Cells In Spinal Cord Research Articles

A rare case of Hirayama disease

Introduction: Hirayama disease is a form of cervical myelopathy common in male adolescents that affects distal upper extremities & anterior horn cells of spinal cord. It is a symmetrical lower motor weakness that is self-contained in hands & forearms.Materials and Methods: Observational type of case study using MRI. 16 years male presented with inability to move his right index finger & decreased sensations for 3 monthsResults: T2 sagittal images showed mild atrophy with flattening of cervical cord & displacement of posterior dura with compression on cord on dynamic flexion. Cord diameter was reduced suggestive of compression.Conclusion: Based on the patient's clinical, electrophysiological, & radiological characteristics,

Dynamic 3D Combinatorial Generation of hPSC-Derived Neuromesodermal Organoids With Diverse Regional and Cellular Identities.

Neuromesodermal progenitors represent a unique, bipotent population of progenitors residing in the tail bud of the developing embryo, which give rise to the caudal spinal cord cell types of neuroectodermal lineage as well as the adjacent paraxial somite cell types of mesodermal origin. With the advent of stem cell technologies, including induced pluripotent stem cells (iPSCs), the modeling of rare genetic disorders can be accomplished in vitro to interrogate cell-type specific pathological mechanisms in human patient conditions. Stem cell-derived models of neuromesodermal progenitors have been accomplished by several developmental biology groups; however, most employ a 2D monolayer format that does not fully reflect the complexity of cellular differentiation in the developing embryo. This article presents a dynamic 3D combinatorial method to generate robust populations of human pluripotent stem cell-derived neuromesodermal organoids with multi-cellular fates and regional identities. By utilizing a dynamic 3D suspension format for the differentiation process, the organoids differentiated by following this protocol display a hallmark of embryonic development that involves a morphological elongation known as axial extension. Furthermore, by employing a combinatorial screening assay, we dissect essential pathways for optimally directing the patterning of pluripotent stem cells into neuromesodermal organoids. This protocol highlights the influence of timing, duration, and concentration of WNT and fibroblast growth factor (FGF) signaling pathways on enhancing early neuromesodermal identity, and later, downstream cell fate specification through combined synergies of retinoid signaling and sonic hedgehog activation. Finally, through robust inhibition of the Notch signaling pathway, this protocol accelerates the acquisition of terminal cell identities. This enhanced organoid model can serve as a powerful tool for studying normal developmental processes as well as investigating complex neurodevelopmental disorders, such as neural tube defects. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Robust generation of 3D hPSC-derived spheroid populations in dynamic motion settings Support Protocol 1: Pluronic F-127 reagent preparation and coating to generate low-attachment suspension culture dishes Basic Protocol 2: Enhanced specification of hPSCs into NMP organoids Support Protocol 2: Combinatorial pathway assay for NMP organoid protocol optimization Basic Protocol 3: Differentiation of NMP organoids along diverse cellular trajectories and accelerated terminal fate specification into neurons, neural crest, and sclerotome derivatives.

Single-cell RNA sequencing reveals dysregulation of spinal cord cell types in a severe spinal muscular atrophy mouse model.

Although spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival of motor neuron (SMN) proteins, there is growing evidence that non-neuronal cells play important roles in SMA pathogenesis. However, transcriptome alterations occurring at the single-cell level in SMA spinal cord remain unknown, preventing us from fully comprehending the role of specific cells. Here, we performed single-cell RNA sequencing of the spinal cord of a severe SMA mouse model, and identified ten cell types as well as their differentially expressed genes. Using CellChat, we found that cellular communication between different cell types in the spinal cord of SMA mice was significantly reduced. A dimensionality reduction analysis revealed 29 cell subtypes and their differentially expressed gene. A subpopulation of vascular fibroblasts showed the most significant change in the SMA spinal cord at the single-cell level. This subpopulation was drastically reduced, possibly causing vascular defects and resulting in widespread protein synthesis and energy metabolism reductions in SMA mice. This study reveals for the first time a single-cell atlas of the spinal cord of mice with severe SMA, and sheds new light on the pathogenesis of SMA.

Liver X Receptor Regulation of Glial Cell Functions in the CNS.

In this review, we discuss the role of liver X receptors (LXRs) in glial cells (microglia, oligodendrocytes and astrocytes) in the central nervous system (CNS). LXRs are oxysterol-activated nuclear receptors that, in adults, regulate genes involved in cholesterol homeostasis, the modulation of inflammatory responses and glutamate homeostasis. The study of LXR knockout mice has revealed that LXRβ plays a key role in maintaining the health of dopaminergic neurons in the substantia nigra, large motor neurons in the spinal cord and retinal ganglion cells in the eye. In the peripheral nervous system (PNS), LXRβ is responsible for the health of the spiral ganglion neurons (SGNs) in the cochlea. In addition, LXRs are essential for the homeostasis of the cerebrospinal fluid (CSF), and in LXRαβ−/− mice, the lateral ventricles are empty and lined with lipid-laden cells. As LXRαβ−/− mice age, lipid vacuoles accumulate in astrocytes surrounding blood vessels. By seven months of age, motor coordination becomes impaired, and there is a loss of motor neurons in the spinal cord of LXRβ−/− mice. During development, migration of neurons in the cortex and cerebellum is retarded in LXRβ−/− mice. Since LXRs are not expressed in dopaminergic or motor neurons in adult mice, the neuroprotective effects of LXRs appear to come from LXRs in glial cells where they are expressed. However, despite the numerous neurological deficits in LXR−/− rodents, multiple sclerosis has the clear distinction of being the only human neurodegenerative disease in which defective LXR signaling has been identified. In this review, we summarize the regulation and functions of LXRs in glial cells and analyze how targeting LXRs in glial cells might, in the future, be used to treat neurodegenerative diseases and, perhaps, disorders caused by aberrant neuronal migration during development.

P13. Transduction of the rat spinal cord using adenoassociated virus serotype 2.5

<h3>BACKGROUND CONTEXT</h3> Gene therapy is a method by which exogenous genes are introduced to treat disease or deliver drugs of interest. This is commonly done through either direct gene delivery in vivo or ex vivo through transduction of cells that are to be transplanted. There are several vectors for delivering genes, including viral vectors, naked plasmids, electroporation and lipid nanoparticles. Adeno-associated viruses (AAVs) are commonly used and considered to be well tolerated by the brain, muscles, nerves and multiple cell types. Rat Schwann cells are transduced efficiently by AAV1 whereas human Schwann cells are better transduced with AAV2 and 6. Rat nerves segments have demonstrated to be efficiently transduced by AAV1, 5, 7 and 9 whereas human nerves prefer AAV2. Many studies also agree that AAV1, 2, 4, 8 and 9 are optimal serotypes for CNS. We have utilized a novel viral vector, AAV serotype 2.5, that is a chimera of AAV2 vector modified with 5 amino acids from AAV1. AAV2.5 has shared characteristics of AAV1 and AAV2 with enhanced transduction for multiple cell types, such as in the muscle and articular tissue. Since AAV2.5 is not a naturally occurring virus, there is lower cross-reactivity to AAV2 antibodies. However, the ability of AAV2.5 to transduce tissues of the central (CNS) and peripheral nervous system (PNS) is not well understood. <h3>PURPOSE</h3> The purpose of this study is to determine the trophism and transduction efficiency of AAV2.5 for CNS and PNS tissue through in vitro transduction of dorsal root ganglion derived neurons, Schwann cells, and in vivo transduction of spinal cord. We predict that since AAV1 and AAV2 have known tropism for CNS and PNS cells to different extents depending on cell type, that the chimera AAV2.5 would be broadly effective. <h3>STUDY DESIGN/SETTING</h3> Cell culture and animal study. <h3>METHODS</h3> The cDNA encoding green fluorescent protein (GFP) was cloned into the conventional AAV packaging vector pTRUF2. For generation of self-complementing AAV vector plasmids, the cDNAs for GFP were directionally inserted into the SacII, NotI sites of pHpa-trs-SK plasmid. Transcription was driven by the cytomegalovirus promoter. Primary Schwann cells were isolated from the sciatic nerves of postnatal day 5 rat pups and dorsal root ganglion (DRG) neurons from embryonic day 15 rat pups. The cells were plated at a density of 125,000 cells per well and infected with 104 viral genomes per cell. For in vivo transduction of spinal cord tissue, 4 µL of 5 × 106– 5 × 1010vg/mL of AAV2.5-GFP was injected into 4 positions at T9. Immunostaining for markers of different spinal cord cell types colabeled with anti-GFP was done. <h3>RESULTS</h3> AAV2.5 was able to successfully infect DRG neurons and Schwann cells. AAV-2.5 vector was much more efficient than the commonly used AAV2 vector at transducing the Schwann cells. Cell survival and gene product production by AAV2.5 was greater than electroporation. When AAV2.5 -GFP was injected into the spinal cord there was a dose dependent increase in GFP expression by neurons and astrocytes up to 4 mm away from the injection site. <h3>CONCLUSIONS</h3> The study presents a novel vector for broad transduction in vivo or ex vivo of cells in the CNS and PNS. AAV2.5 was more efficient than the more commonly used AAV2 vector or electroporation. Since AAV2.5 is presently in phase 1 trials for osteoarthritis (NCT02790723), this vector type presents an interesting clinical translation opportunity for spinal cord and peripheral nerve injuries/diseases. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Intra-amniotic transplantation of brain-derived neurotrophic factor-modified mesenchymal stem cells treatment for rat fetuses with spina bifida aperta

BackgroundSpina bifida aperta (SBA) is a relatively common clinical type of neural tube defect. Although prenatal fetal surgery has been proven to be an effective treatment for SBA, the recovery of neurological function remains unsatisfactory due to neuron deficiencies. Our previous results demonstrated that intra-amniotic transplanted bone marrow mesenchymal stem cells (BMSCs) could preserve neural function through lesion-specific engraftment and regeneration. To further optimize the role of BMSCs and improve the environment of defective spinal cords so as to make it more conducive to nerve repair, the intra-amniotic transplanted BMSCs were modified with brain-derived neurotrophic factor (BDNF-BMSCs), and the therapeutic potential of BDNF-BMSCs was verified in this study.MethodsBMSCs were modified by adenovirus encoding a green fluorescent protein and brain-derived neurotrophic factor (Ad-GFP-BDNF) in vitro and then transplanted into the amniotic cavity of rat fetuses with spina bifida aperta which were induced by all-trans-retinoic acid on embryonic day 15. Immunofluorescence, western blot and real-time quantitative PCR were used to detect the expression of different neuron markers and apoptosis-related genes in the defective spinal cords. Lesion areas of the rat fetuses with spina bifida aperta were measured on embryonic day 20. The microenvironment changes after intra-amniotic BDNF-BMSCs transplantation were investigated by a protein array with 90 cytokines.ResultsWe found that BDNF-BMSCs sustained the characteristic of directional migration, engrafted at the SBA lesion area, increased the expression of BDNF in the defective spinal cords, alleviated the apoptosis of spinal cord cells, differentiated into neurons and skin-like cells, reduced the area of skin lesions, and improved the amniotic fluid microenvironment. Moreover, the BDNF-modified BMSCs showed a better effect than pure BMSCs on the inhibition of apoptosis and promotion of neural differentiation.ConclusionThese findings collectively indicate that intra-amniotic transplanted BDNF-BMSCs have an advantage of promoting the recovery of defective neural tissue of SBA fetuses.

CircRNA3616 knockdown attenuates inflammation and apoptosis in spinal cord injury by inhibiting TLR4/NF-κB activity via sponging miR-137.

The present work focused on exploring the role of circRNA3616 in neuronal inflammation and apoptosis in spinal cord injury (SCI). The SCI mouse model and circRNA3616 knockdown SCI mouse model were established. This work focused on assessing the mouse locomotor function using Basso Mouse Scale (BMS) and BMS subscore. Hematoxylin-eosin (HE) staining and Tunel staining were conducted, while myeloperoxidase (MPO) activity was also detected on spinal cord tissues. We also knocked down circRNA3616 expression in NSC-34 cells. Meanwhile, the SCI cell model was established by oxygen glucose deprivation (OGD) in NSC-34 cells. Moreover, we conducted dual-luciferase reporter gene assay. Flow cytometry (FCM) was conducted to detect SCI cell apoptosis, whereas cell counting kit-8 (CCK-8) assay was performed to analyze cell viability. This study also implemented enzyme-linked immunosorbent assay to detect inflammatory factors in spinal cord tissues, serum, and cells. CircRNA3616 knockdown reduced the damage, inflammatory response, apoptosis, and MPO activity in SCI mouse serum and spinal cord tissues. CircRNA3616 knockdown increased BMS and BMS subscore of SCI mice. CircRNA3616 up-regulated TLR4 expression by sponging miR-137. CircRNA3616 knockdown inhibited the TLR4, p-IkBα, p-p65/p65 protein expression, while promoting IkBα protein expression within SCI mouse spinal cord. TLR4 reversed circRNA3616 knockdown-induced inhibition on NF-κB pathway activity in SCI cells. CircRNA3616 knockdown attenuated neuronal cell inflammation and apoptosis via TLR4/NF-κB pathway after SCI. CircRNA3616 silencing attenuates inflammation and apoptosis in SCI by inhibiting TLR4/NF-κB activity via sponging miR-137. CircRNA3616 is the possible anti-SCI therapeutic target.

Identification of Novel Multipotent Stem Cells in Mouse Spinal Cord Following Traumatic Injury.

We showed that injury-induced multipotent stem cells (iSCs) emerge in the brain after stroke. These brain-derived iSCs (B-iSCs) can differentiate into various lineages, including neurons. This study aimed to determine whether similar stem cells can be induced even after nonischemic injuries, such as trauma to the spinal cord. We characterized these cells, mainly focusing on their stemness, multipotency, and neuronal differentiation activities. Spinal cord injury (SCI) was produced using forceps in adult mice. On day 3 after SCI, samples were obtained from the injured areas. Spinal cord sections were subjected to histological analyses. Cells were isolated and assessed for proliferative activities, immunohistochemistry, reverse transcriptase-polymerase chain reaction, fluorescence-activated cell sorter, and microarray analysis. Although nerve cell morphology was disrupted within the injured spinal cord, our histological observations revealed the presence of cells expressing stem cells, such as nestin and Sox2 in these areas. In addition, cells extracted from injured areas exhibited high proliferative abilities. These cells also expressed markers of both neural stem cells (eg, nestin, Sox2) and multipotent stem cells (eg, Sox2, c-myc, Klf4). They differentiated into adipocytes, osteocytes, and chondrocytes, as well as neuronal cells. Microarray analysis further identified similar properties between spinal cord (SC)-derived iSCs and B-iSCs. However, SC-iSCs revealed specific genes related to the regulation of stemness and neurogenesis. We identified similar features related to multipotency in SC-iSCs compared with B-iSCs, including neuronal differentiation potential. Although the differences between SC-iSCs and B-iSCs remain largely undetermined, this study shows that iSCs can develop even after nonischemic injuries such as trauma. This phenomenon can occur outside the brain within the central nervous system.

In Vivo Chemogenetic Biocompatibility of Mercury as a Specific Hypercalcemia Actuator in Snail's Spinal Cord Cell Manipulation: An Extracellular Field Potential Biosensor.

The in vivo chemogenetic property of mercuric ions (Hg2+) was investigated as a specific hypercalcemia actuator in snail's spinal cord cell manipulation by extracellular field potential biosensing analysis. For this purpose, a three-microelectrode system with working, counter, and pseudo reference electrodes was blindly implanted into the snail's spinal cord to electrically stimulate (triggering) the action potential with a staircase electrical voltage at a very low frequency level, along with measurement of the electrical current, as a detection system. Under optimum conditions, using the one-factor-at-a-time method, a wide linear range between 1.0 × 10-14 and 1.0 × 10-1 mol L-1 with correlation coefficients (R2) >0.98 and a response time (t90) of maximum 10.0 s were approximated. Percentages of relative standard deviation were estimated to be 3.08 (reproducibility, n = 50) and 7.31 (repeatability, n = 15). The detection limit was estimated to be sub 2.1 × 10-16 mol L-1 based on the Xb- + 3Sb definition. The reliability of this phenomenon was evidenced by the estimation of recovery percentages (between 95 and 107%) during spiking Hg2+ standard solutions. The probable mechanism behind this process could be attributed to the following: (i) the neuronal ephaptic coupling during electrical synchronization by a specific brain-triggered wave as a neuronal motor toolkit and (ii) chemical synchronization using a Hg2+ hypercalcemia actuator (biosensor). Linear correlation has been evidenced during interactions between Hg2+ and a calcium ionic channel's protein with a gram molecular weight of 66.2 ± 0.3 KCU. This process, therefore, caused an opening of the Ca2+ channel gates and majorly released the Ca2+ (hypercalcemia) that was detected as the main source of the measured electrical current. At this condition, ultratrace levels of Hg2+ ions not only were considered as nontoxic reagents but also had chemically regulating effects as ephaptic synchronizers to the neuron cells. This report may pave the way for using mercury ions at an ultratrace level for clinical controlling purposes during neuronal spinal cord cell manipulation.

Blocking SP/NK1R signaling improves spinal cord hemisection by inhibiting the release of pro-inflammatory cytokines in rabbits

Objective Incomplete spinal cord injury (SCI) is the most common spinal cord injury in clinic, however its mechanism is still not fully understood. Design We constructed the rabbit spinal cord hemisection (SCH) model and used RT–PCR, western blotting, immunohistochemistry, and immunofluorescence experiments to explore the potential mechanism of SCI. Setting The sham operation (SH) group, the observation (OB, which is the SCH) group, the OB+ substance p (SP) inhibitor group, the OB + NK1R inhibitor group, the OB + NK1R agonist group and the OB + SP inhibitor + NK1R agonist group. Participants New Zealand white rabbits. Interventions Use NK1R inhibitors, NK1R agonists, SP inhibitors to treat the SCH model. Outcome Measures IL-1β, IKKγ, IL-6 and NF-κB. Results The results showed that nissl bodies, inflammatory cells and SP increased notably in the spinal cord cells of the rabbit SCH model. Through in vivo experiments with SP or NK1R inhibitors or NK1R agonists, we found that inhibiting SP/NK1R signaling can help improve SCH by inhibiting the release of pro-inflammatory cytokines IL-1β, IKKγ, IL-6 and NF-κB. Registered trials Animal experiments were approved by Ruijin Hospital, Shanghai Jiaotong University School of Medicine.

转录因子Nkx6.1对脊髓损伤后中间神经元和星形胶质细胞发生的影响

<p indent="0mm">Interneurons are the main local loop connecting sensory and motor neurons and play an important role in the pathological changes after spinal cord injury and the corresponding repair process. After spinal cord injury, astrocyte proliferation and glial scar formation are the main factors hindering the effective regeneration and growth of injured spinal cord axons. In the early stage of neurogenesis, neuroepithelial cells produce astrocytes and intermediate neurons. Therefore, it is likely that astrocytes and intermediate neurons arise from common progenitor cells. In the spinal cord, neuron and glial cell differentiation is regulated by a transcription factor network. In particular, Nkx6.1 is critical for the fate determination of progenitor cells into ventral interneurons. However, the role of Nkx6.1 in the birth of spinal astrocytes after spinal cord injury is still unclear. Herein, we first used the Cre-LoxP system and Rosa26 gene knock-in technology to specifically induce an <italic>Nkx6</italic>.<italic>1</italic> knockout and introduce a lycopene marker gene in different neural precursor cells of adult mice’s spinal cord. Next, we constructed a knockout spinal cord injury model and performed immunofluorescence staining. The staining results showed a substantial number of Nkx6.1⁺ cells in the injured area after spinal cord injury, while <italic>Nkx6</italic>.<italic>1</italic> ablation one week after spinal cord injury would hinder the formation of the glial scar and inhibit the production of En1⁺ intermediate neurons by regulating the expression pattern of Pax6<italic> </italic>in the central neural tube. In conclusion, our study shows that <italic>Nkx6</italic>.<italic>1</italic> plays a crucial role in spinal cord injury, being responsible for effective axon regeneration and motor function recovery.

Comparing effects of microgravity and amyotrophic lateral sclerosis in the mouse ventral lumbar spinal cord

Microgravity (MG) exposure and motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), lead to motor deficits, including muscle atrophy and loss of neuronal activity. Abnormalities in motor neurons and muscles caused by MG exposure can be recovered by subsequent ground exercise. In contrast, the degeneration that occurs in ALS is irreversible. A common phenotype between MG exposure and ALS pathology is motor system abnormality, but the causes may be different. In this study, to elucidate the motor system that is affected by each condition, we investigated the effects of MG and the human SOD1 ALS mutation on gene expression in various cell types of the mouse ventral lumbar spinal cord, which is rich in motor neurons innervating the lower limb. To identify cell types affected by MG or ALS pathogenesis, we analyzed differentially expressed genes with known cell-type markers, which were determined from previous single-cell studies of the spinal cord in MG-exposed and SOD1G93A mice, an ALS mouse model. Differentially expressed genes were observed in MG mice in various spinal cord cell types, including neurons, microglia, astrocytes, oligodendrocytes, oligodendrocyte precursor cells, meningeal cells/Schwann cells, and vascular cells. We also examined neuronal populations in the spinal cord. Gene expression in putative excitatory and inhibitory neurons changed more than that in cholinergic motor neurons of the spinal cord in both MG and SOD1G93A mice. Many putative neuron types, especially visceral motor neurons, and axon initial segments (AIS) were affected in MG mice. In contrast, the effect on neurons and AIS in SOD1G93A mice was slight at P30 but progressed with aging. Interestingly, changes in dopaminergic system-related genes were specifically altered in the spinal cord of MG mice. These results indicate that MG and ALS pathology in various cell types contribute to motor neuron degeneration. Furthermore, there were more alterations in neurons in MG-exposed mice than in SOD1G93A mice. A large number of differentially expressed genes (DEGs) in MG mice represent more than SOD1G93A mice with ALS pathology. Elucidation of MG pathogenesis may provide more insight into the pathophysiology of neurodegenerative diseases.

Radial Glia and Neuronal-like Ependymal Cells Are Present within the Spinal Cord of the Trunk (Body) in the Leopard Gecko (Eublepharis macularius).

As is the case for many lizards, leopard geckos (Eublepharis macularius) can self-detach a portion of their tail to escape predation, and then regenerate a replacement complete with a spinal cord. Previous research has shown that endogenous populations of neural stem/progenitor cells (NSPCs) reside within the spinal cord of the original tail. In response to tail loss, these NSPCs are activated and contribute to regeneration. Here, we investigate whether similar populations of NSPCs are found within the spinal cord of the trunk (body). Using a long-duration 5-bromo-2′-deoxyuridine pulse-chase experiment, we determined that a population of cells within the ependymal layer are label-retaining following a 20-week chase. Tail loss does not significantly alter rates of ependymal cell proliferation within the trunk spinal cord. Ependymal cells of the trunk spinal cord express SOX2 and represent at least two distinct cell populations: radial glial-like (glial fibrillary acidic protein- and Vimentin-expressing) cells; and neuronal-like (HuCD-expressing) cells. Taken together, these data demonstrate that NSPCs of the trunk spinal cord closely resemble those of the tail and support the use of the tail spinal cord as a less invasive proxy for body spinal cord injury investigations.

Differential regulation of L84F SOD1 in motor neurons and skeletal muscles: an insight into ALS pathology

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disorder characterized by progressive deterioration of motor neurons leading to skeletal muscle denervation. Recent evidences have highlighted early involvement of neuromuscular junction (NMJ) in ALS pathogenesis. NMJ is a specialized tripartite chemical synapse which involves a well‐coordinated communication among the presynaptic motor neuron, postsynaptic skeletal muscle and terminal Schwann cells. Despite several evidences, series of events triggering NMJ disassembly in ALS are still obscure.The aim of our study is to investigate the cellular and molecular mechanisms of NMJ dysfunction in an in vitro Superoxide Dismutase 1 (SOD1) induced model of familial ALS. We have generated an ALS cell model based on a novel SOD1 mutation (L84F) identified previously in a family with a history of ALS. This mutation caused decrease in neurite outgrowth which led us to hypothesize that it may be involved in neuromuscular junction disassembly in ALS pathology.We have characterized cellular components of NMJ by differentiating hybrid spinal cord cell line (NSC‐34) into motor neurons and myoblast cell line (C2C12) into myotubes. Expression of mutant SOD1 led to decrease in mitochondrial footprints in axons and synaptic terminals and altered mitochondrial dynamics in motor neurons. However mitochondrial fission/fusion is not altered in muscles suggesting differential consequences of mutant protein. L84F SOD1 mutant readily formed detergent‐resistant aggregates in motor neurons but failed to accumulate in skeletal muscles. Skeletal muscles displayed increased levels of LC3‐II compared to motor neurons suggesting a robust proteasome machinery of muscles is responsible for clearance of mutant protein. Furthermore, motor neurons exhibited increased cell death compared to skeletal muscles when subjected to exogenous stress. Interestingly, we observed that condition media from skeletal muscles was able to rescue motor neurons from exogenous stress. We are currently identifying paracrine neuroprotective factors secreted by the muscles. It is noteworthy that mutant SOD1 led to altered expression of post synaptic acetylcholine receptor complex which might hamper neuromuscular transmission in ALS.To conclude, molecular cues from both neurons and muscles appear to play a role in compromising NMJ integrity in ALS.

Cell type specific isolation of primary astrocytes and microglia from adult mouse spinal cord

BackgroundAstrocytes and microglia are essential cellular elements of the CNS that are critical for normal development, function, and injury responses. Both cell types are highly pleiotropic and respond rapidly to environmental changes, making them challenging to characterize. One approach is to develop efficient isolation paradigms of distinct cell populations, allowing for characterization of their roles in distinct CNS regions and in pathological states. New methodWe have developed an efficient and reliable protocol for isolation of astrocytes and microglia from the adult mouse spinal cord, which can be easily manipulated for immediate or future analyses. This method involves (1) rapid tissue dissociation; (2) cell release after myelin debris removal; (3) magnetic-activated cell sorting; and (4) optional downstream molecular and functional analyses. ResultsHigh levels of viability and purity of the cells were confirmed after isolation. More importantly, characterization of cells verified their ability to proliferate and respond to external stimuli for potential use in downstream molecular and functional assays. Comparison with existing method(s)Long-term culture of cells isolated from neonatal animals and cell type specific isolation from the brain have been successful; however, isolation of spinal cord cells from adult mice has been challenging due to the large amount of myelin and limited size of the tissue compared to the brain. Our method allows for efficient isolation of astrocytes and microglia from spinal cord alone and includes simple modifications to allow for various downstream applications. ConclusionsThis technique will be a valuable tool to better understand the functions of astrocytes and microglia in spinal cord function and pathology.

Mechanism of the Curative Effect of Wen-Shen-Jian-Pi Prescription in the Treatment of Amyotrophic Lateral Sclerosis.

ObjectiveTo study the mechanism of the effect of Wen-Shen-Jian-Pi (WSJP) prescription on an ALS model comprising mice knocked out for an encoding RNA editing, mice (AR2).MethodsTwenty-four transgenic AR2 mice were randomly divided into a vehicle group, a low dose WSJP group (15 mg), a medium-dose WSJP group (30 mg), and a high-dose WSJP group (45 mg) (all n = 6 per group). In the treatment groups, the WSJP prescription was given once a day while the vehicle group was fed the same volume of water. The weekly changes in body weight, rotarod test, and grip strength were used to detect the changes in the AR2 and changes of the number of normal mitochondria, abnormal mitochondria, and autophagosomes in injured spinal cord cells were used to evaluate the pathogenetic effects of WSJP treatment.ResultsThe WSJP-treated AR2 mice gained weight more quickly from 8 weeks, and showed active behavior and displayed significantly better constant rotarod scores and grip strengths during the experiment compared with those of the vehicle AR2 mice. The number of normal mitochondria in the WSJP-treated AR2 mice had significantly more normal mitochondria than the vehicle group, while the numbers of abnormal mitochondria and autophagosomes were greatly decreased compared with those in the vehicle group.ConclusionThe WSJP prescription could delay the decline in motor function of ALS model mice by reducing the degeneration of neurons. The potential of WSJP to treat ALS should be assessed in a clinical trial.

Astrocytes and Microglia Exhibit Cell-Specific Ca2+ Signaling Dynamics in the Murine Spinal Cord.

The spinal cord is the main pathway connecting brain and peripheral nervous system. Its functionality relies on the orchestrated activity of both neurons and glial cells. To date, most advancement in understanding the spinal cord inner mechanisms has been made either by in vivo exposure of its dorsal surface through laminectomy or by acute ex vivo slice preparation, likely affecting spinal cord physiology in virtue of the necessary extensive manipulation of the spinal cord tissue. This is especially true of cells immediately responding to alterations of the surrounding environment, such as microglia and astrocytes, reacting within seconds or minutes and for up to several days after the original insult. Ca2+ signaling is considered one of the most immediate, versatile, and yet elusive cellular responses of glia. Here, we induced the cell-specific expression of the genetically encoded Ca2+ indicator GCaMP3 to evaluate spontaneous intracellular Ca2+ signaling in astrocytes and microglia. Ca2+ signals were then characterized in acute ex vivo (both gray and white matter) as well as in chronic in vivo (white matter) preparations using MSparkles, a MATLAB-based software for automatic detection and analysis of fluorescence events. As a result, we were able to segregate distinct astroglial and microglial Ca2+ signaling patterns along with method-specific Ca2+ signaling alterations, which must be taken into consideration in the reliable evaluation of any result obtained in physiological as well as pathological conditions. Our study revealed a high degree of Ca2+ signaling diversity in glial cells of the murine spinal cord, thus adding to the current knowledge of the astonishing glial heterogeneity and cell-specific Ca2+ dynamics in non-neuronal networks.

Ca2+ signaling–mediated low-intensity pulsed ultrasound–induced proliferation and activation of motor neuron cells

Motor neuron diseases (MND) including amyotrophic lateral sclerosis and Parkinson disease are commonly neurodegenerative, causing a gradual loss of nerve cells and affecting the mechanisms underlying changes in calcium (Ca2+)-regulated dendritic growth. In this study, the NSC-34 cell line, a population of hybridomas generated using mouse spinal cord cells with neuroblastoma, was used to investigate the effect of low-intensity pulsed ultrasound (LIPUS) as part of an MND treatment model. After NSC-34 cells were seeded for 24 h, LIPUS stimulation was performed on the cells at days 1 and 3 using a non-focused transducer at 1.15 MHz for 8 min. NSC-34 cell proliferation and morphological changes were observed at various LIPUS intensities and different combinations of Ca2+ channel blockers. The nuclear translocation of Ca2+-dependent transcription factors was also examined. We observed that the neurite outgrowth and cell number of NSC-34 significantly increased with LIPUS stimulation at days 2 and 4, which may be associated with the treatment’s positive effect on the activation of Ca2+-dependent transcription factors, such as nuclear factor of activated T cells and nuclear factor-kappa B. Our findings suggest that the LIPUS-induced Ca2+ signaling and transcription factor activation facilitate the morphological maturation and proliferation of NSC-34 cells, presenting a promising noninvasive method to improve stimulation therapy for MNDs in the future.

Metformin Protects against Spinal Cord Injury and Cell Pyroptosis via AMPK/NLRP3 Inflammasome Pathway.

Spinal cord injury (SCI) is an extreme neurological impairment with few effective drug treatments. Pyroptosis is a recently found and proven type of programmed cell death that is characterized by a reliance on inflammatory caspases and the release of a large number of proinflammatory chemicals. Pyroptosis differs from other cell death mechanisms such as apoptosis and necrosis in terms of morphological traits, incidence, and regulatory mechanism. Pyroptosis is widely involved in the occurrence and development of SCI. In-depth research on pyroptosis will help researchers better understand its involvement in the onset, progression, and prognosis of SCI, as well as provide new therapeutic prevention and treatment options. Herein, we investigated the role of AMPK-mediated activation of the NLRP3 inflammasome in the neuroprotection of MET-regulated pyroptosis. We found that MET treatment reduced NLRP3 inflammasome activation by activating phosphorylated AMPK and reduced proinflammatory cytokine (IL-1β, IL-6, and TNF-α) release. At the same time, MET improved motor function recovery in rats after SCI by reducing motor neuron loss in the anterior horn of the spinal cord. Taken together, our study confirmed that MET inhibits neuronal pyroptosis after SCI via the AMPK/NLRP3 signaling pathway, which is mostly dependent on the AMPK pathway increase, hence decreasing NLRP3 inflammasome activation.

Exosome-Shuttled miR-672-5p from Anti-Inflammatory Microglia Repair Traumatic Spinal Cord Injury by Inhibiting AIM2/ASC/Caspase-1 Signaling Pathway Mediated Neuronal Pyroptosis.

Traumatic spinal cord injury (TSCI) is a devastating traumatic disease of the central nervous system, which leads to refractory loss of motor and sensory function. So far, there is no effective treatment for TSCI. Recently, however, nano-sized exosomes from various spinal cord cells have shown great prospects in the treatment of various diseases, including TSCI. Microglia are one of the components of the spinal cord microenvironment. Anti-inflammatory microglia (M2) have been shown to inhibit inflammation and promote the functional recovery of spinal cord after TSCI. However, the role micro RNAs (miRNAs) in exosomes derived from M2 microglia in the treatment of TSCI is unclear. In this study, we investigated whether M2 microglial exosomes (M2-Exos) could better promote the functional behavior recovery of mice with TSCI than M0 microglial exosomes (Exos). Compared with Exos, M2-Exos were found to have a better effect in promoting the recovery of functional behavior, promoting axon regeneration and reducing the level of pyroptosis of spinal cord neurons after TSCI. Through a series of experiments, we also confirmed that miR-672-5p is the most critical miRNA associated with M2-Exos, and that its targeting gene is AIM2. M2-Exos rich in miR-672-5p could inhibit the AIM2/ASC/caspase-1 signaling pathway by inhibiting AIM2 activity, so as to inhibit neuronal pyroptosis and finally promote the recovery of functional behavior in mice with TSCI. In conclusion, our study suggests that the application of M2-Exos may be a promising treatment strategy for TSCI.