Caitlin E. Hill

Degeneration and sprouting of identified descending supraspinal axons after contusive spinal cord injury in the rat.

Contusive spinal cord injury (SCI) results in the formation of a chronic lesion cavity surrounded by a rim of spared fibers. Tissue bridges containing axons extend from the spared rim into the cavity dividing it into chambers. Whether descending axons can grow into these trabeculae or whether fibers within the trabeculae are spared fibers remains unclear. The purposes of the present study were (1) to describe the initial axonal response to contusion injury in an identified axonal population, (2) to determine whether and when sprouts grow in the face of the expanding contusion cavity, and (3) in the long term, to see whether any of these sprouts might contribute to the axonal bundles that have been seen within the chronic contusion lesion cavity. The design of the experiment also allowed us to further characterize the development of the lesion cavity after injury. The corticospinal tract (CST) underwent extensive dieback after contusive SCI, with retraction bulbs present from 1 day to 8 months postinjury. CST sprouting occurred between 3 weeks and 3 months, with penetration of CST axons into the lesion matrix occurring over an even longer time course. Collateralization and penetration of reticulospinal fibers were observed at 3 months and were more extensive at later time points. This suggests that these two descending systems show a delayed regenerative response and do extend axons into the lesion cavity and that the endogenous repair can continue for a very long time after SCI.

Labeled Schwann cell transplantation: Cell loss, host Schwann cell replacement, and strategies to enhance survival

Although transplanted Schwann cells (SCs) can promote axon regeneration and remyelination and improve recovery in models of spinal cord injury, little is known about their survival and how they interact with host tissue. Using labeled SCs from transgenic rats expressing human placental alkaline phosphatase (PLAP), SC survival in a spinal cord contusion lesion was assessed. Few PLAP SCs survived at 2 weeks after acute transplantation. They died early due to necrosis and apoptosis. Delaying transplantation until 7 days after injury improved survival. A second wave of cell death occurred after surviving cells had integrated into the spinal cord. Survival of PLAP SCs was enhanced by immunosuppression with cyclosporin; delayed transplantation in conjunction with immunosuppression resulted in the best survival. In all cases, transplantation of SCs resulted in extensive infiltration of endogenous p75+ cells into the injury site, suggesting that endogenous SCs may play an important role in the repair observed after SC transplantation.

Acute transplantation of glial-restricted precursor cells into spinal cord contusion injuries: survival, differentiation, and effects on lesion environment and axonal regeneration

Transplantation of stem cells and immature cells has been reported to ameliorate tissue damage, induce axonal regeneration, and improve locomotion following spinal cord injury. However, unless these cells are pushed down a neuronal lineage, the majority of cells become glia, suggesting that the alterations observed may be potentially glially mediated. Transplantation of glial-restricted precursor (GRP) cells--a precursor cell population restricted to oligodendrocyte and astrocyte lineages--offers a novel way to examine the effects of glial cells on injury processes and repair. This study examines the survival and differentiation of GRP cells, and their ability to modulate the development of the lesion when transplanted immediately after a moderate contusion injury of the rat spinal cord. GRP cells isolated from a transgenic rat that ubiquitously expresses heat-stable human placental alkaline phosphatase (PLAP) were used to unambiguously detect transplanted GRP cells. Following transplantation, some GRP cells differentiated into oligodendrocytes and astrocytes, retaining their differentiation potential after injury. Transplanted GRP cells altered the lesion environment, reducing astrocytic scarring and the expression of inhibitory proteoglycans. Transplanted GRP cells did not induce long-distance regeneration from corticospinal tract (CST) and raphe-spinal axons when compared to control animals. However, GRP cell transplants did alter the morphology of CST axons toward that of growth cones, and CST fibers were found within GRP cell transplants, suggesting that GRP cells may be able to support axonal growth in vivo after injury.

An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat.

Changes in sensory function including chronic pain and allodynia are common sequelae of spinal cord injury (SCI) in humans. The present study documents the extent and time course of mechanical allodynia and cold hyperalgesia after contusion SCI in the rat using stimulation with graded von Frey filaments (4.97–50.45 g force) and ice probes. Fore- and hind-paw withdrawal thresholds to plantar skin stimulation were determined in rats with a range of SCI severities (10-g weight dropped from 6.25, 12.5, or 25 mm using the MASCIS injury device); animals with 25-mm injuries most consistently showed decreased hind-paw withdrawal thresholds to touch and cold, which developed over several weeks after surgery. Stimulation of the torso with graded von Frey hairs was performed at specified locations on the back and sides from the neck to the haunch. Suprasegmental responses (orientation, vocalization, or escape) to mechanical stimulation of these sites were elicited infrequently in the laminectomy control rats and only during the first 3 weeks after surgery, whereas in 25-mm SCI rats, such responses were obtained for the entire 10 weeks of the study. These data suggest that rats with contusion SCI may exhibit sensory alterations relevant to human spinal cord injuries.

Combinatorial strategies with Schwann cell transplantation to improve repair of the injured spinal cord

The immediate effect of spinal cord injury (SCI) is a mechanical trauma that results in direct damage at the lesion site followed by secondary responses leading to loss of adjacent neurons and glia. Consequently, SCI leads to paralysis and loss of sensation below the level of the injury, altered autonomic responses and, frequently, the development of abnormal sensation and pain. Substantial endogenous remodeling of the spinal cord occurs [6] as axons begin to sprout and cells, including inflammatory, endothelial and Schwann cells (SCs), invade the injury site [34], likely contributing to spontaneous improvement observed in humans. Despite this endogenous repair, it is modest and functional improvements are limited [47;55]. Because treatment options are inadequate, additional therapeutic interventions are needed. Some strategies to repair the spinal cord are focusing on neuroprotection, regeneration, and/or tissue replacement. First, strategies should be designed to limit the secondary spread of damage to adjacent axons, neurons and glia. Second, strategies should promote axonal remodeling to maximize the function of spared tissue locally. Lastly, strategies are needed to promote long distance regrowth of damaged axons by reducing inhibition and/or providing permissive substrates and trophic molecules. The development of different injury models to mimic various aspects of human SCI, together with existing procedures to test behavioral recovery, have improved significantly our understanding of the pathophysiology of SCI and, importantly, have enabled investigation of a myriad of therapeutic interventions. Models are utilized to induce complete or incomplete SCI. Standardized devices are used to produce contusion or compression of the spinal cord, resulting in incomplete injuries with varying degrees of sparing depending upon the magnitude of the impact used [4;7;24;81]. Because standardization of the extent of injury is an essential component of these models, careful examination of the injury parameters and the behavioral recovery early after injury is essential to ensure the validity of the results observed in a specific therapeutic paradigm. Whereas contusion/compression models more accurately mimic the human injury, sparing of axons complicates the interpretation of axonal growth versus sparing. As a result, complete transection of the spinal cord is the favored model to study axonal regeneration. A cystic cavity usually forms after SCI, and it is walled off from the surrounding spared rim of white matter by a glial scar [6]. At the margins of the lesion, injured axons terminate in dystrophic endings, indicating thwarted axonal growth [74]. The presence of axons within trabeculae crossing the lesion implies that, when provided with an appropriate substrate, some axons will grow into the injury site despite the scar [6]. The use of permissive substrates such as cells, extracellular matrix proteins or biomaterials appears necessary to span the lesion. Also, cellular transplants can replace lost neurons and/or glia, enhance tissue preservation via neuroprotection and support axonal regeneration. In this chapter, we shall review experiments testing SC transplantation alone or in combination with known neuroprotective and neuroregenerative strategies. SCs from different sources will be described. Results of the use of SCs in three different injury models, complete transection of the spinal cord, lateral hemi-section and contusion, will be discussed. Whereas axonal regeneration is difficult to discern in the contusion model due to fiber sparing, it is relevant to human SCI in which there is usually some remaining tissue. Combinatorial approaches that have been successful to varying degrees will be highlighted; these generally involve combining neurotrophic factors (NTFs), scar modification, elevation of cAMP, reduction in myelin inhibition or olfactory ensheathing cell (OEC) transplantation with SCs. In addition, we shall discuss the rationale behind the success or failure of some combinatorial strategies and promising current treatments that may be amenable to combinatorial therapies.

Early necrosis and apoptosis of Schwann cells transplanted into the injured rat spinal cord

Poor survival of cells transplanted into the CNS is a widespread problem and limits their therapeutic potential. Whereas substantial loss of transplanted cells has been described, the extent of acute cell loss has not been quantified previously. To assess the extent and temporal profile of transplanted cell death, and the contributions of necrosis and apoptosis to this cell death following spinal cord injury, different concentrations of Schwann cells (SCs), lentivirally transduced to express green fluorescent protein (GFP), were transplanted into a 1‐week‐old moderate contusion of the adult rat thoracic spinal cord. In all cases, transplanted cells were present from 10 min to 28 days. There was a 78% reduction in SC number within the first week, with no significant decrease thereafter. Real‐time polymerase chain reaction showed a similar 80% reduction in GFP‐DNA within the first week, confirming that the decrease in SC number was due to death rather than decreased GFP transgene expression. Cells undergoing necrosis and apoptosis were identified using antibodies against the calpain‐mediated fodrin breakdown product and activated caspase 3, respectively, as well as ultrastructurally. Six times more SCs died during the first week after transplantation by necrosis than apoptosis, with the majority of cell death occurring within the first 24 h. The early death of transplanted SCs indicates that factors present, even 1 week after a moderate contusion, are capable of inducing substantial transplanted cell death. Intervention by strategies that limit necrosis and/or apoptosis should be considered for enhancing acute survival of transplanted cells.

Skin Incision Induces Expression of Axonal Regeneration-Related Genes in Adult Rat Spinal Sensory Neurons

UNLABELLED Skin incision and nerve injury both induce painful conditions. Incisional and postsurgical pain is believed to arise primarily from inflammation of tissue and the subsequent sensitization of peripheral and central neurons. The role of axonal regeneration-related processes in development of pain has only been considered when there has been injury to the peripheral nerve itself, even though tissue damage likely induces injury of resident axons. We sought to determine if skin incision would affect expression of regeneration-related genes such as activating transcription factor 3 (ATF3) in dorsal root ganglion (DRG) neurons. ATF3 is absent from DRG neurons of the normal adult rodent, but is induced by injury of peripheral nerves and modulates the regenerative capacity of axons. Image analysis of immunolabeled DRG sections revealed that skin incision led to an increase in the number of DRG neurons expressing ATF3. RT-PCR indicated that other regeneration-associated genes (galanin, GAP-43, Gadd45a) were also increased, further suggesting an injury-like response in DRG neurons. Our finding that injury of skin can induce expression of neuronal injury/regeneration-associated genes may impact how clinical postsurgical pain is investigated and treated. PERSPECTIVE Tissue injury, even without direct nerve injury, may induce a state of enhanced growth capacity in sensory neurons. Axonal regeneration-associated processes should be considered alongside nerve signal conduction and inflammatory/sensitization processes as possible mechanisms contributing to pain, particularly the transition from acute to chronic pain.

Large animal and primate models of spinal cord injury for the testing of novel therapies

Large animal and primate models of spinal cord injury (SCI) are being increasingly utilized for the testing of novel therapies. While these represent intermediary animal species between rodents and humans and offer the opportunity to pose unique research questions prior to clinical trials, the role that such large animal and primate models should play in the translational pipeline is unclear. In this initiative we engaged members of the SCI research community in a questionnaire and round-table focus group discussion around the use of such models. Forty-one SCI researchers from academia, industry, and granting agencies were asked to complete a questionnaire about their opinion regarding the use of large animal and primate models in the context of testing novel therapeutics. The questions centered around how large animal and primate models of SCI would be best utilized in the spectrum of preclinical testing, and how much testing in rodent models was warranted before employing these models. Further questions were posed at a focus group meeting attended by the respondents. The group generally felt that large animal and primate models of SCI serve a potentially useful role in the translational pipeline for novel therapies, and that the rational use of these models would depend on the type of therapy and specific research question being addressed. While testing within these models should not be mandatory, the detection of beneficial effects using these models lends additional support for translating a therapy to humans. These models provides an opportunity to evaluate and refine surgical procedures prior to use in humans, and safety and bio-distribution in a spinal cord more similar in size and anatomy to that of humans. Our results reveal that while many feel that these models are valuable in the testing of novel therapies, important questions remain unanswered about how they should be used and how data derived from them should be interpreted.

A calpain inhibitor enhances the survival of Schwann cells in vitro and after transplantation into the injured spinal cord.

Despite the diversity of cells available for transplantation into sites of spinal cord injury (SCI), and the known ability of transplanted cells to integrate into host tissue, functional improvement associated with cellular transplantation has been limited. One factor potentially limiting the efficacy of transplanted cells is poor cell survival. Recently we demonstrated rapid and early death of Schwann cells (SCs) within the first 24 h after transplantation, by both necrosis and apoptosis, which results in fewer than 20% of the cells surviving beyond 1 week. To enhance SC transplant survival, in vitro and in vivo models to rapidly screen compounds for their ability to promote SC survival are needed. The current study utilized in vitro models of apoptosis and necrosis, and based on withdrawal of serum and mitogens and the application of hydrogen peroxide, we screened several inhibitors of apoptosis and necrosis. Of the compounds tested, the calpain inhibitor MDL28170 enhanced SC survival both in vitro in response to oxidative stress induced by application of H2O2, and in vivo following delayed transplantation into the moderately contused spinal cord. The results support the use of calpain inhibitors as a promising new treatment for promoting the survival of transplanted cells. They also suggest that in vitro assays for cell survival may be useful for establishing new compounds that can then be tested in vivo for their ability to promote transplanted SC survival.

The Interplay of Secondary Degeneration and Self-Repair After Spinal Cord Injury

Trauma to the spinal cord involves the initial damage to the cells at the impact site, followed by expansion of the injury as a result of secondary injury mechanisms. Early after injury, there is substantial cell death, both necrotic and apoptotic. This is followed by the initiation of reparative mechanisms by the spinal cord, including proliferation of precursor cells and ependymal cells, and the infiltration of the lesion by Schwann cells and axons. Although the mammalian spinal cord is not capable of fully repairing itself after injury, it does undergo substantial remodeling. This article discusses the progression of contusive spinal cord injury and the remodeling that occurs. By understanding the endogenous repair responses that occur after injury, it may be possible to enhance them and thus produce more effective therapies.