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Cytological alteration in the rat stomach postburn 1972

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ci CYTOLOGICAL ALTERATION IN THE RAT STOMACH POSTBURN by CURTIS NORMAK HARRIS B. Sc., Oregon State University M.D., Loma Linda University A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of SURGERY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In p resen t i ng t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree tha t the L i b r a r y sha l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy. I f u r t h e r agree t h a t permiss ion f o r ex tens ive copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t is understood tha t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed w i thou t my w r i t t e n permiss ion . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date i ABSTRACT Gastric mucosal erosions were induced in the glandular stomach of rats by scalding. The incorporation of Thymidine- methyl-̂ -H into desoxyribonucleic acid was used to determine changes in gastric epithelial ce l l proliferating abil i ty. Total desoxyribonucleic acid per milligram of gastric tissue was also determined. Sampling was done at twenty-four hours, seven days, and fifteen days postburn. Eighty-nine point two percent of rats with a standard 26,5 t 2% scald burn had developed gastric mucosal erosions by twenty-four hours postburn. Seventeen point eight percent of burned rats had erosions by seven days and the incidence rose to hS,h% by fifteen days postburn. Ten point three percent of control rats in a l l sampling periods developed erosions. The total desoxyribonucleic acid in the gastric samples did not change significantly in any treatment period nor was i t changed by treatment. Uptake of thymldine-methyl^-H was depressed twenty- four hours postburn and renewed so through seven days postburn. The results at the fifteen day sampling were inconclusive. By light microscope, the gastric surface epithelium was lifted from the lamina propria and at times there was complete denudation of this ce l l layer. i i The rat is a satisfactory animal model for gross study of mucosal erosions to at least fifteen day postburn. Because of eschar cannibalization inducing variable secretory status, the rat model was not suitable for thymidine uptake studies past seven days postburn. i i i TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES v LIST OF FIGURES OR ILLUSTRATIONS v i CHAPTER I - Introduction 1 CHAPTER I I - Methods of Experimentation: 3 animals • 3 preparation f o r treatment 3 method of treatment 3 gastric lesions 6 histology 11 nucleic acid studies 11 CHAPTER I I I - Results: 13 incidence of gastric erosions 13 histology, gross and microscopic 13 gross specimens 13 microscopic specimens 18 •a incorporation of Thymidine-methyl -H .... 22 t o t a l c e l l u l a r desoxyribonucleic acid ... 27 CHAPTER IV - Summary and Conclusions: 32 Summary 32 Conclusions 3k iv Page BIBLIOGRAPHY 35 APPENDIX 30 V LIST OF TABLES Page Table I - Frequency Distribution of Number of Erosions per Stomach During Treatment Periods 19 3 Table II - Thymidine-methyl -H uptake counts and total cellular Desoxyribonucleic Acid for twenty-four hour treated and untreated rats 39 Table III - Thymidine-methyl^-H uptake counts and total cellular Desoxyribonucleic Acid for seven day treated and untreated rats • hO 3 Table IV - Thymidine-methyl -H uptake counts and total cellular Desoxyribonucleic Acid for fifteen day treated and untreated rats Ul v i LIST OF ILLUSTRATIONS Page Figure I: Clipped and epilated Wistar rat prior to treatment 5 Figure II: White Wistar rat twenty-four hours postburn 8 Figure III: White Wistar rat seven days postburn 8 Figure IV: White Wistar rat fifteen days postburn . . . 10 Figure V: Percentage of animals developing gastric lesions during treatment periods 15 Figure VI: Opened stomach from uriburned rat with no mucosal lesions 17 Figure VII: Multiple mucosal erosions in stomach of rat twenty-four hours postburn 17 Figure VIII: Uriburned rat skin after clipping and epilation (from original magnification xl20) , 21 Figure IX: Full thickness injury to rat skin after burn treatment at 88°C for twenty seconds (from original magnification xl20) 21 Figure X: Intact surface epithelium of gastric mucosa from unburned rat (from original magnification xl20) 2h v i i Page Figure XI: Extensive sub epithelial vacuolization of gastric mucosa from a burned rat (from original magnification xl20) 2h Figure XII: Absence of surface epithelium of gastric mucosa from a burned rat (from original magnification xl20) / 26 Figure XIII: Specific activity of Desoxyribonucleic Acid following injection of Thyroidine- 3 methyl- H in the glandular stomach in uriburned rats and rats 2h hours, 7 days and 15 days postburn 29 Figure XIV: Total Desoxyribonucleic Acid content of tissue samples from glandular stomachs of uriburned and burned rats after 2h hours, 7 day and 15 day treatment periods 31 1 INTRODUCTION In 1842 Thomas Curling (9) presented ten cases of duodenal ulceration associated with burns before the Medical-Surgical Society of London. Since that time the l e s i o n which bears h i s name has been expanded to include gastric ulcerations as w e l l as duodenal. Other traumatic and disease states have also been associated with gast r o i n t e s t i n a l ulcerations; e.g. i n t r a c r a n i a l l e s i o n (10), trauma (18), coronary occlusion (30), cor pulmonale (15), and these are now c o l l e c t i v e l y termed "STRESS ULCER." In 1965 S e v i t t (UU) reported an incidence of twenty-two percent gastric and duodenal ulcerations i n two-hundred and ninety-one patients autopsied following burns. In 1970, P r u i t t and his group from Brooke Army Hospital reported an o v e r a l l incidence of 11.7 percent "Curling's Ulcer" with approximately 19 percent of those dying of a cause d i r e c t l y related to the ulcer or an operation thereon (38) . This complication i s a s i g n i f i c a n t problem i n burns as w e l l as other forms of trauma. The cause or causes of these ulcerations i s not known. Following Davenport's experiments i n 196Ii (13, 12) there has been an increased impetus to determine what defect occurs i n the gastric mucosal b a r r i e r during episodes of stress. Increasing evidence i n - dicates that there may not be one cause and each different type of stress may have a di f f e r e n t e l l i c i t i n g mechanism. 2 The mucous layer (35, 37, 2, 25, 8), gastric acid (36, 29, 31, 4), gastric epithelial ce l l (29, 49, 40, 26) and electrolyte flux (11, 13, 12, 23, 31) have a l l been subjected to study cl inic- ally and experimentally. In the experimental animal the stress has been most often induced by restraint (29, 31) with the duration therefore being short. This experiment was undertaken to study changes in the pro- liferation of the gastric epithelial ce l l following burn induced stress in the rat. As the cl inical burn is a chronic illness, the duration of the study was extended beyond the usual short 2U-U8 hour treatment. 3 METHODS OF EXPERIMENTATION Animals White male and female Wistar rats weighing from 175 grams to 275 grams were randomly paired. These pairs were then divided into three treatment groups of twenty-eight pairs. The animals were placed in individual cages and given a standard laboratory diet and water ad libitum. Food was withdrawn eighteen hours prior to treatment or sacrifice. Water was allowed ad libitum at a l l times. Preparation for Treatment The rats were anesthetized with Sodium Pentobarbital, 5 milligrams per kilogram intraperitoneally. The backs were clipped with standard animal clippers from behind the ears to the t a i l and well down on the flanks. "MEET", a commercially available depila- tory was next applied to the clipped area, allowed to remain a few minutes and then washed off leaving the skin bare of hair. The animals were then dried with a towel and are shown for treatment in Figure I . Those rats randomly selected to be controls received 20 mi l l i l i tres of physiological saline intraperitoneally and were returned to their cages. Method of Treatment A standard 26.5 + 2 percent f u l l thickness burn was produced Figure I: Clipped and Epilated Wistar rat prior to treatment.  6 on the back of the rat by scalding. This was accomplished by using the weight immersion principle and a machine originally developed by Bailey, Lewis and Blocker (l) and extensively modified by Courtemanche (6). The temperature of the water was maintained at 88° Centigrade and the duration of treatment was twenty seconds. Following burning the rats were dried, given 20 mil l i l i tres of physiological saline intraperitoneally and returned to their cages. The pairs of rats were sacrificed at twenty-four hours, seven days and fifteen days postburn, and the appearance of the burned rats is shown in Figure II on page 8, Figure III on page 8, and Figure IV on page 10 respectively. Twenty-eight pairs of rats were in each treatment-day group. Following an eighteen hour fast, except for water, they were anesthetized with ether and the abdomen opened. The esophagus and pylorus were ligated with black si lk and the stomach excised and placed immediately in ice cold 0.9 Normal saline. The animals were then killed by incising the aorta or femoral artery and allowing exsanguination. Animals dying before sacrifice were discarded. Gastric Lesions The excised stomachs were opened along the greater curvature and the glandular stomach examined with a 10 x power dissecting microscope for mucosal erosions. The presence or absence of lesions was recorded and the significance determined by using the Pearson Chi Square test with continuity correction (17). 7 Figure II: White Wistar rat twenty- four hours postburn. Figure III: White Wistar rat seven days postburn. 6 9 Figure IV: White Wistar rat fifteen days postburn. 10 11 Statistics were computed to test treatment effect, batch and day effects using the University of British Columbia Computing Centre's ANOVAR system (17). Histology Sections of the glandular stomach were removed for histol- ogical examination by hematoxylin and eosin staining (ll;). Nucleic Acid Studies The incorporation of Thymidine-methyl^-H (TdR^-H, 20 c/m Swartz-Mann) into the deoxyribonucleic acid of the gastric epithelial ce l l was measured in control and burned rats. At the completion of the treatment period each rat was injected intraperitoneally with 2̂ 0 pc of TdR -H per kilogram of body weight. Treatment was contin- ued for ninety minutes before sacrifice. Full thickness specimens of glandular stomach of approxim- ately equal size were obtained using a #3 cork borer. Each specimen was weighed and immediately placed in ice-cold 0.01 molar Tris Buffer for total DNA analysis (29) or Hyamine hydroxide for TdR^-H uptake analysis (39). An attempt was made not to include erosions in the specimens. Total DNA was extracted by a method described by Ludwig and Lipkin (29). Two f u l l thickness specimen cores were homogenized with a Teflon pestle in 3 mil l i l i tres of ice-cold 0.01 molar Tris Buffer. The acid insoluble material was precipitated by adding 12 one mi l l i l i tre of cold 2N perchloric acid and the precipitate then washed twice with two mil l i l i tres of 75, 95 and 100 percent alcohol, a one to one mixture of alcohol-ether and ether. The remaining sample was allowed to dry overnight. The next day these samples were hydrolyzed in one mi l l i l i tre of 0.5 N perchloric acid. The diphenylamine procedure of Seibert (Ii3, 5) was used for the f inal coloriraetric deteimination of total DNA. One volume of the hydrolized material was added to two volumes of diphenylamine solution and the mixture heated at 100° C for ten minutes. The absorption curve was read on a Perkin-Elmer spectrophotometer at 650 ji and 595/i (5). Standardization was with highly polymerized calf thymus deoxyribonucleic acid (Sigma Chemical Company). Results were recorded as micrograms of DNA per milligram of tissue. One fu l l thickness core of glandular stomach was placed in a scintillation vial with one mi l l i l i tre of Hyamine Hydroxide and digested at 50° C for I48 hours (hi, 39 , 50). After digestion the sample was decolorized with six drops of 30 percent hydrogen peroxide (19) and acidified to pH 5 with four to six drops of glacial acetic acid (20). Fifteen mil l i l i tres of Aquasol Scintillation fluid (New England Nuclear) were then added, mixed, and the solution counted for ten minutes in the Picker Liquid Scintillation Counter. Results were recorded as counts per minute per milligram of tissue. Final figures were subjected to two way analysis-of-variance for count rate and for total DNA. 13 RESULTS Incidence of Gastric Erosions In Figure V, frequency of erosions of the glandular stomach of burned and uriburned rats is shown. Lesions developed in 10.7 (3 of 28) per cent of uriburned rats in each of the three treatment periods. Eighty-nine point two percent (25 of 28) of burned rats had developed erosions by twenty-four hours post treatment. By day seven the number of burned rats with erosions had declined to 17.8 percent (5 of 28). The frequency of lesions then increased again in the treatment group at day fifteen to U6 .U percent (13 of 28). The significance of these results as analyzed by the Pearson Chi Square test is also shown in Figure V. At one day pJD.OOOl and at fifteen days peO.008. The frequency of gastric ulcerations in burned versus nonburned rats was not of statistical significance at seven days post treatment (p=0.703). The maximum number of erosions occurred in the burned animals during the f irst twenty-four hour treatment period. Histology, Gross and Microscopic Gross Specimens In Figure VI, page 17, are shown the gross characteristics of the opened stomach in an untreated rat. The pink glandular Figure V: Percentage of animals developing gastric lesions during treatment periods. 15 16 Figure VI: Opened stomach from uriburned rat with no mucosal lesions. Figure VII: Multiple mucosal erosions in stomach of rat twenty-four hours postburn. 17 18 portion of the stomach is clearly without lesions. In contrast is the stomach from a burned rat shown in Figure VII, page 17. Multiple erosions of various sizes are present on the glandular portion of this specimen. Some treated animals seemed more likely to develop multiple ulcerations than others, given the same amount of stress. Multiple lesions were more frequent overall than single lesions as shown in Table I . The frequency of multiple lesions did not increase with duration of treatment in this experiment in contrast to the increasing numbers of lesions with duration of treatment shown by Ludwig and Idpkin (29) in their study of restraint stressed guinea pigs. Multiple erosions were not seen in the untreated animals. Microscopic Specimens An hematoxylin and eosin preparation of normal rat skin after clipping and epilation with NEET is shown in Figure VIII, page 21. The epidermis and dermis have been minimally changed by the chemical treatment. Figure IX, page 21, is a representative section after burn treatment demonstrating the f u l l thickness injury imposed by the burn. Disruption of the epidermis with coagulation necrosis of the underlying dermis and subcutaneous tissue are readily discernible. The glandular stomach of the rat is normally covered with columnar epithelial cells with mucous neck cells, parietal and zymogenic cells lining the gastric pits as shown in Figure X, page 2U. Changes following treatment were mainly confined to the surface 19 TABLE I Frequency Distribution of Number of Erosions per Stomach During Treatment Periods. Number of Stomachs Day 1 Day 7 Day 15 1 2 2 5 2-3 9 1 5 4+ lit 2 3 #92 60 61.9 Percent of total stomachs with erosions in which lesions were multiple. 20 Figure VIII: Uriburned rat skin after clipping and epilation (from original magnification xl20). Figure IX: Full thickness injury to rat skin after burn treatment at 88°C for twenty seconds (from original magnification x 120). 21 22 epithelium and sub-epithelial areas. Many histological sections were similar to Figure XI, page 2h, in showing large patches of surface epithelium lifted from the lamina propria. More marked damage is seen in some other areas (Figure XII, page 26), where there was complete destruction of the surface epithelium. Both of these histological changes were more frequent twenty-four hours postburn than later treatment periods. Non-burned rats rarely exhibited subepithelial vacuolization and never denudation. Incorporation of Thymidine-methyl-^H (TdR^-H) The surface epithelium of the rat stomach is a renewing cel l population (35) and does so at a relatively constant rate in the un- stressed animal (3U). The rate of regeneration is not significantly changed by the age of the animal (21) but the secretory rate is a major factor. Hunt demonstrated a marked increase in the gastric epithelial ce l l mitotic activity in the secreting rat over the fast- ing animal (22), thus, the importance of food withdrawal eighteen hours before treatment or sacrifice in this study. Each ce l l nucleus contains desoxyribonucleic acid (DNA). This consists of two long polynucleotide chains made up of pyrimidine bases, phosphoric acid and pentose sugars. With cel l duplication the specific pyrimidine base Thymine is taken up from the circulating pool and i t is at this point TdR^-H can be inserted as a label (U2) to estimate changes in ce l l populations. A close relationship exists 23 Figure X: Intact surface epithelium of gastric mucosa from uriburned rat ( from original magnifica- tion x 120). Figure XI: Extensive sub epithelial vacuo- lization of gastric mucosa from a burned rat (from original magnification xl20). 2k 25 Figure XII: Absence of surface epithelium of gastric mucosa from a burned rat (from original magnification xl20). 26 27 between the uptake of this labeled Thymidine and mitosis (35). Each species has a constant amount of DNA per nucleus (1*8). This serves as a baseline which in conjunction with TdR^-H uptake by the gastric epithelial ce l l allows an accurate estimation of changes in the pro- liferation ability of these cells. The specific activity of TdR^-H is shown in Figure XIII. There is a significant decrease in uptake in burned rats (p 4 .0.0001) at twenty-four hours post treatment. This reduction in uptake continued through day seven (p= . 0 0 1 5 ) . On the fifteenth postburn day there was an apparent increase in uptake in the treated group over the untreated animals. However, in subjecting this to analysis of variance this increase was not statistically significant (p=0.6591). Total Cellular Desoxyribonucleic Acid In Figure XIV, page 31, are shown the relationships of total DNA in the noriburned and burned rats during the fifteen day treat- ment period. The range was 13 to 56 micrograms of DNA per milligram of glandular stomach with a mean of 38.579 micrograms per milligram in untreated animals and a mean of I4O.U36 micrograms per milligram in the treated ones. Computing the statistical significance by the analysis of variance resulted in p=0.0903 at 2k hours, p=O.7010 at seven days and p=O.U376 at fifteen days. 28 Figure XIII: Specific activity of Desoxyribo- nucleic Acid following injection of Thymidine-methyl-^H in the glandular stomach in unburned rats and rats 2h hours, 7 days and 15 days postburn. (THYMIDINE H-3 UPTAKE) DAYS POSTBURN 30 Figure XIV: Total Desoxyribonucleic Acid content of tissue samples from glandular stomachs of uriburned and burned rats after 24 hours, 7 day. and 15 day treatment periods. 31 run mi P=. 7010 iiiiimii = MEAN CONTROL llllllll BURN Infliu P=.0903 P=. 4376 15 I 7 DAYS POSTBURN 32 SUMMARY AND CONCLUSIONS Summary: This experiment has demonstrated the feasibility of using the standard burned rat to study burn induced gastric changes to fifteen days postburn. Gross and microscopic changes are easily followed. The incidence of mucosal lesions during the f irs t twenty-four hours (89.2$) was similar to other reports (31, Ul, 1°, 3). A b i - phasic distribution of erosions with peaks at one day and fifteen days postburn was observed. This resembles the results reported by Sevitt (UU) in his autopsy study of burned humans. The mechanism of this is not known but he suggested that the in i t ia l lesions were as a direct result of the injury with subsequent healing, and then reulceration. Daily sampling of burned animals throughout the treatment period would be helpful in confirming this distribution. The incidence of mucosal lesions in control animals was higher (10.7$) than most studies. The anesthesia, clipping, epilat- ing and washing of these animals apparently produced a significant amount of stress. Variability of the rats in response to stress was also observed. Genetic factors (U5) and environmental factors such as housing and diet (2U, U6) have been implicated. A l l rats in this study were of 33 similar breeding and received identical pre-stress treatment thus minimizing any of these differences. Morphological study demonstrated formation of a subepithelial space. This progressive l i f t ing of the surface epithelium resulted in denudation and probably subsequent digestion of the lamina propria by intra luminal enzymes. These are similar to observations reported by Chiu, McArdle and Brown in a study of low flow rates and intestinal epithelium (7). In their experiment the rats were fasted twenty- four hours after scalding. This fluid deprivation was found to in- crease the incidence of gastric erosion. They postulated that this increase was secondary to a low flow state. In this study rats were allowed ad libitum water before and after burning. In addition, intraperitoneal saline was given immediately after treatment. A high incidence of mucosal erosions s t i l l occurred. This would seem to refute Chiu's postulation but may mean only that a low flow state occurred in spite of the fluid intake, the fluid intake was inadequate for the degree of trauma or other unknown mechanisms were in force. The incorporation of thymidine into desoxyribonucleic acid was depressed at twenty-four hours postburn and remained low through seven days postburn. By fifteen days postburn an overall increase in thymidine uptake appeared to be present in the burned animals. This situation can probably be attributed to the variable secretory rate in this group and which is reflected in the wide range of counts (Appendix A). It was not possible to maintain a fasting state in 34 some of these animals as is required.for basal mitotic activity (22) because many rats would cannibalize their own eschar. This increases secretory rate and thus thymidine uptake. No satisfactory method to prevent this was devised other than to substitute a non-carnivorous animal such as the guinea pig. Total DNA content remained unchanged and in association with the uptake studies indicates reduction in thymidine incorporation into DNA and thus decreased DNA synthesis and ce l l proliferation to day seven postburn. Continued depression of proliferation likely occurs but in view of the above cited difficulties remains unproven. Conclusions: 1. The rat is a satisfactory animal for gross and microscopic study of gastric erosions up to fifteen days postburn. 2. Gastric mucosal cel l proliferation is significantly depressed one day to seven days postburn as measured by thymidine- 's methyl-̂ -II. Eschar cannibalization is the probable cause of variable results at fifteen days postburn. 3. The total DNA content of the stomach mucosa is not sig- nificantly changed within fifteen days postburn despite the severe injury and resulting debility. 4. The exact etiology of mucosal erosions in burns remains unknown but the decreased ability of the surface epithelium to renew adequately seems to be a contributing factor. 35 BIBLIOGRAPHY 1. Bailey, B.N., S.R. Lewis and T.G. Blocken, "Standardization of Experimental Burns i n the Laboratory Rat", Texas Reports on Biology & Medicine, 20: 20, 1962. 2. 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Vaughn, Martha, Daniel Steinberg, & Jane Logan, "Liquid Scintillation Counting of C14 & H3 Labeled Amino Acids & Proteins," Science, Vol. 126: 41*6, 1957. Vendrely, R., & C. Vendrely, "La Teneur du noyau cellulaire en acide de'soxribonucle'ique a. travers les organes, les individus et les especes animales," Experientia, Vol. 4: 434, 1948. Young, Kim S., Robert Kerr, Martin Lipkin, "Cell Proliferation During Development of Stress Erosions in Mouse Stomach," Nature, 215: 1180, 1967. "Protosol the Superior Solubilizer," New England Nuclear Bulletin, Boston, 1972. APPENDIX 39 TABLE II Thymidine-methyl^-H uptake counts and total cellular Desoxyribo- nucleic Acid for twenty-four hour treated and untreated rats. TdR3-H Scintillation Total Micrograms Counts/Milligrams Tissue/Minute DMA/Milligram Tissue Control Rat Burned Rat Control Rat Burned Rat 1. 19.240 12.735 .324 .274 2. 18.639 9.94 .295 .310 3 . 25.116 13.292 .370 .390 4 . 21.165 15.529 .281 .408 5. 15.854 9.48 .401 .522 6. 17.500 12.491 .436 .428 7. 186.790 165.623 .310 .326 8. 182.117 183.015 .232 .322 9. 201.962 159.355 .387 .342 10. 187.077 109.434 .330 .308 11. 160.425 151.671 .309 .366 12. 171.035 I88.338 .330 .460 13. 215.700 209.102 .364 .317 14. 193.954 146.967 .368 .360 15. 169.251 148.147 .282 .406 16. 164.159 153.762 .290 .324 17. 63.960 43.627 .410 .420 18. 61.242 35.257 .422 .406 19. 114.562 37.162 .456 .543 20. 54.661 34.600 .278 .409 21. 57.710 31.682 .365 .356 22. 80.823 30.407 .328 .276 23. 42.892 11.761 .377 .409 24. 84.560 19.406 .337 .260 25. 77.394 17.375 .320 .327 26. 121.310 11.511 .336 .322 27. 48.367 29.050 .373 .420 28. 54.327 18.208 .326 .290 Uo TABLE III Thymidine-raethyl^-H uptake counts and total cellular Desoxyribo- nucleic Acid for seven days treated and untreated rats TcbV-H Scintillation Total Micrograms Counts/Milligrams Tissue/Minute DMA/Milligram Tissue Control Rat Burned Rat Control Rat Burned Rat 1. 205.907 192.939 .Uoi .321 2. 170.249 145.372 .3lU .271 3. 197.106 136.891 .362 .255 U. 25U.672 135.936 .418 .U08 5. 13U.905 141.183 .333 .353 6. 256.275 155.150 .263 .342 7. 217.9U5 277.377 .283 .382 8. 213.464 25U.U75 .328 .336 9. 221.522 231.1U7 .300 .318 10. 276.131 216.909 .326 .333 11. 236.823 227.287 .361 .330 12. 183.3U7 180.0U7 .340 .317 13. 155.022 152.777 .304 .297 lU. 216.861 151.06U .408 .362 15. 172.22U 158.746 .272 .340 16. 170.665 191.603 .297 .240 17. 251.455 179.528 .267 .311 18. 145.328 129.098 .246 .277 19. 71.068 28.692 .155 .159 20. U8.733 27.598 .174 .228 21. 30.U65 28.800 .281 .241 22. 36.313 28.989 .266 .294 23. 6U.156 28.787 .089 .155 2U. 44.785 36.143 .101 .106 25. UU.860 15.066 .276 .318 26. 52.347 23.93U .276 .336 27. 83.788 28.U50 .327 .325 28. 103.061 36.802 .326 .276 TABLE IV Thymidine-methyl^-H uptake counts and total cellular Desoxyribo- nucleic Acid for fifteen days treated and untreated rats. TdR3-H Scintillation Total Micrograms Counts/Milligrams Tissue/Minute DNA/MLlligram Tissue Control Rat Burned Rat Control Rat Burned Rat 1. 230.211 183.297 .339 .331 2. 323.682 229.735 .291 .259 3 . 319.530 463.117 .29U .287 4 . 240.009 910.968 .230 .296 5 . 289.970 535.234 .295 .363 6. 242.601 111.414 .380 .244 7. 198.861 .285 .224 8. 169.715 .261 .215 9. 38.853 22.177 .316 .338 10. 29.849 5U.586 .297 .390 11. 49.106 83.944 .352 .418 12. 72.028 48.413 .319 .323 13. 218.547 238.548 .336 .204 14. 220.316 152.576 .372 .338 15. 194.487 196.343 .371 .340 16. 229.695 152.576 .371 .338 17. 228.085 217.950 .345 .350 18. 257.952 184.940 .385 .440 19. 292.984 223.780 .366 .473 20. 215.026 200.077 .347 .419 21. 202.045 267.961 .370 .471 22. 188.808 145.151 .362 .450 23. 146.461 150.852 .373 .450 24. 222.342 154.588 .391 .383 25. 203.453 163.226 .476 .438 26. 191.102 202.090 .476 .385 27. 311.8̂ 0 267.494 .342 .400 28. 224.290 240.852 .320 .257


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