RU2559935C1 - Method for simulating resected liver regeneration with l-arginine - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: 70% hepatectomy is performed in a laboratory animal on the second experimental day. An agent inducing liver regeneration is L-arginine administered intragastrically in a daily dose of 200.0 mg/kg every 46 hours for the first 7 experimental days.

EFFECT: full reconstruction of the volume, structure and function of the resected liver.

1 ex, 2 tbl

Description

The invention relates to medicine, in particular to experimental pharmacology and experimental surgery, and can be used to stimulate the regeneration of a resected liver.

Closest to the claimed solution is the "Method of post-resection liver regeneration in the experiment" auth. Chikoteeva et al. RU 2232550. In this solution, preliminary xenograft is performed by cryopreserved isolated pig hepatocytes subcutaneously into the anterior abdominal wall. The procedure is carried out 48 hours before the operation. Then, intraoperatively immediately after resection of 90% of the liver, the same cells are transplanted under the spleen capsule.

The disadvantages of this method are the complexity of technical implementation and the high likelihood of complications during xenotransplantation. In addition, 100% mortality in rats of the control group complicates the experiment because of the impossibility of comparing the indicators in the experimental and control groups of animals. According to most researchers, the most optimal for studying liver regeneration in rats is resection of 70% of the organ.

Modern methods of stimulating regeneration of the resected liver should be aimed not only at enhancing the proliferation of hepatocytes, but also at restoring the function of the organ as a whole. The rapid growth of hepatocellular elements disrupts the formation of liver structures. The outcome of areas with excessive accumulation of liver cells, with the absence or insufficiency of the microvasculature is necrosis. However, most studies are of a short-term nature, which does not allow us to trace the fact of restoration of the structure and function of the liver.

Each living organism has complex mechanisms of self-preservation and recovery. The urgent task of medicine is to study and find ways to activate them. In this regard, the phenomenon of ischemic preconditioning deserves the most detailed study. It is known that in the process of preconditioning, the volume of the necrotic zone is not only reduced, but the fundamental genomic reprogramming of cells is also stimulated, activating the reparative and regenerative processes that are so important in surgery. The introduction into practice of pharmacological agents that contribute to the activation and extension of this natural protective mechanism will significantly improve treatment results.

One of the mechanisms for the development of preconditioning is considered to be the stimulation of nitric oxide synthesis. In 1998, R. Bolli proposed the “nitric oxide hypothesis” with respect to the late phase, the so-called “second window” of preconditioning. The author of the “nitric oxide hypothesis” believes that it is the increased activity of the inducible NO synthase detected 24 hours after the ischemic episode that is triggered, which is responsible for the realization of the effects of the “second window” of preconditioning. L-arginine is known as a nitric oxide donor. It can be assumed that the drug is able to activate the preconditioning process. Considering that the genetic reprogramming of the cell, which occurs in the “second window” of preconditioning, plays an important role in the recovery processes, the first injection of the drug was carried out the day before the hepatectomy. Since frequent - daily preconditioning leads to the cancellation of the effect, and an excess of nitric oxide can aggravate tissue damage, subsequent injections of the drug were carried out every 46 hours, it was by this time that the preconditioning effect was weakened. The drug was administered during the first 7 days of the experiment, since it is at this stage that the activation of reparative processes is most effective.

The main advantage of the proposed method is ease of execution. The regeneration of the resected liver was understood as the true regeneration of the organ, including not only the regeneration of hepatocytes, but also the restoration of the volume, structure and function of the liver tissue, observed during the 29 days of the experiment.

The technical result of the invention is the development of a method of stimulating the regeneration of a resected liver using L-arginine. The technical result is achieved by the fact that upon resection of 70% of the liver on the second day of the experiment, L-arginine is intragastrically administered to the laboratory animal in a daily dose of 200.0 mg / kg every 46 hours for the first 7 days of the experiment.

The method is carried out as follows.

The experiments were carried out on white rats of the Wistar line weighing 210-220 g.

Liver resection is performed on the second day in the amount of 70%.

The liver condition is assessed by the rate of mortality of animals, the level of microcirculation, the severity of cytolysis, the indicators of the synthetic function of the liver, and the results of morphological studies in groups of intact animals, falsely operated, with liver resection and in the group of animals with liver resection, which were injected with L-arginine.

The duration of the experiment is 29 days.

The mortality rate is estimated in experimental groups of animals in the first 10 days after surgery.

The level of microcirculation in the liver is determined using Biopac systems equipment: MP 100 polygraph with laser Doppler flowmeter module LDF100C and surface sensor TSD 140. Registration and processing of laser Doppler flowmetry (LDF) results using Acq Knowledge software version 4.2, microcirculation values are expressed in perfusion units (PE). Record the level of microcirculation is carried out sequentially on the surface of the liver, then calculate the average value. Microcirculation level is recorded on the 2nd, 7th, 14th, 21st and 28th day after surgery.

The severity of cytolysis is assessed by the level of AlAT, AsAT, and LDH in the blood of experimental animals on the 2nd, 7th, 14th, 21st, and 28th days after surgery. The indicators are determined by the UV kinetic method on an automated system AU5800 (Beckman Coulter, USA).

Synthetic liver function is evaluated by coagulogram indices (APTT, MHO, fibrinogen) on the 7th, 14th, 21st and 28th days after surgery on a CS-2000i analyzer (Sysmex, Japan).

Morphological research is carried out on the material of standard sections of the liver, taken after removing animals from the experiment. Material processing is carried out according to a standard method with formalin fixation, paraffin embedding, hematoxylin and eosin staining. General morphological and morphometric studies are performed using the MiraxDesk image scanning and archiving system.

L-arginine is administered intragastrically in a daily dose of 200.0 mg / kg every 46 hours for the first 7 days of the experiment.

When statistical data processing is calculated, the average value, the standard deviation. Differences are considered significant at p <0.05.

EXAMPLE OF SPECIFIC PERFORMANCE

Experimental animals were divided into the following groups:

1) intact animals (n = 20);

2) false-operated animals (n = 20);

3) liver resection (n = 50);

4) liver resection + L-arginine (n = 50).

Liver resection was performed by laboratory animals on the second day of the experiment in a volume of 70%. Rats were anesthetized by intraperitoneal administration of a combination of chloral hydrate and zoletil. After anesthesia, the rat was fixed in the supine position. The hair on the abdomen was carefully cut, the skin was treated with a 70% solution of ethyl alcohol. Median laparotomy was performed from a 4 cm long xiphoid process. The liver was mobilized by crossing ligaments. The left and medial lobes were removed after preliminary ligation of the vessels. The laparotomy opening was sutured after the abdominal sanitation and control of hemostasis in layers, with a nodal suture.

Stimulation of liver regeneration was performed in animals of the 4th group by intragastric administration of L-arginine in a daily dose of 200.0 mg / kg every 46 hours for the first 7 days of the experiment.

The mortality rate was evaluated in experimental groups of animals in the first 10 days after surgery. In the group of animals with liver resection, it was 40%, in the group of animals with liver resection and administration of L-arginine, 18%.

The level of microcirculation in the liver was determined using equipment manufactured by Biopac systems: MP 100 polygraph with laser Doppler flow meter LDF100C and surface sensor TSD 140 on the 2nd, 7th, 14th, 21st and 28th days after surgery. The registration and processing of laser Doppler flowmetry (LDF) results was performed using the AcqKnowledge version 4.2 software. Microcirculation values were expressed in perfusion units (PE). The microcirculation level curve was recorded over the entire surface of the liver for 30 seconds at each point. From the obtained values the average was deduced, which was entered into the protocol and was taken as the level of microcirculation in the liver in this animal. From the obtained values, the average was calculated, which was taken as the level of microcirculation in this group of animals at a given period of the study.

In the group of intact animals, the level of microcirculation in the liver was 877 ± 17 PE. The results of assessing the level of microcirculation in animals of the experimental groups are presented in table 1.

The average level of microcirculation in the liver of false-operated animals at all periods has no significant differences from that in intact rats.

After liver resection, the indicator decreases by 50% and remains unchanged for the first 14 days. By 21 days, the indicator begins to grow, but on the 28th day the average microcirculation level in the resected rat liver is significantly lower than that in intact animals.

The introduction of L-arginine contributed to maintaining blood flow in the resected liver at a fairly high level (the indicator decreases by 15%) and restoring it for up to 21 days after resection.

The severity of cytolysis was evaluated by the level of indicators of AlAT, AsAT and LDH in the blood of experimental animals on the 2nd, 7th, 14th and 21st 28th day after surgery. The parameters were determined by UV kinetic method on an automated system AU5800 (Beckman Coulter, USA).

AlAT in the control group on the 2nd day of 108 ± 4 IU / l - decreases to normal by 21 days - 38 ± 2 IU / l, in the group with L-arginine on the 2nd day - 70 ± 2 IU / l - normalizes on the 7th day and remains within normal values for all other periods of the experiment.

AsAT in the control group on the 2nd day 184 ± 7 IU / L - decreases to normal by 21 days, in the group with L-arginine on the 2nd day -97 ± 2 IU / L - normalizes on the 14th day and remains within normal values for all other periods of the experiment.

LDH in the control group on the 2nd day 564 ± 3 IU / l - decreases to normal by 28 days - 250 ± 6 IU / l, in the group with L-arginine on the 2nd day - 320 ± 9 IU / l - normalizes on the 7th day and remains within normal values for all other periods of the experiment.

Synthetic liver function was evaluated by coagulogram indices (APTT, MHO, fibrinogen) on the 7th, 14th, 21st and 28th days after surgery on a CS-2000i analyzer (Sysmex, Japan).

On the 7th day after liver resection, a decrease in synthetic function was noted, manifested by an increase in APTT to 60 ± 1 sec, an increase in MHO to 4 ± 0.2, and a decrease in fibrinogen level to 1.7 ± 0.002 g / l. the greatest changes in indicators were found on the 21st day (APTT - 75 ± 1 sec, MHO - 6 ± 0.1, fibrinogen 0.9 ± 0.003 g / l). On the 28th day, some recovery is noted (APTT - 55 ± 1 sec, MHO - 4 ± 0.1, fibrinogen 1.2 ± 0.002 g / l). Correction with L-arginine contributed to a less pronounced decrease in the synthetic function of the liver after its resection. The most pronounced changes in parameters were recorded on the 7th day (APTT - 42 ± 1 sec, MHO - 2.8 ± 0.02, fibrinogen 1.0 ± 0.01 g / l). By the 14th day, the indicators recovered and remained within the normal range for all other periods of the experiment.

Morphological studies were performed on standard liver sites taken after the animals were removed from the experiment. The material was processed according to a standard method with formalin fixation, paraffin embedding, hematoxylin and eosin staining. General morphological and morphometric studies were performed using the MiraxDesk image scanning and archiving system.

Liver mass in intact animals was 9.2 ± 0.002 g. In the group of false-operated animals, the indicator has no significant differences from that in intact animals. Liver mass immediately after resection 2.8 ± 0.003 g. The average mass of the liver of animals of the experimental groups at different periods of the experiment is presented in table 2.

In the group of animals with liver resection, the liver mass on the 2nd day increases in comparison with the mass immediately after resection by almost 2 times, by the 7th day it decreases slightly, then it begins to increase, but on the 28th day after the resection there is significantly less liver mass in intact rats. In the group of animals with liver resection and the introduction of L-arginine, the liver mass on the 2nd day increases exactly 2 times, continuing to grow and reaching mass in intact animals by 21 days.

Under microscopy, at a long time in the control group, uneven blood filling of vessels of all types remained, foci of granular dystrophy of hepatocytes, foci of sclerosis and inflammatory infiltration of portal tracts were observed. Recovery of hepatic tissue was due to hypertrophy of mononuclear hepatocytes, polyploidy, and amitotic division of binuclear hepatocytes. In the group with the introduction of erythropoietin in the long-term periods, microcirculatory disorders and signs of damage to the hepatocytes were not detected, uniformly expressed hypertrophy of the cytoplasm and hepatocyte nuclei is determined. The restoration of the liver tissue was due to polyploidy, amitotic division of binuclear hepatocytes and mitotic division of mononuclear hepatocytes, i.e. all types of liver regeneration were involved. The main load fell on mononuclear hepatocytes with a core size of 9 μm on the 2nd and 7th day, which indicated the stimulation of the genetic apparatus of the cell.

The introduction of L-arginine contributed to:

- reduction of mortality from postresection liver failure;

- a significant reduction in the severity of symptoms of postresection liver failure in comparison with the control group;

- restoration of the volume, structure and functions of the resected organ up to 28 days after resection, which was not observed in the control group.

The results obtained allow us to ascertain the possibility of stimulating the regeneration of the resected liver in rats with L-arginine, and to conduct clinical trials of the drug. Reducing the recovery time of the resected organ and its lost functions will reduce mortality from postresection liver failure, significantly reduce the severity of symptoms of postresection liver failure, improve the patient's condition in the postoperative period, and shorten the duration of the postoperative rehabilitation period, which is important for public health.

Claims (1)

A method of stimulating regeneration of the resected liver, characterized in that the liver resection is performed on a laboratory animal on the second day of the experiment in a volume of 70%, L-arginine is administered intragastrically at a daily dose of 200.0 mg / kg every 46 hours for the first 7 days of the experiment.