Study of morphological transformation and features of vascular blood flow of the wall of the small and large intestine in the simulation of ischemia in the experiment

Irina Yurevna Karpova; Карпова Ирина Юрьевна; Pyotr Vladimirovich Peretyagin; Перетягин Петр Владимирович; Natalia Yurievna Orlinskaya; Орлинская Наталья Юрьевна; Natalia Yurievna Shirokova; Широкова Наталья Юрьевна; Evgeniya Dmitrievna Pyatova; Пятова Евгения Дмитриевна; Sofia Sergeevna Ptushko; Птушко София Сергеевна

doi:10.18499/2070-478X-2023-16-2-120-129

Study of morphological transformation and features of vascular blood flow of the wall of the small and large intestine in the simulation of ischemia in the experiment

Authors: Karpova I.Y.¹, Peretyagin P.V.², Orlinskaya N.Y.², Shirokova N.Y.², Pyatova E.D.², Ptushko S.S.²
Affiliations:
1. Volga Research Medical University
2. Privolzhsky Research Medical University
Issue: Vol 16, No 2 (2023)
Pages: 120-129
Section: Original articles
URL: https://vestnik-surgery.com/journal/article/view/1677
DOI: https://doi.org/10.18499/2070-478X-2023-16-2-120-129
ID: 1677

Cite item

Full Text

Abstract
Full Text
About the authors
References
Supplementary files
Statistics

Abstract

Introduction. Ischemia of the small and large intestine of various degree was simulated in 45 sexually mature male rats of the Wistar line weighing 150-200 g on the basis of the Department of Experimental Medicine with vivarium at Privolzhsky Research Medical University.
Аim. To present in an experiment the effect of different degrees of occlusive ischemia on the morphological transformation of the intestinal wall and the level of changes in blood flow.
Materials and methods. The anesthetized animals underwent a median laparotomy with subsequent differentiation of the intestinal divisions: the jejunal section was selected in the small intestine, the ascending section was selected in the large intestine. With the help of a nylon thread (5-0), the blood supplying arcades of these zones were ligated and further exposed for 40, 60 and 120 minutes. During the indicated periods of ischemia, the average rate of blood supply in capillary microvessels located at a depth of 0.5-1.0 mm was estimated in relative units on 1.0 mm2-area (LACC-02, NPP Lazma, Russia). After the assessment of vascular blood flow was completed, ischemic intestinal areas were sampled for morphological examination. The study results were processed using Excel application and STADIA statistical package.
Results. In the course of study, the authors registered clear relationships between blood flow parameters in different parts of the intestine and the duration of ischemia. Local trophic disturbance was combined with a transformation in the histoarchitectonics of the intestinal wall. It is noted that adaptive-regenerative mechanisms provide tissue stress reduction in 120 min. due to compensatory mechanisms of blood supply contributing to the restoration of the "villus-crypt" system of the mucous membrane.
Conclusion. Thus, in case of local ischemia in the small and large intestine, the tissue structure is restored due to adaptive mechanisms of blood supply, this preserves the viability and functionality of the intestinal wall.

Keywords

experiment, occlusion, intestinal ischemia, blood flow

Full Text

Relevance
The first mention of mesenteric ischemia dates back to 1507, published in the work of the Italian anatomist A. Bienveni "On some occult and unusual causes of diseases and their treatment" [1].
Acute disorders of mesenteric circulation (ONMC), leading to intestinal infarction, occur in 0.2% of surgical patients. Despite the development of medicine, mortality in this disease remains extremely high, reaching 67-92% [2, 3, 4].
Ischemia of the digestive organs develops due to a variety of
etiological factors and can be caused by both occlusive-stenotic (occlusive) lesion of visceral vessels and non-occlusive disorders of abdominal blood supply caused by a decrease in visceral blood flow at the microcirculatory level [5, 6, 7, 8].
The occlusive type of ONMC, in turn, is divided into a thrombotic type (about 25%), developing as a result of acute arterial thrombosis of the proximal segment of the vessel (most often the mouth of the superior mesenteric artery), and an embolic type (about 50% of all ONMC), developing as a result of occlusion caused by displacement with the blood flow of emboli, initially formed proximally (against the background of atrial fibrillation, parietal thrombosis of the left ventricle after a heart attack, aneurysm of the heart, aorta).
Non-occlusive violation of mesenteric circulation (NONMC) accounts for about 20% of all cases of intestinal infarction. In its pathogenesis, the main role is played by low cardiac output, hypovolemia, hemoconcentration, and a decrease in blood flow in the mesenteric vascular system. These factors most often occur during the prolongation of critical conditions in the intensive care unit, but may occur in patients with a burdened history, for example: with cardiovascular, hematological diseases [2, 4, 9, 10].
The incidence of abdominal ischemic syndrome is quite high: it is detected in 75.5% of cases during autopsy of those who died from coronary heart disease, due to atherosclerosis of the cerebral arteries and/or vessels of the lower extremities, atherosclerosis of the abdominal aorta and its unpaired visceral branches is also detected [11, 12].
The incidence of stenosing lesions of the visceral branches of the abdominal aorta according to autopsy varies from 19.2% to 70%, according to angiography – from 4.1% to 53.5%. [13, 14].
The peculiarities of intestinal blood supply are such that the most vulnerable places are the splenic angle and the left bend of the sigmoid colon, located in the zone of poorly developed anastomoses of the upper and lower mesenteric arteries. Ischemic damage to the rectum due to the efficiency of blood supply is very rare [15].
The aim of the study: to present in an experiment the effect of different degrees of occlusive ischemia on the morphological transformation of the intestinal wall and the level of changes in blood flow.
Materials and methods
On the basis of the Department of Experimental Medicine with vivarium of the Volga Research Medical University, different degrees of ischemia of the small and large intestine were studied on 45 sexually mature male rats of the Wistar line weighing 150-200 g, obtained from the Andreevka branch of the Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies".
All animals were kept in standard vivarium conditions in cages with free access to food and water. The working conditions corresponded to the principles of biological ethics, the requirements of the "International Helsinki Convention on Humane Treatment of Animals" (1972), the rules of the European Convention for the Protection of Vertebrates Used for Experiments or Other Scientific Purposes ET/S 129 (Strasbourg, March 18, 1986) and the Order of the Ministry of Health of the Russian Federation dated 04/01/2016 No. 199n "On approval Rules of good laboratory Practice". Minutes of the meeting of the Ethics Committee of the Federal State Educational Institution "PIMU" of the Ministry of Health of the Russian Federation No. 7 dated 07.04.2021.
Ischemia modeling was performed under general anesthesia (intramuscularly with a solution of Zoletil 100, (60 mg/ kg) in combination with a solution of Xyl, 6 mg/kg). The anesthetized animals underwent median laparotomy followed by differentiation of the intestine sections, the skinny section was selected in the thin one, the ascending one in the thick one. With the help of a nylon thread (5-0), the blood supply arcades of these zones were ligated, with further exposure for 40, 60 and 120 minutes. To compare the results, a control group was formed from animals that did not undergo ischemia (n=15).
After the time of exsanguination of the sites with the help of a laser analyzer of capillary blood flow (LAKK-02, NPP Lazma, Russia), the average rate of blood supply in capillary microvessels located at a depth of 0.5-1.0 mm, on an area of 1.0 mm2 in relative units was estimated (Figure 1).

Fig.1. LAKK-02, NPP Lazma (Russia) with computer software
Fig. 1. LAKK-02, NPP Lazma (Russia) with computer software
The principle of operation of the device is based on the reflection of a probing helium-neon laser beam from moving red blood cells. The microcirculation index (PM) depends on the number of red blood cells in the microvessels, the speed of their movement. The change in the signal frequency (Doppler effect) is associated with the presence of plasma gaps between red blood cells. The installation consists of a unit with a digital indicator and a PM recorder, a laser beam at the tip of the probe, which was installed over the examined area of the intestine. The animal was under anesthesia because the study was carried out during the operation. Registration was performed before the bowel ligation and the formation of the pathological process after 40, 60, 120 minutes.
After the assessment of vascular blood flow was completed, the ischemic areas of the intestine were sampled for morphological examination.
The experimental material was fixed in 10% formalin, after which the preparations were sent to the standard histological wiring on the Excelsior ES (Thermo Scientific) apparatus, immersed in paraffin blocks using the HistoStar filling station (Thermo Scientific), stained with hematoxylin and eosin. For morphometric processing and creation of a video archive of the obtained material, a Leica 2500 microscope and a lens were used ×4, ×10, ×20, ×40, eyepiece × 10 based on the Department of Morphology of NIITO PIMU.
The data of this study were processed using the Excel application and the STADIA statistical package.
The tables "Descriptive statistics" present the basic numerical characteristics of the studied samples: mean, mean-square deviation σ, error of the sample mean m, median, interquartile range and information about the distribution of the sample (N – distribution is almost normal, ≠N – distribution is different from normal). The Excel application and the STADIA package were used for calculations.
To analyze the distributions of samples for proximity to normal, the following methods are recommended: the A and E analysis method (asymmetry and kurtosis), the Kolmogorov method, the w2 method (omega square), the χ2 method (Pearson method) – from the STADIA package.
The vast majority of samples had a distribution different from normal, so at the stage of data comparison, the paired nonparametric Wilcoxon method was used, which studies samples by medians.
The tables indicate the samples being compared, the result of the difference (yes/no), the significance level p at which the No-hypothesis was tested, as well as the name of the numerical characteristic with which the No-hypothesis was tested (But: there is no difference in medians).

Results and their discussion
As part of the experimental modeling, the animals were divided into 4 groups: group I – control, in which the tissues were not subjected to ischemia; group II – ischemia 40 min.; group III – ischemia 60 min.; group IV – ischemia 120 min. (Figure 2).

Fig.2. Study of the indicator of intestinal microcirculation in rats when modeling ischemia using LACC-02
Fig. 2. Study of the indicator of intestinal microcirculation in rats when modeling ischemia using LACC-02
The average rate of blood supply in the capillary microvessels of the small (18.95±2.37) and large intestine (18.77±3.33) animals of group I was identical at p0.05.
Local trophic disturbance after 40 min. (group II) significantly disorganized blood flow in the wall of the small intestine (11.7± 2.8), in the thick PC was 13.2± 2.2, p0.05.
Pathological changes in the structure of the vascular bed in group III (after 60 min.) demonstrated the work of adaptive mechanisms that increased the PC in the small intestine by 1.98, and in the large intestine this indicator exceeded the values of normal blood supply by 1.53.
Ischemia during 120 min. stated a further increase in the blood flow index in the small intestine to 17.3 ± 3.1 and a decrease in PC from 20.3±3.2 to 15.3±4.6 in tolstoy (Tables 1-4).

Table No. 1 Descriptive statistics for the indicator of blood flow of the small intestine

Table No.2 Comparison table (small intestine, paired samples)

Table No. 3 Descriptive statistics for the indicator of colon blood flow

Table No. 4 Comparison table (colon, paired samples)

When analyzing histological sections of the intestine, the unchanged mucous membrane (CO) of the small intestine of rats resembled human CO and was characterized by the presence of high finger-like villi covered with columnar edged epithelial cells with boundaries recognizable at the light-optical level. The nuclei of the latter were located basally, goblet cells were well defined, both in the villi and in the crypts.

About the authors

Irina Yurevna Karpova

Volga Research Medical University

Email: ikarpova73@mail.ru
ORCID iD: 0000-0002-4897-6702
SPIN-code: 8464-8485

Doctor of Medical Sciences, Associate Professor, Department of Pediatric Surgery

Russian Federation, 603005, Nizhny Novgorod, Minin and Pozharsky pl., 10/1