RU2248045C1 - Method for experimental modeling urinary calculosis - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: the present innovation deals with modeling urinary calculosis in rats due to injecting intraperitoneally 60%-glucose solution at 1 ml/100 g animal body weight twice daily for 2 mo. The method is very simple and enables to achieve lithogenesis in 25% experimental animals.

EFFECT: higher efficiency of experimental modeling.

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Description

The invention relates to experimental medicine and relates to the modeling of urolithiasis in rats.

Known methods for modeling diseases in laboratory animals using chemicals introduced in various ways [3, 4].

The purpose of the invention: experimental modeling of urolithiasis.

Description of the method. Male rats kept on a normal food and drink diet were injected 60% glucose at a rate of 1 ml per 100 g of animal weight twice a day (with an interval of 5-6 hours).

An indicator of the presence of stones in the bladder was chosen as a criterion for the development of urolithiasis. Animals were removed from the experiment after two months by decapitation.

This simulation method is simple to implement, does not require expensive reagents and allows stone formation in 25% of experimental animals (Fig. 1, 2).

The starting point for modeling stone formation (urolithiasis) in rats was data on the development of urolithiasis in individuals with severe decompensated diabetes mellitus. It is known that severe hyperglycemia is accompanied by dehydration and significant electrolyte disorders. These disorders lead to an imbalance in the fine regulation of mineral metabolism - the saturation of urine with insoluble substances, their crystallization, followed by aggregation and stone formation [1].

For successful modeling of stone formation in the urinary tract of rats, it was necessary to create certain conditions, in particular, severe and prolonged (up to several months) diabetic, hyperosmolar syndrome, through significant and prolonged hyperglycemia.

The objectives of this study included:

1) disrupt carbohydrate metabolism in experimental rats;

2) cause severe dehydration (hyperosmolar dehydration) of the animal organism with significant and prolonged hyperglycemia.

Using published data, we conducted an experimental search for an effective method of disturbing carbohydrate metabolism in rats that meets the necessary requirements described above. For this, the following methods were used:

1) modeling of alloxan diabetes;

2) keeping animals on a diet rich in fructose;

3) the introduction of a glucose solution using a probe into the stomach (oral way to create artificial hyperglycemia);

4) the introduction of a glucose solution intraperitoneally (parenteral method).

In the experiments, nonlinear animals were used - white male rats weighing 250-300 g, aged 7-8 months. The number of experimental animals in intact and experimental groups ranged from 13 to 25.

A brief report on the results of the methods used.

Method 1. At high values of hyperglycemia, an insufficient duration of experimental diabetes mellitus was noted. Rats with severe diabetes (glycemia - 22 ± 4.8 mmol / l) and severe hyperosmolar syndrome died, usually in the early stages of the onset of the disease - in the first two to three weeks [2], which did not allow fully to achieve the implementation of the process of stone formation.

Method 2. Keeping animals on a fructose diet (60% fructose from the entire diet) for 2 months was accompanied by a sharp decrease in body weight without any significant changes in carbohydrate metabolism. An experimental model of diabetes using this technique could not be obtained.

Method 3. With this method, there was a short-term (no more than 2 hours) and insignificant hyperglycemia (8.6 ± 2.2 mmol / L) (Table 1), which led to the development of a mild severity of hyperosmolar syndrome. The maximum possible dose of carbohydrate load was 600 mg / 100 g of animal weight (or 1 ml of 60% solution per 100 g of animal weight). All attempts to increase the level and duration of glycemia by increasing the concentration and / or volume of the injected glucose solution were unsuccessful. This was explained by the high viscosity of concentrated glucose solutions and the impossibility of their introduction through a probe; osmotic activity of glucose (the development of diarrhea and removal of excess glucose from the digestive tract); limited size of the stomach (the introduction of a significant amount of fluid causes acute expansion of the stomach and death of animals).

Table 1
Glycemic profile with oral carbohydrate load (60% glucose solution 1 ml / 100 g) The carbohydrate load method Glycemia Level (mmol / L) (M ± SD) n on an empty stomach After a carbohydrate load h / w 30 min h / w 1 hour 2 hours 3 hours 4 hours Oral twenty 3.2 ± 0.4 5.0 ± 0.7 8.6 ± 2.2 5.3 ± 1.0 3.7 ± 0.6 -

Method 4 was most optimal for creating severe and prolonged hyperglycemia. This method compares favorably with the previous ones in that it provided great opportunities in selecting the necessary concentration and volume of the injected solution to obtain the required hyperglycemia and, accordingly, the severity of the hyperosmolar syndrome. In addition, the introduction of glucose, bypassing the gastrointestinal tract, provided longer hyperglycemia associated with the absence of a digestive phase of stimulation of insulin release.

By experiment, the maximum possible, non-lethal dose of carbohydrate load was established, which amounted to 600 mg / 100 g. Administration of glucose in the prescribed dose led to the development of severe hyperosmolar syndrome with severe dehydration of animals (Table 2). The duration of the hyperglycemic episode was on average 4 hours.

table 2
Glycemic profile for parenteral carbohydrate loading (600 mg / 100 g) Carbohydrate Load Method n Glycemia Level (mmol / L) (M ± SD) on an empty stomach After a carbohydrate load 30 minutes h / w 1 hour 2 hours 3 hours 4 hours Parenteral twenty 3.4 ± 0.2 23.7 ± 2.9 22.0 ± 3.4 16.8 ± 3.7 12.1 ± 3.1 6.8 ± 2.3

Example 1. It is known that in rats kept under standard vivarium conditions, no stone formation process is observed in the urinary tract.

To obtain a model of urolithiasis in animals, dehydration (hyperosmolar dehydration) was caused by moderate and severe hyperglycemia with intraperitoneal administration of glucose at a dose of 400 mg and 600 mg per 100 g of animal weight, respectively, using different frequency of administration - 1 and 2 times a day.

In an experiment to model urolithiasis, 80 outbred white male rats weighing 250-320 grams were taken, whose age at the beginning of the experiment was 7-8 months. The animals were kept in the vivarium on the usual food and drinking diet in compliance with all nutritional conditions, temperature factors, lighting and humidity of the room.

The experimental animals were divided into two groups of equal size (40 each): the first and second. Each group, in turn, is divided into two subgroups: A and B (10 animals each). In the first group, a carbohydrate load at a dose of 400 mg / 100 g (40% glucose solution 1 ml / 100 g) was used, in the second group - 600 mg / 100 g (60% solution 1 ml / 100 g). In subgroups A, a glucose solution was administered once a day. In subgroups B - 2 times, with an interval between administrations of 6 hours. It was taken into account that the introduction of a concentrated glucose solution in this mode can lead to rapid death of animals from complete dehydration. Therefore, before reintroduction, the severity of the condition was assessed - lethargic and apathetic animals received additional water - ad libitum.

From the experiment, animals were removed by decapitation at the end of the 4th and 8th week. The choice of animals for this was carried out randomly. After slaughter, a macroscopic picture of the internal organs was studied, the lumen of the bladder was opened, and material was taken for histological examination (kidney, bladder).

results

Intraperitoneal administration of a glucose solution at a dose of 400 mg / 100 g led to the development of moderate, moderate hyperglycemia with a total duration of up to 4 hours. An increase in the carbohydrate load to 600 mg / 100 g caused significant and severe hyperglycemia with normalization of blood glucose values by 5 hours after the injection of the solution (Table 3).

Table 3
The glycemic profile of experimental rats with intraperitoneal administration of glucose solutions of various concentrations Groups of experimental animals n Dose of glucose (mg / 100 g) Glycemia Level (mmol / L) (M ± SD) on an empty stomach After a carbohydrate load 30 minutes h / w 1 h h / w 2 h h / w 3 h h / w 4 h 1st group twenty 400 3.2 ± 0.2 18.1 ± 2.6 16.5 ± 3.8 10.1 ± 3.1 7.2 ± 2.7 3.6 ± 0.4 2nd group twenty 600 3.4 ± 0.2 23.7 ± 2.9 22.0 ± 3.4 16.8 ± 3.7 12.1 ± 3.1 6.8 ± 2.3 R = 0.127 = 0.013 = 0.039 = 0.014 = 0.026 = 0.014

The introduction of concentrated glucose solutions was accompanied by thirst (animals licked the metal structures of the cells), polyuria, decreased activity up to complete immobilization and apathy; in the most severe cases, the development of generalized tonic-clonic convulsive seizures was observed. The severity of the manifestation of hyperosmolar syndrome depended on the level and duration of hyperglycemia.

After 4 weeks of the experiment, both in the 1st and 2nd experimental groups, in subgroups A (with a single administration of glucose during the day), in none of the 20 decapitated animals in the urinary tract there were no signs of prolonged excretion of excessively concentrated urine i.e. the formation of crystals and / or stones.

In group 1, with a double administration of glucose, hyperglycemia also did not lead to significant dehydration and crystalluria. None of the 10 animals examined showed stone formation. And only in group 2 B we managed to get the first consequences of severe dehydration - in 3 out of 10 rats in the lumen of the bladder there was a deposition of shapeless and loose masses of white color, which contained single very small light yellow crystals.

At the end of the 8th week of the experiment, the remaining 8 rats (2 rats dropped out of the experiment for various reasons) of group 1A in the bladder did not reveal any pathological formations that could indicate serious violations of the mineral balance of urine. In group 2A, 3 out of 9 rats (1 rat died from severe hyperosmolar syndrome) found stones of a grayish-white color, mainly of irregular round shape, with a rough surface, crumbling with a little pressure, consisting of very small opaque white crystals, as well as transparent crystals, refracting light, some of which had the form of rectangular plates. The number of stones and their sizes were different: from multiple small (up to 12 to 1-2 mm) to single large (up to 10 mm).

Double administration of glucose to animals of group 1B remained as ineffective as before - the level and duration of hyperglycemia were insufficient for the stone formation process.

The largest number of animals with urinary stones was in group 2B - a group in which glycemia reached high numbers (23.7 ± 2.9 mmol / l) with a total duration of about 8 hours during the day. So, out of 7 experimental animals (3 rats died in cases of hyperosmolar syndrome), 4 round-shaped dense calculi of predominantly large sizes (5-12 mm) were found in the bladder that were identical in their external (color and structural) characteristics to those described above.

At the same time, 2 rats having the largest stones (5-12 mm) showed signs of impaired urine outflow. The bladder of such animals was enlarged and contained an excessive amount (from 5 to 10 ml) of turbid urine, straw, and in one case - brown. The walls of the overgrown bladder were thinned, in one rat - with ulceration sites covered with thin overlays - blood clots. The ureters looked like whitish tows with increased clearance. The pelvis of both kidneys was significantly expanded. In two animals with signs of urinary tract obstruction, small multiple subcapsular abscesses were found in the kidneys. Microscopic examination of the kidneys describes the picture of apostematous pyelonephritis - foci of purulent tissue fusion with neutrophilic lymphoid infiltration, dystrophy and atrophy of the tubular epithelium. In the area of the bladder preparation, there is a mucosal defect with hemorrhagic impregnation to the muscle layer of the bladder wall. In the area of the ulceration bottom, in the submucosal layer, banded focal deposits of a dark blue color, similar to calcium, were noted.

Example 2

The second series of experiments was carried out to determine the possibility of stone formation in rats with severe, prolonged (by double glucose administration) hyperglycemia and a longer experiment (up to 16 weeks).

The conditions for severe hyperglycemia and dehydration were left unchanged (glucose dose - 600 mg / 100 g, route of administration - intraperitoneal). In the experiment, 30 nonlinear white male rats were used for 7-8 months (younger animals did not tolerate prolonged hyperglycemia and died quickly), weighing 250-350 g. The animals were divided into two groups - 15 in each. To control stone formation, the slaughter of experimental animals was performed after 8 and 16 weeks from the start of the experiment.

results

After completing the 8-week experiment, the first 15 rats were slaughtered. Of these, 4 in the lumen of the bladder were found to have various (in size and quantity) dense stones of light gray color.

Further administration of glucose in the prescribed dose over the next weeks to the remaining 12 animals changed their tolerance to the hyperosmolar syndrome. After carbohydrate loading, rats started to show motor activity earlier, they began to eat food more willingly; put on weight. Along with this, during the subsequent 10th to 12th weeks of the experiment, in individual animals, during urination, a periodic abundant secretion of curd-like loose whitish-yellow masses was observed, in which, after drying in air, small gray crystals were contained in large quantities. The phenomenon of crystalluria completely stopped by 13 weeks.

The study of the glycemic profile of experimental animals at the time of the end of the experiment (week 16) revealed the following picture. After the introduction of a 60% glucose solution (1 ml / 100 g) in the blood, as before, high glycemic indices of the 30-minute and hour samples (22.1 ± 2.6 and 20.2 ± 3.1 mmol / l, respectively) remained ) However, already 2 hours after the carbohydrate load, a sharp decrease in glycemia was noted to 8.8 mmol / L, which is 1.9 times less than in comparison with the previous indicators of the first weeks of the experiment (16.8 ± 3.7 mmol / L) ( p = 0.006). The duration of hyperglycemia after a single injection of glucose decreased on average by an hour, and the total daily (after a double injection), thus, by two, amounting to 6 hours from the previous 8 (see graph, Fig. 3).

After the end of the 16th experimental week and the slaughter of the remaining 12 animals, no urolithiasis phenomena - accumulation of crystals or the presence of stones in the urinary tract were detected (Table 4).

Table 4
The effect of the dose and duration of the carbohydrate load on the development of crystalluria and stone formation in rats Experimental groups n The total dose of glucose (mg / 100 g) Maximum glycemia (mmol / L) Initial duration of hyperglycemia (h) Experiment duration 4 weeks 8 weeks 12 weeks 16 weeks Signs of urolithiasis (crystalluria, stone formation) 1st group A eighteen 400 18.1 ± 2.6 3 - - IN 19 800 6 - - 2nd group A 19 600 23.7 ± 2.9 4 - + - IN 44 1200 8 + - + + - -

Explanations for the table: (-) lack of signs; (+ -) crystalluria; (+) stone formation. A - a group of animals with a single injection of glucose solution; In - a group of animals with a double introduction of a glucose solution.

Thus, on the basis of the obtained experimental data, it seems quite reasoned that the violation of the balance between the concentration and solubility of substances contained in the urine of rats is possible under the condition of the development of severe and prolonged dehydration of animals, which is achieved by twice daily administration of a glucose solution (600 mg / 100 g) for 8 weeks.

Literature

1. Shulutko B.I. Nephrology 2002. Current status of the problem. - St. Petersburg: Renkor, 2002, - p. 471.

2. Galenok V.A., Gostinskaya E.V., Dicker V.E. Hemorheology in disorders of carbohydrate metabolism. - Novosibirsk .: Nauka, 1987, - p. 63.

3. Modeling, research methods and experimental therapy of pathological processes. - Materials of the All-Union Conference of the Central Research Laboratories of Medical Universities and the Institute of Advanced Medical Doctors of the USSR. / Under the general editorship of A.S. Chechulina / - M., 1973.

4. Modeling of diseases. / Edited by S.V. Andreev. - M.: Medicine, 1973.

Claims (1)

A method for modeling urolithiasis in rats, including the intraperitoneal injection of a chemical substance, characterized in that 60% glucose solution was administered 2 times a day for two months at the rate of 1 ml per 100 g of animal weight.

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