Human induced pluripotent stem cell-derived neural crest stem cells integrate into the injured spinal cord in the fetal lamb model of myelomeningocele.

BACKGROUND/PURPOSE: Neurological function in patients with myelomeningocele (MMC) is limited even after prenatal repair. Neural crest stem cells (NCSCs) can improve neurological function in models of spinal cord injury. We aimed to evaluate the survival, integration, and differentiation of human NCSCs derived from induced pluripotent stem cells (iPSC-NCSCs) in the fetal lamb model of MMC. METHODS: Human iPSCs derived from skin fibroblasts were differentiated into NCSCs in vitro, mixed with hydrogel, and seeded on nanofibrous scaffolds for surgical transplantation. Fetal lambs (n=2) underwent surgical MMC creation and repair with iPSC-NCSC seeded scaffolds. Gross necropsy and immunohistochemistry were performed at term. RESULTS: IPSC-NCSCs expressed NCSC markers, maintained > 95% viability, and demonstrated neuronal differentiation in vitro. Immunohistochemical analysis of repaired spinal cords thirty days after transplantation demonstrated the co-localization of human nuclear mitotic apparatus protein (NuMA) and Neurofilament M subunit (NFM) in the area of spinal cord injury. No gross tumors were identified. CONCLUSIONS: Human iPSC-NCSCs survived, integrated, and differentiated into neuronal lineage in the fetal lamb model of MMC. This is the first description of human stem cell engraftment in a model of fetal MMC and supports the concept of using NCSCs to address spinal cord damage in MMC.

Tumor necrosis factor alpha mediates GABA(A) receptor trafficking to the plasma membrane of spinal cord neurons in vivo.

The proinflammatory cytokine TNFα contributes to cell death in central nervous system (CNS) disorders by altering synaptic neurotransmission. TNFα contributes to excitotoxicity by increasing GluA2-lacking AMPA receptor (AMPAR) trafficking to the neuronal plasma membrane. In vitro, increased AMPAR on the neuronal surface after TNFα exposure is associated with a rapid internalization of GABA(A) receptors (GABA(A)Rs), suggesting complex timing and dose dependency of the CNS's response to TNFα. However, the effect of TNFα on GABA(A)R trafficking in vivo remains unclear. We assessed the effect of TNFα nanoinjection on rapid GABA(A)R changes in rats (N = 30) using subcellular fractionation, quantitative western blotting, and confocal microscopy. GABA(A)R protein levels in membrane fractions of TNFα and vehicle-treated subjects were not significantly different by Western Blot, yet high-resolution quantitative confocal imaging revealed that TNFα induces GABA(A)R trafficking to synapses in a dose-dependent manner by 60 min. TNFα-mediated GABA(A)R trafficking represents a novel target for CNS excitotoxicity.

Animal models of neurologic disorders: a nonhuman primate model of spinal cord injury.

Primates are an important and unique animal resource. We have developed a nonhuman primate model of spinal cord injury (SCI) to expand our knowledge of normal primate motor function, to assess the impact of disease and injury on sensory and motor function, and to test candidate therapies before they are applied to human patients. The lesion model consists of a lateral spinal cord hemisection at the C7 spinal level with subsequent examination of behavioral, electrophysiological, and anatomical outcomes. Results to date have revealed significant neuroanatomical and functional differences between rodents and primates that impact the development of candidate therapies. Moreover, these findings suggest the importance of testing some therapeutic approaches in nonhuman primates prior to the use of invasive approaches in human clinical trials. Our primate model is intended to: 1) lend greater positive predictive value to human translatable therapies, 2) develop appropriate methods for human translation, 3) lead to basic discoveries that might not be identified in rodent models and are relevant to human translation, and 4) identify new avenues of basic research to "reverse-translate" important questions back to rodent models.

Methods for functional assessment after C7 spinal cord hemisection in the rhesus monkey.

BACKGROUND: Reliable outcome measures are essential for preclinical modeling of spinal cord injury (SCI) in primates. MEASURES: need to be sensitive to both increases and decreases in function in order to demonstrate potential positive or negative effects of therapeutics. OBJECTIVES: To develop behavioral tests and analyses to assess recovery of function after SCI in the nonhuman primate. METHODS: In all, 24 male rhesus macaques were subjected to complete C7 lateral hemisection. The authors scored recovery of function in an open field and during hand tasks in a restraining chair. In addition, EMG analyses were performed in the open field, during hand tasks, and while animals walked on a treadmill. Both control and treated monkeys that received candidate therapeutics were included in this report to determine whether the behavioral assays were capable of detecting changes in function over a wide range of outcomes. RESULTS: The behavioral assays are shown to be sensitive to detecting a wide range of motor functional outcomes after cervical hemisection in the nonhuman primate. Population curves on recovery of function were similar across the different tasks; in general, the population recovers to about 50% of baseline performance on measures of forelimb function. CONCLUSIONS: The behavioral outcome measures that the authors developed in this preclinical nonhuman primate model of SCI can detect a broad range of motor recovery. A set of behavioral assays is an essential component of a model that will be used to test efficacies of translational candidate therapies for SCI.

Severity of locomotor and cardiovascular derangements after experimental high-thoracic spinal cord injury is anesthesia dependent in rats.

Anesthetics affect outcomes from central nervous system (CNS) injuries differently. This is the first study to show how two commonly used anesthetics affect continuously recorded hemodynamic parameters and locomotor recovery during a 2-week period after two levels of contusion spinal cord injury (SCI) in rats. We hypothesized that the level of cardiovascular depression and recovery of locomotor function would be dependent upon the anesthetic used during SCI. Thirty-two adult female rats were subjected to a sham, 25-mm or 50-mm SCI at T3-4 under pentobarbital or isoflurane anesthesia. Mean arterial pressure (MAP) and heart rate (HR) were telemetrically recorded before, during, and after SCI. Locomotor function recovered best in the 25-mm-injured isoflurane-anesthetized animals. There was no significant difference in locomotor recovery between the 25-mm-injured pentobarbital-anesthetized animals and the 50-mm-injured isoflurane-anesthetized animals. White matter sparing and extent of intermediolateral cell column loss appeared larger in animals anesthetized with pentobarbital, but this was not significant. There were no differential effects of anesthetics on HR and MAP before SCI, but recovery from anesthesia was significantly slower in pentobarbital-anesthetized animals. At the time of SCI, MAP was acutely elevated in the pentobarbital-anesthetized animals, whereas MAP decreased in the isoflurane-anesthetized animals. Hypotension occurred in the pentobarbital-anesthetized groups and in the 50-mm-injured isoflurane-anesthetized group. In pentobarbital-anesthetized animals, SCI resulted in acute elevation of HR, although HR remained low. Return of HR to baseline was much slower in the pentobarbital-anesthetized animals. Severe SCI at T3 produced significant chronic tachycardia that was injury severity dependent. Although some laboratories monitor blood pressure, HR, and other physiological variables during surgery for SCI, inherently few have monitored cardiovascular function during recovery. This study shows that anesthetics affect hemodynamic parameters differently, which in turn can affect functional outcome measures. This supports the need for a careful evaluation of cardiovascular and other physiological measures in experimental models of SCI. Choice of anesthetic should be an important consideration in experimental designs and data analyses.

Prenatal repair of myelomeningocele with aligned nanofibrous scaffolds-a pilot study in sheep.

BACKGROUND/PURPOSE: Spinal cord damage in myelomeningocele (MMC) results from abnormal cord development and subsequent local trauma. Prenatal surgery prevents additional neural injury. However, existing damage is not reversed. Biodegradable nanofibrous scaffolds (NSs) promote regeneration of neural tissues. They mimic the microtopography of the extracellular matrix and guide tissue formation and organization. The purpose of this pilot study was to evaluate the practicality and safety of using biodegradable NS as a regenerative device in prenatal MMC repair. METHODS: Two fetal lambs underwent a surgically induced MMC defect followed by open fetal repair using aligned biodegradable NS. Lambs were killed at day 138. Spinal cords were examined for inflammation or fibrosis and stained for spinal cord architecture, myelin, and neuron cell bodies. RESULTS: Prenatal repair with NS demonstrated technical feasibility. There was no evidence of a surrounding inflammatory response or foreign-body reaction to the scaffold. CONCLUSION: Biodegradable NS can be used surgically for the prenatal repair of MMC in a large animal model and does not appear to elicit an inflammatory or fibrotic reaction in fetal tissue. Further studies will determine their potential for neural cell infiltration, delivery of growth factors, drugs or stem cells, and functional recovery greater than standard repair.

Glial restricted precursor cell transplant with cyclic adenosine monophosphate improved some autonomic functions but resulted in a reduced graft size after spinal cord contusion injury in rats.

Transplantation of glial restricted precursor (GRP) cells has been shown to reduce glial scarring after spinal cord injury (SCI) and, in combination with neuronal restricted precursor (NRP) cells or enhanced expression of neurotrophins, to improve recovery of function after SCI. We hypothesized that combining GRP transplants with rolipram and cAMP would improve functional recovery, similar to that seen after combining Schwann cell transplants with increasing cAMP. A short term study, (1) uninjured control, (2) SCI+vehicle, and (3) SCI+cAMP, showed that spinal cord [cAMP] was increased 14days after SCI. We used 51 male rats subjected to a thoracic SCI for a 12-week survival study: (1) SCI+vehicle, (2) SCI+GRP, (3) SCI+cAMP, (4) SCI+GRP+cAMP, and (5) uninjured endpoint age-matched control (AM). Rolipram was administered for 2weeks after SCI. At 9days after SCI, GRP transplantation and injection of dibutyryl-cAMP into the spinal cord were performed. GRP cells survived, differentiated, and formed extensive transplants that were well integrated with host tissue. Presence of GRP cells increased the amount of tissue in the lesion; however, cAMP reduced the graft size. White matter sparing at the lesion epicenter was not affected. Serotonergic input to the lumbosacral spinal cord was not affected by treatment, but the amount of serotonin immediately caudal to the lesion was reduced in the cAMP groups. Using telemetric monitoring of corpus spongiosum penis pressure we show that the cAMP groups regained the same number of micturitions per 24hours when compared to the AM group, however, the frequency of peak pressures was increased in these groups compared to the AM group. In contrast, the GRP groups had similar frequency of peak pressures compared to baseline and the AM group. Animals that received GRP cells regained the same number of erectile events per 24hours compared to baseline and the AM group. Since cAMP reduced the GRP transplant graft, and some modest positive effects were seen that could be attributable to both GRP or cAMP, future research is required to determine how cAMP affects survival, proliferation, and/or function of progenitor cells and how this is related to function. cAMP may not always be a desirable addition to a progenitor cell transplantation strategy after SCI.

Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury.

Although axonal regeneration after CNS injury is limited, partial injury is frequently accompanied by extensive functional recovery. To investigate mechanisms underlying spontaneous recovery after incomplete spinal cord injury, we administered C7 spinal cord hemisections to adult rhesus monkeys and analyzed behavioral, electrophysiological and anatomical adaptations. We found marked spontaneous plasticity of corticospinal projections, with reconstitution of fully 60% of pre-lesion axon density arising from sprouting of spinal cord midline-crossing axons. This extensive anatomical recovery was associated with improvement in coordinated muscle recruitment, hand function and locomotion. These findings identify what may be the most extensive natural recovery of mammalian axonal projections after nervous system injury observed to date, highlighting an important role for primate models in translational disease research.

Hypertonic saline attenuates cord swelling and edema in experimental spinal cord injury: a study utilizing magnetic resonance imaging.

OBJECTIVE: To use magnetic resonance imaging (MRI) to characterize secondary injury immediately after spinal cord injury (SCI), and to show the effect of hypertonic saline on MRI indices of swelling, edema, and hemorrhage within the cord. DESIGN: A prospective, randomized, placebo-controlled study. SETTING: Research laboratory. SUBJECTS: Twelve adult Long-Evans female rats. INTERVENTIONS: Rats underwent a unilateral 12.5 mm SCI at vertebral level C5. Animals were administered 0.9% NaCl (n = 6) or 5% NaCl (n = 6) at 1.4 mL/kg intravenously every hour starting 30 minutes after SCI. Immediately after SCI, rats were placed in a 4.7T Bruker MRI system and images were obtained continuously for 8 hours using a home-built transmitter/receiver 3 cm Helmholtz coil. Rats were killed 8 hours after SCI. MEASUREMENTS AND MAIN RESULTS: Quantification of cord swelling and volumes of hypointense and hyperintense signal within the lesion were determined from MRI. At 36 minutes after SCI, significant swelling of the spinal cord at the lesion center and extending rostrally and caudally was demonstrated by MRI. Also, at this time point, a hypointense core was identified on T1, PD, and T2 weighted images. Over time this hypointense core reduced in size and in some animals was no longer visible by 8 hours after SCI, although histopathology demonstrated presence of red blood cells. A prominent ring of T2-weighted image hyperintensity, characteristic of edema, surrounded the hypointense core. At the lesion center, this rim of edema occupied the entire unilateral injured cord and in all animals extended to the contralateral side. Administration of HS resulted in increased serum [Na], attenuation of cord swelling, and decreased volume of hypointense core and edema at the last time points. CONCLUSIONS: We were able to use MRI to detect rapid and acute changes in the evolution of tissue pathophysiology, and show potentially beneficial effects of hypertonic saline in acute cervical SCI.

Longitudinal comparison of two severities of unilateral cervical spinal cord injury using magnetic resonance imaging in rats.

Magnetic resonance imaging (MRI) should be a powerful tool for characterization of spinal cord pathology in animal models. We evaluated the utility of medium-field MRI for the longitudinal assessment of progression of spinal cord injury (SCI) in a rat model. Thirteen adult rats were subjected to a 6.25 or 25 g-cm unilateral cervical SCI, and underwent MRI and behavioral tests during a 3-week study period. MRI was also performed post-mortem. Quantification of cord swelling, hypointense and hyperintense signal, and lesion length were the most valuable parameters to determine and were highly correlated to behavioral and histopathological measures. Immediately after injury, MRI showed loss of gray matter-white matter differentiation, presence of scattered hyperintense signal and local hypointense signal, and cord swelling in both groups. At 7 days after injury, the spinal cord in the 25 g-cm group was significantly larger than that of the 6.25 g-cm group (p = 0.02). Contrast enhancement of the lesion was seen at 24 h in the 6.25 g-cm group, and at 24 h and 7 days in the 25 g-cm group. The volume of hypointense signal, representing hemorrhage, throughout the lesion region was significantly larger in the 25 g-cm compared to the 6.25 g-cm group at both 14 and 21 days after SCI (p,

Novel technique for monitoring micturition and sexual function in male rats using telemetry.

We developed a novel technique to simultaneously monitor micturitions and erections in rats by using pressure monitoring within the corpus spongiosum of the penis (CSP). We present data validating this technique and report pressure waveform characteristics of micturition and erectile events during four different behavioral contexts in 10 awake, freely-moving male rats. Telemetric pressure transducers were implanted in the bulb of the CSP. CSP pressure was monitored while the animals were simultaneously recorded on video for determination of presence and volume (n = 7) of micturitions and while the animals underwent behavioral tests for determination of erections. Observed micturitions and CSP pressure waveforms characteristic of micturitions occurred simultaneously (r = 0.98) at a frequency of 32 +/- 4 micturitions per 24 h and with a volume of 0.95 +/- 0.12 ml/urination. Micturition duration recorded by CSP pressure and volume determined by urine weight were highly correlated (r = 0.82). We found that 100% of visually confirmed erectile events occurred simultaneously with CSP pressure waveforms characteristic of erections during ex copula reflex erection tests. During noncontact erection and mating tests more erections were identified by telemetry than by observation alone. Erections during mating tests had a different appearance than those seen in other contexts; they were shorter in duration (P < 0.05) and typically were characterized by a single suprasystolic CSP pressure peak, highlighting the context-specificity of erections. Quality of recordings remained stable in three of four rats we followed for 8 wk. We demonstrate that telemetric recording of CSP pressure provides a quantitative and qualitative assessment of penile erections and micturition in freely behaving rats.

Alterations in eliminative and sexual reflexes after spinal cord injury: defecatory function and development of spasticity in pelvic floor musculature.

Spinal cord injury often results in loss of normal eliminative and sexual functions. This chapter is focused on defecatory function, although aspects of micturition and erectile function will be covered as well due to the overlap in anatomical organization and response to injury. These systems have both autonomic and somatic components, and are organized in the thoracolumbar (sympathetic), lumbosacral (somatic), and sacral (parasympathetic) spinal cord. Loss of supraspinal descending control and plasticity-mediated alterations at the level of the spinal cord, result in loss of voluntary control and in abnormal functioning of these systems including the development of dyssynergies and spasticity. There are several useful models of spinal cord injury in rodents that exhibit many of the autonomic dysfunctions observed after spinal cord injury in humans. Numerous studies involving these animal models have demonstrated development of abnormalities in bladder, external anal sphincter, and erectile function, such as detrusor-sphincter-dyssynergia and external anal sphincter hyperreflexia. Here we review many of these studies and show some of the anatomical alterations that develop within the spinal cord during the development of these hyperreflexias. Furthermore, we show that spasticity develops in other pelvic floor musculature as well, such as the bulbospongiosus muscle, which results in increased duration and magnitude of pressures developed during erectile events and increased duration of micturition. Advances and continued improvement in the use of current animal models of spinal cord injury should encourage and increase the laboratory work devoted to this relatively neglected area of experimental spinal cord injury.

Telemetric monitoring of corpus spongiosum penis pressure in conscious rats for assessment of micturition and sexual function following spinal cord contusion injury.

Disruption of bladder function and sexual reflexes are major complications following spinal cord injury (SCI). We examined the use of telemetric monitoring of corpus spongiosum penis (CSP) pressures for assessment of micturition and erectile events following SCI in rats. Pressure catheters were implanted in the bulb of the CSP of seven male Long-Evans hooded rats, subjected to a standardized weight drop SCI (10 g x 12.5 mm) at T10. CSP pressures were analyzed for spontaneously occurring micturition and erectile events, and during ex copula reflex erection tests until 25 days after SCI. Urine volume was determined until 21 days after SCI. Results show initial loss of bladder function after SCI with gradual return of reflex micturition. When compared to baseline (BL), micturition pressure characteristics after SCI included prolonged duration, increased area under the curve (AUC), increased mean pressures, increased number of pressure peaks, and increased peak frequency. At 21 days after SCI, the urine volume per micturition was significantly increased. The number of full erectile events decreased significantly following SCI. Pressure wave analyses demonstrated increased AUC, increased maximum pressures, increased suprasystolic peak duration, increased AUC of the suprasystolic peaks, and increased maximum pressures of the suprasystolic peaks during recovery. The number of partial erectile events decreased significantly following SCI. Ex copula reflex erection testing demonstrated significantly decreased latency. The study demonstrates that telemetric monitoring of CSP pressures in conscious rats is a valuable and reliable method for assessing recovery of autonomic function following SCI.

Effect of moderate exercise immediately followed by induced hyperglycemia on gene expression and content of the glucose transporter-4 protein in skeletal muscles of horses.

OBJECTIVE: To determine the effect of a single bout of exercise and increased substrate availability after exercise on gene expression and content of the glucose transporter-4 (GLUT-4) protein in equine skeletal muscle. ANIMALS: 6 healthy adult Thoroughbreds. PROCEDURES: The study was designed in a balanced, randomized, 3-way crossover fashion. During 2 trials, horses were exercised at 45% of their maximal rate of oxygen consumption for 60 minutes after which 1 group received water (10 mL/kg), and the other group received glucose (2 g/kg, 20% solution) by nasogastric intubation. During 1 trial, horses stood on the treadmill (sham exercise) and then received water (10 mL/kg) by nasogastric intubation. Muscle glycogen concentration and muscle GLUT-4 protein and mRNA content were determined before exercise and at 5 minutes and 4, 8, and 24 hours after exercise. RESULTS: Although exercise resulted in a 30% reduction in muscle glycogen concentration, no significant difference was detected in muscle GLUT-4 protein or mRNA content before and after exercise. Glycogen replenishment was similar in both exercised groups and was not complete at 24 hours after exercise. Horses that received glucose had significantly higher plasma glucose and insulin concentrations for 3 hours after exercise, but no effect of hyperglycemia was detected on muscle GLUT-4 protein or mRNA content. CONCLUSION: Under the conditions of this study, neither exercise nor the combination of exercise followed by hyperglycemia induced translation or transcription of the GLUT-4 protein in horses.