RU2567602C1 - Method for simulating localised peritonitis in laboratory non-linear mice - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: 10% faecal suspension from fresh rat faeces in an amount of 20-22 g is introduced intraperitoneally into non-linear laboratory mice through the same prick-in point along a mid-line in an umbilical region at a depth of 2 mm. The suspension is prepared with using sodium chloride solution and filtered once through a double gauze layer. If the model is supposed to be used for more than 13 days, the suspension is introduced in an amount of 0.3-0.4 ml; an amount 0.5 ml is used for the model if used for no more than 13 days. The result is formation of localised abscesses in several anatomical regions of the abdominal cavity.

EFFECT: method enables reproducing a long-standing disease similar in pathogenesis to clinical forms, providing a prevented early animal's death caused by infectious-toxic shock.

4 tbl, 8 dwg

Description

The invention relates to the field of veterinary medicine and medicine, in particular experimental pathology, and is intended for the experimental study of the pathogenesis of peritonitis, testing new means of prevention and treatment of this disease.

A known method for modeling acute peritonitis (RU 2338265, G09B 23/28, 10.11.2008), which consists in the injection into the abdominal cavity of pre-anesthetized rat fecal suspension prepared from the contents of the cecum.

The disadvantage of this method is that relatively large and expensive animals are used. When performing various manipulations in an experiment on rats, additional efforts, time for fixation and preliminary anesthesia of animals are required. Another disadvantage is the injection method of administration, in which the fecal suspension is introduced into the abdominal cavity through one injection point by moving the needle into the right and left hypochondria and into the right and left iliac regions. With this method, the risk of injury to the abdominal organs increases. This significantly changes the course of peritonitis and leads to premature death of animals.

Closest to the claimed solution is a method of reproducing peritonitis in mice, which consists in the fact that animals are injected into the abdominal cavity with 15% fecal suspension prepared from feces of guinea pigs (Resistance to intraperitoneal peritonitis. Inadequacy of experimental peritonitis // URL: http: // medicalplanet.Su/xirurgia/1120.html (accessed 02.02.2014).

A significant disadvantage of this method is the death of animals by the end of the first day after injection. This limits the possibility of conducting lengthy studies on such animals.

The objective of the claimed method is to expand the arsenal of accessible and technically simple methods for creating an experimental model of peritonitis, which is close in pathogenetic relation to the corresponding disease and allows long-term observation of animals in the experiment.

The essence of the invention lies in the fact that in the method for simulating delimited peritonitis in laboratory non-linear mice, including a single intraperitoneal injection of fecal suspension, according to the invention, mice weighing 20-22 g are infected with a 10% fecal suspension of fresh rat feces prepared on isotonic sodium chloride solution and once filtered through a double layer of gauze, through one point of injection along the midline in the umbilical region of the abdomen to a depth of 2 mm in doses of 0.3-0.4 ml for using the model for more than 13 days and 0.5 L for use model is not more than 13 days.

The invention is illustrated by drawings.

In FIG. 1 shows the internal organs of the abdominal cavity of the animal of the control group No. 1 (w / w 0.3 ml of sterile saline solution).

In FIG. 2 shows the pronounced plethora of the omentum, peritoneum, mesentery and testis vessels in an animal from group No. 2 (w / w 0.3 ml of 10% stool suspension from fresh rat feces) on day 3 after injection.

In FIG. 3 shows hyperemia and swelling of the intestine in an animal from group No. 3 (w / w 0.4 ml of 10% stool suspension from fresh rat feces) on day 3 after injection.

In FIG. 4 shows hyperemia and swelling of the intestine in an animal from group No. 4 (w / w 0.5 ml of 10% stool suspension from fresh rat feces) on day 3 after injection.

In FIG. 5 shows abscesses in the abdominal cavity in an animal from group No. 2 (w / w 0.3 ml of 10% stool suspension from fresh rat feces) on day 15 after injection. The upper arrow indicates an abscess located in the left cranial region of the abdominal cavity. The lower arrow indicates an abscess in the left caudal region of the abdominal cavity.

In FIG. 6 shows abscesses in the abdominal cavity of an animal from group No. 3 (w / w 0.4 ml of 10% stool suspension from fresh rat feces) on day 15 after injection. The left arrow indicates a large abscess located on the border between the right and left cranial regions of the abdominal cavity. The right arrow indicates an abscess in the left caudal region.

In FIG. 7 shows abscesses in the abdominal cavity in an animal from group No. 4 (w / w 0.5 ml of 10% stool suspension from fresh rat feces) on day 11 after injection. The upper arrow indicates a large abscess located in the left cranial region of the abdominal cavity. The lower arrows indicate abscesses in the right and left caudal areas of the abdominal cavity.

In FIG. 8 shows the phlegmon of the abdominal wall in an animal from group No. 4 (w / w 0.5 ml of 10% stool suspension from fresh rat feces) on day 13 after injection.

The method is as follows.

Animals are selected weighing 20-22 g, age 45-55 days without signs of disease. Mice are kept under specialized vivarium conditions in cages with a solid floor, which receive granulated food and water of full value in the public domain.

10% suspension in isotonic sodium chloride solution is prepared from fresh rat feces and filtered once through a double layer of gauze.

Intraperitoneal injection to mice is carried out once with a 26 G × 1/2 needle (for intradermal injection) through one point of injection along the midline in the umbilical region of the abdomen to a depth of 2 mm. Pre-injection site is treated with 70% ethanol.

After intraperitoneal injection to mice of 10% fecal suspension from fresh rat feces in doses of 0.3-0.4 ml, the observation period for animals will be more than 13 days, after injection at a dose of 0.5 ml - not more than 13 days.

The parameters of the method were determined as follows.

In the proposed method, the fecal suspension from fresh rat feces was selected as the infectious mixture. Laboratory rats are one of the most common laboratory animals. Compared to rat feces, mice are much smaller and dry quickly, which is unacceptable, because they dramatically reduced the number of bacteria. Therefore, preference was given to larger feces of rats.

Determination of the optimal weight and age of mice for infection of the abdominal cavity with rat fecal suspension.

The determination of the optimal weight and age of mice was carried out on three groups of laboratory non-linear mice, which were injected intraperitoneally with 10% fecal suspension from fresh rat feces in an amount of 0.6 ml. The evaluation criterion was the death of animals. The first group included mice weighing 15-18 g, age 30-40 days, the second group included animals weighing 20-22 g, age 45-55 days, the third group included mice weighing 30-35 g, age 75-85 days. The death of animals is presented in table 1.

From table 1 it is seen that the mortality of mice in groups No. 1 and 3 on the first day after injection was 70 and 100%, respectively. In group No. 2, the death of animals began on day 4 after the injection. Consequently, mice weighing 20-22 g, age 45-55 days, are more resistant to infection of the abdominal cavity with 10% fecal suspension from fresh rat feces. Therefore, the use of laboratory nonlinear mice weighing 20-22 g, age 45-55 days, when modeling peritonitis will allow for longer studies.

Justification of the route of administration

The parameters of the intraperitoneal method of administering fecal suspension were tested on two groups of laboratory non-linear mice. In order to assess the nature of the distribution of fluid in the abdominal cavity after injection through one point of injection along the midline in the umbilical region of the abdomen to a depth of 2 mm, 0.1 ml of dye (black mascara) was injected into the animals instead of fecal suspension. In the first group, the diagnosis was carried out immediately after administration. At autopsy, it was determined that in all mice black mascara was evenly distributed and was found in all anatomical regions of the abdominal cavity.

In the second group, the diagnosis was performed on day 5 after intraperitoneal injection of the dye. At autopsy, diffuse impregnation with the dye of the abdominal wall at the injection site was noted. It was also determined that the largest amount of dye accumulated in the gland. Small oval carcass fragments freely lying in the abdominal cavity up to 2 × 2.5 mm in size were found in the subhepatic space, in the right and left lateral canals, between the loops of the intestine and in the pelvic cavity. With this method of administration, signs of a violation of the integrity of the internal organs in the abdominal cavity were not detected in any animal. Therefore, immersion of the needle to a depth of not more than 2 mm avoids damage to internal organs, and also provides a diffuse distribution of injection material in the abdominal cavity.

Determination of the optimal concentration and dose of fecal suspension from fresh rat feces

Determining the effect of various doses and concentrations of fecal suspension from fresh rat feces on the occurrence of peritonitis in laboratory non-linear mice, animals were divided into 4 groups, each of 6 subgroups. In control group No. 1, mice were injected into the abdominal cavity according to the method described above with sterile saline in amounts from 0.1 to 0.6 ml. Animals of groups No. 2, 3 and 4 were injected into the abdominal cavity in the same way with 5%, 10% and 20% filtered suspension from fresh rat feces in amounts from 0.1 to 0.6 ml, respectively.

The observation period was 15 days. The evaluation criteria were the death of animals (table 2) and pathological changes at autopsy (table 3).

The presence of changes in the abdominal cavity visible at autopsy greatly facilitates the diagnosis and determination of the severity of the disease. Therefore, the optimal concentrations and doses of fecal suspension causing peritonitis were considered to be those with the introduction of which macroscopic pathological changes characteristic of peritonitis were found in the abdominal cavity of mice and with the introduction of which the period of observation of animals in the experiment would be longer.

In order to describe the localization of abdominal changes, the abdominal cavity of mice was divided into topographic areas according to N.V. Sadovsky (1953). In this case, four anatomical areas were distinguished: right and left cranial (front) areas of the abdomen, right and left caudal (rear) areas of the abdomen.

From the data given in tables 2 and 3, it is seen that for modeling peritonitis with characteristic pathological changes and with a long period of observation of animals, it is optimal to infect the abdominal cavity with 10% fecal suspension in an amount of 0.3-0.5 ml (subgroups 3- 5 groups No. 3). As a result, peritonitis develops in 100% of animals, which is prone to delimiting the inflammatory process. The life expectancy of animals after the introduction of 0.3 and 0.4 ml of fecal suspension is more than 13 days. The life expectancy of animals after the introduction of 0.5 ml of fecal suspension is not more than 13 days.

In animals of the control group No. 1, subgroups 1-2 of group No. 2 (0.1-0.2 ml of 5% fecal suspension) and subgroup 1 of group No. 3 (0.1 ml of 10% fecal suspension) at the opening of any changes in the abdominal cavity is not detected.

In animals of subgroups 3-6 of group No. 2 (0.3-0.6 ml of 5% fecal suspension), subgroups of group 2 No. 3 (0.2 ml of 10% fecal suspension), intraperitoneal administration of fecal suspension in more animals leads to to the formation of small abscesses (up to 7 mm in diameter) in only one anatomical region of the abdominal cavity.

Therefore, a 5% concentration of fecal suspension in an amount of 0.1-0.6 ml (subgroups 1-6 of group No. 2) and 10% concentration of fecal suspension in doses of 0.1 and 0.2 ml (subgroups of groups 1 and 2 No. 3) are insufficient, because either they do not contribute to the formation of clearly expressed pathological and anatomical changes, or they contribute to the formation of delimited abscesses mainly in the same anatomical region.

Excessive are 10% concentration of fecal suspension in an amount of 0.6 ml (subgroup 6 of group No. 3) and 20% concentration in doses of 0.1-0.6 ml (subgroup 3-6 of group No. 4), since they do not allow long-term monitoring animals in the experiment.

The method for modeling delimited peritonitis was tested on 4 groups of laboratory nonlinear mice (9 animals in each group). In control group No. 1, mice were injected into the abdominal cavity through a single point of injection along the midline in the umbilical region of the abdomen to a depth of 2 mm with 0.3 ml of sterile physiological saline, previously the injection site was treated with 70% ethanol. Animals of experimental groups No. 2, 3, and 4 were injected in the same way with 10% fecal suspension from fresh rat feces in the amount of 0.3, 0.4, and 0.5 ml, respectively.

The observation period was 15 days. On day 3 after injection of fecal suspension, a diagnostic autopsy was performed on 2 animals from each group. The weight of the mice was determined on days 1, 2, 3, 8, and 15 after the injection of fecal suspension, the dead animals were weighed on the day of death. Within 15 days, the general condition of the animals was assessed by external examination. At the end of the observation period, the mice were killed by an overdose of anesthesia and pathological changes at autopsy were evaluated. Pathological and anatomical study of dead animals was performed on the day of death.

On the first day after injection in animals of experimental groups No. 2, 3 and 4, the rigidity of the abdominal wall was determined. Mice predominantly lay on their stomach, while periodically stretching their hind legs back. Animals grouped in the corner of the cage, refused food and water. The mice in the control group looked healthy after the injection.

On the second day, the activity of the animals of the experimental groups was restored, the mice received food and water. The rigidity of the abdominal wall was not determined.

In the second half of the experiment, periodic inhibition was observed in animals of groups 2 and 3, mice were reluctant to eat food, six became dull and disheveled, and an increase in the size of the abdomen was noted. Within 15 days in groups No. 2 and 3 there were no animals.

An external examination of group 4 mice showed more pronounced inhibition. Animals reluctantly consumed food, were less mobile compared to animals of groups No. 1-3. The coat became dull and disheveled. The animals showed marked exhaustion, sunken in the iliac region of the side while the size of the abdomen was increased. The death of mice occurred on the 11-13th day after an intraperitoneal injection of fecal suspension.

Animals of the control group remained healthy.

The groups of animals and the results of determining their body mass indices (M ± m) are shown in table 4. From the data given in table 4, it can be seen that on the second day of observation, a significant decrease in the body weight of mice in the experimental groups (the maximum this indicator reaches values in the 4th group) relative to the control (group No. 1). With an increase in the observation period, the weight of animals in the control group increases, which is associated with the growth of young mice. This indicator is lower in groups 2 and 3 relative to control, but in group 4 it does not increase.

At autopsy in animals of the control group, pathological and anatomical changes characteristic of peritonitis were not detected (Fig. 1).

On the third day in the animals of the experimental groups in the abdominal cavity, pronounced plethora of the omentum, peritoneum, mesentery and testis vessels (Fig. 2), hyperemia and intestinal edema (Fig. 3, Fig. 4) were noted. Exudation in the abdominal cavity was absent.

In animals of the experimental groups for 11-15 days in the abdominal cavity there was sharp plethora of the vessels of the peritoneum, omentum and mesentery, hyperemia and edema of the intestinal wall, gas formation in the intestine and its paresis.

In group No. 2, abscesses were found in 100% of the animals, located mainly in the left cranial and left caudal areas of the abdominal cavity (Fig. 5) with a diameter of up to 11 mm. Viscero-visceral and viscero-parietal adhesions were found at the location of the abscesses.

In group No. 3, in 100% of the animals in the abdominal cavity, abscesses were noted in the cranial and caudal regions of the abdominal cavity (Fig. 6) with a diameter of up to 12 mm. In 51.7% of the animals, intestinal abscesses with a diameter of 4-8 mm in an amount of up to 3 were found. Viscero-visceral and visceroparietal adhesions were found at the location of the abscesses. In 42.8% of animals, abscesses were found on the surface of the liver with a diameter of 2-3 mm in an amount of up to 3 pcs. Noted the absence of effusion.

In group No. 4, abscesses up to 13 mm in diameter located in the cranial and caudal regions of the abdominal cavity were found in 100% of the animals (Fig. 7). In intestinal abscesses with a diameter of 3-7 mm in an amount of up to 5 pcs. Were found in 71.4% of mice. Viscero-visceral and visceroparietal adhesions and turbid serous-fibrinous exudate in an amount of 0.1-0.15 ml were found in the abdominal cavity. In 51.7% of animals, abscesses were found on the surface of the liver with a diameter of 2-3 mm in an amount of up to 4 pcs. Two animals were diagnosed with phlegmon of the abdominal wall (Fig. 8).

Thus, the technical result of our proposed method is the development in nonlinear mice of delimited peritonitis lasting at least 10 days with the formation of abscesses in the abdominal cavity. The technical result is achieved by infection of nonlinear mice weighing 20-22 g through one point of injection along the midline in the umbilical region of the abdomen to a depth of not more than 2 mm with 10% fecal suspension from fresh rat feces in doses of 0.3-0.5 ml.

Claims (1)

A method for simulating delimited peritonitis in non-linear laboratory mice, including a single intraperitoneal administration of fecal suspension, characterized in that mice weighing 20-22 g are infected with 10% fecal suspension from fresh rat feces prepared on an isotonic solution of sodium chloride and filtered once through a double layer of gauze, through one point of injection along the midline in the umbilical region of the abdomen to a depth of 2 mm, in doses of 0.3-0.4 ml - for using the model for more than 13 days and 0.5 ml - for using the model for no more than 13 days .

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