Rat Model Of Spinal Cord Injury Research Articles

Inhibition of Ferroptosis by Mesenchymal Stem Cell-Derived Exosomes in Acute Spinal Cord Injury: Role of Nrf2/GCH1/BH4 Axis.

The therapeutic benefits of exosomes obtained from mesenchymal stem cells (MSCs) in acute spinal cord injury (SCI) have been demonstrated in recent years, but the precise mechanisms remain unknown. In this study, the efficacy and mechanisms of MSC-derived exosomes (MSC-Exo) in acute SCI were investigated. By utilizing a BV2 ferroptosis cellular model and an SCI rat model, we investigated the effects of MSC-Exo on iron death related indicators and NF-E2 related factor 2 (Nrf2)/GTP cyclolase I (GCH1)/5,6,7,8-tetrahydrobiopterin (BH4) signaling axis, as well as their therapeutic effects on SCI rats. The results revealed that MSC-Exo effectively inhibited the production of ferrous iron, lipid peroxidation products malonaldehyde and reactive oxygen species, and ferroptosis-promoting factor prostaglandin-endoperoxide synthase 2. Concurrently, they upregulated ferroptosis suppressors FTH-1 (ferritin heavy chain 1), SLC7A11 (solute carrier family 7 member 11), FSP1 (ferroptosis suppressor protein 1), and GPX4 (glutathione peroxidase 4), contributing to enhanced neurological recovery in SCI rats. Further analysis showed the Nrf2/GTP/BH4 signaling pathway's critical role in suppressing ferroptosis. Additionally, MSC-Exo was found to inhibit lipopolysaccharide-induced ferroptosis in BV2 cells and SCI rats by activating the Nrf2/GCH1/BH4 axis. In summary, the study demonstrates that MSC-Exo mitigates microglial cell ferroptosis via the Nrf2/GCH1/BH4 axis, showing potential for preserving and restoring neurological function post-SCI.

Combination of induced pluripotent stem cell-derived motor neuron progenitor cells with irradiated brain-derived neurotrophic factor over-expressing engineered mesenchymal stem cells enhanced restoration of axonal regeneration in a chronic spinal cord injury rat model

BackgroundSpinal cord injury (SCI) is a disease that causes permanent impairment of motor, sensory, and autonomic nervous system functions. Stem cell transplantation for neuron regeneration is a promising strategic treatment for SCI. However, selecting stem cell sources and cell transplantation based on experimental evidence is required. Therefore, this study aimed to investigate the efficacy of combination cell transplantation using the brain-derived neurotrophic factor (BDNF) over-expressing engineered mesenchymal stem cell (BDNF-eMSC) and induced pluripotent stem cell-derived motor neuron progenitor cell (iMNP) in a chronic SCI rat model.MethodA contusive chronic SCI was induced in Sprague-Dawley rats. At 6 weeks post-injury, BDNF-eMSC and iMNP were transplanted into the lesion site via the intralesional route. At 12 weeks post-injury, differentiation and growth factors were evaluated through immunofluorescence staining and western blot analysis. Motor neuron differentiation and neurite outgrowth were evaluated by co-culturing BDNF-eMSC and iMNP in vitro in 2-dimensional and 3-dimensional.ResultsCombination cell transplantation in the chronic SCI model improved behavioral recovery more than single-cell transplantation. Additionally, combination cell transplantation enhanced mature motor neuron differentiation and axonal regeneration at the injured spinal cord. Both BDNF-eMSC and iMNP played a critical role in neurite outgrowth and motor neuron maturation via BDNF expression.ConclusionsOur results suggest that the combined transplantation of BDNF- eMSC and iMNP in chronic SCI results in a significant clinical recovery. The transplanted iMNP cells predominantly differentiated into mature motor neurons. Additionally, BDNF-eMSC exerts a paracrine effect on neuron regeneration through BDNF expression in the injured spinal cord.Graphical

Repeated trans-spinal magnetic stimulation promotes microglial phagocytosis of myelin debris after spinal cord injury through LRP-1

Spinal cord injury (SCI) is a serious trauma of the central nervous system. The clearance of myelin debris is a critical step in the functional recovery following spinal cord injury (SCI). Recent studies have begun to reveal critical roles for professional phagocytes in the central nervous system, microglia, and their receptors in the control of myelin debris in neurodegenerative disease. Repeated trans-spinal magnetic stimulation (rTSMS) has been demonstrated as a noninvasive SCI treatment that enhances tissue repair and functional recovery. In this study, we investigated the role and molecular mechanism of rTSMS on microglial phagocytosis of myelin debris in a rat SCI model. In our studies, we found that rTSMS significantly promoted the motor function recovery of SCI rats associated with the inhibition the neuroinflammation and glia scar formation. Immunofluorescence results further showed that the rTSMS promotes the clearance of myelin debris by microglia in vivo and in vitro. Additionally, receptor-associated protein (RAP), a Low-density lipoprotein receptor-related protein-1 (LRP-1) inhibitor, could cancel the accelerated microglial phagocytosis of myelin debris after rTSMS in vitro experiments. Simultaneously, Elisa's results and western blotting respectively showed that rTSMS significantly decreased the levels of soluble LRP-1(sLRP-1) and the LRP-1 splicing enzyme of ADAM17. In conclusion, rTSMS could promote the clearance of myelin debris by microglia through LRP-1 to improve the functional recovery of SCI rats.

Dysregulated MiR-223-5p Modulates Inflammation and Oxidative Stress in Traumatic Spinal Cord Injury

ABSTRACT Aim This study aimed to evaluate the miR-223-5p expression in patients with spinal cord injury (SCI) and to determine its role in the pathogenesis of SCI. Methods The serum miR-223-5p levels were analyzed using quantitative real-time polymerase chain reaction. The diagnostic accuracy of miR-223-5p was evaluated using the receiving operating characteristic curves. LPS-induced PC12 cells were established as an in vitro inflammatory cell model. Cell apoptosis, inflammation and oxidative stress were examined. The SCI rat model was constructed to evaluate the effects of miR-223-5p on inflammatory response and motor function in rats. Results MiR-223-5p expression was upregulated in SCI patients. MiR-223-5p expression in the complete SCI group was significantly higher than that in incomplete SCI group. ROC analysis showed that miR-223-5p can distinguish SCI patients from healthy volunteers. In vitro experiments demonstrated that LPS upregulated apoptosis and inflammation in PC12 cells. Treatment with miR-223-5p inhibitor alleviated the changes in LPS-induced PC12 cells . Inhibition of miR-223-5p can alleviate the activation of inflammatory response and the effects of SCI on the motor function in rats. Conclusions MiR-223-5p is a potential diagnostic marker for SCI, and it can promote the SCI progression by regulating nerve cell survival, inflammation, and oxidative stress.

Enhancing motor functional recovery in spinal cord injury through pharmacological inhibition of Dickkopf-1 with BHQ880 antibody

BackgroundMounting experimental evidence has underscored the remarkable role played by the Wnt family of proteins in the spinal cord functioning and therapeutic potential in spinal cord injury (SCI). We aim to provide a therapeutic prospect associated with the modulation of canonical Wnt signaling, examining the spatio-temporal expression pattern of Dickkopf-1 (Dkk1) and its neutralization after SCI. We employ an intraparenchymal injection of the clinically validated Dkk1-blocking antibody, BHQ880, to elucidate its effects in SCI. MethodsA rat model of contusion SCI was used. Histological analyses were performed, wherein Dkk1 protein was sought, and ELISA analyses were employed for Dkk1 detection in cerebrospinal fluid and serum. To ascertain the BHQ880 therapeutic effect, rats were subjected to SCI and then injected with the antibody in the lesion epicenter 24 hours post-injury (hpi). Subsequent evaluation of motor functional recovery extended up to 56 days post-injury (dpi). qRT-PCR and histological analyses were conducted. ResultsWe demonstrate the presence of Dkk1 in the healthy rat spinal cord, with pronounced alterations observed following injury, primarily concentrated in the epicenter regions. Notably, a significative upregulation of Dkk1 was detected at 24 hpi, peaking at 3 dpi and remaining elevated until 42 dpi. Moreover, we revealed that early administration of BHQ880 considerably improved motor functional recovery, promoted preservation of myelinated tissue, and reduced astroglial and microglia/macrophage reactivity. Furthermore, there was a decrease in the acute expression of different inflammatory genes. ConclusionsCollectively, our findings highlight the therapeutic potential of BHQ880 treatment in the context of SCI.

Systematic analysis of co-expression network and gene expression profile of differentially expressed lncRNA-mRNA one week after spinal cord injury

Spinal cord injury (SCI) is a severe event that occurs in the central nervous system (CNS). Long noncoding RNAs (lncRNAs) have already been proven to play key roles in different diseases of the CNS. Nevertheless, the roles of messenger RNAs (mRNAs) and lncRNAs one week after SCI remain to be elucidated. We established a rat model of SCI and examined the differential levels of mRNAs and lncRNAs, and then, the differentially expressed mRNAs (DE mRNAs) and lncRNAs (DE lncRNAs) were investigated. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DE lncRNAs and mRNAs were subsequently carried out to explore crucial biological processes and signaling pathways. Moreover, we constructed protein–protein interaction (PPI) networks, the STRING database, and identified the 10 high degree core nodes. We also established a co-expression network by calculating the correlation between lncRNA expression profile and mRNA expression profile. The identified DE lncRNAs and DE mRNAs may potentially be critical targets for the diagnosis and treatment of SCI in the future. Abbreviations: SCI: spinal cord injury; CNS: central nervous system; YLDs: years of life lived with disability; lncRNAs: long noncoding RNAs; mRNAs: messenger RNAs; DE: differentially expressed; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: gene set enrichment analysis; RIN: Renewable Identification Number; PCA: principal component analysis; PPI: protein–protein interaction; MF, molecular function; BP: biological process; CC: cellular component; MCODE: molecular complex detection

Pre-clinical evaluation of clinically relevant iPS cell derived neuroepithelial stem cells as an off-the-shelf cell therapy for spinal cord injury.

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.

Stem cell exosome-loaded Gelfoam improves locomotor dysfunction and neuropathic pain in a rat model of spinal cord injury.

Spinal cord injury (SCI) is a debilitating illness in humans that causes permanent loss of movement or sensation. To treat SCI, exosomes, with their unique benefits, can circumvent limitations through direct stem cell transplantation. Therefore, we utilized Gelfoam encapsulated with exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-EX) in a rat SCI model. SCI model was established through hemisection surgery in T9 spinal cord of female Sprague-Dawley rats. Exosome-loaded Gelfoam was implanted into the lesion site. An in vivo uptake assay using labeled exosomes was conducted on day 3 post-implantation. Locomotor functions and gait analyses were assessed using Basso-Beattie-Bresnahan (BBB) locomotor rating scale and DigiGait Imaging System from weeks 1 to 8. Nociceptive responses were evaluated through von Frey filament and noxious radiant heat tests. The therapeutic effects and potential mechanisms were analyzed using Western blotting and immunofluorescence staining at week 8 post-SCI. For the in vivo exosome uptake assay, we observed the uptake of labeled exosomes by NeuN+, Iba1+, GFAP+, and OLIG2+ cells around the injured area. Exosome treatment consistently increased the BBB score from 1 to 8 weeks compared with the Gelfoam-saline and SCI control groups. Additionally, exosome treatment significantly improved gait abnormalities including right-to-left hind paw contact area ratio, stance/stride, stride length, stride frequency, and swing duration, validating motor function recovery. Immunostaining and Western blotting revealed high expression of NF200, MBP, GAP43, synaptophysin, and PSD95 in exosome treatment group, indicating the promotion of nerve regeneration, remyelination, and synapse formation. Interestingly, exosome treatment reduced SCI-induced upregulation of GFAP and CSPG. Furthermore, levels of Bax, p75NTR, Iba1, and iNOS were reduced around the injured area, suggesting anti-inflammatory and anti-apoptotic effects. Moreover, exosome treatment alleviated SCI-induced pain behaviors and reduced pain-associated proteins (BDNF, TRPV1, and Cav3.2). Exosomal miRNA analysis revealed several promising therapeutic miRNAs. The cell culture study also confirmed the neurotrophic effect of HucMSCs-EX. Implantation of HucMSCs-EX-encapsulated Gelfoam improves SCI-induced motor dysfunction and neuropathic pain, possibly through its capabilities in nerve regeneration, remyelination, anti-inflammation, and anti-apoptosis. Overall, exosomes could serve as a promising therapeutic alternative for SCI treatment.

Design and synthesis of sulfonamide phenothiazine derivatives as novel ferroptosis inhibitors and their therapeutic effects in spinal cord injury

Ferroptosis is a novel style of cell death, and studies have shown that ferroptosis is strongly associated with spinal cord injury (SCI). A large number of ferroptosis inhibitors have been reported, but so far no ferroptosis inhibitor has been used clinically. Therefore there is an urgent need to discover a better inhibitor of ferroptosis. In this study, 24 novel sulfonamide phenothiazine ferroptosis inhibitors were designed and synthesized, followed by structure–activity relationship studies on these compounds. Among them, compound 23b exhibited the best activity in Erastin-induced PC12 cells (EC50 = 0.001 μM) and demonstrated a low hERG inhibition activity (IC50 > 30 μM). Additionally, compound 23b was identified as a ROS scavenger and showed promising therapeutic effects in an SD rat model of SCI. Importantly, 23b did not display significant toxicity in both in vivo and in vitro experiments and show good pharmacokinetic properties. These findings suggest that compound 23b, a novel ferroptosis inhibitor, holds potential as a therapeutic agent for spinal cord injury and warrants further investigation.

Conductive Hydrogel Restores Electrical Conduction to Promote Neurological Recovery in a Rat Model.

Spinal cord injury (SCI), caused by significant physical trauma, as well as other pathological conditions, results in electrical signaling disruption and loss of bodily functional control below the injury site. Conductive biomaterials have been considered a promising approach for treating SCI, owing to their ability to restore electrical connections between intact spinal cord portions across the injury site. In this study, we evaluated the ability of a conductive hydrogel, poly-3-amino-4-methoxybenzoic acid-gelatin (PAMB-G), to restore electrical signaling and improve neuronal regeneration in a rat SCI model generated using the compression clip method. Gelatin or PAMB-G was injected at the SCI site, yielding three groups: Control (saline), Gelatin, and PAMB-G. During the 8-week study, PAMB-G, compared to Control, had significantly lower proinflammatory factor expression, such as for tumor necrosis factor -α (0.388 ± 0.276 for PAMB-G vs. 1.027 ± 0.431 for Control) and monocyte chemoattractant protein (MCP)-1 (0.443 ± 0.201 for PAMB-G vs. 1.662 ± 0.912 for Control). In addition, PAMB-G had lower astrocyte and microglia numbers (35.75 ± 4.349 and 40.75 ± 7.890, respectively) compared to Control (50.75 ± 6.5 and 64.75 ± 10.72) and Gelatin (48.75 ± 4.787 and 71.75 ± 7.411). PAMB-G-treated rats also had significantly greater preservation and regeneration of remaining intact neuronal tissue (0.523 ± 0.059% mean white matter in PAMB-G vs 0.377 ± 0.044% in Control and 0.385 ± 0.051% in Gelatin) caused by reduced apoptosis and increased neuronal growth-associated gene expression. All these processes stemmed from PAMB-G facilitating increased electrical signaling conduction, leading to locomotive functional improvements, in the form of increased Basso-Beattie-Bresnahan scores and steeper angles in the slope test (76.667 ± 5.164 for PAMB-G, vs. 59.167 ± 4.916 for Control and 58.333 ± 4.082 for Gelatin), as well as reduced gastrocnemius muscle atrophy (0.345 ± 0.085 for PAMB-G, vs. 0.244 ± 0.021 for Control and 0.210 ± 0.058 for Gelatin). In conclusion, PAMB-G injection post-SCI resulted in improved electrical signaling conduction, which contributed to lowered inflammation and apoptosis, increased neuronal growth, and greater bodily functional control, suggesting its potential as a viable treatment for SCI.

Iridoids rich fraction from Valeriana jatamansi Jones promotes axonal regeneration and motor functional recovery after spinal cord injury through activation of the PI3K/Akt signaling pathway.

Valeriana jatamansi Jones (VJJ), renowned for its extensive history in traditional Chinese medicine and ethnomedicine within China, is prevalently utilized to alleviate ailments such as epigastric distension and pain, gastrointestinal disturbances including food accumulation, diarrhea, and dysentery, as well as insomnia and other diseases. Moreover, the Iridoid-rich fraction derived from Valeriana jatamansi Jones (IRFV) has demonstrated efficacy in facilitating the recuperation of motor functions after spinal cord injury (SCI). This study is aimed to investigate the therapeutic effect of IRFV on SCI and its underlying mechanism. Initially, a rat model of SCI was developed to assess the impact of IRFV on axonal regeneration. Subsequently, employing the PC12 cell model of oxidative damage, the role and mechanism of IRFV in enhancing axonal regeneration were explored using the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway inhibitor LY294002. Ultimately, the same inhibitor was administered to SCI rats to confirm the molecular mechanism through which IRFV promotes axonal regeneration by activating the PI3K/Akt signaling pathway. The results showed that IRFV significantly enhanced motor function recovery, reduced pathological injury, and facilitated axonal regeneration in SCI rats. In vitro experiments revealed that IRFV improved PC12 cell viability, augmented axonal regeneration, and activated the PI3K/Akt signaling pathway. Notably, the inhibition of this pathway negated the therapeutic benefits of IRFV in SCI rats. In conclusion, IRFV promote promotes axonal regeneration and recovery of motor function after SCI through activation of the PI3K/Akt signaling pathway.

Injectable Hydrogel Loaded with CDs and FTY720 Combined with Neural Stem Cells for the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is an incurable and disabling event that is accompanied by complex inflammation-related pathological processes, such as the production of excessive reactive oxygen species (ROS) by infiltrating inflammatory immune cells and their release into the extracellular microenvironment, resulting in extensive apoptosis of endogenous neural stem cells. In this study, we noticed the neuroregeneration-promoting effect as well as the ability of the innovative treatment method of FTY720-CDs@GelMA paired with NSCs to increase motor function recovery in a rat spinal cord injury model. Carbon dots (CDs) and fingolimod (FTY720) were added to a hydrogel created by chemical cross-linking GelMA (FTY720-CDs@GelMA). The basic properties of FTY720-CDs@GelMA hydrogels were investigated using TEM, SEM, XPS, and FTIR. The swelling and degradation rates of FTY720-CDs@GelMA hydrogels were measured, and each group's ability to scavenge reactive oxygen species was investigated. The in vitro biocompatibility of FTY720-CDs@GelMA hydrogels was assessed using neural stem cells. The regeneration of the spinal cord and recovery of motor function in rats were studied following co-treatment of spinal cord injury using FTY720-CDs@GelMA hydrogel in combination with NSCs, utilising rats with spinal cord injuries as a model. Histological and immunofluorescence labelling were used to determine the regeneration of axons and neurons. The recovery of motor function in rats was assessed using the BBB score. The hydrogel boosted neurogenesis and axonal regeneration by eliminating excess ROS and restoring the regenerative environment. The hydrogel efficiently contained brain stem cells and demonstrated strong neuroprotective effects in vivo by lowering endogenous ROS generation and mitigating ROS-mediated oxidative stress. In a follow-up investigation, we discovered that FTY720-CDs@GelMA hydrogel could dramatically boost NSC proliferation while also promoting neuronal regeneration and synaptic formation, hence lowering cavity area. Our findings suggest that the innovative treatment of FTY720-CDs@GelMA paired with NSCs can effectively improve functional recovery in SCI patients, making it a promising therapeutic alternative for SCI.

Oxygen Therapy After Spinal Cord Injury Reduces Spinal Microglial Inflammatory Signalling

Spinal cord injury (SCI) interrupts blood flow, causing O2 partial pressure near the site of injury to decrease to very low levels. Spinal hypoxia contributes to tissue necrosis and neuroinflammation, leading to secondary injury. The majority of prior O2 therapy studies after SCI use hyperbaric O2 (HBO), and considerable evidence suggests HBO decreases spinal inflammation and neurodegeneration. HBO consists of 100% O2 (hyperoxia), which is easy to implement, at elevated pressure (hyperbaria), which is challenging to implement. Using an adult rat model of cervical SCI (C2 hemilesion), we compared the impact of daily HBO vs. normobaric hyperoxia therapy on the pro-inflammatory profile of spinal microglia isolated via immunomagnetic cell separation. Rats (n=3 per group) were given C2 hemilesion, followed by 14 days of normobaric hyperoxia (100% O2, 1 hour/day) or HBO (100% O2, 3 ATA, 1 hour/day). The C2-C6 spinal cord was harvested for immunomagnetic microglial isolation using CD11b antibodies. We screened pro-inflammatory gene expression with a PCR array (Rat Toll-Like Receptor Signaling Pathway, Qiagen). Both types of O2 therapy impacted suppressed microglial pro-inflammatory gene expression. In particular, subunit expression of the canonical transcription factor orchestrating microglial inflammatory responses, NFkB2, was significantly reduced. Downregulation of NFkB after HBO has been previously reported, but this is the first evidence for downregulation of NFkB after normobaric O2, and the first evidence for a microglia-specific response. Other pro-inflammatory genes with reduced expression after O2 therapy include: Clec4e, IL1β and CD14. Clec4e is as a transmembrane pattern recognition receptor and “necrotic cell sensor”. Thus, Clec4e downregulation after O2 therapy suggests a shift towards reduced necrosis after SCI. These initial results indicate that both HBO and normobaric hyperoxia treatments reduce the spinal microglia pro-inflammatory responses to cervical SCI, possibly leading to increased cell survival. HL153140 (DDF), HL147554 (GSM), HL149800 (DDF), McKnight Brain Institute (DDF, GSM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Jisuikang formula promotes spinal cord injury repair in rats by activating the YAP/PKM2 signaling axis in astrocytes

To investigate the effect of Jisuikang formula-medicated serum for promoting spinal cord injury (SCI) repair in rats and explore the possible mechanism. Thirty adult SD rats were randomized into sham-operated group, SCI (induced using a modified Allen method) model group, and Jisuikang formula-medicated serum treatment group. After the operations, the rats were treated with normal saline or Jisuikang by gavage on a daily basis for 14 days, and the changes in hindlimb motor function of the rats was assessed with Basso-Beattie-Bresnahan (BBB) scores and inclined-plate test. The injured spinal cord tissues were sampled from the SCI rat models for single-cell RNA sequencing, and bioinformatics analysis was performed to identify the target genes of Jisuikang, spinal cord injury and glycolysis. In the cell experiment, cultured astrocytes from neonatal SD rat cortex were treated with SOX2 alone or in combination with Jisuikang-medicated serum for 21 days, and the protein expressions of PKM2, p-PKM2 and YAP and colocalization of PKM2 and YAP in the cells were analyzed with Western blotting and immunofluorescence staining, respectively. The SCI rats with Jisuikang treatment showed significantly improved BBB scores and performance in inclined-plate test. At the injury site, high PKM2 expression was detected in various cell types. Bioinformatic analysis identified the HIPPO-YAP signaling pathway as the target pathway of Jisuikang. In cultured astrocytes, SOX2 combined with the mediated serum, as compared with SOX2 alone, significantly increased PKM2, p-PKM2 and YAP expressions and entry of phosphorylated PKM2 into the nucleus, and promoted PKM2 and YAP co-localization in the cells. Jisuikang formula accelerates SCI repair in rats possibly by promoting aerobic glycolysis of the astrocytes via activating the PKM2/YAP axis to induce reprogramming of the astrocytes into neurons.

AAV6 mediated Gsx1 expression in neural stem progenitor cells promotes neurogenesis and restores locomotor function after contusion spinal cord injury

Genomic screened homeobox 1 (Gsx1 or Gsh1) is a neurogenic transcription factor required for the generation of excitatory and inhibitory interneurons during spinal cord development. In the adult, lentivirus (LV) mediated Gsx1 expression promotes neural regeneration and functional locomotor recovery in a mouse model of lateral hemisection spinal cord injury (SCI). The LV delivery method is clinically unsafe due to insertional mutations to the host DNA. In addition, the most common clinical case of SCI is contusion/compression. In this study, we identify that adeno-associated virus serotype 6 (AAV6) preferentially infects neural stem/progenitor cells (NSPCs) in the injured spinal cord. Using a rat model of contusion SCI, we demonstrate that AAV6 mediated Gsx1 expression promotes neurogenesis, increases the number of neuroblasts/immature neurons, restores excitatory/inhibitory neuron balance and serotonergic neuronal activity through the lesion core, and promotes locomotor functional recovery. Our findings support that AAV6 preferentially targets NSPCs for gene delivery and confirmed Gsx1 efficacy in clinically relevant rat model of contusion SCI.

Effect of Myelin Debris on the Phenotypic Transformation of Astrocytes after Spinal Cord Injury in Rats

After spinal cord injury (SCI), the accumulation of myelin debris can serve as proinflammatory agents, hindering axon regrowth and exacerbating damage. While astrocytes have been implicated in the phagocytosis of myelin debris, the impact of this process on the phenotypic transformation of astrocytes and their characteristics following SCI in rats is not well understood. Here, we demonstrated that the conditioned medium of myelin debris can trigger apoptosis in rat primary astrocytes in vitro. Using a compressional SCI model in rats, we observed that astrocytes can engulf myelin debris through ATP-binding cassette transporter sub-family A member 1 (ABCA1), and these engulfed cells tend to transform into A1 astrocytes, as indicated by C3 expression. At 4 days post-injury (dpi), astrocytes rapidly transitioned into A1 astrocytes and maintained this phenotype from 4 to 28 dpi, while A2 astrocytes, characterized by S100, were only detected at 14 and 28 dpi. Reactive astrocytes, identified by Nestin, emerged at 4 and 7 dpi, whereas scar-forming astrocytes, marked by N-cadherin, were evident at 14 and 28 dpi. This study illustrates the distribution patterns of astrocyte subtypes and the potential interplay between astrocytes and myelin debris after SCI in rats. We emphasize that myelin debris can induce astrocyte apoptosis in vitro and promote the transformation of astrocytes into A1 astrocytes in vivo. These two classification methods are not mutually exclusive, but rather complementary.

Analgesic Mechanism of Dexmedetomidine and Esketamine in Rats with Spinal Cord Injury.

Spinal cord injury (SCI) is usually caused by external direct or indirect factors, and with a high morbidity and mortality rate. The aim of this study was to observe the effects of Dexmedetomidine (DEX) combined with Esketamine (ESK) on pain behavior and potential analgesic mechanisms in rats with SCI. The goal was to provide a reliable multimodal analgesic medication regimen for SCI. Thirty rats were divided into five groups with six rats in each group: Sham group, SCI group, DEX group, ESK group, and DEX+ESK group. The SCI model in rats was constructed, and the motor function of hind limbs of rats was measured using Basso Beattie Bresnahan (BBB) locomotor rating scale and inclined plate test. The levels of interleukin 18 (IL-18), interleukin 1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the spinal cord were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of substance P (SP), neurokinin-1 receptor (NK-1R), B cell lymphoma-2 (Bcl-2), and Bcl2-associated X protein (Bax) in the rats' spinal cord were measured by Western blot assay. The viability of spinal astrocytes was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 7 days, the BBB scores were significantly higher in the DEX, ESK, and DEX+ESK groups compared to the SCI group (p < 0.01). Additionally, the DEX+ESK group had significantly higher scores than both the DEX and ESK groups (p < 0.01). The maximum angle of the DEX (p < 0.05), ESK (p < 0.05), and DEX+ESK groups (p < 0.01) were higher than the SCI group, and the maximum angle of DEX+ESK group was higher than DEX and ESK groups (p < 0.05). The levels of IL-18, IL-1β, and TNF-α in the DEX, ESK, and DEX+ESK groups were lower than the SCI group (p < 0.01), while the DEX+ESK group had significantly lower IL-18, IL-1β, and TNF-α levels than the DEX and ESK groups (p < 0.01). The levels of SP (p < 0.01) and NK-1R (p < 0.05) were lower in the DEX, ESK, and DEX+ESK groups compared to the SCI group, and the levels of SP and NK-1R were lower in the DEX+ESK group compared to the DEX and ESK groups (p < 0.01). The DEX and ESK groups suppressed the activity of spinal astrocytes (p < 0.01), however, the DEX+ESK group had larger effects on spinal astrocytes than the ESK group (p < 0.05). Treatment using DEX combined with ESK improves the motor function, inhibits inflammation and astrocyte activity, and exerts analgesic effects on rats with SCI. These findings can serve as a reference for the selection of multi-modal analgesics.

465 Focused Ultrasound-Mediated Blood-Spinal Cord Barrier Permeabilization: A Promising Targeted Approach for Treating Chronic Spinal Cord Injury in Rat Models

INTRODUCTION: Focused ultrasound (FUS) with microbubble contrast agents can temporarily disrupt the blood-brain barrier (BBB), enabling drug delivery. FUS may also offer a minimally invasive method for delivering therapeutic agents to the affected area in chronic spinal cord injury (SCI) by transiently opening the blood-spinal cord barrier (BSCB). METHODS: Nine female Sprague-Dawley rats underwent a T9-T11 laminectomy with contusion SCI and were randomly allocated into FUS or control group. Three weeks later, FUS was administered intravenously with microbubbles. A pressure sweep was performed until ultra-harmonics were captured using a wideband hydrophone. We administered 2% Evans blue (EB) immediately after FUS confirmed the BSCB permeability. Transcardial perfusion was performed 2 hours later, and spinal cords were collected. Spectrophotometry was used to determine the EB concentration. Statistical analysis was performed using a student t-test. RESULTS: Five rats were treated with FUS while four were used as controls. Successful feedback control was achieved with FUS, and ultra-harmonics were observed in the FUS group at a mean peak pressure of 0.5 MPa. Macroscopic identification of EB in the spinal cord sections of animals exposed to FUS confirmed the localized opening of the BSCB. Spectrophotometry revealed an increase in EB concentration in the FUS group compared to controls: Mean EB concentration was 0.03 μg in the FUS group versus 0.016 μg in controls at 1-cm above the injury (p &lt; 0.001); 0.27 μg versus 0.093 μg at the injury site (p &lt; 0.001); and 0.25 μg versus 0.18 μg at 1-cm below the injury(p &lt; 0.001). CONCLUSIONS: FUS increases the permeability of the BSCB in a chronic contusion SCI rat model. This suggests that FUS-mediated BSCB permeabilization may hold promise as a targeted approach for improving therapeutic outcomes in the treatment of chronic SCI.

Establishment of a bio-industry-need hemisection of spinal cord rat model

Spinal cord injury (SCI) is a severe central nervous system disorder, and due to its complex pathophysiology, effective treatment strategies are currently lacking, posing a significant challenge in the field of medicine. While research into the fundamentals of SCI continues to advance and innovate, there exists a gap between basic research and clinical applications, hindering the translation of basic research findings into clinical practice. One of the reasons for this gap is the use of non-standardized animal models for SCI, which leads to inaccuracies in research results and reduces the guidance value of basic research for clinical treatment. This study provides a SCI rat model for R&amp;D of SCI medicine target research and therapeutic strategy designs.

Hesperetin ameliorates spinal cord injury in rats through suppressing apoptosis, oxidative stress and inflammatory response

Spinal cord injury (SCI), a fatal condition, is characterized by progressive tissue degradation and extreme functional deficits with limited treatment options. Hesperetin, a natural flavonoid with potent antioxidant, antiapoptotic and anti-inflammatory properties, has yet to be systematically investigated for its therapeutic effects on neurological damage in rat models of SCI. In this study, rats were given oral hesperetin once daily for 28 days, and their locomotion and histopathological changes were assessed. The findings demonstrated that hesperetin alleviates neurological damage caused by SCI. The observed behavioral improvement could be due to an increase in the survival rate of neurons and oligodendrocytes. This improvement further boosted the ability to repair tissue and form myelin after SCI, ultimately resulting in better neurological outcomes. Furthermore, the present study revealed that hesperetin possesses potent antioxidant capabilities in the context of SCI, reducing the levels of harmful oxygen free radicals and increasing the activity of antioxidant enzymes. Additionally, hesperetin markedly inhibited injury-induced apoptosis, as assessed by caspase-3 immunofluorescence staining and the expression level of caspase-3, indicating the ability of hesperetin to prevent cell death after SCI. Finally, after SCI, hesperetin treatment effectively reduced the expression of inflammatory factors, including IL-1β, TNFα, and NF-kB, demonstrating the anti-inflammatory effect of hesperetin. Together, our results suggest that hesperetin should be considered a valuable therapeutic aid following SCI, as its positive effects on the nervous system, including antioxidant, anti-inflammatory and antiapoptotic effects, may be crucial mechanisms through which hesperetin exerts neuroprotective effects against SCI.