Experimental intravitreous cysticercosis

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Graefe's Arch Clin Exp Ophthalmol (1996) 234:515-520 © Springer-Verlag 1996

Arturo Santos Jos6 A. Paczka Juan M. Jim6nez-Sierra Patricia Ch6vez Cecilio Velasco Ana Flisser Hugo Quiroz-Mercado

Received: 6 February 1995 Revised version received: 14 March 1996 Accepted: 18 April 1996 Presented in part at the ARVO Annual Meeting, May 3, 1990. A. Santos - J.A.Paczka J.M. Jim6nez-Sierra • R Ch6vez C. Velasco • H. Quiroz-Mercado Laboratorio de Cirugfa Experimental, Asociaci6n para Evitar la Ceguera en M6xico, Hospital "Dr. Luis Sfinchez Bulnes", Vicente Garcia Torres 46, 04030 Coyoac~n DF, Mexico A. Santos ( ~ ) C.U.C.S, Universidad de Guadalajara RO. Box 5-902, Guadalajara, Jalisco 45042, Mexico Tel. +52-3-1212706 A. Flisser Instituto de Investigaciones Biom~dicas, Universidad Nacional Aut6noma de M6xico, Ciudad Universitaria, 04510 Mexico, DF, Mexico

Experimental intravitreous cysticercosis

Abstract • Background: Cysticercosis is one of the parasitic diseases that most frequently affects the eye. The most common and severe manifestations of ocular infection are secondary to posterior segment involvement, which often leads to blindness and atrophy of the eye. The pathogenesis of ocular injury in this disease is poorly understood. The authors have developed an experimental animal model for intravitreous cysticercosis using New Zealand rabbits and Taenia crassiceps cysticerci. • Methods: Twelve rabbits were divided into two groups. Rabbits in group I were inoculated with one living cysticercus in the vitreous cavity. Rabbits in group II received an intramuscular dose of steroids prior to inoculation of parasites • Results: An intense inflammatory reaction, which lead

Introduction Human cysticercosis is caused by infection with the larval stage of the porcine tapeworm, Taenia solium [5, 10, 15, 17, 20, 25]. Infection and disease are increasingly recognized not only in many areas of the developing world but also in industrialized nations without endemic disease, mainly because of the migration of those who are infected [17]. Cysticercosis is particularly common in Mexico and other parts of Central and South America, Africa, India, China, Eastern Europe, and Indonesia [14]. The eye is one of the organs which is frequently affected by cysticerci [15]. The most common and severe

to a severe ocular injury, was observed in rabbits of group I, while rabbits ing roup II had minimal inflammatory changes. Histopathological studies showed a severe histiocytic infiltrate with generalized retinal damage in group I, and a mild inflammatory infiltrate, limited to the area of direct contact with the parasite in group II. The ocular lesions found in rabbits which did not receive steroids (group I) resembled those found in human ocular cysticercosis. • Conclusion: These observations indicate that ocular damage in this parasitic disease might be directly related to inflammatory changes produced by the presence of cysticerci. This model appears to be useful for future investigations.

manifestations of ocular infection are secondary to posterior segment involvement, which often leads to blindness and atrophy of the eye [2, 3, 9, 13, 15, 21, 26, 28]. The pathogenesis, host immune response, and effective medical treatment in this entity are still unclear [3, 11, 19]. It is difficult to obtain infected human eyes for histopathological study because this blinding eye disease is not fatal. Often, enucleated eyes represent endstage changes in which the presence of secondary pathologies is common. It is important to study earlier stages and to follow the sequence of changes not available in autopsy cases; thus, the pathogenesis of ocular damage in cysticercosis cannot be easily determined from human samples. The histopathological study of this ocular parasitic

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disease has s h o w n that the cysticercus or its debris are surrounded by a g r a n u l o m a t o u s i n f l a m m a t o r y process w h i c h usually contains abundant eosinophils [7, 27]. T h e development o f an experimental m o d e l is useful to provide valuable information for the study o f the pathogenesis o f ocular cysticercosis. Taenia solium eggs can be obtained only f r o m h u m a n feces and are difficult to obtain in large amounts. The use o f a closely related species, Taenia crassiceps, appears to be a g o o d alternative for the development o f an experimental model. T h e p r o c e d u r e to obtain Taenia crassiceps cysticerci has been previously d e s c r i b e d [6]. Cfirdenas and c o - w o r k e r s successfully used Taenia crassiceps cysticerci in the development o f an experimental animal m o d e l for anterior c h a m b e r cysticercosis [4]. The p r e s e n c e o f Taenia crassiceps in the h u m a n eye has been r e p o r t e d [1, 22]. Since intravitreous cysticercosis is one o f the most frequent locations o f this parasitic eye disease, the p u r p o s e o f the present study was to establish an experimental animal m o d e l for posterior s e g m e n t cysticercosis by intravitreal inoculation o f Taenia crassiceps cysticerci in rabbits. In addition, this m o d e l was used to evaluate clinically and histopathologically the ocular r e s p o n s e to the p r e s e n c e o f the parasite in normal and steroid-treated rabbits.

Materials and methods Parasites and animals

Taenia crassiceps cysticerci (ORF) were kept by passage in the peritoneal cavity of BALB/c mice using the procedure previously reported by Freeman [6]. Four weeks after peritoneal inoculation, a mouse was killed and parasites were obtained, carefully washed with sterile balanced salt solution and used immediately for intravitreal inoculation. Healthy white New Zealand rabbits of both sexes, weighing 2.5-3 kg, were used; all procedures were carried out under xylazine and ketamine anesthesia. Animal care and procedures were strictly performed in accordance with the guidelines of the ARVO Resolution on the Use of Animals in Research.

Clinical evaluation Clinical slit-lamp examination, posterior segment photography and fluorescein angiography were performed by the usual methods. Echography was carried out according to the method described by Moragrega for the diagnosis of free intravitreous cysticerci [16]. All these procedures were performed in both experimental and control eyes, 1 day prior to the inoculation of cysticerci and on days 2, 7 and 14 after inoculation. Ocular inflammation was graded daily according to the method of Hogan and co-workers [8]. Histopathological study All animals were killed on day 14 after intravitreal inoculation of the parasite, and the eyes were enucleated, fixed using 10% formaldehyde, embedded in paraffin and sectioned. Microscopic sections were then deparaffinized and stained by hematoxylin and eosin (H & E) and periodic acid-Schiff (PAS) for light microscopy.

Results All parasitized eyes o f rabbits in group I showed a similar intense and rapidly evolving inflammation characterized by ciliary injection, pupillary miosis, keratic precipitates, m a r k e d anterior c h a m b e r flare, abundant cells in the aqueous humor, posterior synechiae with iris bomb6, peripheral anterior synechiae, elevated intraocular pressure and lens opacities; these c h a n g e s p r e c l u d e d viewing o f the fundus. Posterior segment s o n o g r a p h y c o n d u c t e d on day 14 after inoculation o f the parasite disclosed a cystic i m a g e surrounded by highly e c h o g e n i c images indicating the parasite surrounded by a severe i n f l a m m a t o r y process (Fig. 1). The histopathological study showed a severe i n f l a m m a t o r y reaction, characterized by the p r e s e n c e o f an extensive histiocytic infiltrate c o m p r i s i n g an intravitreous abscess. The cysticercus wall debris was surrounded by p o l y m o r p h o n u c l e a r

Experimental design Twelve rabbits were used and divided into two groups. Rabbits in group I (n=6) received an intravitreal injection of 0.1 ml of sterile balanced salt solution containing several fragments of one living cysticercus in the right eye. This was performed using a l-ml tuberculin syringe equipped with a 23-gauge needle inserted through the sclera, 4 mm posterior to the corneoscleral junction in the superotemporal quadrant under stereomicroscopic control. Anterior chamber paracentesis was performed to reduce the intraocular pressure. Rabbits in group II (n=6) received an intramuscular dose of 0.5 mg of dipropionate betamethasone and 0.2 mg of disodium phosphate betamethasone in 0.1 ml 3 h previous to inoculation of parasites. The latter was performed using the same procedure carried out in group I. At the end of the procedure a snbconjunctival betamethasone injection (same combination of dose and volume used for the intramuscular injection) was administered. In both groups, left eyes were considered controls and were injected only with 0.1 ml of sterile balanced salt solution.

Fig. 1 Sonogram obtained by contact method on day 14 showing a free intravitreous cysticercus surrounded by a severe inflammatory reaction (group I, subject 2)

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Discussion

Fig. 2 From upper to lower sections of photograph: V vitreous abscess, R necrotic retina infiltrated by lymphocytes and plasma cells, RPE residual retinal pigment epithelium, C choroid, S sclera (PAS, magnification ×44; group I, subject 2)

leukocytes, macrophages and eosinophils. Generalized retinal destruction was noted. The necrotic retina was infiltrated by lymphocytes and plasma cells. The presence of inflammatory cells in the choroid was also observed (Figs. 2-4). Anterior segment findings included moderate iris atrophy, both posterior and peripheral anterior synechiae and lens epithelial proliferation. Intravitreal injection of cysticerci in all rabbits of group II resulted in a mild inflammatory reaction in the posterior segment which consisted of a few scattered and coarse opacities, and a few cells present in the anterior vitreous at slit-lamp examination. The fundus was clearly seen despite these changes. No inflammatory changes were detected in the anterior segment. Fluorescein angiography showed no abnormalities in retinal and choroidal vasculature. The parasites were noted in the vitreous cavity immediately after inoculation. Posterior segment photographic and sonographic follow-up demonstrated parasite motility and growth (Figs. 5, 6). The histopathological study showed localized areas of retinal atrophy, which corresponded to the areas of direct contact with the parasite, infiltrated by a few inflammatory cells. These areas were associated with the presence of inflammatory cells in the vitreous and lymphocytes and plasma cells infiltrating the choroid. Surrounding these areas a normal retina with a few macrophages and eosinophils was observed (Figs. 7, 8).

An experimental animal model for anterior chamber cysticercosis has been previously reported elsewhere [4]; however, to our knowledge, there are no reports in the literature of the development of an experimental model for intravitreous cysticercosis. Since the vitreous is one of the most frequent locations of ocular cysticercosis, the establishment of an experimental model for this variety appears to be important. Taenia crassiceps occurs in foxes and rodents [6]. The presence of Taenia crassiceps in the human eye has been reported. Shea and associates [22] reported a 17-yearold girl with an intraocular parasite which was removed by surgical intervention from the subretinal space and positively identified as Taenia crassiceps. Arocker-Mettinger and co-workers described a case in which a contractable living parasite was removed from the anterior chamber of an otherwise healthy 15-year-old girl. The tapeworm was found to be too young to have developed the characteristic scolex. Western immunoblotting gave indirect evidence for infection with Taenia crassiceps [1]. Taenia crassiceps undergoes asexual reproduction by budding [6]. This fact represents an important technical advantage in the development of an experimental animal model for intravitreous cysticercosis. Thus it is possible to inoculate multiple parasite buds originated from a fragmented living cysticercus after its passage through a fine needle into the recipient eye. The presence of living cysticerci in the vitreous cavity of those rabbits not receiving steroids (group I) produced ocular lesions with a resulting intense inflammatory reaction which resembled those observed in human ocular cysticercosis. The typical histopathological findings in endstage intraocular cysticercosis reported by G6mezLeal [7] include the presence of a central eosinophilic mass with parasite elements, surrounded by a polymorphonuclear sheath which in turn is surrounded by a granulomatous inflammatory process. These structures are encompassed by a fibrous capsule that is infiltrated by chronic inflammatory cells. Intraocular tissues are disorganized; the choroid is infiltrated by lymphocytes and plasma cells; the retina is detached, and presents atrophy and gliois in some areas; the crystalline lens is opacified and is adherent to the inflammatory process of the vitreous cavity through a cyclitic membrane; the iris is atrophic, and peripheral anterior synechiae occlude the angle; the ciliary body also presents some degree of atrophy. Most of these findings were present in the infected eyes of rabbits in group I. In contrast, the infected eyes of rabbits which did receive steroids (group II) showed no ocular lesions; the inflammatory reaction was mild or absent, and the viability of cysticerci was preserved. When an inflammatory reaction was present, it was limited to the area in direct

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Fig. 3 Vitreous abscess; there is cysticercus wall debris (C) in the central portion and eosinophilic exudate in the upper portion (PAS, magnification ×44; group I, subject 1) Fig. 4 Inflammatory infiltrate composed of polymorphonuclear leukocytes, macrophages, and some eosinophils which surround the parasite wall debris (C) (PAS, magnification X 176; group I, subject 1)

Fig. 5 A Fundus photograph taken on day 2 showing multiple cysticerci in vitreous cavity without inflammatory reaction. B Cysticerci (arrows) have grown on day 14; inflammation remains absent. Bar 1 mm (group II, subject 2) Fig. 6 Sonogram obtained by contact method on day 14 demonstrating a free intravitreous cysticercus just in front of the retina without inflammatory reaction (group II, subject 2) Fig. 7 Area of retinal atrophy (R) with a few inflammatory cells. Lymphocytes and plasma cells infiltrating choroid (C) (H & E, magnification ×88; group II, subject 2)

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Fig. 8 On the left, an area of retinal atrophy with inflammatory cells in vitreous (V). On the right, a normal retina (R) with a few macrophages and eosinophils (H & E, magnification x88, group II, subject 2)

c o n t a c t w i t h the p a r a s i t e , as d e m o n s t r a t e d c l i n i c a l l y and histopathologically. T h e s e o b s e r v a t i o n s i n d i c a t e that o c u l a r d a m a g e in this p a r a s i t i c d i s e a s e m i g h t b e d i r e c t l y r e l a t e d to the i n f l a m m a t o r y c h a n g e s p r o d u c e d b y the p r e s e n c e o f c y s t i c e r c i .

F u r t h e r m o r e , s i n c e s y s t e m i c s t e r o i d s have b e e n c o n s i d e r e d i m p o r t a n t in l i m i t i n g or d i m i n i s h i n g the i n f l a m m a t o r y r e a c t i o n p r o d u c e d b y s o m e o f the t h e r a p e u t i c m o d a l i t i e s u s e d in i n t r a o c u l a r c y s t i c e r c o s i s (e.g., p h o t o c o a g u l a t i o n and s u r g e r y [12, 18, 24], and c h e m o t h e r a p y in n e u r o c y s t i c e r c o s i s [23]), it is v e r y i m p o r t a n t to e l u c i d a t e the role that s t e r o i d s p l a y in e x p e r i m e n t a l and hum a n c y s t i c e r c o s i s . Thus, the e x p e r i m e n t a l m o d e l r e p o r t ed h e r e i n m a y b e u s e f u l to s t u d y the e f f e c t o f e n h a n c e d or c o n t r o l l e d i m m u n e r e s p o n s e as w e l l as the p a t h o g e n i c m e c h a n i s m s o f o c u l a r d a m a g e in c y s t i c e r c o s i s . In this e x p e r i m e n t a l m o d e l , the e n t r y p o r t a l o f the p a r a s i t e and the t i m e c o u r s e o f the d i s e a s e are d i s s i m i l a r to h u m a n c y s t i c e r c o s i s ; however, the m o d e l r e p r e s e n t s a f e a s i b l e m e t h o d o f infection, gives a v a l u a b l e e x p e r i m e n t a l setting o f i n t r a v i t r e o u s c y s t i c e r c o s i s and m a y f a c i l i t a t e the e x p e r i m e n t a l e v a l u a t i o n o f d r u g and s u r g i c a l t h e r a pies.

Acknowledgements The authors wish to acknowledge and thank Mr. Ricardo Montoya for technical assistance in the development of photographic material and Mr. Enrique Santana for help in the care of laboratory animals.

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