AN EXPERIMENTAL MODEL OF LIVER ECHINOCOCCOSIS IN LABORATORY RATS TO STUDY THE EFFECTIVENESS OF ANTHELMINTIC DRUGS

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Every year, more than 2 million new cases of echinococcosis infection are registered in the world, which ranges from 0.05 to 1.5% in the structure of infectious pathology, while in recent years there has been a significant increase in the incidence of this helminthiasis, which in a number of countries ranges from 30 to 95%. Among them, the number of cases with complicated forms is growing, the proportion of which reaches 35 - 40% [1, 2, 3] with a mortality rate of 2-5% [4]. In this regard, by the decision of the WHO, echinococcosis is included in the list of the most common human helminthiasis requiring priority elimination [5, 6].

The epidemiological situation of parasitic diseases in Russia, including echinococcosis, remains difficult. Every year, over 500 cases of this larval helminthiasis are registered in the country for the first time. In 2021, the incidence increased by 18.75% compared to 2020 and amounted to 0.19 per 100 thousand population [7].

Clinical diagnosis of echinococcosis is difficult due to its long-term latent course, in this regard, instrumental (ultrasound, CT, etc.) and serological methods currently play a leading role in making a final diagnosis, the diagnostic significance of which increases when they are used together [8, 9].

To date, the main method of treating echinococcosis remains surgical, the possibilities of which are severely limited in cases of multiple invasion or inoperable [10]. The first attempts to develop a method of specific chemotherapy for echinococcosis based on mebendazole, a drug of the carbamatbenzimidazole group, were made back in the 80s of the last century. In the late 90s, the structural analogue of mebendazole, albendazole, became widely used. The insufficient effectiveness of both oral drugs is largely due to their relatively low bioavailability [11]. However, many researchers have shown that mebendazole showed greater toxicity and lower efficacy in cystic echinococcosis and alveococcosis, therefore, albendazole is currently most widely used in clinical practice [12]. Attempts to increase the bioavailability of these drugs by creating experimental oil dosage forms did not yield the expected results [13, 14]. The use of praziquantel as a tissue anthelmintic drug is limited by the peculiarities of its pharmacokinetics, in particular, a short half-life, as well as a significant range of adverse reactions with prolonged use [15].

To date, surgeons, infectious disease specialists and doctors of other specialties do not have unified approaches to the therapeutic tactics of managing patients with echinococcosis. The introduction into clinical practice of drug therapy with anthelmintic drugs from the carbamatbenzimidazole group has reduced the need for aggressive surgical interventions at the initial stages of the development of parasitic cysts. However, there is still no consensus on in which cases and at what sizes of cysts the use of carbamate benzimidazole monotherapy will be sufficient, and in which cases a combination of surgical and therapeutic methods of treatment is necessary. According to a number of authors, the main reasons for the ineffectiveness of monotherapy is the inability to conduct a full course of albendazole due to the pronounced polymorphism of genes encoding albendazole biotransformation enzymes, as well as various side effects of the drug [16]. It is also known that after surgery, which is not accompanied by subsequent carbamate benzimidazole therapy, relapses of the disease often develop.

Currently, the data of clinical studies and the proposed experimental models on laboratory animals do not allow us to fully assess the histological changes in the structure of echinococcal cysts and the viability of the parasite against the background of etiotropic therapy in dynamics.

In the scientific literature, there are works on experimental modeling of echinococcal cysts by introducing germ elements intraperitoneally, directly into the liver parenchyma or into vessels bringing blood to the organ [17, 18, 19], however, their disadvantages are: multi-stage technique, the need for repeated surgery to remove the balloon and implantation of a daughter bladder, the possibility of obstruction of large vessels and organ infarction during administration embryonic elements of echinococcus in the liver parenchyma, long-term modeling of an echinococcal cyst, high mortality among experimental animals.

In this regard, we have developed a new, technically simpler model of liver echinococcosis in experimental animals, which allows us to assess the dynamics of histological changes against the background of anthelmintic therapy.

The purpose of this study was, based on the developed experimental model of liver echinococcosis in laboratory animals, to study the dynamics of histological changes in echinococcal cysts during therapy with anthelmintic drugs albendazole and praziquantel in order to assess their effectiveness.

Materials and methods

Experimental work on animals, approved by the independent Ethical Committee at the Military Medical Academy named after S.M. Kirov (VMedA) (Protocol No. 258 dated 12/21/2021), was performed on the basis of the vivarium of the Department of Military Toxicology and Medical Protection of VMEDA. The maintenance and care of experimental animals were carried out in accordance with the requirements of the Law of the Russian Federation "On Veterinary Medicine" No. 4979-1 dated 05/14/1993 [20] and "Recommendations of the Ethics Committee conducting expertise in biomedical research" [21].

The study included 30 male Wistar rats weighing 250±50 g, in which echinococcosis of the liver was modeled according to the developed technique. The formation of experimental liver echinococcosis was performed as follows. In the operating room, the general anesthetic of dissociative action Zoletil 100 (tiletamine hydrochloride and zolepam hydrochloride 250 mg each) was administered at a dose of 20 mg /kg body weight. After the onset of anesthesia, the animal's fur was shaved off in the abdomen and chest area, after which fixation was carried out on the operating table. The surgical field was treated with an antiseptic solution according to Filonchikov-Grossich. The xiphoid process and the edge of the right costal arch were palpated. A median laparotomy was performed using a scalpel. A part of the wall of the echinococcal bladder obtained from sheep affected by echinococcus was placed on the capsule of the right lobe of the liver, so that the germinal layer adjoined the capsule. This part of the parasite, about 10x10 mm in size, was fixed to the liver using a ligature with a single suture. Then the wound of the abdominal cavity was tightly sewn up.

On the 60th day of the experiment, the formation of a liver cyst was confirmed by ultrasound in all animals. After that, the rats were randomly divided into three groups: the first control group (10 rats) and two experimental groups of 10 rats each. For 28 days, albendazole suspension in a daily dose of 2.5 mg was intragastrically administered to animals of the second group through a probe, which was divided into two doses. To increase bioavailability, 100 mg of liquid butter was added to the drug. Animals of the third group received praziquantel in the form of a suspension, which was prepared from a tablet form of the drug. The drug was administered using a gastric tube at a daily dose of 15 mg in two doses for 15 days. The design of the study is shown in Figure 1.

Figure 1. Research design.

During the experiment, all animals received a standard rodent feed (Nuvilab CR1s Nuvital S.A., Colombo-PR, Brazil), which consisted of 22% protein, 4% fat, 4% crude fiber, which corresponded to 290 kcal/100 g. Each animal received 12 g of feed during the day.

At the end of the experiment, the animals were killed, the liver was extracted for further study. Its fragments with parasitic cysts were fixed in a solution of 10% neutral formalin for 24 hours. After fixation, they were dehydrated by incubation in isopropyl alcohols, and then soaked in paraffin according to a generally accepted method. Tissue sections with a thickness of 4 microns were made from paraffin blocks, which were stained with hematoxylin-eosin and picrofuxin according to Van Gieson, after which they were placed under cover glasses [22]. Histological preparations were studied using a binocular microscope in transmitted light with a total magnification of ×50, ×200, ×1000,

The results of the study

In animals of the first group (n=10, control), which were not prescribed an anthelmintic drug, in all cases, the development of typical echinococcal cysts in the form of acephalocysts (without protoscolexes) was observed. Microscopy of histological sections revealed an external fibrous membrane of a specific structure with an organ-like structure containing blood vessels that transport nutrients to the parasitic cyst. In the immediate vicinity of the fibrous membrane, inflammatory polymorphocellular infiltration was detected in liver tissue, including segmented neutrophils, macrophages and fibroblasts with single lymphocytes (Fig. 2 a, b).

Figure 2. a is a quinococcal cyst (magnification x50). b is a part of the cyst with inner membranes (magnification x1000). 1 is the germinal membrane, 2 is the cuticular membrane.

Figure 2. a - echinococcal cyst (magnification x50). b - part of the cyst with inner membranes (magnification x1000). 1 - germinative shell, 2 - suticle shell.

In echinococcal liver cysts in experimental animals of the second group (n=10) treated with albendazole on the 88th day of the experiment (28th day of therapy), destructive changes developed in the cuticle and in the germinal membranes. The histological picture was characterized by detachment and fragmentation of the germinal layer, and an accumulation of detritical masses was observed in the cyst cavity, which indicated processes indicating the death of the parasite. In addition, perifocal cell infiltration was observed in the fibrous membrane and adjacent tissues, represented by lymphocytes, neutrophils and eosinophilic leukocytes, macrophages and mast cells (Fig. 3 a, b).

In echinococcal liver cysts in experimental animals of the third group (n=10) treated with praziquantel, destructive changes from the cuticle and germinal layers were also observed on the 75th day of the experiment (15th day of therapy). However, these changes were more significantly pronounced than in rats treated with albendazole, which was manifested by massive leukocyte infiltration and cellular fragmentation of the germinal membrane (Fig.3 c, d).

 Figure 3. Destructive changes in the cuticle and germinal membrane of an echinococcal cyst during therapy with albendazole (a, b) and praziquantel (c, d). 1. Destructive changes in the germinal membrane. 2. Destructive changes in the cuticle membrane 3.  Massive leukocyte infiltration.

When using albendazole, significant destructive changes in the cuticle and germinal membranes of echinococcal cysts were noted on the 28th day of therapy. When using praziquantel, as early as day 15, the histological picture was characterized by a more powerful cellular infiltration of all layers of the cyst. However, taking this drug in animals was accompanied by a complex of negative external signs indicating poor tolerability of therapy, and possibly the development of significant adverse reactions, which was manifested by a decrease in motor activity and hair loss.

Thus, the new model of liver echinococcosis in rats developed by us provides high survival in experimental animals with guaranteed formation of parasitic cysts, which allows it to be used in experimental studies to evaluate the effectiveness of various anthelmintic therapy regimens taking into account the dynamics of histological changes.

 

 

About the authors

Timofey Gavrilyuk

S. M. Kirov Military Medical Academy, Saint Petersburg

Author for correspondence.
Email: Gtv-25@mail.ru
ORCID iD: 0000-0001-7102-0672
SPIN-code: 9515-3727

Associate Professor of the Department of Infectious Diseases (with a course in medical Parasitology and Tropical Diseases)

Russian Federation, 6 Akademika Lebedeva str., Saint Petersburg, 194044,

Andrey Saulevich

S. M. Kirov Military Medical Academy, Saint Petersburg

Email: saulevich_andrei@mail.ru
ORCID iD: 0000-0001-6756-3105
SPIN-code: 9356-8410

Candidate of Medical Sciences, Lecturer at the Department of Infectious Diseases (with a course in medical Parasitology and Tropical Diseases)

Russian Federation, 6 Akademika Lebedeva str., Saint Petersburg, 194044,

Sergey Kozlov

S. M. Kirov Military Medical Academy, Saint Petersburg;
St. Petersburg State Medical Pediatric University of the Ministry of Health of the Russian Federation, St. Petersburg;
Saint Petersburg Children's Scientific and Clinical Center of Infectious Diseases of the FMBA, Saint Petersburg.

Email: infectology@mail.ru
ORCID iD: 0000-0003-0632-7306
SPIN-code: 5519-9190

Doctor of Medical Sciences, Professor of the Department of Infectious Diseases (with a course in medical Parasitology and Tropical Diseases)

Russian Federation, 6 Akademika Lebedeva str., Saint Petersburg, 194044; St. Petersburg, Litovskaya str., 2A; St. Petersburg, St. Petersburg, Professor Popov str., 9.

Yuri Zaharkiv

S. M. Kirov Military Medical Academy, Saint Petersburg

Email: zufbiology@gmail.com
ORCID iD: 0000-0002-3453-7557
SPIN-code: 6541-9803

Candidate of Medical Sciences, Associate Professor of the Department of Biology

Russian Federation, 6 Akademika Lebedeva str., Saint Petersburg, 194044

Konstantin Kozlov

S. M. Kirov Military Medical Academy, Saint Petersburg

Email: kosttiak@mail.ru
ORCID iD: 0000-0002-4398-7525
SPIN-code: 7927-9076

Doctor of Medical Sciences, Associate Professor of the Department of Infectious Diseases (with a course in medical Parasitology and Tropical Diseases)

Russian Federation, 6 Akademika Lebedeva str., Saint Petersburg, 194044

Vladimir Turicin

S. M. Kirov Military Medical Academy, Saint Petersburg;
Saint Petersburg State Agrarian University, Saint Petersburg

Email: turicin_spb@mail.ru
ORCID iD: 0000-0001-9066-0026
SPIN-code: 2022-1869

Candidate of Biological Sciences, Associate Professor of the Department of Aquatic Bioresources and Aquaculture

Russian Federation, 6 Akademika Lebedeva str., Saint Petersburg, 194044; 196601, St. Petersburg, Pushkin Peterburgskoe shosse, 2, lit. A

Vadim Karev

Saint Petersburg Children's Scientific and Clinical Center of Infectious Diseases of the FMBA, Saint Petersburg

Email: vadimkarev@yandex.ru
ORCID iD: 0000-0002-7972-1286
SPIN-code: 7503-3253

Doctor of Medical Sciences

Russian Federation, St. Petersburg, St. Petersburg, Professor Popov str., 9.

References

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