Aquaporins in spinal cord injury: the janus face of aquaporin 4.

Although malfunction of spinal cord water channels (aquaporins, AQP) likely contributes to severe disturbances in ion/water homeostasis after spinal cord injury (SCI), their roles are still poorly understood. Here we report and discuss the potential significance of changes in the AQP4 expression in human SCI that generates glial fibrillary acidic protein (GFAP)-labeled astrocytes devoid of AQP4, and GFAP-labeled astroglia that overexpress AQP4. We used a rat model of contusion SCI to study observed changes in human SCI. AQP4-negative astrocytes are likely generated during the process of SCI-induced replacement of lost astrocytes, but their origin and role in SCI remains to be investigated. We found that AQP4-overexpression is likely triggered by hypoxia. Our transcriptional profiling of injured rat cords suggests that elevated AQP4-mediated water influx accompanies increased uptake of chloride and potassium ions which represents a protective astrocytic reaction to hypoxia. However, unbalanced water intake also results in astrocytic swelling that can contribute to motor impairment, but likely only in milder injuries. In severe rat SCI, a low abundance of AQP4-overexpressing astrocytes was found during the motor recovery phase. Our results suggest that severe rat contusion SCI is a better model to analyze AQP4 functions after SCI. We found that AQP4 increases in the chronic post-injury phase are associated with the development of pain-like behavior in SCI rats, while possible mechanisms underlying pain development may involve astrocytic swelling-induced glutamate release. In contrast, the formation and size of fluid-filled cavities occurring later after SCI does not appear to be affected by the extent of increased AQP4 levels. Therefore, the effect of therapeutic interventions targeting AQP4 will depend not only on the time interval after SCI or animal models, but also on the balance between protective role of increased AQP4 in hypoxia and deleterious effects of ongoing astrocytic swelling.

Xenografts of expanded primate olfactory ensheathing glia support transient behavioral recovery that is independent of serotonergic or corticospinal axonal regeneration in nude rats following spinal cord transection.

Transplantation of olfactory ensheathing glial cells (OEG) may improve the outcome from spinal cord injury. Proof-of-principle studies in primates are desirable and the feasibility and efficacy of using in vitro expanded OEG should be tested. An intermediate step between the validation of rodent studies and human clinical trials is to study expanded primate OEG (POEG) xenografts in immunotolerant rodents. In this study the time course to generate purified POEG was evaluated as well as their survival, effect on damaged axons of the corticospinal and serotonergic systems, tissue sparing, and chronic locomotor recovery following transplantation. Fifty-seven nude rats underwent T9/10 spinal cord transection. Thirty-eight rats received POEG, 19 controls were injected with cell medium, and 10 received lentivirally-GFP-transfected POEG. Histological evaluation was conducted at 6 weeks, 8 weeks, 14 weeks and 23-24 weeks. Of these 57 rats, 18 were studied with 5-HT immunostaining, 16 with BDA anterograde CST labeling, and six were used for transmission electron microscopy. In grafted animals, behavioral recovery, sprouting and limited regeneration of 5-HT fibers, and increased numbers of proximal collateral processes but not regeneration of CST fibers was observed. Grafted animals had less cavitation in the spinal cord stumps than controls. Behavioral recovery peaked at three months and then declined. Five POEG-transplanted animals that had shown behavioral recovery underwent retransection and behavioral scores did not change significantly, suggesting that long tract axonal regeneration did not account for the locomotor improvement. At the ultrastructural level presumptive POEG were found to have direct contacts with astrocytes forming the glia limitans, distinct from those formed by Schwann cells. At 6 weeks GFP expression was detected in cells within the lesion site and within nerve roots but did not match the pattern of Hoechst nuclear labeling. At 3.5 months only GFP-positive debris in macrophages could be detected. Transplanted POEG support behavioral recovery via mechanisms that appear to be independent of long tract regeneration.

Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP panel: clinical trial design.

The International Campaign for Cures of Spinal Cord Injury Paralysis established a panel tasked with reviewing the methodology for clinical trials for spinal cord injury (SCI), and making recommendations on the conduct of future trials. This is the fourth of four papers. Here, we examine the phases of a clinical trial program, the elements, types, and protocols for valid clinical trial design. The most rigorous and valid SCI clinical trial would be a prospective double-blind randomized control trial utilizing appropriate placebo control subjects. However, in specific situations, it is recognized that other trial procedures may have to be considered. We review the strengths and limitations of the various types of clinical trials with specific reference to SCI. It is imperative that the design and conduct of SCI clinical trials should meet appropriate standards of scientific inquiry to insure that meaningful conclusions about efficacy and safety can be achieved and that the interests of trial subjects are protected. We propose these clinical trials guidelines for use by the SCI clinical research community.

Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP Panel: clinical trial inclusion/exclusion criteria and ethics.

The International Campaign for Cures of Spinal Cord Injury Paralysis established a panel tasked with reviewing the methodology for clinical trials for spinal cord injury (SCI), and making recommendations on the conduct of future trials. This is the third of four papers. It examines inclusion and exclusion criteria that can influence the design and analysis of clinical trials in SCI, together with confounding variables and ethical considerations. Inclusion and exclusion criteria for clinical trials should consider several factors. Among these are (1) the enrollment of subjects at appropriate stages after SCI, where there is supporting data from animal models or previous human studies; (2) the severity, level, type, or size of the cord injury, which can influence spontaneous recovery rate and likelihood that an experimental treatment will clinically benefit the subject; and (3) the confounding effects of various independent variables such as pre-existing or concomitant medical conditions, other medications, surgical interventions, and rehabilitation regimens. An issue of substantial importance in the design of clinical trials for SCI is the inclusion of blinded assessments and sham surgery controls: every effort should be made to address these major issues prospectively and carefully, if clear and objective information is to be gained from a clinical trial. The highest ethical standards must be respected in the performance of clinical trials, including the adequacy and clarity of informed consent.

Guidelines for the conduct of clinical trials for spinal cord injury (SCI) as developed by the ICCP panel: clinical trial outcome measures.

An international panel reviewed the methodology for clinical trials of spinal cord injury (SCI), and provided recommendations for the valid conduct of future trials. This is the second of four papers. It examines clinical trial end points that have been used previously, reviews alternative outcome tools and identifies unmet needs for demonstrating the efficacy of an experimental intervention after SCI. The panel focused on outcome measures that are relevant to clinical trials of experimental cell-based and pharmaceutical drug treatments. Outcome measures are of three main classes: (1) those that provide an anatomical or neurological assessment for the connectivity of the spinal cord, (2) those that categorize a subject's functional ability to engage in activities of daily living, and (3) those that measure an individual's quality of life (QoL). The American Spinal Injury Association impairment scale forms the standard basis for measuring neurologic outcomes. Various electrophysiological measures and imaging tools are in development, which may provide more precise information on functional changes following treatment and/or the therapeutic action of experimental agents. When compared to appropriate controls, an improved functional outcome, in response to an experimental treatment, is the necessary goal of a clinical trial program. Several new functional outcome tools are being developed for measuring an individual's ability to engage in activities of daily living. Such clinical end points will need to be incorporated into Phase 2 and Phase 3 trials. QoL measures often do not correlate tightly with the above outcome tools, but may need to form part of Phase 3 trial measures.

Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP panel: spontaneous recovery after spinal cord injury and statistical power needed for therapeutic clinical trials.

The International Campaign for Cures of Spinal Cord Injury Paralysis (ICCP) supported an international panel tasked with reviewing the methodology for clinical trials in spinal cord injury (SCI), and making recommendations on the conduct of future trials. This is the first of four papers. Here, we examine the spontaneous rate of recovery after SCI and resulting consequences for achieving statistically significant results in clinical trials. We have reanalysed data from the Sygen trial to provide some of this information. Almost all people living with SCI show some recovery of motor function below the initial spinal injury level. While the spontaneous recovery of motor function in patients with motor-complete SCI is fairly limited and predictable, recovery in incomplete SCI patients (American spinal injury Association impairment scale (AIS) C and AIS D) is both more substantial and highly variable. With motor complete lesions (AIS A/AIS B) the majority of functional return is within the zone of partial preservation, and may be sufficient to reclassify the injury level to a lower spinal level. The vast majority of recovery occurs in the first 3 months, but a small amount can persist for up to 18 months or longer. Some sensory recovery occurs after SCI, on roughly the same time course as motor recovery. Based on previous data of the magnitude of spontaneous recovery after SCI, as measured by changes in ASIA motor scores, power calculations suggest that the number of subjects required to achieve a significant result from a trial declines considerably as the start of the study is delayed after SCI. Trials of treatments that are most efficacious when given soon after injury will therefore, require larger patient numbers than trials of treatments that are effective at later time points. As AIS B patients show greater spontaneous recovery than AIS A patients, the number of AIS A patients requiring to be enrolled into a trial is lower. This factor will have to be balanced against the possibility that some treatments will be more effective in incomplete patients. Trials involving motor incomplete SCI patients, or trials where an accurate assessment of AIS grade cannot be made before the start of the trial, will require large subject numbers and/or better objective assessment methods.

Demyelination and Schwann cell responses adjacent to injury epicenter cavities following chronic human spinal cord injury.

The natural history of post-traumatic demyelination and myelin repair in the human spinal cord is largely unknown and has remained a matter of speculation. A wealth of experimental studies indicate that mild to moderate contusive injuries to the mammalian spinal cord evolve into a cavity with a preserved rim of white matter in which a population of segmentally demyelinated axons persists. It is believed that such injured axons have abnormal conduction properties. Theoretically, such axons might show improved function if myelin repair occurred. Schwann cells can remyelinate axons affected by multiple sclerosis, but little evidence exists that such repair can occur spontaneously following traumatic human SCI. Therefore, it is important to determine if chronic demyelination is present following human spinal cord injury. There are no previous reports that have conclusively demonstrated demyelination in the human spinal cord following traumatic spinal cord injury using immunohistochemical techniques. Immunohistochemical methods were used to study the distribution of peripheral and central myelin proteins as well as axonal neurofilament at the injury epicenter in 13 postmortem chronically injured human spinal cords 1-22 years following injury. Of these seven could be assessed by our methods. We found that some axonal demyelination can be detected even a decade following human SCI and indirect evidence that invading Schwann cells contributed to restoration of myelin sheaths around some spinal axons.

Thoracic disc herniation presenting with transient anterior spinal artery syndrome. A case report.

SUMMARY: This report is a clinical and radiologic correlation of anterior spinal arterial distribution ischemia with a thoracic disc herniation affecting the artery of Adamkiewicz. We could only find one other similar reported case. A 38-year-old woman developed sudden onset of severe back pain and radiculopathy, followed by rapidly evolving paraparesis. The neurological examination was consistent with a deficit caused by anterior spinal artery ischemia. MRI revealed T2 signal change in the thoracolumbar spinal cord and a laterally placed, non-calcified disc herniation. Selective spinal angiography performed 30 hours after onset revealed displacement of the left T9 radicular feeding artery by the disc herniation; at this time the artery was patent. The patient experienced some resolution of symptoms within the first 24 hours and was managed conservatively and made a significant recovery within two weeks. Appropriately located thoracic disc herniations can disturb the blood supply to the thoracolumbar spinal cord.

Influence of IN-1 antibody and acidic FGF-fibrin glue on the response of injured corticospinal tract axons to human Schwann cell grafts.

Two strategies have been shown by others to improve CST regeneration following thoracic spinal cord injury: 1) the administration of a monoclonal antibody, IN-1, raised against a myelin-associated, neurite growth inhibitory protein, and 2) the delivery of acidic fibroblast growth factor (aFGF) in fibrin glue in association with peripheral nerve grafts. Because autologous transplantation of human Schwann cells (SCs) is a potential strategy for CNS repair, we evaluated the ability of these two molecular agents to induce CST regeneration into human SC grafts placed to span a midthoracic spinal cord transection in the adult nude rat, a xenograft tolerant strain. IN-1 or control (HRP) antibodies were delivered to the injury/graft region by encapsulated hybridoma cells ("IN-1 ravioli") or daily infusion of hybridoma culture supernatant; aFGF-fibrin glue was placed in the same region in other animals. Anterograde tracing from the motor cortex using the dextran amine tracers, Fluororuby (FR) and biotinylated dextran amine (BDA), was performed. Thirty-five days after grafting, the CST response was evaluated qualitatively by looking for regenerated CST fibers in or beyond grafts and quantitatively by constructing camera lucida composites to determine the sprouting index (SI), the position of the maximum termination density (MTD) rostral to the GFAP-defined host/graft interface, and the longitudinal spread (LS) of bulbous end terminals. The latter two measures provided information about axonal die-back. In control animals (graft only), the CST did not enter the SC graft and underwent axonal die-back [SI = 1.4 +/- 0.1, MTD = 2.0 +/- 0.2, LS = 1.3 +/- 0.3, (n = 3)]. Results of IN-1 delivery from ravioli did not differ from controls, but injections of IN-1-containing supernatant resulted in a significant degree of sprouting but did not prevent axonal die-back [SI = 1.9 +/- 0.1, MTD = 1.5 +/- 0.2, LS = 1.1 +/- 0.1, (n = 7)] and traced fibers did not enter grafts. Acidic FGF dramatically reduced axonal die-back and caused sprouting [SI = 2.0 +/- 0.1 (n = 5), MTD = 0.5 +/- 0.04 (n = 6), LS = 0.4 +/- 0.1 (n = 6)]. Some traced fibers entered SC grafts and in 2/6 cases entered the distal interface. We conclude that 1) human SC grafts alone do not support the regeneration of injured CST fibers and do not prevent die-back, 2) grafts plus IN-1 antibody-containing supernatant support some sprouting but die-back continues, and 3) grafts plus aFGF-fibrin glue support regeneration of some fibers into the grafts and reduce die-back.

The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord.

Advances in the purification and expansion of Schwann cells (SCs) from adult human peripheral nerve, together with biomaterials development, have made the construction of unique grafts with defined properties possible. We have utilized PAN/PVC guidance channels to form solid human SC grafts which can be transplanted either with or without the channel. We studied the ability of grafts placed with and without channels to support regeneration and to influence functional recovery; characteristics of the graft and host/graft interface were also compared. The T9-T10 spinal cord of nude rats was resected and a graft was placed across the gap; methylprednisolone was delivered acutely to decrease secondary injury. Channels minimized the immigration of connective tissue into grafts but contributed to some necrotic tissue loss, especially in the distal spinal cord. Grafts without channels contained more myelinated axons (x = 2129 +/- 785) vs (x = 1442 +/- 514) and were larger in cross-sectional area ( x = 1.53 +/- 0.24 mm2) vs (x = 0.95 +/- 0.86 mm2). The interfaces formed between the host spinal cord and the grafts placed without channels were highly interdigitated and resembled CNS-PNS transition zones; chondroitin sulfate proteoglycans was deposited there. Whereas several neuronal populations including propriospinal, sensory, motoneuronal, and brainstem neurons regenerated into human SC grafts, only propriospinal and sensory neurons were observed to reenter the host spinal cord. Using combinations of anterograde and retrograde tracers, we observed regeneration of propriospinal neurons up to 2.6 mm beyond grafts. We estimate that 1% of the fibers that enter grafts reenter the host spinal cord by 45 days after grafting. Following retrograde tracing from the distal spinal cord, more labeled neurons were unexpectedly found in the region of the dextran amine anterograde tracer injection site where a marked inflammatory reaction had occurred. Animals with bridging grafts obtained modestly higher scores during open field [(x = 8.2 +/- 0.35) vs (x = 6.8 +/- 0.42), P = 0.02] and inclined plane testing (x = 38.6 +/- 0. 542) vs (x = 36.3 +/- 0.53), P = 0.006] than animals with similar grafts in distally capped channels. In summary, this study showed that in the nude rat given methylprednisolone in combination with human SC grafts, some regenerative growth occurred beyond the graft and a modest improvement in function was observed.