Posthypoxic Changes in Rat Offspring under the Intestinal Wall Transformation

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Introduction
For many decades, there has been an active search for risk factors, the presence of which directly correlates with the formation of congenital malformations and surgical diseases of the newborn period [1].
It is proved that hypoxia is one of the leading predictors of the development of many pathological processes, such as various forms of intestinal atresia, Hirschsprung's disease, necrotizing enterocolitis [2, 3].
In the domestic and foreign literature, a large number of experimental works are presented, which, from the point of view of evidence-based medicine, confirm the severity of the effects of hypoxia on the body, both in the antenatal and postnatal periods.
Structural changes in the vascular and neuro-regulatory links are a priority in the formation of pathological processes in the gastrointestinal tract [4-7].
It is noted that the intestinal nervous system is not fully developed at the time of birth and continues to form neurons in the early postnatal stages. The ratio of the cytoplasmic region of the immunoreactive centers of the brain to the area of the mesenteric ganglia in the intestinal tube is significantly reduced in the group of patients with necrotizing enterocolitis, in contrast to newborns with intestinal atresia. Experimental transplantation of nerve stem cells with their subsequent differentiation into functional neurons leads to an improvement in intestinal transit [8].
When occluding the vessels of the mesenteric arcade of the distal ileum of low-weight full-term piglets for 48 hours, it was shown that there is a wide range of changes in the morphological picture: from minimal in the form of erosion of the mucous membrane and desquamation of single epithelial cells, to pronounced with necrosis, ulceration, pneumatosis, perforation of the intestinal wall and the development of peritonitis [9, 10].
The aim of the work is to study the posthypoxic transformation of the intestinal wall on an experimental model of rat offspring using light and electron microscopy.

Materials and methods
Research design
In the experimental work, 24 female white mongrel rats with a weight of 156-230g, aged from 4 to 10 months, were used. The laboratory animals were divided into 4 groups of 6 females each. Group I underwent hypoxia in the first trimester of pregnancy (week 1), group II-in the second trimester (week 2), group III – in the third trimester (week 3), and Group IV-during the entire antenatal period (21 days). In parallel with the hypoxia, the control group V was planted, in which the individuals were not subjected to oxygen starvation.
To simulate chronic hypobaric hypoxia, rats were placed in a vacuum flow pressure chamber, in conditions corresponding to the ascent to a height of 5000 m at a speed of 25 m / s. for 40 minutes. by group, daily for 7 days.
After delivery, the offspring were examined, subjected to hexenal anesthesia, and then decapitated out of the experiment.
The experimental study was conducted in accordance with the legislation of the Russian Federation ("Rules for the Humane Treatment of Laboratory Animals", "Deontology of Medical and Biological Experiment") and the ethical principles established by the European Convention for the Protection of Vertebrates Used for Experimental and Other Scientific Purposes (adopted in Strasbourg on 18.03.1986 and confirmed in Strasbourg on 15.06.2006) with the permission of the Ethics Committee of the Nizhny Novgorod State Medical Academy (Protocol No. 16, 2013).
For light microscopy, a resected section of the small, large intestine was taken, fixed in a 10% solution of neutral formalin, dehydrated in alcohols and poured into paraffin. Sections prepared on a Leica SM 2000 R microtome, 5-7 microns thick, were stained with hematoxylin-eosin-overview staining.
Subsequent microscopy was performed on Topic microscopes (Belgium, 2000) and Bio Blue microscopes (Netherlands, 2013) using a 10x eyepiece and 40x and 100x lenses.
A morphometric study of all the structures of the intestinal wall was performed – the number of villi per unit perimeter of the intestinal tube, the height of the epithelium, the height of the villi, the depth of the crypts, the thickness of the muscle membrane, as well as the area of the microcirculatory bed of the serous membrane. The area of the vascular bed per unit area of the mesentery, the thickness of the walls of the blood vessels of the arterial system of the mesentery were studied.
Electron microscopic analysis of intestinal samples was performed according to the standard method: fixation in a 2.5 % solution of glutaraldehyde on a phosphate buffer (pH = 7.4), followed by additional fixation with a 1% solution of osmium tetrachloride and pouring into a mixture of araldite and epon epoxy resins. Semitone and ultrathin sections were obtained on the Leica UC7 ultramicrotome (Austria). The sections were contrasted with an alcoholic solution of uranyl acetate and lead citrate according to Reynolds, then analyzed in a Morgagni 268D transmission electron microscope (FEI, USA).

Results and discussion
Histological examination of the intestine in rats of all groups showed that the structure of the intestinal wall does not differ from the control group, and is represented by the presence of four layers – mucosal, submucosal, muscular and serous.
At the same time, the study of the wall revealed a significant decrease in the thickness of the mucosa and thickening of the muscle membranes (Fig. 1).

 

Fig. 1. The mucous membrane of a newborn rat of the first group of studies. Thinning of the mucous membrane (indicated by an arrow) (hematoxylin-eosin, uv. - vol. 40x, approx. 15x)
Fig. 1. The mucous membrane of a newborn rat of the first group of studies. Thinning of the mucous membrane (indicated by an arrow) (hematoxylin-eosin, uv. - volume 40x, approx. 15x)

Chronic hypoxia, modeled in the first trimester of pregnancy, led to the development of secondary tissue hypoxia in the intestinal wall, resulting in a decrease in the epithelial component, manifested by a decrease in the thickness of the mucous membrane and the growth of connective tissue in the muscle layer (Fig. 2). An increase in connective tissue instead of smooth muscle fibers, as a rule, leads to a violation of the contractility of the wall and the development of dynamic obstruction.

2. 2. The intestinal wall of a newborn rat of group I in chronic hypoxia. Thickening of the muscle layer (indicated by an arrow) (hematoxylin-eosin, uv. - vol. 40x, approx. 10x)
Fig. 2. The intestinal wall of a newborn rat of group I in conditions of chronic hypoxia. Thickening of the muscle layer (indicated by an arrow) (hematoxylin-eosin, uv. - volume 40x, approximately 10x)

Electron microscopic examination of the intestinal tissue showed that interepithelial lymphocytes were found in the mucous membrane among columnar epithelial cells, goblet cells and endocrinocytes. Mitotic activity was detected in epithelial cells (Fig. 3a), while myelin-like structures were detected (Fig. 3b). Hypertrophied Golgi complex was found in goblet cells (Fig. 3b).
Cytoplasmic cleavage, plasmalemma homogenization, visual reduction in the number of ribosomes, expansion of the cisterns of the granular endoplasmic reticulum, mitochondrial matrix cleavage, and crist destruction were observed in the fibroblasts in the mucosal lamina proper. Macrophages, single red blood cells, eosinophils, and lymphocytes were found in the submucosal base. Connective tissue cells in the state of mitosis, nerve fibers with signs of impaired ultrastructure were found. Thus, vacuolated mitochondria and axial cylinders with electron-transparent cytoplasm were found in the Schwann cell (Fig. 3b).
The microcirculatory bed was represented by hemocapillaries containing plasma. Part of the vessels included mature and young forms of red blood cells (Fig. In the basal membrane of the vessels, there were homogenized loci or areas of dislocation. In the endothelium, there were numerous vesicles, mitochondria with matrix enlightenment. Quantitative analysis showed that 36% of the hemocapillaries showed a violation of the ultrastructure in the form of swelling or thinning of the endothelial lining, vacuolization, and membrane structures in the lumen, leading to microcirculation disorders.

 

 

3. Ultrastructure of the wall of the descending colon of the rat offspring (prenatal hypoxia of group 1). a-mitosis of epithelial cells (arrow), x 4500; b-myelin-like structures in the epithelial cell (arrow), x 11000; c-Schwann cell with vacuolated mitochondria (arrow), x 8900; d-hemocapillary, x 5600
3. Ultrastructure of the wall of the descending colon of the offspring of rats (prenatal hypoxia of group 1). a-mitosis of epithelial cells (arrow), x 4500; b-myelin-like structures in the epithelial cell (arrow), x 11000; c-Schwann cell with vacuolated mitochondria (arrow), x 8900; d-hemocapillary, x 5600

Hypoxia formed in the second and third periods of pregnancy did not lead to such a significant increase in the fibrous tissue in the intestinal wall, but the material showed the involvement of the epithelial component in the pathological process. Thus, hypoxia of the second period of pregnancy was accompanied by atrophy of the mucous membrane mainly due to shortening and reducing the number of villi (Fig. 4).

Fig. 4. The intestinal wall of a newborn rat of group II. Shortening of the villi (1) and reducing their number (hematoxylin-eosin, uv. - vol. 10x, approx. 10x)
Fig. 4. Intestinal wall of a newborn rat of group II. Shortening of the villi (1) and reducing their number (hematoxylin-eosin, uv. - volume 10x, approximately 10x)

The integumentary epithelium remained prismatic, although flattened, with areas of atrophy, an increase in the number of goblet-shaped cells with apocrine secretion, filled with vacuolated cytoplasm, whereas in the animals of the control group, the latter had a clear, even contour, and the cytoplasm of the cells looked uniformly eosin-positive (Fig. 5).

5. 5. Intestinal villi of animals of group III of the study. Vacuolation of epithelial cells (hematoxylin-eosin, uv. - vol. 40x, approx. 15x)
Fig. 5. Intestinal villi of animals of group III of the study. Vacuolation of epithelial cells (hematoxylin-eosin, uv. - volume 40x, approx. 15x)

Electron microscopic examination of the intestinal wall of the offspring of group II rats revealed a similar morphological picture with group I animals. It was noted that the epithelial layer contains a significant number of goblet cells, columnar epithelial cells in the state of division, and endocrinocytes. The intercellular spaces in the basal part of the cells were also enlarged, while the dense contacts in the apical part were preserved. Edema of interstitial tissue and swelling of individual fibroblasts were detected in the own plate of the mucosa and in the submucosal base (Fig. 6a). Neutrophils were found in the intercellular space.
Plasma capillaries were observed in the microcirculatory bed (Fig. 6b). Mature and young red blood cells, platelets, and neutrophils were detected in vessels containing shaped blood elements. Pinocytic vesicles were found in the capillary endothelium (Fig.6b), and transformed crystals and partial matrix leaching were found in the mitochondria. The basal membrane was loosened over a longer length. Quantitative analysis showed that 33% of the vessels had a violation of the ultrastructure.

 

6. Ultrastructure of the descending colon wall of rat offspring (prenatal hypoxia of group II): a-fibroblasts with hyaloplasma swelling, x 8900; b-hemocapillary, x 11000
Fig.6. Ultrastructure of the descending colon wall of rat offspring (prenatal hypoxia of group II): a-fibroblasts with hyaloplasma swelling, x 8900; b-hemocapillary, x 11000

Chronic hypoxia, modeled in the third period of pregnancy (group III), was accompanied by a decrease in the thickness of the mucous membrane. However, the change in the thickness of the intestinal wall in animals was associated with the effect of peeling of the integumentary-pit epithelium and the formation of" bare "villi (Fig. 7, 8). Among them, many thickened" stem-like " forms with a flattened surface were noted (Figure 9).

 

Fig. 7. Epithelium of the intestinal villi of animals of the control group (hematoxylin-eosin, uv. - vol. 40x, approx. 15x)
Fig. 7. Epithelium of the intestinal villi of animals of the control group (hematoxylin-eosin, uv. - vol. 40x, approx. 15x)

 

Figure 8. Gut wall of group III animals. Exfoliation of the epithelium of the villi (hematoxylin-eosin, uv. - vol. 20x, approx. 15x)
Fig. 8. Gut wall of group III animals. Exfoliation of the epithelium of the villi (hematoxylin-eosin, uv. - vol. 20x, approx. 15x)

Fig. 9. Intestinal villi of group III animal, thickening and shortening of villi (indicated by arrows) (hematoxylin-eosin, uv. - vol. 40x, approx. 15x)
Fig. 9. Intestinal villi of group III animal, thickening and shortening of villi (indicated by arrows) (hematoxylin-eosin, uv. - vol. 40x, approx. 15x)

Electron microscopic examination of the intestinal wall of group III animals showed that both adaptive and destructive processes were detected in the tissues.
Violations of the ultrastructure in 50% of the vessels were revealed in the form of the presence of membrane structures in the lumen, thinning of the endothelial layer, vacuolation of mitochondria, which contributed to the violation of permeability and pericapillary edema. In some vessels, there was an aggregation of red blood cells (Fig. 10a, b).

 

10. Ultrastructure of the descending colon wall of rat offspring (prenatal hypoxia of the third trimester): a-pericapillary edema (arrow), x11000; b – aggregation of red blood cells in the hemocapillary (arrow), x5600
Fig. 10. Ultrastructure of the descending colon wall of rat offspring (prenatal hypoxia of the third trimester): a-pericapillary edema (arrow), x11000; b – aggregation of red blood cells in the hemocapillary (arrow), x5600

A change in the ratio between the layers of the intestinal wall led to a narrowing of the lumen of the latter, due to fibrosis of the middle shell, which was found in animals of the first group. In the offspring of the second and third groups, the expansion of the lumen contributed to the violation of the dynamic properties of the intestinal tube and the development of its dysfunction (Fig. 11).

11. Lumen of the intestine of group I animals. Narrowing of the intestinal lumen (1) (hematoxylin-eosin, uv. - vol. 20x, approx. 10x)
Fig. 11. Lumen of the intestine of group I animals. Narrowing of the intestinal lumen (1) (hematoxylin-eosin, uv. - vol. 20x, approx. 10x)

To assess the adequacy of the blood supply to the intestine, a morphometric study of the area of the arterial and venous fragments of the bloodstream, as well as the wall thickness of the arteries and veins was performed (table).

Table/ Table
Characteristics of the structure of the intestinal microcirculatory bed
Characteristics of the structure of the intestinal microcirculatory bed

Indicators / Indicators Control group
(n = 14) /
Control group (n = 14) Group I
(n = 14)/
Group I
(n = 14) Group II
(n =28)/
Group II
(n = 28) Group III
(n = 19)/
Group III
(n = 19)
Area of 50-and arterial vessels of the mesentery (mm2)/Area of 50-and arterial vessels of the mesentery (m2)

305,59±18,44

520,97±26,84*

420,73±22,94*

265,25±15,92*

Arteriole wall thickness (microns)/Arteriole wall thickness (microns)
3,58±0,23

6,02±0,32*

3,80±0,33

3,78±0,23

The area of 50 capillaries of the serous lining of the intestine
(mm2)/The area of 50 capillaries of the serous lining of the intestine
(mm2)

156,39±19,60

344,84±37,07*

424,46±21,33*

465,89±24,54*

The significance of the differences is shown in comparison with the control group.
* - p 0.001 / Reliability of differences is given in comparison with the control group
* - p 0.001

The characteristics of the microcirculatory bed indicated that chronic hypoxia, modeled in the first period of pregnancy, leads to a compensatory expansion of the arterial locus in the fetus with the subsequent development of chronic venous fullness, while the area of the venous part is doubled. Hypoxia, created in the late stages of pregnancy, does not lead to the development of compensatory processes of the arterial bed and is accompanied by venous stagnation with an increase in the area of blood vessels by three times. This condition increases hypoxia in the intestinal wall, against which atrophic and dystrophic changes in the epithelium of the mucous membrane develop.

Conclusion
Thus, chronic hypoxia, modeled in the first trimester of pregnancy, leads to significant changes in the intestinal mucosa in combination with the growth of the connective tissue component in the muscle layer. The risk of developing dynamic intestinal obstruction occurs in offspring of all groups, which is confirmed by an increase in fibrous tissue in the intestinal wall.
It was experimentally proved that pronounced changes in the microcirculatory bed were detected in groups II and III, since there is a predisposition to the development of pre-ulcer and ulcerative defects in the wall of the small intestine.
At the submicroscopic level, it is shown that adaptive and destructive processes occur in the ultrastructure of the intestinal wall of rat offspring after hypoxic exposure in the prenatal period in all experimental groups. In the microcirculatory bed, an increase in venous congestion was detected, and the most pronounced changes were found in the offspring of group III.

About the authors

Irina Yuryevna Karpova

Privolzhsky Research Medical University

Email: ikarpova73@mail.ru
ORCID iD: 0000-0002-4897-6702

M.D., Associate Professor, Professor of the Department of Pediatric Surgery of the Privolzhsky Research Medical University

Russian Federation, N. Novgorod, Russian Federation

Marina Leonidovna Bugrova

Privolzhsky Research Medical University

Email: marysmir@mail.ru

Doctor of Biological Sciences, Associate Professor, Professor of the Department of Histology with Cytology and Embryology of the Privolzhsky Research Medical University (main place of work), Head. Department of Electron Microscopy of the Central Research Institute (combination)

Russian Federation, 10/1 Minin and Pozharsky Square, N. Novgorod, 603005, Russian Federation

Tayiana Ivanovna Vasyagina

Privolzhsky Research Medical University

Email: marysmir@mail.ru

Candidate of Biological Sciences, Senior Researcher of the Department of Electron Microscopy of the Central Research Institute of the Privolzhsky Research Medical University

Russian Federation, 10/1 Minin and Pozharsky Square, N. Novgorod, 603005, Russian Federation

Daria Vasilyevna Karpeeva

Privolzhsky Research Medical University

Author for correspondence.
Email: dashenka8723@mail.ru

Assistant of the Department of Pediatric Surgery of the Privolzhsky Research Medical University

Russian Federation, 10/1 Minin and Pozharsky Square, N. Novgorod, 603005, Russian Federation

References

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