Method for experimental modeling urinary calculosis

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.

2 dwg, 2 ex, 4 tbl

 

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

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

The purpose of the invention: experimental simulation of urolithiasis.

Description of the method. Rats-males contained on regular food and drinking diet twice a day (every 5-6 hours) were injected intraperitoneally 60% glucose solution at the rate of 1 ml per 100 g weight of the animal.

As a criterion for the development of urolithiasis was chosen as the indicator of the presence of stones in the bladder. The animals were sacrificed experiment two months later by decapitation.

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

The starting point for modeling lithogenesis (urolithiasis) in rats served as data on the development of urolithiasis in patients with severe decompensated diabetes. It is known that severe hyperglycemia is accompanied by dehydration of the organism and significant electrolyte disorders. These violations lead to an imbalance in the delicate regulation of mineral metabolism - oversaturation of urine Neretva Imami substances, their crystallization with subsequent aggregation and formation of stone [1].

For a successful simulation of stone formation in the urinary tract of rats required the creation of certain conditions, in particular heavy and prolonged (several months) diabetogenic, hyperosmolar syndrome, through significant and prolonged hyperglycemia.

The objectives of this study were:

1) to impair carbohydrate metabolism in experimental rats;

2) to cause severe dehydration (hyperosmolar dehydration) of animals significant and prolonged hyperglycemia.

Using the literature data, we conducted a pilot search of an effective method of carbohydrate metabolism in rats that meets the requirements described above. This applies to the following methods:

1) modeling of alloxan diabetes;

2) the keeping of animals on a diet enriched with fructose;

3) glucose solution using a probe into the stomach (oral method of creating artificial hyperglycemia);

4) glucose solution intraperitoneally (parenteral).

In experiments were used nonlinear animals white rats-males weighing 250-300 g at the age of 7-8 months. The number of animals tested in the intact and experimental groups was the 13 to 25.

A brief report on the results of the used methods.

Method 1. At high values of hyperglycemia noted the insufficient duration of experimental diabetes. Rats with severe diabetes (glycemia - 224.8 mmol/l) and severe hyperosmolar syndrome was killed, usually in the early stages of the disease onset in the first two or three weeks [2], it was not possible to fully implement the process of stone formation.

Method 2. The keeping of animals on the fructose diet (60% fructose from whole food diet) for 2 months was accompanied by a dramatic weight loss without any significant changes in carbohydrate metabolism. Experimental model of diabetes with the help of this technique could not be obtained.

Method 3. With this method was briefly (no more than 2 hours), and mild hyperglycemia (8,62.2 mmol/l) (table 1), which led to the development of light on the severity of the hyperosmolar syndrome. The maximum possible dose to carbohydrate load was 600 mg/100 g weight of the animal (or 1 ml of 60% solution per 100 g of weight). All attempts to increase the level and duration of blood glucose by increasing the concentration and/or volume injected glucose solution was unsuccessful. This was attributed to the higher viscosity of concentri avannah solutions of glucose and the impossibility of their insertion through the probe; the osmotic activity of the glucose (the development of diarrhea and the removal of excess glucose from the digestive tract); the limited size of the stomach (the introduction of a significant amount of fluid causes acute gastric dilatation and destruction of animals).

Table 1

The glycemic profile in oral carbohydrate load (60% glucose, 1 ml/100 g)
The way carbohydrate loadThe level of glycemia (mmol/l) (M±SD)
non an empty stomachAfter a carbohydrate load
h/C 30 minh/C 1 hourh/C 2 hoursh/C 3 hoursh/C 4 hours
Oral203,20,45,0±0,78,6±2,25,3±1,03,7±0,6-

Method 4 was the most optimal for creating a severe and prolonged hyperglycemia. This method was distinguished from the previous one that provided great opportunities for selecting the desired concentration and volume of the injected solution to obtain the desired magnitude of hyperglycemia and therefore the severity of hyperosmolar syndrome. It is the same glucose, bypassing the gastrointestinal tract, provided more long-term hyperglycemia, resulting from a lack of digestive phase stimulate the release of insulin.

Empirically was installed as possible, not lethal dose of carbohydrate load, which amounted to 600 mg/100 g of a glucose in the prescribed dose has led to the development of severe hyperosmolar syndrome with severe dehydration, animals (table 2). However, the duration of hyperglycemic episode averaged 4 hours.

Table 2

The glycemic profile of parenteral method of carbohydrate loading (600 mg/100 g)
The way carbohydrate loadnThe level of glycemia (mmol/l) (M±SD)
on an empty stomachAfter a carbohydrate load
h/C 30 minh/C 1 hourh/C 2 hoursh/C 3 hoursh/C 4 hours
Parenteral203,40,223,72,922,03,416,83,712,13,16,82,3

Example 1. It is known that in rats contained in standard vivarium conditions, urinary tract nenablyudaemo process of stone formation.

To obtain a model of urolithiasis in animals caused dehydration (hyperosmolar dehydration) hyperglycemia moderate and severe severity intraperitoneal injection of glucose at a dose of 400 mg and 600 mg per 100 g weight of the animal, respectively, using different multiplicity of 1 and 2 times a day.

In the experiment on modeling of urolithiasis were taken 80 outbred white rats-males weighing 250-320 g, age at the beginning of the experiment was 7-8 months. The animals were housed in the vivarium at the usual food and drinking diet with all conditions of food, temperature factors, light and humidity.

Experimental animals were divided into two equal groups (40 in each): the first and the second. Each group, in turn, divided into two groups: a and b (10 animals). In the first group was used for carbohydrate loading dose of 400 mg/100 g (40% glucose, 1 ml/100 g), the second group of 600 mg/100 g (60% solution 1 ml/100 g). In the subgroups And the glucose solution was administered once a day. In subgroups In 2 times, with an interval between doses 6 hours. It was considered that the introduction of this mode of concentrated glucose solution can lead to rapid death from complete dehydration. Therefore, before re-introduction was estimated tegest the state - dull and listless animals received additional water ad libitum.

From the experiment the animals were sacrificed by decamethonium at the end of the 4th and 8th weeks. Selection of animals for this purpose was carried out randomly. After slaughter, were studied macroscopic picture of the internal organs, uncovered the lumen of the bladder was performed biopsy specimens for histological examination (kidney, bladder).

Results

Intraperitoneal injection of glucose solution at a dose of 400 mg/100 g resulted in the development of mild, moderate hyperglycemia for a total duration of up to 4 hours. Increased carbohydrate load up to 600 mg/100 g caused a significant and severe hyperglycemia with variables normalization of blood glucose to 5 hours after administration of the solution (table 3).

Table 3

The glycemic profile of Wistar rats with intraperitoneal injection of glucose solutions of different concentrations
Groups of experimental animalsnThe dose of glucose (mg/100 g)The level of glycemia (mmol/l) (MSD)
on an empty stomachAfter a carbohydrate load
h/C 30 minh/C 1 hh/C 2 hthe/C 3 h h/C 4 h
1st group204003,20,218,12,616,53,810,13,17,2±2,73,6±0,4
2nd group206003,40,223,72,922,03,416,83,712,13,16,82,3
P  =to 0.127=0,013=0,039=0,014=0,026=0,014

The introduction of concentrated solutions of glucose was accompanied by thirst (animals licked the metal construction of the cells), polyuria, decreased activity until full Immobilise and apathy; in the most severe cases, observed the development of generalized tonic-clonic seizures. The severity of the symptoms of hyperosmolar syndrome depended on the level and duration of hyperglycemia.

After 4 weeks of the experiment, as in the 1st and in the 2nd experimental groups, subgroups And (with a single glucose during the day), none of the 20 decapitating animals in the urinary tract was not detected signs of long-term allocation overly concentrated urine, i.e. the formation of crystals and/or stones.

In the 1st group is e, with double glucose, hyperglycemia did not cause significant dehydration and crystalluria. None of the 10 test animals were observed stone formation. And only in group 2 In we managed to get the first consequences of severe dehydration, 3 of 10 rats in the lumen of the urinary bladder occurred deposition shapeless and loose masses of white color, which contained a single very fine pale-yellow crystals.

Upon completion of the 8 weeks of the experiment the remaining 8 rats (2 rats were eliminated from the experiment for various reasons) 1A group in the bladder was never discovered any pathological lesions that could indicate serious violations of the mineral balance of the urine. In 2A group 3 rats of 9 (1 rat died from severe hyperosmolar syndrome) were found stones of grayish-white color, mostly irregular rounded shape, with a rough surface, crumbling at low pressure, consisting of very small opaque white crystals and clear crystals, the refractive light, some of which were in the form of rectangular plates. The number of stones and their sizes were different: multiple small (up to 12 1-2 mm) to a single large (up to 10 mm).

Twice a glucose animals 1V groups remained the same, the same ineffective, as before, the level and duration of hyperglycemia were insufficient for carrying out the process of stone formation.

The greatest number of animals with urinary stones were in group 2B group, in which glycemia reached high numbers (23,7±2.9 mmol/l) with a total duration during the day, about 8 hours. So from 7 experimental animals (3 rats died with symptoms of hyperosmolar syndrome) 4 in the urinary bladder were found in dense rounded concretions of mostly large sizes (5-12 mm), exterior (colour and structural) characteristics identical to those described above.

In 2 rats, which had the largest stones (5-12 mm), showed signs of impaired outflow of urine. The bladder such animals was increased in size and contained excessive amounts (5 to 10 ml) turbid urine, straw, and in one case - brown color. Wall perekrestnogo bladder was thinning, one of the rat, with areas of ulceration, covered with a thin overlays and blood clots. The ureters had the appearance of whitish harness with increased ground clearance. Pelvis of both kidneys were significantly expanded. Two animals with signs of obstruction of the urinary tract in the kidneys found multiple small subcapsular ulcers. Microscopic examination of the kidneys is described card is on aposematism pyelonephritis - the foci of purulent fusion fabric with neutrophile-lymphoid infiltration, degeneration and atrophy of the tubular epithelium. In the area of drug bladder - defect of mucous membrane with hemorrhagic soaking up the muscle layer of the bladder wall. In the bottom of ulceration, submucosal layer was observed banded focal deposits of dark blue color, similar to calcium.

Example 2.

The second series of experiments was conducted to determine the possibility of stone formation in rats with severe, extended (using a double injection of glucose) hyperglycemia and greater duration of the experiment (up to 16 weeks).

Conditions for obtaining severe hyperglycemia and dehydration were left unchanged (dose glucose - 600 mg/100 g, the route of administration, intra - abdominal). In the experiment used 30 nonlinear white rats-males 7-8 months (younger animals poorly tolerated prolonged hyperglycemia and quickly died), weighing 250-350 g Animals were divided into two groups of 15 each. For control of lithogenic slaughter experimental animals were performed after 8 and 16 weeks from the beginning of the experiment.

Results

After completing the 8-week experiment were scored the first 15 rats. 4 of them in the lumen of the urinary bladder were found different (in size and quantity) dense QAM and gray.

Further introduction of glucose in the prescribed dose over the next weeks remaining 12 animals changed their portability hyperosmolar syndrome. Rats after a carbohydrate load started earlier to demonstrate locomotor activity, were more willing to eat food; gained weight. Along with this over the next 10 - 12 weeks of the experiment in individual animals during urination was observed periodic plentiful allocation torontotoday friable whitish-yellow mass, which after drying in air contained in a large number of small gray crystals. The phenomenon of crystalluria completely stopped for 13 weeks.

The study of the glycemic profile in experimental animals by the end of the experiment (16 weeks) showed the following picture. After the introduction of 60% glucose solution (1 ml/100 g) in the blood, as before, remained high glycemic 30-minute and hour samples (22,12,6 and 20.23.1 mmol/l, respectively). However, 2 hours after a carbohydrate load was a sharp decrease of glycemia to 8.8 mmol/l, in comparison with the same indicators of the first weeks of the experiment (16,83.7 mmol/l) less than 1.9 times (p=0.006). The duration of hyperglycemia after a single injection of glucose decreased on average by the hour, and total day (after two introduction the tion), thus, two, accounting for 6 hours from the previous 8 (see graph 3).

After completion of the 16-th experimental week and slaughter the remaining 12 animals of any phenomena of urolithiasis - clusters of crystals or the presence of stones in the urinary tract, were not detected (table 4).

Table 4

Influence of dose and duration of carbohydrate loading on the development of crystalluria and stone formation in rats
The experimental groupnThe total dose of glucose (mg/100 g)Maximum glycemia (mmol/l)The initial duration of hyperglycemia (h)The duration of the experiment
to 4 weeks.8 weeks.12 weeks.16 weeks.
Signs of urolithiasis (crystalluria, present)
1st groupAnd1840018,12,63--  
In198006--  
2-I g is the SCP And1960023,72,94-+-  
In4412008+-++--

Notes to table: (-) absence of signs; (+ -) crystalluria; (+) present. A group of animals with a single glucose solution; In the group of animals with two glucose solution.

Thus, on the basis of the obtained experimental data seems to be well-reasoned conclusion that the violation of the balance between concentration and solubility of the substances contained in the urine of rats, is possible if the development of a severe and prolonged dehydration animals that achieved twice daily glucose solution (600 mg/100 g) for 8 weeks.

Literature

1. Soludo B. I. Nephrology 2002. Current status of the problem. - SPb.: Rancor, 2002, - s.

2. Galenic C. A., Gostynska E.V., Dicker VE Hemorheology disorders of carbohydrate metabolism. - Novosibirsk.: Science, 1987, - p.63.

3. Modeling, methods of study and experimental treatment of pathological processes. Materials of all-Union conference of the Central research laboratory is thorium medical universities and institutes of improvement of doctors of the USSR. /Under the General editorship of ALEXANDER Chechulin/), 1973.

4. Modeling of diseases. /Edited Svendita. - M.: Medicine, 1973.

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



 

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