Minocycline Administration Does Not Have an Effect on Retinal Ganglion Cell Survival in a Murine Model of Ocular Hypertension.

This study aims to investigate two key aspects in a mouse model of ocular hypertension (OHT): first, the time course of retinal ganglion cell (RGC) death and the parallel activation of caspase-3 (a-Casp3+ cells) to narrow the therapeutic window; and second, the effect of caspase-3 and microglia inhibition by minocycline on RGC rescue in this model. RGC loss after OHT induction was significant at day 7 and progressed to 30 days. However, anatomical RGC death was preceded by significant Casp3 activation on day 3. Microglial inhibition by minocycline did not alter the course of OHT or rescue RGCs but resulted in a decrease in a-Casp3+ cells and phagocytic and total microglia. Therefore, RGC death commitment occurs earlier than their loss of Brn3a expression, microglial cells do not exacerbate RGC loss, and while this death is primarily apoptotic, apoptosis inhibition does not rescue RGCs, suggesting that alternative death pathways play a role in glaucomatous injury.

Attitudes and knowledge of myopia management by Spanish optometrists.

PURPOSE: To investigate the knowledge, training and clinical practice of Spanish optometrists about preventing and controlling myopia progression. METHODS: A web-based questionnaire was distributed to Spanish optometrists through social networks, optometric professional bodies and one of the major Spanish optometrists' associations to assess practitioner perception, understanding, and self-reported clinical practice behavior related to myopia diagnosis and management. RESULTS: A total of 534 optometrists with a mean age of 40.8 ± 10.3 years completed the survey. Most respondents have been practicing optometry for more than 20 years (89.8%), report having actively treated childhood myopia (82.4%), and are very concerned about the increasing frequency of pediatric myopia in their daily practice (85.3%). Almost all of the respondents (97.3%) agreed that the efficacy of treatment is related to the age at which it is prescribed, and more than half (53.6%) considered a progression higher than - 0.50 and up to - 1.00D as the minimum necessary to consider a myopia management option. Respondents who reported actively managing childhood myopia considered orthokeratology, atropine and soft-defocus contact lenses the most effective myopia control interventions. However, the most frequently prescribed form of myopia correction by Spanish optometrists was single-vision spectacles, followed by orthokeratology and soft-defocus contact lenses. CONCLUSIONS: Spanish optometrists are very active in the management of myopia, especially by fitting orthokeratology lenses or dual-focus soft contact lenses for myopia control, but there is still potential for improvement in the methodology they follow for both the diagnosis and management of myopia.

Methods to Identify Rat and Mouse Retinal Ganglion Cells in Retinal Flat-Mounts.

The identification of distinct retinal ganglion cell (RGC) populations in flat-mounted retinas is key to investigating pathological or pharmacological effects in these cells. In this chapter, we review the main techniques for detecting the total population of RGCs and various of their subtypes in whole-mounted retinas of pigmented and albino rats and mice, four of the animal strains most studied by the scientific community in the retina field. These methods are based on the studies published by the Vidal-Sanz's laboratory.

Computer vision syndrome-related symptoms in presbyopic computer workers.

PURPOSE: To evaluate the prevalence of computer vision syndrome (CVS)-related symptoms in a presbyopic population using the computer as the main work tool, as well as the relationship of CVS with the electronic device use habits and the ergonomic factors. METHODS: A sample of 198 presbyopic participants (aged 45-65 years) who regularly work with a computer completed a customised questionnaire divided into: general demographics, optical correction commonly used and for work, habits of electronic devices use, ergonomic conditions during the working hours and CVS-related symptoms during work performance. A total of 10 CVS-related symptoms were questioned indicating the severity with which they occurred (0-4) and the median total symptom score (MTSS) was calculated as the sum of the symptoms. RESULTS: The MTSS in this presbyopic population is 7 ± 5 symptoms. The most common symptoms reported by participants are dry eyes, tired eyes and difficulties in refocusing. MTSS is higher in women (p < 0.05), in laptop computer users (p < 0.05) and in teleworkers compared to office workers (p < 0.05). Regarding ergonomic conditions, MTSS is higher in participants who do not take breaks while working (p < 0.05), who have an inadequately lighting in the workspace (p < 0.05) and in the participants reporting neck (p < 0.01) or back pain (p < 0.001). CONCLUSION: There is a relationship between CVS-related symptoms, the use of electronic devices and the ergonomic factors, which indicates the importance of adapting workplaces, especially for home-based teleworkers, and following basic visual ergonomics rules.

Systemic taurine treatment affords functional and morphological neuroprotection of photoreceptors and restores retinal pigment epithelium function in RCS rats.

The aim of our work was to study whether taurine administration has neuroprotective effects in dystrophic Royal College of Surgeons (RCS) rats, suffering retinal degeneration secondary to impaired retinal pigment epithelium phagocytosis caused by a MERTK mutation. Dystrophic RCS-p + female rats (n = 36) were divided into a non-treated group (n = 16) and a treated group (n = 20) that received taurine (0.2 M) in drinking water from postnatal day (P)21 to P45, when they were processed. Retinal function was assessed with electroretinogram. Retinal morphology was assessed in cross-sections using immunohistochemical techniques to label photoreceptors, retinal microglial and macroglial cells, active zones of conventional and ribbon synaptic connections, and oxidative stress. Retinal pigment epithelium function was examined using intraocular fluorogold injections. Our results document that taurine treatment increases taurine plasma levels and photoreceptor survival in dystrophic rats. The number of photoreceptor nuclei rows at P45 was 3-5 and 6-11 in untreated and treated animals, respectively. Electroretinograms showed increases of 70% in the rod response, 400% in the a-wave amplitude, 30% in the b-wave amplitude and 75% in the photopic b-wave response in treated animals. Treated animals also showed decreased numbers of microglial cells in the outer retinal layers, decreased glial fibrillary acidic protein (GFAP) expression in Müller cells, decreased oxidative stress in the outer and inner nuclear layers and improved maintenance of synaptic connections. Treated animals showed increased FG phagocytosis in the retinal pigment epithelium cells. In conclusion, systemic taurine treatment decreases photoreceptor degeneration and increases electroretinographic responses in dystrophic RCS rats and these effects may be mediated through various neuroprotective mechanisms.

University students fail to comply with contact lens care.

PURPOSE: To assess the level of compliance related to contact lens (CL) wear in university students in Spain. METHODS: A web-based questionnaire was distributed to university students through their representatives to assess general demographic information, questions related to CL history, level of compliance with CL care and CL-related complications. RESULTS: A total of 266 participants with an average age of 22 (±4.5) years completed the online questionnaire. Only 39.1 % of respondents indicated that they always replace their CLs within the recommended schedule, and 63.6 % indicated that they usually wear their CLs more hours per day than recommended. Surprisingly, 64.9 % of participants reported that they had not been informed about the potential risks of CL wear, and only 20 % indicated that they always comply with follow-up visits, whereas 42.1 % of respondents expose their CL to water frequently. Participants who received proper CL education were more likely to attend aftercare visits (X2(2) = 9.104, p < 0.05). Participants with a longer history of CL wear had a greater tendency to expose their CLs to water (X2(6) = 18.768, p < 0.05) and suffer CL-related problems (X2(3) = 12.183, p < 0.05). There was also a relationship between an increased frequency of CL exposure to water and an increased tendency to experience CL-related adverse events (X2(2) = 10.864, p < 0.05). CONCLUSION: A relatively high percentage of university CL wearers displayed some degree of non-compliance, which emphasises the importance of providing accurate and comprehensive CL care guidelines and attending aftercare visits to minimise potential CL-related complications. CL wearers should be provided with clear and unambiguous guidelines to avoid any exposure of CL's and CL cases to water.

Assessment of dry eye symptoms among university students during the COVID-19 pandemic.

CLINICAL RELEVANCE: The synchronous hybrid learning environment is associated with increased time spent by students working with VDT and increased prevalence of dry eye symptoms in a university-based population. BACKGROUND: To assess the prevalence of dry eye symptoms using the ocular surface disease index (OSDI) questionnaire in university students and to identify whether factors such as the synchronous hybrid learning environment as a preventive measure of COVID-19, video display terminal use, gender or contact lens wear influence dry eye symptomatology. METHODS: This study was performed using a web-based questionnaire that was distributed to university students to assess questions related to class attendance, to the use of video display terminals, the need for optical correction and, finally, the OSDI questionnaire. RESULTS: A total of 676 university students with an average age of 20.7 ± 2.9 years completed the questionnaire, of which 72.6% (491) were females and 27.4% (185) were males. Only 10.2% of the participants attended face to face classes. Of the participants, 35.5% were contact lens wearers. The mean OSDI score of the study population was 27.68 ± 20.09 and the prevalence of symptomatic dry eye disease (OSDI score above 22) was 51.8%. Female gender (X2(3) = 38.605, p < 0.001), online class attendance (X2(1) = 20.31; p < 0.001), increased hours of online class attendance (X2(2) = 26.84, p < 0.001) and contact lens wear (X2(2) = 15.264, p < 0.05) were associated with a higher incidence of symptomatic dry eye disease. CONCLUSION: The synchronous hybrid learning environment increases the time students spend working with video display terminals and the prevalence of dry eye symptoms. Female gender and contact lens wear were also associated with a higher prevalence of dry eye symptoms. It should not be ignored that dry eye could also affect academic performance.

Animal Models of LED-Induced Phototoxicity. Short- and Long-Term In Vivo and Ex Vivo Retinal Alterations.

Phototoxicity animal models have been largely studied due to their degenerative communalities with human pathologies, e.g., age-related macular degeneration (AMD). Studies have documented not only the effects of white light exposure, but also other wavelengths using LEDs, such as blue or green light. Recently, a blue LED-induced phototoxicity (LIP) model has been developed that causes focal damage in the outer layers of the superior-temporal region of the retina in rodents. In vivo studies described a progressive reduction in retinal thickness that affected the most extensively the photoreceptor layer. Functionally, a transient reduction in a- and b-wave amplitude of the ERG response was observed. Ex vivo studies showed a progressive reduction of cones and an involvement of retinal pigment epithelium cells in the area of the lesion and, in parallel, an activation of microglial cells that perfectly circumscribe the damage in the outer retinal layer. The use of neuroprotective strategies such as intravitreal administration of trophic factors, e.g., basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or pigment epithelium-derived factor (PEDF) and topical administration of the selective alpha-2 agonist (Brimonidine) have demonstrated to increase the survival of the cone population after LIP.

Neuroprotection and Axonal Regeneration Induced by Bone Marrow Mesenchymal Stromal Cells Depend on the Type of Transplant.

Mesenchymal stromal cell (MSC) therapy to treat neurodegenerative diseases has not been as successful as expected in some preclinical studies. Because preclinical research is so diverse, it is difficult to know whether the therapeutic outcome is due to the cell type, the type of transplant or the model of disease. Our aim here was to analyze the effect of the type of transplant on neuroprotection and axonal regeneration, so we tested MSCs from the same niche in the same model of neurodegeneration in the three transplantation settings: xenogeneic, syngeneic and allogeneic. For this, bone marrow mesenchymal stromal cells (BM-MSCs) isolated from healthy human volunteers or C57/BL6 mice were injected into the vitreous body of C57/BL6 mice (xenograft and syngraft) or BALB/c mice (allograft) right after optic nerve axotomy. As controls, vehicle matched groups were done. Retinal anatomy and function were analyzed in vivo by optical coherence tomography and electroretinogram, respectively. Survival of vision forming (Brn3a+) and non-vision forming (melanopsin+) retinal ganglion cells (RGCs) was assessed at 3, 5 and 90 days after the lesion. Regenerative axons were visualized by cholera toxin ß anterograde transport. Our data show that grafted BM-MSCs did not integrate in the retina but formed a mesh on top of the ganglion cell layer. The xenotransplant caused retinal edema, detachment and folding, and a significant decrease of functionality compared to the murine transplants. RGC survival and axonal regeneration were significantly higher in the syngrafted retinas than in the other two groups or vehicle controls. Melanopsin+RGCs, but not Brn3a+RGCs, were also neuroprotected by the xenograft. In conclusion, the type of transplant has an impact on the therapeutic effect of BM-MSCs affecting not only neuronal survival but also the host tissue response. Our data indicate that syngrafts may be more beneficial than allografts and, interestingly, that the type of neuron that is rescued also plays a significant role in the successfulness of the cell therapy.

Short- and Long-Term Study of the Impact of Focal Blue Light-Emitting Diode-Induced Phototoxicity in Adult Albino Rats.

BACKGROUND: In adult rats we study the short- and long-term effects of focal blue light-emitting diode (LED)-induced phototoxicity (LIP) on retinal thickness and Iba-1+ activation. METHODS: The left eyes of previously dark-adapted Sprague Dawley (SD) rats were photoexposed to a blue LED (20 s, 200 lux). In vivo longitudinal monitoring of retinal thickness, fundus images, and optical retinal sections was performed from 1 to 30 days (d) after LIP with SD-OCT. Ex vivo, we analysed the population of S-cone and Iba-1+ cells within a predetermined fixed-size circular area (PCA) centred on the lesion. RESULTS: LIP resulted in a circular focal lesion readily identifiable in vivo by fundus examination, which showed within the PCAs a progressive thinning of the outer retinal layer, and a diminution of the S-cone population to 19% by 30 d. In parallel to S-cone loss, activated Iba-1+ cells delineated the lesioned area and acquired an ameboid morphology with peak expression at 3 d after LIP. Iba-1+ cells adopted a more relaxed-branched morphology at 7 d and by 14-30 d their morphology was fully branched. CONCLUSION: LIP caused a progressive reduction of the outer retina with loss of S cones and a parallel dynamic activation of microglial cells in the lesioned area.

Computer Vision Syndrome in the Spanish Population during the COVID-19 Lockdown.

SIGNIFICANCE: After 6 to 8 weeks of mandatory lockdown due to coronavirus disease 2019 (COVID-19) in Spain, the encouraged change in daily habits resulted in a significant increase in electronic device use. Computer vision syndrome-related symptoms were reported more often in participants who used electronic device for more time and spent less time outdoors. PURPOSE: The main purpose of this study was to evaluate computer vision syndrome-related eye symptoms due to the use of electronic devices during COVID-19 lockdown decreed in Spain in 2020. METHODS: After 6 to 8 weeks of strict lockdown, a total of 730 participants (18 to 73 years old) filled in a customized questionnaire divided into three sections: (1) general demographics, (2) usage habits of electronic devices during this period, and (3) computer vision syndrome-related ocular and visual symptoms associated with their use and with ergonomic practices. RESULTS: The daily duration of use of electronic devices increased an average of 3.1 ± 2.2 h/d during the lockdown, with computer use increasing the most. The main symptoms reported by the participants were headache (36.7%), dry eye (31.1%), irritation (24.1%), blurred vision (21.2%), and ocular pain (14.9%). There was a significant relationship between computer vision syndrome-related symptoms and age (greater in participants between 18 and 30 years old than in those older than 45 years, P < .001), primary activity (greater in studying from home and remote working, P < .001), and extended periods of electronic device use (greater when used more than 10 h/d, P = .05). Symptoms were also associated with time spent outdoors (greater in participants with <1 h/d, P = .02). CONCLUSIONS: The lockdown due to COVID-19 showed an increase in the electronic device use. Participants who spent more time with electronic devices and less time outdoors reported more computer vision syndrome-related eye symptoms.

An in vivo model of focal light emitting diode-induced cone photoreceptor phototoxicity in adult pigmented mice: Protection with bFGF.

PURPOSE: To develop a model of focal injury by blue light-emitting diode (LED)-induced phototoxicity (LIP) in pigmented mouse retinas and to study the effects on cone, Iba-1+ cells and retinal pigment epithelium (RPE) cell populations after administration of basic fibroblast growth factor (bFGF) and minocycline, alone or combined. METHODS: In anesthetized dark-adapted adult female pigmented C57BL/6 mice, left pupils were dilated and the eye exposed to LIP (500 lux, 45 s). The retina was monitored longitudinally in vivo with SD-OCT for 7 days (d). Ex vivo, the effects of LIP and its protection with bFGF (0.5 µg) administered alone or combined with minocycline (45 mg/kg) were studied in immunolabeled arrestin-cone outer segments (a+OS) and quantified within a predetermined fixed-size circular area (PCA) centered on the lesion in flattened retinas at 1, 3, 5 or 7d. Moreover, Iba-1+ cells and RPE cell morphology were analysed with Iba-1 and ZO-1 antibodies, respectively. RESULTS: LIP caused a focal lesion within the superior-temporal retina with retinal thinning, particularly the outer retinal layers (116.5 ± 2.9 µm to 36.8 ± 6.3 µm at 7d), and with progressive diminution of a+OS within the PCA reaching minimum values at 7d (6218 ± 342 to 3966 ± 311). Administration of bFGF alone (4519 ± 320) or in combination with minocycline (4882 ± 446) had a significant effect on a+OS survival at 7d and Iba-1+ cell activation was attenuated in the groups treated with minocycline. In parallel, the RPE cell integrity was progressively altered after LIP and administration of neuroprotective components had no restorative effect at 7d. CONCLUSIONS: LIP resulted in progressive outer retinal damage affecting the OS cone population and RPE. Administration of bFGF increased a+OS survival but did not prevent RPE deterioration.

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-ß 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-1ß 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.

Ly6c as a New Marker of Mouse Blood Vessels: Qualitative and Quantitative Analyses on Intact and Ischemic Retinas.

Ly6c is an antigen commonly used to differentiate between classical and non-classical monocytes/macrophages. Here we show its potential as a marker of the mouse vasculature, particularly of the retinal vascular plexuses. Ly6c was immunodetected in several tissues of C57BL/6 mice using isolectin IB4 as the control of vasculature staining. In the retina, Ly6c expression was analyzed qualitatively and quantitatively in intact, ischemic, and contralateral retinas from 0 to 30 days after the insult. Ly6c expression was observed in all organs and tissues tested, with a brighter signal and more homogeneous staining than the IB4. In the retinas, Ly6c was well expressed, allowing a detailed study of their anatomy. The three retinal plexuses were morphologically different, and from the superficial to the deep one occupied 15 ± 2, 24 ± 7, and 38 ± 1.4 percent of the retinal surface, respectively. In the injured retinas, there was extravasation of the classically activated monocyte/macrophages (Ly6chigh) and the formation of new vessels in the superficial plexus, increasing the area occupied by it to 25 ± 1%. In the contralateral retinas, the superficial plexus area decreased gradually, reaching significance at 30 days, and Ly6c expression progressively disappeared in the intermediate and deep plexuses. Although the role of Ly6c in vascular endothelial cell function is still not completely understood, we demonstrate here that Ly6c can be used as a new specific marker of the mouse vasculature and to assess, qualitatively and quantitatively, vascular changes in health and disease.

Glial Cell Activation and Oxidative Stress in Retinal Degeneration Induced by ß-Alanine Caused Taurine Depletion and Light Exposure.

We investigate glial cell activation and oxidative stress induced by taurine deficiency secondary to ß-alanine administration and light exposure. Two months old Sprague-Dawley rats were divided into a control group and three experimental groups that were treated with 3% ß-alanine in drinking water (taurine depleted) for two months, light exposed or both. Retinal and external thickness were measured in vivo at baseline and pre-processing with Spectral-Domain Optical Coherence Tomography (SD-OCT). Retinal cryostat cross sections were immunodetected with antibodies against various antigens to investigate microglial and macroglial cell reaction, photoreceptor outer segments, synaptic connections and oxidative stress. Taurine depletion caused a decrease in retinal thickness, shortening of photoreceptor outer segments, microglial cell activation, oxidative stress in the outer and inner nuclear layers and the ganglion cell layer and synaptic loss. These events were also observed in light exposed animals, which in addition showed photoreceptor death and macroglial cell reactivity. Light exposure under taurine depletion further increased glial cell reaction and oxidative stress. Finally, the retinal pigment epithelial cells were Fluorogold labeled and whole mounted, and we document that taurine depletion impairs their phagocytic capacity. We conclude that taurine depletion causes cell damage to various retinal layers including retinal pigment epithelial cells, photoreceptors and retinal ganglion cells, and increases the susceptibility of the photoreceptor outer segments to light damage. Thus, beta-alanine supplements should be used with caution.

Influence of the COVID-19 pandemic on contact lens wear in Spain.

PURPOSE: To investigate the behaviour of contact lens (CL) wearers in Spain during the COVID-19 pandemic. METHODS: An anonymized web-based questionnaire was used to assess demographics, CL history, and activity, CL wear habits and perceived risk of infection due to CL wear during the COVID-19 pandemic. RESULTS: A total of 737 participants with an average age of 27.4 (±9.3) years completed the online questionnaire. The vast majority of respondents were soft CL wearers and reported at least two years of CL wear. Patients concerns about the increased risk of SARS-CoV-2 infection due to CL wear (40.6 % of participants) were significantly related (χ2(1) = 11.195, p < 0.05) to CL discontinuation (46 % of participants) during the COVID-19 pandemic. This fact joins the significant changes in the frequency of CL wear during the COVID-19 pandemic (χ2(4) = 31.982, p < 0.05), with a tendency to increase occasional CL wear from 29.1 % to 61.8 %. Interestingly, the majority of respondent (87.9 %) indicated that no professional had offered them information related to CL wear and COVID-19, and that they had not sought it on their own (82.2 %). CONCLUSION: There is a relationship between the perceived risk of infection and CL dropout during the COVID-19 pandemic, and a tendency to change the CL frequency of wear, with an increase in occasional CL wear. During the ongoing pandemic, eye care practitioners should reinforce CL patient education to minimize the risk of SARS-CoV-2 infection and CL-related complications requiring clinical care.

Pigment Epithelium-Derived Factor (PEDF) Fragments Prevent Mouse Cone Photoreceptor Cell Loss Induced by Focal Phototoxicity In Vivo.

Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor.

Tracing the retina to analyze the integrity and phagocytic capacity of the retinal pigment epithelium.

We have developed a new technique to study the integrity, morphology and functionality of the retinal neurons and the retinal pigment epithelium (RPE). Young and old control albino (Sprague-Dawley) and pigmented (Piebald Virol Glaxo) rats, and dystrophic albino (P23H-1) and pigmented (Royal College of Surgeons) rats received a single intravitreal injection of 3% Fluorogold (FG) and their retinas were analyzed from 5 minutes to 30 days later. Retinas were imaged in vivo with SD-OCT and ex vivo in flat-mounts and in cross-sections. Fifteen minutes and 24 hours after intravitreal administration of FG retinal neurons and the RPE, but no glial cells, were labeled with FG-filled vesicles. The tracer reached the RPE 15 minutes after FG administration, and this labeling remained up to 30 days. Tracing for 15 minutes or 24 hours did not cause oxidative stress. Intraretinal tracing delineated the pathological retinal remodelling occurring in the dystrophic strains. The RPE of the P23H-1 strain was highly altered in aged animals, while the RPE of the RCS strain, which is unable to phagocytose, did not accumulate the tracer even at young ages when the retinal neural circuit is still preserved. In both dystrophic strains, the RPE cells were pleomorphic and polymegathic.

Time course of bilateral microglial activation in a mouse model of laser-induced glaucoma.

Microglial activation is associated with glaucoma. In the model of unilateral laser-induced ocular hypertension (OHT), the time point at which the inflammatory process peaks remains unknown. Different time points (1, 3, 5, 8, and 15 d) were compared to analyze signs of microglial activation both in OHT and contralateral eyes. In both eyes, microglial activation was detected in all retinal layers at all time points analyzed, including: i) increase in the cell number in the outer segment photoreceptor layer and plexiform layers (only in OHT eyes) from 3 d onward; ii) increase in soma size from 1 d onward; iii) retraction of the processes from 1 d in OHT eyes and 3 d in contralateral eyes; iv) increase in the area of the retina occupied by Iba-1+ cells in the nerve fiber layer/ganglion cell layer from 1 d onward; v) increase in the number of vertical processes from 1 d in contralateral eyes and 3 d in OHT eyes. In OHT eyes at 24 h and 15 d, most Iba-1+ cells were P2RY12+ and were down-regulated at 3 and 5 d. In both eyes, microglial activation was stronger at 3 and 5 d (inflammation peaked in this model). These time points could be useful to identify factors implicated in the inflammatory process.

ß-alanine supplementation induces taurine depletion and causes alterations of the retinal nerve fiber layer and axonal transport by retinal ganglion cells.

To study the effect of taurine depletion induced by ß-alanine supplementation in the retinal nerve fiber layer (RNFL), and retinal ganglion cell (RGC) survival and axonal transport. Albino Sprague-Dawley rats were divided into two groups: one group received ß-alanine supplementation (3%) in the drinking water during 2 months to induce taurine depletion, and the other group received regular water. After one month, half of the rats from each group were exposed to light. Retinas were analyzed in-vivo using Spectral-Domain Optical Coherence Tomography (SD-OCT). Prior to processing, RGCs were retrogradely traced with fluorogold (FG) applied to both superior colliculi, to assess the state of their retrograde axonal transport. Retinas were dissected as wholemounts, surviving RGCs were immunoidentified with Brn3a, and the RNFL with phosphorylated high-molecular-weight subunit of the neurofilament triplet (pNFH) antibodies. ß-alanine supplementation decreases significantly taurine plasma levels and causes a significant reduction of the RNFL thickness that is increased after light exposure. An abnormal pNFH immunoreactivity in some RGC bodies, their proximal dendrites and axons, and a further diminution of the mean number of FG-traced RGCs compared with Brn3a+RGCs, indicate that their retrograde axonal transport is affected. In conclusion, taurine depletion causes RGC loss and axonal transport impairment. Finally, our results suggest that care should be taken when ingesting ß-alanine supplements due to the limited understanding of their potential adverse effects.