Nitrergic Enteric Neurons in Health and Disease-Focus on Animal Models.

Nitrergic enteric neurons are key players of the descending inhibitory reflex of intestinal peristalsis, therefore loss or damage of these neurons can contribute to developing gastrointestinal motility disturbances suffered by patients worldwide. There is accumulating evidence that the vulnerability of nitrergic enteric neurons to neuropathy is strictly region-specific and that the two main enteric plexuses display different nitrergic neuronal damage. Alterations both in the proportion of the nitrergic subpopulation and in the total number of enteric neurons suggest that modification of the neurochemical character or neuronal death occurs in the investigated gut segments. This review aims to summarize the gastrointestinal region and/or plexus-dependent pathological changes in the number of nitric oxide synthase (NOS)-containing neurons, the NO release and the cellular and subcellular expression of different NOS isoforms. Additionally, some of the underlying mechanisms associated with the nitrergic pathway in the background of different diseases, e.g., type 1 diabetes, chronic alcoholism, intestinal inflammation or ischaemia, will be discussed.

Netrin-1-like-immunoreactivity Coexpresses With DCC and Has a Differential Level in the Myenteric Cholinergic and Nitrergic Neurons of the Adult Mouse Colon.

Netrin-1 is a potent axonal and neuronal guidance cue in the developing nervous system. Netrin-1 functions are mediated by its receptors, such as deleted in colorectal cancer (DCC) present on axons and neurons. Localization of DCC and Netrin-1 on various types of enteric neurons and their role in the mature enteric nervous system is unknown. The results of our study revealed that almost all enteric neurons and processes express DCC and Netrin-1 in the adult mice. Netrin-1-like-immunoreactivity (IR) was detected in the cytoplasm of neurons with some showing strong or weak staining. The majority of Netrin-1-like-immunoreactive enteric neurons were choline acetyltransferase (ChAT)-positive. However, ~19% of neurons were strongly Netrin-1-like-positive but ChAT-negative while ~8% of neurons were Netrin-1-like-negative but strongly ChAT-positive. In contrast, almost all nitric oxide synthase (nNOS)-positive enteric neurons displayed strong Netrin-1-like-IR. This differential intensity of Netrin-1 expression in the myenteric neurons might determine major neuronal subtypes regulating intestinal motility, ChAT-IR excitatory, and nNOS-IR inhibitory muscle motor and interneurons. This is the first study demonstrating the localization of DCC and Netrin-1 in the colonic myenteric plexus of the adult mice and their expression level determining two major neuronal subtypes regulating intestinal motility.

Balanced Caloric Restriction Minimizes Changes Caused by Aging on the Colonic Myenteric Plexus.

Aging can promote significant morphofunctional changes in the gastrointestinal tract (GIT). Regulation of GIT motility is mainly controlled by the myenteric neurons of the enteric nervous system. Actions that aim at decreasing the aging effects in the GIT include those related to diet, with caloric restriction (CR). The CR is achieved by controlling the amount of food or by manipulating the components of the diet. Therefore, the objective of this study was to evaluate different levels of CR on the plasticity of nicotinamide adenine dinucleotide phosphate- (NADPH-) reactive myenteric neurons in the colon of Wistar rats during the aging process using ultrastructural (transmission electron microscopy) and morphoquantitative analysis. Wistar male rats (Rattus norvegicus) were distributed into 4 groups (n = 10/group): C, 6-month-old animals; SR, 18-month-old animals fed a normal diet; CRI, 18-month-old animals fed a 12% CR diet; CRII, 18-month-old animals fed a 31% CR diet. At 6 months of age, animals were transferred to the laboratory animal facility, where they remained until 18 months of age. Animals of the CRI and CRII groups were submitted to CR for 6 months. In the ultrastructural analysis, a disorganization of the periganglionar matrix with the aging was observed, and this characteristic was not observed in the animals that received hypocaloric diet. It was observed that the restriction of 12.5% and 31% of calories in the diet minimized the increase in density and cell profile of the reactive NADPH neurons, increased with age. This type of diet may be adapted against gastrointestinal disturbances that commonly affect aging individuals.

Octodon degus, a new model to study the agonist and plexus-induced response in the urinary bladder.

Urinary bladder function consists in the storage and controlled voiding of urine. Translational studies require animal models that match human characteristics, such as Octodon degus, a diurnal rodent. This study aims to characterize the contractility of the detrusor muscle and the morphology and code of the vesical plexus from O. degus. Body temperature was measured by an intra-abdominal sensor, the contractility of detrusor strips was evaluated by isometric tension recording, and the vesical plexus was studied by electrical field stimulation (EFS) and immunofluorescence. The animals showed a diurnal chronotype as judged from core temperature. The myogenic contractile response of the detrusor muscle to increasing doses of KCl reached its maximum (31.04 mN/mm2) at 60 mM. In the case of cumulative dose-response of bethanecol, the maximum response (37.42 mN/mm2) was reached at 3.2 × 10-4 M. The response to ATP was clearly smaller (3.8 mN/mm2). The pharmacological dissection of the EFS-induced contraction identified ACh and sensory fibers as the main contributors to this response. The neurons of the vesical plexus were located mainly in the trigone area, grouped in big and small ganglia. Out of them, 48.1 % of the neurons were nitrergic and 62.7 % cholinergic. Our results show functional and morphological similarities between the urinary bladder of O. degus and that of humans.

Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion.

Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED.

Cytoarchitecture of nitrergic neurons in the human striatum and subthalamic nucleus.

BACKGROUND: Nitric oxide (NO) is a gaseous molecule that modulates several physiological processes, including signal transmission in the central nervous system. There is evidence supporting NO as a major neurotransmitter involved in motor and emotion/behavior control. We investigated the distribution and morphology of nitrergic neurons in the two main input structures of the basal ganglia of human brain: the striatum and subthalamic nucleus. METHODS: We studied samples of striatum (caudate and putamen) and subthalamic nucleus of 20 human brains from subjects without neurological/psychiatric diseases. The tissues were stained by histochemistry for nicotinamide adenine dinucleotide phosphate diaphorase activity and by immunohistochemistry for neuronal NO synthase (nNOS). Subsequently, we analyzed the nitrergic neuronal profile and its morphometric parameters. RESULTS: Our data corroborate that approximately 2% of neurons in striatum express nNOS and these exhibited morphology characteristic of interneurons. Posterior regions of the striatum have a higher nitrergic neuronal profile than anterior regions of this nucleus suggesting an anteroposterior gradient of nitrergic neurons. Posterior limbic-associated areas of the striatum have a higher nitrergic neuronal profile compared to other functional subdivisions. Also, approximately 90% of neurons in the subthalamic nucleus express nNOS. CONCLUSIONS: A remarkable presence of nitrergic neurons in the human striatum and subthalamic nucleus suggests that NO may play a critical role in the physiology of these nuclei.

Neurovascular Interface in Porcine Small Intestine: Specific for Nitrergic rather than Nonnitrergic Neurons.

In the 1970s, by using classic histological methods, close topographical relationships between special areas of enteric ganglia and capillaries were shown in the pig. In this study, by application of double and triple immunohistochemistry, we confirmed this neurovascular interface and demonstrated that these zones are mainly confined to nitrergic neurons in the myenteric and the external submucosal plexus. In the upper small intestine of the pig, the respective neurons display type III morphology, i.e. they have long, slender and branched dendrites and a single axon. In another set of experiments, we prepared specimens for electron-microscopical analysis of these zones. Both ganglia and capillaries display continuous basement membranes, the smallest distances between them being 1,000 nm at the myenteric and 300 nm at the external submucosal level. The capillary endothelium was mostly continuous but, at the external submucosal level, scattered fenestrations were observed. This particular neurovascular relationship suggests that nitrergic neurons may require a greater amount of oxygen and/or nutrients. In guinea pig and mouse, previous ischemia/reperfusion experiments showed that nitrergic neurons are selectively damaged. Thus, a preferential blood supply of enteric nitrergic neurons may indicate that these neurons are more vulnerable in ischemia.

[A COMPARATIVE STUDY OF CATECHOLAMINERGIC AND NITROXIDERGIC VASOMOTOR NEURONS IN THE NUCLEI OF THE CAUDAL PART OF THE BRAINSTEM OF THE RAT].

Immunohistochemical methods for the demonstration of tyrosine hydrolase (TH) and neuronal form of nitric oxide synthase (nNOS) were used to study the distribution of catecholaminergic and nitroxidergic vasomotor neurons respectively, in the nuclei of the medulla oblongata and the pons of 12 Wistar rats. Most often the expression of TG was found in neurons located in the nucleus and several reticular nuclei (gigantocellular, paragigantocellular, caudal pons nucleus), but the proportion of immunoreactive neurons did not exceed 8-14%. In the other nuclei (reticular parvocellular nucleus and oral pons nucleus, spinal nucleus of the trigeminal nerve) the value of this parameter ranged from 1 to 3%. In a large group of nuclei with proven vasomotor function such neurons were constantly not detected. In the structures with high content of catecholaminergic neurons, nNOS-positive cells were found, as a rule, in fewer numbers than in the nuclei with a limited number of TH-positive neurons.

[THE DISTRIBUTION OF CHOLINERGIC AND NITROXIDERGIC NEURONS IN THE SPINAL CORD OF NEWBORN AND ADULT RATS].

The aim of this study was to examine the distribution of cholinergic and nitroxidergic neurons in the spinal cord (SC) of adult and newborn rats. Using immunohistochemical demonstration of choline acetyltransferase (ChAT) and nitric oxide synthase (NOS), cervical portions of SC were studied in newborn (n=5) and adult (n=5) Wistar rats. It was found that ChAT-positive neurons were localized in the anterior horns of the SC, while individual cells were located in of SC posterior horns, in the central gray matter and at the boundary of VI-VII Rexed laminae. Nitroxidergic neurons were located in the superficial layers of SC posterior horns of grey matter, in the central gray matter and in the area of VI-VII Rexed laminae. It is found that SC of newborn and adult rats contained cholinergic neurons expressing NOS. Detection of cells containing both enzymes already at postnatal Day 1, suggests that they were formed in rat SC during prenatal ontogenesis

Vitamin A and amygdala: functional and morphological consequences.

Intake of vitamin A is essential for correct embryonic development of the central nervous system (CNS). Its increased intake during gravidity can cause various malformations and dysfunctions of the CNS. In our work, we intended to investigate the effect of vitamin A on emotional behavior and morphology of nitrergic neurons in basolateral nucleus of the rat amygdala. For this purpose, we have administered retinoic acid (RA), a metabolite of vitamin A, to females on 14-16 days of pregnancy at a dose 1 mg RA/kg body weight. Adult progeny of these mothers were tested in elevated plus maze test, the most widely used test for measuring anxiety-like behavior. After the test, brains of the rats were processed for reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry, which is commonly used to mark cells containing nitric oxide synthase. Our results have shown that RA applied during the sensitive phase of intrauterine development influences emotional behavior of adult rats. Animals exposed to RA had increased levels of fear and anxiety, which has been manifested by reducing the time spent in the open arms of plus maze test. Interestingly, detected behavioral changes do not correlate with the result of our morphological study. The number and morphology of nitrergic neurons in amygdala were very similar in experimental and control rats. Our results demonstrate that prenatal exposure to RA has no effect on morphological structure of amygdala, but influences its function.

Nitrergic neurons during early postnatal development of the prefrontal cortex in the rat: histochemical study.

The presence of nitrergic cells in the prefrontal cortex has been confirmed, however little is known about the postnatal development of these cells. Nitrergic neurons were studied histochemically by using NADPH-diaphorase staining in the prefrontal cortex of male Wistar rats from postnatal day 7-21 (P7-21). Neuronal NADPH-diaphorase is a nitric oxide synthase that provides a specific histochemical marker for neurons producing nitric oxide (NO). NO acts as a neurotransmitter and intracellular signaling molecule in the nervous system. We observed in 7 day old rats NADPH-d containing neurons that were intensely stained. These neurons were bipolar with a short dendrite with average length of 23 µm. During the second postnatal week, the neurons were mainly bipolar and were rarely multipolar. By P14 the cells were located primarily in cortical layers III-VI. Nitrergic neurons of the 21 day old rats were histochemically identified as multipolar cells with long radial extending dendrites. Dendrites of neurons in 14 and 21 day old rats were a similar length with an average of 57 µm. These results suggest that nitrergic neurons differentiate during a relatively short period of time and reach their structural maturity by the end of the second week of postnatal development.

Nitrergic neurons in the spinal cord of the bottlenose Dolphin (Tursiops truncatus).

Nitric oxide (NO) is a freely diffusible gaseous neurotransmitter generated by a selected population of neurons and acts as a paracrine molecule in the nervous system. NO is synthesized from l-arginine by means of the neuronal nitric oxide synthase (nNOS), an enzyme requiring nicotine adenine dinucleotide phosphate (NADPH) as cofactor. In this study, we used histochemical and immunohistochemical techniques to investigate the distribution of NADPH-diaphorase (NADPH-d) and nNOS in the spinal cord of the bottlenose dolphin (Tursiops truncatus). Cells with a fusiform-shaped somata were numerous in the laminae I and II. The intermediolateral horn showed darkly-stained cells with a multipolar morphology. Neurons with a multipolar or fusiform morphology were observed in the ventral horn. Multipolar and fusiform neurons were the most common cell types in lamina X. Nitrergic fibers were numerous especially in the dorsal and intermediolateral horns. The presence of nitrergic cells and fibers in different laminae of the spinal cord suggests that NO may be involved in spinal sensory and visceral circuitries, and potentially contribute to the regulation of the complex retia mirabilia.

Regulation of myometrial circulation and uterine vascular tone by constitutive nitric oxide.

Pregnancy is a physiological state that involves an increase in uterine blood flow, which is mediated in part by nitric oxide (NO) liberated from the endothelium and nitrergic neurons. The main focus of this review article is to provide information about how endogenous NO regulates uterine and placental blood flow and vascular tone in experimental animals and humans in vivo or in vitro in non-pregnant and pregnant states as well as pregnancy with pre-eclampsia. Uterine arteries from non-pregnant women respond to NO liberated from the endothelium and nitrergic nerves with relaxations, and the release of endothelial NO is influenced by the phase of the estrous cycle, with its enhanced release at the follicular phase when the estrogen level is high. NO bioavailability in the uteroplacental circulatory system is gradually increased during pregnancy. Pre-eclamptic pregnancies with or without intrauterine growth restriction show impaired uteroplacental blood flow accompanied by reduced NO synthesis due to down-regulation of eNOS as well as asymmetric dimethylarginine accumulation and by augmented NO degradation by oxidative stress. Further studies are expected to provide new mechanistic insights into the fascinating process of maternal uterine adaptation in humans and novel prophylactic and therapeutic measures against pre-eclampsia.

Morphological demonstration of a vagal inhibitory pathway to the lower esophageal sphincter via nitrergic neurons in the rat esophagus.

BACKGROUND: The involvement of vagal parasympathetic efferents in esophageal myenteric neurons in vagal inhibitory pathways to the lower esophageal sphincter (LES) is not clear. Thus, this study was performed to demonstrate morphologically the presence of vagal inhibitory pathways to the LES via esophageal neurons. METHODS: Fast Blue (FB) was injected into the LES of Wistar rats, and 3 days after injection, the animals were subjected to electrical stimulation of the vagus nerve. The esophagus was processed for immunohistochemistry for Fos that was an immediate-early gene as a marker of neuronal activity, nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). The immunoreactivities were then compared with the FB labeling in esophageal neurons. KEY RESULTS: Fast Blue-labeled neurons were observed within an esophageal area of 30 mm oral to the LES, with the highest frequency in the esophagus just above the LES. Most of the FB-labeled neurons were positive for NOS and VIP, but a few for ChAT. Following vagal-electrical stimulation, one fourth of the FB-labeled neurons presented nuclei expressing Fos and most of these Fos/FB neurons were NOS-positive. CONCLUSIONS & INFERENCES: A majority of the FB-labeled esophageal neurons appeared to be descending motor neurons innervating the LES. Moreover, the colocalization of VIP and NOS in most of the LES-projecting neurons suggests that VIP and NO released from these neurons induce LES relaxation, and the innervation of the vagal efferents to the LES-projecting esophageal neurons in the distal esophagus implies a vagal inhibitory pathway responsible for LES relaxation.

The expression of neuronal nitric oxide synthase in the brain of the mouse during embryogenesis.

The distribution of neuronal nitric oxide synthase (nNOS) in the process of normal mouse brain growth from embryonic (E) Day 11 to postnatal (P) Day 1 was investigated by means of immunohistochemical and immunofluorescent methods. Our results demonstrated that nNOS positive neurons appeared early in superficial cortex at E11. At E13, nNOS positive neurons were located in lateral hypothalamus and amygdala, and temporarily in medullar and ventral hypothalamic neuroepithelia. From E15 to P0, nNOS positive neurons were distributed in superior and inferior colliculi, positive staining could also be seen in superior and inferior tectal neuroepithelium at E15. From E17 to birth, the medial geniculate nucleus had a high density of nNOS labeling. The distribution of nNOS gradually increased and extended laterally in embryo brain, which in turn implies that NO might be involved in the development of mouse brain.

Development of the human corpus striatum and the presence of nNOS and 5-HT2A receptors.

This study focussed on the development of the corpus striatum in the fetus, using silver impregnation and immunohistochemistry. For the latter, we looked for nNOS positive cells and 5-HT(2A) receptors positive cells in the corpus striatum during development. During the initial formation of the corpus striatum, there was migration cells of the ganglionic eminence toward the putamen by 15-17 weeks of gestation. Process formation in the neurons started by week 17 and became very complex before term (31/32 weeks of gestation). By 25-27 gestational weeks, the globus pallidus already had two parts and the corpus striatum was similar to the adult in configuration. The nNOS positive cells appeared early (21-23 weeks in gestation) while 5-HT(2A) receptors positive cells were not observed until 31/32 weeks gestation. The number of positive cells in both groups was relatively small. It is anticipated that further developmental changes would occur in the postnatal/neonatal phases.

[The application of a "pixel method" for the quantitative assessment of the results of the histochemical studies].

The results of the application of a "pixel method" for the automated measurement of the intensity of histochemical reaction in the neurons are presented. The method is based on measuring principle, which includes the automatic counting the sum of brightnesses of all the pixels forming the image with the help of standard computer programs. The potential of this method is demonstrated on the example of NADPH-diaphorase activity study in the nitroxidergic neurons of sensory and motor nuclei of the medulla of healthy rats.

NADPH diaphorase expression in superior colliculus of developing, aging and visually deafferented rats.

We have studied the development of NADPH-diaphorase activity in the retinorecipient layers of the superior colliculus (SC) in rats from embryonic day 17 to adulthood, during aging, and following neonatal tetrodotoxin injection or unilateral eye removal in the neonatal or in the adult animal. In the superficial SC, NADPH-d activity is first seen in neurons on postnatal day (P) 4; over the next two weeks, enzyme expression increases gradually, in cells as well as in the neuropil. By P12-14, around the time of eye opening, NADPH-d reactivity increases dramatically. In parallel, the dendrites of many NADPH-d-positive neurons in the superficial gray layer, more or less randomly distributed at first, gradually align their orientation relative to the dorsoventral axis. The pattern of NADPH-d activity in the superficial layers of the SC (i.e. stratum griseum superficiale and stratum opticum) is adult-like by the fourth week of age. Deafferentation of the superficial SC, both in the neonatal and adult rat, and block of retinal activity lead to reduction in the size of the SC and changes in NADPH-d-positive neurons, including dendrite misorientation, decreased cell size and reduced number. Some of these changes are seen also in the aging animal. These results document a protracted and progressive increase in the development of NADPH-d expression in the SC. Our results suggest a strong influence of retinal afferents and activity on the development and maintenance of NAPHD-positive neurons in the retinorecipient layers of the SC, where NO can act as a retrograde signal to carve the terminal arbors of retinal axons.

Partial, selective survival of nitrergic neurons in chagasic megacolon.

One frequent chronic syndrome of Chagas' disease is megacolon, an irreversible dilation of a colonic segment. Extensive enteric neuron loss in the affected segment is regarded as key factor for deficient motility. Here, we assessed the quantitative balance between cholinergic and nitrergic neurons representing the main limbs of excitatory and inhibitory colonic motor innervation, respectively. From surgically removed megacolonic segments of four patients, each three myenteric wholemounts (from non-dilated oral, megacolonic and non-dilated anal parts) was immunohistochemically triple-stained for choline acetyltransferase, neuronal nitric oxide synthase (NOS) and the panneuronal human neuronal protein Hu C/D. Degenerative changes were most pronounced in the megacolonic and anal regions, e.g. bulked, honeycomb-like ganglia with few neurons which were partly enlarged or atrophic or vacuolated. Neuron counts from each 15 ganglia of 12 megacolonic wholemounts were compared with those of 12 age- and region-matched controls. Extensive neuron loss, mainly in megacolonic and anal wholemounts, was obvious. In all three regions derived from megacolonic samples, the proportion of NOS-positive neurons (control: 55%) was significantly increased: in non-dilated oral parts to 61% (p = 0.003), in megacolonic regions to 72% (p < 0.001) and in non-dilated anal regions to 78% (p < 0.001). We suggest the chronic dilation of megacolonic specimens to be due to the preponderance of the nitrergic, inhibitory input to the intestinal muscle. However, the observed neuronal imbalance was not restricted to the dilated regions: the non-dilated anal parts may be innervated by ascending, cholinergic axons emerging from less affected, more anally located regions.