Depression of Phagocytic Activity of The Immune System Following Traditional Cautery in Experimental Animals

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1 Depression of Phagocytic Activity of The Immune System Following Traditional Cautery in Experimental Animals Abdulaziz N. Al-Nasser, MBBS, MSc, MRCGP; Abdul Kareem Al-Aska, FACHARTZ; Ali Al-Twaijri, PhD From the Department of Family and Community Medicine (Dr. Al-Nasser), Department of Medicine (Dr. Al Aska), and Department of Physiology (Dr. Al Twaijri), King Saud University, College of Medicine, Riyadh. Address reprint requests and correspondence to: Dr. Al-Nasser: Department of Family and Community Medicine, College of Medicine, King Saud University, P.O. Box No. 2925, Riyadh 11461, Saudi Arabia. Accepted for publication 27 March Traditional cautery, which is practiced widely in Saudi Arabia and some other countries, has not been exposed to detailed scientific investigation. In an attempt to elucidate some of its physiological aspects, we assessed the effect of cautery on a normal animal's nonspecific immune system. Male Wistar rats and guinea pigs were cauterized to simulate traditional cautery in size and percentage of cauterized area. Using radioactive sulfur colloid uptake and clearance, the effect of cautery on the mononuclear phagocyte system was evaluated in rats. The respiratory burst in peripheral polymorphonuclear leukocytes (PMNs) after cautery was measured in the guinea pigs using the chemiluminescence technique. The results showed marked reduction in the intravascular clearance of the colloid with prolonged clearance time following cautery. In addition, the liver uptake of the colloid was reduced in cauterized animals compared with control animals and the white cell count was also significantly reduced. The study showed marked inhibition of phagocytic function in guinea pigs in both whole blood and isolated PMNs. These results indicate that traditional cautery reduces the physiological function of the phagocytic system in normal experimental animals. The influence of traditional cautery on infected animals deserves further investigation. AN. Al-Nasser, A K Al-Aska, A Al-Twaijri, Depression of Phagocytic Activity of The Immune System Following Traditional Cautery in Experimental Animals. 1991; 11(1): Traditional medicine is practiced widely in various countries in keeping with social and cultural traditions. To promote such traditional self-reliance, the World Health Organization established programs to effect the incorporation of useful traditional practices into the general health care delivery system of such countries by the year Atleast two centers for research on traditional medicine will have been identified in each region [1]. Cautery, which consists of treatment by thermal injury or heat, is one of the most common methods used by traditional healers in Saudi Arabia, and is used in the treatment of a wide variety of disorders including disk prolapse, gastroenteritis in children, pneumonia and pleurisy, facial palsy, and some mental or psychological

2 problems. Although there are some differences in the methods used by various healers, there is general agreement on the size and site of cautery for specific problems; for example, for disk prolapse all healers perform it with a circular shaped iron rod applied around the lateral malleolus. We are unaware of a study that examines the mechanism by which cautery cures or relieves symptoms of disease. However, as some of the conditions for which cautery is commonly used are infectious or inflammatory, it is possible that cautery may affect the body's defense mechanisms. The aim of this study, which was done on experimental animals, was to assess the effect of traditional cautery on the nonspecific immune system (i.e., the phagocyte system). Experimental Animals Material and Methods Male Wistar rats were used as the experimental animals to evaluate the influence of traditional cautery on the mononuclear phagocyte system (MPS) function, as determined by sulfur colloid uptake and clearance. The peripheral polymorphonuclear leukocyte respiratory burst after cautery was measured in guinea pigs using a chemiluminescence technique [2 4]. The two different sets of animals were selected on the basis of technical reasons only. Both animal species were obtained from the Animal House, College of Medicine, King Saud University, Riyadh. Table 1 summarizes the experimental design. Cauterization The size of the cautery surface area is important, as in burned animals when more than 15% of surface area is involved there is marked inhibition of phagocytosis [5 8]. Thus the size of the cautery area was kept minimal to conform with the common practice of the traditional healers. The average diameter is about 0.2 cm. Two sizes were used: 1 point (0.2 cm) and 3 points (3 0.2 cm). The back of the animal's neck was shaved and the animal anesthetized with ether. Once the animal was under deep and complete anesthesia and no longer responded to painful stimulation, a metal rod (diameter, 0.2 cm) was heated on a Bunsen burner until it glowed red (Figure 1). This was then applied for 3 seconds to the selected site on the animal. This method is similar to that used by traditional healers, although they do not use anesthesia or painkillers. The percentage of the cauterized area was calculated as follows: Percentage of cauterized area = a/b 100, where a is the surface area and b is the total surface area for rats, which is approximately 0.07 m 2. Phagocytic function was determined by the intravascular clearance rate of colloid, tissue distribution, white blood cell count (WBC), and chemiluminescence peak response. The radiolabeled sulfur colloid was prepared according to the method described by Larson and Nelp [9] and Saad et al [10].

3 Table 1. Experimental design for assessing MPS function in rats and guinea pigs. Item Study I (3 spots) Study II(1 spot) Cautery area 1.7% 0.58% Rats: Phagocytosis MPS organ distribution White blood cell & differential count 99m Tc-S colloid clearance rate 99m Tc-S colloid uptake by liver, spleen, kidneys, and lungs 0, 6, 12 days 99m Tc-S colloid clearance rate 99m Tc-S colloid uptake by liver, spleen, kidneys, and lungs Days 0, 2, 6, 12 days 0, 2, 6, 12 days Number of 5 per group 5 per group animals used Guinea pigs: Chemiluminescence Whole blood and Not done PMNs Days 0, 6, 12 days Not done Number of animals 5 per group MPS = mononuclear phagocyte system; 99m Tc-S = technetium 99m labeled sulfur; PMN = polymorphhonuclear leukocytes. Determination of MPS Phagocytic Function Male Wistar rats weighing 300 to 500 g were used. These rats were anesthetized with ether prior to injection of the colloid. A 23-gauge butterfly cannula was inserted via the tail vein. The upper thigh was then exposed through a detector and the other parts of the body covered by lead. This detector was attached to the two-channel scintillation detector interface. The rate of clearance of colloid was then read every 30 seconds for 20 minutes at 10-second counts. All readings were plotted using logarithmic paper. Figure 1. Samples of rods commonly used for cauterization by traditional healers. The distribution of the labeled colloid was also measured in the individual MPS organs. Total percentages of activity in the body taken up by each organ were calculated [9,10]. Assessment of Phagocyte Respiratory Burst To study the influence of traditional cautery on peripheral phagocytes, whole blood or isolated polymorphonuclear leukocytes (PMNs) were obtained from cauterized guinea pigs before cautery, and at 2, 6 and 12 days thereafter. The phagocyte respiratory burst activity was monitored in vitro by lumino-dependent chemiluminescence [2 4]. Venous blood samples were obtained from cauterized as well as noncauterized guinea pigs. Heparin was used as an anticoagulant (10 U/ml). PMNs were isolated as described by Al Tuwaijri et al [11]. The PMNs were then diluted in phosphate-buffered saline to a concentration of cells/ml. Cell viability was tested by trypan blue (0.2% W/V) exclusion.

4 Chemiluminescence Assay A Luminometer (LKB-Wallac 125) with a constant-temperature (37 C) sample holder was used for measurement of chemiluminescence. It records the light emission (in millivolts) on a digital display which is well correlated with the amount of oxygen produced from phagocyte cells and ultimately the degree of phagocytosis. This allows us to examine precisely the phagocytic activity in vitro even in whole blood, using a small quantity of blood (100 μl). Phorbol myristate acetate was used as a soluble phagocyte stirhulus. Statistical Analysis All values were expressed as the mean ± standard error. Student's t test was used to assess the differences between the measurements at different time points of cauterization. The results were considered significant at P <0.05. Results The influence of cautery on the general health of both rats and guinea pigs was minimal with no mortality, and the thermal burns healed within 7 to 10 days following cautery. The average size of the cautery was 0.58% of the total surface area in the group of animals receiving one-spot cauterization and 1.7% in the three-spot group. There was no change in the animals' body weight, or in the weight of the liver, spleen, or lungs. The cauterized rats showed a marked reduction in the intravascular clearance of colloid. Both the slow and fast clearance rates were significantly reduced (P <0.01) compared with those in the control group (Table 2, Figure 2). The cauterized rats showed a significantly prolonged clearance time in the fast rate compared with that in the control group (P <0.05). Similarly, the intravascular clearance slow rate was tremendously prolonged in cauterized animals compared with that in the control group (Figure 2, Table 2). The rate of clearance was markedly reduced in all cauterized rats regardless of the time intervals following cautery. The ability of MPS organs (liver, spleen, lungs, and kidneys) to take up radioactive colloid, as an indicator of phagocytic activity in cauterized animals, was markedly changed following cautery. The percentage of 99m Tc sulfur colloid in the liver was significantly reduced compared with that in control animals at 2, 6, and 12 days following cautery. In the other organs, colloid uptake was either increased (lungs) or not markedly affected (spleen) (Table 3). The results also showed that there was no significantly different effect on MPS function associated with the two sizes of cautery area used (0.58 or 1.7% of the total body surface area).

5 Figure 2. A biexponential plot of 99m T-sulfur colloid intravascular clearance in (A) control rats, (B) cauterized rats at 6 days post cautery, and (C) at 12 days post cautery. Table 2. Influence of traditional injury on phagocyte function measured by 99m Tc-sulfur colloid intravascular clearance in rats. Intravascular clearance rate (min) Group Days Fast rate Slow rate Control ± ± 1.8 Cautery ± 8.5* 63.3 ± 2.4 Cautery ± ± 21.5 All values are expressed as the mean ± S.E. 0.5 ml of 99m Tc-sulfur colloid was injected via the tail vein; three spots of cauterization were done at 6 and 12 days prior to injection of the colloid. * P <0.003; P <0.001; P <0.05; P < Table 3. Percentage uptake of 99m Tc-sulfur colloid distribution in the mononuclear phagocyte system organs of male

6 Wistar rats. Study group Liver Lungs Spleen Kidneys Control ± ± ± ± 0.66 Cauterized Day ± 5.2* ± 3.9* 5.29 ± ± 4.1 Day ± ± 2.5* 3.00 ± ± 1.5 Day ± ± ± ± 1.1 All values are expressed as the mean ± S.E. 0.5 ml of 99 m Tc-sulfur colloid was injected in the tall vein. Three spots of cauterization were done 2, 6, and 12 days prior to injection of the colloid. * P<0.01; P < In rats, the total WBC count was significantly reduced at 2, 6, and 12 days after cautery in cauterized groups compared with that in control animals (Table 4). A similar reduction was found, in the values of neutrophils, lymphocytes, and monocytes in the differential count. There was a significant inhibition of phagocytic function of both whole blood and isolated PMNs in cauterized guinea pigs, as measured by chemiluminescence, compared with that in control animals. The maximum peak response of PMNs measured by chemiluminescence was markedly reduced to 63.6 and 8.8 mv in cauterized animals at 6 and 12 days, respectively, following cautery, compared with 68.7 mv in control animals (Figure 3, Table 5). The same pattern of inhibition was shown using whole blood, although the response was relatively lower. Discussion In the last few decades, depression of experimental animal host defense system following thermal injury (burn) has been widely reported [12 15]. Several experimental studies have shown that the activity of the hepatic phagocytic cell of the MPS is markedly inhibited after thermal injury and circulatory shock [5,16 19] and increases the animal's susceptibility to infection and reduces microorganism clearance by the MPS in animals infected with bacteria [6, 12]. Another study found a selective depression of the peritoneal macrophage function in burned mice [20]. The mechanism by which thermal injury induces the MPS depression has not been clarified. Several questions remain, including why is there a marked depression in the MPS after thermal injury? More specifically, what is the factor (or factors) responsible for the inhibition of the MPS following thermal injury? Also, which steps of the phagocytic process are altered? Table 4. Influence o f traditional cautery on total and differential white blood cell and differential counts in guinea pigs at different time intervals post cautery. Group Day Total white blood cells Neutrophils Lymphocytes Monocytes Eosinophils Control ± ± ± ± ± 0.00 Cauterized ± ± ± * 111 ± ± Cauterized ± 230* 713 ± 75.98* 4910 ± 63.22* 127 ± ± 0.00 Cauterized ± 460* 649 ± * 5115 ± 70.80* 100 ± 32.20* 59 ± 0.00 All values are expressed as the mean ± S.E. Cauterization was done 2, 6, and 12 days prior to measurements. Cautery was donein three locations. * P < 0.01.

7 Table 5. Phagocytic function of whole blood and PMNs of normal and cauterized guinea pigs stimulated with PMA at different time intervals post cautery and measured by chemiluminescence. Group Whole blood PMNs Max. Integral Max. Integral peak (mv) (mv/min) peak (mv) (mv/min) Control Day Day * 2.02* 8.80* 89.29* PMNs count: ml. Incubation temperature: 37 C. Cauterization was done inthree locations. Chemil uminescence was measured at 0, 6, and12 days post cautery. Phorbol myristate acetate concentratewas 10 5 M and luminal concentrate was 10 4 M. * P < PMN = polymorphonuclear leukocytes. Figure 3. Chemiluminescence profile of polymorphonuclear leukocytes obtained from normal guinea pigs (heavy solid black line), and at 6 days (broken line) and 12 days (thin solid black line) after cauterization. Thermal injury was shown to reduce the clearance of erythrocyte debris, and was also found to be associated with a depression of MPS phagocyte function. Loegering et al [21 23] have studied the possibility that hemolysis following thermal injury plays the key role in this process. Moreover, studies have demonstrated that experimental thermal injury induces a depressed intravascular clearance rate of test particles that bind to undefined receptors on hepatic macrophages [8,14,22]. In vitro, thermal injury has also been observed to reduce the phagocytosis of yeast particles by alveolar and peritoneal macrophages [7]. Recently, Cuddy et al [15], who studied the effect of thermal injury on complement receptors in vivo, found a significant depression in the in vivo clearance function of hepatic complement receptors following thermal injury. This reduction may contribute to the susceptibility to infection associated with thermal injury. Lastly, a profound inhibition of LTB4 and oxygen release in PMNs was noted in patients who suffered total body surface area burns of 40 to 80% [24]. Several points can be drawn based on the findings of these earlier studies. First, a correlation exists between the severity of thermal injury and the degree of impairment of MPS. Second, all the earlier findings, whether obtained from experimental animals or human studies, were obtained in cases of a relatively high percentage of burn injury (more than 15%) of total body surface area. Third, there are no studies in which thermal injury was applied to a small surface area (1 to 2%). In the Kingdom of Saudi Arabia, a large number of Arabic traditional healers practice traditional cautery in large cities, villages, and the desert. Patients with a wide variety of diseases resort to this type of treatment, especially if modern treatments fail. These native Arabic healers believe that traditional cautery is a nonspecific

8 treatment for a broad spectrum of diseases, both organic and nonorganic. They apply cautery to treat bacterial infections such as pneumonia and viral infections such as viral hepatitis. Traditional cautery is also used to treat neurological diseases, and may be supplemented by medicinal plants and herbs. The size of the traditional thermal cautery area (percentage of burned skin) varies, depending on the practitioner, but is usually less than 2% of the total body surface area. The average sizes we have seen in cauterized patients were between 1 to 2 cm in diameter. The number and sites of cautery marks also vary depending on the location of pain. The traditional healers believe in a specific site for a specific disease. The results of the present study indicate that traditional thermal injury (cautery), although usually involving a small area, causes significant depression of MPS phagocytosis in normal experimental animals. This is indicated by the depressed blood clearance capability of the hepatic phagocytic cells of the MPS. The concentration of radioactive labeled colloid in the liver, the major port of the MPS, is significantly reduced. This finding agrees with those in earlier reports [6,8,13,17,25,26]. Conversely, Ikeuchi et al [27] reported activated macrophages in the spleens of burned mice, and Guo and Gu [28] showed that peritoneal macrophages are activated in the early period after theburn, then they begin to be depressed thereafter. In contrast to previous studies in which there was more extensive thermal injury (26 28%), the thermal injury (cautery) inflicted in our study was minimal (less than 2% of the total body surface area). The size of thermal injury may play an important role in a diseased host, especially if he or she suffers from an infection. Since this study was conducted on healthy experimental animals, one cannot conclude that the minimal thermal injury (less than 2% of the total body surface area) inhibits disease. However, the traditional cautery we performed in normal rats caused a depression in the MPS phagocytic capacity and inability to clear the foreign particles from the blood stream. In addition, using chemiluminescence, a marked inhibition of respiratory burst function was observed in the neutrophils of animals subjected to thermal injury. Total WBC and differential count were also significantly altered after thermal injury. Interestingly, endocytosis in hepatic macrophages, represented by a fast clearance rate, was markedly depressed in cauterized compared with noncauterized rats. The slow phase of the blood intravascular clearance rate of colloid was also reduced, yielding a prolonged half life in cauterized animals compared with normals. Acknowledgment We acknowledge the support and funding for this work given by King Abdulaziz City for Science and Technology. References 1. Seventh General Programme of work covering the period World Health Organization, Health for All Series No. 8, Geneva Ernst M, Fisher H. Chemiluminescence measurements of immune cells. A tool in immunology and clinical research. J Clin Chem Clin Biochem 1977;2: De Sole P, Lippa S, et al. Whole blood for chemiluminescence: a new technical approach to assess oxygen-dependent microbial activity of the granulocytes. J Clin Lab Immunol 1977;3: Tonooka T, Ueno N, Matsumoto T, et al. Chemiluminescence of whole blood. 1. A simple andrapid method for the estimation of phagocytic function of granulocyte and opsonic activity in whole blood. Clin Immunol Immunopathol 1983;26: Altura BM, Hershey SG. Reticuloendothelial function in experimental injury and tolerance to shock. Adv Exp Med Biol 1972;33: Di Miao A, Di Miao D, Jacques L. Phagocytosis in experimental burns. J Surg Res 1976;21: Loose LD, Turinsky J. Macrophage dysfunction after burn injury. Infect Immunol 1979;26: Rittenburg MS, Handback LD. Phagocytic depression in thermal injuries. J Trauma 1976;7: Larson SM, Nelp WB. Radiopharmacology of a simplified technetium 99m colloid preparation for photo-scanning. J Nucl Med 1966;7: Saad AH, Rutishauser SC, Williams AR. Computer simulations and the use of radiolabelled sulfur colloid to measure the efficiency of the mononuclear phagocyte system. J Immunol Methods 1986;83: Al Tuwaijri A, Al Mofleh I, Mahmoud A. Effect of Leishmania major on human polymorphonuclear leucocyte function in vitro. J Med Microbiol (in press).

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