RESUMO
A challenge for central nervous system (CNS) tissue analysis in neuroscience research has been the difficulty to codetect and colocalize gene and protein expression in the same tissue. Given the importance of identifying gene expression relative to proteins of interest, for example, cell-type specific markers, we aimed to develop a protocol to optimize their codetection. RNAscope fluorescent in situ hybridization (FISH) combined with immunohistochemistry (IHC) in fixed (CNS) tissue sections allows for reliable quantification of gene transcripts of interest within IHC-labeled cells. This paper describes a new method for simultaneous visualization of FISH and IHC in thicker (14-µm), fixed tissue samples, using spinal cord sections. This method's effectiveness is shown by the cell-type-specific quantification of two genes, namely the proinflammatory cytokine interleukin-1beta (IL-1b) and the inflammasome NLR family pyrin domain containing 3 (NLRP3). These genes are challenging to measure accurately using immunohistochemistry (IHC) due to the nonspecificity of available antibodies and the hard-to-distinguish, dot-like visualizations of the labeled proteins within the tissue. These measurements were carried out in spinal cord sections after unilateral chronic constriction injury of the sciatic nerve to induce neuroinflammation in the spinal cord. RNAscope is used to label transcripts of genes of interest and IHC is used to label cell-type specific antigens (IBA1 for microglia, NeuN for neurons). This combination allowed for labeled RNA transcripts to be quantified within cell-type specific boundaries using confocal microscopy and standard image analysis methods. This method makes it easy to answer empirical questions that are intractable with standard IHC or in situ hybridization alone. The method, which has been optimized for spinal cord tissue and to minimize tissue preparation time and costs, is described in detail from tissue collection to image analysis. Further, the relative expression changes in inflammatory genes NLRP3 and IL-1b in spinal cord microglia vs. neurons of somatotopically relevant laminae are described for the first time.
RESUMO
Diabetic neuropathic pain is associated with elevated plasma levels of methylglyoxal (MGO). MGO is a metabolite of glycolysis that causes mechanical hypersensitivity in mice by inducing the integrated stress response (ISR), which is characterized by phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of antioxidant proteins that neutralize MGO. We hypothesized that activating Nrf2 using diroximel fumarate (DRF) would alleviate MGO-induced pain hypersensitivity. We pretreated male and female C57BL/6 mice daily with oral DRF prior to intraplantar injection of MGO (20 ng). DRF (100 mg/kg) treated animals were protected from developing MGO-induced mechanical and cold hypersensitivity. Using Nrf2 knockout mice we demonstrate that Nrf2 is necessary for the anti-nociceptive effects of DRF. In cultured mouse and human dorsal root ganglion (DRG) sensory neurons, we found that MGO induced elevated levels of p-eIF2α. Co-treatment of MGO (1 µM) with monomethyl fumarate (MMF, 10, 20, 50 µM), the active metabolite of DRF, reduced p-eIF2α levels and prevented aberrant neurite outgrowth in human DRG neurons. Our data show that targeting the Nrf2 antioxidant system with DRF is a strategy to potentially alleviate pain associated with elevated MGO levels.
RESUMO
Up to 92% of patients suffering from multiple sclerosis (MS) experience pain, most without adequate treatment, and many report pain long before motor symptoms associated with MS diagnosis. In the most commonly studied rodent model of MS, experimental autoimmune encephalomyelitis (EAE), motor impairments/disabilities caused by EAE can interfere with pain testing. In this study, we characterize a novel low-dose myelin-oligodendrocyte-glycoprotein (MOG)-induced Sprague-Dawley (SD) model of EAE-related pain in male rats, optimized to minimize motor impairments/disabilities. Adult male SD rats were treated with increasing doses of intradermal myelin-oligodendrocyte-glycoprotein (MOG1-125) (0, 4, 8, and 16 µg) in incomplete Freund's adjuvant (IFA) vehicle to induce mild EAE. Von Frey testing and motor assessments were conducted prior to EAE induction and then weekly thereafter to assess EAE-induced pain and motor impairment. Results from these studies demonstrated that doses of 8 and 16 µg MOG1-125 were sufficient to produce stable mechanical allodynia for up to 1 month in the absence of hindpaw motor impairments/disabilities. In the follow-up studies, these doses of MOG1-125, were administered to create allodynia in the absence of confounded motor impairments. Then, 2 weeks later, rats began daily subcutaneous injections of the Toll-like receptor 2 and 4 (TLR2-TLR4) antagonist (+)-naltrexone [(+)-NTX] or saline for an additional 13 days. We found that (+)-NTX also reverses EAE-induced mechanical allodynia in the MOG-induced SD rat model of EAE, supporting parallels between models, but now allowing a protracted timecourse to be examined completely free of motor confounds. Exploring further mechanisms, we demonstrated that both spinal NOD-like receptor protein 3 (NLRP3) and interleukin-17 (IL-17) are necessary for EAE-induced pain, as intrathecal injections of NLRP3 antagonist MCC950 and IL-17 neutralizing antibody both acutely reversed EAE-induced pain. Finally, we show that spinal glial immunoreactivity induced by EAE is reversed by (+)-NTX, and that spinal demyelination correlates with the severity of motor impairments/disabilities. These findings characterize an optimized MOG-induced SD rat model of EAE for the study of pain with minimal motor impairments/disabilities. Finally, these studies support the role of TLR2-TLR4 antagonists as a potential treatment for MS-related pain and other pain and inflammatory-related disorders.
