Clinical Hemorheology and Microcirculation 60 (2015) 405–411 DOI 10.3233/CH-141860 IOS Press

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Hemorheological parameters and their correlations in OXYS rats: A new model of hyperviscosity syndrome Mikhail Y. Maslova , Galina A. Chernyshevab , Vera I. Smol’jakovab , Oleg I. Alievb,d , Natalia G. Kolosovac and Mark B. Plotnikovb,d,∗ a

Steward St. Elizabeth’s Medical Center/Tufts University School of Medicine, Department of Anesthesiology, Critical Care, and Pain Medicine, Boston, MA, USA b E.D. Goldberg Institute of Pharmacology, Russian Academy of Medical Sciences, Tomsk, Russia c Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia d Tomsk State University, Tomsk, Russia

Abstract. Rheohaemapheresis aims to normalize major rheological parameters and is used to treat patients with dry age-related macular degeneration (AMD). While effective, this approach is invasive and requires specially trained personnel. Therefore, the search for novel effective compounds with hemorheological properties that can be taken orally to treat AMD is justified. The use of a robust rodent model of AMD with high blood viscosity is crucial to test the efficacy of potential hemorheological drugs to treat this disease. The objective of this study was to investigate whether OXYS rats, generally used as an animal model of AMD, have hyperviscosity syndrome. The results of this study show that blood viscosity in OXYS rats at low (3–10 s−1 ) and high (45–300 s−1 ) shear rates were 14–20% and 7–10% higher than in Wistar rats, while hematocrit and plasma viscosity were not different. Red blood cells (RBCs) in OXYS rats were more prone to aggregation as shown by 39% shorter half-time than in Wistar rats. RBCs were also more rigid in OXYS than in Wistar rats as shown by 21–33% lower index of elongation at the shear stress of 1–7 Pa. These data indicate that OXYS rats have hyperviscosity syndrome as the result of abnormal RBC deformability and aggregation. We propose to use OXYS rats as an animal model for preclinical studies to test compounds with hemorheological properties aimed to treat AMD. Keywords: OXYS rats, hyperviscosity syndrome, whole blood viscosity, plasma viscosity, RBC aggregation, RBC deformability

1. Introduction Dry age-related macular degeneration (AMD) is a debilitating disease which accounts for 90% of all AMD cases and frequently leads to the loss of vision in elderly population [2, 40]. Even though the pathogenesis of this disease is still vague [3, 23], the role of metabolic, hemodynamic, genetic, environmental and other factors has been suggested [10, 21, 37, 38]. While conventional therapies for AMD exist, they are either invasive [3] or the efficacy is poor [5]. One of the possible reasons for low efficacy of pharmacotherapy is that it targets pathological processes that may play only a minor role in the development of the disease. Of interest, while high blood viscosity and subsequently inefficient retinal macular blood perfusion has been suggested as one of the crucial contributing factors in the development ∗ Corresponding author: Mark B. Plotnikov, E.D. Goldberg Institute of Pharmacology, Siberian Branch, Russian Academy of Medical Sciences, Lenin St., 3, Tomsk 634028, Russia. Tel./Fax: +7 3822 418373; E-mail: [email protected].

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of degenerative changes leading to dry AMD [6, 7], none of the pharmacotherapies aim to improve rheological properties of blood. Hyperviscosity syndrome in patients with AMD is formed by increased whole blood and plasma viscosity, fibrinogen level, RBC count, aggregation and reduced deformability [17, 21]. Of note, the severity of AMD has been directly related to the increased fibrinogen level and plasma viscosity [17]. Rheohaemopheresis, as one of the invasive means to improve hemorheological parameters, has been shown to prevent the progression of the symptoms and, in some cases, improve vision [3]. Klingel et al. [13] also showed that the removal of high molecular weight proteins from the bloodstream decreases whole blood and plasma viscosity, improves microcirculation and recovers retinal function. Unfortunately, rheohaemopheresis is one of the most complex procedures among therapeutic apheresis as it is invasive, requires specially trained personnel to carry out and special equipment, has adverse effects and needs to be repeated to maintain the improvement [3]. On the other hand, these encouraging data open up a venue for the potential use of hemorheological drugs that can be administered orally to treat AMD. Preclinical studies of novel compounds with hemorheological properties for AMD treatment require the use of robust animal models. Senescence-accelerated OXYS rats have been suggested as a good fit for a model of AMD [19, 42] that can potentially be used for evaluation of the efficacy of the retina-protective pharmacotherapy [22]. In this study, we evaluated and characterized hemorheological parameters and interplay between them in OXYS rats. 2. Material and methods 2.1. Experimental animals All studies were approved by the Institutional Animal Care and Use Committee at the Institute of Pharmacology at the Russian Academy of Medical Sciences (Tomsk, Russia). Experiments were carried out on 8 adult male OXYS rats and 8 adult male Wistar rats (400–475 grams) obtained from the Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia). The pups were weaned at the age of 4 weeks, housed in groups of five animals per cage (57 × 36 × 20 cm) in standard laboratory conditions (ambient temperature of 22 ± 2◦ C, relative humidity of 60%, light/dark period 12/12 hours a day) in cages with sawdust bedding and provided with standard rodent feed (PK-120-1, Ltd., Laboratorsnab, Russia),) and ad libitum water access. After delivery to the animal facility at the location where experiments were conducted (Institute of Pharmacology, Tomsk, Russia), rats were quarantined for 2 weeks. 2.2. Blood sampling Animals were anesthetized with sodium thiopental (60 mg/kg, ip). Blood was sampled from the catheterized right carotid artery accessed through a 2.5 cm midline vertical incision in the neck and lateral reflection of the neck muscles, stabilized with aqueous solution of sodium citrate, and stored at 37◦ C in “Teflon”-coated tubes for immediate analysis. 2.3. Hemorheologic measurements Whole blood and plasma viscosity was measured using rotational viscometer (LVDV-II+P, CP40, Brookfield Engineering Labs Inc., USA). Hematocrit (Ht) was measured by gravimetric technique by

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centrifuging blood samples at 650 g for 20 min. The ratio of hematocrit to blood viscosity at high shear rates (45–300 s−1 ) was used as a measure of oxygen availability for tissues [37]. RBC aggregation and deformability were measured using RheoScan AnD-300 (RheoMeditech Inc., Korea). Half-time (T1/2 ) aggregation was used to characterize RBC aggregation, and elongation index (EI) was used to characterize RBC deformability [34]. 2.4. Data analysis and statistics Data is presented as mean ± S.E.M. A non-parametric Wilcoxon-Mann-Whitney test was used to compare hemorheological parameters between groups (STATISTICA 6.0.). Data were considered statistically distinct if the p-value was less than 0.05. 3. Results OXYS rats had a hyperviscosity syndrome as parameters of blood rheology significantly deviated from normal values in Wistar rats. Whole blood viscosity at low (3–10 s−1 ) and high shear rates (45–300 s−1 ) was elevated by 14–20% and 7–10% compared with Wistar rats (Fig. 1). RBC half-time aggregation was 39% shorter suggesting an increased rate of aggregation. EI within shear stress of 3–300 s−1 was 21–33% lower than in healthy animals indicating RBCs’ rigidity (Table 1). Macrorheological parameters, such as Ht and plasma viscosity were similar in OXYS and Wistar rats (Table 1). These data suggest that hyperviscosity syndrome in OXYS rats was attributable to negative changes in RBC rheological parameters. Rigid and prone to high aggregation, RBCs also had a lower capacity for oxygen delivery to tissues as shown by 10–13% decreased index of oxygen availability (Fig. 1). Correlation analysis showed that relationships between macro- and microhemorheological parameters seen in Wistar rats were disrupted in OXYS rats. Specifically, while whole blood viscosity at high shear rates (90–300 s−1 ) inversely correlated with RBC deformability (r = –0.79 to –0.92, p < 0.02), and RBC half-time aggregation inversely correlated with Ht (r = –0.72, p < 0.03) in Wistar rats, these correlations were weak and statistically insignificant in OXYS rats. Also, an inverse correlation between whole blood viscosity at low shear rates (3 and 10 s−1 ) and RBC half-time aggregation (r = –0.72 to –0.73, p < 0.03) observed in Wistar rats was insignificant in OXYS rats. Nevertheless, positive correlation between whole blood viscosity at low and high shear rates and Ht was preserved in OXYS rats (r = 0.86 to 0.89, p < 0.03 for both, low and high shear rates) and was very close to that in Wistar rats (r = 0.71 to 0.84, p < 0.03 and r = 0.70 to 0.74, p < 0.04, at low and high shear rates accordingly) (Fig. 2). Table 1 Hematocrit (Ht, %), plasma viscosity (PV, mPa·s), half-period of RBC aggregation (T1/2 , s), RBC elongation index (EI) (EI, rel. units) on different share stress in Wistar rats and OXYS rats Group of rats

Ht

PV

Wistar rats (n = 8) 45 ± 1 1.3 ± 0.1 OXYS rats (n = 8) 43 ± 1 1.4 ± 0.1

T1/2

19.0 ± 1.6 11.5 ± 1.5*

*–p < 0.05 compared to values in Wistar rats.

EI 1 Pa

2 Pa

3 Pa

7 Pa

0.204 ± 0.010 0.136 ± 0.003*

0.257 ± 0.011 0.186 ± 0.008*

0.313 ± 0.009 0.248 ± 0.007*

0.375 ± 0.009 0.294 ± 0.008*

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Fig. 1. Whole blood viscosity (A, left scale, mPa·s) and availability of O2 for tissues (B, right scale, rel. units) in Wistar rats (1) and OXYS rats (2). Along the abscissa axis – share rates, s–1 . *–p < 0.05 compared to values in Wistar rats.

–0.79 - –0.92

–0.72 - –0.73 Half-time of RBC aggregation

+0.72

Whole blood viscosity

+0.70-0.84

Hematocrit

Wistar rats

RBC elongation index

Half-time of RBC aggregation

RBC elongation index

Whole blood viscosity

+0.86-0.89

Hematocrit

OXYS rats

Fig. 2. Correlation between hemorheogical parameters in Wistar rats and OXYS rats (indicated only significant values of correlation coefficient).

4. Discussion The OXYS rat strain [33] used in this study was created at the Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia) from the Wistar line by selection and inbreeding of animals prone to cataractogenic effects induced by a galactose-rich diet [14, 24, 35]. OXYS rats are predisposed to early chorioretinal dystrophy with most pronounced changes occurring in the choroid vessels and hematoretinal barrier [19, 42]. Morphological analysis of the retina in OXYS rats shows abnormality in the vascular bed with 2.5-fold decrease of the specific area of functioning capillaries compared to Wistar rats. It is

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also common for OXYS rats to have wide areas with choriocapillaries with stasis and thrombosis already by 6 months of age [19]. Morphological analysis of semithin sections of the posterior wall of the eye in 5 month old OXYS rats also shows that choroid vessels, pigmented epithelium, and radial glia are most vulnerable to injury [42]. Retinal hypoxia and destruction of the pigmented epithelium associated with circulatory disorders in the choroid vessels are likely causes of injury to the neurosensory cells (mainly the external segments) and the 3.5-fold increase in the fraction of photoreceptors with nuclear pyknosis compared to Wistar rats [42]. Compromised resistance of OXYS rats to oxidative stress is linked to the pathological processes leading to early aging and progression of age-related diseases such as cataract, macular dystrophy, osteoporosis, cognitive and behavioral dysfunctions, and also lung and liver pathologies [12, 15]. In this study we, for the first time, demonstrate that OXYS rats have hyperviscosity syndrome due to microrheological abnormalities such as increased RBC aggregation and poor deformability. Impaired microcirculation, as a result of hyperviscosity syndrome, may cause retinal hypoxia and exacerbate the damaging effects of oxidative stress on the retina [12, 15, 18]. Even though the role of oxidative stress in triggering processes leading to hemorheological disturbances has been suggested [4], there is no commonly accepted view on the causative role of the oxidative stress in the development of hyperviscosity syndrome [1, 16, 39, 41]. While this issue still needs to be addressed, in the meantime, antioxidant therapy has been shown to be effective in mitigating hyperviscosity syndrome. For instance, treatment with epigallocatechin-3-gallate, the most potent antioxidant from green tea, reduces the level of malondialdehyde in RBC membrane and, as a result, improves deformability and reduces aggregation of RBCs and normalizes blood viscosity at all shear rates [4]. We have previously reported that antioxidants of natural and synthetic origin improve rheological properties of blood in models of hypervisosity syndrome with underlying oxidative stress [27, 28, 30, 31]. While there is a strong correlation between whole blood viscosity and major rheological parameters such as hematocrit, plasma viscosity, RBC deformability and aggregation [25] in Wistar rats, in this study in OXYS rats, we demonstrate the correlation only between blood viscosity and hematocrit. Probably the damaging impact of oxidative stress on the RBC rheology in OXYS rats is so significant that it disrupts the correlation between RBC aggregation and deformability and blood viscosity. We have previously reported that a similar phenomenon is common for models of hyperviscosity syndrome developed as a result of acute cerebral ischemia, ovarietomy and in spontaneously hypertensive rats [26, 29, 32]. The loss of correlations between parameters is frequently referred to as the “general biological law manifestation” observed during pathological conditions [9]. Even though clinical observations showed correlations between basic hemorheological parameters in patients with AMD, these correlations are rather weak or non-significant, except for correlations between fibrinogen, plasma viscosity, and RBC aggregation [21]. Importantly, abnormal capillary geometry and configuration, such as sharply narrowed lumen by bulged out cytoplasm or nucleated part of endotheliocytes and ectasia of lumen commonly observed in the retina of OXYS rats [42], may contribute to microcirculatory disorders due to increased turbulence in blood flow and activation of blood cells [20] that can trigger formation of RBC sludges and impedance to blood flow and rheological occlusion [8]. This notion is supported by the data that 25% of all microvessels in OXYS rats have stasis, sludges and thrombosis, in contrast to 1.5% in Wistar rats [19]. In summary, we suggest the use of OXYS rats (6 months of age) as a reliable AMD model of hyperviscosity syndrome with disrupted correlations between major hemorheological parameters for preclinical studies of compounds with hemorheological activity as potential drugs with retina-protective effects for AMD treatment.

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References [1] M. Andriollo-Sanchez, I. Hininger-Favier, N. Meunier, E. Venneria, J.M. O’Connor, G. Maiani, C. Coudray and A.M. Roussel, Age-related oxidative stress and antioxidant parameters in middle-aged and older European subjects: The ZENITH study, Eur J Clin Nutr 59(Suppl 2) (2005), 58–62. [2] A. Bird, Age-related macular disease, Br J Ophthalmol 80 (1996), 2–3. [3] M. Bl´aha, E. Rencov´a, H. Langrov´a, M. L´ansk´a, V. Bl´aha, J. Studniˇcka, P. Rozs´ıval, R. Mal´y, I. F´atorov´a, S. Filip, J. Drˇsata, L. Hejsek and J. Mal´y, The importance of rheological parameters in the therapy of the dry form of age-related macular degeneration with rheohaemapheresis, Clin Hemorheol Microcirc 50 (2012), 245–255. [4] H.C. Cheng, C.M. Chan, H.S. Tsay, H.J. Liang, Y.C. Liang and D.Z. Liu, Improving effects of epigallocatechin-3-gallate on hemorheological abnormalities of aging Guinea pigs, Circ J 71 (2007), 597–603. [5] G.C. Chiou, Pharmacological treatment of dry age-related macular degeneration (AMD), Taiwan J Ophthalmol 1 (2011), 2–5. [6] T.A. Ciulla, A. Harris, H.S. Chung, R.P. Danis, L. Kagemann, L. McNulty, L.M. Pratt and B.J. Martin, Color Doppler imaging discloses reduced ocular blood flow velocities in nonexudative age-related macular degeneration, Am J Ophthalmol 128 (1999), 75–80. [7] T.A. Ciulla, A. Harris and B.J. Martin, Ocular perfusion and age-related macular degeneration, Acta Ophthalmol Scand 79 (2001), 108–115. [8] L. Dintenfass, Red cell aggregation in cardiovascular diseases and crucial role in inversion phenomena, Angiology 36 (1985), 315–326. [9] E.D. Goldberg, A.M. Dygai, V.V. Udut, S.A. Naumov and I.A. Khlusov, Patterns of structural organization of the life support systems in normal and pathological process development, Izd. Tom. un-ta, Tomsk, 1976 [in Russian]. [10] J. Goldberg, G. Flowerdew, E. Smith, J.A. Brodyand and M.O.M. Tso, Factors associated with age-related macular degeneration, Am J Epidemiol 128 (1988), 700–710. [11] T. Kato, Relation between blood viscosity and hematocrit in rabbits subjected to repeated treatment with endotoxin, Resuscitation 21 (1991), 61–66. [12] E.A. Kemeleva, O.I. Sinitsyna, K.A. Conlon, M. Berrios, N.G. Kolosova, D.O. Zharkov, E.A. Vasyunina and G.A. Nevinsky, Oxidation of guanine in liver and lung DNA of prematurely aging OXYS rats, Biochemistry (Mosc) 71 (2006), 612–618. [13] R. Klingel, C. Fassbender, I. Fischer, L. Hattenbach, H. G¨umbel, J. Pulido and F. Koch, Rheopheresis for age-related macular degeneration: A novel indication for therapeutic apheresis in ophthalmology, Ther Apher 6 (2002), 271–281. [14] N.G. Kolosova, P.A. Lebedev, S.V. Aidagulova and T.S. Morozkova, OXYS rats as a model of senile cataract, Bull Exp Biol Med 136 (2003), 415–419. [15] N.G. Kolosova, T.V. Shcheglova, T.G. Amstislavskaya and L.V. Loskutova, Comparative analysis of LPO products in brain structures of Wistar and OXYS rats of different age, Bull Exp Biol Med 135 (2003), 593–596. [16] K. Kujawski, K. Kedziora-Kornatowska, M. Koter, P. Duchnowicz, J. Rysz, L. Markuszewski, R. Błaszczak, T. Kornatowski, G. Grze´sk, J. Kedziora and R. Olszewski, Evaluation of some selected structural and functional parameters of red blood cells as oxidative stress markers in elderly people, Pol Merkur Lekarski 19 (2005), 774–778. [17] P.L. Lip, A.D. Blann, M. Hope-Ross, J.M. Gibson and G.Y. Lip, Age-related macular degeneration is associated with increased vascular endothelial growth factor, hemorheology and endothelial dysfunction, Ophthalmology 108 (2001), 705–710. [18] L. Lu, S.F. Hackett, A. Mincey, H. Lai and P.A. Campochiaro, Effects of different types of oxidative stress in RPE cells, J Cell Physiol 206(1) (2006), 119–125. [19] A.M. Markovets, V.B. Saprunova, A.A. Zhdankina, A.Zh. Fursova, L.E. Bakeeva and N.G. Kolosova, Alterations of retinal pigment epithelium cause AMD-like retinopathy in senescence-accelerated OXYS rats, Aging (Albany NY) 3(1) (2011), 44-54. [20] G.I. Mchedlishvili, Disturbances of the normal structuring of the blood flow in the microvessels as the cause of hemorheological disorders, Fiziol Zh Im I.M. Sechenova 82 (1996), 41–47 [in Russian]. [21] K. Michalska-Malecka, L. Slowinska, M. Dorecka and W. Romaniuk, Correlations in some pathogenetic factors and values of hemorheological parameters in age-related macular degeneration, Clin Hemorheol Microcirc 38 (2008), 209–216. [22] V.V. Neroev, M.M. Archipova, L.E. Bakeeva, AZh Fursova, E.N. Grigorian, A.Y. Grishanova, E.N. Iomdina, Zh, N. Ivashchenko, L.A. Katargina, I.P. Khoroshilova-Maslova, O.V. Kilina, N.G. Kolosova, E.P. Kopenkin, S.S. Korshunov, N.A. Kovaleva, Y.P. Novikova, P.P. Philippov, D.I. Pilipenko, O.V. Robustova, V.B. Saprunova, I.I. Senin, M.V. Skulachev,

M.Y. Maslov et al. / Hemorheological parameters and their correlations in OXYS rats

[23] [24] [25] [26] [27]

[28] [29]

[30]

[31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42]

411

L.F. Sotnikova, N.A. Stefanova, N.K. Tikhomirova, I.V. Tsapenko, A.I. Shchipanova, R.A. Zinovkin and V.P. Skulachev, Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals, Biochemistry (Mosc) 73 (2008), 1317–1328. J.Z. Nowak, Age-related macular degeneration (AMD): Pathogenesis and therapy, Pharmacol Rep 58 (2006), 353–363. L.A. Obukhova, V.P. Skulachev and N.G. Kolosova, Mitochondria-targeted antioxidant SkQ1 inhibits age-dependent involution of the thymus in normal and senescence-prone rats, Aging (Albany NY) 1 (2009), 389–401. G. Pasquini, B. Albanese, P.G. Manescalchi and R. Morini, Relation of blood viscosity, plasma viscosity and hematocrit, Ric Clin Lab 13(Suppl 3) (1983), 327–331. M.B. Plotnikov, O.I. Aliev, A.A. Koltunov and M.Y. Maslov, Increased blood viscosity syndrome in SHR rats: Analysis of the model adequacy, Biull Eksp Biol Med 126 (1998), 150–151 [in Russian]. M.B. Plotnikov, O.I. Aliev, N.A. Tyukavkina and T.M. Plotnikova, Hemorheological Effects of Antioxidant Complex Comprising Dihydroquercetin and Ascorbic Acid (Ascovertin). A New Aspect of Antioxidants Application, in: Frontiers in Antioxidant Research, H.V. Panglossi, ed., Nova Science Publishers, New York, 2006, pp. 103–132. M.B. Plotnikov, G.A. Chernysheva, V.I. Smol’yakova, M.Y. Maslov, I.V. Cherkashina, A.P. Krysin, I.V. Sorokina and T.G. Tolstikova, Effect of n-tyrosol on blood viscosity and platelet aggregation, Bull Exp Biol Med 143 (2007), 61–63. M.B., Plotnikov, A.A., Koltunov, O.I. Aliev and B. Baskakova, Model of blood hyperviscosity syndrome in rats: Relationship between hemorheological indices, their informativeness during brain ischemia, Biull Eksp Biol Med 122 (1996), 274–275 [in Russian]. M.B. Plotnikov, V.I. Smolyakova, I.S. Ivanov, G.A. Chernisheva, A.V. Kuchin, I.J. Chukicheva and E.A. Krasnov, Hemorheological effects of ortho-isobornyl phenol derivative under conditions of brain ischemia in rats, Bull Exp Biol Med 149 (2010), 721–723. M.B. Plotnikov, O.I. Aliev, M.Yu. Maslov, A.S. Vasiliev and N.A. Tjukavkina, Correction of haemorheological disturbances in myocardial infarction by diquertin and ascorbic acid, Phytother Res 17 (2003), 86–88. A.M. Plotnikova, Z.T. Shulgau, T.M. Plotnikova, O.I. Aliev and T.G. Borovskaya, Hyperviscosity syndrome in ovariectomized rats, Bull Exp Biol Med 146 (2008), 92–95. Rat genome database: http://rgd.mcw.edu S. Shin, J.X. Hou, J.S. Suh and M. Singh, Validation and application of a microfluidic ektacytometer (RheoScan-D) in measuring erythrocyte deformability, Clin Hemorheol Microcirc 37 (2007), 319–328. N.A. Solovyeva, T.S. Morozkova and R.I. Salganik, Development of a rat subline with symptoms of hereditary galactosemia and study of its biochemical characteristics, Genetika 11 (1975), 63–71 [in Russian] J.F. Stoltz, M. Donner, S. Muller and A. Larcan, Hemorheology in clinical practice. Introduction to the notion of hemorheologic profile, J Mal Vasc 16 (1991), 261–270. The Eye Disease Case-Control Study Group Risk factors for neovascularage-related macular degeneration, Arch Ophthalmol 110 (1992), 1701–1708. M.O.M. Tso, Pathogenetic factors of aging macular degeneration, Ophthalmology 92 (1985), 628–635. V.Iu. Lishnevskaia, The role of free radicals oxidation in the deterioration of haemovascular homeostasis in aging, Adv Gerontol 13 (2004), 52–57. J.R. Vingerling, I. Dielemans, M.L. Bots, A. Hofman, D.E. Grobbee and P.T. de Jong, Age-related macular degeneration is associated with atherosclerosis. The Rotterdam Study, Am J Epidemiol 142 (1995), 404–409. H. Watanabe, A. Kobayashi, T. Yamamoto, S. Suzuki, H. Hayashi and N. Yamazaki, Alteration of human erythrocytes membrane fluidity by oxygen derived free radicals and calcium, Free Radical Biol Med 8 (1990), 507–514. A.A. Zhdankina, A.Zh. Fursova, S.V. Logvinov and N.G. Kolosova, Clinical and morphological characteristics of chorioretinal degeneration in early aging OXYS rats, Bull Exp Biol Med 146 (2008), 455–458 [in Russian].