Dysregulation of the OGF–OGFr pathway correlates with elevated serum OGF and ocular surface complications in the diabetic rat

Abstract

<jats:p> Diabetes often presents with ocular surface complications including dry eye, keratopathy, and altered sensitivity, along with systemic disorders. A common theme associated with corneal surface defects is decreased cellular proliferation. The opioid growth factor (OGF)–OGF receptor (OGFr) regulatory axis maintains epithelial homeostasis and can be modulated by naltrexone, an opioid receptor antagonist, to block OGF–OGFr interaction and increase cellular replication. Complete blockade using naltrexone accelerates cell proliferation, increases the rate of re-epithelialization in corneal surface abrasions, reverses dry eye, and restores corneal surface sensitivity in animal models of type 1 and type 2 diabetes. Data on the efficacy of naltrexone in these models suggest that the OGF–OGFr axis is dysregulated in diabetes. In the present study, we investigated the OGF–OGFr axis by assessing serum and tissue levels of OGF and OGFr during the development of streptozotocin-induced hyperglycemia and postulated a mechanism of action. We correlated the dysregulation of the OGF–OGFr axis with the onset and magnitude of corneal surface complications (e.g. tear fluid production, corneal surface sensitivity) in type 1 diabetes (T1D). Serum levels of OGF increased in both uncontrolled T1D and insulin-controlled (T1D-INS) male rats within four weeks of streptozotocin injection. Serum OGFr levels were significantly reduced in diabetic rats on weeks 3 and 8 post streptozotocin. Tear production was significantly reduced, and corneal sensitivity measurements were abnormal in both T1D and T1D-INS animals within four weeks of streptozotocin. Corneal re-epithelialization was delayed in T1D rats, but not in T1D-INS animals; however, expression levels of the inhibitory growth factor OGF and its receptor, OGFr, were elevated in the corneal epithelium more than 2-fold in both diabetic groups. These data demonstrate for the first time that dysregulation of the OGF–OGFr axis in the diabetic cornea is associated with the onset and magnitude of ocular surface complications. </jats:p><jats:sec><jats:title>Impact statement</jats:title><jats:p> This research extends our knowledge about the presence and role of the OGF–OGFr regulatory axis in type 1 diabetes (T1D) and demonstrates specific targets within the pathway that are dysregulated. Serum levels of OGF, an inhibitory growth factor, are significantly elevated in male T1D rats, and OGFr serum values are increased in T1D. The onset of elevated OGF corresponds to the onset of ocular surface complications including dry eye, delayed corneal epithelial repair, and abnormal corneal surface sensitivity in T1D. Systemic insulin does not protect against elevated OGF levels or the onset of dry eye and sensitivity. These data are the first to associate some ocular surface defects in T1D with alterations in the OGF–OGFr pathway. </jats:p></jats:sec>

Blockade of OGFr delays the onset and reduces the severity of diabetic ocular surface complications

“…However, when the changes in OGF expression following topical application are viewed in concert with serum-based effects of systemic NTX, there is strong evidence that OGF expression is correlated with ocular surface defects. As discussed earlier, 22 systemic insulin does not prevent or protect against dry eye and altered corneal surface sensitivity in T1D, whereas the OGF-OGFr pathway appears to have a significant role. In this study, it was demonstrated that two different methods of establishing receptor blockade of OGF from interaction with OGFr resulted in some level of protection from the ocular surface complications of diabetes.…”

“…Body weights and blood glucose levels were monitored periodically throughout the eight-week period. 22 Multiple cohorts of animals were required for systemic and topical NTX studies, and at least 8–15 rats were included in each treatment group. Any rat that did not convert to hyperglycemia within four days following STZ injection was removed from the study.…”

“…The methodology for induction of hyperglycemia followed published procedures. 14,19,22 Briefly, six-week-old fasted male Sprague-Dawley rats (Charles River) were injected intraperitoneally (i.p.) with 55 mg/kg streptozotocin (STZ; Sigma-Aldrich) on two consecutive days.…”

“…Blood was collected at four and eight weeks and isolated serum was stored at À80 C. OGF and OGFr protein expression levels were determined by immunohistochemistry. Corneal epithelial histologic sections were stained with validated antibodies using published procedures 22 ; sections stained with secondary antibody only were negative controls. Positive controls included ocular tissue from earlier investigations.…”

Ocular surface complications result from dysregulation of the OGF‑OGFr signaling pathway in female diabetic rats

“…The eyes were removed from the orbital cavity, processed and immunohistochemically stained with OGF (Penn State University, 1:200) and OGFr (MyBioSource MBS2124207, 1:150) antibodies as described previously ( 16 ). Frozen corneal sections (6-8 µm) from each group were stained following previously published protocols ( 7 , 11 , 12 , 16 ); control sections were stained with the secondary antibody only (Alexa Flour568; Invitrogen; Thermo Fisher Scientific, Inc.). Stained sections were imaged using a Keyence Scope (KEYENCE, Ltd.) and further quantified using ImageJ 1.8.0_172 (National Institutes of Health) to quantify optical density (OD).…”