Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.     

KU0063794, a Dual mTORC1 and mTORC2 Inhibitor,Reduces Neural Tissue Damage and Locomotor Impairment After Spinal Cord Injury in Mice

Autophagy is an intracellular catabolic mechanism for the degradation of cytoplasmic constituents in the autophagosomal–lysosomal pathway. This mechanism plays an important role in homeostasis and it is defective in certain diseases. Preceding studies have revealed that autophagy is developing as an important moderator of pathological responses associated to spinal cord injury (SCI) and plays a crucial role in secondary injury initiating a progressive degeneration of the spinal cord. Thus, based on this evidence in this study, we used two different selective inhibitors of mTOR activity to explore the functional role of autophagy in an in vivo model of SCI as well as to determine whether the autophagic process is involved in spinal cord tissue damage. We treated animals with a novel synthetic inhibitor temsirolimus and with a dual mTORC1 and mTORC2 inhibitor KU0063794 matched all with the well-known inhibitor of mTOR the rapamycin. Our results demonstrated that mTOR inhibitors could regulate the neuroinflammation associated to SCI and the results that we obtained evidently demonstrated that rapamycin and temsirolimus significantly diminished the expression of iNOS, COX2, GFAP, and re-established nNOS levels, but the administration of KU0063794 is able to blunt the neuroinflammation better than rapamycin and temsirolimus. In addition, neuronal loss and cell mortality in the spinal cord after injury were considerably reduced in the KU0063794-treated mice. Accordingly, taken together our results denote that the administration of KU0063794 produced a neuroprotective function at the lesion site following SCI, representing a novel therapeutic approach after SCI.     

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.     

Effects of retrograde gene transfer of brain-derived neurotrophic factor in the rostral spinal cord of a compression model in rat

Recovery after spinal cord injury (SCI) is rare in humans and experimental animals. Following SCI in adults, changes in gene expression and the regulation of these genes are associated with the pathological development of the injury. High levels of brain-derived neurotrophic factor (BDNF) in the injury area during the post-injury period contribute to enhanced neuroprotection and axonal regeneration. Intervention at the level of gene regulation has the potential to promote SCI repair. In this study, the injection of adenovirus-mediated BDNF in the lesion area (rostral spinal cord) up-regulated the expression of BDNF in the injury zone of a compression model in rat, thereby protecting neurons and enhancing behavioral function.     

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.¹ Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5° (animals rotated at 22.5°), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya''s staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.     

Cervical spinal cord deformation during simulated head-first impact injuries

The relationship between bony spinal column and spinal cord injury during an injury event is not well understood. While several studies have measured spinal canal occlusion during axial impact, there has been limited work done to quantify the spinal cord compression or deformation during simulated injury. Because the cord is a viscoelastic solid it may provide resistance to bone fragments, ligaments or other elements that move into the canal and impinge it during column injury. This would differentiate the measurement of cord compression from the measurement of occlusion of an empty canal. In the present study, a novel method of visualizing and quantifying spinal cord deformation during dynamic head-first impact of ex vivo human cervical spine specimens (N=6) was developed. A radiodense, biofidelic surrogate spinal cord was imaged in the spinal canal using high speed cineradiography at 1000 frames per second. The dorsal-ventral diameter of the cord was measured at 1.5mm increments along its length for each frame of the radiographic footage. The resulting cord deformations were used to determine the theoretical neurological outcome of the impact based on published in vivo ferret studies. The corresponding probability of recovery for the spinal cord deformations in these tests ranged between 8% for atlantoaxial dislocation injury and 95% for mid-cervical spine hyperextension injury (based on the ferret data). Clinically relevant spinal column fracture patterns were produced in this study.     

Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation

Spinal cord injury (SCI) is a severe neurological disease; however, few drugs have been proved to treat SCI effectively. Neuroinflammation is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Salidroside (Sal) has been reported to exert anti‐inflammatory effects in airway, adipose and myocardial tissue; however, the role of Sal in SCI therapeutics has not been clarified. In this study, we showed that Sal could improve the functional recovery of spinal cord in rats as revealed by increased BBB locomotor rating scale, angle of incline, and decreased cavity of spinal cord injury and apoptosis of neurons in vivo. Immunofluorescence double staining of microglia marker and M1/M2 marker demonstrated that Sal could suppress M1 microglia polarization and activate M2 microglia polarization in vivo. To verify how Sal exerts its effects on microglia polarization and neuron protection, we performed the mechanism study in vitro in microglia cell line BV‐2 and neuron cell line PC12. The results showed that Sal prevents apoptosis of PC12 cells in coculture with LPS‐induced M1 BV‐2 microglia, also the inflammatory secretion phenotype of M1 BV‐2 microglia was suppressed by Sal, and further studies demonstrated that autophagic flux regulation through AMPK/mTOR pathway was involved in Sal regulated microglia polarization after SCI. Overall, our study illustrated that Sal could promote spinal cord injury functional recovery in rats, and the mechanism may relate to its microglia polarization modulation through AMPK‐/mTOR‐mediated autophagic flux stimulation.     

Omega-3 fatty acids and neurological injury

Studies with omega-3 polyunsaturated fatty acids (PUFA) have shown that these compounds have therapeutic potential in several indications in neurology and psychiatry. Acute spinal cord injury (SCI) is an event with devastating consequences, and no satisfactory treatment is available at present. The pathogenetic mechanisms associated with SCI include excitotoxicity, increased oxidation and inflammation. We review here our recent studies, which suggest that omega-3 PUFA have significant neuroprotective potential in spinal cord trauma. In a first study, we administered an intravenous bolus of alpha-linolenic acid (LNA) or docosahexaenoic acid (DHA) 30 min after spinal cord hemisection injury in adult rats. The omega-3 PUFA led to increased neuronal and glial survival, and a significantly improved neurological outcome. In subsequent studies, we tested DHA in a more severe compression model of SCI. We also explored a combined acute and chronic treatment regime using DHA. Saline or DHA was administered intravenously 30 min after compression of the spinal cord. After injury, the saline group received a standard control diet, whereas DHA-injected animals received either a control or a DHA-enriched diet for 6 weeks following injury. We assessed locomotor recovery and analysed markers for cell survival and axonal damage, and we also investigated the effects of the treatment on the inflammatory reaction and the oxidative stress that follow SCI. We showed that the acute DHA treatment is neuroprotective after compression SCI, even if the treatment is delayed up to an hour after injury. The DHA injection led to an increased neuronal and glial cell survival, and the effect of the DHA injection was amplified by addition of DHA to the diet. Rats treated with a DHA injection and a DHA-enriched diet performed significantly better at 6 weeks in terms of neurological outcome. The analysis of the tissue after DHA administration showed that the fatty acid significantly reduced lipid peroxidation, protein oxidation and RNA/DNA oxidation, and the induction of COX-2. Parallel studies in a facial nerve injury model in mice also showed pro-regenerative effects of chronic dietary administration of DHA after nerve lesion. These observations suggest that treatment with omega-3 PUFA could represent a promising therapeutic approach in the management of neurological injury.     

Metformin ameliorates BSCB disruption by inhibiting neutrophil infiltration and MMP‐9 expression but not direct TJ proteins expression regulation

Blood‐spinal cord barrier (BSCB) disruption is a major process for the secondary injury of spinal cord injury (SCI) and is considered to be a therapeutic target for SCI. Previously, we demonstrated that metformin could improve functional recovery after SCI; however, the effect of metformin on BSCB is still unknown. In this study, we found that metformin could prevent the loss of tight junction (TJ) proteins at day 3 after SCI in vivo, but in vitro there was no significant difference of these proteins between control and metformin treatment in endothelial cells. This indicated that metformin‐induced BSCB protection might not be mediated by up‐regulating TJ proteins directly, but by inhibiting TJ proteins degradation. Thus, we investigated the role of metformin on MMP‐9 and neutrophils infiltration. Neutrophils infiltration is the major source of the enhanced MMP‐9 in SCI. Our results showed that metformin decreased MMP‐9 production and blocked neutrophils infiltration at day 1 after injury, which might be related to ICAM‐1 down‐regulation. Also, our in vitro study showed that metformin inhibited TNF‐α‐induced MMP‐9 up‐regulation in neutrophils, which might be mediated via an AMPK‐dependent pathway. Together, it illustrated that metformin prevented the breakdown of BSCB by inhibiting neutrophils infiltration and MMP‐9 production, but not by up‐regulating TJ proteins expression. Our study may help to better understand the working mechanism of metformin on SCI.     

Combined nonlinear metrics to evaluate spontaneous EEG recordings from chronic spinal cord injury in a rat model: a pilot study

Spinal cord injury (SCI) is a high-cost disability and may cause permanent loss of movement and sensation below the injury location. The chance of cure in human after SCI is extremely limited. Instead, neural regeneration could have been seen in animals after SCI, and such regeneration could be retarded by blocking neural plasticity pathways, showing the importance of neural plasticity in functional recovery. As an indicator of nonlinear dynamics in the brain, sample entropy was used here in combination with detrended fluctuation analysis (DFA) and Kolmogorov complexity to quantify functional plasticity changes in spontaneous EEG recordings of rats before and after SCI. The results showed that the sample entropy values were decreased at the first day following injury then gradually increased during recovery. DFA and Kolmogorov complexity results were in consistent with sample entropy, showing the complexity of the EEG time series was lost after injury and partially regained in 1 week. The tendency to regain complexity is in line with the observation of behavioral rehabilitation. A critical time point was found during the recovery process after SCI. Our preliminary results suggested that the combined use of these nonlinear dynamical metrics could provide a quantitative and predictive way to assess the change of neural plasticity in a spinal cord injury rat model.     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.     

Photobiomodulation by diffusing optical fiber on spinal cord: A feasibility study in piglet model

Previous studies on spinal cord injury (SCI) have confirmed that percutaneous photobiomodulation (PBM) therapy can ameliorate immunoinflammatory responses at sites of injury, accelerate nerve regeneration, suppress glial scar formation and promote the subsequent recovery of locomotor function. The current study was performed to evaluate a large‐animal model employing implanted optical fibers to accurately irradiate targeted spinal segments. The method's feasibility and irradiation parameters that do not cause phototoxic reaction were determined, and the methodology of irradiating the spinal cord with near‐infrared light was investigated in detail. A diffusing optical fiber was implanted above the T9 spinal cord of Bama miniature pigs and used to transfer near‐infrared light (810 nm) onto the spinal cord surface. After daily irradiation with 200, 300, 500 or 1000 mW for 14 days, both sides of the irradiated area of the spinal cord were assessed for temperature changes. The condition of the spinal cord and the position of optical fiber were investigated by magnetic resonance imaging (MRI), and different parameters indicating temperature increases or phototoxicity were measured on the normal spinal cord surface due to light irradiation (ie, heat shock responses, inflammatory reactions and neuronal apoptosis), and the animals' lower‐limb neurological function and gait were assessed during the irradiation process. The implanted device was stable inside the freely moving animals, and light energy could be directly projected onto the spinal cord surface. The screening of different irradiation parameters preliminary showed that direct irradiation onto the spinal cord surface at 200 and 300 mW did not significantly increase the temperature, stress responses, inflammatory reactions and neural apoptosis, whereas irradiation at 500 mW slightly increased these parameters, and irradiation at 1000 mW induced a significant temperature increase, heat shock, inflammation and apoptosis responses. HE staining of spinal cord tissue sections did not reveal any significant structural changes of the tissues compared to the control group, and the neurological function and gait of all irradiated animals were normal. In this study, we established an in‐vivo optical fiber implantation method, which might be safe and stable and could be used to directly project light energy onto the spinal cord surface. This study might provide a new perspective for clinical applications of PBM in acute SCI.     

Mechanical indicators of injury severity are decreased with increased thecal sac dimension in a bench-top model of contusion type spinal cord injury

The cerebrospinal fluid (CSF) is thought to protect the spinal cord from physiologic loading; however, it is unclear whether this protective role extends to traumatic events in which bone fragments enter the canal at high velocity. A synthetic model of the spinal neural anatomy, with mechanical properties similar to native tissues, was constructed to determine if the thickness of the CSF layer (0, 12.8, 19.2 and 24.8 mm, 10 mm cord) and the velocity (1.2, 2.4, 3.7 and 4.8 m/s) of a 20 g impactor affect mechanical predictors of spinal cord injury (SCI) severity. Cord compression was directly proportional to impact velocity, inversely proportional to CSF dimension and zero for the largest dura size. The cord was compressed by more than 18% of its original diameter for the "no CSF" condition and the small dura size for all velocities. Impact loads were directly proportional to velocity, and inversely proportional to the thickness of the CSF layer. Peak cord tension increased with dura size and velocity. Peak CSF pressure decreased with distance from the impact epicenter for all dura sizes; attenuation was proportional to the velocity and greatest for the smallest dura. Increased CSF dimension led to reduced CSF pressure near the impact epicenter but had little effect at the remote sites. The results suggest that a thicker CSF layer may reduce the stress induced in the cord, and therefore metrics of SCI risk may be improved by incorporating thecal sac dimensions. Computational, synthetic, cadaveric and animal models may better simulate the biomechanics of human SCI if fluid interaction is incorporated.     

Effects of photobiomodulation combined with MSCs transplantation on the repair of spinal cord injury in rat

Stem cell transplantation has shown promising regenerative effects against neural injury, and photobiomodulation (PBM) can aid tissue recovery. This study aims to evaluate the therapeutic effect of human umbilical cord mesenchymal stem cells (hUCMSCs) and laser alone or combined on spinal cord injury (SCI). The animals were divided into SCI, hUCMSCs, laser treatment (LASER) and combination treatment (hUCMSCs + LASER) groups. Cell‐enriched grafts of hUCMSCs (1 × 10⁶ cells/ml) were injected at the site of antecedent trauma in SCI model rats. A 2 cm² damaged area was irradiated with 630 nm laser at 100 mW/cm² power for 20 min. Locomotion was evaluated using Basso–Beattie–Bresnahan (BBB) scores, and neurofilament repair were monitored by histological staining and diffusion tensor imaging (DTI). First, after SCI, the motor function of each group was restored with different degrees, the combination treatment significantly increased the BBB scores compared to either monotherapy. In addition, Nissl bodies were more numerous, and the nerve fibers were longer and thicker in the combination treatment group. Consistent with this, the in situ expression of NF‐200 and glial fibrillary acidic protein in the damaged area was the highest in the combination treatment group. Finally, DTI showed that the combination therapy optimally improved neurofilament structure and arrangement. These results may show that the combination of PBM and hUCMSCs transplantation is a feasible strategy for reducing secondary damage and promoting functional recovery following SCI.     

Mesenchymal stem cell graft improves recovery after spinal cord injury in adult rats through neurotrophic and pro-angiogenic actions

Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue.     

Involvement of acidic fibroblast growth factor in spinal cord injury repair processes revealed by a proteomics approach

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.     

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/ref-1) and DNA damage in the caudal region of acute and chronic spinal cord injured rats treated by embryonic neural stem cells

The oxidative mechanisms of injury-induced damage of neurons within the spinal cord are not very well understood. We used a model of T8-T9 spinal cord injury (SCI) in the rat to induce neuronal degeneration. In this spinal cord injury model, unilateral avulsion of the spinal cord causes oxidative stress of neurons. We tested the hypothesis that apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1) regulates this neuronal oxidation mechanism in the spinal cord region caudal to the lesion, and that DNA damage is an early upstream signal. The embryonic neural stem cell therapy significantly decreased DNA-damage levels in both study groups - acutely (followed up to 7 days after SCI), and chronically (followed up to 28 days after SCI) injured animals. Meanwhile, mRNA levels of APE/Ref-1 significantly increased after embryonic neural stem cell therapy in acutely and chronically injured animals when compared to acute and chronic sham groups. Our data has demonstrated that an increase of APE/Ref-1 mRNA levels in the caudal region of spinal cord strongly correlated with DNA damage after traumatic spinal cord injury. We suggest that DNA damage can be observed both in lesional and caudal regions of the acutely and chronically injured groups, but DNA damage is reduced with embryonic neural stem cell therapy.     

Sustained Calpain Inhibition Improves Locomotor Function and Tissue Sparing Following Contusive Spinal Cord Injury

Following contusive spinal cord injury (SCI), calpain activity is dramatically increased and remains elevated for days to weeks. Although calpain inhibition has previously been demonstrated to be neuroprotective following spinal cord injury, most studies administered the calpain inhibitor at a single time point. We hypothesized that sustained calpain inhibition would improve functional and pathological outcomes, as compared to the results obtained with a single postinjury administration of the calpain inhibitor. Contusion SCI was produced in female Long-Evans rats using the Infinite Horizon spinal cord injury impactor at the 200 kdyn force setting. Open-field locomotor function was evaluated until 6 weeks postinjury. Histological assessment of lesion volume and tissue sparing was performed at 6 weeks after SCI. Calpain inhibitor MDL28170 administered as a single postinjury i.v. bolus (20 mg/kg) or as a daily i.p. dose (1 mg/kg) improved locomotor function, but did not increase tissue sparing. Combined i.v. and daily i.p. MDL28170 administration resulted in significant improvement in both functional and pathological outcome measures, supporting the calpain theory of SCI proposed by Dr. Banik and colleagues. Special issue in honor of Naren Banik.