RESUMEN
While in clinical research epidemiological data indicate a higher prevalence of chronic pain in women, preclinical research has long focused on male animal models. Recently, in the study of neuropathic pain, it was found that mechanisms responsible for pain behavior after a peripheral nerve injury in male differed from the ones in female. At a time when gender equality is being claimed, science is reviewing its copy and has to find the sex differences that could lead to new and appropriate therapeutic targets.
Alors qu'en recherche clinique, les données épidémiologiques indiquent une plus forte prévalence de douleur chronique chez la femme, la recherche préclinique s'est longtemps concentrée sur des modèles animaux uniquement mâles. Récemment, une équipe a démontré que les mécanismes menant au comportement douloureux après une lésion nerveuse périphérique chez le mâle différaient de ceux de la femelle. A l'heure où l'égalité des sexes est revendiquée, la science doit revoir sa copie et chercher les différences de mécanismes de la douleur entre homme et femme, afin d'espérer aboutir à de nouvelles thérapies ciblées.
RESUMEN
The immune system is involved in the development of neuropathic pain. In particular, the infiltration of T-lymphocytes into the spinal cord following peripheral nerve injury has been described as a contributor to sensory hypersensitivity. We used the spared nerve injury (SNI) model of neuropathic pain in Sprague Dawley adult male rats to assess proliferation, and/or protein/gene expression levels for microglia (Iba1), T-lymphocytes (CD2) and cytotoxic T-lymphocytes (CD8). In the dorsal horn ipsilateral to SNI, Iba1 and BrdU stainings revealed microglial reactivity and proliferation, respectively, with different durations. Iba1 expression peaked at D4 and D7 at the mRNA and protein level, respectively, and was long-lasting. Proliferation occurred almost exclusively in Iba1 positive cells and peaked at D2. Gene expression observation by RT-qPCR array suggested that T-lymphocytes attracting chemokines were upregulated after SNI in rat spinal cord but only a few CD2/CD8 positive cells were found. A pronounced infiltration of CD2/CD8 positive T-cells was seen in the spinal cord injury (SCI) model used as a positive control for lymphocyte infiltration. Under these experimental conditions, we show early and long-lasting microglia reactivity in the spinal cord after SNI, but no lymphocyte infiltration was found.
Asunto(s)
Microglía/fisiología , Traumatismos de los Nervios Periféricos/complicaciones , Traumatismos de la Médula Espinal/etiología , Linfocitos T/fisiología , Animales , Antígenos CD2/genética , Antígenos CD8/genética , Proteínas de Unión al Calcio/genética , Proliferación Celular , Quimiocinas/inmunología , Modelos Animales de Enfermedad , Expresión Génica , Masculino , Proteínas de Microfilamentos/genética , Microglía/metabolismo , Microglía/patología , Neuralgia , Traumatismos de los Nervios Periféricos/inmunología , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/fisiopatología , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
BACKGROUND: The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. RESULTS: We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. CONCLUSIONS: In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.