Adult Mouse Spinal Cord Research Articles

A method for selective and efficient isolation of gray matter astrocytes from the spinal cord of adult mice

A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2+ cells were divided into two distinct populations, ACSA-2high and ACSA-2low. By analyzing the expression of cell-type marker genes, the ACSA-2high and ACSA-2low populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2high cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.

Evidence for Two Subpopulations of Cerebrospinal Fluid-Contacting Neurons with Opposite GABAergic Signaling in Adult Mouse Spinal Cord.

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl-) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.3%) express the Na+-K+-Cl- cotransporter 1 (NKCC1) allowing intracellular Cl- accumulation. However, we did not find expression of the K+-Cl- cotransporter 2 (KCC2) responsible for Cl- efflux in any CSF-cNs. The lack of KCC2 expression results in low Cl- extrusion capacity in CSF-cNs under high Cl- load in whole-cell patch clamp. Using cell-attached patch clamp allowing recordings with intact intracellular Cl- concentration, we found that the activation of ionotropic GABAA receptors (GABAA-Rs) induced both depolarizing and hyperpolarizing responses in CSF-cNs. Moreover, depolarizing GABA responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. Blocking NKCC1 with bumetanide inhibited the GABA-induced calcium transients in CSF-cNs. Finally, we show that metabotropic GABAB receptors have no hyperpolarizing action on spinal CSF-cNs as their activation with baclofen did not mediate outward K+ currents, presumably due to the lack of expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline subpopulations of spinal CSF-cNs expressing inhibitory or excitatory GABAA-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit.

A Disintegrin And Metalloprotease 10 expression within the murine central nervous system

A Disintegrin And Metalloprotease 10 (ADAM10), is able to control several important physiopathological processes through the shedding of a large number of protein substrates. Although ADAM10 plays a crucial role in the central nervous system (CNS) development and function, its protein distribution in the CNS has not been fully addressed. Here, we described the regional and cellular ADAM10 protein expression in C57BL/6 mice examined by immunofluorescence 1) throughout the adult mouse brain, cerebellum and spinal cord in vivo and 2) in different cell types as neurons, astrocytes, oligodendrocytes and microglia in vitro. We observed ADAM10 expression through the whole CNS, with a strong expression in the hippocampus, in the hypothalamus and in the cerebral and piriform cortex in the brain, in the Purkinje and in granular cell layers in the cerebellum and in the spinal cord to a lower extent. In vivo, ADAM10 protein expression was mainly found in neurons and in some oligodendroglial cell populations. However, in primary cultures we observed ADAM10 expression in neurons, oligodendrocytes, astrocytes and microglia. Interestingly, ADAM10 was not only found in the membrane but also in cytoplasmic vesicles and in the nucleus of primary cultured cells. Overall, this work highlights a wide distribution of ADAM10 throughout the CNS. The nuclear localization of ADAM10, probably due to its intracellular domain, emphasizes its role in cell signalling in physiological and pathological conditions. Further investigations are required to better elucidate the role of ADAM10 in glial cells.

Isolation and monoculture of functional primary astrocytes from the adult mouse spinal cord.

Astrocytes are a widely heterogenic cell population that play major roles in central nervous system (CNS) homeostasis and neurotransmission, as well as in various neuropathologies, including spinal cord injury (SCI), traumatic brain injury, and neurodegenerative diseases, such as amyotrophic lateral sclerosis. Spinal cord astrocytes have distinct differences from those in the brain and accurate modeling of disease states is necessary for understanding disease progression and developing therapeutic interventions. Several limitations to modeling spinal cord astrocytes in vitro exist, including lack of commercially available adult-derived cells, lack of purchasable astrocytes with different genotypes, as well as time-consuming and costly in-house primary cell isolations that often result in low yield due to small tissue volume. To address these issues, we developed an efficient adult mouse spinal cord astrocyte isolation method that utilizes enzymatic digestion, debris filtration, and multiple ACSA-2 magnetic microbead purification cycles to achieve an astrocyte monoculture purity of ≅93-98%, based on all markers assessed. Importantly, the isolated cells contain active mitochondria and express key astrocyte markers including ACSA-1, ACSA-2, EAAT2, and GFAP. Furthermore, this isolation method can be applied to the spinal cord of male and female mice, mice subjected to SCI, and genetically modified mice. We present a primary adult mouse spinal cord astrocyte isolation protocol focused on purity, viability, and length of isolation that can be applied to a multitude of models and aid in targeted research on spinal-cord related CNS processes and pathologies.

Spatial transcriptomics and single-nucleus RNA sequencing reveal a transcriptomic atlas of adult human spinal cord

Despite the recognized importance of the spinal cord in sensory processing, motor behaviors, and neural diseases, the underlying organization of neuronal clusters and their spatial location remain elusive. Recently, several studies have attempted to define the neuronal types and functional heterogeneity in the spinal cord using single-cell or single-nucleus RNA sequencing in animal models or developing humans. However, molecular evidence of cellular heterogeneity in the adult human spinal cord is limited. Here, we classified spinal cord neurons into 21 subclusters and determined their distribution from nine human donors using single-nucleus RNA sequencing and spatial transcriptomics. Moreover, we compared the human findings with previously published single-nucleus data of the adult mouse spinal cord, which revealed an overall similarity in the neuronal composition of the spinal cord between the two species while simultaneously highlighting some degree of heterogeneity. Additionally, we examined the sex differences in the spinal neuronal subclusters. Several genes, such as SCN10A and HCN1, showed sex differences in motor neurons. Finally, we classified human dorsal root ganglia (DRG) neurons using spatial transcriptomics and explored the putative interactions between DRG and spinal cord neuronal subclusters. In summary, these results illustrate the complexity and diversity of spinal neurons in humans and provide an important resource for future research to explore the molecular mechanisms underlying spinal cord physiology and diseases.

Spatial atlas of the mouse central nervous system at molecular resolution

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

A new Hoxb8FlpO mouse line for intersectional approaches to dissect developmentally defined adult sensorimotor circuits.

Improvements in the speed and cost of expression profiling of neuronal tissues offer an unprecedented opportunity to define ever finer subgroups of neurons for functional studies. In the spinal cord, single cell RNA sequencing studies support decades of work on spinal cord lineage studies, offering a unique opportunity to probe adult function based on developmental lineage. While Cre/Flp recombinase intersectional strategies remain a powerful tool to manipulate spinal neurons, the field lacks genetic tools and strategies to restrict manipulations to the adult mouse spinal cord at the speed at which new tools develop. This study establishes a new workflow for intersectional mouse-viral strategies to dissect adult spinal function based on developmental lineages in a modular fashion. To restrict manipulations to the spinal cord, we generate a brain-sparing Hoxb8FlpO mouse line restricting Flp recombinase expression to caudal tissue. Recapitulating endogenous Hoxb8 gene expression, Flp-dependent reporter expression is present in the caudal embryo starting day 9.5. This expression restricts Flp activity in the adult to the caudal brainstem and below. Hoxb8FlpO heterozygous and homozygous mice do not develop any of the sensory or locomotor phenotypes evident in Hoxb8 heterozygous or mutant animals, suggesting normal developmental function of the Hoxb8 gene and protein in Hoxb8FlpO mice. Compared to the variability of brain recombination in available caudal Cre and Flp lines, Hoxb8FlpO activity is not present in the brain above the caudal brainstem, independent of mouse genetic background. Lastly, we combine the Hoxb8FlpO mouse line with dorsal horn developmental lineage Cre mouse lines to express GFP in developmentally determined dorsal horn populations. Using GFP-dependent Cre recombinase viruses and Cre recombinase-dependent inhibitory chemogenetics, we target developmentally defined lineages in the adult. We show how developmental knock-out versus transient adult silencing of the same ROR𝛃 lineage neurons affects adult sensorimotor behavior. In summary, this new mouse line and viral approach provides a blueprint to dissect adult somatosensory circuit function using Cre/Flp genetic tools to target spinal cord interneurons based on genetic lineage.

Neuropeptide Y-expressing dorsal horn inhibitory interneurons gate spinal pain and itch signalling

Somatosensory information is processed by a complex network of interneurons in the spinal dorsal horn. It has been reported that inhibitory interneurons that express neuropeptide Y (NPY), either permanently or during development, suppress mechanical itch, with no effect on pain. Here, we investigate the role of interneurons that continue to express NPY (NPY-INs) in the adult mouse spinal cord. We find that chemogenetic activation of NPY-INs reduces behaviours associated with acute pain and pruritogen-evoked itch, whereas silencing them causes exaggerated itch responses that depend on cells expressing the gastrin-releasing peptide receptor. As predicted by our previous studies, silencing of another population of inhibitory interneurons (those expressing dynorphin) also increases itch, but to a lesser extent. Importantly, NPY-IN activation also reduces behavioural signs of inflammatory and neuropathic pain. These results demonstrate that NPY-INs gate pain and itch transmission at the spinal level, and therefore represent a potential treatment target for pathological pain and itch.

Preparation of Primary Mixed Glial Cell Cultures from Adult Mouse Spinal Cord Tissue.

Central nervous system glial cells are known to mediate many neurocognitive/neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Similar glial responses have been recognized as critical factors contributing to the development of diseases in the peripheral nervous system, including various types of peripheral neuropathies, such as peripheral nerve injury-induced neuropathic pain, diabetic neuropathy, and HIV-associated sensory neuropathy. Investigation of the central mechanisms of these peripherally-manifested diseases often requires the examination of spinal cord glial cells at cellular/molecular levels in vitro. When using rodent models to study these diseases, many investigators have chosen to use neonatal cerebral cortices to prepare glial cultures or immortalized cell lines in order to obtain sufficient numbers of cells for assessment. However, differences in responses between cell lines versus primary cultures, neonatal vs. adult cells, and brain vs. spinal cord cells may result in misleading data. Here, we describe a protocol for preparing mixed glial cells from adult mouse spinal cord that can be used for direct in vitro evaluations or further preparation of microglia-enriched and microglia-depleted cells. In this protocol, spinal cord tissue is enzymatically dissociated and adult mixed glial cells are ready to be used between 12 and 14 days after the establishment of the culture. This protocol may be further refined to prepare spinal cord glial cells from spinal cord tissues of adult rats and potentially other species. Mixed glial cultures can be prepared from animals of different strains or post-in vivo manipulations and therefore are suitable for studying a variety of diseases/disorders that involve spinal cord pathological changes, such as amyotrophic lateral sclerosis and multiple sclerosis, as well as toxin-induced changes. © 2023 Wiley Periodicals LLC. Basic Protocol: Preparation of primary mixed glial cell cultures from adult mouse spinal cord tissue.

An ependymal cell census identifies heterogeneous and ongoing cell maturation in the adult mouse spinal cord that changes dynamically on injury.

The adult spinal cord stem cell potential resides within the ependymal cell population and declines with age. Ependymal cells are, however, heterogeneous, and the biological diversity this represents and how it changes with age remain unknown. Here, we present a single-cell transcriptomic census of spinal cord ependymal cells from adult and aged mice, identifying not only all known ependymal cell subtypes but also immature as well as mature cell states. By comparing transcriptomes of spinal cord and brain ependymal cells, which lack stem cell abilities, we identify immature cells as potential spinal cord stem cells. Following spinal cord injury, these cells re-enter the cell cycle, which is accompanied by a short-lived reversal of ependymal cell maturation. We further analyze ependymal cells in the human spinal cord and identify widespread cell maturation and altered cell identities. This in-depth characterization of spinal cord ependymal cells provides insight into their biology and informs strategies for spinal cord repair.

GABAergic regulation of cell proliferation within the adult mouse spinal cord

Manipulation of neural stem cell proliferation and differentiation in the postnatal CNS is receiving significant attention due to therapeutic potential. In the spinal cord, such manipulations may promote repair in conditions such as multiple sclerosis or spinal cord injury, but may also limit excessive cell proliferation contributing to tumours such as ependymomas. We show that when ambient γ-aminobutyric acid (GABA) is increased in vigabatrin-treated or decreased by GAD67 allele haplodeficiency in glutamic acid decarboxylase67-green fluorescent protein (GAD67-GFP) mice of either sex, the numbers of proliferating cells respectively decreased or increased. Thus, intrinsic spinal cord GABA levels are correlated with the extent of cell proliferation, providing important evidence for manipulating these levels. Diazepam binding inhibitor, an endogenous protein that interacts with GABA receptors and its breakdown product, octadecaneuropeptide, which preferentially activates central benzodiazepine (CBR) sites, were highly expressed in spinal cord, especially in ependymal cells surrounding the central canal. Furthermore, animals with reduced CBR activation via treatment with flumazenil or Ro15-4513, or with a G2F77I mutation in the CBR binding site had greater numbers of Ethynyl-2′-deoxyuridine positive cells compared to control, which maintained their stem cell status since the proportion of newly proliferated cells becoming oligodendrocytes or astrocytes was significantly lower. Altering endogenous GABA levels or modulating GABAergic signalling through specific sites on GABA receptors therefore influences NSC proliferation in the adult spinal cord. These findings provide a basis for further study into how GABAergic signalling could be manipulated to enable spinal cord self-regeneration and recovery or limit pathological proliferative activity.

MIF homolog d-dopachrome tautomerase (D-DT/MIF-2) does not inhibit accumulation and toxicity of misfolded SOD1

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurons. About 20% of familial ALS cases are caused by dominant mutations in SOD1. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is unclear why misfolded SOD1 accumulates within specific tissues. We have demonstrated that macrophage migration inhibitory factor (MIF), a multifunctional protein with cytokine/chemokine and chaperone-like activity, inhibits the accumulation and aggregation of misfolded SOD1. Although MIF homolog, D-dopachrome tautomerase (D-DT/MIF-2), shares structural and genetic similarities with MIF, its biological function is not well understood. In the current study, we investigated, for the first time, the mechanism of action of D-DT in a model of ALS. We show that D-DT inhibits mutant SOD1 amyloid aggregation in vitro, promoting the formation of amorphous aggregates. Moreover, we report that D-DT interacts with mutant SOD1, but does not inhibit misfolded mutant SOD1 accumulation and toxicity in neuronal cells. Finally, we show that D-DT is expressed mainly in liver and kidney, with extremely low expression in brain and spinal cord of adult mice. Our findings contribute to better understanding of D-DT versus MIF function in the context of ALS.

Sectioning and Counting of Motor Neurons in the L3 to L6 Region of the Adult Mouse Spinal Cord.

Histology is the study of the microscopic structure of tissues. This protocol permits the generation of frozen transverse sections of lumbar spinal cord regions L3 to L6. It enables counting of murine ventral horn lumbar motor neurons in a reproducible manner. Methods include spinal column dissection, hydraulic extrusion, and histological processing. The preparation for cryo-sectioning includes embedding lumbar spinal cord in optimal cutting temperature (OCT) medium. The correct orientation of the tissue is critical as calculating the amount of tissue to discard saved time overall. Specific details regarding section thickness and mounting are described. These requirements not only allow optimum coverage of specific regions but also ensure that no individual motor neuron was counted twice. The Nissl bodies of the motor neurons were stained using gallocyanin. The sections obtained are all of a comparable area and quality assurance is consistent. The specificity of the staining enables the scientist to identify and reliably quantify lumbar motor neurons. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Euthanasia of mouse and isolation of spinal cord Basic Protocol 2: Hydraulic extrusion of the spinal cord Basic Protocol 3: Identification of the lumbar region Basic Protocol 4: Embedding cord in OCT Basic Protocol 5: Collection of frozen sections onto slides Basic Protocol 6: Gallocyanin staining Basic Protocol 7: Motor neuron counting.

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.

Temporal and spatial cellular and molecular pathological alterations with single-cell resolution in the adult spinal cord after injury

Spinal cord injury (SCI) involves diverse injury responses in different cell types in a temporally and spatially specific manner. Here, using single-cell transcriptomic analyses combined with classic anatomical, behavioral, electrophysiological analyses, we report, with single-cell resolution, temporal molecular and cellular changes in crush-injured adult mouse spinal cord. Data revealed pathological changes of 12 different major cell types, three of which infiltrated into the spinal cord at distinct times post-injury. We discovered novel microglia and astrocyte subtypes in the uninjured spinal cord, and their dynamic conversions into additional stage-specific subtypes/states. Most dynamic changes occur at 3-days post-injury and by day-14 the second wave of microglial activation emerged, accompanied with changes in various cell types including neurons, indicative of the second round of attacks. By day-38, major cell types are still substantially deviated from uninjured states, demonstrating prolonged alterations. This study provides a comprehensive mapping of cellular/molecular pathological changes along the temporal axis after SCI, which may facilitate the development of novel therapeutic strategies, including those targeting microglia.

Phosphoinositide-3-kinase regulatory subunit 4 participates in the occurrence and development of amyotrophic lateral sclerosis by regulating autophagy.

The development of amyotrophic lateral sclerosis (ALS) may be related to the abnormal alterations of multiple proteins. Our previous study revealed that the expression of phosphoinositide-3-kinase regulatory subunit 4 (PIK3R4) was decreased in ALS. However, the role of PIK3R4 in ALS pathogenesis remains unknown. This study was the first to find that transfection of PC12 cells with small interfering RNA against the PIK3R4 gene significantly decreased the expression levels of PIK3R4 and the autophagy-related proteins p62 and LC3. Additionally, in vivo experiments revealed that the PIK3R4 protein was extensively expressed in the anterior horn, posterior horn, central canal, and areas surrounding the central canal in cervical, thoracic, and lumbar segments of the spinal cord in adult mice. PIK3R4 protein was mainly expressed in the neurons within the spinal lumbar segments. PIK3R4 and p62 expression levels were significantly decreased at both the pre-onset and onset stages of ALS disease in Tg(SOD1*G93A)1Gur mice compared with control mice, but these proteins were markedly increased at the progression stage. LC3 protein expression did not change during progression of ALS. These findings suggest that PIK3R4 likely participates in the prevention of ALS progression. This study was approved by the Ethics Committee for Animal Care and Use of Jiangxi Provincial People's Hospital, Affiliated People's Hospital of Nanchang University (approval No. 2020025) on March 26, 2020.

Downstream projection of Barrington's nucleus to the spinal cord in mice.

Barrington's nucleus (Bar), which controls micturition behavior through downstream projections to the spinal cord, contains two types of projection neurons, BarCRH and BarESR1, that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus, whereas BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory postsynaptic currents (oEPSCs) in 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI)-labeled lumbosacral preganglionic neurons (LS-PGNs) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGNs, whereas BarESR1 neurons also elicited smaller-amplitude glutamatergic polysynaptic oEPSCs or polysynaptic opto-evoked inhibitory postsynaptic currents (oIPSCs) in some LS-PGNs. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting to either the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous postsynaptic currents in LS-PGNs. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.NEW & NOTEWORTHY Photostimulation of BarCRH or BarESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

MicroRNA-7 promotes motor function recovery following spinal cord injury in mice

Spinal cord injury (SCI) is a devastating neurological condition for which there are no effective therapies. Following an initial injury, there is a cascade of multiple downstream events termed secondary injury. Thus, therapeutic approaches targeting a single pathway may not offer the best solution for treating SCI. One of the most attractive properties of microRNAs (miR) as potential therapeutics is that they are highly effective in regulating complex biological pathways by targeting multiple genes and pathways. The current study investigated the role of miR-7-5p (miR-7), which was previously shown to have neuroprotective functions, in promoting motor function recovery following SCI. We used an adeno-associated virus 1 (AAV1) vector to deliver the gene encoding miR-7 to the spinal cord of adult mice and found that this virus was mainly transduced into the neurons of the spinal cord. Transduction of AAV1-miR-7 improved hindlimb locomotor function following SCI over an 8-week observation period. This improvement was accompanied by reduced neuronal loss in the lesion. In addition, the beneficial effect of miR-7 was associated with enhanced levels of TH-positive axons in the lesion. Taken together, we suggest that miR-7 improves motor function recovery after SCI by protecting neuronal death and increasing axon levels. These findings suggest that miR-7 could be developed as a potential treatment for SCI in human.

Nkx6.1 enhances neural stem cell activation and attenuates glial scar formation and neuroinflammation in the adult injured spinal cord

Nkx6.1 plays an essential role during the embryonic development of the spinal cord. However, its role in the adult and injured spinal cord is not well understood. Here we show that lentivirus-mediated Nkx6.1 expression in the adult injured mouse spinal cord promotes cell proliferation and activation of endogenous neural stem/progenitor cells (NSPCs) at the acute phase of injury. In the chronic phase, Nkx6.1 increases the number of interneurons, reduces the number of reactive astrocytes, minimizes glial scar formation, and represses neuroinflammation. Transcriptomic analysis reveals that Nkx6.1 upregulates the sequential expression of genes involved in cell proliferation, neural differentiation, and Notch signaling pathway, downregulates genes and pathways involved in neuroinflammation, reactive astrocyte activation, and glial scar formation. Together, our findings support the potential role of Nkx6.1 in neural regeneration in the adult injured spinal cord.

Gucy2d selectively marks inhibitory dynorphin neurons in the spinal dorsal horn but is dispensable for pain and itch sensitivity.

Inhibitory neurons in the spinal dorsal horn can be classified based on expression of neurochemical marker genes. However, these marker genes are often expressed throughout the central nervous system, which poses challenges for manipulating genetically identified spinal neurons without undesired off-target effects. We investigated whether Gucy2d, previously identified as a highly selective marker of dynorphin-lineage neurons in the dorsal horn, is expressed in other locations within the adult mouse spinal cord, dorsal root ganglia (DRG), or brain. In addition, we sought to molecularly characterize Gucy2d-expressing dorsal horn neurons and investigate whether the disruption of Gucy2d gene expression affects sensitivity to itch or pain. In situ hybridization experiments assessed Gucy2d mRNA expression in the adult mouse spinal cord, DRG, and brain, and its colocalization with Pax2, Bhlhb5, and Pde2a in dorsal horn neurons. Knockout mice lacking Gucy2d expression were compared with littermate controls to assess sensitivity to chloroquine-induced itch and dry skin-mediated chronic itch, as well as heat, cold, or mechanical stimuli. Gucy2d is selectively expressed in dynorphin-lineage neurons in lamina I-III of the adult mouse spinal cord but not in the brain or DRG. Spinal Gucy2d-expressing neurons are inhibitory neurons that also express the transcription factor Bhlhb5 and the cGMP-dependent phosphodiesterase Pde2a. Gucy2d knockout mice did not exhibit altered responses to itch or pain. The selective expression of Gucy2d within a subpopulation of inhibitory dorsal horn neurons may yield a means to selectively manipulate inhibitory signaling at the level of the spinal cord without effects on the brain.