Ouabain-Sensitive Na-K Atpase Response in the Rabbit Iris-Ciliary Body After Lensectomy-Vitrectomy

Ouabain-Sensitive Na-K ATPase Response in the Rabbit Iris-Ciliary Body After Lensectomy-Vitrectomy

Takezo Mito, Sei-ichi Ishiguro, and Makoto Tomai

We studied ouabain-sensitive Na-K adenosine triphosphatase (ATPase) activities in the iris-ciliary body of rabbit eyes after lensectomy-vitrectomy. Changes in enzyme activities were quantitatively investigated in the plasma membrane of iris-ciliary body at 0 or 7 hours and at days 1,3,7, and 14. The specific activity of Na-K ATPase rose to significantly higher levels than the control value at 7 hours following surgery, but returned to the baseline value after 7 days. In addition, we evaluated enzyme

activities after lensectomy-vitrectomy during which SF6 or silicone oil was injected. The specific activity of Na-K ATPase following the injection of SF6 or silicone oil was significantly higher than the control value at 7 hours and did not return to the normal value even after 14 days. Consensual reaction, demonstrated by increased Na-K ATPase activity, also was found in the contralateral unoperated eyes

of SF6- and silicone oil-injected rabbits. The increased Na-K ATPase activity in the iris-ciliary body after experimental surgery may play an important role in restoring swollen tissues. Invest Ophthalmol Vis Sci 33:172-177,1992

Ouabain-sensitive Na-K adenosine triphosphatase lateral unoperated eyes after surgery. Moreover, we (ATPase) may contribute to the regulation of cell vol- compared those findings with the results of lensec- ume and the maintenance and regulation of the intra- tomy-vitrectomy during which SF6 or silicone oil was cellular and extracellular ionic environment.1 Further- injected. more, it has been proposed that the secretion of aqueous humor results from active Na transport by Materials and Methods Na-K ATPase.2 The ciliary body contains a high con- centration of ouabain-sensitive Na-K ATPase,3 and Domestic albino rabbits, weighing about 2 kg each, the enzyme has been localized on the plasma mem- were obtained from a local breeder and were housed brane of ciliary body epithelial cells histochemi- under the same conditions. Rabbits were anesthetized cally.4"10 with an intravenous dose of xylazine and ketamine Mechanical trauma such as paracentesis causes hydrochloride. A lensectomy-vitrectomy was performed using the Peyman Vitrophage 9000 in the breakdown of the blood-aqueous barrier in rabbits'' 14 1213 12 13 right eye, as described by Cottingham and Forster and primates. Bartels et al and Okisaka showed 15 that the ciliary body, particularly in the anterior pars and Kaplan and coworkers. Ringer's lactate plus so- plicata region, is a source of secondary aqueous after lution (Ringer's lactate supplemented with 5.0 raM glucose and 18.3 mM bicarbonate) was used during paracentesis. 16 19 The iris-ciliary body also is affected during vitreous the operation. " After the lensectomy-vitrectomy, surgery by mechanical trauma, prolonged intraocular 30% SF6/fluid and silicone oil/air exchanges were per- irrigation, and various methods of tamponade. In this formed. Air was passed through a cellulose nitrate study, we evaluated changes in the iris-ciliary body filter (pore size = 0.02 /im; Toyo Roshi, Tokyo). Sili- through the biochemically measured activity of Na-K cone oil (1000 centistokes, specific gravity 0.98) was ATPase after experimental lensectomy-vitrectomy. purchased from Koken Co., Tokyo. Erythromycin We found increased specific activity of Na-K ATPase ointment was applied topically to the eye immedi- activity in the iris-ciliary body of operated and contra- ately afterward. Postoperative examinations were made at 1 hour and at 1, 3, and 14 days. Eyes were excluded from analysis if they had hemorrhage, reti- From the Department of Ophthalmology, Tohoku University nal detachment, or glaucoma postoperatively. The School of Medicine, Sendai 980, Japan. left eye served as the untreated control for each ani- Submitted for publication: November 20, 1990; accepted July mal. The care and treatment of animals in this inves- 25, 1991. Reprint requests: Sei-ichi Ishiguro, Department of Ophthalmol- tigation were in compliance with the ARVO Resolu- ogy, Tohoku University School of Medicine, Sendai, Miyagi 980, tion on the Use of Animals in Research. Japan. The time course of the biochemical study was mea-

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E 10.0-

Fig. 1. Specific activity of Na-K ATPase in the final pellet of iris-ciliary body after surgery in experimental (closed squares) and control (open squares) eyes. Values indicate mean 5.0- ± SD (n = 4). *P < 0.01 (paired t-test).

1 0- I 14 days

sured from completion of the operation: 0 and 7 Chifflet et al.22 For the assay, the incubation time was hours, and 1, 3, 7, and 14 days. Rabbits were killed no more than 30 min, which was in a linear range. with a lethal injection of sodium pentobarbital at each The specific activity of Na-K ATPase was expressed indicated time. The eyes were rapidly enucleated and in IJM inorganic phosphate liberated/mg protein/ hemisected posterior to the ora serrata. The ciliary hour. Ouabain was used in parallel samples, so data body and iris were dissected from the anterior half. shown reflect differences between Na-K ATPase activ- Ciliary body and iris were homogenized, as previously ity measured ± ouabain in the incubation. described by Mittag et al,20 in 0.32 M sucrose-contain- For light microscopy, the enucleated eyeballs were ing histidine (30 mM) and EGTA (1 mM) with use of fixed with periodate-lysine-paraformaldehyde23 and Biotron's homogenizer, and centrifuged at 1,000 X g were embedded in paraffin. The 3-/im-thick sections for 10 min. The supernatant was decanted and recen- were sliced and stained with hematoxylin-eosin. trifuged at 12,000 X g for 60 min. The pellet was resuspended with 0.1% deoxycholate in 0.25 M sucrose Results and the extract was centrifuged at 100,000 X g for 60 min. The final pellet was resuspended in water. Pro- Figure 1 shows the specific activities of Na-K ATP- tein determinations were done by the method of ase. Immediately (0 hour) following surgery, the Na- 21 Lowry et al. K ATPase activity corresponded to the control value. The Na-K ATPase assay was performed at 37°C at At 7 hours after surgery, the activity was 2.3 times pH 7.5, according to the methods of Mittag et al20 and higher than the control value. On days 1 and 3, the

«> 10.0-

Fig. 2. The effect of SF6 (open squares) and silicone oil (open circles) on specific activities of Na-K ATPase in the final pellet of iris- ciliary body was compared with that of vitrectomy (Ringer's lactate plus solution, closed squares) alone. Values indicate mean ± SD (n = 4). *P < 0.01; **P < 0.05 (paired t-test).

0J 0 7h 1 14 days

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Fig. 3. Na-K ATPase response in the contralateral, unoperated eyes after lensectomy-vi- 1 0- trectomy. Enzyme preparation is described in Materials and Methods. Iris-ciliary body in the contralateral unoperated eyes of lensectomy-vitrectomy alone (Ringer's lactate plus solution, open squares) shows no consensual o « reaction. Conversely, consensual reaction, 2 6- demonstrated by increased Na-K ATPase specific activity in the iris-ciliary body, is observed in the contralateral unoperated eyes of (0 *-* 4 - silicone oil- (closed squares) and SF6-injected (open circles) rabbits compared with those that underwent lensectomy-vitrectomy alone. 2 - Prolonged increase of the specific activity in the contralateral eyes of SF6-injected rabbits is observed. Values indicate mean ± SD (n = 4). J 0 *P < 0.01; **P < 0.05 (paired t-test). DAYS 1 4

activities were 1.5 times higher than the control value. 4c). Conversely, when SF6 injection (Fig. 4d) or sili- However, after day 7, activity decreased and corre- cone oil injection (Fig. 4e) was performed, the ciliary sponded to the control value. body remained edematous even after day 14. No swell- The results of specific activities of Na-K ATPase on ing was observed in the ciliary body of the contralat- the effect of SF6 and silicone oil are summarized in eral eyes on day 14 after SF6 injection (Fig. 4f) nor in Figure 2. In the beginning, the specific activity of Na- the iris on day 1 after lensectomy-vitrectomy alone K ATPase after the injection of SF6 or silicone oil (Fig. 4g). corresponded to the specific activities of Ringer's lactate plus solution. However, these increased specific Discussion activities of the enzyme did not return to the control value even after 14 days. In the rabbit, the lens is disproportionately large, Consensual reaction, that is, increased Na-K ATP- and we found it technically impossible to perform a ase specific activity, was observed in the iris-ciliary pars plicata vitrectomy without simultaneously re- body of contralateral unoperated eyes (Fig. 3). The moving the lens. Therefore, a lensectomy was in- specific activity in unoperated eyes of silicone oil-in- cluded in our experiment.1424 Technically, removing jected rabbits was significantly higher than that of residual lens capsule and cortex completely near the Ringer's lactate plus solution-substituted rabbits on ciliary body using the Peyman Vitrophage 9000 also day 1 (P < 0.01). Thereafter, it returned to the basal was difficult. These remaining substances seemed to level. On the other hand, prolonged increase of the be stimuli for cellular infiltration of the iris-ciliary specific activity in the contralateral eyes of SF6-in- body after vitreous surgery. jected rabbits was observed 7 hours (P < 0.01), 3 days The biochemical results of this experiment revealed (P < 0.05), 7 days (P < 0.05), and 14 days (P < 0.01) remarkable changes in ouabain-sensitive Na-K ATP- after surgery. ase activity in the rabbit iris-ciliary body, which was Light microscopy showed an extensive edema and examined at several time intervals during the 2 weeks vessel dilation of the ciliary body at day 1 after lensec- after the surgical procedure. tomy-vitrectomy (Fig. 4a). A single layer of pig- Two results confirmed that the preparation method mented ciliary epithelia was observed in a tip of ante- of Mittag et al20 was reasonable for assessing Na-K rior pars plicata of the ciliary body (Fig. 4b). At day ATPase response in the iris-ciliary body. First, we re- 14, the edema and vessel dilation were decreased (Fig. covered more than 85% of Na-K ATPase activity in

Fig. 4. Photomicrographs of the ciliary body and the iris after vitreous surgery, (a) Extensive edema (*) and vessel dilation of the ciliary body are noted on day 1 after lensectomy-vitrectomy. (b) A photomicrograph at higher magnification than in (a) shows a single layer of pigmented epithelial cells (arrowhead) in a tip of anterior pars plicata region on day 1 after surgery. Dark, smaller, and slightly flat nuclei of pigmented epithelial cells can be seen, (c) On day 14, edema (*) and vessel dilation of the ciliary body are decreased, (d) The ciliary body after injection of SF6 is still edematous (*) on day 14. (e) The ciliary body after injection of silicone oil is still edematous (*) on day 14. (f) The ciliary body of the contralateral eyes on day 14 after SF6 injection demonstrates no swelling in the ciliary processes, (g) A photomicrograph of the iris on day 1 after lensectomy-vitrectomy discloses no appreciable swelling. PC: posterior chamber. Bars = 100 /xm.

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the final pellet from crude homogenate. Second, we of vitreous surgery through the breakdown of blood- removed blood cells through anterior ciliary arteries aqueous barrier and resulting serum protein leakage by a perfusion technique with homogenization buffer and edema. Berggren28-29 observed that the swollen using enucleated, unoperated eyes and compared the ciliary processes in vitro gradually shrunk during the specific activity of the final pellet from the iris-ciliary course of an experiment, but that the shrinkage was body with that of nonperfused eyes. We found no sig- inhibited by ouabain. The mechanism of the Na-K nificant difference in specific activity between per- ATPase response after lensectomy-vitrectomy is un- fused and nonperfused pellets. We concluded, there- known, but increased Na-K ATPase activity in the fore, that with this method blood cells did not affect iris-ciliary body may play an important role in the specific activity of Na-K ATPase of iris-ciliary body. recovery of edematous tissues. In the present study, we obtained markedly in- Miyake et al30 reported significant disruption of the creased Na-K ATPase activity in iris-ciliary body. If blood-aqueous barrier in the contralateral eyes of pa- there were some contamination of red blood cells, the tients undergoing cataract extraction and lens im- specific activity of Na-K ATPase in iris-ciliary body plantation surgery. They demonstrated that topical should have been decreased because the specific activ- indomethacin effectively inhibited the disruption of ity of red blood cells is markedly lower than that of the barrier in the surgically treated eyes but did not iris-ciliary body.3 Thus, it seems unlikely that we stifle the reaction in the contralateral eyes. Kottow overestimated the Na-K ATPase response in iris-cil- and Seligman31 also demonstrated that the consen- iary body. sual reaction to paracentesis in the rabbit eye was

As SF6 and silicone oil are widely used intraopera- more efficiently inhibited by nerve-blocking agents tively as vitreous substitutes, we determined the ef- than by the prostaglandin inhibitor, aspirin. We fects of these materials on the same enzyme activities. showed increased Na-K ATPase activity in the contra- Even at postoperative day 14, the specific activities of lateral unoperated eyes of silicone oil- and SF6-in- Na-K ATPase were not restored. The delayed return jected rabbits (Fig. 3). The mechanism of the consen- to control values may indicate that extensive damage sual Na-K ATPase response also is unknown, but the occurred to the iris-ciliary body after the injection of response is still useful for assessing the effects of vitre- SF6 or silicone oil. ous substitutes. Our results show that the increased Durlu et al19 showed that markers of Miiller cells Na-K ATPase activity in the contralateral unoperated associated with glycogenolysis and gluconeogenesis, eyes of silicone oil-injected rabbits remains until day 7 glutamate-glutamine cycle, and cytoskeletal protein after surgery but that the activity in the contralateral metabolism were affected by the experimental lensec- eyes of SF6-injected rabbits is still high on day 14. tomy-vitrectomy. In these experiments, the widely These findings indicate that vitreous substitute SF6 used vitreous substitute, silicone oil, did not apprecia- stimulates more iris-ciliary body edema and Na-K bly change these enzyme activities in retinal Miiller ATPase activity than silicone oil. cells when compared postoperatively with Ringer's We concluded that ouabain-sensitive Na-K ATP- lactate plus solution. However, in the present study, ase activities of iris-ciliary body showed a prominent we found that silicone oil and SF6 apparently affected time-dependent response that might be related to envi- Na-K ATPase activity in the iris-ciliary body. These ronmental changes of iris-ciliary body after experi- substitutes are not inert for the iris-ciliary body cells. mental lensectomy-vitrectomy and may play an im- Prostaglandins have been implicated in the irrita- portant role in restoring the edematous tissues. tive response after mechanical trauma to the eye, re- Key words: experimental vitrectomy, iris-ciliary body, Na- sulting in miosis, vasodilation, increased protein lev- K ATPase, SF , silicone oil, consensual reaction els in the aqueous, and increased intraocular pres- 6 sure.2 Prostaglandins applied topically to the eye Acknowledgments result in a breakdown of the tight junctions of nonpigmented epithelium.2526 Breakdown of the blood- The authors thank Ms. Maxine A. Gere for her helpful aqueous barrier occurs after paracentesis of the ante- review of this manuscript. rior chamber.""13 Fragmentation of the tight junctions, particularly in the anterior pars plicata region of References the ciliary body, occurs after paracentesis, with subse- 1. Stryer L: Membrane transport. In Biochemistry, 3rd ed. New quent leakage of plasma protein into the aqueous hu- York, WH Freeman and Company, 1988, pp. 949-955. mor.12-27 In the present study, we observed the swollen 2. Caprioli J: The ciliary epithelia and aqueous humor. In Adler's Physiology of the Eye, 8th ed., Moses RA and Hart WM Jr, anterior pars plicata of the ciliary body after lensec- editors. St. Louis, The CV Mosby Company, 1987, pp. 204- tomy-vitrectomy. Substances such as prostaglandins 222. probably affect iris-ciliary body by an irritative effect 3. Bonting SL, Simon KA, and Hawkins NM: Studies on sodium-

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