RESUMO
Neuropathic pain is a frequent complication of spinal cord injury (SCI), still refractory to conventional treatment. The presence and biological activity of steroidogenic regulatory proteins and enzymes in the spinal cord suggests that neurosteroids locally generated could modulate pain messages. In this study we explored temporal changes in the spinal expression of the 18kDa translocator protein TSPO, the steroidogenic acute regulatory protein (StAr) and the steroidogenic enzyme 5α-reductase (5α-RI/II) in an experimental model of central chronic pain. Male Sprague-Dawley rats were subjected to a SCI and sacrificed at different time points (1, 14 or 28days). The development of mechanical and cold allodynia was assessed. Injured animals showed an early increase in the mRNA levels of TSPO and 5α-RII, whereas in the chronic phase a significant decrease in the expression of 5α-RI and 5α-RII was observed, coinciding with the presence of allodynic behaviors. Furthermore, since we have shown that progesterone (PG) administration may offer a promising perspective in pain modulation, we also evaluated the expression of steroidogenic proteins and enzymes in injured animals receiving daily injections of the steroid. PG-treated did not develop allodynia and showed a marked increase in the mRNA levels of TSPO, StAR, 5α-RI and 5α-RII 28days after injury. Our results suggest that in the acute phase after SCI, the increased expression of TSPO and 5α-RII may represent a protective endogenous response against tissue injury, which is not maintained in the chronic allodynic phase. PG may favor local steroidogenesis and the production of its reduced metabolites, which could contribute to the antiallodynic effects observed after PG treatment.
Assuntos
Proteínas de Transporte/metabolismo , Colestenona 5 alfa-Redutase/metabolismo , Neuralgia/metabolismo , Progesterona/administração & dosagem , Receptores de GABA-A/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Hiperalgesia/enzimologia , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Masculino , Neuralgia/enzimologia , Neuralgia/etiologia , Neuralgia/prevenção & controle , Limiar da Dor/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/enzimologiaRESUMO
Spinal cord injury (SCI) is a condition producing irreversible damage to the neurological function. Among the leading mechanisms associated to cell death after SCI, excitotoxicity, oxidative stress, inflammatory response and apoptosis are considered potential targets to prevent tissue damage. We recently reported that dapsone an anti-inflammatory drug, decreases the activity of myeloperoxidase, lipid peroxidation, improve neurological function and increase the amount of spared tissue after SCI in rats. In this study, we characterized the anti-apoptotic effect of dapsone administered at 12.5 mg/kg/24 h dose, starting at 3 and 5 h after SCI. We monitored the activity of caspases-8, 9, and 3 and quantitated Annexin V and TUNEL positive cells in the core of the lesion. Results showed increased activities of caspase-8, 9 and 3 at 72 h by SCI to reach increments of 69, 143 and 293 %, respectively, as compared to sham group. Meanwhile, dapsone, administered at 3 and 5 after SCI, reduced caspase-8 activity by 36 and 44 % respectively, whereas the activity of caspase-9 was diminished by 37 %. Likewise, the activity of caspase-3 showed a decrease of 38 %. Finally, both Annexin V and TUNEL-positive cells were significantly reduced by DDS as compared to untreated SCI animals. Results showed that dapsone exerted anti-apoptotic effect after SCI.
Assuntos
Apoptose/efeitos dos fármacos , Dapsona/farmacologia , Dapsona/uso terapêutico , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Animais , Caspases/metabolismo , Feminino , Marcação In Situ das Extremidades Cortadas , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/enzimologiaRESUMO
Subjects with spinal cord injury (SCI) exhibit impaired left ventricular (LV) diastolic function, which has been reported to be attenuated by regular physical activity. This study investigated the relationship between circulating matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) and echocardiographic parameters in SCI subjects and the role of physical activity in this regard. Forty-two men with SCI [19 sedentary (S-SCI) and 23 physically-active (PA-SCI)] were evaluated by clinical, anthropometric, laboratory, and echocardiographic analysis. Plasmatic pro-MMP-2, MMP-2, MMP-8, pro-MMP-9, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay and zymography. PA-SCI subjects presented lower pro-MMP-2 and pro-MMP-2/TIMP-2 levels and improved markers of LV diastolic function (lower E/Em and higher Em and E/A values) than S-SCI ones. Bivariate analysis showed that pro-MMP-2 correlated inversely with Em and directly with E/Em, while MMP-9 correlated directly with LV mass index and LV end-diastolic diameter in the whole sample. Following multiple regression analysis, pro-MMP-2, but not physical activity, remained associated with Em, while MMP-9 was associated with LV mass index in the whole sample. These findings suggest differing roles for MMPs in LV structure and function regulation and an interaction among pro-MMP-2, diastolic function and physical activity in SCI subjects.
Assuntos
Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/diagnóstico , Disfunção Ventricular Esquerda/sangue , Disfunção Ventricular Esquerda/diagnóstico , Adulto , Biomarcadores/sangue , Estudos Transversais , Humanos , Masculino , Metaloproteinases da Matriz/sangue , Atividade Motora/fisiologia , Traumatismos da Medula Espinal/enzimologia , Inibidor Tecidual de Metaloproteinase-2/sangue , Disfunção Ventricular Esquerda/enzimologia , Função Ventricular Esquerda/fisiologiaRESUMO
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
Characterization of auto-destructive mechanisms, leading to cell death after spinal cord injury (SCI) is important to prevent further damage to tissue. Heme oxygenase (HO) catalyzes the oxidation of heme to biliverdin and carbon monoxide (CO), as a response to cell damage. Products of HO action have biological effects, as antioxidant biliverdin. We evaluated the changes of HO activity after injury, and the effect of pharmacological treatments with hemin (an inducer) and (Sn)-protoporphyrin (an inhibitor, Sn-PPIX) of HO, upon motor recovery after SCI. Female Wistar rats were submitted to SCI by trauma and sacrificed at several times (2, 4, 8, 12 and 24h) after injury to evaluate HO activity. Additional groups of rats were treated with either hemin or Sn-PPIX, to evaluate motor recovery, spared spinal cord tissue and HO activity. Results showed that HO control activity was increased by effect of SCI, at all times evaluated, as compared to sham group values. Twenty-four hours after injury, HO activity was increased 7.2-fold by hemin treatment, as compared to SCI plus vehicle group values. In addition, animals treated with hemin 2 and 8h after SCI, showed a better motor recovery and higher spared cord tissue, as compared to control group values. Our findings indicate that activation of HO is a beneficial mechanism when attained during the acute phase after SCI.
Assuntos
Heme Oxigenase (Desciclizante)/metabolismo , Destreza Motora , Traumatismos da Medula Espinal/fisiopatologia , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática , Indução Enzimática , Feminino , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Hemina/farmacologia , Metaloporfirinas/farmacologia , Protoporfirinas/farmacologia , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologia , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/patologia , Fatores de TempoRESUMO
Nitric oxide is generated from l-arginine by a family of three distinct nitric oxide synthase (NOS) enzymes playing a crucial role in the physiopathology of spinal cord injury (SCI). Cyclosporin-A (CsA), an immunosupressive agent, may be used to inhibit the activity of iNOS and perhaps to protect against neural tissue destruction. Rats were submitted to SCI by contusion, and killed 4, 24 and 72 h after lesion. Results showed an increase in the activity of iNOS at 72 h after the SCI, inhibited by CsA (2.5 mg/kg) administered 12 h after trauma. iNOS Western blot assay showed an increase in the expression of iNOS after trauma, also antagonized by CsA administration.
Assuntos
Ciclosporina/farmacologia , Degeneração Neural/tratamento farmacológico , Degeneração Neural/prevenção & controle , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico/biossíntese , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Proteínas I-kappa B/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Imunossupressores/farmacologia , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Degeneração Neural/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/fisiopatologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
Nitric oxide (NO) plays a role in the secondary damage after spinal cord (SC) injury. NO is produced by the activity of two classes of enzymes: calcium-dependent constitutive nitric oxide synthase (NOS) and calcium-independent inducible NOS. To determine the time course of both NOS activities after SC injury, 50 Wistar rats were submitted to severe SC contusion. NOS activities were assayed at the site of SC injury at several times after lesion. Results showed a significant increase of 138 and 96% in the constitutive NOS activity at 4 and 8 h after the lesion, respectively, as compared to sham-operated rats. iNOS activity was increased 72 h after lesion by 103% (P<0.05). In conclusion, both isoforms of NOS increase their activity at different time periods after SC injury.
Assuntos
Contusões/enzimologia , Óxido Nítrico Sintase/metabolismo , Traumatismos da Medula Espinal/enzimologia , Medula Espinal/enzimologia , Animais , Feminino , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Ratos , Ratos Wistar , Fatores de TempoRESUMO
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
Previously, we have shown that immediately after an experimental spinal cord injury (SCI) in anaesthetized rats, there is a large fall in mean arterial pressure (MAP) and heart rate (HR), followed by an abrupt increase in MAP. To evaluate the participation of nitric oxide (NO), we evaluated the activity of nitric oxide synthase (NOS) using Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry in sections of atria at several post-injury time-intervals. Staining increased at 3 min, reached a maximum at 9 min and diminished 30 min after injury. Pretreatment with atropine prevented changes in MAP, HR and NADPH-d staining suggesting that such modifications result from an increased vagal stimulation. In conclusion, the NOS activity is transiently elevated in the atrial intramural arteries of rats subjected to an SCI.