Transient ischemia increases neuronal nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase co-expression in rat striatal neurons

In neurodegenerative diseases, an increased number of neuronal nitric oxide synthase (nNOS)-positive neurons was reported, but nothing is known on which are the neurons induced to express nNOS. Argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and nNOS act in the L-arginine-NO-L-citrulline cycle permitting a correct NO production. In the brain, nNOS-positive neurons co-expressing ASS were known, while those co-expressing ASL were not demonstrated. We investigated by immunohistochemistry the presence of these types of neurons in the rat striatum to verify whether there was a correlation between their changes due to neurotoxic insults and animal survival. Transient ischemia, a neurodegenerative insult model, was induced in rat brain by 2 h of middle cerebral artery occlusion. The striatum, the core of ischemia, was examined at 24, 72 and 144 h after reperfusion and compared with that of rats in normal condition. ASS, ASL and nNOS-positive neurons, some of the latter also expressing ASS and ASL, were present both in normal and ischemic conditions. At 24 h after reperfusion, the number of the nNOS-positive neurons and the percentage of those co-expressing ASS and ASL were significantly increased in the animals with a longer survival and at 144 h after ischemia there was an almost complete restore of the number and/or percentage of these neurons. We hypothesize that the neurons induced to express nNOS were the ASS- and ASL-positive ones and that the neurons co-expressing nNOS, ASS and ASL, since having the enzymes necessary to maintain a correct NO production, might protect from neurotoxic insults.     

心脏骤停复苏犬脑组织iNOS mRNA nNOS mRNA的表达及中药的干预作用

目的：探讨复苏饮对犬心脏骤停复苏后iNOSmRNA、nNOSmRNA表达的影响。方法：在自主循环恢复180min,以逆转录-聚合酶链反应法（RT-PCR）检测各组海马组织iNOSmRNA、nNOSmRNA表达的情况。结果：iNOSmRNA在假手术组未发现表达,nNOSmRNA有表达,模型组、复苏饮组iNOSmRNA、nNOSmRNA的表达均比假手术组的表达明显增高（P〈0.01,P〈0.05）,模型组iNOSmRNA、nNOSmRNA表达又明显高于复苏饮（P〈0.05）。结论：复苏饮抑制iNOSmRNA、nNOSmRNA的表达,是其脑保护作用机制之一。     

Diabetes only affects nitric oxide synthase-containing myenteric neurons that do not contain heme oxygenase 2

It has been demonstrated that subpopulations of myenteric neurons are differentially susceptible to the development of neuropathy in diabetes. Within the myenteric plexus are neurons that contain neuronal nitric oxide synthase (nNOS). However, these are not a homogeneous population. Some of the nNOS-containing neurons also contain heme oxygenase 2 (HO2). Therefore, the aim of this study was to compare the effects of diabetes on HO2- and nNOS-containing neurons within the myenteric plexus of the rat ileum. Diabetes was induced in male Wistar rats (350-400 g) by a single i.p. injection of buffered streptozotocin (65 mg/kg). After 12 weeks, immunostaining of wholemount preparations of ileum revealed that diabetes induced a significant shift (P < 0.001, chi-squared test for trend) towards increased neuronal cell body size in nNOS-immunoreactive neurons while HO2-immunoreactive neurons remained unaffected. Double-labeling studies revealed that approximately 50% of nNOS-containing neurons also contained HO2 and that the diabetes-induced change in size was confined to nNOS-immunoreactive neurons that did not contain HO2 (P < 0.01). No change in the size distribution occurred in neurons in which nNOS and HO2 were colocalized. Differences in the response of these two subpopulations of nNOS-containing neurons to diabetes could occur because they supply different targets within the gastrointestinal tract or indicate that the antioxidant, HO2, protects those nNOS-containing neurons in which it is colocalized, against oxidative stress that occurs in diabetes.     

Expression and colocalization of NADPH-diaphorase and Fos in the subnuclei of the parabrachial nucleus in rats following visceral noxious stimulation

To investigate whether neural nitric oxide synthase (nNOS) in the parabrachial nucleus (PB) is involved in processing visceral noxious stimulation, we mapped the distribution of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nNOS, and immunohistochemical staining for Fos, a neuronal activity marker, in the subnuclei of the PB following 2% formalin injection into the stomach of rats. NADPH-d and noxious-stimuli induced Fos staining were also examined in tissue containing PB cells labeled by the retrograde transport of fluogold (FG) injected into the central nucleus of the amygdala (CeA). We found that the number of Fos immunoreactive (Fos-IR) neurons was significantly increased in the dorsal lateral (dl), external lateral (el) and K?lliker-Fuse (KF) subnuclei of the PB. We observed that intensely labeled (type 1) NADPH-d positive neurons were mainly located in the rostral part of the PB; they extended long processes adjacent Fos-IR neurons, but no Fos/type 1 NADPH-d double-labeled neurons were seen. In contrast, lightly labeled (type 2) NADPH-d positive neurons were principally localized in the dl of the PB, in which a few Fos/type 2 NADPH-d double-labeled neurons were detected. Additionally, a large number of FG/Fos double-labeled neurons were observed to be surrounded closely by the intensive NADPH-d staining in the el of the PB. These results suggest that neurons in the el of the PB that project to the CeA are activated by visceral noxious stimulation and could be indirectly influenced by nitric oxide in the PB.     

Modulation of sensory neuron‐specific receptors in the development of morphine tolerance and its neurochemical mechanisms

Prevention of opiate tolerance is a critical issue in pain management. The present study was designed to characterize the pharmacological properties of sensory neuron‐specific receptors (SNSR; also known as Mas‐related gene receptors, or Mrg) for their modulation in the development of morphine tolerance and to investigate the underlying mechanism(s). Daily coadministration of the SNSR agonist BAM8‐22 at a dose of 0.01 or 0.001, but not 1.0, nmol with morphine (intrathecally, or i.t., 20 μg/day) for 6 days significantly decreased the development of morphine tolerance. Coadministration of BAM8‐22 (i.t., 1.0 nmol) on days 1, 3, and 5 completely blocked tolerance to morphine‐induced analgesia. Intermittent coadministration of the structurally dissimilar SNSR agonist (Tyr⁶)‐2‐MSH‐6‐12 (MSH; 5 nmol) also produced similar modulation. Chronic administration of morphine (20 μg, i.t.) increased expression of neuronal nitric oxide synthase (nNOS) and calcitonin gene‐related peptide (CGRP) in superficial layers of the spinal cord and dorsal root ganglia. All these increases were abolished when BAM8‐22 or MSH was intermittently coadministered. Furthermore, intermittent administration of BAM8‐22 inhibited morphine‐induced increase in protein kinase Cγ (PKCγ) in both membrane and cytosol of spinal dorsal horn neurons. These results suggest that moderate activation of SNSR modulated morphine tolerance by inhibition of the PKC signaling pathway, leading to abolishment of enhancement of nNOS and CGRP. As SNSR are uniquely located ina subset of small‐sized neurons in dorsal root and trigeminal ganglia, intermittent combination of SNSR agonist could be a promising adjunct for sustained use of opiates without central nervous system side effects. © 2010 Wiley‐Liss, Inc.     

Effects of Insulin Treatment on HuC/HuD, NADH Diaphorase, and nNOS-Positive Myoenteric Neurons of the Duodenum of Adult Rats with Acute Diabetes

We carried out this investigation with the purpose of verifying whether insulin treatment prevents changes in the density of myoenteric neurons of the duodenum of Wistar rats with streptozotocin short-term diabetes. The animals from the diabetic group (D) lost more weight than the controls (group C), while the insulin treatment (group T) prevented weight loss in three animals and increased visceral fat in all of the animals of this group. Insulin treatment did not prevent the early loss of HuC/HuD myoenteric neurons. The density of nNOS-positive neurons did not change significantly in groups D and T. The density of NADHd-positive neurons in these groups was greater than in group C, indicating that short-term diabetes increases the activity of respiratory chain enzymes.     

红霉素预适应对局部缺血再灌注大鼠皮层神经型一氧化氮合酶影响

目的：探讨缺血再灌注红霉素预适应对神经型一氧化氮合酶的影响。方法：制备SD大鼠大脑中动脉缺血再灌注模型,用苏木精伊红染色观察缺血后脑纽织形态学改变,用免疫组织化学法显示神经型一氧化氮合酶变化。结果：神经型一氧化氮合酶在大脑皮层红霉素缺血组表达高于缺血组（P〈0．05）,红霉素组的表达高于正常对照组（P〈0．05）。结论：红霉素预适应促进神经型一氧化氮舍酶表达,保护脑组织,避免损伤。     

Lack of iNOS induction in a severe model of transient focal cerebral ischemia in rats

Calcium-independent nitric oxide synthase (NOS) activity has been reported in ischemic brains and usually attributed to the inducible isoform, iNOS. Because calcium-independent mechanisms have recently been shown to regulate the constitutive calcium-dependent NOS, we proposed to confirm the presence of iNOS activity in our model of transient focal cerebral ischemia in rats. Our initial results showed that, in our model, ischemia induced an important increase in brain calcium concentration. Consequently, the determination of calcium-independent NOS activity required a higher concentration of calcium chelator than classically used in the NOS assay. In these conditions, calcium-independent NOS activity was not observed after ischemia. Moreover, our ischemia was associated with neither iNOS protein expression, measured by Western blotting, nor increased NO production, evaluated by its metabolites (nitrate/nitrite). Our results demonstrate that iNOS activity may be overestimated due to increased brain calcium concentration in ischemic conditions and also that iNOS is not systematically induced after cerebral ischemia.     

脑溢安对大鼠舌下神经压榨伤后神经元型一氧化氮合酶nNOS表达的影响

目的：观察大鼠舌下神经压榨伤后,舌下神经核内nNOS的表达变化及中药脑溢安对其表达的影响。方法：建立大鼠舌下神经压榨伤模型,脑溢安治疗组用脑溢安药液灌胃,正常对照组和实验对照组用灭菌生理盐水灌胃。采用NADPH-d组化＋中性红复染组织化学方法,分别于第1、4、7、14天检测舌下神经核内nNOS表达变化。结果：正常对照组大鼠两侧舌下神经核内均未检测到nNOS免疫阳性细胞;与生理盐水对照组相比,脑溢安治疗组损伤后第4、7、14天损伤侧舌下神经核内阳性细胞数少,细胞成活率高,有统计学差异（P〈0.05）。结论：脑溢安降低大鼠舌下神经压榨伤后神经元胞体nNOS的表达。     

Cavernous smooth muscle hyperplasia in a rat model of hyperlipidaemia-associated erectile dysfunction

Study Type – Aetiology (case control) Level of Evidence 3b What’s known on the subject? and what does the study add? Increased cavernous smooth muscle content has been repeatedly observed in rat models of hyperlipidaemia – associated erectile dysfunction. This study shows that the increased smooth muscle content is due to hyperplasia.

OBJECTIVE

? To investigate the structural changes, including possible smooth muscle hyperplasia, in the penis of a hyperlipidaemia‐associated erectile dysfunction (ED) animal model.

MATERIALS AND METHODS

? Hyperlipidaemia was induced in rats through a high‐fat diet. ? Penile tissues of normal and hyperlipidaemic rats were stained with Alexa‐488‐conjugated phalloidin and/or with antibodies against rat endothelial cell antigen, neuronal nitric oxide synthase (nNOS), and collagen type IV (Col‐IV) before image and statistical analyses were carried out. ? The main outcome measures were the smooth muscle, endothelial, Col‐IV and nNOS content of the corpus cavernosum.

RESULTS

? Phalloidin intensely stained all smooth muscle in the penis, revealing the circular and longitudinal components of cavernous smooth muscle (CSM). ? The CSM content was significantly higher in the hyperlipidaemic than in the normal rats (P < 0.05). ? Cell numbers in both circular and longitudinal CSM were significantly higher in the hyperlipidaemic than in the normal rats (P < 0.05). ? Cavernous endothelial content was significantly lower in hyperlipidaemic than in normal rats (P < 0.05). ? nNOS‐positive nerves within the dorsal nerves, around the dorsal arteries, and in the corpora cavernosa were all significantly lower in the hyperlipidaemic than in the normal rats (P < 0.05).

CONCLUSIONS

? Hyperlipidaemia is associated with reduced nNOS‐positive nerves, reduced endothelium, and increased CSM in the penis. ? The increased CSM is attributable to hyperplasia. ? These structural changes may explain why hyperlipidaemic men are more likely to develop ED.