Cord Hemisection In Rats Research Articles

3D Gelatin Microsphere Scaffolds Promote Functional Recovery after Spinal Cord Hemisection in Rats.

Spinal cord injury (SCI) damages signal connections and conductions, with the result that neuronal circuits are disrupted leading to neural dysfunctions. Such injuries represent a serious and relatively common central nervous system condition and current treatments have limited success in the reconstruction of nerve connections in injured areas, especially where sizeable gaps are present. Biomaterial scaffolds have become an effective alternative to nerve transplantation in filling these gaps and provide the foundation for simulating the 3D structure of solid organs. However, there remain some limitations with the application of 3D bioprinting for preparation of biomaterial scaffolds. Here, the approach in constructing and testing mini-tissue building blocks and self-assembly, solid 3D gelatin microsphere (GM) scaffolds with multiple voids as based on the convenient preparation of gelatin microspheres by microfluidic devices is described. These 3D GM scaffolds demonstrate suitable biocompatibility, biodegradation, porosity, low preparation costs, and relative ease of production. Moreover, 3D GM scaffolds can effectively bridge injury gaps, establish nerve connections and signal transductions, mitigate inflammatory microenvironments, and reduce glial scar formation. Accordingly, these 3D GM scaffolds can serve as a novel and effective bridging method to promote nerve regeneration and reconstruction and thus recovery of nerve function after SCI.

Analysis of Movements in Spinal Cord Hemisection Treatment with Amniotic Membrane – Preclinical Study

Aims: This study aimed to evaluate the efficacy of Amniotic Membrane application in rats with Spinal Cord Injury induced by transverse hemisection using kinematic analysis and Sciatic Functional Index.
 Study design: True experimental research design.
 Place and Duration of Study: Instiuto de Pesquisa e Desenvolvimento (IP&D) of Universidade do Vale do Paraíba (UNIVAP), between September 2016 and December 2017.
 Methodology: Fifteen adult male rats were used, allocated into three equal groups: Control (the spinal cord injury and Amniotic Membrane application were simulated), Lesion (spinal cord injury not treated), Amniotic membrane (spinal cord injury treated by amniotic membrane). All animals underwent surgical procedures. A transverse hemisection was performed in groups Lesion and Amniotic Membrene. A fragment of the biomaterial was applied in group AM covering the hemisection area.
 Results: Sciatic Functional Index and motion analysis were performed by comparing images taken at pre- and postoperative time at 7, 14, 21, and 28 days. The kinematic analysis showed a significant difference between groups Control and Lesion at 7 days (p = 0.023) and 14 days (p = 0.015), and between groups Lesion and Amniotic Membrane at 14 days (p = 0.039), comparing the postoperative periods. The Sciatic Functional Index revealed significant differences between Groups Control and Lesion at 7 (p = -0.002), 14 (p = 0.003), and 21 days (p = 0.009), between Groups Control and Amniotic Membrane at 7 (p = 0.014), 14 (p = 0.007), and 28 days (p = 0; 013), and between Groups Lesion and Amniotic Membrane only at 14 days (p = 0.039).
 Conclusion: Application of amniotic membrane in spinal cord hemisection in rats induced gait recovery and improvement in SFI compared to the untreated group.

Effects of Chronic High-Frequency rTMS Protocol on Respiratory Neuroplasticity Following C2 Spinal Cord Hemisection in Rats.

Simple SummaryHigh spinal cord injuries (SCIs) are known to lead to permanent diaphragmatic paralysis, and to induce deleterious post-traumatic inflammatory processes following cervical spinal cord injury. We used a noninvasive therapeutic tool (repetitive transcranial magnetic stimulation (rTMS)), to harness plasticity in spared descending respiratory circuit and reduce the inflammatory processes. Briefly, the results obtained in this present study suggest that chronic high-frequency rTMS can ameliorate respiratory dysfunction and elicit neuronal plasticity with a reduction in deleterious post-traumatic inflammatory processes in the cervical spinal cord post-SCI. Thus, this therapeutic tool could be adopted and/or combined with other therapeutic interventions in order to further enhance beneficial outcomes.High spinal cord injuries (SCIs) lead to permanent diaphragmatic paralysis. The search for therapeutics to induce functional motor recovery is essential. One promising noninvasive therapeutic tool that could harness plasticity in a spared descending respiratory circuit is repetitive transcranial magnetic stimulation (rTMS). Here, we tested the effect of chronic high-frequency (10 Hz) rTMS above the cortical areas in C2 hemisected rats when applied for 7 days, 1 month, or 2 months. An increase in intact hemidiaphragm electromyogram (EMG) activity and excitability (diaphragm motor evoked potentials) was observed after 1 month of rTMS application. Interestingly, despite no real functional effects of rTMS treatment on the injured hemidiaphragm activity during eupnea, 2 months of rTMS treatment strengthened the existing crossed phrenic pathways, allowing the injured hemidiaphragm to increase its activity during the respiratory challenge (i.e., asphyxia). This effect could be explained by a strengthening of respiratory descending fibers in the ventrolateral funiculi (an increase in GAP-43 positive fibers), sustained by a reduction in inflammation in the C1–C3 spinal cord (reduction in CD68 and Iba1 labeling), and acceleration of intracellular plasticity processes in phrenic motoneurons after chronic rTMS treatment. These results suggest that chronic high-frequency rTMS can ameliorate respiratory dysfunction and elicit neuronal plasticity with a reduction in deleterious post-traumatic inflammatory processes in the cervical spinal cord post-SCI. Thus, this therapeutic tool could be adopted and/or combined with other therapeutic interventions in order to further enhance beneficial outcomes.

Longitudinal Analysis of Gut Microbiome Composition After Experimental Cervical Spinal Cord Injury

Following an experimental C2 spinal cord hemisection in rats, there is a gradual spontaneous recovery of breathing function that can take place over time. Additionally, interventions at later time points are more effective after injury. What is not known is the mechanism mediating this observation. To begin to answer this question, we investigated the role of the gut microbiome after injury. Recent studies have emerged suggesting that the gut microbiome has critical implications on the proper functioning of the central nervous system (CNS). Indeed, gut dysbiosis, or a microbiome imbalance, can occur which can negatively impact the CNS. Neurotrauma, including spinal cord injury (SCI), can lead to acute gut dysbiosis and impaired recovery. It is our hypothesis that the composition of the gut microbiome is dynamic after injury with dysbiosis improving over time from an acute post‐SCI state. In this study, we build upon these initial studies and investigate the impact of cervical SCI on the gut microbiome over time and up to chronic timepoints. In order to test our hypothesis, we collected fecal samples before and up to 12 weeks after a C2 hemisection in adult female rats, in order to assess microbiome composition at various timepoints post injury. Preliminary results suggest that following cervical SCI, gut dysbiosis occurs immediately after injury but recovers by two months post injury. Future studies will classify bacterial identities and assess the impact of the post‐injury gut microbiome on recovery of respiratory motor function and plasticity.

The Landscape of Gene Expression and Molecular Regulation Following Spinal Cord Hemisection in Rats

Spinal cord injury (SCI) is a challenging clinical problem worldwide. The cellular state and molecular expression in spinal cord tissue after injury are extremely complex and closely related to functional recovery. However, the spatial and temporal changes of gene expression and regulation in various cell types after SCI are still unclear. Here, we collected the rostral and caudal regions to the lesion at 11 time points over a period of 28 days after rat hemisection SCI. Combining whole-transcriptome sequencing and bioinformatic analysis, we identified differentially expressed genes (DEGs) between spinal cord tissue from injured and sham-operated animals. Significantly altered biological processes were enriched from DEGs in astrocytes, microglia, oligodendrocytes, immune cells, and vascular systems after SCI. We then identified dynamic trends in these processes using the average expression profiles of DEGs. Gene expression and regulatory networks for selected biological processes were also constructed to illustrate the complicate difference between rostral and caudal tissues. Finally, we validated the expressions of some key genes from these networks, including α-synuclein, heme oxygenase 1, bone morphogenetic protein 2, activating transcription factor 3, and leukemia inhibitory factor. Collectively, we provided a comprehensive network of gene expression and regulation to shed light on the molecular characteristics of critical biological processes that occur after SCI, which will broaden the understanding of SCI and facilitate clinical therapeutics for SCI.

Evaluation of Respiratory and Cardiovascular Parameters by Ultrasound Echography Following C2 Spinal Cord Hemisection in Rat

High cervical spinal cord injuries induce several permanent deficits of vital functions, often leading to the death of the patient. The injury immediately disrupts the bulbospinal pathways innervating phrenic motoneurons that connect to the diaphragm and also causes damages to the sympathetic innervation of the vascular system. The subsequent functional deficits are respiratory failure and cardiovascular dysfunction. In this study, we use a preclinical model of surgical cervical C2 partial section in the rat that mimic these respiratory and cardiovascular impairments. The physiopathology of the injury is assessed by the adaptation of a non‐invasive medical ultrasound echography device (Logic 9, GE, France) to our model.Seven days post‐injury, we observed a live passive displacement of the diaphragm by ultrasound on the injured side in anesthetized animals, corresponding to an absence of hemidiaphragm contraction (−0,37 ± 0,17 mm) compared to contralateral side (3,27 ± 0,44 mm; p<0,01) and to control group (3,79 ± 0,27 mm; p<0,01). These results have been confirmed by in vivo diaphragmatic electromyography, with low electromyographic activity recorded on the ipsilateral side (0,06 ± 0,03 μV.s.s) compared to the contralateral side (0,53 ± 0,08 μV.s.s; p<0,01) and to control group (0,33 ± 0,08 μV.s.s; p = 0,013). We also observed a reduction of the thickness of the rectus abdominis muscle (accessory expiratory muscle) on both sides (injured and non‐injured) at 7 days post‐injury (non‐injured side = 1,78 ± 0,10 mm; injured side = 1,80 ± 0,10 mm) compared to control (2,14 ± 0,08 mm; p = 0,009 and p = 0,013 respectively), suggesting a role for this muscle in active exhalation following the injury.The doppler echocardiography for the cardiovascular evaluation showed a statistical reduction of the left ventricular ejection fraction (LVEF = 59 ± 3%) compared to control (LVEF = 70 ± 2%; p = 0,01), which corresponds to a systolic dysfunction. However, no diastolic dysfunction has been observed. The mean arterial pressure is getting close to non‐injured levels at 7 days post‐injury (68 ± 7 mmHg) compared to control group (84 ± 10 mmHg). These results suggest that the reduction of the sympathetic innervation caused by the spinal cord injury conduct to a cardiovascular dysfunction partly corrected at 7 days post‐injury.Altogether, we demonstrate that medical ultrasound machine could be a reliable non‐invasive method to evaluate chronic respiratory and cardiovascular dysfunction following high cervical spinal cord injury and for studying chronically the evolution of the eventual spontaneous restoration that can occur in this model.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

O2-6-08. Mechanism of forelimb motor function restoration after cervical spinal cord hemisection in rats: Electorophysiological verification

The objective of this study was to electrophysiologically assess corticospinal tracts of adult rats and recovery of motor function of their forelimbs after cervical cord hemisection. Of 39 adult rats used, compound muscle action potentials (CMAPs) of forelimbs of 15 rats were evaluated, before they received left C5 segmental hemisection of the spinal cord, by stimulating the pyramid of medulla oblongata on one side using an exciting microelectrode. All 15 rats exhibited contralateral electrical activity, but their CMAPs disappeared after hemisection. The remaining 24 rats received hemisection first, and CMAPs of 12 rats were assessed over time to study their recovery time. All of them exhibited electrical activity of forelimbs in four weeks after surgery. The remaining 12 rats received additional right C2 segmental hemisection, and variation of CMAPs between before and after surgery was examined. The right side of the 12 rats that received the additional hemisection exhibited no electrical activity in response to the stimulation of pyramids on both sides. These results suggest that changes in path between the resected and healthy sides, activation of ventral corticospinal tracts, and propriospinal neurons were involved in the recovery of motor function after cervical cord injury.

Thermogelling chitosan lactate hydrogel improves functional recovery after a C2 spinal cord hemisection in rat.

The present study was designed to provide an appropriate micro-environment for regenerating axotomized neurons and proliferating/migrating cells. Because of its intrinsic permissive properties, biocompatibility and biodegradability, we chose to evaluate the therapeutic effectiveness of a chitosan-based biopolymer. The biomaterial toxicity was measured through in vitro test based on fibroblast cell survival on thermogelling chitosan lactate hydrogel substrate and then polymer was implanted into a C2 hemisection of the rat spinal cord. Animals were randomized into three experimental groups (Control, Lesion and Lesion + Hydrogel) and functional tests (ladder walking and forelimb grip strength tests, respiratory assessment by whole-body plethysmography measurements) were used, once a week during 10 weeks, to evaluate post-traumatic recoveries. Then, electrophysiological examinations (reflexivity of the sub-lesional region, ventilatory adjustments to muscle fatigue known to elicit the muscle metaboreflex and phrenic nerve recordings during normoxia and temporary hypoxia) were performed. In vitro results indicated that the chitosan matrix is a non-toxic biomaterial that allowed fibroblast survival. Furthermore, implanted animals showed improvements of their ladder walking scores from the 4th week post-implantation. Finally, electrophysiological recordings indicated that animals receiving the chitosan matrix exhibited recovery of the H-reflex rate sensitive depression, the ventilatory response to repetitive muscle stimulation and an increase of the phrenic nerve activity to asphyxia compared to lesioned and nonimplanted animals. This study indicates that hydrogel based on chitosan constitute a promising therapeutic approach to repair damaged spinal cord or may be used as an adjuvant with other treatments to enhance functional recovery after a central nervous system damage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2004-2019, 2017.

Anatomical mechanism of spontaneous recovery in regions caudal to thoracic spinal cord injury lesions in rats

BackgroundThe nerve fibre circuits around a lesion play a major role in the spontaneous recovery process after spinal cord hemisection in rats. The aim of the present study was to answer the following question: in the re-control process, do all spinal cord nerves below the lesion site participate, or do the spinal cord nerves of only one vertebral segment have a role in repair?MethodsFirst we made a T7 spinal cord hemisection in 50 rats. Eight weeks later, they were divided into three groups based on distinct second operations at T7: ipsilateral hemisection operation, contralateral hemisection, or transection. We then tested recovery of hindlimbs for another eight weeks. The first step was to confirm the lesion had role or not in the spontaneous recovery process. Secondly, we performed T7 spinal cord hemisections in 125 rats. Eight weeks later, we performed a second single hemisection on the ipsilateral side at T8–T12 and then tested hindlimb recovery for another six weeks.ResultsIn the first part, the Basso, Beattie, Bresnahan (BBB) scores and the electrophysiology tests of both hindlimbs weren’t significantly different after the second hemisection of the ipsilateral side. In the second part, the closer the second hemisection was to T12, the more substantial the resulting impairment in BBB score tests and prolonged latency periods.ConclusionsThe nerve regeneration from the lesion area after hemisection has no effect on spontaneous recovery of the spinal cord. Repair is carried out by all vertebrae caudal and ipsilateral to the lesion, with T12 being most important.

Mechanism of Restoration of Forelimb Motor Function after Cervical Spinal Cord Hemisection in Rats: Electrophysiological Verification.

The objective of this study was to electrophysiologically assess the corticospinal tracts of adult rats and the recovery of motor function of their forelimbs after cervical cord hemisection. Of 39 adult rats used, compound muscle action potentials (CMAPs) of the forelimbs of 15 rats were evaluated, before they received left C5 segmental hemisection of the spinal cord, by stimulating the pyramid of the medulla oblongata on one side using an exciting microelectrode. All 15 rats exhibited contralateral electrical activity, but their CMAPs disappeared after hemisection. The remaining 24 rats received hemisection first, and CMAPs of 12 rats were assessed over time to study their recovery time. All of them exhibited electrical activity of the forelimbs in 4 weeks after surgery. The remaining 12 rats received additional right C2 segmental hemisection, and variation of CMAPs between before and after surgery was examined. The right side of the 12 rats that received the additional hemisection exhibited no electrical activity in response to the stimulation of the pyramids on both sides. These results suggest that changes in path between the resected and healthy sides, activation of the ventral corticospinal tracts, and propriospinal neurons were involved in the recovery of motor function after cervical cord injury.

Mechanism of Forelimb Motor Function Restoration after Cervical Spinal Cord Hemisection in Rats: A Comparison of Juveniles and Adults

The aim of this study was to investigate forelimb motor function after cervical spinal cord injury in juvenile and adult rats. Both rats received a left segmental hemisection of the spinal cord after C3-C4 laminectomy. Behavioral evaluation of motor function was monitored and assessed using the New Rating Scale (NRS) and Forelimb Locomotor Scale (FLS) and by measuring the range of motion (ROM) of both the elbow and wrist. Complete left forelimb motor paralysis was observed in both rats. The NRS showed motor function recovery restored to 50.2 ± 24.7% in juvenile rats and 34.0 ± 19.8% in adult rats. FLS was 60.4 ± 26.8% in juvenile rats and 46.5 ± 26.9% in adult rats. ROM of the elbow and wrist were 88.9 ± 20.6% and 44.4 ± 24.1% in juvenile rats and 70.0 ± 29.2% and 40.0 ± 21.1% in adult rats. Thus, the NRS and ROM of the elbow showed a significant difference between age groups. These results indicate that left hemisection of the cervical spinal cord was not related to right-sided motor functions. Moreover, while motor paralysis of the left forelimb gradually recovered in both groups, the improvement was greater in juvenile rats.

Ipsilateral inspiratory intercostal muscle activity after C2 spinal cord hemisection in rats

BackgroundUpper cervical spinal cord hemisection causes paralysis of the ipsilateral hemidiaphragm; however, the effect of C2 hemisection on the function of the intercostal muscles is not clear. We hypothesized that C2 hemisection would eliminate inspiratory intercostal activity ipsilateral to the injury and that some activity would return in a time-dependent manner.MethodsFemale Sprague Dawley rats were anesthetized with urethane and inspiratory intercostal electromyogram (EMG) activity was recorded in control rats, acutely injured C2 hemisected rats, and at 1 and 16 weeks post C2 hemisection.ResultsBilateral recordings of intercostal EMG activity showed that inspiratory activity was reduced immediately after injury and increased over time. EMG activity was observed first in rostral spaces followed by recovery occurring in caudal spaces. Theophylline increased respiratory drive and increased intercostal activity, inducing activity that was previously absent.ConclusionThese results suggest that there are crossed, initially latent, respiratory connections to neurons innervating the intercostal muscles similar to those innervating phrenic motor neurons.

Functional reorganization of the forepaw cortical representation immediately after thoracic spinal cord hemisection in rats

Spinal cord injury may produce long-term reorganization of cortical circuits. Little is known, however, about the early neurophysiological changes occurring immediately after injury. On the one hand, complete thoracic spinal cord transection of the spinal cord immediately decreases the level of cortical spontaneous activity and increases the cortical responses to stimuli delivered to the forepaw, above the level of the lesion. On the other hand, a thoracic spinal cord hemisection produces an immediate cortical hyperexcitability in response to preserved spinothalamic inputs from stimuli delivered to the hindpaw, below the level of the lesion. Here we show that a thoracic spinal cord hemisection also produces a bilateral increase of the responses evoked in the forepaw cortex by forepaw stimuli, associated with a bilateral decrease of cortical spontaneous activity. Importantly, the increased cortical forepaw responses are immediate in the cortex contralateral to the hemisection (significant within 30min after injury), but they are progressive in the cortex ipsilateral to the hemisection (reaching significance only 2.5h after injury). Conversely, the decreased cortical spontaneous activity is progressive both ipsilaterally and contralaterally to the hemisection (again reaching significance only 2.5h after injury). In synthesis, the present work reports a functional reorganization of the forepaw cortical representation immediately after thoracic spinal cord hemisection, which is likely important to fully understand the mechanisms underlying long-term cortical reorganization after incomplete spinal cord injuries.

The Use of Poly(N-[2-Hydroxypropyl]-Methacrylamide) Hydrogel to Repair a T10 Spinal Cord Hemisection in Rat: A Behavioural, Electrophysiological and Anatomical Examination

There have been considerable interests in attempting to reverse the deficit because of an SCI (spinal cord injury) by restoring neural pathways through the lesion and by rebuilding the tissue network. In order to provide an appropriate micro-environment for regrowing axotomized neurons and proliferating and migrating cells, we have implanted a small block of pHPMA [poly N-(2-hydroxypropyl)-methacrylamide] hydrogel into the hemisected T10 rat spinal cord. Locomotor activity was evaluated once a week during 14 weeks with the BBB rating scale in an open field. At the 14th week after SCI, the reflexivity of the sub-lesional region was measured. We also monitored the ventilatory frequency during an electrically induced muscle fatigue known to elicit the muscle metaboreflex and increase the respiratory rate. Spinal cords were then collected, fixed and stained with anti-ED-1 and anti-NF-H antibodies and FluoroMyelin. We show in this study that hydrogel-implanted animals exhibit: (i) an improved locomotor BBB score, (ii) an improved breathing adjustment to electrically evoked isometric contractions and (iii) an H-reflex recovery close to control animals. Qualitative histological results put in evidence higher accumulation of ED-1 positive cells (macrophages/monocytes) at the lesion border, a large number of NF-H positive axons penetrating the applied matrix, and myelin preservation both rostrally and caudally to the lesion. Our data confirm that pHPMA hydrogel is a potent biomaterial that can be used for improving neuromuscular adaptive mechanisms and H-reflex responses after SCI.

Effects of Human Mesenchymal Stem Cell Transplantation Combined with Polymer on Functional Recovery Following Spinal Cord Hemisection in Rats

The spontaneous axon regeneration of damaged neurons is limited after spinal cord injury (SCI). Recently, mesenchymal stem cell (MSC) transplantation was proposed as a potential approach for enhancing nerve regeneration that avoids the ethical issues associated with embryonic stem cell transplantation. As SCI is a complex pathological entity, the treatment of SCI requires a multipronged approach. The purpose of the present study was to investigate the functional recovery and therapeutic potential of human MSCs (hMSCs) and polymer in a spinal cord hemisection injury model. Rats were subjected to hemisection injuries and then divided into three groups. Two groups of rats underwent partial thoracic hemisection injury followed by implantation of either polymer only or polymer with hMSCs. Another hemisection-only group was used as a control. Behavioral, electrophysiological and immunohistochemical studies were performed on all rats. The functional recovery was significantly improved in the polymer with hMSC-transplanted group as compared with control at five weeks after transplantation. The results of electrophysiologic study demonstrated that the latency of somatosensory-evoked potentials (SSEPs) in the polymer with hMSC-transplanted group was significantly shorter than in the hemisection-only control group. In the results of immunohistochemical study, β-gal-positive cells were observed in the injured and adjacent sites after hMSC transplantation. Surviving hMSCs differentiated into various cell types such as neurons, astrocytes and oligodendrocytes. These data suggest that hMSC transplantation with polymer may play an important role in functional recovery and axonal regeneration after SCI, and may be a potential therapeutic strategy for SCI.

Role of spinal cholecystokinin in neuropathic pain after spinal cord hemisection in rats

In the present study we determined whether spinal cholecystokinin (CCK) or the cholecystokinin receptor is involved in below-level neuropathic pain of spinal cord injury (SCI). The effect of the CCKB receptor antagonist, CI-988 on mechanical allodynia and the expression level of CCK and CCKB receptor were investigated. Spinal hemisection was done at the T13 level in rats under enflurane anesthesia. CI-988 was administered intraperitoneally and intrathecally and behavioral tests were conducted. After systemic injection, mechanical allodynia was reduced by higher doses of CI-988 (10 and 20mg/kg). Intrathecal CI-988 (100, 200 and 500μg) dose-dependently increased the paw withdrawal threshold in both paws. Following spinal hemisection, CCK mRNA expression increased on the ipsilateral side at the spinal segments caudal to the injury and both sides of the spinal L4-5 segments without any significant changes in CCKB receptor mRNA levels. These results suggest that up-regulation of spinal CCK may contribute to maintenance of mechanical allodynia following SCI and that clinical application of CI-988 or similar drugs may be useful therapeutic agents for management of central neuropathic pain.

Neuroimmunophilin Ligands Improve Functional Recovery and Increase Axonal Growth after Spinal Cord Hemisection in Rats

We have previously shown that FK506 accelerates the rate of nerve regeneration in the peripheral nervous system (PNS) and increases regeneration of central nervous system (CNS) axons into a peripheral nerve graft. In the present study, we examined whether FK506 and a nonimmunosuppressive derivative (FK1706) improve functional recovery and long distance regeneration following a hemisection lesion of spinal cord at T10/T11. Rats were given daily subcutaneous injections of either FK506 (2 mg/kg/day), FK1706 (2 mg/kg/day), an equivalent volume of saline or 30% DMSO as vehicle, respectively. Functional recovery was assessed using a modified Tarlov/Klinger scale, walking along progressively narrower wooden beams (7.7-1.7 cm widths), and analysis of footprints obtained during walking. Compared to both control groups, FK506 and FK1706-treated animals demonstrated significant functional recovery 4 days (beam walking), 2 weeks (footprints), and 4 weeks (Tarlov/Klinger scale). By 11 weeks, FK506-treated and FK1706-treated animals were able to walk, albeit poorly, along even the narrowest (1.7 cm) beam. At 11 weeks, the spinal cords were re-exposed and a small piece of gel foam-soaked Fluoro-Gold was placed on the injured side 2-cm caudal to the first injury. Five days later, the animals were perfused and tissues prepared for fluorescence microscopy. FK506-treated and FK1706-treated rats demonstrate a significantly greater number of retrogradely labeled neurons in the red nucleus. The results implicate a nonimmunosuppressant mechanism in FK506's action and suggest that FK506 or a nonimmunosuppressant derivative may be useful for treatment of spinal cord injuries.

Calpain Inhibitor Inhibits p35-p25-Cdk5 Activation, Decreases Tau Hyperphosphorylation, and Improves Neurological Function after Spinal Cord Hemisection in Rats

Aberrant calpain activation is a key mediator of neuron death. We examined the cell-permeable calpain inhibitor MDL28170 in the pathophysiological processes after spinal cord injury (SCI) including p35-p25- cyclin-dependent kinase-5 (Cdk5) activation, tau hyperphosphorylation, neuron cell death, calpain I activation, astrogliosis, and microglia activation. Our study showed that intrathecal administration of MDL28170 improved neurologic dysfunction, prevented neuron loss, decreased the number of apoptotic cells, and abated astrogliosis and microglia activation 7 days after spinal cord hemisection in rats. Reverse transcription polymerase chain reaction demonstrated calpain inhibition significantly attenuated the ratio of proapoptotic Bax/anti-apoptotic Bcl-2 mRNA in the lesion and penumbra after SCI. Calpain, the calcium-activated proteolytic enzyme, was found to digest p35 to its truncated product, p25. Moreover, abnormal Cdk5 activation by p25 and subsequent tau hyperphosphorylation triggers pathologic events leading to neurodegeneration and neurofibrillary tangles. We found p35-p25-Cdk5 activation and tau hyperphosphorylation in SCI, and then we showed that intrathecal MDL28170 treatment could diminish p35 truncation, and abrogate aberrant tau phosphorylation. Double labeling of calpain I and phosphorylated tau (AT8) in the same cells of spinal cord lesion further implicated pathogenesis of SCI. In conclusion, MDL28170 abated calpain I activation, inhibited apoptosis and neuron loss, quenched microglia and astrocyte activation, and significantly improved neurologic deficit one week after spinal cord hemisection. The neuroprotective mechanisms of calpain inhibitor in SCI could be attenuating upregulation of Bax/Bcl-2 ratio, preventing p35 truncation in the lesion and penumbra, and abrogating tau hyperphosphorylation.

Intraspinal transplantation of CD34+ human umbilical cord blood cells after spinal cord hemisection injury improves functional recovery in adult rats.

The present study was designed to compare the functional outcome of the intraspinal transplantation of CD34+ human umbilical cord blood (CB) cells with that of human bone marrow stromal (BMS) cells in adult rats with spinal cord injury. Sixty adult Wistar rats were subjected to left spinal cord hemisection, and then divided into three groups randomly. The control group received an injection of PBS without cells, while the two other groups of rats received a transplantation of 5 x 10(5) CD34+ CB or BMS cells, respectively. Functional outcome was measured using the modified Tarlov score at days 1, 7, 14, 21, and 28 after transplantation. A statistically significant improvement in functional outcome and survival rate in the experimental groups of rats was observed compared with the control group. Rats that received CD34+ CB cells achieved a better improvement in functional score than those that received BMS cells at days 7 and 14 after transplantation. Histological evaluation revealed that bromodeoxyuridine (BrdU)-labeled CD34+ CB and BMS cells survived and migrated into the injured area. Some of these cells expressed glial fibriliary acidic protein (GFAP) or neuronal nuclear antigen (NeuN). Our data demonstrate for the first time that intraspinal transplantation of human CD34+ CB cells provides benefit in function recovery after spinal cord hemisection in rats and suggest that CD34+ CB cells may be an excellent choice of cells as routine starting material of allogenic and autologous transplantations for the treatment of spinal cord injury.

Efficient reinnervation of hindlimb muscles by thoracic motor neurons after nerve cross-anastomosis in rats.

Peripheral motor axons can regenerate through motor endoneurial tubes of foreign nerves to reinnervate different target muscles. This regenerative capacity has been brought to clinical applications for restorative surgery after nerve or root injury. In this study the authors explore the extent to which nerve cross-anastomosis between lower intercostal nerves and lumbar ventral roots would be effective in inducing reinnervation of paralyzed hindlimb muscles after spinal cord hemisection at the thoracolumbar boundary in rats. The proximal extremities of sectioned intercostal nerves T10-12 were surgically connected to the distal extremities of sectioned ipsilateral lumbar ventral roots L3-5, respectively. Motor activity reappeared 2 months postsurgery; however, locomotion was not restored and inappropriate motor patterns persisted at 9 months postsurgery. At that time, data from electrophysiological and histological studies and horseradish peroxidase retrograde labeling demonstrated efficient regrowth of thoracic motor neuron axons that reached hindlimb muscles. They also revealed a persistent maturation defect of regrown fibers, as shown by size heterogeneity and presumable extensive axonal branching. These features are consistent with reduced neural activity subsequent to continuing inappropriate motor patterns. These results indicate that cross-anastomosis of intercostal nerves with lumbar ventral roots allows efficient reinnervation of paralyzed hindlimb muscles after spinal cord hemisection in rats. Stimulating the reorganization of the neuronal circuitry in the central nervous system by locomotion training or other methods would presumably result in both functional and anatomical improvements. This experimental setting provides a convenient animal model to investigate these processes.