Project description:RNA-seq analysis of microglia following spinal cord injury demonstrated that microglia response is driven by time after injury but not lesion severity.
Project description:We investigated the gene expression profile of monocyte-derived macrophages and microglia following spinal cord injury. Moreover, we investigated the gene expression profole of M-CSF induced macrophages and new-born derived microglia following TGFb1 treatment. monocyte-derived macrophages and microglia following spinal cord injury M-CSF induced macrophages and new-born derived microglia following TGFb1 treatment
Project description:Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unknown whether distinct microglia subpopulations or states contribute to different stages of pain development and maintenance. Here, we demonstrate, using single-cell RNA-sequencing, that microglia transcriptional states differ at early and late time points following nerve injury. Male microglia show more proliferation and distinct transcriptional changes in response to nerve injury comparing to females. Apolipoprotein E (Apoe) was the top upregulated gene in microglia at chronic time points after nerve injury in mice and polymorphisms in the APOE in humans are associated with chronic pain. Single-cell analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia and identify a previously unrecognized role for ApoE in neuropathic pain.
Project description:Mice lacking the developmental axon guidance molecule EphA4 have previously been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury, a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated or otherwise altered expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wild-type spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage / microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages / activated microglia in injured EphA4 knockout compared to wild-type spinal cords at two, four or fourteen days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages / activated microglia was observed in EphA4 knockout spinal cords at four days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury. Comparison was made between gene expression in wild-type and knockout samples both before and after injury. 3 replicates per group.
Project description:Purpose: We purified spinal cord microglia utilizing percoll gradients and magnetic beads, followed by transcriptome profiling (RNA-seq) to define microglia expression profiles against other neural, immune cell-types. We next observed how the microglial transcriptomes change during activation in the SOD1-G93A mouse model of motor neuron degeneration at 3 time points. We also compared these profiles with that induced by LPS injection. Results and conclusions: ALS microglia were found to differ substantially from those activated by LPS and from M1/M2 macrophages by comparison with published datasets. These ALS microglia showing substantial induction of a neurodegeneration-tailored phenotype, with induction of lysosomal, RNA splicing, and Alzheimer's disease pathway genes. Overall they express a mixture of neuroprotective and neurotoxic factors during activation in ALS mice, showing that neuro-immune activation in the spinal cord is a double-edged sword. We also detected the transcriptional nature of surface marker expression in microglia (CD11b, CD86, CD11c), and substantial T-cell microglia cross-talk using correlative microglia transcriptome/FACS analysis. 42 total RNA samples from purified spinal cord microglia were subjected to paired-end RNA-sequencing. Parallel flow cytometry data was collected from the same spinal cords.
Project description:Summary: Spinal cord injury (SCI) is a damage to the spinal cord induced by trauma or disease resulting in a loss of mobility or feeling. SCI is characterized by a primary mechanical injury followed by a secondary injury in which several molecular events are altered in the spinal cord often resulting in loss of neuronal function. Analysis of the areas directly (spinal cord) and indirectly (raphe and sensorimotor cortex) affected by injury will help understanding mechanisms of SCI. Hypothesis: Areas of the brain primarily affected by spinal cord injury are the Raphe and the Sensorimotor cortex thus gene expression profiling these two areas might contribute understanding the mechanisms of spinal cord injury. Specific Aim: The project aims at finding significantly altered genes in the Raphe and Sensorimotor cortex following an induced moderate spinal cord injury in T9.
Project description:Combining proteomics and systems biology analyses, we demonstrated that neonatal microglial cells derived from two different CNS locations (cortex and spinal cord) displayed different phenotypes upon different physiological or pathological conditions. These cells also exhibited great variability in terms of both cellular and small extracellular vesicles (sEVs) protein contents and levels. Bioinformatics data analysis showed that the cortical microglia had anti-inflammatory and neurogenesis/tumorigenesis properties, while the spinal cord microglia was rather involved in inflammatory response process. Of interest, while both sEVs microglia sources enhanced growth of DRGs axons, only the spinal cord-derived sEVs microglia under LPS stimulation significantly attenuated glioma proliferation. These results were confirmed through neurite outgrowth assays in DRGs cell line and glioma proliferation analysis in 3D spheroid cultures. Results from these in vitro assays indicated that the microglia localized at different CNS regions can ensure different biological functions. Together, these works indicate that neonatal microglia locations regulate their physiological and pathological functional fates, and could explain the high prevalence of brain vs. spinal cord glioma in adults.
Project description:Mice lacking the developmental axon guidance molecule EphA4 have previously been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury, a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated or otherwise altered expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wild-type spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage / microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages / activated microglia in injured EphA4 knockout compared to wild-type spinal cords at two, four or fourteen days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages / activated microglia was observed in EphA4 knockout spinal cords at four days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury.
Project description:To analyse lesion-induced gene regulation in macrophage and microglia at single cell resolution, we performed single cell RNAseq on FACS-isolated mpeg1.1:GFP cells from the spinal cord at 24 hours post-lesion (hpl) after spinal injury at 3 days post-fertilisation (dpf), compared to age-matched uninjured animals.