{"paper_id":"26cebbea-7d09-416d-ade9-58d1a146acbe","body_text":"Postoperative peritoneal adhesions (PA), defined as fibrous band formation between\nintra-abdominal organs, is one of the most important problems of surgery. It is\nstated that the frequency of PA increases up to 90% in some sources \n 1 \n . Abdominal reoperation is more difficult and riskier when there are\nadhesions. Increased enterotomy risk, longer dissection times and longer operation\ntimes are serious consequences of intraabdominal adhesions \n 2 \n \n , \n \n 3 \n .\nPAs are caused by the normal peritoneal healing not being limited, due to an increase\nin vascular permeability as a result of peritoneal damage and release of fibrin-rich\nexudate. Various drugs, medical agents and surgical techniques have been tried to\nprevent PPA formation, but there is currently no active and accepted treatment\nmodality. Experimental animal studies are preferred for the studies on prevention of\nPPAs, mainly because it is difficult and risky to apply to humans, and most of these\nstudies are performed on rats \n 4 \n \n – \n \n 7 \n .\nInflammation and infection secondary to intraabdominal operations may cause adhesions\nand may be responsible for the etiopathogenesis of lymphadenopathy secondary to\ninflammation \n 7 \n \n , \n \n 8 \n . In animal models, many products have shown to reduce postoperative adhesion\nformation, such as corticosteroids, non-steroidal anti-inflammatory drugs, reactive\noxygen species (ROS) scavengers, fibrinolytic agents, flotation agents and semisolid\nbarriers and mechanical barriers \n 8 \n \n – \n \n 11 \n .\nAllium sativum has been used as a medicine in traditional medicine for centuries and\nis a well-known substance with anti-inflammatory, antibacterial, fibrinolytic and\nwound healing properties \n 8 \n \n , \n \n 12 \n . Considering that if allium sativum has an effect on preventing peritoneal\nadhesion, it can be used in routine practice because it can be found cheap and easy\nto prevent postoperative adhesion, in addition to examining its effectiveness in\npreventing intraabdominal adhesions, we aimed to investigate the effects of free\noxygen radicals immunohistochemically and the number and size of reactive lymph\nnodes.\n\nThis study was performed in the laboratory of Hamidiye Turkey, with the approval\nof the university's Laboratory Animals Ethics Committee (No:\n15.05.2019/2019-05/07).\nRats were obtained from SBU experimental animals center and kept in special cages\nunder appropriate feeding conditions during the study. The rats were housed in\nspecial metal cages with controlled heat (between 19-22C) and illumination\n(08-20 light, 20-08 dark). Rats had normal water and standard foods access\nwithout restriction. Prior to the test procedure, all rats were weighed with an\nanalytical balance and their body weights (BW) recorded. Sprague dawley rats\nweighing 250-300g were divided into three groups (n=8, in each group).\nAll rats were anesthetized with intramuscular (IM) ketamine (Ketalar 500 mg, 35\nmg/kg body weight [BW]; Pfizer) and xylazine (Kepro Xylazine 20, 15 mg/kg BW;\nBiopharm Veterinary Drugs). After anesthesia, they were cleaned with 10%\npovidone-iodine solution. A 3 cm median laparotomy was performed. After the\nprocedure, the incision was closed with continuous 3-0 polypropylene sutures\n(Prolene; Bicakcilar Co., Istanbul, Turkey).\nGroup 1 (sham): After laparotomy, the abdomen of the rats was closed again\nwithout performing any procedure. On postoperative day 7, rats were\nrelaparotomized under anesthesia; peritoneal tissue surrounding the cecum was\nsampled and rats were sacrificed.\nGroup 2 (control group: physiological saline solution): After laparotomy in rats,\nabrasion, punctate hemorrhage with sterile gauze was created in cecal fat tissue\nand intraperitoneal 2 cc saline solution was applied. On postoperative day 7,\nrats were relaparotomized under anesthesia; peritoneal tissue surrounding the\ncecum was sampled and rats were sacrificed ( Fig.\n1 ).\nGroup 3 (experiment group: allium sativum): After laparotomy in rats, abrasion,\npunctate hemorrhage with sterile gauze was created in cecal fat tissue and\nintraperitoneal 5 ml/kg (Oleum Allii SativiR; Arifoglu, Firuzkoy Esenyurt Road\nNo. 32 Avcilar, Istanbul) derivative was applied. On postoperative day 7, rats\nwere relaparotomized under anesthesia; peritoneal tissue surrounding the cecum\nwas sampled and rats were sacrificed.\nAll materials were fixed with 10% buffered formaldehyde. Samples embedded in\nparaffin blocks were slit sections of 4-5 micron thickness and stained with\nhematoxylin & eosin (H&E). In addition to H&E, the samples were\nsectioned with a thickness of 3-4 microns and stained with Masson's trichrome\n(MT) histochemically. In addition, 3 micron sections were taken from paraffin\nblocks and immunohistochemical staining with GSTP1, GLUT-red, SOD1, CAT was\nperformed using LEICA kits in LEICA automatic staining device.\nHistopathological effects were evaluated according to the parameters listed below\n( Table 1 ):\nFibrosis\nInflammation\nNumber of lymph nodes\nSize of largest lymph node (mm)\nFibrosis Scores with H&E were evaluated as:\nNo effect: 0\nLight effect: 1\nModerate effect: 2\nIntense effect: 3\nImmunohistochemical staining scores for GSTP1, SOD1, CAT and Glut Red were\nevaluated as:\n0: no staining\n1: focal weak positivity\n2: moderate positivity\n3: intense strong positivity\nHistochemical fibrosis evaluation scores with Masson Trichrome (MT) were\nevaluated as:\n0: no staining\n1: focal weak positivity\n2: moderate positivity\n3: intense strong positivity\nData were analyzed by SPSS 24 IBM SPSS Statistics for Windows, version 24 (IBM\nCorp., Armonk, N.Y., USA). The normal distribution of the variables was examined\nby analytical methods (Kolmogorow Smirnow test) considering visual (histogram\nand probability graphs) and sample size. Kruskal-Wallis test was used for\ncomparisons between the groups since not all variables had normal distribution.\nDescriptive statistics are given as arithmetic mean ± standard deviation and\nmedian (quarters). One-Way ANOVA test was used for binary comparisons.\nDifferences between the groups and changes in the number of lymph nodes over\ntime and total score variables were analyzed by two-way variance analysis.\nBonferroni test was used for differences between the groups. Statistical\nsignificance level was accepted as P <0.05.\n\nFrom statistical analyses, the results of specimens in Groups 1, 2, and 3 were not\nnormally distributed; therefore, they were compared using the Kruskal-Wallis\ntest.\nIn Kruskal-Wallis analysis, there was statistical difference between the groups in\nterms of inflammation, fibrosis, lymph node size and histochemical fibrosis. ( Fig. 2 ). They were higher in Group 3 compared to\nother groups (p<0.05) ( Table 2 ).\nIn the paired group analysis, there was a statistical significance for fibrosis when\nGroup 1 and Group 2 (p=0.018) and Group 1 and Group 3 (p=0.003) were compared,\nwhereas when Group 2 and Group 3 were compared, it was not statistically significant\n(p=0.483) ( Table 3 ).\nP  <0.05, pair-group analysis was evaluated via the\nMann-Whitney U test.\n** Group 1: sham group, group 2: control group, group 3: experimental\ngroup.\n*** Lymph node size was microscopically measured in the histopathological\nexamination in mm.\nFor fibrosis with Masson's Trichrome, there was a statistical significance when Group\n1 and Group 2 (p=0.018) and Group 1 and Group 3 (p=0.003) were compared, whereas\nwhen Group 2 and Group 3 were compared, it was not statistically significant\n(p=0.483).\nFor inflammation, there was a statistical significance when Group 1 and Group 2\n(p=0.026) and Group 1 and Group 3 (p=0.003) were compared, whereas when Group 2 and\nGroup 3 were compared, it was not statistically significant (p=0.179).\nFor lymph node size, there was a statistical significance when Group 1 and Group 2\n(p=0.01) and Group 1 and Group 3 (p=0.007) were compared, whereas when Group 2 and\nGroup 3 were compared, it was not statistically significant (p=1.000).\nThe total immunohistochemical score and number of lymph nodes were normally\ndistributed, and statistical evaluation between the three groups was done using\none-way ANOVA.\nWhen the groups were compared in terms of immunohistochemical score, the mean values\nwere 1.75 in Group 1, 5.25 in Group 2, and 4.33 in Group 3. The P-value was 0.0001\n(P < 0.005), and there was a significant difference ( Table 4 ).\nIn the paired group analysis in terms of immunohistochemical score, there was a\nstatistical significance between Group 1 and Group 2 (p=0.004) and Group 1 and Group\n3 (p=0.001), whereas the difference between Group 2 and Group 3 was not\nstatistically significant (p=1.000) ( Table\n5 ).\nThe mean difference is significant at the level of 0.15.\n** Post hoc pair-group analysis was performed using Bonferroni\ncorrection.\n*** Statistical evaluation between the three groups was assessed with\none-way ANOVA.\n**** Each immunohistochemical marker was individually scored, but\nstatistical evaluation was performed for each rat on the basis of the\ntotal score (SOD-1, CAT, GSTP-1, Glut Red).\n***** In the histopathological examination, the number of lymph nodes was\nmicroscopically counted for each rat.\nEach immunohistochemical marker was individually scored, but statistical evaluation\nwas performed for each rat on the basis of the total score (SOD-1, CAT, GSTP-1, Glut\nRed). Statistical evaluation between the three groups was done using one-way\nANOVA.\nIn immunohistochemical analyses (SOD-1, Glut Red, CAT, GSTP-1) on free oxygen\nradicals, allium sativum statistically increased the production of free oxygen\nradicals in the tissues ( Fig. 3 ).\nIn terms of the number of lymph nodes, the mean values were 3.1111 in Group 1, 3.6667\nin Group 2, and 1.4444 in Group 3. P value was 0.934 (P> 0.005) and there was no\nstatistically significant difference ( Table\n6 ).\n* The mean difference is significant at the level of 0.15.\n** Post hoc pair-group analysis was performed using Bonferroni\ncorrection.\n*** Statistical evaluation between the three groups was assessed with\none-way ANOVA.\n**** Each immunohistochemical marker was individually scored, but\nstatistical evaluation was performed for each rat on the basis of the\ntotal score (SOD-1, CAT, GSTP-1, Glut Red).\n***** In the histopathological examination, the number of lymph nodes was\nmicroscopically counted for each rat.\nIn the paired group analysis for the number of lymph nodes, the P value was 1.0\nbetween Groups 1 and 2, 1.0 between Groups 1 and 3, and 1.0 between Groups 2 and 3\n( Table 5 ).\nPost-hoc paired group analyses were evaluated using Bonferroni correction *** for\ntotal immunohistochemical score and number of lymph nodes (Mean difference was\nsignificant at 0.15.).\n\nPeritoneal adhesions (PA) remain a major health problem and, according to some\nsources, have been reported to be seen at very high rates, especially after\nabdominal surgery \n 1 \n . Intestinal obstruction, abdominal pain due to visceral peritoneal traction,\ninfertility due to extraluminal compression, urinary dysfunction due to traction of\nureters may be seen secondary to PA \n 13 \n .\nPA may be congenital or especially acquired in the postoperative period \n 13 \n . In a postmortem study, approximately 1/3 of the cases with peritoneal\nadhesion were found to be non-surgical cases and endometriosis, peritoneal dialysis\nand radiotherapy were among the events that triggered intraabdominal inflammation,\nbut the majority of cases were reported in the postoperative period \n 14 \n .\nIn case of a possible re-operation in PA cases, prolongation of operation and\ndissection times and increased risk of surgical complications can be mentioned \n 2 \n \n , \n \n 3 \n .\nMultiple strategies have been proposed to eliminate PA, but there are varying\nresults \n 15 \n .\nAfter any intervention in the peritoneal tissue (surgery, inflammation of the trauma,\ninfection or placement of foreign bodies in the peritoneal cavity, etc.), the\ndevelopment of adhesion may begin.\nAfter injury/damage to the mesothelial cells covering the peritoneal surface, the\nhealing process begins. Vasoactive amines such as histamine and quinine contribute\nto the accumulation of fibrin-rich exudates around the damaged area by increasing\nvascular permeability.\nAs the fibrin polymers in the exudate interact with the fibronectin to form the\nfibrin gel matrix, which produces fibrin bands between the injured area,\nsimultaneous fibrinolytic activity begins. Fibrinolytic activity is more dominant in\nareas where healing occurs without adhesion. In contrast, if there is a defect or\nimbalance in fibrinolytic activity, there is persistence of fibrinous material in\nthese areas. Secondary to this, proliferative fibroblasts migrate to this area and\naccumulate extracellular matrix material that contains collagen that contributes to\nadhesion formation. Thus, the different mechanical steps, some of which are\ndescribed above, regulate the healing process and the presence of imbalances in any\nof them potentially contributes to the development of adhesion \n 7 \n \n , \n \n 16 \n .\nFree oxygen radicals are molecules that are released from polymorphic leukocytes and\nmacrophages and show their effects in the early stage of inflammation. Free radicals\ntarget DNA, proteins and lipids, attack membrane lipids and cause peroxidation. This\ncauses damage to the cell membranes and in this case increases the formation of\nmicrovascular edema. In recent years, it has been shown that oxygen-derived free\nradicals and metabolites play a role in increased cell and tissue damage in\nleukocyte-dependent inflammatory reactions and that these radicals cause secondary\ndamage in many tissues \n 17 \n \n , \n \n 18 \n . As a result, inflammation and fibrosis are observed histopathologically on\na microscopic level.\nAdhesions have shown to be a result of incorrect healing caused by injured peritoneal\ntissues associated with oxidative stress. In addition to parameters such as fibrosis\nand inflammation, GSTP-1, GLUT Red, SOD-1 and CAT were used to determine oxidative\nstress parameters immunohistochemically in tissues. On postoperative day 7, it was\nreported that these adhesive strips progressed to permanent fibrous adhesions, and\nthat hypoxia directly promoted the production of free oxygen radicals in\ntissues \n 7 \n \n , \n \n 8 \n .\nVarious antioxidant agents have been used to prevent adhesion formation by preventing\ntissue damage caused by free oxygen radicals. Nitric oxide (NO) can prevent adhesion\nby decreasing free oxygen radicals due to its superoxide binding properties.\nPhosphodiesterase inhibitors such as sildenafil citrate increasing NO levels have\nexperimentally shown to inhibit adhesion \n 19 \n .\nAnti-inflammatory properties of Allium sativum have been previously reported. Allium\nsativum has been reported to inhibit anti-inflammatory activity by inhibiting\nactivation of nuclear factor kappa-B (NF-kB) caused by oxidative stress \n 20 \n . In addition, it is reported in the literature that allium sativum inhibits\nthe production of T helper 1 cells and inflammatory cytokines, increasing\ncorticosteroid levels, and acts as an anti-inflammatory and immunomodulating\nagent \n 21 \n .\nRassoul  et al . \n 22 \n , and Kuo  et al . \n 23 \n  showed that Allium sativum extract showed an anti-inflammatory effect by\ninhibiting the expression of IL-1 alpha-derived vascular cell adhesion molecule-1\n(VCAM-1) and intracellular adhesion molecule-1 (ICAM-1).\nIn a study by Şahbaz  et al . \n 8 \n  Allium sativum was shown to be effective in preventing peritoneal adhesion\non postoperative day 10 due to its fibrinolytic, antithrombotic, anti-inflammatory\nand antioxidant effects.\nIn addition to inflammation and fibrosis, many reactive lymphadenopathies of various\nsizes were detected in almost all groups. Reactive lymphadenopathy usually occurs\ndue to infections, inflammation or malignancy. Lymph nodes have functions such as\nmicroorganism filtration and antibody production. Lymph nodes grow in the presence\nof microorganisms, malignant cells, or antigenic reactions, which cause the\nproliferation of lymphocytes or macrophage hyperplasia \n 24 \n .\nIn a study similar to ours, Goret  et al . \n 7 \n  found in their 2018 study that picnogenol-induced foreign body reaction and\nlymph node number increased in peritoneal adhesion model. In their study, they\nreported that the presence of acute inflammation, which later became chronic, played\na role in the etiology of lymphoid hyperplasia, which was statistically significant,\nespecially in the group receiving picnogenol.\nHowever, contrary to the results in the literature, we could not detect any positive\neffects of Allium sativum in addition to daily routine procedures on inflammation,\nfibrosis or lymphoid hyperplasia. In addition, no benefit of allium sativum was\ndetected in immunohistochemical analysis to detect oxidative stress.\nAlthough the positive effect of Allium sativum on peritoneal adhesion has been\nreported in the literature, with the early period findings in our study, it was\nfound that this substance did not provide an additional contribution to the current\nprotocol.\nVarious studies are needed to test and investigate the effects of this type of\ntraditional medicine used in PA prevention.\n\nIn this study, no positive results were found that Allium sativum had positive\neffects on fibrosis, inflammation or reactive lymph node size/number which are among\nthe intraabdominal adhesion parameters; in addition, immunohistochemical analysis to\ndetect oxidative stress at the cellular level did not show a positive decrease in\nfree oxygen radicals.","source_license":"CC-BY-4.0","license_restricted":false}