{"paper_id":"269ebbb6-bc3f-430a-bf51-065138087e60","body_text":"A direct method for imaging gradient levels of retinal hypoxia in a model of retinopathy of prematurity (ROP) | 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 A direct method for imaging gradient levels of retinal hypoxia in a model of retinopathy of prematurity (ROP) MD Imam Uddin, Sara Jamal, John S. Penn This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7247191/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 Background: Retinal hypoxia may contribute to the development of preretinal neovascularization in patients with retinopathy of prematurity (ROP). Ciliary bodies compensate oxygen delivery to the retina, and the levels of hypoxia may vary across the peripheral avascular area in ROP. In this study, we have investigated a direct method for imaging gradient levels of retinal hypoxia at the peripheral avascular retina using a model ROP. Methods. The rat 50/10 oxygen-induced retinopathy (OIR) model was generated by exposing the newly born Brown-Norway rat pups to a 24 hours alternate cycles of 50% and 10% oxygen for 14 days. We also confirmed the development of neovascularization in this model. HYPOX4 was used as a direct method for imaging gradient levels of retinal hypoxia at the peripheral avascular retina. A separate group of rat OIR pups were used to confirm gradient levels of retinal hypoxia using pimonidazole immunostaining. Gradient levels of retinal hypoxia was analyzed using ImageJ software from fluorescence intensities of HYPOX-4 and Pimonidazole immunostaining. Results : Retinal hypoxia was observed in the peripheral avascular retinas in rat OIR. Based on fluorescence intensity measurements, retinal hypoxia was at minimal levels near the ciliary bodies. Retinal hypoxia was at its maximum levels towards the avascular-vascular transition zones. Interestingly, we observed hemiretinal avascular retina temporal to the optic nerve in this OIR model, similar to human ROP retinas. In the retinal cross-section, hypoxia was not detectable near the ora serrata in rat OIR may be due to oxygen delivery by the ciliary bodies. Both pimonidazole and HYPOX-4 showed similar patterns of retinal hypoxia at the peripheral avascular retina in this model. As expected, preretinal neovascularization was observed at the avascular-vascular transition zones arising from the existing retinal vascular structures in this OIR model in Brown-Norway rats. Conclusions : In this study, we have characterized gradient levels of retinal hypoxia in the rat model of 50/10 OIR using a direct method from HYPOX-4 fluorescence. We observed minimal levels of retinal hypoxia near the ciliary bodies in this model and increased towards the avascular-vascular transition zones. In addition, we observed that the central vascularized retina remains gradient hypoxic in this model which could be detected using HYPOX-4. This study may clarify our understanding of retinal hypoxia in the ROP patient at the peripheral retinas. Retinopathy of prematurity ROP molecular imaging retinal hypoxia HYPOX-4 optical imaging fluorescence imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 CLINICAL IMPLICATIONS Imaging gradient levels of retinal hypoxia is an important indicator of severity of ROP pathogenesis including neovascularization. We used HYPOX-4 as a direct method for imaging gradient levels of retinal hypoxia in a model of ROP. We observed minimal levels of retinal hypoxia near the ciliary bodies in this rat 50/10 OIR model and increased levels of hypoxia towards the avascular-vascular transition zones. Even though the central area of the rat 50/10 OIR retina is vascularized, the vascularized retina remains gradient hypoxic which could be detected using HYPOX-4. This study may clarify our understanding of levels of retinal hypoxia in the ROP patient at the peripheral avascular retina; and also, at the vascularized areas of the retina which is significant and needs future clinical investigations. INTRODUCTION Retinopathy of prematurity (ROP) is a leading cause of vision loss in premature infants and its pathogenesis has been described as consisting of two phases, Phase I and II ( 1 , 2 ). Phase I culminates in an ischemia-induced retinal hypoxia ( 3 – 5 ). Preterm infants with an immature retinal vasculature are administered supplemental oxygen to compensate for underdeveloped lung function, which may cause systemic oxygen levels to rise periodically. However, due to systemic maladies such as patent ductus arteriosus (PDA) that are associated with prematurity and the necessary manipulations required for the care of the infant, episodes of low oxygen tension may also occur. Due to the combination of aforementioned as well as other treatments, conditions and events, the premature infant experiences variable oxygen levels throughout the course of oxygen treatment. Variable oxygen attenuates normal retinal vascular development, and when the oxygen therapy is discontinued, the infant is left with a large peripheral avascular retina (ischemia) that rapidly becomes hypoxic. Molecular studies have shown that retinal hypoxia increases the expression of proangiogenic growth factors and cytokines; the most important of these is vascular endothelial growth factor (VEGF)( 6 , 7 ). VEGF triggers the onset of the vasoproliferative phase (Phase II) of ROP, resulting in the formation of pre-retinal dysplastic structures commonly referred to as neovascular tufts ( 3 , 8 ). These structures are leaky, fragile, prone to hemorrhage, predisposing the affected infant to tractional retinal detachment and blindness. Upon considering the integral role of hypoxia in ROP pathogenesis, it becomes evident that a reliable non-invasive method for detecting, measuring and imaging retinal hypoxia in premature infants would offer great clinical utility. For example, infants could be screened for retinal hypoxia as a predictor of progression to phase II, perhaps guiding the clinician to initiate a prophylactic therapy. Assessment of retinal hypoxia may also indicate the severity of retinopathy and it could also be used as a benchmark to gauge the efficacy of therapy against established neovascular disease. Though, methods have been developed for the measurement of oxygen tension levels in tissues; these include nuclear magnetic resonance ( 5 , 6 ), retinal oximetry ( 9 ), phosphorescence lifetime imaging ( 10 ), doppler optical coherence tomography (D-OCT) ( 11 ), and visible-light OCT ( 12 ). Their application has provided a clearer understanding of the vascular oxygen supply and metabolism in the retina, none of these imaging methods have been used successfully to measure retinal hypoxia. Pimonidazole-mediated immunohistochemistry is the most common method to study retinal hypoxia, but this technique is limited for its method of examination and not suitable for clinical in vivo applications ( 13 ). Our laboratory has developed HYPOX-4, a hypoxia sensitive fluorescent molecular imaging probe to detect retinal hypoxia in the living retina ( 14 – 16 ). In the current study, we have investigated the application of HYPOX-4, as a direct method to detect and measure retinal hypoxia in the 50/10 oxygen induced retinopathy (OIR) using Brown-Norway rats. This model faithfully recapitulates several of the pathologic features of human ROP ( 17 , 18 ). Previously, we have demonstrated the development of HYPOX-4 for the assessment of retinal hypoxia in mouse OIR ( 14 ), another model with an ischemia-induced hypoxia pathologic component. In this approach, the systemically administered HYPOX-4 is delivered successfully to the hypoxic avascular retina where it is presumably retained by the reduction of hypoxia-regulated nitro-reductases, thus allowing real time in vivo hypoxia-dependent fluorescence imaging ( 19 ). In the current study, we tried to overcome the challenges to deliver the HYPOX-4 after intraperitoneal injections to a remote peripheral avascular retina in the rat OIR model. We have used HYPOX-4 to characterize the levels and distribution of hypoxia in this rat 50/10 OIR model as a direct method to detect hypoxia in the peripheral retina. Furthermore, HYPOX-4-dependent imaging of gradient levels of retina hypoxia was compared to the profiles obtained using pimonidazole-adduct immunostaining method. Herein we report our results. MATERIALS AND METHODS Synthesis of HYPOX-4 The HYPOX-4 was synthesized according to our previously reported methods( 14 ). Chemical structure of HYPOX-4 is shown in Fig. 1 . This in vivo molecular imaging probe contains a hypoxia sensitive, pimonidazole compound conjugated to a clinically compatible fluorescent dye via an amide linkage. HYPOX-4 is water soluble and has no residual toxicity to the retinal cells. Animals Multi-timed pregnant Brown Norway Female rats were purchased from Charles River Laboratories; Chicago, Illinois. All animal procedures used in this study were approved by the Vanderbilt University Institutional Animal Care and Use Committee (Institutional approval number M1600260-01) and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with ARRIVE guidelines. Animals were group-housed according to their randomly assigned experimental groups in ventilated cages maintained under a 12 hours light and dark cycle at 22 ± 2°C within an institutional animal care facility. Animals were provided with clean water (Nashville Metro Water Services, Nashville, TN) and a standard diet consisting of 4.5% fat (PicoLab Rodent Diet 5L0D; LabDiet, St. Louis, MO) ad libitum. Rats were sacrificed by CO 2 -induced asphyxiation followed by cervical dislocation. Rat 50/10 oxygen-induced retinopathy model in Brown Norway To generate Brown Norway rat 50/10 oxygen-induced retinopathy model (50/10 OIR), newborn rat pups were treated with alternating episodes of 50% oxygen for 24 hours then 10% oxygen for 24 hours for a total of 14 days. After the oxygen treatments, rat pups with their nursing mother return from the 50/10 OIR chamber to the normal room air. This treatment protocol mimics the variable systemic oxygen levels observed in cohorts of premature infants treated in intensive care units. At the end of the oxygen-treatment there is a peripheral avascular retina similar to that observed in human ROP whereas both are the exact opposite to the pattern observed in mouse OIR post oxygen-treatment. Direct method for imaging retinal hypoxia HYPOX-4 was used as the direct method, and pimonidazole-adducts immunoassaying was used as indirect method to detect retinal hypoxia. After returning from the 50/10 OIR chamber to room air, the rat pups were intraperitoneally injected with 60 mg/kg HYPOX-4. Eighteen hours post-intraperitoneal injection, animals were sacrificed and retinas were dissected, and stained with isolectin B4 conjugated to AF647 to visualize the retinal vasculature by confocal microscopy. To evaluate the presence of hypoxia at the peripheral avascular retina, pimonidazole-adducts immunostaining method was used. Pimonidazole hydrochloride was injected intraperitoneally at a dose of 60 mg/Kg on day-14 (P14) at two hours after removal to room air. Two hours after the pimonidazole injection, animals were sacrificed. Retinas were dissected, immunoassayed for pimonidazole-adducts using antibody against pimonidazole-adducts (Hypoxyprobe, Burlington, MA, USA) was used to stain retinal hypoxia. ICAM-2 conjugated to AF-647 was used to counter stain for retinal blood vessels. In addition, retinal cross sections were also immunoassayed for pimonidazole-adducts. ImageJ software was used to quantify the levels of retinal hypoxia. RESULTS Direct method for imaging retinal hypoxia in rat 50/10 OIR using HYPOX-4 The rat 50/10 OIR model of ROP was developed in our laboratory to study the pathogenesis of neovascular retinopathy in premature infants ( 20 – 22 ). Among the models of ROP, it most closely recapitulates a majority of the pathologic features observed in human ROP patients. Among the disease pathologies, neovascularization is the most severe consequence of this disease condition. Levels of retinal hypoxia is an important indicator of severity of neovascularization. In this study, we have investigated the levels of retinal hypoxia in rat 50/10 OIR model. In this model, newborn rat pups were exposed to episodes of 50% oxygen for 24 hours then 10% oxygen for 24 hours for a total of 14 days (Fig. 1 ). At postnatal day 14 pups are returned to room air. Retinal hypoxia was monitored at P14. We used HYPOX-4 as a direct method for imaging gradient levels of retinal hypoxia in this model. Retinal hypoxia was observed in the peripheral avascular retinas in this model. Based on fluorescence intensity measurements, retinal hypoxia was at minimal levels near the ciliary bodies (Fig. 2 ). Retinal hypoxia was at its maximum levels towards the avascular-vascular transition zones. Interestingly, we observed hemiretinal avascular retina temporal to the optic nerve in this model, similar to human ROP retinas. In addition, we observed that the central retina is vascularized in this rat 50/10 OIR model; however the vascularized retina remains gradient hypoxic which could be detected using HYPOX-4 as shown in Fig. 2 D-F. These observations may clarify the levels of retinal hypoxia in the ROP patient at the peripheral avascular retina and also at the vassalized areas of the ROP retinas. Characterization of retinal hypoxia in rat 50/10 OIR using pimonidazole-immunostaining We have further characterized retinal hypoxia at the peripheral avascular retinal using the standard pimonidazole-adduct immunostaining technique (Fig. 3 ). Retinal hypoxia was detected at the peripheral avascular retina. Gradient levels of retinal hypoxia were analyzed from fluorescence intensity measurements using ImageJ software (Fig. 3 B). Both pimonidazole and HYPOX-4 showed similar patterns of retinal hypoxia at the peripheral avascular retina in this model. In addition, retinal hypoxia was observed at the vascularized area of the retina as shown in Fig. 3 D. These results further confirmed the presence of retinal hypoxia even at the vascularized areas of the retina in this rat OIR model. In the retinal cross-section, hypoxia was not detectable near the ora serrata in the rat OIR model, may be due to oxygen delivery by the ciliary bodies (Fig. 4 ). In addition, retinal hypoxia was observed mostly at the inner retinal layers including retinal ganglion cell layer (RGC), inner plexiform layer (IPL) and inner nuclear layer (INL). As expected, preretinal neovascularization was observed at the avascular-vascular transition zones arising from the existing retinal vascular structures in this OIR model in Brown-Norway rats (Fig. 5 ). Thus, future clinical studies focusing on these findings could improve the diagnosis and treatment options for patients with hypoxic retinopathies. DISCUSSIONS Retinal-hypoxia is associated with both early (phase I) and proliferative stage (phase II) of ROP, however the precise relationship between its onset, evolution and resolution, to other pathologic events, such as increased expression of proangiogenic growth factors and cytokines and ROP morphometrics (e.g. retinal vessel tortuosity, peripheral avascular area and severity of neovascular tuft formation etc.) is largely unknown ( 23 ). Therefore, the ability to reliably detect, measure and image retinal hypoxia would offer great advantages to the management of ROP. For example, infants could be screened for retinal hypoxia as a predictor for transition into phase II. Quantification of retinal hypoxia may help to establish ROP severity and could also be used as a benchmark to gauge the efficacy of therapy against neovascular disease. Furthermore, accurate measurement of retinal hypoxia would be of great benefit to the researcher investigating ROP pathogenesis in experimental models of ROP-like disease, leading to a better understanding of the role of hypoxia in ischemic retinopathies and the development of new drugs. In summary hypoxia plays an integral role in ROP pathogenesis and currently its relationship to other events in the ROP pathogenic cascade is largely unknown. An understanding of this relationship would be of great benefit in the management of ROP. Current methods that are routinely used to measure levels of tissue oxygen tension suffer from drawbacks that limit their application to real time in vivo imaging of retinal hypoxia. Our newly developed direct imaging method using HYPOX-4 is safe, effective and reliable to detect retinal hypoxia to achieve these goals. CONCLUSION In this study, we have characterized the gradient levels of retinal hypoxia in the peripheral avascular retina in rat 50/10 OIR model of ROP. We have utilized HYPOX-4 as a direct method to detect gradient levels of retinal hypoxia in this model. We found that HYPOX-4 is a clinically relevant molecular imaging probe to detect retinal hypoxia and could be used in future studies to improve the diagnosis and treatments for patients with hypoxic retinopathies. Abbreviations BM basement membrane CC choriocapillaris DCP deep capillary plexus FA Fluorescein Angiography GCL ganglion cell layer INL inner nuclear layer IPL inner plexiform layer LPS Lipopolysaccharide MCP middle capillary plexus NV neovascularization OCT optical coherence tomography ONH optic nerve head ONL outer nuclear layer OPL outer plexiform layer PDR proliferative diabetic retinopathy ROP retinopathy of prematurity SCP superficial capillary plexus VEGF vascular endothelial growth factor Declarations FUNDING This research project was supported by grants from the National Institutes of Health (NIH; Bethesda, MD; R01EY029693 to MIU and R01EY023397 to MIU and JSP), a grant from American Diabetes Association, ADA Grant ref #11-22-IBSPM-12 (to MIU), a grant from Carl Marshall Reeves & Mildred Almen Reeves Foundation, Inc. (Fenton, MO; to MIU), a support provided in part by funding to the Vanderbilt Eye Institute from the International Retinal Research Foundation (Birmingham, AL), and a grant from Vanderbilt Vision Research Center NEI Core (P30-EY008126) and an unrestricted Grant from Research to Prevent Blindness (RPB). The funding organizations had no role in the design or conduct of this research. Author Contributions: SJ performed the experiments, collected and analyzed the data. JSP helped revise the manuscript. MIU conceived and supervised the project, designed, and synthesized the compound HYPOX-4, designed and performed experiments, and wrote the manuscript. All authors contributed to the article and approved the submitted version. ACKNOWLEDGEMENTS We thank Prof Dr. Gary W. McCollum (Vanderbilt University School of Medicine, Nashville, TN 37232, USA) for helpful discussions in this study. DISCLOSURES MD Imam Uddin is an inventor on a Patent US10695446B2 (issued) that includes the discovery of HYPOX-4 and its applications. AVAILABILITY OF DATA AND MATERIALS All data that support the results of this study are available within the article or upon request to the corresponding author (MIU). Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare that they have no conflicts of interest to report. References Hartnett ME, Penn JS. Mechanisms and Management of Retinopathy of Prematurity. New Engl J Med. 2012;367(26):2515–26. 10.1056/Nejmra1208129 . PubMed PMID: WOS:000312714200009. Gariano RF, Gardner TW. Retinal angiogenesis in development and disease. Nature. 2005;438(7070):960–6. 10.1038/nature04482 . PubMed PMID: WOS:000233934600055. Sapieha P, Joyal JS, Rivera JC, Kermorvant-Duchemin E, Sennlaub F, Hardy P, Lachapelle P, Chemtob S. Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life. J Clin Invest. 2010;120(9):3022–32. PubMed PMID: 20811158; PMCID: PMC2929716. Fevereiro-Martins M, Marques-Neves C, Guimaraes H, Bicho M. Retinopathy of prematurity: A review of pathophysiology and signaling pathways. Surv Ophthalmol. 2023;68(2):175–210. PubMed PMID: 36427559. Selvam S, Kumar T, Fruttiger M. Retinal vasculature development in health and disease. Prog Retin Eye Res. 2018;63:1–19. PubMed PMID: 29129724. Penn JS, Madan A, Caldwell RB, Bartoli M, Caldwell RW, Hartnett ME. Vascular endothelial growth factor in eye disease. Prog Retin Eye Res. 2008;27(4):331–71. 10.1016/j.preteyeres.2008.05.001 . Epub 2008/07/26. Alon T, Hemo I, Itin A, Pe'er J, Stone J, Keshet E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med. 1995;1(10):1024–8. 10.1038/nm1095-1024 . Epub 1995/10/01. Sapieha P, Hamel D, Shao Z, Rivera JC, Zaniolo K, Joyal JS, Chemtob S. Proliferative retinopathies: angiogenesis that blinds. Int J Biochem Cell Biol. 2010;42(1):5–12. 10.1016/j.biocel.2009.10.006 . Epub 2009/10/20. Hardarson SH, Harris A, Karlsson RA, Halldorsson GH, Kagemann L, Rechtman E, Zoega GM, Eysteinsson T, Benediktsson JA, Thorsteinsson A, Jensen PK, Beach J, Stefansson E. Automatic retinal oximetry. Invest Ophth Vis Sci. 2006;47(11):5011–6. 10.1167/iovs.06-0039 . PubMed PMID: WOS:000241557500053. Shahidi M, Shakoor A, Blair NP, Mori M, Shonat RD. A method for chorioretinal oxygen tension measurement. Curr Eye Res. 2006;31(4):357–66. PubMed PMID: WOS:000237274400009. Dai CX, Liu XJ, Zhang HF, Puliafito CA, Jiao SL. Absolute Retinal Blood Flow Measurement With a Dual-Beam Doppler Optical Coherence Tomography. Invest Ophth Vis Sci. 2013;54(13):7998–8003. 10.1167/iovs.13-12318 . PubMed PMID: WOS:000328133800021. Soetikno BT, Yi J, Shah R, Liu W, Purta P, Zhang HF, Fawzi AA. Inner retinal oxygen metabolism in the 50/10 oxygen-induced retinopathy model. Sci Rep. 2015;5:16752. 10.1038/srep16752 . PubMed PMID: 26576731; PMCID: 4649746. Varia MA, Calkins-Adams DP, Rinker LH, Kennedy AS, Novotny DB, Fowler WC Jr., Raleigh JA. Pimonidazole: a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in cervical carcinoma. Gynecol Oncol. 1998;71(2):270–7. 10.1006/gyno.1998.5163 . PubMed PMID: 9826471. Uddin MI, Evans SM, Craft JR, Capozzi ME, McCollum GW, Yang R, Marnett LJ, Uddin MJ, Jayagopal A, Penn JS. In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy. Sci Rep. 2016;6:31011. 10.1038/srep31011 . Epub 2016/08/06. Uddin MI, Jayagopal A, McCollum GW, Yang R, Penn JS. In Vivo Imaging of Retinal Hypoxia Using HYPOX-4-Dependent Fluorescence in a Mouse Model of Laser-Induced Retinal Vein Occlusion (RVO). Invest Ophthalmol Vis Sci. 2017;58(9):3818–24. 10.1167/iovs.16-21187 . Epub 2017/07/28. Jamal SZ, Dieckmann BW, McCollum GW, Penn JS, Jayagopal A, Imam Uddin MD. Imaging Hypoxia to Predict Primary Neuronal Cell Damage in Branch Retinal Artery Occlusion. Microcirculation. 2024;31(7):e12883. 10.1111/micc.12883 . Epub 2024/08/31. Scott A, Fruttiger M. Oxygen-induced retinopathy: a model for vascular pathology in the retina. Eye. 2010;24(3):416–21. 10.1038/eye.2009.306 . PubMed PMID: WOS:000275447200003. Di Fiore JM, Kaffashi F, Loparo K, Sattar A, Schluchter M, Foglyano R, Martin RJ, Wilson CG. The relationship between patterns of intermittent hypoxia and retinopathy of prematurity in preterm infants. Pediatr Res. 2012;72(6):606–12. 10.1038/pr.2012.132 . PubMed PMID: 23037873; PMCID: 4433009. Hodgkiss RJ. Use of 2-nitroimidazoles as bioreductive markers for tumour hypoxia. Anti-Cancer Drug Des. 1998;13(6):687–702. PubMed PMID: WOS:000077238500010. Penn JS, Tolman BL, Henry MM. Oxygen-induced retinopathy in the rat: relationship of retinal nonperfusion to subsequent neovascularization. Invest Ophthalmol Vis Sci. 1994;35(9):3429–35. Epub 1994/08/01. PubMed PMID: 8056518. Penn JS, Henry MM, Tolman BL. Exposure to alternating hypoxia and hyperoxia causes severe proliferative retinopathy in the newborn rat. Pediatr Res. 1994;36(6):724–31. 10.1203/00006450-199412000-00007 . Epub 1994/12/01. Penn JS, Tolman BL, Lowery LA. Variable oxygen exposure causes preretinal neovascularization in the newborn rat. Invest Ophthalmol Vis Sci. 1993;34(3):576–85. Epub 1993/03/01. PubMed PMID: 8449677. Mowat FM, Luhmann UF, Smith AJ, Lange C, Duran Y, Harten S, Shukla D, Maxwell PH, Ali RR, Bainbridge JW. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia. PLoS ONE. 2010;5(6):e11103. 10.1371/journal.pone.0011103 . PubMed PMID: 20559438; PMCID: 2885428. 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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-7247191\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":500168779,\"identity\":\"6145e3e1-d58e-4fc3-896e-f107361d5fcf\",\"order_by\":0,\"name\":\"MD Imam Uddin\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYFCCBIYDDAzMjG0MzAdI1sKWQLwWBpCWBgYeA+I06LYnMB5gbLOW7ZPu+SbNm2PHwC99/AJeLWZnHjAAtaQbt8mc3SbNuy2ZQbIvpwC/lhsJIC2HE9skcjcb8247wGBwhieBWC05j0nXwvgYooX9AAG/PGw4kHAO6BeJNMOHc7cl80j28ODVwWB2PPnwhw9l1rLzZyQ/OPB2m50cPw/7A/x6GIAxgux2oBXERhASIGjLKBgFo2AUjDAAAH0VRvLBMK0iAAAAAElFTkSuQmCC\",\"orcid\":\"https://orcid.org/0000-0001-5611-9666\",\"institution\":\"Vanderbilt University School of Medicine\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"MD\",\"middleName\":\"Imam\",\"lastName\":\"Uddin\",\"suffix\":\"\"},{\"id\":500168780,\"identity\":\"31dd4000-e087-44ec-8285-9555a9a9c458\",\"order_by\":1,\"name\":\"Sara Jamal\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Vanderbilt University School of Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sara\",\"middleName\":\"\",\"lastName\":\"Jamal\",\"suffix\":\"\"},{\"id\":500168781,\"identity\":\"214ebe6d-e294-4224-9894-0e1eca939c1d\",\"order_by\":2,\"name\":\"John S. Penn\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Vanderbilt University School of Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"John\",\"middleName\":\"S.\",\"lastName\":\"Penn\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-07-30 00:52:28\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7247191/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7247191/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":89594345,\"identity\":\"f4a0877d-4310-48da-bbe7-a72916eee747\",\"added_by\":\"auto\",\"created_at\":\"2025-08-21 16:28:17\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":28706,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDirect method for imaging gradient levels of hypoxia in rat 50/10 oxygen-induced retinopathy (OIR) model. (A) Chemical structure of HYPOX-4. This in vivo imaging probe contains hypoxia sensitive compound, pimonidazole conjugated via an amide linkage to dye compatible with clinically used fluorescence imaging equipment. HYPOX-4 is water soluble and has no residual toxicity to the retinal cells. (B) Graph of inspired oxygen treatment to develop 50/10 OIR model in Brown Norway rat pups. In this model, newborn rat pups experience alternating episodes of 50% oxygen for 24 hours then 10% oxygen for 24 hours for a total of 14 days. At postnatal day-14 (P14) pups are returned to room air. Retinal hypoxia was monitored at P14. This model recapitulates features of neovascularization and intensifies at P19, similar to infants with severe ROP.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7247191/v1/49af4ffff335eb3bb47415e7.png\"},{\"id\":89592368,\"identity\":\"fcf14ebf-d38c-4ec1-b004-72a222ea3b21\",\"added_by\":\"auto\",\"created_at\":\"2025-08-21 16:12:17\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":633321,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImaging of retinal hypoxia using HYPOX-4 in 50/10 rat OIR. Isolectin B4 (IB4) was used to counter stain the vascular structures. (A, B) HYPOX-4 was localized largely in the avascular OIR retina. (C, D) Magnification of A and B respectively. (E, F) ImageJ software was used to analyze gradient levels of retinal hypoxia from HYPOX-4 fluorescence intensities across the 50/10 OIR retina as shown in F. Total of 12 eyes were analyzed for this study.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7247191/v1/06b3f83d824695c14bc99366.png\"},{\"id\":89592372,\"identity\":\"c7813587-2b2b-48a2-be77-e5f47827cbef\",\"added_by\":\"auto\",\"created_at\":\"2025-08-21 16:12:17\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":387486,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRetinal hypoxia was characterized in peripheral avascular retina in rat 50/10 OIR model. Pimonizadole hydrochloride was injected intraperitoneally at a dose of 60 mg/Kg on day-14, 2 hours after removal to room air. Rat pups were sacrificed two hours after the pimonidazole injection (which is four hours after removal to room air). Retinas were dissected, flat-mounted and immunoassayed for pimonidazole-adducts. (A, B) Gradient levels of retinal hypoxia were analyzed from fluorescence intensity measurements. ImageJ software was used to analyze the fluorescence intensities across the peripheral avascular 50/10 OIR retina. (C, D) Intense retinal hypoxia was observed at the peripheral avascular retina in this model. In addition, mild hypoxia was also observed at the central retina as shown in D. Total of 12 eyes were analyzed for this study.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7247191/v1/11f33e53e17a9180ef47c59b.png\"},{\"id\":89594346,\"identity\":\"3e1544a0-9ad7-4bb7-a708-da396dee22fc\",\"added_by\":\"auto\",\"created_at\":\"2025-08-21 16:28:17\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":688543,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImaging retinal hypoxia in 50/10 OIR retinal cross sections. Pimonidazole immunostaining (HP-1) was used to visualize the retinal hypoxia and ICAM-2 was used to counterstain retinal vascular structures. (A-E) Retinal hypoxia was observed at different depth of the 50/10 OIR retina, mostly in the inner retina. (F-G) Retinal hypoxia was also monitored in 50/10 rat retinal cross sections. Retinal hypoxia was observed at different layers including retinal ganglion cell layer (RGC), inner plexiform layer (IPL) and inner nuclear layer (INL). DAPI was used to counterstain the nuclei to localize HP-1 fluorescence in retinal layers.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7247191/v1/65c55bfa174529f34a8b1905.png\"},{\"id\":89592369,\"identity\":\"e45575fb-d336-414e-8f79-350a2159e9c3\",\"added_by\":\"auto\",\"created_at\":\"2025-08-21 16:12:17\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":474386,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCharacterization of preretinal neovascularization in Brown Norway 50/10 OIR model. (A) IB4 staining showing the neovascularization at the border of vascular/avascular area. Hypoxia may contribute to the development of neovascularization observed at P19. (B) Magnification view of A. (C) Preretinal neovascular tufts were localized in retinal cross section at P19.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7247191/v1/033d55e5c52204a9a1ecbeea.png\"},{\"id\":92905430,\"identity\":\"df5b69af-24b1-4dff-888d-db7f3b7dd904\",\"added_by\":\"auto\",\"created_at\":\"2025-10-07 00:15:20\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2557679,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7247191/v1/738b4a16-9913-4bea-8342-ce6b2ff5082c.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"A direct method for imaging gradient levels of retinal hypoxia in a model of retinopathy of prematurity (ROP)\",\"fulltext\":[{\"header\":\"CLINICAL IMPLICATIONS\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eImaging gradient levels of retinal hypoxia is an important indicator of severity of ROP pathogenesis including neovascularization. We used HYPOX-4 as a direct method for imaging gradient levels of retinal hypoxia in a model of ROP. \\u0026nbsp;\\u0026nbsp;\\u003c/li\\u003e\\n\\u003c/ol\\u003e\\n\\u003col start=\\\"2\\\"\\u003e\\n \\u003cli\\u003eWe observed minimal levels of retinal hypoxia near the ciliary bodies in this rat 50/10 OIR model and increased levels of hypoxia towards the avascular-vascular transition zones.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\\n\\u003col start=\\\"3\\\"\\u003e\\n \\u003cli\\u003eEven though the central area of the rat 50/10 OIR retina is vascularized, the vascularized retina remains gradient hypoxic which could be detected using HYPOX-4.\\u0026nbsp;\\u003c/li\\u003e\\n\\u003c/ol\\u003e\\n\\u003col start=\\\"4\\\"\\u003e\\n \\u003cli\\u003eThis study may clarify our understanding of levels of retinal hypoxia in the ROP patient at the peripheral avascular retina; and also, at the vascularized areas of the retina which is significant and needs future clinical investigations. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eRetinopathy of prematurity (ROP) is a leading cause of vision loss in premature infants and its pathogenesis has been described as consisting of two phases, Phase I and II (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). Phase I culminates in an ischemia-induced retinal hypoxia (\\u003cspan additionalcitationids=\\\"CR4\\\" citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e). Preterm infants with an immature retinal vasculature are administered supplemental oxygen to compensate for underdeveloped lung function, which may cause systemic oxygen levels to rise periodically. However, due to systemic maladies such as patent ductus arteriosus (PDA) that are associated with prematurity and the necessary manipulations required for the care of the infant, episodes of low oxygen tension may also occur. Due to the combination of aforementioned as well as other treatments, conditions and events, the premature infant experiences variable oxygen levels throughout the course of oxygen treatment. Variable oxygen attenuates normal retinal vascular development, and when the oxygen therapy is discontinued, the infant is left with a large peripheral avascular retina (ischemia) that rapidly becomes hypoxic. Molecular studies have shown that retinal hypoxia increases the expression of proangiogenic growth factors and cytokines; the most important of these is vascular endothelial growth factor (VEGF)(\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e). VEGF triggers the onset of the vasoproliferative phase (Phase II) of ROP, resulting in the formation of pre-retinal dysplastic structures commonly referred to as neovascular tufts (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e). These structures are leaky, fragile, prone to hemorrhage, predisposing the affected infant to tractional retinal detachment and blindness.\\u003c/p\\u003e\\u003cp\\u003eUpon considering the integral role of hypoxia in ROP pathogenesis, it becomes evident that a reliable non-invasive method for detecting, measuring and imaging retinal hypoxia in premature infants would offer great clinical utility. For example, infants could be screened for retinal hypoxia as a predictor of progression to phase II, perhaps guiding the clinician to initiate a prophylactic therapy. Assessment of retinal hypoxia may also indicate the severity of retinopathy and it could also be used as a benchmark to gauge the efficacy of therapy against established neovascular disease. Though, methods have been developed for the measurement of oxygen tension levels in tissues; these include nuclear magnetic resonance (\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e), retinal oximetry (\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e), phosphorescence lifetime imaging (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e), doppler optical coherence tomography (D-OCT) (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e), and visible-light OCT (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e). Their application has provided a clearer understanding of the vascular oxygen supply and metabolism in the retina, none of these imaging methods have been used successfully to measure retinal hypoxia. Pimonidazole-mediated immunohistochemistry is the most common method to study retinal hypoxia, but this technique is limited for its method of examination and not suitable for clinical \\u003cem\\u003ein vivo\\u003c/em\\u003e applications (\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eOur laboratory has developed HYPOX-4, a hypoxia sensitive fluorescent molecular imaging probe to detect retinal hypoxia in the living retina (\\u003cspan additionalcitationids=\\\"CR15\\\" citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e). In the current study, we have investigated the application of HYPOX-4, as a direct method to detect and measure retinal hypoxia in the 50/10 oxygen induced retinopathy (OIR) using Brown-Norway rats. This model faithfully recapitulates several of the pathologic features of human ROP (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e). Previously, we have demonstrated the development of HYPOX-4 for the assessment of retinal hypoxia in mouse OIR (\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e), another model with an ischemia-induced hypoxia pathologic component. In this approach, the systemically administered HYPOX-4 is delivered successfully to the hypoxic avascular retina where it is presumably retained by the reduction of hypoxia-regulated nitro-reductases, thus allowing real time \\u003cem\\u003ein vivo\\u003c/em\\u003e hypoxia-dependent fluorescence imaging (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e). In the current study, we tried to overcome the challenges to deliver the HYPOX-4 after intraperitoneal injections to a remote peripheral avascular retina in the rat OIR model. We have used HYPOX-4 to characterize the levels and distribution of hypoxia in this rat 50/10 OIR model as a direct method to detect hypoxia in the peripheral retina. Furthermore, HYPOX-4-dependent imaging of gradient levels of retina hypoxia was compared to the profiles obtained using pimonidazole-adduct immunostaining method. Herein we report our results.\\u003c/p\\u003e\"},{\"header\":\"MATERIALS AND METHODS\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eSynthesis of HYPOX-4\\u003c/h2\\u003e\\u003cp\\u003eThe HYPOX-4 was synthesized according to our previously reported methods(\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e). Chemical structure of HYPOX-4 is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. This in vivo molecular imaging probe contains a hypoxia sensitive, pimonidazole compound conjugated to a clinically compatible fluorescent dye via an amide linkage. HYPOX-4 is water soluble and has no residual toxicity to the retinal cells.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eAnimals\\u003c/h3\\u003e\\n\\u003cp\\u003eMulti-timed pregnant Brown Norway Female rats were purchased from Charles River Laboratories; Chicago, Illinois. All animal procedures used in this study were approved by the Vanderbilt University Institutional Animal Care and Use Committee (Institutional approval number M1600260-01) and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with ARRIVE guidelines. Animals were group-housed according to their randomly assigned experimental groups in ventilated cages maintained under a 12 hours light and dark cycle at 22\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2\\u0026deg;C within an institutional animal care facility. Animals were provided with clean water (Nashville Metro Water Services, Nashville, TN) and a standard diet consisting of 4.5% fat (PicoLab Rodent Diet 5L0D; LabDiet, St. Louis, MO) ad libitum. Rats were sacrificed by CO\\u003csub\\u003e2\\u003c/sub\\u003e-induced asphyxiation followed by cervical dislocation.\\u003c/p\\u003e\\n\\u003ch3\\u003eRat 50/10 oxygen-induced retinopathy model in Brown Norway\\u003c/h3\\u003e\\n\\u003cp\\u003eTo generate Brown Norway rat 50/10 oxygen-induced retinopathy model (50/10 OIR), newborn rat pups were treated with alternating episodes of 50% oxygen for 24 hours then 10% oxygen for 24 hours for a total of 14 days. After the oxygen treatments, rat pups with their nursing mother return from the 50/10 OIR chamber to the normal room air. This treatment protocol mimics the variable systemic oxygen levels observed in cohorts of premature infants treated in intensive care units. At the end of the oxygen-treatment there is a peripheral avascular retina similar to that observed in human ROP whereas both are the exact opposite to the pattern observed in mouse OIR post oxygen-treatment.\\u003c/p\\u003e\\n\\u003ch3\\u003eDirect method for imaging retinal hypoxia\\u003c/h3\\u003e\\n\\u003cp\\u003eHYPOX-4 was used as the direct method, and pimonidazole-adducts immunoassaying was used as indirect method to detect retinal hypoxia. After returning from the 50/10 OIR chamber to room air, the rat pups were intraperitoneally injected with 60 mg/kg HYPOX-4. Eighteen hours post-intraperitoneal injection, animals were sacrificed and retinas were dissected, and stained with isolectin B4 conjugated to AF647 to visualize the retinal vasculature by confocal microscopy. To evaluate the presence of hypoxia at the peripheral avascular retina, pimonidazole-adducts immunostaining method was used. Pimonidazole hydrochloride was injected intraperitoneally at a dose of 60 mg/Kg on day-14 (P14) at two hours after removal to room air. Two hours after the pimonidazole injection, animals were sacrificed. Retinas were dissected, immunoassayed for pimonidazole-adducts using antibody against pimonidazole-adducts (Hypoxyprobe, Burlington, MA, USA) was used to stain retinal hypoxia. ICAM-2 conjugated to AF-647 was used to counter stain for retinal blood vessels. In addition, retinal cross sections were also immunoassayed for pimonidazole-adducts. ImageJ software was used to quantify the levels of retinal hypoxia.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eDirect method for imaging retinal hypoxia in rat 50/10 OIR using HYPOX-4\\u003c/h2\\u003e\\u003cp\\u003eThe rat 50/10 OIR model of ROP was developed in our laboratory to study the pathogenesis of neovascular retinopathy in premature infants (\\u003cspan additionalcitationids=\\\"CR21\\\" citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). Among the models of ROP, it most closely recapitulates a majority of the pathologic features observed in human ROP patients. Among the disease pathologies, neovascularization is the most severe consequence of this disease condition. Levels of retinal hypoxia is an important indicator of severity of neovascularization. In this study, we have investigated the levels of retinal hypoxia in rat 50/10 OIR model. In this model, newborn rat pups were exposed to episodes of 50% oxygen for 24 hours then 10% oxygen for 24 hours for a total of 14 days (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). At postnatal day 14 pups are returned to room air. Retinal hypoxia was monitored at P14.\\u003c/p\\u003e\\u003cp\\u003eWe used HYPOX-4 as a direct method for imaging gradient levels of retinal hypoxia in this model. Retinal hypoxia was observed in the peripheral avascular retinas in this model. Based on fluorescence intensity measurements, retinal hypoxia was at minimal levels near the ciliary bodies (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Retinal hypoxia was at its maximum levels towards the avascular-vascular transition zones. Interestingly, we observed hemiretinal avascular retina temporal to the optic nerve in this model, similar to human ROP retinas. In addition, we observed that the central retina is vascularized in this rat 50/10 OIR model; however the vascularized retina remains gradient hypoxic which could be detected using HYPOX-4 as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD-F. These observations may clarify the levels of retinal hypoxia in the ROP patient at the peripheral avascular retina and also at the vassalized areas of the ROP retinas.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eCharacterization of retinal hypoxia in rat 50/10 OIR using pimonidazole-immunostaining\\u003c/h3\\u003e\\n\\u003cp\\u003eWe have further characterized retinal hypoxia at the peripheral avascular retinal using the standard pimonidazole-adduct immunostaining technique (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Retinal hypoxia was detected at the peripheral avascular retina. Gradient levels of retinal hypoxia were analyzed from fluorescence intensity measurements using ImageJ software (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). Both pimonidazole and HYPOX-4 showed similar patterns of retinal hypoxia at the peripheral avascular retina in this model. In addition, retinal hypoxia was observed at the vascularized area of the retina as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD. These results further confirmed the presence of retinal hypoxia even at the vascularized areas of the retina in this rat OIR model.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn the retinal cross-section, hypoxia was not detectable near the ora serrata in the rat OIR model, may be due to oxygen delivery by the ciliary bodies (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). In addition, retinal hypoxia was observed mostly at the inner retinal layers including retinal ganglion cell layer (RGC), inner plexiform layer (IPL) and inner nuclear layer (INL). As expected, preretinal neovascularization was observed at the avascular-vascular transition zones arising from the existing retinal vascular structures in this OIR model in Brown-Norway rats (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Thus, future clinical studies focusing on these findings could improve the diagnosis and treatment options for patients with hypoxic retinopathies.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"DISCUSSIONS\",\"content\":\"\\u003cp\\u003eRetinal-hypoxia is associated with both early (phase I) and proliferative stage (phase II) of ROP, however the precise relationship between its onset, evolution and resolution, to other pathologic events, such as increased expression of proangiogenic growth factors and cytokines and ROP morphometrics (e.g. retinal vessel tortuosity, peripheral avascular area and severity of neovascular tuft formation etc.) is largely unknown (\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e). Therefore, the ability to reliably detect, measure and image retinal hypoxia would offer great advantages to the management of ROP. For example, infants could be screened for retinal hypoxia as a predictor for transition into phase II. Quantification of retinal hypoxia may help to establish ROP severity and could also be used as a benchmark to gauge the efficacy of therapy against neovascular disease. Furthermore, accurate measurement of retinal hypoxia would be of great benefit to the researcher investigating ROP pathogenesis in experimental models of ROP-like disease, leading to a better understanding of the role of hypoxia in ischemic retinopathies and the development of new drugs.\\u003c/p\\u003e\\u003cp\\u003eIn summary hypoxia plays an integral role in ROP pathogenesis and currently its relationship to other events in the ROP pathogenic cascade is largely unknown. An understanding of this relationship would be of great benefit in the management of ROP. Current methods that are routinely used to measure levels of tissue oxygen tension suffer from drawbacks that limit their application to real time \\u003cem\\u003ein vivo\\u003c/em\\u003e imaging of retinal hypoxia. Our newly developed direct imaging method using HYPOX-4 is safe, effective and reliable to detect retinal hypoxia to achieve these goals.\\u003c/p\\u003e\"},{\"header\":\"CONCLUSION\",\"content\":\"\\u003cp\\u003eIn this study, we have characterized the gradient levels of retinal hypoxia in the peripheral avascular retina in rat 50/10 OIR model of ROP. We have utilized HYPOX-4 as a direct method to detect gradient levels of retinal hypoxia in this model. We found that HYPOX-4 is a clinically relevant molecular imaging probe to detect retinal hypoxia and could be used in future studies to improve the diagnosis and treatments for patients with hypoxic retinopathies.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cdiv class=\\\"DefinitionList\\\"\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eBM\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003ebasement membrane\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eCC\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003echoriocapillaris\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eDCP\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003edeep capillary plexus\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eFA\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eFluorescein Angiography\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eGCL\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eganglion cell layer\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eINL\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003einner nuclear layer\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eIPL\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003einner plexiform layer\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eLPS\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eLipopolysaccharide\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eMCP\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003emiddle capillary plexus\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eNV\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eneovascularization\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eOCT\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eoptical coherence tomography\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eONH\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eoptic nerve head\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eONL\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eouter nuclear layer\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eOPL\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eouter plexiform layer\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003ePDR\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eproliferative diabetic retinopathy\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eROP\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eretinopathy of prematurity\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eSCP\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003esuperficial capillary plexus\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eVEGF\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003evascular endothelial growth factor\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eFUNDING\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis research project was supported by grants from the National Institutes of Health (NIH; Bethesda, MD; R01EY029693 to MIU and R01EY023397 to MIU and JSP), a grant from American Diabetes Association, ADA Grant ref #11-22-IBSPM-12 (to MIU), a grant from Carl Marshall Reeves \\u0026amp; Mildred Almen Reeves Foundation, Inc. (Fenton, MO; to MIU), a support provided in part by funding to the Vanderbilt Eye Institute from the International Retinal Research Foundation (Birmingham, AL), and a \\u0026nbsp;grant from Vanderbilt Vision Research Center NEI Core (P30-EY008126) and an unrestricted Grant from Research to Prevent Blindness (RPB). The funding organizations had no role in the design or conduct of this research. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions:\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSJ performed the experiments, collected and analyzed the data. JSP helped revise the manuscript. MIU conceived and supervised the project, designed, and synthesized the compound HYPOX-4, designed and performed experiments, and wrote the manuscript. All authors contributed to the article and approved the submitted version.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eACKNOWLEDGEMENTS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Prof Dr. Gary W. McCollum (Vanderbilt University School of Medicine, Nashville, TN 37232, USA) for helpful discussions in this study. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDISCLOSURES \\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMD Imam Uddin is an inventor on a Patent US10695446B2 (issued) that includes the discovery of HYPOX-4 and its applications. \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAVAILABILITY OF DATA AND MATERIALS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll data that support the results of this study are available within the article or upon request to the corresponding author (MIU). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate:\\u003c/strong\\u003e Not applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication:\\u003c/strong\\u003e Not applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests:\\u003c/strong\\u003e The authors declare that they have no conflicts of interest to report.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eHartnett ME, Penn JS. Mechanisms and Management of Retinopathy of Prematurity. New Engl J Med. 2012;367(26):2515\\u0026ndash;26. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1056/Nejmra1208129\\u003c/span\\u003e\\u003cspan address=\\\"10.1056/Nejmra1208129\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: WOS:000312714200009.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGariano RF, Gardner TW. Retinal angiogenesis in development and disease. Nature. 2005;438(7070):960\\u0026ndash;6. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1038/nature04482\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/nature04482\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: WOS:000233934600055.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSapieha P, Joyal JS, Rivera JC, Kermorvant-Duchemin E, Sennlaub F, Hardy P, Lachapelle P, Chemtob S. Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life. J Clin Invest. 2010;120(9):3022\\u0026ndash;32. PubMed PMID: 20811158; PMCID: PMC2929716.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFevereiro-Martins M, Marques-Neves C, Guimaraes H, Bicho M. Retinopathy of prematurity: A review of pathophysiology and signaling pathways. Surv Ophthalmol. 2023;68(2):175\\u0026ndash;210. PubMed PMID: 36427559.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSelvam S, Kumar T, Fruttiger M. Retinal vasculature development in health and disease. Prog Retin Eye Res. 2018;63:1\\u0026ndash;19. PubMed PMID: 29129724.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePenn JS, Madan A, Caldwell RB, Bartoli M, Caldwell RW, Hartnett ME. Vascular endothelial growth factor in eye disease. Prog Retin Eye Res. 2008;27(4):331\\u0026ndash;71. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1016/j.preteyeres.2008.05.001\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.preteyeres.2008.05.001\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. Epub 2008/07/26.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAlon T, Hemo I, Itin A, Pe'er J, Stone J, Keshet E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med. 1995;1(10):1024\\u0026ndash;8. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1038/nm1095-1024\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/nm1095-1024\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. Epub 1995/10/01.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSapieha P, Hamel D, Shao Z, Rivera JC, Zaniolo K, Joyal JS, Chemtob S. Proliferative retinopathies: angiogenesis that blinds. Int J Biochem Cell Biol. 2010;42(1):5\\u0026ndash;12. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1016/j.biocel.2009.10.006\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.biocel.2009.10.006\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. Epub 2009/10/20.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eHardarson SH, Harris A, Karlsson RA, Halldorsson GH, Kagemann L, Rechtman E, Zoega GM, Eysteinsson T, Benediktsson JA, Thorsteinsson A, Jensen PK, Beach J, Stefansson E. Automatic retinal oximetry. Invest Ophth Vis Sci. 2006;47(11):5011\\u0026ndash;6. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1167/iovs.06-0039\\u003c/span\\u003e\\u003cspan address=\\\"10.1167/iovs.06-0039\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: WOS:000241557500053.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eShahidi M, Shakoor A, Blair NP, Mori M, Shonat RD. A method for chorioretinal oxygen tension measurement. Curr Eye Res. 2006;31(4):357\\u0026ndash;66. PubMed PMID: WOS:000237274400009.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eDai CX, Liu XJ, Zhang HF, Puliafito CA, Jiao SL. Absolute Retinal Blood Flow Measurement With a Dual-Beam Doppler Optical Coherence Tomography. Invest Ophth Vis Sci. 2013;54(13):7998\\u0026ndash;8003. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1167/iovs.13-12318\\u003c/span\\u003e\\u003cspan address=\\\"10.1167/iovs.13-12318\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: WOS:000328133800021.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSoetikno BT, Yi J, Shah R, Liu W, Purta P, Zhang HF, Fawzi AA. Inner retinal oxygen metabolism in the 50/10 oxygen-induced retinopathy model. Sci Rep. 2015;5:16752. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1038/srep16752\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/srep16752\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: 26576731; PMCID: 4649746.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eVaria MA, Calkins-Adams DP, Rinker LH, Kennedy AS, Novotny DB, Fowler WC Jr., Raleigh JA. Pimonidazole: a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in cervical carcinoma. Gynecol Oncol. 1998;71(2):270\\u0026ndash;7. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1006/gyno.1998.5163\\u003c/span\\u003e\\u003cspan address=\\\"10.1006/gyno.1998.5163\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: 9826471.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eUddin MI, Evans SM, Craft JR, Capozzi ME, McCollum GW, Yang R, Marnett LJ, Uddin MJ, Jayagopal A, Penn JS. In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy. Sci Rep. 2016;6:31011. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1038/srep31011\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/srep31011\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. Epub 2016/08/06.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eUddin MI, Jayagopal A, McCollum GW, Yang R, Penn JS. In Vivo Imaging of Retinal Hypoxia Using HYPOX-4-Dependent Fluorescence in a Mouse Model of Laser-Induced Retinal Vein Occlusion (RVO). Invest Ophthalmol Vis Sci. 2017;58(9):3818\\u0026ndash;24. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1167/iovs.16-21187\\u003c/span\\u003e\\u003cspan address=\\\"10.1167/iovs.16-21187\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. Epub 2017/07/28.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eJamal SZ, Dieckmann BW, McCollum GW, Penn JS, Jayagopal A, Imam Uddin MD. Imaging Hypoxia to Predict Primary Neuronal Cell Damage in Branch Retinal Artery Occlusion. Microcirculation. 2024;31(7):e12883. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1111/micc.12883\\u003c/span\\u003e\\u003cspan address=\\\"10.1111/micc.12883\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. Epub 2024/08/31.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eScott A, Fruttiger M. Oxygen-induced retinopathy: a model for vascular pathology in the retina. Eye. 2010;24(3):416\\u0026ndash;21. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1038/eye.2009.306\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/eye.2009.306\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: WOS:000275447200003.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eDi Fiore JM, Kaffashi F, Loparo K, Sattar A, Schluchter M, Foglyano R, Martin RJ, Wilson CG. The relationship between patterns of intermittent hypoxia and retinopathy of prematurity in preterm infants. Pediatr Res. 2012;72(6):606\\u0026ndash;12. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1038/pr.2012.132\\u003c/span\\u003e\\u003cspan address=\\\"10.1038/pr.2012.132\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: 23037873; PMCID: 4433009.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eHodgkiss RJ. Use of 2-nitroimidazoles as bioreductive markers for tumour hypoxia. Anti-Cancer Drug Des. 1998;13(6):687\\u0026ndash;702. PubMed PMID: WOS:000077238500010.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePenn JS, Tolman BL, Henry MM. Oxygen-induced retinopathy in the rat: relationship of retinal nonperfusion to subsequent neovascularization. Invest Ophthalmol Vis Sci. 1994;35(9):3429\\u0026ndash;35. Epub 1994/08/01. PubMed PMID: 8056518.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePenn JS, Henry MM, Tolman BL. Exposure to alternating hypoxia and hyperoxia causes severe proliferative retinopathy in the newborn rat. Pediatr Res. 1994;36(6):724\\u0026ndash;31. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1203/00006450-199412000-00007\\u003c/span\\u003e\\u003cspan address=\\\"10.1203/00006450-199412000-00007\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. Epub 1994/12/01.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePenn JS, Tolman BL, Lowery LA. Variable oxygen exposure causes preretinal neovascularization in the newborn rat. Invest Ophthalmol Vis Sci. 1993;34(3):576\\u0026ndash;85. Epub 1993/03/01. PubMed PMID: 8449677.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMowat FM, Luhmann UF, Smith AJ, Lange C, Duran Y, Harten S, Shukla D, Maxwell PH, Ali RR, Bainbridge JW. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia. PLoS ONE. 2010;5(6):e11103. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1371/journal.pone.0011103\\u003c/span\\u003e\\u003cspan address=\\\"10.1371/journal.pone.0011103\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e. PubMed PMID: 20559438; PMCID: 2885428.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"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\":\"info@researchsquare.com\",\"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\":\"Retinopathy of prematurity, ROP, molecular imaging, retinal hypoxia, HYPOX-4, optical imaging, fluorescence imaging\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7247191/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7247191/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground:\\u003c/strong\\u003e Retinal hypoxia may contribute to the development of preretinal neovascularization in patients with retinopathy of prematurity (ROP). Ciliary bodies compensate oxygen delivery to the retina, and the levels of hypoxia may vary across the peripheral avascular area in ROP. In this study, we have investigated a direct method for imaging gradient levels of retinal hypoxia at the peripheral avascular retina using a model ROP.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethods. \\u003c/strong\\u003eThe rat 50/10 oxygen-induced retinopathy (OIR) model was generated by exposing the newly born Brown-Norway rat pups to a 24 hours alternate cycles of 50% and 10% oxygen for 14 days. We also confirmed the development of neovascularization in this model. HYPOX4 was used as a direct method for imaging gradient levels of retinal hypoxia at the peripheral avascular retina. A separate group of rat OIR pups were used to confirm gradient levels of retinal hypoxia using pimonidazole immunostaining. Gradient levels of retinal hypoxia was analyzed using ImageJ software from fluorescence intensities of HYPOX-4 and Pimonidazole immunostaining.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults\\u003c/strong\\u003e: Retinal hypoxia was observed in the peripheral avascular retinas in rat OIR. Based on fluorescence intensity measurements, retinal hypoxia was at minimal levels near the ciliary bodies. Retinal hypoxia was at its maximum levels towards the avascular-vascular transition zones. Interestingly, we observed hemiretinal avascular retina temporal to the optic nerve in this OIR model, similar to human ROP retinas. In the retinal cross-section, hypoxia was not detectable near the ora serrata in rat OIR may be due to oxygen delivery by the ciliary bodies. Both pimonidazole and HYPOX-4 showed similar patterns of retinal hypoxia at the peripheral avascular retina in this model. As expected, preretinal neovascularization was observed at the avascular-vascular transition zones arising from the existing retinal vascular structures in this OIR model in Brown-Norway rats.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusions\\u003c/strong\\u003e: In this study, we have characterized gradient levels of retinal hypoxia in the rat model of 50/10 OIR using a direct method from HYPOX-4 fluorescence. We observed minimal levels of retinal hypoxia near the ciliary bodies in this model and increased towards the avascular-vascular transition zones. In addition, we observed that the central vascularized retina remains gradient hypoxic in this model which could be detected using HYPOX-4. This study may clarify our understanding of retinal hypoxia in the ROP patient at the peripheral retinas.\\u003c/p\\u003e\",\"manuscriptTitle\":\"A direct method for imaging gradient levels of retinal hypoxia in a model of retinopathy of prematurity (ROP)\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-08-21 16:12:13\",\"doi\":\"10.21203/rs.3.rs-7247191/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"4639ddbe-037f-45ed-a57d-cf1191242edb\",\"owner\":[],\"postedDate\":\"August 21st, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-10-07T00:07:12+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-08-21 16:12:13\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7247191\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7247191\",\"identity\":\"rs-7247191\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}