Local Microglial Activation Induced and Labeled in the Retina in a Novel Subretinal Hemorrhage Mouse Model

Local Microglial Activation Induced and Labeled in the Retina in a Novel Subretinal Hemorrhage Mouse Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Local Microglial Activation Induced and Labeled in the Retina in a Novel Subretinal Hemorrhage Mouse Model Boglárka Balogh, Marietta Zille, Gergely Szarka, Loretta Péntek, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5788846/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Subretinal hemorrhage (SRH) is caused by the accumulation of blood between the neurosensory retina and the retinal pigment epithelium or between the retinal pigment epithelium and the choroid. It often arises from age-related macular degeneration, traumas, and may occur spontaneously caused by other diseases like hypertension and diabetes. Here, we developed a novel technique by co-injection of blood and a dye-coupled tracer protein, Cholera toxin subunit B (CtB), to better localize and understand the disease and how it can cause microglial activation, inflammation, and partial vision loss. Our results show that microglia are activated in the inner retinal regions and also in the zones neighboring the blood injection. In contrast, the non-affected zone of the affected eye showed no microglial activation. Based on the results, we provide evidence of inner retinal microglial activation and the appearance of microglia and macrophages in the pigment epithelium. Using advanced imaging techniques, we were able to localize better the affected area that comprises not only the retinal area over the blood clot but the neighboring regions as well. These findings will provide the basis for novel therapeutic interventions targeting neuroinflammation in the retina after subretinal hemorrhage and other diseases affecting the eye. bleeding blood cholera toxin experimental ophthalmology eye inflammation retinal pigment epithelium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background The retina is a gateway to all visual information. It is, however, one of the most exposed parts of the central nervous system (CNS) ( 1 ), and thus susceptible to diverse retinal diseases and environmental insults ( 2 ). Bleeding in the eye can arise from various etiologies, including trauma, systemic diseases, and ocular disorders, and in most cases, leads to major vision loss or blindness ( 3 – 6 ). Retinal hemorrhages can be classified based on their location within the retinal layers: preretinal, intraretinal, and subretinal. Subretinal hemorrhage (SRH) is the accumulation of blood between the retina and the retinal pigment epithelium (RPE) or between the RPE and the choroid ( 7 ) (Fig. 1 ). The presence of blood in this space is toxic to retinal cells, and rapid tissue damage is induced starting with photoreceptor and RPE degradation ( 8 ). If the clot is left untreated, scarring processes can be initiated potentially leading to visual loss in the affected area. If the hemorrhage occurs in the macular region, it usually leads to severe and irreversible vision loss ( 9 ). However, the visual prognosis in all cases depends on the initial visual acuity, the size of the hemorrhage, the time interval between the onset of SRH and treatment, and the concomitant involvement of different retinal segments. The development of SRH may be associated with several pathologies such as age-related macular degeneration, hypertension, diabetic retinopathy, or even blunt head trauma ( 10 ). The retinal tissue damage is facilitated by three main pathological pathways: 1) The clot acts as a diffusion barrier, which prevents the transfer of nutrients from the choroid to the photoreceptors by the RPE and the return of by-products to the choroid ( 10 , 11 ). 2) Mechanical effects are caused by the contraction of the fibrin network formed after the clot, which can tear the photoreceptor outer segment from the retina in sheets ( 10 ). 3) Lysis of erythrocytes results in the release of hemoglobin, which is no longer protected, and the iron in the heme is oxidized from the Fe 2+ state to the Fe 3+ state ( 12 ). Oxidized heme and other blood breakdown products have been demonstrated to induce cell death of the RPE and photoreceptors ( 8 ). In addition, other cellular changes are observed, including activation and infiltration of microglia and macrophages, Müller cell disposition, presence and activation of migrating RPE cells, suggestive of dedifferentiation and signs of gliosis ( 13 ). As a result, nerve cells will suffer from severe inflammation and cell death. Microglia are the resident immune cells in the central nervous system, including the retina. In the human retina, they form a heterogeneous population; perivascular microglia filter substances that enter the retina through the vasculature, whereas parenchymal microglia have high motility, constantly monitoring the microenvironment to remove possible metabolic products and cellular debris and mediate synaptic remodeling ( 14 ). Microglia are also programmed for immunological tolerance and possess an anti-inflammatory phenotype, which is essential for retinal immune competence ( 15 ). Because of their monocytic origin, these cells can respond incredibly quickly to pathological stimuli, therefore, they are excellent indicators of retinal pathologies, including inflammation. Microglia protect neurons from cell death and promote tissue regeneration but can also induce secondary tissue damage by triggering chronic inflammation ( 16 – 19 ). They can take different morphological states; each morphotype is associated with different activation states and has unique functions to maintain a physiologically normal state ( 20 – 23 ). In this research, we use microglia to show the retinal activation of SRH based on their morphotypes and movement in the tissue. For this, we developed a novel co-injection model because available SRH models did not allow for direct fluorescent labeling of the injection site, not to mention microglial activation through labeling with phagocytosis of the dye-toxin-subunit complex. Materials and methods Animals and preparation Adult, male, P30-90 old C57BL/6J (n = 9) mice were maintained in a 12/12-hours dark/light cycle. Max. 4 mice were placed in a cage with food and water ad libitum . Animal housing, handling, and all utilized experimental procedures were approved by the ethical committee of the University of Pécs (BA02/2000-27/2024). All animals were treated following the ARVO Statement for the ‘Use of Animals in Ophthalmic and Vision Research’. All efforts were made to minimize pain and discomfort during the experiments and all procedures were done by obeying the 3R law. To create the mouse model of SRH, animals were deeply anesthetized via intraperitoneal injection of pentobarbital (80x dilution from stock (400 mg/ml) → for mice 180 µl/10 g). Autologous blood was collected from the tail vein with a 27-gauge needle and immediately used for subretinal injections. Before the subretinal injections, a temporal tunnel incision posterior to the limbus was made in the sclera using a 27-gauge needle under a dissecting microscope. The subretinal injections of autologous blood were achieved with a glass injector needle connected to a 1 ml syringe. Glass injector needles were pulled with a Sutter P-87 (program: Cycle1: Heat = 760, Pull = 250, Vel.=50, Time = 200; Cycle2: Heat = 760, Pull = 250, Vel.=0, Time = 200) from 1.5mm capillaries (World Precision Instruments, Inc. Item Nr. 1B150F-4, Lot Nr. 2605323). For the simple SRH group, we injected 2 µl blood into the subretinal space. During the co-injection experiments, we loaded the glass injector with 0.25 µl of CtB conjugated with Alexa-555 solution (cc.: 1µg/µl in PBS, Invitrogen, Lot Nr. 201622) followed by 2 µl of blood (Fig. 2 A). We performed the SRH injections only on the left eyes, while the right eyes were used as controls. After 24-hour survival, mice were sedated with Forane (isoflurane 4%, 200 µl/l) and then sacrificed using cervical dislocation. Eyes were immediately removed with fine forceps. Eyeballs were cut at the ora serrata, and the lens and vitreous body were removed. The retinas and the attached pigment epithelium sheets were fixed in 4% paraformaldehyde on filter papers for 30 min at room temperature (Fig. 2 B). Immunohistochemistry Flat-mounted retinas and RPEs were blocked in blocking solution (CTA, containing 5% Chemiblocker, 0.5% TritonX-100, 0.05% Na-azide in PBS) for 2 hours at room temperature, humidified. After blocking, the retinas and RPEs were treated with the primary antibodies (Table 1 ; diluted in CTA) for 48 hours at room temperature (RT). After washing three times in PBS, secondary antibodies (Table 1 ) were applied in CTA, and incubated overnight at RT. After washing three times for 10 min in PBS, they were coverslipped facing ganglion cell layer (GCL) up, or RPE up with Vectashield (Vector Laboratories, Peterborough, UK). Table 1 Antibodies Primary Antibodies Name Abbr. Dilution Source Code RRID Ionized calcium-binding adapter molecule 1 gp-Iba1 2000x SySy 234004 AB_2924932 Junctional adhesion molecule B gt-JAM-B 1000x R&D sys AF988 AB_355767 Secondary Antibodies Name Abbr. Dilution Source Code Anti-guinea pig - Alexa647 a-gp-A647 500x Invitrogen A21450 Anti-mouse - Alexa405 a-ms-A405 500x AbCam ab175658 Anti-goat - Alexa647 a-gt-A647 500x AbCam AB150131 Anti-guinea pig - DyLight405 a-gp-DL405 500x Jackson 706-475-148 Microscopy Retinas and RPEs were inspected using a Zeiss LSM 710 confocal laser scanning microscope (with Plan Apochromat 10x, 20x, and 63x objectives (NA: 0.45, 0.8, 1.4, Carl Zeiss Inc., Jena, Germany)) with normalized laser power and filter settings making 1.5- and 0.5-µm thin optical sections. Image analysis All measurements were performed using FIJI (ImageJ, NIH, Bethesda, MD, USA). First, we performed two z-merges from consecutive optical stacks for the superficial and deep regions of microglia from 3 areas from the SRH-treated eyes (Z1-3) and one area from the control eyes (Z4). We chose the injection site (Z1) based on the presence of blood albumin (a-albumin staining). The size was variable, depending on the injection. Z2 was determined as an area (700x700 µm) in connection with the injection site but with no extravasal albumin present. The Z3 was the distal-most relative to the injection site (contralateral retinal hemisphere) in the sample (700x700 µm). We used the 20x magnification for clustering microglia activation. Microglia were divided into superficial (SL) and deep layers (DL) defining their positions in the retina, represented by the z-stack regions from GCL, inner plexiform layer, neuronal layers, and the superficial layer of the inner retinal vasculature when merging images for SL-microglia and the outer plexiform layer/DL neuronal layers together with the deep layer of inner retinal vasculature to merge the microglia images for the DL from the same Z-stack. Microglia were separated from the other signals based on their expression of ionized calcium-binding adapter molecule 1 (Iba1). Cells were manually grouped one by one according to their morphologies into activated and non-activated (see the details of cell classification in ( 24 ), using the “Cell-counter” plugin in FIJI. Only fully visible cells were included in the analyses, while we omitted those that fell on the edges. Motion was detected on 15 sec. time-lapse images. We decoded the change in Z with ImageJ ΔF/F script (by ( 25 )). Statistics Data was curated in MS Excel. Non-parametric Kruskal-Wallis and Dunn’s posthoc analyses were performed using IBM SPSS Statistics Version 25.0 (IBM Corp., Armonk NY). Normality tests were performed before the analyses, because some of the data sets showed non-normal distribution, we chose to perform non-parametric tests on all data. Results A novel SRH model Based on previous studies (see details in Table 2 ), we have developed a modified model for SRH, where compared to previous methods, we used freshly pulled glass injectors to avoid complications after injection and labeled the injection site with the co-injection of CtB-A555 toxin subunit conjugate to allow better visualization of the injection site and the phagocytosis of the dye in the live retina. Table 2 Previous SRH models. Animal Method Result Limitations Mouse ( 26 ) Injections via transscleral route with a 33G needle 1.6 µl autologous blood (from tail vein); survival times 6 hours to 10 days Increased expression of inflammatory cytokines, chemokines and adhesion molecules; microglial migration, increased density; effects of minocycline usage of metal needle Rat ( 27 – 29 ) heparinized and diluted blood (one part blood to ten parts saline) injected subretinally The retina overlying the hemorrhage became intensely degenerated over a period of months Heparin is an anticoagulant, clotting process differs from fresh blood injections Rabbit ( 30 ) 0.1 ml fresh blood from ear; specially constructed 30G needle tip encased in a 22G spinal needle; clot prevention 0.03cc of air in the needle tip (also injected in the subretinal space); survival times:1 hour to 28 days Signs of degeneration after 1 day in the outer retinal layer usage of plus air bubble, can make retinal detachment worse Rabbit ( 31 ) 0.1 ml fresh blood from ear; 30G needle; Clot prevention: air bubble Intravitreal injection of tPA 1 day after subretinal injection of blood in rabbits facilitated more rapid lysis of the clotted blood, however, retinal damage was not prevented usage of plus air bubble, can make retinal detachment worse Rabbit ( 32 ) 100 µl autologous blood from ear vein in a nasal juxtapapillary location along the myelinated streak; An angled 1-inch, 30-gauge needle was introduced transsclerally 3–4 mm posterior to the limbus Photoreceptor toxicity caused by SRH occurs at least in part by apoptosis and is associated with iron migration to the photoreceptor layer usage of metal needle Cat ( 33 ) the tip of a 20-gauge surgical knife or a 25-gauge needle was passed transsclerally into the bleb and then withdrawn, allowing choroidal blood to extend under the retina into the area centralis; Lesions were observed 25 minutes to 14 days In 6 of 9 clots more than 1 hour old, fibrin was associated with tearing of sheets of photoreceptor inner and outer segments. Later degeneration progressed to involve all retinal layers overlying the densest areas of fibrin in the clots; hemorrhages into subretinal blebs containing tPA did not form fibrin strands or cause photoreceptor tearing usage of metal needle Cat ( 34 ) the tapetal or nasal retina with a neodymium:YAG (Nd:YAG) laser focused through a preformed retinal bleb; 2–25 laser shots (mean, 14) at between 20 and 25 mJ were fired until one created a large subretinal hemorrhage Removing experimental SRHs within 7 days of their occurrence with the assistance of rt-PA and an ultra-microsurgical approach may reduce outer retinal degeneration Laser Injection sites of all injected mice healed entirely without any complications or infection. The entry site on the eyeballs were closed in 24 hours with only mild post-operational scar visible only in the stereo microscope after careful examination, where we were able to detect and validate the injection site and the presence of subretinal blood in the eyecup before fixing with 4% paraformaldehyde or removing the retina for live imaging (Fig. 2 ). For the first time, we used CtB to label the injection site in the subretinal space and the overlying cells, therefore, the retinal localization of the injection can be precisely outlined. By using this technique, we have defined three zones in the injected eye, including the injection site (Z1), the neighboring site, in direct contact with the injection site (Z2), and the contralateral same-retinal zone (Z3). We added an endogenous control zone from the contralateral, non-injected eye (Z4). We observed that the CtB-A555 not only overlapped with the blood-related extravasal albumin (Fig. 3 ) but the label displays better Z1 vs. Z2 contrast superior to the albumin signal based on laser intensities and gain, therefore, it is easier to demarcate the SRH site in comparison to the non-co-injected retina, where only intravasal albumin was present (Fig. 3 a). We could not identify any blood-related extravasal albumin deposits in the contralateral side (Z4) retinas, used as an endogenous negative control (Fig. 3 a). By using higher magnification (20x) each area can be easily outlined with the CtB-A555, therefore each of the microglia could be classified into the 4 zones (Z1-4) without a problem. Based on this our measurements accurately represent the microglial states in each of the zones (Fig. 4 ) As the example images show the microglial cells from the SL, that lie on the NFL side of the GCL, show massive activation that can be seen also in the DL, at the border of the inner nuclear layer and outer plexiform layer (Fig. 4 a). The two layers of MGs shown in the healthy retina broke up and became miss-ordered in Z1 while in Z2 the SL-DL division was still intact while more activated MGs were present (Fig. 4 b) compared to both Z3 and Z4. Retinal microglial activation after SRH Based on morphological classification (previously described in ( 24 )) of microglia (Fig. 5 A), SRH induced microglial activation in Z1 and Z2 was present in both the SL and DL (Fig. 5 B). We observed a 3.66-fold increase of microglia count in Z1 (from a median of 6 ± 3 to 22 ± 16) as well as a 3.33-fold increase in Z2 (from 6 ± 3 to 20 ± 8) when compared to the control Z4 sample. In addition, we detected an increase in the number of microglia between the Z1 and the Z3 and between the Z2 and Z3 (Z3-Z1: 3.14-fold, from 7 ± 5 to 22 ± 16; Z3-Z2: 2.86-fold, from 7 ± 5 to 20 ± 8). A similar comparison of Z3 and Z4, did not result in a change in the number of the activated microglia numbers, meaning that these microglia lack activating signals, retaining their non-activated morphologies. We did not see any significant difference between the total microglia numbers in the different zones, however, we observed an increased variability of the total microglia counts across retinas in Z1 (Fig. 5 C, n = 3202 cells in 17 retinas). Interestingly, when we examined the microglial activation in the SL and DL, we observed a less pronounced change. We only detected a significant difference between Z1 and Z3 in the number of activated microglia in the SL (Fig. 6 A, 3 .57-fold, from 7 ± 6 to 25 ± 15). On the other hand, we found that the counts of the non-activated microglia in the SL were significantly less in Z2 when compared to either Z3 or Z4 (Fig. 6 B, Z4-Z2: 2.47-fold, from 42 ± 6 to 17 ± 9; Z3-Z2: 2.24-fold, from 38 ± 8 to 17 ± 9). When examining the DL, we found a significant increase in the number of activated microglia in Z2 compared to both Z3 and Z4 (Fig. 6 C, Z4 - Z2: 5.5-fold, from 4 ± 2 to 22 ± 8; Z3 -Z2: 3.14-fold, from 7 ± 5 to 22 ± 8). Examining the number of non-activated microglia, there was a significant decrease of Z1 and Z2 microglia counts when compared to Z4 (Fig. 6 D, Z4 - Z2: 3.06-fold, from 52 ± 9 to 17 ± 6; Z4-Z1: 2.26-fold, from 52 ± 9 to 23 ± 19). Microglia in the subretinal space In healthy animals, microglia are normally only present in the inner retina. We examined if SRH attracted inner retinal microglia to the affected subretinal site or induced a macrophage infiltration. Based on the morphological divergence, we regularly observed microglia in direct contact with the RPE layer with only a sparse presence of macrophage-like Iba1 + cells (6 macrophage-like morphologies out of 27 Iba1 + cells, n = 2; Fig. 7 ). By using the Junctional Adhesion Molecule B (JamB), we labeled RPE tight junctions to outline the microglia on the retinal side. Although these cells were less numerous, all of them showed an activated morphology, including enlarged, disorganized soma, relative soma size increase, amoeboid and leaf-like structures on the dendritic endfeet, and aggregated occurrence of these activated microglia morphologies. We were unable to find Iba1 + cells attached to the RPE in healthy, control retinas, with only the JamB labeling of RPE cells being clearly visible ( Suppl. Figure 1 ). CtB labeling on and inside MGs Previous studies suggested CtB can form direct contact with microglial cell membranes ( 35 ). By using high magnification microscopy, we observed the internalization of CtB-A555 in infiltrated microglia, in the vicinity of the RPE (Fig. 8 ). Interestingly, this was not the case with the rest of the inner retinal microglia as we did not find microglia internalizing CtB particles. These latter microglia on the other hand still showed that their endfeet moved in the direction of dye particles (Fig. 8 B). This observation was reinforced by the ex vivo live imaging, where we were able to follow the microglia in real-time and observed their change in motility that increased due to inflammatory signals. Microglia appeared highly motile, and they also started breaking down CtB plaques by phagocytosis and transporting them (Fig. 9 ). Discussion Novel SRH model In the present study, a modified model was established for SRH in the mouse eye. In contrast to previous models (Table 2 ), no air or heparin was used to stop the rapid clotting. Instead, the sterile and inert glass injector gives enough working time to perform the SRH injection. The use of smaller, pulled, and therefore heat-sterilized needles protect against reflux (blood or blood-CtB mix) at the injection site ( 36 ). There were no signs of infections, such as redness, purulent discharge, swelling, discomfort, irritation indicated by scratching, photophobia. The use of a thin, hollow microneedle helps the delivery method to be minimally invasive because the microneedle only penetrates some hundreds of micrometers into the eyeball and narrows down to 8–12 µm at the tip, thereby protecting the retina from mechanical damage and also reducing the risk of infection ( 37 ). Besides using pulled glass micropipettes to deliver blood into subretinal space, our method is novel due to the utilization of CtB-A555 co-injection. CtB-conjugates generally serve as retrograde neuronal tracers ( 38 ) that, following endocytosis, are transported back to the Golgi network and the endoplasmic reticulum. In our labeling scheme, CtB-A555 has been used for the first time to help identify the injection site. In microglia, CtB is colocalized either by binding to GM1 ganglioside glycolipid molecules in lipid rafts or was engulfed by microglia and stored intracellularly ( 35 ). In our experiments, we show that the A555 conjugate was similarly localized intracellularly. Previous studies have shown that activated microglial cells can engulf various dyes due to their tissue-specific clearing phagocytic activity ( 39 , 40 ). Apparently, microglia can internalize CtB molecules as well via similar mechanisms and thus its injection combined with fluorescent fundoscopy can be used to visualize cells even in the human eye when needed ( 41 ). Although the recently developed adaptive optics fundoscopy also offers a dye-free solution to visualize microglia and thus a minimally invasive examination of both the mouse and the human eye is possible, allowing a direct examination of the live retina ( 42 – 47 ) our CtB-conjugate coinjection method still provides a cost-efficient alternative for laboratories with limited or no access to such equipment with much higher detail, even allowing super-resolution reconstruction of the cells. Finally, even if our study has different aims, CtB can have anti-inflammatory effects as well ( 48 , 49 ) thereby further decreasing the risk of inflammation in the experiments. Microglia activation in SRH Microglia are resident macrophages of the retina that perform surveillance and neuroprotection against pathophysiological insults ( 50 ). In the healthy retina, they are quiescent and confined to the GCL and the plexiform layers. When microglia get activated and migrate to the location of the insult, they may appear among photoreceptor outer segments or RPE in retinitis pigmentosa and age-related macular degeneration, respectively ( 51 ). Similarly, we observed that after SRH, microglia appear in the vicinity of the RPE and most of these pathologically located cells displayed morphological characteristics of the activated microglia phenotype. However, previous studies limited the examinations to the outer retina (RPE and photoreceptors) whereas possible parallel inner retinal changes have rarely been described. In this present study we expanded our observations to the inner retinal areas as well and identified a widespread activation of microglia due to SRH treatment. This suggests that SRH initiated a complex chemotactic activation of blood-related factors, including fibrin and albumin. When these factors appear in the extravasal space ( 52 ) and diffuse to inner tissue compartments, they activate microglia, which then migrate towards the blood clot to facilitate clearance. By performing this radial migration, microglia must pass photoreceptor inner and outer segments and eventually reach the RPE. In our study, we observed both microglia that internalized CtB-A555 (Fig. 8 ) and also microglia with no sign of internalized particles. This indicates that a population of microglia participated in the clearance of the dye through phagocytosis while the rest of them were only activated without transforming into phagocytic cells. However, it is uncertain whether these two functionally different populations exist or phagocytotic transformation was just delayed for some of the cells. In a previous study, the accumulation of fluorescent carbocyanine vital dyes in microglia demonstrated the phagocytosis of dying neurons ( 53 ). Here, we showed that CtB-A555 molecules can be endocytosed by microglia from the extracellular milieu (Fig. 8 ). CtB has potential anti-inflammatory effects as previously shown by Zhang et al ( 48 ). 24 hours after ischemia, CtB downregulated the levels of proinflammatory cytokines and microglia/macrophage transformation. Based on microglia morphological characteristics, we did not observe any indication of this latter CtB-induced effect as microglia counts of simple blood injections and CtB/blood coinjected samples appeared similar (data not shown). Microglia activated by SRH can release a number of proinflammatory cytokines that can induce inflammation and corresponding tissue degeneration later on ( 26 ). Therefore, any inflammatory changes observed in our experiments are rather induced by the SRH and corresponding microglia activation and largely independent of the presence of coinjected CtB. Bordering zones and localization According to our results, SRH is well maintained within an outlined Z1 including the injection site with the primary insult induced by the blood clot. Surprisingly, microglia in the Z2 area were activated as well contrary to the fact that they were not in direct contact with the blood clot itself. Z2 microglia may get informed about the insult through proinflammatory factors ( 26 ) either originating from the clot and/or released by activated microglia of the Z1 site. Contrary, we have not detected activation or changes in the number of microglia in the non-affected Z3 and Z4 sites (Figs. 5 and 6 ). Therefore, we hypothesize that SRH is limited to the Z1 and Z2 sites while the rest of the tissue remains intact. Conclusions SRH can be a devastating disease affecting vision. Our new model can help identify new treatment methods while enabling the monitoring of microglia with dye-conjugated CtB. The results show that overlaying inner retinal microglia are activated even if blood is injected between the photoreceptors and the RPE. Activated microglia bind CtB-A555 but are less likely to phagocyte it while microglial and infiltrated macrophages at the RPE will internalize the dye. This internalized CtB dye, and the morphological changes of microglia can be visualized in the living tissue for the monitoring of the microglial activation process. Moreover, in this model, the borders of the SRH site can be precisely demarcated thus allowing for the development of future solutions to avoid retinal cell death or to use microglia as biomarkers during pathological changes. Abbreviations A555 Alexa-555 (fluorescent dye) Am Amacrine Cell AMD age-related macular degeneration AO adaptive optics AS Astrocyte BC Bipolar Cell C Cone cLSM confocal laser scanning microscope CNS central nervous system CTA Blocking solution containing 5% Chemiblocker, TritonX-100, 0.05% Na-azide in PBS CtB Cholera toxin subunit B DL Deep Layer (of microglia) ERK1/2 Extracellular signal-regulated kinase 1/2 GC Ganglion Cell GCL Ganglion Cell Layer HC Horizontal Cell IBA1 Ionized calcium-binding adapter molecule 1 IL-1ß interleukin-1ß IL-6 interleukin-6 INL Inner Nuclear Layer IPL Inner Plexiform Layer ISI injection site JAM-B Junctional adhesion molecule B MG Microglia NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells NFL Neurofilament Layer OCT optical coherence tomography ONL Outer Nuclear Layer OPL Outer Plexiform Layer OSL Outer Segment Layer p65 NF-kB p65 subunit, encoded by RELA gene PBS Phosphate buffered saline solution RPE retinal pigment epithelium RT room temperature SL Superficial Layer (of microglia) SRH Subretinal hemorrhage TGF-ß transforming growth factor-ß Z1-4 zone 1–4 Declarations Ethics approval and consent to participate Animal housing, handling, and all experimental procedures were approved by the ethical committee of the University of Pécs under the number BA02/2000-27/2024. All animals were treated following the ARVO Statement for the ‘Use of Animals in Ophthalmic and Vision Research’. All efforts were made to minimize pain and discomfort during the experiments and all procedures were done by obeying the 3R law. Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was financially supported by NKFIH, MOH and BMBF under the action of the ERA-NET COFUND (2019-2.1.7-ERANET-2021–00018; NEURON (NEURON-066 Rethealthsi) to BV. This research was also supported by the NKFI (OTKA NN128293) (B.V.) from the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TKP2020 IKA-07 National Excellence Program (B.V.). In addition, this research was also financed by the Thematic Excellence Program 2021 Health Sub-program of The Ministry of Innovation and Technology (Hungary), within the framework of the EGA-16 project of the University of Pécs. Supported by the EKÖP-24-4-I-PTE-11 (G.S.), EKÖP-24-3-I-PTE-129 (B.B.) University Research Grant Programme of the Ministry of Culture and Innovation, National Fund for Research, Development, and Innovation. This work was carried out using the Nano-Bio-Imaging and SzKK Imaging Core Facilities of the Szentágothai Research Centre, University of Pécs. Authors' contributions Conceptualization, TKO and BB; methodology, TKO and BB; validation, TKO, BB, GS, MZ, LP, VB; formal analysis, BB, TKO; investigation, BB, TKO, GS, LP, AF ; resources, BV, MZ, BB, GS, TKO; data curation, TKO, BB, GS, MZ, LP, VB; writing—original draft preparation, TKO and BB; writing—review and editing, TKO, BB, GS, MZ, LP, AF, VB; visualization, BB, TKO, GS; funding acquisition, BV, MZ, BB, GS, TKO. All authors contributed to the article and approved the submitted version. Acknowledgements This work was carried out using the Imaging Core Facility and the Animal Facility of the Szentágothai Research Centre, University of Pécs. References London A, Benhar I, Schwartz M. The retina as a window to the brain—from eye research to CNS disorders. Nature Reviews Neurology 2012 9:1 [Internet]. 2012 Nov 20 [cited 2024 Sep 18];9(1):44–53. Available from: https://www.nature.com/articles/nrneurol.2012.227 Rosenblatt TR, Vail D, Saroj N, Boucher N, Moshfeghi DM, Moshfeghi AA. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5788846","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":402541933,"identity":"4314e524-2fc1-40d5-8269-cd7ba10296a2","order_by":0,"name":"Boglárka Balogh","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Boglárka","middleName":"","lastName":"Balogh","suffix":""},{"id":402541934,"identity":"d8b9b7a0-5157-4004-85ec-f09df4c36a1a","order_by":1,"name":"Marietta Zille","email":"","orcid":"","institution":"University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Marietta","middleName":"","lastName":"Zille","suffix":""},{"id":402541935,"identity":"e200decc-ac11-48d9-a506-7aec47dc006f","order_by":2,"name":"Gergely Szarka","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Gergely","middleName":"","lastName":"Szarka","suffix":""},{"id":402541936,"identity":"95b8f536-38a0-481f-b9e3-715238132d16","order_by":3,"name":"Loretta Péntek","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Loretta","middleName":"","lastName":"Péntek","suffix":""},{"id":402541937,"identity":"67db9012-4903-415f-9d44-aa9a698f5e7f","order_by":4,"name":"Anett Futácsi","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Anett","middleName":"","lastName":"Futácsi","suffix":""},{"id":402541938,"identity":"55b56ed1-fd69-4a32-a091-b0272a95ae8f","order_by":5,"name":"Béla Völgyi","email":"","orcid":"","institution":"University of Pécs","correspondingAuthor":false,"prefix":"","firstName":"Béla","middleName":"","lastName":"Völgyi","suffix":""},{"id":402541939,"identity":"6c29c0d4-d7d4-407f-9ba6-78c08581581b","order_by":6,"name":"Tamás Kovács-Öller","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYBACCRiDjx0uxnwARCYQ1MLGDFfHlsBwgEQtPAZ4tUi29xh++PGHQY6NmfnYB8YfdtH8/We+Pf5QwZBncAC7FmmeM8aSvW0MxmzMbMkzGBKSc2fcyN1ucOAMQzEuLXISaQnSjA3/E9uYeYyBrmHObbjBu03iYBtD4gbcWpJ/M/xhAGrh/wzUUp87//yZZxIH/+HWIi2RfEyagQ2khQcYAAmHczccyGGTONiAW4tkz+FjllC/GDMkpB3P3XgjzdzgzDGJxJk4tEgcb2y+AQoxfvbmxwwfbKpz550//OxBRY1NYh8OLaggAUKxMSBFGHGAjTTlo2AUjIJRMNwBALvCWJ/RnyOkAAAAAElFTkSuQmCC","orcid":"","institution":"University of Pécs","correspondingAuthor":true,"prefix":"","firstName":"Tamás","middleName":"","lastName":"Kovács-Öller","suffix":""}],"badges":[],"createdAt":"2025-01-08 12:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5788846/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5788846/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73939771,"identity":"88d0aa56-1053-4eaf-9c7e-8ee4efd0ac7f","added_by":"auto","created_at":"2025-01-16 07:47:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":482614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSubretinal hemorrhage. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA)\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eSchematic represents the human eye and B) the retinal layers with the respective cell types. Dashed red boxes show microglia layers in the healthy retina. Abbr. Astrocyte (AS), Ganglion Cell (GC), Microglia (MG), Amacrine Cell (Am), Bipolar Cell (BC), Horizontal Cell (HC), Cone (C), Rod (R). Neurofilament Layer (NFL), Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner Nuclear Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Outer Segment Layer (OSL), Retinal Pigment Epithelium (RPE), Subretinal Hemorrhage (SRH) Superficial Layer (SL), Deep Layer (DL) of microglia.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/2acf2ff72b236eafeea1f01f.png"},{"id":73939755,"identity":"4e173283-b0c4-4a1c-9b51-d394330984ad","added_by":"auto","created_at":"2025-01-16 07:47:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":611942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSubretinal injection in the mouse. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA) SRH injections. B) Stereo-microscopic images during the eye dissection validating the injection site and the presence of subretinal blood (*). Red stars show the SRH sites, demonstrating the healing of the injury at the ora serrata.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/f769cef5f44627b23deacdf7.png"},{"id":73939757,"identity":"583ad699-d36a-430c-b735-aaa3c481a892","added_by":"auto","created_at":"2025-01-16 07:47:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1228359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eControl, SRH - blood, SRH - CtB. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA) Whole mount control retina in low (10x, top) and high (20x, bottom) magnification from the same sample. B) Blood injected whole mount SRH retina. The white circle in (top) shows the injection site (ISI), while the image underneath (B) focuses on the ISI in 20x magnification of the same sample. C) Blood/CtB-A555 co-injected whole mount SRH retina. Again, the white circle marks the ISI (top), which then is represented in a higher magnification (bottom) from the same sample.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/0ccce653d4fe98b149ea0200.png"},{"id":73939760,"identity":"23a6d99d-51ef-41a7-b619-394162289161","added_by":"auto","created_at":"2025-01-16 07:47:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1201294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eExample microscopic images of the SRH site and the surrounding areas. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA) SRH site (Z1) at the level of the superficial layer (left) and the deep layer (right). B) Neighboring zone (Z2) superficial layer (left) and the deep layer (right). C) Z3, the farthest zone from the SRH site at the level of the superficial layer (left) and the deep layer (right). D) Control retina (Z4; contralateral eye) at the level of the superficial layer (left) and the deep layer (right). All images are 20x magnification confocal images.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/1f34a1f374c6a106c5613e17.png"},{"id":73941198,"identity":"600b3d8c-f965-4707-ad9e-ba194b7b5098","added_by":"auto","created_at":"2025-01-16 07:55:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":402192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChange in the number of Activated Microglia due to SRH. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA) Typical morphological changes from our dataset, ranging from non-activated (top row), ramified (middle row) to activated, amoeboid (bottom) scale bar: 10 μm. B) The SRH (Z1) site showed a pronounced increase in the activated microglia count in comparison to all other zones, including neighboring (Z2), off-site (Z3), and contralateral retinal (Z4). C) After 24 hours, we detected a small but non-significant fluctuation in the total number of microglia in Z1-4. Kruskal Wallis, Dunn's post hoc **p\u0026lt;0.01 o=outlier x=mean.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/d4e6646a33d3006acba3e629.png"},{"id":73941197,"identity":"3fca7479-ac63-4557-a7d3-f4c62ccbb5b0","added_by":"auto","created_at":"2025-01-16 07:55:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":212071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChange of activated MG counts in the superficial (SL) and deep layers (DL) of the SRH inner retina\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. A-B) SL microglia activation states in the different zones. C-D) DL microglia distribution in the different zone and activation states. Kruskal Wallis, Dunn's post hoc **p\u0026lt;0.01 *p\u0026lt;0.05 o=outliers x=mean.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/5aa2fb5e6bed26ebda074d09.png"},{"id":73939765,"identity":"9c992d46-8e9a-44b5-9d36-cded5d50e5e6","added_by":"auto","created_at":"2025-01-16 07:47:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1061739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIba1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e cells infiltrated into the subretinal space showed activated morphology\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. A) RPE from SRH-treated animals. B-C) High-magnification of the CtB\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Iba1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells. The cells on B) show ellipsoid macrophage morphology, possibly infiltrated macrophages, while most of the Iba1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells were classified as activated microglia as on panel C). D) The cells marked in panel C are shown Y-angle rotated in a position to the RPE.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/8342553e777970b11b55d512.png"},{"id":73939813,"identity":"86073d40-0d85-4969-9fa4-b343235c5789","added_by":"auto","created_at":"2025-01-16 07:47:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":678559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eHigh-resolution images from microglia in the inner retina (A; GCL) and RPE (B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. CtB is only internalized by RPE microglia (B) but not inner retinal ramified microglia contrary to the direct contact with the CtB-A555 particles of these cells (A). In the RPE, Iba1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells internalized the dye and clearly expressed JamB tight junction protein (blue arrowheads)(\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eSuppl. Video 1\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e for rotation of B); JamB is normally expressed only by RPE cells (blue, highlighted with light blue dots).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/ee7d57ce6c1bfdcffe758905.png"},{"id":73941217,"identity":"276eeab3-aa55-4982-b66b-d67fbb2a9b49","added_by":"auto","created_at":"2025-01-16 07:55:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1841766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSRH-induced microglial motility in the live retina. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA) Ex vivo confocal images from the live imaging sample area (the dashed area outlines the visible area in B). B) Image created by collapsing 81 frames of a 20-minute-long time-lapse recording along the time domain, where colors represent the frames (see the color scale on top). This image sequence was recorded at the SRH injection site, 24 hours post-injection with CtB-A555. The presence of rainbow-colored structures reflects the time-dependent change in the location of structures; when the location of a structure is unchanged, the corresponding pixels contain all colors thus they appear white. See also the time-lapse \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eSuppl. Video 2.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e C) Motion detected in the inset during the 20-minutes timelapse video (15 sec/frame time-lapse, 4xZ-merged). Each image represents the accumulated movement in 2 min. Color code shows movement activity across frames.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/e3530391ff3248e965e70d88.png"},{"id":73943157,"identity":"140dee93-5387-4ba2-94ff-0e120c13b393","added_by":"auto","created_at":"2025-01-16 08:19:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9329923,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/e2868903-cbfd-40ae-a316-cd594c493465.pdf"},{"id":73939753,"identity":"cdc4cca3-e60a-4a7f-9595-da8045d5bac8","added_by":"auto","created_at":"2025-01-16 07:47:02","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":738735,"visible":true,"origin":"","legend":"","description":"","filename":"SupVid1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/ff9b2e2ad9185bb56501e8f1.mp4"},{"id":73939792,"identity":"2182fba2-4396-4b31-b47e-c0b5ee6bbe67","added_by":"auto","created_at":"2025-01-16 07:47:04","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5773089,"visible":true,"origin":"","legend":"","description":"","filename":"SupVid2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/3847e52ad74be07592842d9e.mp4"},{"id":73939761,"identity":"f87aa530-ba24-415c-9e2a-f16dc5d275b6","added_by":"auto","created_at":"2025-01-16 07:47:02","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1141137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSuppl. Fig.\u0026nbsp;1.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Control RPE without the presence of Iba1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"supfig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5788846/v1/0cfd4c073ddbd275a8ec2af5.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Local Microglial Activation Induced and Labeled in the Retina in a Novel Subretinal Hemorrhage Mouse Model","fulltext":[{"header":"Background","content":"\u003cp\u003eThe retina is a gateway to all visual information. It is, however, one of the most exposed parts of the central nervous system (CNS) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), and thus susceptible to diverse retinal diseases and environmental insults (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Bleeding in the eye can arise from various etiologies, including trauma, systemic diseases, and ocular disorders, and in most cases, leads to major vision loss or blindness (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Retinal hemorrhages can be classified based on their location within the retinal layers: preretinal, intraretinal, and subretinal.\u003c/p\u003e \u003cp\u003eSubretinal hemorrhage (SRH) is the accumulation of blood between the retina and the retinal pigment epithelium (RPE) or between the RPE and the choroid (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The presence of blood in this space is toxic to retinal cells, and rapid tissue damage is induced starting with photoreceptor and RPE degradation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). If the clot is left untreated, scarring processes can be initiated potentially leading to visual loss in the affected area. If the hemorrhage occurs in the macular region, it usually leads to severe and irreversible vision loss (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, the visual prognosis in all cases depends on the initial visual acuity, the size of the hemorrhage, the time interval between the onset of SRH and treatment, and the concomitant involvement of different retinal segments. The development of SRH may be associated with several pathologies such as age-related macular degeneration, hypertension, diabetic retinopathy, or even blunt head trauma (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe retinal tissue damage is facilitated by three main pathological pathways: 1) The clot acts as a diffusion barrier, which prevents the transfer of nutrients from the choroid to the photoreceptors by the RPE and the return of by-products to the choroid (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). 2) Mechanical effects are caused by the contraction of the fibrin network formed after the clot, which can tear the photoreceptor outer segment from the retina in sheets (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). 3) Lysis of erythrocytes results in the release of hemoglobin, which is no longer protected, and the iron in the heme is oxidized from the Fe\u003csup\u003e2+\u003c/sup\u003e state to the Fe\u003csup\u003e3+\u003c/sup\u003e state (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Oxidized heme and other blood breakdown products have been demonstrated to induce cell death of the RPE and photoreceptors (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In addition, other cellular changes are observed, including activation and infiltration of microglia and macrophages, M\u0026uuml;ller cell disposition, presence and activation of migrating RPE cells, suggestive of dedifferentiation and signs of gliosis (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). As a result, nerve cells will suffer from severe inflammation and cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMicroglia are the resident immune cells in the central nervous system, including the retina. In the human retina, they form a heterogeneous population; perivascular microglia filter substances that enter the retina through the vasculature, whereas parenchymal microglia have high motility, constantly monitoring the microenvironment to remove possible metabolic products and cellular debris and mediate synaptic remodeling (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Microglia are also programmed for immunological tolerance and possess an anti-inflammatory phenotype, which is essential for retinal immune competence (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBecause of their monocytic origin, these cells can respond incredibly quickly to pathological stimuli, therefore, they are excellent indicators of retinal pathologies, including inflammation. Microglia protect neurons from cell death and promote tissue regeneration but can also induce secondary tissue damage by triggering chronic inflammation (\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). They can take different morphological states; each morphotype is associated with different activation states and has unique functions to maintain a physiologically normal state (\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this research, we use microglia to show the retinal activation of SRH based on their morphotypes and movement in the tissue. For this, we developed a novel co-injection model because available SRH models did not allow for direct fluorescent labeling of the injection site, not to mention microglial activation through labeling with phagocytosis of the dye-toxin-subunit complex.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and preparation\u003c/h2\u003e \u003cp\u003eAdult, male, P30-90 old C57BL/6J (n\u0026thinsp;=\u0026thinsp;9) mice were maintained in a 12/12-hours dark/light cycle. Max. 4 mice were placed in a cage with food and water \u003cem\u003ead libitum\u003c/em\u003e. Animal housing, handling, and all utilized experimental procedures were approved by the ethical committee of the University of P\u0026eacute;cs (BA02/2000-27/2024). All animals were treated following the ARVO Statement for the \u0026lsquo;Use of Animals in Ophthalmic and Vision Research\u0026rsquo;. All efforts were made to minimize pain and discomfort during the experiments and all procedures were done by obeying the 3R law.\u003c/p\u003e \u003cp\u003eTo create the mouse model of SRH, animals were deeply anesthetized via intraperitoneal injection of pentobarbital (80x dilution from stock (400 mg/ml) \u0026rarr; for mice 180 \u0026micro;l/10 g). Autologous blood was collected from the tail vein with a 27-gauge needle and immediately used for subretinal injections. Before the subretinal injections, a temporal tunnel incision posterior to the limbus was made in the sclera using a 27-gauge needle under a dissecting microscope. The subretinal injections of autologous blood were achieved with a glass injector needle connected to a 1 ml syringe. Glass injector needles were pulled with a Sutter P-87 (program: Cycle1: Heat\u0026thinsp;=\u0026thinsp;760, Pull\u0026thinsp;=\u0026thinsp;250, Vel.=50, Time\u0026thinsp;=\u0026thinsp;200; Cycle2: Heat\u0026thinsp;=\u0026thinsp;760, Pull\u0026thinsp;=\u0026thinsp;250, Vel.=0, Time\u0026thinsp;=\u0026thinsp;200) from 1.5mm capillaries (World Precision Instruments, Inc. Item Nr. 1B150F-4, Lot Nr. 2605323). For the simple SRH group, we injected 2 \u0026micro;l blood into the subretinal space. During the co-injection experiments, we loaded the glass injector with 0.25 \u0026micro;l of CtB conjugated with Alexa-555 solution (cc.: 1\u0026micro;g/\u0026micro;l in PBS, Invitrogen, Lot Nr. 201622) followed by 2 \u0026micro;l of blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We performed the SRH injections only on the left eyes, while the right eyes were used as controls.\u003c/p\u003e \u003cp\u003eAfter 24-hour survival, mice were sedated with Forane (isoflurane 4%, 200 \u0026micro;l/l) and then sacrificed using cervical dislocation. Eyes were immediately removed with fine forceps. Eyeballs were cut at the ora serrata, and the lens and vitreous body were removed. The retinas and the attached pigment epithelium sheets were fixed in 4% paraformaldehyde on filter papers for 30 min at room temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFlat-mounted retinas and RPEs were blocked in blocking solution (CTA, containing 5% Chemiblocker, 0.5% TritonX-100, 0.05% Na-azide in PBS) for 2 hours at room temperature, humidified. After blocking, the retinas and RPEs were treated with the primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; diluted in CTA) for 48 hours at room temperature (RT). After washing three times in PBS, secondary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were applied in CTA, and incubated overnight at RT. After washing three times for 10 min in PBS, they were coverslipped facing ganglion cell layer (GCL) up, or RPE up with Vectashield (Vector Laboratories, Peterborough, UK).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibodies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003ePrimary Antibodies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbbr.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRRID\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIonized calcium-binding adapter molecule 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egp-Iba1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2000x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSySy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e234004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_2924932\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJunctional adhesion molecule B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egt-JAM-B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u0026amp;D sys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAF988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAB_355767\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSecondary Antibodies\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eName\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAbbr.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eDilution\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eSource\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003eCode\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-guinea pig - Alexa647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ea-gp-A647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eA21450\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-mouse - Alexa405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ea-ms-A405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbCam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eab175658\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-goat - Alexa647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ea-gt-A647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbCam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eAB150131\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-guinea pig - DyLight405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ea-gp-DL405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJackson\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e706-475-148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eMicroscopy\u003c/h3\u003e\n\u003cp\u003eRetinas and RPEs were inspected using a Zeiss LSM 710 confocal laser scanning microscope (with Plan Apochromat 10x, 20x, and 63x objectives (NA: 0.45, 0.8, 1.4, Carl Zeiss Inc., Jena, Germany)) with normalized laser power and filter settings making 1.5- and 0.5-\u0026micro;m thin optical sections.\u003c/p\u003e\n\u003ch3\u003eImage analysis\u003c/h3\u003e\n\u003cp\u003eAll measurements were performed using FIJI (ImageJ, NIH, Bethesda, MD, USA). First, we performed two z-merges from consecutive optical stacks for the superficial and deep regions of microglia from 3 areas from the SRH-treated eyes (Z1-3) and one area from the control eyes (Z4). We chose the injection site (Z1) based on the presence of blood albumin (a-albumin staining). The size was variable, depending on the injection. Z2 was determined as an area (700x700 \u0026micro;m) in connection with the injection site but with no extravasal albumin present. The Z3 was the distal-most relative to the injection site (contralateral retinal hemisphere) in the sample (700x700 \u0026micro;m).\u003c/p\u003e \u003cp\u003eWe used the 20x magnification for clustering microglia activation. Microglia were divided into superficial (SL) and deep layers (DL) defining their positions in the retina, represented by the z-stack regions from GCL, inner plexiform layer, neuronal layers, and the superficial layer of the inner retinal vasculature when merging images for SL-microglia and the outer plexiform layer/DL neuronal layers together with the deep layer of inner retinal vasculature to merge the microglia images for the DL from the same Z-stack. Microglia were separated from the other signals based on their expression of ionized calcium-binding adapter molecule 1 (Iba1). Cells were manually grouped one by one according to their morphologies into activated and non-activated (see the details of cell classification in (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), using the \u0026ldquo;Cell-counter\u0026rdquo; plugin in FIJI. Only fully visible cells were included in the analyses, while we omitted those that fell on the edges.\u003c/p\u003e \u003cp\u003eMotion was detected on 15 sec. time-lapse images. We decoded the change in Z with ImageJ ΔF/F script (by (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)).\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eData was curated in MS Excel. Non-parametric Kruskal-Wallis and Dunn\u0026rsquo;s posthoc analyses were performed using IBM SPSS Statistics Version 25.0 (IBM Corp., Armonk NY). Normality tests were performed before the analyses, because some of the data sets showed non-normal distribution, we chose to perform non-parametric tests on all data.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eA novel SRH model\u003c/h2\u003e \u003cp\u003eBased on previous studies (see details in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we have developed a modified model for SRH, where compared to previous methods, we used freshly pulled glass injectors to avoid complications after injection and labeled the injection site with the co-injection of CtB-A555 toxin subunit conjugate to allow better visualization of the injection site and the phagocytosis of the dye in the live retina.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrevious SRH models.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnimal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLimitations\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMouse\u003c/b\u003e (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInjections via transscleral route with a 33G needle 1.6 \u0026micro;l autologous blood (from tail vein);\u003c/p\u003e \u003cp\u003esurvival times 6 hours to 10 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIncreased expression of inflammatory cytokines, chemokines and adhesion molecules; microglial migration, increased density; effects of minocycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eusage of metal needle\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRat\u003c/b\u003e (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eheparinized and diluted blood (one part blood to ten parts saline) injected subretinally\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThe retina overlying the hemorrhage became intensely degenerated over a period of months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeparin is an anticoagulant, clotting process differs from fresh blood injections\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRabbit\u003c/b\u003e (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1 ml fresh blood from ear;\u003c/p\u003e \u003cp\u003especially constructed 30G needle tip encased in a 22G spinal needle;\u003c/p\u003e \u003cp\u003eclot prevention 0.03cc of air in the needle tip (also injected in the subretinal space);\u003c/p\u003e \u003cp\u003esurvival times:1\u0026nbsp;hour to 28\u0026nbsp;days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSigns of degeneration after 1\u0026nbsp;day in the outer retinal layer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eusage of plus air bubble, can make retinal detachment worse\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRabbit\u003c/b\u003e (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1 ml fresh blood from ear;\u003c/p\u003e \u003cp\u003e30G needle;\u003c/p\u003e \u003cp\u003eClot prevention: air bubble\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntravitreal injection of tPA 1 day after subretinal injection of blood in rabbits facilitated more rapid lysis of the clotted blood, however, retinal damage was not prevented\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eusage of plus air bubble, can make retinal detachment worse\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRabbit\u003c/b\u003e (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u0026nbsp;\u0026micro;l autologous blood from ear vein in a nasal juxtapapillary location along the myelinated streak;\u003c/p\u003e \u003cp\u003eAn angled 1-inch, 30-gauge needle was introduced transsclerally 3\u0026ndash;4 mm posterior to the limbus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhotoreceptor toxicity caused by SRH occurs at least in part by apoptosis and is associated with iron migration to the photoreceptor layer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eusage of metal needle\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCat\u003c/b\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ethe tip of a 20-gauge surgical knife or a 25-gauge needle was passed transsclerally into the bleb and then withdrawn, allowing choroidal blood to extend under the retina into the area centralis;\u003c/p\u003e \u003cp\u003eLesions were observed 25 minutes to 14 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIn 6 of 9 clots more than 1 hour old, fibrin was associated with tearing of sheets of photoreceptor inner and outer segments. Later degeneration progressed to involve all retinal layers overlying the densest areas of fibrin in the clots; hemorrhages into subretinal blebs containing tPA did not form fibrin strands or cause photoreceptor tearing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eusage of metal needle\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCat\u003c/b\u003e (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ethe tapetal or nasal retina with a neodymium:YAG (Nd:YAG) laser focused through a preformed retinal bleb; 2\u0026ndash;25 laser shots (mean, 14) at between 20 and 25 mJ were fired until one created a large subretinal hemorrhage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRemoving experimental SRHs within 7 days of their occurrence with the assistance of rt-PA and an ultra-microsurgical approach may reduce outer retinal degeneration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLaser\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eInjection sites of all injected mice healed entirely without any complications or infection. The entry site on the eyeballs were closed in 24 hours with only mild post-operational scar visible only in the stereo microscope after careful examination, where we were able to detect and validate the injection site and the presence of subretinal blood in the eyecup before fixing with 4% paraformaldehyde or removing the retina for live imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the first time, we used CtB to label the injection site in the subretinal space and the overlying cells, therefore, the retinal localization of the injection can be precisely outlined. By using this technique, we have defined three zones in the injected eye, including the injection site (Z1), the neighboring site, in direct contact with the injection site (Z2), and the contralateral same-retinal zone (Z3). We added an endogenous control zone from the contralateral, non-injected eye (Z4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe observed that the CtB-A555 not only overlapped with the blood-related extravasal albumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) but the label displays better Z1 vs. Z2 contrast superior to the albumin signal based on laser intensities and gain, therefore, it is easier to demarcate the SRH site in comparison to the non-co-injected retina, where only intravasal albumin was present (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). We could not identify any blood-related extravasal albumin deposits in the contralateral side (Z4) retinas, used as an endogenous negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eBy using higher magnification (20x) each area can be easily outlined with the CtB-A555, therefore each of the microglia could be classified into the 4 zones (Z1-4) without a problem. Based on this our measurements accurately represent the microglial states in each of the zones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) As the example images show the microglial cells from the SL, that lie on the NFL side of the GCL, show massive activation that can be seen also in the DL, at the border of the inner nuclear layer and outer plexiform layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The two layers of MGs shown in the healthy retina broke up and became miss-ordered in Z1 while in Z2 the SL-DL division was still intact while more activated MGs were present (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) compared to both Z3 and Z4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRetinal microglial activation after SRH\u003c/h3\u003e\n\u003cp\u003eBased on morphological classification (previously described in (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)) of microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), SRH induced microglial activation in Z1 and Z2 was present in both the SL and DL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We observed a 3.66-fold increase of microglia count in Z1 (from a median of 6\u0026thinsp;\u0026plusmn;\u0026thinsp;3 to 22\u0026thinsp;\u0026plusmn;\u0026thinsp;16) as well as a 3.33-fold increase in Z2 (from 6\u0026thinsp;\u0026plusmn;\u0026thinsp;3 to 20\u0026thinsp;\u0026plusmn;\u0026thinsp;8) when compared to the control Z4 sample. In addition, we detected an increase in the number of microglia between the Z1 and the Z3 and between the Z2 and Z3 (Z3-Z1: 3.14-fold, from 7\u0026thinsp;\u0026plusmn;\u0026thinsp;5 to 22\u0026thinsp;\u0026plusmn;\u0026thinsp;16; Z3-Z2: 2.86-fold, from 7\u0026thinsp;\u0026plusmn;\u0026thinsp;5 to 20\u0026thinsp;\u0026plusmn;\u0026thinsp;8). A similar comparison of Z3 and Z4, did not result in a change in the number of the activated microglia numbers, meaning that these microglia lack activating signals, retaining their non-activated morphologies. We did not see any significant difference between the total microglia numbers in the different zones, however, we observed an increased variability of the total microglia counts across retinas in Z1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, n\u0026thinsp;=\u0026thinsp;3202 cells in 17 retinas).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, when we examined the microglial activation in the SL and DL, we observed a less pronounced change. We only detected a significant difference between Z1 and Z3 in the number of activated microglia in the SL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cb\u003e3\u003c/b\u003e.57-fold, from 7\u0026thinsp;\u0026plusmn;\u0026thinsp;6 to 25\u0026thinsp;\u0026plusmn;\u0026thinsp;15). On the other hand, we found that the counts of the non-activated microglia in the SL were significantly less in Z2 when compared to either Z3 or Z4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Z4-Z2: 2.47-fold, from 42\u0026thinsp;\u0026plusmn;\u0026thinsp;6 to 17\u0026thinsp;\u0026plusmn;\u0026thinsp;9; Z3-Z2: 2.24-fold, from 38\u0026thinsp;\u0026plusmn;\u0026thinsp;8 to 17\u0026thinsp;\u0026plusmn;\u0026thinsp;9). When examining the DL, we found a significant increase in the number of activated microglia in Z2 compared to both Z3 and Z4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Z4 - Z2: 5.5-fold, from 4\u0026thinsp;\u0026plusmn;\u0026thinsp;2 to 22\u0026thinsp;\u0026plusmn;\u0026thinsp;8; Z3 -Z2: 3.14-fold, from 7\u0026thinsp;\u0026plusmn;\u0026thinsp;5 to 22\u0026thinsp;\u0026plusmn;\u0026thinsp;8). Examining the number of non-activated microglia, there was a significant decrease of Z1 and Z2 microglia counts when compared to Z4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, Z4 - Z2: 3.06-fold, from 52\u0026thinsp;\u0026plusmn;\u0026thinsp;9 to 17\u0026thinsp;\u0026plusmn;\u0026thinsp;6; Z4-Z1: 2.26-fold, from 52\u0026thinsp;\u0026plusmn;\u0026thinsp;9 to 23\u0026thinsp;\u0026plusmn;\u0026thinsp;19).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicroglia in the subretinal space\u003c/h2\u003e \u003cp\u003eIn healthy animals, microglia are normally only present in the inner retina. We examined if SRH attracted inner retinal microglia to the affected subretinal site or induced a macrophage infiltration. Based on the morphological divergence, we regularly observed microglia in direct contact with the RPE layer with only a sparse presence of macrophage-like Iba1\u003csup\u003e+\u003c/sup\u003e cells (6 macrophage-like morphologies out of 27 Iba1\u003csup\u003e+\u003c/sup\u003e cells, n\u0026thinsp;=\u0026thinsp;2; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). By using the Junctional Adhesion Molecule B (JamB), we labeled RPE tight junctions to outline the microglia on the retinal side. Although these cells were less numerous, all of them showed an activated morphology, including enlarged, disorganized soma, relative soma size increase, amoeboid and leaf-like structures on the dendritic endfeet, and aggregated occurrence of these activated microglia morphologies. We were unable to find Iba1\u003csup\u003e+\u003c/sup\u003e cells attached to the RPE in healthy, control retinas, with only the JamB labeling of RPE cells being clearly visible (\u003cb\u003eSuppl. Figure\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCtB labeling on and inside MGs\u003c/h2\u003e \u003cp\u003ePrevious studies suggested CtB can form direct contact with microglial cell membranes (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). By using high magnification microscopy, we observed the internalization of CtB-A555 in infiltrated microglia, in the vicinity of the RPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Interestingly, this was not the case with the rest of the inner retinal microglia as we did not find microglia internalizing CtB particles. These latter microglia on the other hand still showed that their endfeet moved in the direction of dye particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). This observation was reinforced by the \u003cem\u003eex vivo\u003c/em\u003e live imaging, where we were able to follow the microglia in real-time and observed their change in motility that increased due to inflammatory signals. Microglia appeared highly motile, and they also started breaking down CtB plaques by phagocytosis and transporting them (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNovel SRH model\u003c/h2\u003e \u003cp\u003eIn the present study, a modified model was established for SRH in the mouse eye. In contrast to previous models (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), no air or heparin was used to stop the rapid clotting. Instead, the sterile and inert glass injector gives enough working time to perform the SRH injection. The use of smaller, pulled, and therefore heat-sterilized needles protect against reflux (blood or blood-CtB mix) at the injection site (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). There were no signs of infections, such as redness, purulent discharge, swelling, discomfort, irritation indicated by scratching, photophobia. The use of a thin, hollow microneedle helps the delivery method to be minimally invasive because the microneedle only penetrates some hundreds of micrometers into the eyeball and narrows down to 8\u0026ndash;12 \u0026micro;m at the tip, thereby protecting the retina from mechanical damage and also reducing the risk of infection (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBesides using pulled glass micropipettes to deliver blood into subretinal space, our method is novel due to the utilization of CtB-A555 co-injection. CtB-conjugates generally serve as retrograde neuronal tracers (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) that, following endocytosis, are transported back to the Golgi network and the endoplasmic reticulum. In our labeling scheme, CtB-A555 has been used for the first time to help identify the injection site.\u003c/p\u003e \u003cp\u003eIn microglia, CtB is colocalized either by binding to GM1 ganglioside glycolipid molecules in lipid rafts or was engulfed by microglia and stored intracellularly (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In our experiments, we show that the A555 conjugate was similarly localized intracellularly. Previous studies have shown that activated microglial cells can engulf various dyes due to their tissue-specific clearing phagocytic activity (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Apparently, microglia can internalize CtB molecules as well via similar mechanisms and thus its injection combined with fluorescent fundoscopy can be used to visualize cells even in the human eye when needed (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Although the recently developed adaptive optics fundoscopy also offers a dye-free solution to visualize microglia and thus a minimally invasive examination of both the mouse and the human eye is possible, allowing a direct examination of the live retina (\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) our CtB-conjugate coinjection method still provides a cost-efficient alternative for laboratories with limited or no access to such equipment with much higher detail, even allowing super-resolution reconstruction of the cells. Finally, even if our study has different aims, CtB can have anti-inflammatory effects as well (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) thereby further decreasing the risk of inflammation in the experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicroglia activation in SRH\u003c/h2\u003e \u003cp\u003eMicroglia are resident macrophages of the retina that perform surveillance and neuroprotection against pathophysiological insults (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). In the healthy retina, they are quiescent and confined to the GCL and the plexiform layers. When microglia get activated and migrate to the location of the insult, they may appear among photoreceptor outer segments or RPE in retinitis pigmentosa and age-related macular degeneration, respectively (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Similarly, we observed that after SRH, microglia appear in the vicinity of the RPE and most of these pathologically located cells displayed morphological characteristics of the activated microglia phenotype.\u003c/p\u003e \u003cp\u003eHowever, previous studies limited the examinations to the outer retina (RPE and photoreceptors) whereas possible parallel inner retinal changes have rarely been described. In this present study we expanded our observations to the inner retinal areas as well and identified a widespread activation of microglia due to SRH treatment. This suggests that SRH initiated a complex chemotactic activation of blood-related factors, including fibrin and albumin. When these factors appear in the extravasal space (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) and diffuse to inner tissue compartments, they activate microglia, which then migrate towards the blood clot to facilitate clearance. By performing this radial migration, microglia must pass photoreceptor inner and outer segments and eventually reach the RPE.\u003c/p\u003e \u003cp\u003eIn our study, we observed both microglia that internalized CtB-A555 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) and also microglia with no sign of internalized particles. This indicates that a population of microglia participated in the clearance of the dye through phagocytosis while the rest of them were only activated without transforming into phagocytic cells. However, it is uncertain whether these two functionally different populations exist or phagocytotic transformation was just delayed for some of the cells. In a previous study, the accumulation of fluorescent carbocyanine vital dyes in microglia demonstrated the phagocytosis of dying neurons (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Here, we showed that CtB-A555 molecules can be endocytosed by microglia from the extracellular milieu (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCtB has potential anti-inflammatory effects as previously shown by Zhang et al (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). 24 hours after ischemia, CtB downregulated the levels of proinflammatory cytokines and microglia/macrophage transformation. Based on microglia morphological characteristics, we did not observe any indication of this latter CtB-induced effect as microglia counts of simple blood injections and CtB/blood coinjected samples appeared similar (data not shown). Microglia activated by SRH can release a number of proinflammatory cytokines that can induce inflammation and corresponding tissue degeneration later on (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Therefore, any inflammatory changes observed in our experiments are rather induced by the SRH and corresponding microglia activation and largely independent of the presence of coinjected CtB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBordering zones and localization\u003c/h2\u003e \u003cp\u003eAccording to our results, SRH is well maintained within an outlined Z1 including the injection site with the primary insult induced by the blood clot. Surprisingly, microglia in the Z2 area were activated as well contrary to the fact that they were not in direct contact with the blood clot itself. Z2 microglia may get informed about the insult through proinflammatory factors (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) either originating from the clot and/or released by activated microglia of the Z1 site. Contrary, we have not detected activation or changes in the number of microglia in the non-affected Z3 and Z4 sites (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Therefore, we hypothesize that SRH is limited to the Z1 and Z2 sites while the rest of the tissue remains intact.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eSRH can be a devastating disease affecting vision. Our new model can help identify new treatment methods while enabling the monitoring of microglia with dye-conjugated CtB. The results show that overlaying inner retinal microglia are activated even if blood is injected between the photoreceptors and the RPE. Activated microglia bind CtB-A555 but are less likely to phagocyte it while microglial and infiltrated macrophages at the RPE will internalize the dye. This internalized CtB dye, and the morphological changes of microglia can be visualized in the living tissue for the monitoring of the microglial activation process. Moreover, in this model, the borders of the SRH site can be precisely demarcated thus allowing for the development of future solutions to avoid retinal cell death or to use microglia as biomarkers during pathological changes.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eA555\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAlexa-555 (fluorescent dye)\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAm\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAmacrine Cell\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAMD\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eage-related macular degeneration\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAO\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eadaptive optics\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAS\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAstrocyte\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eBC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eBipolar Cell\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eCone\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ecLSM\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003econfocal laser scanning microscope\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCNS\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ecentral nervous system\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCTA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eBlocking solution containing 5% Chemiblocker, TritonX-100, 0.05% Na-azide in PBS\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCtB\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eCholera toxin subunit B\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eDL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eDeep Layer (of microglia)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eERK1/2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eExtracellular signal-regulated kinase 1/2\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eGC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eGanglion Cell\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eGCL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eGanglion Cell Layer\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eHC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eHorizontal Cell\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eIBA1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eIonized calcium-binding adapter molecule 1\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eIL-1\u0026szlig;\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003einterleukin-1\u0026szlig;\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eIL-6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003einterleukin-6\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eINL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eInner Nuclear Layer\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eIPL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eInner Plexiform Layer\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eISI\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003einjection site\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eJAM-B\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eJunctional adhesion molecule B\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eMG\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eMicroglia\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNF-kB\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eNuclear factor kappa-light-chain-enhancer of activated B cells\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNFL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eNeurofilament Layer\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOCT\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eoptical coherence tomography\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eONL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eOuter Nuclear Layer\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOPL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eOuter Plexiform Layer\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOSL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eOuter Segment Layer\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ep65\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eNF-kB p65 subunit, encoded by RELA gene\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePBS\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePhosphate buffered saline solution\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRPE\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eretinal pigment epithelium\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRT\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eroom temperature\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSL\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSuperficial Layer (of microglia)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSRH\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSubretinal hemorrhage\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eTGF-\u0026szlig;\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003etransforming growth factor-\u0026szlig;\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eZ1-4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ezone 1\u0026ndash;4\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eAnimal housing, handling, and all experimental procedures were approved by the ethical committee of the University of P\u0026eacute;cs under the number BA02/2000-27/2024. All animals were treated following the ARVO Statement for the \u0026lsquo;Use of Animals in Ophthalmic and Vision Research\u0026rsquo;. All efforts were made to minimize pain and discomfort during the experiments and all procedures were done by obeying the 3R law.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was financially supported by NKFIH, MOH and BMBF under the action of the ERA-NET COFUND (2019-2.1.7-ERANET-2021\u0026ndash;00018; NEURON (NEURON-066 Rethealthsi) to BV. This research was also supported by the NKFI (OTKA NN128293) (B.V.) from the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TKP2020 IKA-07 National Excellence Program (B.V.). In addition, this research was also financed by the Thematic Excellence Program 2021 Health Sub-program of The Ministry of Innovation and Technology (Hungary), within the framework of the EGA-16 project of the University of P\u0026eacute;cs. Supported by the EK\u0026Ouml;P-24-4-I-PTE-11 (G.S.), EK\u0026Ouml;P-24-3-I-PTE-129 (B.B.) University Research Grant Programme of the Ministry of Culture and Innovation, National Fund for Research, Development, and Innovation. This work was carried out using the Nano-Bio-Imaging and SzKK Imaging Core Facilities of the Szent\u0026aacute;gothai Research Centre, University of P\u0026eacute;cs.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eConceptualization, TKO and BB; methodology, TKO and BB; validation, TKO, BB, GS, MZ, LP, VB; formal analysis, BB, TKO; investigation, BB, TKO, GS, LP, AF ; resources, BV, MZ, BB, GS, TKO; data curation, TKO, BB, GS, MZ, LP, VB; writing\u0026mdash;original draft preparation, TKO and BB; writing\u0026mdash;review and editing, TKO, BB, GS, MZ, LP, AF, VB; visualization, BB, TKO, GS; funding acquisition, BV, MZ, BB, GS, TKO. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was carried out using the Imaging Core Facility and the Animal Facility of the Szent\u0026aacute;gothai Research Centre, University of P\u0026eacute;cs.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLondon A, Benhar I, Schwartz M. The retina as a window to the brain\u0026mdash;from eye research to CNS disorders. Nature Reviews Neurology 2012 9:1 [Internet]. 2012 Nov 20 [cited 2024 Sep 18];9(1):44\u0026ndash;53. 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Trends Neurosci. 1994;17(5):177\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"bleeding, blood, cholera toxin, experimental ophthalmology, eye, inflammation, retinal pigment epithelium","lastPublishedDoi":"10.21203/rs.3.rs-5788846/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5788846/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSubretinal hemorrhage (SRH) is caused by the accumulation of blood between the neurosensory retina and the retinal pigment epithelium or between the retinal pigment epithelium and the choroid. It often arises from age-related macular degeneration, traumas, and may occur spontaneously caused by other diseases like hypertension and diabetes. Here, we developed a novel technique by co-injection of blood and a dye-coupled tracer protein, Cholera toxin subunit B (CtB), to better localize and understand the disease and how it can cause microglial activation, inflammation, and partial vision loss. Our results show that microglia are activated in the inner retinal regions and also in the zones neighboring the blood injection. In contrast, the non-affected zone of the affected eye showed no microglial activation. Based on the results, we provide evidence of inner retinal microglial activation and the appearance of microglia and macrophages in the pigment epithelium. Using advanced imaging techniques, we were able to localize better the affected area that comprises not only the retinal area over the blood clot but the neighboring regions as well. These findings will provide the basis for novel therapeutic interventions targeting neuroinflammation in the retina after subretinal hemorrhage and other diseases affecting the eye.\u003c/p\u003e","manuscriptTitle":"Local Microglial Activation Induced and Labeled in the Retina in a Novel Subretinal Hemorrhage Mouse Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-16 07:46:57","doi":"10.21203/rs.3.rs-5788846/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"797f6059-8ef5-4b39-ba85-fd09591436e9","owner":[],"postedDate":"January 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-21T07:53:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-16 07:46:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5788846","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5788846","identity":"rs-5788846","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}