Rat Peripheral Nervous System Research Articles

Peripheral sensory neurons and non-neuronal cells express functional Piezo1 channels.

Here, we present evidence showing Piezo1 protein expression in the primary sensory neurons (PSNs) and non-neuronal cells of rat peripheral nervous system. Using a knockdown/knockout validated antibody, we detected Piezo1 immunoreactivity (IR) in ∼60% of PSNs of rat dorsal root ganglia (DRG) with higher IR density in the small- and medium-sized neurons. Piezo1-IR was clearly identified in DRG perineuronal glia, including satellite glial cells (SGCs) and Schwann cells; in sciatic nerve Schwann cells surrounding the axons and cutaneous afferent endings; and in skin epidermal Merkel cells and melanocytes. Neuronal and non-neuronal Piezo1 channels were functional since various cells (dissociated PSNs and SGCs from DRGs, isolated Schwann cells, and primary human melanocytes) exhibited a robust response to Piezo1 agonist Yoda1 by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses were abolished by non-specific Piezo1 antagonist GsMTx4. Immunoblots showed elevated Piezo1 protein in DRG proximal to peripheral nerve injury-induced painful neuropathy, while PSNs and SGCs from rats with neuropathic pain showed greater Yoda1-evoked elevation of [Ca2+]i and an increased frequency of cells responding to Yoda1, compared to controls. Sciatic nerve application of GsMTx4 alleviated mechanical hypersensitivity induced by Yoda1. Overall, our data show that Piezo1 is widely expressed by the neuronal and non-neuronal cells in the peripheral sensory pathways and that painful nerve injury appeared associated with activation of Piezo1 in PSNs and peripheral glial cells.

Experimental unilateral intracerebral hemorrhage induces delayed bilateral neurodegeneration of sciatic nerve fibres in rats.

Pathological processes, such as inflammatory effects, oxidative stress, apoptosis and cytotoxicity of blood after an intracerebral hemorrhage (ICH), generally contribute to a secondary injury. One of the secondary ICH consequences in the nervous system may be delayed neurodegeneration of the peripheral nerves. Therefore, the aim of our study was to investigate possible structural changes in the sciatic nerve and changes in the conduction velocity via this nerve at different terms after experimental ICH in male Wistar rats. Intracerebral hemorrhage was provided by direct injection of autologous blood into the capsula interna. On the 10th day after ICH mean conduction velocity in sciatic nerve was 15% smaller compared to the control. On the 30th and 90th days after ICH, highly significant decreases in the conduction velocity by 62% and 60%, respectively in comparison with the control group of animals were observed. The data of morphometric analysis demonstrated significant decreases in the mean diameter and density of myelinated fibres at all examined terms after ICH. A number of the myelin sheaths were swollen and lost their regular laminations. Axoplasmic and myelin degenerations were the most frequent events in these nerve fibres; reductions of the diameter of the axis cylinders were also observed. In the contralateral nerve (related to the hemisphere with ICH), negative changes were greater, while the ipsilateral nerve was also subjected to those. Our data demonstrate that the consequences of unilateral ICH in the capsula interna induce bilateral negative changes in the peripheral nervous system of rats.

Modulatory effects of photobiomodulation in the anterior cingulate cortex of diabetic rats.

Anterior Cingulate Cortex (ACC) has a crucial contribution to higher order pain processing. Photobiomodulation (PBM) has being used as integrative medicine for pain treatment and for a variety of nervous system disorders. This study evaluated the effects of PBM in the ACC of diabetic rats. Type 1 diabetes was induced by a single dose of streptozotocin (85mg/Kg). A total of ten sessions of PBM (pulsed gallium-arsenide laser, 904nm, 9500Hz, 6.23J/cm2) was applied to the rat peripheral nervous system. Glial fibrillary acidic protein (GFAP), mu-opioid receptor (MOR), glutamate receptor 1 (GluR1), and glutamic acid decarboxylase (GAD65/67) protein level expression were analyzed in the ACC of diabetic rats treated with PBM. Our data revealed that PBM decreased 79.5% of GFAP protein levels in the ACC of STZ rats. Moreover, STZ + PBM rats had protein levels of MOR increased 14.7% in the ACC. Interestingly, STZ + PBM rats had a decrease in 70.7% of GluR1 protein level in the ACC. Additionally, PBM decreased 45.5% of GAD65/67 protein levels in the ACC of STZ rats.

Proliferative and Nonproliferative Lesions of the Rat and Mouse Central and Peripheral Nervous Systems: New and Revised INHAND Terms.

Harmonization of diagnostic terminology used during the histopathologic analysis of rodent tissue sections from nonclinical toxicity studies will improve the consistency of data sets produced by laboratories located around the world. The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a cooperative enterprise of 4 major societies of toxicologic pathology to develop a globally accepted standard vocabulary for proliferative and nonproliferative lesions in rodents. A prior manuscript (Toxicol Pathol 2012;40[4 Suppl]:87S-157S) defined multiple diagnostic terms for toxicant-induced lesions, common spontaneous and age-related changes, and principal confounding artifacts in the rat and mouse central nervous system (CNS) and peripheral nervous system (PNS). The current article defines 9 new diagnostic terms and updates 2 previous terms for findings in the rodent CNS and PNS, the need for which has become evident in the years since the publication of the initial INHAND nomenclature for findings in rodent neural tissues. The nomenclature presented in this document is also available electronically on the Internet at the goRENI website (http://www.goreni.org/).

Gadolinium Retention in the Central and Peripheral Nervous System: Implications for Pain, Cognition, and Neurogenesis.

Background Despite the wide use of gadolinium-based contrast agents (GBCAs) for enhanced MRI, their neurochemical and behavioral consequences, if any, remain poorly understood. Purpose To investigate the effect of repeated exposure to a linear or macrocyclic GBCA on gadolinium retention in the central and peripheral nervous system of rats and to assess the functional implications of such retention on hippocampal neurogenesis and sensory and cognitive processing. Materials and Methods Seventy male Sprague-Dawley rats (4 weeks old) received intraperitoneal injections of gadoterate meglumine (0.6 or 2.5 mmol per kilogram of body weight), gadodiamide (0.6 or 2.5 mmol/kg), or saline daily for 20 days (February 2018-March 2019). The 5-bromo-2'-deoxyuridine injections were administered every 3 days to determine the number of proliferating cells and the number of newly maturing neurons in the hippocampus. Sensory and cognitive behavioral tests were performed to assess the effect of GBCAs on pain sensitivity and spatial working memory function, respectively. Finally, inductively coupled plasma mass spectrometry analysis was used to quantify gadolinium retention in the brain, spinal cord, and peripheral nerves 24 hours after the last GBCA administration. One-way and mixed-design analyses of variance were used for statistical analysis. Results All GBCAs resulted in significant gadolinium retention in central and peripheral nervous tissues (1.8-333.2 nmol Gd/g tissue). Pain hypersensitivity to thermal and mechanical stimuli (P < .001) was observed after gadodiamide exposure in rats but not after gadoterate meglumine exposure. Rats injected with both GBCAs showed no changes in spatial working memory or in hippocampal cell proliferation and maturation. Conclusion Gadolinium was retained in the spinal cord and peripheral nerves in rats exposed to multiple administrations of linear and macrocyclic contrast agents. Gadodiamide (linear contrast agent) but not gadoterate meglumine (macrocyclic contrast agent) led to pain hypersensitivity, but neither affected spatial working memory performance, hippocampal cellular proliferation, or hippocampal neurogenesis. © RSNA, 2020 See also the editorial by Radbruch in this issue.

Exposure to Acute, High Dose Cadmium Significantly Downregulates Postnatal Expression of the Vascular Endothelial Barrier Antigen in the Rat Central Nervous System as Quantitated by Immunofluorescent Microscopy

Clinical and experimental studies have demonstrated the neurotoxic and behavioral effects of cadmium. The exact mechanism(s) of cadmium neurotoxicity on the human central nervous system is not completely understood. It has been suggested that the metal exerts its effect via oxidative stress‐induced morphological and functional disruption of blood‐brain barrier (BBB) structures. A sensitive and specific rat BBB marker, the endothelial barrier antigen (EBA), has been previously identified and characterized by lymphocyte hybridoma technology. Alterations in EBA expression have been reported to occur during infectious and non‐infectious central and peripheral nervous system (CNS and PNS) pathologies. We have previously demonstrated that in the rat, EBA first becomes detectable during the first week postnatally and is progressively expressed along with the biochemical and hemodynamic changes that accompany normal brain maturation and barriergenesis. We have also shown that this anti‐EBA IgG1 antibody exclusively recognizes barrier‐competent microvessels in rat central and peripheral nervous system (CNS and PNS). Endothelial cells of peripheral tissues such as the liver and kidney and brain circumventricular organs possessing fenestrated microvascular endothelia, do not display immunoreactivity for EBA.In the present study, a double, sequential immunofluorescent protocol was employed to colocalize EBA‐specific microvessels and astrocytes, using anti‐EBA and anti‐GFAP antibodies, respectively. Sections were examined with an Olympus BX63 Fluorescent microscope outfitted with a DP80 Camera and cellSense software. The EBA‐immunoreactive microvessels were quantitated by measuring the areal fraction of the microvessels per field using an algorithm that allowed for the microvessels to be segmented from the surrounding background within the region of interest (ROI) using a global threshold. The grand mean areal fractions of all microscopic fields captured from the normal control brain sections were compared for statistical significance with the areal fraction grand mean for the cadmium‐treated brain sections using a two‐sample t‐test at a 95% confidence interval. Positive staining was not detected in any of the tissue sections in which the primary antibodies were omitted. Also, EBA expression was not detected in sections from the kidney, known to be vascularized by fenestrated capillaries lacking a barrier function. Statistical analysis (two‐sample t‐test) of the Grand Mean Areal Fractions (μm2) for all microscopic fields evaluated for control and experimental groups yielded a p‐value of less than 0.001 (t = 5.8507, df = 1789, p‐value = 5.808e‐09; p &lt; 0.001).This study indicates that acute exposure to a single, high dose of cadmium significantly downregulates the expression of EBA in the developing rat CNS. The results suggest that EBA is involved in the pathogenesis of cadmium neurotoxicity and could serve as a useful tool for studying the extent of cadmium‐induced impact on BBB structures of the central nervous system in the rat model system.Support or Funding InformationWe express our thanks and sincere appreciation of the research funding provided by the Alabama College of Osteopathic Medicine.

FERM domain\u2013containing protein 6 identifies a subpopulation of varicose nerve fibers in different vertebrate species

FERM domain–containing protein 6 (FRMD6) is a member of the FERM protein superfamily, which is evolutionary highly conserved and has recently been identified as an upstream regulator of the conserved growth-promoting Hippo signaling pathway. In clinical studies, the FRMD6 gene is correlated with high significance to Alzheimer’s disease and cognitive impairment implicating a wider role of this protein in the nervous system. Scare data are available on the localization of endogenous FRMD6 in neural tissues. Using a FRMD6-directed antiserum, we detected specific immunoreactivity in varicose nerve fibers in the rat central and peripheral nervous system. FRMD6-immunoreactive (-ir) neurons were found in the sensory ganglia of cranial nerves, which were marked by a pool of labeled cytoplasmic granules. Cross-species comparative studies detected a morphologically identical fiber population and a comparable fiber distribution in tissues from xenopus and human cranial nerves and ganglia. In the spinal cord, FRMD6-ir was detectable in the terminal endings of primary afferent neurons containing substance P (SP). In the rat diencephalon, FRMD6-ir was co-localized with either SP- or arginine vasopressin–positive fibers in Broca’s diagonal band and the lateral septum. Dense fiber terminals containing both FRMD6-ir and growth hormone-releasing hormone were found in the median eminence. The intimate association of FRMD6 with secretory vesicles was investigated in vitro. Induction of exocytotic vesicles in cultured cells by ectopic expression of the SP precursor molecule preprotachykinin A led to a redistribution and co-localization of endogenous FRMD6 with secretory granules closely mimicking the observations in tissues.

Association of Acute, High-dose Cadmium Exposure with Alterations in Vascular Endothelial Barrier Antigen Expression and Astrocyte Morphology in the Developing Rat Central Nervous System

Clinical and experimental studies have demonstrated the neurotoxic and behavioural effects of cadmium. However, the exact pathophysiological mechanism(s) of cadmium neurotoxicity on the human central nervous system (CNS) is not completely understood. A rat blood-brain barrier (BBB) endothelial marker, the endothelial barrier antigen (EBA), has been identified and we have shown previously that an anti-EBA IgG1 antibody exclusively recognizes barrier-competent microvessels in the rat CNS and peripheral nervous system (PNS). Endothelial cells of peripheral tissues or brain regions possessing fenestrated microvascular endothelia do not display immunoreactivity for EBA. Here, we describe the application of sequential indirect immunofluorescence with anti-EBA, and an antibody directed against glial fibrillary acidic protein (GFAP), to evaluate the immunoreactivity patterns and morphological alterations in BBB microvessels and astrocytes, following a single, high dose of cadmium in normal, term-delivered young rats. We detected a moderate reduction in immunoreactivity and number of microvessels labelled by the anti-EBA in the forebrain, cerebellum and midbrain in cadmium-exposed rats compared with normal controls. We observed weakly GFAP-reactive astrocytes displaying cell bodies with ill-defined borders and blurred cytoplasm within the white and grey matter of cadmium-exposed brains. The astrocyte nuclei were markedly enlarged, intensely hyperchromatic and exhibited chromatin condensation with nuclear fragmentation. This study indicates for the first time that EBA is involved in, and could serve as a potentially useful marker for studying, cadmium neurotoxicity in the rat model system.

Synergistic Effect of Asiatic Acid and Madecassic Acid against Antioxidant Deficit in Rat Peripheral Nervous System

Synergistic Effect of Asiatic Acid and Madecassic Acid against Antioxidant Deficit in Rat Peripheral Nervous System

A brand-new generation of fluorescent nano-neural tracers: biotinylated dextran amine conjugated carbonized polymer dots.

The anterograde neuroanatomical tracing technique plays a crucial role in studying and charting the complex interconnections of the nervous system. But there are several major limitations for traditional neural tracers, such as complex immunohistochemical staining procedures, low fluorescence intensity and quick fluorescence quenching. Carbon dots (CDs) as fluorescent bio-probes have been widely used in the biological studies due to their superior optical properties, excellent chemical stability, low toxicity, and easy modifications. In this study, biotinylated dextran amine (BDA) and red fluorescent carbonized polymer dots (CPDs) are successfully conjugated to develop a brand-new generation of fluorescent nano-neural tracers: BDA-CPDs. They are successfully applied as fluorescent probes for in vitro and in vivo bioimaging. In vitro biodistribution of BDA-CPDs shows that they distribute mainly in lysosomes and endoplasmic reticulum. Moreover, two metabolic pathways of BDA-CPDs are found through the investigation of in vivo biodistribution of BDA-CPDs. Furthermore, they can be taken up and anterogradely transported by neurons within the peripheral nervous system of rats. Our results suggest that BDA-CPDs have many advantages over traditional tracers, such as low toxicity, high photoluminescence intensity, excellent photostability and easy procedures. Anterograde pathway tracing with BDA-CPDs is a simple, direct and economical way of studying the connections in the nervous system. Therefore, BDA-CPDs are reliable and valuable fluorescent anterograde neural tracers.

Enhanced Pain Sensitivity with Systemic Ultrastructural Changes of the Nervous Systems after Cobra Venom Injection is Reversed by Electroacupuncture Treatment

Electroacupuncture (EA) has been proved to be effective in treating certain neuropathic pain conditions. The mechanisms of pain relief by EA are not fully understood. There have been sporadic reports of damage in the peripheral nervous system (PNS) and regions of the central nervous system (CNS) at the ultrastructural level following peripheral nerve injury. However, information about possible systemic changes in the PNS and CNS after nerve injury is scarce. The goal of this study was to examine the ultrastructural changes of the nervous system induced by a local injection of cobra venom into the sciatic nerve and to compare the ultrastructural changes in rats with or without treatment with EA or pregabalin. An experimental study. Department of Anesthesiology, Pain Medicine, and Critical Care Medicine, Aviation General Hospital of China Medical University. In this study, using an established model of sciatic neuralgia induced by local injection of cobra venom into the sciatic nerve, we examined ultrastructural changes of the PNS and CNS and how they respond to EA and pregabalin treatment. EA and pregabalin were given daily from postoperative day (POD) 14 to 36. Based on previous works, the frequency of EA stimulation of the ST36 and GB34 acupoints was held to 2/100 Hz variable. Pain sensitivity in the sciatic neuralgia rats with and without treatments was assessed using the von Frey test. Ultrastructural alterations were examined bilaterally in the prefrontal cortex, hippocampus, medulla oblongata; and the cervical, thoracic, and lumbar spinal cords on PODs 14, 40, and 60. Ultrastructural examinations were also carried out on the bilateral sciatic nerves and dorsal root ganglion (DRG) at the cervical, thoracic and lumbar levels. In rats treated with EA or pregabalin, the ultrastructure was examined on PODs 40 and 60. Behavioral signs of pain and systemic ultrastructural changes including demyelination were observed at all levels of the PNS and CNS in rats with sciatic neuralgia. After intervention, the mechanical withdrawal thresholds of the EA group and pregabalin group were significantly higher than that of the cobra venom group (P < 0.05). Both EA and pregabalin treatments partially reversed increased cutaneous sensitivity to mechanical stimulation. However, only the EA treatment was able to repair the ultrastructural damages caused by cobra venom. The results confirm that peripheral nerve injury led to the ultrastructural damage at different levels of the CNS as demonstrated with electron microscopy; however, we need to further verify this at both the molecular level and in light microscope level. Sciatic neuralgia induced by cobra venom is a chemical injury, and whether this exactly mimics a peripheral nerve mechanical injury is still unclear. Local cobra venom injection leads to systemic neurotoxicity. EA and pregabalin alleviate pain via different mechanisms. Sciatic neuralgia, cobra venom, demyelination, electroacupuncture, pregabalin, rat model.

A novel approach to 32-channel peripheral nervous system myelin imaging in vivo, with single axon resolution.

OBJECTIVE Intravital spectral imaging of the large, deeply situated nerves in the rat peripheral nervous system (PNS) has not been well described. Here, the authors have developed a highly stable platform for performing imaging of the tibial nerve in live rodents, thus allowing the capture of high-resolution, high-magnification spectral images requiring long acquisition times. By further exploiting the qualities of the topically applied myelin dye Nile red, this technique is capable of visualizing the detailed microenvironment of peripheral nerve demyelination injury and recovery, while allowing us to obtain images of exogenous Schwann cell myelination in a living animal. METHODS The authors caused doxorubicin-induced focal demyelination in the tibial nerves of 25 Thy-1 GFP rats, of which 2 subsets (n = 10 each) received either BFP-labeled SKP-SCs or SCs to the zone of injury. Prior to acquiring images of myelin recovery in these nerves, a tibial nerve window was constructed using a silicone hemitube, a fast drying silicone polymer, and a small coverslip. This construct was then affixed to a 3D-printed nerve stage, which in turn was affixed to an external fixation/microscope stage device. Myelin visualization was facilitated by the topical application of Nile red. RESULTS The authors reliably demonstrated intravital peripheral nerve myelin imaging with micron-level resolution and magnification, and minimal movement artifact. The detailed microenvironment of nerve remyelination can be vividly observed, while exogenously applied Schwann cells and skin-derived precursor Schwann cells can be seen myelinating axons. CONCLUSIONS Topically applied Nile red enables intravital study of myelin in the living rat PNS. Furthermore, the use of a tibial nerve window facilitates stable intravital peripheral nerve imaging, making possible high-definition spectral imaging with long acquisition times.

Functional and structural characterization of axonal opioid receptors as targets for analgesia.

BackgroundOpioids are the gold standard for the treatment of acute pain despite serious side effects in the central and enteric nervous system. µ-opioid receptors (MOPs) are expressed and functional at the terminals of sensory axons, when activated by exogenous or endogenous ligands. However, the presence and function of MOP along nociceptive axons remains controversial particularly in naïve animals. Here, we characterized axonal MOPs by immunofluorescence, ultrastructural, and functional analyses. Furthermore, we evaluated hypertonic saline as a possible enhancer of opioid receptor function.ResultsComparative immunolabeling showed that, among several tested antibodies, which all provided specific MOP detection in the rat central nervous system (CNS), only one monoclonal MOP-antibody yielded specificity and reproducibility for MOP detection in the rat peripheral nervous system including the sciatic nerve. Double immunolabeling documented that MOP immunoreactivity was confined to calcitonin gene-related peptide (CGRP) positive fibers and fiber bundles. Almost identical labeling and double labeling patterns were found using mcherry-immunolabeling on sciatic nerves of mice producing a MOP-mcherry fusion protein (MOP-mcherry knock-in mice). Preembedding immunogold electron microscopy on MOP-mcherry knock-in sciatic nerves indicated presence of MOP in cytoplasm and at membranes of unmyelinated axons. Application of [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) or fentanyl dose-dependently inhibited depolarization-induced CGRP release from rat sciatic nerve axons ex vivo, which was blocked by naloxone. When the lipophilic opioid fentanyl was applied perisciatically in naïve Wistar rats, mechanical nociceptive thresholds increased. Subthreshold doses of fentanyl or the hydrophilic opioid DAMGO were only effective if injected together with hypertonic saline. In vitro, using β-arrestin-2/MOP double-transfected human embryonic kidney cells, DAMGO as well as fentanyl lead to a recruitment of β-arrestin-2 to the membrane followed by a β-arrestin-2 reappearance in the cytosol and MOP internalization. Pretreatment with hypertonic saline prevented MOP internalization.ConclusionMOPs are present and functional in the axonal membrane from naïve animals. Hypertonic saline acutely decreases ligand-induced internalization of MOP and thereby might improve MOP function. Further studies should explore potential clinical applications of opioids together with enhancers for regional analgesia.

Immunocytochemical Localization of Monoamine Oxidase Type B in Rat'sPeripheral Nervous System.

Immunohistochemistry is used to investigate subcellular localization of monoamine oxidase type B (MAOB) in the axon of the rat's peripheral nervous system. Through light and electron microscopy, the presence of MAOB-immunoreactive structures in the propria lamina of tongue and on the outer membranes of mitochondria in both myelinated and unmyelinated axons can be detected. As a result, MAOB may potentially play a crucial role in the axons of the rat's peripheral nervous system and may be closely associated with both axonal transport and nerve conduction.

A novel fluorescent retrograde neural tracer: cholera toxin B conjugated carbon dots.

The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde tracers.

Genetic dissection of the Mss4 locus mediating sex-biased cancer resistance in the rat peripheral nervous system.

The incidence of neural tumors is sexually dimorphic in both, humans and rodents. The identification of genetic determinants contributing to sex-biased tumor development is an essential prerequisite for differential tumor prevention in males and females. F2 hybrids of inbred BDIV and BDIX rats, resistant and susceptible, respectively, to ethylnitrosourea-induced malignant peripheral nerve sheath tumors (MPNST) display a marked sex bias regarding tumor risk. Homozygous BDIV alleles at the Mss4 locus (90.9-111.2 Mb, chromosome 6) mediate MPNST resistance exclusively in female F2 rats according to a genome wide association analysis. This locus was functionally confirmed and fine mapped through MPNST induction in males and females of three congenic rat strains (BDIX.BDIV-Mss4a, b, d). As a consequence, it could be subdivided in Mss4.1 (98.8-99.7 Mb) mediating cancer resistance, and Mss4.2 (99.7-111.2 Mb) enhancing sex specificity. Positional candidate genes were selected through DNA sequencing and expression profiling using RNAs from trigeminal nerve tissue of parental and congenic male and female animals. The transregulatory fingerprint of BDIV or BDIX alleles at Mss4.1 and/or Mss4.2, respectively, provided insight into the processes influencing cancer risk in a sex-biased way. A group of genes, a fraction of which involved in Schwann cell differentiation, showed low, male-biased expression in nerve tissues under the control of BDIX susceptibility alleles, but high, female-biased transcript levels when controlled by BDIV resistance alleles at Mss4. The Esr2 gene located in Mss4.1 constitutes an interesting functional candidate together with a yet unidentified gene/enhancer in Mss4.2.

Epigenetic Regulation of Genes That Modulate Chronic Stress-Induced Visceral Pain in the Peripheral Nervous System

Chronic stress alters the hypothalamic-pituitary-adrenal axis, increases gut motility, and increases the perception of visceral pain. We investigated whether epigenetic mechanisms regulate chronic stress-induced visceral pain in the peripheral nervous systems of rats. Male rats were subjected to 1 hour of water avoidance stress each day, or given daily subcutaneous injections of corticosterone, for 10 consecutive days. L4-L5 and L6-S2 dorsal root ganglia (DRG) were collected and compared between stressed and control rats (placed for 1 hour each day in a tank without water). Levels of cannabinoid receptor 1 (CNR1), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), transient receptor potential vanilloid type 1 (TRPV1), and EP300 were knocked down in DRG neurons in situ with small interfering RNAs. We measured DNA methylation and histone acetylation at genes encoding the glucocorticoid receptor (NR3C1), CNR1, and TRPV1. Visceral pain was measured in response to colorectal distention. Chronic stress was associated with increased methylation of the Nr3c1 promoter and reduced expression of this gene in L6-S2, but not L4-L5, DRGs. Stress also was associated with up-regulation in DNMT1-associated methylation of the Cnr1 promoter and down-regulation of glucocorticoid-receptor-mediated expression of CNR1 in L6-S2, but not L4-L5, DRGs. Concurrently, chronic stress increased expression of the histone acetyltransferase EP300 and increased histone acetylation at the Trpv1 promoter and expression of the TRPV1 receptor in L6-S2 DRG neurons. Knockdown of DNMT1 and EP300 in L6-S2 DRG neurons of rats reduced DNA methylation and histone acetylation, respectively, and prevented chronic stress-induced increases in visceral pain. Chronic stress increases DNA methylation and histone acetylation of genes that regulate visceral pain sensation in the peripheral nervous system of rats. Blocking epigenetic regulatory pathways in specific regions of the spinal cord might be developed to treat patients with chronic abdominal pain.

Localization of SSeCKS in unmyelinated primary sensory neurons

BackgroundSSeCKS (Src SupprEssed C Kinase Substrate) is a proposed protein kinase C substrate/A kinase anchoring protein (AKAP) that has recently been characterized in the rat peripheral nervous system. It has been shown that approximately 40% of small primary sensory neurons contain SSeCKS-immunoreactivity in a population largely separate from substance P (95.2%), calcitonin gene related peptide (95.3%), or fluoride resistant acid phosphatase (55.0%) labeled cells. In the spinal cord, it was found that SSeCKS-immunoreactive axon collaterals terminate in the dorsal third of lamina II outer in a region similar to that of unmyelinated C-, or small diameter myelinated Aδ-, fibers. However, the precise characterization of the anatomical profile of the primary sensory neurons containing SSeCKS remains to be determined. Here, immunohistochemical labeling at the light and ultrastructural level is used to clarify the myelination status of SSeCKS-containing sensory neuron axons and to further clarify the morphometric, and provide insight into the functional, classification of SSeCKS-IR sensory neurons.MethodsColocalization studies of SSeCKS with myelination markers, ultrastructural localization of SSeCKS labeling and ablation of largely unmyelinated sensory fibers by neonatal capsaicin administration were all used to establish whether SSeCKS containing sensory neurons represent a subpopulation of unmyelinated primary sensory C-fibers.ResultsDouble labeling studies of SSeCKS with CNPase in the dorsal horn and Pzero in the periphery showed that SSeCKS immunoreactivity was observed predominantly in association with unmyelinated primary sensory fibers. At the ultrastructural level, SSeCKS immunoreactivity was most commonly associated with axonal membrane margins of unmyelinated fibers. In capsaicin treated rats, SSeCKS immunoreactivity was essentially obliterated in the dorsal horn while in dorsal root ganglia quantitative analysis revealed a 43% reduction in the number of SSeCKS-labeled cells. This attenuation is concomitant with a decrease in fluoride-resistant acid phosphatase labeled fibers in the spinal cord dorsal horn and small neuronal somata in sensory ganglia.ConclusionThese results demonstrate that SSeCKS is primarily localized within a distinct subpopulation of small diameter, largely unmyelinated C-fiber primary sensory neurons putatively involved in nociception.

Sox2 in the adult rat sensory nervous system

Sex-determining region Y (SRY)-box 2 (Sox2) is a member of the Sox family transcription factors. In the central nervous system, Sox2 is expressed in neural stem cells from neurogenic regions, and regulates stem cell proliferation and differentiation. In the peripheral nervous system, Sox2 is found only in the immature and dedifferentiated Schwann cells, and is involved in myelination inhibition or N-cadherin redistribution. In the present immunohistochemical study, we found that Sox2 is also expressed in other cells of the adult rat peripheral nervous system. Nuclear Sox2 was observed in all satellite glial cells, non-myelinating Schwann cells, and the majority of terminal Schwann cells that form lamellar corpuscles and longitudinal lanceolate endings. Sox2 was not found in myelinating Schwann cells and terminal Schwann cells of subepidermal free nerve endings. Satellite glial cells exhibit strong Sox2 immunoreactivity, whereas non-myelinating Schwann cells show weak immunoreactivity. RT-PCR confirmed the presence of Sox2 mRNA, indicating that the cells are likely Sox2 expressors. Our findings suggest that the role of Sox2 in the peripheral nervous system may be cell-type-dependent.

Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry.

Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TOF) MS. The protocol requires ∼2 h for sample preparation, neuron isolation and metabolite extraction, and 1 h for metabolic measurement. We used the approach to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25-500 μm in diameter) isolated from the sea slug (Aplysia californica) central and rat (Rattus norvegicus) peripheral nervous systems. We found that a subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains.