Lentiviral vector delivery of short hairpin RNA to NgR1 promotes nerve regeneration and locomotor recovery in injured rat spinal cord.
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ABSTRACT:
SUBMITTER: Zhao X
PROVIDER: S-EPMC5882972 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Spinal cord stimulation has become an important modality in pain treatment especially for neuropathic pain conditions refractory to pharmacotherapy. However, the molecular control of inhibitory and excitatory mechanisms observed after spinal cord stimulation are poorly understood. Here, we used RNA-seq to identify differences in the expression of genes and gene networks in spinal cord tissue from nerve-injured rats with and without repetitive conventional spinal cord stimulation treatment. Five weeks after chronic constrictive injury to the left sciatic nerve, male and female rats were randomized to receive repetitive spinal cord stimulation or no treatment. Rats receiving spinal cord stimulation underwent epidural placement of a miniature stimulating electrode and received seven sessions of spinal cord stimulation (50 Hz, 80% motor threshold, 0.2 ms, constant current bipolar stimulation, 120 min/session) over four consecutive days. Within 2 h after the last spinal cord stimulation treatment, the L4-L6 spinal segments ipsilateral to the side of nerve injury were harvested and used to generate libraries for RNA-seq. Our RNA-seq data suggest further increases of many existing upregulated immune responses in chronic constrictive injury rats after repetitive spinal cord stimulation, including transcription of cell surface receptors and activation of non-neuronal cells. We also demonstrate that repetitive spinal cord stimulation represses transcription of several key synaptic signaling genes that encode scaffold proteins in the post-synaptic density. Our transcriptional studies suggest a potential relationship between specific genes and the therapeutic effects observed in patients undergoing conventional spinal cord stimulation after nerve injury. Furthermore, our results may help identify new therapeutic targets for improving the efficacy of conventional spinal cord stimulation and other chronic pain treatments.
| S-EPMC6293371 | biostudies-literature
Project description:Because there currently is no treatment for spinal cord injury, most patients are living with long-standing injuries. Therefore, strategies aimed at promoting restoration of function to the chronically injured spinal cord have high therapeutic value. For successful regeneration, long-injured axons must overcome their poor intrinsic growth potential as well as the inhibitory environment of the glial scar established around the lesion site. Acutely injured axons that regenerate into growth-permissive peripheral nerve grafts (PNGs) reenter host tissue to mediate functional recovery if the distal graft-host interface is treated with chondroitinase ABC (ChABC) to cleave inhibitory chondroitin sulfate proteoglycans in the scar matrix. To determine whether a similar strategy is effective for a chronic injury, we combined grafting of a peripheral nerve into a highly relevant, chronic, cervical contusion site with ChABC treatment of the glial scar and glial cell line-derived neurotrophic factor (GDNF) stimulation of long-injured axons. We tested this combination in two grafting paradigms: (1) a peripheral nerve that was grafted to span a chronic injury site or (2) a PNG that bridged a chronic contusion site with a second, more distal injury site. Unlike GDNF-PBS treatment, GDNF-ChABC treatment facilitated axons to exit the PNG into host tissue and promoted some functional recovery. Electrical stimulation of axons in the peripheral nerve bridge induced c-Fos expression in host neurons, indicative of synaptic contact by regenerating fibers. Thus, our data demonstrate, for the first time, that administering ChABC to a distal graft interface allows for functional axonal regeneration by chronically injured neurons.
| S-EPMC2824589 | biostudies-literature
Project description:Promoting the combination of robust regeneration of damaged axons and synaptic reconnection of these growing axon populations with appropriate neuronal targets represents a major therapeutic goal following spinal cord injury (SCI). A key impediment to achieving this important aim includes an intrinsic inability of neurons to extend axons in adult CNS, particularly in the context of the chronically-injured spinal cord. We tested whether an inhibitory peptide directed against phosphatase and tensin homolog (PTEN: a central inhibitor of neuron-intrinsic axon growth potential) could restore inspiratory diaphragm function by reconnecting critical respiratory neural circuitry in a rat model of chronic cervical level 2 (C2) hemisection SCI. We found that systemic delivery of PTEN antagonist peptide 4 (PAP4) starting at 8 weeks after C2 hemisection promoted substantial, long-distance regeneration of injured bulbospinal rostral Ventral Respiratory Group (rVRG) axons into and through the lesion and back toward phrenic motor neurons (PhMNs) located in intact caudal C3-C5 spinal cord. Despite this robust rVRG axon regeneration, PAP4 stimulated only minimal recovery of diaphragm function. Furthermore, re-lesion through the hemisection site completely removed PAP4-induced functional improvement, demonstrating that axon regeneration through the lesion was responsible for this partial functional recovery. Interestingly, there was minimal formation of putative excitatory monosynaptic connections between regrowing rVRG axons and PhMN targets, suggesting that (1) limited rVRG-PhMN synaptic reconnectivity was responsible at least in part for the lack of a significant functional effect, (2) chronically-injured spinal cord presents an obstacle to achieving synaptogenesis between regenerating axons and post-synaptic targets, and (3) addressing this challenge is a potentially-powerful strategy to enhance therapeutic efficacy in the chronic SCI setting. In conclusion, our study demonstrates a non-invasive and transient pharmacological approach in chronic SCI to repair the critically-important neural circuitry controlling diaphragmatic respiratory function, but also sheds light on obstacles to circuit plasticity presented by the chronically-injured spinal cord.
| S-EPMC8197742 | biostudies-literature
Project description:We report that prospectively isolated, human CNS stem cells grown as neurospheres (hCNS-SCns) survive, migrate, and express differentiation markers for neurons and oligodendrocytes after long-term engraftment in spinal cord-injured NOD-scid mice. hCNS-SCns engraftment was associated with locomotor recovery, an observation that was abolished by selective ablation of engrafted cells by diphtheria toxin. Remyelination by hCNS-SCns was found in both the spinal cord injury NOD-scid model and myelin-deficient shiverer mice. Moreover, electron microscopic evidence consistent with synapse formation between hCNS-SCns and mouse host neurons was observed. Glial fibrillary acidic protein-positive astrocytic differentiation was rare, and hCNS-SCns did not appear to contribute to the scar. These data suggest that hCNS-SCns may possess therapeutic potential for CNS injury and disease.
| S-EPMC1216836 | biostudies-literature
Project description:BackgroundPrior studies have applied driver mutations targeting the RTK/RAS/PI3K and p53 pathways to induce the formation of high-grade gliomas in rodent models. In the present study, we report the production of a high-grade spinal cord glioma model in pigs using lentiviral gene transfer.MethodsSix Gottingen Minipigs received thoracolumbar (T14-L1) lateral white matter injections of a combination of lentiviral vectors, expressing platelet-derived growth factor beta (PDGF-B), constitutive HRAS, and shRNA-p53 respectively. All animals received injection of control vectors into the contralateral cord. Animals underwent baseline and endpoint magnetic resonance imaging (MRI) and were evaluated daily for clinical deficits. Hematoxylin and eosin (H&E) and immunohistochemical analysis was conducted. Data are presented using descriptive statistics including relative frequencies, mean, standard deviation, and range.Results100% of animals (n = 6/6) developed clinical motor deficits ipsilateral to the oncogenic lentiviral injections by a three-week endpoint. MRI scans at endpoint demonstrated contrast enhancing mass lesions at the site of oncogenic lentiviral injection and not at the site of control injections. Immunohistochemistry demonstrated positive staining for GFAP, Olig2, and a high Ki-67 proliferative index. Histopathologic features demonstrate consistent and reproducible growth of a high-grade glioma in all animals.ConclusionsLentiviral gene transfer represents a feasible pathway to glioma modeling in higher order species. The present model is the first lentiviral vector induced pig model of high-grade spinal cord glioma and may potentially be used in preclinical therapeutic development programs.
| S-EPMC7093438 | biostudies-literature
Project description:Traumatic insults to the spinal cord induce both immediate mechanical damage and subsequent tissue degeneration leading to a substantial physiological, biochemical, and functional reorganization of the spinal cord. Various spinal cord injury (SCI) models have shown the adaptive potential of the spinal cord and its limitations in the case of total or partial absence of supraspinal influence. Meaningful recovery of function after SCI will most likely result from a combination of therapeutic strategies, including neural tissue transplants, exogenous neurotrophic factors, elimination of inhibitory molecules, functional sensorimotor training, and/or electrical stimulation of paralyzed muscles or spinal circuits. Peripheral nerve grafts provide a growth-permissive substratum and local neurotrophic factors to enhance the regenerative effort of axotomized neurons when grafted into the site of injury. Regenerating axons can be directed via the peripheral nerve graft toward an appropriate target, but they fail to extend beyond the distal graft-host interface because of the deposition of growth inhibitors at the site of SCI. One method to facilitate the emergence of axons from a graft into the spinal cord is to digest the chondroitin sulfate proteoglycans that are associated with a glial scar. Importantly, regenerating axons that do exit the graft are capable of forming functional synaptic contacts. These results have been demonstrated in acute injury models in rats and cats and after a chronic injury in rats and have important implications for our continuing efforts to promote structural and functional repair after SCI.
| S-EPMC3101823 | biostudies-other
Project description:Transplantation of neural stem cells (NSCs) is a potential strategy for the treatment of spinal cord transection (SCT). Here we investigated whether transplanted NSCs would improve motor function of rats with SCT and explored the underlying mechanism. First, the rats were divided into sham, SCT, and NSC groups. Rats in the SCT and NSC groups were all subjected to SCT in T10, and were administered with media and NSC transplantation into the lesion site, respectively. Immunohistochemistry was used to label Nestin-, TUNEL-, and NeuN-positive cells and reveal the expression and location of type I insulin-like growth factor receptor (IGF-1 R). Locomotor function of hind limbs was assessed by Basso, Beattie, Bresnahan (BBB) score and inclined plane test. The conduction velocity and amplitude of spinal nerve fibers were measured by electrophysiology and the anatomical changes were measured using magnetic resonance imaging. Moreover, expression of IGF-1 R was determined by real-time polymerase chain reaction and Western blotting. The results showed that NSCs could survive and differentiate into neurons in vitro and in vivo. SCT-induced deficits were reduced by NSC transplantation, including increase in NeuN-positive cells and decrease in apoptotic cells. Moreover, neurophysiological profiles indicated that the latent period was decreased and the peak-to-peak amplitude of spinal nerve fibers conduction was increased in transplanted rats, while morphological measures indicated that fractional anisotropy and the number of nerve fibers in the site of spinal cord injury were increased after NSC transplantation. In addition, mRNA and protein level of IGF-1 R were increased in the rostral segment in the NSC group, especially in neurons. Therefore, we concluded that NSC transplantation promotes motor function improvement of SCT, which might be associated with activated IGF-1 R, especially in the rostral site. All of the above suggests that this approach has potential for clinical treatment of spinal cord injury.
| S-EPMC6767897 | biostudies-literature
Project description:The failure of the spinal cord to regenerate can be attributed both to a lack of trophic support for regenerating axons and to upregulation of inhibitory factors such as chondroitin sulphate proteoglycans including NG2 following injury. Lentiviral vector-mediated gene therapy is a possible strategy for treating spinal cord injury (SCI). This study investigated the effect of lentiviral vectors expressing Neurotrophin-3 (NT-3) and short-hairpin RNA against NG2 (NG2 sh) to enhance neurite outgrowth in in vitro and ex vivo transection injury models. Conditioned medium from cells transduced with NT-3 or shNG2 lentiviruses caused a significant increase in neurite length of primary dorsal root ganglia neurons compared to the control group in vitro. In an ex vivo organotypic slice culture (OSC) transduction with Lenti-NT-3 promoted axonal growth. Transducing OSCs with a combination of Lenti-NT-3/NG2 sh lead to a further increase in axonal growth but only in injured slices and only within the region adjacent to the site of injury. These findings suggest that the combination of lentiviral NT-3 and NG2 sh reduced NG2 levels and provided a more favourable microenvironment for neuronal regeneration after SCI. This study also shows that OSCs may be a useful platform for studying glial scarring and potential SCI treatments.
| S-EPMC7150802 | biostudies-literature
Project description:ObjectiveSpinal cord injury (SCI) affects a quarter million individuals in the United States, and there is currently no clinical treatment. Both fresh and acellular peripheral nerve grafts can induce spinal axon regeneration and support functional recovery in experimental injury models. Nonetheless, a scaffold that can be injected into a spinal contusion would be far less invasive to apply. We aimed to develop the first injectable acellular nerve graft for promoting repair after contusion SCI.ApproachWe report a method to enzymatically solubilize optimized acellular (OA) nerve-a decellularized peripheral nerve graft developed in our laboratory and currently used clinically-to obtain an injectable solution that undergoes thermal gelation under physiological conditions. We quantified multiple physical and compositional properties of this novel material as well as tested its efficacy at acute and chronic time points following cervical contusion SCI.Main resultsThis injectable optimized acellular (iOA) nerve graft retains native chemical cues such as collagens and glycosaminoglycans. By varying hydrogel concentration, the rheological properties and compressive modulus of iOA were similar to that previous reported for rat central nervous tissue. iOA solution was compatible with rat Schwann cells in culture, and hydrogel injection into a rat cervical contusion model significantly reduced the ratio of M1:M2 macrophages after one week, favoring regenerative phenotypes (p < 0.05). Furthermore, while iOA treatment did not affect locomotor or respiratory recovery over an eight week period, the percentage of axonal coverage increased at the distal tissue interface (p < 0.05), suggesting enhanced axonal extension within this region.SignificanceOur data indicate that this novel injectable form of acellular nerve grafts is amenable for use after contusion SCI and may bolster a simultaneous therapy by acutely modulating the inflammatory milieu and supporting axonal growth.
| S-EPMC5911159 | biostudies-literature
Project description:Oscillating field stimulation (OFS) is a potential method for treating spinal cord injury. Although it has been used in spinal cord injury (SCI) therapy in basic and clinical studies, its underlying mechanism and the correlation between its duration and nerve injury repair remain poorly understood. In this study, we established rat models of spinal cord contusion at T10 and then administered 12 weeks of OFS. The results revealed that effectively promotes the recovery of motor function required continuous OFS for more than 6 weeks. The underlying mechanism may be related to the effects of OFS on promoting axon regeneration, inhibiting astrocyte proliferation, and improving the linear arrangement of astrocytes. This study was approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (supplemental approval No. AEEI-2021-204) on July 26, 2021.
| S-EPMC8643069 | biostudies-literature