The Role of Opioid Growth Factor in Enhancing the Success of Trabeculectomy
Open Access
- Author:
- Klocek, Matthew Stephen
- Graduate Program:
- Anatomy
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 02, 2009
- Committee Members:
- Ian Stuart Zagon, Dissertation Advisor/Co-Advisor
Ian Stuart Zagon, Committee Member
Patricia J Mc Laughlin, Committee Chair/Co-Chair
Joseph W Sassani, Committee Member
H Paul Ehrlich, Committee Member
Alphonse E Leure Dupree, Committee Member - Keywords:
- Opioid Growth Factor
Glaucoma
Wound Healing - Abstract:
- Over 2.2 million people have glaucoma in the U.S. Trabeculectomy surgery is commonly used to prevent complications of glaucoma, and the success of the trabeculectomy depends on the reaction of Tenon's Capsule Fibroblasts (TCFs) to the surgical trauma. The studies depicted in this thesis were aimed at establishing the role of OGF and OGFr in the modulation of cell proliferation of rabbit TCFs (RTCFs) in vitro as well as in vivo. RTCFs were isolated and established in culture. The presence of opioid growth factor (OGF) and its receptor (OGFr) was determined by immunoreactivity. The kinetics of OGFr were established in receptor binding assays. The ability of OGF to inhibit RTCF proliferation was assessed with dose response growth curves. Receptor-mediated effects of OGF were ascertained by treatment with OGF and naloxone (NAL). The toxicity of OGF action was examined in reversibility experiments. Interference of OGF-OGFr interaction by opioid antagonists or OGF antibody was conducted. The mechanism of action of the OGF/OGFr axis on the survival of RTCFs was evaluated with siRNA, BrdU, and TUNEL techniques. Treating RTCFs with OGF over a 10 million-fold range of dosages revealed that OGF works to inhibit proliferation of RTCFs in a dose dependent manner. Through receptor binding assays, OGF was seen to work in a one-site model of binding. When treating RTCFs with the long-acting opioid antagonist, naltrexone (NTX), RTCFs proliferated at a much higher rate than control or OGF treated cells. Cell counts of RTCFs treated with mitomycin-C (MMC) were significantly lower than control, OGF, and NTX treatment groups. MMC and MMC/OGF treated RTCFs did not differ in regard to cell number. The unique ability of OGF to alter cell proliferation was not evident when RTCFs were treated with an array of traditional opioids. OGF works in a receptor-mediated manner. When treated with the short-acting opioid antagonist naloxone, RTCF cell numbers were not significantly elevated compared to controls or RTCFs treated with OGF and naloxone simultaneously. Specificity of OGF was determined through treatment of RTCFs with an antibody to OGF. Cell counts of antibody treated RTCFs were significantly higher than control counterparts, thus indicating that endogenous OGF plays an integral role in the regulation of cell proliferation. Reversibility experiments showed that the inhibition of cell proliferation characteristic to OGF is not permanent. This is an important factor in demonstrating the non-toxic inhibitory effects of OGF compared to other anti-proliferative therapies. OGFr siRNA experiments, demonstrated that OGFr is necessary for OGF to inhibit RTCF proliferation. OGF also decreases DNA synthesis in RTCFs compared to NTX, MMC, and control groups. MMC did not alter DNA synthesis. OGF and NTX did not induce apoptosis as evident through caspase 3 and TUNEL assays indicting that both compounds displayed non-toxic characteristics. MMC treated RTCFs had significantly higher caspase 3 activity and TUNEL labeling when compared to control, OGF, and NTX treatment groups. TUNEL labeling revealed that MMC/OGF treated RTCFs possessed significantly less TUNEL labeling compared to MMC alone. This decrease in late apoptotic activity suggests that OGF has some protective effects. In the in vivo studies, a modified Scheie procedure was performed on New Zealand White (NZW) rabbits. Animals were treated intraoperatively with PBS (control), Hydron + OGF, MMC, or Hydron alone. Intraocular pressure was measured daily by a tonometer. Daily measurements of bleb height, area, and vascularity were assessed via a slit lamp microscope. H&E, trichrome, Giemsa, and Sirius Red staining were used to assess cellularity, blood vessels, inflammatory cells, and collagen fiber orientation. Cell proliferation and apoptosis assays were also performed. Filtering blebs of the Hydron + OGF group survived more than twice as longer as controls, while also maintaining a reduced IOP for a significantly longer period of time. Filtering blebs of Hydron + OGF, and MMC animals had comparable survival times. Bleb height and area of the MMC and Hydron + OGF treated animals were comparable throughout the study. IOPs of surviving blebs did not differ between any treatment group, while vascularity of control, Hydron, and Hydron + OGF group was significantly increased compared to MMC. Histopathology evaluation revealed that Hydron and Hydron + OGF specimens had developed exuberant granulation tissue inside of the surgical bleb at their respective late time points, while the MMC specimen did not. Also, the Hydron + OGF specimen had less small and large collagen fibers within the bleb area compared to the Hydron specimen. DNA synthesis was markedly increased in the Hydron specimen in the areas of the scleral fistula and Tenon’s capsule compared to control, MMC, and Hydron + OGF groups, whereas the Hydron + OGF specimen had significantly less DNA synthesis than all other treatment groups in the scleral fistula and Tenon’s capsule areas. MMC specimens had an increased amount of TUNEL labeling in the scleral fistula and conjunctiva areas compared to control, Hydron + OGF, and Hydron groups. This finding illustrates that treatment with MMC results in more toxic side effects than OGF, which has been documented to be safe and non-toxic. In summary these data indicate that OGF is effective at inhibiting RTCFs in a safe and non-toxic manner. However, an optimal vehicle for OGF administration is needed to improve delivery of OGF, and thus optimize the inhibitory effects of OGF in vivo.