current body composition screening procedures for the general population fail to identify individuals with SCI who are obese or have SLOP. Muscle contractions provide physiological loads on bone; thereby a muscle-bone relationship is proposed with proportional declines in muscle and bone after SCI. In addition, both positive and negative relationships have been proposed between adipose tissue and bone; increased skeletal load bearing from excess adipose tissue mass may account for the positive associations reported to date. Due to a lack of load bearing activity after SCI, there should be a negative association between adipose tissue and bone. Objectives: The primary objective is to characterize body composition among adults with chronic SCI using valid, reliable, and interpretable measures, and to suggest screening procedures for the detection of obesity and SLOP in this population. The secondary objectives are to explore the associations between: 1) muscle and bone, and 2) adipose tissue and bone. Design and Setting: Cross sectional observational. Population: A sample of 16 individuals (13 men, 3 women) with chronic SCI participated in this study. The neurological level of lesion ranged from C3-T12, with 9 motor complete and 7 incomplete SCI. Average±standard deviation for age was 51.12±12.37 years, and duration of injury 16.5±7.87 years. An additional 29 individuals with chronic SCI were included when exploring the relationship between muscle and bone. Forty-one individuals (31 men, 9 women) were included in this analysis; the neurological level of lesion ranged from C2-T12, with 13 motor complete and 28 incomplete SCI. Average±standard deviation for age was 48.7±13.36 years, and duration of injury 114.22±10.4 years. Methods: Lean tissue, adipose tissue, and bone tissue were measured via surrogates of body adiposity, as well as two different scanning technologies. Lean tissue was assessed via muscle cross sectional area (CSA) (mm2) and muscle density (mg/cm3), and measured using peripheral quantitative computed tomography (pQCT). Adipose tissue was assessed via body mass index (BMI) (kg/m2), waist circumference (WC) (cm), and % body fat, and measured using a floor scale, tape measure, and dual energy x-ray absorptiometry (DXA), respectively. Bone tissue was assessed via hip, distal femur, and proximal tibia areal bone mineral density (aBMD) (g/cm2) using DXA, as well as cortical thickness (mm) and total volumetric bone mineral density (vBMD) (mg/cm3) at the 1/3 proximal tibia, and trabecular vBMD (mg/cm3) and total vBMD (mg/cm3) at the distal tibia using pQCT. The relationships between muscle and bone, and adipose tissue and bone, were determined by correlating muscle CSA with indices of bone strength, and indices of obesity with indices of SLOP, respectively. Results: The majority of participants had lean tissue values below able-bodied norms (67-100%). When using the able-bodied definition of BMI >30 kg/m2, 19% of individuals were obese, whereas 63% and 81% were obese when using SCI-specific definitions of BMI >25 kg/m2 or >22 kg/m2, respectively. One hundred percent of individuals had SLOP using distal femur Z-score, and over 50% were at risk of fracture using distal femur fracture threshold of <0.78 g/cm2. Weak (r=0.42) to moderate (r=0.57) correlations were found between muscle CSA and indices of bone strength, supporting the theory of a muscle-bone unit. No correlations were found between adipose tissue and bone. Conclusions: Based on the cohort data, we propose that individuals with ≥2 risk factors (female, ≥60 years of age, duration of injury (DOI) ≥10, tetraplegia, motor complete) should be screened for obesity using % body fat from DXA as well as a combination of carefully interpreted SCI-specific BMI and WC. In addition, these same individuals should be screened for SLOP using a distal femur Z-score and fracture threshold from DXA. It is clear that due to the prevalence of obesity and SLOP in this population, intervention for prevention or treatment is essential. The presence of a muscle-bone unit indicates that muscle atrophy contributes to a reduction in bone strength; this is clinically important, as muscle strength is potentially amenable to rehabilitation intervention. No correlation was found between adipose tissue and bone. Future work should continue to explore these relationships using appropriate technology.

Background: Persons who sustain a spinal cord injury (SCI) experience a dramatic loss of muscle and bone, and a dramatic increase in adipose tissue. It has been suggested that the muscle atrophy, obesity, and sublesional osteoporosis (SLOP) that occurs after SCI is due in part to the loss of voluntary control of the skeletal muscles in the lower extremities, impaired energy metabolism below the level of the lesion, and cessation of sufficient mechanical strain on bone. The prevalence of obesity and SLOP after SCI leads to increased cardiovascular disease and fracture risk, respectively. Current body composition screening procedures for the general population fail to identify individuals with SCI who are obese or have SLOP. Muscle contractions provide physiological loads on bone; thereby a muscle-bone relationship is proposed with proportional declines in muscle and bone after SCI. In addition, both positive and negative relationships have been proposed between adipose tissue and bone; increased skeletal load bearing from excess adipose tissue mass may account for the positive associations reported to date. Due to a lack of load bearing activity after SCI, there should be a negative association between adipose tissue and bone. Objectives: The primary objective is to characterize body composition among adults with chronic SCI using valid, reliable, and interpretable measures, and to suggest screening procedures for the detection of obesity and SLOP in this population. The secondary objectives are to explore the associations between: 1) muscle and bone, and 2) adipose tissue and bone. Design and Setting: Cross sectional observational. Population: A sample of 16 individuals (13 men, 3 women) with chronic SCI participated in this study. The neurological level of lesion ranged from C3-T12, with 9 motor complete and 7 incomplete SCI. Average±standard deviation for age was 51.12±12.37 years, and duration of injury 16.5±7.87 years. An additional 29 individuals with chronic SCI were included when exploring the relationship between muscle and bone. Forty-one individuals (31 men, 9 women) were included in this analysis; the neurological level of lesion ranged from C2-T12, with 13 motor complete and 28 incomplete SCI. Average±standard deviation for age was 48.7±13.36 years, and duration of injury 114.22±10.4 years. Methods: Lean tissue, adipose tissue, and bone tissue were measured via surrogates of body adiposity, as well as two different scanning technologies. Lean tissue was assessed via muscle cross sectional area (CSA) (mm2) and muscle density (mg/cm3), and measured using peripheral quantitative computed tomography (pQCT). Adipose tissue was assessed via body mass index (BMI) (kg/m2), waist circumference (WC) (cm), and % body fat, and measured using a floor scale, tape measure, and dual energy x-ray absorptiometry (DXA), respectively. Bone tissue was assessed via hip, distal femur, and proximal tibia areal bone mineral density (aBMD) (g/cm2) using DXA, as well as cortical thickness (mm) and total volumetric bone mineral density (vBMD) (mg/cm3) at the 1/3 proximal tibia, and trabecular vBMD (mg/cm3) and total vBMD (mg/cm3) at the distal tibia using pQCT. The relationships between muscle and bone, and adipose tissue and bone, were determined by correlating muscle CSA with indices of bone strength, and indices of obesity with indices of SLOP, respectively. Results: The majority of participants had lean tissue values below able-bodied norms (67-100%). When using the able-bodied definition of BMI >30 kg/m2, 19% of individuals were obese, whereas 63% and 81% were obese when using SCI-specific definitions of BMI >25 kg/m2 or >22 kg/m2, respectively. One hundred percent of individuals had SLOP using distal femur Z-score, and over 50% were at risk of fracture using distal femur fracture threshold of <0.78 g/cm2. Weak (r=0.42) to moderate (r=0.57) correlations were found between muscle CSA and indices of bone strength, supporting the theory of a muscle-bone unit. No correlations were found between adipose tissue and bone. Conclusions: Based on the cohort data, we propose that individuals with ≥2 risk factors (female, ≥60 years of age, duration of injury (DOI) ≥10, tetraplegia, motor complete) should be screened for obesity using % body fat from DXA as well as a combination of carefully interpreted SCI-specific BMI and WC. In addition, these same individuals should be screened for SLOP using a distal femur Z-score and fracture threshold from DXA. It is clear that due to the prevalence of obesity and SLOP in this population, intervention for prevention or treatment is essential. The presence of a muscle-bone unit indicates that muscle atrophy contributes to a reduction in bone strength; this is clinically important, as muscle strength is potentially amenable to rehabilitation intervention. No correlation was found between adipose tissue and bone. Future work should continue to explore these relationships using appropriate technology.

Spinal cord injury occurs at a rate of 11.5 - 53.4 per million in developed countries with great emotional and financial consequences. The damage caused by the initial injury is followed by secondary damage, a complex cascade of mechanisms including ischemia, oxidative stress, inflammation and apoptosis. Although nothing can be done to reverse the initial damage to the spinal cord once it occurs, the secondary damage can be targeted by therapeutics to improve recovery. Following injury, concentrations of the potent antioxidant glutathione (GSH) are decreased in the spinal cord which potentiates mechanisms of secondary damage. In an attempt to maintain the GSH concentrations, the non-essential amino acid glutamine was tested as it was shown to increase GSH concentrations both in vivo and in vitro. Glutamine is being used extensively in clinical research in an expansive number of physiological and pathological conditions including brain trauma. To examine the therapeutic potential of glutamine after spinal cord trauma, two compression injury models, the modified aneurysm clip and the modified forceps, were used to induce an injury in male Wistar rats. We have demonstrated the ability of glutamine treatment (1 mmol/kg), given 1 hour after a 30 g aneurysm clip injury to increase GSH not only in whole blood samples but within the spinal tissue at the site of injury. Increasing GSH in this way also resulted in improved locomotor scores and maintenance of white matter tissue at the injury epicenter. Experiments using the forceps model were then performed to determine if the potency of glutamine treatment would be carried over to a different model and at a variety of severities. Glutamine, again, demonstrated the ability to improve maintenance of whole blood GSH, locomotor scores and tissue histology. In our experiments, glutamine has proven to be a potent therapeutic for spinal cord injury with an effect that is matched by few compounds currently being studied and well exceeding the standard therapeutic, methylprednisolone. Given the breadth of knowledge regarding the effects of glutamine clinically in numerous paradigms and the potency of the therapeutic effect seen in these studies, we believe that glutamine is fit for clinical trial and has a high potential for success.

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.