Mechanical allodynia (MA), including punctate and dynamic forms, is a common and debilitating symptom suffered by millions of chronic pain patients. Some peripheral injuries result in the development of bilateral MA, while most injuries usually led to unilateral MA. To date, the control of such laterality remains poorly understood. Here, to study the role of microglia in the control of MA laterality, we used genetic strategies to deplete microglia and tested both dynamic and punctate forms of MA in mice. Surprisingly, the depletion of central microglia did not prevent the induction of bilateral dynamic and punctate MA. Moreover, in dorsal root ganglion-dorsal root-sagittal spinal cord slice preparations we recorded the low-threshold Aβ-fiber stimulation-evoked inputs and outputs of superficial dorsal horn neurons. Consistent with behavioral results, microglial depletion did not prevent the opening of bilateral gates for Aβ pathways in the superficial dorsal horn. This study challenges the role of microglia in the control of MA laterality in mice. Future studies are needed to further understand whether the role of microglia in the control of MA laterality is etiology-or species-specific.
Mice ; Animals ; Hyperalgesia/metabolism* ; Microglia/metabolism* ; Disease Models, Animal ; Spinal Cord/metabolism* ; Spinal Cord Dorsal Horn/metabolism* ; Ganglia, Spinal/metabolism*

Dorsal root ganglia (DRG) is an essential part of the peripheral nervous system and the hub of the peripheral sensory afferent. The dynamic changes of neuronal cells and their gene expression during the development of dorsal root ganglion have been studied through single-cell RNAseq analysis, while the dynamic changes of non-neuronal cells have not been systematically studied. Using single cell RNA sequencing technology, we conducted a research on the non-neuronal cells in the dorsal root ganglia of rats at different developmental stage. In this study, primary cell suspension was obtained from using the dorsal root ganglions (DRGs, L4-L5) of ten 7-day-old rats and three 3-month-old rats. The 10×Genomics platform was used for single cell dissociation and RNA sequencing. Twenty cell subsets were acquired through cluster dimension reduction analysis, and the marker genes of different types of cells in DRG were identified according to previous researches about DRG single cell transcriptome sequencing. In order to find out the non-neuronal cell subsets with significant differences at different development stage, the cells were classified into different cell types according to markers collected from previous researches. We performed pseudotime analysis of 4 types Schwann cells. It was found that subtype Ⅱ Schwann cells emerged firstly, and then were subtype Ⅲ Schwann cells and subtype Ⅳ Schwann cells, while subtype Ⅰ Schwann cells existed during the whole development procedure. Pseudotime analysis indicated the essential genes influencing cell fate of different subtypes of Schwann cell in DRG, such as Ntrk2 and Pmp2, which affected cell fate of Schwann cells during the development period. GO analysis of differential expressed genes showed that the up-regulated genes, such as Cst3 and Spp1, were closely related to biological process of tissue homeostasis and multi-multicellular organism process. The down regulated key genes, such as Col3a1 and Col4a1, had close relationship with the progress of extracellular structure organization and negative regulation of cell adhesion. This suggested that the expression of genes enhancing cell homestasis increased, while the expression of related genes regulating ECM-receptor interaction pathway decreased during the development. The discovery provided valuable information and brand-new perspectives for the study on the physical and developmental mechanism of Schwann cell as well as the non-neuronal cell changes in DRG at different developmental stage. The differential gene expression results provided crucial references for the mechanism of somatosensory maturation during development.
Rats ; Animals ; Ganglia, Spinal/metabolism* ; Rats, Sprague-Dawley ; Transcriptome ; Neurons/metabolism* ; Schwann Cells/physiology*

OBJECTIVE: To determine the expression and significance of p75NTR in the neuron and glia of dorsal root ganglia (DRG) in different injury models. METHODS: The models of sciatic nerve injury, spinal cord injury, and combined injury (sciatic nerve injury one week prior to spinal cord injury) were established. The rats were randomly divided into a normal group,a sciatic nerve injury group,a spinal cord injury group, and a combined injury group. The sensory neurons in the DRG were labeled by fast blue (FB) injected in the dorsal column of spinal cord 0.5mm rostral to the transection site. The expression of p75NTR in the neurons and glia of the DRG was examined with immunofluorescence histochemistry after different kinds of injury and its expression in the FB positive neurons was further observed with immunofluorescence histochemistry combined with FB retrograde labeling. RESULTS: The expression of p75NTR was increased in the glia, but was downregulated in sensory neurons in the sciatic nerve injury group compared with the normal group. p75NTR immunoreactive products were downregulated in the glia in the spinal cord injury group compared with the sciatic nerve injury group or the combined injury group. In the combined lesion animals, the expression of p75NTR was similar to that of the sciatic nerve injury group. Its expression in the sensory neurons of DRG was downregulated,but was upregulated in the glia. The majority of sensory neurons labeled by FB in the combined injury group were p75NTR-negative, but surrounded by p75NTR-positive glia. CONCLUSION p75NTR immunoreactive products in the glia and neurons of DRG have significant discrepancy after injury. The glial p75NTR in the DRG may play a role in the enhanced regeneration of acsending tract in the injured spinal cord after combined injury.
Animals ; Ganglia, Spinal ; metabolism ; pathology ; Male ; Neuroglia ; metabolism ; Neurons ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Nerve Growth Factor ; metabolism ; Sciatic Nerve ; injuries ; metabolism ; Spinal Injuries ; metabolism

OBJECTIVETo establish the co-culture model of cancer cells and nerve, and to study the influence of esophageal cancer on nerve fibers.

METHODSMouse dorsal root ganglion (DRG) was cultured in sterile conditions by primary isolation. Co-culture model was established using matrigel matrix-embedded DRG and EC109 (esophageal cancer cell line) transfected with green fluorescent protein. Morphological changes of DRG, number and area of neurites were quantified with microscopy and image analysis. Furthermore, the mRNA expression of nerve growth factor(NGF) and brain derived neurotrophic factor(BDNF) was detected by real-time PCR.

RESULTSIn mixture cultivation model of EC109 and DRG cells, directional outgrowth of neurite projecting to EC109 was observed, and the length of neurite was markedly longer in proximal field compared to distal field. The number and area of neurite were 87 and 346 μm(2) in proximal field respectively, and 23 and 141 μm(2) in distal field on the 7th day. The expressions of NGF and BDNF were up-regulated in esophageal cancer cells.

CONCLUSIONSThe esophageal cancer may play an important role in nerve fiber growth and guidance, which may be associated with the up-regulation of NGF and BDNF expressions.

Animals ; Coculture Techniques ; Esophageal Neoplasms ; metabolism ; Ganglia, Spinal ; Humans ; Neurites ; Rats, Sprague-Dawley ; Up-Regulation

OBJECTIVETo investigate the effects of glucocorticoid receptor antagonist-M8046 on the behavior and the cyclooxygenase-2/prostaglandin E2( COX-2/PGE2) expression in spinal cord dorsal horn and dorsal root ganglia (DRG) in chronic constrictive injury (CCI) rats.

METHODSOne hundred and forty-four male SD rats were randomly divided into 4 groups, 36 rats in each group: Sham operation group (Sham), chronic constrictive group (CCI), M8046 treated group (M8046) and solvent controlled group (Sc). M8046 3 mg/(kg x d) intraperitoneal injection was given after operation in group M8046. Paw thennal withdrawal (PTWL) and paw mechanical withdrawal threshold (PMWT) of rats were measured on 2 pre-operative and 1, 3, 7, 10, 14 post-operative days. The spinal cord and L15 DRG of the operated side was removed at 3, 7, 14 days after surgery. The change of COX-2 and PGE2 expression was determined by immunohistochemical staining and ELISA separately.

RESULTSPTWL and PMWT in CCI group were significantly lower than those in Sham group on every post-operative day (P < 0.05). PTWL and PMWT in M8046 group were significantly higher than those in CCI group on 7, 10, 14 post-operative day (P < 0.05). In spinal dorsal horn, the level of COX-2 and PGE2 expression in CCI group was significantly higher than that in Sham group (P < 0.05). M8046 could significantly attenuate the activation of COX-2 and PGE2 induced by CCI (P < 0.05). The expression of COX-2 and PGE2 in DRG was similar to that in spinal dorsal horn.

CONCLUSIONThe effects of M8046 ameliorate the CCI-induced neuropathic pain may be related to attenuate the expression of COX-2 and PGE2 in spinal cord and DRG.

Animals ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; metabolism ; Ganglia, Spinal ; drug effects ; metabolism ; Male ; Neuralgia ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Glucocorticoid ; antagonists & inhibitors ; Spinal Cord ; drug effects ; metabolism

In the present study, the intracellular free calcium concentration ([Ca(2+)](i)) in acutely isolated rat dorsal root ganglia (DRG) neurons modulated by loureirin B, an active component of "dragon's blood" which is a kind of Chinese herbal medicine, was determined by the means of Fura-2 based microfluorimetry. It was found that loureirin B could evoke the elevation of [Ca(2+)](i) in a dose-dependent manner. However, the elevation of [Ca(2+)](i) evoked in the calcium free solution was much smaller than that in the standard external cell solution, suggesting that most change of [Ca(2+)](i) was generated by the influx of extracellular Ca(2+), not by the activities of intracellular organelles like Ca(2+) stores and mitochondria. In addition, the mixture of loureirin B and caffeine also induced [Ca(2+)](i) rise, but the peak of [Ca(2+)](i) rise induced by the mixture was significantly lower than that by caffeine alone, which means the triggering pathway and the targets of caffeine are probably involved in loureirin B-induced [Ca(2+)](i) rise. Moreover, compared to the transients induced by caffeine, KCl and capsaicin, the loureirin B-induced [Ca(2+)](i) rise is much slower and more stable. These results indicate that the capability of loureirin B of inducing the [Ca(2+)](i) rise is solid and unique.
Animals ; Caffeine ; pharmacology ; Calcium ; metabolism ; Ganglia, Spinal ; drug effects ; metabolism ; Neurons, Afferent ; drug effects ; metabolism ; Rats ; Resins, Plant ; pharmacology

Due to the complex circuitry and plethora of cell types involved in somatosensation, it is becoming increasingly important to be able to observe cellular activity at the population level. In addition, since cells rely on an intricate variety of extracellular factors, it is important to strive to maintain the physiological environment. Many electrophysiological techniques require the implementation of artificially-produced physiological environments and it can be difficult to assess the activity of many cells simultaneously. Moreover, imaging Ca transients using Ca-sensitive dyes often requires in vitro preparations or in vivo injections, which can lead to variable expression levels. With the development of more sensitive genetically-encoded Ca indicators (GECIs) it is now possible to observe changes in Ca transients in large populations of cells at the same time. Recently, groups have used a GECI called GCaMP to address fundamental questions in somatosensation. Researchers can now induce GCaMP expression in the mouse genome using viral or gene knock-in approaches and observe the activity of populations of cells in the pain pathway such as dorsal root ganglia (DRG), spinal neurons, or glia. This approach can be used in vivo and thus maintains the organism's biological integrity. The implementation of GCaMP imaging has led to many advances in our understanding of somatosensation. Here, we review the current findings in pain research using GCaMP imaging as well as discussing potential methodological considerations.
Afferent Pathways ; physiology ; Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; genetics ; Ganglia, Spinal ; metabolism ; Humans ; Pain ; metabolism ; pathology

The Baimai Ointment with the effect of relaxing sinew and activating collaterals demonstrates a definite effect on Baimai disease with pain, spasm, stiffness and other symptoms, while the pharmacodynamic characteristics and mechanism of this agent remain unclear. In this study, a rat model of chronic compression of L4 dorsal root ganglion(CCD) was established by lumbar disc herniation, and the efficacy and mechanism of Baimai Ointment in the treatment of CCD were preliminarily explored by behavioral tests, side effect evaluation, network analysis, antagonist and molecular biology verification. The pharmacodynamic experiment indicated that Baimai Ointment significantly improved the pain thresholds(mechanical pain, thermal pain, and cold pain) and gait behavior of CCD model rats without causing tolerance or obvious toxic and side effects. Baimai Ointment inhibited the second-phase nociceptive response of mice in the formalin test, increased the hot plate threshold of normal mice, and down-regulated the expression of inflammatory cytokines in the spinal cord. Network analysis showed that Baimai Ointment had synergistic effect in the treatment of CCD and was related to descending inhibition/facilitation system and neuroinflammation. Furthermore, behavioral tests, Western blot, and immunofluorescence assay revealed that the pain-relieving effect of Baimai Ointment on CCD may be related to the regulation of the interaction between neuroactive ligand and receptors(neuroligands) such as CHRNA7, ADRA2A, and ADRB2, and the down-regulation of the expression of NOS2/pERK/PI3K, the core regulatory element of HIF-1 signaling pathway in spinal microglia. The findings preliminarily reveal the mechanism of relaxing sinew and activating collaterals of Baimai Ointment in the treatment of Baimai disease, providing a reference for the rational drug use and further research of this agent.
Rats ; Mice ; Animals ; Chronic Pain/metabolism* ; Rats, Sprague-Dawley ; Ganglia, Spinal/metabolism* ; Ligands ; Signal Transduction ; Hyperalgesia/metabolism* ; Drugs, Chinese Herbal

Different physical and chemical stimuli are detected by the peripheral sensory receptors of dorsal root ganglion (DRG) neurons, and the generated inputs are transmitted via afferent fibers into the central nervous system. The gene expression profiles of DRG neurons contribute to the generation, transmission, and regulation of various somatosensory signals. Recently, the single-cell transcriptomes, cell types, and functional annotations of somatosensory neurons have been studied. In this review, we introduce our classification of DRG neurons based on single-cell RNA-sequencing and functional analyses, and discuss the technical approaches. Moreover, studies on the molecular and cellular mechanisms underlying somatic sensations are discussed.
Animals ; Ganglia, Spinal ; cytology ; Gene Regulatory Networks ; Humans ; Pain ; genetics ; metabolism ; pathology ; Sensory Receptor Cells ; metabolism ; Sequence Analysis, RNA ; Transcriptome

BACKGROUNDParvalbumin (PV), as a mobile endogenous calcium buffer, plays an important role in affecting temporospatial characteristics of calcium transients and in modulating calcium homeostasis. PV is expressed in neurons in the dorsal root ganglion (DRG) and spinal dorsal horn and may be involved in synaptic transmission through regulating cytoplasm calcium concentrations. But the exact role of PV in peripheral sensory neurons remains unknown. Microtubule-associated protein 2 (MAP-2), belonging to structural microtubule-associated protein family, is especially vulnerable to acute central nervous system (CNS) injury, and there will be rapid loss of MAP-2 at the injury site. The present study investigated the changes of PV expressing neurons and the MAP-2 neurons in the DRG after an operation for chronic constriction injury to the unilateral sciatic nerve (CCI-SN), in order to demonstrate the possible roles of PV and MAP-2 in transmission and modulation of peripheral nociceptive information.

METHODSSeventy-two adult male Sprague-Dawley (SD) rats, weighing 180 - 220 g, were randomly divided into two groups (36 rats in each group), the sham operation group and chronic constriction injury (CCI) group. Six rats in each group were randomly selected to receive mechanical and thermal sensitivity tests at one day before operation and 1, 3, 5, 7, and 14 days after surgery. After pain behavioral test, ipsilateral lumbar fifth DRGs were removed and double immunofluorescence staining was performed to assess the expression changes of PV and of MAP2 expressing neurons in the L5 DRG before or after surgery.

RESULTSThe animals with CCI-SN showed obvious mechanical allodynia and thermal hyperalgesia (P < 0.05). Both the thermal and mechanical hyperalgesia decreased to their lowest degree at 7 days after surgery compared to the baseline before surgery (P < 0.01). In normal rats before surgery, a large number of neurons were MAP-2 single labeled cells, and just a small number of PV-expressed neurons were found. PV-positive neurons, PV-positive nerve fibers and PV-negative neurons, formed a direct or close contact for cross-talk. We used immunocytochemical staining to quantify the time course of changes to PV and MAP-2 expressing neurons in tissue, and found that the number of PV expressing neurons began to slightly decrease at 3 days after surgery, and had a significant reduction at CCI day 5, day 7 (P < 0.05). But MAP-2 neurons significantly decreased on just the 3rd day after CCI (P < 0.05). No changes in PV and MAP-2 expression were almost found in sham operated rats. The number of PV positive neurons, was positively correlated with the hyperalgesia threshold.

CONCLUSIONSA sharp decline in MAP-2 neurons may be the early response to surgical injury, and PV positive neurons were much more effective at affecting the changes of pain behaviors, indicating that the down-regulation of PV protein could participate in, at least in part, the modulation of nociceptive transmission.

Animals ; Constriction, Pathologic ; Ganglia, Spinal ; metabolism ; pathology ; Immunohistochemistry ; Male ; Microtubule-Associated Proteins ; metabolism ; Neurons ; metabolism ; Parvalbumins ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sciatic Neuropathy ; metabolism ; pathology