Modulation of iris sphincter and ciliary muscles by urocortin 2.

Urocortin 2 (UCN2) is a peptide related to corticotropin-releasing factor, capable of activating CRF-R2. Among its multisystemic effects, it has actions in all 3 muscle subtypes. This study's aim was to determine its potential role in two of the intrinsic eye muscle kinetics. Strips of iris sphincter (rabbit) and ciliary (bovine) muscles were dissected and mounted in isometric force-transducer systems filled with aerated-solutions. Contraction was elicited using carbachol (10(-6) M for iris sphincter, 10(-5) M for ciliary muscle), prior adding to all testing substances. UCN2 induced relaxation in iris sphincter muscle, being the effect maximal at 10(-7) M concentrations (-12.2 % variation vs. control). This effect was abolished with incubation of indomethacin, antisauvagine-30, chelerytrine and SQ22536, but preserved with L-nitro-L-arginine. In carbachol pre-stimulated ciliary muscle, UCN2 (10(-5) M) enhanced contraction (maximal effect of 18.2 % increase vs. control). UCN2 is a new modulator of iris sphincter relaxation, dependent of CRF-R2 activation, synthesis of prostaglandins (COX pathway) and both adenylate cyclase and PKC signaling pathways, but independent of nitric oxide production. Regarding ciliary muscle, UCN2 enhances carbachol-induced contraction, in higher doses.

A Critical Analysis of the Available In Vitro and Ex Vivo Methods to Study Retinal Angiogenesis.

Angiogenesis is a biological process with a central role in retinal diseases. The choice of the ideal method to study angiogenesis, particularly in the retina, remains a problem. Angiogenesis can be assessed through in vitro and in vivo studies. In spite of inherent limitations, in vitro studies are faster, easier to perform and quantify, and typically less expensive and allow the study of isolated angiogenesis steps. We performed a systematic review of PubMed searching for original articles that applied in vitro or ex vivo angiogenic retinal assays until May 2017, presenting the available assays and discussing their applicability, advantages, and disadvantages. Most of the studies evaluated migration, proliferation, and tube formation of endothelial cells in response to inhibitory or stimulatory compounds. Other aspects of angiogenesis were studied by assessing cell permeability, adhesion, or apoptosis, as well as by implementing organotypic models of the retina. Emphasis is placed on how the methods are applied and how they can contribute to retinal angiogenesis comprehension. We also discuss how to choose the best cell culture to implement these methods. When applied together, in vitro and ex vivo studies constitute a powerful tool to improve retinal angiogenesis knowledge. This review provides support for researchers to better select the most suitable protocols in this field.

Identification of the ghrelin-GHSR 1 system and its influence in the modulation of induced ocular hypertension in rabbit and rat eyes.

Recent studies evidenced a decrease in ghrelin's aqueous humor levels in patients with glaucoma. The goal of our investigation was to study the effect of the ghrelin-GHSR-1a system in the modulation of intraocular pressure in acute ocular hypertension models and its expression and distribution in ocular tissues. Two animal models of acute ocular hypertension were used to study the effect of the ghrelin-GHSR-1a system in the modulation of intraocular pressure: the rabbit and the rat. Ocular hypertension was induced by an intravitreal injection of 20% NaCl. Ghrelin or des-acyl ghrelin were delivered subconjunctivally and the intraocular pressure was assessed by a rebound tonometer that was calibrated for each species. In addition, we have studied the influence of nitric oxide and prostaglandins on ghrelin's effect in the rabbit animal model. Finally, we determined by immunofluorescence the expression of ghrelin and GHSR-1 in the rat's ocular tissue. Ghrelin decreased the intraocular pressure in both animal models (maximum decrease: 43.8±12.0% in the rabbit and 29.0±7.46% in the rat). In the rabbit, this effect was blunted in the presence of l-NAME and ketorolac. Des-acyl ghrelin only decreased the intraocular pressure in the rat (maximum decrease: 34.9±8.15%). Ghrelin expression was detected in the ciliary processes and GHSR-1 expression was detected in the trabecular meshwork and ciliary body. The ghrelin-GHSR-1 system is expressed in the anterior segment of the eye. Ghrelin and des-acyl ghrelin are responsible for a hypotensive effect in acute ocular hypertension animal models.

Congenital Aniridia: Clinic, Genetics, Therapeutics, and Prognosis.

Congenital aniridia is a rare condition related to a deficiency in the PAX6 gene expression, which may occur as a result of a family inheritance or a sporadic occurrence. Additionally, this condition may occur as an isolated ocular phenotype or in association with a systemic syndrome. The most common abnormality is iris hypoplasia; however, a panocular disease which also affects the cornea, anterior chamber of the eye, lens, and the posterior segment with presence of optic nerve and foveal hypoplasia is also evident. The development of keratopathy, glaucoma, and cataract is frequent and its presence has implications in the patient's visual acuity. Managing aniridia is challenging since the focus is on treating the previously mentioned disorders, and the outcomes are often disappointing. In this paper, we shall review the epidemiology, pathophysiology, and clinical characteristics of patients with aniridia. We shall also make a review of the therapeutic options for the several conditions affecting this syndrome and consider the genetics and prognostic factors.

New therapeutic targets for intraocular pressure lowering.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible and preventable blindness and ocular hypertension is the strongest known risk factor. With current classes of drugs, management of the disease focuses on lowering intraocular pressure (IOP). Despite of their use to modify the course of the disease, none of the current medications for POAG is able to reduce the IOP by more than 25%-30%. Also, some glaucoma patients show disease progression despite of the therapeutics. This paper examines the new described physiological targets for reducing the IOP. The main cause of elevated IOP in POAG is thought to be an increased outflow resistance via the pressure-dependent trabecular outflow system, so there is a crescent interest in increasing trabecular meshwork outflow by extracellular matrix remodeling and/or by modulation of contractility/TM cytoskeleton disruption. Modulation of new agents that act mainly on trabecular meshwork outflow may be the future hypotensive treatment for glaucoma patients. There are also other agents in which modulation may decrease aqueous humour production or increase uveoscleral outflow by different mechanisms from those drugs available for glaucoma treatment. Recently, a role for the ghrelin-GHSR system in the pathophysiology modulation of the anterior segment, particularly regarding glaucoma, has been proposed.

ETB2 receptor subtype stimulation relaxes the iris sphincter muscle.

Effects of ET(B) receptor stimulation and its subcellular pathways were evaluated in carbachol pre-contracted rabbit iris sphincter muscles (n=51). ET(B) stimulation with sarafotoxin (SRTX-c; 10(-10)-10(-6) M) was tested in the absence (n=7) or presence of 10(-5) M of: BQ-788 (ET(B2) receptor antagonist; n=6), L-NA (NOS inhibitor; n=7) or indomethacin (cyclooxygenase inhibitor; n=10). Effects of ET(B) stimulation by endothelin-1 (ET-1; 10(-10)-10(-7) M) in the presence of an ET(A) receptor antagonist (BQ-123; 10(-5) M; n=7) and of ET(B1) stimulation by IRL-1620 (10(-10)-10(-7) M; n=7) were also tested. Finally, the effects of SRTX-c (10(-9)-10(-7) M) in electric field stimulation (EFS) contraction were evaluated (n=7). ET(B) receptor stimulation by SRTX-c or ET-1 in presence of BQ-123 promoted a concentration-dependent relaxation of the rabbit iris sphincter muscle by 10.8+/-2.0% and 9.4+/-1.8%, respectively. This effect was blocked by BQ-788 (-2.3+/-2.0 %), L-NA (4.5+/-2.3 %) or indomethacin (2.3+/-2.9 %). Selective ET(B1) stimulation by IRL-1620 did not relax the iris sphincter muscle (0.9+/-5.4 %). EFS elicited contraction was not altered by SRTX-c. In conclusion, ET(B) receptor stimulation relaxes the carbachol precontracted iris sphincter muscle, an effect that is mediated by the ET(B2) receptor subtype, through NO and the release of prostaglandins.

The obestatin/ghrelin system as a novel regulatory mechanism of iris muscle contraction.

PURPOSE: To evaluate obestatin and ghrelin effects on iris muscle contraction. MATERIALS AND METHODS: Obestatin (10(-5) M) or ghrelin (10(-5) M) were tested on two consecutive carbachol-or epinephrine-elicited contractions of iris rabbit sphincter or dilator muscles. Ghrelin and obestatin effects on iris muscles basal tension were also tested, and their effects on iris sphincter EFS-elicited contraction were evaluated. RESULTS: Compared with the first, tension of the second carbachol-induced contraction of the iris sphincter decreased 11.5+/-5.5% in the vehicle group, increased 19.0+/-10.2% in presence of obestatin, and remained unchanged by ghrelin. Epinephrine-induced contractions were not affected by obestatin or ghrelin. EFS-elicited contractions were decreased 9.3+/-3.2% by ghrelin. Basal tension of the iris sphincter decreased 21.7+/-3.7% in presence of ghrelin (10(-5) M), while that of the dilator decreased 14.1+/-5.0% in presence of obestatin (10(-5) M). CONCLUSION: This study suggests that obestatin potentiates the cholinergic contraction of the iris sphincter and relaxes the iris dilator muscles.

Ghrelin as a novel locally produced relaxing peptide of the iris sphincter and dilator muscles.

Ghrelin is a recently described acylated peptide, which works as a somatosecretagogue and has described effects on the smooth, skeletal and cardiac muscle. We examined the production and effects of ghrelin on relaxation of the iris muscles. Contractile effects of 1-5 human ghrelin (frGhr, 10(-9)-6 x 10(-5)M) and 1-5 human des-octanoyl-ghrelin (d-frGhr; 10(-9)-6 x 10(-5)M) were tested on iris rabbit sphincter (n=11 frGhr; n=7 d-frGhr), dilator (n=6 frGhr; n=6 d-frGhr) and rat sphincter (n=6 frGhr; n=8 d-frGhr) precontracted muscles. On rabbit sphincter the effect of frGhr was also tested in presence of: i) L-NA (10(-5)M; n=7); ii) indomethacin (10(-5)M; n=7); iii) DLys(3)GHRP6 (10(-4)M; n=6); and iv) apamin+carybdotoxin (10(-6)M; n=6). Furthermore, on rabbit dilator the effect of frGhr was tested in presence of DLys(3)GHRP6 (10(-4)M; n=7). Finally, ghrelin mRNA production was assessed by "in situ" hybridization in Wistar rat eyes (n=8). In all muscles, frGhr promoted a concentration-dependent relaxation, maximal at 6 x 10(-5)M, 1.5-3 min after its addition, decreasing tension by 34.1+/-12.1%, 25.8+/-4.8% and 52.1+/-10.3% in the rabbit sphincter, dilator and rat sphincter, respectively. In the rabbit sphincter the relaxing effects of frGhr were: (i) enhanced in presence of DLys(3)GHRP6 (118.1+/-21.1%); (ii) blunted by indomethacin; and (iii) not altered by apamin+carybdotoxin (36.4+/-14.4%) or L-NA (52.4+/-11.4%). Relaxing effects of d-frGhr in rabbit (43.3+/-5.2%) and rat (77.1+/-15.3%) sphincter muscles were similar to those of frGhr. In rabbit dilator muscle, d-frGhr did not significantly alter active tension and the relaxing effect of frGhr was blunted by GHSR-1a blockage. Ghrelin mRNA was identified in iris posterior epithelium. In conclusion, ghrelin is a novel, locally produced, relaxing agent of iris dilator and sphincter muscles, an effect that is mediated by GHSR-1a in the former, but not in the latter. Furthermore, in the sphincter it seems to be mediated by prostaglandins, but not by NO or K(Ca) channels.

Identification of XLRS1 gene mutation (608C > T) in a Portuguese family with juvenile retinoschisis.

PURPOSE: To characterize electroretinogram (ERG) and molecular genetic findings in a family with XLRS1 mutation. The authors present two cases of a Portuguese family with juvenile retinoschisis with a mutation in exon 6. METHODS: Two brothers and their parents, grandmother, and uncle underwent a full ophthalmic examination. The two brothers with ophthalmic disease were evaluated with color fundus photography, fluorescein angiography, optical coherence tomography (OCT), molecular genetic study (Group VI of Retinoschisis Consortium), pattern visual evoked potential (PVEP), and full field ERG. RESULTS: Both patients presented funduscopic manifestations of vitre o retinal degeneration. They presented peripheral schisis and retinal detachment. However, foveal schisis had never been observed at funduscopy. A negative ERG was recorded in both. Six months after that, the younger brother showed a typical foveal schisis at fundus examination. A retinoschisis gene (XLRS1) mutation with transition of cytosine by thymine at position 608 (608C > T) had been identified in both. CONCLUSIONS: Negative ERG is the most secure clinical marker to establish the diagnosis of juvenile retinoschisis. XLRS1 gene 608C > T mutation was described for the first time in a Portuguese family.

Identification of XLRS1 gene mutation (608C>T) in a Portuguese family with juvenile retinoschisis.

PURPOSE: To characterize electroretinogram (ERG) and molecular genetic findings in a family with XLRS1 mutation. The authors present two cases of a Portuguese family with juvenile retinoschisis with a mutation in exon 6. METHODS: Two brothers and their parents, grandmother, and uncle underwent a full ophthalmic examination. The two brothers with ophthalmic disease were evaluated with color fundus photography, fluorescein angiography, optical coherence tomography (OCT), molecular genetic study (Group VI of Retinoschisis Consortium), pattern visual evoked potential (PVEP), and full field ERG. RESULTS: Both patients presented funduscopic manifestations of vitre o retinal degeneration. They presented peripheral schisis and retinal detachment. However, foveal schisis had never been observed at funduscopy. A negative ERG was recorded in both. Six months after that, the younger brother showed a typical foveal schisis at fundus examination. A retinoschisis gene (XLRS1) mutation with transition of cytosine by thymine at position 608 (608C>T) had been identified in both. CONCLUSIONS: Negative ERG is the most secure clinical marker to establish the diagnosis of juvenile retinoschisis. XLRS1 gene 608C>T mutation was described for the first time in a Portuguese family. (Eur J Ophthalmol 2005; 15: 638-40 ).

Modern sports eye injuries.

AIMS: To determine the severity and long term sequelae of eye injuries caused by modern sports that could be responsible for significant ocular trauma in the future. METHODS: Prospective observational study of 24 (25 eyes) athletes with sports related ocular injuries from health clubs, war games, adventure, radical and new types of soccer, presenting to an eye emergency department between 1992 and 2002 (10 years). RESULTS: Modern sports were responsible for 8.3% of the 288 total sports eye injuries reported. Squash (29.2%) was the most common cause, followed by paintball (20.8%) and motocross (16.6%). The most common diagnosis during the follow up period was retinal breaks (20%). 18 (75%) patients sustained a severe injury. The final visual acuity remained <20/100 in two paintball players. CONCLUSIONS: Ocular injuries resulting from modern sports are often severe. Adequate instruction of the participants in the games, proper use of eye protectors, and a routine complete ophthalmological examination after an eye trauma should be mandatory.

[Mechanisms underlying endothelin-1 effects on myocardial function]. / Mecanismos subjacentes aos efeitos da endotelina-1 sobre a função miocárdica.

INTRODUCTION: Endothelin-1 (ET-1) has potent vasoconstrictor, growth promoting and positive inotropic properties. Its effects on the intrinsic properties of the myocardium were recently described. The present study investigated the mechanisms underlying those effects. METHODS: The myocardial effects of 1 and 10 nM of ET-1 were evaluated in isolated rabbit papillary muscles (n = 9) and human atrial trabecula from CABG patients (Krebs-Ringer; 1.8 mM CaCl2; 35 degrees C). In papillary muscles the effects of 1 nM ET-1 were also studied in the presence of: (i) a selective ETA receptor antagonist, BQ-123 (0.1 microM; n = 9); (ii) a selective ETB receptor antagonist, BQ-788 (0.1 microM; n = 6); and (iii) an Na+/H+ exchanger inhibitor, methyl-isobutyl-amiloride (MIA; 1 microM; n = 6). Only significant results (mean +/- SE, p < 0.05) are given, expressed as delta % baseline. RESULTS: In AT by papillary muscles, 1 nM of ET-1 increased 64 +/- 16%, dT/dtmin 39 +/- 13% and decreased PT by 11 +/- 2%. The analysis of atrial strip contractions yielded similar results. In papillary muscles the effects of ET-1 were not affected by BQ-788, yet they were abolished by BQ-123, and reduced by 44% by MIA. CONCLUSIONS: The action of ET-1 on myocardial function is similar in human and non-human myocardium. The myocardial effects observed in the present study are mediated by the binding to ETA receptors, and partially dependent on Na+/H+ exchanger activation.

[The effects of endothelin-1 on myocardial function]. / Efeitos da endotelina-1 sobre a função miocárdica.

INTRODUCTION: Endothelin-1 (ET-1) has potent vasoconstrictor, growth promoting and positive inotropic properties, with increased plasma levels in heart failure (HF). With regard to the cardiac effects of ET-1, most studies have not been able to differentiate its effects on the intrinsic properties of the myocardium from the secondary effects resulting from load changes and coronary vasoconstriction. This study investigated the myocardial effects of ET-1. METHODS: The study was performed on isolated rabbit papillary muscles (n = 9), before and after the addition of ET-1 (10 nM) to the superfusing solution (Krebs-Ringer; 1.25 nM Ca2+; 35 degrees C). One isotonic, one isometric and two afterloaded-isotonic twitches were recorded and analyzed. Only significant results (mean +/- SE, p < 0.05) are given, expressed as delta % baseline. RESULTS: ET-1 induced an increase of AT (147 +/- 33%), dT/dtmax (154 +/- 39%) and dT/dtmin (145 +/- 38%), while the duration of the twitch did not vary significantly. In addition, after ET-1, RT at the end of the isometric twitch decreased by 19 +/- 3%, when compared with the control and to its value at the beginning of the twitch. CONCLUSIONS: This study showed that, in addition to the well-known positive inotropic effect, ET-1 improves diastolic function by accelerating relaxation rate (dT/dtmin) and decreasing RT. These results may have important implications in the pathophysiology of HF.