Spinal Cord Hemisection In Rats Research Articles

METTL3 Affects Spinal Cord Neuronal Apoptosis by Regulating Bcl-2 m6A Modifications After Spinal Cord Injury.

Spinal cord injury (SCI) is a severe type of neurological trauma. N6-methyladenosine (m6A) modification is one of the most common internal modifications of RNA. The role of METTL3, the predominant methylation enzyme of m6A modification, in SCI remains unclear. This study aimed to investigate the role of methyltransferase METTL3 in SCI. After establishing the oxygen-glucose deprivation (OGD) model of PC12 cells and rat spinal cord hemisection model, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. The m6A modification was identified on B-cell lymphoma 2 (Bcl-2) messenger RNA (mRNA) by bioinformatics analysis, and m6A-RNA immunoprecipitation and RNA immunoprecipitation. In addition, METTL3 was blocked by the specific inhibitor STM2457 and gene knockdown, and then apoptosis levels were measured. In different models, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. After inducing OGD, inhibition of METTL3 activity or expression increased the mRNA and protein levels of Bcl-2, inhibited neuronal apoptosis, and improved neuronal viability in the spinal cord. Inhibition of METTL3 activity or expression can inhibit the apoptosis of spinal cord neurons after SCI through the m6A/Bcl-2 signaling pathway.

Fidgetin impacts axonal growth and branching in a local mTOR signal dependent manner.

Neurons require a constant increase in protein synthesis during axonal growth and regeneration. AKT-mTOR is a central pathway for mammalian cell survival and regeneration. Fidgetin (Fign) is an ATP-dependent microtubule (MT)-severing enzyme whose functions are associated with neurite outgrowth, axon regeneration and cell migration. Although most previous studies have indicated that depletion of Fign is involved in those biological activities by increasing labile MT mass, it remains unknown whether mTOR activation contributes to this process. Here, we showed that depletion of Fign enhanced p-mTOR/p-S6K activation, and the mTOR inhibitor Rapamycin inhibited axon outgrowth and p-rpS6 activation. We then investigated the effects of neuronal-specific Fign deletion in a rat spinal cord hemisection model by injecting syn-GFP Fign shRNA virus. BBB values revealed an improvement in functional recovery. The p-mTOR was activated along with neuronal Fign depletion. The syn-mCherry virus showed more sprouting neurites entering the injury region, which was confirmed by immunostaining GAP43 protein. Further, we showed that Fign siRNA treatment promoted axon outgrowth and branching, whose underlying mechanism was firstly attributed to local activation of the mTOR pathway, and increased MT dynamicity. Finally, considering L-leucine, promotes axonal growth and neuronal survival, we applied L-leucine with Fign depletion after spinal cord injury or in chondroitin sulfate proteoglycan inhibitory molecules. The phenomenon of synergistically augmented axon regeneration was observed. In summary, our results indicated a novel local mTOR pathway for fidgetin to impact axon growth and provided a combined strategy in SCI.

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.

Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro.

Apelin is a 77-amino-acid peptide that is an endogenous ligand for the G protein-coupled receptor APJ (Apelin receptor, APJ). Apelin-13, as the most bioactive affinity fragment of apelin, plays a role in energy metabolism, myocardial ischemia‒reperfusion injury, and the regulation of the inflammatory response during oxidative stress, but its role in spinal cord injury is still unclear. This research identified and verified the differential expression of apelin in rat spinal cord injured tissues and normal spinal cord tissues by transcriptome sequencing in vivo and proved that apelin-13 protects neurons by strengthening autophagy and attenuating early-stage postspinal cord injury apoptosis in vitro. After constructing the model concerning a rat spinal cord hemisection damage, transcriptome sequencing was performed on the injured and normal spinal cord tissues of rats, which identified the differentially expressed gene apelin, with qRT-PCR detecting the representative level of apelin. The oxygen-glucose deprivation (OGD) model of PC12 cells was constructed in vitro to simulate spinal cord injury. The OGD injury times were 2 h, 4 h, 6 h, 8 h, and 12 h, and the non-OGD injury group was used as the control. The expression of apelin at each time point was observed by Western blotting. The expression of apelin was the lowest in the 6 h OGD injury group (p < 0.05). Therefore, the OGD injury time of 6 h was used in subsequent experiments. The noncytotoxic drug concentration of apelin-13 was determined with a Cell Counting Kit-8 (CCK-8) assay. An appropriate dose of apelin-13 (1 μM) significantly improved cell survival (p < 0.05). Thus, subsequent experiments selected a concentration of 1 μM apelin-13 as it significantly increased cell viability. Finally, we divided the experimental groups into four groups according to whether they received drugs (1 μM apelin-13, 24 h) or OGD (6 h): (1) control group: without apelin-13 or OGD injury; (2) apelin-13 group: with apelin-13 but no OGD injury; (3) OGD group: with OGD injury but without apelin-13; and (4) OGD + apelin-13 group: with apelin-13 and OGD injury. The TUNEL assay and flow cytometry results showed that compared with the OGD group, apoptosis in the OGD+Apelin-13 group was significantly reduced (p < 0.001). Determination of cell viability under different conditions by CCK-8 assay results displays that Apelin-13 can significantly improve the cell viability percentage under OGD conditions (p < 0.001). Western blotting results showed that apelin-13 decreased the expression ratios of apoptosis-related proteins Bax/Bcl-2 and cleaved-caspase3/caspase3 (p < 0.05), increasing the key to Beclin1-dependent autophagy pathway expression of the protein Beclin1. This finding indicates that apelin-13 protects neurons by strengthening autophagy and attenuating early-stage postspinal cord injury apoptosis in vitro.

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.

Cryogen spray cooling mitigates inflammation and injury-induced CISD2 decline in rat spinal cord hemisection model.

Therapeutic strategies for traumatic spinal cord injury generally involve rectifying concomitant destruction to the spinal cord from inflammation, mitochondrial dysfunction, and eventual neuronal apoptosis. Elevating the expression of spinal cord injury-attenuated CDGSH iron-sulfur domain-2 has been shown to mitigate the pathologies above. In the current work, hypothermia was induced via continuous cryogen spray cooling in a rat spinal cord hemisection model. Spinal cord injury was shown to elevate the mRNA expression of proinflammatory mediators, including NFκB, iNOS, TNF-α, and regulated upon activation, normal T-cell expressed and secreted as well as lower CDGSH iron-sulfur domain-2 expression. Cryogen spray cooling treatment was shown to attenuate inflammatory reactions and elevate CDGSH iron-sulfur domain-2 expression. Immunohistochemical analysis of the glial fibrillary acidic protein, caspase-3 and NeuN in spinal cord injured rats that underwent cryogen spray cooling treatment revealed notable reductions in injury-induced astrocytic activation, apoptosis, neuronal loss, and decline in CDGSH iron-sulfur domain-2 expression. These results demonstrate the CDGSH iron-sulfur domain-2 preserving effects of cryogen spray cooling, which could contribute to the prevention of astrocytic activation, astrocyte-mediated neuroinflammation, apoptosis, and neuron loss.

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.

Inhibitory effect of hyaluronidase-4 in a rat spinal cord hemisection model

Objective: In this study, the effects of anti-hyaluronidase-4 (Hyal-4) antibody on the histological changes of Hyal-4 and the corresponding chondroitin sulfate proteoglycan (CSPG) expression in the rat spinal cord hemisection model were examined. Methods: After creating a rat spinal cord hemisection injury, experiments were conducted by administering anti-Hyal-4 antibody or control immunoglobulin G by intraspinal injection as a single dose, or intrathecal administration, using osmotic pumps, as multiple doses. Frozen sections of the injured spinal cord were made after a single-dose administration on days 1 and 4 and 1 week or at 1, 2, 3, and 4 weeks after the start of pump-aided injections. Immunofluorescence studies were then conducted using CS56 for CSPGs and anti-glial fibrillary acidic protein antibody for reactive astrocytes. Results: No difference was observed between the test and control groups in the single-dose administration of the antibody. In pump-aided administration, CSPGs in the control group decreased at 4 weeks, but those in the anti-Hyal-4 antibody administered group did not. Conclusion: Persistent suppression of Hyal-4 allowed CSPGs to remain and also increase in the rat spinal cord hemisection model, confirming Hyal-4 as an endogenous digestive enzyme of CSPGs.

PHBV/PLA/Col-Based Nanofibrous Scaffolds Promote Recovery of Locomotor Function by Decreasing Reactive Astrogliosis in a Hemisection Spinal Cord Injury Rat Model.

Biomaterials are used to aid in the regeneration of damaged tissue and in promotion of axonal guidance following spinal cord injury (SCI). In the present study, electrospun composite poly(hydroxybutyrate-cohydroxyvalerate) (PHBV), poly(lactic acid) (PLA), and collagen (Col) nanofibrous scaffolds were applied to determine their roles in neural regeneration and recovery in a rat model of SCI. The morphological and chemical properties of the electrospun scaffolds were investigated. The growth and proliferation of astrocytes on the scaffolds were assessed by MTT assay. The differentiation and gene expression of astrocytes on the scaffolds were measured by immunofluorescence and quantitative real-time polymerase chain reaction (q-PCR) assays. In a rat spinal cord hemisection model with 3-mm defects, 80 Sprague-Dawley rats were randomly divided into five groups: Sham group, SCI group, SCI+PHBV/PLA group, SCI+PHBV/PLA/Col (70:30) group, and SCI+PHBV/PLA/Col (50:50) group. The Basso-Beattie-Bresnahan (BBB) scores were evaluated every week postsurgery, and (immuno) histological and protein analyses were performed on specimens at 8 weeks. PHBV/PLA/Col scaffolds strongly inhibited the activation of astrocytes without decreasing their proliferation. qPCR assays revealed significant increases in the expression of brain lipid-binding protein (BLBP), glutamate transporter 1 (GLT-1) and S100 calcium-binding protein B (S100-β), but decreases in the expression of glial fibrillary acidic protein (GFAP), chondroitin sulphate sulfate proteoglycan (CSPG), neurocan, and phosphacan in the PHBV/PLA/Col scaffold group. In a series of in vivo experiments, PHBV/PLA/Col scaffold-treated SCI groups showed significant reductions in the numbers of CD68- and GFAP-immunopositive astrocytes within the interface of the remodeled tissue layer, but increased expression of NF-200 in residual neurons with better locomotor functional recovery. However, there were no significant differences between the PHBV/PLA/Col (70:30) and PHBV/PLA/Col (50:50) groups, except in BBB scores. PHBV/PLA/Col nanofibrous scaffolds were biocompatible and significantly promoted astrocyte differentiation but decreased astrocyte activation. The topographic structures of the PHBV/PLA/Col (70:30 and 50:50) nanofibers were favorable for neural regeneration due to a decrease in astrogliosis in SCI rats.

Human menstrual blood-derived stem cells promote functional recovery in a rat spinal cord hemisection model

Spinal cord injury (SCI) is associated with a dismal prognosis including severe voluntary motor and sensory deficits in the presence of the current therapies, thus new and efficient treatment strategies are desperately required. Along with several advantages, such as easy accessibility, high-yield, potential of enormous proliferation, menstrual blood-derived mesenchymal stem cells (MenSCs) have been proposed as a promising strategy in regeneration medicine. In this study, the MenSCs were transplanted into incomplete thoracic (T10) spinal cord injury (SCI) rats, all rats were sacrificed at 7, 14, and 28 days after surgery. Based on the results, we found that MenSCs transplantation improved the hind limb motor function. Besides, H&E staining showed that MenSCs treatment markedly reduced cavity formation in the lesion site. Furthermore, treatment by MenSCs showed more MAP2-positive mature neurons, as well as axonal regeneration manifested by NF-200 and less expression of chondroitin sulfate proteoglycans (CSPGs) than the non-treatment in the lesion site. Additionally, immunofluorescence, Western blot, and qRT-PCR methods showed that levels of brain-derived neurotrophic factor (BDNF) were significantly higher in the injured spinal cord after implantation of MenSCs. Results of qRT-PCR indicated that inflammatory factors, including TNF-α and IL-1β were inhibited after MenSCs transplantation. The improved motor function of hind limb and the increased cell body area of motor neurons were suppressed by blocking of the BDNF-TrkB signaling. It was eventually revealed that MenSCs implantation had beneficial therapeutic effects on the rehabilitation of the rat spinal cord hemisection model, mainly by enhancing the expression of BDNF. MenSCs transplantation may provide a novel therapeutic strategy for patients with SCI in the future.

A cellular spinal cord scaffold seeded with rat adipose‑derived stem cells facilitates functional recovery via enhancing axon regeneration in spinal cord injured rats.

Spinal cord injury (SCI), usually resulting in severe sensory and motor deficits, is a major public health concern. Adipose-derived stem cells (ADSCs), one type of adult stem cell, are free from ethical restriction, easily isolated and enriched. Therefore, ADSCs may provide a feasible cell source for cell-based therapies in treatment of SCI. The present study successfully isolated rat ADSCs (rADSCs) from Sprague-Dawley male rats and co-cultured them with acellular spinal cord scaffolds (ASCs). Then, a rat spinal cord hemisection model was built and rats were randomly divided into 3 groups: SCI only, ASC only, and ASC + ADSCs. Furthermore, behavioral tests were conducted to evaluate functional recovery. Hematoxylin & Eosin staining and immunofluorence were carried out to assess histopathological remodeling. In addition, biotinylated dextran amines anterograde tracing was employed to visualize axon regeneration. The data demonstrated that harvested cells, which were positive for cell surface antigen cluster of differentiation (CD) 29, CD44 and CD90 and negative for CD4, detected by flow cytometry analysis, held the potential to differentiate into osteocytes and adipocytes. Rats that received transplantation of ASCs seeded with rADSCs benefited greatly in functional recovery through facilitation of histopathological rehabilitation, axon regeneration and reduction of reactive gliosis. rADSCs co-cultured with ASCs may survive and integrate into the host spinal cord on day 14 post-SCI.

Acellular Spinal Cord Scaffold Implantation Promotes Vascular Remodeling with Sustained Delivery of VEGF in a Rat Spinal Cord Hemisection Model.

Promoting angiogenesis provides a possible therapeutic approach in treating spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) is a pro-angiogenic substance that is involved in endothelial cell (EC) proliferation, migration, and survival. Exogenous administration of VEGF to the lesion epicenter of the spinal cord has been recently revealed as a potential method for promoting the blood vessel sprouting. Spinal cord hemisection in a rat model was established and angiogenesis was studied through implant of an acellular spinal cord scaffold (ASCS) with sustained delivery of VEGF<sub>165</sub>. The poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating VEGF<sub>165</sub> were fabricated on basis of an emulsion and solvent evaporation method and conjugated to ASCS by a Genipin (GP) crosslinking technology. The resultant scaffolds were marked as V-ASCS. VEGF<sub>165</sub> entrapment efficiency (EE) and released kinetics were determined by an ultraviolet absorption measurement. Angiogenesis and vascular remodeling were observed via a high-resolution micro-CT and analyzed quantitatively by vascular morphometric parameters. Spinal cord histology and Basso, Beattie, and Bresnahan (BBB) locomotor rating scale were further studied. VEGF<sub>165</sub> was entrapped with high efficiency (90.8±3.1) %. In vitro VEGF<sub>165</sub> release kinetics study showed an initial burst of 1.966 μg mg NPs-1 and 1.045μg mg V-ASCS-1 respectively in the first 24 hours. In the phase of sustained release, approximately 0.040μg mg NPs-1 and 0.022μg mg V-ASCS-1 per day was on-going until 720h. In the rat spinal cord hemisection model, implant of V-ASCS at the injured site showed a promotion of angiogenesis and vascular remodeling following SCI. A better outcome can be confirmed histologically. However, functional improvement is limited in the animal model. The results indicate that progress of vascular reconstruction is accelerated in the V-ASCS implanted SCI rats.

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.

Therapeutic Effect of Neurotrophin-3 Treatment in an Injectable Collagen Scaffold Following Rat Spinal Cord Hemisection Injury.

Spinal cord injury (SCI) patients display varying quantities of spinal cord tissue damage with injuries that present as complete, incomplete or compressive. One theory proposed to repair the injured spinal cord and regain motor control is to regenerate axons through the lesion site. This study was designed to quantify the impact of a local injectable in situ forming hydrogel reservoir therapy following rat hemisection SCI. We investigated the effect of hydrogel only treatment following SCI in addition to hydrogels loaded with a neurotrophic factor, Neurotrophin-3 (NT-3), immediately following SCI. Functional recovery, assessed by Basso Beattie Bresnahan (BBB) locomotor test, and local healing mechanisms, including neuronal growth, glial scar formation, inflammation and collagen deposition were investigated one and 6 weeks postsurgery. Delivery of an injectable hydrogel significantly increased functional recovery at four and 6 weeks post injury. In addition, a significant reduction in the inhibitory glial scar and in inflammation was observed at the injury site. Similarly hydrogel + NT-3 delivered directly into the injury site significantly reduced glial scarring and collagen deposition. The hydrogel + NT-3 also resulted in a significant increase in neurons at 6 weeks post injury. This study represents a novel and effective therapy combining growth factor and a biomaterial based therapy following SCI.