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Khurshidakhon Abduboriyevna Rasulova, PhD, Senior Researcher of Department of Neurology, pediatric neurology and medical genetics, Head of Department of coordination of research activity of Tashkent Pediatric Medical Institute. E-mail: [email protected] Botir Turgunpulatovich Daminov, Doctor of Medical Sciences, Professor, rector of Tashkent Pediatric Medical Institute.
Marina Ilyinichna Chiniyeva, Junior Researcher of Tashkent Pediatric Medical Institute.
Morphological changes in kidneys after experimental acute brain ischemia in rats
Abstract: The morphological study of the kidneys and the brain after acute brain ischemia in rats proved the existence of angio-cerebro-renal relationships due to hemodynamic disorders and endothelial dysfunction. Considering the secondariness and the mediation of renal injury in experimental ischemic stroke, angio-cerebro-renal dysfunction in ischemic stroke may be caused by violation of central regulation, vascular-hemodynamic disorders and general systemic inflammatory response. Keywords: ischemic stroke, kidneys, rats, experiment.
Introduction. Stroke is the leading cause of death and disability among the working population in most countries of the world. In the United States annually about 700 thousand people suffer an ischemic stroke, while in Russia this rate is more than 450 thousand people, and in Uzbekistan — more than 40 thousand people. About 15% of stroke patients die within first weeks of the disease, the majority of survivors after stroke lose the ability to work and in need of constant care [7; 9; 19]. This makes stroke an urgent problem of not only medical, but also socio-economic significance.
The role of kidneys in stroke has not been well studied. It is known that the pathogenesis of stroke is closely associated with hypertension, heart diseases and atherosclerosis, which are among the most important causes of acute disorders of cerebral hemodynamics [2]. In turn, renal disorders are the important risk factor for cardiac and cerebrovascular complications [6]. Population-based and epidemiological studies have shown that even the earliest subclinical disorders in renal function are suggested as an independent risk factor for cardiac and cerebrovascular complications, as well as for repeated violations and mortality. To date, several main controlled trials have been conducted that assessed the relationship of the severity of chronic kidney disease (CKD) with risk of development of cardiovascular complications and mortality [5; 18].
The brain, heart and kidneys in the human body function independently from each other. However, high consumption of oxygen and energy is common for them that determines their high sensitivity to hypoxia [12; 16]. Vascular system performs a communicative function, and any changes of the vascular wall (endothelium), changing the indicators of central and peripheral hemodynamics, would contribute to the changes of transcapillary exchange, development of hypoxia and hypotrophy of tissues [8]. In turn, at local necrotic processes in brain ischemia, decay products, entering the blood, circulate, contributing to the development of response of the endothelium, and are excreted by the kidneys that can lead to changes of the renal parenchyma, i. e. worsening the course of both primary and secondary damage of organs and tissues [2]. At the same time, polypharmacy and a large number of drugs used in stroke, their inadequate use, complicate the kidneys function that, in general, affects the state of stroke patients and impairs the prognosis.
It is known that the study of mechanisms of disease in various experimental models contributes to solving medico-social problems arising in connection with the prevalence of stroke. However, there is no sufficient data concerning morphological changes in the kidney after ischemic stroke in the available literature that prompted us to conduct the present study.
The purpose of the study was to investigate the morphological status ofkidneys in the model of experimental ischemic stroke in rats. Materials and Methods Production of experimental acute brain ischemia Forty-two male outbred white rats weighing 220-280 g. at the age of 4-7 months were taken for the experiments. Rats were kept in vivarium conditions with free access to food and water. 21 rats (main group) were anesthetized with etaminal (50 mg/kg, i. p.). Anesthetized rats were laid on their back and a midline neck incision made. The left common carotid artery was carefully exposed and isolated. Then, the artery was doubly ligated with 3-0 silk suture for 40 minutes with subsequent reperfusion and full restoration of cerebral blood flow by elimination of the ligature and wound closure. In 10 rats under the same anesthesia, skin incision of the neck over the carotid artery was made followed by suturing of the skin (false-operated group). 11 rats were intact. After the surgical operation, the rats were maintained under an infrared heat lamp until awake to avoid a decline in body temperature.
The animals care and the experiments were conducted in accordance with the requirements of International rules for the humane treatment of animals "Guide for the Care and Use of Laboratory Animals" as contained in the Sanitary rules for equipment and maintenance of experimental biological clinics (vivariums) and conducted in compliance with the rules adopted in the European Convention for the protection of laboratory animals used for experimental and other scientific purposes (ETS N 123), Strasbourg (18.03.1986). All experimental procedures involving animals were approved by the Institutional rules for laboratories, vivariums, animal care and use.
Assessment of neurological state of animals The animals were observed daily for 7 days. The Stroke-index MC Graw scale was used to assess the neurological status of animals
at 1-2 hours after surgery and in dynamics at the 1st, 3rd and 7th days of the experiment.
Morphology, microscopic preparations and images taking At 1st, 3rd and 7th days after acute brain ischemia, the animals decapitated under ether anesthesia. Then, the brains and kidneys were removed. In accordance with the periods of observation, the main group of rats was divided into 3 groups of 7 animals each. The pieces of brain and kidney tissues no more 1x1 cm were immediately immersed in 2.5% solution of glutaraldehyde for fixation with subsequent wiring according to standard methods in alcohols of increasing concentration. After embedding in Epon-Araldite mixture (Fluka AG, Araldite Accelerator 964 + Epon Hardener DDSA, CH-9470, "Buchs", Switzerland). Ultrathin sections were cut on the ultramicrotome LKB-V ("Broma", Sweden). These were used for methylene blue and fuchsin staining for morphological evaluation. After staining, formed microscopic images of the studied tissues were taken with the aid of light-optical microscopy on the microscope "Mikromed-2" (Russia) with fixed digital camera Scope Tek DCM-510 (USB 2.0) ("Leitz", Germany) connected to a computer Pentium-4 with the pre-installed software "Scope Photo". The images were printed on a color printer "Epson". Results of the study and their discussion Neuronal cell death and neurological state of the rats after acute brain ischemia
At the day of surgery, in rats were objectively observed weakness and hypotonia of the limbs on the contralateral side to the lesion, narrowing of the palpebral fissure (voluptas) on the side of the lesion. The animals could not rest on the feet, did not respond to the injections on the side of paresis, lost the appetite, became sloppy, careless and aggressive. One week after the operation, 9.52% of rats died and the surviving animals showed neuronal deficit of different severity.
At the first day after acute brain ischemia, the evaluation of neurological state in rats showed that nearly all (90-100%) animals had moderate neurological deficit in the form of sluggishness and slowness of movements. Significant neurological impairment, manifested in the form of manege movements in a circle and paralysis of the limbs, was observed in 30-40% of cases. Neurological deficit gradually increased in a time-dependent manner by the 7th day after the experiment.
It is reported that in the absence of hypotension, occlusion of common carotid artery generally does not cause neuronal cell death. However, other studies show that common carotid artery occlusion in Slc/Wistar rats induces neuronal cell death due to the patency of the posterior communicating arteries [11]. Thus, acute cerebrovascular occlusion, even if subsequent reperfusion made, induced failure of the cerebral circulation and acute neuronal cell death in the brains of adult rats.
Morphological investigations showed that at the first hours after acute brain ischemia pathological changes of neurons in the brain were characterized by polymorphism. Chromolysis of varying severity was noted. Brain edema, manifested by swelling and increase in the sizes and pallor ofneurons, as well as by occurrence ofpale extracellular fields of neuroglia, was revealed. Changes in the brain affect mainly separate nerve cells and blood vessels. These changes fit into the conventional morphological pattern of experimental ischemic stroke, the model of which was reproduced by the common standard methodology [1]. Morphological signs of the kidneys function after acute brain ischemia Morphological structure of the kidneys after acute brain ischemia differed from that of the intact and false-operated rats. Macroscopic study of false-operated animals marked that kidneys visu-
ally were bean-shaped, capsule was smooth, shiny, easily removed, exposing a smooth surface of kidneys. After cut, renal tissue was reddish-brown, the boundary between the layers of the kidneys was clear. There was a picture of glomerulonephritis and pyelonephritis as the result of an inflammatory response in 90% of rats in the main group.
Microscopically, already at the 1st day of acute brain ischemia, hypertrophy of the glomeruli, focal or segmental proliferation of mesangial cells was revealed in the kidneys (Image 1). This, perhaps, indicates inflammatory reaction in response to hemodynamic disturbances.
Changes in the tubulointerstitial apparatus were not less important. In 100% of cases the lumens of the proximal and distal tubules contained infiltrates ofunknown etiology.
Image 1. Kidney of rats at the 1st day of acute brain ischemia. Hypertrophy of glomeruli, focal and segmental proliferation of mesangial cells. Staining with methylene blue and fuchsin. Magnification: Ob. 40. Oc. 10
Production of cytokines (activating fibroblasts) with lymphocytes infiltration of the glomerulus, the erosion of the basal membranes of tubules and glomerulus and the ingress of infiltration of the glomerulus into the urinary space were observed (Image 2). We hypothesize that the appearance of infiltration may be due to cross-reactive antibodies to the basal membrane of tubules and glomeruli. This proves the involvement of immunological mechanisms in the kidney damage.
Image 2. Kidney of rats at the 1st day of acute brain ischemia. The violation of the proliferation of mesangial cells (2). Production of cytokines (activating fibroblasts) with lymphocytes infiltration of the glomerulus (1). The erosion of the basal membranes of tubules and glomerulus and the ingress of infiltration of the glomerulus into the urinary space (3). Staining with methylene blue and fuchsin. Magnification: Ob. 40. Oc. 10
The glomerulus damaged in 100% of animals at the 1st day after acute brain ischemia. In mesangial area were observed deposition of fibrin. This further leads to damage of the epithelium of the capillaries. The capillaries were significantly enlarged, erythrocytes were visible. There was vascular damage in the intertubular region (Image 3).
Image 3. Kidney of rats at the 1st day of acute brain ischemia. Glomerular damage. Deposition of fibrin in mesangial area (2). Significant dilation of the capillaries of the glomerulus with visible erythrocytes (1). Vascular damage the intertubular region (3). Staining with methylene blue and fuchsin.
Magnification: Ob. 40. Oc. 10
At this period, there was marked vasodilation of the capillaries of the glomerulus with erythrocytes aggregation like "coin columns" (Image 4). This was an evidence of the violation of blood rheology in the vascular system, particularly in the microvasculature. Erythrocytes aggregation creates favorable conditions for thrombus formation and deceleration of blood flow. On the other hand, erythrocytes aggregation is a compensatory response and lead to a faster delivery of oxygen to those tissues, which are in need of its presence. Because particularly erythrocytes carry oxygen, and their aggregation promotes oxygen rapid transportation to extensive system of capillaries. Slowing of blood flow at erythrocytes aggregation was noted in venules, whereas in the arterioles the resistance to blood flow was much less that promotes more rapid movement of the conglomerate of erythrocytes (columns) with oxygen in the desired direction. Under these conditions, the aggregation abilities of erythrocytes have a positive effect on the outcome of the disease.
Image 4. Kidney of rats at the 1st day of acute brain ischemia. Vasodilation of the capillaries of the glomerulus with erythrocyte aggregation like "coin columns". Staining with methylene blue and fuchsin. Magnification: Ob. 40. Oc. 10
At the 3rd day of the experiment, we found that after acute brain ischemia in the kidneys were observed dissolution of the
basal membrane of the glomerulus and the migration of macrophages into the urinary space (Image 5). This lead to the appearance of the lymphocytic-macrophage infiltration in the capsule of Shumlyansky-Bowman.
Image 5. Kidney of rats at the 3rd day of acute brain ischemia. Infiltration in the urinary space (1). Dissolution of the basal membrane and migration of macrophages into the urinary space (2). Staining with methylene blue and fuchsin.
Magnification: Ob. 40. Oc. 10
Simultaneously, there was destruction of the vascular glomerulus, significant gaps of the basal membrane of the capillaries of the glomerulus. There were determined zones of fibroid necrosis in the loops of the glomerulus (Image 6).
Image 6. Kidney of rats at the 3rd day of acute brain ischemia. Destruction of the vascular glomerulus. Significant
gaps of the basal membrane of the capillaries of the glomerulus (a). Zones of fibroid necrosis in the loops of the glomerulus (a). Staining with methylene blue and fuchsin.
Magnification: Ob. 40. Oc. 10 At the 7th day, there was complete disappearance of the glomerulus and its replacement by fibrin (Image 7). 70% of animals observed a picture of acute renal failure (shock kidney), which was associated with impaired blood supply, ischemia of the kidneys with subsequent necrosis of the epithelium of the renal tubules and development of acute renal failure — uremia. At shock kidney, disorders of blood circulation and reduction of water filtration in the renal tubules cause oliguria and anuria, followed by uremia.
Thus, morphological changes in the kidneys after acute brain ischemia demonstrated the picture of nephrosclerosis. The most typical changes were in the blood vessels and the tubuloin-terstitial apparatus. In the glomeruli, changes were characterized by ischemic disorders (thickening and wrinkling of the capillary walls with a gradual loss of permeability and formation of ischemic
wrinkling), proliferation of mesangial matrix with subsequent formation of focal and segmental hyalinosis and sclerosis. These structural changes were considered by several authors as morphological markers of hypertensive renal damage [3; 10; 13; 14; 15]. There were infiltrates of different origin and changes of peritubular capillaries in the tubules. Scarring, atrophy of prostaglandinsynthase cells of the renal medulla were almost always present in the interstices. Analyzing the data of the literature [10; 17] and our own observations, we can conclude that sclerosis identified in the renal intersti-tium reflects the loss by the kidney its depressant properties and is essential morphological substrate of hypertension and ischemia.
Image 7: Kidney of rats at the 7th day of acute brain ischemia. Global defeat of the glomerulus and its replacement by fibrous formation. Staining with methylene blue and
fuchsin. Magnification: Ob. 40. Oc. 10 The mechanism of structural damage of the kidneys at the present stage appear to be consistent with the following processes in the kidney. Increased hydrostatic intraglomerular pressure, which is transmitted uniformly in all directions, leads to the loss of negative charge and violation of the permeability of the basal membrane of glomeruli [13; 14]. Prolonged exposure of the increased hydrostatic intraglomerular pressure on the mesangium area leads to the deposition of low molecular weight proteins and albumin in it. This causes expansion of the mesangium and proliferation of mesangial cells, as well as destruction of small processes of the podocytes. Fur-
ther, this leads to the development of focal and segmental hyalinosis and sclerosis [10].
The studies ofAmerican researchers [4] have shown that hemo-dynamic changes lead to hyperfiltration, which is a mechanism of adaptation when reducing the number of functioning nephrons of different origin. Despite the considerable smaller mass of functioning renal parenchyma, the kidneys retain their essential functions and maintain homeostasis a certain time. It should be noted that the state of hyperfiltration characterizes not only extremely high values of glomerular filtration rate (GFR), but also no increase or decrease of GFR in response to stimulation. Hyperfiltration is an early sign of violations of intraglomerular hemodynamics. On the one hand, this process has a compensatory nature, on the other — damaging effect on glomerular structure and accelerating the development of pathological process in the kidney [10].
Conclusions
Thus, the harmful effect of brain ischemia on the kidneys is of no doubt. The kidney may act as a direct initiator and "behind the scenes director" of the development of acute brain ischemia or be on-target. Glomerular hyperfiltration, microalbuminuria, proteinuria, focal-segmental glomerulosclerosis, sclerosis of the renal in-terstitium may serve as morphological markers of the development of hyperperfusional renal damage. In this regard, the need for the use of pharmacological drugs that improve renal hemodynamics, preventing or slowing the progression of chronic renal failure, is obvious. Nephrosclerosis is the end point of continuous ischemia and hemodynamic disturbances. Severe damages of vital organstargets in stroke, particularly the kidneys, and difficulties of correcting their functions determine the urgency of the problem of effective nephroprotection in stroke patients.
Our data support the hypothesis of clear angio-cerebro-renal relationships and the similarity of structural changes in brain and renal tissues in rats after acute brain ischemia that caused by he-modynamic disorders and endothelial dysfunction. Considering the secondariness and the mediation of renal injury in experimental ischemic stroke, angio-cerebro-renal dysfunction in ischemic stroke may be caused by violation of central regulation, vascular-he-modynamic disorders and general systemic inflammatory response that is manifested as morphologically, as functionally.
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Shamsutdinova Elvira Faridovna, Republican Specialized Scientific Medical Center of Obstetrics and Gynecology E-mail: [email protected]
Sexual development and biochemical values of hepatic function in dynamics with background various methods of hormonal therapy in adolescent girls with congenital estrogen deficit
Abstract: The results of the performed research confirm the possibility of administration of both 17-beta estradiol and tefestrol in the complex of hormonal therapy for teenager girls with hypo gonadism. Patients with diseases of hepatic-billiary system and its chronic forms in history should prefer tefestrol in combination with didrogesterone Keywords: hypogonadism, female teenagers, hormone replacement therapy.
Disorder of sexual development can be caused by a wide range of pathological states, where, together with various congenital diseases of reproductive system, an important role is played by primary or secondary deficiency of ovarian function. Ovarian deficiency (hypo gonadism) is a pathological state, conditioned by decrease of estrogen level in organism (or weakening of effect in tissue), manifested by underdevelopment of internal and external sexual organs, no secondary sexual traits, and disorder of fertility [1]. We can isolate primary (hyper gonadotropic) hypo gonadism, caused by lesion of ovaries; and secondary (hypo gonadotropic) hypo gonadism, conditioned by decrease of gonadoliberin and\or gonadotropin secretion [3].
In spite of the diversity of clinical forms of estrogen deficiency states in girls today it is generally accepted that hyper gonadotropic and stable hypo gonadotropic hypo gonadism, conditioned by congenital diseases of hypothalamus and pituitary, requires long-term hormonal therapy, the aim of which is compensation of congenital deficit of sexual hormones. At least feminization of appearance and development of secondary sexual traits should be achieved [2].
The therapy with sexual hormones and similar agents in children and teenagers is peculiar, and it is preconditioned by good knowledge of pediatric and adolescent physiology, pharmacology, and pharmacodynamics ofvarious estrogen-containing agents. It is compulsory for optimal choice and success of the therapy. Hormonal therapy started in adolescence requires long term application on
one hand, and following safety rules, on the other. In the modern time in hormonal therapy in teenagers herbal agents are preferred due to softer effect, in comparison with its predecessors — synthetic estrogens. Prescription of these agents lead to significant improvement of psycho-emotional status of teenagers, formation of more complete self-perception of a person, it promotes correction of clinical symptoms of hypo gonadism. The choice of an agent for long-term administration should be done using forms with minimal amount of side-effects [4].
The objective of the research was assessment of the parameters of sexual development and several biochemical values of functional metabolic activity of liver in girls with hypo gonadism together with various methods of compensatory hormonal therapy.
Materials and methods of the research. We examined 42 patients in age groups from 13 to 15 and from 16 to 18 years old with hypo gonadotropic and hyper gonadotropic hypo gonadism, the parameters of sexual development of which had 3 years and more retardation (II-III degree of sexual development retardation). Patients of the 1 group (64 teenagers) administered an agent for traditional hormonal therapy, containing 17-beta-estradiol and didrogesterone (14 tablets, containing 2mg 17-beta-estradiol, 14 tablets containing 2mg of 17-beta-estradiol and 10mg of didrogesterone). Patients of the 2 group (60 teenagers) administered estrogen-like agent tefestrol in combination with 10mg of didrogesterone. All examined patients before and together with the therapy had clinical
