Animal Studies

Neuropathic pain is one of the key features of the classical phenotype of Fabry disease (FD). Acid sensing ion channels (ASICs) are H-gated cation channels, which belong to the epithelial sodium channel/DeGenerin superfamily, sensitive to the diuretic drug Amiloride. Molecular cloning has identified several distinct ASIC subunits. In particular the ASIC1a subunit has been associated to pain and its upregulation has been documented in animal models of pain. We analyzed the expression of ASIC1a channels in cellular models that mimic the accumulation of glycosphingolipids in FD (FD-GLs) like Gb3, and LysoGb3. We used mouse primary neurons from brain cortex and hippocampus -supraspinal structures that accumulate FD-GLs-, as well as HEK293 cells. Incubation with Gb3, lysoGb3 and the inhibitor (1-deoxy-galactonojirymicin, DJG) of the enzyme α-galactosidase A (Gla) lead to the upregulation of ASIC1a channels. In addition, activation of ASIC1a results in the activation of the MAPK ERK pathway, a signaling pathway associated with pain. Moreover, accumulation of glycosphingolipids results in activation of ERK, an effect that was prevented by blocking ASIC1a channels with the specific blocker Psalmotoxin. Our results suggest that FD-GLs accumulation and triggering of the ERK pathway via ASIC channels might be involved in the mechanism responsible for pain in FD, thus providing a new therapeutic target for pain relief treatment.

Many HIV patients develop chronic pain and use opioid-derived medicine as primary analgesics. Emerging clinical evidence suggests that chronic use of opioid analgesics paradoxically heightens pain states in patients. This side effect of opioid analgesics has a significant negative impact on clinical practice, but the underlying pathogenic mechanism remains elusive. Using a mouse model of HIV-associated pain, we simulated the development of morphine exacerbation on pain and investigated potential underlying cellular and molecular pathways. We found that repeated morphine treatment promoted astrocyte activation in the spinal dorsal horn (SDH) and up-regulation of pro-inflammatory cytokines IL-1β and TNF-α. Furthermore, we observed that morphine administration potentiated mitochondrial reactive oxygen species (ROS) in the SDH of the HIV pain model, especially on astrocytes. Systemic application of the ROS scavenger phenyl-N-t-butyl nitrone (PBN) not only blocked the enhancement of gp120-induced hyperalgesia by morphine but also astrocytic activation and cytokine up-regulation. These findings suggest a critical role of ROS in mediating the exacerbation of gp120-induced pain by morphine. Graphical abstract.

Chronic constriction injury (CCI) is widely used as an animal neuropathic pain model. Neuropathic pain is considered to exist when withdrawal latency to thermal stimulation is decreased after inducing a CCI to the sciatic nerve. However, it is known that CCI leads to changes in skin temperature and that skin temperature can affect withdrawal latency. Aim of this study was to compare withdrawal latencies of constricted and contralateral hind limbs, to thermal stimulation, at the same artificially-induced skin temperatures.

Activation of the trigeminal system causes the release of various neuropeptides, cytokines, and other immune mediators. Calcitonin gene-related peptide (CGRP), which is a potent algogenic mediator, is expressed in the peripheral sensory neurons of trigeminal ganglion (TG). It affects the inflammatory responses and pain sensitivity by modulating the activity of glial cells. The primary aim of this study was to use array analysis to investigate the effect of CGRP on the glial cells of TG in regulating nuclear factor kappa B (NF-κB) signaling genes and to further check if CGRP in the TG can affect neuron-glia activation in the spinal trigeminal nucleus caudalis. The glial cells of TG were stimulated with CGRP or Minocycline (Min) + CGRP. The effect on various genes involved in NF-κB signaling pathway was analyzed compared to no treatment control condition using a PCR array analysis. CGRP, Min + CGRP or saline was directly injected inside the TG and the effect on gene expression of , and and protein expression of cleaved Caspase3 (cleav Casp3) in the TG, and c-Fos and glial fibrillary acidic protein (GFAP) in the spinal section containing trigeminal nucleus caudalis was analyzed. Results showed that CGRP stimulation resulted in the modulation of several genes involved in the interleukin 1 signaling pathway and some genes of the tumor necrosis factor pathway. Minocycline pre-treatment resulted in the modulation of several genes in the glial cells, including anti-inflammatory genes, and neuronal activation markers. A mild increase in cleav Casp3 expression in TG and c-Fos and GFAP in the spinal trigeminal nucleus of CGRP injected animals was observed. These data provide evidence that glial cells can participate in neuroimmune interaction due to CGRP in the TG via NF-κB signaling pathway.

Organic electronic devices implemented on flexible thin films are attracting increased attention for biomedical applications because they possess extraordinary conformity to curved surfaces. A neuronal device equipped with an organic light-emitting diode (OLED), used in combination with animals that are genetically engineered to include a light-gated ion channel, would enable cell type-specific stimulation to neurons as well as conformal contact to brain tissue and peripheral soft tissue. This potential application of the OLEDs requires strong luminescence, well over the neuronal excitation threshold in addition to flexibility. Compatibility with neuroimaging techniques such as MRI provides a method to investigate the evoked activities in the whole brain. Here, we developed an ultrathin, flexible, MRI-compatible OLED device and demonstrated the activation of channelrhodopsin-2-expressing neurons in animals. Optical stimulation from the OLED attached to nerve fibers induced contractions in the innervated muscles. Mechanical damage to the tissues was significantly reduced because of the flexibility. Owing to the MRI compatibility, neuronal activities induced by direct optical stimulation of the brain were visualized using MRI. The OLED provides an optical interface for modulating the activity of soft neuronal tissues.

Viscera receive innervation from sensory ganglia located adjacent to multiple levels of the brainstem and spinal cord. Here we examined whether molecular profiling could be used to identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and nodose ganglia (NG) in male and female mice. Profiling of TL and LS bladder afferents was also done. Visceral afferents were back-labeled using retrograde tracers injected into proximal and distal regions of colon or bladder, followed by single cell RT-qPCR and analysis via an automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for colon) based on their established role in stimulus detection, regulation of sensitivity/function or neuroimmune interaction. A total of 132 colon afferents (from NG, TL, and LS) and 128 bladder afferents (from TL and LS) were analyzed. Retrograde labeling from the colon showed NG and TL afferents innervate proximal and distal regions of the colon whereas 98% of LS afferents only project to distal regions. There were clusters of colon and bladder afferents, defined by mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was found. In addition, transcriptionally, NG colon afferents were almost completely segregated from colon TL and LS neurons. Furthermore, colon and bladder afferents expressed genes at similar levels, although different gene combinations defined the clusters. These results indicate that genes implicated in both homeostatic regulation and conscious sensations are found at all anatomic levels, suggesting afferents from different portions of the neuraxis have overlapping functions.Visceral organs are innervated by sensory neurons whose cell bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, where sensation and pain is mediated by spinal afferents and fear and anxiety (the affective aspects of visceral pain) are the domain of nodose afferents. Transcriptomic analysis performed here reveals that genes implicated in both homeostatic regulation and pain are found in afferents across all ganglia types, suggesting that conscious sensation and homeostatic regulation is the result of convergence, and not segregation, of sensory input.

Highly active antiretroviral therapy (HAART) works effectively in inhibiting HIV replication in patients. However, the use of nucleoside reverse transcriptase inhibitors (NRTIs) often causes side effects of neuropathic pain, and its mechanism remains to be elucidated. Therefore, we aim to explore the mechanism of NRTIs-induced neuropathic pain at the transcriptome level. C57BL/6 J mice were given intraperitoneal injection of zalcitabine (ddC) or saline (control) for 2 weeks, during which the mechanical pain threshold of the mice was detected by von Frey test. Then the L3~L5 spinal segments of the mice were isolated and subsequently used for RNA sequencing (RNA-seq) on the last day of treatment. The mechanical pain threshold of mice given ddC decreased significantly. Compared with the control group, ddC caused significant changes in the expression of 135 genes, of which 66 upregulated and 69 downregulated. Enrichment analysis showed that the functions of these genes are mainly enriched in regulation of transcription, multicellular organism development, and cell differentiation, and the pathway is mainly enriched in the cGMP-PKG signaling pathway and AMPK signaling pathway. Furthermore, key genes such as Gabrd, Kcnd3, Npcd, Insr, Lypd6, Scd2, and Mef2d were also identified. These may serve as drug targets for the prevention or treatment of NRTI-induced neuropathic pain.