The Utility of 3D Ultramicroscopy for Evaluating Cellular Therapies After Spinal Cord Injury.

Cell therapies have shown promise for repairing the injured spinal cord in experimental models and are now being evaluated in clinical trials for the treatment of human spinal cord injury (SCI). To date, experimental evaluation of implanted cell survival, migration, and integration within the injured central nervous system (CNS) of animals has been technically demanding, requiring tissue sectioning, staining, imaging, and manual reconstruction of 2-dimensional (2D) specimens in 3 dimensions (3D). Not only are these histological procedures laborious and fraught with processing artifacts during manual 3D reconstruction, but they are time-intensive. Herein we describe the utility of 3D ultramicroscopy for assessment of cell therapies after SCI, a new state-of-the-art imaging modality in which whole brain and spinal cord samples are optically sectioned to allow evaluation of intact, macroscopic specimens with microscopic resolution.

Transplantation and repair: combined cell implantation and chondroitinase delivery prevents deterioration of bladder function in rats with complete spinal cord injury.

STUDY DESIGN: Additional examination. In this study, we report changes in bladder function after a combined treatment that was designed to study axonal regeneration after complete spinal cord injury (SCI) in rats. OBJECTIVES: To report effects on bladder function following the administration of a combined treatment for complete SCI. SETTING: University of Alberta, Faculty of Rehabilitation Medicine, Edmonton, Canada. METHODS: Eight rats received Schwann cells in Matrigel-filled guidance channels, olfactory ensheathing glia and chondroitinase ABC at the lesion site following complete thoracic SCI. Controls (n=7) received Matrigel only. Daily bladder examinations were performed. Analysis of bladder size, wall thickness, actin and collagen type III was performed after 14 weeks. RESULTS: Following SCI, both groups regained bladder voiding after 3 weeks. However, 2 weeks later, incontinence was observed in all untreated rats and two treated rats. Post-mortem examination of bladders revealed enlarged bladder sizes. Thicker bladder walls were found in untreated rats, which were composed of disorganized bundles of smooth muscle fibers surrounded by high amounts of collagen (type III). CONCLUSION: We show that the combined treatment prevents collagen deposition in bladder walls and maintains the rat's ability to void efficiently. Although the mechanism responsible for this improvement is unclear, our study shows that the present combinatory therapy can influence bladder function, thus expanding their utility as a broad reparative approach for SCI.

Stem and progenitor cell therapies: recent progress for spinal cord injury repair.

Mechanical trauma to the spinal cord is often accompanied by irreversible tissue damage, limited endogenous repair and permanent loss of motor, sensory and autonomic function. The implantation of exogenous cells or the stimulation of endogenous cells, to repopulate and replace or to provide a conducive environment for repair, offers a promising therapeutic direction for overcoming the multitude of obstacles facing successful recovery from spinal cord injury. Although relatively new to the scene of cell based therapies for reparative medicine, stem cells and their progenitors have been labeled as the 'cell of the future' for revolutionizing the treatment of CNS injury and neurodegenerative disorders. The following review examines the different types of stem cells and their progenitors, their utility in experimental models of spinal cord injury and explores the outstanding issues that still need to be addressed before they move towards clinical implementation.

Schwann cell transplantation improves reticulospinal axon growth and forelimb strength after severe cervical spinal cord contusion.

Schwann cell (SC) implantation alone has been shown to promote the growth of propriospinal and sensory axons, but not long-tract descending axons, after thoracic spinal cord injury (SCI). In the current study, we examined if an axotomy close to the cell body of origin (so as to enhance the intrinsic growth response) could permit supraspinal axons to grow onto SC grafts. Adult female Fischer rats received a severe (C5) cervical contusion (1.1 mm displacement, 3 KDyn). At 1 week postinjury, 2 million SCs ex vivo transduced with lentiviral vector encoding enhanced green fluorescent protein (EGFP) were implanted within media into the injury epicenter; injury-only animals served as controls. Animals were tested weekly using the BBB score for 7 weeks postimplantation and received at end point tests for upper body strength: self-supported forelimb hanging, forearm grip force, and the incline plane. Following behavioral assessment, animals were anterogradely traced bilaterally from the reticular formation using BDA-Texas Red. Stereological quantification revealed a twofold increase in the numbers of preserved NeuN+ neurons rostral and caudal to the injury/graft site in SC implanted animals, corroborating previous reports of their neuroprotective efficacy. Examination of labeled reticulospinal axon growth revealed that while rarely an axon was present within the lesion site of injury-only controls, numerous reticulospinal axons had penetrated the SC implant/lesion milieu. This has not been observed following implantation of SCs alone into the injured thoracic spinal cord. Significant behavioral improvements over injury-only controls in upper limb strength, including an enhanced grip strength (a 296% increase) and an increased self-supported forelimb hanging, accompanied SC-mediated neuroprotection and reticulospinal axon growth. The current study further supports the neuroprotective efficacy of SC implants after SCI and demonstrates that SCs alone are capable of supporting modest supraspinal axon growth when the site of axon injury is closer to the cell body of the axotomized neuron.

Increased spinal c-Fos expression with noxious and non-noxious peripheral stimulation after severe spinal contusion.

The effects of severe contusive spinal cord injury (SCI), at thoracic level 8 (T8), on lumbar c-Fos expression in the spinal cord was investigated. As hypothesized, chronic SCI has a significant effect on expression of c-Fos in the dorsal spinal sensory areas with noxious and innocuous peripheral stimulation of the sciatic nerve. This alteration to stimulation effects was measured using counts of c-Fos immunoreactive cells in the dorsal horn of the L5 lumbar spinal cord in injured animals at 90 days post-injury and in uninjured controls. The number of c-Fos immunoreactive cells increased in SCI rats only after noxious peripheral stimulation (electrical and chemical) suggesting a general increase in excitability in spinal pathways (central sensitization) associated with chronic SCI. These altered responses may represent a functional anatomical reorganization of spinal cord circuitry leading to increased dorsal horn c-Fos expression as a response to severe chronic contusive damage to the spinal cord sensory pathways.

Designing cell- and gene-based regeneration strategies to repair the injured spinal cord.

There is an array of new and promising strategies being developed to improve function after spinal cord injury (SCI). The targeting of a diversity of deleterious processes within the tissue after SCI will necessitate a multi-factorial intervention, such as the combination of cell- and gene-based approaches. To ensure proper development and design of these experiments, many issues need to be addressed. It is the purpose of this review to consider the strategies involved in testing the efficacy of these new combinations to improve axonal regeneration. For cell-based therapy, issues are choosing a SCI model, the time of cell implantation, placement of cells and their subsequent migration, fluid versus solid grafts, use of agents to prevent immune rejection, and tracking of implanted cells. Grafting is also discussed in view of improving function, reducing secondary damage, bridging the injured spinal cord, supporting axonal regrowth, replacing lost neurons, facilitating myelination, and promoting axonal growth from the implant into the cord. The choice of a gene delivery system, gene-based therapies in vivo to provide chemoattractant and guidance cues, altering the intrinsic regenerative capacity of neurons, enhancing endogenous non-neuronal cell functions, and targeting the synthesis of growth inhibitory molecules are also discussed, as well as combining ex vivo gene and cell therapies.

Survival, integration, and axon growth support of glia transplanted into the chronically contused spinal cord.

Due to an ever-growing population of individuals with chronic spinal cord injury, there is a need for experimental models to translate efficacious regenerative and reparative acute therapies to chronic injury application. The present study assessed the ability of fluid grafts of either Schwann cells (SCs) or olfactory ensheathing glia (OEG) to facilitate the growth of supraspinal and afferent axons and promote restitution of hind limb function after transplantation into a 2-month-old, moderate, thoracic (T8) contusion in the rat. The use of cultured glial cells, transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP), permitted long-term tracking of the cells following spinal cord transplantation to examine their survival, migration, and axonal association. At 3 months following grafting of 2 million SCs or OEG in 6 microl of DMEM/F12 medium into the injury site, stereological quantification of the three-dimensional reconstructed spinal cords revealed that an average of 17.1 +/- 6.8% of the SCs and 2.3 +/- 1.4% of the OEG survived from the number transplanted. In the OEG grafted spinal cord, a limited number of glia were unable to prevent central cavitation and were found in patches around the cavity rim. The transplanted SCs, however, formed a substantive graft within the injury site capable of supporting the ingrowth of numerous, densely packed neurofilament-positive axons. The SC grafts were able to support growth of both ascending calcitonin gene-related peptide (CGRP)-positive and supraspinal serotonergic axons and, although no biotinylated dextran amine (BDA)-traced corticospinal axons were present within the center of the grafts, the SC transplants significantly increased corticospinal axon numbers immediately rostral to the injury-graft site compared with injury-only controls. Moreover, SC grafted animals demonstrated modest, though significant, improvements in open field locomotion and exhibited less foot position errors (base of support and foot rotation). Whereas these results demonstrate that SC grafts survive, support axon growth, and can improve functional outcome after chronic contusive spinal cord injury, further development of OEG grafting procedures in this model and putative combination strategies with SC grafts need to be further explored to produce substantial improvements in axon growth and function.

Histopathological and behavioral characterization of a novel cervical spinal cord displacement contusion injury in the rat.

Cervical contusive trauma accounts for the majority, of human spinal cord injury (SCI), yet experimental use of cervical contusion injury models has been limited. Considering that (1) the different ways of injuring the spinal cord (compression, contusion, and transection) induce very different processes of tissue damage and (2) the architecture of the spinal cord is not uniform, it is important to use a model that is more clinically applicable to human SCI. Therefore, in the current study we have developed a rat model of contusive, cervical SCI using the Electromagnetic Spinal Cord Injury Device (ESCID) developed at Ohio State University (OSU) to induce injury by spinal cord displacement. We used the device to perform mild, moderate and severe injuries (0.80, 0.95, and 1.1 mm displacements, respectively) with a single, brief displacement of <20 msec upon the exposed dorsal surface of the C5 cervical spinal cord of female (180-200 g) Fischer rats. Characterization of the model involved the analysis of the temporal histopathological progression of the injury over 9 weeks using histochemical stains to analyze white and gray mater integrity and immunohistochemistry to examine cellular changes and physiological responses within the injured spinal cord. Accompanying the histological analysis was a comprehensive determination of the behavioral functionality of the animals using a battery of motor tests. Characterization of this novel model is presented to enable and encourage its future use in the design and experimental testing of therapeutic strategies that may be used for human SCI.

Comparison of iNOS inhibition by antisense and pharmacological inhibitors after spinal cord injury.

Inducible nitric oxide synthase (iNOS) is a key mediator of inflammation during pathological conditions. We examined, through the use of selective iNOS inhibitors, the role of iNOS in specific pathophysiological processes after spinal cord injury (SCI), including astrogliosis, blood-spinal cord barrier (BSCB) permeability, polymorphonuclear leukocyte infiltration, and neuronal cell death. Administration of iNOS antisense oligonucleotides (ASOs) (intraspinally at 3 h) or the pharmacological inhibitors, N-[3(Aminomethyl) benzyl] acetamidine (1400 W) (i.v./i.p. 3 and 9 h) or aminoguanidine (i.p. at 3 and 9 h) after moderate contusive injury decreased the number of iNOS immunoreactive cells at the injury site by 65.6% (iNOS ASOs), 62.1% (1400 W), or 59% (aminoguanidine) 24 h postinjury. iNOS activity was reduced 81.8% (iNOS ASOs), 56.7% (1400 W), or 67.9% (aminoguanidine) at this time. All iNOS inhibitors reduced the degree of BSCB disruption (plasma leakage of rat immunoglobulins), with iNOS ASO inhibition being more effective (reduced by 58%). Neutrophil accumulation within the injury site was significantly reduced by iNOS ASOs and 1400 W by 78.8% and 20.9%, respectively. Increased astrogliosis was diminished with iNOS ASOs but enhanced following aminoguanidine. Detection of necrotic and apoptotic neuronal cell death by propidium iodide and an FITC-conjugated Annexin V antibody showed that iNOS inhibition could significantly retard neuronal cell death rostral and caudal to the injury site. These novel findings indicate that acute inhibition of iNOS is beneficial in reducing several pathophysiological processes after SCI. Furthermore, we demonstrate that the antisense inhibition of iNOS is more efficacious than currently available pharmacological agents.

Jun, Fos and Krox in the thalamus after C-fiber stimulation: coincident-input-dependent expression, expression across somatotopic boundaries, and nucleolar translocation.

Expression of the inducible transcription factors Jun, Fos and Krox is commonly used to map neurons in the brain that are activated by sensory inputs. However, some neurons known to be electrically excited by such inputs do not always express these factors. In particular, stimulation of hindlimb sensory nerve C-fibers induces expression of c-Fos in the medial thalamus (the mediodorsal, intermediodorsal, centrolateral and centromedial), but not in the lateral thalamus (the ventroposterolateral, ventroposteromedial and posterior group). We hypothesized that c-Fos expression might only occur in these lateral areas after more complex stimulation patterns, or that only other transcription factors can be induced in these areas by such stimuli. Thus we examined the effects of single, repeated and coincident C-fiber inputs on expression of six inducible transcription factors in the medial, lateral and reticular thalamus of the rat. A weak C-fiber input caused by noxious mechanical stimulation of the skin of one hindpaw did not induce expression of c-Fos, FosB, Krox-20 or Krox-24; but it did reduce the basal expressions of c-Jun and JunD in both the medial and lateral areas. An intense input produced by electrical stimulation of all the C-fibers in one sciatic nerve also failed to induce expression of c-Fos, FosB, Krox-20 or Krox-24 in the medial or lateral areas. However, in the medial thalamus it increased c-Jun and reduced the basal expression of JunD, whereas in the lateral thalamus it had no effect on c-Jun but again reduced the basal expression of JunD. With repeated stimulation, i.e. when the noxious stimulus was applied to the contralateral hindpaw 6 h after the sciatic stimulation, there was again no induction of c-Fos, FosB or Krox-20 in the medial thalamus; but there was an increase in c-Jun and Krox-24, and a decrease in JunD levels. In the lateral thalamus the repeated stimulation again failed to induce c-Fos, but the expressions of FosB, c-Jun and Krox-24 were increased, and that of JunD was again reduced. With coincident stimulation, i.e. when a stimulus was applied to each hindpaw simultaneously, c-Fos and Krox-24 remained absent; but there was a marked induction of FosB and Krox-20, a strong repression of c-Jun, and no effect or a reduction of the basal levels of JunD. This coincident stimulation also caused FosB to appear in the nucleolus of many thalamic neurons. MK-801, but not L-NAME, blocked all these changes. In summary, noxious stimulation affects the expression of all transcription factors in the medial, lateral and reticular thalamus in a complex manner depending upon the inducible transcription factor considered, the thalamic nucleus, and the stimulation paradigm. The expression of some transcription factors uniquely after simultaneous inputs suggests they act as coincidence detectors at the gene level.