DOI 10.1007/s10517-015-2900-2
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Experimental Modeling of Proliferative Vitreoretinopathy. An Experimental Morphological Study I. P. Khoroshilova-Maslova, N. L. Leparskaya, M. M. Nabieva, and L. D. Andreeva Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 159, No. 1, pp. 112-114, January, 2015 Original article submitted January 29, 2014 A model of proliferative vitreoretinopathy induced by simultaneous intravitreal injection of recombinant IL-1β and platelet concentrate is created and its main morphological manifestations are studied on Chinchilla rabbits. The model reflects pathogenesis of proliferative vitreoretinopathy: epiretinal membrane with the formation of retinal plication, traction detachment of the retina; moderate inflammatory reaction in the uveal tract, in the optic nerve infundibulum, in the vitreous body; intact structural elements of the retina, dissociation of the retinal pigmented epithelium cells with their subsequent migration. The model is adequate to the clinical picture of proliferative vitreoretinopathy in humans, which recommends it for experimental studies of the efficiency of drug therapy and prevention of this disease. Key Words: proliferative vitreoretinopathy; interleukine-1β; platelet concentrate; epiretinal membrane Proliferative vitreoretinopathy (PVR) is a complex heterogeneous disease characterized by the formation of cell membranes in the inner surface of the retina [7]. A specific feature of these membranes is their capacity to traction leading to traction detachment of the retina with loss of visual functions. As a multifactorial pathological process, PVR most often develops as a complication in such clinical conditions as rheumatogenic and traumatic retinal detachment. The development of PVR after surgery for retinal detachment causes disease relapses – retinal nonadhesion [1,8]. The formation of PVR is based on the proliferation of the retinal pigmented epithelium (RPE) cells and retinal glial cells [2,5]. The therapy of PVR is difficult, because of process location mainly in the posterior segment of the eye [8]. Hence, an adequate experimental model is needed for objective evaluation of drug efficiency. Numerous studies aimed at PVR simulation have been carried out over the recent 25 years. The fibroHelmholtz Moscow Research Institute of Ocular Diseases, Ministry of Health of the Russian Federation, Moscow, Russia. Address for correspondence: [email protected]. N. L. Leparskaya
blastic model is most often used; cultured fibroblasts serve as the model material in this model. In another model, platelet model, the platelet concentrate serves as the model material [3]. However, both models differ significantly from the natural clinical course of PVR: manifestations of the proliferative and inflammatory reactions are inadequate, the morphological signs of pathological process are not universal. All these facts necessitate the creation of a standard model reflecting the pathogenesis of PVR and corresponding to the natural clinical course of the process. The most important factor in the development of PVR is the inflammatory reaction inducing various cell–cell interactions. Cytokines, polypeptides regulating the immune and inflammatory reaction, play an important role in this process [2,4-6,9]. The key cytokine in this process is IL-1β characterized by a wide spectrum of activities in inflammatory reaction. In addition to the inflammatory reaction, hemorrhages, in which platelets play the leading role, are essential for PVR etiology and pathogenesis. The aim of our study is the creation of a model of PVR induced by cytokine, with limited dose of
0007-4888/15/15910100 © 2015 Springer Science+Business Media New York
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platelets as an accessory factor stimulating the proliferative process. The model is expected to reflect the pathogenetic characteristics of PVR cytopathogenesis.
MATERIALS AND METHODS Two components served as the model material in our study: recombinant IL-1β in a previously determined dose of 2000 U [2] and a low dose of heterogeneous platelet concentrate (25,000 platelets/ml). The study was carried out on Chinchilla rabbits (2.5-3 kg; 8 rabbits, 16 eyes). The animals were kept at standard illumination, temperature, and humidity on standard fodder. After instillation of a local anesthetic (alcaine 0.5%), all animals received an intravitreal injection through the flat part of the ciliary body with a 27G needle. Three series of experiments were carried out. Series I: 2 rabbits, 4 eyes; 0.1 ml recombinant IL-1β (2000 U) injected into the vitreous body. Series II: 2 rabbits, 4 eyes; 0.1 ml platelet concentrate (25,000/ml) injected into the vitreous body. Series III: 4 rabbits, 8 eyes; IL-1β (0.1 ml, 2000 U) and platelet concentrate (25,000/ml) injected simultaneously into the vitreous body. After 1 month, the animals were sacrificed by air embolism. The eyes were enucleated, fixed in 10% buffered formalin, and histological analysis was carried out. Series I and II served as control, series III was experimental. Methods of ophthalmologic studies were used: side illumination, biomicroscopy, and direct microscopy of the fundus oculi. The main conclusive method was morphological identification of PVR. Paraffin sections (12-14 sections from each eye), 5-6 μ thick, stained with hematoxylin and eosin, picrofuchsin, and by van Gieson’s method, were examined under a Leica microscope system.
Fig. 1. Experimental PVR: IL-1β (2000 U), 4 weeks after experimental simulation. Formation of fine fibrous ERM. Hematoxylin and eosin staining, ×200.
RESULTS Clinical studies in experimental series I showed no changes in the anterior segment of the eye, just slight “dimness” of the retinal surface and depigmentation zones were detected. Morphological studies showed the development of PVR in the form of a fine fibrous epiretinal membrane (ERM) on the inner surface of the retina (Fig. 1). This membrane was focal, tightly adhering to the internal limiting membrane in some places and in some places rather distant from it at a considerable length. The retina in the PVR foci was plicated and retained its common lamellar structure. Minor manifestation of the inflammatory process was worthy of note. Solitary lymphocytes were scattered in the vitreous body around the ciliary processes. Changes in the RPE were regular: cell dissociation
Fig. 2. Experimental PVR model: IL-1β (2000 U)+platelet concentrate (25,000/ml), 4 weeks after simulation. a) Formation of funnel-shaped retinal detachment. Macroscopic picture of enucleated eye; b) formation of ERM with pronounced inflammatory infiltration in the inner surface of retina. Hematoxylin and eosin staining, ×100.
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and migration. This series was characterized by mild inflammatory reaction and proliferative processes, protracted development of pathological changes, and the absence of traction retinal detachment. Clinical picture of anterior and posterior active uveitis was observed in series II. Morphological studies showed the predominating process: active inflammatory infiltration presented by lymphocytes, macrophages in the ciliary body, choroid, and optic nerve infundibulum. The inflammatory process was paralleled by manifest destructive changes with foci of lysis in the uveal tract and retina. The proliferative processes were poorly discernible. Clinical observations of experimental series III (IL-1β+platelet concentrate) showed no changes in the anterior segment; ophthalmoscopy showed formation of traction detachment of the retina. The morphological picture (in 8 eyes) was characterized by polymorphism, with predominance of inflammatory infiltration and proliferative processes with the development of traction detachment of the retina (Fig. 2, a). A stronger ERM was forming on the inner retinal surface in comparison with series I ERM (Fig. 2, b). Pronounced changes were observed in RPE: active dissociation and migration of cells. The modified PVR model is based on combined use of recombinant IL-1β (2000 U) and low dose of platelets (25,000/ml). Pathohistological studies show that addition of platelets to the cytokine model stimulates the proliferative processes with the development of PVR. A more compact ERM forms on the inner retinal surface in comparison with fine fibrous membranes in the cytokine model. Activation of the proliferative processes is closely related to more pronounced infil-
tration. Involvement of the retina in our model is rare and is due to excessively high counts of platelets in the platelet concentrate of the model material; hence, the dose of injected platelets should be precisely measured. Hence, the morphological identification of the standard PVR model is characterized by a combination of certain morphological signs detected 4 weeks after the start of the experiment: ERM with the formation of retinal plication; moderate inflammatory reaction in the uveal tract, optic nerve infundibulum, vitreous body; intact structural elements of the retina; and RPE cell dissociation and subsequent migration. The regularity of these changes recommends this model for experimental studies of the efficiency of drug therapy for PVR and prevention of this condition.
REFERENCES 1. E. V. Boiko, M. M. Shishkin, E. A. Ryabous, and S. V. Lepneva, Vestn. Oftalmol., 126, No. 4, 25-30 (2010). 2. I. P. Khoroshilova-Maslova, N. L. Leparskaya, M. M. Nabieva, and L. D. Andreeva, Ross. Oftalm. Zh., 6, No. 1, 78-80 (2013). 3. C. Baudouin, M. Ettaiche, F. Imbert, et al., Exp. Eye Res., 59, No. 6, 697-706 (1994). 4. I. A. El-Ghrably, H. S. Dua, G. M. Orr, et al., Brit. J. Ophthalmol., 85, No. 5, 461-470 (2005). 5. G. J. Jaffe, W. L. Roberts, H. L. Wong, et al., Exp. Eye Res., 60, No. 5, 533-543 (1995). 6. C. H. Kon, N. L. Occleston, G. W. Aylward, and P. T. Khaw, Invest. Ophthalmol., 40, No. 3, 705-712 (1999). 7. J. C. Pastor, Surv. Ophthalmol., 43, No. 1, 3-18 (1998). 8. A. Sadaka and G. P. Giuliari, Clin. Ophthalmol., 6, 1325-1333 (2012). 9. P. Wiedemann, Surv. Ophthalmol., 36, No. 5, 373-384 (1992).
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Experimental Modeling of Proliferative Vitreoretinopathy. An Experimental Morphological Study I. P. Khoroshilova-Maslova, N. L. Leparskaya, M. M. Nabieva, and L. D. Andreeva Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 159, No. 1, pp. 112-114, January, 2015 Original article submitted January 29, 2014 A model of proliferative vitreoretinopathy induced by simultaneous intravitreal injection of recombinant IL-1β and platelet concentrate is created and its main morphological manifestations are studied on Chinchilla rabbits. The model reflects pathogenesis of proliferative vitreoretinopathy: epiretinal membrane with the formation of retinal plication, traction detachment of the retina; moderate inflammatory reaction in the uveal tract, in the optic nerve infundibulum, in the vitreous body; intact structural elements of the retina, dissociation of the retinal pigmented epithelium cells with their subsequent migration. The model is adequate to the clinical picture of proliferative vitreoretinopathy in humans, which recommends it for experimental studies of the efficiency of drug therapy and prevention of this disease. Key Words: proliferative vitreoretinopathy; interleukine-1β; platelet concentrate; epiretinal membrane Proliferative vitreoretinopathy (PVR) is a complex heterogeneous disease characterized by the formation of cell membranes in the inner surface of the retina [7]. A specific feature of these membranes is their capacity to traction leading to traction detachment of the retina with loss of visual functions. As a multifactorial pathological process, PVR most often develops as a complication in such clinical conditions as rheumatogenic and traumatic retinal detachment. The development of PVR after surgery for retinal detachment causes disease relapses – retinal nonadhesion [1,8]. The formation of PVR is based on the proliferation of the retinal pigmented epithelium (RPE) cells and retinal glial cells [2,5]. The therapy of PVR is difficult, because of process location mainly in the posterior segment of the eye [8]. Hence, an adequate experimental model is needed for objective evaluation of drug efficiency. Numerous studies aimed at PVR simulation have been carried out over the recent 25 years. The fibroHelmholtz Moscow Research Institute of Ocular Diseases, Ministry of Health of the Russian Federation, Moscow, Russia. Address for correspondence: [email protected]. N. L. Leparskaya
blastic model is most often used; cultured fibroblasts serve as the model material in this model. In another model, platelet model, the platelet concentrate serves as the model material [3]. However, both models differ significantly from the natural clinical course of PVR: manifestations of the proliferative and inflammatory reactions are inadequate, the morphological signs of pathological process are not universal. All these facts necessitate the creation of a standard model reflecting the pathogenesis of PVR and corresponding to the natural clinical course of the process. The most important factor in the development of PVR is the inflammatory reaction inducing various cell–cell interactions. Cytokines, polypeptides regulating the immune and inflammatory reaction, play an important role in this process [2,4-6,9]. The key cytokine in this process is IL-1β characterized by a wide spectrum of activities in inflammatory reaction. In addition to the inflammatory reaction, hemorrhages, in which platelets play the leading role, are essential for PVR etiology and pathogenesis. The aim of our study is the creation of a model of PVR induced by cytokine, with limited dose of
0007-4888/15/15910100 © 2015 Springer Science+Business Media New York
I. P. Khoroshilova-Maslova, N. L. Leparskaya, et al.
101
platelets as an accessory factor stimulating the proliferative process. The model is expected to reflect the pathogenetic characteristics of PVR cytopathogenesis.
MATERIALS AND METHODS Two components served as the model material in our study: recombinant IL-1β in a previously determined dose of 2000 U [2] and a low dose of heterogeneous platelet concentrate (25,000 platelets/ml). The study was carried out on Chinchilla rabbits (2.5-3 kg; 8 rabbits, 16 eyes). The animals were kept at standard illumination, temperature, and humidity on standard fodder. After instillation of a local anesthetic (alcaine 0.5%), all animals received an intravitreal injection through the flat part of the ciliary body with a 27G needle. Three series of experiments were carried out. Series I: 2 rabbits, 4 eyes; 0.1 ml recombinant IL-1β (2000 U) injected into the vitreous body. Series II: 2 rabbits, 4 eyes; 0.1 ml platelet concentrate (25,000/ml) injected into the vitreous body. Series III: 4 rabbits, 8 eyes; IL-1β (0.1 ml, 2000 U) and platelet concentrate (25,000/ml) injected simultaneously into the vitreous body. After 1 month, the animals were sacrificed by air embolism. The eyes were enucleated, fixed in 10% buffered formalin, and histological analysis was carried out. Series I and II served as control, series III was experimental. Methods of ophthalmologic studies were used: side illumination, biomicroscopy, and direct microscopy of the fundus oculi. The main conclusive method was morphological identification of PVR. Paraffin sections (12-14 sections from each eye), 5-6 μ thick, stained with hematoxylin and eosin, picrofuchsin, and by van Gieson’s method, were examined under a Leica microscope system.
Fig. 1. Experimental PVR: IL-1β (2000 U), 4 weeks after experimental simulation. Formation of fine fibrous ERM. Hematoxylin and eosin staining, ×200.
RESULTS Clinical studies in experimental series I showed no changes in the anterior segment of the eye, just slight “dimness” of the retinal surface and depigmentation zones were detected. Morphological studies showed the development of PVR in the form of a fine fibrous epiretinal membrane (ERM) on the inner surface of the retina (Fig. 1). This membrane was focal, tightly adhering to the internal limiting membrane in some places and in some places rather distant from it at a considerable length. The retina in the PVR foci was plicated and retained its common lamellar structure. Minor manifestation of the inflammatory process was worthy of note. Solitary lymphocytes were scattered in the vitreous body around the ciliary processes. Changes in the RPE were regular: cell dissociation
Fig. 2. Experimental PVR model: IL-1β (2000 U)+platelet concentrate (25,000/ml), 4 weeks after simulation. a) Formation of funnel-shaped retinal detachment. Macroscopic picture of enucleated eye; b) formation of ERM with pronounced inflammatory infiltration in the inner surface of retina. Hematoxylin and eosin staining, ×100.
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and migration. This series was characterized by mild inflammatory reaction and proliferative processes, protracted development of pathological changes, and the absence of traction retinal detachment. Clinical picture of anterior and posterior active uveitis was observed in series II. Morphological studies showed the predominating process: active inflammatory infiltration presented by lymphocytes, macrophages in the ciliary body, choroid, and optic nerve infundibulum. The inflammatory process was paralleled by manifest destructive changes with foci of lysis in the uveal tract and retina. The proliferative processes were poorly discernible. Clinical observations of experimental series III (IL-1β+platelet concentrate) showed no changes in the anterior segment; ophthalmoscopy showed formation of traction detachment of the retina. The morphological picture (in 8 eyes) was characterized by polymorphism, with predominance of inflammatory infiltration and proliferative processes with the development of traction detachment of the retina (Fig. 2, a). A stronger ERM was forming on the inner retinal surface in comparison with series I ERM (Fig. 2, b). Pronounced changes were observed in RPE: active dissociation and migration of cells. The modified PVR model is based on combined use of recombinant IL-1β (2000 U) and low dose of platelets (25,000/ml). Pathohistological studies show that addition of platelets to the cytokine model stimulates the proliferative processes with the development of PVR. A more compact ERM forms on the inner retinal surface in comparison with fine fibrous membranes in the cytokine model. Activation of the proliferative processes is closely related to more pronounced infil-
tration. Involvement of the retina in our model is rare and is due to excessively high counts of platelets in the platelet concentrate of the model material; hence, the dose of injected platelets should be precisely measured. Hence, the morphological identification of the standard PVR model is characterized by a combination of certain morphological signs detected 4 weeks after the start of the experiment: ERM with the formation of retinal plication; moderate inflammatory reaction in the uveal tract, optic nerve infundibulum, vitreous body; intact structural elements of the retina; and RPE cell dissociation and subsequent migration. The regularity of these changes recommends this model for experimental studies of the efficiency of drug therapy for PVR and prevention of this condition.
REFERENCES 1. E. V. Boiko, M. M. Shishkin, E. A. Ryabous, and S. V. Lepneva, Vestn. Oftalmol., 126, No. 4, 25-30 (2010). 2. I. P. Khoroshilova-Maslova, N. L. Leparskaya, M. M. Nabieva, and L. D. Andreeva, Ross. Oftalm. Zh., 6, No. 1, 78-80 (2013). 3. C. Baudouin, M. Ettaiche, F. Imbert, et al., Exp. Eye Res., 59, No. 6, 697-706 (1994). 4. I. A. El-Ghrably, H. S. Dua, G. M. Orr, et al., Brit. J. Ophthalmol., 85, No. 5, 461-470 (2005). 5. G. J. Jaffe, W. L. Roberts, H. L. Wong, et al., Exp. Eye Res., 60, No. 5, 533-543 (1995). 6. C. H. Kon, N. L. Occleston, G. W. Aylward, and P. T. Khaw, Invest. Ophthalmol., 40, No. 3, 705-712 (1999). 7. J. C. Pastor, Surv. Ophthalmol., 43, No. 1, 3-18 (1998). 8. A. Sadaka and G. P. Giuliari, Clin. Ophthalmol., 6, 1325-1333 (2012). 9. P. Wiedemann, Surv. Ophthalmol., 36, No. 5, 373-384 (1992).
