Echothiophate-Induced Structural Alterations in the Anterior Chamber Angle of the Cynomolgus Monkey
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Echothiophate-induced structural alterations in the anterior chamber angle of the cynomolgus monkey Elke Liitjen-Drecoll and Paul L. Kaufman Four cynomolgus monkeys were treated topically twice daily in one eye with echothiophate iodide (PI) doses of 63, 75, or 250 fjig per treatment for 7.7 weeks to 7 months. The opposite eyes of two monkeys received a control solution (diluent). The anterior ocular segments of all six eyes were studied by light and transmission electron microscopy during treatment. In the Pl-treated eyes, the cribriform and outer corneoscleral meshwork were unusually dense. The trabecular meshwork was collapsed and the lamellae showed thickened basement membranes and thickened sheaths of elastic-like material. Most endothelial cells were enlarged and activated. Some contained many glycogen particles; others showed evidence of degeneration. The crib- riform meshwork contained much more extracellular fine fibrillar material than normal, and the endothelium of the inner wall of Schlemm's canal was damaged. The Pi-contracted ciliary muscle had a more rectangular shape than normally contracted muscles, and the inner edge extended so far anteriorly that in some areas it overlapped and occluded the trabecular meshwork. The muscle cells appeared damaged, and their basement membranes were thickened. The nonpigmented epithelial cells of the ciliary processes showed signs of degenera- tion, and within the pars plana some contained large, weakly osmiophilic inclusions. The basement membrane of the ciliary epithelium was thickened everywhere. The stromal vessels of the pars plana were dilated, and signs of inflammation were present. The sphincter iridis was damaged and there were iridocorneal adhesions. The 5 month diluent-treated eye demonstrated mild structural abnormalities in the meshwork. Key words: angle-closure glaucoma, anterior chamber angle, canal of Schlemm, cholinesterase inhibitors, ciliary muscle, ciliary processes, echothiophate, iris, monkey eye, trabecular meshwork Long-acting cholinesterase (ChE) inhibitors From the Department of Anatomy, University of are applied topically to the human eye for the Erlangen-Niirnberg, Erlangen, West Germany; the treatment of some cases of glaucoma. These Department of Medical Pharmacology, University of drugs lower intraocular pressure (IOP) at Uppsala, Uppsala, Sweden; and the Department of least initially by increasing the facility of Ophthalmology, University of Wisconsin Medical aqueous humor outflow. Although ChE in- School, Madison, Wise. Supported by grants from the Deutsche Forschungsge- hibitors may provide long-term lowering of meinschaft, Bad Godesberg (Ro 81/14, to Prof. J. W. IOP in many cases, some eyes eventually be- Rohen) and the National Institutes of Health (EY00231, come refractory to these drugs.1 A possible IF03 EY55678, 5 SOI RR05435). reason for this is decreased sensitivity of Submitted for publication Aug. 15, 1978. outflow facility to cholinomimetic com- Reprint requests: Dr. Elke Liitjen-Drecoll, Anatom- 2 isches Institut der Universitat Erlangen-Niirnberg, pounds, presumably due to acetylcholine- 8520 Erlangen, Krankenhausstrasse 9, West Germany. induced cholinergic subsensitivity of the 918 0146-0404/79/090918 + 12$01.20/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc. Downloaded from iovs.arvojournals.org on 09/26/2021 Volume 18 Number 9 Echothiophate-induced angle changes 919 A IP 4, -20 © © © © © -40 PV PV AV AV g r -30 c < 300 ^ ® PI 2.5 % m -12 m z ©DILUENT H -20^ NO CATARACT »1 ® DILUENT O NO CATARACT 10 5 > © PI 0.5% CYNO 110 CYNO 225 35 DAYS DAYS 200- © © © © © © •40 C PV PV PV PV PV iA A A >- I 50- 1 1 1 I 30 < O © PI 0.5% -o ANIRIDIC CYNO 280 70 105 140 210 DAYS Fig. 1. Treatment protocols. Control eyes (110 R and 225 L) received same volume of diluent, containing NaCl in place of PI, as fellow experimental eyes. Cynomolgi (CYNO) 110 and 225 received virtually all treatments while fully conscious; cynomolgus 280 received alternate treatments under CI-744 anesthesia for the first 3 months. Aniridic, Iris totally removed8 previously; R, right eye; L, left eye; j, slit-lamp examination; P, posterior subcapsular lens opacities; A, anterior subcapsular opacities; V, lens vacuoles; ±, questionable lens opacities; C, clear lens; f, procedure or event other than slit-lamp examination (refraction not indicated); Eu enucleation following death by intracardiac pentobarbital; E2, enucleation in vivo under pentobarbital anesthesia. ciliary muscle.2 4 We report here another sodium phosphate, mannitol, and potassium ace- possible reason for loss of the IOP-lowering tate necessary to give concentrations equal to effect of ChE inhibitor treatment: ChE those in the corresponding PI solution. The os- inhibitor-induced structural alterations in the molality of the diluent was adjusted to approxi- anterior chamber angle. mate that of the corresponding PI solution by add- ing sodium chloride (1.8 or 9.0 mg/ml). The 0.5% Materials and methods PI solution had pH = 6.39 and osmolality = 435 Animals. Four adolescent or young adult cyno- mOsm; the corresponding diluent had pH = 6.30 molgus monkeys (Macaca fascicularis), weighing and osmolality = 425 mOsm. The 2.5% PI so- 1.8 to 2.4 kg, were studied. lution had pH = 6.97 and osmolality = 1900 Drugs. Ophthalmic PI was purchased com- mOsm; the corresponding diluent had pH = 6.91 mercially. To the drug powder was added the ac- and osmolality = 1820 mOsm. companying diluent to yield either a 0.5% or a Topical drug treatment. The eyes were treated 2.5% solution of echothiophate iodide (PI). Dilu- topically twice daily on weekdays (7:15 to 7:45 ent was made by adding to distilled water the A.M. and 4:15 to 4:45 P.M.) and once daily on quantities of boric acid, chlorobutanol, dibasic weekends (9:00 to 9:30 A.M.) with either PI or Downloaded from iovs.arvojournals.org on 09/26/2021 Invest. Ophthalmol. Visual Set. 920 Liitjen-Drecoll and Kaufman September 1979 ¥ K . Fig, 2. For legend see facing page. Downloaded from iovs.arvojournals.org on 09/26/2021 Volume 18 Number 9 Echothiophate-induced angle changes 921 diluent. The conscious animal was removed from treated with 1% periodic acid for 5 min to demon- its cage and restrained supine by a gloved assis- strate glycogen particles.10 tant. The upper lid was retracted digitally, and Results with a specially prepared microdropper, a 2.5 /xl drop of drug solution was applied to the central The morphological appearances of the PI cornea every 20 to 30 sec until the desired drug and diluent-treated eyes were compared with dose had been administered. Blinking was pre- each other and with the appearances of a large vented between drops and for 20 to 30 sec after number of non-PI-, non-fZi/«en£-treated the last drop. The lids were then wiped dry and cynomolgus eyes.11"15 Total iridectomy8 itself the animal returned to its cage. Treatments under caused no abnormalities visible by LM or general anesthesia were given in a similar manner, TEM in the ciliary muscle, trabecular but without assistance. Separate droppers were meshwork, or Schlemm's canal.8' 15 used for each of the four drug solutions. PI-treated eyes Slit-lamp examination. This was performed pe- Trabecular meshwork and Schlemm's canal riodically on the anesthetized monkey.5'6 Anesthesia. General anesthesia for topical 54 DAYS PI. The cribriform meshwork adja- treatments and slit-lamp examination was by in- cent to Schlemm's canal was thicker and tramuscular CI-744 (Tilazol; Parke-Davis), 5 to 10 more dense than normal (Fig. 2, B). The mg/kg.7 cribriform endothelial cells were enlarged Fig. 1 gives additional details of treatment and and exhibited enlarged nuclei, prominent enucleation. Treatment was started with smaller nucleoli, and large amounts of rough surfaced doses (same solutions, fewer drops), and the dos- endoplasmic reticulum. Beneath the inner age was gradually increased over several weeks to wall endothelium and within the cribriform 2 minimize the risk of systemic toxicity. meshwork there was more fine fibrillar ma- Tissue processing terial than normal (Fig. 3, B). In the Cynomolgus 275. Immediately following enu- corneoscleral and uveal meshwork, the tra- cleation, postequatorial scleral windows were cut, and the eye was immersed in phosphate-buflfered becular endothelial cells were also activated. (pH 7.2) 2.5% glutaraldehyde for 1 hr. The eye 138 AND 155 DAYS PI. The changes were in was then opened equatorially, the posterior half general similar to those seen after 54 days but discarded, and the anterior half cut into small were much more pronounced. In some re- sagittal wedges. The wedges were processed for gions there was so much fine fibrillar material light microscopy (LM) and transmission electron that the inner canal wall was separated from 9 microscopy (TEM). the first subendothelial cell layer (Figs. 2, C, Cynomolgi 110, 225, and 280. Immediately fol- and 3, C). In the inner portion of the lowing enucleation, the eye was opened equato- corneoscleral meshwork and in the uveal rially, and the posterior half discarded. Under an meshwork, some of the trabecular endothe- operating microscope, the zonule was cut all lial cells were degenerating (evidenced by around with a fine Vannas scissors, with care being taken not to touch the lens or ciliary processes. swollen mitochondria and vacuolization The lens was lifted free by a stainless steel ring and within the cytoplasm), and in some places a glass rod and processed for other studies. Small they were separated from the trabecular sagittal wedges of the anterior ocular segment beams. However, in the subopercular12 re- were then cut and processed for LM and TEM.9 gion of the anterior part of the meshwork Some ultrathin sections from each eye were there were clusters of activated cells, perhaps Fig. 2. Trabecular meshwork and Schlemm's canal. A, Monkey 225 R, 54 days diluent.