Rat Spinal Cord Tissue Research Articles

MALDI TIMS IMS of Disialoganglioside Isomers─GD1a and GD1b in Murine Brain Tissue.

Gangliosides are acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or more sialic acid residue(s) and are highly diverse isomeric structures with distinct biological roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, directly from tissue sections. Integrating trapped ion mobility spectrometry with MALDI IMS allows for the analysis of isomeric lipid structures in situ. Here, we demonstrate the gas-phase separation and identification of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in multiple samples, including a standard mixture of both isomers, a biological extract, and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) isomers were determined in rat hippocampus and spinal cord tissue sections, demonstrating the ability to structurally characterize and spatially map gangliosides based on both the carbohydrate chain and ceramide moieties.

Sp1 Inhibits PGC-1α via HDAC2-Catalyzed Histone Deacetylation in Chronic Constriction Injury-Induced Neuropathic Pain.

Our previous study has illuminated that PGC-1α downregulation promoted chronification of pain after burn injury. RNA-seq analysis predicted association between Sp1 and chronic constriction injury (CCI)-provoked neuropathic pain. Further ChIP-Atlas data investigation suggested the binding to Sp1 to PGC-1α. Thereby, we performed this study to illustrate the functional relevance of the Sp1/PGC-1α axis in neuropathic pain. Neuropathic pain was induced by CCI in vivo in rats, followed by assessment of neuropathic pain-like behaviors. The expression of Sp1 and correlated genes was determined in CCI rat spinal cord tissues. Furthermore, microglia were exposed to lipopolysaccharide (LPS) to mimic inflammation and then cocultured with neurons. Knockdown and ectopic expression methods were used in vivo and in vitro to define the role the Sp1/HDAC2/PGC-1α axis. Sp1 expression was upregulated in spinal cord tissues of CCI rats. Silencing Sp1 ameliorated CCI-induced neuropathic pain, as reflected by elevated paw withdrawal threshold and paw withdrawal latency, as well as alleviated microglia activation, neuronal dysfunction, inflammatory responses, mitochondrial dysfunction, and oxidative stress in spinal cord tissues. Sp1 knockdown also reversed LPS-induced microglial inflammation and neuronal dysfunction. Sp1 promoted histone deacetylation in the PGC-1α promoter and inhibited PGC-1α expression via recruiting HDAC2. PGC-1α overexpression diminished CCI-induced neuropathic pain and LPS-induced inflammation and mitochondrial dysfunction, based on which Sp1 aggravated microglial inflammation and neuronal dysfunction in neuropathic pain. This study elucidated the promoting effects of Sp1 on CCI-induced neuropathic pain via the HDAC2/PGC-1α axis.

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.

Inhibition of microRNA-19a-3p alleviates the neuropathic pain (NP) in rats after chronic constriction injury (CCI) via targeting KLF7

Background/purposeNeuropathic pain(NP) is derived from the dysfunctions of nerve system. The current research is to explore the impact and mechanism of miR-19a-3p in neuropathic pain in rats. MethodsThe NP was induced through the chronic constriction injury (CCI) surgery in rats. The pro-inflammatory factors (IL-1β, IL-6, TNF-α) in spinal cord tissues from rats were measured using Elisa kits. Moreover, the different levels of thermal hyperalgesia and mechanical allodynia in rats were examined through paw withdrawal latency (PWL) and paw withdrawal threshold (PWT). To investigate into the role of miR-19a-3p and KLF7 in NP of rats, the knockdown of miR-19a-3p alone or along with KLF7 downregulation in rats were achieved through lentivirus injection. The miR-19a-3p and KLF7 expression in spinal cord of rats on Day 3,7,14 after CCI were detected using RT-qPCR. The protein expression of KLF7 were measured by Western blot. Bioinformatics and luciferase assays were used for the prediction and verification of bindings between KLF7 and miR-19a-3p. ResultsCCI surgery caused neuropathic pain in rats with the levels of inflammatory cytokines increased and PWL and PWT decreased. Moreover, miR-19a-3p expression was increased while the protein and mRNA levels were decreased in spinal cord tissues in rats after CCI surgery. In rat microglial cells, miR-19a-3p downregulation could promote the KLF7 in both mRNA and protein expression. In spinal cord tissues of rats, the inhibition of miR-19a-3p enhanced the KLF7 expression. Furthermore, miR-19a-3p downregulation suppressed the IL-1β, IL-6 and TNF-α concentrations, and could decrease the NP but inhibition of KLF7 could partially reverse this in CCI rats. ConclusionmiR-19a-3p inhibition may alleviate NP via KLF7 in CCI rats.

MiR-30b-5p attenuates the inflammatory response and facilitates the functional recovery of spinal cord injury by targeting the NEFL/mTOR pathway.

Neurofilament light chain (NEFL) has been identified as a biomarker for spinal cord injury (SCI), but its effect and underlying mechanism in SCI remain unclear. SCI rat models were established for in vivo studies. Lipopolysaccharide (LPS)-induced cell models were used for in vitro studies. The protein and mRNA expression levels of genes were evaluated by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The pathological changes in rats after SCI were subjected to histological examinations. The interaction of NEFL and upstream miRNAs was explored using dual-luciferase reporter gene assays. NEFL was highly expressed in SCI rat spinal cord tissues and LPS-stimulated PC12 cells. NEFL silencing showed an inhibitory effect on the morphological changes of SCI rats and the secretion of inflammatory factors and facilitated functional recovery of SCI rats. MiR-30b-5p was demonstrated to target NEFL and negatively regulate NEFL mRNA and protein levels. Downregulation of miR-30b-5p in SCI cell and rat models was demonstrated. MiR-30b-5p alleviated the inflammatory response in SCI rat models and LPS-stimulated PC12 cells and promoted functional recovery in rats by targeting NEFL. NEFL activated mTOR signaling. MiR-30b-5p inactivated mTOR signaling by negatively regulating NEFL. MiR-30b-5p alleviated the inflammatory response and facilitated the functional recovery of SCI rats by targeting NEFL to inactivate the mTOR pathway.

Resveratrol Glycosides Impede Microglial Apoptosis and Oxidative Stress in Rats for Spinal Cord Injury

Spinal cord injury (SCI) usually occurs after severe trauma, which can lead to detrimental and unpredictable secondary diseases, including dyskinesia, paraplegia and even quadriplegia, resulting in seriously reduced quality of life among these patients. Oxidative damage is one of the major pathogenic factors of SCI. Resveratrol biologically exerts a significant antioxidant activity by increasing the levels of antioxidants and sequentially scavenging free radicals, so as to protect multiple organs from damage. This study investigates whether resveratrol can function as a protective mediator in SCI and the underlying mechanisms, aiming to provide a theoretical hint for the treatment of SCI. After establishment of SCI model in rats, serial doses of resveratrol were administrated. Afterwards, the therapeutic effects of resveratrol glycosides were evaluated by analyzing the motor function, spinal cord edema, cellular apoptosis and oxidative reaction in rats. Eventually, the potential mechanisms of resveratrol glycosides were studied via Western blotting. Our results showed that the pro-apoptosis proteins were highly expressed in the spinal cord tissue of rats after SCI. In comparison with healthy rats, those with SCI exhibited significant widespread dead neurons, glial cell apoptosis, oxidative stress and more serious functional defects. Nevertheless, resveratrol glycosides can ameliorate oxidative stress, inhibit the apoptosis of glial cells and neuronal death after SCI. Importantly, it can induce the activation of the Nrf2/HO-1 signal transduction pathway that mediated the alleviation of SCI in rats. Resveratrol can improve motor dysfunction after SCI, which may be a result of its anti-oxidation and anti-apoptotic effects via modulating the Nrf2 signal transduction pathway of microglia, which provides a new idea for the treatment of SCI.

Single-cell sequencing reveals microglia induced angiogenesis by specific subsets of endothelial cells following spinal cord injury.

Spinal cord injury (SCI) results in dynamic alterations of the microenvironment at the lesion site, which inevitably leads to neuronal degeneration and functional impairment. The destruction of the spinal vascular system leads to a significant deterioration of the milieu, which exacerbates inflammatory response and deprives cells of nutrient support in the lesion. Limited endogenous angiogenesis occurs after SCI, but the cellular events at the lesion site during this process are unclear so far. Here, we performed single-cell RNA sequencing (scRNA-seq) on spinal cord tissues of rats at different time points after SCI. After clustering and cell-type identification, we focused on vascular endothelial cells (ECs), which play a pivotal role in angiogenesis, and drew the cellular and molecular atlas for angiogenesis after SCI. We found that microglia and macrophages promote endogenous angiogenesis by regulating EC subsets through SPP1 and IGFsignaling pathways. Our results indicate that immune cells promote angiogenesis by regulating specific subsets of vascular ECs, which provides new clues for exploring SCI intervention.

FTO knockdown alleviates hypoxia-induced PC12 cell injury by stabilizing GADD45B in an IGF2BP2-dependent manner

RNA N6-methyladenosine (m6A) level is closely associated with neurodevelopment and central nervous system dysfunctions including spinal cord injury (SCI). M6A level can be dynamically regulated by m6A methyltransferases and demethylases. In this text, the roles of m6A demethylase FTO alpha-ketoglutarate dependent dioxygenase (FTO) in SCI development along with its m6A-dependent regulatory mechanisms were investigated in hypoxia-induced PC12 cell injury model. The results showed that FTO was low expressed in spinal cord tissues of rats after contusive SCI and hypoxia-treated PC12 cells. FTO knockdown alleviated hypoxia-induced PC12 cell injury. FTO loss increased GADD45B expression and m6A level in PC12 cells. GADD45B knockdown weakened the protective effects of FTO depletion on hypoxia-treated PC12 cells. FTO regulated GADD45B expression in an IGF2BP2-dependent manner. In conclusion, FTO knockdown mitigated the injury of hypoxia-induced PC12 cells by up-regulating GADD45B in an IGF2BP2-dependent manner.

Structural changes of spinal cord tissue of rats with experimental spinal cord injury after implantation of fibrin matrix associated with neural and mesenchymal progenitor cells

Structural changes of spinal cord tissue of rats with experimental spinal cord injury after implantation of fibrin matrix associated with neural and mesenchymal progenitor cells

Tetrahydropalmatine alleviated diabetic neuropathic pain by inhibiting activation of microglia via p38 MAPK signaling pathway

Neuropathic pain is one of the common complications of diabetes. Tetrahydropalmatine(THP) is a main active component of Corydalis Rhizoma with excellent anti-inflammatory and pain-alleviating properties. This study aims to investigate the therapeutic effect of THP on diabetic neuropathic pain(DNP) and the underlying mechanism. High-fat and high-sugar diet(4 weeks) and streptozotocin(STZ, 35 mg·kg~(-1), single intraperitoneal injection) were employed to induce type-2 DNP in rats. Moreover, lipopolysaccharide(LPS) was used to induce the activation of BV2 microglia in vitro to establish an inflammatory cellular model. Fasting blood glucose(FBG) was measured by a blood glucose meter. Mechanical withdrawal threshold(MWT) was assessed with von Frey filaments, and thermal withdrawal latency(TWL) with hot plate apparatus. The protein expression levels of OX42, inducible nitric oxide synthase(iNOS), CD206, p38, and p-p38 were determined by Western blot, the fluorescence expression levels of OX42 and p-p38 in the dorsal horn of the rat spinal cord by immunofluorescence, the mRNA content of p38 and OX42 in rat spinal cord tissue by qRT-PCR, and levels of nitric oxide(NO), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), and serum fasting insulin(FINS) by enzyme-linked immunosorbent assay(ELISA). RESULTS:: showed that the mo-del group demonstrated significant decrease in MWT and TWL, with pain symptoms. THP significantly improved the MWT and TWL of DNP rats, inhibited the activation of microglia and p38 MAPK signaling pathway in rat spinal cord, and ameliorated its inflammatory response. Meanwhile, THP promoted the change of LPS-induced BV2 microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, suppressed the activation of the p38 MAPK signaling pathway, decreased the expression levels of inflammatory factors NO, IL-1β, IL-6, and TNF-α, and increased the expression level of anti-inflammatory factor IL-10. The findings suggested that THP can significantly ameliorate the pain symptoms of DNP rats possibly by inhibiting the inflammatory response caused by M1 polarization of microglia via the p38 MAPK pathway.

Erodible thermogelling hydrogels for localized mitochondrial transplantation to the spinal cord

We developed a thermal-gelling, erodible hydrogel system for localized delivery of viable mitochondria in vivo, as well as labeled transplanted mitochondria with specific dyes and/or genetically modified mitochondria tagged with red fluorescence protein (RFP). We also employed cell lines to optimize a hydrogel composed of methylcellulose and hyaluronic acid designed to preserve bioenergetics while facilitating mitochondrial release. We further investigated how transplantation of allogeneic or xenogeneic mitochondria into respective cell lines affects host cellular metabolism, as measured by MTS assay. We found that 70% of mitochondria are released from the hydrogel within 20 min at 37 °C, that the respiratory capacity of hydrogel-released mitochondria over 60 min was greater than those without gel, and that MTR-labeling of mitochondria is not indelible. RFP-tagged transgenic mitochondria isolated from modified SH-SY5Y human neuroblastoma cells showed effective uptake into both naïve SH-SY5Y cells and rat PC-12 cells, notably when released from hydrogel. The hydrogel both protected the mitochondria at physiological conditions in vitro while solidifying and diffusing within 60 min locally in situ. To assess metabolic effects, both cell lines were transplanted with different concentrations of SH-SY5Y or PC-12 cell line-derived mitochondria and all resulted in significant increases in metabolism at 6- and 24-hour after transplantation. Alternatively, transplanted mitochondria at highest concentration from rat brain and spinal cord tissues reduced metabolic activities after 24-hour. Along with hydrogel refinements, we are further investigating whether such metabolic changes are due to alterations in cell proliferation or the number of exogenous mitochondria incorporated into individual host cells.

Human Epidural AD-MSC Exosomes Improve Function Recovery after Spinal Cord Injury in Rats.

Spinal cord injury (SCI) interferes with the normal function of the autonomic nervous system by blocking circuits between the sensory and motor nerves. Although many studies focus on functional recovery after neurological injury, effective neuroregeneration is still being explored. Recently, extracellular vesicles such as exosomes have emerged as cell-free therapeutic agents owing to their ability of cell-to-cell communication. In particular, exosomes released from mesenchymal stem cells (MSCs) have the potential for tissue regeneration and exhibit therapeutic effectiveness in neurological disorders. In this study, we isolated exosomes from human epidural adipose tissue-derived MSCs (hEpi AD–MSCs) using the tangential flow filtration method. The isolated exosomes were analyzed for size, concentration, shape, and major surface markers using nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. To evaluate their effect on SCI recovery, hEpi AD–MSC exosomes were injected intravenously in SCI-induced rats. hEpi AD–MSC exosomes improved the locomotor function of SCI-induced rats. The results of histopathological and cytokine assays showed that hEpi AD–MSC exosomes regulated inflammatory response. Genetic profiling of the rat spinal cord tissues revealed changes in the expression of inflammation-related genes after exosome administration. Collectively, hEpi AD–MSC exosomes are effective in restoring spinal functions by reducing the inflammatory response.

MiR-185-5p alleviates CCI-induced neuropathic pain by repressing NLRP3 inflammasome through dual targeting MyD88 and CXCR4

MicroRNAs (miRNAs) are important modulators in the evolvement and progression of neuropathic pain (NP). According to reports, miR-185-5p contributes to various diseases and inflammatory responses. However, it is not clear whether miR-185-5p mediates neuroinflammation and NP following chronic constrictive injury (CCI). The CCI model was constructed in rats to induce NP. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were employed to evaluate pain threshold in CCI rats. The expression of miR-185-5p, GFAP, Iba1, Caspase-3-positive cells, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-labeled apoptotic neurons, inflammatory mediators, including interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) in lumbar portion (L4-L6) of CCI rats were determined. Furthermore, the targets of miR-185-5p were predicted by the Starbase, and the binding association between miR-185-5p and MyD88, miR-185-5p and CXCR4 was verified by the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. As shown by the data, miR-185-5p was distinctly reduced in L4-L6 spinal cord tissues of rats after CCI. Up-regulating miR-185-5p alleviated mechanical and thermal hyperalgesia, inactivated microglia and astrocytes accumulation, and abated the contents of IL-1β, IL-6 and TNF-α in L4-L6 spinal cord tissues of CCI rats. Bioinformatics analysis suggested that MyD88 and CXCR4 were potential target genes of miR-185-5p. Increasing miR-185-5p expression notably impeded the expression of MyD88, CXCR4 and NLRP3 inflammasome in BV2 microglia, while attenuating miR-185-5p expression exerted the opposite effects. Notably, down-regulating MyD88 and CXCR4 significantly enhanced the miR-185-5p-mediated anti-inflammatory effects, and reversed miR-185-5p inhibitor-mediated proinflammatory effects. Additionally, up-regulating miR-185-5p repressed BV2-induced neuronal apoptosis and increased neuronal viability. In conclusion, this study suggested that miR-185-5p chokes CCI-induced NP and neuroinflammation by targeting MyD88 and CXCR4, indicating that miR-186-5p is an underlying therapeutic target for NP.

Cotransplantation with RADA16-PRG-Self-Assembled Nanopeptide Scaffolds, Bone Mesenchymal Stem Cells and Brain-Derived Neurotrophic Factor-Adeno-Associated Virus Promote Functional Repair After Acute Spinal Cord Injury in Rats.

We transplanted RADA16-PRG self-assembled nanopeptide scaffolds (SAPNSs), bone mesenchymal stem cells (BMSCs), and a brain-derived neurotrophic factor (BDNF)-expressing adeno-associated virus (AAV) into rats subjected to acute spinal cord injury (SCI) to investigate the effects of these transplantations on acute SCI repair and explore their mechanisms. Forty-eight SCI rats were randomly divided into four groups: BBR, BR, B, and NC groups. Seven and 28 days after SCI, evoked potentials (EPs) and BBB scores were assessed to evaluate the recovery of rats' motor behavior and sensory function after injury. HE and toluidine blue staining were performed to investigate the histological structure of the spinal cord tissue of rats from each group, and immunofluorescence staining was used to observe the red fluorescent protein (RFP) intensity of BMSCs and glial fibrillary acidic protein (GFAP) and neurofilament (NF) in the damaged area in each group. RT-PCR was utilized to detect the expression levels of the BDNF, GFAP, and neuron-specific enolase (NSE) genes in the injured area in each group. The results showed that cotransplantation of RADA16-PRG-SAPNs, BMSCs, and BDNF-AVV promoted the spinal cord's motor and sensory function of SCI rats; increased levels of BMSCs, inhabited glial cells proliferation, and promoted neurons proliferations in the injured area; and increased NF, BDNF, and NSE levels and decreased its GFAP in the injured area. Thus, cotransplantation of RADA16-PRG-SAPNS, BMSCs, and BDNF-AAV can prolong the survival time of BMSCs in rats, reduce the postoperative scarring caused by glial proliferation, and promote the migration and proliferation of neurons in the injured area, resulting in the promotion of functional repair after acute SCI.

Minocycline-Loaded Poly(α-Lipoic Acid)-Methylprednisolone Prodrug Nanoparticles for the Combined Anti-Inflammatory Treatment of Spinal Cord Injury.

PurposeTraumatic spinal cord injury (TSCI) induces a powerful inflammatory response that can significantly exacerbate the extent and severity of neural damage (termed as “secondary injury”). Thus, the suppression of inflammation is crucial for reducing neurological dysfunction following TSCI. However, the conventional anti-inflammatory drugs show limited efficacy because of poor penetration and release kinetics at the injury site. This study describes the design, synthesis, release kinetics, biosafety, and preclinical efficacy of minocycline (MC)-loaded poly(α-lipoic acid)–methylprednisolone (PαLA-MP) prodrug nanoparticles (NPs) for the combined anti-inflammatory treatment of TSCI.MethodsNPs were produced by conjugating MP to PαLA and then loading MC. The NP structure was confirmed through 1H nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy, ultraviolet–visible absorption spectroscopy, gel permeation chromatography, dynamic light scattering, and transmission electron microscopy. Drug-loading content and efficacy were measured using high-performance liquid chromatography (HPLC) or 1H NMR and release kinetics through HPLC. Biosafety was examined using the MTT assay, cell penetration efficiency using confocal microscopy, and flow cytometry using Cyanine5 (Cy5)-labeled MC-PαLA-MP NPs, effects on injury-induced pro-inflammatory cytokine release using enzyme-linked immunosorbent assays and immunofluorescence, and treatment efficacy by measuring motor recovery in a rat model of TSCI.ResultsThe MC-PαLA-MP NPs exhibited high biocompatibility and released 81% MC and 54% MP within 24 h under TSCI-like conditions, effectively reducing 40% of pro-inflammatory cytokine release both in cultures and injured rat spinal cord tissues. Systemic injection increased the Basso, Beattie, Bresnahan score of TSCI rats from 2.33 ± 0.52 to 8.83 ± 1.83 in 8 weeks, providing effective neuroprotection and enhanced exercise recovery in the TSCI rats.ConclusionThe MC-PαLA-MP NPs can mitigate secondary inflammation and preserve motor function following experimental TSCI, which suggests their potential for clinical application.

Autophagy of Spinal Microglia Affects the Activation of Microglia through the PI3K/AKT/mTOR Signaling Pathway

Delayed paralysis occurs within some patients suffered from ischemic spinal cord injury (ISCI) due to the aorta occlusion during the repair surgery of thoracic and thoracoabdominal aortic aneurysms. Although mild hypothermia has been reported to improve ISCI and prolong the tolerance of rats to ISCI without inducing immediate paralysis, the mechanism remains unclear. Herein, the study revealed that the mild hypothermia treatment indeed partially improved the ISCI in rats caused by cross-clamping at the descending aorta. ISCI induced the excessive activation of microglia and moderate autophagy in the spinal cord tissues of rats, while mild hypothermia significantly induced autophagy and reversed the excessive activation of microglia in the spinal cord tissues of rats. In OGD-stimulated mouse microglia BV-2 cells, the excessive activation of microglia and moderate autophagy were also observed; in the rapamycin-treated OGD model in BV-2 cells, autophagy was significantly enhanced whereas the excessive activation of microglia was reversed. In both in vivo ISCI model in rats and in vitro OGD model in BV-2 cells, the PI3K/AKT/mTOR pathway showed to be inhibited, whereas the PI3K/AKT/mTOR pathway was further inhibited by mild hypothermia in ISCI rats or rapamycin treatment in OGD-stimulated BV-2 cells. In conclusion, enhanced autophagy might be the mechanism of inhibited microglia activation by hypothermia treatment in ISCI rats and by rapamycin treatment in OGD-stimulated BV-2 cells. Autophagy could be enhanced through inhibiting the PI3K/AKT/mTOR pathway.

Bioinformatics analysis of the wheel treadmill test on motor function recovery after spinal cord injury.

This study aimed to explore the possible target and mechanism of the wheel treadmill (WTM) test for motor function recovery of spinal cord injury (SCI). Rats were divided into sham, control and WTM groups to establish an SCI mode. Rats in the WTM group were trained on the WTM test, and Basso-Beattie-Bresnahan (BBB) scores were determined. The samples were collected, and mRNA sequencing was conducted to determine the changes in gene expression. The coexpressed genes were screened to construct a protein-protein interaction (PPI), followed by the Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology function enrichment analysis, and the differentially expressed genes (DEGs) volcano map and hub gene expression heat map were constructed using R language. The BBB scores in the control and WTM groups increased with time, with the WTM group scoring higher than the control group. The results of rat spinal cord tissue sequencing showed that a total of 1679 DEGs were screened in the sham and control groups; 928 DEGs and 731 overlapping genes were screened in the WTM and control groups. The key genes were identified by PPI analysis. One hundred and thirty-three genes were found to be overlapping by combined analysis of spinal cord sequencing data and BBB scores of rats at Week 7. The top 10 DEGs from high to low were Tyrobp, Rac2, Cd68, C1qb, Aif1, Cd74, Spi1, Fcer1g, RT1-DA, and Ccl4. The terms with the highest enrichment scores were microglia-mediated positive regulation of cytotoxicity and major histocompatibility complex class II protein complexes. Treatment with the WTM test promotes recovery of motor function after SCI in rats by modulating intercellular communication and immune function.

Insulin growth factor-1 enhances proliferation and inhibits apoptosis of neural progenitor cells by phosphorylation of Akt/mTOR/p70S6K molecules and triggering intrinsic apoptosis signaling pathway

Neural progenitor cells (NPCs) transplantation is known as a potential strategy for treating spinal cord injury (SCI). This study aimed to investigate effects of insulin growth factor-1 (IGF-I) on NPCs proliferation and clarify associated mechanisms. NPCs isolated from T8-T10 segmental spinal cord tissues of rats were cultured and identification. Then, lentivirus packing plasmids containing IGF-I was constructed and used for NPCs infection. Cell proliferation was evaluated by detecting 5-Bromodeoxyuridine (BrdU) expression in NPCs, cell differentiation was detected using double-labeling immunofluorescence staining while cell apoptosis was detected using TUNEL assay. In addition, the signal expression of Akt/mTOR/p70S6K in NPCs cells were investigated using immunofluorescence staining and western blot assay. The experimental group was defined as pCMV-IGF-I group, while the negative control group was defined as pCMV-LacZ group. Cells infected with pCMV-IGF-I lentivirus followed by addition of 100mg/ml rapamycin were defined as pCMV-IGF-I + Rapa group. NPCs were successfully isolated, identified and cultured. IGF-I overexpression significantly inhibited cell apoptosis and enhanced cell migration. Akt/mTOR/ p70S6K signaling cascade was proved to be present in NPCs, IGF-I overexpression significantly activated Akt/mTOR/p70S6K signaling cascade, while rapamycin addition inhibited its expression. Also, the activated Akt/mTOR/p70S6K signal cascade induced by IGF-I significantly enhanced BrdU expression and inhibited cell apoptosis, and promoted the differentiation of NPC into the neuronal system. However, the rapamycin addition inhibited the cell response induced by IGF-I overexpression. IGF-I overexpression could enhance cell proliferation, inhibit cell apoptosis and promote their differentiation into neuronal systems by activating Akt/mTOR/p70S6K signaling cascade in vitro, indicating that the Akt/mTOR/p70S6K signaling cascade may be the potentially mechanism for the endogenous repair and remodeling of spinal cord after injury.

Repair of spinal cord injury in rats via exosomes from bone mesenchymal stem cells requires sonic hedgehog.

ObjectiveThe loss of neural ability leading to subsequent diminishing of motor function and the impairment below the location of the injury is a result of the SCI (Spinal Cord Injury). Among the many therapeutic agents for SCI, the exosomes considered as extracellular vesicles seem to be the most promising. Sonic Hedgehog (Shh) is an exosome-carrying protein. This Study's purpose was to identify whether Shh is required for exosomes from BMSCs (mesenchymal stem cells of the bone) and plays a protective effect on SCI.MethodsSpinal cord injection with shRNA Shh-adeno associated virus (sh-Shh-AAV) were used to silence Shh. Exosomes were extracted from BMSCs. Rats that had suffered SCI were given intravenous injections of exosomes through the veins of the tail. Immunohistochemistry was used to identify the expression of Shh glycoprotein molecule as well as the expression of Gli-1 (glioma-associated oncogene homolog 1) in the rat spinal cord tissues. Western blot was performed to measure the levels of growth associated protein-43 (GAP-43). The BBB (Basso Beattie Bresnahan) score was used to assess the motor functions of the hind legs. In the same manner, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling or TUNEL and Nissl Staining was deployed to assess the level of regeneration of neurons and assess the level of histopathological damage in the tissues of the Spinal Cord.ResultsIn the case of the rats with SCI, the levels of display of Gli-1 and Shh showed dramatic improvement after the BMSCs exosome injections. In comparison to rats with SCI, the subjects of BMSCs exosomes group showed an improvement in their SCI, including a higher BBB score and Nissl body count, increasing GAP-43 expression, along with a much-decreased number of cells that suffered apoptosis. While the exosome effect on Spinal Cord Injury was completely ineffective in rats that had Shh silencing.ConclusionsExosomes secreted from BMSCs showed great effectiveness in the SCI healing with a vital involvement of Shh in this repair.

Characterization of ultrasound-mediated delivery of trastuzumab to normal and pathologic spinal cord tissue

Extensive studies on focused ultrasound (FUS)-mediated drug delivery through the blood–brain barrier have been published, yet little work has been published on FUS-mediated drug delivery through the blood-spinal cord barrier (BSCB). This work aims to quantify the delivery of the monoclonal antibody trastuzumab to rat spinal cord tissue and characterize its distribution within a model of leptomeningeal metastases. 10 healthy Sprague–Dawley rats were treated with FUS + trastuzumab and sacrificed at 2-h or 24-h post-FUS. A human IgG ELISA (Abcam) was used to measure trastuzumab concentration and a 12 ± fivefold increase was seen in treated tissue over control tissue at 2 h versus no increase at 24 h. Three athymic nude rats were inoculated with MDA-MB-231-H2N HER2 + breast cancer cells between the meninges in the thoracic region of the spinal cord and treated with FUS + trastuzumab. Immunohistochemistry was performed to visualize trastuzumab delivery, and semi-quantitative analysis revealed similar or more intense staining in tumor tissue compared to healthy tissue suggesting a comparable or greater concentration of trastuzumab was achieved. FUS can increase the permeability of the BSCB, improving drug delivery to specifically targeted regions of healthy and pathologic tissue in the spinal cord. The achieved concentrations within the healthy tissue are comparable to those reported in the brain.