Uveitis Patterns and Severity: An Epidemiologic Study from a Tertiary Care Private Referral Center in Buenos Aires, Argentina.

PURPOSE: To report uveitis' spectrum in a private practice cohort in the city of Buenos Aires, Argentina. METHODS: Retrospective review at Instituto de la Visión (November 2011-October 2015). Standard demographics, ethnicity and Native American aboriginal ancestry were recorded. RESULTS: Among 212 patients, median age 45 (6-97), 10% pediatric, 35% bilateral, 72% non-idiopathic, 36% infectious. Anterior uveitis presented in 50%, followed by posterior (32%), intermediate (9%) and panuveitis (8%). Frequent visits (≥ 6 per year) needed by 29%: posterior, non-idiopathic disease with 79% systemic immunosuppression requirement was their main presentation. Native American aboriginal ancestry was reported by 22.64% of the whole cohort and 37% of frequent visits' subgroup. CONCLUSIONS: Unilateral, non-idiopathic, non-infectious anterior uveitis was the most frequent presentation, in agreement with reports coming from western developed cities. The multi-racial Argentinian population with specific Native American aboriginal ancestry might contribute to certain forms of posterior uveitis and their response to treatment.

Development of the endothelium.

Our understanding of the regulation of vascular development has exploded over the past decade. Prior to this time, our knowledge of vascular development was primarily based on classic descriptive studies. The identification of stem cells, lineage markers, specific growth factors and their receptors, and signalling pathways has facilitated a rapid expansion in information regarding details of the mechanisms that govern development of the vascular system.

Interleukin-1beta regulates nitric oxide production and gamma-glutamyl transpeptidase activity in sertoli cells.

Several cytokines have been involved in the regulation of Sertoli cell function. Further investigations are required to elucidate the role of interleukin-1beta (IL1beta) in Sertoli cell physiology. Twenty-day-old rat Sertoli cell cultures were used to investigate a possible role of IL1beta in the regulation of gamma-glutamyl transpeptidase (gammaGTP) and to elucidate the signaling pathway utilized by this cytokine. GammaGTP is a membrane-bound enzyme that has been involved in amino acid transport across the plasma membrane and in protection from oxidative stress through its importance in the regulation of glutathione levels. Previous studies suggested that IL1beta stimulates NO biosynthesis in other cell types. Therefore, we investigated whether IL1beta modified the level of nitrite, a stable metabolite of NO, in Sertoli cells. Dose-response curves to IL1beta for gammaGTP activity and nitrite production were observed. The increments observed in gammaGTP activity and nitrite production were partially and completely blocked by simultaneous treatment with the NO synthase inhibitor aminoguanidine. Treatment of Sertoli cell cultures with the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine resulted in an increase in gammaGTP activity. The presence of neural, endothelial, and inducible isoforms of NO synthase (NOS) was investigated by a immunohistochemical technique using specific antibodies. The 2 constitutive isoforms were present under basal conditions, and the inducible protein appeared in IL1beta-treated cultures. Finally, translocation of NF-kappaB p65 subunit to the nucleus in IL1beta-treated cultures was observed. These findings suggest that the action of IL1beta on Sertoli cell gammaGTP activity is partially mediated via activation of NF-kappaB and increments in iNOS and cellular production of NO.

Lipopolysaccharide directly affects pancreatic acinar cells: implications on acute pancreatitis pathophysiology.

We have explored whether lipopolysaccharide (LPS, endotoxin) induces pancreatic injury on pancreatic acinar cells both in vivo and in vitro. Wistar male rats were treated with four intraperitoneal injections of 10 mg/kg LPS, and AR4-2J cells were exposed to increasing doses of LPS. Expression of pancreatitis-associated-protein (PAP) mRNA was strongly induced in AR4-2J cells exposed to LPS, while amylase mRNA was reduced. LPS also induced apoptosis and expression of TNF-alpha, IL-1beta, and IL-8 mRNA in AR4-2J cells. The in vivo effect of LPS showed structural signs of cellular damage, including numerous cytoplasmic vacuoles, severe nuclear alterations, and high expression of PAP mRNA. This study demonstrated that LPS induced pancreatic damage by directly affecting the pancreatic acinar cells. The role of LPS in the pathophysiology of acute pancreatitis may be partly due to the effect LPS has on the acinar cell.

Control of salivary secretion by nitric oxide and its role in neuroimmunomodulation.

In many in vivo systems exposure to endotoxins (LPS) leads to the co-induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which is important to the regulation of the function of different systems during infection. In submandibular glands (SMG) neural (n)NOS is localized in neural terminals and in striated, granular convoluted and excretory ducts, endothelial (e)NOS in vascular endothelium and ducts, and iNOS in macrophages and in tubules and ducts. In normal adult male rats, injection of an inhibitor of NOS decreased the stimulated salivary secretion and a donor of NO potentiated it, indicating that NO exerts a stimulatory role. A single high dose of LPS (5 mg/kg, i.p.) induced an increase in NOS activity measured by the 14C-citrulline method, increased PGE content almost 100% as measured by RIA, and blocked stimulated salivary secretion. The administration of a specific iNOS inhibitor, aminoguanidine (AG), with LPS not only decreased NOS activity but significantly decreased PGE content, indicating that NO triggered the activation of COX-2. LPS increased conversion of labeled arachidonate to prostaglandins (PGs) showing that COX was induced. Since a PGE1 analogue blocked stimulated salivation, the LPS-induced inhibition of salivation is probably due to release of PGs. Therefore, the use of inhibitors of iNOS and COX-2 could be very useful to increase salivation during infection since saliva has antimicrobial actions.

Structural specializations of the eye in the vizcacha (Lagostomus maximus maximus).

Vizcachas (Lagostomus maximus maximus, Chinchillidae) are nocturnal rodents living in burrows in many regions of Argentina, Bolivia, and Chile. We have studied the eye of the vizcacha using several light and electron microscopic procedures, with the purpose of understanding the role of vision in the behavior of this species. Our observations demonstrated an avascular, rod-rich retina, with a specialized region spanning through most of the equator of the eye. In this central band, all neural retinal layers exhibited a high cell density, whereas the photoreceptor layer was characterized by the presence of very long rods. In addition, the central region was associated with a distinct pigmentation pattern, including scarce granulation of the pigment epithelium, low pigmentation of the choroid, and the selective attachment of suprachoroidal cells to the inner scleral surface. These central modifications probably form the structural basis of a reflecting tapetum. The eye of the vizcacha received both long and short ciliary vessels, and a specialized cilio-sclero-choroidal vascular network appeared at the equatorial region. Our findings suggest that the equatorial region of the eye of the vizcacha could be a highly sensitive light detector related to foraging behaviors during crepuscular or nocturnal hours.

Differential effects of superior and inferior spermatic nerves on testosterone secretion and spermatic blood flow in cats.

It has been postulated that testosterone secretion is partially regulated by signals from the spermatic nerves. To further examine this hypothesis in vivo, the superior (SSN) or the inferior (ISN) spermatic nerves were stimulated electrically (varying intensity, 25 Hz, 0.2 msec, 10 min) in anesthetized cats, determining the testosterone concentration and the blood flow in the spermatic vein. In some additional experiments arterial blood was sampled, and norepinephrine (NE) output was calculated. Stimulation of the SSN (25-35 V) increased the testosterone concentration in spermatic vein blood (P < 0.01 compared with prestimulation levels). The response varied among animals, reaching a 50-100% increase in some animals, whereas in others it ranged from almost undetectable to more than 10 ng/100 g x min. Under the same experimental conditions, the NE output increased from 135.4 +/- 99 to 1614.2 +/- 347 pg/ml (P < 0.01), and spermatic blood flow decreased from 24.1 +/- 1.42 to 20.2 +/- 1.65 ml/min x 100 g (P < 0.05) during nerve stimulation. By contrast, stimulation of the ISN (25-35 V) modified neither the testosterone concentration, the NE output, nor the blood flow in the spermatic vein. High intensity stimulation (36-70 V) of each spermatic nerve evoked different vascular and hormonal effects. SSN activation induced a marked decrease in spermatic blood flow during stimulation and an increase in the testosterone response, whereas ISN activation resulted only in an enhanced spermatic blood flow. Our results suggest that testosterone secretion, although mainly dependent on gonadotropin secretion, could be further regulated by neural inputs from the SSN acting directly or alternatively through changes in blood flow. It would appear that the SSN mainly supplies the vasoconstrictor fibers to the testis, whereas the ISN provides vasodilator fibers.

Innervation of blood vessels in the vomeronasal complex of the rat.

We have made an immunohistochemical study of the vomeronasal (VN) complex of 12-day-old rats to characterize the innervation of its blood vessels. The VN complex can be subdivided into rostral, middle and caudal segments, each one with a particular vascularization pattern. Several small vessels were associated with the rostral segment, whereas a large venous sinus ran along the middle and caudal segments. Immunostaining for alpha-smooth muscle actin demonstrated that the muscular sheath was asymmetric, with more cells layers in its lateral than in its medial walls. Nerves were demonstrated with antisera against protein gene product 9.5 (PGP), and against several molecules associated with specific classes of nerve fibers: the C-terminal peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (NOS). The latter, was also studied with NADPH-diaphorase. Vascular associated fibers exhibited NOS-, CPON-, GAL-, CGRP-, SP- and VIP-immunoreactivity. Only the vessels of the rostral segment showed VIP-immunoreactive fibers. Each wall of the venous sinus exhibited different types of nerve fibers. CPON-, GAL-, CGRP- and SP-immunoreactive fibers concentrated in the medial wall, whereas NOS-immunoreactive ones concentrated in the lateral wall. This distribution of vascular fibers, plus the presence of sensory fibers exhibiting CGRP-, SP- and GAL-immunoreactivity within the pseudostratified epithelium of the VN tube, would be relevant to understand the operation of the pumping mechanism regulating influx and efflux from the VN tube.

Role of nitric oxide in salivary secretion.

Since nitric oxide has been found to control the function of many organs of the body by the non-adrenergic, non-cholinergic branch of the autonomic nervous system, we hypothesized that it might play a role in salivary secretion. Therefore, we investigated the distribution of nitric oxide synthase (NOS) throughout the submaxillary gland and also studied the ability of inhibitors of NOS to interfere with salivation induced by a cholinergic agonist, metacholine, and by a polypeptide, substance P. The secretory responses were determined in rats anesthetized with chlorolose following intravenous injection of the various pharmacological agents. There was no basal flow of saliva and dose-response curves were obtained by sequential intravenous injection of increasing doses of the drugs. Then, in the same animal, the same dose-response curves were performed in the presence of NOS inhibitors. L-Nitro-arginine-methyl-ester (L-NAME; 20 mg/kg) produced an over 50% inhibition of the dose-related salivation induced by metacholine. Similar results were produced with L-NG-monomethyl-L-arginine (L-NMMA; 5 mg/kg). The salivation induced by much lower molar doses of substance P was dramatically greater than that obtained with metacholine. The response to substance P was almost completely inhibited by L-NMMA at the lowest dose (0.3 mg/kg), but at higher doses (1 mg/kg), the inhibition was only around 60% and at the highest dose (3 mg/kg) only about 20%. In control rats, there were roughly equal amounts of calcium-dependent and calcium-independent NOS in the gland at this time. At the end of the experiment, the effect of the inhibitor of NOS, L-NMMA, on the NOS activity in the submandibular gland was determined. At this time, the Ca2+-dependent NOS was decreased and the Ca2+-independent NO was increased. The prior injection of L-NMMA reduced calcium-dependent NOS activity by approximately 70% but calcium-independent activity by only 30%. These results indicate that, at least at the end of the experiment, the blockade of NOS imposed by NMMA was incomplete. This could account in part for the failure of the inhibitors to block completely the stimulatory effect of the two secretagogues. Analysis of the distribution of NOS in the salivary gland revealed that it was not present in the acinar cells, but in neural terminals within the gland and also in the ductile system which contained neural (n) NOS in the apical membrane of the excretory and striated ducts, the cytoplasm of granular convoluted tubules and, to a lesser extent, in the cytoplasm of excretory and striated ducts. Macrophage (inducible) NOS was also found not only in the macrophages, but also in the tubules and ducts. Since drugs were used that would act on the receptors in the gland, the role of NO in our conditions is probably mediated by nNOS and iNOS in the ductile and tubular structures. Since iNOS would already be active, it is unlikely to play a role in this acute secretory activity. Rather the nNOS in these non-neural cells is probably activated by muscarinic or K1 receptors by metacholine and substance P, respectively, leading to an increase in intracellular free calcium that activates NOS leading to the generation of cGMP that opens ion channels to initiate the secretory process.

Innervation of nasal turbinate blood vessels in rhinitic and nonrhinitic children.

An immunohistochemical study of the nasal mucosa was done in pediatric patients attending an otorhinolaringology (ORL) clinic. The goal was a comparison between vascular innervation in patients with or without symptoms of chronic rhinitis. All patients had an indication for tonsillectomy prior to their inclusion in this study. Samples were obtained under general anesthesia at the time of programmed surgery and fixed in a paraformaldehyde-picric acid mixture. Cryostat sections were immunostained for the following neuronal markers: protein-gene product 9.5 (PGP), calcitonin gene- related peptide (CGRP), substance P (SP), and C-terminal peptide of neuropeptide Y (CPON). The following classes of vessels were identified: arteries, sinusoids, veins, and arteriovenous anastomoses (AVAs). As shown by immunostaining with the general neuronal marker PGP, each vessel type had a characteristic innervation pattern, differing in the amount of fibers and their distribution within the adventitial and muscle layers. Evaluation of PGP, CPON, and CGRP immunoreactivity patterns indicated that rhinitic arteries and AVAs displayed a richer innervation than did nonrhinitic blood vessels. Quantification of vascular PGP immunostaining confirmed the difference of vascular innervation between nonrhinitic and rhinitic patients. Fibers immunostained by CPON partially accounted for the rhinitic arterial hyperinnervation.

Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP.

Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release.

Monoaminergic and peptidergic contributions of the superior and the inferior spermatic nerves to the innervation of the testis in the rat.

Sections of the rat testis and whole-mounts of the testicular capsule were studied microscopically using the glyoxylic acid-induced fluorescence method, to detect monoamines, and immunohistochemical procedures for the detection of immunoreactivities to protein gene-product 9.5 (PGP 9.5), the C-terminal accompanying peptide of neuropeptide Y (CPON), and vasoactive intestinal polypeptide (VIP). Monoaminergic nerves were only observed around the intracapsular blood vessels: the initial segment of the testicular artery and the superior venous plexus, and in the anterior aspect of the upper and lower testicular poles. These capsular nerve networks were associated with the superior and inferior ligaments of the testis. Nerves displaying PGP 9.5 and CPON immunoreactivity appeared in the same sites and followed the same distribution as monoaminergic nerves. By contrast, VIP-immunoreactive fibers were only found in the nerve network of the lower pole. Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole. On the other hand, fibers from the inferior spermatic nerve were restricted to the nerve network of the lower pole.

Distribution of neuronal and non-neuronal NADPH diaphorases and nitric oxide synthases in rat uterine horns under different hormonal conditions.

Since pharmacological evidence indicates that nitric oxide (NO) operates in the control of uterine motility, we have studied the distribution of NADPH diaphorase and NO synthases in the rat uterus using histochemical and immunohistochemical methods. Numerous nerve fibers displayed NADPH diaphorase activity and immunoreactivity to antisera raised against neuronal NO synthase. Nerve fibers appeared in all stages of the estrous cycle and also after ovariectomy. NADPH diaphorase activity was also present in endothelia and cells dispersed in the different uterine layers. Most NADPH diaphorase-positive (ND) cells had eosinophilic granules with occasional cells expressing the ED1 macrophage-monocyte marker. Immunoreactivity for an inducible NO synthase was found in a small number of macrophage-like cells without NADPH diaphorase activity. Thus, ND cells may express another NO synthase isoform not detected by the available antisera. In normal cycling rats, ND cells were most abundant during proestrus, and their number further increased after estrogen treatment. ND cells were not observed after ovariectomy but were present after estrogen replacement therapy. ND cells could be involved in the estrogenic control of in vivo and in vitro uterine.

Role of nitric oxide in eicosanoid synthesis and uterine motility in estrogen-treated rat uteri.

Cholinergic stimulation of vascular endothelin activates NO synthase (NOS), leading to generation of NO from arginine. This NO diffuses to the overlying vascular smooth muscle and causes vasodilatation. NOS has also been found in the central and peripheral nervous systems and it is clear now that NO plays an important role as a neurotransmitter. Here we investigate the role of NO in controlling contraction of uterine smooth muscle. Our previous work showed that NO activates the cyclooxygenase enzyme in the hypothalamus, leading to production of prostaglandin E2 (PGE2). We began by determining whether NO was involved in production of arachidonic acid metabolites in the uterus. Uteri were removed from female rats that had been treated with estrogen (17 beta-estradiol). Control animals were similarly injected with diluent. Tissues were incubated in vitro in the presence of [14C]arachidonic acid for 60 min. Synthesis of PGs and thromboxane B2 (TXB2) was markedly stimulated by sodium nitroprusside (NP), the releaser of NO. The effect was greatest on TXB2; there were no significant differences in increases of different PGs. The response to NP was completely prevented by Hb, a scavenger of NO. The inhibitor of NOS, NG-monomethyl-L-arginine (NMMA), significantly decreased synthesis of PGE2 but not the other prostanoids (6-keto-PGF1 alpha and PGF2 alpha). Addition of Hb to scavenge the spontaneously released NO inhibited synthesis of 6-keto-PGF1 alpha, PGE2, and PGF2 alpha, but not TXB2. There was a much lesser effect on products of lipoxygenase, such that only 5-hydroxy-5,8,11,14-eicosatetraenoic acid (5-HETE) synthesis was increased by NP, an effect that was blocked by Hb; there was no effect of NMMA or Hb on basal production of 5-HETE. Thus, NO stimulates release of the various prostanoids and 5-HETE; blockade of NOS blocked only PGE2 release, whereas Hb to scavenge the NO released also blocked synthesis of 6-keto-PFG1 alpha, PGE2, and PGF2 alpha, indicating that basal NO release is involved in synthesis of all these PGs, especially PGE2. Presumably, NMMA did not block NOS completely, whereas Hb completely removed released NO. This may explain the different responses of the various prostanoids to NMMA and Hb. To determine the role of these prostanoids and NO in control of spontaneous in vitro uterine contractility in the estrogen-treated uterus, the effect of blocking NOS with NMMA and of scavenging NO produced by Hb on the time course of spontaneous uterine contractility was studied. Surprisingly, blockade of NOS or removal of NO by Hb prevented the spontaneous decline in uterine motility that occurs over 40 min of incubation. We interpret this to mean that NO was released in the preparation and activated guanylate cyclase in the smooth muscle, resulting in production of cGMP, which reduces motility and induces relaxation. When the motility had declined to minimal levels, the effect of increased NO provided by NP was evaluated; apparently by stimulating the release of prostanoids, a rapid increase in motility that persisted for 10 min was produced. This effect was completely blocked by Hb. The action of NO was also blocked by indomethacin, indicating that it was acting via release of PGs. Apparently, when motility is low, activation of PG synthesis by NO to activate the cyclooxygenase enzyme causes a rapid induction of contraction, whereas, when motility is declining, NO acts primarily via guanylate cyclase to activate cGMP release; the action of the prostanoids released at this time is in some manner blocked.

Distribution of the inositol 1,4,5-trisphosphate receptor, P400, in adult rat brain.

The distribution of the inositol 1,4,5-trisphosphate receptor protein, P400, was investigated in adult rat brain by immunocytochemistry with the monoclonal antibody 4C11 raised against mouse cerebellar inositol 1,4,5-trisphosphate receptor protein. Immunoreactive neuronal cell bodies were detected in the cerebral cortex, the claustrum, the endopiriform nucleus, the corpus callosum, the anterior olfactory nuclei, the olfactory tubercle, the nucleus accumbens, the lateral septum, the bed nucleus of the stria terminalis, the hippocampal formation, the dentate gyrus, the caudate-putamen, the fundus striatum, the amygdaloid complex, the thalamus, the caudolateral part of the hypothalamus, the supramammillary nuclei, the substantia nigra, the pedunculopontine tegmental nucleus, the ventrotegmental area, the Purkinje cells in the cerebellum, the dorsal cochlear nucleus, the subnucleus oralis and caudalis of trigeminal nerve, and the dorsal horn of the spinal cord. Immunoreactive fibres were found in the medial forebrain bundle, the globus pallidus, the stria terminalis, the pyramidal tract, the spinal tract of trigeminal nerve, and the ventral horn of spinal cord. Nerve fibres forming a dense plexus ending in terminal-like boutons were detected in relation to nonimmunoreactive neurons of the dentate, interpositus, and fastigial nuclei of the cerebellum and around neurons of the vestibular nuclei. This receptor protein binds a specific second messenger, inositol 1,4,5-trisphosphate, which produces a mobilization of intracellular Ca2+ and a modulation of transmitter release.

Immunohistochemical localization of the inositol 1,4,5-trisphosphate receptor in the human nervous system.

A monoclonal antibody raised against the mouse cerebellar inositol trisphosphate receptor was used to study the immunohistochemical localization of this protein in the human central nervous system. As in the brain of rodents, strong immunoreactivity was found in dendrites, axon and cell bodies of Purkinje cells, as well as in nerve endings in the cerebellar and vestibular nuclei. Cerebellar efferent fibres were the only positive structures demonstrated in the brainstem and no immunostaining could be detected in the spinal cord or dorsal root ganglia. By contrast, numerous immunoreactive neurons were present in several telencephalic and diencephalic structures, including the brain cortex, hippocampus, basal ganglia, basal forebrain, amygdala and thalamus. Immunostaining of these brain neurons was weaker than that found in Purkinje cells and was evident in cell bodies and dendrites. Thus, the human brain contains a molecule cross-reacting with the mouse inositol trisphosphate receptor protein that is expressed in a pattern similar to that found in rodents. These findings can be of great importance for understanding the function of this protein in normal brain and its modifications in neuropathological disorders.