Haining Dai

Remodeling of synaptic structures in the motor cortex following spinal cord injury.

After spinal cord injury (SCI), structural reorganization occurs at multiple levels of the motor system including the motor cortex, and this remodeling may underlie recovery of motor function. The present study determined whether SCI leads to a remodeling of synaptic structures in the motor cortex. Dendritic spines in the rat motor cortex were visualized by confocal microscopy in fixed slices, and their density and morphology were analyzed after an overhemisection injury at C4 level. Spine density decreased at 7 days and partially recovered by 28 days. Spine head diameter significantly increased in a layer-specific manner. SCI led to a higher proportion of longer spines especially at 28 days, resulting in a roughly 10% increase in mean spine length. In addition, filopodium-like long dendritic protrusions were more frequently observed after SCI, suggesting an increase in synaptogenic events. This spine remodeling was accompanied by increased expression of polysialylated neural cell adhesion molecule, which attenuates adhesion between the pre- and postsynaptic membranes, in the motor cortex from as early as 3 days to 2 weeks after injury, suggesting a decrease in synaptic adhesion during the remodeling process. These results demonstrate time-dependent changes in spine density and morphology in the motor cortex following SCI. This synaptic remodeling seems to proceed with a time scale ranging from days to weeks. Elongation of dendritic spines may indicate a more immature and modifiable pattern of synaptic connectivity in the motor cortex being reorganized following SCI.

Neutralization of ciliary neurotrophic factor reduces astrocyte production from transplanted neural stem cells and promotes regeneration of corticospinal tract fibers in spinal cord injury

Transplantation of neural stem cells (NSC) into lesioned spinal cord offers the potential to increase regeneration by replacing lost neurons or oligodendrocytes. The majority of transplanted NSC, however, typically differentiate into astrocytes that may exacerbate glial scar formation. Here we show that blocking of ciliary neurotrophic factor (CNTF) with anti‐CNTF antibodies after NSC transplant into spinal cord injury (SCI) resulted in a reduction of glial scar formation by 8 weeks. Treated animals had a wider distribution of transplanted NSC compared with the control animals. The NSC around the lesion coexpressed either nestin or markers for neurons, oligodendrocytes, or astrocytes. Approximately 20% fewer glial fibrillary acidic protein‐positive/bromodeoxyuridine (BrdU)‐positive cells were seen at 2, 4, and 8 weeks postgrafting, compared with the control animals. Furthermore, more CNPase+/BrdU+ cells were detected in the treated group at 4 and 8 weeks. These CNPase+ or Rip+ mature oligodendrocytes were seen in close proximity to host corticospinal tract (CST) and 5HT+ serotonergic axon. We also demonstrate that the number of regenerated CST fibers both at the lesion and at caudal sites in treated animals was significantly greater than that in the control animals at 8 weeks. We suggest that the blocking of CNTF at the beginning of SCI provides a more favorable environment for the differentiation of transplanted NSC and the regeneration of host axons.

Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices

Visualization of dendritic spines is an important tool for researches on structural synaptic plasticity. Fluorescent labeling of the dendrites and spines followed by confocal microscopy permits imaging a large population of dendritic spines with a higher resolution. We sought to establish an optimal protocol to label neurons in cortical slices with the carbocyanine dye DiI for confocal microscopic imaging of dendritic spines. DiI finely labeled dendrites and spines in slices prefixed (by cardiac perfusion) with 1.5% paraformaldehyde to the similar extent that could be achieved in live preparation. In contrast, fixation with 4% paraformaldehyde severely compromised dye diffusion. Confocal microscopy showed that structural integrity of dendrites and spines was preserved much better in lightly (1.5%) fixed slices than those prepared without fixation. Quantitative measurement revealed that spine density was lower in live slices than that counted in lightly fixed slices, suggesting that fixation is necessary for an adequate evaluation of spine density. The quality of confocal microscopic images obtained from lightly fixed slices allowed us to observe distinctive morphologies such as branched spines and dendritic filopodium, which may be indicative of structural changes at synapses. This method will thus be useful for studying structural synaptic plasticity.

Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells

Given the involvement of post-mitotic neurons, long axonal tracts and incompletely elucidated injury and repair pathways, spinal cord injury (SCI) presents a particular challenge for the creation of preclinical models to robustly evaluate longitudinal changes in neuromotor function in the setting in the presence and absence of intervention. While rodent models exhibit high degrees of spontaneous recovery from SCI injury, animal care concerns preclude complete cord transections in non-human primates and other larger vertebrate models. To overcome such limitations a segmental thoracic (T9-T10) spinal cord hemisection was created and characterized in the African green monkey. Physiological tolerance of the model permitted behavioral analyses for a prolonged period post-injury, extending to predefined study termination points at which histological and immunohistochemical analyses were performed. Four monkeys were evaluated (one receiving no implant at the lesion site, one receiving a poly(lactide-co-glycolide) (PLGA) scaffold, and two receiving PLGA scaffolds seeded with human neural stem cells (hNSC)). All subjects exhibited Brown-Séquard syndrome 2 days post-injury consisting of ipsilateral hindlimb paralysis and contralateral hindlimb hypesthesia with preservation of bowel and bladder function. A 20-point observational behavioral scoring system allowed quantitative characterization of the levels of functional recovery. Histological endpoints including silver degenerative staining and Iba1 immunohistochemistry, for microglial and macrophage activation, were determined to reliably define lesion extent and correlate with neurobehavioral data, and justify invasive telemetered electromyographic and kinematic studies to more definitively address efficacy and mechanism.

Transplantation of embryonic spinal cord-derived neurospheres support growth of supraspinal projections and functional recovery after spinal cord injury in the neonatal rat

Great interest exists in using cell replacement strategies to repair the damaged central nervous system. Previous studies have shown that grafting rat fetal spinal cord into neonate or adult animals after spinal cord injury leads to improved anatomic growth/plasticity and functional recovery. It is clear that fetal tissue transplants serve as a scaffold for host axon growth. In addition, embryonic Day 14 (E14) spinal cord tissue transplants are also a rich source of neural‐restricted and glial‐restricted progenitors. To evaluate the potential of E14 spinal cord progenitor cells, we used in vitro‐expanded neurospheres derived from embryonic rat spinal cord and showed that these cells grafted into lesioned neonatal rat spinal cord can survive, migrate, and differentiate into neurons and oligodendrocytes, but rarely into astrocytes. Synapses and partially myelinated axons were detected within the transplant lesion area. Transplanted progenitor cells resulted in increased plasticity or regeneration of corticospinal and brainstem‐spinal fibers as determined by anterograde and retrograde labeling. Furthermore, transplantation of these cells promoted functional recovery of locomotion and reflex responses. These data demonstrate that progenitor cells when transplanted into neonates can function in a similar capacity as transplants of solid fetal spinal cord tissue.

Modulation of Dendritic Spine Remodeling in the Motor Cortex Following Spinal Cord Injury: Effects of Environmental Enrichment and Combinatorial Treatment with Transplants and Neurotrophin-3

Incomplete spinal cord injury (SCI) elicits structural plasticity of the spared motor system, including the motor cortex, which may underlie some of the spontaneous recovery of motor function seen after injury. Promoting structural plasticity may become an important component of future strategies to improve functional outcomes. We have recently observed dynamic changes in the density and morphology of dendritic spines in the motor cortex following SCI. The present study sought to test whether SCI‐induced changes in spine density and morphology could be modulated by potential strategies to enhance functional recovery. We examined the effects of enriched environment, transplants, and neurotrophin‐3 on the plasticity of synaptic structures in the motor cortex following SCI. Housing rats in an enriched environment increased spine density in the motor cortex regardless of injury. SCI led to a more slender and elongated spine morphology. Enriched housing mitigated the SCI‐induced morphological alterations, suggesting that the environmental modification facilitates maturation of synaptic structures. Transplantation of embryonic spinal cord tissue and delivery of neurotrophin‐3 at the injury site further increased spine density when combined with enriched housing. This combinatorial treatment completely abolished the injury‐induced changes, restoring a preinjury pattern of spine morphology. These results demonstrated that remodeling of dendritic spines in the motor cortex after SCI can be modulated by enriched housing, and the combinatorial treatment with embryonic transplants and neurotrophin‐3 can potentiate the effects of enriched housing. We suggest that synaptic remodeling processes in the motor cortex can be targeted for an intervention to enhance functional recovery after SCI. J. Comp. Neurol. 507:473–486, 2008.

Quantifying changes following spinal cord injury with velocity dependent locomotor measures

Many locomotor measures commonly used to assess functional deficits following neurological injury are velocity dependent. This makes the comparison of faster pre-injury walking to slower post-injury walking a challenging process. In lieu of calculating mean values at specific velocities, we have employed the use of nonlinear regression techniques to quantify locomotor measures across all velocities. This enables us to assess more accurately the locomotor recovery of rats after a cervical spinal cord injury. For example, while the mean stride length of the hindlimbs decreased following injury, regression analysis revealed that the change was due to the reduction in walking speed and not a functional deficit. A significant difference in the percent of the right forelimb step cycle that was spent in stance phase, or duty factor, was found across all velocities, however this deficit spontaneously recovered after 6 weeks. Conversely, no differences were initially found in hindlimb stride length, but abnormal compensatory techniques were found to have developed 3 weeks after injury.

Opiate-Free Pain Therapy Using Carbamazepine-Loaded Microparticles Provides Up to 2 Weeks of Pain Relief in a Neuropathic Pain Model

Opioids remain a mainstay in the treatment of acute and chronic pain, despite numerous and potentially dangerous side effects. There is a great unmet medical need for alternative treatments for patients suffering from pain that do not result in addiction or adverse side effects. Anticonvulsants have been shown to be effective in managing pain, though high systemic levels and subsequent side effects limit their widespread usage. Our goal was to determine if the incorporation of an anticonvulsant, carbamazepine, into a biodegradable microparticle for local sustained perineural release would be an efficacious analgesic following a peripheral injury.