Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

Endostatin, a naturally cleaved fragment of type XVIII collagen with antiangiogenic activity, has been involved in the regulation of neovascularization during diabetic retinopathy. Here, the intracellular distribution of endostatin in healthy mouse and human neuroretinas has been analyzed. In addition, to study the effect of experimental hyperglycemia on retinal endostatin, the db/db mouse model has been used. Endostatin protein expression in mouse and human retinas was studied by immunofluorescence and Western blot, and compared with db/db mice. Eye fundus angiography, histology, and immunofluorescence were used to visualize mouse retinal and intravitreal vessels. For the first time, our results revealed the presence of endostatin in neurons of mouse and human retinas. Endostatin was mainly expressed in bipolar cells and photoreceptors, in contrast to the optic disc, where endostatin expression was undetectable. Diabetic mice showed a reduction of endostatin in their retinas associated with the appearance of intravitreal vessels at the optic disc in 50% of db/db mice. Intravitreal vessels showed GFAP positive neuroglia sheath, basement membrane thickening by collagen IV deposition, and presence of MMP-2 and MMP-9 in the vascular wall. All together, these results point that decreased retinal endostatin during experimental diabetes is associated with optic disc intravitreal vascularization. Based on their phenotype, these intravitreal vessels could be neovessels. However, it cannot be ruled out the possibility that they may also represent persistent hyaloid vessels.

TIM2 modulates retinal iron levels and is involved in blood-retinal barrier breakdown.

Careful control of iron availability in the retina is central to maintenance of iron homeostasis, as its imbalance is associated with oxidative stress and the progression of several retinopathies. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-transporter protein, through its binding to Scara5 and TIM2 membrane receptors. In this study, the presence and iron-related functions of TIM2 in the mouse retina were investigated. Our results revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly in Müller cells. Experimental TIM2 downregulation in the mouse retina promoted, probably due to a compensatory mechanism, Scara5 overexpression that increased retinal ferritin uptake and induced iron overload. Consecutive reactive oxygen species (ROS) overproduction and vascular endothelial growth factor (VEGF) overexpression led to impaired paracellular and transcellular endothelial transport characterized by tight junction degradation and increased caveolae number. In consequence, blood-retinal barrier (BRB) breakdown and retinal edema were observed. Altogether, these results point to TIM2 as a new modulator of retinal iron homeostasis and as a potential target to counteract retinopathy.

Vascular Interstitial Cells in Retinal Arteriolar Annuli Are Altered During Hypertension.

Purpose: It has been suggested that arteriolar annuli localized in retinal arterioles regulate retinal blood flow acting as sphincters. Here, the morphology and protein expression profile of arteriolar annuli have been analyzed under physiologic conditions in the retina of wild-type, ß-actin-Egfp, and Nestin-gfp transgenic mice. Additionally, to study the effect of hypertension, the KAP transgenic mouse has been used. Methods: Cellular architecture has been studied using digested whole mount retinas and transmission electron microscopy. The profile of protein expression has been analyzed on paraffin sections and whole mount retinas by immunofluorescence and histochemistry. Results: The ultrastructural analysis of arteriolar annuli showed a different cell population found between endothelial and muscle cells that matched most of the morphologic criteria established to define interstitial Cajal cells. The profile of protein expression of these vascular interstitial cells (VICs) was similar to that of interstitial Cajal cells and different from the endothelial and smooth muscle cells, because they expressed ß-actin, nestin, and CD44, but they did not express CD31 and α-SMA or scarcely express F-actin. Furthermore, VICs share with pericytes the expression of NG2 and platelet-derived growth factor receptor beta (PDGFR-ß). The high expression of Ano1 and high activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase observed in VICs was diminished during hypertensive retinopathy suggesting that these cells might play a role on the motility of arteriolar annuli and that this function is altered during hypertension. Conclusions: A novel type of VICs has been described in the arteriolar annuli of mouse retina. Remarkably, these cells undergo important molecular modifications during hypertensive retinopathy and might thus be a therapeutic target against this disease.

Correction: L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy.

[This corrects the article DOI: 10.1371/journal.pone.0106974.].

Animal Models of Depression and Drug Delivery with Food as an Effective Dosing Method: Evidences from Studies with Celecoxib and Dicholine Succinate.

Multiple models of human neuropsychiatric pathologies have been generated during the last decades which frequently use chronic dosing. Unfortunately, some drug administration methods may result in undesirable effects creating analysis confounds hampering model validity and preclinical assay outcomes. Here, automated analysis of floating behaviour, a sign of a depressive-like state, revealed that mice, subjected to a three-week intraperitoneal injection regimen, had increased floating. In order to probe an alternative dosing design that would preclude this effect, we studied the efficacy of a low dose of the antidepressant imipramine (7 mg/kg/day) delivered via food pellets. Antidepressant action for this treatment was found while no other behavioural effects were observed. We further investigated the potential efficacy of chronic dosing via food pellets by testing the antidepressant activity of new drug candidates, celecoxib (30 mg/kg/day) and dicholine succinate (50 mg/kg/day), against standard antidepressants, imipramine (7 mg/kg/day) and citalopram (15 mg/kg/day), utilizing the forced swim and tail suspension tests. Antidepressant effects of these compounds were found in both assays. Thus, chronic dosing via food pellets is efficacious in small rodents, even with a low drug dose design, and can prevail against potential confounds in translational research within depression models applicable to adverse chronic invasive pharmacotherapies.

L-ferritin binding to scara5: a new iron traffic pathway potentially implicated in retinopathy.

Iron is essential in the retina because the heme-containing enzyme guanylate cyclase modulates phototransduction in rods and cones. Transferrin endocytosis is the classical pathway for obtaining iron from the blood circulation in the retina. However, the iron storage protein ferritin has been also recently proposed as an iron carrier. In this study, the presence of Scara5 and its binding to L-ferritin was investigated in the retina. Our results showed that Scara5, the specific receptor for L-ferritin, was expressed in mouse and human retinas in many cell types, including endothelial cells. Furthermore, we showed that intravenously injected ferritin crossed the blood retinal barrier through L-ferritin binding to Scara5 in endothelial cells. Thus, suggesting the existence of a new pathway for iron delivery and trafficking in the retina. In a murine model of photoreceptor degeneration, Scara5 was downregulated, pointing out this receptor as a potential player implicated in retinopathy and also as a possible therapeutic target.

Altered emotionality, hippocampus-dependent performance and expression of NMDA receptor subunit mRNAs in chronically stressed mice.

N-Methyl-D-aspartate receptor (NMDAR)-mediated neurotransmission in the hippocampus is implicated in cognitive and emotional disturbances during stress-related disorders. Here, using quantitative RT-PCR, we investigated the hippocampal expression of NR2A, NR2B and NR1 subunit mRNAs in a mouse stress paradigm that mimics clinically relevant conditions of simultaneously affected emotionality and hippocampus-dependent functions. A 2-week stress procedure, which comprised ethologically valid stressors, exposure to a rat and social defeat, was applied to male C57BL/6J mice. For predation stress, mice were introduced into transparent containers that were placed in a rat home cage during the night; social defeat was applied during the daytime using aggressive CD1 mice. This treatment impaired hippocampus-dependent performance during contextual fear conditioning. A correlation between this behavior and food displacement performance was demonstrated, suggesting that burrowing behavior is affected by the stress procedure and is hippocampus-dependent. Stressed mice (n = 22) showed behavioral invigoration and anomalous anxiolytic-like profiles in the O-maze and brightly illuminated open field, unaltered short-term memory in the step-down avoidance task and enhanced aggressive traits, as compared to non-stressed mice (n = 10). Stressed mice showed increased basal serum corticosterone concentrations, hippocampal mRNA expression for the NR2A subunit of the NMDAR and in the NR2A/NR2B ratio; mRNA expression of NR2B and NR1 was unchanged. Thus, stress-induced aberrations in both hippocampal-dependent performance and emotional abnormalities are associated with alterations in hippocampal mRNA NR2A levels and the NR2A/NR2B ratio and not with mRNA expression of NR2B or NR1.

Intercapillary bridging cells: immunocytochemical characteristics of cells that connect blood vessels in the retina.

Intervascular bridges are fibrous strands that connect neighboring capillaries. These strands present associated cells, intervascular bridging cells (IBCs), whose nature and functional significance remains controversial. The aim of this study was to characterize the immunophenotype of IBCs, and contribute to understand their mechanical and intercellular communication properties in the retina. Quantification and retinal distribution of IBCs were also determined. For this purpose, C57BL/6N and nestin-GFP transgenic mice, as well as human retinas, were used. Whole-mount retinas were studied by means of immunohistochemistry and cytochemistry, and isolation of retinal vasculature was achieved by trypsin/pepsin digest technique. PAS reaction and the immunolabeling with anti-collagen IV and laminin antibodies revealed that IBCs were completely surrounded by a basement membrane, connecting two or more neighboring capillaries. IBCs were scarce and their number decreased with age. They were preferentially localized in the deep vascular plexus. In a murine model of experimental glaucoma, methylcellulose injected eyes showed retinal neovascularization and increased number of IBCs in the deep vascular plexus. IBCs were marked with anti-NG2, anti-PDGFR-ß and anti-CD34 antibodies, and with tomato lectin, and were negative for PECAM-1. IBCs expressed nestin and filamentous actin, but desmin and α-smooth muscle actin were not detected. Moreover, these cells expressed the gap junction protein connexin 43. These results showed that IBCs had a pericytic nature since they expressed NG2 and the receptor for PDGF-B, and they were negative for PECAM-1. However, they were marked with CD34 and the tomato lectin, suggesting that they constitute a special subtype of pericytes, sharing characteristics with endothelial cells. IBCs presumably present mechanical functions due to the presence of filamentous actin. Connexin 43 was found in IBCs, suggesting that these cells allow intercellular communication between adjacent capillaries. This may represent an advantage for vasomotor tone integration and coordination in blood vessels without innervation, such as those of the retina.