Immunotherapeutic modulation of intraperitoneal adhesions by Asparagus racemosus.NN Rege, HM Nazareth, AA Isaac, SM Karandikar, SA Dahanukar
The hypothesis that macrophages appear to play a pivotal role in the development of intraperitoneal adhesions and that modulation of macrophage activity, therefore, is likely to provide a tool for prevention of adhesions, was tested in the present study. Effect of Asparagus racemosus, an indigenous agent with immunostimulant properties, was evaluated in an animal model of intraperitoneal adhesions induced by caecal rubbing. Animals were sacrificed 15 days following surgery. The peritoneal macrophages were collected to assess their activity. At the same time, peritoneal cavity was examined for the presence of adhesions, which were graded. A significant decrease was observed in the adhesion scores attained by animals receiving Asparagus racemosus. This was associated with significant increase in the activity of macrophages (70.1 +/- 2.52), compared to that in surgical controls (53.77 +/- 10.8). These findings support our hypothesis and provide a novel approach for the prevention and management of post-operative adhesions.
Keywords: Adhesions, Adjuvants, Immunologic, pharmacology,Animal, Cecal Diseases, prevention &control,Female, Macrophage Activation, drug effects,Male, Peritoneal Diseases, prevention &control,Plants, Rats,
Serious morbidity due to intraperitoneal adhesions is frequently encountered in surgical practice., Various etiological factors viz., bleeding, trauma to viscera, serosal injuries, infection, foreign body deposition like talc powder and ischaemia are incriminated in development of adhesions. Apparently though these etiologies appear diverse, deposition of fibrin is always an essential step in the pathophysiology of adhesion formation. Whether the fibrinous exudate will undergo complete dissolution or will be converted to fibrous strand, entirely depends on the concentration of endogenous proteolytic enzymes and interaction of cells e.g., mast cells, eosinophils, macrophages and fibroblasts.,, The key cells regulating the complex process of fibrosis appear to be the macrophages. These cells not only phagocytose the cellular debris and modulate the nonspecific, early inflammatory phase of adhesion formation, but also affect the fibroproliferative response. The secretory products of these cells are capable of degrading the collagen and proteoglycan matrix.
Modulation of activity of macrophages, therefore could be an important means of preventing adhesions. The present study was carried out to investigate the state of activity of macrophages in an animal model of intraperitoneal adhesions. The macrophages can be activated by a number of exogenously administered biological products and chemical substances. To evaluate whether modulation of macrophage activity prevents adhesion formation, Asparagus racemosus, a plant with rasayana properties was selected. This plant has been shown to increase the activity of rat peritoneal macrophages. It was, therefore, of interest to study its effect on peritoneal adhesions.
The study was carried out in albino rats of either sex, weighing between 100 and 120 gm. These animals were divided into 5 groups as follows:
Group I: Eight animals received only vehicle and served as normal controls.
Group II: Eight rats were given 200 mg/ kg of Asparagus racemosus as total extract, orally every day for 15 days.
Group III: Surgical procedure was carried out in 12 animals to induce intraperitoneal adhesions. These animals received only vehicle following surgery and served as surgical controls.
Group IV: Nine animals were pretreated with Asparagus racemosus for 15 days, following which intraperioneal adhesions were induced. The treatment was continued after surgery.
Group V: Seven animals belonging to this group received Asparagus racemosus from the day of surgery. The therapy was continued for 15 days.
Intraperitoneal adhesions were induced as per the method described by Sachdev et al. Rats were anaesthetized with pentobarbitone sodium; laparotomy was performed. Caecum was delivered out gently through the incision and approximately an area of 1 cm diameter over the caccal wall was rubbed with a cotton gauze piece, wrapped around an index finger. Rubbing was continued till the punctuate haemorrhages appeared. Caecum was replaced back into the abdominal cavity and abdominal incision was sutured in two layers with chromic catgut (No. 4.0) and black silk (No. 3.0 respectively.
Evaluation of macrophage activity
Animals from Gr. I and II were sacrificed after 15 days of vehicle or drug therapy. The rest of the animals were sacrificed on the 15th day after surgery. The abdominal cavity was opened through an another incision made in the left flank. A lavage with minimum essential medium (MEM) was given to collect peritoneal macrophages. Activity of the harvested macrophages was determined with the help of a phagocytic assay technique using S. aureus as the test organism. The phagocytic activity was expressed as lye phagocytosis.
Assessment of adhesions
After the collection of macrophages, the incision was further extended in Gr. III, IV and V. A search was made for the presence of adhesions. Severity of adhesions was decided by attributing the grades to number, character and area involved as shown in[Table - 1].
A score was developed for each animal by adding up the grades assigned for individual attribute. The maximum score that could be obtained by an animal was 9. Assessment was done by a research fellow, who was unaware of treatments received by different groups.
The data was analysed using Student's `t' test and Mann Whitney test.
The results of the study are illustrated in Fig. 1. The peritoneal macrophages obtained from the normal rats exhibited 32 ± 1.77% phagocytosis. The animals receiving Asparagus racemosus (Gr. II) showed a significant increase in the phagocytic activity (53 ± 5.78%) of macrophages (p < 0.05).
The animals which underwent surgery (Gr. III) developed considerable adhesions on the 15th day after surgery. These adhesions were scored and the pattern of score distribution for this group is depicted in Fig. 1 [Figure - 1]. The macrophages isolated from the peritoneal cavity of these animals also showed on increase in the activity (53.77 ±10.8%) as compared to normal (p<0.05).
Pretreatment with Asparagus racemosus (Gr. IV), however, decreased the incidence of adhesion formation. The adhesions when encountered in these animals, were thin and involved smaller area. The distribution pattern of score attained by this group corroborates this fact [Figure - 1]. When compared with the Gr. III, all the three attributes of adhesion viz., number, character and area involved were found to be significantly less (a = 0.05). The percentage phagocytosis of macrophages was 70.1 ± 2.52. This activity was greater than that of Gr. III (p < 0.05).
The animals which received treatment following induction of adhesions (Gr. V) also exhibited similar response. The development of adhesions was significantly inhibited (a = 0.05) and this was associated with increased phagocytic activity of peritoneal macrophages (68.5 ± 4.2; ) (p < 0.05).
Precise knowledge of key events in the pathogenesis of disease and selection of a suitable animal model which reproduces most of the features of the condition, are the prerequisites for evaluation of any therapeutic agent, intraperitoneal adhesions being no exception. In the present study, the adhesions were induced by rubbing the wall of the caecum by a gauze piece. This causes drying of serosa and oozing of blood, simulating the two relatively minor, yet common accidents, that may occur during abdominal surgery in humans, leading to development of adhesions. Three phases are discernible in the evolution of adhesions viz., (1) degeneration and desquamation of mesothelial cells (0-7 hours), (2) exudation and deposition of fibrin (7 hours-10 days) and (3) transformation of fibrinous bands to fibrous tissue (10 days-1 month) It has been reported that macrophages infiltrate the serofibrinous exudate within the first 24 hours of trauma, increase in number and mature during following days and a few of them may remain at the site 15 days later, inspite of conversion of fibrinous strands to fibrous ones.
In our model, the activity of macrophages isolated from the peritoneal cavity, 15 days following surgery, was found to be higher than in the normal animals. However, the presence of adhesions in this group suggests that this increase in the activity may be a result of an early inflammation which precedes fibrous tissue deposition. The macrophages digest the cellular debris during the process of inflammation and undergo stimulation by the mediators released by other cells at the site e.g. lymphocytes. The proliferation of fibroblasts and collagen deposition is the end result of this increased activity.,
The animals receiving Asparagus racemosus showed a significant increase in macrophage phagocytosis and when these pretreated animals underwent surgery, further increase in the activity of macrophages was observed on the 15th post-operative day. At the same time, the adhesions were significantly less. Same picture was seen with the group which received post-operative therapy. This indicates that macrophages stimulated beyond certain limits, can prevent adhesion formation.
Such bidirectional regulation of collagen deposition by macrophages is possible and has been demonstrated previously for alveolar macrophages.
It seems that following administration of Asparagus racemosus, the ability of macrophages to function efficiently increases. In other words, the macrophages undergo 'activation'. This activation was assessed in the present study in terms of phagocytic activity of cells, which is one of the indices of activation. The activated macrophages are known to secrete wide range of biologically active products; which are barely released by the unstimulated cells. These compounds are capable of affecting the various steps in pathogenesis of adhesions [Figure - 2].
The protection offered by Asparagus racemosus may be due to the amplification of secretory activities of macrophages. This must have provided suppressive signal to fibroblasts and have attenuated the process of fibrosis.
[Figure - 1], [Figure - 2][Table - 1]