The Contribution of TSLP Activation to Hyperalgesia in Dorsal Root Ganglia Neurons of a Rat

Peripheral nerve injury involves divergent alterations within dorsal root ganglia (DRG) neurons sensitized by persistent inflammation. Thymic stromal lymphopoietin (TSLP) production is crucial in the development of chronic inflammatory responses. Herein, we investigate the changes of TSLP expression in rats’ DRG neurons between injured and uninjured sides in the same rat. Linalyl acetate (LA) was served as a TSLP inhibitor and given intraperitoneally. Rats were assigned to be group of chronic constriction injury (CCI) of the sciatic nerve and the group of CCI of the sciatic nerve administrated with LA. Over 14 days, the rats were measured for paw withdrawal thresholds. DRGs were collected to assess morphological changes via immunofluorescence study. After receiving CCI, the rats rapidly developed mechanical hyperalgesia. TSLP expression at DRG, on the ipsilateral injured side, was consistent with changes in pain behaviors. TSLP appeared in nerve fibers with both small diameters and large diameters. Additionally, TSLP was expressed mostly in transient receptor potential vanilloid-1 (TRPV1)-positive nociceptive neurons. Administration with LA can attenuate the pain behaviors and expression of TSLP in DRG neurons, and in apoptotic neurons at the injured side, but not in the contra-lateral uninjured side. Overall, these results imply that altered expressions of TSLP in nociceptive DRG neurons contributed to mechanical hyperalgesia in a CCI rat model.     

Macrophage Activation in the Dorsal Root Ganglion in Rats Developing Autotomy after Peripheral Nerve Injury

Autotomy, self-mutilation of a denervated limb, is common in animals after peripheral nerve injury (PNI) and is a reliable proxy for neuropathic pain in humans. Understanding the occurrence and treatment of autotomy remains challenging. The objective of this study was to investigate the occurrence of autotomy in nude and Wistar rats and evaluate the differences in macrophage activation and fiber sensitization contributing to the understanding of autotomy behavior. Autotomy in nude and Wistar rats was observed and evaluated 6 and 12 weeks after sciatic nerve repair surgery. The numbers of macrophages and the types of neurons in the dorsal root ganglion (DRG) between the two groups were compared by immunofluorescence studies. Immunostaining of T cells in the DRG was also assessed. Nude rats engaged in autotomy with less frequency than Wistar rats. Autotomy symptoms were also relatively less severe in nude rats. Immunofluorescence studies revealed increased macrophage accumulation and activation in the DRG of Wistar rats. The percentage of NF200+ neurons was higher at 6 and 12 weeks in Wistar rats compared to nude rats, but the percentage of CGRP+ neurons did not differ between two groups. Additionally, macrophages were concentrated around NF200-labeled A fibers. At 6 and 12 weeks following PNI, CD4+ T cells were not found in the DRG of the two groups. The accumulation and activation of macrophages in the DRG may account for the increased frequency and severity of autotomy in Wistar rats. Our results also suggest that A fiber neurons in the DRG play an important role in autotomy.     

Ca2+ Signalling Induced by NGF Identifies a Subset of Capsaicin-Excitable Neurons Displaying Enhanced Chemo-Nociception in Dorsal Root Ganglion Explants from Adult pirt-GCaMP3 Mouse

Nociceptors sense hazards via plasmalemmal cation channels, including transient receptor potential vanilloid 1 (TRPV1). Nerve growth factor (NGF) sensitises TRPV1 to capsaicin (CAPS), modulates nociceptor excitability and induces thermal hyperalgesia, but cellular mechanisms remain unclear. Confocal microscopy was used to image changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) across neuronal populations in dorsal root ganglia (DRG) explants from pirt-GCaMP3 adult mice, which express a fluorescent reporter in their sensory neurons. Raised [Ca²⁺]_i was detected in 84 neurons of three DRG explants exposed to NGF (100 ng/mL) and most (96%) of these were also excited by 1 μM CAPS. NGF elevated [Ca²⁺]_i in about one-third of the neurons stimulated by 1 μM CAPS, whether applied before or after the latter. In neurons excitable by NGF, CAPS-evoked [Ca²⁺]_i signals appeared significantly sooner (e.g., respective lags of 1.0 ± 0.1 and 1.9 ± 0.1 min), were much (>30%) brighter and lasted longer (6.6 ± 0.4 vs. 3.9 ± 0.2 min) relative to those non-responsive to the neurotrophin. CAPS tachyphylaxis lowered signal intensity by ~60% but was largely prevented by NGF. Increasing CAPS from 1 to 10 μM nearly doubled the number of cells activated but only modestly increased the amount co-activated by NGF. In conclusion, a sub-population of the CAPS-sensitive neurons in adult mouse DRG that can be excited by NGF is more sensitive to CAPS, responds with stronger signals and is further sensitised by transient exposure to the neurotrophin.     

17β-Estradiol Exacerbated Experimental Occlusal Interference-Induced Chronic Masseter Hyperalgesia by Increasing the Neuronal Excitability and TRPV1 Function of Trigeminal Ganglion in Ovariectomized Rats

Pain symptoms in temporomandibular disorders (TMD) predominantly affect reproductive women, suggesting that estrogen regulates pain perception. However, how estrogen contributes to chronic TMD pain remains largely unclear. In the present study, we performed behavioral tests, electrophysiology, Western blot and immunofluorescence to investigate the role and underlying mechanisms of estrogen in dental experimental occlusal interference (EOI)-induced chronic masseter mechanical hyperalgesia in rats. We found that long-term 17β-estradiol (E2) replacement exacerbated EOI-induced masseter hyperalgesia in a dose-dependent manner in ovariectomized (OVX) rats. Whole-cell patch-clamp recordings demonstrated that E2 (100 nM) treatment enhanced the excitability of isolated trigeminal ganglion (TG) neurons in OVX and OVX EOI rats, and EOI increased the functional expression of transient receptor potential vanilloid-1 (TRPV1). In addition, E2 replacement upregulated the protein expression of TRPV1 in EOI-treated OVX rats. Importantly, intraganglionic administration of the TRPV1 antagonist AMG-9810 strongly attenuated the facilitatory effect of E2 on EOI-induced masseter mechanical sensitivity. These results demonstrate that E2 exacerbated EOI-induced chronic masseter mechanical hyperalgesia by increasing TG neuronal excitability and TRPV1 function. Our study helps to elucidate the E2 actions in chronic myogenic TMD pain and may provide new therapeutic targets for relieving estrogen-sensitive pain.     

Activation of Inflammatory Networks in the Lungs Caused by Chronic Cold Stress Is Moderately Attenuated by Glucose Supplementation

Mammals that live in cold climates endure months of exposure to low temperature in the winter. The incidence of respiratory diseases has increased. The goal of this study was to investigate the effects of chronic cold stress on lung inflammatory networks, apoptosis, and mitochondrial function via Yorkshire pig models, as well as the ameliorative effect of glucose as energy supplements. Here, two trials were conducted (chronic cold stress and glucose supplementation). The results showed that chronic cold stress induced obvious inflammatory cell infiltration in the lungs and damaged the lung tissue structure. Compared with the Y-Con group, the expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), high mobility group box 1 (HMGB1), nucleotide-binding domain, and leucine-rich repeat protein 3 (NLRP3), IL-1β, IL-2, IL-6, and IFN-γ in the lungs of the Y-CS group was enhanced by chronic cold stress (p < 0.05). Moreover, chronic cold stress promoted the expression of the Bax and Mfn2 in lungs of Y-CS group (p < 0.05). Interestingly, dietary glucose supplementation significantly reduced inflammatory cell infiltration in the lungs. Moreover, glucose supplementation inhibited the expression of TLR4, MyD88, HMGB1, NLRP3, IL-1β, IL-2, IL-6, IFN-γ, and Bax during chronic cold stress. In conclusion, chronic cold stress promoted inflammatory networks, apoptosis, and mitochondrial fusion in the lungs. Dietary glucose supplementation inhibited the inflammatory network during chronic cold stress.     

Differential Effects of High-Fish Oil and High-Lard Diets on Cells and Cytokines Involved in the Inflammatory Process in Rat Insulin-Sensitive Tissues

Dietary fat sources may differentially affect the development of inflammation in insulin-sensitive tissues during chronic overfeeding. Considering the anti-inflammatory properties of ω-3 fatty acids, this study aimed to compare the effects of chronic high-fish oil and high-lard diets on obesity-related inflammation by evaluating serum and tissue adipokine levels and histological features in insulin-sensitive tissues (white adipose tissue, skeletal muscle and liver). As expected, a high-lard diet induced systemic and peripheral inflammation and insulin resistance. Conversely, compared with a high-lard diet, a high-fish oil diet resulted in a lower degree of systemic inflammation and insulin resistance that were associated with a lower adipocyte diameter as well as lower immunoreactivity for transforming growth factor β 1 (TGFβ1) in white adipose tissue. A high-fish oil diet also resulted in a lower ectopic lipid depot, inflammation degree and insulin resistance in the skeletal muscle and liver. Moreover, a high-fish oil diet attenuated hepatic stellate cell activation and fibrogenesis in the liver, as indicated by the smooth muscle α-actin (α-SMA) and TGFβ1 levels. The replacement of lard (saturated fatty acids) with fish oil (ω-3 fatty acids) in chronic high-fat feeding attenuated the development of systemic and tissue inflammation.     

Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.     

Molecular Effects of Low-Intensity Shock Wave Therapy on L6 Dorsal Root Ganglion/Spinal Cord and Blood Oxygenation Level-Dependent (BOLD) Functional Magnetic Resonance Imaging (fMRI) Changes in Capsaicin-Induced Prostatitis Rat Models

Neurogenic inflammation and central sensitization play a role in chronic prostatitis/chronic pelvic pain syndrome. We explore the molecular effects of low-intensity shock wave therapy (Li-ESWT) on central sensitization in a capsaicin-induced prostatitis rat model. Male Sprague–Dawley rats underwent intraprostatic capsaicin (10 mM, 0.1 cm³) injections. After injection, the prostate received Li-ESWT twice, one day apart. The L6 dorsal root ganglion (DRG)/spinal cord was harvested for histology and Western blotting on days 3 and 7. The brain blood oxygenation level-dependent (BOLD) functional images were evaluated using 9.4 T fMRI before the Li-ESWT and one day after. Intraprostatic capsaicin injection induced increased NGF-, BDNF-, and COX-2-positive neurons in the L6 DRG and increased COX-2, NGF, BDNF, receptor Trk-A, and TRPV1 protein expression in the L6 DRG and the dorsal horn of the L6 spinal cord, whose effects were significantly downregulated after Li-ESWT on the prostate. Intraprostatic capsaicin injection increased activity of BOLD fMRI responses in brain regions associated with pain-related responses, such as the caudate putamen, periaqueductal gray, and thalamus, whose BOLD signals were reduced after Li-ESWT. These findings suggest a potential mechanism of Li-ESWT on modulation of peripheral and central sensitization for treating CP/CPPS.     

Phylogenetic and Transcriptional Analyses of the HSP20 Gene Family in Peach Revealed That PpHSP20-32 Is Involved in Plant Height and Heat Tolerance

The heat shock protein 20 (HSP20) proteins comprise an ancient, diverse, and crucial family of proteins that exists in all organisms. As a family, the HSP20s play an obvious role in thermotolerance, but little is known about their molecular functions in addition to heat acclimation. In this study, 42 PpHSP20 genes were detected in the peach genome and were randomly distributed onto the eight chromosomes. The primary modes of gene duplication of the PpHSP20s were dispersed gene duplication (DSD) and tandem duplication (TD). PpHSP20s in the same class shared similar motifs. Based on phylogenetic analysis of HSP20s in peach, Arabidopsis thaliana, Glycine max, and Oryza sativa, the PpHSP20s were classified into 11 subclasses, except for two unclassified PpHSP20s. cis-elements related to stress and hormone responses were detected in the promoter regions of most PpHSP20s. Gene expression analysis of 42 PpHSP20 genes revealed that the expression pattern of PpHSP20-32 was highly consistent with shoot length changes in the cultivar ‘Zhongyoutao 14’, which is a temperature-sensitive semi-dwarf. PpHSP20-32 was selected for further functional analysis. The plant heights of three transgenic Arabidopsis lines overexpressing PpHSP20-32 were significantly higher than WT, although there was no significant difference in the number of nodes. In addition, the seeds of three over-expressing lines of PpHSP20-32 treated with high temperature showed enhanced thermotolerance. These results provide a foundation for the functional characterization of PpHSP20 genes and their potential use in the growth and development of peach.