Chronic spinal cord injury (SCI) is a devastating medical condition. In the acute phase after injury, there is cell loss resulting in chronic axonal damage and loss of sensory and motor function including loss of oligodendrocytes that results in demyelination of axons and further dysfunction. In the chronic phase, the inhibitory environment within the lesion including the glial scar can arrest axonal growth and regeneration and can also potentially affect transplanted cells. We hypothesized that glial scar ablation (GSA) along with cell transplantation may be required as a combinatorial therapy to achieve functional recovery, and therefore we proposed to examine the survival and fate of human induced pluripotent stem cell (iPSC) derived pre-oligodendrocyte progenitor cells (pre-OPCs) transplanted in a model of chronic SCI, whether this was affected by GSA, and whether this combination of treatments would result in functional recovery. In this study, chronically injured athymic nude (ATN) rats were allocated to one of three treatment groups: GSA only, pre-OPCs only, or GSA+pre-OPCs. We found that human iPSC derived pre-OPCs were multi-potent and retained the ability to differentiate into mainly oligodendrocytes or neurons when transplanted into the chronically injured spinal cords of rats. Twelve weeks after cell transplantation, we observed that more of the transplanted cells differentiated into oligodendrocytes when the glial scar was ablated compared with no GSA. Further, we also observed that a higher percentage of transplanted cells differentiated into V2a interneurons and motor neurons in the pre-OPCs only group when compared with GSA+pre-OPCs. This suggests that the local environment created by ablation of the glial scar may have a significant effect on the fate of cells transplanted into the injury site.

There are no effective therapies available currently to ameliorate loss of function for patients with spinal cord injuries (SCIs). In addition, proposed treatments that demonstrated functional recovery in animal models of acute SCI have failed almost invariably when applied to chronic injury models. Glial scar formation in chronic injury is a likely contributor to limitation on regeneration. We have removed existing scar tissue in chronically contused rat spinal cord using a rose Bengal-based photo ablation approach. In this study, we compared two chemically modified rose bengal derivatives to unmodified rose bengal, both confirming and expanding on our previously published report. Rats were treated with unmodified rose bengal (RB1) or rose bengal modified with hydrocarbon (RB2) or polyethylene glycol (RB3), to determine the effects on scar components and spared tissue post-treatment. Our results showed that RB1 was more efficacious than RB2, while still maintaining minimal collateral effects on spared tissue. RB3 was not taken up by the cells, likely because of its size, and therefore had no effect. Treatment with RB1 also resulted in an increase in serotonin eight days post-treatment in chronically injured spinal cords. Thus, we suggest that unmodified rose Bengal is a potent candidate agent for the development of a therapeutic strategy for scar ablation in chronic SCI.

Functional recovery is the final goal in the treatment of spinal cord injury. However, to date, few treatment strategies have demonstrated significant locomotor improvement in animal experiments. By using tail nerve electrical stimulation (TANES) as an open-field locomotor training method combined with glial scar ablation and cell transplantation, we have successfully promoted locomotor recovery in rats with chronic spinal cord contusion injury. The purpose of the present study is to further investigate the mechanism of TANES and its effect on electrophysiology. Spinal cord segment T10 of female, adult Long-Evans rats was contused using the NYU impactor device with 25 mm height setting. After injury, rats were randomly divided into three groups. Group I was used as a control without any treatment, group II and group III were subjected to basic treatment including glial scar ablation and transplantation of olfactory lamina propria 6 weeks after injury, and group III received TANES-induced open-field locomotor training weekly after basic treatment. All animals were allowed to survive 22 weeks, except some rats which were transected. Basso, Beattie, and Bresnahan (BBB) open-field locomotor rating scale, horizontal ladder rung walking test, and electrophysiological tests were used to assess the restoration of functional behavior and conduction. Results showed that TANES significantly improves locomotor recovery and spinal cord conduction, reflex, as well as significantly reduces the occurrence of autophagia. Additionally, after transection, trained rats still maintained higher BBB score than that of control rats. This may be related to the activity-dependent plasticity promoted by TANES-induced locomotor training.

This is a randomized controlled prospective trial with two parallel groups. The objective of this study was to determine whether early application of tail nerve electrical stimulation (TANES)-induced walking training can improve the locomotor function. This study was conducted in SCS Research Center in Colorado, USA. A contusion injury to spinal cord T10 was produced using the New York University impactor device with a 25 -mm height setting in female, adult Long–Evans rats. Injured rats were randomly divided into two groups (n=12 per group). One group was subjected to TANES-induced walking training 2 weeks post injury, and the other group, as control, received no TANES-induced walking training. Restorations of behavior and conduction were assessed using the Basso, Beattie and Bresnahan open-field rating scale, horizontal ladder rung walking test and electrophysiological test (Hoffmann reflex). Early application of TANES-induced walking training significantly improved the recovery of locomotor function and benefited the restoration of Hoffmann reflex. TANES-induced walking training is a useful method to promote locomotor recovery in rats with spinal cord injury.

Schwann cells are glial cells of peripheral nervous system, responsible for axonal myelination and ensheathing, as well as tissue repair following a peripheral nervous system injury. They are one of several cell types that are widely studied and most commonly used for cell transplantation to treat spinal cord injury, due to their intrinsic characteristics including the ability to secrete a variety of neurotrophic factors. This mini review summarizes the recent findings of endogenous Schwann cells after spinal cord injury and discusses their role in tissue repair and axonal regeneration. After spinal cord injury, numerous endogenous Schwann cells migrate into the lesion site from the nerve roots, involving in the construction of newly formed repaired tissue and axonal myelination. These invading Schwann cells also can move a long distance away from the injury site both rostrally and caudally. In addition, Schwann cells can be induced to migrate by minimal insults (such as scar ablation) within the spinal cord and integrate with astrocytes under certain circumstances. More importantly, the host Schwann cells can be induced to migrate into spinal cord by transplantation of different cell types, such as exogenous Schwann cells, olfactory ensheathing cells, and bone marrow-derived stromal stem cells. Migration of endogenous Schwann cells following spinal cord injury is a common natural phenomenon found both in animal and human, and the myelination by Schwann cells has been examined effective in signal conduction electrophysiologically. Therefore, if the inherent properties of endogenous Schwann cells could be developed and utilized, it would offer a new avenue for the restoration of injured spinal cord.

To date, few treatment strategies applying cellular transplantation to the chronically injured spinal cord have yielded significant functional improvement in animal experiments. Here we report that significant improvement of locomotor function was achieved in rats with chronic spinal cord injury (SCI) by the application of combination treatments with tail nerve electrical stimulation (TANES), which can activate the central pattern generator, inducing active weight-supported stepping. Contusion injury (25 mm) to spinal cord T10 was produced by using the NYU impactor device in female, adult Long-Evans rats. Rats in 2 of 4 groups with SCI received basic treatments (scar ablation followed by transplantation of lamina propria of olfactory mucosa and cultured olfactory ensheathing cells into the lesion cavity) 6 weeks after SCI. Rats both with and without basic treatments were subjected to TANES one week after secondary surgery or 7 weeks after SCI. Sixteen weeks after secondary surgery or 22 weeks after SCI rats in two groups receiving TANES significantly improved their functional recovery compared with those without TANES, when evaluated with BBB open field rating scale (p<0.01). Among them, however, rats with basic treatments performed better than those without basic treatments. TANES may contribute to the activity-dependent plasticity below the injury level, which is critical for functional recovery. Additionally, TANES may promote axonal regeneration, including those from supraspinal level. Since TANES demonstrated considerable potential for achieving improvement of functional recovery in rat model, it would suggest a new strategy for chronic SCI.

Somatosensory evoked potentials (SSEPs) are a sensitive quantitative measure of conduction in somatosensory pathways of the central nervous system and are increasingly used in both clinical trials and animal experiments. SSEPs can be recorded in non-sedated rodents by magnetic stimulation (MS) of peripheral nerves. To overcome some disadvantages caused by using anesthesia and implanted recording electrodes, we used subdermal needle electrodes located on the midline of the skull to successfully record SSEPs in non-sedated rats, elicited by stimulating the tibial nerve with a magnetic stimulator. The wave form contains a typical P1 peak and N1 peak. Although there is a variation of P1 latency, N1 latency, and P1-N1 amplitude between right side and left side, it was not statistically significant. In addition, there is a significantly positive relationship between P1-N1 amplitude and MS strength, suggesting that the increase in magnetic stimulating strength resulted in the increase in P1-N1 amplitude. Results in the present study demonstrate that our modified method is a reliable and feasible paradigm for recording SSEPs in non-sedated rats. (C) 2012 Elsevier B.V. All rights reserved.

We have successfully removed an existing glial scar in chronically contused rat spinal cord using a rose Bengal-based phototoxic method. The purpose of this study is to examine if scar ablation benefits the anatomical recovery by cell/tissue transplantation, and thus provides a more permissive physical and biochemical environment for axonal growth, which may lead to functional recovery. Immediately after scar ablation, we transplanted lamina propria (LP) of the olfactory mucosa alone or in combination with cultured olfactory ensheathing cells (OEC) into the lesion cavity 6weeks after contusion injury (NYU impactor device, 25mm height setting) at spinal cord segment T10 of adult female Long–Evans rats. Sixteen weeks after scar ablation and transplantation, we found that the initial repaired tissue significantly expanded, companied by remarkable reduction or disappearance of the lesion cavity and integration of repaired tissue with the spared tissue, thus resulting in histological repair of damaged cord tissue at the injury epicenter. Glial scar reformation was effectively prevented after ablation due to the tissue repair. In addition, at the injury epicenter P0 (myelin glycoprotein P-zero)-positive myelination formed by Schwann cells, which are known to myelinate regenerating and demyelinated axons, were significantly increased in number compared with the control animals. However, when evaluated with BBB open-field scale a significant improvement of locomotor function was not observed in this study; the possible reasons were discussed.

The spinal cord has an intrinsic, limited ability of spontaneous repair; the endogenous repair of damaged tissue starts a few days after spinal cord injury (SCI). To date, however, detailed observation in histology at the injury site has not been well documented. In the present study we analyzed the histological structure of the repaired tissue from injury site of rats 6 or 14 weeks after contusion injury (NYU impactor device, 25 mm height setting) on T10, and rats 8 weeks after transplantation of lamina propria (LP) or acellular lamina propria. We found that the initial repaired tissue can be histologically divided into three different zones, i.e., fibrotic, cellular and axonal. The fibrotic zone consists of invading connective tissue, while the cellular zone is composed of invading, densely compacted Schwann cells. Schwann cells migrate from dorsal roots laterally toward and merge underneath the fibrotic zone, forming the U-shape shell of the cellular zone. The major component of the axonal zone is regenerating axons. Schwann cells myelinate regenerating axons in all three zones. In rats with combination treatments including scar ablation and LP transplantation, both cellular and axonal zones significantly expand in size, resulting in the disappearance of the lesion cavity and the integration of repaired tissue with spared tissue. Olfactory ensheathing cells from transplanted LP may promote the expansion of the cellular and axonal zones through stimulating host Schwann cells, indirectly contributing to tissue repair and axonal regeneration. The ependyma-derived cells may be directly involved in tissue repair, but not contribute to the formation of myelin sheaths.

Intrathecal infusion has been widely used to directly deliver drugs or neurotrophins to a lesion site following spinal cord injury. Evidence shows that intrathecal infusion is efficient for 7 days but is markedly reduced after 14 days, due to time dependent occlusion. In addition, extensive fibrotic scarring is commonly observed with intrathecal infusion. These anomalies need to be clearly elucidated in histology. In the present study, all adult Long-Evans rats received a 25 mm contusion injury on spinal cord T10 produced using the NYU impactor device. Immediately after injury, catheter tubing with an outer diameter of 0.38 mm was inserted through a small dural opening at L3 into the subdural space with the tubing tip positioned near the injury site. The tubing was connected to an Alzet mini pump, which was filled with saline solution and was placed subcutaneously. Injured rats without tubing served as control. Rats were behaviorally tested for 6 weeks using the BBB locomotor rating scale and histologically assessed for tissue scarring. Six weeks later, we found that the intrathecal tubing caused extensive scarring and inflammation, related to neutrophils, macrophages and plasma cells. The tubing's tip was occluded by scar tissue and inflammatory cells. The scar tissue surrounding the tubing consists of 20-70 layers of fibroblasts and densely compacted collagen fibers, seriously compressing and damaging the cord tissue. BBB scores of rats with intrathecal tubing were significantly lower than control rats (p<0.01) from 2 weeks after injury, implying serious impairment of functional recovery caused by the scarring.

Walking or stepping has been considered the result from the activation of the central pattern generator (CPG). In most patients with spinal cord injury (SCI) the CPG is undamaged. To date, there are no noninvasive approaches for activating the CPG. Recently we developed a noninvasive technique, tail nerve electrical stimulation (TANES), which can induce positive hind limb movement of SCI rats. The purpose of this study is to introduce the novel technique and examine the effect of TANES on CPG activation. A 25mm contusion injury was produced at spinal cord T10 of female, adult Long-Evans rats by using the NYU impactor device. Rats received TANES (∼40mA at 4kHz) 7 weeks after injury. During TANES all injured rats demonstrated active body weight-supported stepping of hind limbs with left–right alternation and occasional front–hind coordination, resulting in significant, temporary increase in BBB scores (p<0.01). However, there is no response to TANES from rats with L2 transection, consistent with other reports that the CPG may be located at L1–2. S1 transection negatively implies the key role of TANES in CPG activation. The TANES not only renders paralyzed rats with a technique-induced ability to walk via activating CPG, but also is likely to be used for locomotor training. It has more beneficial effects for physical training over other training paradigms including treadmill training and invasive functional electrical stimulation. Therefore the TANES may have considerable potential for achieving improvement of functional recovery in animal models and a similar method may be suggested for human study.

Statins elicit numerous favorable effects on central nervous system (CNS) injury, including inhibition of the rhoA/ROCK pathway. In the present study, we show that statins decrease acute astrocyte activation in CNS injury, and decrease chondroitin sulfate proteoglycan (CSPG) levels in astrocyte Cultures as well as CNS injury. CSPG levels decreased by LIP to 45% in simvastatin-treated astrocyte Cultures compared to control Cultures. In simvastatin-treated animals, CSPG levels declined by 60% 8 days after brain stab injury, and by 62-64% 4 weeks after spinal Cord injury (SCI). Glial fibrillary acid protein (GFAP) levels decreased in brain stab at 8 clays after surgery/intervention, suggesting that statins produce a decrease in astrocyte activation. Attenuation of astrocyte activation may contribute to the decline in CSPG levels. However, there are likely other contributing factors, since GFAP levels were not a contributing factor in the decline of CSPG levels in astrocyte cultures. Robust locomotor improvements were not observed with any treatment. The numerous beneficial effects of statins on CNS injury render them an attractive candidate in the treatment of CNS injury. (C) 2008 Elsevier Inc. All rights reserved.

Extensive dendritic beading of MAP2 (microtubule-associated protein 2) immunoreactivity has previously been observed in the contused rat spinal cord. However, we have also observed dendritic beading in occasional uninjured animals. The purpose of this study was to examine the possibility that perfusion conditions contributed to the dendritic beading. Under deep anesthesia, uninjured rats (adult female Long-Evans, 200-225 g) were transcardially perfused with 0.9% saline solution followed by 4% paraformaldehyde at cold (4 degrees C) or warm (20 degrees C) temperature, and at a low (20 ml/min) or high (50 ml/min) flow rate. Dendrites were visualized by MAP2 immunoreactivity. The results demonstrate that perfusion with cold solutions at a high flow rate induces pronounced dendritic beading, and when perfused at a low flow rate, results in moderate dendritic beading. Warm perfusates did not induce dendritic beading when administered at a low flow rate, but occasional beading was observed with a high flow rate. Western blots revealed spectrin breakdown, but not MAP2 loss, in rats perfused with cold saline solution at a high flow rate, conditions that also resulted in dendritic beading. These findings demonstrate that dendritic morphology is sensitive to both temperature and flow rate of the perfusate. Warm fixative and a low perfusion flow rate minimized the perfusion-induced dendritic beading.

Statins (3-hydroxy-3-methylglutaryl-CoA [HMG-CoA] reductase inhibitors) inhibit the rate-limiting step in the mevalonate pathway, conversion of HMG-CoA to mevalonate, by competitive inhibition with the enzyme HMG-CoA reductase. Statins not only lower cholesterol levels, but are also thought to exert neuroprotective and neurogenic effects that may be beneficial in treating brain and spinal cord injuries. Data presented here illustrate that simvastatin enables neurite outgrowth in the presence of growth-inhibitory molecules commonly found at central nervous system (CNS) injury sites. To assess the effect of simvastatin on neurite outgrowth in the presence of inhibitory molecules present at CNS injury sites, rat embryonic cortex explants or postnatal spinal cord explants were grown on membrane filters prepared with alternating stripes of laminin and myelin/laminin. Immunostaining indicated that myelin stripes contain myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and Nogo, but do not contain chondroitin sulfate proteoglycan (CSPG). When control explants were grown in the presence of alternating stripes, neurite outgrowth preferentially extended in regions containing laminin only. In contrast, neurite outgrowth from explants grown in the presence of simvastatin was significantly less selective for laminin regions and was able to extend into regions containing myelin (p < 0.01). Simvastatin-induced effects were reversed by addition of mevalonate. Isoprenyl transferase inhibitors GGTI-286 and FTI-277, inhibitors of biochemical steps subsequent to HMG-CoA conversion to mevalonate, mimicked simvastatin- induced effects. These data suggest that simvastatin counteracts myelin-associated neurite outgrowth inhibition signals via mevalonate pathway inhibition, and may be beneficial in promoting axon regeneration in brain and spinal cord injury.

Glial scar represents a physical and molecular barrier to axonal regeneration and has become an important target for regeneration research in chronic spinal cord injury. Although many methods have been proven useful for the prevention of scar formation in an acute injury model, to date no effective method has been described to remove an existing glial scar in a chronic injury. The chronic lesion possesses an irregular shaped scar that lines the entire perimeter of the cavity. In the present study, we used rose bengal, a molecule commonly used for biological staining, injected into the cavity at the injury site of Long-Evans rat spinal cord (5 weeks after 25-mm contusion injury). Visible light was used to illuminate the injury site. Histological observation illustrates that at least partial glial scar tissue is ablated by rose bengal/illumination. The lack of glial fibrillary acidic protein (GFAP) immunoreactivity at the glial scar coupled with the reduction of GFAP density surrounding spared tissue suggests that this photochemical scar ablation preferentially kills astrocytes at the scar tissue but also reacts, to a lesser degree, in the spared tissue. There is an observed reduction of Basso, Beattie, and Bresnahan (BBB) scale scores after scar ablation, but it is not statistically significant from stabilized behavioral scoring prior to the scar ablation treatment. Our findings indicate that the rose bengal/illumination is feasible for ablation of the glial scar which surrounds an irregular lesion cavity in shape. The scar ablation might provide a permissive environment for the regenerating axons when enriched by cellular or drug therapy.

Spinal cord injury (SCI) results in cell death and tissue destruction, and ultimately cavitation followed by the formation of lesion scars at the injury site. The lesion scars include an astrocytic component (glial scar) and a fibroblastic component (connective tissue scar). The purpose of the present study is to determine if X-irradiation could minimize the formation of lesion scars and reduce the levels of chondroitin sulfate proteoglycans (CSPGs) in the contusion SCI model of the adult rat. Two weeks after SCI, a connective tissue scar formed at the injury site consisting primarily of fibroblasts and exhibits strong CSPG immunoreactivity. The fibroblasts might originate from the connective tissue of pia mater or arachnoid mater. At the same time, reactive astrocytes in the spared tissue accumulate surrounding the lesion cavity to form a thick glial scar with significant enhancement of glial fibrillary acidic protein (GFAP) and CSPG immunoreactivity. After X-irradiation (40 Gy) of the injury site 2 days post-injury, that results in an attenuated dose to the lesion, the connective tissue scar was not observed, and accordingly, almost no CSPG immunoreactivity was detected at this area. Meanwhile, the glial scar and its CSPG immunoreactivity were prominently reduced. X-irradiation did not show significant improvement in locomotor recovery, but resulted in a slight delay of body weight recovery following injury. This preparative treatment could be used to reduce secondary scarring in the lesion resulting in an enriched site for further treatment such as growth related transplantation.

Liposomes can deliver plasmid DNA, viruses, antisense oligonucleotides, and pharmacological agents to the central nervous system. Conjugation of antibodies to liposomes increases delivery specificity. Immunoliposomes created with Thy 1.1 antibody have previously been shown to be effective for neuronal delivery. The intracellular delivery of these immunoliposomes is evaluated by light and electron microscopy. Thy 1.1 conjugated liposomes were loaded with horseradish peroxidase and stereotactically injected into rat striatum. On light microscopy, immunoliposomes were concentrated within 0.2 mm of the injection site 8 h following delivery but, 24 h post-operatively, had diffused more than 0.5 mm from the injection site. With transmission electron microscopy, immunoliposomes were observed entering numerous neurons and some astrocytes in a process distinct from the clathrin-coated pit mechanism. These findings suggest that Thy 1.1 immunoliposomes are effective for intracellular delivery in vivo and their endocytosis occurs independently of a coated pit process. The research has helped to elucidate alternative mechanisms for immunoliposomal delivery. A more fundamental understanding of these attributes is needed to achieve the therapeutic potential of immunoliposomes.