Резюме
Пасєчнікова Н.В., Науменко В.О., Віт В.В., Пількевич Т.С. Структурні зміни сітківки залежно від концентрації еритропоетину в склоподібному тілі і периферичній крові щурів, через 3 і 6 місяців після відтворення стрептозотоцинового цукрового діабету.
Цукровий діабет (ЦД) - одне з найбільш поширених захворювань. Поширеність діабетичної ретинопатії (ДРП) серед хворих на ЦД становить 10-90%. Мета: вивчити особливість структурних змін сітківки залежно від концентрації еритропоетину в склоподібному тілі і периферичній крові щурів через 3 і 6 місяців після відтворення стрептозотоцинового цукрового діабету. Експериментальне дослідження проведено на 50 щурах (100 очей), породи Вістар, масою 240,5-270,0 гр. Всі тварини були розподілені на 3 групи: 1-а група - інтактні тварини (10 щурів), друга - тварини з модельованим цукровим діабетом (20 щурів), третя група - тварини, з модельованим цукровим діабетом, які отримували рекомбінантний еритропоетин (рЕПО) (20 щурів). ЦД у щурів моделювали одноразовим введенням внутрішньочеревно стрептозотоцина (SIGMA, США ) в дозі 65 мг на 1 кг ваги тіла. Починаючи з 10 -ї діби щурам третьої групи вводили підшкірно рЕПО 3 рази на тиждень по 6 ОД на 100 г маси тіла протягом 6 місяців. Введення тваринам з стрептозотоциновим діабетом протягом 3 і 6 місяців рЕПО призводить до статистично значимого підвищення концентрації еритропоетину в порівнянні з інтактними тваринами і з тваринами з цукровим діабетом, як в периферичної крові, так і в склоподібному тілі. Через 3 і 6 місяців при відтворенні стрептозотоцинового діабету у всіх тварин визначаються грубі структурні зміни сітчастої оболонки, істотно порушується гістоархітектоніка сітківки, виражені явища набряку внутрішнього плексиформного шару і атрофія сітківки. Через 3 місяці після початку експерименту при введенні рЕПО тільки у 4 щурів з 10 визначається незначний набряк гангліозних клітин, у решти 6 щурів структурних змін сітчастої оболонки не виявлено. При цьому порушення гістоархітектонікі всіх шарів сітківки не виявляється , а через 6 місяців після початку експерименту тільки у 6 щурів з 10 структурні зміни сітківки зводяться лише до набряку і вакуольної дегенерації частини гангліозних клітин , а у 4 щурів структурних змін сітчастої оболонки не виявлено. Через 3 і 6 місяців після початку експерименту при введенні рЕПО у всіх тварин судинна система сітківки збережена, а у всіх тварин з стрептозотоциновим діабетом з боку судинної системи сітківки виявляються гліальні проліферати, а також облітерація частини судин.
Ключові слова: стрептозотоциновий діабет, рекомбинантний еритропоетин, діабетична ретинопатія.
Резюме
Пасечникова Н.В., Науменко В.А., Вит В.В., Пилькевич Т.С. Структурные изменения сетчатой оболочки в зависимости от концентрации эритропоэтина в стекловидном теле и периферической крови крыс, спустя 3 и 6 месяцев после воспроизведения стрептозотоцинового сахарного диабета.
Сахарный диабет (СД) — одно из наиболее распространенных заболеваний. Распространенность диабетической ретинопатии (ДРП) среди больных СД составляет 10–90 %. Цель: изучить особенность структурных изменений сетчататой оболочки в зависимости от концентрации эритропоэтина в стекловидном теле и периферической крови крыс спустя 3 и 6 месяцев после воспроизведения стрептозотоцинового сахарного диабета. Экспериментальное исследование проведено на 50 крысах (100 глаз), породы Вистар, массой 240,5-270,0 гр. Все животные были распределены на 3 группы: 1-я группа – интактные животные (10 крыс), вторая – животные с моделированным сахарным диабетом (20 крыс), третья группа - животные, с моделированным сахарным диабетом, получавшие рекомбинантный эритропоэтин (рЭПО) (20 крыс). СД у крыс моделировали одноразовым введением внутрибрюшинно стрептозотоцина (SIGMA, США) в дозе 65 мг на 1 кг веса тела. Начиная с 10-х суток (период стабильной гипергликемии) крысам третьей группы вводили подкожно рЭПО 3 раза в неделю по 6 ЕД на 100 г массы тела в течении 6 месяцев. Введение животным со стрептозотоциновым диабетом на протяжении 3 и 6 месяцев рЭПО приводит к статистически значимому повышению концентрации эритропоэтина по сравнению с интактными животными и с животными с сахарным диабетом, как в периферической крови, так и в стекловидном теле. Через 3 и 6 месяцев при воспроизведении стрептозотоцинового диабета у всех животных определяются грубые структурные изменения сетчатой оболочки, существенно нарушается гистоархитектоника сетчатки, выражены явления отека внутреннего плексиформного слоя и атрофия сетчатки. Через 3 месяца после начала эксперимента при введении рЭПО только 4 крыс из 10 определяется незначительный отек ганглиозных клеток, у остальных 6 крыс структурных изменений сетчатой оболочки не выявлено. При этом нарушения гистоархитектоники всех слоев сетчатки не обнаруживается, а через 6 месяцев после начала эксперимента только у 6 крыс из 10 структурные изменения сетчатки сводятся лишь к отеку и вакуольной дегенерации части ганглиозных клеток, а у 4 крыс структурных изменений сетчатой оболочки не выявлено. Через 3 и 6 месяцев после начала эксперимента при введении рЭПО у всех животных сосудистая система сетчатки сохранена, а у всех животных со стрептозотоциновым диабетом со стороны сосудистой системы сетчатки обнаруживаются глиальные пролифераты, а также облитерация части сосудов.
Ключевые слова: стрептозотоциновый диабет, рекомбинантный эритро-поэтин, диабетическая ретинопатия.
Summary
Pasechnikova N.V, Naumenko V.A, Vit V.V., Pilkevich T.S. Structural changes in the retina, depending on the concentration of erythropoietin in the vitreous body and the peripheral blood of rats after 3 and 6 months after streptozotocin-induced diabetes simulation.
Diabetes mellitus (DM) is one of the most common diseases. The prevalence of diabetic retinopathy among diabetic patients is 10-90%. To study the feature of structural changes in the retina depending on the concentration of erythropoietin in the vitreous humor and peripheral blood of rats after 3 and 6 months after streptozotocin-induced diabetes simulation. Experimental study was performed on 50 Wistar  rats (100 eyes), weighted 240.5-270.0. All animals were divided into 3 groups: Group 1 - intact animals (10 rats), Group 2 - diabetes mellitus simulated animals (20rats), Group 3 - animals with simulated diabetes treated with recombinant erythropoietin (rEPO) (20 rats). SD rats simulated disposable intraperitoneal administration of streptozotocin (SIGMA, USA) at a dose of 65 mg per 1 kg of body weight. Starting from the 10th day (the period of stable hyperglycemia) the rats of the Group 3 were injected subcutaneously rEPO 6 units per 100 g of body weight three times per week for 6 months. REPO administration to streptozotocin-induced diabetic animals for 3 and 6 months resulted in a statistically significant increase in the concentration of erythropoietin compared to the intact animals and animals with diabetes mellitus in the peripheral blood and in the vitreous. After 3 and 6 months after streptozotocin-induced diabetes simulation there was determined serious structural changes in the retina in all animals, histoarchitectonics of the retina was significantly disrupted, there were expressed signs of swelling of the internal plexiform layer and retinal atrophy. 3 months after the experiment had began only in 4 of 10 rats receiving rEPO there was determined mild swelling of ganglion cells, in other 6 rats the structural changes of the retina were not identified. Herewith histoarchitectonics violations in all layers of the retina were not noted. And 6 months after the start of the experiment only 6 out of 10 rats had retinal structural changes reduced only to edema and vacuolar degeneration of the ganglion cells, and in 4 rats the structural changes of the retina were not identified. After 3 and 6 months after the beginning of the experiment when administered rEPO all animals kept the retinal vascular system, and all streptozotocin-induced diabetic animals had vascular retinal glial proliferates and obliteration of blood vessels.
Key words: streptozotocin-induced diabetes, recombinant erythropoietin, diabetic retinopathy.
Рецензент: д.мед.н., проф. А.М. Петруня

УДК 617.735:616-076.4:616.379-008.64-085-092

ГУ "Институт глазных болезней и тканевой терапии им. В.П.Филатова НАМН Украины"

SI “The Filatov Institute of Eye Diseases and Tissue Therapy of the National Academy of Medical Sciences of Ukraine”

tassya1979@yandex.ua

Introduction: Diabetes mellitus (DM) and its complications is one of the most important medical, social and economic problems of modern health care. The prevalence of diabetic retinopathy (DRP) among diabetic patients is 10-90% [1]. The literature shows conflicting information about the role of erythropoietin (EPO ) in the development and progression of the DRP. Erythropoietin (EPO) is produced mainly in the kidney and to a lesser degree in the liver (from 5% to 15%), but today, and other lesions identified erythropoietin production: brain, uterus, retina, in which the expression of EPO has also tissue specificity [3,4,5,6]. The main factor governing the production of EPO is hypoxia, hypoxia in the amount of circulating plasma EPO increases approximately 1000 times [3,4,7]. It is proved that the use of EPO has a protective effect on ischemia - reperfusion in various organs and tissues, including the brain, spinal cord, kidney, muscle tissue vessels, heart [ 8,9,10,11,12,13 ]. According to some authors increased production of EPO in the aqueous humor of patients with diabetes and the presence of diabetic retinopathy has a protective nature due to the powerful antioxidant and neuroprotective effects and anti-ischemic action [15]. But there are other data showing that EPO may be an inducer of angiogenesis and thereby worsen diabetic retinopathy [2].

Based on our previous studies it has been shown that rEPO administration subcutaneously into streptozotocin-induced diabetic animal for one month resulted in a significant increase in the concentration of EPO in the peripheral blood as well as in the vitreous body, as compared to animals with simulated diabetes mellitus and intact animals. When administrating erythropoietin, changes of the sensory retina consist only in a slight swelling of the ganglion cells, while violations of histoarchitectonics in all layers of the retina are not detected.

Objective: To study the features of structural changes of the retina depending on the concentration of erythropoietin in the vitreous humor and peripheral blood of rats after 3 and 6 months after streptozotocin-induced diabetes simulation.

Material and methods. Experimental study was conducted on 270 rats. The Group 1 of intact animals included 10 rats. The Group 2 (rats with diabetes simulated) consisted of 141 rats. The Group 3 (streptozotocin-induced diabetic rats with rEPO administration) included 119 rats. DM was simulated in rats by disposable intraperitoneal administration of streptozotocin (SIGMA, USA) at a dose of 65 mg per 1 kg of body weight.

First symptoms of diabetes appeared three days after streptozotocin administration in all rats of the second and third group. There was an increase in blood glucose levels to an average of 10.0 mmol / l, and increased water intake with a constant increase in diuresis. Starting from the 10th day (the period of stable hyperglycemia) [14] the rats of the third group were injected subcutaneously rEPO 6 units per 100 g of body weight three times per week for 6 months.

As shown in our earlier published articles within the first 3 months the death loss in the second and the third groups was 79 and 38 animals, respectively. 40 animals were euthanized. After 3 months the death loss in the second and the third groups was 18 rats each and 20 rats were euthanized. After 5 months death loss in the second and third groups was 4 and 23 animals, respectively (that we can explain by the side effects of erythropoietin, namely thrombosis due to increased amount of red blood cells). Before the experiment animals were kept in quarantine for two weeks. Animals were kept under standard vivarium conditions. The experiment was conducted with the implementation of ethical standards stipulated by the international principles of the European Convention "On Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" (Strasbourg, 1985) norms of bio- medical ethics approved by the First National Congress of Ukraine on Bioethics (2001) as well as the Law of Ukraine № 3447 -IV «About Zahist Tvaryny od zhorstokogo povodzhennya" (Kiev, 2006).

This part of the experimental study conducted on 50 Wistar  rats (100 eyes) weighted 240.5-270.0 g, which were divided as follows: the Group 1 - intact animals (10 rats), the Group 2 - streptozotocin-induced diabetic animals (20 rats), the Group 3 -  streptozotocin-induced diabetic animals receiving recombinant EPO (rEPO) (20 rats).

Animals were taken out of the experiment at 3 and 6 months after the beginning of the experiment by decapitation under ether anesthesia, after which the minimum period conducted fence 5 ml of blood and eye enucleation. Excretion of experimental animals was conducted simultaneously.

The eyeballs of animals were fixed in 10% formalin for 24-48 hours, then standard processing technique generally produces a material (material conclusion paraffin, staining with hematoxylin and eosin sections). Ultrastructural studies were carried out on the retina of 4 experimental animals (2 retinas when observing the animals at 3 months and 2 - six months). For electron microscopic examination tissue pieces were fixed in 2.5% glutaraldehyde solution in a ph osphate buffer at pH - 7.4 with subsequent fixation 1% osmic acid solution with the same buffer solution. The samples were then dehydrated in alcohols of increasing strength. Impregnation of the material and its conclusion was made in a mixture of Epon - Araldite. Then ultrathin sections were contrasted by the Reynolds method. Sections were examined and photographed with an electron microscope TEM -100- 01. Histomorphological and ultrastructural studies were carried out on the basis of Laboratory of Pathomorphology and Electron Microscopy using a microscope Jenamed 2.

Erythropoietin levels in plasma and vitreous humor were determined by enzyme immunoassay. For this purpose, the plates for the quantitative measurement of erythropoietin in serum, plasma and other biological fluids of rats (E90028 Ra 96 Tests Enzym-Linked Immunosorbent Assay Kit for Erythropoetin, made in USA) were used.

Statistical processing of data was performed using the statistical software Statistica 9.0. To analyze the quantitative differences in the two groups we used a parametric analysis of variance (ANOVA) with a preliminary estimate of normality by the Shapiro-Wilks. Multiple Newman-Keylse comparison was used after conducting an analysis of variance test.

Results. In the study of the concentration of EPO in the peripheral blood of  streptozotocin-induced diabetic rats after 3 and 6 months, we found a significant increase in its level compared to intact animals (31.8 ± 1.3 pg / ml) and p = 0.002 (29.0 ± 0.96 pg / ml) p = 0.003, respectively (tab.1).

Table 1

The concentration of EPO in the peripheral blood and the vitreous body in rats with diabetes of 3 and 6 months duration

As seen from Table 1, the level of erythropoietin in the study of the vitreous in streptozotocin-induced diabetic rats after 3 and 6 months from the start of the experiment, we found a significant increase in its concentration (405.8 ± 12.1 pg / ml) and p = 0.001 (389.1 ± 10.04 pg / ml), p = 0.003, respectively, compared to intact animals (331.6 ± 21.8). However, it should be noted that there was no significant increase of the concentration of EPO in the peripheral blood and in the vitreous humor in the group of animals with respect 3 and 6 months.

In streptozotocin-induced diabetic rats with rEPO administration after both 3 and 6 months in the peripheral blood there was a significant increase in erythropoietin level (49.4 ± 0.7 pg / ml) and p = 0.000 (43.0 ± 1.43 pg / ml) p = 0.0001, respectively, compared to the intact animals and animals with diabetes simulated (tab.1).

When investigating the level of erythropoietin in the vitreous in streptozotocin diabetic rats receiving  rEPO after both 3 and 6 months from the start of the experiment , there was a significant increase in its concentration (566.3 ± 5.3 pg / ml) p = 0.001 (476.9 ± 4.65 pg / ml), p = 0.0001, respectively, compared to intact animals and animals with diabetes simulated (table 1). When comparing the concentration of EPO in the peripheral blood of rats with streptozotocin diabetes receiving rEPO relatively 3 and 6 months, a significant increase in the concentration of EPO in the peripheral blood was not noted. However, in this group during the same period there was a statistically significant increase in the EPO concentration in the vitreous body.

Structural changes in the retina

3 months after the DM simulation without rEpo administration there was microscopically detected focal swelling and thinning of the inner plexiform layer, vacuolar degeneration of the ganglion cells and a decrease in their number in the retina of all animals examined. Part of ganglion cells had pyknosis. Noteworthy is thinning of the nerve fiber layer, focal destruction of the internal limiting membrane (fig. 1 and 2).

Fig. 1. Light microscopy of structural changes in the retina 3 months after DM simulation without rEPO. Hematoxylin and eosin. x 200

Fig. 2. Electron microscopy of structural changes in the retina 3 months after DM simulation without rEPO. x 2000

Certain structural changes were detected in the retina of the vascular system. They were reduced to the degeneration of the endothelial lining of the capillary vessels, some disintegration of the wall of the capillary vessels and diapedesis of blood into the surrounding tissue (Fig. 3).

Fig. 3. Electron microscopy of the capillary vessels of the retina after 3 months of playing without rEPO model.

After 6 months, all animals with experimental diabetes, untreated rEPO had deepening processes of neuronal degeneration of the retina, especially the layer of ganglion cells (Fig.4,5). Histoarchitectonics of the retina was significantly disrupted and there were expressed signs of swelling of inner plexiform layer and retinal atrophy. Part of the vessels were obliterated and glial proliferates were noted.

Fig. 4. Light microscopy of the retina after 6 months after MD simulation without rEPO. Hematoxylin and eosin. x 120.

Fig. 5. Electron microscopy of the retina after 6 months after MD simulation without rEPO. x 2000.

3 months after simulation of experimental streptozotocin-induced diabetes and recombinant EPO administration all animals had retinal histoarchitectonics preserved. Of the 10 animals only four had moderate edema of inner layers of retina. Insignificant part of the ganglion cells was in a state of vacuolar degeneration of moderate severity. And the cells increased in size, nuclei were light and vesicular (Fig.6,7). The other 6 animals did not have changes in the retina. It should be noted that the structure of blood vessels in all 10 animals was without significant structural changes (Fig. 8).

Fig. 6. Light microscopy of the retina 3 months after streptozotocin-induced diabetes simulation and rEPO administration. Hematoxylin and eosin. x 200.

Figure 7. Electron microscopy of the retina 3 months after streptozotocin-induced diabetes simulation and rEPO administration. x 1800

Fig. 8. Electron microscopy of the retina of 3 months after streptozotocin-induced diabetes simulation and rEPO administration. x 5000.

6 months after the beginning of the experiment, 4 animals injected rEPO did not have any changes in the retina, and 6 of 10 animals had only edema and vacuolar degeneration of the ganglion cells (Fig. 9, 10). It should be noted that the vascular system of the retina was preserved in all ten animals (Fig. 11).

Fig. 9. Light microscopy of the retina 6 months after streptozotocin-induced diabetes simulation and rEPO administration. Hematoxylin and eosin. x 120.

Fig. 10. Electron microscopy of the retina of 6 months after streptozotocin-induced diabetes simulation and rEPO administration x 4000.

Fig. 11 Electron microscopy of the retina of 6 months after streptozotocin-induced diabetes simulation and rEPO administration x 8000.

Discussion: In streptozotocin-induced diabetic rats within follow up period of 3 and 6 months, we observe a significant increase in the concentration of EPO in the peripheral blood as well as in the vitreous body, it must be noted that we have not noted the increase in hemoglobin concentration in the peripheral blood during the entire experiment, and the number of erythrocytes is even reduced. After 3 months, the concentration of hemoglobin in the second group of animals was 145 g / l, in 6 months - 145.7 g / l. The erythrocyte amount after 3 months in this group of animals was 4.9 × 10¹², after 6 months – 3.7 × 10¹².

In streptozotocin-induced diabetic animals receiving rEPO there was a statistically significant increase in the concentration of EPO in the peripheral blood as well as in the vitreous body during the whole experiment. It should be noted that the concentration of reduced hemoglobin is already at 3 months after the start of the experiment, only increasing the amount of red blood cells throughout the experiment, but a reduced amount of erythrocytes contained hemoglobin and their size was smaller than normal. This phenomenon is attributed to the insufficient intake of iron, so the red blood cells are untenable. After 3 months, the concentration of hemoglobin in the group 3 animals reached 193.8 g / l, in 6 months - 197.1 g / liter. The erythrocyte amount after 3 months in this group of animals was 10.6 × 10¹², after 6 months – 11.9 × 10¹².

There is a direct link between the maintenance of the concentration of EPO in the blood and vitreous and safety of the retina of the structural elements. Thus, as a  result of our experiment, we can assume that erythropoietin may have a direct effect on cell preservative elements of the retina, resulting in reducing apoptosis that was detected by Z. Cai, DJ Manalo, G. Wei (2003) in studying brain neurons.

Conclusions.

1. In animals with streptozotocin-induced diabetes at 3 and 6 months after the beginning of the experiment, there is a statistically significant increase in the concentration of erythropoietin, as compared to intact animals, both in the peripheral blood and in the vitreous.
2. Recombinant erythropoietin administration into animals with streptozotocin-induced diabetes for 3 and 6 months leads to statistically significant increase in the concentration of erythropoietin compared to the intact animals and animals with diabetes mellitus in the peripheral blood as well as in the vitreous.
3. 3 months after the experiment start when administrating rEpo in 6 animals of 10 structural changes in the retina are not detected and only 4 rats have mild swelling of ganglion cells. Herewith violation of histoarchitectonics in all layers of the retina is detected, and 6 months after the beginning of the experiment, 4 of 10 rats do not have structural changes in the retina and only 6 rats have. The structural changes in the retina are reduced only to edema and vacuolar degeneration of the ganglion cells.
4. After 3 and 6 3 months after streptozotocin-induced diabetes simulation and rEPO administration in all animals there are serious structural changes in the retina: histoarchitectonics of the retina is significantly disrupted and there are expressed signs of swelling in inner plexiform layer and retinal atrophy.
5. After 3 and 6 months after the beginning of the experiment and rEPO administration in all animals the retinal vascular system was preserved, and all animals with streptozotocin-induced diabetes had glial proliferates of vascular retinal system and obliteration of blood vessels.

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