Regional differences in peripapillary scleral fibroblast cytoskeleton and nuclear morphology after intraocular pressure elevation in mouse eyes

Regional differences in peripapillary scleral fibroblast cytoskeleton and nuclear morphology after intraocular pressure elevation in mouse eyes | 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 Article Regional differences in peripapillary scleral fibroblast cytoskeleton and nuclear morphology after intraocular pressure elevation in mouse eyes Ann Mozzer, Joe Gerald Jesu Raj, Nushrat Yasmin, Edward Esposito, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5397173/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract These studies aimed to study the mechanisms of glaucomatous peripapillary scleral (PPS) remodeling by investigating IOP-induced changes in fibroblast actin-collagen alignment and nuclear morphology in mouse PPS. Cryosections from the optic nerve heads (ONH) of eyes isolated 1- and 6-weeks after bead-induced IOP elevation were imaged for nuclei, fibrillar actin (FA), and collagen (second harmonic generation, SHG). Nuclear morphology was analyzed using VAMPIRE machine-learning image analysis and FA-collagen alignment was determined by comparing vector fields of FA and SHG images. Nuclear morphology was regionally defined with the inner pial PPS (pPPS) containing nuclei with higher aspect ratios than the peripheral PPS (outer PPS, oPPS) and peripheral sclera. FA-collagen alignment was higher in the PPS than in the peripheral sclera (7.1±2.5° versus 10.0±1.4°, p = 0.05, n = 6). One and six weeks after BI, there were nuclear morphologic changes reflecting a transition to a rounder shape in all scleral regions and persistently reduced FA-collagen alignment in the PPS regions. This study therefore concludes that chronic IOP elevation is associated with persistent alterations in nuclear morphology and FA-collagen alignment that indicate sustained cellular responses to tissue stress. These changes could be a precursor to more chronic scleral remodeling that underlies glaucoma pathogenesis. Biological sciences/Cell biology/Cell migration Health sciences/Diseases/Eye diseases/Ocular hypertension Health sciences/Diseases/Eye diseases/Scleral diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Glaucoma, a leading cause of irreversible blindness worldwide, is a multifactorial disease characterized by progressive vision loss due to retinal ganglion cell death that is initiated at the optic nerve head. 1 – 3 Risk factors for glaucoma pathogenesis and progression include genetic contributions, aging, high IOP levels, vascular insufficiency, and neuroinflammation. IOP fluctuations generate mechanical strain in optic tissues that contributes to glaucoma progression. 4 – 11 The structural and mechanical properties of the optic nerve head (ONH) region, which includes cellular and fibrous elements of the peripapillary sclera (PPS) and the lamina cribrosa (LC), play a critical role in development and progression of glaucoma by translating IOP-mediated mechanical strain to ONH axons, astrocytes, and microglia. 3 , 12 – 14 PPS stromal fibroblasts tune the mechanical properties of the ONH by remodeling and maintaining scleral tissue. Their prominent role in glaucoma pathogenesis was highlighted by genome wide association studies (GWAS) of glaucoma patients and single nucleus transcriptomics studies of ocular tissues. 15 The PPS fibroblast response to IOP elevation, however, is not yet fully characterized. Scleral fibroblasts reside within different extracellular environments that are designed to meet diverse functional requirements. 10 Collagen fiber lamellae within the PPS are circumferentially arranged around the optic nerve and have a relatively high elastin content – features that protect the optic nerve from IOP-generated mechanical strain. This circumferential microarchitectural organization contrasts with the peripheral sclera in which collagen lamellae have a basket weave organization and the limbal sclera in which downstream aqueous humor outflow pathways are present. In glaucoma, PPS mechanical qualities are altered - reduced collagen alignment, stiffening, loss of elastin organization - which shifts the biomechanical response to IOP variations to cause axonal damage at the ONH. 16 – 20 Mechanical alterations that occur in glaucomatous sclera do not arise passively, but rather through the combined remodeling actions of scleral cells – the majority of which are resident fibroblasts. Fibroblast function, in turn, is influenced by extracellular matrix (ECM) composition, organization, and biomechanical stimuli that are transmitted through the ECM. Due to the complexity of the scleral extracellular environment, this iterative process in which ECM and fibroblasts communicate to remodel tissue cannot be modeled in cell culture. There is a recognized need to develop techniques to study fibroblasts in their tissue environment. Scleral fibroblasts are exposed to IOP-generated mechanical strains that influence their structure, gene transcription, and phenotypic behavior. 10 These alternations include changes in cell morphology, proliferation, migration, extracellular matrix synthesis, and cell differentiation. 21 – 24 Cells can detect and respond to diverse external stimuli including matrix stiffness, surface topography, ligand presentation, and compressive, tensile, and shear strain. 25 Actin filaments, as a major component of the cytoskeleton, are particularly sensitive to extracellular cues and undergo dynamic reorganization in response to mechanical stimuli. Actin rearrangements, in turn, influence cell adhesion, migration, and differentiation. Mechanical forces transmitted through the extracellular matrix are communicated to nuclei which not only impacts nuclear morphology but also gene expression. 26 – 28 Dysregulation of the cellular response to mechanical stimuli can lead to pathologic conditions such as organ fibrosis. 29 , 30 Mechanotransduction pathways, such as the Rho/ROCK, integrin, and MAPK signaling cascades, have been implicated in mediating these fibroblast responses to mechanical strain; however, their role in glaucomatous scleral remodeling is not fully known. 31 – 34 We previously demonstrated a close association between cellular structures and collagenous ECM in normal human PPS 22 , finding that filamentous actin (FA) was closely aligned with surrounding collagen fibrils in the PPS. Additionally, the nuclear aspect ratio varied regionally, with rounder PPS nuclei and more elongate nuclei in the sclera further from the ONH. To investigate these phenomena under experimental conditions, we used a mouse model of sustained IOP elevation to quantify changes in PPS fibroblast cytoskeletal alignment with extracellular collagen fibers and nuclear morphology. 2. Materials and Methods 2.1 Bead-injection All animal experimentation was conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Johns Hopkins University and according to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Experimental design and reporting are in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE, 2.0) guidelines. We used 18 eight to twelve-week old male and female CD1 mice (Charles River, Inc., Wilmington, MA) in total. Twelve mice underwent unilateral intracameral bead injection as described previously and six were used as naïve controls. 35 For bead-induced ocular hypertension (BI), following sedation with a mixture of ketamine (Fort Dodge Animal Health, Fort Dodge, IA), xylazine (VedCo Inc., Saint Joseph, MO), and acepromazine (Phoenix Pharmaceuticals, Burlingame, CA) polystyrene microspheres were injected into the anterior chamber using a glass cannula with a 50 µM tip diameter connected to a Hamilton syringe (Hamilton, Inc. ,Reno, NV). We injected a mixture of Polybead Microspheres® (Polysciences, Inc.,Warrington, PA, USA) of 6 µM and 1 µM diameter followed by 10 mg/ml sodium hyaluronate (Healon PRO; Advanced Medical Optics Inc., Santa Ana, CA). IOPs were measured using the TonoLab rebound tonometer (iCare, Vantaa, Finland) according to the manufacturer’s instructions with the magnetic probe in the horizontal position. IOPs were taken under anesthesia through inhalation of isoflurane administered by a RC2- Rodent Circuit Controller (VetEquip, Inc., Pleasanton, CA). Mice spent two minutes in an induction chamber and 1 minute on a nose cone before measurements were taken. The mean of two to three recordings per eye, with each recording from the tonometer resulting from six readings, was used as the final IOP measurement. 2.2 Specimen preparation and imaging After euthanasia by carbon dioxide inhalation and verification of death, eyes were enucleated under a dissecting microscope and prefixed for 1–3 hours with 4% paraformaldehyde in Sorenson's phosphate buffer. Extraocular tissue was removed, posterior eye cups were isolated and cryopreserved in a 2:1 mixture of Sucrose and TissueTek ® O.C.T. compound (Sakura Finetek USA, Inc., Torrance, CA) solution respectively. Eyes were then cryo-sectioned at 12–14 µm thickness. Section preparation and imaging was performed as previously described. 22 Sections were incubated in Hanks’ balanced salt solution (HBSS) with 0.5% Triton X-100 (HBT) and 10% normal goat serum (NGS; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) overnight at 4°C. Slides were then washed with HBT with NGS, SYTOX Green at 1:30,000 (S7020; Thermo Fisher Scientific, Waltham, MA, USA) and phalloidin Alexa Fluor 568 (A1280, Thermo). After washing with HBT and mounting with Dako mounting media (Dako, Carpenteria, CA, USA), imaging was performed on a Zeiss confocal laser scanning microscope 710 (Carl Zeiss Microscopy, Thornwood, NY, USA). SHG imaging was performed using a coherent Chameleon Ultra II laser tuned to 780 nm and a 390 to 410 band pass filter. Laser power (2–4%) was adjusted to minimize oversaturated pixels. Images were stitched using ZEN 2.3 software (Carl Zeiss Microscopy) and converted to .tiff for image analysis. 2.3 Filamentous actin (FA)-collagen alignment analysis We previously described a method to quantify alignment of FA with extracellular collagen fibrils in human sclera and demonstrated a high level of FA-collagen alignment in the PPS and peripheral sclera. 22 Here, we modified this approach to assess alignment in mice. Briefly, vector fields of FA and SHG images were created by a a masked investigator using the OrientationJ plugin of ImageJ using a Cubic Spline analysis with 64 µm 2 grid size and a 2.5 µm local window. Orientation J provided an output for FA and SHG images that included orientation (in degrees) and an intensity value for each vector. Vector fields were overlaid and difference in orientation between FA and collagen vectors was calculated in degrees using the OrientationJ output and MATLAB (The MathWorks Inc., Natick, MA) with 0˚ indicated completely parallel alignment and 90˚ indicating perpendicular vectors. Using this method, each image analysis produced three outputs: (1) an alignment heatmap with light blue indicating close alignment and dark blue indicating poor alignment, (2) a mean alignment value for each image that represented the overall FA-collagen alignment, and (3) a histogram showing the alignment distribution of windows within each image. In this analysis we examined the PPS region and peripheral scleral region adjacent to the PPS. This method was modified from out previous studies in human tissues in two ways ( Supplemental Fig. 1 ). First, we increased the resolution of the analysis by decreasing the vector window size from 178 µm 2 to 64 µm 2 . This change increased the vector number from 1024 to 4096 per image. Second, we performed a study to determine the best local window (σ) to use in our vector field analysis. The σ is a parameter that determines the scale of the analysis and should have a value that close to the size of structures of interest which in this case are collagen fibrils and actin fibrils. We found that small σ values excluded windows from analysis due to poor signal strength (black regions in heatmaps) and large σ values polished over any regional variations in alignment creating uniform heatmaps. We therefore proceeded with these studies using a σ value of 2.5 µm which allowed analysis at a subcellular level while preserving data points. 2.4 Analysis of nuclear morphology Vampire analysis of nuclei was described previously. 36 Briefly, all images of nuclei were first segmented using CellProfiler and these segmented images were used to build a model using the VAMPIRE program. 36 , 37 Inputs into the model build include a cohort of segmented images of interest (control and glaucomatous sclera, n = 6 for control and n = 6 and 6 for 7D and 6W glaucoma respectively), the number of shape modes that VAMPIRE should create (10), and the number of coordinates to use to establish the nuclei boundary (30). To perform this analysis, the VAMPIRE algorithm was trained on cross-sectional images of CD-1 mice. Overall, 23,000 individual nuclei were used in training to develop 10 shape modules. This model is then applied to segmented images from the training cohort and produces an output that includes the percentage of each shape mode within a given image segment, aspect ratio, and the location of each nucleus examined. 2.5 Statistics All values are mean ± standard deviation (SD). Unless otherwise noted, means were compared using analysis of variance test (ANOVA) with Dunnett post-hoc tests to correct for multiple comparisons. A probability value of < 0.05 was considered significant. 2.6 Data Availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. 3. Results 3.1 The PPS is divided into two discreet regions To define the anatomy of the peripapillary sclera, we first examined nerve sections cut parallel to the optic axis through optic nerve and peripapillary region. Sections were labeled for nuclei (green) and actin (fibrillar actin, red). After imaging nuclei and actin, each section was imaged again using SHG to visualize collagen fibrils (Fig. 1). Low magnification images demonstrated a split in the peripheral sclera as it approached the optic nerve that created two previously described layers: a layer that is continuous with the globe’s contour (the scleral flange) and the dural struts (DS) that split from the globe 270±60 µm from the optic nerve and fuse with the pial layer of the posterior optic nerve. 38 We next examined these structures in relation to previously defined regions relative to Bruch’s membrane opening (BMO). From BMO, 3 regions were marked: unmyelinated optic nerve (UMy, 200 µm posterior from BMO), myelin transition zone (MTZ, 200–350 µm from BMO), and myelinated optic nerve (My, > 350 µm from BMO). 13 , 39 These markers verified that the scleral flange contacts the optic nerve within the UMy, while the dural struts extend to the pia mater in the My. We next created serial axial sections (cross-sections perpendicular to the optic axis) through the optic nerve and PPS starting from the distal optic nerve and progressing towards the ONH and PPS (Fig. 2). The distal optic nerve was round and surrounding with a collagenous pial layer, peripheral dural struts, and vascular structures. As sections approached the globe and PPS, the nerve became bean-shaped and the pial collagen layer was surrounded by a wider circumferentially organized layer that strongly labelled for fibrillar actin. Peripheral scleral outside this layer contained lamellae organized in a bask-weave pattern. Like previously described structures in the rat eye, vessels surrounded the ONH with a greater concentration inferior to the nerve where the retinal vein and artery penetrate the sclera. 40 Our goal in these studies was to examine the PPS region that mechanically affects optic nerve function - the scleral flange region that contacts the optic nerve. Therefore, for downstream analyses, we selected PPS regions in the UON that had bordering scleral tissue that surrounded the optic nerve. In examining the scleral flange, we separated it into 3 separate regions. The PPS could be separated into two distinct regions (1) the inner pial region that opposed the optic nerve (pPPS) and (2) the outer PPS (oPPS) that was immediately adjacent to the pPPS but still retained circumferentially arranged collagen and actin fibrils. The third region comprised sclera peripheral to the PPS and contained collagen lamellae organized in a basket weave rather than circumferential pattern (Fig. 3). 3.2 Nuclear morphology is distinct between sclera regions Nuclei play a pivotal role in sensing extracellular cues. We previously described discreet PPS regions in normal human eyes with nuclear aspect ratio increasing as distance increased from the optic nerve. To investigate whether there were regional nuclear differences in mouse PPS, we analyzed nuclear shape in the pPPS, oPPS, and peripheral sclera of control eyes using the VAMPIRE algorithm (Fig. 4). The VAMPIRE algorithm is an unsupervised machine-learning method that identifies shape morphologies in an image set and can be used to quantify the frequency (%) of each morphology in a given image region. 36 Using this analysis, we found that PPS regions had a higher frequency of shape modes with a high aspect ratio (g-j) than the peripheral scleral which had a higher frequency of round nuclei (e). 3.3 Filamentous actin is highly aligned with surrounding collagen fibers Mean FA-collagen alignment was greater in PPS (pPPS and oPPS combined; 7.1±2.5°) than peripheral scleral (10.0±1.4°; p = 0.05, n = 6), with notably reduced alignment in superior peripheral sclera as evident on heat maps. To further examine FA-collagen alignment, we examined the frequency of highly aligned image windows ( 40° of misalignment) in each image. Using these criteria, the PPS had a higher percentage of highly aligned windows than the peripheral sclera (85.7±8.4% versus 75.9±5.9%, p = 0.04, n = 6) and a lower percentage of poorly aligned windows (6.1±3.6% versus 17.7±2.9%, p = 0.0001, n = 6) (Fig. 5). 3.4 IOP elevation minimally alters nuclear morphology. We then examined eyes 1 and 6 weeks and bead injection for chronic IOP elevation ( Supplemental Fig. 2 ). For the 1 week study, average IOP compared to fellow non-injected eyes was elevated by 10.4±4.6 mmHg at 3 days and 7.0±4.0 mmHg at 7 days. Peak IOPs ranged from 15 to 30 mmHg. For the 6 week study, average IOP compared to fellow non-injected eyes was elevated 10.6±3.8 mmHg at 3 days and 5.4±3.5 mmHg at 7 days with BI IOPs normalizing by 14 days. BI increased axial length in glaucomatous compared to fellow eyes in the 1 (10.7±6.6%) and 6 (10.1±6.2%) week studies. VAMPIRE analysis of BI eyes compared to control eyes revealed only minimal changes in nuclear morphology (Fig. 6). In the pPPS, oPPS, and peripheral sclera there was a small increase in rounder nuclear morphologies (shape modes e and f) with IOP elevation which corresponded with a decrease in morphologies with a higher aspect ratio (shape modes h through j). These changes were small in value, however, with no changes in exceeding 5% in frequency. 3.5 IOP elevation reduces FA-collagen alignment Using these images, we compared FA-collagen alignment of control eyes and eyes 1 and 6 weeks after BI. At 1 week, heat maps of glaucomatous eyes showed a high level of FA-collagen alignment with focal areas of decreased alignment superiorly and pPPS (Fig. 7). Our initial analysis of mean alignment in control eyes was not significantly different from 1 week glaucoma eyes in the PPS region (7.1±2.5° versus 14.9±11.4°, p = 0.17), however, there was an outlier in the glaucomatous data that significantly altered the results towards poor alignment. With this outlier eliminated from the analysis, there was a small but significant reduction in average alignment between control (7.1±2.5°) and 1 week glaucoma (10.4±1.6°) eyes (p = 0.02). Glaucomatous PPS had 73.2±3.9% of windows with 40% misalignment was observed in 13.6±3.4% of windows in the PPS of glaucomatous eyes; this increase was significant compared to control eyes (6.1±3.6%, p = 0.002). In the peripheral sclera, unlike the PPS, there was no reduction in average alignment between control (10.0±1.4°) and 1 week glaucoma (12.0±1.6°)(p = 0.05), but there was a significant reduction in windows with 40° of misalignment (12.9±3.9° versus 18.1±2.9°, p = 0.01) in control versus glaucomatous peripheral sclera. Reduced FA-collagen alignment persisted at six weeks after BI. Again, there was a significant reduction in average alignment between control (7.1±2.5°) and 6 week glaucoma (11.5±1.2°) eyes (p = 0.004) in the PPS but not the peripheral sclera (p = 0.12). In the PPS there were fewer windows with 40° of misalignment (14.0±3.7%, p = 0.003) compared to control eyes. In the peripheral sclera, the average alignment was not significantly reduced (p = 0.12) and there was no significant difference in windows with 40° of misalignment (p = 0.08). 4. Discussion The goal of these studies was to develop methods to assess scleral fibroblast structure within the complex tissue environment of the mouse sclera and to evaluate the fibroblast response to IOP elevation. Here, we describe four main findings. First, we describe easily discernable zones of the mouse ONH sclera that include pial PPS, outer PPS, and peripheral regions. Second, we used AI-based image analysis to identify variations in nuclear morphology between these regions. Third, we demonstrated that like human sclera, FA was highly aligned with surrounding collagen fibers. Lastly, we showed that IOP elevation subtly altered PPS nuclear morphology and significantly reduced FA-collagen alignment. Together, these findings demonstrate that the mouse PPS scleral fibroblast response to IOP elevation can be observed and quantified; and that IOP elevation leads to significant changes in the alignment of PPS fibroblast FA to surrounding collagen fibrils that is sustained for at least 6 weeks. Cells within the scleral stroma reside within a complex environment that cannot be adequately modeled using in vitro systems. We and others have studied scleral fibroblasts isolated from different regions (peripapillary and peripheral) in culture and have shown transdifferentiation from fibroblast to myofibroblast after exposure to glaucomatous stimuli such as TGFβ exposure or cyclic mechanical strain. 22 , 24 , 31 , 41 – 45 In vitro studies fall short of modeling complex extracellular architecture of the sclera and the connections that native cells make with the ECM. Therefore, to better understand the response(s) of scleral stromal cells to glaucomatous stimuli, cellular behavior must be studied in its native environment. Here, we apply unbiased image analysis techniques to better define regional cellular heterogeneity and the relationships between cellular cytoskeletal elements and their surrounding ECM. These studies extend on previous work from our lab and others that described cellular niches within the sclera. We showed previously that human ONH sclera was divided into the inner PPS (iPPS), outer PPS (oPPS), and peripheral sclera. 22 Nuclei within these regions varied in morphology and density – with the iPPS and oPPS having a greater density of nuclei and rounder nuclei that the peripheral sclera. Additional proof of scleral fibroblast heterogeneity was provided by single cell transcriptomic studies of mouse and human sclera. 46 – 48 In human sclera, single cell transcriptomic studies noted variations in the transcriptional profiles of fibroblast populations in the PPS, peripheral sclera, choroid, and optic nerve. Significant overlaps between the transcriptomic profiles of PPS fibroblasts and single nucleotide polymorphisms (SNPs) uncovered in glaucoma GWAS studies supports the hypothesis that PPS fibroblasts populations play a role in glaucoma. 15 Therefore, studying regional fibroblast populations will be an important future step in understanding glaucoma pathogenesis. Similar to our previous studies in the organization of the human PPS, here, we found the mouse ONH was divided into two PPS regions (the pPPS and oPPS) based on cell morphology and ECM density. Thus, regional variations in cellular and ECM organization are conserved between human and mouse PPS, and these findings support the use of the mouse as a model organism to understand the PPS cellular behavior. IOP elevation altered cytoskeletal FA alignment with surrounding collagen fibers. At baseline IOP, like in human sclera, collagen fibrils and cytoskeletal FA structures were highly aligned, and alignment was greater in the circumferentially organized PPS tissue than in peripheral sclera. IOP elevation, however, altered several measures of FA-collagen alignment: it decreased mean FA-collagen alignment, it decreased the number of highly aligned regions, and it increased the amount of poorly aligned regions. This effect was present in both the PPS and peripheral sclera at 1 week after IOP elevation, but only present in the PPS at 6 weeks after IOP elevation. These findings have implications in the process of glaucomatous scleral remodeling. FA-collagen alignment with surrounding collagen fibers is mediated by focal adhesion contact points between the cell membrane and the ECM as well as topographic cues that regulate cytoskeletal organization, nuclear structure, and gene expression. 49 , 50 Mechanical extracellular signals are transmitted through these contact points and cells then adapt to a changing extracellular environment in an iterative process that can either maintain tissue homeostasis or cause pathologic ECM remodeling. In glaucomatous sclera and PPS, extracellular signals lead to decreased collagen alignment, scleral stiffening, and changes in scleral viscoelasticity. Loss of FA-collagen alignment that we demonstrated after IOP elevation could potentially represent one facet of that process, however, the significance of alignment loss and the mechanism(s) that underlie it are not yet known. Nuclear morphology was minimally affected by IOP elevation. The frequency of any one of the 10 individual shape modes did not change by greater than 5% at 1 or 6 weeks after IOP elevation. The most notable changes were in shape mode “e” which increased following IOP elevation in the pPPS, oPPS, and peripheral sclera. The small frequency increase in this rounder shape mode was balanced by small decreases in the frequency of the higher aspect ratio shape modes (h, I, and j) in the oPPS and peripheral sclera. Nuclei are mechanoresponsive organelles that are connected to the ECM through cytoskeletal bridges. In fact, Bonnevvie et al demonstrated that nuclear morphology of cultured cells can be used to predict the biomechanical state (stiffness) of the culture environment. 51 Therefore, the relatively small changes in nuclear morphology following IOP elevation were surprising in light of the persistent cytoskeletal remodeling induced by IOP elevation and the ECM changes (alterations in stiffness and ECM microarchitecture) that occur with chronic IOP elevation. While this analysis did not appreciate a significant change following IOP elevation, it is possible that our analysis was not appropriately designed to detect differences. Alternatively, examining alternate nuclear features such as nuclear envelope invaginations could have distinguished control and glaucomatous scleral cell nuclei. These studies and techniques have several recognized limitations. The scleral flange region of the mouse PPS is an extremely thin structure (< 50 µm); it is technically challenging obtain sections through this region and the number of sections from each that can be used in the analysis is small. Due to these challenges, individual sections can contain scleral regions of varying depth that could potentially affect the analysis. Additionally, all analyses were conducted on 2D images. This aspect of the analysis has the greatest potential to affect the nuclear morphology analysis in which any alterations in morphology detected could not only indicate a change in morphology but also could indicate a change in nuclear orientation that occurred with cell migration. Therefore, even the small changes in nuclear morphology observed could be due to changes in nuclear orientation rather than change in the overall nuclear shape. 5. Conclusions In summary we describe imaging studies of the cellular architecture of the mouse PPS and peripheral sclera. Our studies identified regions within the PPS and peripheral sclera that mirror the organization of the human PPS. Examination of FA-collagen alignment showed a high degree of alignment of extracellular collagen and cytoskeletal FA. IOP elevation reduced this alignment in the PPS and peripheral sclera at the 1 week timepoint and at the 6 week timepoint in the PPS. The studies provide insight into the cellular organization of the PPS and the mechanoresponse of scleral PPS fibroblasts to IOP elevation. Declarations Funding /Acknowledgements: IP was supported by a grant from the BrightFocus foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation. Author Contribution I.P, A.M, J.R., and N.Y. conducted experiments and performed data analysisE.E. performed data analysisI.P wrote the main manuscript and prepared figuresAll authors reviewed the manuscriptThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Data Availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. References Tham, Y. C. et al. Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology 121 (11), 2081–2090. 10.1016/j.ophtha.2014.05.013 (2014). Epub 2014/07/01. Quigley, H. A., Addicks, E. M., Green, W. R. & Maumenee, A. E. Optic nerve damage in human glaucoma. II. The site of injury and susceptibility to damage. Arch. Ophthalmol. 99 (4), 635–649. 10.1001/archopht.1981.03930010635009 (1981). PubMed PMID: 6164357. Quigley, H. & Anderson, D. R. 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Matrix stiffness-induced myofibroblast differentiation is mediated by intrinsic mechanotransduction. Am. J. Respir Cell. Mol. Biol. 47 (3), 340–348. 10.1165/rcmb.2012-0050OC (2012). Epub 2012/03/31. Follonier, L., Schaub, S., Meister, J. J. & Hinz, B. Myofibroblast communication is controlled by intercellular mechanical coupling. J. Cell. Sci. 121 (Pt 20), 3305–3316. 10.1242/jcs.024521 (2008). Epub 2008/10/02. Hu, D. et al. Mechanical Strain Regulates Myofibroblast Differentiation of Human Scleral Fibroblasts by YAP. Front. Physiol. 12 , 712509. 10.3389/fphys.2021.712509 (2021). Epub 20210930. Yuan, Y. et al. The Role of the RhoA/ROCK Signaling Pathway in Mechanical Strain-Induced Scleral Myofibroblast Differentiation. Invest. Ophthalmol. Vis. Sci. 59 (8), 3619–3629. 10.1167/iovs.17-23580 (2018). Epub 2018/07/22. Oglesby, E. et al. Losartan inhibits myofibroblast transdifferentiation of human scleral fibroblasts. Investig. Ophthalmol. Vis. Sci. 58 (8), 4604 (2017). Quigley, H. A. et al. Losartan Treatment Protects Retinal Ganglion Cells and Alters Scleral Remodeling in Experimental Glaucoma. PLoS One . 10 (10), e0141137. 10.1371/journal.pone.0141137 (2015). Epub 2015/10/28. Cone, F. E., Gelman, S. E., Son, J. L., Pease, M. E. & Quigley, H. A. Differential susceptibility to experimental glaucoma among 3 mouse strains using bead and viscoelastic injection. Exp. Eye Res. 91 (3), 415–424. 10.1016/j.exer.2010.06.018 (2010). Epub 20100626. Phillip, J. M., Han, K. S., Chen, W. C., Wirtz, D. & Wu, P. H. A robust unsupervised machine-learning method to quantify the morphological heterogeneity of cells and nuclei. Nat. Protoc. 16 (2), 754–774. 10.1038/s41596-020-00432-x (2021). Epub 20210111. Stirling, D. R. et al. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinform. 22 (1), 433. 10.1186/s12859-021-04344-9 (2021). Epub 20210910. Korneva, A., Kimball, E. C., Jefferys, J. L., Quigley, H. A. & Nguyen, T. D. Biomechanics of the optic nerve head and peripapillary sclera in a mouse model of glaucoma. J. R Soc. Interface . 17 (173), 20200708. 10.1098/rsif.2020.0708 (2020). Epub 20201216. Korneva, A. et al. A method to quantify regional axonal transport blockade at the optic nerve head after short term intraocular pressure elevation in mice. Exp. Eye Res. 196 , 108035. 10.1016/j.exer.2020.108035 (2020). Epub 2020/04/27. Pazos, M. et al. Rat optic nerve head anatomy within 3D histomorphometric reconstructions of normal control eyes. Exp. Eye Res. 139 , 1–12. 10.1016/j.exer.2015.05.011 (2015). Epub 20150526. Chow, A. et al. Dasatinib inhibits peripapillary scleral myofibroblast differentiation. Exp. Eye Res. 194 , 107999. 10.1016/j.exer.2020.107999 (2020). Epub 2020/03/18. Pitha, I. F. et al. The multikinase inhibitor dasatinib inhibits scleral myofibroblast differentiation. Investig. Ophthalmol. Vis. Sci. 60 (9), 6474 (2019). Pitha, I. et al. Rho-Kinase Inhibition Reduces Myofibroblast Differentiation and Proliferation of Scleral Fibroblasts Induced by Transforming Growth Factor beta and Experimental Glaucoma. Transl Vis. Sci. Technol. 7 (6), 6. 10.1167/tvst.7.6.6 (2018). Epub 2018/11/28. Totaro, A., Panciera, T. & Piccolo, S. YAP/TAZ upstream signals and downstream responses. Nat. Cell. Biol. 20 (8), 888–899. 10.1038/s41556-018-0142-z (2018). Epub 2018/07/28. Mozzer, A. & Pitha, I. Cyclic strain alters the transcriptional and migratory response of scleral fibroblasts to TGFbeta. Exp. Eye Res. 244 , 109917. 10.1016/j.exer.2024.109917 (2024). Epub 20240430. Monavarfeshani, A. et al. Transcriptomic Analysis of the Ocular Posterior Segment Completes a Cell Atlas of the Human Eye. bioRxiv. Epub 20230427. doi: (2023). 10.1101/2023.04.26.538447 . PubMed PMID: 37162855; PMCID: PMC10168356. Gautam, P. et al. Multi-species single-cell transcriptomic analysis of ocular compartment regulons. Nat. Commun. 12 (1), 5675. 10.1038/s41467-021-25968-8 (2021). Epub 20210928. Wu, H. et al. Scleral hypoxia is a target for myopia control. Proc. Natl. Acad. Sci. U S A . 115 (30), E7091–E100. 10.1073/pnas.1721443115 (2018). Epub 2018/07/11. Tabdanov, E. D., Puram, V., Zhovmer, A. & Provenzano, P. P. Microtubule-Actomyosin Mechanical Cooperation during Contact Guidance Sensing. Cell. Rep. 25 (2). 10.1016/j.celrep.2018.09.030 (2018). PubMed PMID: 30304674; PMCID: PMC6226003328 – 38 e5Epub 2018/10/12. Zhou, J. et al. Mechanism of Focal Adhesion Kinase Mechanosensing. PLoS Comput. Biol. 11 (11), e1004593. 10.1371/journal.pcbi.1004593 (2015). Epub 20151106. Bonnevie, E. D. et al. Cell morphology and mechanosensing can be decoupled in fibrous microenvironments and identified using artificial neural networks. Sci. Rep. 11 (1), 5950. 10.1038/s41598-021-85276-5 (2021). Epub 20210315. Additional Declarations No competing interests reported. Supplementary Files SuppFig1.tiff SuppFig2.tiff Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 28 Jan, 2025 Reviews received at journal 23 Jan, 2025 Reviews received at journal 23 Jan, 2025 Reviews received at journal 08 Jan, 2025 Reviewers agreed at journal 07 Jan, 2025 Reviewers agreed at journal 02 Dec, 2024 Reviewers agreed at journal 22 Nov, 2024 Reviewers invited by journal 22 Nov, 2024 Editor assigned by journal 22 Nov, 2024 Editor invited by journal 22 Nov, 2024 Submission checks completed at journal 21 Nov, 2024 First submitted to journal 05 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-5397173","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":384967830,"identity":"74d0be06-7347-4c71-aad4-24f0ed680aec","order_by":0,"name":"Ann Mozzer","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Ann","middleName":"","lastName":"Mozzer","suffix":""},{"id":384967831,"identity":"f242a6d7-f9f8-491d-be99-383cedaa7f48","order_by":1,"name":"Joe Gerald Jesu Raj","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Joe","middleName":"Gerald Jesu","lastName":"Raj","suffix":""},{"id":384967832,"identity":"aecb9962-51cc-4a78-a6d1-9ff3abae633c","order_by":2,"name":"Nushrat Yasmin","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Nushrat","middleName":"","lastName":"Yasmin","suffix":""},{"id":384967833,"identity":"500bb9a8-4538-4eb9-bc2a-e792e30edb9e","order_by":3,"name":"Edward Esposito","email":"","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":false,"prefix":"","firstName":"Edward","middleName":"","lastName":"Esposito","suffix":""},{"id":384967834,"identity":"ccd5c12f-7205-4989-a02a-5a988124c8a5","order_by":4,"name":"Ian Pitha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACNjBpwJDAwN7AwMAIxAwHiNNikMDAc4CxgSgtUADUIpFApBY+9rMHmAsK/uTxz3xj/uDnDgY5vhsJBBzGk5fAPMPAoFjido5hY+8ZBmNJgloYcgyYeQwMEhuAWhp42xgSNxDUwv8GomX+zTOGjX/bGOoJa5GA2rLhBo9hM9CWBAPCWt4YHJ5hYJy48Uxa4WzZNgnDmWce4Nci359j+Ljgj1zivOOHN3x822Yjz3ecgC0gcBiJLUFYOQgwE6dsFIyCUTAKRiwAAJI+RHi5BgfQAAAAAElFTkSuQmCC","orcid":"","institution":"Johns Hopkins University","correspondingAuthor":true,"prefix":"","firstName":"Ian","middleName":"","lastName":"Pitha","suffix":""}],"badges":[],"createdAt":"2024-11-05 16:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5397173/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5397173/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-08251-4","type":"published","date":"2025-07-01T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71890575,"identity":"da24452d-dc3e-432e-80c8-00195c4b90e3","added_by":"auto","created_at":"2024-12-19 13:00:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":533627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eONH anatomy.\u003c/strong\u003eLow (A-D, scale bar = 100 µm) and high (E-H, scale bar = 50 µm) magnification transverse sections through the ONH with fibrillar actin (FA, red) and nuclear (green) labels. (B,F) SHG imaging for collagen visualization, and (C,G) merged images. (D,H) An illustration outlining scleral (black), retinal (blue), and choroidal (brown) boundaries. White and red lines demarcate the basement membrane opening (BMO), unmyelinated (UMy), and myelinated (My) landmarks. Arrows demarcate region of the scleral flange.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/77177c737039b441162c0ebd.png"},{"id":71891136,"identity":"cc9fae96-60f7-4398-8eab-785abd6a1b82","added_by":"auto","created_at":"2024-12-19 13:08:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":640731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerial sections though the ONH. \u003c/strong\u003eSerial sections of a single mouse eye progressing from the optic nerve (A,E) to the PPS and scleral flange (L,P) labelled for FA (red) and nuclear structures (green)(A-D and I-L). The inferior nerve (left of each image) contains numerous blood vessels. SHG imaging of the same sections for collagen visualization (E-H and M-P). The collagenous pPPS can be followed through the images to the PPS (arrows). The Asterix in (P) demarcates the level of the scleral flange.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/1db612b964233762d8920093.png"},{"id":71890585,"identity":"0aa1cb3a-baed-47f2-bf12-6a419dd35791","added_by":"auto","created_at":"2024-12-19 13:00:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":567442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScleral flange regions. \u003c/strong\u003eImages of a single scleral flange section with nuclei and FA labelling (A) and SHG/collagen (B). The optic nerve (inside the white border), pPPS (green border), and oPPS (yellow border) regions are outlined with the peripheral sclera outside the yellow border.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/f98476b91d052eb12a717e1f.png"},{"id":71890594,"identity":"633f3d5e-60e2-4dc7-a1be-93a62e8483d5","added_by":"auto","created_at":"2024-12-19 13:00:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVAMPIRE analysis of PPS and peripheral scleral nuclei.\u003c/strong\u003e (A) Images of ONH nuclei shown prior to segmentation (green), after nuclear segmentation, and after segmentation into pPPS (green border), oPPS (yellow border), and peripheral sclera (red border) regions. Nuclei within the white border were from the optic nerve and excluded from analysis. (B) Overlay of 20 randomly selected raw shapes in each shape mode after VAMPIRE training. (C) Dendrogram of shape modes with a heatmap showing the frequency (%) of each shape mode in the pPPS, oPPS, and peripheral sclera (n=number of nuclei).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/a862515ff12a04a09ef35801.png"},{"id":71890601,"identity":"2a7a264b-58b8-47a3-9f71-c632f663287c","added_by":"auto","created_at":"2024-12-19 13:00:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":245331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFilamentous actin-collagen alignment in the PPS and peripheral sclera. \u003c/strong\u003e(A) 64 x 64 vector fields of FA and SHG images from the same ONH section. (B) Magnified view of the red box in each region with nine FA (black) and SHG (green) vectors overlaid, calculation of alignment in degrees, and alignment heatmap of the nine regions. (C) Alignment heatmap of FA and SHG images shown in (A) with 4,096 regions of alignment. The orange circle demarcates the separation of PPS (inside) and peripheral (outside) regions. (D) Histogram of alignment values (\u003cem\u003en\u003c/em\u003e=6 eyes from 6 animals).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/6c5787689b467ff474573999.png"},{"id":71890590,"identity":"291da9b1-a59f-43f2-8e9c-43ea489c2fc7","added_by":"auto","created_at":"2024-12-19 13:00:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":191339,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVAMPIRE analysis after IOP elevation. \u003c/strong\u003e\u0026nbsp;Dendrogram and shape modes with frequency (%) of each mode in the pPPS, oPPS, and peripheral sclera under baseline conditions and following one and six weeks of IOP elevation (n=number of nuclei).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/dbb126e1097f3508ef3e257e.png"},{"id":71891120,"identity":"b5827bf4-8ed7-4223-8fd9-f2cb8ae51656","added_by":"auto","created_at":"2024-12-19 13:08:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":171569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFA-collagen alignment after IOP elevation. \u003c/strong\u003e(A) FA, SHG, and alignment heatmap images from 2 eyes after one and six weeks of IOP elevation with the optic nerve region blacked out in the heatmap images. (B, C) Histogram of alignment values in peripheral sclera (B) and PPS (C) (n=6 control; n=5 1W glaucoma; n=6 6W glaucoma).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/b35a8d99dfe8eb420f1eeab1.png"},{"id":86178976,"identity":"1b297640-1110-4870-9a54-df581f655c5d","added_by":"auto","created_at":"2025-07-07 16:13:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3199850,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/838a7cc5-f7cd-41ac-9b83-d5060eff3c3b.pdf"},{"id":71890596,"identity":"7756195a-1584-4031-a757-9cc23b4ed6d3","added_by":"auto","created_at":"2024-12-19 13:00:25","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":172646,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFig1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/94eb7b4a152bc6929ad60279.tiff"},{"id":71890577,"identity":"741ae44a-92f8-44a2-8f05-249d24dea755","added_by":"auto","created_at":"2024-12-19 13:00:23","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":36107,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFig2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5397173/v1/30619a8334e4713b328b8577.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regional differences in peripapillary scleral fibroblast cytoskeleton and nuclear morphology after intraocular pressure elevation in mouse eyes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlaucoma, a leading cause of irreversible blindness worldwide, is a multifactorial disease characterized by progressive vision loss due to retinal ganglion cell death that is initiated at the optic nerve head.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Risk factors for glaucoma pathogenesis and progression include genetic contributions, aging, high IOP levels, vascular insufficiency, and neuroinflammation. IOP fluctuations generate mechanical strain in optic tissues that contributes to glaucoma progression.\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9 CR10\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e The structural and mechanical properties of the optic nerve head (ONH) region, which includes cellular and fibrous elements of the peripapillary sclera (PPS) and the lamina cribrosa (LC), play a critical role in development and progression of glaucoma by translating IOP-mediated mechanical strain to ONH axons, astrocytes, and microglia.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e PPS stromal fibroblasts tune the mechanical properties of the ONH by remodeling and maintaining scleral tissue. Their prominent role in glaucoma pathogenesis was highlighted by genome wide association studies (GWAS) of glaucoma patients and single nucleus transcriptomics studies of ocular tissues.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The PPS fibroblast response to IOP elevation, however, is not yet fully characterized.\u003c/p\u003e \u003cp\u003eScleral fibroblasts reside within different extracellular environments that are designed to meet diverse functional requirements.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Collagen fiber lamellae within the PPS are circumferentially arranged around the optic nerve and have a relatively high elastin content \u0026ndash; features that protect the optic nerve from IOP-generated mechanical strain. This circumferential microarchitectural organization contrasts with the peripheral sclera in which collagen lamellae have a basket weave organization and the limbal sclera in which downstream aqueous humor outflow pathways are present. In glaucoma, PPS mechanical qualities are altered - reduced collagen alignment, stiffening, loss of elastin organization - which shifts the biomechanical response to IOP variations to cause axonal damage at the ONH.\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Mechanical alterations that occur in glaucomatous sclera do not arise passively, but rather through the combined remodeling actions of scleral cells \u0026ndash; the majority of which are resident fibroblasts. Fibroblast function, in turn, is influenced by extracellular matrix (ECM) composition, organization, and biomechanical stimuli that are transmitted through the ECM. Due to the complexity of the scleral extracellular environment, this iterative process in which ECM and fibroblasts communicate to remodel tissue cannot be modeled in cell culture. There is a recognized need to develop techniques to study fibroblasts in their tissue environment.\u003c/p\u003e \u003cp\u003eScleral fibroblasts are exposed to IOP-generated mechanical strains that influence their structure, gene transcription, and phenotypic behavior.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e These alternations include changes in cell morphology, proliferation, migration, extracellular matrix synthesis, and cell differentiation.\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Cells can detect and respond to diverse external stimuli including matrix stiffness, surface topography, ligand presentation, and compressive, tensile, and shear strain.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Actin filaments, as a major component of the cytoskeleton, are particularly sensitive to extracellular cues and undergo dynamic reorganization in response to mechanical stimuli. Actin rearrangements, in turn, influence cell adhesion, migration, and differentiation. Mechanical forces transmitted through the extracellular matrix are communicated to nuclei which not only impacts nuclear morphology but also gene expression.\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Dysregulation of the cellular response to mechanical stimuli can lead to pathologic conditions such as organ fibrosis.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Mechanotransduction pathways, such as the Rho/ROCK, integrin, and MAPK signaling cascades, have been implicated in mediating these fibroblast responses to mechanical strain; however, their role in glaucomatous scleral remodeling is not fully known.\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWe previously demonstrated a close association between cellular structures and collagenous ECM in normal human PPS\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, finding that filamentous actin (FA) was closely aligned with surrounding collagen fibrils in the PPS. Additionally, the nuclear aspect ratio varied regionally, with rounder PPS nuclei and more elongate nuclei in the sclera further from the ONH. To investigate these phenomena under experimental conditions, we used a mouse model of sustained IOP elevation to quantify changes in PPS fibroblast cytoskeletal alignment with extracellular collagen fibers and nuclear morphology.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bead-injection\u003c/h2\u003e \u003cp\u003e All animal experimentation was conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Johns Hopkins University and according to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Experimental design and reporting are in accordance with Animal Research: Reporting of \u003cem\u003eIn\u003c/em\u003e Vivo Experiments (ARRIVE, 2.0) guidelines. We used 18 eight to twelve-week old male and female CD1 mice (Charles River, Inc., Wilmington, MA) in total. Twelve mice underwent unilateral intracameral bead injection as described previously and six were used as na\u0026iuml;ve controls.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e For bead-induced ocular hypertension (BI), following sedation with a mixture of ketamine (Fort Dodge Animal Health, Fort Dodge, IA), xylazine (VedCo Inc., Saint Joseph, MO), and acepromazine (Phoenix Pharmaceuticals, Burlingame, CA) polystyrene microspheres were injected into the anterior chamber using a glass cannula with a 50 \u0026micro;M tip diameter connected to a Hamilton syringe (Hamilton, Inc. ,Reno, NV). We injected a mixture of Polybead Microspheres\u0026reg; (Polysciences, Inc.,Warrington, PA, USA) of 6 \u0026micro;M and 1 \u0026micro;M diameter followed by 10 mg/ml sodium hyaluronate (Healon PRO; Advanced Medical Optics Inc., Santa Ana, CA).\u003c/p\u003e \u003cp\u003eIOPs were measured using the TonoLab rebound tonometer (iCare, Vantaa, Finland) according to the manufacturer\u0026rsquo;s instructions with the magnetic probe in the horizontal position. IOPs were taken under anesthesia through inhalation of isoflurane administered by a RC2- Rodent Circuit Controller (VetEquip, Inc., Pleasanton, CA). Mice spent two minutes in an induction chamber and 1 minute on a nose cone before measurements were taken. The mean of two to three recordings per eye, with each recording from the tonometer resulting from six readings, was used as the final IOP measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Specimen preparation and imaging\u003c/h2\u003e \u003cp\u003eAfter euthanasia by carbon dioxide inhalation and verification of death, eyes were enucleated under a dissecting microscope and prefixed for 1\u0026ndash;3 hours with 4% paraformaldehyde in Sorenson's phosphate buffer. Extraocular tissue was removed, posterior eye cups were isolated and cryopreserved in a 2:1 mixture of Sucrose and TissueTek \u0026reg; O.C.T. compound (Sakura Finetek USA, Inc., Torrance, CA) solution respectively. Eyes were then cryo-sectioned at 12\u0026ndash;14 \u0026micro;m thickness.\u003c/p\u003e \u003cp\u003eSection preparation and imaging was performed as previously described.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Sections were incubated in Hanks\u0026rsquo; balanced salt solution (HBSS) with 0.5% Triton X-100 (HBT) and 10% normal goat serum (NGS; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) overnight at 4\u0026deg;C. Slides were then washed with HBT with NGS, SYTOX Green at 1:30,000 (S7020; Thermo Fisher Scientific, Waltham, MA, USA) and phalloidin Alexa Fluor 568 (A1280, Thermo). After washing with HBT and mounting with Dako mounting media (Dako, Carpenteria, CA, USA), imaging was performed on a Zeiss confocal laser scanning microscope 710 (Carl Zeiss Microscopy, Thornwood, NY, USA). SHG imaging was performed using a coherent Chameleon Ultra II laser tuned to 780 nm and a 390 to 410 band pass filter. Laser power (2\u0026ndash;4%) was adjusted to minimize oversaturated pixels. Images were stitched using ZEN 2.3 software (Carl Zeiss Microscopy) and converted to .tiff for image analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Filamentous actin (FA)-collagen alignment analysis\u003c/h2\u003e \u003cp\u003eWe previously described a method to quantify alignment of FA with extracellular collagen fibrils in human sclera and demonstrated a high level of FA-collagen alignment in the PPS and peripheral sclera.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Here, we modified this approach to assess alignment in mice. Briefly, vector fields of FA and SHG images were created by a a masked investigator using the OrientationJ plugin of ImageJ using a Cubic Spline analysis with 64 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e grid size and a 2.5 \u0026micro;m local window. Orientation J provided an output for FA and SHG images that included orientation (in degrees) and an intensity value for each vector. Vector fields were overlaid and difference in orientation between FA and collagen vectors was calculated in degrees using the OrientationJ output and MATLAB (The MathWorks Inc., Natick, MA) with 0˚ indicated completely parallel alignment and 90˚ indicating perpendicular vectors. Using this method, each image analysis produced three outputs: (1) an alignment heatmap with light blue indicating close alignment and dark blue indicating poor alignment, (2) a mean alignment value for each image that represented the overall FA-collagen alignment, and (3) a histogram showing the alignment distribution of windows within each image. In this analysis we examined the PPS region and peripheral scleral region adjacent to the PPS.\u003c/p\u003e \u003cp\u003eThis method was modified from out previous studies in human tissues in two ways (\u003cb\u003eSupplemental Fig.\u0026nbsp;1\u003c/b\u003e). First, we increased the resolution of the analysis by decreasing the vector window size from 178 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e to 64 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This change increased the vector number from 1024 to 4096 per image. Second, we performed a study to determine the best local window (σ) to use in our vector field analysis. The σ is a parameter that determines the scale of the analysis and should have a value that close to the size of structures of interest which in this case are collagen fibrils and actin fibrils. We found that small σ values excluded windows from analysis due to poor signal strength (black regions in heatmaps) and large σ values polished over any regional variations in alignment creating uniform heatmaps. We therefore proceeded with these studies using a σ value of 2.5 \u0026micro;m which allowed analysis at a subcellular level while preserving data points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analysis of nuclear morphology\u003c/h2\u003e \u003cp\u003eVampire analysis of nuclei was described previously.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Briefly, all images of nuclei were first segmented using CellProfiler and these segmented images were used to build a model using the VAMPIRE program.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Inputs into the model build include a cohort of segmented images of interest (control and glaucomatous sclera, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 for control and \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 and 6 for 7D and 6W glaucoma respectively), the number of shape modes that VAMPIRE should create (10), and the number of coordinates to use to establish the nuclei boundary (30). To perform this analysis, the VAMPIRE algorithm was trained on cross-sectional images of CD-1 mice. Overall, 23,000 individual nuclei were used in training to develop 10 shape modules. This model is then applied to segmented images from the training cohort and produces an output that includes the percentage of each shape mode within a given image segment, aspect ratio, and the location of each nucleus examined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistics\u003c/h2\u003e \u003cp\u003eAll values are mean \u0026plusmn; standard deviation (SD). Unless otherwise noted, means were compared using analysis of variance test (ANOVA) with Dunnett post-hoc tests to correct for multiple comparisons. A probability value of \u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Data Availability\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The PPS is divided into two discreet regions\u003c/h2\u003e \u003cp\u003eTo define the anatomy of the peripapillary sclera, we first examined nerve sections cut parallel to the optic axis through optic nerve and peripapillary region. Sections were labeled for nuclei (green) and actin (fibrillar actin, red). After imaging nuclei and actin, each section was imaged again using SHG to visualize collagen fibrils (Fig.\u0026nbsp;1). Low magnification images demonstrated a split in the peripheral sclera as it approached the optic nerve that created two previously described layers: a layer that is continuous with the globe\u0026rsquo;s contour (the scleral flange) and the dural struts (DS) that split from the globe 270\u0026plusmn;60 \u0026micro;m from the optic nerve and fuse with the pial layer of the posterior optic nerve.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e We next examined these structures in relation to previously defined regions relative to Bruch\u0026rsquo;s membrane opening (BMO). From BMO, 3 regions were marked: unmyelinated optic nerve (UMy, 200 \u0026micro;m posterior from BMO), myelin transition zone (MTZ, 200\u0026ndash;350 \u0026micro;m from BMO), and myelinated optic nerve (My, \u0026gt;\u0026thinsp;350 \u0026micro;m from BMO).\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e These markers verified that the scleral flange contacts the optic nerve within the UMy, while the dural struts extend to the pia mater in the My.\u003c/p\u003e \u003cp\u003eWe next created serial axial sections (cross-sections perpendicular to the optic axis) through the optic nerve and PPS starting from the distal optic nerve and progressing towards the ONH and PPS (Fig.\u0026nbsp;2). The distal optic nerve was round and surrounding with a collagenous pial layer, peripheral dural struts, and vascular structures. As sections approached the globe and PPS, the nerve became bean-shaped and the pial collagen layer was surrounded by a wider circumferentially organized layer that strongly labelled for fibrillar actin. Peripheral scleral outside this layer contained lamellae organized in a bask-weave pattern. Like previously described structures in the rat eye, vessels surrounded the ONH with a greater concentration inferior to the nerve where the retinal vein and artery penetrate the sclera.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur goal in these studies was to examine the PPS region that mechanically affects optic nerve function - the scleral flange region that contacts the optic nerve. Therefore, for downstream analyses, we selected PPS regions in the UON that had bordering scleral tissue that surrounded the optic nerve. In examining the scleral flange, we separated it into 3 separate regions. The PPS could be separated into two distinct regions (1) the inner pial region that opposed the optic nerve (pPPS) and (2) the outer PPS (oPPS) that was immediately adjacent to the pPPS but still retained circumferentially arranged collagen and actin fibrils. The third region comprised sclera peripheral to the PPS and contained collagen lamellae organized in a basket weave rather than circumferential pattern (Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Nuclear morphology is distinct between sclera regions\u003c/h2\u003e \u003cp\u003eNuclei play a pivotal role in sensing extracellular cues. We previously described discreet PPS regions in normal human eyes with nuclear aspect ratio increasing as distance increased from the optic nerve. To investigate whether there were regional nuclear differences in mouse PPS, we analyzed nuclear shape in the pPPS, oPPS, and peripheral sclera of control eyes using the VAMPIRE algorithm (Fig.\u0026nbsp;4). The VAMPIRE algorithm is an unsupervised machine-learning method that identifies shape morphologies in an image set and can be used to quantify the frequency (%) of each morphology in a given image region.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Using this analysis, we found that PPS regions had a higher frequency of shape modes with a high aspect ratio (g-j) than the peripheral scleral which had a higher frequency of round nuclei (e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Filamentous actin is highly aligned with surrounding collagen fibers\u003c/h2\u003e \u003cp\u003eMean FA-collagen alignment was greater in PPS (pPPS and oPPS combined; 7.1\u0026plusmn;2.5\u0026deg;) than peripheral scleral (10.0\u0026plusmn;1.4\u0026deg;; p\u0026thinsp;=\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;6), with notably reduced alignment in superior peripheral sclera as evident on heat maps. To further examine FA-collagen alignment, we examined the frequency of highly aligned image windows (\u0026lt;\u0026thinsp;20\u0026deg; of misalignment) and poor aligned windows (\u0026gt;\u0026thinsp;40\u0026deg; of misalignment) in each image. Using these criteria, the PPS had a higher percentage of highly aligned windows than the peripheral sclera (85.7\u0026plusmn;8.4% versus 75.9\u0026plusmn;5.9%, p\u0026thinsp;=\u0026thinsp;0.04, n\u0026thinsp;=\u0026thinsp;6) and a lower percentage of poorly aligned windows (6.1\u0026plusmn;3.6% versus 17.7\u0026plusmn;2.9%, p\u0026thinsp;=\u0026thinsp;0.0001, n\u0026thinsp;=\u0026thinsp;6) (Fig.\u0026nbsp;5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 IOP elevation minimally alters nuclear morphology.\u003c/h2\u003e \u003cp\u003eWe then examined eyes 1 and 6 weeks and bead injection for chronic IOP elevation (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e). For the 1 week study, average IOP compared to fellow non-injected eyes was elevated by 10.4\u0026plusmn;4.6 mmHg at 3 days and 7.0\u0026plusmn;4.0 mmHg at 7 days. Peak IOPs ranged from 15 to 30 mmHg. For the 6 week study, average IOP compared to fellow non-injected eyes was elevated 10.6\u0026plusmn;3.8 mmHg at 3 days and 5.4\u0026plusmn;3.5 mmHg at 7 days with BI IOPs normalizing by 14 days. BI increased axial length in glaucomatous compared to fellow eyes in the 1 (10.7\u0026plusmn;6.6%) and 6 (10.1\u0026plusmn;6.2%) week studies.\u003c/p\u003e \u003cp\u003eVAMPIRE analysis of BI eyes compared to control eyes revealed only minimal changes in nuclear morphology (Fig.\u0026nbsp;6). In the pPPS, oPPS, and peripheral sclera there was a small increase in rounder nuclear morphologies (shape modes e and f) with IOP elevation which corresponded with a decrease in morphologies with a higher aspect ratio (shape modes h through j). These changes were small in value, however, with no changes in exceeding 5% in frequency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 IOP elevation reduces FA-collagen alignment\u003c/h2\u003e \u003cp\u003eUsing these images, we compared FA-collagen alignment of control eyes and eyes 1 and 6 weeks after BI. At 1 week, heat maps of glaucomatous eyes showed a high level of FA-collagen alignment with focal areas of decreased alignment superiorly and pPPS (Fig.\u0026nbsp;7). Our initial analysis of mean alignment in control eyes was not significantly different from 1 week glaucoma eyes in the PPS region (7.1\u0026plusmn;2.5\u0026deg; versus 14.9\u0026plusmn;11.4\u0026deg;, p\u0026thinsp;=\u0026thinsp;0.17), however, there was an outlier in the glaucomatous data that significantly altered the results towards poor alignment. With this outlier eliminated from the analysis, there was a small but significant reduction in average alignment between control (7.1\u0026plusmn;2.5\u0026deg;) and 1 week glaucoma (10.4\u0026plusmn;1.6\u0026deg;) eyes (p\u0026thinsp;=\u0026thinsp;0.02). Glaucomatous PPS had 73.2\u0026plusmn;3.9% of windows with \u0026lt;\u0026thinsp;20\u0026deg; of misalignment. This value was significantly less than non-glaucomatous PPS (85.7\u0026plusmn;8.6%, p\u0026thinsp;=\u0026thinsp;0.008). Further, \u0026gt;\u0026thinsp;40% misalignment was observed in 13.6\u0026plusmn;3.4% of windows in the PPS of glaucomatous eyes; this increase was significant compared to control eyes (6.1\u0026plusmn;3.6%, p\u0026thinsp;=\u0026thinsp;0.002). In the peripheral sclera, unlike the PPS, there was no reduction in average alignment between control (10.0\u0026plusmn;1.4\u0026deg;) and 1 week glaucoma (12.0\u0026plusmn;1.6\u0026deg;)(p\u0026thinsp;=\u0026thinsp;0.05), but there was a significant reduction in windows with \u0026lt;\u0026thinsp;20\u0026deg; of misalignment (75.6\u0026plusmn;5.1\u0026deg; versus 68.1\u0026plusmn;4.0\u0026deg;, p\u0026thinsp;=\u0026thinsp;0.008) and an increase in windows with \u0026gt;\u0026thinsp;40\u0026deg; of misalignment (12.9\u0026plusmn;3.9\u0026deg; versus 18.1\u0026plusmn;2.9\u0026deg;, p\u0026thinsp;=\u0026thinsp;0.01) in control versus glaucomatous peripheral sclera.\u003c/p\u003e \u003cp\u003eReduced FA-collagen alignment persisted at six weeks after BI. Again, there was a significant reduction in average alignment between control (7.1\u0026plusmn;2.5\u0026deg;) and 6 week glaucoma (11.5\u0026plusmn;1.2\u0026deg;) eyes (p\u0026thinsp;=\u0026thinsp;0.004) in the PPS but not the peripheral sclera (p\u0026thinsp;=\u0026thinsp;0.12). In the PPS there were fewer windows with \u0026lt;\u0026thinsp;20\u0026deg; of misalignment (72.9\u0026plusmn;5.6%, p\u0026thinsp;=\u0026thinsp;0.01) and more windows with \u0026gt;\u0026thinsp;40\u0026deg; of misalignment (14.0\u0026plusmn;3.7%, p\u0026thinsp;=\u0026thinsp;0.003) compared to control eyes. In the peripheral sclera, the average alignment was not significantly reduced (p\u0026thinsp;=\u0026thinsp;0.12) and there was no significant difference in windows with \u0026lt;\u0026thinsp;20\u0026deg; of misalignment (p\u0026thinsp;=\u0026thinsp;0.06) or \u0026gt;\u0026thinsp;40\u0026deg; of misalignment (p\u0026thinsp;=\u0026thinsp;0.08).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe goal of these studies was to develop methods to assess scleral fibroblast structure within the complex tissue environment of the mouse sclera and to evaluate the fibroblast response to IOP elevation. Here, we describe four main findings. First, we describe easily discernable zones of the mouse ONH sclera that include pial PPS, outer PPS, and peripheral regions. Second, we used AI-based image analysis to identify variations in nuclear morphology between these regions. Third, we demonstrated that like human sclera, FA was highly aligned with surrounding collagen fibers. Lastly, we showed that IOP elevation subtly altered PPS nuclear morphology and significantly reduced FA-collagen alignment. Together, these findings demonstrate that the mouse PPS scleral fibroblast response to IOP elevation can be observed and quantified; and that IOP elevation leads to significant changes in the alignment of PPS fibroblast FA to surrounding collagen fibrils that is sustained for at least 6 weeks.\u003c/p\u003e \u003cp\u003eCells within the scleral stroma reside within a complex environment that cannot be adequately modeled using \u003cem\u003ein vitro\u003c/em\u003e systems. We and others have studied scleral fibroblasts isolated from different regions (peripapillary and peripheral) in culture and have shown transdifferentiation from fibroblast to myofibroblast after exposure to glaucomatous stimuli such as TGFβ exposure or cyclic mechanical strain.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42 CR43 CR44\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eIn vitro\u003c/em\u003e studies fall short of modeling complex extracellular architecture of the sclera and the connections that native cells make with the ECM. Therefore, to better understand the response(s) of scleral stromal cells to glaucomatous stimuli, cellular behavior must be studied in its native environment. Here, we apply unbiased image analysis techniques to better define regional cellular heterogeneity and the relationships between cellular cytoskeletal elements and their surrounding ECM.\u003c/p\u003e \u003cp\u003eThese studies extend on previous work from our lab and others that described cellular niches within the sclera. We showed previously that human ONH sclera was divided into the inner PPS (iPPS), outer PPS (oPPS), and peripheral sclera.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Nuclei within these regions varied in morphology and density \u0026ndash; with the iPPS and oPPS having a greater density of nuclei and rounder nuclei that the peripheral sclera. Additional proof of scleral fibroblast heterogeneity was provided by single cell transcriptomic studies of mouse and human sclera.\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e In human sclera, single cell transcriptomic studies noted variations in the transcriptional profiles of fibroblast populations in the PPS, peripheral sclera, choroid, and optic nerve. Significant overlaps between the transcriptomic profiles of PPS fibroblasts and single nucleotide polymorphisms (SNPs) uncovered in glaucoma GWAS studies supports the hypothesis that PPS fibroblasts populations play a role in glaucoma.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Therefore, studying regional fibroblast populations will be an important future step in understanding glaucoma pathogenesis. Similar to our previous studies in the organization of the human PPS, here, we found the mouse ONH was divided into two PPS regions (the pPPS and oPPS) based on cell morphology and ECM density. Thus, regional variations in cellular and ECM organization are conserved between human and mouse PPS, and these findings support the use of the mouse as a model organism to understand the PPS cellular behavior.\u003c/p\u003e \u003cp\u003eIOP elevation altered cytoskeletal FA alignment with surrounding collagen fibers. At baseline IOP, like in human sclera, collagen fibrils and cytoskeletal FA structures were highly aligned, and alignment was greater in the circumferentially organized PPS tissue than in peripheral sclera. IOP elevation, however, altered several measures of FA-collagen alignment: it decreased mean FA-collagen alignment, it decreased the number of highly aligned regions, and it increased the amount of poorly aligned regions. This effect was present in both the PPS and peripheral sclera at 1 week after IOP elevation, but only present in the PPS at 6 weeks after IOP elevation. These findings have implications in the process of glaucomatous scleral remodeling. FA-collagen alignment with surrounding collagen fibers is mediated by focal adhesion contact points between the cell membrane and the ECM as well as topographic cues that regulate cytoskeletal organization, nuclear structure, and gene expression.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e Mechanical extracellular signals are transmitted through these contact points and cells then adapt to a changing extracellular environment in an iterative process that can either maintain tissue homeostasis or cause pathologic ECM remodeling. In glaucomatous sclera and PPS, extracellular signals lead to decreased collagen alignment, scleral stiffening, and changes in scleral viscoelasticity. Loss of FA-collagen alignment that we demonstrated after IOP elevation could potentially represent one facet of that process, however, the significance of alignment loss and the mechanism(s) that underlie it are not yet known.\u003c/p\u003e \u003cp\u003eNuclear morphology was minimally affected by IOP elevation. The frequency of any one of the 10 individual shape modes did not change by greater than 5% at 1 or 6 weeks after IOP elevation. The most notable changes were in shape mode \u0026ldquo;e\u0026rdquo; which increased following IOP elevation in the pPPS, oPPS, and peripheral sclera. The small frequency increase in this rounder shape mode was balanced by small decreases in the frequency of the higher aspect ratio shape modes (h, I, and j) in the oPPS and peripheral sclera. Nuclei are mechanoresponsive organelles that are connected to the ECM through cytoskeletal bridges. In fact, Bonnevvie et al demonstrated that nuclear morphology of cultured cells can be used to predict the biomechanical state (stiffness) of the culture environment.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e Therefore, the relatively small changes in nuclear morphology following IOP elevation were surprising in light of the persistent cytoskeletal remodeling induced by IOP elevation and the ECM changes (alterations in stiffness and ECM microarchitecture) that occur with chronic IOP elevation. While this analysis did not appreciate a significant change following IOP elevation, it is possible that our analysis was not appropriately designed to detect differences. Alternatively, examining alternate nuclear features such as nuclear envelope invaginations could have distinguished control and glaucomatous scleral cell nuclei.\u003c/p\u003e \u003cp\u003eThese studies and techniques have several recognized limitations. The scleral flange region of the mouse PPS is an extremely thin structure (\u0026lt;\u0026thinsp;50 \u0026micro;m); it is technically challenging obtain sections through this region and the number of sections from each that can be used in the analysis is small. Due to these challenges, individual sections can contain scleral regions of varying depth that could potentially affect the analysis. Additionally, all analyses were conducted on 2D images. This aspect of the analysis has the greatest potential to affect the nuclear morphology analysis in which any alterations in morphology detected could not only indicate a change in morphology but also could indicate a change in nuclear orientation that occurred with cell migration. Therefore, even the small changes in nuclear morphology observed could be due to changes in nuclear orientation rather than change in the overall nuclear shape.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary we describe imaging studies of the cellular architecture of the mouse PPS and peripheral sclera. Our studies identified regions within the PPS and peripheral sclera that mirror the organization of the human PPS. Examination of FA-collagen alignment showed a high degree of alignment of extracellular collagen and cytoskeletal FA. IOP elevation reduced this alignment in the PPS and peripheral sclera at the 1 week timepoint and at the 6 week timepoint in the PPS. The studies provide insight into the cellular organization of the PPS and the mechanoresponse of scleral PPS fibroblasts to IOP elevation.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003e/Acknowledgements: IP was supported by a grant from the BrightFocus foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eI.P, A.M, J.R., and N.Y. conducted experiments and performed data analysisE.E. performed data analysisI.P wrote the main manuscript and prepared figuresAll authors reviewed the manuscriptThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTham, Y. C. et al. 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Epub 20210315.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5397173/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5397173/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThese studies aimed to study the mechanisms of glaucomatous peripapillary scleral (PPS) remodeling by investigating IOP-induced changes in fibroblast actin-collagen alignment and nuclear morphology in mouse PPS. Cryosections from the optic nerve heads (ONH) of eyes isolated 1- and 6-weeks after bead-induced IOP elevation were imaged for nuclei, fibrillar actin (FA), and collagen (second harmonic generation, SHG). Nuclear morphology was analyzed using VAMPIRE machine-learning image analysis and FA-collagen alignment was determined by comparing vector fields of FA and SHG images. Nuclear morphology was regionally defined with the inner pial PPS (pPPS) containing nuclei with higher aspect ratios than the peripheral PPS (outer PPS, oPPS) and peripheral sclera. FA-collagen alignment was higher in the PPS than in the peripheral sclera (7.1±2.5° versus 10.0±1.4°, p = 0.05, n = 6). One and six weeks after BI, there were nuclear morphologic changes reflecting a transition to a rounder shape in all scleral regions and persistently reduced FA-collagen alignment in the PPS regions. This study therefore concludes that chronic IOP elevation is associated with persistent alterations in nuclear morphology and FA-collagen alignment that indicate sustained cellular responses to tissue stress. These changes could be a precursor to more chronic scleral remodeling that underlies glaucoma pathogenesis.\u003c/p\u003e","manuscriptTitle":"Regional differences in peripapillary scleral fibroblast cytoskeleton and nuclear morphology after intraocular pressure elevation in mouse eyes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 13:00:18","doi":"10.21203/rs.3.rs-5397173/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-28T06:50:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-24T04:51:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-24T04:19:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-08T17:40:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49026148379201177894545379179586881462","date":"2025-01-07T14:40:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77044462838141025663376367844312883163","date":"2024-12-02T06:39:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105517757644593202649387136200477489669","date":"2024-11-22T17:33:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-22T16:17:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-22T12:51:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-22T12:47:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-21T14:10:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-11-05T16:50:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9dcb8720-a437-4e6d-9c54-08bdd00965c9","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41005784,"name":"Biological sciences/Cell biology/Cell migration"},{"id":41005785,"name":"Health sciences/Diseases/Eye diseases/Ocular hypertension"},{"id":41005786,"name":"Health sciences/Diseases/Eye diseases/Scleral diseases"}],"tags":[],"updatedAt":"2025-07-07T16:01:54+00:00","versionOfRecord":{"articleIdentity":"rs-5397173","link":"https://doi.org/10.1038/s41598-025-08251-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:56:54","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-12-19 13:00:18","video":"","vorDoi":"10.1038/s41598-025-08251-4","vorDoiUrl":"https://doi.org/10.1038/s41598-025-08251-4","workflowStages":[]},"version":"v1","identity":"rs-5397173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5397173","identity":"rs-5397173","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}