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Expression profiling of spinal cord dorsal horn in a rat model of complex regional pain syndrome type-I uncovers potential mechanisms mediating pain and neuroinflammation responses.

ABSTRACT: BACKGROUND:Complex regional pain syndrome type-I (CRPS-I) is a progressive and devastating pain condition. The mechanisms of CRPS-I still remain poorly understood. We aim to explore expression profiles of genes relevant to pain and neuroinflammation mechanisms involved in CRPS-I. METHODS:The rat chronic post-ischemic pain (CPIP) model that mimics human CRPS-I was established. RNA-sequencing (RNA-Seq), qPCR, Western blot, immunostaining, and pharmacological studies were used for profiling gene changes in ipsilateral spinal cord dorsal horn (SCDH) of CPIP model rat and further validation. RESULTS:CPIP rats developed persistent mechanical allodynia in bilateral hind paws, accompanied with obvious glial activation in SCDH. RNA-Seq identified a total of 435 differentially expressed genes (DEGs) in ipsilateral SCDH of CPIP rats. qPCR confirmed the expression of several representative genes. Functional analysis of DEGs identified that the most significantly enriched biological processes of upregulated genes include inflammatory and innate immune response. We further identified NLRP3 inflammasome expression to be significantly upregulated in SCDH of CPIP rats. Pharmacological blocking NLRP3 inflammasome reduced IL-1? overproduction, glial activation in SCDH as well as mechanical allodynia of CPIP rats. CONCLUSION:Our study revealed that immune and inflammatory responses are predominant biological events in SCDH of CPIP rats. We further identified NLRP3 inflammasome in SCDH as a key contributor to the pain and inflammation responses in CPIP rats. Thus, our study provided putative novel targets that may help to develop effective therapeutics against CRPS-I.

SUBMITTER: Chen R

PROVIDER: S-EPMC7245895 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Expression profiling of spinal cord dorsal horn in a rat model of complex regional pain syndrome type-I uncovers potential mechanisms mediating pain and neuroinflammation responses.

Chen Ruixiang R Yin Chengyu C Hu Qimiao Q Liu Boyu B Tai Yan Y Zheng Xiaoli X Li Yuanyuan Y Fang Jianqiao J Liu Boyi B

Journal of neuroinflammation 20200523 1

<h4>Background</h4>Complex regional pain syndrome type-I (CRPS-I) is a progressive and devastating pain condition. The mechanisms of CRPS-I still remain poorly understood. We aim to explore expression profiles of genes relevant to pain and neuroinflammation mechanisms involved in CRPS-I.<h4>Methods</h4>The rat chronic post-ischemic pain (CPIP) model that mimics human CRPS-I was established. RNA-sequencing (RNA-Seq), qPCR, Western blot, immunostaining, and pharmacological studies were used for pr ...[more]

PMID: 32446302

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Project description:The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent's model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses.

Dataset Information

Expression profiling of spinal cord dorsal horn in a rat model of complex regional pain syndrome type-I uncovers potential mechanisms mediating pain and neuroinflammation responses.

Publications

Expression profiling of spinal cord dorsal horn in a rat model of complex regional pain syndrome type-I uncovers potential mechanisms mediating pain and neuroinflammation responses.

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