Cells In Spinal Cord Research Articles

The active contribution of OPCs to neuroinflammation is mediated by LRP1.

Oligodendrocyte progenitor cells (OPCs) account for about 5% of total brain and spinal cord cells, giving rise to myelinating oligodendrocytes that provide electrical insulation to neurons of the CNS. OPCs have also recently been shown to regulate inflammatory responses and glial scar formation, suggesting functions that extend beyond myelination. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted phagocytic receptor that is highly expressed in several CNS cell types, including OPCs. Here, we have generated an oligodendroglia-specific knockout of LRP1, which presents with normal myelin development, but is associated with better outcomes in two animal models of demyelination (EAE and cuprizone). At a mechanistic level, LRP1 did not directly affect OPC differentiation into mature oligodendrocytes. Instead, animals lacking LRP1 in OPCs in the demyelinating CNS were characterized by a robust dampening of inflammation. In particular, LRP1-deficient OPCs presented with impaired antigen cross-presentation machinery, suggesting a failure to propagate the inflammatory response and thus promoting faster myelin repair and neuroprotection. Our study places OPCs as major regulators of neuroinflammation in an LRP1-dependent fashion.

Neural stem cell plasticity of radial glial cells in zebrafish spinal cord

Neural stem cell plasticity of radial glial cells in zebrafish spinal cord

Infiltration of Blood-Derived Macrophages Contributes to the Development of Diabetic Neuropathy.

Background and Objective Diabetic neuropathic pain (DNP) is a common complication associated with diabetes. Currently, its underlying pathomechanism remains unknown. Studies have revealed that the recruitment of blood monocyte-derived macrophages (MDMs) to the spinal cord plays a pivotal role in different models of central nervous system injury. Therefore, the present study aimed at exploring the infiltration and function of MDMs in DNP using a mice model. Methods Diabetes was induced using streptozotocin in male A/J mice. Mechanical withdrawal thresholds were measured weekly to characterize neuropathy phenotype. Quantitative analysis of CD11b was performed and visualized by immunofluorescence. Spinal cord cells were isolated from myelin and debris by Percoll gradient. Flow cytometry was used to label CD11b and CD45 antibodies to differentiate MDMs (CD45highCD11b+) from resident microglia (CD45lowCD11b+). Mice were injected with clodronate liposomes to investigate the role of MDMs in DNP. The successful depletion of monocytes was determined by flow cytometry. Results The DNP mice model was successfully established. Compared with nondiabetic mice, diabetic mice displayed a markedly higher level of CD11b immunofluorescence in the spinal cord. The number of CD11b-positive microglia/macrophages gradually increased over the 28 days of testing after STZ injection, and a significant increase was observed on Day 14 (P < 0.01) and 28 (P < 0.01). Further analysis by flow cytometry showed that the infiltration of peripheral macrophages began to increase in 2 weeks (P < 0.001) and reached a maximum at 4 weeks (P < 0.001) post-STZ injection compared to the control. The depletion of MDMs by clodronate liposomes alleviated diabetes-induced tactile allodynia (P < 0.05) and reduced the infiltration of MDMs (P < 0.001) as well as the expression of IL-1β and TNF-α in the spinal cord (P < 0.05). Conclusions The infiltration of blood MDMs in the spinal cord may promote the development of painful neuropathy in diabetes.

Adeno-associated viral vector serotype 9-based gene therapy for Niemann-Pick disease type A.

Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders.

Abstract 872: Inhibition of Spinal Astrocytes Activation Attenuates Myocardial Ischemia-Reperfusion Injury by Regulating NGF/TRPV1 Nociceptive Signaling

Background and Aim: Spinal astrocytes activation are implicated in nociception and pain processing after noxious stimuli, while its role in cardiac ischemic nociception is unknown. We recently reported spinal nerve growth factor (NGF)-sensitized transient receptor potential vanilloid 1 (TRPV1) nociceptive signaling following cardiac ischemia and reperfusion (I/R). This study was design to investigate whether spinal astrocytes are activated following myocardial I/R and its effects on spinal NGF-TRPV1 signaling. Methods and Results: Myocardial I/R injury was induced by 30 min occlusion of the left coronary artery followed by 120 min of reperfusion in rats. Glial fibrillary acidic protein (GFAP), an astrocyte marker, was found up-regulated in thoracic spinal cord during ischemia and reperfusion. Intrathecally pre-injection of fluorocitrate (FC), an astrocyte inhibitor, reduced infarct size (IS/AAR, area at risk), from 48.4±2.67 to 29.7±3.13 (P&lt;0.05, Fig.1 A). The high level of cardiac troponin T caused by I/R was decreased by FC injection as well (from 530±65 to 129±50 μg/μl, P&lt;0.01, Fig.1 B). Upon immunofluorescence staining, GFAP immunopositive cells in spinal cord were found markedly increased after I/R, while the staining was suppressed by intrathecal FC administration (Fig.1 C). Moreover, the up-regualtion of NGF and TRPV1 protein levels in spinal cord were both inhibited by FC pretreatment (Fig.1 D-F). Conclusion: Myocardial I/R induced the activation of spinal astrocytes, inhibition of which reduces infarct size and cTnT level, as well as NGF-TRPV1 signaling, which suggests a novel therapeutic target for preventing myocardial ischemic injury.

Reactive astrocytes increase expression of proNGF in the mouse model of contused spinal cord injury

Astrocytes are major glial cells critically in maintaining stability of the central nervous system and functional activation of astrocytes occurs rapidly in various diseased or traumatic events. We are interested in functional changes of astrocytes during the spinal cord injury, and studied expression of nerve growth factor (NGF) in activated astrocytes by mouse model of contused spinal cord injury and cell culture experiment. It revealed that the spinal cord injury resulted in apparent activation of astrocytes and microglial cells and decreased BMS scores. A larger number of astrocytes showed immunoreactivity to proNGF in the injured spinal cord areas, and proNGF expression increased and remained high level at 7 to 14dpi, which was coincided with upregulation of glial fibrillary acidic protein. The proNGF was clearly localized in both exosome-like vesicles and cytoplasm of astrocytes in culture. Electron microscopy confirmed exosome-like vesicles with proNGF-immunoreactivity in diameter sizes of 50–100 nm. Finally, cell culture with lipopolysaccharide (LPS) experiment indicated increasing expression and release of proNGF in the astrocytes with LPS exposure. This study demonstrated that reactive astrocytes increased proNGF expression after spinal cord injury, also suggesting involvement of exosome-like proNGF transport or release in triggering neuronal apoptosis and aggravating progression of spinal cord injury.

Effects of mu opioid receptors in paraventricular nucleus on ejaculation through mediating sympathetic nerve system activity

To investigate the roles of mu opioid receptors (MORs) in paraventricular nucleus of the hypothalamus (PVN) on ejaculation and its underlying mechanism in the rats, we performed copulation behavioral testing and acute experiments. During the acute experiments, mean arterial pressure (MAP), heart rate (HR), bulbospongiosus muscle-electromyogram (BSM-EMG) and pressure of vas deferens (PVD) were all recorded. The expression levels and distributions of opioid receptors were also assessed in PVN of male rats. Moreover, adeno-associated virus type 1 (AAV1) was microinjected into PVN to demonstrate whether there are direct projections from PVN to lumbar spinothalamic (LSt) cells. We found that microinjection of MOR agonist, D-A1a2-NM9-Phe4-Gly(ol)5enkephalin (DAGO), into the PVN prolonged the intromission latency and inhibited ejaculation (P = 0.0241, P = 0.0473, respectively), while the opposed results appeared in CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, MOR antagonist) group (P = 0.0021, P = 0.0286, respectively). Moreover, DAGO caused a significant decrease in MAP and HR (P = 0.0065, P = 0.0030, respectively), and PVD decreased significantly after DAGO microinjection in PVN (P = 0.0383). CTAP not only blocked the effect of DAGO but also significantly increased MAP, HR and PVD (P = 0.0003, P = 0.0010, P = 0.0074, respectively). Meanwhile, a significant increase was observed in BSM-EMG activity after microinjecting of CTAP (P = 0.0022), accompanied by visible BSM contraction. Additionally, anterograde monosynaptic transneuronal tracer AAV1 labeling revealed that neurons in PVN projected directly to LSt cells in L3-4 spinal cord. These results indicate that MORs in PVN centrally mediate ejaculation by regulating the sympathetic outflow, which may be treated as a therapeutic target for ejaculation disorders in the future.

Phenotypic and functional changes in gamma delta T lymphocytes from HTLV-1 carriers.

Human T-cell lymphotropic virus type-1 (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic inflammatory disease that leads to gradual loss of motor movement as a result of the death of spinal cord cells through immune mediated mechanisms. The risk to develop HAM/TSP disease positively correlates with the magnitude of HTLV-1 proviral load. Gamma-delta T lymphocytes have been recognized as important players in a variety of infectious diseases. Therefore, we have investigated interactions between HTLV-1 infection and γδ T lymphocytes during HAM/TSP. Similar frequencies of total γδ T lymphocytes and their Vγ9δ2+ and Vγ9δ2neg subpopulations were observed in HAM/TSP patients. However, T lymphocytes obtained from HTLV-1 carriers displayed significantly higher rates of spontaneous proliferation and NKp30 expression when compared to cells from uninfected donors. In addition, an important decrease in the frequency of granzyme B+ γδ T lymphocytes (approximately 50%) was observed in HAM/TSP patients. Higher proportion of IFN-γ+ γδ T lymphocytes was found in HTLV-1-infected patients, which positively correlated with the HTLV-1 proviral load in peripheral blood mononuclear cells. Collectively, our data indicates that HTLV-1 infection leads to phenotypic and functional changes in the population of γδ T lymphocyte population, suggesting that HTLV-1 infection modulates functions associated to these cells, which might be involved in controlling the infection or in the development of HTLV-1-associated diseases.

Calbindin-D28k expression in spinal electromotoneurons of the weakly electric fish Apteronotus leptorhynchus during adult development and regeneration.

Additive neurogenesis, the net increase in neuronal numbers by addition of new nerve cells to existing tissue, forms the basis for indeterminate spinal cord growth in brown ghost knifefish (Apteronotus leptorhynchus). Among the cells generated through the activity of adult neural stem cells are electromotoneurons, whose axons constitute the electric organ of this weakly electric fish. Electromotoneuron development is organized along a caudo-rostral gradient, with the youngest and smallest of these cells located near the caudal end of the spinal cord. Electromotoneurons start expressing calbindin-D28k when their somata have reached diameters of approximately 10μm, and they continue expression after they have grown to a final size of about 50μm. Calbindin-D28k expression is significantly increased in young neurons generated in response to injury. Immunohistochemical staining against caspase-3 revealed that electromotoneurons in both intact and regenerating spinal cord are significantly less likely to undergo apoptosis than the average spinal cord cell. We hypothesize that expression of calbindin-D28k protects electromotoneurons from cell death; and that the evolutionary development of such a neuroprotective mechanism has been driven by the indispensability of electromotoneurons in the fish's electric behavior, and by the high size-dependent costs associated with their production or removal upon cell death.

LINGO-1 deficiency promotes nerve regeneration through reduction of cell apoptosis, inflammation, and glial scar after spinal cord injury in mice

Leucine-rich repeat and immunoglobulin domain-containing protein 1 (LINGO-1) is a transmembrane protein that negatively regulates neural regeneration in the central nervous system. LINGO-1 expression is up-regulated after central nerve injury, and is accompanied by cell death. Both LINGO-1 and cell death in the injury microenvironment are thought to limit neural regeneration, but the relationship between LINGO-1 and cell death has not been characterized. To investigate whether LINGO-1 deletion improves the spinal cord microenvironment after spinal cord injury (SCI) and contributes to cell survival, we generated LINGO-1 knockout (KO) mice. These mice and wild-type control mice were subjected to spinal cord transection. Fourteen days after spinal cord transection, cell apoptosis, inflammation, glial scar, and growth of nerve fibers were evaluated by immunostaining. The results showed that LINGO-1 KO mice demonstrated a profound reduction in expression of caspase-3, transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL), ionized calcium binding adapter molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and chondroitin sulfate proteoglycans (CSPGs) compared to controls. In contrast, expression of neurofilament (NF) at the SCI site in LINGO-1 KO mice was markedly increased compared to that in wild-type mice. These results suggested that LINGO-1 plays a critical role in the injury microenvironment in processes such as cell death, inflammatory response, and glial scar formation. Importantly, LINGO-1 deletion and a positive microenvironment may exert synergistic effects to promote nerve fiber regeneration. Therefore, inhibition of LINGO-1 may be a therapeutic strategy to promote neural regeneration following SCI.

Subclinical lipopolysaccharide from Salmonella Enteritidis induces neuropeptide dysregulation in the spinal cord and the dorsal root ganglia

BackgroundDespite increasing evidence that lipopolysaccharide (LPS) affects the biological active substances of dorsal root ganglia (DRG) we have limited knowledge of the influence of a single low dose of LPS, which does not result in any clinical symptoms of disease (subclinical LPS) on neuropeptides connected with the sensory pathway. Accordingly, in this work, we investigated the influence of subclinical LPS from Salmonella Enteritidis on selected neuropeptides: substance P (SP), galanin (GAL), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM) in the cervical, thoracic, lumbar and sacral regions of the DRG and spinal cord.MethodsThis study was performed on immature female pigs of the Pietrain × Duroc breed. Seven days after the intravenous injection of saline solution for control animals (n = 5) and 5 μg/kg b.w. LPS from S. Enteritidis for the experimental group (n = 5), the DRG and the spinal cord were collected to extract the neuropeptides using solid-phase extraction technology.ResultsOur results demonstrated that subclinical LPS in DRG was able to change the levels of all studied neuropeptides except SOM, whereas in the spinal cord it down-regulated all studied neuropeptides in the sacral spinal cord, maintaining the concentration of all studied neuropeptides in other regions similar to that observed in the control animals. The significant differences in the intensity and character of observed changes between particular regions of the DRG suggest that the exact functions of the studied neuropeptides and mechanisms of responses to subclinical LPS action depend on specific characteristics and functions of each examination region of DRG.ConclusionsThe mechanisms of observed changes are not fully understood and require further study of the molecular interactions between subclinical LPS from S. Enteritidis and neuronal and non-neuronal cells of DRG and spinal cord. The peripheral and central pain pathways must be analysed with the aspect of unknown long-term consequences of the influence of subclinical LPS from S. Enteritidis on neuropeptides in the spinal cord and the dorsal root ganglia.

The specialised pro-resolving lipid mediator maresin 1 reduces inflammatory pain with a long-lasting analgesic effect.

Maresin 1 (MaR1) is a specialised pro-resolving lipid mediator with anti-inflammatory and analgesic activities. In this study, we addressed the modulation of peripheral and spinal cord cells by MaR1 in the context of inflammatory pain. Mice were treated with MaR1 before intraplantar injection of carrageenan or complete Freund's adjuvant (CFA). Mechanical hyperalgesia was assessed using the electronic von Frey and thermal hyperalgesia using a hot plate. Spinal cytokine production and NF-κB activation were determined by ELISA and astrocytes and microglia activation by RT-qPCR and immunofluorescence. CGRP release by dorsal root ganglia (DRG) neurons was determined by EIA. Neutrophil and macrophage recruitment were determined by immunofluorescence, flow cytometry, and colorimetric methods. Trpv1 and Nav1.8 expression and calcium imaging of DRG neurons were determined by RT-qPCR and Fluo-4AM respectively. MaR1 reduced carrageenan- and CFA-induced mechanical and thermal hyperalgesia and neutrophil and macrophage recruitment proximal to CGRP+ fibres in the paw skin. Moreover, MaR1 reduced NF-κB activation, IL-1β and TNF-α production, and spinal cord glial cells activation. In the DRG, MaR1 reduced CFA-induced Nav1.8 and Trpv1 mRNA expression and calcium influx and capsaicin-induced release of CGRP by DRG neurons. MaR1 reduced DRG neurons activation and CGRP release explaining, at least in part, its analgesic and anti-inflammatory effects. The enduring analgesic and anti-inflammatory effects and also post-treatment activity of MaR1 suggest that specialised pro-resolving lipid mediators have potential as a new class of drugs for the treatment of inflammatory pain.

New roles for Wnt and BMP signaling in neural anteroposterior patterning.

During amphibian development, neural patterning occurs via a two-step process. Spemann's organizer secretes BMP antagonists that induce anterior neural tissue. A subsequent caudalizing step re-specifies anterior fated cells to posterior fates such as hindbrain and spinal cord. The neural patterning paradigm suggests that a canonical Wnt-signaling gradient acts along the anteroposterior axis to pattern the nervous system. Wnt activity is highest in the posterior, inducing spinal cord, at intermediate levels in the trunk, inducing hindbrain, and is lowest in anterior fated forebrain, while BMP-antagonist levels are constant along the axis. Our results in Xenopus laevis challenge this paradigm. We find that inhibition of canonical Wnt signaling or its downstream transcription factors eliminates hindbrain, but not spinal cord fates, an observation not compatible with a simple high-to-low Wnt gradient specifying all fates along the neural anteroposterior axis. Additionally, we find that BMP activity promotes posterior spinal cord cell fate formation in an FGF-dependent manner, while inhibiting hindbrain fates. These results suggest a need to re-evaluate the paradigms of neural anteroposterior pattern formation during vertebrate development.

A-109 A novel multi-cytokine inhibitory strategy in treating HTLV-1 diseases

Human T-cell leukemia virus-I (HTLV-1) is the most oncogenic human virus and arguably the most carcinogenic agent to humans (Gallo, Willems and Tagaya, 2017, Martin et al, 2018). Nevertheless, HTLV-1 is not receiving the deserving attention. We have been advocating for HTLV-1 to enhance the visibility of its research and to make the public aware of its biological and social importance for the past few years as a part of HTLV-1 task force of Global Virus Network (GVN), and we believe we are seeing some promising changes. Despite nearly 40 years of extensive research on the mechanisms by which HTLV-1 causes multiple human diseases including adult T-cell leukemia (ATL), HAM/TSP (HTLV-1 associate myelopathy/Tropical Spastic Paraparesis), and immunosuppressive condition leading to bacterial infections, no reliable cures are available for these diseases. HTLV-1 infection to CD4 T-cells enhances the production of multiple cytokines by CD4 T-cells through the transactivating nature of virus genes such as Tax. This causes a by-stander activation of CD8 T-cells and turn them into inflammatory cells, which contributes to worsening the disease burden in HTLV-1 diseases. For example, the damages to spinal cord cells in HAM/TSP is caused by the inflammatory CD8 T-cells. We have shown that such activation of CD8 T-cells is mainly caused by the cooperation of IL-2 and IL-15, 2 sibling cytokines belonging to the common gamma (γc)-family (Nata et al 2015, Massoud et al 2015) and that only the co-inhibition of these 2 cytokines, but not the inhibition of either of them, effectively suppresses the activation of CD8 T-cells isolated from HAM/TSP patients in an ex vivo assay system. The co-inhibition of 2 cytokines is a clinical challenge because currently available modalities do not allow to accomplish the goal without adverse effects. For example, monoclonal antibodies need to be combined, but is infeasible in the clinic from the viewpoint of cost. Bispecific antibodies do not exist to co-inhibit these 2 cytokines. Signal inhibitors such as Jak kinase inhibitors will have profound side effects by broadly blocking non-γc cytokines such as Erythropoietin and Thrombopoietin and causing severe anemia and bleeding tendency. Even specific JAk3 inhibitors, if available, will cause immunodeficiency by blocking IL-7 and significantly reducing the number of T-cells when used chronically. We thus developed a novel technology to develop inhibitors which block only desired members of the γc-family cytokines. We noted that all γc-tyokines (eg, IL-2, -4, -7, -9-, 15, and −21) use their D-helices to interact with the shared γc-receptor subunit. Accordingly, we found that the D-helices from 6 γc-cytokine show modest conservation in their primary structure. We postulated that a short peptide mimicking these D-helices might inhibit multiple γc-cytokines by competing with them in the binding to the γc, and that leveraging the conserved and non-conserved positions in this region across all γc-cytokines might allow us to synthesize a specific inhibitor to a given choice of γc-cytokines. We in fact successfully synthesized several peptides showing inhibition to only selected γc-cytokines, but not all γc-cytokines with different target spectrums. Among them is the BNZ-1 peptide which blocks IL-2, IL-15 and IL-9. In an ex vivo assay using T-cells derived from HAM/TSP patients, BNZ-1 effectively blocked the proliferation and activation of HAM/TSP T-cells whereas mAb for IL-2 or IL-15 only showed marginal inhibition, suggesting that this compound represents a novel therapeutic candidate for HAM/TSP. It is likely that BNZ-1 could treat chronic ATL as well since chronic ATL has been shown to pathogenically involve IL-2 and IL-15 produced by HTLV-1 infected CD4 T-cells. We have tested the safety, pharmacokinetics and pharmacodynamics of the PEGylated form of the BNZ-1 peptide (BNZ-1/PEG) in animal models (mouse and non-human primate) and in healthy human volunteers (Phase I study) and observed that it blocks IL-2 and IL-15 activity effectively in humans, and that a weekly or biweekly administration maintains the drug level above the minimum effective dose. BNZ-1/PEG has been approved as an IND for treating HAM/TSP and T-cell malignancies (CD8 type LGL-leuekmia/cutaneous T-cell Lymphoma (CTCL)). Currently we are conducting a phase II clinical trial involving LGL-leukemia/CTCL (see a poster presented by J Geh et al) and are aiming at starting a trial involving HAM/TSP in the near future.

The origin, fate, and contribution of macrophages to spinal cord injury pathology.

Virtually all phases of spinal cord injury pathogenesis, including inflammation, cell proliferation and differentiation, as well as tissue remodeling, are mediated in part by infiltrating monocyte-derived macrophages. It is now clear that these infiltrating macrophages have distinct functions from resident microglia and are capable of mediating both harmful and beneficial effects after injury. These divergent effects have been largely attributed to environmental cues, such as specific cytokines, that influence the macrophage polarization state. In this review, we also consider the possibility that different macrophage origins, including the spleen, bone marrow, and local self-renewal, may also affect macrophage fate, and ultimately their function that contribute to the complex pathobiology of spinal cord injury.

Single cell transcriptomics reveals spatial and temporal dynamics of gene expression in the developing mouse spinal cord.

ABSTRACTThe coordinated spatial and temporal regulation of gene expression in the vertebrate neural tube determines the identity of neural progenitors and the function and physiology of the neurons they generate. Progress has been made deciphering the gene regulatory programmes that are responsible for this process; however, the complexity of the tissue has hampered the systematic analysis of the network and the underlying mechanisms. To address this, we used single cell mRNA sequencing to profile cervical and thoracic regions of the developing mouse neural tube between embryonic days 9.5-13.5. We confirmed that the data accurately recapitulates neural tube development, allowing us to identify new markers for specific progenitor and neuronal populations. In addition, the analysis highlighted a previously underappreciated temporal component to the mechanisms that generate neuronal diversity, and revealed common features in the sequence of transcriptional events that lead to the differentiation of specific neuronal subtypes. Together, the data offer insight into the mechanisms that are responsible for neuronal specification and provide a compendium of gene expression for classifying spinal cord cell types that will support future studies of neural tube development, function and disease.

The short interference RNA (siRNA) targeting NMUR2 relieves nociception in a bone cancer pain model of rat through PKC-ERK and PI3K-AKT pathways

Background/aimBone cancer pain (BCP) causes troubles and burdens to patients globally. Increasing evidence proved that neuromedin U receptor 2 (NMUR2) was involved in pains. Our study was performed to investigate the role of NMUR2 on BCP and the underlying mechanism. MethodsThe rats were raised and BCP rat model was established by injection with Walker 256 cells. The RNA and protein expression levels of NMUR2 in rat neurons-dorsal spinal cord cells, RNdsc cells were detected by qRT-PCR and western blot. The administration with NMUR2 was via intrathecal injection with siRNA to silence NMUR2. The tolerance of rat to pain was measured by mechanical allodynia test and presented by paw withdrawal threshold (PWT) value. The effects on protein kinase C (PKC)/extracellular regulated protein kinases (ERK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signal pathways were examined by western blot. ResultsThe expression of NMUR2 in both mRNA and protein levels was upregulated in BCP rat model. In addition, siRNA injection significantly decreased the expression of NMUR2 on the 3rd, 7th and 14th day. BCP group revealed lower PWT value compared with control while NMUR2 silence increased the PWT value compared with negative control. The phosphorylation of PKC, ERK, PI3K and AKT was increased in BCP model while was decreased by si-NMUR2. PKC/ERK and PI3K/AKT inhibitor administration increased the PWT value compared with BCP group. Conclusionsi-NMUR2 alleviates BCP via inactivation of PKC/ERK and PI3K/AKT signal pathways.

The Impact of High Salt Diet on the Nociceptive Pain Thresholds and Functional Phenotype of Myeloid Cells

Poster Introduction/Aim: Though several studies have shown that high salt diet (HSD) is related to the development of chronic diseases, the effect of long-term high-salt intake on the immune system and pain behavior remains elusive. Here, we aimed to investigate whether and how HSD affected mouse pain thresholds and innate immunity, especially myeloid cells.Methods: Healthy C57BL/6 male and female mice were fed with HSD (containing 4% NaCl in chow and 1% NaCl in water) right from weaning period (3-week after birth) for 1–3 months.Results: Behavioral analysis demonstrated that HSD-fed mice were hypersensitive to mechanical stimuli. Moreover, HSD induced an expansion of circulating monocytes, especially the proinflammatory monocyte (Ly6Chigh, CCR2+) subsets. HSD also increased the density of Iba-1+ microglia and the number of FcγII/III receptor (CD16/32) expressing cells in the lumbar spinal cord. Prolonged HSD feeding, meanwhile, resulted in increased infiltration of monocyte to the peripheral nerves, contributing to the monocyte-derived macrophage (CD11b+, F4/80+) population. Finally, shifting the HSD back to the regular laboratory chow (with 0.3% NaCl) cannot reverse the HSD-induced mechanical hypersensitivity myeloid cell expansion and polarization towards the inflammatory subsets in the periphery. However, treating HSD mice with CCR2 antagonist effectively normalized the pain threshold and myeloid cell profile in the peripheral blood and nerves.Discussion/Conclusions: Long term HSD feeding decreased mouse mechanical thresholds, in parallel with an expansion and polarization of myeloid cells towards inflammatory status. Such changes were not reversible when HSD-fed mice back to normal diet. However, blocking CCR2 signaling successfully attenuated HSD-induced hypersensitivity.

Tet1 Overexpression and Decreased DNA Hydroxymethylation Protect Neurons Against Cell Death After Injury by Increasing Expression of Genes Involved in Cell Survival

Spinal cord and neuron injury result in loss of muscle function, sensation, or autonomic function in the body. Tet1 produces 5-hydroxymethylcytosin. The conversion was proposed as the initial step of deoxyribonucleic acid demethylation in mammals. However, effects of Tet1 expression and hydroxymethylation status on neuron injury remain unclear. Therefore the current study was designed to explore effects of Tet1 expression and hydroxymethylation status on cell survival and gene expression after neuron injury. Mouse models of spinal cord injury and cell model of neuron injury were created. Animals were sacrificed, and injured spinal cord tissue was harvested. Neuron-like cells were cultured after scratch injury. Hydroxymethylated deoxyribonucleic acid concentration was detected, and Tet1 expression was examined by qPCR. Neuron-like cells were divided into 3 groups: control, injury, and azacytidine+ injury (before injury, cells were pretreated with azacytidine) groups. Culture supernatant was collected, and lactate dehydrogenase concentration was detected. Meanwhile, injured neuron-like cells were divided into 3 groups: negative control, Tet1 overexpression, and Tet1 interference. Relative expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2 in neuron-like cells was detected by qPCR. In addition, neuron-like cells were divided into 7 groups. Tet1 expression and deoxyribonucleic acid hydroxymethylation increased initially and decreased thereafter after neuron injury in both animal and cell models. Percentages of dead cells increased significantly in neuron-like cells after injury. The percentages of dead cells markedly decreased in injured neuron-like cells that were pretreated with azacytidine. The percentages of dead cells increased markedly in the Tet1 interference group and decreased significantly in the Tet1 overexpression group. Expression of Tet1, BDNF, A20, FLIP, HSP70, HSP90, and Bcl2 decreased significantly after injury. Azacytidine pretreatment in injured neuron-like cells markedly increased expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2. Moreover, Tet1 interference significantly decreased the expression of Tet1, BDNF, A20, FLIP, HSP70, and HSP90 in neuron-like cells, whereas Tet1 overexpression markedly increased the expression of Tet1, BDNF, NTF3, A20, FLIP, HSP70, HSP90, HSP27, and Bcl2. BDNF interference significantly increased percentages of dead cells after injury. BDNF interference also markedly decreased the protection of azacytidine and Tet1 overexpression against cell death. Tet1 overexpression and demethylation caused by azacytidine protect neurons against cell death after injury by increasing expression of genes involved in cell survival.

Diameters of Perikarya in Different Regions of Rat Central Nervous System after Streptomycin and Kanamycin Intoxication

Introduction: Streptomycin and Kanamycin, an aminoglycosidic antibiotic, is known to destroy the ventral cochlear nuclei of brainstem in man. Ototoxicity is well known side effect of kanamycin, the effect on central nervous system in general and central auditory pathway in particular is still unclear. Subjects and Methods: Thirty albino rats were divided into three groups I, II and III of ten animals each. Group III was control. Group I and II received Streptomycin (30mg/Kg body weight) and kanamycin (400mg/kg body weight) intramuscular injections, daily for 3 weeks. Paraffin embedded sections of cerebellum, spinal cord, dorsal cochlear nucleus, inferior colliculus nucleus and auditory cortex were stained with haematoxylin and eosin stains and perikarya measured using slide and ocular micrometres. Results: Size of cerebellar Purkinje cells increased significantly in control rats for streptomycin and kanamycin intoxicated animals respectively. Ventral horn cells of spinal cord are affected highly significantly only by streptomycin. Vestibular nucleus also showed similar results i.e. neuronal body. Neurons of dorsal cochlear nucleus is affected significantly by both the drugs i.e. streptomycin and kanamycin. Conclusion: Streptomycin causes an increase in diameter of auditory cortical cells on other hand kanamycin led to highly significant decrement in size of cells of same region. Preferential affinity and differential effects were noticed. The latter throws some clues about mechanism of action.