Post-Treatment at 12 or 18 Hours with 3-Aminobenzamide Ameliorates Retinal Ischemia–Reperfusion Damage

Abstract

purpose. The window of protection afforded by 3-aminobenzamide (3-ABA), a poly-(ADP-ribose) polymerase (PARP) inhibitor, against apoptotic loss of inner retinal elements after ischemia–reperfusion insult in rats was examined.

methods. Ischemia–reperfusion injury to the retinas in albino Lewis rats was induced by elevated intraocular pressure (IOP) through cannulation of the anterior chamber with a needle connected to a saline column delivering a pressure of 110 mm Hg. The ischemic period was held at 60 minutes, and reperfusion was established immediately afterward. 3-Aminobenzamide (3-ABA) was administered intravitreally at 0, 4, 8, 12, 18, or 24 hours after reperfusion and its effect evaluated by morphology and morphometry of the inner retinas at 7 days after reperfusion. Immunohistochemistry of poly-(ADP-ribose), a product of PARP activity, and Western blot analysis for PARP were performed on retinas at 0, 4, 8, 12, 18, and 24 hours after reperfusion.

results. Morphology and morphometry showed significantly better preserved inner retinas in animals receiving 3-ABA between 12 and 18 hours after reperfusion. Immunohistochemical study of poly-(ADP-ribose) showed elevated levels at the retinal ganglion cell layer and the inner nuclear layer at 12 and 18 hours after reperfusion. Western blot analysis of PARP showed a notable increase in the 116-kDa band (PARP) from 4 to 18 hours after reperfusion.

conclusions. Administration of 3-ABA at 12 or 18 hours after ischemia, when there was accumulation of poly-(ADP-ribose) in the inner retina, significantly ameliorated retinal ischemia–reperfusion injury. These findings, together with earlier reports from our laboratory, are consistent with a late and pivotal role of PARP in apoptotic loss of inner retinal elements after ischemia–reperfusion insult to the retina.

Poly-(ADP-ribose) polymerase (PARP), a 116-kDa nuclear enzyme activated by single-strand DNA breaks and implicated in DNA repair, ¹ may modulate a variety of enzyme functions and activities by poly(ADP)-ribosylating nuclear histones, ² enzymes including PARP itself, ³ and nuclear matrix proteins using nicotine adenine dinucleotide (NAD). ³ Kaufmann ⁴ first demonstrated the cleavage of PARP during apoptosis. PARP was later demonstrated to be one of the substrates for caspase 3, an analogue of Ced-3 and a key enzyme in apoptosis, suggesting a likely role of PARP in apoptosis. ⁵ It has been postulated that in excitotoxic death of neurons and other systems, activation of PARP may play a pivotal role by depleting the cell of adenosine triphosphate as it uses NAD, resulting in cell death. ⁶  

Inhibitor studies have shown contradictory results, with some showing a beneficial effect in ameliorating cell loss, ^{7 8} and others showing exacerbation of cell death and loss. ⁹ It is possible that the role of PARP may be different in different tissues and with different triggering signals. On the contrary, PARP knockout mouse studies have shown that PARP may not be essential for apoptosis. ^{10 11} In ischemia–reperfusion injury to the heart, PARP has been shown to regulate the levels of extracellular matrix P-selectin and intercellular adhesion molecule-1, ¹² proteins known to modulate the inflammatory response and therefore tissue damage and the injury outcome. In short, the exact role(s) of PARP in cell death and apoptosis remains unclear. 

Previously, we demonstrated apoptosis in retinal ischemia–reperfusion injury by histologic and ultrastructural criteria, detection of internucleosomal DNA fragmentation at 12 and 18 hours after reperfusion, and in situ TdT-mediated dUTP-biotin nick-end labeling (TUNEL) of cells at the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) between 8 and 18 hours after reperfusion. ^{13 14} A protective effect by YVAD, an inhibitor of caspases, when administered between 0 and 4 hours, and an elevated immunoreactivity of caspases 3 in the same period after reperfusion indicate a possible involvement of caspases in the early phase of the apoptotic process. ¹⁴ We also reported a dose-dependent ameliorative effect of 3-aminobenzamide (3-ABA), an inhibitor of PARP, on ischemia–reperfusion injury to the retina and that 3-ABA works by inhibiting the apoptotic pathway. ¹⁵ It is not clear whether PARP is similar to caspases that are involved in the early phase of the apoptotic process. To further examine the role of PARP in apoptotic death of inner retinal elements in retinal ischemia–reperfusion, we studied the window of protection by 3-ABA treatment under the same retinal reperfusion–ischemia injury conditions as in previous studies and performed in situ immunohistochemistry of poly-(ADP ribose), the product of PARP activity, and Western blot analysis with an antibody to the C terminus of PARP at various times after reperfusion. 

Materials and Methods

Induction of Retinal Ischemia by Elevated Intraocular Pressure

A previously published procedure to generate an ischemia–reperfusion insult in rats was used. ¹⁴ Briefly, male albino Lewis rats (55–60 days of age) were anesthetized with intraperitoneal injections of chloral hydrate (400 mg/kg). After instillation of 0.5% proparacaine hydrochloride, the anterior chamber was cannulated with a 25-gauge needle connected to a normal saline reservoir. The intraocular pressure (IOP) in the experimental eyes was controlled by the height of the saline reservoir to maintain a pressure of 110 mm Hg for 60 minutes. Retinal ischemia was confirmed by the whitening of the anterior segment of the eye and the blanching of retinal arteries by ophthalmoscopic examination during the experimental period. At the end of the experiment, the needle was removed, and reperfusion of the retinal vasculature was confirmed by ophthalmoscopic examination. Tobramycin ointment was applied at the end of the procedure for prophylaxis. Groups of animals were killed at 0, 4, 8, 12, 18, or 24 hours or 7 days after reperfusion. All use and handling of the animals adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and procedures were approved by the institutional Animal Ethics Committee. 

Immunohistochemistry and Western Blot Analysis of PARP

The enucleated eyes were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C. The anterior segments of the eyes were removed. The posterior segments were processed and embedded in paraffin. Immunostaining of poly-(ADP-ribose) was then performed using a standard immunohistochemistry protocol on paraffin sections with a 1:200 dilution of anti-poly(ADP-ribose) from a poly (ADP-ribose) detection kit (Trevigen, Gaithersburg, MD). 

Retinas were isolated from animals and lysed for 30 minutes in ice-cold 0.1-M phosphate-buffered saline (PBS) with 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 μl/ml aprotinin, and 1 mM sodium orthovanadate (all from Sigma, St. Louis, MO). The suspension was carefully homogenized with a loose pestle, followed by sonication. Total cell lysate was obtained by centrifugation at 8000g for 30 minutes at 4°C. Lysates in 20 μg protein per lane were separated in 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a nitrocellulose membrane. The membrane was hybridized with PARP antibody (SC-7150; Santa Cruz Biotechnology, CA) against the carboxyl-terminus of PARP. Positive binding was visualized by Western blot luminol reagent (SC-2048; Santa Cruz Biotechnology). An identical gel and blot were performed with aliquots of the lysates, with an antibody to actin (A-4700; Sigma) serving as the control. 

Administration of 3-ABA

An aliquot of 2 μl 100 μM 3-ABA in 0.1 M PBS (pH 7.4) was injected intravitreally at 0, 4, 8, 12, 18, or 24 hours after reperfusion. The same volume of 0.1 M PBS was administered similarly to animals after reperfusion as a control. The animals were killed 7 days after reperfusion, and their eyes were processed for morphologic and morphometric evaluations. 

Morphologic and Morphometric Studies

The enucleated eyes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4°C. The anterior segments were removed, and the eyecups were divided into four strips: one each from the nasal, inferior, temporal and superior quadrants. The tissue samples were than osmificated, dehydrated, and embedded in epoxy resin for sectioning into 1-μm sections for light microscopy and morphometry of inner retinal thickness (IRT). 

Ischemia–reperfusion’s effect on the inner retinal layers was evaluated quantitatively by measuring the thickness between the inner limiting membrane to the interface of the outer plexiform layer (OPL) and the outer nuclear layer (ONL) in Epon-embedded sections, as described previously. ¹⁴ Measurements were performed using an image processing workstation (Leica, Deerfield, IL) and taken from the posterior pole to the ora serrata, excluding the optic nerve area. Readings from all available quadrants of the same eye were averaged to obtain the value in one eye. 

Results

Figure 1 is a composite micrograph showing sections from similar regions of the normal and the ischemic-reperfused retinas at 7 days of reperfusion with 3-ABA administered at various times after the initial insult. Compared with the normal retina (Fig. 1A) , the ischemic-reperfused retina without 3-ABA treatment (treated with PBS vehicle; Fig. 1B ) showed typical inner retinal losses: disappearance of cells from the RGCL, and thinning of the inner plexiform layer (IPL) and the inner nuclear layer (INL). The OPL was vacuolated. Retinas treated with 3-ABA at 0, 4, 8, or 12 hours showed changes similar to the vehicle-treated ones except a slightly better preserved INL with 3-ABA treatment administered at 4 (Fig. 1C) , 8 (not shown), or 12 hours (Fig. 1D) after ischemia. With treatment at 18 hours, the retina showed a notably better preserved RGCL, IPL, and INL (Fig. 1E) . Retinas with intravitreal injection of 3-ABA at 24 hours (Fig. 1F) were similar to those treated at 12 hours, showing slightly better preserved IPL and INL. 

Figure 2 depicts the effects of 3-ABA administration at various times after reperfusion on IRT of the ischemic-reperfused retinas measured at 7 days after reperfusion. In the ischemic-reperfused retinas, intravitreal injection of 3-ABA (200 micromoles) at 0, 4, 8, or 24 hours after reperfusion showed slight but nonsignificant ameliorative effect. However, significant ameliorative effects on IRT were noted when 3-ABA was administered at 12 or 18 hours (P < 0.05, Tukey’s test) after reperfusion. 

Immunohistochemistry of poly-(ADP-ribose), the reaction product of PARP, showed positive immunoreactivity in cells in the RGCL and INL (especially the inner part of the INL) at 12 (Fig. 3C ) and 18 (Fig. 3D) hours after reperfusion. Other than those two time points, the normal retina (Fig. 3A) and the retinas from earlier (Figs. 3B 4H) or later time points (not shown) showed no immunoreactivity. Western blot analysis (Fig. 4) showed elevated levels of PARP protein (116 kDa) at 4 to 18 hours after reperfusion, with an antibody to the C terminus of PARP (Fig. 4) . Control blot actin showed no change in protein levels (Fig. 4) . 

Discussion

Previously, we demonstrated the involvement of apoptosis and caspases in retinal ischemia–reperfusion injury. ¹⁴ In a separate study, we reported a dose-dependent ameliorative effect of 3-ABA, a PARP inhibitor, on apoptotic loss of inner retinal elements after ischemia–reperfusion injury to the retina. ¹⁵ In the same report, we demonstrated that the ameliorative effect of 3-ABA is inhibition of apoptosis. In this study, we explored the window of protection of 3-ABA after the initial insult and showed that it is most effective after treatment at 12 or 18 hours. In addition, this window of protection corresponds to a transient elevation of poly-(ADP-ribose), an indication of PARP activity, in retinas after the ischemia–reperfusion injury. These results indicate an elevated activity of PARP together with elevated PARP protein levels after ischemia–reperfusion injury to the retina. In addition, these observations, together with findings in our earlier studies, ^{14 15} are supportive of a pivotal, downstream postcaspase role of PARP in apoptotic loss of inner retinal element after ischemia–reperfusion injury to the retina and the feasibility of exploring the large window of treatment with apoptotic modulators in retinal neuronal death. 

Our earlier study shows a dose-dependent effect of 3-ABA on ischemia–reperfusion insult to the retina when 3-ABA is infused into the retina during the ischemic period, ¹⁵ whereas in this report, a single intravitreal injection was used. Therefore, the actual amount of 3-ABA delivered to the vitreous and retina would be significantly higher in the previous study than the present one. It follows that the absence of significant effect by morphometry when 3-ABA was administered at 0, 4, or 8 hours after reperfusion in the present study may have been due to pharmacodynamic and/or pharmacokinetic factors, such as differences in the amount of 3-ABA and/or clearance from retina, resulting in minimal effect. 

Results of other studies suggest that during apoptosis, caspase 3 activates endogenous PARP by proteolytic action generating an 89-kDa (C terminus) and a 24-kDa (N terminus) fragment. ⁴ The activation of caspases is believed to be an irreversible step of apoptosis. Our immunohistochemical detection of poly-(ADP-ribose) indicates an elevated activity of PARP in selected cells of the inner retina at 12 and 18 hours after the ischemia–reperfusion insult. This time course of elevated activity corresponds to that of internucleosomal DNA degradation as determined by the characteristic appearance of ladder in DNA analysis showing a maximum at 18 hours after ischemic insult. ¹⁴ Our Western blot analysis showed a notable level of PARP in the normal retina, together with a low enzyme activity but an elevated level of the parent PARP with no 89-kDa fragment after the injury. The increased protein levels of the parent 116-kDa PARP after the insult suggests either an increased synthesis or decreased degradation of PARP. It is not clear whether our observation is unique in retina. Further studies into the transcription, translation, activation, and fragmentation of PARP are needed to understand its roles in apoptotic death of retinal cells. 

Similar to our caspase inhibitor study, ¹⁴ this report further confirms the feasibility of using apoptotic modulators to modify tissue responses to ischemia–reperfusion insult or other pathologic conditions of retina involving apoptosis, such as glaucoma. In addition, PARP inhibitors appear to provide an even larger window of protection, in that PARP appears closer to the later steps such as internucleosomal fragmentation in the apoptotic pathway in retinal ischemia–reperfusion injury. 

 Supported by Research Grant Council grant CUHK295/96M and Direct Grant 2040693 from the Medicine Panel, Chinese University of Hong Kong.

 Submitted for publication June 17, 1999; revised December 14, 1999 and April 24, 2000; accepted April 27, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Retinas at 7 days of reperfusion with 3-ABA administered at various times after ischemia. (A) Normal retina, (B) ischemic-reperfused retina without 3-ABA treatment, and retinas treated with 3-ABA at (C) 4 hours, (D) 12 hours, (E) 18 hours, and (F) 24 hours. Note better preserved retinas when 3-ABA was administered at 12 (D), 18 (E), and 24 (F) hours. Toluidine blue. Scale bar, 20 μm.

Figure 2.

 

Morphometry of average IRT between the inner limiting membrane and the OPL 7 days after reperfusion. Note significant improvement in IRT with the administration of 3-ABA at 12 or 18 hours (*P < 0.05, Tukey’s test) after ischemia.

Figure 3.

 

Immunoreactivity of poly-(ADP-ribose) in rat retinas after reperfusion. (A) Normal retina, (B) 4 hours, (C) 12 hours, and (D) 18 hours after reperfusion. Positive immunoreactivity (arrows) was noted in the RGCL and the INL at 12 (C) and 18 (D) hours after reperfusion. Scale bar, 20 μm.

Figure 4.

 

Western blot analysis of total cell lysates from rat retinas at various time after reperfusion using antibody to the C terminus of PARP. Top left to right: standard (std), normal (N) retina, and retinas at 0, 4, 8, 12, and 18 hours after reperfusion. Note the significant increase of the 116-kDa band at 4 to 18 hours. Actin showed no change in protein levels.