Effect of quetiapine on morphologic manifestations of traumatic inflammation during mechanical impact on the blood-ophthalmic barrier

. Mechanical trauma of the eye is an example of a limited injury in which systemic changes in the immune system occur. The aim of the study was to investigate the dynamics of morphological manifestations of traumatic inflammation in experimental mechanical trauma of the eye with the inclusion of quercetin in the schemes of experimental therapy. The experiment was conducted on male mongrel rats of six-month age, weighing 220-240 g in the number of 120 animals. All animals were given a penetrating wound of both eyes. Group 1 animals were not treated for mechanical ocular trauma. Group 2 rats received standard therapy for eye injury, group 3 animals received standard therapy with the addition of quercetin injections intraperitoneally, and group 4 animals received quercetin injections only. To obtain macro preparations of the eyeball, adjacent tissues and subsequent preparation of histologic preparations of both eyes, the head of each animal was fixed in a solution of 10% neutral formalin. Histologic sections were examined using the ocular stereometric grid of G.G. Avtandilov. Conclusions: addition of quercetin injections to the standard therapy of eye trauma promotes intensification of reparative processes, reduction of purulent inflammation intensity and phagocytosis in the remote period, increase in the number of immunocompetent cells in the peri-circumferential zones.


Introduction
Due to the unique physiological characteristics of the eye and its special protection by the immune system, the therapy of penetrating eye injuries and their treatment is a complex problem, requiring a multidisciplinary approach [1][2][3][4][5][6][7][8][9].Mechanical trauma of the eye is an example of a limited injury in which not only local reactions with impaired immunosuppressive mechanisms directly in the organ as a whole are disturbed, but also systemic changes in the immune system [9][10][11][12][13][14][15][16][17][18].All of this is accompanied by oxidative stress.
Thus, the aim of our study was to investigate the dynamics of morphological manifestations of traumatic inflammation in experimental mechanical ocular trauma with the inclusion of quercetin in the schemes of experimental therapy.

Materials and methods of research
The experiment was performed on male outbred rats, six months old and weighing 220-240 g, in a total of 120 animals.All animals were equally divided into 4 groups of 30 rats each.All animals received penetrating wounds in both eyes.Group 1 animals received no treatment for mechanical eye injury.Group 2 rats received standard therapy for eye injury, group 3 animals received standard therapy with the addition of intraperitoneal quercetin injections, and group 4 received quercetin injections only.The detailed methodology of the experiment is presented in our previously published work [19].Animals were maintained throughout the experiment on standard vivarium chow with free access to water and food [20].
Eyes of intact and experimental animals were examined to assess cell division activity in the stratified squamous epithelium (SSE) of the cornea.
To obtain macroscopic specimens of the eyeball and adjacent tissues for subsequent histological preparation of both eyes, the head of each animal was fixed in 10% neutral formalin solution.After extraction and macroscopic examination, the ocular structures of the animals were dehydrated in a series of increasing alcohol concentrations.The material was then embedded in paraffin blocks and serial 3-5 micron thick sections were prepared and stained with haematoxylin and eosin, Van Giezon's to identify collagen fibres and Weigert's to identify elastic fibres.
Histological sections were examined using an ocular stereometric grid developed by G. Avtandilov [21].Measurements were made at 10 random overlaps of the grid on the structures of the scar and peri-scar tissue.The number of cells and fibre density per unit area were determined.0.01 mm2 of G. Avtandilov's net was used as the unit of area.The following indices were estimated: the number (density) of collagen and elastic fibres, fibroblasts in the scar; the number of macrophages, lymphocytes, eosinophilic and neutrophilic granulocytes, plasma cells in the tissue surrounding the scar.
The mitotic activity of the SSE of the cornea in the control and experimental groups of animals was evaluated by the mitotic index, expressed in o/oo.
The digital material obtained was subjected to statistical processing by non-parametric statistical analysis using the Mann-Whitney U test.

Results of the study
Tables 1 and 2 contain information on the quantitative characteristics of the structural elements of the scar and damaged ocular tissues.Comments.In this table, the differences are reliable at P<0,05: 1 -in comparison with the indices of group 1 on day 7 of the experiment; 2 -in comparison with the indices of the same group on day 7 of the experiment; 3 -in comparison with the index of the first group on day 14 of the experiment.
On day 7 of the experiment, the number of collagen and elastic fibres in the scar zone of the injured eye of animals in groups 2 and 4 was virtually indistinguishable from that of rats in group 1 without treatment for mechanical ocular trauma.At the same time, the number of collagen and elastic fibres in the scar zone of the injured eye of animals of group 3 decreased by 23.3% compared to rats in group 1.On day 14, the number of collagen and elastic fibres in the scar zone increased by 6.0%, 4.4% and 9.9% in animals in groups 1, 2 and 3, respectively, and decreased by 10.1% in animals in group 4 in comparison with the day 7 of the experiment.It should be noted that the number of collagen and elastic fibres in the scar of animals in groups 3 and 4 was significantly different from that of animals in group 1 by 16.3% and 20.5%, respectively, on day 14 of the experiment.
The number of fibroblasts in the scar zone of the injured eye in animals of groups 2, 3 and 4 was significantly higher than in group 1 by 68.5%, 20.8% and 59.8%, respectively.On day 14, the number of fibroblasts in the scar zone increased in animals of groups 1, 2 and 3 by 8.0%, 22.9% and 32.6%, respectively, and in animals of group 4, the number of fibroblasts in the scar zone decreased insignificantly compared to day 7 of the experiment.It should be noted that in animals of groups 2, 3 and 4, the number of fibroblasts in the scar on day 14 of the experiment differed significantly from that of group 1 by 91.8%, 48.3% and 44.7%, respectively.
On day 7 of the experiment, the number of granulocytes in the scar zone of the injured eye in animals in groups 2, 3 and 4 was 44.1%, 157.0% and 15.1% higher, respectively, than in rats in group 1 without treatment for mechanical eye trauma.On day 14 of the experiment, the number of granulocytes in the scar zone of the injured eye continued to increase in animals in groups 1, 2 and 3 by 215, 1 %, 30.6% and 134.3%, respectively, and decreased by 34.3% in animals in group 3.It was found that in animals in groups 2, 3 and 4, the number of granulocytes in the scar on day 14 of the experiment differed significantly from that of animals in group 1 in a smaller direction by 40.2%, 46.5% and 14.4%, respectively.On day 7 of the experiment, the number of macrophages in the scar zone of the injured eye was 116.3%, 18.5% and 89.1% higher in animals in groups 2, 3 and 4 than in rats in group 1 without treatment for mechanical eye trauma.On the 14th day of the experiment, in animals in groups 1, 2 and 4, the number of macrophages in the scar zone of the injured eye decreased by 14.1%, 7.6% and 24.7%, respectively, from the value on the 7th day, and in animals in groups 3, there was an opposite tendency: an increase in the number of macrophages in the scar zone of the injured eye by 25.7%.In general, the number of macrophages in the scar zone of the injured eye on day 14 in animals in groups 2, 3 and 4 was significantly higher than in animals in group 1 by 130.4%, 73.4% and 65.8%, respectively.
On day 7 of the experiment, the number of lymphocytes in the scar zone of the injured eye in animals in groups 2, 3 and 4 was 6.7 times, 5.0 times and 4.5 times higher, respectively, than in rats in group 1 without treatment of mechanical eye trauma.On day 14 of the experiment, the number of lymphocytes in the scar zone of the injured eye increased in animals in groups 1, 2 and 4 by 290.7%, 14.0% and 27.9%, respectively, compared with the value on day 7, and the opposite tendency was observed in animals in group 3 -the number of lymphocytes decreased by 6.5%.In animals in groups 2, 3 and 4, the number of lymphocytes in the scar zone of the injured eye on day 14 was significantly higher than in animals in group 1 by 94.0%, 20.8% and 47.0%, respectively.
On day 7 of the experiment, the number of plasma cells in the scar zone of the injured eye in animals in groups 2, 3 and 4 was 61.5%, 123.1% and 30.8% higher, respectively, than in rats in group 1 without treatment for mechanical eye trauma.After 14 days, the number of plasma cells in the scar zone of the injured eye was 176.9%, 57.1%, 531.7% and 94.1% higher in animals in groups 1, 2, 3 and 4 compared to day 7 number.In animals in groups 2 and 4, the number of plasma cells in the scar zone of the injured eye on day 14 of the experiment was reliably 8.3% lower than in animals in group 1, and in animals in group 3 it was 405.6% higher.
The mitotic activity of the multilayered corneal squamous epithelium in the scar zone in animals of different groups is shown in Table 3.On day 7 of the experiment, the mitotic index of the stratified squamous epithelium of the cornea in the area of the injury in animals in groups 2, 3 and 4 was 68.8%, 135.4% and 5.8% higher, respectively, than in rats in group 1 without treatment of mechanical trauma to the eye.On day 14 of the experiment in animals of groups 1, 3 and 4 the increase of the mitotic index of the stratified squamous epithelium of the cornea in the area of the injury was registered by 99.0%, 44.0% and 5.4%, respectively, in comparison with the value on day 7, and in group 2 the decrease of the mitotic index was noted by 16.1%.On day 14 of the experiment, the mitotic index of the stratified squamous epithelium of the cornea in the area

Discussion of results
As a result of the conducted studies, the characteristics of the morphological structure of corneal structures in the dynamics of the recovery process at mechanical eye trauma in animals on 7 and 14 days at different methods of therapy were established.Standard ocular trauma therapy and standard therapy with quercetin injections increased the proliferative activity of fibroblasts and the synthesis of collagen and elastic fibres in the corneal scar zone to different degrees.On the contrary, treatment with quercetin alone stimulates the synthetic activity of fibroblasts in the corneal scar with the formation of intercellular matrix components, reduces the production of collagen fibres and dynamically modulates the inflammatory response in the scar and peri-scar tissue, increasing the number of immunocompetent cells.
In animals with mechanical trauma of the eye in conditions of application of quercetin in addition to standard therapy and in contrast to it in early and late terms after trauma the morphofunctional state of corneal epithelium in the scar zone is activated due to the increase of mitotic activity of its cells, indicating intensive reparative processes in the cornea, there is an increase in the number of immunocompetent cells in the peri-scar zones.
At the same time with quercetin injection as monotherapy a decrease in the intensity of purulent inflammation and phagocytosis in the remote period after mechanical eye trauma is also observed.

Conclusions
The addition of quercetin injections to the standard therapy of ocular trauma promotes the intensification of reparative decreases the intensity of purulent inflammation and phagocytosis in the remote period, increases the number of immunocompetent cells in the peri-scar zones.

Table 1 .
Quantitative characteristics of structural elements of the scar of the injured eye at 7 and 14 days after injury with different treatment methods (in 0.01 mm 2 ) https://doi.org/10.1051/e3sconf/202343101048

Table 2 .
Number of granulocytes, macrophages, lymphocytes, and plasma cells in the ocular tissues adjacent to the scar with different therapies (in 0.01 mm2)

Table 3 .
Mitotic activity of corneal stratified squamous epithelium in the lesion area under different therapies ( 0 /00) ://doi.org/10.1051/e3sconf/202343101048 of the injury in animals in groups 2 and 4 was reliably lower than in animals in group 1 by 28.9% and 18.4%, respectively, and in animals in group 3 by 70.3%. https