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PUBLISHED DATE: - 30-10-2024
https://doi.org/10.37547/TAJMSPR/Volume06Issue10-05
PAGE NO.: - 27-36
MORPHOLOGICAL SUBSTANTIATION OF THE
EFFECTIVENESS OF PHOTODYNAMIC
THERAPY IN CASE OF WIDESPREAD
PERITONITIS IN AN EXPERIMENT
Аlisher Sabirmatov
PhD, Tashkent Pediatric Medical Institute, Uzbekistan
INTRODUCTION
Despite the achievements of modern medical
science, the issues of prevention, diagnosis and
treatment of peritonitis remain relevant. Mortality
in acute peritonitis varies from 32.4-45.5% to 50-
90% [2,7,9]. Various microorganisms play an
essential role in the etiology of peritonitis. In the
vast majority of cases, peritonitis is the result of
autoinfection, which is usually polymicrobial in
nature [3,8,10]. It is known that the successful
result of treatment of diffuse purulent peritonitis
depends only 15-20% on the effectiveness of
antibacterial therapy, and the remaining 80% are
associated with adequate surgical tactics,
including full sanitation of the abdominal cavity
[8]. It should be noted that the question of the
choice of the sanitizing solution remains one of the
most controversial, the discussion about the
methods of intraoperative sanitation of the
abdominal cavity continues to this day [6,7], and
according to Gostishchev G, it is advisable to use
those solutions that can be administered
parenterally [5]. In recent years, the method of
photodynamic therapy has been introduced into
surgical practice in connection with the
bactericidal properties of photosensitizers
associated with the formation of singlet oxygen
against the background of photochemical
reactions. There are publications on the use of
photodithiazine, chlorophyllipt, dimegin, etc. as
RESEARCH ARTICLE
Open Access
Abstract
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photosensitizers. The problem of the choice of a
photosensitizer for PDT [10], as well as the study
of their effect on the ultrastructural elements of the
peritoneum in peritonitis [4,7], remains urgent.
However, studies on the effect of methylene blue
(MS) on the ultrastructure of the healthy
peritoneum, and in experimental peritonitis on the
inflamed peritoneum, we have not seen. In this
regard, the purpose of our study was: to study the
effect on the ultrastructural elements of the
visceral and parietal peritoneum of methylene
blue, 0.02% chlorhexidine, LED radiation and
photodynamic therapy in normal conditions and in
experimental peritonitis.
METHODS
The experiments were divided into 3 stages: 1) to
study the histological structure of the visceral and
parietal peritoneum in healthy experimental
animals; 2) to study the effects of methylene blue,
chlorhexidine and LED radiation on the intact
peritoneum separately; 3) to study the effect of
0.02% chlorhexidine solution and photodynamic
therapy, i.e., LED radiation and methylene blue at a
concentration of 0.05% per peritoneum in
experimental peritonitis in a comparative aspect.
To create a model of acute experimental
widespread purulent peritonitis, we used the
technique of Yu.Yu. Blinkov, (RF Patent No.
2338265) [1]. In the group of animals with
experimental peritonitis during the operation, the
abdominal cavity was drained with suction, then
washed with saline. 3 ml of 0.05% methylene blue
solution was poured into the abdominal cavity
with a syringe and left for 5 minutes, then the
solution was removed by suction and PDT was
started. The laparotomic wound was sutured
through all layers with silk, the animals were
marked and placed in standard vivarium
conditions. The animals were taken out of the
experiment by decapitation immediately after
their introduction into ether anesthesia. As a light
source for the PDT session, a domestic-made LED
installation "FDU-1" with an output power of 100
mW / cm2, a wavelength of 630 ± 20 nm in a
continuous red optical range was used. The energy
density during the LED irradiation session was 25-
35 J / cm2, the exposure time was 3-5 minutes. In
the control group, the abdominal cavity was
sanitized by a similar method as in the main group,
then 0.02% chlorhexidine solution was poured
into the abdominal cavity, draining. The
laparotomic wound was sutured through all layers
with silk, the animals were marked and placed in
standard vivarium conditions. To study the
ultrastructural elements of the visceral and
parietal peritoneum, the intestine with the visceral
peritoneum in rats was taken in the area of the
ileocecal junction (with the visceral peritoneum),
the parietal peritoneum in the lateral surface of the
abdomen. Pieces of 0.5 1.0 cm in size of the small
and large intestines of all experimental animals
were fixed in 10-12% neutral formalin with a
volume of 0.5 cm3, then washed in running water,
dehydrated
in
alcohols
of
increasing
concentration, impregnated with xelol and
embedded in paraffin. Sections were stained with
hematoxylin and eosin using a conventional
method, fixed with Canadian balsam, and then
covered with glass. When viewing and describing
the micro preparations, an electronic binocular
microscope CYAN was used. Model DN-30OM.
With 34 MP MICROSCOPE Camera attachment,
ocular objective magnification from x = 4x10x0.5, x
= 10x0.25x0.5, x = 40x0.65x0.5.
RESULT AND DISCUSSION
At the first stage of our research, we studied the
ultrastructural structure of the parietal and
visceral peritoneum of healthy outbred white rats.
The histological visceral layer consists of
mesothelial cells (they originate from a single layer
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squamous epithelium). Mesothelial cells produce a
secretion - serous fluid (synovial fluid). It provides
free sliding between organs and ensures normal
intestinal motility. Figure 1A shows the small
intestine.
Figure 1.A. Normal small intestine with all layers.
Serous
membrane,
muscle
and
mucous
membranes (with its own plate). G / E coloring.
Magnification 10X10. Figure 1B. Normal small
intestine. Mesothelial cells of serous foliage (1),
muscular membrane in the longitudinal and
transverse directions (2), intrinsic plastic of the
mucous membrane (3), villi of the mucous
membrane (4). G / E coloring. Magnification 10X10
Figure 1.B shows a normal small intestine with
mesothelial cells; the muscularis is presented in
the longitudinal and transverse directions. In the
striated muscle of the anterior abdominal wall,
uneven interstitial edema of the muscular plexuses
is noted (Fig. 2A).
А В
Figure 2A. The striated muscle of the anterior
abdominal wall. Uneven interstitial plexus edema.
The cross-striping of muscle cells is preserved.
Some cores are enlarged. G / E coloring.
Magnification 10X10.
Figure 2B. Small intestine. Serous membrane of
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different thickness. The blood vessels are full
blooded. The mesothelial cells of the serous
membrane have different textures. Desquamation
of focal mesothelial cells. G / E coloring.
Magnification 10X10.
At stage 2, the effect of 0.05% MS solution on
healthy peritoneum (visceral and parietal) of
intact animals was studied. MS was injected into
the abdominal cavity by puncture of the abdomen
in its lower floor with a 3 ml syringe. Due to the fact
that MS is used in medicine as an antiseptic and is
also an oxidant, it causes some changes in organs
and tissues. So, when MS is injected into the
abdominal cavity, the mucopolysaccharides of the
serous fluid interact and promotes the formation
of acidic glycosaminoglycans. In this regard, the
number of mesothelial cells decreases, the latter
are labile cells (actively multiplying). As a result,
MS begins to exert cytostatic and cytotoxic effects.
These changes are presented in Figures 2B, 3A.
А В
Figure 3A. The impact of MS on the serous
membrane of the small intestine is marked by
desquamation and necrosis of mesothelial cells, in
defective places, proliferation and uneven
arrangement of mesothelial cells are observed.
Figure 3B. Erosive-necrotic changes in the serous
membrane of the small intestine under the
influence of MS. G / E coloring. Magnification
60X10. There are multiple recovery processes in
the foci of desquamation (3B, 4A). They are
indicated by arrows. In the patient's own plastics,
uneven edema is noted, in plastics of the
connective tissue, fibrinoid swelling and focal
fibrinoid necrosis. G / E coloring. Magnification
40X10.
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А В
Figure 4A. In the bare surfaces of the serous
membrane of the small intestine, under the
influence of MS, scar surfaces of transformed
fibroblasts are formed. They are shown by arrows.
Figure 4B. The striated muscle of the anterior
abdominal wall. On the side of the parietal
peritoneum, there are multiple foci of fibrinoid
necrosis. Indicated by an arrow. Dystrophic
changes are observed in the striated muscles, the
transverse is lost. G / E coloring. Magnification
10X10.
Figures 4B - 5A show that MS on the parietal
peritoneum of healthy animals cause fibrinoid
necrosis; however, below ultrastructural changes
in animals with peritonitis will be presented, when
MS, on the contrary, enhances proliferative
processes.
А В
Figure 5A. In the parietal peritoneum (in the
structures of the basal layer of fine-fibrous
connective
tissue),
fibrinoid
swelling,
desquamation of mesothelial cells are noted, they
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are indicated by arrows, there are interstitial
edema of different sizes between the muscle
bundles, different staining of the dystrophic
altered cytoplasm. G / E coloring. Magnification
40X10. Figure 5B. Colon after exposure to PDT
with MS. The serous membrane contains many
tortuous and hyperplastic mesothelial cells (1).
The muscular membrane of the same texture, not
pronounced interstitial edema (2). The mucous
membrane and its own plate are full-blooded. In
the general background, the histtioarchitectonics
of the tissue is absolutely unchanged.
Thus, in healthy experimental animals, when MS is
injected into the abdominal cavity, a focal one is
observed on the surface of the serous membrane,
or massive foci of desquamation are determined in
places of a large accumulation of MS. In the blood
vessels of the visceral peritoneum there is uneven
plethora, on the basal plate there are bare areas,
the transformation of macrophages into
fibroblasts and the formation of adhesions, in the
vessels of muscles there is uneven plethora,
dystrophic changes in the muscles, in the lamina
propria there is interstitial edema, foci of mucoid
swelling in the vasculature. At the 3rd stage of the
study, we studied morphological changes in the
parietal and visceral peritoneum under the
influence of PDT with MS in experimental
peritonitis (Fig. 5B, 6A).
А В
Figure 6A. Condition after PDT with MS. On the cut
of the small intestine. The visceral peritoneum is of
varying thickness, clearly basophilic. Interstitial
edema to varying degrees (1). Full-blooded
capillaries (2). Increased proliferation of (3)
mesothelial cells. Basement membrane of uneven
thickness (4). There is obvious plethora in the
muscle layer and in the lamina propria (4). Figure
6B. Visceral peritoneum after PDT with MS. There
is plethora in the vessels of the visceral peritoneum
(1). Intensive proliferation of mesothelial cells of
the visceral peritoneum (2). A group of not yet
formed mesothelial cells (3). Congestion in the
lamina propria of the muscular and mucous
membranes (4). Due to proliferation, the surface is
tortuous. G / E coloring. Magnification 10X10.
Thus, under the influence of PDT with MS in
experimental peritonitis, the following changes
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were revealed: firstly, due to the release of singlet
oxygen during a photochemical reaction, it can
damage the membranes of any cells and cause a
necrotic process. If a cell contains a large number
of mitochondria, singlet oxygen is involved in the
Krebs cycle and utilized by mitochondria. These
properties are mainly observed in organs formed
(parenchymatosis) from the epithelial cell. It is due
to the fact that mesothelial cells by origin are
epithelial in nature, they are not significantly
damaged and due to the increase in the
proliferative properties of the mesothelial layer
cell, proliferative processes occur. Their number is
increasing, they are multi-row and one-story (Fig.
6A, 6B); Secondly, due to the release of singlet
oxygen from the sensitized molecule, the
membranes of the infectious agent are damaged
and, as a result, the inflammatory process fades
away; Thirdly, fibroblasts, the main component of
which is connective tissue, by origin, which are
mesenchymal tissues, containing a small number
of mitochondria, their resistance to PDT and a
decrease in proliferation, the plate of mesothelial
cells thickens (Fig. 6A); Fourthly, there is a
decrease in the activity of the inflammatory
process; in the lamina propria of the muscular and
mucous membrane, a reaction of macrophageal
infiltration is noted (Fig. 6A, 6B). The accumulation
of basophilic stained cells is determined; fifthly,
the exudative phase of inflammation fades away,
since reparative processes (restoration of
damaged tissues) are enhanced under the
influence on the surface of damaged tissues.
Morphological changes in the parietal and visceral
peritoneum after the introduction of 0.02%
chlorhexidine solution into the abdominal cavity
were as follows (Fig. 7A, 7B):
А В
Figure 7A. Part of the colon. Serous membrane
(visceral leaf), the surface is uneven, tortuous (1).
There are many foci of neutrophilic infiltration
between the muscular membrane (2). There is
swelling in the lamina propria of the mucous
membrane (3). In the mucous and submucosal
layers vascular congestion. Figure 7B. The striated
muscle and parietal peritoneum after exposure to
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0.02% chlorhexidine solution. Components of the
parietal peritoneum: destruction in fiber
structures,
disorganization,
necrosis
(1).
Congestion in the vessels (2). In the interstitium of
muscle tissue, uneven edema (3), in Andy's muscle
plexuses, foci of emerging foci of fragmentation
and decomplexation are determined (4), myocytes
are focally chromophobically stained (5), zones of
forming thin-fibrous connective tissue are
determined in interstitial spaces (6).
Figure 8. Changes after exposure to chlorhexidine.
Disorganization
and
destruction
of
the
components of the parietal peritoneum. The
muscular plexuses lose their striation. The cells are
chromophobic stained. Incession is unevenly
edematous. In the interstitium, fine-fibrous
connective tissue connective tissue is formed.
Myocyte nuclei are reduced. The foci of
lymphocytic infiltration are determined. Rom the
presented figures 7B, 8 it follows that 0.02%
chlorhexidine solution in healthy animals in the
parietal peritoneum causes destruction in fiber
structures, disorganization and necrosis. In the
muscular plexuses, the zones of forming foci of
fragmentation and decomposition are determined.
Rom the presented figures 7B, 8 it follows that
0.02% chlorhexidine solution in healthy animals in
the parietal peritoneum causes destruction in fiber
structures, disorganization and necrosis. In the
muscular plexuses, the zones of forming foci of
fragmentation and decomposition are determined.
The muscular plexuses are likely to be affected due
to the absorption of chlorhexidine. It should be
noted that in the work of S.A. Pusty. it was shown
about the toxic effect of 0.05% chlorhexidine
solution on the parietal and visceral peritoneum,
even on the functional state of the liver and spleen
[5]. Thus, in healthy experimental animals, when
MS is injected into the abdominal cavity, a focal one
is noted on the surface of the serous membrane,
especially in places of a large accumulation of MS,
massive foci of desquamation are determined.
Meanwhile, with experimental peritonitis, i.e. with
a damaged peritoneum, due to the formation of
singlet oxygen against the background of a
photochemical reaction, cell membranes can be
damaged, however, if the cell contains a
largenumber of mitochondria, oxygen is involved
in the Krebs cycle and is utilized by mitochondria.
These properties are mainly observed in organs
formed from the epithelial cell. Mesothelial cells
are epithelial in origin and contain a large number
of mitochondria, and in this regard, proliferative
processes in the cells of the mesothelial layer are
activated. Consequently, mesothelial cells are less
susceptible to damage. The results of
morphological studies clearly confirm the
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differences in the results of intraoperative
sanitation of the abdominal cavity and treatment of
experimental fecal peritonitis in rats with PDT
using MS in comparison with the group with
sanitation with 0.02% chlorhexidine solution. The
histological picture of the peritoneum in rats after
abdominal sanitation by PDT at all stages of the
study was qualitatively better than in the
representatives of the group after abdominal
sanitation with 0.02% chlorhexidine solution.
Moreover, dystrophic and destructive changes in
the mesothelial layer, edema and vascular
disorders in the main group are less pronounced.
Findings
1. Methylene blue and LED radiation in the red
range with a wavelength of 630 ± 20 nm, with a
power density of 100 mW / cm2, an energy density
of 25-35 J / cm2, an exposure duration of 3-5
minutes, individually and in combination, cause
desquamation, plethora vessels of the visceral and
parietal peritoneum of intact animals, but the
mesothelial cells contain a large number of
mitichondria and they utilize singlet oxygen, and
therefore the damage is not significant. Moreover,
proliferative processes are induced in the cells of
the mesothelial layer.
2. Aqueous 0.02% chlorhexidine solution, when
administered into the abdominal cavity of animals,
has a damaging effect on the visceral and parietal
peritoneum. This is expressed in the following:
destruction of fiber structures, disorganization,
necrosis, plethora in the vessels, uneven edema in
the muscular plexuses, and in the plexuses of Andy,
foci of emerging foci of fragmentation and
decomplexation are determined.
3. In conditions of acute experimental widespread
purulent peritonitis, photodynamic sanitation of
the abdominal cavity with methylene blue at a
concentration of 0.05% with LED radiation with a
wavelength of 630 ± 20 nm, with a power density
of 100 mW / cm2, an energy density of 25-35 J /
cm2, duration
4. Exposure 3-5 minutes showed that the
histological picture of the peritoneum in dynamics
is better with PDT in comparison with 0.02%
chlorhexidine
solution.The
availability
of
methylene blue, the reliability, the simplicity of the
method, the exclusion of the damaging effect of the
combination of photodynamic action with MS
allows us to conclude that the method of
photodynamic sanitation of the abdominal cavity
can be introduced into clinical practice.
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