Abstract
Objective—To assess the intraoperative and postoperative clinical effects and histologic effects of intracameral administration of α-chymotrypsin in clinically normal dogs undergoing standard intracapsular lens extraction (ICLE).
Animals—6 young adult male dogs without evidence of systemic or ocular disease.
Procedures—All dogs underwent bilateral ICLE 7 minutes following injection of 75 U of α-chymotrypsin or an identical volume (0.5 mL) of a commercially available balanced saline solution (BSS) into the posterior chamber of the eye. Ease of lens extraction was subjectively assessed and intraoperative intraocular hemorrhage and fibrin accumulation scored. For 27 days after surgery, ocular hyperemia and discharge, chemosis, corneal edema, hyphema, and aqueous flare were scored, and intraocular pressure (IOP) was measured. Thirty days after surgery, histologic evidence of anterior synechia, collapse of and inflammation within the iridocorneal angle, and iritis were scored.
Results—In 5 of 6 dogs, the surgeon was able to correctly identify the eye treated with α-chymotrypsin on the basis of ease of lens extraction. Mean intraoperative intraocular hemorrhage and fibrin scores for BSS-treated eyes were significantly higher than for α-chymotrypsin-treated eyes. Postoperatively, there were no significant differences between treatments for any clinical variables, including IOP Histologic scores were not significantly different between treatments for any variable. Vision was lost as a result of glaucoma in 1 α-chymotrypsin-treated eye and 1 BSS-treated eye.
Conclusions and Clinical Relevance—Intracameral administration of 75 U of α-chymotrypsin 7 minutes before ICLE facilitated lensectomy without apparent adverse effects in clinically normal dogs.
The canine lens is suspended from the ciliary body by a complex of lens zonules. Rupture of this suspensory apparatus leads to lens dislocation, which may be further classified as partial (lens subluxation) or complete (lens luxation), anterior or posterior (depending on whether the lens is displaced in front of or behind the iris, respectively), and primary or secondary Primary lens dislocation is a result of a presumed heritable weakness of the zonular fibers1,2 and has been described in many dog breeds.3–9 By contrast, secondary lens dislocation may be seen in any breed and is typically a sequela to uveitis, glaucoma, cataract formation, or intraocular neoplasia.10,11 Regardless of the primary insult, a dislocated lens frequently causes secondary intraocular diseases, the most painful and vision-threatening of which is glaucoma.10,12 Although medical management delays anterior lens dislocation in eyes with lens instability miotic treatment does not prolong time to onset of glaucoma or time to loss of vision in eyes with an unstable lens.13 This finding, along with reported outcomes of ICLE, has led to recommendations for urgent removal of anteriorly luxated lenses before secondary glaucoma ensues and for prompt removal of subluxated or posteriorly luxated lenses before they become anteriorly luxated.7,14–16 The preferred surgical technique for patients with lens dislocation is ICLE; however, this procedure can be associated with intraoperative or postoperative intraocular hemorrhage and fibrin formation, uveitis, vitreous prolapse, and retinal detachment, especially when some zonular fibers remain attached to the lens capsule, as is the case with lens subluxation. Therefore, a safe means of inducing zonulolysis would be an important adjunct to ICLE and expected to improve the prognosis following this procedure.
α-Chymotrypsin is an endopeptidase with proteolytic action similar to that of trypsin but with a narrow specificity of action.17 Because of its relatively specific dissolution of lens zonules, α-chymotrypsin was used widely during the 1960s and 1970s as an adjunct to ICLE in humans with cataracts. Typically, the enzyme was injected into the posterior chamber (the space between the iris and the lens) to ensure adequate access to the lens zonules. Beginning in the late 1960s, phacoemulsification largely replaced ICLE for treatment of cataracts, and reports of the use of α-chymotrypsin in humans became infrequent. However, as recently as 1995, α-chymotrypsin was recommended as an inexpensive and socially responsible means for treatment of people who were blind as a result of cataracts but who did not have access to phacoemolsification.18 Most of the 20 million people in the world who are blind as a result of cataracts live outside of developed countries.19
Zonulolysis with α-chymotrypsin has also been assessed as a sole or adjunctive treatment for cataracts in dogs.16,20–25 Initial failures led authors to propose that canine lens zonules were poorly lysed by this enzyme because of presumed resistance of the canine zonules to lysis23–26 or differences in anatomy of the suspensory apparatus in dogs23,25,27 relative to that in humans. This led to the use of doses of α-chymotrypsin in dogs up to 17-fold higher than had been used in humans.20,21,23–25,28,29 Unfortunately, many of these experiments were associated with unacceptable intraocular pathological changes, including elevated IOP, iridocyclitis, retinal detachment, retinal degeneration, and optic nerve degeneration.21,28,29 However, closer review of these studies suggests that there may have been other factors that led to such toxic effects. Some investigators injected the drug into ocular compartments other than the posterior chamber, such as the anterior chamber23,24 or vitreous body21,28 where contact with the lens zonules may have been inadequate and access to the retina, cornea, iris, and ciliary body may have been enhanced. Others used doses up to 17-fold higher than those used in humans,23 and many used α-chymotrypsin as a sole means of inducing lens luxation rather than as an adjunctive measure to aid ICLE.21,23,24,28–30 Therefore, we hypothesized that doses of α-chymotrypsin necessary to weaken the lens zonules would be lower than those required to cause lens luxation. We tested this hypothesis by use of a linear actuator and force transducer in enucleated canine eyes and determined that as little as 75 U of α-chymotrypsin allowed to remain in contact with the zonules for 7 minutes significantly reduced (by 58%) the force required for lens extraction.31 On the basis of these ex vivo data, we hypothesized that α-chymotrypsin could be used in the same manner to facilitate ICLE in clinically normal dogs without adverse intraocular effects. The study reported here was designed to test that hypothesis. Specifically, the purpose of the study reported here was to assess intraoperative and postoperative clinical effects and histologic effects of intracameral administration of α-chymotrypsin in clinically normal dogs undergoing ICLE.
Materials and Methods
Animals—Six young adult male hound-type dogs that ranged in body weight from 14.8 to 17.2 kg were assessed to be free of systemic and ocular disease on the basis of results of complete physical and ophthalmic examinations. Before surgery, ophthalmic examination was conducted on all dogs between 6 am and 8 am on the same day (baseline values). This examination consisted of assessment of direct and consensual pupillary light reflexes and menace response, an STT, and complete globe and adnexal evaluation by use of diffuse illumination with a Finoff transilluminator, slit-lamp biomicroscopy, applanation tonometrya gonioscopy, and, after pupil dilation, binocular indirect ophthalmoscopy. All aspects of the study were approved by the Institutional Animal Care and Use Committee of the University of Missouri, Columbia.
Surgical procedures—Bleeding tendency was assessed before surgery on the basis of results of a CBC (including platelet count), prothrombin time, activated partial thromboplastin time, and buccal mucosal bleeding time. On the day of surgery, all eyes were treated by administration of 1 drop of tropicamide ophthalmic solution every 15 minutes until adequate pupil dilation was detected (4 to 7 applications) and 1 drop of neomycin-polymyxin B-dexamethasone ophthalmic suspension every 30 minutes (2 to 4 applications); each dog also was administered flunixin meglumine (0.66 mg/kg, IV). Each dog was premedicated by IM administration of glycopyrrolate (0.01 mg/kg), morphine (0.4 to 0.5 mg/kg), and acepromazine maleate (0.02 to 0.03 mg/kg). Anesthesia was induced with thiopental (administered to effect) and maintained with isoflurane in oxygen. Neuromuscular blockade was induced with atracurium (0.2 mg/kg, IV). Dogs were mechanically ventilated during neuromuscular blockade by use of a time-cycled, volume-limited ventilator. Settings were adjusted to maintain end-tidal partial pressure of carbon dioxide between 36 and 40 mm Hg, peak inspiratory pressure between 15 and 22 cm H2O, and respiratory rate between 8 and 12 breaths/min. Spontaneous ventilation was allowed to resume after recovery from neuromuscular blockade, which was determined by use of a peripheral nerve stimulator placed over the tibial nerve.
Standard intracapsular lens cryoextraction was performed in all dogs through a 160° clear corneal incision.32 One eye from each dog was assigned via a randomization procedure (coin toss) to receive 75 U (0.5 mL) of α-chymotrypsin.b In accordance with the manufacturer's recommendations, the α-chymotrypsin was diluted with 0.9% NaCl solution to a final dilution of 1:5,000 immediately before injection. The α-chymotrypsin was infused via a 27-gauge irrigation cannula placed through a small stab incision in the cornea and directed throughout a 360° arc immediately posterior to the iris leaflet. The α-chymotrypsin was allowed to incubate in this location for 7 minutes prior to further globe manipulation. The contralateral eye was treated in an identical manner but with infusion of 0.5 mL of sterile BSS.c Following incubation with α-chymotrypsin or BSS for 7 minutes, the corneal incision was extended to a length of approximately 3.2 mm by use of a slit blade, and the anterior chamber was filled with viscoelastic. Although eyes were selected randomly for α-chymotrypsin treatment, the surgical procedures were always performed first on the right eye of each dog. All eyes received an intracameral infusion of approximately 0.1 mL of epinephrine (dilution, 1:10,000) during ICLE to maintain pupil dilation and reduce intraocular hemorrhage. When the anterior vitreous could not be gently swept from the posterior lens capsule with closed Westcott scissors, or when vitreous extruded through the corneal incision during closure, manual vitrectomy was performed by use of a cellulose spear and Westcott tenotomy scissors. The minimum amount of vitreous was resected to facilitate closure of the incision. All eyes were reformed by infusion of viscoelastic prior to closure of the incision, and corneal incisions were closed with 8-polyglactin in a simple continuous suture pattern. Sutures were not removed. All surgeries were performed by 1 investigator (MPN) who was not aware of the treatment assignment of each eye. Intraoperative assessment with respect to intraocular hemorrhage and fibrin formation, as well as ease of lens extraction, was recorded. After surgery, all eyes were treated twice daily by administration of 1 drop of neomycin-polymyxin B-dexamethasone ophthalmic suspension.
Data collection—Regular ophthalmic examinations consisting of evaluation by use of diffuse illumination, slit-lamp biomicroscopy, indirect binocular ophthalmoscopy, and applanation tonometry were performed by 1 investigator (RMM) who was unaware of the treatment for each eye. Examinations were performed approximately 3, 6, 18, and 27 hours after surgery and then at least twice weekly for 27 days after surgery. The IOP was measured, and ocular discharge, ocular hyperemia, chemosis, corneal edema, hyphema, and aqueous flare were scored by use of published scales (Appendix).33,34
All dogs were euthanatized 30 days after ICLE. Both eyes were immediately enucleated, placed in Davidson's fixative, and routinely processed for histologic examination. The left eye was always enucleated first. All histologic sections were assessed by 1 investigator (RMM) who was not aware of the treatment for each eye. General histologic observations were recorded for all eyes examined. In addition, anterior synechia, mononuclear cell and protein accumulation within the ICA, iritis, and PIFM formation were all scored on a semiquantitative scale (0 = absent, 1 = mild, 2 = moderate, and 3 = severe). Collapse of the ICA was recorded as present or absent in the sections examined.
Statistical analysis—Baseline IOP values and STT results were compared between eyes treated with intracameral administration of α-chymotrypsin and those infused with BSS and between left and right eyes by use of a paired t test. Incidence of intraoperative intraocular hemorrhage and fibrin formation and the need for vitrectomy was compared between groups via the Fisher exact test. Severity of intraoperative intraocular hemorrhage and fibrin formation was compared between groups by use of the Mann-Whitney rank sum test. A repeated-measures ANOVA was used to assess the effect of treatment, time, and the treatment-by-time interaction for all clinical data (IOP, chemosis, ocular hyperemia, ocular discharge, corneal edema, hyphema, and aqueous flare). A paired t test was used to compare scores for histologic lesions (anterior synechia, mononuclear cell infiltration and protein accumulation in the ICA, iritis, and PIFM formation) between treatments. The McNemar test for paired binary data was used to compare the number of eyes with collapse of the ICA between treatments. All normally distributed data were expressed as mean ± SD; data with a nonnormal distribution were expressed as the median. For all analyses, a value of P < 0.05 was considered significant.
Results
Baseline results for general physical and ophthalmic examinations as well as results of a CBC, platelet count, prothrombin time, activated partial thromboplastin time, and buccal mucosal bleeding time revealed only 4 abnormalities. In 1 eye of each of 2 dogs, the STT result was low (10 and 14 mm in 1 minute, respectively). Both of these eyes received intracameral administration of BSS. In 1 dog, the baseline IOP was 30 mm Hg in the right eye and 24 mm Hg in the left eye; the right eye received BSS, and the left eye received α-chymotrypsin. Randomization resulted in intracameral administration of α-chymotrypsin in the left eye of 4 dogs and the right eye of 2 dogs.
Mean ± SD IOP at baseline did not differ significantly (P = 0.8) between right (19.2 ± 5.6 mm Hg) and left (19.5 ± 2.6 mm Hg) eyes or between eyes subsequently treated with α-chymotrypsin (19.5 ± 2.5 mm Hg) or BSS (19.2 ± 5.6 mm Hg). Similarly mean ± SD STT results at baseline did not differ significantly between right (17.7 ± 4.4 mm in 1 minute) and left (17.8 ± 3.8 mm in 1 minute) eyes (P = 0.9) or between eyes subsequently treated with α-chymotrypsin (18.2 ± 3.4 mm in 1 minute) or BSS (17.3 ± 4.6 in 1 minute [P = 0.8]).
No anesthetic complications were recorded for any dog. Surgical time (defined as the interval from induction to extubation) ranged from 63 to 117 minutes. In 5 of 6 dogs, the surgeon correctly identified the eye treated with α-chymotrypsin on the basis of the increased ease with which the lens was extracted. Intraoperative intraocular hemorrhage was rated on a scale from 0 to 3 as absent (2 α-chymotrypsin-treated and 0 BSS-treated eyes), mild (4 α-chymotrypsin-treated and 2 BSS-treated eyes), moderate (0 α-chymotrypsin-treated and 3 BSS-treated eyes), or severe (0 α-chymotrypsin-treated and 1 BSS-treated eye). The median intraoperative intraocular hemorrhage score was significantly (P = 0.026) higher for BSS-treated eyes (score = 2) than for α-chymotrypsin-treated eyes (score = 1). Intraoperative intraocular fibrin formation was rated on a scale from 0 to 3 as absent (3 α-chymotrypsin-treated and 0 BSS-treated eyes), mild (3 α-chymotrypsin-treated and 2 BSS-treated eyes), moderate (0 α-chymotrypsin-treated and 3 BSS-treated eyes), or severe (0 α-chymotrypsin-treated and 1 BSS-treated eye). The mean intraoperative intraocular fibrin score was significantly (P = 0.006) higher for BSS-treated eyes (score =1.8) than for α-chymotrypsin-treated eyes (score = 0.5). Anterior manual vitrectomy was required in 1 α-chymotrypsin-treated and 3 BSS-treated eyes. There was no significant difference between the treatment groups with respect to incidence of intraoperative intraocular hemorrhage (P = 0.46), fibrin formation (P = 0.18), or frequency of vitrectomy (P = 0.55).
After surgery, there was a significant increase (P < 0.001) in IOP of all eyes, irrespective of treatment group (Figure 1). For all 12 eyes, the mean ± SD peak IOP was 42.9 ± 12.8 mm Hg and was detected 5 to 7 hours after surgery. However, at no time did the mean IOP of eyes treated with α-chymotrypsin differ significantly (P = 0.5) from that of eyes treated with BSS. Mean scores for all clinical markers of ocular inflammation (chemosis, ocular hyperemia, ocular discharge, corneal edema, hyphema, and aqueous flare) also increased significantly (P < 0.001) after surgery; however, no significant (P = 0.27 to 0.83) difference was detected between α-chymotrypsin-treated and BSS-treated eyes for any variable (Figure 2). Other clinical lesions detected during the postoperative period but not scored were a suture line staphyloma with subsequent buphthalmos, corneal vascularization, and phthisis in 2 eyes (1 α-chymotrypsin-treated and 1 BSS-treated eye) and dyscoria in 3 eyes (1 α-chymotrypsin-treated eye and 2 BSS-treated eyes).
Mean ± SD IOP before (time 0) and at various times for the first 27 hours after ICLE (A) and for days 1 through 27 after ICLE (B) in 12 eyes of 6 dogs treated by intracameral administration of 75 U of α-chymotrypsin (black circles) or an identical volume (0.5 mL) of BSS (white circles) 7 minutes prior to lensectomy. In each dog, 1 eye was assigned via a randomization procedure to receive α-chymotrypsin, and the other eye received BSS.
Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1475
Mean ± SD scores for ocular hyperemia (A), chemosis (B), ocular discharge (C), corneal edema (D), hyphema (E), and aqueous flare (F) before (time 0) and at various times for 27 days after ICLE in 12 eyes of 6 dogs treated by intracameral administration of 75 U of α-chymotrypsin (black circles) or an identical volume of BSS (white circles) 7 minutes prior to lensectomy. In each dog, 1 eye was assigned via a randomization procedure to receive α-chymotrypsin, and the other eye received BSS. See Appendix for scoring system.
Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1475
At the termination of the study, 2 of 12 eyes (1 α-chymotrypsin-treated right eye and 1 BSS-treated right eye) were judged to have lost vision on the basis of ophthalmic examination findings and lack of a menace response. The clinical and histologic diagnosis for vision loss in both eyes was glaucomatous retinopathy and optic neuropathy with retinal detachment. The BSS-treated eye that became glaucomatous was the eye with the elevated IOP (30 mm Hg) at baseline. All eyes had histologically detectable anterior synechia at the incision site; however, this varied in magnitude among eyes. Collapse of the adjacent ICA was seen less commonly (2 α-chymotrypsin–treated and 3 BSS-treated eyes). Mononuclear cells were detected in the ICA of all but 2 α-chymotrypsin-treated and 1 BSS-treated eye. By contrast, proteinaceous debris in the ICA was an uncommon finding and was evident in only 3 eyes, all of which were treated with BSS. Iritis of varying degrees accompanied by PIFM formation was detected in all eyes. All retinas, except for the 2 with glaucomatous degeneration, were histologically normal, with the inner nuclear layer having a thickness of 3 to 4 cells and the outer nuclear layer having a thickness of 10 to 14 cells. No evidence of drug-related toxicosis was detected in any ocular tissue examined. No significant (P = 0.08 to 1.0) difference in mean histologic scores was detected between α-chymotrypsin-treated and BSS-treated eyes for any variable assessed (Figure 3). However, protein was not detected in the anterior chamber of BSS-treated eyes, whereas it was detected in α-chymotrypsin-treated eyes. This difference may have been significant if a larger number of eyes were assessed. No significant (P = 0.56) difference was detected between the number of α-chymotrypsin-treated (n = 2) or BSS-treated (3) eyes in which collapse of the ICA was detected.
Mean ± SD histologic score for ocular conditions in tissue samples obtained 30 days after ICLE in 12 eyes of 6 dogs treated by intracameral administration of 75 U of α-chymotrypsin (black bars) or an identical volume of BSS (white bars) 7 minutes prior to lensectomy. In each dog, 1 eye was assigned via a randomization procedure to receive α-chymotrypsin, and the other eye received BSS. Scores were assigned by use of the following scale: 0 = absent, 1 = mild, 2 = moderate, and 3 = severe.
Citation: American Journal of Veterinary Research 71, 12; 10.2460/ajvr.71.12.1475
Discussion
Analysis of data from the study reported here revealed that administration of 75 U of α-chymotrypsin into the posterior chamber of clinically normal dogs for 7 minutes greatly facilitated ICLE without apparent adverse effects. Although this outcome provides in vivo verification of our findings obtained by use of enucleated dog eyes,31 it contrasts with results of earlier in vivo studies that suggested toxicosis21,24,29 or unreliable efficacy.23,24,26–29 There are many possible explanations for the lack of adverse effects in the present study relative to other studies. First, all of the previous studies, except for one,29 were performed prior to 1967. Since that time, there have been substantial improvements in surgical instruments and techniques, perioperative pharmaceuticals (most notably, mydriatic, anti-inflammatory antimicrobial, and viscoelastic agents), and anesthetic equipment and techniques (most notably, neuromuscular blockade). These improvements were all used in the present study and may have reduced the cumulative magnitude or number of insults on the eyes or protected them from otherwise adverse effects of α-chymotrypsin. Additionally, studies to date have involved the use of remarkably variable delivery methods for α-chymotrypsin. These have included administration of α-chymotrypsin into the posterior chamber at 420,23 or an unstated number17,22,25,29 of sites, directly onto the zonules in an eye devoid of aqueous humor,23,24,30 into the anterior chamber,23,24 intravitreally21,28 or via an unstated route.24 Subsequently, irrigation of the α-chymotrypsin was performed in some studies22,24,25 but not in others.20,23,24,28–30 In the present study, we administered α-chymotrypsin into the posterior chamber through a full 360° without subsequent irrigation of the enzyme to ensure that it contacted as many zonules as possible, as directly as possible, but with minimal contact with other potentially vulnerable intraocular structures. Finally, in other studies,20,22–25,29,30 the α-chymotrypsin dose (when identified) ranged from 150 to 2,500 U with incubation times of up to 10 minutes, whereas in the study reported here, we infused 75 U of α-chymotrypsin with a 7-minute incubation period. Assuming that toxicosis is related to delivered drug dose and contact time, this may explain the lack of adverse effects that we detected in the present study.
Although α-chymotrypsin is relatively specific with respect to the amino acid bonds that it cleaves, these bonds are commonly found throughout ocular tissues; thus, the specificity and safety of this enzyme are only relative.17,35 This is likely to explain some of the adverse effects attributed to intraocular administration of α-chymotrypsin. Eyes of dogs receiving intraocularly administered α-chymotrypsin via various routes, at various doses, and for various incubation times have developed a variety of clinical, functional, and histologic adverse effects, sometimes in the absence of desired lens dislocation. These have included iridocyclitis, ocular hypertension and glaucoma, vitritis, vitreous hemorrhage and liquefaction, retinal detachment, and retinal and optic nerve degeneration.21,28,29 Although many of these signs were evident to some degree in the present study, none were more severe in α-chymotrypsin-treated eyes than in BSS-treated eyes, which suggested that the regimen for α-chymotrypsin administration used in the present study was safe. However, these data should be interpreted conservatively because our inability to detect a significant difference may have been a result of a small sample size for both treatment groups.
Of the signs commonly ascribed to α-chymotrypsin administration in dogs, perhaps the best understood is ocular hypertension, which also has been detected in humans,36 primates,37 and rabbits.38 In fact, ocular hypertension is so reliably induced by intraocular administration of α-chymotrypsin in rabbits that this has been used as a method of inducing elevated IOP in rabbits to enable the study of glaucoma.39 Ocular hypertension following intraocular administration of α-chymotrypsin is believed to be attributable to accumulation of lens zonular fibers in the ICA.39 This mechanism may partially explain the elevated IOP detected in α-chymotrypsin-treated eyes during the immediate postoperative period in the present study; however, it cannot fully explain the elevated IOP because ocular hypertension was equivalent in timing and degree in α-chymotrypsin-treated and BSS-treated eyes. Certainly, there are many other mechanisms by which ocular hypertension could have been induced in this study, including anterior uveitis from surgical manipulation and globe decompression, obstruction of the ICA by intraoperative injection of viscoelastic, collapse of the deeper aspects of the ICA as a result of globe decompression, and lens luxation.10,40
Retinal degeneration is another frequently reported consequence of intracameral administration of α-chymotrypsin. This is especially true when α-chymotrypsin is administered intravitreally, in which case the degeneration is reported to be most severe at sites directly exposed to the enzyme.21,28,29 In the study reported here, 2 eyes (1 α-chymotrypsin-treated eye and 1 BSS-treated eye) developed glaucomatous retinopathy, optic neuropathy, and retinal detachment after lensectomy However, no other funduscopic, behavioral, or histologic signs of retinal or optic nerve degeneration were detected in any other eyes. By contrast, in a study37 in primates, an extremely rapid toxic effect on the retina was attributed to α-chymotrypsin. This was detected by use of electroretinography when as little as 75 U of α-chymotrypsin was administered intravitreally or intracamerally.37 Although these functional changes were detected prior to histologic lesions becoming visible in some animals, these animals ultimately developed histologically detectable retinal degeneration by 40 days after α-chymotrypsin injection. In the present study, we did not perform electroretinography, and our methods of visual assessment were relatively coarse. Therefore, it is possible that the dose of α-chymotrypsin we used may have had some mild toxic effects on the canine retina. However, if this were true, these did not become histologically visible by 30 days after α-chymotrypsin infusion. Because changes in the posterior segment in the study reported here were detected equally in α-chymotrypsin-treated and BSS-treated eyes, it seems likely that they resulted from surgical manipulations rather than from administration of α-chymotrypsin. If so, it is possible that preoperative retinopexy or automated rather than manual vitrectomy may have reduced the incidence of retinal detachment in the 2 eyes in which it was detected in the present study.
Although it has been suggested that α-chymotrypsin can delay or reduce corneal wound healing in humans,41 effects of α-chymotrypsin in other species are less clear. In dogs, infrequent and variable stromal keratitis and scleritis adjacent to the injection site, sometimes with ectasia, were seen in 3 of 22 eyes injected intravitreally with α-chymotrypsin.28 By contrast, in a controlled assessment of the effect of α-chymotrypsin on corneal wound healing, rabbit corneas exposed to α-chymotrypsin for 30 minutes and assayed for wound and suture strength and histologic differences 14 days later revealed no significant effects.42 In the present study, we detected clinical or histologic evidence suggestive of altered wound healing in only 2 eyes (1 α-chymotrypsin-treated eye and 1 BSS-treated eye). Both of these eyes developed a suture line staphyloma associated with glaucoma and subsequent buphthalmos. Given that an equal number of eyes treated with α-chymotrypsin or BSS were affected and because the elevated IOP was the likely cause of this change,40 it seems unlikely that this was an adverse effect of α-chymotrypsin administration.
It is also important to consider explanations for the observation that the relatively low dose of α-chymotrypsin used in the study reported here was highly effective at achieving zonulolysis. This is particularly intriguing because doses previously used in dogs often were higher than those used in humans (usually 150 to 300 U) and higher than that used in the present study and yet were still sometimes unsuccessful at achieving zonulolysis. Certainly incubation time appears important for efficacy. In a study30 involving the use of enucleated canine eyes, the rate of dissolution of lens zonules was continuously observed following application of an unstated volume of α-chymotrypsin (75 U/mL). No apparent change in zonule morphology was detected during the first 4 minutes. Between 4 and 8 minutes, all zonular bundles became less distinct and more homogenous and then they completely dissolved and disappeared. Data from the present study were consistent with this rate of zonulolysis and perhaps provided 1 reason for the lack of zonulolysis detected in studies in which shorter incubation times were used. Although not mentioned in veterinary reports, it is also possible that chemical factors in canine aqueous humor may inhibit or otherwise consume α-chymotrypsin, as has been reported for human aqueous humor.43 Given the increased porosity of the canine blood-aqueous barrier relative to that in humans,44 it is possible that leakage of plasma constituents into canine aqueous humor as a result of a preexisting uveitis or during surgical entry into the anterior chamber reduced α-chymotrypsin activity in some studies. By contrast, eyes in the present study were determined to be free of inflammation at baseline, were pretreated with a topical corticosteroid and a systemic nonsteroidal agent, and were then injected with α-chymotrypsin prior to any intraocular surgical manipulations, thus guaranteeing that the enzyme was exposed to a relatively protein-free aqueous humor. Perhaps the major reason that the present study revealed a clinically useful degree of zonulolysis at low doses of α-chymotrypsin relates to the clinical outcome we used to measure success. Investigators in many other studies21,23,24,28–30 have used lens dislocation without surgical manipulation as the measured outcome. By contrast, we used the more clinically relevant measure of reduction in surgical traction required for lensectomy, which presumably was achieved at lower doses of α-chymotrypsin than those required to cause purely enzymatic lens dislocation.
Although we did not intend to quantify the degree of zonulolysis achieved, there are many indicators that suggest it was substantial and of a clinically useful magnitude. First, the zonulolytic effect in α-chymotrypsin-treated eyes was of sufficient magnitude that the surgeon correctly detected the treated eye in 5 of 6 dogs on the basis of the ease with which the lens was removed. Second, the force required for lensectomy can be inferred from the degree of intraoperative intraocular hemorrhage and fibrin formation, both of which were significantly reduced in α-chymotrypsin-treated eyes. These data are consistent with results of another study31 conducted by our laboratory group in which we detected a 58% reduction in force required for lensectomy in enucleated eyes receiving an identical dose of α-chymotrypsin with an identical incubation time to those used in the study reported here. It appears likely that the dose of α-chymotrypsin used in the present study may be even more effective if this enzyme were to be used to facilitate ICLE of a subluxated lens in dogs. As a result of pathological conditions and age, the lens zonules in those dogs would be expected to be less robust than those in the young healthy dogs used in the present experiment and other experiments.2,12,45,46 Collectively, data from the present study and our previous study31 suggest that 75 U of α-chymotrypsin injected into the posterior chamber and allowed to remain in contact with the lens zonules for 7 minutes causes a clinically important reduction in the force required to extract the lens in clinically normal dogs, without detectable adverse effects. Although application of these data to dogs with subluxated lenses is required, administration of α-chymotrypsin may permit earlier removal of subluxated lenses. However, α-chymotrypsin is currently not commercially available; thus, development of a product that is safe and efficacious for intracameral use is essential.
Abbreviations
| BSS | Balanced saline solution |
| ICA | Iridocorneal angle |
| ICLE | Intracapsular lens extraction |
| IOP | Intraocular pressure |
| PIFM | Preiridal fibrovascular membrane |
| STT | Schirmer tear test |
Tono-Pen XL, Mentor Ophthalmics, Norwell, Mass.
Zonulysin, Henley's Medical, Brownfields, Welwyn Garden City, Hertfordshire, England; generously provided by Dr. Pip Boydell, Animal Medical Centre Referral Services, Chorlton-cum-Hardy Manchester, England.
BSS, Alcon Laboratories Inc, Fort Worth, Tex.
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Appendix
Scoring system used to score signs of ocular inflammation33,34 before and after bilateral ICLE after 7 minutes of incubation with intracamerally administered α-chymotrypsin or BSS in 6 clinically normal dogs. In each dog, 1 eye was assigned via a randomization procedure to receive α-chymotrypsin, and the other eye received BSS.
| Clinical sign | Score |
|---|---|
| Ocular discharge | 1 = Absent |
| 2 = Slight | |
| 3 = Moderate (wet hair evident only on eyelids) | |
| 4 = Severe (wet hair evident on large surface around eye) | |
| Ocular hyperemia | 1 = Absent |
| 2 = Slight (slight vascular injection) | |
| 3 = Moderate (vessels distinguished with difficulty) | |
| 4 = Severe (dark red conjunctiva) | |
| Chemosis | 1 = Absent |
| 2 = Slight | |
| 3 = Moderate (no eyelid protrusion) | |
| 4 = Severe (eyelid protrusion) | |
| Corneal edema | 1 = Absent |
| 2 = Slight (localized, mild, or both) | |
| 3 = Moderate (slight masking of iris detail) | |
| 4 = Severe (obvious masking of iris detail) | |
| Hyphema | 1 = Absent |
| 2 = Slight | |
| 3 = Moderate (filling ventral portion of the anterior chamber) | |
| 4 = Complete hyphema | |
| Aqueous flare | 0 = Absent |
| 1 = Faint (barely detectable) | |
| 2 = Moderate (iris and lens details clear) | |
| 3 = Marked (iris and lens details hazy) | |
| 4 = Intense (fixed coagulated aqueous with considerable fibrin) |
