Bilateral early activation of retinal microglial cells in a mouse model of unilateral laser-induced experimental ocular hypertension

The immune system plays an important role in glaucomatous neurodegeneration. Retinal microglial reactivation associated with ganglion cell loss could reportedly contribute to the glaucoma progression. Recently we have described signs of microglia activation both in contralateral and ocular hypertension (OHT) eyes involving all retinal layers 15 days after OHT laser induction in mice. However, no works available have analyzed the microglial activation at earliest time points after OHT induction (24 h) in this experimental model. Thus, we seek to describe and quantify signs of microglia activation and differences depending on the retinal layer, 24 h after unilateral laser-induced OHT. Two groups of adult Swiss mice were used: age-matched control (naïve) and lasered. In the lasered animals, OHT eyes as well as contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1 and MHC-II. We quantified the number of microglial cells in the photoreceptor layer (OS), outer plexiform layer (OPL), and inner plexiform layer (IPL); the number of microglial vertical processes connecting the OPL and OS; the area of the retina occupied by Iba-1+ cells (Iba1-RA) in the nerve fiber layer-ganglion cell layer (NFL-GCL), the total arbor area of microglial cells in the OPL and IPL and; Iba-1+ cell body area in the OPL, IPL and NFL-GCL. In contralateral and OHT eyes the morphological features of Iba-1+ cell activation were: migration, enlargement of the cell body, higher degree of branching and reorientation of the processes, radial disposition of the soma and processes toward adjacent microglial plexuses, and presence of amoeboid cells acting as macrophages. These signs were more pronounced in OHT eyes. Most of Iba-1+ cells did not express MHC-II; rather, only dendritic and rounded cells expressed it. In comparison with naïve eyes, in OHT eyes and contralateral eyes no significant differences were found in the microglial cell number; but there was a significant increase in Iba1-RA. The total arbor area of microglial cells was significantly decreased in: i) OHT eyes with respect contralateral eyes and naïve-eyes in IPL; ii) OHT eyes with respect to naïve eyes in OPL. The number of microglial vertical processes connecting the OPL and OS were significantly increased in contralateral eyes compared with naïve-eyes and OHT eyes. In OPL, IPL and NFL-GCL, the cell body area of Iba-1+ cells was significantly greater in OHT eyes than in naïve and contralateral eyes, and greater in contralateral eyes than in naïve eyes. A non-proliferative microglial reactivation was detected both in contralateral eyes and in OHT eyes in an early time after unilateral laser-induced OHT (24 h). This fast microglial activation, which involves the contralateral eye, could be mediated by the immune system

Retinal Molecular Changes Are Associated with Neuroinflammation and Loss of RGCs in an Experimental Model of Glaucoma

Signaling mediated by cytokines and chemokines is involved in glaucoma-associated neuroinflammation and in the damage of retinal ganglion cells (RGCs). Using multiplexed immunoassay and immunohistochemical techniques in a glaucoma mouse model at different time points after ocular hypertension (OHT), we analyzed (i) the expression of pro-inflammatory cytokines, anti-inflammatory cytokines, BDNF, VEGF, and fractalkine; and (ii) the number of Brn3a+ RGCs. In OHT eyes, there was an upregulation of (i) IFN-γ at days 3, 5, and 15; (ii) IL-4 at days 1, 3, 5, and 7 and IL-10 at days 3 and 5 (coinciding with downregulation of IL1-β at days 1, 5, and 7); (iii) IL-6 at days 1, 3, and 5; (iv) fractalkine and VEGF at day 1; and (v) BDNF at days 1, 3, 7, and 15. In contralateral eyes, there were (i) an upregulation of IL-1β at days 1 and 3 and a downregulation at day 7, coinciding with the downregulation of IL4 at days 3 and 5 and the upregulation at day 7; (ii) an upregulation of IL-6 at days 1, 5, and 7 and a downregulation at 15 days; (iii) an upregulation of IL-10 at days 3 and 7; and (iv) an upregulation of IL-17 at day 15. In OHT eyes, there was a reduction in the Brn3a+ RGCs number at days 3, 5, 7, and 15. OHT changes cytokine levels in both OHT and contralateral eyes at different time points after OHT induction, confirming the immune system involvement in glaucomatous neurodegeneration

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

A Chronic Ocular-Hypertensive Rat Model induced by Injection of the Sclerosant Agent Polidocanol in the Aqueous Humor Outflow Pathway

Background: To induce a moderate chronic ocular hypertension (OHT) by injecting polidocanol, a foamed sclerosant drug, in the aqueous humor outflow pathway. Methods: Intraocular pressure (IOP) was monitored for up to 6 months. Pattern and full-field electroretinogram (PERG and ERG) were recorded and retinal ganglion cells (RGC) and retinal nerve fiber layer (RNFL) thickness were assessed in vivo with optical coherence tomography (OCT) and ex vivo using Brn3a immunohistochemistry. Results: In the first 3 weeks post-injection, a significant IOP elevation was observed in the treated eyes (18.47 3.36 mmHg) when compared with the control fellow eyes (12.52 2.84 mmHg) (p 25% over the baseline. PERG responses were seen to be significantly reduced in the hypertensive eyes (2.25 0.24 V) compared to control eyes (1.44 0.19 V) (p < 0.01) at week 3, whereas the ERG components (photoreceptor a-wave and bipolar cell b-wave) remained unaltered. By week 24, RNFL thinning and cell loss in the ganglion cell layer was first detected (2/13, 15.3%) as assessed by OCT and light microscopy. Conclusions: This novel OHT rat model, with moderate levels of chronically elevated IOP, and abnormal PERG shows selective functional impairment of RGC.Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020 (RD16/0008/0026; RD16/0008/0020; FIS/PI 18-00754)Ministerio de Economía y Competitividad (SAF2015-67643, CIBER-BBN)Fundación Séneca, Agencia de Ciencia y Tecnología Región de Murcia (19881/GERM/15)4.556 JCR (2019) Q1, 74/297 Biochemistry & Molecular Biology1.317 SJR (2019) Q1, 8/77 SpectroscopyNo data IDR 2019UE

Potential role of P2X7 receptor in neurodegenerative processes in a murine model of glaucoma

Glaucoma is a common cause of visual impairment and blindness, characterized by retinal ganglion cell (RGC) death. The mechanisms that trigger the development of glaucoma remain unknown and have gained significant relevance in the study of this neurodegenerative disease. P2X7 purinergic receptors (P2X7R) could be involved in the regulation of the synaptic transmission and neuronal death in the retina through different pathways. The aim of this study was to characterize the molecular signals underlying glaucomatous retinal injury. The time-course of functional, morphological, and molecular changes in the glaucomatous retina of the DBA/2J mice were investigated. The expression and localization of P2X7R was analysed in relation with retinal markers. Caspase-3, JNK, and p38 were evaluated in control and glaucomatous mice by immunohistochemical and western-blot analysis. Furthermore, electroretinogram recordings (ERG) were performed to assess inner retina dysfunction. Glaucomatous mice exhibited changes in P2X7R expression as long as the pathology progressed. There was P2X7R overexpression in RGCs, the primary injured neurons, which correlated with the loss of function through ERG measurements. All analyzed MAPK and caspase-3 proteins were upregulated in the DBA/2J retinas suggesting a pro-apoptotic cell death. The increase in P2X7Rs presence may contribute, together with other factors, to the changes in retinal functionality and the concomitant death of RGCs. These findings provide evidence of possible intracellular pathways responsible for apoptosis regulation during glaucomatous degeneration

Microglia in mouse retina contralateral to experimental glaucoma exhibit multiple signs of activation in all retinal layers

Background: Glaucomatous optic neuropathy, a leading cause of blindness, can progress despite control of intraocular pressure - currently the main risk factor and target for treatment. Glaucoma progression shares mechanisms with neurodegenerative disease, including microglia activation. In the present model of ocular hypertension (OHT), we have recently described morphological signs of retinal microglia activation and MHC-II upregulation in both the untreated contralateral eyes and OHT eyes. By using immunostaining, we sought to analyze and quantify additional signs of microglia activation and differences depending on the retinal layer. Methods: Two groups of adult Swiss mice were used: age-matched control (nai¨ve, n = 12), and lasered (n = 12). In the lasered animals, both OHT eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1, MHC-II, CD68, CD86, and Ym1. The Iba-1+ cell number in the plexiform layers (PL) and the photoreceptor outer segment (OS), Iba-1+ arbor area in the PL, and area of the retina occupied by Iba-1+ cells in the nerve fiber layer-ganglion cell layer (NFL-GCL) were quantified. Results: The main findings in contralateral eyes and OHT eyes were: i) ameboid microglia in the NFL-GCL and OS; ii) the retraction of processes in all retinal layers; iii) a higher level of branching in PL and in the OS; iv) soma displacement to the nearest cell layers in the PL and OS; v) the reorientation of processes in the OS; vi) MHC-II upregulation in all retinal layers; vii) increased CD68 immunostaining; and viii) CD86 immunolabeling in ameboid cells. In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL. In addition, rounded Iba-1+ CD86+ cells in the NFL-GCL, OS and Ym1+ cells, and rod-like microglia in the NFL-GCL were restricted to OHT eyes. Conclusions: Several quantitative and qualitative signs of microglia activation are detected both in the contralateral and OHT eyes. Such activation extended beyond the GCL, involving all retinal layers. Differences between the two eyes could help to elucidate glaucoma pathophysiology

Rod-Like Microglia Are Restricted to Eyes with Laser-Induced Ocular Hypertension but Absent from the Microglial Changes in the Contralateral Untreated Eye

In the mouse model of unilateral laser-induced ocular hypertension (OHT) the microglia in both the treated and the normotensive untreated contralateral eye have morphological signs of activation and up-regulation of MHC-II expression in comparison with naive. In the brain, rod-like microglia align to less-injured neurons in an effort to limit damage. We investigate whether: i) microglial activation is secondary to laser injury or to a higher IOP and; ii) the presence of rod-like microglia is related to OHT. Three groups of mice were used: age-matched control (naive, n=15); and two lasered: limbal (OHT, n=15); and non-draining portion of the sclera (scleral, n=3). In the lasered animals, treated eyes as well as contralateral eyes were analysed. Retinal whole-mounts were immunostained with antibodies against, Iba-1, NF-200, MHC-II, CD86, CD68 and Ym1. In the scleral group (normal ocular pressure) no microglial signs of activation were found. Similarly to naive eyes, OHT-eyes and their contralateral eyes had ramified microglia in the nerve-fibre layer related to the blood vessel. However, only eyes with OHT had rod-like microglia that aligned end-to-end, coupling to form trains of multiple cells running parallel to axons in the retinal surface. Rod-like microglia were CD68+ and were related to retinal ganglion cells (RGCs) showing signs of degeneration (NF-200+ RGCs). Although MHC-II expression was up-regulated in the microglia of the NFL both in OHT-eyes and their contralateral eyes, no expression of CD86 and Ym1 was detected in ramified or in rod-like microglia. After 15 days of unilateral lasering of the limbal and the non-draining portion of the sclera, activated microglia was restricted to OHT-eyes and their contralateral eyes. However, rod-like microglia were restricted to eyes with OHT and degenerated NF-200+ RGCs and were absent from their contralateral eyes. Thus, rod-like microglia seem be related to the neurodegeneration associated with HTO

ERG changes in albino and pigmented mice after optic nerve transection

AbstractOptic nerve transection (ONT) triggers retinal ganglion cell (RGC) death. By using this paradigm, we have analyzed for the first time in adult albino and pigmented mice, the effects of ONT in the scotopic threshold response (STR) components (negative and positive) of the full-field electroretinogram. Two weeks after ONT, when in pigmented mice approximately 18% of the RGC population survive, the STR-implicit time decreased and the p and nSTR waves diminished approximately to 40% or 55%, in albino or pigmented, respectively, with respect to the values recorded from the non-operated contralateral eyes. These changes were maintained up to 12weeks post-ONT, demonstrating that the ERG–STR is a useful parameter to monitor RGC functionality in adult mice

Assessment of inner retina dysfunction and progressive ganglion cell loss in a mouse model of glaucoma

The DBA/2J mouse is a model of ocular hypertension and retinal ganglion cell (RGC) degeneration, the main features of which are iris pigment dispersion (IPD) and iris stromal atrophy (ISA). These animals also experience glaucomatous changes, including an increase in intraocular pressure (IOP) beginning at about 9-12 months of age and sectorial RGC death in the retina. The aim of this study was to determine the onset of functional changes exhibited by DBA/2J mice in the inner retina. This was performed by means of electroretinographic recordings (scotopic threshold response, STR) and their correlation with morphological changes (loss of RGCs). To this end, we recorded the scotopic threshold response in control C57BL/6J and in DBA/2J mice at different ages. The RGCs, in both DBA/2J and C57BL/6J animals, were identified at 15 months of age by retrograde tracing with an analogue of fluorogold, hydroxystilbamidine methanesulfonate (OHSt), applied on the superior colliculi. Whole mount retinas were processed to quantify the population of RGCs identified by fluorogold tracing and Brn3a immunodetection, and were counted using image analysis software; an isodensity contour plot was generated for each retina. DBA/2J mice showed a significant reduction in the positive STR (pSTR) amplitudes at 12 months of age, as compared to control C57BL/6J mice of the same age. The pSTR mean amplitude decreased to approximately 27.82% of the values recorded in control mice (p=0.0058). STR responses decreased in both strains as a result of the natural process of aging, but the decrease was more pronounced in DBA/2J mice. Furthermore, quantification of the total number of RGCs identified by OHSt and Brn3a expression showed a reduced population of RGCs in DBA/2J mice as compared to control mice. Regression analysis revealed significant correlations between the decrease in pSTR and a non-homogeneous reduction in the number of RGCs throughout the retina. Our results indicate the existence of a correlation between retinal function impairment and RGC loss. This functional and morphological analysis allows a reliable assessment of the progression of the disease