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Histopathological and Immunohistochemical Features of Vitreoretinopathy in Shih Tzu Dogs
J. Comp. Path. 2013, Vol. 148, 230e235
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SPONTANEOUSLY ARISING DISEASE
Histopathological and Immunohistochemical Features of Vitreoretinopathy in Shih Tzu Dogs Nikolaos G. Papaioannou* and Richard R. Dubielzig† *Department of Pathology, School of Veterinary Medicine, Aristotle University of Thessaloniki, Greece and † Comparative Ocular Pathology Laboratory of Wisconsin, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, Madison, WI, USA
Summary Fifty cases of Shih Tzu ocular vitreoretinopathy were selected from the database of the Comparative Ocular Pathology Laboratory of Wisconsin. Cases with severe coexisting conditions (e.g. corneal disease, uveitis or endophthalmitis) were excluded. Microscopical changes were evaluated and immunohistochemistry was used to define spindle cells, gliosis and the presence of basement membranes in the vitreous. Expression of glial fibrillary acidic protein, vimentin and smooth muscle actin was also performed. The mean age of the 50 cases was 10.1 years (range 2.5e15 years). The most characteristic microscopical abnormalities (50/50 cases) were retinal detachment and extensive retinal tear. Additionally, extracellular, eosinophilic matrix material admixed with few spindle cells, and pre-iridal fibrovascular membrane, goniodysgenesis, secondary glaucoma, hypermature and subcapsular cataract were detected. The spindle cells within the collagen matrix were strongly labelled for expression of vimentin, with weaker expression of smooth muscle actin. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: dog; immunohistochemistry; Shih Tzu; vitreoretinopathy
Introduction Over the last decade the Shih Tzu has become a very popular breed, but these dogs are affected by a variety of medical problems, especially ocular diseases (Christmas, 1992; Gough and Thomas, 2004; Itoh et al., 2004). Common ocular problems of this breed include medial canthal entropion, distichiasis, various trichiasis problems, dermoids, proptosis, third eyelid gland prolapse, chronic and pigmentary keratitis, refractory corneal ulceration and keratoconjuctivitis sicca. However, the major ocular diseases of the Shih Tzu are progressive retinal atrophy, vitreal synaeresis and retinal detachment (RD). Vitreoretinopathy in this breed is often not recognized until the onset of neovascular glaucoma secondary to RD (Christmas, 1992; Gough and Thomas, 2004; Itoh et al., 2004; Vainisi and Wolfer, 2004; Dubielzig et al., 2010). Vitreal diseases are poorly understood in the dog; however, it is known that vitreous liquefaction predisCorrespondence to: N. Papaioannou (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.05.014
poses to RD, especially in certain breeds, including the Shih Tzu, Brussels griffon, chihuahua, Chinese crested dog, Havanese, Italian greyhound, lowchen, papillon and whippet (Manschot, 1958; Vainisi et al., 2007). The vitreous plays a major role in retinal attachment and so any changes in the vitreous can lead to RD (Boeve and Stabes, 2007). RD involves separation of the neurosensory retina from the underlying retinal pigment epithelium (RPE). RD can be either rhegmatogenous (RRD) or non-rhegmatogenous (non-RRD). Primary RRD is preceded by an alteration or degeneration of the vitreous (Vainisi et al., 2007), while secondary RRD is the result of trauma, glaucoma, lens surgery or surgery involving the ciliary body or the ora ciliaris retinae, the most prominent of these being cataract surgery (Hendrix et al., 1993). The term ‘vitreous degeneration’ is used to indicate changes that are consistent with breakdown of the vitreous hydrogel (Boeve and Stabes, 2007). Signs of vitreous degeneration include liquefaction (e.g. synaeresis) or opacities (e.g. vitreous ‘floaters’, asteroid hyalosis and synchysis scintilans). Normal canine vitreous has Ó 2012 Elsevier Ltd. All rights reserved.
231
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a range of different consistencies. Synaeresis is degenerative breakdown of the vitreous gel with separation of the liquid and solid components, resulting in liquefaction and development of fluid-filled cavities within the vitreous (Manschot, 1958; Vainisi et al., 2007). This breakdown may occur with age, but it may also be the result of inflammation or unknown causes (Ladbeter et al., 2004; Vainisi et al., 2007). In primary vitreal degeneration in the Shih Tzu, vitreous material may sometimes leak into the anterior chamber. In this instance, the animal is at greater risk of developing glaucoma due to iridocorneal cleft obstruction by the vitreous material (Rubin, 1989). The aim of this study was to describe the histopathological and immunohistochemical features of vitreoretinopathy in Shih Tzu dogs and to propose possible pathogenic mechanisms underlying this ocular disease.
Materials and Methods Fifty cases of ocular vitreoretinopathy in Shih Tzus were selected from the database of the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW). The database also included 47 other cases of vitreoretinopathy in several other canine breeds. The signalment and presenting complaints were obtained from the submission reports. The only clinical feature reported routinely was the presence or absence of glaucoma. Most of the globes were removed because of glaucoma. All globes were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4e5 mm) were stained with haematoxylin and eosin (HE), alcian blueeperiodic acideShiff (PAS; PAS stains carbohydrate-rich proteins such as basement membranes and alcian blue stains the hyalouronic acid of the vitreous), Masson’s trichrome (to distinguish between collagen and other protein deposits), sirius red stain for collagen and Perl’s Prussian blue reaction (for haemosiderin and free iron). Immunohistochemistry (IHC) was performed to identify vimentin, a-smooth muscle actin (SMA) and glial fibrillary acidic protein (GFAP) using the avidinebiotin immunoperoxidase method (ABC kit, peroxidase standard Vectastain, Vector Laboratories, Burlingame, California, USA). Sections for IHC (3 mm) were mounted on positively-charged slides (Probe-On Plus; Fisher Scientific, Pittsburg, Pennsylvania, USA) and dried at 60 C for 2 h. The slides were dewaxed through xylene and graded alcohols and rinsed in tap water. Endogenous peroxidase activity was blocked using H2O2 3% in methanol for 15 min at room temperature (RT). Antigen retrieval for vimentin and a-SMA was achieved by heating the sections in citrate buffer (pH 6.0) and then incubating them in com-
mercial retrieval solution (Decloaker solution; Biocare Medical, Walnut Creek, California, USA) allowing them to reach a temperature of 125 C and 24e29 lb of pressure for 30 sec and then 90 C for 10 sec. No antigen retrieval was required for GFAP. Slides were incubated with Dako Ready-to-Use Protein Serum (Dako, Carpinteria, California, USA) for 10 min at 37 C in order to block non-specific protein binding. Polyclonal rabbit anti-GFAP (Dako, catalogue number Z0334, dilution 1 in 1,500), monoclonal mouse antivimentin (clone V9, Dako, catalogue number M0725, dilution 1 in 200) and monoclonal mouse anti-a-SMA (Dako, catalogue number M0851, dilution 1 in 100) were applied to the slides in a humid chamber at RT for 60 min. The secondary reagent for the antibodies to vimentin and a-SMA was biotinylated horse antimouse IgG (Vector Laboratories, dilution 1 in 125) applied for 30 min at RT, and for anti-GFAP was biotinylated goat anti-rabbit IgG (dilution 1 in 250) applied for 30 min at RT. Between incubations, rinsing steps were performed using Tris-buffered saline (TBS). Finally, all sections were incubated with ABC (Vector Laboratories) and rinsed with TBS. ‘Visualization’ was performed by incubation with 3,30 -diaminobenzidine solution (DAB, Sigma, St Louis, Missouri, USA) for 10 min and counterstaining was with Harris’ haematoxylin. Sections were dehydrated and coverslipped with permanent mounting media (Polymount, PolyScientific, Bayshore, New York, USA). Negative controls were processed in the same way, using buffer solution in place of the primary antibody. The positive control for GFAP was canine brain, and for vimentin and a-SMA it was canine small intestine.
Results The mean age of the 50 dogs was 10.1 3.42 years (range 2.5e15 years). Twenty-five dogs were male (of which 23 were neutered) and 23 were female (of which 20 were neutered). Two dogs were of unknown gender. All eyes had similar gross appearance including enlargement of the globe associated with complete RD, tearing, buphthalmous, lens subluxation, liquefaction and haemorrhage in the vitreous as well as optic nerve head cupping (Fig. 1). The most characteristic microscopical abnormalities, seen in all cases, were the presence of extensive RD associated with retinal atrophy and extensive retinal tear (Table 1. Extracellular eosinophilic matrix material, admixed with some spindle cells, phagocytes and erythrocytes, was present in the vitreous attached to the posterior lens capsule in 31 cases. The presence of collagen and proteoglycan matrix within this eosinophilic matrix material was confirmed by alcian blueePAS, sirius red and Perl’s Prussian blue staining (Fig. 2).
232
N.G. Papaioannou and R.R. Dubielzig Table 1 Histopathological features of vitreoretinopathy in Shih Tzu dogs Total number of cases: 50 RD Extracellular, eosinophilic matrix material in the vitreous PIFM Goniodysgenesis Secondary glaucoma Cataract Keratitis
50/50 31/50
34/50 4/50 26/50 13/50 30/50
secondary glaucoma, characterized by diminished ganglion cells, optic nerve head cupping and gliosis, was noted in 26/50 globes. In 13/50 globes, hypermature and subcapsular cataracts were detected. Histologically, the lens capsule was often wrinkled, with liquefaction of cortical lens fibres, and the lens epithelium had migrated posteriorly with few bladder cells present in the posterior cortex. In 5/50 globes a cyclitic membrane was detected, which was also confirmed by alcian blueePAS and Masson’s trichrome staining (Fig. 5). In 30/50 globes chronic superficial keratitis, characterized by the presence of neutrophils and lymphocytes with evidence of prior ulceration, was also detected. The spindle cells within the collagen matrix in the vitreous were labelled strongly for expression of vimentin (15/50) and less strongly for expression of a-SMA (13/50), indicating that they were myofibroblasts (Fig. 6). The pattern of GFAP expression in the retina (15/50) consisted of fibres extending from the inner to the outer retinal layers, suggestive of M€ uller cells, and fibres which formed a tangle in the inner retina, interpreted as being astrocytes (Fig. 7).
Discussion
Fig. 1. An affected globe showing complete RD (a, b) and tearing (arrowhead, a), haemorrhage and collagen matrix in the vitreous (star, a), haemorrhage in the anterior and posterior chamber (star, b) and nerve head cupping (arrow, b). HE.
A pre-iridal fibrovascular membrane (PIFM) was identified in 34/50 globes. These PIFMs were characterized by a proliferation of spindle cells and blood vessels on the surface of the iris and were also stained with alcian blueePAS and Masson’s trichrome (Fig. 3). Goniodysgenesis, characterized by the extension of iris-like tissue from the base of the iris to the termination of Descemet’s membrane, was detected in 4/50 globes (Fig. 4). Typical
The pathology and pathogenesis of vitreoretinopathy in Shih Tzu dogs has not been extensively investigated previously. The results of the present study indicate that the major pathological changes in this condition are RD and extensive retinal tears as well as collagen deposition in the vitreous body. Affected dogs were middle-aged to older, consistent with previous descriptions of vitreous degeneration in Shih Tzu dogs, which is referred to as an age-related condition (Gough and Thomas, 2004; Labruyere et al., 2008; Dubielzig et al., 2010). This observation suggests a similarity with retinal tear associated with vitreous degeneration in humans, where loss of vitreous gel and the separation of the vitreous body from the retina increase the risk of retinal tear, RD and agerelated nuclear cataract (Ambati et al., 2003).
Vitreoretinopathy in Shih Tzu Dogs
233
Fig. 2. Presence of collagen and proteoglycan matrix within the eosinophilic material admixed with occasional spindle cells (aec) and erythrocytes with free iron deposits (b). (a) Sirius red, (b) Perl’s Prussian blue and (c) alcian blueePAS.
RD and retinal tear were seen in all of the globes evaluated. Abnormal levels of collagen and spindle cells expressing a-SMA were seen in 13/50 of the affected globes. Portions of the vitreous were also liquefied in affected dogs. The partially liquid vitreous created uneven stress and support for the retina. It has been shown that in RD, a form of traumatic injury in which the retina becomes separated from the underlying retinal pigment epithelial layer, the M€ uller
Fig. 3. Iris. Presence of a PIFM (arrowhead). Alcian blueePAS.
cell, generally considered a specialized radial astrocyte, is the predominant glial cell type involved (Lewis et al., 2010). Following RD, M€ uller cells proliferate actively and hypertrophy within the retina and onto the retinal surface where they form structures similar to those formed by reactive astrocytes in the brain and spinal cord (Fisher and Lewis, 2003). GFAP is the main component of astrocytic intermediate filaments and it is present in astrocytes and other cells with these protein components. In the present study, GFAP was expressed strongly by M€ uller cells and fibres, forming a tangle in the inner retina that was suggestive of astrocytes. Additionally, the increased expression of GFAP with increased organization of vitreal membranes is consistent with the intermediate filament changes seen in chronic glial responses (Zeiss and Dubielzig, 2004). The extracellular eosinophilic matrix material admixed with spindle cells expressing a-SMA in the vitreous represents collagen formation in the vitreous. This matrix material may create traction, which contributes subsequently to the detachment and tearing of the retina. Vascular endothelial growth factor (VEGF) is expressed in normal human and canine retina, iris and cornea (Kim et al., 1999; Phillip et al., 2000; SaintGeniez et al., 2008; Zafross et al., 2010). Studies of
234
N.G. Papaioannou and R.R. Dubielzig
Fig. 4. Goniodysgenesis. A solid sheet of iris-like tissue extending from the base of the iris to the termination of Descemet’s membrane (arrow). HE.
human patients indicated that VEGF could be a vascular survival factor in normal ocular tissue and may also facilitate pathological neovascularization in disease states or when produced in excess (Kim et al., 1999; Saint-Geniez et al., 2008). We hypothesize that RD leads to retinal degeneration and hypoxia causing release of angiogenic factors such as VEGF and fibroblast growth factor (FGF). These factors, when released in the eye, cause the formation of PIFM with subsequent haemorrhage and neovascular glaucoma. Oxidative stress plays a role in the pathogenesis of human age-related macular degeneration and in diseases of the central nervous system of man and dogs (Stone et al., 1979; Alder and Cringle, 1985; Bazan, 1985; Uscida et al., 1993; Ando et al., 1997; Beatty et al., 2000; Papaioannou et al., 2001; Ambati et al., 2003). Many age-related lesions have been attributed to cumulative oxidative damage caused by reactive
Fig. 5. A cyclitic membrane located along the central axis of the eye, just posterior to the lens (arrow) and consisting predominantly of collagen fibres. Alcian blueePAS.
Fig. 6. Expression of vimentin by spindle cells within the collagen matrix in the vitreous. IHC.
oxygen intermediates (ROIs) such as hydroxen peroxide, singlet oxygen, superoxide anion and hydroxyl radical. ROIs arise as byproducts of cellular metabolism or photochemical reactions. The retina is particularly susceptible to oxidative damage because of high oxygen tension, a high proportion of polyunsaturated fatty acids in the photoreceptor outer segments, numerous chromophores in the retina and the fact that RPE phagocytosis of photoreceptor discs generates ROIs (Stone et al., 1979; Alder and Cringle, 1985; Bazan, 1985; Gaillard et al., 1995; Ambati et al., 2003). With age, the human Bruch’s membrane undergoes numerous changes that impede normal function. The progressive increase in lipid content in Bruch’s membrane leads to the development of lesions in the RPE and retina (Holz et al., 1994). Furthermore, oxidative damage is an important step in the pathogenesis of primary open-angle glaucoma in people (Sacca et al., 2005). We propose that these mechanisms could help to explain the pathogenesis of RD in
Fig. 7. Expression of GFAP by M€ uller cells and astrocytes in the retina. IHC.
Vitreoretinopathy in Shih Tzu Dogs
the Shih Tzu dog. However, further studies are needed to determine the influence of ROI and especially the effect of lipid peroxidation in canine ocular disorders.
References Alder VA, Cringle SJ (1985) The effect of the retinal circulation on vitreal oxygen tension. Current Eye Research, 4, 121e129. Ambati J, Ambati BK, Yoo SH, Ianchulev S, Adamis AP (2003) Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies. Survey of Ophthalmology, 48, 257e293. Ando Y, Nyhlin N, Suhr O, Holmgren G, Uchida K et al. (1997) Oxidative stress is found in amyloid deposits in systemic amyloidosis. Biochemical and Biophysical Research Communications, 232, 497e502. Bazan NG (1985) The metabolism of omega-3 polyunsaturated fatty acids in the eye: the possible role of docosahexaenoic acid and docosanoids in retinal physiology and ocular pathology. Progress in Clinical Biological Research, 312, 95e112. Beatty SP, Koh H, Phil M, Henson D, Boulton M (2000) The role of oxidative stress in the pathogenesis of agerelated macular degeneration. Survey of Ophthalmology, 45, 115e134. Boeve MH, Stabes FC (2007) Diseases and surgery of the canine vitreous. In: Veterinary Ophthalmology, KN Gelatt, Ed., Blackwell Publishing, Ames, pp. 932e943. Christmas RE (1992) Common ocular problems of ShihTzu dogs. Canadian Veterinary Journal, 33, 390e393. Dubielzig RR, Ketring KL, McLellan GJ, Albert DM (2010) Abnormalities associated with specific animal breeds. In: Veterinary Ocular Pathology: A Comparative Review, Vol. 3, Saunders Elsevier, St. Louis, pp. 34e49. Fisher SK, Lewis GP (2003) Muller cell and neuronal remodeling in retinal detachment and reattachment and their potential consequences for visual recovery: a review and reconsideration of recent data. Vision Research, 43, 887e897. Gaillard ER, Anterton SJ, Eldred G, Dillon J (1995) Photophysical studies on human reatinal lipofuscin. Photochemistry and Photobiology, 61, 448e453. Gough A, Thomas A (2004) Breed Predispositions to Disease in Dogs and Cats. Blackwell Publishing, Oxford, pp. 143e145. Hendrix DV, Nasisse MP, Cowen P, Davidson MG (1993) Clinical signs, concurrent diseases, and risk factors associated with retinal detachments in dogs. Progress in Veterinary Comparative Ophthalmology, 3, 87e91. Holz FG, Sheraidach G, Pauleikhoff D, Bird AC (1994) Analysis of lipid deposits extracted from human macular and peripheral Bruch’s membrane. Archives of Ophthalmology, 112, 402e406. Itoh Y, Maehara S, Yamasaki A, Tsuzuki K, Izumisawa Y (2004) Investigation of fellow eye of unilateral retinal detachment in Shih-Tzu. Veterinary Ophthalmology, 13(5), 289e293. Kim I, Ryan AM, Rohan R, Amanos S, Agular S et al. (1999) Constitutive expression of VEGF, VEGFR-1 and VEGFR-2 in normal eyes. Investigative Ophthalmology and Visual Science, 40, 2115e2121.
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Labruyere JJ, Hartley C, Rogers K, Wetherill G, McConnell JF et al. (2008) Vitreous degeneration, especially mild vitreal syneresis is not uncommon in normal dogs. Veterinary Radiology and Ultrasound, 49, 165e171. Ladbeter EC, Millichamp NJ, Dziezyc J (2004) Microbial contamination of the anterior chamber during cataract phacoemulsification and intraocular lens implantation in dogs. Veterinary Ophthalmology, 7, 327e334. Lewis GP, Chapin EA, Luna G, Linberg KA, Fisher SK (2010) The fate of Muller’s glia following experimental retinal detachment: nuclear migration, cell division, and subretinal glial scar formation. Molecular Vision, 16, 1361e1372. Manschot WA (1958) Persistent hyperplastic primary vitreous. Archives of Ophthalmology, 59, 188e203. Papaioannou N, Tooten PCJ, vanEderen AM, Bohl JRE, Rofina J et al. (2001) Immunohistochemical investigation of the brain of aged dogs. I. Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, an oxidative damage product, in senile plaques. Amyloid Journal: Protein Folding Disorders, 8, 11e21. Phillip W, Speicher L, Humpel C (2000) Expression of vascular growth factor and its receptors in inflamed and vascularized human corneas. Investigative Ophthalmology and Visual Science, 41, 2514e2522. Rubin LF (1989) Inherited Eye Diseases in Purebred Dogs. Williams and Wilkins, Baltimore, pp. 266e268. Sacca SC, Pascoto A, Camicione P, Capris P, Izztti A (2005) Oxidative DNA damage in the human trabecular meshwork: clinical correlation in patients with primary open-angle glaucoma. Archives of Ophthalmology, 123, 458e463. Saint-Geniez M, Maharaj AS, Walshe TE, Tucker BA, Sekiyama E et al. (2008) Endogenous VEGF is required for visual function: evidence for a survival role on Muller cells and photoreceptors. PLoS ONE, 3, 3554. Stone WL, Farnsworth CC, Dratz EA (1979) A reinvestigation of the fatty acid content of bovine, rat and frog retinal rod outer segments. Experimental Eye Research, 28, 387e397. Uscida K, Szweda LI, Chae HZ, Stadtman ER (1993) Immunohistochemical detection of 4-hydroxynonenal protein adducts in oxidized hepatocytes. Proceedings of the National Academy of Sciences USA, 90, 8742e8746. Vainisi SJ, Wolfer JC (2004) Canine retinal surgery. Veterinary Ophthalmology, 7, 291e306. Vainisi SJ, Wolfer JC, Smith PJ (2007) Surgery of the canine posterior segment. In: Veterinary Ophthalmology, 4th Edit., KN Gelatt, Ed., Blackwell Publishing, Ames, pp. 1026e1058. Zafross MK, Breaux CB, Whiteley HE, Hamor RE, Flaws JA et al. (2010) Canine pre-iridal fibrovascular membranes: morphologic and immunohistochemical investigations. Veterinary Ophthalmology, 13, 4e13. Zeiss C, Dubielzig RR (2004) A morphologic study of intravitreal membranes associated with intraocular hemorrhage in the dog. Veterinary Ophthalmology, 7, 239e242. February 29th, 2012 ½ Received, Accepted, May 21st, 2012
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
SPONTANEOUSLY ARISING DISEASE
Histopathological and Immunohistochemical Features of Vitreoretinopathy in Shih Tzu Dogs Nikolaos G. Papaioannou* and Richard R. Dubielzig† *Department of Pathology, School of Veterinary Medicine, Aristotle University of Thessaloniki, Greece and † Comparative Ocular Pathology Laboratory of Wisconsin, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, Madison, WI, USA
Summary Fifty cases of Shih Tzu ocular vitreoretinopathy were selected from the database of the Comparative Ocular Pathology Laboratory of Wisconsin. Cases with severe coexisting conditions (e.g. corneal disease, uveitis or endophthalmitis) were excluded. Microscopical changes were evaluated and immunohistochemistry was used to define spindle cells, gliosis and the presence of basement membranes in the vitreous. Expression of glial fibrillary acidic protein, vimentin and smooth muscle actin was also performed. The mean age of the 50 cases was 10.1 years (range 2.5e15 years). The most characteristic microscopical abnormalities (50/50 cases) were retinal detachment and extensive retinal tear. Additionally, extracellular, eosinophilic matrix material admixed with few spindle cells, and pre-iridal fibrovascular membrane, goniodysgenesis, secondary glaucoma, hypermature and subcapsular cataract were detected. The spindle cells within the collagen matrix were strongly labelled for expression of vimentin, with weaker expression of smooth muscle actin. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: dog; immunohistochemistry; Shih Tzu; vitreoretinopathy
Introduction Over the last decade the Shih Tzu has become a very popular breed, but these dogs are affected by a variety of medical problems, especially ocular diseases (Christmas, 1992; Gough and Thomas, 2004; Itoh et al., 2004). Common ocular problems of this breed include medial canthal entropion, distichiasis, various trichiasis problems, dermoids, proptosis, third eyelid gland prolapse, chronic and pigmentary keratitis, refractory corneal ulceration and keratoconjuctivitis sicca. However, the major ocular diseases of the Shih Tzu are progressive retinal atrophy, vitreal synaeresis and retinal detachment (RD). Vitreoretinopathy in this breed is often not recognized until the onset of neovascular glaucoma secondary to RD (Christmas, 1992; Gough and Thomas, 2004; Itoh et al., 2004; Vainisi and Wolfer, 2004; Dubielzig et al., 2010). Vitreal diseases are poorly understood in the dog; however, it is known that vitreous liquefaction predisCorrespondence to: N. Papaioannou (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.05.014
poses to RD, especially in certain breeds, including the Shih Tzu, Brussels griffon, chihuahua, Chinese crested dog, Havanese, Italian greyhound, lowchen, papillon and whippet (Manschot, 1958; Vainisi et al., 2007). The vitreous plays a major role in retinal attachment and so any changes in the vitreous can lead to RD (Boeve and Stabes, 2007). RD involves separation of the neurosensory retina from the underlying retinal pigment epithelium (RPE). RD can be either rhegmatogenous (RRD) or non-rhegmatogenous (non-RRD). Primary RRD is preceded by an alteration or degeneration of the vitreous (Vainisi et al., 2007), while secondary RRD is the result of trauma, glaucoma, lens surgery or surgery involving the ciliary body or the ora ciliaris retinae, the most prominent of these being cataract surgery (Hendrix et al., 1993). The term ‘vitreous degeneration’ is used to indicate changes that are consistent with breakdown of the vitreous hydrogel (Boeve and Stabes, 2007). Signs of vitreous degeneration include liquefaction (e.g. synaeresis) or opacities (e.g. vitreous ‘floaters’, asteroid hyalosis and synchysis scintilans). Normal canine vitreous has Ó 2012 Elsevier Ltd. All rights reserved.
231
Vitreoretinopathy in Shih Tzu Dogs
a range of different consistencies. Synaeresis is degenerative breakdown of the vitreous gel with separation of the liquid and solid components, resulting in liquefaction and development of fluid-filled cavities within the vitreous (Manschot, 1958; Vainisi et al., 2007). This breakdown may occur with age, but it may also be the result of inflammation or unknown causes (Ladbeter et al., 2004; Vainisi et al., 2007). In primary vitreal degeneration in the Shih Tzu, vitreous material may sometimes leak into the anterior chamber. In this instance, the animal is at greater risk of developing glaucoma due to iridocorneal cleft obstruction by the vitreous material (Rubin, 1989). The aim of this study was to describe the histopathological and immunohistochemical features of vitreoretinopathy in Shih Tzu dogs and to propose possible pathogenic mechanisms underlying this ocular disease.
Materials and Methods Fifty cases of ocular vitreoretinopathy in Shih Tzus were selected from the database of the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW). The database also included 47 other cases of vitreoretinopathy in several other canine breeds. The signalment and presenting complaints were obtained from the submission reports. The only clinical feature reported routinely was the presence or absence of glaucoma. Most of the globes were removed because of glaucoma. All globes were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4e5 mm) were stained with haematoxylin and eosin (HE), alcian blueeperiodic acideShiff (PAS; PAS stains carbohydrate-rich proteins such as basement membranes and alcian blue stains the hyalouronic acid of the vitreous), Masson’s trichrome (to distinguish between collagen and other protein deposits), sirius red stain for collagen and Perl’s Prussian blue reaction (for haemosiderin and free iron). Immunohistochemistry (IHC) was performed to identify vimentin, a-smooth muscle actin (SMA) and glial fibrillary acidic protein (GFAP) using the avidinebiotin immunoperoxidase method (ABC kit, peroxidase standard Vectastain, Vector Laboratories, Burlingame, California, USA). Sections for IHC (3 mm) were mounted on positively-charged slides (Probe-On Plus; Fisher Scientific, Pittsburg, Pennsylvania, USA) and dried at 60 C for 2 h. The slides were dewaxed through xylene and graded alcohols and rinsed in tap water. Endogenous peroxidase activity was blocked using H2O2 3% in methanol for 15 min at room temperature (RT). Antigen retrieval for vimentin and a-SMA was achieved by heating the sections in citrate buffer (pH 6.0) and then incubating them in com-
mercial retrieval solution (Decloaker solution; Biocare Medical, Walnut Creek, California, USA) allowing them to reach a temperature of 125 C and 24e29 lb of pressure for 30 sec and then 90 C for 10 sec. No antigen retrieval was required for GFAP. Slides were incubated with Dako Ready-to-Use Protein Serum (Dako, Carpinteria, California, USA) for 10 min at 37 C in order to block non-specific protein binding. Polyclonal rabbit anti-GFAP (Dako, catalogue number Z0334, dilution 1 in 1,500), monoclonal mouse antivimentin (clone V9, Dako, catalogue number M0725, dilution 1 in 200) and monoclonal mouse anti-a-SMA (Dako, catalogue number M0851, dilution 1 in 100) were applied to the slides in a humid chamber at RT for 60 min. The secondary reagent for the antibodies to vimentin and a-SMA was biotinylated horse antimouse IgG (Vector Laboratories, dilution 1 in 125) applied for 30 min at RT, and for anti-GFAP was biotinylated goat anti-rabbit IgG (dilution 1 in 250) applied for 30 min at RT. Between incubations, rinsing steps were performed using Tris-buffered saline (TBS). Finally, all sections were incubated with ABC (Vector Laboratories) and rinsed with TBS. ‘Visualization’ was performed by incubation with 3,30 -diaminobenzidine solution (DAB, Sigma, St Louis, Missouri, USA) for 10 min and counterstaining was with Harris’ haematoxylin. Sections were dehydrated and coverslipped with permanent mounting media (Polymount, PolyScientific, Bayshore, New York, USA). Negative controls were processed in the same way, using buffer solution in place of the primary antibody. The positive control for GFAP was canine brain, and for vimentin and a-SMA it was canine small intestine.
Results The mean age of the 50 dogs was 10.1 3.42 years (range 2.5e15 years). Twenty-five dogs were male (of which 23 were neutered) and 23 were female (of which 20 were neutered). Two dogs were of unknown gender. All eyes had similar gross appearance including enlargement of the globe associated with complete RD, tearing, buphthalmous, lens subluxation, liquefaction and haemorrhage in the vitreous as well as optic nerve head cupping (Fig. 1). The most characteristic microscopical abnormalities, seen in all cases, were the presence of extensive RD associated with retinal atrophy and extensive retinal tear (Table 1. Extracellular eosinophilic matrix material, admixed with some spindle cells, phagocytes and erythrocytes, was present in the vitreous attached to the posterior lens capsule in 31 cases. The presence of collagen and proteoglycan matrix within this eosinophilic matrix material was confirmed by alcian blueePAS, sirius red and Perl’s Prussian blue staining (Fig. 2).
232
N.G. Papaioannou and R.R. Dubielzig Table 1 Histopathological features of vitreoretinopathy in Shih Tzu dogs Total number of cases: 50 RD Extracellular, eosinophilic matrix material in the vitreous PIFM Goniodysgenesis Secondary glaucoma Cataract Keratitis
50/50 31/50
34/50 4/50 26/50 13/50 30/50
secondary glaucoma, characterized by diminished ganglion cells, optic nerve head cupping and gliosis, was noted in 26/50 globes. In 13/50 globes, hypermature and subcapsular cataracts were detected. Histologically, the lens capsule was often wrinkled, with liquefaction of cortical lens fibres, and the lens epithelium had migrated posteriorly with few bladder cells present in the posterior cortex. In 5/50 globes a cyclitic membrane was detected, which was also confirmed by alcian blueePAS and Masson’s trichrome staining (Fig. 5). In 30/50 globes chronic superficial keratitis, characterized by the presence of neutrophils and lymphocytes with evidence of prior ulceration, was also detected. The spindle cells within the collagen matrix in the vitreous were labelled strongly for expression of vimentin (15/50) and less strongly for expression of a-SMA (13/50), indicating that they were myofibroblasts (Fig. 6). The pattern of GFAP expression in the retina (15/50) consisted of fibres extending from the inner to the outer retinal layers, suggestive of M€ uller cells, and fibres which formed a tangle in the inner retina, interpreted as being astrocytes (Fig. 7).
Discussion
Fig. 1. An affected globe showing complete RD (a, b) and tearing (arrowhead, a), haemorrhage and collagen matrix in the vitreous (star, a), haemorrhage in the anterior and posterior chamber (star, b) and nerve head cupping (arrow, b). HE.
A pre-iridal fibrovascular membrane (PIFM) was identified in 34/50 globes. These PIFMs were characterized by a proliferation of spindle cells and blood vessels on the surface of the iris and were also stained with alcian blueePAS and Masson’s trichrome (Fig. 3). Goniodysgenesis, characterized by the extension of iris-like tissue from the base of the iris to the termination of Descemet’s membrane, was detected in 4/50 globes (Fig. 4). Typical
The pathology and pathogenesis of vitreoretinopathy in Shih Tzu dogs has not been extensively investigated previously. The results of the present study indicate that the major pathological changes in this condition are RD and extensive retinal tears as well as collagen deposition in the vitreous body. Affected dogs were middle-aged to older, consistent with previous descriptions of vitreous degeneration in Shih Tzu dogs, which is referred to as an age-related condition (Gough and Thomas, 2004; Labruyere et al., 2008; Dubielzig et al., 2010). This observation suggests a similarity with retinal tear associated with vitreous degeneration in humans, where loss of vitreous gel and the separation of the vitreous body from the retina increase the risk of retinal tear, RD and agerelated nuclear cataract (Ambati et al., 2003).
Vitreoretinopathy in Shih Tzu Dogs
233
Fig. 2. Presence of collagen and proteoglycan matrix within the eosinophilic material admixed with occasional spindle cells (aec) and erythrocytes with free iron deposits (b). (a) Sirius red, (b) Perl’s Prussian blue and (c) alcian blueePAS.
RD and retinal tear were seen in all of the globes evaluated. Abnormal levels of collagen and spindle cells expressing a-SMA were seen in 13/50 of the affected globes. Portions of the vitreous were also liquefied in affected dogs. The partially liquid vitreous created uneven stress and support for the retina. It has been shown that in RD, a form of traumatic injury in which the retina becomes separated from the underlying retinal pigment epithelial layer, the M€ uller
Fig. 3. Iris. Presence of a PIFM (arrowhead). Alcian blueePAS.
cell, generally considered a specialized radial astrocyte, is the predominant glial cell type involved (Lewis et al., 2010). Following RD, M€ uller cells proliferate actively and hypertrophy within the retina and onto the retinal surface where they form structures similar to those formed by reactive astrocytes in the brain and spinal cord (Fisher and Lewis, 2003). GFAP is the main component of astrocytic intermediate filaments and it is present in astrocytes and other cells with these protein components. In the present study, GFAP was expressed strongly by M€ uller cells and fibres, forming a tangle in the inner retina that was suggestive of astrocytes. Additionally, the increased expression of GFAP with increased organization of vitreal membranes is consistent with the intermediate filament changes seen in chronic glial responses (Zeiss and Dubielzig, 2004). The extracellular eosinophilic matrix material admixed with spindle cells expressing a-SMA in the vitreous represents collagen formation in the vitreous. This matrix material may create traction, which contributes subsequently to the detachment and tearing of the retina. Vascular endothelial growth factor (VEGF) is expressed in normal human and canine retina, iris and cornea (Kim et al., 1999; Phillip et al., 2000; SaintGeniez et al., 2008; Zafross et al., 2010). Studies of
234
N.G. Papaioannou and R.R. Dubielzig
Fig. 4. Goniodysgenesis. A solid sheet of iris-like tissue extending from the base of the iris to the termination of Descemet’s membrane (arrow). HE.
human patients indicated that VEGF could be a vascular survival factor in normal ocular tissue and may also facilitate pathological neovascularization in disease states or when produced in excess (Kim et al., 1999; Saint-Geniez et al., 2008). We hypothesize that RD leads to retinal degeneration and hypoxia causing release of angiogenic factors such as VEGF and fibroblast growth factor (FGF). These factors, when released in the eye, cause the formation of PIFM with subsequent haemorrhage and neovascular glaucoma. Oxidative stress plays a role in the pathogenesis of human age-related macular degeneration and in diseases of the central nervous system of man and dogs (Stone et al., 1979; Alder and Cringle, 1985; Bazan, 1985; Uscida et al., 1993; Ando et al., 1997; Beatty et al., 2000; Papaioannou et al., 2001; Ambati et al., 2003). Many age-related lesions have been attributed to cumulative oxidative damage caused by reactive
Fig. 5. A cyclitic membrane located along the central axis of the eye, just posterior to the lens (arrow) and consisting predominantly of collagen fibres. Alcian blueePAS.
Fig. 6. Expression of vimentin by spindle cells within the collagen matrix in the vitreous. IHC.
oxygen intermediates (ROIs) such as hydroxen peroxide, singlet oxygen, superoxide anion and hydroxyl radical. ROIs arise as byproducts of cellular metabolism or photochemical reactions. The retina is particularly susceptible to oxidative damage because of high oxygen tension, a high proportion of polyunsaturated fatty acids in the photoreceptor outer segments, numerous chromophores in the retina and the fact that RPE phagocytosis of photoreceptor discs generates ROIs (Stone et al., 1979; Alder and Cringle, 1985; Bazan, 1985; Gaillard et al., 1995; Ambati et al., 2003). With age, the human Bruch’s membrane undergoes numerous changes that impede normal function. The progressive increase in lipid content in Bruch’s membrane leads to the development of lesions in the RPE and retina (Holz et al., 1994). Furthermore, oxidative damage is an important step in the pathogenesis of primary open-angle glaucoma in people (Sacca et al., 2005). We propose that these mechanisms could help to explain the pathogenesis of RD in
Fig. 7. Expression of GFAP by M€ uller cells and astrocytes in the retina. IHC.
Vitreoretinopathy in Shih Tzu Dogs
the Shih Tzu dog. However, further studies are needed to determine the influence of ROI and especially the effect of lipid peroxidation in canine ocular disorders.
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