Effects of ascorbic acid supplementation in ileum myenteric neurons of streptozotocin-induced diabetic rats

The exacerbation of the oxidative stress and of the polyol pathway which impair damage myenteric plexus are metabolic characteristics of diabetes. The ascorbic acid (AA) is an antioxidant and an aldose reductase inhibitor, which may act as neuroprotector. The effects of AA supplementation on the density and cellular body profile area (CP) of myenteric neurons in STZ-induced diabetes in rats were assessed. Four groups with five animals each were formed: normoglycemic (C); diabetic (D); AA-treated diabetic (DS) and AA-treated normoglycemic (CS). Dosagen of 50mg of AA were given, three times a week, for each animal (group DS and CS). Ninety days later and after euthanasia, the ileum was collected and processed for the NADPH-diaphorase technique. There were no differences (P>0.05) in the neuronal density among the groups. The CP area was lower (P<0.05) in the DS and CS groups, with a higher incidence of neurons with a CP area exceeding 200µm² for groups C and D. The AA had no influence on the neuronal density in the ileum but had a neuroprotective effect, preventing the increase in the CP area and allowing a higher number of neurons with a CP area with less than 200µm².

TRAINING ALTERS CARDIAC NEURON SIZES IN WISTAR RATS

The action of the parasympathetic nerves on the heart is made through a group of neurons located on the surface of the atria. This study evaluated the effect of a chronic training protocol on the number and sizes of the cardiac neurons of Wistar rats. Whole mount preparations of the atria of 12-month old male sedentary and trained rats (40 weeks of running on a treadmill 3 times a week, 16 m/min) were assessed for number and size (maximal cellular profile area) of the cardiac neurons. The cardiac neurons were ascertained by using the NADH-diaphorase technique that stains the cell bodies of the neurons in dark blue. The, number of cardiac neurons in the trained rats (P>0.05) did not change significantly. In the sedentary group there were small, medium sized and large neurons. However there was a notable increase in the percentage of small neurons in the rats submitted to the training compared to the sedentary group (P<0.05). Previous studies have shown that electrophysiologically, the small neurons are more easily excitable than the large neurons. It is possible that the results of the present work reflect an adaptation mechanism of the cardiac neurons presumably with the objective of increasing the excitability of the neurons for the vagal action and resulting facilitation of the sinusal bradycardia observed at rest and in the exercise. We concluded that the training affects significantly the size of the cardiac neurons in Wistar rats. (Biol.Sport 26.245-254, 2009)

Central nitrergic system regulation of neuroendocrine secretion, fluid intake and blood pressure induced by angiotensin-II

Background: Nitric oxide (NO) synthesis has been described in several circumventricular and hypothalamic structures in the central nervous system that are implicated in mediating central angiotensin-II (ANG-II) actions during water deprivation and hypovolemia. Neuroendocrine and cardiovascular responses, drinking behavior, and urinary excretions were examined following central angiotensinergic stimulation in awake freely-moving rats pretreated with intracerebroventricular injections of N omega-nitro-L-arginine methyl ester (L-NAME, 40 mu g), an inhibitor of NO synthase, and L-arginine (20 ug), a precursor of NO. Results: Injections of L-NAME or ANG-II produced an increase in plasma vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) levels, an increase in water and sodium intake, mean arterial blood pressure and sodium excretion, and a reduction of urinary volume. L-NAME pretreatment enhanced the ANG-II response, while L-arginine attenuated VP and OT release, thirst, appetite for sodium, antidiuresis, and natriuresis, as well as pressor responses induced by ANG-II. Discussion and conclusion: Thus, the central nitrergic system participates in the angiotensinergic responses evoked by water deprivation and hypovolemia to refrain neurohypophysial secretion, hydromineral balance, and blood pressure homeostasis.

Effects of perinatal protein deprivation and recovery on esophageal myenteric plexus

AIM: To evaluate effects of pre- and postnatal protein deprivation and postnatal recovery on the myenteric plexus of the rat esophagus. METHODS: Three groups of young Wistar rats (aged 42 d) were studied: normal-fed (N42), protein-deprived (D42), and protein-recovered (R42). The myenteric neurons of their esophagi were evaluated by histochemical reactions for nicotinamide adenine dinucleotide (NADH), nitrergic neurons (NADPH)-diaphorase and acetylcholinesterase (AChE), immunohistochemical reaction for vasoactive intestinal polypeptide (VIP), and ultrastructural analysis by transmission electron microscopy. RESULTS: The cytoplasms of large and medium neurons from the N42 and R42 groups were intensely reactive for NADH. Only a few large neurons from the D42 group exhibited this aspect. NADPH detected in the D42 group exhibited low reactivity. The AChE reactivity was diffuse in neurons from the D42 and R42 groups. The density of large and small varicosities detected by immunohistochemical staining of VIP was low in ganglia from the D42 group. In many neurons from the D42 group, the double membrane of the nuclear envelope and the perinuclear cisterna were not detectable. NADH and NADPH histochemistry revealed no group differences in the profile of nerve cell perikarya (ranging from 200 to 400 mu m(2)). CONCLUSION: Protein deprivation causes a delay in neuronal maturation but postnatal recovery can almost completely restore the normal morphology of myenteric neurons. (C) 2010 Baishideng. All rights reserved.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Tolerance to the cataleptic effect that follows repeated nitric oxide synthase inhibition may be related to functional enzymatic recovery

Systemic or intra-striatal acute administration of nitric oxide synthase (NOS) inhibitors causes catalepsy in rodents. This effect disappears after sub-chronic treatment. The aim of the present study was to investigate if this tolerance is related to changes in the expression of NOS or dopamine-2 (D(2)) receptor or to a recovery of NOS activity. Male albino Swiss mice (25-30 g) received single or sub-chronic (once a day for 4 days) i.p. injections of saline or L-nitro-arginine (L-NOARG, 40 mg/kg), a non-selective inhibitor of neuronal nitric oxide synthase (nNOS). Twenty-four hours after the last injection, the animals were killed and their brains were removed for immunohistochemistry assay to detect the presence of nNOS or for `in-situ` hybridisation study using (35)S-labeled oligonucleotide probe complementary to D(2) receptor mRNA. The results were analysed by computerised densitometry. Independent groups of animals received the same treatment, but were submitted to the catalepsy test and had their brain removed to measure nitrite and nitrate (NOx) concentrations in the striatum. Acute administration of L-NOARG caused catalepsy that disappeared after sub-chronic treatment. The levels of NOx were significantly reduced after acute L-NOARG treatment. The decrease in NOx after drug injection suffered a partial tolerance after sub-chronic treatment. The catalepsy time after acute or sub-chronic treatment with L-NOARG was negatively (r = -0.717) correlated with NOx levels. Animals that received repeated L-NOARG injections also showed an increase in the number of nNOS-positive neurons in the striatum. No change in D(2) receptor mRNA expression was found in the dorsal striatum, nucleus accumbens and substantia nigra. Together, these results suggest that tolerance to L-NOARG cataleptic effects do not depend on changes in D(2) receptors. They may depend, however, on plastic changes in nNOS neurons resulting in partial recovery of NO formation in the striatum.

A nitric oxide synthase inhibitor decreases 6-hydroxydopamine effects on tyrosine hydroxylase and neuronal nitric oxide synthase in the rat nigrostriatal pathway

There is evidence that nitric oxide plays a role in the neurotransmitter balance within the basal ganglia and in the pathology of Parkinson`s disease. In the present work we investigated in striatal 6-hydroxydopamine (6-OHDA) lesioned rats the effects of a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NOARG), given systemically on both the dopaminergic (DA) neuronal loss and the neuronal NOS cell density. We analyzed the DA neuronal loss through tyrosine hydroxylase immunohistochemistry (TH). The nitrergic system was evaluated using an antibody against the neuronal NOS (nNOS) isoform. Treatment with the L-NOARG significantly reduced 6-OHDA-induced dopaminergic damage in the dorsal striatum, ventral substantia nigra and lateral globus pallidus, but had no effects in the dorsal substantia nigra and in the cingulate cortex. Furthermore, L-NOARG reduced 6-OHDA-induced striatal increase, and substantia nigra compacta decrease, in the density of neuronal nitric oxide synthase positive cells. These results suggest that nitric oxide synthase inhibition may decrease the toxic effects of 6-OHDA on dopaminergic terminals and on dopamine cell bodies in sub-regions of the SN and on neuronal nitric oxide synthase cell density in the rat brain. (c) 2008 Elsevier B.V. All rights reserved.

Marine biotoxins in the Catalan littoral: could biosensors be integrated into monitoring programmes?

Aquest article descriu els sensors enzimàtics i immunosensors electroquímics que s’han desenvolupat als nostres grups per a la detecció de la biotoxina marina àcid okadaic (OA), i discuteix la possibilitat d’integrar-los en programes de seguiment. Els sensors enzimàtics per a OA que es presenten es basen en la inhibició de la proteïna fosfatasa (PP2A) per aquesta toxina i la mesura electroquímica de l’activitat enzimàtica mitjançant l’ús de substrats enzimàtics apropiats, electroquímicament actius després de la seva desfosforació per l’enzim. Els immunosensors electroquímics descrits en aquest article es basen en un enzimoimmunoassaig sobre fase sòlida competitiu indirecte (ciELISA), amb fosfatasa alcalina (ALP) o peroxidasa (HRP) com a marcatges, i un sistema de reciclatge enzimàtic amb diaforasa (DI). Els biosensors presentats aquí s’han aplicat a l’anàlisi de dinoflagel·lats, musclos i ostres. Les validacions preliminars amb assaigs colorimètrics i LC-MS/MS han demostrat la possibilitat d’utilitzar les bioeines desenvolupades per al cribratge preliminar de biotoxines marines en mostres de camp o de cultiu, que ofereixen informació complementària a la cromatografia. En conclusió, tot i que encara cal optimitzar alguns paràmetres experimentals, la integració dels biosensors a programes de seguiment és viable i podria proporcionar avantatges respecte a altres tècniques analítiques pel que fa al temps d’anàlisi, la simplicitat, la selectivitat, la sensibilitat, el fet de poder ser d’un sol ús i l’efectivitat de cost. This article describes the electrochemical enzyme sensors and immunosensors that have been developed by our groups for the detection of marine biotoxin okadaic acid (OA), and discusses the possibility of integrating them into monitoring programmes. The enzyme sensors for OA reported herein are based on the inhibition of immobilised protein phosphatase 2A (PP2A) by this toxin and the electrochemical measurement of the enzyme activity through the use of appropriate enzyme substrates, which are electrochemically active after dephosphorylation by the enzyme. The electrochemical immunosensors described in this article are based on a competitive indirect Enzyme- Linked ImmunoSorbent Assay (ciELISA), using alkaline phosphatase (ALP) or horseradish peroxidase (HRP) as labels, and an enzymatic recycling system with diaphorase (DI). The biosensors presented herein have been applied to the analysis of dinoflagellates, mussels and oysters. Preliminary validations with colorimetric assays and LC-MS/MS have demonstrated the possibility of using the developed biotools for the preliminary screening of marine biotoxins in field or cultured samples, offering complementary information to chromatography. In conclusion, although optimisation of some experimental parameters is still required, the integration of biosensors into monitoring programmes is viable and may provide advantages over other analytical techniques in terms of analysis time, simplicity, selectivity, sensitivity, disposability of electrodes and cost effectiveness.

The number and profile of reactive NADH-d and NADPH-d neurons of myenteric plexus of six-month-old rats are different in the cecum portions

Whole-mount preparations were prepared and submitted to NADH-diaphorase and NADPH-diaphorase histochemistry techniques. The myenteric plexus arrangement and the number of neurons were comparatively evaluated among the different portions of the cecum. The neurons from the apical and basal regions were distributed in classes at intervals of 100µm², the means of the corresponding intervals being compared. The ganglia, in both techniques, were often connected by fine bundles, which became thicker in the mesenteric region and in the region next to the cecal ampulla. The number of positive NADH-d neurons was higher than that of NADPH-d neurons in all portions, from both regions. The numbers of reactive NADH-d e NADPH-d neurons were significantly different among the different portions of the cecum, except for the antimesenteric basal and intermediate basal regions, considering the NADH-d neurons. The profile area for the reactive NADH-d e NADPH-d neurons was higher in the apical region than in the basal area. Differences in arrangement, distribution and size of positive NADH-d e NADPH-d neurons in the different cecum portions evidenced the importance of the subdivision of the analyzed organ.

Effects of ascorbic acid supplementation in ileum myenteric neurons of streptozotocin-induced diabetic rats

The exacerbation of the oxidative stress and of the polyol pathway which impair damage myenteric plexus are metabolic characteristics of diabetes. The ascorbic acid (AA) is an antioxidant and an aldose reductase inhibitor, which may act as neuroprotector. The effects of AA supplementation on the density and cellular body profile area (CP) of myenteric neurons in STZ-induced diabetes in rats were assessed. Four groups with five animals each were formed: normoglycemic (C); diabetic (D); AA-treated diabetic (DS) and AA-treated normoglycemic (CS). Dosagen of 50mg of AA were given, three times a week, for each animal (group DS and CS). Ninety days later and after euthanasia, the ileum was collected and processed for the NADPH-diaphorase technique. There were no differences (P>0.05) in the neuronal density among the groups. The CP area was lower (P<0.05) in the DS and CS groups, with a higher incidence of neurons with a CP area exceeding 200µm² for groups C and D. The AA had no influence on the neuronal density in the ileum but had a neuroprotective effect, preventing the increase in the CP area and allowing a higher number of neurons with a CP area with less than 200µm².

Horizontal projections of area 17 in Cebus monkeys: metric features, and modular and laminar distribution

Metric features and modular and laminar distributions of intrinsic projections of area 17 were studied in Cebus apella. Anterogradely and retrogradely labeled cell appendages were obtained using both saturated pellets and iontophoretic injections of biocytin into the operculum. Laminar and modular distributions of the labeled processes were analyzed using Nissl counterstaining, and/or cytochrome oxidase and/or NADPH-diaphorase histochemistry. We distinguished three labeled cell types: pyramidal, star pyramidal and stellate cells located in supragranular cortical layers (principally in layers IIIa, IIIb a, IIIb ß and IIIc). Three distinct axon terminal morphologies were found, i.e., Ia, Ib and II located in granular and supragranular layers. Both complete and partial segregation of group I axon terminals relative to the limits of the blobs of V1 were found. The results are compatible with recent evidence of incomplete segregation of visual information flow in V1 of Old and New World primates

Effect of the injection of an extract of Ascaris suum on macrophage activation during the early phase of Mycobacterium bovis BCG infection in C57Bl/6 mice

Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-g-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7%, BCG + Asc = 13%, and Asc = 4%; 7 days: control = 4, BCG = 13%, BCG + Asc = 21%, and Asc = 4.5%) and TNF-a levels (mean ± SD; 2 days: control = 0, BCG = 169 ± 13, BCG + Asc = 202 ± 37, and Asc = 0; 7 days: control = 0, BCG = 545 ± 15.5, BCG + Asc = 2206 ± 160.6, and Asc = 126 ± 26; 14 days: control = 10 ± 1.45, BCG = 9 ± 1.15, BCG + Asc = 126 ± 18, and Asc = 880 ± 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean ± SD; 2 days: control = 0, BCG = 3.7 ± 1.59, BCG + Asc = 0.82 ± 0.005, Asc = 0.48 ± 0.33; 7 days: control = 0, BCG = 2.78 ± 1.54, BCG + Asc = 3.07 ± 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 ± 0.53, BCG + Asc = 9.61 ± 0.81, Asc = 10.5 ± 0.2 (2 x 106) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean ± SD; 2 days: BCG = 1.13 ± 0.07 and BCG + Asc = 0.798 ± 0.305; 7 days: BCG = 1.375 ± 0.194 and BCG + Asc = 0.548 ± 0.0226; 14 days: BCG = 0.473 ± 0.184 and BCG + Asc = 0.675 ± 0.065 (x 102) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection.

Expression of neuronal nitric oxide synthase in the developing superficial layers of the rat superior colliculus

We investigated the level of expression of neuronal nitric oxide synthase (nNOS) in the retinorecipient layers of the rat superior colliculus during early postnatal development. Male and female Lister rats ranging in age between the day of birth (P0) and the fourth postnatal week were used in the present study. Two biochemical methods were used, i.e., in vitro measurement of NOS specific activity by the conversion of [³H]-arginine to [³H]-citrulline, and analysis of Western blotting immunoreactive bands from superior colliculus homogenates. As revealed by Western blotting, very weak immunoreactive bands were observed as early as P0-2, and their intensity increased progressively at least until P21. The analysis of specific activity of NOS showed similar results. There was a progressive increase in enzymatic activity until near the end of the second postnatal week, and a nonsignificant tendency to an increase until the end of the third week was also observed. Thus, these results indicated an increase in the amount of nNOS during the first weeks after birth. Our results confirm and extend previous reports using histochemistry for NADPH-diaphorase and immunocytochemistry for nNOS, which showed a progressive increase in the number of stained cells in the superficial layers during the first two postnatal weeks, reaching an adult pattern at the end of the third week. Furthermore, our results suggested that nNOS is present in an active form in the rat superior colliculus during the period of refinement of the retinocollicular pathway.

Diversity among satellite glial cells in dorsal root ganglia of the rat

Peripheral glial cells consist of satellite, enteric glial, and Schwann cells. In dorsal root ganglia, besides pseudo-unipolar neurons, myelinated and nonmyelinated fibers, macrophages, and fibroblasts, satellite cells also constitute the resident components. Information on satellite cells is not abundant; however, they appear to provide mechanical and metabolic support for neurons by forming an envelope surrounding their cell bodies. Although there is a heterogeneous population of neurons in the dorsal root ganglia, satellite cells have been described to be a homogeneous group of perineuronal cells. Our objective was to characterize the ultrastructure, immunohistochemistry, and histochemistry of the satellite cells of the dorsal root ganglia of 17 adult 3-4-month-old Wistar rats of both genders. Ultrastructurally, the nuclei of some satellite cells are heterochromatic, whereas others are euchromatic, which may result from different amounts of nuclear activity. We observed positive immunoreactivity for S-100 and vimentin in the cytoplasm of satellite cells. The intensity of S-100 protein varied according to the size of the enveloped neuron. We also noted that vimentin expression assumed a ring-like pattern and was preferentially located in the cytoplasm around the areas stained for S-100. In addition, we observed nitric oxide synthase-positive small-sized neurons and negative large-sized neurons equal to that described in the literature. Satellite cells were also positive for NADPH-diaphorase, particularly those associated with small-sized neurons. We conclude that all satellite cells are not identical as previously thought because they have different patterns of glial marker expression and these differences may be correlated with the size and function of the neuron they envelope.

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% 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% when compared to the contralateral SC of the same animals and by 64% 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.