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This study compared refractive and axial length (AL) changes in children wearing dual-focus soft contact lenses for myopia control (MiSight®) with myopic children wearing spectacles one year from the start of lockdown during the COVID-19 pandemic.This retrospective, descriptive, parallel-group, observational study reviewed the charts of 11 children who began treatment for myopia control with dual-focus soft contact lenses for myopia control (MiSight®) in March 2020 and 11 matched spectacle-wearing controls. The mean increase in spherical equivalent refraction (SER) and AL from the beginning of the lockdown and up to 1 year later were compared. The parents of the children were asked about the average time spent on near work, contact lens wearing time both during and after the strict confinement and whether they had discontinued contact lens wear during lockdown.During this first year of preventive COVID-19 measures (March 2020-March 2021), for the contact lens group the average SER and AL increased -0.14 ± 0.09D and 0.13 ± 0.05 mm, respectively. For the spectacle wearers, the corresponding increases were -0.54 ± 0.16D and 0.25 ± 0.08 mm, respectively. A significant difference was found between the groups for both SER (p 0.001) and AL (p 0.05). The average time spent outdoors was restricted for both groups during lockdown and increased after. However, statistically significant changes in the time spent outdoors during and after lockdown were only found for the spectacle group (p 0.05; t-test), whereas this change was not significant for the contact lens group (p = 0.08).Over the observed time period, dual-focus soft contact lenses for myopia control were effective despite the decreased time spent outdoors during the COVID-19 pandemic.

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 p p p p p p 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.

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

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

To study and compare effects of syngeneic bone marrow mononuclear stem cells (BM-MNCs) transplants on inherited retinal degeneration in two animal models with different etiologies: the RCS and the P23H-1 rats. To compare the safety and efficacy of two methods of intraocular delivery: subretinal and/or intravitreal.A suspension of BM-MNCs was injected subretinally or intravitreally in the left eyes of P23H-1 and RCS rats at post-natal day (P) 21. At different survival intervals after the injection: 7, 15, 30 or 60 days, the retinas were cross-sectioned, and photoreceptor survival and glial cell responses were investigated using immunodetection of cones (anti-cone arrestin), synaptic connections (anti-bassoon), microglia (anti-Iba-1), astrocytes and Müller cells (anti-GFAP). Electroretinographic function was also assessed longitudinally.Intravitreal injections (IVIs) or subretinal injections (SRIs) of BM-MNCs did not produce adverse effects. The transplanted cells survived for up to 15 days but did not penetrate the retina. Both IVIs and SRIs increased photoreceptor survival, decreased synaptic degeneration and glial fibrillary acidic protein (GFAP) expression in Müller cells but did not modify microglial cell activation and migration or the electroretinographic responses.Intravitreal and subretinal syngeneic BM-MNCs transplantation decreases photoreceptor degeneration and shows anti-gliotic effects on Müller cells but does not ameliorate retinal function. Moreover, syngeneic BM-MNCs transplants are more effective than the xenotransplants of these cells. BM-MNC transplantation has potential therapeutic effects that merit further investigation.

International audience; We investigate glial cell activation and oxidative stress induced by taurine deficiency secondary to β-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% β-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.

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 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.

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 (&asymp, 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.

Inherited photoreceptor degenerations are not treatable diseases and a frequent cause of blindness in working ages. In this study we investigate the safety, integration and possible rescue effects of intravitreal and subretinal transplantation of adult human bone-marrow-derived mononuclear stem cells (hBM-MSCs) in two animal models of inherited photoreceptor degeneration, the P23H-1 and the Royal College of Surgeons (RCS) rat. Immunosuppression was started one day before the injection and continued through the study. The hBM-MSCs were injected in the left eyes and the animals were processed 7, 15, 30 or 60 days later. The retinas were cross-sectioned, and L- and S- cones, microglia, astrocytes and M&uuml, ller cells were immunodetected. Transplantations had no local adverse effects and the CD45+ cells remained for up to 15 days forming clusters in the vitreous and/or a 2&ndash, 3-cells-thick layer in the subretinal space after intravitreal or subretinal injections, respectively. We did not observe increased photoreceptor survival nor decreased microglial cell numbers in the injected left eyes. However, the injected eyes showed decreased GFAP immunoreactivity. We conclude that intravitreal or subretinal injection of hBM-MSCs in dystrophic P23H-1 and RCS rats causes a decrease in retinal gliosis but does not have photoreceptor neuroprotective effects, at least in the short term. However, this treatment may have a potential therapeutic effect that merits further investigation.

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

Background and Aims Insulin resistance (IR)-related metabolic disorders, such as diabetes type 2 and obesity, are linked to renal and cardiovascular risk. Unfortunately, they lack a better prognosis before their full establishment. An important mechanism associated to the IR development is the dysregulation of lipolysis in white adipose tissue (WAT), which is driven by the excessive lipid accumulation, together with inflammation and the excessive deposition of ECM components such as collagens and fibronectin, known as fibrosis. The intracellular lipid adjustment is affected by changes in the expression and activity of the hormone-sensitive lipase (HSL) and the intracellular and circulating balance of the lipolysis products glycerol and free fatty acids. We published that transgenic depletion of the scaffold intracellular protein Integrin-linked kinase (ILK), one of the adhesome translators of the ECM signals, modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017]. We also published that ILK downregulation in WAT is an early event in a short-term high fat diet (HFD) mice model that correlates with a faster insulin sensitivity loss, accompanied with an early increase of body weight and the erratic expression of WAT adipokines and metabolites transporters. [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Here we used in vivo and in vitro approaches to decode the role of ILK downregulation during WAT expansion and altered lipolysis. Method Adult mice with global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. We determined the weight changes on body and WAT depots, epidydimal (eWAT) and subcutaneous (scWAT), and the circulating lypolisis product glycerol. The expression of ECM component fibronectin (FN), fibrosis marker TGF-beta and the glycerol transporter AQP3 were determined in eWAT by RT-qPCR or Western blot. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). We determined the expression of ILK, total and active HSL isoforms (HSL, p-HSL s660) by RT-qPCR or Western blot and the intracellular fat droplet content (by adipo-red dye) Results HFD increased progressively CT eWAT, scWAT and whole body weight gains. HFD-fed cKD-ILK have an earlier increase of body and eWAT weights. However, the scWAT weight ratio against eWAT was inverted in the same cKD-ILK animals. This early depot-specific response to HFD coincide with increase in IR [Wajchenberg BL. Endocr Rev 2000]. HFD-fed cKD-ILK WAT early expressed altered levels of fibrotic markers (TGF-beta and Fibronectin). HFD-fed cKD-ILK shown a temporary increase of circulating glycerol and its WAT transporter AQP3. In cultured adipocytes with downregulated expression of ILK, the intracellular fat droplet was increased and the HSL contain and its lipolysis activity (measured as levels of p-HSL isoform) were reduced. Conclusion We suggest ILK as a modulator during the obesity establishment. Considering the observed relation between ILK expression with the altered lipolysis and fibrosis patterns observed in visceral WAT, ILK downregulation may be a predictive value and probably its pharmacological upregulation may be a novel therapeutic strategy against obesity and IR.

Background and Aims Patients with CKD have a higher risk of developing cardiovascular diseases. Protein-bound uremic toxins, such as p-cresol (pc) and indoxyl-sulfate (IS), are retention solutes, poorly removed during hemodialysis. They lead to endothelial dysfunction inducing the expression of adhesion molecules and leucocyte adhesion to endothelium. Monocyte extravasation can be carried out by dynamic adhesion structures called podosomes. Integrin-linked kinase (ILK), a kinase and an intracellular scaffold protein, links the cell-adhesion receptors, integrins and growth factors to the actin cytoskeleton and to a range of signalling pathways. We demonstrate the role of ILK in the accumulation of integrin-associated proteins inside the podosomes. The aim of this study was to investigate if ILK is involved in uremic toxin-induced leucocyte podosome formation and adhesion under uremic conditions that simulate CKD. Method In vitro experiments were carried out in human cell line of leukemic monocytes, THP-1. We tested the effect of uremic toxins on cell viability and ILK expression levels or activation by performing dose and time-response experiments. Cells were exposed to IS (25-100 µg ml-1) plus pc (10-100 µg ml-1) both combined, for different times. ILK expression levels were determined by Western Blot and RT-qPCR and its kinase activity was tested by its downstream effector GSK-3β phosphorylated levels. Cell adhesion of THP-1 cells stained with cell tracker to a monolayer of human endothelial cells (EA.hy926) and podosome formation and cell adhesion of THP-1 cells stained with phalloidin to a fibronectin extracellular matrix, was determined by fluorescence confocal microscopy. ILK co-localization with podosome specific protein cortactin was assessed by fluorescence confocal microscopy. For ex vivo experiments in ILK conditional-knockdown mice (cKD-ILK), male CRE-LOX mice were injected with tamoxifen (cKD-ILK) or vehicle (wild-type, WT), to induce ILK deletion. Peripheral blood mononuclear cells (PBMCs) were obtained and treated with uremic toxins. Cell adhesion to a fibronectin extracellular matrix was determined by cell count of the percentage of attached cells against the total cells collected. Results Our data suggests that uremic toxins did not induce toxicity in THP-1 cells. Furthermore, ILK was activated by uremic toxins both at low and high concentrations, without changes in the protein expression. In the cell adhesion assay to the endothelium, uremic toxins induces an increase of THP-1 cells adhesion, which is completely abolished when ILK is knocked down by ILK siRNA, both at low and high toxins concentrations. Similar results were obtained from the cell adhesion assay to the fibronectin extracellular matrix. Moreover, uremic toxins induced podosome formation in THP-1 cells, even at low concentrations, compared to the control, while ILK knockdown abrogated almost completely fibronectin adhesion and podosome formation in uremic toxin-treated cells. Interestingly, we tested that ILK co-localize with cortactin in podosomes, which confirmed the implication of ILK in podosome structures formation induced by uremic toxins. In cKD-ILK PBMC mice the transgenic ILK depletion significantly decrease non-excised ILK mRNA levels. These cKD-ILK PBMC mice exhibited a lower adhesion to the fibronectin extracellular matrix compared to WT. By last, uremic toxins induced a significant increase of mice PBMCs adhesion in WT animals compared to the control that was significantly lower in PBMCs of cKD-ILK animals. Conclusion These data suggest that ILK plays a critical role in the required processes for leukocyte extravasation. ILK deletion may prevent podosome formation and adhesion of leucocytes, decreasing the endothelial and vascular damage caused by the accumulation of uremic toxins in CKD. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.

Background and Aims Renal ILK transgenic deletion on mice (cKD-ILK) address basal polyuria compatible with nephrogenic diabetes insipidus (NDI), due to disrupted tubular water channel aquaporin 2 (AQP2) AQP2 expression and vesicular trafficking to the apical membrane [Cano-Peñalver JL et al. FASEB J. 2014; Mamuya FA et al. Am J Physiol Renal Physiol. 2016]. An early symptom of chronic renal diseases (CKD) is NDI. ILK depletion also have a nephron-protective effect in renal damage in mice due to the increase in cGMP [Cano-Peñalver JL et al. Mol Med. 2016; de Frutos S et al. Biochim Biophys Acta Mol Basis Dis. 2019], the latter an alternative anti-NDI mediator. Here we demonstrate that ILK-mediated regulation of AQP2, under basal or CKD contexts, does not depend on cGMP. Method cKD-ILK and controls (WT) were treated orally with a NO donor, as precursor of cGMP formation (IDN, 300mg / Kg / day) for 24 hours. Urine volume was determined as a measure of tubular functionality. Fresh kidneys from non-treated mice were cultured ex vivo with precursors of cGMP (NO donor SNP, 1 µM or cGMP analogue 8Br-cGMP, 0.2 mM) for 30 minutes. AQP2 in the apical membrane of the tubules was quantified by immunohistochemistry. More cKD-ILK and WT were subjected for 6 weeks to a diet supplemented with 0.2% adenine (A) as CKD inducer or baseline standard diet. Urine volume and total ranal AQP2 levels were determined. Results Compared to WT, cGMP axis activation in vivo did not compensate the basal NDI present in cKD-ILK and the activation ex vivo did not improve the apical presence of AQP2 in cKD-ILK. Both WT and cKD-ILK subjected to CKD showed exacerbated polyuria and decreased levels of renal AQP2, but no differences were observed between groups. Conclusion ILK regulates AQP2, both in a basal and pathological context (CKD) independent of cGMP which otherwise is nephron-protective. Our results probably explain the co-existence of cGMP increase together with AQP2 decrease observed in the cKD-ILK kidneys.

Background and Aims Important mechanism associated to the development of Insulin resistance (IR) are dysregulation of lipid adjustment and excessive deposition of extracellular matrix (ECM) in the visceral white adipose tissue (WAT). The transgenic depletion of the ECM-to-cell scaffold intracellular protein Integrin-linked kinase (ILK) modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017] and body weight gain and adipose malfunction correlates with early downregulation of ILK expression in WAT during a short-term high fat diet (HFD) mice model [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Thus, we demonstrated that transcriptional regulation of ILK might govern the expansion of adipocytes. Between several transcriptional factors implicated in lipid metabolism, the activation of the transcription factor peroxisome proliferator activated receptor (PPAR) family member PPARβ/δ ameliorates IR. Taking in consideration that it modulates the expression of ILK in other contexts aside metabolism [Di-Poï N et al. J Steroid Biochem Mol Biol. 2003, Zhu B et al. Oncogene. 2014], here we activated PPARβ/δ in vivo and in vitro to upregulate adipose ILK expression and prevent WAT expansion. Method Adult mice were fed with either standard (STD) or HFD diets during 4 weeks and divided into groups treated with PPARβ/δ activator L-165041 (i.p. 2 mg/kg b.w per day) or vehicle for 2 additional weeks with maintained diet challenges [Lim HJ et al. Eur J Pharmacol. 2009]. Body and epidydimal WAT depot (eWAT) weights changes were compared and ILK expression was determined by RT-qPCR. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). After cells were treated with L-165041 (1 microM, 24h), we determined the intracellular triglyceride content (by adipo-red dye), the expression of ILK and its downstream substrate AKT (total and active p-AKT in ser473) by RT-qPCR or Western blot. Results HFD increased progressively eWAT and whole body weight gains, which coincide with increase in IR. PPARβ/δ-activated mice under STD or HFD have preventive reduction of weight gains and increased expression of ILK in eWAT. Downregulated ILK expression in culture adipocytes coincide with increased triglyceride content, whereas the activation of PPARβ/δ reduced it at the same time that increased ILK expression and activity. Conclusion We suggest ILK modulation as a novel therapeutic strategy during the obesity establishment. The pharmacological activation of PPARβ/δ increases ILK expression and activity, which correlates with a weight loss in IR-based models in vivo. Although the increased presence of ILK induces the phosphorylation of AKT, in turn one of the main mediators during insulin signalling, we cannot conclude the role of ILK-mediated AKT modulation during the reduction of the adipocyte expansion observed

Background and Aims Calpains are intracellular cysteine proteases that play a critical role in cell remodeling, being involved in multiple biological processes linked to tissue damage and repair mechanisms. In addition, they are released into the circulation, being able to carry out systemic actions with pathological consequences. The aim of this study was to investigate the role of calpains in the progression of chronic kidney disease (CKD) in an experimental model of chronic renal damage induced by adenine. Method We induced an experimental model of CKD, in mice fed for 2 weeks with an adenine-supplemented diet (0.2% adenine) (A). Animals receiving this diet develop a tubulointerstitial damage resembling that is observed in human CKD. Mice with standard diet were used as controls (C). Renal function was assessed by measuring serum blood urea nitrogen (BUN) and creatinine (mg/dl). Fibrosis markers (collagen type I and fibronectin) were determined by RT-qPCR. Changes in the renal content of calpains 1 and 2 were analyzed by western blot (protein content), and RT-qPCR (mRNA expression). Results Our results show functional and structural changes at renal level in the adenine-fed mice, with increased BUN (A: 72 mg/dl, C: 28 mg/dl, p < 0.05), creatinine (A: 0.58 mg/dl, C: 0.25 mg/dl, p < 0.05), collagen type I mRNA expression (A: 12.9 units, C: 1.2 units, p < 0.05) and fibronectin mRNA expression (A: 3.46 units, C: 1.3 units, p < 0.05). Furthermore, protein content of calpains 1 (A: 1.27 units, C: 0.78 units, p < 0.05) and 2 (A: 1.30 units, C: 0.66 units, p < 0.05) was significantly higher in adenine-fed mice when compared to control. At the same time, we observed a significant increase in gene expression of both calpain 1 (A: 4.21 units, C: 0.51 units, p < 0.05) and 2 (A: 4.93 units, C: 0.56 units, p < 0.05) in the adenine model regarding to mice with standard diet. Our results demonstrate that calpain 1 and 2 expression in renal tissue increases as CKD progresses. Interestingly, we found statistically significant correlations between renal calpains 1 and 2 protein and mRNA content and plasma BUN and creatinine (p < 0.05, r between 0.79 and 0.92), as well as protein expression of calpain 2 and mRNA expression of collagen type I (p < 0.05, r = 0.76). These data suggest a potential direct relationship between renal calpain 1 and 2 content and loss of renal function, in part due probably to the modulation of the fibrotic changes, in adenine fed mice. Conclusion We suggest an implication of calpains 1 and 2 in the development of CKD. Thus, effective calpain blockade or downregulation could be useful as a therapeutic strategy to prevent CKD. Further experiments will be necessary to establish the relationship between these factors.

Purpose To analyze the role of microglial and Muller cells in the formation of rings of photoreceptor degeneration caused by phototoxicity. Methods Two-month-old Sprague-Dawley rats were exposed to light and processed 1, 2, or 3 months later. Retinas were dissected as whole-mounts, immunodetected for microglial cells, Muller cells, and S- and L/M-cones and analyzed using fluorescence, thunder imaging, and confocal microscopy. Cone populations were automatically counted and isodensity maps constructed to document cone topography. Results Phototoxicity causes a significant progressive loss of S- and L/M-cones of up to 68% and 44%, respectively, at 3 months after light exposure (ALE). One month ALE, we observed rings of cone degeneration in the photosensitive area of the superior retina. Two and 3 months ALE, these rings had extended to the central and inferior retina. Within the rings of cone degeneration, there were degenerating cones, often activated microglial cells, and numerous radially oriented processes of Muller cells that showed increased expression of intermediate filaments. Between 1 and 3 months ALE, the rings coalesced, and at the same time the microglial cells resumed a mosaic-like distribution, and there was a decrease of Muller cell gliosis at the areas devoid of cones. Conclusions Light-induced photoreceptor degeneration proceeds with rings of cone degeneration, as observed in inherited retinal degenerations in which cone death is secondary to rod degeneration. The spatiotemporal relationship of cone death microglial cell activation and Muller cell gliosis within the rings of cone degeneration suggests that, although both glial cells are involved in the formation of the rings, they may have coordinated actions and, while microglial cells may be more involved in photoreceptor phagocytosis, Muller cells may be more involved in cone and microglial cell migration, retinal remodeling and glial seal formation.

Background/Aims: Diabetes type 2, metabolic syndrome or non-alcoholic fatty liver disease are insulin resistance-related metabolic disorders, which lack a better prognosis before their full establishment. We studied the importance of the intracellular scaffold protein integrin linked kinaes (ILK) as a key modulator in the initial pathogenesis and the early progression of those insulin resistance- related disorders. Methods: Adult mice with a global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. Weights, blood glucose and other systemic biochemical parameters were determined in animals under fasting conditions and after glucose or pyruvate intraperitoneal injections to test their tolerance. In RNA or proteins extracted from insulin-sensitive tissues, we determined by reverse transcription?quantitative PCR and western blot the expression of ILK, metabolites transporters and other metabolism and inflammatory markers. Glucose uptake capacity was studied in freshly isolated tissues. Results: HFD feeding was able to early and progressively increase glycaemia, insulinemia, circulating glycerol, body weight gain, liver-mediated gluconeogenesis along this time lapse, but cKD-ILK have all these systemic misbalances exacerbated compared to CT in the same HFD time lapse. Interestingly, the tisular expression of ILK in HFD-fed CT was dramatically downregulated in white adipose tissue (WAT), skeletal muscle and liver at the same extent of the original ILK downregulation of cKD-ILK. We previously published that basal STD-fed cKD-ILK compared to basal STD-CT have different expression of glucose transporters GLUT4 in WAT and skeletal muscle. In the same STD-fed cKD-ILK, we observed here the increased expressions of hepatic GLUT2 and WAT pro-inflammatory cytokines TNF-? and MCP-1. The administration of HFD exacerbated the expression changes in cKD-ILK of these and other markers related to the imbalanced metabolism observed, such as WAT lipolysis (HSL), hepatic gluconeogenesis (PCK-1) and glycerol transport (AQP9). Conclusion: ILK expression may be taken as a predictive determinant of metabolic disorders establishment, because its downregulation seems to correlate with the early imbalance of glucose and glycerol transport and the subsequent loss of systemic homeostasis of these metabolites., Instituto de Salud Carlos III-ISCIII, Comunidad de Madrid, Fondo Europeo de Desarrollo Regional-FEDER, Instituto Ramon y Cajal de Investigación Sanitária-IRYCIS, Fundación Renal Iñigo Álvarez de Toledo-FRIAT

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. This study was supported by the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional “Una manera de hacer Europa” (PI16/00031, PI16/00380, PI19/00071, PI19/00203, SAF2015-67643-P, RD16/0008, RD16/0008/0026 and RD16/0008/0016) and by the Fundación Séneca, Agencia de Ciencia y Tecnología Región de Murcia (19881/GERM/15).

Retinal degenerative diseases affecting the outer retina in its many forms (inherited, acquired or induced) are characterized by photoreceptor loss, and represent currently a leading cause of irreversible vision loss in the world. At present, there are very few treatments capable of preventing, recovering or reversing photoreceptor degeneration or the secondary retinal remodeling, which follows photoreceptor loss and can also cause the death of other retinal cells. Thus, these diseases are nowadays one of the greatest challenges in the field of ophthalmological research. Bone marrow derived-mononuclear stem cell transplantation has shown promising results for the treatment of photoreceptor degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore should be applied in the early stages of photoreceptor degenerations. Furthermore, because of their possible paracrine effects, they may have a wide range of clinical applications, since they can potentially impact on several retinal cell types at once and photoreceptor degenerations can involve different cells and/or begin in one cell type and then affect adjacent cells. The intraocular injection of bone marrow derived-mononuclear stem cells also enhances the outcomes of other treatments aimed to protect photoreceptors. Therefore, it is likely that future investigations may combine bone marrow derived-mononuclear stem cell therapy with other systemic or intraocular treatments to obtain greater therapeutic effects in degenerative retinal diseases.

In this work we study the effects of an acute light-induced retinal degeneration on the population of melanopsin positive retinal ganglion cells (m+RGCs) and the expression of the melanopsin protein in the retina. The m+RGCs may be more resistant than other RGCs to lesion, but the effects of an acute light exposure in this population are unknown. Albino rats were exposed to white light (3000 lux) continuously for 48 h and processed 0, 3, 7 or 30 days after light exposure (ALE). Whole-mounted retinas were immunodetected with antibodies against melanopsin, Brn3a, and rhodopsin to study the populations of m+RGC, Brn3a+RGC and rods (which are the most abundant photoreceptors in the rat retina). Three days ALE there was substantial rod loss in an arciform area of the superior retina and with time this loss expanded in the form of rings all throughout the retina. Light exposure did not affect the number of Brn3a+RGCs but diminished the numbers of m+RGCs. Immediately ALE there was a significant decrease in the mean number of immunodetected m+RGCs that was more marked in the superior retina. Later, the number of m+RGCs increased progressively and reached normal values one month ALE. Western blot analysis showed that melanopsin expression down-regulates shortly ALE and recovers thereafter, in accordance with the anatomical data. This study demonstrates that there is a transient downregulation of melanopsin expression in the RGCs during the first month ALE. Further studies would be needed to clarify the long-term effect of light exposure on the m+RGC population.

Purpose To develop a focal photoreceptor degeneration model by blue light-emitting diode (LED)-induced phototoxicity (LIP) and investigate the protective effects of topical brimonidine (BMD) or intravitreal brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (bFGF). Methods In anesthetized, dark-adapted, adult female Swiss mice, the left eye was dilated and exposed to blue light (10 seconds, 200 lux). After LIP, full-field electroretinograms (ERG) and spectral-domain optical coherence tomography (SD-OCT) were obtained longitudinally, and reactive-Iba-1+monocytic cells, TUNEL+ cells and S-opsin+ cone outer segments were examined up to 7 days. Left eyes were treated topically with BMD (1%) or vehicle, before or right after LIP, or intravitreally with BDNF (2.5 μg), CNTF (0.2 μg), bFGF (0.5 μg), or corresponding vehicle right after LIP. At 7 days, S-opsin+ cone outer segments were counted within predetermined fixed-size areas (PFA) centered on the lesion in both flattened retinas. Results SD-OCT showed a circular region in the superior-temporal left retina with progressive thinning (207.9 ± 5.6 μm to 160.7 ± 6.8 μm [7 days], n = 8), increasing TUNEL+ cells (peak at 3 days), decreasing S-opsin+ cone outer segments, and strong microglia activation. ERGs were normal by 3 days. Total S-opsin+ cones in the PFA for LIP-treated and fellow-retinas were 2330 ± 262 and 5601 ± 583 (n = 8), respectively. All neuroprotectants (n = 7-11), including topical BMD pre- or post-LIP, or intravitreal BDNF, CNTF, and bFGF, showed significantly greater S-opsin+ cone survival than their corresponding vehicle-treated groups. Conclusions LIP is a reliable, quantifiable focal photoreceptor degeneration model. Topical BMD or intravitreal BDNF, CNTF, or bFGF protect against LIP-induced cone-photoreceptor loss. Translational relevance Topical BMD or intravitreal BDNF, CNTF, or bFGF protect cones against phototoxicity.

International audience; To study the effect of taurine depletion induced by β-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 β-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 RFNL with phosphorylated high-molecular-weight subunit of the neurofilament triplet (pNFH) antibodies. β-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 β-alanine supplements due to the limited understanding of their potential adverse effects.

Purpose The purpose of this study was to study the effect of minocycline and several neurotrophic factors, alone or in combination, on photoreceptor survival and macro/microglial reactivity in two rat models of retinal degeneration. Methods P23H-1 (rhodopsin mutation), Royal College of Surgeon (RCS, pigment epithelium malfunction), and age-matched control rats (Sprague-Dawley and Pievald Viro Glaxo, respectively) were divided into three groups that received at P10 for P23H-1 rats or P33 for RCS rats: (1) one intravitreal injection (IVI) of one of the following neurotrophic factors: ciliary neurotrophic factor (CNTF), pigment epithelium-derived factor (PEDF), or basic fibroblast growth factor (FGF2); (2) daily intraperitoneal administration of minocycline; or (3) a combination of IVI of FGF2 and intraperitoneal minocycline. All animals were processed 12 days after treatment initiation. Retinal microglial cells and cone photoreceptors were immunodetected and analyzed qualitatively in cross sections. The numbers of microglial cells in the different retinal layers and number of nuclei rows in the outer nuclear layer (ONL) were quantified. Results IVI of CNTF, PEDF, or FGF2 improved the morphology of the photoreceptors outer segment, but only FGF2 rescued a significant number of photoreceptors. None of the trophic factors had qualitative or quantitative effects on microglial cells. Minocycline treatment reduced activation and migration of microglia and produced a significant rescue of photoreceptors. Combined treatment with minocycline and FGF2 had higher neuroprotective effects than each of the treatments alone. Conclusions In two animal models of photoreceptor degeneration with different etiologies, minocycline reduces microglial activation and migration, and FGF2 and minocycline increase photoreceptor survival. The combination of FGF2 and minocycline show greater neuroprotective effects than their isolated effects.

International audience; Purpose: To examine if light exposure exacerbates retinal neuronal loss induced by taurine depletion.Methods: Albino rats received β-alanine in the drinking water to induce taurine depletion. One month later, half of the animals were exposed to white light (3000 lux) continuously for 48 hours and the rest remained in normal environmental conditions. A control group of animals nontreated with β-alanine also was prepared, and half of them were exposed to light using the same protocol. All the animals were processed 2 months after the beginning of the experiment. Retinas were dissected as wholemounts and immunodetected with antibodies against Brn3a, melanopsin, S-opsin, and L-opsin to label different retinal populations: Brn3a+ retinal ganglion cells (RGCs) (image-forming RGCs), m+RGCs (non-image-forming RGCs), and S- and L/M-cones, respectively.Results: Light exposure did not affect the numbers of Brn3a+RGCs or m+RGCs but diminished the numbers of S- and L/M-cones and caused the appearance of rings devoid of cones, mainly in an "arciform" area in the superotemporal retina. Taurine depletion caused a diminution of all the studied populations, with m+RGCs the most affected, followed by S-cones. Light exposure under taurine depletion increased photoreceptor degeneration but did not seem to increase Brn3a+RGCs or m+RGCs loss.Conclusions: Our results document that taurine is necessary for cell survival in the rat retina and even more under light-induced photoreceptor degeneration. Thus, taurine supplementation may help to prevent retinal degenerations, especially those that commence with S-cone degeneration or in which light may be an etiologic factor, such as inherited retinal degenerations, AMD, or glaucoma.

Light causes damage to the retina (phototoxicity) and decreases photoreceptor responses to light. The most harmful component of visible light is the blue wavelength (400–500 nm). Different filters have been tested, but so far all of them allow passing a lot of this wavelength (70%). The aim of this work has been to prove that a filter that removes 94% of the blue component may protect the function and morphology of the retina significantly. Three experimental groups were designed. The first group was unexposed to light, the second one was exposed and the third one was exposed and protected by a blue-blocking filter. Light damage was induced in young albino mice (p30) by exposing them to white light of high intensity (5,000 lux) continuously for 7 days. Short wavelength light filters were used for light protection. The blue component was removed (94%) from the light source by our filter. Electroretinographical recordings were performed before and after light damage. Changes in retinal structure were studied using immunohistochemistry, and TUNEL labeling. Also, cells in the outer nuclear layer were counted and compared among the three different groups. Functional visual responses were significantly more conserved in protected animals (with the blue-blocking filter) than in unprotected animals. Also, retinal structure was better kept and photoreceptor survival was greater in protected animals, these differences were significant in central areas of the retina. Still, functional and morphological responses were significantly lower in protected than in unexposed groups. In conclusion, this blue-blocking filter decreases significantly photoreceptor damage after exposure to high intensity light. Actually, our eyes are exposed for a very long time to high levels of blue light (screens, artificial light LED, neons…). The potential damage caused by blue light can be palliated. This study was supported by project grants #SAF2010-21879 from Ministerio de Ciencia e Innovación (http://www.idi.mineco.gob.es), FIS #PI13-02098 and RETICS #RD12-0034-0006 from Instituto de Salud Carlos III (http://www.isciii.es) to PdV; #BES-2005-8339 from Ministerio de Educación y Ciencia to JV-T; #PTQ-10-03588 from Ministerio de Ciencia e Innovación (http://www.idi.mineco.gob.es) to CS-R.

To investigate the long-term effects of laser-photocoagulation (LP)-induced ocular hypertension (OHT) in the innermost and outermost (outer-nuclear and outer segment)-retinal layers (ORL). OHT was induced in the left eye of adult rats. To investigate the ganglion cell layer (GCL) wholemounts were examined at 1, 3 or 6 months using Brn3a-immunodetection to identify retinal ganglion cells (RGCs) and DAPI-staining to detect all nuclei in this layer. To study the effects of LP on the ORL up to 6 months, retinas were: i) fresh extracted to quantify the levels of rod-, S- and L-opsin; ii) cut in cross-sections for morphometric analysis, or; iii) prepared as wholemounts to quantify and study retinal distributions of entire populations of RGCs (retrogradely labeled with fluorogold, FG), S- and L-cones (immunolabeled). OHT resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3a+RGCs but with large numbers of DAPI+nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of ORL degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the S- and L-cone populations diminished to 65% and 80%, or to 20 and 35% at 1 or 6 months, respectively. In conclusion, LP induces in the GCL selective RGCs loss that does not progress after 1 month, and S- and L-cone loss that progresses for up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL.