A artrite reumatoide afeta o sistema nervoso entérico? / Does the rheumatoid arthritis affect the enteric nervous system?

ABSTRACT BACKGROUND: Few studies regarding arthritic diseases have been performed to verify the presence of the neurodegeneration. Given the increased oxidative stress and extra-articular effects of the rheumatoid arthritis, the gastrointestinal studies should be further investigated aiming a better understanding of the systemic effects the disease on enteric nervous system. OBJECTIVE: To determine whether the rheumatoid arthritis affects the nitrergic density and somatic area of the nNOS- immunoreactive (IR) myenteric neurons, as well as the morphometric areas of CGRP and VIP-IR varicosities of the ileum of arthritic rats. METHODS: Twenty 58-day-old male Holtzmann rats were distributed in two groups: control and arthritic. The arthritic group received a single injection of the Freund's Complete Adjuvant in order to induce arthritis model. The whole-mount preparations of ileum were processed for immunohistochemistry to VIP, CGRP and nNOS. Quantification was used for the nitrergic neurons and morphometric analyses were performed for the three markers. RESULTS: The arthritic disease induced a reduction 6% in ileal area compared to control group. No significant differences were observed in nitrergic density comparing both groups. However, arthritic group yielded a reduction of the nitrergic neuronal somatic area and VIP-IR varicosity areas. However, an increase of varicosity CGRP-IR areas was also observed. CONCLUSION: Despite arthritis resulted in no alterations in the number of nitrergic neurons, the retraction of ileal area and reduction of nitrergic somatic and VIP-IR varicosity areas may suggest a negative impact the disease on the ENS.

RESUMO CONTEXTO: Poucos estudos sobre doenças artríticas têm sido realizados para verificar a presença de neurodegeneração. Diante do aumento do estresse oxidativo e dos efeitos extra-articulares da artrite reumatoide, estudos gastrointestinais devem ser investigados visando uma melhor compreensão dos efeitos sistêmicos da doença no sistema nervoso entérico. OBJETIVO: Determinar se a artrite reumatoide afeta a densidade nitrérgica e a área somática dos neurônios mioentéricos imunorreativos ao nNOS (nNOS-IR), bem como para as áreas morfométricas das varicosidades CGRP-IR e VIP-IR do íleo de ratos artríticos. MÉTODOS: Vinte ratos Holtzmann, com 58 dias de idade, foram distribuídos em dois grupos: controle e artrítico. O grupo artrítico recebeu uma única injeção do adjuvante completo de Freund para induzir o modelo de artrite. Os preparados totais de íleo foram processados para imuno-histoquímica ao VIP, CGRP e nNOS. A quantificação foi utilizada para os neurônios nitrérgicos e as análises morfométricas foram realizadas para os três marcadores. RESULTADOS: A doença artrítica induziu uma redução de 6% na área ileal em relação ao grupo controle. Não foram observadas diferenças significativas na densidade nitrérgica comparando os dois grupos. No entanto, o grupo artrítico produziu uma redução da área somática neuronal nitrérgica e da área das varicosidades do VIP-IR. Entretanto, foi observado um aumento das áreas das viricosidades CGRP-IR. CONCLUSÃO: Apesar da artrite não resultar em alterações no número de neurônios nitrérgicos, a retração da área ileal e a redução das áreas somática nitrérgica e das varicosidades do VIP-IR podem sugerir um impacto negativo da doença no sistema nervoso entérico.

Penile alterations at early stage of type 1 diabetes in rats

ABSTRACT Objective Diabetes affects the erectile function significantly. However, the penile alterations in the early stage of diabetes in experimental animal models have not been well studied. We examined the changes of the penis and its main erectile components in diabetic rats. Materials and methods Male Sprague-Dawley rats were divided into 2 groups: streptozotocin (STZ)-induced diabetics and age-matched controls. Three or nine weeks after diabetes induction, the penis was removed for immunohistochemical staining of smooth muscle and neuronal nitric oxide synthase (nNOS) in midshaft penile tissues. The cross-sectional areas of the whole midshaft penis and the corpora cavernosa were quantified. The smooth muscle in the corpora cavernosa and nNOS in the dorsal nerves were quantified. Results The weight, but not the length, of the penis was lower in diabetics. The cross-sectional areas of the total midshaft penis and the corpora cavernosa were lower in diabetic rats compared with controls 9 weeks, but not 3 weeks after diabetes induction. The cross-sectional area of smooth muscle in the corpora cavernosa as percentage of the overall area of the corpora cavernosa was lower in diabetic rats than in controls 9 weeks, but not 3 weeks after diabetes induction. Percentage change of nNOS in dorsal nerves was similar at 3 weeks, and has a decreased trend at 9 weeks in diabetic rats compared with controls. Conclusions Diabetes causes temporal alterations in the penis, and the significant changes in STZ rat model begin 3-9 weeks after induction. Further studies on the reversibility of the observed changes are warranted.

Neurobiological effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, in experimental paradigm of Alzheimer’s disease.

Nitric oxide plays a role in a series of neurobiological functions, underlying behaviour and memory. The functional role of nNOS derived nitric oxide in cognitive functions is elusive. The present study was designed to investigate the effect of specific neuronal nitric oxide synthase inhibitor, 7-nitroindazole, against intracerebroventricular streptozotocin-induced cognitive impairment in rats. Learning and memory behaviour was assessed using Morris water maze and elevated plus maze. 7-nitroindazole (25 mg/kg, ip) was administered as prophylactically (30 min before intracerebroventricular streptozotocin injection on day 1) and therapeutically (30 min before the assessment of memory by Morris water maze on day 15). Intracerebroventricular streptozotocin produced significant cognitive deficits coupled with alterations in biochemical indices.These behavioural and biochemical changes were significantly prevented by prophylactic treatment of 7-nitroindazole. However, therapeutic intervention of 7-nitroindazole did not show any significant reversal. The results suggests that 7-nitroindazole can be effective in the protection of dementiainduced by intracerebroventricular streptozotocin only when given prophylactically but not therapeutically.

Depressed nNOS expression during spine transition in the developing hippocampus of FMR1 KO mice

Nitric oxide (NO), synthesized as needed by NO synthase (NOS), is involved in spinogenesis and synaptogenesis. Immature spine morphology is characteristic of fragile X syndrome (FXS). The objective of this research was to investigate and compare changes of postnatal neuronal NOS (nNOS) expression in the hippocampus of male fragile X mental retardation 1 gene knockout mice (FMR1 KO mice, the animal model of FXS) and male wild-type mice (WT) at postnatal day 7 (P7), P14, P21, and P28. nNOS mRNA levels were analyzed by real-time quantitative PCR (N = 4-7) and nNOS protein was estimated by Western blot (N = 3) and immunohistochemistry (N = 1). In the PCR assessment, primers 5’-GTGGCCATCGTGTCCTACCATAC-3’ and 5’-GTTTCGAGGCAGGTGGAAGCTA-3’ were used for the detection of nNOS and primers 5’-CCGTTTCTCCTGGCTCAGTTTA-3’ and 5’-CCCCAATACCACATCATCCAT-3’ were used for the detection of β-actin. Compared to the WT group, nNOS mRNA expression was significantly decreased in FMR1 KO mice at P21 (KO: 0.2857 ± 0.0150, WT: 0.5646 ± 0.0657; P < 0.05). Consistently, nNOS immunoreactivity also revealed reduced staining intensity at P21 in the FMR1 KO group. Western blot analysis validated the immunostaining results by demonstrating a significant reduction in nNOS protein levels in the FMR1 KO group compared to the WT group at P21 (KO: 0.3015 ± 0.0897, WT: 1.7542 ± 0.5455; P < 0.05). These results suggest that nNOS was involved in the postnatal development of the hippocampus in FXS and impaired NO production may retard spine maturation in FXS.

Involvement of neuronal NO synthase in collateral artery growth.

To evaluate the role of neuronal nitric oxides synthase (nNOS) in collateral artery growth (arteriogenesis), we analyzed the expression pattern of nNOS at distinct time points on RNA and protein levels in a rabbit and a murine model of peripheral arteriogenesis. In the rabbit model, Northern blot analyses revealed a significant upregulation of nNOS at 6 h (1.6-fold), 12 h (2.2-fold) and 24 h (2.0-fold) after induction of arteriogenesis via femoral artery ligation, when compared to the sham operated side. In mice, an upregulation of nNOS was also detected using Northern blot (at 6 h, 12 h) and qRT-PCR (12 h: 2.4-fold). On the protein level, nNOS was found to be upregulated 24 h after femoral artery ligation. Immunohistochemical staining showed that nNOS was localized in endothelial and smooth muscle cells of collateral arteries, as well as in skeletal muscle and nerves. In summary, our data provide evidence that nNOS is not constitutively expressed, but is induced during arteriogenesis, playing a role in supplying reactive oxygen species such as H2O2 and low levels of NO.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48 percent by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469 percent). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20 percent), reaching the greatest increase (60 percent) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Endogenous angiotensin II modulates nNOS expression in renovascular hypertension

Nitric oxide (NO) influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS). Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C) hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight) divided into 2K1C (N = 19) and sham-operated (N = 19) groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9) was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan (10 mg·kg-1·day-1; N = 5) or the superoxide scavenger tempol (0.2 mmol·kg-1·day-1; N = 5), which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

NMDA receptor blockade alters the intracellular distribution of neuronal nitric oxide synthase in the superficial layers of the rat superior colliculus

Nitric oxide (NO) is a molecular messenger involved in several events of synaptic plasticity in the central nervous system. Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR) triggers the synthesis of NO by activating the enzyme neuronal nitric oxide synthase (nNOS) in postsynaptic densities. Therefore, NMDAR and nNOS are part of the intricate scenario of postsynaptic densities. In the present study, we hypothesized that the intracellular distribution of nNOS in the neurons of superior colliculus (SC) superficial layers is an NMDAR activity-dependent process. We used osmotic minipumps to promote chronic blockade of the receptors with the pharmacological agent MK-801 in the SC of 7 adult rats. The effective blockade of NMDAR was assessed by changes in the protein level of the immediate early gene NGFI-A, which is a well-known NMDAR activity-dependent expressing transcription factor. Upon chronic infusion of MK-801, a decrease of 47 percent in the number of cells expressing NGFI-A was observed in the SC of treated animals. Additionally, the filled dendritic extent by the histochemical product of nicotinamide adenine di-nucleotide phosphate diaphorase was reduced by 45 percent when compared to the contralateral SC of the same animals and by 64 percent when compared to the SC of control animals. We conclude that the proper intracellular localization of nNOS in the retinorecipient layers of SC depends on NMDAR activation. These results are consistent with the view that the participation of NO in the physiological and plastic events of the central nervous system might be closely related to an NMDAR activity-dependent function.

Defecto en la homeostasis del óxido nítrico: Mecanismo común subyacente de la insulino-resistencia, la hiperactividad simpática y la morbi-mortalidad cardiovascular / Defective nitric oxide homeostasis: Common underlying mechanism between insulin resistance, sympathetic overactivity and cardiovascular morbidity and mortality

La incidencia de la obesidad y de la resistencia a la insulina con sus complicaciones asociadas, como la hipertensión arterial y el aumento de la morbi-mortalidad cardiovascular, alcanzan hoy en día proporciones epidémicas y representan un problema mayor de salud pública. En los últimos años se ha demostrado que la administración de insulina, además de sus efectos metabólicos, posee efectos cardiovasculares importantes. El sistema nervioso simpático y el sistema L-arginina - óxido nítrico son los mediadores centrales de estas acciones cardiovasculares de la insulina. Mostramos, gracias a estudios realizados en animales y en humanos, que no sólo un déficit de la síntesis del óxido nítrico (NO), sino también un aumento exagerado en su producción representan un defecto subyacente central de las anomalías metabólicas, cardiovasculares y del sistema nervioso simpático que caracterizan a la insulino resistencia. Mostramos cómo estos resultados establecen el fundamento científico para la utilización de sustancias farmacológicas capaces de liberar de manera prolongada cantidades fisiológicas de NO o de inhibidores de su sobreproducción como futuros tratamientos para la resistencia a la insulina y sus complicaciones asociadas.

Obesity, insulin resistance and associated cardiovascular complications are reaching epidemic proportions worldwide and represent a major public health problem. Over the past decade, evidence has accumulated indicating that insulin administration, in addition to its metabolic effects, also has important cardiovascular actions. The sympathetic nervous system and the L-arginine-nitric oxide pathway are the central players in the mediation of insulin's cardiovascular actions. Based on recent animal and human research, we demonstrate that both defective and augmented NO synthesis represent a central defect triggering many of the metabolic, vascular and sympathetic abnormalities characteristic of insulin-resistant states. These observations provide the rationale for the use of pharmaceutical drugs releasing small and physiological amounts of NO and/or inhibitors of NO overproduction as a future treatment for insulin resistance and associated comorbidities.

Papel do óxido nítrico na regulação da circulação pulmonar: implicações fisiológicas, fisiopatológicas e terapêuticas / Role of nitric oxide in the control of the pulmonary circulation: physiological, pathophysiological, and therapeutic implications

O nitric oxide (NO, óxido nítrico) é um mediador endógeno vasoativo que contribui para a homeostase vascular pulmonar. O NO é produzido por três isoformas das nitric oxide synthases (NOS, óxido nítrico sintases)-NOS neuronial (nNOS); NOS induzida (iNOS); e NOS endotelial (eNOS)-estando as três presentes no pulmão. Estudos que utilizaram inibidores farmacológicos ou camundongos knockout têm demonstrado que o NO derivado da eNOS desempenha importantes papéis ao modular o tônus vascular pulmonar e atenuar a hipertensão pulmonar. Por outro lado, estudos focados no papel da iNOS têm mostrado que essa isoforma contribui para a fisiopatologia da lesão pulmonar aguda e da síndrome do desconforto respiratório agudo. Esta revisão objetivou delinear o papel desempenhado pelo NO no controle da circulação pulmonar, tanto em condições fisiológicas como fisiopatológicas. Além disso, revisamos as evidências de que a via L-arginina-NO-guanosina monofosfato cíclico seja um importante alvo farmacológico para a terapia de doenças vasculares pulmonares.

Nitric oxide (NO) is an endogenous vasoactive compound that contributes to pulmonary vascular homeostasis and is produced by three nitric oxide synthase (NOS) isoforms-neuronal NOS (nNOS); inducible NOS (iNOS); and endothelial NOS (eNOS)-all three of which are present in the lung. Studies using pharmacological inhibitors or knockout mice have shown that eNOS-derived NO plays an important role in modulating pulmonary vascular tone and attenuating pulmonary hypertension. However, studies focusing on the role of iNOS have shown that this isoform contributes to the pathophysiology of acute lung injury and acute respiratory distress syndrome. This review aimed at outlining the role played by NO in the control of pulmonary circulation, both under physiological and pathophysiological conditions. In addition, we review the evidence that the L-arginine-NO-cyclic guanosine monophosphate pathway is a major pharmacological target in the treatment of pulmonary vascular diseases.

Expression of neuronal nitric oxide synthase in the hippocampal formation in affective disorders

Hippocampal output is increased in affective disorders and is mediated by increased glutamatergic input via N-methyl-D-aspartate (NMDA) receptor and moderated by antidepressant treatment. Activation of NMDA receptors by glutamate evokes the release of nitric oxide (NO) by the activation of neuronal nitric oxide synthase (nNOS). The human hippocampus contains a high density of NMDA receptors and nNOS-expressing neurons suggesting the existence of an NMDA-NO transduction pathway which can be involved in the pathogenesis of affective disorders. We tested the hypothesis that nNOS expression is increased in the human hippocampus from affectively ill patients. Immunocytochemistry was used to demonstrate nNOS-expressing neurons in sections obtained from the Stanley Consortium postmortem brain collection from patients with major depression (MD, N = 15), bipolar disorder (BD, N = 15), and schizophrenia (N = 15) and from controls (N = 15). nNOS-immunoreactive (nNOS-IR) and Nissl-stained neurons were counted in entorhinal cortex, hippocampal CA1, CA2, CA3, and CA4 subfields, and subiculum. The numbers of Nissl-stained neurons were very similar in different diagnostic groups and correlated significantly with the number of nNOS-IR neurons. Both the MD and the BD groups had greater number of nNOS-IR neurons/400 µm² in CA1 (mean ± SEM: MD = 9.2 ± 0.6 and BD = 8.4 ± 0.6) and subiculum (BD = 6.7 ± 0.4) when compared to control group (6.6 ± 0.5) and this was significantly more marked in samples from the right hemisphere. These changes were specific to affective disorders since no changes were seen in the schizophrenic group (6.7 ± 0.8). The results support the current view of the NMDA-NO pathway as a target for the pathophysiology of affective disorders and antidepressant drug development.

Role of nitiic oxide and nitric oxide synthases in ischemia-reperfusion injury in rat organotypic hippocampus slice / 华中科技大学学报（医学）（英德文版）

To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthase isoforms (nNOS and iNOS), rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen-glucose deprivation (OGD) for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD (P < 0.05). The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase (LDH) efflux into the incubation solution. The results suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.