Nitric Oxide Synthase Immunoreactivity Research Articles

The Effects of Irisin on L-Name Induced Hypertension in Rats

Background:The cause of about 95% of hypertension, an important public health problem, is unknown. Intensive studies are underway to understand the physiopathology of hypertension. Irisin, a newly discovered hormone, has been reported to dilate vascular smooth muscle and lower blood pressure acutely.Aims:To investigate the effects of chronic irisin treatment on blood pressure and renal functions in a hypertension model established by nitric oxide synthase inhibition by treatment with Nω-nitro-L-arginine methyl ester hydrochloride.Study Design:Animal experimentation.Methods:Male Sprague−Dawley rats were divided into four groups (n=8). Control and irisin groups received an intravenous saline injection, hypertension and hypertension + irisin (hypertension + irisin) groups received 1.5 mg/100 g Nω-nitro-L-arginine methyl ester hydrochloride. Nω-nitro-L-arginine methyl ester hydrochloride (150 mg/L) was added to the drinking water of rats in groups hypertension and hypertension + irisin for three weeks. In the second week of the experiment, irisin (50 nmol/day) was given to rats in groups irisin and hypertension + irisin, and saline was administered to rats in groups control and hypertension for two weeks through subcutaneously placed osmotic minipumps. Blood pressure was measured by the tail-cuff plethysmography method. On the twenty-first day of the experiment, 24-hour urine, blood, and both kidneys of the rats were collected.Results:The hypertension group had elevated systolic, diastolic, and mean arterial blood pressure values compared with the control group, with decreased glutathione levels in tissue and serum, but an increase in serum oxidized glutathione level (p<0.05). Histopathologically, increased tubular injury, cast formation, glomerular sclerosis, and peritubular fibrosis levels were observed (p<0.05). Irisin treatment did not cause any significant change in blood pressure, renal functions, and injury scores. However, renal nitric oxide levels significantly increased, and endothelial nitric oxide synthase immunoreactivity was determined to be reduced (p<0.05).Conclusion:Treatment with chronic irisin at a physiological dose does not reduce blood pressure in an experimental model of hypertension. In different models of experimental hypertension, the effects of irisin administration at different doses and at different periods should be thoroughly investigated.

Distribution of neuronal nitric oxide synthase immunoreactivity in adult male Sprague-Dawley rat brain

Neuronal NOS (nNOS) accounts for most of the NO production in the nervous system that modulates synaptic transmission and neuroplasticity. Although previous studies have selectively described the localisation of nNOS in specific brain regions, a comprehensive distribution profile of nNOS in the brain is lacking. Here we provided a detailed morphological characterization on the rostro-caudal distribution of neurons and fibres exhibiting positive nNOS-immunoreactivity in adult Sprague-Dawley rat brain. Our results demonstrated that neurons and fibres in the brain regions that exhibited high nNOS immunoreactivity include the olfactory-related areas, intermediate endopiriform nucleus, Islands of Calleja, subfornical organ, ventral lateral geniculate nucleus, parafascicular thalamic nucleus, superior colliculus, lateral terminal nucleus, pedunculopontine tegmental nucleus, periaqueductal gray, dorsal raphe nucleus, supragenual nucleus, nucleus of the trapezoid body, and the cerebellum. Moderate nNOS immunoreactivity was detected in the cerebral cortex, caudate putamen, hippocampus, thalamus, hypothalamus, amygdala, and the spinal cord. Finally, low NOS immunoreactivity were found in the corpus callosum, fornix, globus pallidus, anterior commissure, and the dorsal hippocampal commissure. In conclusion, this study provides a comprehensive view of the morphology and localisation of nNOS immunoreactivity in the brain that would contribute to a better understanding of the role played by nNOS in the brain.

Histoloical and Biochemichal Study on the Toxic Effects of Bisphenol A on the Thyroid Gland of Adult Male Albino Rats and the Possible Protection by Selenium

Background: The worldwide used bisphenol A (BPA) which is incorporated in many plastic industries is considered as an endocrine disruptor. Warnings raised by many agencies against the excessive use of such substances because of its confirmed hazardous effects. Selenium (Se) is known to have antioxidant role in living systems.Aim of the work: The aim of the current study was to evaluate the extent to which BPA can affect the thyroid gland structure and function and if there is any protective role for selenium. Material and Methods: Twenty four adult male rats divided equally into three groups. Group I as a control, group II included rats that received 50 mg/kg/ day bisphenol orally for eight weeks and group III that received bisphenol in the same dose for the same duration concomitantly with selenium in a dose of 0.5 mg/kg. At the end of the experiment, blood samples were taken for hormonal essay and statistical analysis. Thyroid gland tissue samples were processed for histological and immunohistochemical study.Results: BPA-treated rats showed degenerative changes in the form of decreased or absent colloid and fusion of the follicles. Other follicles showed signs of hyperactivity as microfollicles, follicular epithelial cell stratification and many lysosomes. Strong positive inducible nitric oxide synthase immunoreactivity and highly significant increase of serum TSH with decreased T3 and T4 were recorded. BPA+selenium treated group revealed nearly normal appearance of thyroid gland architecture. Conclusion: BPA had deleterious effects on thyroid structure and function. Concomitant administration of selenium preserved to a great extent the thyroid gland architecture.

Characterization of projections of longitudinal muscle motor neurons in human colon.

The enteric nervous system contains inhibitory and excitatory motor neurons which modulate smooth muscle contractility. Cell bodies of longitudinal muscle motor neurons have not been identified in human intestine. We used retrograde tracing ex vivo with DiI, with multiple labeling immunohistochemistry, to characterize motor neurons innervating tenial and inter-tenial longitudinal muscle of human colon. The most abundant immunohistochemical markers in the tertiary plexus were vesicular acetylcholine transporter, nitric oxide synthase (NOS), and vasoactive intestinal polypeptide (VIP). Of retrogradely traced motor neurons innervating inter-tenial longitudinal muscle, 95% were located within 6mm oral or anal to the DiI application site. Excitatory motor neuron cell bodies, immunoreactive for choline acetyltransferase (ChAT), were clustered aborally, whereas NOS-immunoreactive cell bodies were distributed either side of the DiI application site. Motor neurons had small cell bodies, averaging 438+18µm2 in cross-sectional area, similar for ChAT- and NOS-immunoreactive subtypes. Motor neurons innervating the tenia had slightly longer axial projections, with 95% located within 9mm. ChAT-immunoreactive excitatory motor neurons to tenia were clustered aborally, whereas NOS-immunoreactive inhibitory motor neurons had both ascending and descending projections. VIP immunoreactivity was rarely present without NOS immunoreactivity in motor neurons. Tenial and inter-tenial motor neurons innervating the longitudinal muscle have short projections. Inhibitory motor neurons have less polarized projections than cholinergic excitatory motor neurons. Longitudinal and circular muscle layers are innervated by distinct local populations of excitatory and inhibitory motor neurons. A population of human enteric neurons that contribute significantly to colonic motility has been characterized.

Ghrelin as a Novel Pharmacological Treatment for Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the leading cause of mortality from gastrointestinal (GI) disease in preterm neonates. Its precise pathogenesis is unknown, but early stages of NEC are thought to be characterized by damage to the intestinal mucosa; while full intestinal necrosis and perforation occur during the later stages.The high‐frequency power spectra of heart rate variability (HF‐HRV) is a non‐invasive measure of vagal tone, our laboratory has shown that decreases in HF‐HRV may predict which neonates are at increased risk for NEC before the onset of clinical signs (Doheny et al. Neurogastroenterol. Motil., 2014). Furthermore, experimental NEC in newborn rat pups reduces HF‐HRV and increases NOS immunoreactivity in the myenteric neurons of the lower GI tract (Meister et al., Neurogastroenterol. Motil., 2018). Ghrelin is a GI hormone known to increase gastric motility via actions at several sites, including an excitation of dorsal vagal motoneurons upon brainstem microinjection, hence leading to an increase in vagal output to the GI tract.The aim of the present study was to test the hypotheses that ghrelin administration in a rodent model of NEC will: i) increase HF‐HRV values, and ii) restore the neurochemical phenotype of myenteric neurons of the lower GI tract.Newborn Sprague‐Dawley rats were randomly separated in the following groups: i) control, ii) mild NEC (&lt;grade 2) induced via exposure to cold stress and hypoxia twice daily, and iii) mild NEC and anterior subdiaphragmatic vagotomy (grade &gt;2). All groups underwent twice daily intraperitoneal injections of saline or ghrelin (0.05mg/kg) on postnatal days (PD)‐5 to PD‐10. Electrocardiogram recordings using bilateral electrodes to measure HF‐HRV were conducted on PD‐1, ‐5, and ‐10, prior to sacrifice on PD12. Upon sacrifice, GI tissues were collected to confirm NEC grade via H&amp;E staining, and to assess the neurochemical phenotype of myenteric neurons through fluorescence immunohistochemistry.Mild NEC was confirmed via grade &gt;2 histological examination; these pups did not display typical developmental increases in HF‐HRV. Ghrelin, but not saline, treatment reduced the NEC histological scale, increased HF‐HRV values and restored NOS‐immunoreactivity (NOS‐IR) in neurons of the myenteric plexus to levels similar to controls. Data are summarized in the Table below.Our data indicate that ghrelin administration in rodent model of NEC: i) decreased the histological severity of NEC, ii) increased HF‐HRV values, and iii) restored the neurochemical phenotype of myenteric neurons of the lower GI tract. We suggest that ghrelin may be a promising treatment to attenuate, or even reverse, the impaired HF‐HRV and altered neurochemical phenotype observed after NEC induction.Support or Funding InformationAmerican Physiological Society grant to C.R.B. &amp; NIH grant DK99350 to R.A.T. &amp; K.K.D.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distribution, projections, and association with calbindin baskets of motor neurons, interneurons, and sensory neurons in guinea-pig distal colon.

Normal gut function relies on the activity of the enteric nervous system (ENS) found within the wall of the gastrointestinal tract. The structural and functional organization of the ENS has been extensively studied in the guinea pig small intestine, but less is known about colonic circuitry. Given that there are significant differences between these regions in function, observed motor patterns and pathology, it would be valuable to have a better understanding of the colonic ENS. Furthermore, disorders of colonic motor function, such as irritable bowel syndrome, are much more common. We have recently reported specialized basket-like structures, immunoreactive for calbindin, that likely underlie synaptic inputs to specific types of calretinin-immunoreactive neurons in the guinea-pig colon. Based on detailed immunohistochemical analysis, we postulated the recipient neurons may be excitatory motor neurons and ascending interneurons. In the present study, we combined retrograde tracing and immunohistochemistry to examine the projections of circular muscle motor neurons, myenteric interneurons, and putative sensory neurons. We focused on neurons with immunoreactivity for calbindin, calretinin and nitric oxide synthase and their relationship with calbindin baskets. Retrograde tracing using indocarbocyanine dye (DiI) revealed that many of the nerve cell bodies surrounded by calbindin baskets belong to motor neurons and ascending interneurons. Unique functional classes of myenteric neurons were identified based on morphology, neuronal markers and polarity of projection. We provide evidence for three groups of ascending motor neurons based on immunoreactivity and association with calbindin baskets, a finding that may have significant functional implications.

Exacerbation of blood-brain barrier breakdown, edema formation, nitric oxide synthase upregulation and brain pathology after heat stroke in diabetic and hypertensive rats. Potential neuroprotection with cerebrolysin treatment.

There is a growing trend of hypertension among military and civilian populations due to lifetime stressful situations. If hypertension is uncontrolled it leads to development of diabetes and serious neurological complications. Most of the World populations live in temperate zone across the World. Thus, a possibility exists that these hypertensive and diabetic people may have external heat as potential risk factors for brain damage. We have seen brain edema and brain damage following exposure to heat stress at 38°C for 4h. A possibility exists that heat exposure in diabetic-hypertensive (DBHY) cases exacerbates exacerbation of brain pathology and edema formation. This hypothesis is examined in a rat model. The role of nitric oxide (NO) in exacerbation of HS-induced brain pathology was also evaluated using nitric oxide synthase (NOS) immunoreactivity. Hypertensive rats (produced by two-kidney one clip (2K1C) method) were made diabetic with streptozotocine (50mg/kg, i.p./day for 3days) treatment. After 6weeks, DBHY rats show 20-30mM/L Blood Glucose and hypertension (180-200mmHg). Subjection of these rats to 4h HS resulted in six- to eightfold higher BBB breakdown, brain edema formation and brain pathology. At this time, neuronal or inducible NOS expression was four- to sixfold higher in DBHY rats compared to controls. Interestingly, iNOS expression was higher than nNOS in DBHY rats. Cerebrolysin in high doses (10-mL/kg, i.v. instead of 5-mL/kg) induced significant neuroprotection and downregulation of nNOS and iNOS in DBHY animals whereas normal animals need only 5-mL/kg doses for this purpose. Our observations demonstrate that co-morbidly factors exacerbate brain damage in HS through NOS expression and require double dose of cerebrolysin for neuroprotection as compared to normal rats, not reported earlier.

Necrotizing enterocolitis attenuates developmental heart rate variability increases in newborn rats.

We have shown previously that a decreased high-frequency spectrum of heart rate variability (HF-HRV), indicative of reduced vagal tone, shows promise in predicting neonates likely to develop necrotizing enterocolitis (NEC) before its clinical onset. We hypothesized that NEC induction in rat pups decreases HF-HRV power; subdiaphragmatic vagotomy worsens the severity of the NEC phenotype, increases levels of pro-inflammatory cytokines, and alters the myenteric phenotype. Newborn Sprague-Dawley rats, representative of preterm human neonates, were subjected to 7-8days of brief periods of cold stress and hypoxia to induce NEC with or without unilateral subdiaphragmatic vagotomy. HRV was measured at postnatal days one and five, pups were sacrificed at day 8/9, and gastrointestinal tissues and blood were collected for immunohistochemical, corticosterone, and cytokine analysis. Compared to control, NEC-induced rats showed the following: (a) typical histological signs of grade 2 NEC, which were more severe in rats that underwent vagotomy; (b) reduced developmental increases in time (RMSSD) and frequency (HF) HRV spectra when combined with the stress of laparotomy/vagotomy; (c) increases in nitric oxide synthase-immunoreactivity in the myenteric plexus of jejunum and ileum; furthermore, compared to mild NEC and controls, vagotomized NEC rats had increased plasma values of pro-inflammatory cytokines IL-1β and IL-6. Our data suggest that in rodents, similar to neonatal observations, NEC induction attenuated developmental HF-HRV increases, furthermore, subdiaphragmatic vagotomy worsened the histological severity, increased pro-inflammatory cytokines, and altered the nitrergic myenteric phenotype, suggesting a role of the vagus in the development of NEC pathology.

Development of nNOS-positive preganglionic sympathetic neurons in the rat thoracic spinal cord

To gain a better understanding of the neuroplasticity of sympathetic neurons during postnatal ontogenesis, the distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity was studied in sympathetic preganglionic neurons (SPN) in the spinal cord (Th2 segment) of female Wistar rats at different ages (newborn, 10-, 20-, 30-day-old; 2-, 6-month-old; 3-year-old). In all age groups, the majority of nNOS-immunoreactive (IR) neurons was observed in the nucleus intermediolateralis thoracolumbalis pars principalis. In the first month, the proportion of nNOS-IR neurons decreased significantly from 92 ± 3.4% in newborn to 55 ± 4.6% in 1-month-old, while the number of choline acetyltransferase (ChAT)-IR neurons increased from 74 ± 4.2% to 99 ± 0.3% respectively. Decreasing nNOS expression in the first 10days of life was also confirmed by western blot analysis. Some nNOS-IR SPN also colocalized calbindin (CB) and cocaine and amphetamine-regulated transcript (CART). The percentage of NOS(+)/CB(-) SPN increased from 23 ± 3.6% in 10-day-old to 36 ± 4.2% in 2-month-old rats. Meanwhile, the proportion of NOS(+)/CART(-) neurons decreased from 82 ± 4.7% in newborn to 53 ± 6.1% in 1-month-old rats. The information provided here will also serve as a basis for future studies investigating the mechanisms of autonomic neuron development.

Changes in neuromuscular structure and functions of human colon during ageing are region-dependent

ObjectiveTo determine if human colonic neuromuscular functions decline with increasing age.DesignLooking for non-specific changes in neuromuscular function, a standard burst of electrical field stimulation (EFS) was used to evoke neuronally...

Self-protection of type III fibrocytes against severe 3-nitropropionic-acid-induced cochlear damage in mice.

After intense sound exposure, the lack of obvious degeneration in type III fibrocytes suggests that they might protect themselves against acoustic trauma. However, it is unknown whether and how type III fibrocytes play this role in other cochlear damage models. In this study, we investigated the self-protection of type III fibrocytes against severe cochlear energy failure induced by local administration of 3-nitropropionic acid to the inner ear. We detected that the type III fibrocytes did not degenerate significantly after 500 mM 3-nitropropionic acid application, and showed increased expression of proliferation marker Ki67. Moreover, low immunoreactivity for inducible nitric oxide synthase and cleaved caspase-3 was observed in type III fibrocytes 2 days after damage. These results indicate that after severe cochlear energy failure type III fibrocytes possess obvious proliferation activity, as well as strong antioxidant and antiapoptotic capacity, which can protect them from degeneration.

Distribution of nitric oxide synthase in the rock cavy (Kerodon rupestris) brain I: The diencephalon

Nitric oxide (NO) is a highly soluble and membrane-permeable neurotransmitter, so it does not need to be packed in vesicles or have a membrane receptor. In the nervous system, NO is synthesized by the neuronal form of the nitric oxide synthase (NOS) enzyme and has been considered as a local neurotransmitter. NOS distribution is widespread in the nervous system of various vertebrate species, which may explain its participation in many functions such as memory, blood pressure regulation and sexual behavior. Here we used immunohistochemistry against NOS and NADPH diaphorase histochemistry to map the distribution of NO in the diencephalon of the rock cavy (Kerodon rupestris), a rodent endemic to the Brazilian Northeast. Rock cavy has crepuscular habits and is adapted to ecological conditions such as heat and scarcity of water and food. This study found that NOS distribution was more concentrated in the hypothalamus of this animal. Among the hypothalamic nuclei, the median preoptic, supraoptic, paraventricular nucleus of the hypothalamus, ventromedial nucleus of the hypothalamus, ventral and dorsal premammillary nucleus, supramammillary nucleus, lateral mammillary nucleus and dorsal hypothalamic nucleus had the largest collections of NOS immunoreactive (NOS-ir) neurons. Some nuclei of the thalamus and epithalamus such as the paraventricular nucleus of the thalamus, the ventral lateral geniculate nucleus, the medial geniculate nucleus and the lateral habenula showed NOS-ir neurons. This distribution is similar to that described in other rodents, indicating that NO also has an important role in rock cavy's physiology.

Assessing the therapeutic effect of resveratrol in heart failure following blunt chest trauma and the potential role of endocan as a biomarker of inflammation using rats.

The present study investigated the therapeutic effect of resveratrol on cardiac injury resulting from blunt chest trauma and the utility of endocan as a biomarker of the inflammation process using rats. The rats were randomly divided into the following four groups (n=7 in each group): a control group (no treatment or trauma); trauma-induced group (trauma group); resveratrol group (resveratrol 0.3 mg/kg administered via the intraperitoneal [i.p.] route group); and resveratrol + trauma group (resveratrol 0.3 mg/kg administered via the i.p. route 1 hour prior to the induction of trauma). The immunoreactivity of tumor necrosis factor-α and inducible nitric oxide synthase in the trauma group was increased, whereas the reaction intensity in resveratrol + trauma group was deceased. The mean endocan values of the differed between the groups (p<0.001). The mean endocan value in the resveratrol + trauma group was higher than that of the other groups. Resveratrol exhibited anti-inflammatory and antioxidant effects in lung injury after blunt chest trauma and contributed favorably to the treatment process. We believe that there is a need for further studies on the clinical use of endocan as a biomarker of inflammation in cardiac injury after blunt chest trauma.

Effects of stem cells applications on oxidative stress and apoptosis during implantation

Objective: To investigate the effects of bone marrow derived mesenchymal stem cell (BMSC) application into the rat endometrium on oxidative stress, cell proliferation and apoptosis. Methods: The female rats selected in estrous cycle were divided into three groups (saline, media and BMSC group). The intrauterine and intraperitoneal injections were performed using the saline (200 μL), culture media (200 μL) and 1×106 BMSCs/200 μL culture media, and then they were mated with male rats. On the 7th day of the pregnancy, uterine samples were harvested and dyed with heamatoxylin-eosin histochemically, anti-endothelial nitric oxide synthase and anti-inducible nitric oxide synthase, and anti-proliferating cell nuclear antigen immunohistochemically, with terminal deoxynucleotidyl transferas dUTP nick end labeling for apoptosis. The stainings were evaluated by H-score and the results were analyzed using one-way ANOVA test statistically. Results: It was found that BMSCs increased the endometrial thickness, endometrial epithelium thickness and number of endometrial glands compared to control and sham groups. The intrauterine BMSC application decreased both anti-endothelial nitric oxide synthase and anti-inducible nitric oxide synthase immunoreactivities and the number of apoptotic cells compared to the intraperitoneal applications whereas the immunoreactivity of proliferating cell nuclear antigen was increased. Conclusions: In current study, we define that stem cells do not cause any structural damages. Also they change the distribution of oxidative stress and cell proliferation marker. These findings support the reliability of stem cells in clinical use in the case of infertility.

Mitoprotection attenuates myocardial vascular impairment in porcine metabolic syndrome.

Metabolic syndrome (MetS) leads to cardiac vascular injury, which may reflect in increased retention of endothelial progenitor cells (EPCs). Coronary endothelial cell (EC) mitochondria partly regulate vascular function and structure. We hypothesized that chronic mitoprotection would preserve EC mitochondria and attenuate coronary vascular injury and dysfunction in swine MetS. Pigs were studied after 16 wk of diet-induced MetS, MetS treated for the last 4 wk with the mitochondria-targeted peptide elamipretide (ELAM; 0.1 mg/kg sc once daily), and lean controls ( n = 6 each). Cardiac remodeling and function were assessed in vivo by multidetector-computed tomography (CT), and coronary artery and sinus blood samples were collected. EC mitochondrial density, apoptosis, oxidative stress, endothelial nitric oxide synthase immunoreactivity, myocardial microvascular density (three-dimensional microcomputed tomography), and coronary endothelial function (organ bath) were assessed ex vivo. The number and arteriovenous gradient of CD34+/KDR+ EPCs were calculated by FACS (a negative net gradient indicating EPC retention). MetS and MetS + ELAM pigs developed similar MetS (obesity, hyperlipidemia, insulin resistance, and hypertension). EC mitochondrial density decreased in MetS animals compared with lean animals but normalized in MetS + ELAM animals. ELAM also attenuated EC oxidative stress and apoptosis and improved subendocardial microvascular density. ELAM-induced vasculoprotection was reflected by decreased coronary retention of EPCs. ELAM also partly improved endothelial nitric oxide synthase immunoreactivity, coronary endothelial function, and vessel maturity, whereas myocardial perfusion was unaffected. Chronic mitoprotection improved coronary EC mitochondrial density and decreased vascular remodeling and dysfunction. However, additional mitochondria-independent mechanisms likely contribute to MetS-induced cardiac vascular injury. NEW & NOTEWORTHY The present study shows that chronic mitoprotection preserved coronary endothelial cell mitochondria and decreased vascular injury, subendocardial microvascular loss, coronary retention of endothelial progenitor cells, and release of markers of vascular injury. However, myocardial perfusion remained blunted, suggesting that additional mitochondria-independent mechanisms likely contribute to metabolic syndrome-induced cardiac vascular injury.

Comparison of Changes in the Interstitial Cells of Cajal and Neuronal Nitric Oxide Synthase-positive Neuronal Cells With Aging Between the Ascending and Descending Colon of F344 Rats.

Background/AimsNeuronal degeneration and changes in interstitial cells of Cajal (ICCs) are important mechanisms of age-related constipation. This study aims to compare the distribution of ICCs and neuronal nitric oxide synthase (nNOS) with regard to age-related changes between the ascending colon (AC) and descending colon (DC) in 6-, 31-, and 74-week old and 2-year old male Fischer-344 rats.MethodsThe amount of fecal pellet and the bead expulsion times were measured. Fat proportion in the muscle layer of the colon was analyzed by hematoxylin and eosin staining. Proto-oncogene receptor tyrosine kinase (KIT) and neuronal nitric oxide synthase (nNOS) expression were analyzed with Western blotting and immunohistochemistry. Isovolumetric contractile measurements and electrical field stimulation were used to assess smooth muscle contractility.ResultsColon transit and bead expulsion slowed with senescence. Fat in the muscle layer accumulated with age in the AC, but not in the DC. The proportion of KIT-immunoreactive ICCs in the submucosal and myenteric plexus was higher in the DC than in the AC, and it declined with age, especially in the AC. In contrast, the proportion of NOS-immunoreactive neurons in the myenteric plexus was higher in the AC than in the DC, and both decreased in older rats. Nitric oxide levels declined with age in the DC. Muscle strip experiments showed that the inhibitory response mediated by nitric oxide in the circular direction of the DC was reduced in 2-year old rats.ConclusionThe AC and DC differ in their distribution of ICCs and nNOS, and age-related loss of nitrergic neurons more severely affects the DC than the AC.

Synaptic activation of putative sensory neurons by hexamethonium-sensitive nerve pathways in mouse colon.

The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene-related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modeling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator fluo 4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5-10 mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300 μM) and by low-calcium Krebs solution. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways. NEW & NOTEWORTHY Myenteric neurons in isolated mouse colon were recorded using calcium imaging and then neurochemically defined. Short-latency calcium transients were detected in >90% of calcitonin gene-related peptide-immunoreactive neurons to electrical stimulation of hexamethonium-sensitive pathways. Putative sensory Dogiel type II calcitonin gene-related peptide-immunoreactive myenteric neurons may receive widespread fast synaptic inputs in mouse colon.

Comparative investigation of neuronal nitric oxide synthase immunoreactivity in rat and human claustrum

We compared the distribution, density and morphological characteristics of nitric oxide synthase-immunoreactive (NOS-ir) neurons in the rat and human claustrum. These neurons were categorized by diameter into three main types: large, medium and small. In the human claustrum, large neurons ranged from 26 to 40μm in diameter, medium neurons from 20 to 25μm and small neurons from 13 to 19μm. In the rat claustrum, large neurons ranged from 19 to 23μm in diameter, medium neurons from 15 to 18μm and small neurons from 10 to 14μm. The cell bodies of large and medium neurons varied broadly in shape – multipolar, elliptical, bipolar and irregular, consistent with a projection neuron phenotype. The small neurons were most seen as being oval or elliptical in shape, resembling an interneuron phenotype. Based on a quantitative comparison of their dendritic characteristics, the NOS-ir neurons of humans and rats displayed a statistically significant difference.

Oxidative Injury and Iron Redistribution Are Pathological Hallmarks of Marmoset Experimental Autoimmune Encephalomyelitis.

Oxidative damage and iron redistribution are associated with the pathogenesis and progression of multiple sclerosis (MS), but these aspects are not entirely replicated in rodent experimental autoimmune encephalomyelitis (EAE) models. Here, we report that oxidative burst and injury as well as redistribution of iron are hallmarks of the MS-like pathology in the EAE model in the common marmoset. Active lesions in the marmoset EAE brain display increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p22phox, p47phox, and gp91phox) and inducible nitric oxide synthase immunoreactivity within lesions with active inflammation and demyelination, coinciding with enhanced expression of mitochondrial heat-shock protein 70 and superoxide dismutase 1 and 2. The EAE lesion-associated liberation of iron (due to loss of iron-containing myelin) was associated with altered expression of the iron metabolic markers FtH1, lactoferrin, hephaestin, and ceruloplasmin. The enhanced expression of oxidative damage markers in inflammatory lesions indicates that the enhanced antioxidant enzyme expression could not counteract reactive oxygen and nitrogen species-induced cellular damage, as is also observed in MS brains. This study demonstrates that oxidative injury and aberrant iron distribution are prominent pathological hallmarks of marmoset EAE thus making this model suitable for therapeutic intervention studies aimed at reducing oxidative stress and associated iron dysmetabolism.

Neuronal nitric oxide synthase immunoreactivity in the respiratory tract of the frog, Rana temporaria

Neuronal nitric oxide synthase immunoreactivity in the respiratory tract of the frog, Rana temporaria