RESUMO
BACKGROUND: Spinal cord injury (SCI) results in the development of chronic pain that is refractory to conventional treatment. Progesterone, a neuroprotective steroid, may offer a promising perspective in pain modulation after central injury. Here, we explore the impact of progesterone administration on the post-injury inflammatory cascade involving the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) at the spinal cord level. We also analyse pain behaviours, the profile of glial cell activation, and IκB-α mRNA levels, as an index of NF-κB transactivation. METHODS: We used biochemical, immunohistochemical and molecular techniques, as well as behavioural studies, to investigate the effects of progesterone in a well-characterized model of central neuropathic pain. RESULTS: Injured animals receiving progesterone presented reduced mRNA levels of the proinflammatory enzymes, as well as decreased COX-2 activity and nitrite levels, as compared to vehicle-treated injured rats. Further, animals receiving the steroid exhibited lower levels of IκB-α mRNA, suggesting decreased NF-κB transactivation. Progesterone administration also attenuated the injury-induced increase in the number of glial fibrillary acidic protein and OX-42 positive cells both at early and late time points after injury, and prevented the development of mechanical and thermal allodynia. Further, when injured rats received early progesterone administration for a critical period of time after injury, they did not display allodynic behaviours even after the treatment had stopped. CONCLUSIONS: Our results suggest that progesterone, by modulating early neuroinflammatory events triggered after SCI, may represent a useful strategy to prevent the development of central chronic pain.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/metabolismo , Progesterona/uso terapêutico , Medula Espinal/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Hiperalgesia/enzimologia , Hiperalgesia/etiologia , Masculino , Neuralgia/enzimologia , Neuralgia/etiologia , Medição da Dor , Progesterona/farmacologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/enzimologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/enzimologiaRESUMO
Progesterone provides neuroprotection after spinal cord injury, but the molecular mechanisms involved in this effect are not completely understood. In this work, expression of two binding proteins for progesterone was studied in intact and injured rat spinal cord: the classical intracellular progesterone receptor (PR) and 25-Dx, a recently discovered progesterone membrane binding site. RT-PCR was employed to determine their relative mRNA levels, whereas cellular localization and relative protein levels were investigated by immunocytochemistry. We observed that spinal cord PR mRNA was not up-regulated by estrogen in contrast to what is observed in many brain areas and in the uterus, but was abundant as it amounted to a third of that measured in the estradiol-stimulated uterus. In male rats with complete spinal cord transection, levels of PR mRNA were significantly decreased, while those of 25-Dx mRNA remained unchanged with respect to control animals. When spinal cord-injured animals received progesterone treatment during 72 h, PR mRNA levels were not affected and remained low, whereas 25-Dx mRNA levels were significantly increased. Immunostaining of PR showed its intracellular localization in both neurons and glial cells, whereas 25-Dx immunoreactivity was localized to cell membranes of dorsal horn and central canal neurons. As the two binding proteins for progesterone differ with respect to their response to lesion, their regulation by progesterone, their cellular and subcellular localizations, their functions may differ under normal and pathological conditions. These observations point to a novel and potentially important role of the progesterone binding protein 25-Dx after injury of the nervous system and suggest that the neuroprotective effects of progesterone may not necessarily be mediated by the classical progesterone receptor but may involve distinct membrane binding sites.
Assuntos
Proteínas de Transporte/metabolismo , Progesterona/farmacologia , Receptores de Progesterona/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Estradiol/farmacologia , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Proteínas de Membrana , Regeneração Nervosa/fisiologia , Progesterona/sangue , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Progesterona/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologiaRESUMO
Glucocorticoids (GC) provide neuroprotection and early recovery after spinal cord injury (SCI). While several mechanisms were proposed to account for these effects, limited information exists regarding GC actions in sensory areas of the spinal cord. Presently, we studied the time course of Fos expression, and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical staining to monitor neuronal responses to SCI with or without GC treatment. Rats with sham-operation or transection at the thoracic level (T7-T8) received vehicle or 5 mg/kg of the GC dexamethasone (DEX) at 5 min post-lesion and were sacrificed 2 or 4 h after surgery. Another group of SCI rats received vehicle or intensive DEX treatment (5 min, 6 h, 18 h and 46 h post-lesion) and were sacrificed 48 h after surgery. The number of NADPH-d positive neurons or Fos immunoreactive nuclei was studied by computer-assisted image analysis in superficial dorsal horn (Laminae I-III) and central canal area (Lamina X) below the lesion. While constitutive Fos immunoreactive nuclei were sparse in controls, SCI increased Fos expression at 2 and 4 h after injury. DEX treatment significantly enhanced the number of Fos positive nuclei in Laminae I-III by 4 h after transection, although the response was not maintained by intensive steroid treatment when tested at 48 h after SCI. NADPH-d positive neurons in Laminae I-III increased at 2 and 4 h after SCI while a delayed increased was found in central canal area (Lamina X). DEX treatment decreased NADPH-d positive neurons to sham-operated levels at all time points examined. Thus, while GC stimulation of Fos suggests activation of neurons involved in sympathetic outflow and/or pain, down-regulation of NADPH-d indicates attenuation of nociceptive outflow, considering the role of enzyme-derived nitric oxide in pain-related mechanisms. Differential hormonal effects on these molecules agree with their localization in different cell populations.
Assuntos
Glucocorticoides/farmacologia , NADPH Desidrogenase/efeitos dos fármacos , Óxido Nítrico/metabolismo , Dor/tratamento farmacológico , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Substância Gelatinosa/efeitos dos fármacos , Animais , Contagem de Células , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Esquema de Medicação , Imuno-Histoquímica , Masculino , NADPH Desidrogenase/metabolismo , Dor/enzimologia , Dor/fisiopatologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/fisiopatologia , Substância Gelatinosa/citologia , Substância Gelatinosa/enzimologia , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Progesterone (P4) can be synthesized in both central and peripheral nervous system (PNS) and exerts trophic effects in the PNS. To study its potential effects in the spinal cord, we investigated P4 modulation (4 mg/kg/day for 3 days) of two proteins responding to injury: NADPH-diaphorase, an enzyme with nitric oxide synthase activity, and glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. The proteins were studied at three levels of the spinal cord from rats with total transection (TRX) at T10: above (T5 level), below (L1 level) and caudal to the lesion (L3 level). Equivalent regions were dissected in controls. The number and area of NADPH-diaphorase active or GFAP immunoreactive astrocytes/0.1 mm(2) in white matter (lateral funiculus) or gray matter (Lamina IX) was measured by computerized image analysis. In controls, P4 increased the number of GFAP-immunoreactive astrocytes in gray and white matter at all levels of the spinal cord, while astrocyte area also increased in white matter throughout and in gray matter at the T5 region. In control rats P4 did not change NADPH-diaphorase activity. In rats with TRX and not receiving hormone, a general up-regulation of the number and area of GFAP-positive astrocytes was found at all levels of the spinal cord. In rats with TRX, P4 did not change the already high GFAP-expression. In the TRX group, instead, P4 increased the number and area of NADPH-diaphorase active astrocytes in white and gray matter immediately above and below, but not caudal to the lesion. Thus, the response of the two proteins to P4 was conditioned by environmental factors, in that NADPH-diaphorase activity was hormonally modulated in astrocytes reacting to trauma, whereas up-regulation of GFAP by P4 was produced in resting astrocytes from non-injured animals.
Assuntos
Astrócitos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , NADPH Desidrogenase/metabolismo , Progesterona/fisiologia , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/citologia , Animais , Astrócitos/enzimologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Medula Espinal/enzimologia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/patologiaRESUMO
Using the KC 146 monoclonal antibody recognizing the B-form of the progesterone receptor (PR) and immunocytochemical techniques, we investigated if PR-immunoreactive cells are present in the rat spinal cord. Neurons from ventral horn Lamina IX, glial cells in gray and white matter and ependymal cells were PR-positive. Evidence for estrogen-inducibility of PR in ovariectomized rats was not observed. There were no significant gender differences in neuronal PR immunostaining intensity in the spinal cord, measured by computerized image analysis. In pituitary and uterus from estrogenized female rats, PR showed a strict nuclear localization, whereas in neurons and glial cells of the spinal cord, PR localized in cytoplasm and/or nucleus and in some cell processes. This receptor may be implicated in some of the biological effects of progesterone described in the spinal cord.