Müller glial cells located in the peripheral retina are more susceptible to high pressure: implications for glaucoma

Pereiroetal. Cell & Bioscience (2024) 14:5 https://doi.org/10.1186/s13578-023-01186-1 RESEARCH Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cell & Bioscience Müller glial cells located intheperipheral retina are more susceptible tohigh pressure: implications forglaucoma Xandra Pereiro1,3* , Noelia Ruzafa1, Mikel Azkargorta2, Félix Elortza2, Arantxa Acera1, António Francisco Ambrósio3,4,5,6, Ana Raquel Santiago3,4,5,6 and Elena Vecino1* Abstract Background Glaucoma, a progressive neurodegenerative disease, is a leading cause of irreversible vision loss worldwide. This study aims to elucidate the critical role of Müller glia (MG) in the context of retinal ganglion cell (RGC) death, particularly focusing on the influence of peripheral MG sensitivity to high pressure (HP). Methods Co-cultures of porcine RGCs with MG were isolated from both the central and peripheral regions of pig retinas and subjected to both normal and HP conditions. Mass spectrometry analysis of the MG-conditioned medium was conducted to identify the proteins released by MG under all conditions. Results Peripheral MG were found to secrete a higher quantity of neuroprotective factors, effectively promoting RGC survival under normal physiological conditions. However, under HP conditions, co-cultures with peripheral MG exhibited impaired RGC survival. Moreover, under HP conditions, peripheral MG significantly upregulated the secretion of proteins associated with apoptosis, oxidative stress, and inflammation. Conclusions This study provides robust evidence suggesting the involvement of MG in RGC death in glaucoma, thus paving the way for future therapeutic investigations. Keywords Müller glia, Retinal ganglion cells, Glaucoma, Retina, Neurodegeneration Background Retinal ganglion cells (RGCs) are the neurons that transmit visual information from the eye to the brain and thus, their survival is critical for vision. These neurons are very sensitive to insults, and damage to RGC axons in the optic nerve may lead to rapid RGC death in acute diseases like ischemic optic neuropathy or optic neuritis, or in chronic diseases like glaucoma [1]. Different RGC subtypes respond distinctly to stimuli and insults, and not as a single entity, such that their type-specific vulnerability has been studied extensively. As such, early functional alterations to certain subtypes of RGCs have been proposed [2–5]. In glaucoma, there is a greater loss of large RGCs in the peripheral retina, which also occurs *Correspondence: Xandra Pereiro [email protected] Elena Vecino [email protected] 1 Experimental Ophthalmo-Biology Group, Department of Cell Biology and Histology, University of the Basque Country UPV/EHU, 48940 Leioa, Spain 2 Proteomics Platform, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), CIBERehdProteoRed-ISCIII, Bizkaia Science and Technology Park, 48160 Derio, Spain 3 Faculty of Medicine, Coimbra Institute for Clinical and Biomedical Research (iCBR), University of Coimbra, Coimbra, Portugal 4 Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Coimbra, Portugal 5 Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal 6 Association for Innovation and Biomedical Research on Light and Image (AIBILI), Coimbra, Portugal Page 2 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 in animal models of glaucoma that resemble the features described in glaucoma patients [6, 7]. Müller Glia (MG) are the principal macroglial cells in the retina. They are radially oriented and span the entire thickness of the retina. These cells are responsible for the homeostatic and metabolic support of retinal neurons, making them essential for neuronal survival [8]. Reactive MG can maintain extracellular homeostasis during retinal insult or damage, thereby protecting retinal neurons [9]. These neuroprotective effects of MG may be mediated by diverse mechanisms, enhancing physiological functions like glutamate or potassium uptake. Moreover, neuronal survival can be further preserved through the release of neuroprotective factors by MG [10–12]. MG also represent a heterogeneous population, in which some cells participate more closely in retinal repair than others. Although little is known about the heterogeneity of MG, only one subset of MG express the Chx10 transcription factor [13], while 30% of MG express class II MHC antigen invitro, suggesting that they might be involved in immune reactions [14]. In terms of their distribution in the retina, MG morphology varies in relation to retinal topography [15]. The central retina is thicker than the periphery, with a greater density of neurons and MG. Moreover, MG from the central retinal are longer and thinner than those in the periphery, and they have a smaller volume but a higher surface to volume ratio [16]. In the chick retina, the region in which proliferating MG accumulate in response to retinal damage becomes mainly confined to the periphery [17]. Two types of MG were distinguished in the chick retina, referred to as type I cells with numerous thin processes, and type II cells with fewer and thicker processes [18, 19]. When characterized further, the type I cells were seen to be in the majority across the retina, whereas type II MG were mainly found in the peripheral retina [20]. Moreover, while mammalian MG lose the capacity to proliferate and regenerate, unlike other vertebrates as fish, peripheral MG express proteins characteristic of stem cells after damage, such as CD44 [21] and the neural progenitor marker, nestin [22]. In the human retina, the microenvironment of the central retina not only has specific characteristics with respect to the periphery but also, it is more susceptible to certain diseases [23]. Phosphoglycerate dehydrogenase, the rate-limiting enzyme in serine synthesis, is expressed more in MG localized in the macula than in peripheral MG, suggesting higher susceptibility of macular cells to oxidative stress than peripheral cells [24]. Macular MG also express more aquaporin-4 than peripheral MG [25]. Furthermore, in a mouse model of laser-induced glaucoma, the strongest activation of macroglial cells was in the intermediate and peripheral retina relative to the central retina [26]. MG isolated from the central or peripheral retina consistently respond in a different manner to the conditions to which they are exposed, indicating molecular differences between the MG from these two different locations [24]. The differences in protein expression among MG at distinct retinal locations may indicate that they fulfill different functions, although this still needs to be extensively studied. MG are considered the principal retinal sensor that can respond promptly to mechanical changes [27]. The heterogeneity of MGs and their ability to sense pressure could explain the differences in susceptibility to death of peripheral RGCs in glaucoma. As such, MGs may play an important role and could offer insights into the potential causes of the onset of glaucomatous damage. It is important to know if the MG located in the retinal area where the RGCs die first in glaucoma behave differently to those in other retinal areas and if they are also more vulnerable to changes in intraocular pressure (IOP). To address this issue and based on our experience in handling MG and RGCs in culture, we first studied, using co-cultures, the interaction of MG isolated from the peripheral and central retina with RGCs, focusing on the survival of these cells. Subsequently, we examined the effect of HP on these cocultures using central and peripheral MG, focusing on the susceptibility to death of both MG and RGC. Finally, we compared the proteome of MG conditioned medium in the experimental conditions tested, exposure or not to HP, to identify proteins secreted by MG from the central and peripheral retina. Thus, here we examined the possible different protective effect of MG located in the central or peripheral retina on RGCs, identifying proteins that may be involved in these events. The data obtained led us to suggest that peripheral MG may be involved in the initiation of glaucomatous damage. Methods Animals This study was carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals. All the experimental protocols complied with the European (2010/63/UE) and Spanish (RD53/2013) regulations for the protection of experimental animals, and they were approved by the Ethical Committee for Animal Welfare at the University of Basque country. All animal experimentation adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For MG cultures, adult porcine eyes were obtained from a local abattoirs and transported to the laboratory in cold CO2-independent medium (Life Technologies, Carlsbad, CA, USA) containing 0.1% gentamicin (Life Technologies, Carlsbad, CA, USA). For pure RGC cultures, eyes were obtained from adult female Page 3 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 Sprague–Dawley rats (200–250 g) housed on a 12 h light–dark cycle with adlibitum access to food and water, and they were sacrificed humanely by exposure to CO2. Porcine retinal Müller glia cultures MG cultures were prepared as described previously [28]. First, dissecting adult porcine eyes within 2 h of enucleation. Briefly, the major blood vessels were removed and the retina was washed in CO2-independent medium. The retinas were dissected out and two different areas of the retina were obtained with an 8mm diameter dissecting trephine (Biomedical Research Instruments, Silver Spring, MD, USA): central and peripheral. The retinal tissue was dissociated for 30min at 37°C in 0.2% activated papain (Worthington, Lakewood, NJ, USA) with 10% DNAse I (Worthington, Lakewood, NJ, USA). Enzyme activity was stopped by the addition of MG medium (see below) and DNase I. The tissue was then disaggregated by gentle trituration using pipette tips of decreasing diameter. Purified MG were cultured in DMEM (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS: Life Technologies, Carlsbad, CA, USA). MG and RGCs were co-cultured in Neurobasal A medium (NBA: Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS and 2% B27 (Life Technologies, Carlsbad, CA, USA). In addition, 1% l-glutamine (2 mM: Life Technologies, Carlsbad, CA, USA) and 0.1% gentamicin (50mg/mL: Life Technologies, Carlsbad, CA, USA) were added to the culture media of purified MG and to co-cultures. Dissociated cells were pelleted by centrifugation (1200 rpm, 5 min), resuspended in MG medium and plated on poly-l-lysine (100μg/mL: Sigma-Aldrich, St. Louis, MO, USA) and laminin (10μg/mL: Sigma-Aldrich, St. Louis, MO, USA) coated 13 mm diameter glass coverslips in 24-well plates. The cells were maintained in a humidified incubator at 37°C in an atmosphere of 5% CO2. Cells in suspension were removed after 24h by changing the medium. For maintenance, half of the medium was replaced every 2–3days. Conditioned medium (CM) from MG cultures was collected when the cultures reached confluence at day 7. First carefully washing the wells three times with NBA medium, and subsequently adding fresh NBA medium to each well for 3h before the medium was changed to eliminate the rest of the FBS and B27. Fresh NBA medium was added and conditioned over 2days before it was collected sterilized by passing through a 0.22μm filter and frozen in aliquots at − 20°C. Finally, the MG were fixed for 10min with methanol at − 20°C. At least three replicates of each culture were set-up and the procedure was performed at least in triplicate. Purified RGC cultures RGC cultures were prepared as described previously [29] from a mixed suspension of retinal cells obtained from pig or rat retinas. Pig RGCs were obtained from the center and periphery of the retina, and rat RGCs were obtained from the entire retina. Retinal tissue was dissociated enzymatically using the Papain Dissociation Kit (Worthington Biochemical, Lakewood, NJ, USA), following the manufacturer’s instructions, namely digesting the tissue for 90min at 37°C in 0.2% activated papain with 10% DNAse I. After gentle trituration using pipette tips of decreasing diameter to disaggregate the tissue, and after using a density gradient, the purified RGCs were plated on 13 mm poly-l-lysine (100 μg/ mL, Sigma-Aldrich, St. Louis, MO, USA) and laminin (10μg/mL: Sigma-Aldrich, St. Louis, MO, USA) coated glass coverslips in 24‐well plates. The pig and rat cells were then seeded at 1 × 105 viable cells per well and the cultures were maintained in Neurobasal A medium (Life Technologies, Carlsbad, AC, USA) supplemented with 2% B27, 1% l-glutamine (2mm: Life Technologies, Carlsbad, CA, USA) and 0.1% gentamicin (50mg/mL: Life Technologies, Carlsbad, CA, USA). Rat RGCs were seeded on confluent MG cultures from the center or periphery pig retina. In addition, RGCs were also cultivated adding either CM obtained from cultures of central or peripheral MG, collected in NBA plus NBA/B27 (1:1). As control, rat RGCs were cultured in NBA/B27. All the media used contained 1% l-glutamine and 0.1% gentamicin. RGC cultures were maintained for 6days at 37°C in a humidified atmosphere containing 5% CO2, and the medium was changed every 3days. Finally, the RGCs were fixed for 10min with methanol at − 20°C on day 6. At least four were performed for each analysis described, repeating each independent experiment a minimum of three times. Cultures exposed tohigh pressure Cultures were subjected to high pressure (HP) using a custom-made humidified pressure chamber equipped with a pressure gauge and a pressure regulator as described previously with minor changes [30]. This set-up as allowed a constant pressure to be maintained with an air mixture of 95% air, 5% CO2 and 2%O2. The pressure chamber was placed in an oven at 37°C and the cultures were maintained under HP for 72h. The magnitude of pressure elevation (70 mmHg above atmospheric pressure) was chosen in accordance with previous studies [31]. Control cultures were kept at normal atmospheric pressure in a standard cell incubator, and at least three experimental replicates Page 4 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 were analyzed, performed in triplicate independent experiments. Immunocytochemistry After fixation in methanol and washing with PBS (phosphate buffered saline, pH 7.0), the binding of nonspecific antigens was blocked with blocking buffer (3% BSA and 0.1% Triton X-100 in PBS) before incubating the cells overnight at 4°C with the primary antibodies (see Table1) diluted in blocking buffer. After washing, the binding of these antibodies were detected with Alexa Fluor 555 or 488 conjugated goat anti-mouse and goat anti-rabbit antibodies (diluted 1:1000: Life Technologies, Carlsbad, CA, USA). Cells were finally counterstained with the nuclear marker DAPI (diluted 1:10,000: Life Technologies, Carlsbad, CA, USA). After washing, the coverslips were mounted using Fluor-save Reagent (Sigma-Aldrich, St. Louis, MO, USA). Quantification ofRGCs andMG RGCs were observed in an epifluorescence microscope (Zeiss, Jena, Germany) coupled to a digital camera (Zeiss Axiocam MRM, Zeiss, Jena, Germany). All images were obtained under the same conditions of intensity and exposure time. At least three coverslips were analyzed for each experimental condition and from a minimum of three independent experiments. The density of the RGC cultures was quantified. In addition, the RGCs were classified as: (1) cells with no neurites; (2) cells with a longest neurite < 50μm; (3) cells with the longest neurite between 50 and 200μm; and (4) cells with neurites longer than 200μm. The total number of RGCs surviving in each condition was counted. The MG present in the cultures were also analyzed and counted in images. Semiautomatic Zen software (Zeiss, Jena, Germany) was used to count the number of nuclei stained with DAPI, taking into consideration the limits of the axis of the MG nuclei to obtain more accurate measurements. As such, we used a macro designed to specifically measure the limits of the axes (55–70μm), which was corrected manually for each image. Mass spectrometry analysis oftheCM anddata processing The proteomic analysis of the CM obtained of the central and peripheral MG cultures under control and HP conditions was carried out at the CIC bioGUNE Proteomics Service (Derio, Bizkaia, Spain), using the Filter Aided Sample Preparation (FASP) protocol [32], with minor modifications. After solution digestion, the proteins were extracted in a sample containing 7M urea, 2 M Thiourea, 4% CHAPS and 5mM DTT. Trypsin was added at a trypsin: protein ratio of 1:20, and the mixture was incubated overnight at 37°C, dried in a RVC2 25 speedvac concentrator (Christ, Osterode am Harz, Germany), and resuspended in 0.1% formaldehyde (FA). Peptides were desalted and resuspended in 0.1% FA using C18 stage tips (Merck Millipore, Burlington, MA, USA). The samples were analyzed in a timsTOF Pro with PASEF (Bruker, Billerica, MA, USA) apparatus coupled online to a Evosep ONE liquid chromatograph (Evosep Biosystems, Odense, Denmark), loading 200ng directly onto the Evosep ONE and employing a 60 samples-per-day protocol. Protein identification and quantification was carried out using PEAKS X software (Bioinformatics solutions, Waterloo, Canada), carrying out searches against a database consisting of Sus scrofa entries from UniProt (https:// www. unipr ot. org/), with precursor and fragment tolerances of 20ppm and 0.05Da. Only proteins identified with at least two peptides at a False Detection Rate (FDR) of 1% were considered for further analysis. Protein abundances inferred from PEAKS were loaded onto the Perseus platform, log2 transformed and imputed before analyzing with a Student’s t-test. Proteins that were considered significantly different between the groups were those with a p-value < 0.05, and also those that exceeded that value, up to a p-value of 0.1, but with a fold change > 2 for each comparison analyzed. The proteins listed were ordered according to the fold change obtained, and the proteins selected, out of a total of 893 proteins, were categorized based on their functions attributed in the UniProt database. In addition, for some proteins of particular interest in this study, an in silico analysis was carried out using the freely available STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database (https:// stringdb. org/). The number of protein–protein interactions registered in the database was determined for the proteins that were differentially overexpressed. For visualization, a diagram was assembled linking the proteins depicted by nodes Table 1 Primary antibodies used αSMA α-smooth muscle actin, TRPV4 transient receptor potential cation channel subfamily V member 4 Antigen Target Host Dilution Supplier α-SMA Dedifferentiation Mouse 1:1000 Abcam β-III-Tubulin RGCs Rabbit 1:2000 Promega β-Catenin Dedifferentiation Rabbit 1:2000 Abcam CD-133 Dedifferentiation Rabbit 1:200 Abcam Nestin Dedifferentiation Rabbit 1:500 Sigma OCT4 Dedifferentiation Rabbit 1:200 Abcam p75NTR Müller glia Rabbit 1:2000 Abcam Piezo1 Mechanosensor Rabbit 1:100 Life Technologies Trpv4 Mechanosensor Rabbit 1:500 Life Technologies Vimentin Müller glia Mouse 1:10,000 Dako Page 5 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 based on recognized connections with the proteins identified. Statistical analysis The experimental procedures were replicated at least three times to ensure the reliability and consistency of the findings. The cell density was defined as the mean number of cells per cm2, and the mean and standard error of mean (SEM) are presented for each condition. Statistical analyses were carried out using the IBM SPSS Statistical software v.24-0. The data from the different experimental conditions were compared using the nonparametric Mann–Whitney U test. When more than two independent groups were compared, a Kruskal–Wallis non-parametric test was used, and if the Kruskal–Wallis test was significant, a post-hoc Dunn test was performed in order to determine which groups differ from the others. Differences were considered significant for all tests at a p-value < 0.05. Results The survival of pig RGCs from the center or from the periphery of the retina was analyzed in purified cultures. Cultured RGCs from the central retina survived significantly better (173 ± 27 RGCs/cm2) than those from the peripheral retina (87 ± 11 RGCs/cm2, Fig.1A). When the effect of MG, isolated from the center or periphery of the pig retina, on RGC survival was analyzed in co-cultures, the survival of peripheral RGCs and MG (3389 ± 471 and 61,876 ± 10,509 cells/cm2, respectively) was significantly increased comparing to co-cultures with MG and RGCs isolated from the central retina (1566 ± 484 RGCs/cm2 and 33,232 ± 3181 MG/cm2: Fig.1B). To confirm the more robust neuroprotective effect of the MG isolated from the peripheral retina, purified rat RGCs were seeded onto a monolayer of MG from the central and peripheral retina already cultured for 7days invitro (DIV). The survival of the rat RGCs increased significantly (1914 ± 176 RGCs/cm2) when they were seeded on peripheral MG rather than on central retinal MG (987 ± 139 RGCs/cm2) after 6days in co-culture (Fig.1C). To assess whether factors secreted by peripheral MG may have the same effect on RGC survival as MG monolayers, rat RGCs were cultured in CM from the central and peripheral MG. RGCs were cultured in NBA/B27 (control) or in NBA/ B27 medium:CM (1:1) obtained from either central or peripheral MG in culture. More RGCs survived when the cells were maintained in CM from peripheral MG (249 ± 48 RGCs/cm2) than in the CM from central retinal MG (114 ± 25 RGCs/cm2), or control RGCs without CM (71 ± 2 RGCs/cm2) (Fig.1D). The RGC neurite length was assessed to determine whether the different CM affected these parameters (Fig.1D). When RGCs were maintained in CM secreted by peripheral MG, there were more RGCs with short neurites (< 50μm, 147 ± 32 RGCs/cm2) than when RGCs were maintained in CM by MG from the central retina (61 ± 13 RGCs with short neurites/cm2). To determine if the central or peripheral MG behave differently in culture, the expression of certain stem cell-like markers was analyzed in purified MG cultures: vimentin (MG specific marker), β-III-Tubulin (neural marker), α-SMA (dedifferentiation marker), CD133 (glial stem cell marker), OCT4 (important in MG reprogramming during retinal regeneration in zebrafish), nestin (marker for glial and neuronal progenitors), and β-catenin (stem cell-like marker). Although the MG specific marker vimentin was expressed strongly in both types of cultures, β-III-Tubulin, α-SMA, CD133, OCT4, nestin, and β-catenin were expressed more by MG from the peripheral retina than by central MG. Hence, peripheral MG appear to be more dedifferentiated towards a stem cell-like phenotype (Fig.2). The analysis of the proteome of CM obtained from the central and peripheral MG was performed, identifying the expression of more stem cell-like markers in the CM of peripheral MG, and also more neuroprotective molecules. The proteome of the MG CMs contained differentially expressed proteins (DEPs), with 22 more DEPs in the central MG CM and 113 proteins more strongly expressed in the peripheral CM. All proteins more strongly expressed in the CM of peripheral MG, along with their associated functions: “Cytoskeleton, cell adhesion and cell shape”; “Inflammation and immune response”; “Survival and homeostasis”; “Neuroprotection and neurite outgrowth”; “Proliferation”; “Ubiquitination”; “Angiogenesis”; “Dedifferentiation”; “Transport”; “Apoptosis and proliferation inhibition”; “Oxidative stress and stress response”; and “Other functions” (Table2). When the proteins sorted by function were compared between central and peripheral CM (Fig.3A), the proportion of proteins in the peripheral CM related to “Proliferation” and “Cytoskeleton, cell adhesion, cell shape” were 14.2% and 24.8% of total proteins identified, respectively, while in central CM only 4.6% of proteins were related to these functions. Another interesting function for the present study is “Neuroprotection and neurite outgrowth” and as expected, the proteins related to this role were more strongly expressed in the peripheral CM, representing 13.3% of the proteins. Another function worth highlighting is “Dedifferentiation” and although the proportion of these proteins was not very high (3.5%), they only appeared in the peripheral CM, consistent with the expression of the stem cell-like markers detected (Fig.2). A STRING analysis was also performed on some of the proteins from the “Proliferation”, “Neuroprotection and Page 6 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 neurite outgrowth” and “Dedifferentiation” categories identified in our proteomic analysis. Again, some of the proteins identified in the STRING analysis were more strongly represented in the peripheral CM (Fig.3B). The effect of HP on RGC and MG survival was analyzed in co-cultures from the central and peripheral retina. After 72h of HP, central RGC survival decreased to 65.5 ± 8.0%. Surprisingly, the survival of peripheral RGCs was more strongly affected, decreasing to 41.2 ± 5.3% relative to the control. The same applied to the MG, with 82.7 ± 15. % of the central MG surviving after 72h of HP, and decreasing to 69.6 ± 13.6% when the MG were from the periphery of the retina (Fig.4). To confirm the effect of HP on the susceptibility of peripheral MG, purified control rat RGCs were seeded on a MG monolayer from the central or peripheral pig retina at 7 DIV, and exposed to HP for 72h. The survival of the rat RGCs, comparing to the control, decreased Fig. 1 A The survival of central or peripheral pig RGCs in purified cultures. Images of purified RGC cultures from the central and the peripheral retina. The same number of RGCs were seeded in both cases. Histogram of the number of RGCs in the cultures from each retinal region. B Effect of MG from the central and peripheral retina on the survival of RGCs from the central and peripheral retina, respectively, in co-cultures. Images of co-cultures of pig RGCs and MG from the central and peripheral retina. The MG and RGC in the co-cultures were both isolated from the same area of the retina (center and periphery). Number of RGCs in co-cultures from the center and periphery of the retina and number of MG in co-cultures from the center and periphery of the retina were represented. C The survival of rat RGCs seeded on confluent pig MG isolated from the central and peripheral retina. Images of rat RGCs seeded on pig MG from the central and peripheral retina. The same initial number of rat RGC were seeded on pig MG cultures. The number of rat RGCs present on pig MG cultures from the central or peripheral retina. D Survival and neuritogenesis of rat RGCs when maintained in CM secreted by MG from the central and peripheral retina. Images of rat RGCs maintained with conditioned medium (CM) from central and peripheral pig MG. The same number of RGCs were seeded in all conditions. Number of surviving rat RGCs maintained with CM secreted by MG from the central or peripheral pig retina was represented. To analyze neuritogenesis, the RGCs were classified as RGCs without neurites (blue), RGCs with the longest neurite < 50 μm (orange), RGCs with the longest neurite between 50 and 200 μm (grey), and those with neurites longer than 200 μm (yellow). The number of RGCs in each category is shown for those maintained in the presence of both types of CM. The RGCs were labelled with antibodies against β-III-Tubulin (red) and the MG with vimentin (green). Nuclei were stained with DAPI (blue): *p-value < 0.05. Scale bar: 50 µm Page 7 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 dramatically when they were seeded on peripheral MG under conditions of HP (16.4 ± 4.5% RGCs) as opposed to MG from the central retina (52.6 ± 30.1% RGCs) (Fig.5). Having observed distinct susceptibility of both RGCs and MG to HP depending on their position in the retina, central or peripheral, we analyzed the expression of two common pressure receptors, PIEZO1 and TRPV4 in purified MG cultures under control conditions and after exposure to HP for 72h. At 7 DIV, both central and peripheral MG express PIEZO1 in response to HP (Fig.6E, G). Likewise, TRPV4 is also more expressed in central and peripheral MG when they are exposed to HP. In addition, in control condition, is more strongly expressed in peripheral MG than in the central MG (Fig.6F, H). The proteome of the CM obtained from the central and peripheral pig MG exposed to HP and under control condition was compared. When the CM from central MG exposed to HP was compared to control, 18 proteins were more significantly expressed in the control CM while 75 proteins were significantly more expressed in the EH CM. When the proteins were sorted by function, there were more proteins related to “Oxidative stress, stress response” (11.8%) and “Survival, Homeostasis” (17.1%) in the EH CM comparing to the control CM, in which the percentage of proteins related to “Oxidative Fig. 2 Immunolabelling of vimentin, CD133, OCT4, nestin, α-SMA, β-catenin and β-III Tubulin in pure cultures of MG isolated from the central and peripheral pig retina. Images of cultured MG isolated from the center (A, C, E, G, I) or periphery (B, D, F, H, J) of the retina. The cells were labelled with antibodies against CD133 (A, B, red), OCT4 (C, D, red), nestin (E, F, red), β-catenin (G, H, red), β-III Tubulin (I, J, red), α-SMA (E, F, green), or vimentin (A–J, green). Nuclei were stained with DAPI (blue). Note the increase in the expression of all stem cell-like markers and β-III-Tubulin in the peripheral MG. Scale bar: 50 µm Page 8 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 Table 2 Proteins more strongly represented in the CM from peripheral MG comparing to the central MG, and their functions Gene symbol Entrez gene name Fold change P-valueBiological functions SFRP2Secreted frizzled related protein 2 49.99 0.049 Proliferation, Dedifferentiation PLOD2Procollagen-lysine 5-dioxygenase 28.82 0.0010 Cytoskeleton, cell adhesion, cell shape COL11A1Collagen type XI alpha 1 chain 23.55 0.004 Cytoskeleton,cell adhesion, cell shape CLUClusterin 22.78 0.0003 Inflammation, Immune response, Survival, Homeostasis APOE Apolipoprotein E 19.98 0.005 Neuroprotection, Neurite Outgrowth, Survival, Homeostasis LMNB1Lamin B1 18.39 0.010 Cytoskeleton, cell adhesion, cell shape ATP5PB ATP synthase F(0) complex subunit B1 mitochondrial15.67 0.041 Transport DKK3Dickkopf WNT signalling pathway inhibitor 314.77 0.011 Dedifferentiation CRYAB Alpha-crystallin B chain 13.28 0.05 Survival, Homeostasis EFEMP1 EGF containing fibulin extracellular matrix protein 110.88 0.016 Cytoskeleton, cell adhesion, cell shape, Proliferation CLN5 Ceroid-lipofuscinosis neuronal protein 5 10.75 0.034 Neuroprotection, Neurite Outgrowth GLRX3Glutaredoxin 3 10.70 0.06 Survival, Homeostasis, Proliferation SCIN Adseverin 10.17 0.08 Apoptosis, Proliferation inhibition, Dedifferentiation EFEMP2 EGF containing fibulin extracellular matrix protein 210.12 0.009 Cytoskeleton, cell adhesion, cell shape COX5ACytochromec oxidase polypeptide Va 10.01 0.06 Oxidative Stress HSPA13 Heat shock 70 kDa protein 13 9.57 0.05 Ubiquitination ESM1 Endothelial cell specific molecule 19.040.042 Angiogenesis LPLLipoprotein lipase 8.85 0.05 Inflammation, Immune response DAG1 Dystroglycan 8.54 0.013 Survival, Homeostasis, Cytoskeleton, cell adhesion, cell shape SPARCSecreted protein acidic and cysteine rich 8.45 0.007 Cytoskeleton, cell adhesion, cell shape, Proliferation, Inflammation, Immune response MYH11Myosin-11 8.26 0.09 Cytoskeleton, cell adhesion, cell shape MSTN Growth/differentiation factor 87.430.025 Inflammation, Immune response APLP2Amyloid beta precursor like protein 27.390.015 Survival, Homeostasis C1QC Complement C1q C chain7.190.035 Inflammation, Immune response SDC2 Syndecan 27.150.0009 Cytoskeleton, cell adhesion, cell shape, Neuroprotection, Neurite Outgrowth, Proliferation C5 Complement C5a anaphylatoxin 6.84 0.05 Inflammation, Immune response, Angiogenesis NRCAMNeuronal cell adhesion molecule 6.74 0.010 Neuroprotection, Neurite Outgrowth FBLN2Fibulin 26.670.07Cytoskeleton, cell adhesion, cell shape APP Amyloid-beta A4 protein6.500.08 Neuroprotection, Neurite Outgrowth, Oxidative Stress, Proliferation, Inflammation, Immune response TUFM Elongation factor Tu 6.32 0.023 Inflammation, Immune response PSME1 Proteasome activator complex subunit 16.300.019 Inflammation, Immune response CFIComplement factor I isoform 1 preproprotein6.22 0.05 Inflammation, Immune response SETProtein SET6.130.015 Neuroprotection, Neurite Outgrowth PTK7 Inactive tyrosine-protein kinase 76.120.014 Dedifferentiation, Neuroprotection, Neurite Outgrowth FTLFerritin light chain (fragment) 5.93 0.06 Survival, Homeostasis TIMP2Metalloproteinase inhibitor 25.680.05Inflammation, Immune response SDHA Succinate dehydrogenase [ubiquinone] flavoprotein subunit mitochondrial5.65 0.020 Oxidative Stress FN1Fibronectin 15.550.010 Cytoskeleton, cell adhesion, cell shape, Angiogenesis CDH2 Cadherin-2 5.54 0.015 Cytoskeleton, cell adhesion, cell shape HSP90AB1 HATPase_c domain-containing protein5.180.07 Neuroprotection, Neurite Outgrowth, Survival, Homeostasis, Inflammation, Immune response ISLR Immunoglobulin superfamily containing leucine rich repeat 5.14 0.0007 Neuroprotection, Neurite Outgrowth COL5A1 Collagen type V alpha 1 chain5.130.011 Cytoskeleton, cell adhesion, cell shape Page 9 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 Table 2 (continued) CDH11Cadherin 11 4.90 0.0002 Cytoskeleton, cell adhesion, cell shape SPARCL1SPARC like 14.890.06Cytoskeleton, cell adhesion, cell shape TAGLN2 Transgelin 24.770.010 Cytoskeleton, cell adhesion, cell shape OTUB1Ubiquitin thioesterase4.750.004 Inflammation, Immune response, Ubiquitination HNRNPU Heterogeneous nuclear ribonucleoprotein U4.640.09Proliferation FMOD Fibromodulin 4.54 0.004 Cytoskeleton, cell adhesion, cell shape ARSB Arylsulfatase B4.530.05Neuroprotection, Neurite Outgrowth ANXA7Annexin 4.46 0.09 Angiogenesis, Transport CLIC4Chloride intracellular channel protein4.440.047 Proliferation FSTL1Follistatin-like protein 14.380.016 Angiogenesis, Inflammation, Immune response, Proliferation, Survival, Homeostasis RDXRadixin 4.27 0.06 Proliferation PHBProhibitin4.260.06Apoptosis, Proliferation inhibition RPS4X 40S ribosomal protein S4 4.23 0.08 Cytoskeleton, cell adhesion, cell shape ANXA2Annexin A2 4.22 0.09 Cytoskeleton, cell adhesion, cell shape, Neuroprotection, Neurite Outgrowth IL4I1Amine oxidase4.160.027 Oxidative Stress, Inflammation, Immune response, Proliferation PCMT1Protein-L-isoaspartate (D-aspartate) Omethyltransferase 4.12 0.012 Survival, Homeostasis RNH1 Ribonuclease inhibitor4.040.07Angiogenesis LAMB2Laminin subunit beta 23.940.010 Cytoskeleton, cell adhesion, cell shape, Neuroprotection, Neurite Outgrowth PGD6-Phosphogluconate dehydrogenase decarboxylating 3.91 0.045 Oxidative Stress MDH2 Malate dehydrogenase mitochondrial3.910.07Oxidative Stress SERPINE1 Plasminogen activator inhibitor 13.810.06Inflammation, Immune response, Angiogenesis LTBP1Latent transforming growth factor beta binding protein 13.61 0.043 Proliferation CDH6 Cadherin-6 3.59 0.025 Cytoskeleton, cell adhesion, cell shape GAPDHGlyceraldehyde-3-phosphate dehydrogenase 3.24 0.09 Apoptosis, Proliferation inhibition PDGFRB Platelet-derived growth factor receptor beta 3.12 0.09 Angiogenesis, Inflammation, Immune response, Proliferation, Survival, Homeostasis CAP1 Adenylyl cyclase-associated protein3.100.08Cytoskeleton, cell adhesion, cell shape SPON1 F-spondin3.100.045 Cytoskeleton, cell adhesion, cell shape, Neuroprotection, Neurite Outgrowth DNASE2 Deoxyribonuclease-2-alpha 2.97 0.06 Apoptosis, Proliferation inhibition CST3 Cystatin C2.920.040 Survival, Homeostasis BMP1 Metalloendopeptidase 2.77 0.10 Cytoskeleton, cell adhesion, cell shape WDR1 WD repeat domain 12.700.09Cytoskeleton, cell adhesion, cell shape, Proliferation DPYSL2Dihydropyrimidinase-related protein 22.590.030 Neuroprotection, Neurite Outgrowth CKAP4Cytoskeleton associated protein 42.370.09Proliferation COL18A1Collagen type XVIII alpha 1 chain2.360.09Angiogenesis, Proliferation, Cytoskeleton, cell adhesion, cell shape RO60 Ro60 Y RNA binding protein2.190.032 Inflammation, Immune response ACTB Actin cytoplasmic 12.190.06Neuroprotection, Neurite Outgrowth, Survival, Homeostasis LAMC1Laminin subunit gamma 12.100.004 Cytoskeleton, cell adhesion, cell shape AKR1A1 Aldo-keto reductase family 1 member A1 2.08 0.08 Oxidative Stress VTNVitronectin 1.82 0.027 Cytoskeleton, cell adhesion, cell shape HTRA1Serine protease HTRA11.810.013 Apoptosis, Proliferation inhibition F2 Prothrombin1.490.015 Proliferation The proteins mentioned in the table are a selection of more than 890 proteins obtained in the proteomic analysis. Proteins considered significantly different between groups were those with a p-value less than 0.05 and that exceeded that value but had a > twofold change in each comparison analyzed, up to a p-value of 0.1. Proteins listed were ordered by the fold change observed Page 16 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 with increased β-catenin expression by peripheral MGs in vitro. The STRING analysis highlights protein– protein interaction networks, which is important for the system-level understanding of cellular processes. This analysis is based on physical interactions and on the interactions of different molecular pathways. The STRING analyses represented here focused on proteins that are more strongly represented in the CM of the peripheral MG, highlighting interactions with proteins mainly represented in this CM and supporting the results obtained in the proteomic analysis. These signaling pathways may underlie the differences between the two types of MG and their effect on RGC survival. Furthermore, elevated IOP is the main risk factor for the onset and progression of glaucoma. Although there has been considerable research in the field of glaucoma, the pathological mechanisms underlying the disease onset and development are still not fully understood. Neuronal degeneration in glaucoma might be due to a combination of factors, among which the RGC and MG interactions. The effect of HP mimics the effect of elevated IOP in the retina [59]. In primary cultures of MG and RGCs exposed to HP, the cell death is enhanced [60]. Moreover, retinal astrocytes and microglia have a differential effect on the pressure-induced death of RGCs [61]. The obtained results demonstrate that peripheral and central MG have different susceptibility to HP, and this impacts on the type of secreted factors, consequently affecting RGC survival. It is known that MG are sensors of pressure within the retina, as demonstrated invivo [27] and invitro [62]. We studied how conditions of HP might alter the expression of the pressure receptor channels TRPV4 and Piezo1, demonstrating that both receptors were overexpressed in MG exposed to HP. Piezo proteins play important roles in touch sensing pressure, respiration, angiogenesis and stem cell differentiation, and their activation increases calcium influx raising the intracellular calcium ion concentration ([Ca2+]i), which may trigger apoptosis [63]. Therefore, changes in cell stiffness and pressure acting through Piezo1 mechanosensitive channels could contribute to neurodegeneration [64]. TRPV4 activation can also provoke an increase in [Ca2+]i and continued channel activation induces MG gliosis in the mouse retina, as well as apoptosis in cultured mouse RGCs and adult porcine RGCs [65–67], which is in accordance with the data presented here. Indeed, the TRPV4 expression in peripheral MG could explain why these glial cells are more susceptible to HP. The proteomic analyses of the central and peripheral CM from control and HP conditions identified a series of DEPs. When these were sorted by function, an increase of proteins related to “Oxidative stress and stress response”, “Inflammation and immune response” and “Apoptosis and inhibition of proliferation” was evident in the CM from the peripheral MG exposed to HP. However, in the CM of central MG there was an increase of the proteins related to “Survival and homeostasis”. These results could explain why central MG and RGCs are more resistant to HP than the peripheral cells. Among the proteins overexpressed in peripheral MG under exposed to HP as opposed to the control peripheral MG are: ACTN4, a member of the actin binding protein family that interacts with DNaseY and mediates DNA fragmentation during apoptosis [68]; Epha2, from the Eph receptor tyrosine kinase (RTK) family, the largest group of tyrosine kinases in the genome [69], an RTK regulated by p53 proteins that induces apoptosis [70]; FABP3, member of a family of binding proteins that inhibits proliferation and promotes apoptosis when overexpressed [71]; TXN is a key element in the elimination of reactive oxygen species [72]; and PSMA4 that interacts with proteins with a strong immune response [73]. The increase in the proportion of proteins related to these functions suggests that the peripheral MG are more sensitive to HP, and consequently they negatively affect the survival of RGCs and of themselves. In addition, a STRING analysis of several of these proteins show that they interact with proteins also present in the CM of peripheral MG exposed to HP, suggesting that these pathways may be involved in the increased susceptibility of MG to HP and consequently decrease RGC survival. Conclusion This study (summarized in Fig.8), demonstrates a clear heterogeneity between MG from the periphery and central retina, based on their behavior and secretion of specific factors invitro. In control cultures, peripheral MG is more neuroprotective to RGCs, which could be due to being in a more dedifferentiated state. However, peripheral MG are more susceptible to pressure, which causes the secretion of proteins related to apoptosis, oxidative stress and inflammation, which may be implicated in RGC death at the early stages of glaucoma. Better understanding the different factors secreted by subpopulations of MG could identify potential therapeutic targets to enhance retinal neuroprotection and confirm the role of MG in the death of RGCs. We conclude that MG are important sensors of pressure changes in the eye, and they can influence the survival of their neighboring RGCs by secreting different proteins. Page 17 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 Abbreviations RGCs Retinal ganglion cells MG Müller glia HP High pressure IOP Intraocular pressure FBS Fetal bovine serum NBA Neurobasal A CM Conditioned medium PBS Phosphate buffered saline BSA Bovine serum albumin DAPI 4′,6-Diamidino-2-phenylindole αSMA α-Smooth muscle actin OCT4 Octamer-binding transcription factor 4 p75NTR P75 neurotrophin receptor TRPV4 Transient receptor potential cation channel subfamily V member 4 FASP Filter aided sample preparation DTT DL-Dithiothreitol FA Formaldehyde FDR False detection rate STRING Search tool for the retrieval of interacting genes/proteins SEM Standard error of mean DIV Days in vitro DEP Differentially expressed protein DKK Dickkopf RTK Receptor tyrosine kinase Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s1357802301186-1. Additional file1: Complete proteomic analysis and comparisons of Müller glia Conditioned Media (CM). Acknowledgements We would like to express our sincere gratitude to Stefanie M. Hauck for their invaluable contribution in the preliminary analysis of the proteomic study. Author contributions Conceptualization: XP, EV. Methodology: XP, NR, MA, FE. Investigation: XP, NR, MA, FE, AA, AFA, ARS, EV. Visualization: XP, EV. Supervision: EV, AFA, ARS. Writing—original draft: XP. Writing—review and editing: XP, NR, AA, AFA, ARS, EV. Funding This research was supported by: Grupos Consolidados Gobierno Vasco IT1510-22 (EV), ELKARTEK KK-2019/00086 (EV), MINECO-Retos PID2019111139RB-I00 (EV), PIBA 2020_1_0026 (EV), Gobierno Vasco postdoctoral grant POS_2022_2_0007 (XP). UPV/EHU postdoctoral grant (NR). Availability of data and materials All datasets generated are included in this article. Declarations Ethics approval and consent to participate This study does not contain any human data. All the experimental protocols complied with the European (2010/63/UE) and Spanish (RD53/2013) regulations for the protection of experimental animals, and they were approved by the Ethical Committee for Animal Welfare at the University of Basque country. Consent for publication All authors agreed to publish. Competing interests All authors declare no competing interests. Fig. 8 Graphical overview. Visual summary illustrating the key findings and main conclusions of the study Page 18 of 19 Pereiroetal. Cell & Bioscience (2024) 14:5 Received: 20 September 2023 Accepted: 13 December 2023 References 1. Goldberg JL, Corredor RG. Retinal ganglion cell life and death— mechanism and implications for ophthalmology. Eur Ophthalmic Rev. 2009;3:109–12. 2. Ou Y, Jo RE, Ullian EM, Wong RO, Della Santina L. Selective vulnerability of specific retinal ganglion cell types and synapses after transient ocular hypertension. J Neurosci. 2016;36:9240–52. 3. Zhang N, He X, Xing Y, Yang N. 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