ABSTRACT COMPARTMENTAL BLOOD FLOW IN THE CANINE JEJUNUM WITH FOOD OR 50% GLUCOSE IN THE LUMEN BY Ya-Mei Tung Yu The blood flow to the small intestine increases after meals or after‘ingestion of hypertonic glucose solution. It is not clear however, whether this increased flow is distributed equally to the three tissue layers of the intestine. The present study was designed to investigate the effects of luminal placement of digested dog food and 50% glucose on blood flow and its distribution in the three layers of the canine jejunum. The three layers were the mucosa, submucosa, and muscle-serosa of the jejunal wall. The radioactive microsphere method was used for this purpose. The validity of the method was also evaluated by using two types of microspheres, one labeled with ceriumrl41 and the other with strontiumPBS. Isotonic polyethylene glycol (PEG), digested dog food, or 50% glucose was placed into the lumen of naturally perfused in situ jejunal segments in anesthetized dogs. PEG was used as the control of food and glucose. Twenty minutes after the luminal placement of these substances, two types of microspheres, 15 :15 u in size, were injected into the left ventricle. These two types of microspheres were injected alternately at 3 minute intervals to each dog. In another group of dogs, 0.4% Ya—Mei Tung Yu dibucaine was placed into the lumen for 20 minutes before the placement of 50% glucose. The purpose was to see if local mucosal anesthesia would alter the response of blood flow to luminal placement of 50% glucose. The segments containing food or 50% glucose had higher radioactiv- ities in the whole wall and the mucosa than the control segments. The radioactivities in the submucosa and muscle-serosa of the segments containing food were not significantly different from those in the control segments. But in the segments containing 50% glucose, the radio- activity in the submucosa was slightly higher than or similar to that in the control segments and the radioactivity in the muscle-serosa was not different from that in the control segments. 0n the basis of per- cent distribution of total jejunal blood flow, the mucosa of the seg- ments containing food or 50% glucose received the greater while the submucosa and muscle-serosa received the smaller shares of the total flow as compared to those in the control segments. The segments which were treated with dibucaine prior to placement of 50% glucose still had higher radioactivities in the whole wall and the mucosa than the segw ments containing PEG. But the segments had lower radioactivities in the whole wall and the mucosa than the segments containing 50% glucose. The results obtained from microspheres labeled with Ce-l4l corre— lated well with those labeled with Sr-85. This indicates that one type of microspheres may be used as a control for the other type. Ya-Mei Tung Yu It is concluded that luminal placement of food or 50% glucose in the jejunum increased total blood flow to the jejunum and caused redistribution of blood flow in the jejunal wall. Luminal placement of food caused an increase in mucosal flow, but no change in flow to the submucosa or muscle-serosa. Luminal placement of 50% glucose caused an increase in mucosal flow, a slight increase or no change in submucosal flow and no change in flow to the muscle-serosa. The in- creased blood flow caused by luminal placement of 50% glucose could be partly blocked by exposing.the mucosa to a local anesthetic, dibucaine. COMPARTMENTAL BLOOD FLOW IN THE CANINE JEJUNUM WITH FOOD OR 50% GLUCOSE IN THE LUMEN BY YaéMei Tung Yu A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1974 ‘1 ACKNOWLEDGMENTS The author would like to thank Dr. C. C. Chou for his invalu- able encouragement and guidance throughout the course of my training. A special appreciation is extended to Dr. J. M. Dabney and Dr. B. H. Selleck for their guidance and service on the examination com- mittee. The author also wishes to express her special thanks to Dr. Luke C. Yu, my husband and best assistant, without whose help this would have been impossible. ii Chapter TABLE OF CONTENTS LIST OF TABLES O O O O O O O O O O O I O O O O O O O O O 0 LIST OF FIGURES I O O O O O O O O 0 O O O O O O O O O O O I 0 INTRODUCTION 0 I O O O O O O O O O O O O O O O O O O O O C II 0 REVIEW OF LITERATURE O O O O O O O O O O O O O O O O O O O A. B. Feeding and Blood Flow . . . . . . . . . . . . . . Methods for the Measurement of Compartmental Blood Flow in the Intestine . . . . . . . . . . I I I 0 MATERIALS AND METHODS . O C C I O O Q C I O O O O O O O O l. 2. 3. 4. 5. 6. 7. 8. Preparation of Radioactive Microspheres. . . . . . Preparation of Digested Dog Food . . . . . . . . . Surgical Procedures. . . . . . . . . . . . . . . . Experimental Procedures. . . . . . . . . . . . . . Measurement of Radioactivity . . . . . . . . . . . Calculation and Expression of the Results. . . . Weight Distribution of the Canine Jejunal Wall . . Statistical Analysis of the Results. . . . . . . . IV 0 RESULTS 0 O O O O O O O O O O O O O O O I O O O O O O O 0 Mean Weight Distribution in the Jejunal Wall . . . Effects of Luminal Placement of PEG, Food, and 50% Glucose.. . . . . . . . . . . . . . . . . . Effects of Mucosal Anesthesia on Blood Flow Responses to 50% Glucose. . . . . . . . . . . . Comparison of the Results Obtained from.the Micro- spheres Labeled with Ce-l4l and Those Labeled with Sr-85. . . . . . . . . . . . . . . . . . . V. DISCUSSION. 0 O O O O O O O O O O O O O O O O O O O O O 0 VI. SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . BIBLIOGRAPHY O C O O O O O O O C O O * O O O O O O O O O O O Page iv 21 21 21 22 23 24 27 28 29 3O 30 36 43 46 54 67 70 LIST OF TABLES Weight distribution in the jejunal wall (%) (mean;t S.E.) (N - 12) O O O O O O O O O O O O O O O O O O O O O O O O O The average radioactivity (cpm/gm tissue) of Ce-l4l of the whole wall, the mucosa, submucosa, and muscle-serosa of the segments which were empty or containing PEG, food, or 50% glucose. (mean i;S.E.) (N = 10) . . . . . . . . . Average percentage distribution of blood flow in the wall of the jejunal segments which were empty or containing PEG, food, or 50% glucose as determined by Ce-l4l labeled microspheres. (mean.i;S.E.) (N - 10) . . . . . . . . . . The average radioactivity (cpm/gm tissue) of Sr-85 of the whole wall, the mucosa, submucosa, and muscle-serosa of the segments which were empty or containing PEG, food, or 50% glucose. (mean : S.E.) (N - 10). . . . . . . . . . . Average percentage distribution of blood flow in the wall of the jejunal segments which were empty or containing PEG, food, or 50% glucose as determined by Sr-85 labeled microspheres. (mean i S.E.) (N - 10) . . . . . . . . . . The average radioactivity (cpm/gm tissue) of Ce-l41 of the whole wall, the mucosa, submucosa, and muscle-serosa of the segments containing PEG, 50% glucose, or dibu- caine-50% glucose. (mean i_S.E.) (N = 8) . . . . . . . . The average radioactivity (cpm/gm tissue) of Sr—85 of the whole wall, the mucosa, submucosa, and muscle—serosa of the segments containing PEG, 50% glucose, or dibucaine- 50% glucose. (mean i_S.E.) (N - 8) . . . . . . . . . . . iv Page 35 37 39 41 42 44 45 LIST OF FIGURES Figure 1. Photomicrograph of the mucosa of the canine jejunum after separating it from the submucosa and muscle- serosa. O O O O O O O O O O O O O O O O O O O O O O O O Photomicrograph of the submucosa of the canine jejunum after separating it from the mucosa and muscle-serosa . . Photomicrograph of the muscle-serosa of the canine jejunum after separating it from the mucosa and sub- mucosa. O O O O O O O O O O O O O O O O O O I O O O O O 0 Comparison of the mucosal blood flows, expressed as per- cent of total blood flow in the jejunal wall, estimated by two types of the microspheres, one labeled with Ce-l4l and the other with Sr-85. The linear regression line was Y = -4.806 + 1.065 X. . . . . . . . . . . . . . . . . . . Comparison of the submucosal blood flows, expressed as percent of total blood flow in the jejunal wall, esti- mated by two types of the microspheres, one labeled with Ce-l4l and the other with Sr-85. The linear regression line was Y = 0.525 + 1.052 X. . . . . . . . . . . . . . . Comparison of.the muscle-serosal blood flows, expressed as percent of total blood flow in the jejunal wall, esti- mated by two types of the microspheres, one labeled with Ce—l4l and the other with Sr-85. The linear regression line was Y = 1.393 + 0.911 X. . . . . . . . . . . . . . . Page 32 32 34 48 50 52- CHAPTER I INTRODUCTION The relation between intestinal blood flow and function is not only of interest to physiologists, but also-of clinical importance in conditions such as the dumping syndrome. The majority of evidence indicates that splanchnic or the superior mesenteric arterial blood flow increases after meals (2, 3, 15, 20, 54). Some investigators proposed that the hyperemia results from increased cardiac output and involves all organs of the body (20). Recent studies (2, 3, 15, 54), however, indicate that following a meal the blood flow increases in the superior mesenteric artery and decreases in brachiocephalic and iliac arteries. The cardiac output is not changed. Thus, there is a redis- tribution of cardiac output favoring the vascular bed supplied by superior mesenteric artery at the expense of blood flow to other areas. It has been also shown that the increased splanchnic blood flow following a meal is localized in the area exposed to chyme or in the area performing digestive and/or absorptive functions (10). Furthermore, the increased flow appears to occur only in the segment containing food or glucose (7). These studies thus appear to indicate that the increased flow following meals is a local phenomenon. A review of the literature reveals that the information in regard to the effects of feeding on the compartmental blood flows of the jejunal wall is scarce. The present study was, therefore, designed to investigate the effects of luminal placement of digested dog food and 50% glucose on blood flow and its distribution in the wall of canine jejunum. CHAPTER II REVIEW'OF‘LITERATURE A. Feeding and Blood Flow The majority of evidence indicates that mesenteric blood flow increases after meals. Herrick et al., in 1934 (20), used the thermo— stromuhr to measure blood flow in intact dogs“and'found that the‘ superior mesenteric blood flow began‘to increase within 10 minutes after ingestion of'a meal consisting of'a'milkeegghglucose mixture and a dog food composed of meat and cereals. 'It reached a maximum (60% above the fasting level)"90*minute3‘after ingestion and then gradually declined and returned to nearly thE'original level four and one half hours after ingestion.~ They also found that"the increase in the superior mesenteric arterial flow occurred concurrently with an in- creased flow in the femoral and carotid‘arteries and they surmised that an increase in‘cardiac output occurred"postprandially. Using electromagnetic flowmeters, Huse and Hinshaw in 1960 (22) showed, in dogs, an average increase of 45% of mesenteric blood flow when 50% glucose was injected into the proximal jejunum. The maximal effect was seen between 20 and 35 minutes after the glucose injection. In contradistinction, there was an average decrease of 34% in renal flow, 36% in carotid flow, and 32% in femoral flow. Cardiac outputs showed an average decrease of 20%. From these results, they suggested that there was a redistribution of the cardiac output favoring the expanded splanchnic vascular bed at the expense of blood flow to other areas. By means of chronically implanted electromagnetic flow probes, Fronek and Stahlgren, in 1968 (15), showed in dogs that at l and 3 hours after ingestion of standard canned dog food there were no sig- nificant changes in cardiac output, heart rate, and mean arterial blood pressure. The flow in the superior mesenteric artery, however, in- creased to 133% and mesenteric regional resistance decreased to 82% of the control. Contrarily, the flows in the brachiocephalic and iliac arteries decreased to 86.5% and 74.6% of their controls, respectively. The ratio of flow in the superior mesenteric artery to cardiac output increased from 8.9% to 12.5% in the third hour. They concluded that there was a redistribution of blood flow during digestion with a pref- erence for the vascular bed of the superior mesenteric artery. Using the same method, Burns et al., in 1960 (2), found that the mesenteric blood flow in conscious dogs began to increase within 5 minutes after feeding of horsemeat and reached a maximum around 50 minutes. Although the flow gradually declined thereafter, the mean flow was still 50% above the fasting level 3 hours after feeding. There was no detectable rise in cardiac output in response to feeding except for occasional transient changes during ingestion. These results were simi- lar to those reported by the same investigator in 1967 (3). With the use of flowmeters, Vatner, Franklin, and VanCitters, in 1970 (54), found that in conscious dogs following an initial decrease of 10% during the anticipation of food, the mesenteric blood flow began to increase within 5—15 minutes and reached a maximum (from 115 to 300% of control) within 30-90 minutes after eating. The flow gradually returned to control levels within 3-7 hours. Transient increases in cardiac output, heart rate, and aortic blood pressure during anticipa- tion and ingestion of food were followed by a return to control levels after 10-30 minutes and remained there throughout the experiments. In 1972, by collecting local venous outflow, Chou and Dabney (7) showed an increase of 6%, 8%, 11% and 23% in blood flow of canine jejunum when 2.5%, 5.4%, 20% and 50% glucose solutions were placed into the jejunal lumen, respectively. Following the luminal placement of any glucose solution, venous outflow of the jejunal segment usually increased in the first three minutes and remained at the same level or increased slightly in the next eight minutes. Brandt et al., in 1955 (1), studied the effect of oral protein and glucose feeding on splanchnic blood flow in normal and cirrhotic subjects by means of bromsulphalein (BSP) clearance method. They found that protein feeding induced an increase in splanchnic blood flow in both normal and cirrhotic subjects. The increased flow occurred sooner and was of greater magnitude in the normal subjects than the cirrhotic patients. They also found that glucose feeding resulted in minimal change in splanchnic blood flow in both normal and cirrhotic subjects. Using the same BSP method, Castenfors, Eliasch, and Hultman, in 1961 (4), found a 25% increase in splanchnic blood flow in normal subjects and a 70% increase in patients who were subjected to partial gastrectomy 19 minutes after the ingestion of hyperosmotic glucose solution. This appears to indicate that gastric emptying and thus the amount and/or duration of the presence of food in the small intestine is a significant factor influencing increased splanchnic flow. From these studies, it can be seen that blood flow to the splanchnic viscera or small intestine increases after meals in dogs as well as in men. In general, the flow increases within 5-15 minutes and reaches a maximum within 20-90 minutes after ingestion of food. The flow then gradually returns to control within 4-7 hours. Although Herrick in 1934 suggested that no redistribution of cardiac output occurred post-prandially, recent studies, using electromagnetic flow- meters, has shown that there is redistribution of cardiac output in favor of the vascular bed of superior mesenteric artery. In the above studies, only total splanchnic blood flow, superior mesenteric arterial flow, or blood flow to an intestinal segment was measured. The effects of digestion and absorption of food or glucose solution on blood flow distribution in the different compartments of the intestinal wall, however, have not been studied. Although Grim and Lindseth (16) made attempts to estimate the blood flow distribution in the intestinal wall during fasting and digestive states, they only showed the flow rate per 100 grams of each compartment. Percentage distribution of blood flow in the intestinal wall was not calculated. In order to determine the blood flow in the small intestine during di- gestive phase, the dogs were fed twice, 6 hours and 2 hours before the experiments. Whether the digested food was actually present in the intestinal loop at the time of'sampiing was not mentioned in their paper. In addition, they used glass microspheres labeled with Na—24 for measurement of blood flow. 'The'specific gravity~of'these glass microspheres differs greatly from that’of”blood'componentsz' Also, they injected microspheres into*the local artery of the isolated loop of intestine, instead of the left ventricle; ‘These“tw6'factor8'cou1d reduce the possibility of even distribution of microspheres with‘blood components and minimize the accuracy of‘the’technique.v B. Methods for the Measurement of Compartmental Blood Flow in the Intestine Three methods, namely, radioactive inert gas wash out technique, Kr42 clearance technique, and radioactive microsphere technique, have been used to measure the compartmental blood flow in the intestine. 1. Radioactive Inert Gas (Kr-85 or Xe-l33) Wash out Technique The estimation of blood flow from clearance of radioactive tracers injected into the region of interest was first described by Kety in 1949 (28), and has been applied chiefly to the determination of cerebral (23) or myocardial (40) blood flow. Kampp et al. in 1966 (25) and Selkurt et al. in 1967 (45) applied the same technique to the small intestine. They measured and analyzed the multiple exponential curves obtained following intra-arterial injection of Kr-85 or Xe-l33 to esti- mate total blood flow and flow distribution in the small intestine. Theoretical considerations (26, 27, 29, 30) After intra-arterial injection of radioactive inert gas, Kr-85 or Xe-l33, multiple exponential curve was registered by a scintillation detector placed 4-6 cm from the intestine. The multiple exponential curve is composed of several components representing different elimi- nation rates of injected substance. The relative amount of blood flow distributed to each tissue compartment which corresponds to each elimi- nation rate can be estimated. The elimination of an inert gas from a constantly and uniformly perfused tissue can be described by the following equation provided that the arterial concentration of the gas is zero or negligibly low during the wash out period: where Ct and Co denote the tissue concentration of the gas at times t and o, and k denotes the clearance constant. The constant, k, which is closely related to the blood flow can be determined from the following equation: 1n 2 k t 2 35 where t k is the half time of decay in min. and is calculated from the straight line when plotting equation 1 on semilogarithmic paper. If the disappearance of gas from two or more parallel-coupled, homogenously perfused tissues with different rates of clearance is simultaneously registered, the resulting elimination curve is the sum of the single exponentials according to the general formula A =A1 .e'k1t+A2 .e_k2t+A3 .e-k3t+...+An .e‘k“t 3 t O O O 0 Where At is total counts per min. at time t; A10, A20, A30, ... , Ano are the number of counts per min. initially present in each component; and kl, k2, k3, ..., kn are the clearance constants of the different components. An arched curve is obtained when At is plotted semilog- arithemically against time. Provided n (the number of components) is low and the k values sufficiently differ from each other, this composite curve can be resolved into its different components by means of the method originally described by Dobson and Warner in 1957 (14). Briefly, a straight line is drawn through the terminal straight portion of the curve and extrapolated to time zero. A new curve is constructed by sub- tracting this straight line from the original curve. A straight line is again drawn through the terminal straight portion of the constructed curve. The procedure is repeated until a final straight line is ob- tained. These straight lines represent the rates of clearance in dif- ferent compartments. In the small intestine, Kampp et a1. resolved their curves into 4 and Selkurt et a1. resolved into 3 components. The k values are ob— tained from equation 2. Correct estimations of the respective k values of the different components can be obtained only if the diffusion of the tracer between the different compartments is negligible. The calculation of the blood flow is based on the assumption that the gas leaves the tissue compartment exclusively via the blood and the equilibrium of gas between blood and tissue is reached in a frac- tion of a second. The blood flow (f) in different compartments can be calculated in m1/min/100 gm from the following formula: f = k . s . 100 4 10 where 8 denotes the tissue blood partition coefficient of the gas di— vided by the specific weight of the tissue. Kampp et a1. (25) studied the blood flow and flow distribution within the denervated jejunum of the cat by injecting Kr-85 intra- arterially and monitoring its clearance with a scintillation detector placed 4-6 cm from the intestine. They analyzed the multiexponential curve they obtained into four components based on the theoretical considerations described above. They proposed that component I (half time value 0.05 - 0.20 min; percent initial activity 35-50) reflects short circuit of Kr-85 via countercurrent exchange in villi, component 11 (half time value 1-2 min; percent initial activity 30-40) reflects blood flow within the mucosa, component III (half time value 4-9 min; percent initial activity 25) reflects blood flow of the muscularis, and component IV (half time value 20-60 min) is located outside the intes- tinal wall, probably in the perivascular fat of the mesentery. Flow values in ml/min/lOO gm of musoca and muscularis were 30—70 and 10-20 respectively. They have used four independent methods to prove their impression of four different components in their registered curves. These methods are registration of the elimination of B—activity, local injections of Kr—85 into different parts of the intestinal segment, autoradiography and comparisons of weights of the compartments. Based on the same principles, Selkurt et a1. (45) estimated the compartmental blood flow in canine small intestine with the use of the Xe-l33 wash out technique. They resolved the wash out curve into three components which reflected the blood flow to the epithelial glandular 11 tissue, to smooth muscle, and to connective and supportive tissue. The average values per minute per 100 gm of these tissues were, respec- tively, 148.8, 35.7, and 3.4 m1. Applying these values to the anatomic- ally distinct compartments gave a total flow in the wall of 64.2 m1/min/ 100 gm. The total blood flow measured simultaneously with the electro- magnetic flow probe was 63.1 ml/min/100 gm. 2. K—42 Clearance Technique In 1958, Sapirstein (43) used the clearance of K-42 or Rb-86 to estimate blood flow in various organs. Delaney and Grim, in 1964 (12) and 1965 (13), used the same technique to estimate the canine gastric blood flow and its distribution. Theoretical consideration (12, 43) This method is based upon the assumption that the extraction ratio of tracers in any organ in the body is the same and also all tissues in an organ have the same extraction ratio. It is also assumed that the K-42 contained in an organ or tissue 30-60 sec. after intravenous injec- tion of a small bolus of the isotope is proportional to the blood flow perfusing that organ or tissue. With this method, the cardiac output can be estimated from the isotope dilution curve by the conventional Stewart- Hamilton equation: Cardiac output = fig; 5 o where I is the amount of isotope injected, c is the arterial radioactiv- ity and t is time. Blood flow to any tissue or compartment, or to the whole organ, can be calculated as follows: Regional flowa -if- ' cardiac output 6 12 where IR is the amount of isotope recovered in the tissue at sacrifice. By combining equation 5 and 6, the formula for the calculation of regional flow becomes: Regional flow - 717-{%75; 7 o In other words, regional flow equals the K-42 content in the tissue divided by the area under the arterial dilution curve. Furthermore, the compartmental blood flow distribution, e.g., that in the intestinal wall, can be calculated simply by dividing the amount of isotope in each tissue layer by the amount of isotope in the whole intestinal wall. Delaney and Grim (12) used this technique and found the distribu- tion of blood flow among tissue layers of gastric corpus in intact anesthetized dogs to be mucosa 72%, submucosa 13%, and muscularis 15%, and that in intact unanesthetized dogs mucosa 74%, submucosa 14%, and muscularis 12%. 3. Radioactive Microsphere Method The first application of this method to measure gastrointestinal blood flow was reported by Grim and Lindseth in 1958 (16). They used glass microspheres labeled with Na-24 to study the distribution of blood flow in different layers of the small intestinal wall of dogs. In 1964, Delaney and Grim (12) used the same method to determine the blood flow distribution in the wall of canine stomach. Historical development of microspheres Prinzmetal (38), the first investigator to use microspheres in the study of the circulation, used glass spheres to detect arterio- arterial and arteriovenous anastomoses in human hearts in 1947. 13 Subsequently, he and several other investigators (34, 37, 47, 51) in- jected glass microspheres of various sizes into the inflow of a number of organs of several species, and recovered the outflowing glass spheres. Although most of the studies were interpreted as demonstrating arteriovenous shunting, their conclusions have not been generally accepted (19). The problems with glass microspheres are their high density, the necessity of using large amounts and non-quantitative. Ryan prepared radioactive microspheres by neutron bombardment of glass microspheres to convert some of the sodium in the glass to Na-24 (55). This greatly improved the previous technique and was adopted by others. Ceramic microspheres labeled with different isotopes were developed subsequently by Lahy and Ryan. By 1966, ceramic microspheres with density of 3.0 gm/cc and plastic microspheres with density of 1.3 gm/cc were available labeled with any of 27 different radionuclides (55). The plastic microspheres, supplied by the Nuclear Product Division of the Minnesota Mining and Manufacturing Company, are referred to as "carbonized microspheres". They are black in appearance and consist of carbon, hydrogen, oxygen and a trace amount of the nuclide of interest. The nuclide is incorporated in the microspheres and is not merely a coating on the surface. The microspheres resist temperatures up to 400°C, above which they begin to disintegrate. They are quite insoluble in all common organic or inorganic solvents at room temperature but can be dissolved by boiling in concentrated acids or bases. Theoretical consideration (55) The application of microspheres to the study of the circulation rests on four basic assumptions: 1) the microspheres are adequately 14 mixed with the blood and have essentially the same rheology as red blood cells; 2) the microspheres themselves do not alter the circula- tion; 3) the radioactivity remains bound to the microspheres during the period of observation; and 4) the microspheres are sufficiently large to be removed from the circulation by lodging in the capillary beds. If the above assumptions are all true, the microspheres are mixed with the inflowing blood and distributed in the same manner as blood. The microspheres, however, are entrapped in the capillaries of the perfused tissues and are completely cleared from the blood in a single passage. The number of microspheres in the organ or tissue thus can be calcu- lated from the following equation: t q = f foc(t)dt 8 where q is the number of microspheres in the organ or tissue of inter- est, f is blood flow (ml/min) to the organ or tissue and c is the con— centration of microspheres in the blood (microspheres/ml). Since the radioactivity remains bound to the microspheres during the period of observation, the distribution of the radioactivity is a measure of the relative distribution of blood perfusing the region at the time of in- jection of the microspheres. Therefore, the blood flow distribution in the intestinal wall can be estimated from the distribution of radio— activity among its three compartments, i.e., the mucosa, submucosa, and muscle-serosa. Experiments which support the assumptions 1. The adequacy of mixing of the microspheres: 15 Shibata and MacLean, in 1966 (55), demonstrated uniform distribu- tion of microspheres in the lung by obtaining multiple small tissue samples after an intravenous injection of radioactive microspheres. Phibbs et al. in 1967 "(36) and 1970 (35)“and‘1feutze'et al. in 1968 (33) studied the distribution of radioactive microspheres within flowing blood by ultra-rapid freezing of femoral arteries in rabbits. Using this technique, blood flowing through a segment of the artery was immobi- lized in less than 0.05 seconds. The serial cross sections were examined microscopically. They found that the majority of the microspheres were in the more central three-fourths of the arterial lumen, where flow is faster. The finding suggests that the distribution of microspheres is at least similar, if not identical to that of the flowing blood cells. If axial streaming were important, there could be a difference in the concentration of microspheres in carotid and femoral arterial blood samples after left ventricular injection. Neutze et a1. (33) compared the amount of radioactivity in carotid and femoral arterial blood with- drawn simultaneously during left ventricular injection of microspheres. They found that there was no difference between the radioactivities in blood samples collected from these two arteries. Therefore, significant preferential streaming to either artery is excluded. Rudolph and Haymann, in 1967 (42), measured the flows in the two umbilical veins of fetus of sheep and goats with electromagnetic flow- meters and simultaneously measured the radioactivity in the placenta following injection of radioactive microspheres. They found that the ratio of flows in the two umbilical veins as determined by flowmeters 16 was similar to the ratio of radioactivities in each part of the placenta drained by each vein. In addition, they used a physical model to prove this principle. The physical model was composed of a rotary pump and a system of four branching tubes, the flow through each of these tubes could be regulated. They also found satisfactory agreement between the measured flow and the distribution of radioactive microspheres. Neutze et al., in 1968 (33), simultaneously collected the blood flow from the right and left femoral arteries during an injection of radioactive microspheres into the left ventricle. The volume and radioactivity of the blood from each artery were measured. They found that the ratio of the flows of the two femoral arteries determined by direct collection was in close agreement with the ratio of the radioactivities in the col- lected blood. These studies indicate that the microspheres are dis- tributed in direct proportion to blood flow. Kaihara et al., in 1968 (24), injected microspheres labeled with two different radionuclides, Sc-46 or Yb-169, successively into the left atrium, left ventricle, or at the origin of the aorta to test the completeness of mixing of the microspheres after injection. The inter- val between the two injections was 5 to 10 minutes. If mixing of micro- spheres was complete, the distribution of the two differently labeled microspheres would be the same. They found that when microspheres were injected into the left atrium, there was no difference in the distribu- tion of the two radionuclides. When microspheres were injected into the left ventricle or at the origin of the aorta, all organs showed the same distribution except the heart. These results suggested that when microspheres were injected into the left ventricle or aorta, mixing was 17 not complete at the origin of the coronary artery. 0n the other hand, when they were injected into the left atrium, mixing was complete before microspheres left the heart. Therefore, they concluded that the micro- spheres should be injected into the left atrium if the distribution of flow in the cardiac tissues is to be studied. If the relative blood flow to areas other than the heart is of interest, injection at the origin of the aorta or into the left ventricle is satisfactory. 2. The effect of microspheres on the circulation: The experiments described above, done by Kaihara et al. in 1968 (24), also demonstrated that injection of microspheres per se has no effect on the circulation. They injected one type of radioactive micro- pheres into the canine left heart soon after another type of microspheres labeled with a different nuclide. The two injections produced the same distribution of cardiac output suggesting that the first injection did not alter the distribution of blood flow in the body. Hoffbrand and Forsyth, in 1969 (21), made duplicate determinations at an interval of 24 hours. They showed that the presence of micro- spheres in the tissues does not disturb the circulation at rest since the monkey had a satisfactorily constant distribution of cardiac output, organ flow and resistance. The more acute effects of microsphere infu- sions were also studied in conscious monkeys. Four separate batches of radioactive microspheres were given over a two hour period. The changes in the distribution of cardiac output as compared to the results obtained from the first batch of microspheres were generally not sig- nificant. There was, however, a small but statistically significant increase in total peripheral resistance with the third batch. 18 3. The fate of radioactive carbonized microspheres fallowing injections: If the radioactivity remains bound to the microspheres, the dis- tribution of the radioactivity in the body will not be altered during the period of observation. To test this, Kaihara et a1. (24) injected radioactive carbonized microspheres, 50 u in diameter, labeled with Sc-46, into the left atrium of dogs. The concentration of radioactivity in various organs was then measured over a tw0dweek period by means of an external detector. In the first 5 days, the radioactivity in the urine and feces was measured. They found there was no change in the organ content of radioactivity over the two~week period. The radio- activity found in the urine and feces during the first 5 days was negligible. They concluded that the carbonized microspheres were not metabolized and stayed in the capillaries almost indefinitely. 4. The extraction efficiency of microspheres by capillary beds: Kaihara et a1. (24) found that no recirculation of microspheres of either 15 u or 50 u in diameter following intravenous injections. All of the microspheres, i.e., 100% of radioactivity, injected were recovered in the lungs and no radioactivity was detected in the liver, kidneys and spleen. When microspheres with 15 u in diameter were in— jected into the internal carotid artery, the common carotid artery, or the abdominal aorta, about 5-10% of microspheres passed through the systemic vascular beds and appeared in the lungs. But no detectable radioactivity was found in the lungs when the microspheres of 50 u in diameter were injected into a systemic artery. Thus, small microspheres 19 (15 D) are not completely removed by some peripheral vascular beds. Since the lungs can completely remove the microspheres of 15 u diameter (24), recirculation will not be a problem if the microspheres are in- jected into the systemic arterial system. Applications of radioactive microsphere method to the study of gastrointestinal blood flow and its distribution Grim and Lindseth, in 1958 (16), used radioactive microsphere method to study the distribution of blood flow in different tissue layers of the small intestine of dogs. They injected 12 u glass microspheres, labeled with Na-24, into the local artery of an isolated 100p of small intestine to estimate capillary blood flow. Venous outflow of the loop was also simultaneously measured by collection of venous outflow. The loop was then removed from the animal and separated into four tissue fractions, i.e., the mucosa, submucosa, muscle, and mesentery. The radio— activity of each tissue sample and venous blood was determined. They found that the capillary flows per unit weight of the mucosa, submucosa, muscle, and mesentery in fasted jejunum and ileum were about the same (40-50 m1/min/100 gm). A possible exception was flow to the ileal muscle which was slightly higher (55 ml/min/lOO gm). By using different sizes of microspheres, they also showed that the arteriovenous anastomotic flow was very low in the intestine (2-4%). Delaney and Grim, in 1964 (12), estimated the canine gastric blood flow distribution by using two different methods, K-42 clearance and microsphere methods. Radioactive microspheres, 16 u or 20 u in diameter, labeled with Na-24, were injected into the celiac artery. The distribu- tion of capillary blood flow among tissue layers of gastric body 20 determined by the radioactive microsphere technique was mucosa 68%, submucosa 11%, and muscle 21%. These data were in close agreement with those determined by K942 method which showed mucosa 72%, submucosa 13%, and muscle 15%. They concluded that both methods provide reasonably accurate measures of blood flow distribution in the stomach. CHAPTER III MATERIALS AND METHODS 1. Preparation of Radioactive Microspheres Carbonized microspheres labeled with either cerium-141 or strontium-85 were obtained from the Nuclear Product Division of the Minnesota Mining and Manufacturing Company (3M Center, St. Paul, Minne- sota). The size of the microspheres was 15 u in diameter with a vari- ation of i_5 u. The specific activity was 10 millicuries/gm. One milligram of the microspheres contained about 440,000 microspheres. The stock microspheres were suspended in a solution of 10% dextran (l millicurie/lO ml). A drop of Tween 80 (polyoxyethylene sorbitan mono-oleate) was added to the stock solution to prevent aggregation of the microspheres. Microsphere suspensions to be used for experiments were prepared from the stock solution a few minutes before use. Four- tenth ml of the stock microspheres was added to 2 m0 of 20% dextran. The suspension was then treated with an ultrasonic sonifier cell dis- ruptor (Branson Instrument 00., Long Island, N.Y.) to achieve uniform dispersion of the microspheres. 2. Preparation of DigestedlDog‘FOOd One can of dog food (Alpo liver or beef, Allen Products Co., Allentown, Pa.) was mixed in an electric blender until its consistency was that of a thick, smooth milk shake. To the blended food was added 21 22 0.75 grams of pancreatin (whole porcine pancreas containing protease, amylase, lipase, esterases, peptidases, nucleases, elastase, ViokaseR, Viobin Co., Monticello, Ill.). The pH was adjusted to about 7.0 by adding sodium bicarbonate. Then, the mixture was stirred for 5 hours with a magnetic stirrer (Thomas stirrer, Model 15, Arthur H. Thomas 00., Philadelphia, Pa.) at room temperature. 3. Surgical Procedures A. Group A Ten mongrel dogs of either sex, fasted for 24 hours, weighing be- tween 13 and 15 kilograms were used. They were anesthetized with sodium pentobarbital (30 mg/kg body weight) and ventilated with a positive pressure respiration pump (Harvard, Model No. 607, Dover, Mass.) via an endotrachael tube. A polyethylene tube, filled with heparinized saline, was inserted into a femoral artery and connected to a Statham pressure transducer (Model No. P 23 Gb) for monitoring the systemic arterial pressure. The abdominal cavity was opened through a midline incision. A loop of the jejunum about 20 cm aboral to the ligament of Treitz was exteriorized and divided into four segments according to the natural vascular pattern. A rubber tube, with an outer diameter of 0.5 cm, was placed into the lumen of each segment for introduction of the substances to be tested. Both ends of each segment were tired and the mesentery was cut to exclude collateral flow. Thus, four separate and naturally perfused in situ segments were formed. The segments were kept moist and warm during the experiment by covering them with a sheet of plastic film and by a heating lamp. 23 B. Group B In another eight dogs, the same surgical procedures were performed as described above except that 3 segments of jejunum were prepared. Following the above procedures, in the group A or B, a special catheter used in angiography (Bardic deseret Angiocath, catheter 16 G x 2%", needle 19 G x 2 3/4") was inserted into the left ventricle of the heart through the chest wall. The needle inside the catheter was then removed. The presence of the catheter in the left ventricle was con- firmed by connecting the catheter to a Statham.pressure transducer (Model No. P 23 Gb) to record the pressure. The catheter was then fixed to the chest wall with a suture to prevent it from moving out of the left ventricle. This catheter was used for injecting radioactive micro- spheres. 4. Experimental Procedures A. Group A One segment of the four prepared jejunal segments was left empty but each of the other three segments received 10 m1 of either isotonic nonabsorbable polyethylene glycol (PEG), digested dog food or 50% glu- cose. PEG was used as the control of food and 50% glucose. The test solutions were placed into the four segments in random sequence. B. Group B The experiment in this group was designed to see if local mucosal anesthesia vdJJ. alter the response of blood flow to luminal placement of 50% glucose. One jejunal segment was pretreated by luminal placement of 10 m1 of 0.4% dibucaine for 20 minutes. Dibucaine was then withdrawn 24 and 10 ml of 50% glucose was introduced into the lumen. At the same time, each of the other two segments received 10 ml of either PEG or 50% glucose. PEG was again used as a control. In both groups, the test solutions remained in the lumen for 20 minutes. Two types of the prepared microspheres (Ce—141 and Sr-85) were injected into the left ventricle at 3 minute intervals through the preinserted catheter in each dog. The purpose of injecting two types of microspheres in the same dog was to see if they would produce similar results. The order of the injections was thus alternated in the experi- ments. The dog was then sacrificed by an intracardiac injection of the saturated potassium chloride solution. The segments were removed and the mesentery was trimmed off from the segments. Each segment was separated into three portions, i.e., the mucosa, submucosa, and muscle plus serosa. This dissection was accomplished easily, the mucosa and muscle were simply scraped off the submucosa with a blunt instrument. Each tissue sample, in duplicate, was placed into the preweighed plastic counting tube. The actual weight of each tissue sample was calculated after reweighing the counting tube. In order to get an accurate count- ing, each tissue sample was placed in the bottom of the counting tube not exceeding 2 cm in height and care was taken to avoid sticking the tissue on the wall of the tube. The amount of radioactivity of each nuclide was measured with a scintillation gamma spectrometer (Parkard Instrument Co., Model 3002 Tri-carb scintillation spectrometer). 5. Measurement of Radioactivity Separation of two isotopes in each tissue sample was performed 25 according to the following steps. The windows of the spectrometer were set so that the window A included the main energy peaks of gamma spectrum of Ce-l4l and the window B included that of Sr-85. The energy peaks were determined by counting a small amount of the stock micro- spheres labeled with Ce-l41 or Sr—85 with the spectrometer. The deter- minations were done for each batch of the microspheres. The channels 1 and 2 of the spectrometer recorded the radioactivities counted in the windows A and B respectively. Since the window A would count not only the radioactivity of Ce-l4l, but also some radioactivity of Sr-85, the latter should be excluded to obtain the real radioactivity of Ce-l4l counted in window A. The same phenomenon would also occur in window B. The real radioactivity of each isotope was calculated as follows: Ce-l4l (cpm) = b (Total cmp - BG. cpm) Ch 1 - (Total cpm - BG. cpm)Ch 2 b - a 9 Sr-85 (cpm) = b [(TotalJcpm - BG. ch)Ch.2 - a (Total cpm - BG. ch)Ch. 1] 10 b - a where: BG. = background, Ch. 1 = channel 1, Ch. 2 = channel 2. a = (Ce cpm)Ch.2/(Ce ch)Ch. 1. b ‘ (Sr Cpm)Ch. 2/(Sr ch)Ch. 1 The cpm of Ce-l41 or Sr—85 in each channel was obtained by counting the blood sampled from a femoral artery immediately following the injection of each type of microspheres. Since there was no recirculation of microspheres in the systemic arterial blood, the sampled blood contained 26 only one type of microspheres. The tissue samples, however, contained both types of isotopes. Therefore, the data obtained from blood samples as calculated as a and b were used to obtain and real radio- activity of Ce-l4l and Sr-85 in the tissues. Formulas 9 and 10 shown above were derived from the following steps: In both channel 1 and channel 2, Total cpm 8 Ce cpm + Sr cpm + BG. cpm (Ce cpm) = (Total cpm) Ch. 1 Ch. 1 ’ (3r ch)Ch. 1 ‘ (35° cpm)Ch. 1 since b = (Sr cpm)Ch 2/(Sr cpm)Ch 1 - (Sr cpm)ch. 2 c m) Ch. 1 b P Ch. 1 _ (Total ch)Ch. 2- (Ce ch)Ch. 2 - Ch. 1 b (Ce cpm) = (Total cpm) - (BG. Ch. 1 = (Total cpm) (BG. cpm)Ch. 2 - (BG. ch)Ch. 1 since a = (Ce cpm) /(Ce cpm) Ch. 2 Ch. 1 - (Total cpm)Ch. 2-a(Ce cpm)Ch. 1 - Ch. 1 b (Ce ch)Ch. 1= (Total cpm) (BG. cpm)Ch.2 - (3G ' Cpm)Ch. 1 (b - a)(Ce cpm) = b (Total cpm - BG. cpm) - (Total cpm - Ch. 1 Ch. 1 BG. cpm)Ch 2 by moving (b - a) to the right side of the equation, the equation becomes formula 9. (Sr cpm) = (Total cpm) - (Ce cpm) - (BG. cpm) Ch. 2 Ch. 2 Ch. 2 Ch. 2 since a = (Ce cpm) /(Ce cpm) Ch. 2 Ch. 1 27 (Sr cpm) = (Total cpm)Ch. 2 - a (Ce cpm)Ch. 1 - (BG. = (Total Cpm)Ch. 2 - a [(Total ch)Ch. 1 - (Sr Cpm)Ch. 1 - since b = (Sr cpm)Ch. 2/(Sr ch)Ch. 1 (Sr cpm)Ch. 2 = (Total ch)Ch. 2 - a [(Total Cpm)Ch. 1 ' (St cpm)ChI 2 - (BG c m) ] - (BG m) b ' P Ch. 1 ' °P Ch. 2 (b - a) (SI cpm)Ch. 2 = b [(Total cpm - BG. ch)Ch. 2 - a (Total cpm - BG. ch)Ch. 1] by moving (b - a) to the right side of the equation, the equation becomes formula 10. 6. Calculation and.Expression of.the Results A. Total radioactivity in the whole wall of the jejunum Since total radioactivity in the whole wall of the jejunum was technically difficult to measure, the total radioactivity in the whole wall was calculated from the radioactivity of each tissue layer (cpm/gm) and the weight distribution among the three tissue layers (as % of the whole wall). The weight distribution in the jejunal wall was obtained from randomly selected 12 dogs (see below). The formula for calculation of the total radioactivity in the whole wall was: Total radioactivity in the whole wall (cpm/gm) = (cpm/gm.mucosa x mucosa weight % + cpm/gm submucosa x submucosa weight % + cpm/gm muscle-serosa x muscle-serosa weight %) x 10-2 ll 28 B. Flgw distribution among three tissue layers of thejejunum Since the microspheres were well mixed with the inflowing blood and distributed in the same manner as blood and entrapped in the capil- laries of the perfused tissues, the distribution of the radioactivity in the tissues is a measure of the distribution of blood flow. The flow distributed to each layer in % of the total blood flow of the jejunum was determined from the following formula: Radioactivity in one compartment (% of total radioactivity) - Blood flow to the compartment (% of total blood flow) = radioactivity of the compartment (cmegp) x weightgpercentagg radioactivity of the whole wall (cpm/gm) of the compartment 1%) 12 7. Weight Distribution of the Canine Jejunal Wall Twelve mongrel dogs of either sex, weighing between 13 to 15 kilo- grams were anesthetized with sodium pentobarbital (30 mg/kg body weight). The abdominal cavity was opened through a midline incision and the jejunum was exposed. One segment of the jejunum was randomly selected and removed. After the mesentery was trimmed off, the jejunal wall was separated into the three portions, i.e., the mucosa, submucosa, and muscle plus serosa in the same way as described above. The dissected three layers were checked microscopically to make sure that the sep- aration was complete. All of the tissue samples obtained from each layer were weighed. The weight distribution in the jejunal wall in each dog was calculated by dividing the weight of each tissue layer of a segment by the total weight of the segment. 29 8. Statistical Analysis of the Results Student's t test for paired comparison, correlation coefficient, and regression analysis were employed in statistical analysis of the results (49). CHAPTER IV RESULTS In all experiments, the systemic arterial pressure remained con- stant throughout the experiments. The average systemic arterial pressure was 120 mm Hg. The systemic arterial pressure was not altered following injections of radioactive microspheres or following placement of test solutions into the lumens of the jejunal segments. Since good separation of the three tissue layers of the jejunal wall is essential for reliable results, many preliminary studies were performed to refine the technique of the separation. During these studies the dissected three layers were examined microscopically. As can be seen in Figures 1, 2, and 3, separation of the three tissue ' layers was perfected by these studies. 1. Mean Weight Distribution in the Jejunal Wall Table 1 shows the mean weight distribution in the jejunal wall determined in 12 randomly selected mongrel dogs. The standard errors for the means were all small. This indicates that the relative weights in the jejunal wall varies little from animal to animal. In this study, these means were used to calculate the radioactivity, in cpm per gram tissue weight, of the whole wall as shown in the following formula (see Formula 11 on page 27). 3O Figure 1. Figure 2. 31 Photomicrograph of the mucosa of the canine jejunum after separating it from the submucosa and muscle-serosa. Photomicrograph of the submucosa of the canine jejunum after separating it from the mucosa and muscle-serosa. h ,1, 32 Figure l Figure 2 }: Figure 3. 33 Photomicrograph of the muscle-serosa of the canine jejunum.after separating it from the mucosa and submucosa. _———d'_—‘-— 34 Figure 3 35 Table l.--Weight distribution in the jejunal wall (%). (mean -_|-_ S.E.) (N - 12) Tissue layer Weight, % Mucosa 63.1 i 0.9 Submucosa 11.9 i 0.5 Muscle-Serosa 25.0 i 0.7 36 Total radioactivity in the whole wall (cpm/gm) = cpm/gm mucosa x 0.631 + cpm/gm submucosa x 0.119 13 + cpm/gm muscle-serosa x 0.250 These means were also used to calculate the percentage distribution of the radioactivity in the wall and thus the percentage distribution of the total blood flow among the three tissue layers of the jejunal wall as shown in the following formulas (see Formula 12 on page 28). Radioactivity in mucosa (% of total radioactivity) = Blood flow to mucosa (% of total blood flow) cpmigm mucosa x 0.631 cpm/gm whole wall x 100% 14 Radioactivity in submucosa (% of total radioactivity) - Blood flow to submucosa (% of total blood flow) a cpmlgmpsubmucosa x 0.119 cpm/gm whole wall x 100% 15 Radioactivity in muscle-serosa (% of total radioactivity) a Blood flow to muscle-serosa (% of total blood flow) _ cmegm muscle—serosa x 0.250 cpm/gm whole wall 3 10074 . 16 2. Effects of.Luminal.Placement of PEG, Food, and 50% Glucose A. Results obtained from the microspheres labeled with Ce-141: The average radioactivities of Ce-l4l of the whole wall, the mucosa, submucosa, and muscle-serosa of the empty segments and the seg- ments containing-PEG, food,-and 50% glucose are shown in Table 2. As can be seen in Table 2, the radioactivities of the whole wall, the mucosa, submucosa, and muscle-serosa of the segments containing PEG were 37 .Ho.ouvm um 0mm mo moam> wnanommmHuoo as» scum .mo.ouvm on own mo oodm> onvnommouuoo ogu aoum unmuommHv hHHmoHumHHMum mH opawb man uwau mouooon «a unouomev eaaonumHumum mH osam> onu umnu monocon « R: H is :3 H some :2 H omam :3 H ~13 383.30%: as. H 82 is: H are 2m H 8: on H 33 $82.58 was H 3% .332 H «$3 «.32 H 23 a: H 82 38:2 8m H .23 3.32 H on: «as H 38 mm... H 23 2...: 325 Beam 38:8 Now soon 0mm 8H .. 5 Add H :35 .omoooaw Non no .voom .umm onnHmuooo no house ouoB Snga musoawom as» no mwouomloaomaa use .mmooasnsm .mmooaa oau .HHmB oaons osu mo Heauoo mo AoammHu aw\aoov huH>Huom0vau owmuo>w onHII.N manna 38 not significantly different from those of the empty segments. These results thus indicate that luminal placement of 10 ml of PEG for 20 minutes did not significantly alter the total blood flow and compart- mental blood flow of the jejunum. The segments containing food, on the other hand, had significantly higher radioactivities in the whole wall and the mucosa as compared to the segments containing PEG. The radio- activity in the submucosa or muscle-serosa of the segments containing food however was not significantly different from that of the segments containing PEG.. The segments containing 50% glucose had significantly higher radioactivities in the whole wall, the mucosa, and submucosa as compared to the segments containing PEG. The radioactivity in the muscle—serosa of the segments containing 50% glucose was not signifi- cantly different from that of the segments containing PEG. Table 3 shows the percentage distribution of the blood flow among the three tissue layers of the jejunal wall following luminal placement of various solutions. The percentage distribution in the jejunal seg- ments containing PEG was similar to that in the empty segments. In the segments containing food, the mucosa had, on the average, a greater share of the total blood flow than that in the segments containing PEG. Although the difference between. these two segments was not statistical- ly significant at p values less than 0.05, it was statistically signifi- cant at p values less than 0.1. In the segments containing 50% glucose, the mucosa had significantly greater and the submucosa or muscle-serosa had significantly smaller shares of the total blood flow than those in the segments containing PEG. 39 28.0 ve um 0mm mo moan; wafiunoemonnoo men aonu noonommfiu kHHmoHumHuwum 3 and; 05 non» mononon . .. «e .35 vm um 33 mo moan; wdwvnommonnoo 93 Sony uaonommdo kHHmoHanuwnm mH and; on”. n93 won—once 1.. .H.o vm no 9: mo and; waHvaommonnoo on”. aonm unonommflo haaonumHuonm mH endear on”. use» mouoaonw T... 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Effects of Mucosal Anesthesia on Blggd Flow Responses to 50% Glucose A. Results obtained from the microspheres labeled with Ce-l41: As can be seen in Table 6, the segments which were treated with luminal placement of 0.4% dibucaine prior to placement of 50% glucose still had significantly higher radioactivities in the whole wall and the mucosa as compared to the segments containing PEG. But the segments had significantly lower radioactivities in the whole wall and the mucosa as compared to the segments containing 50% glucose without prior exposure to dibucaine. The radioactivities in the submucosa and muscle-serosa were not significantly different from those of the segments containing PEG. B. Results obtained from the microspheres labeled with Sr-85: The results (Table 7) were similar to those obtained from the microspheres labeled with Ce-l4l. The segments containing 50% glucose with prior exposure to 0.4% dibucaine had significantly higher radio- activities in the whole wall and the mucosa than the segments containing PEG. But the segments had significantly lower radioactivities in the whole wall and the mucosa than the segments containing 50% glucose with- out prior exposure to 0.4% dibucaine. The radioactivities in the sub- mucosa and muscle-serosa were not significantly different from those of the segments containing PEG. 44 28.0 vAH no omoosaw N0m mo 05Hm> wnHvaommonnoo onn Bonn noonCMMHv mHHmoHannmum mH o=Hm> men omen monononfie .H0.0uvm um 0mm mo o=Hm> wannommonnoo can Bonn uaonCMMHv haamoHumHuMnm mH oaam> any wean mononon as .omoosaw Non mo ucmamomam onowon nHa 0N now oGHmoanv No.0 mo HE 0H on vomomxm onus mucoawom omonu mo amend osaa as + $3 33 + .33 as + Se. ooooomuonoooz own H ..moN oNo H 38 on H on: $838... a. 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I I I «.52 + 33 .353 + $3 2: + .82 2a.. 295 ooooono nomuoonoooono ooooono now one H 8 u a To...“ H oooao .owoonaw NomloonoanHv no .omooaaw Now .omm waHnHMnaoo munoamow man mo moonomIoHomda was .mmooaansm .mmoosa man .Hamz maonB man no mwlnm mo AmammHn aw\amov muH>Hnomovan owmno>m osaII.n manna 46 4. Comparison of the Results Obtained from the Microspheres Labeled with Ce-l4l and Those Labeled with Sr-85 To test if the results obtained from the microspheres labeled with Ce—l4l and those obtained from the microspheres labeled with Sr-85 were similar, the results from these two types of microspheres were statis- tically compared. Both correlation and paired comparison methods were used for this purpose. All data from group A, i.e., four segments of 10 dogs, were included in the comparison. In Figure 4, the mucosal blood flows, as percent of total jejunal blood flow, obtained from Sr-85 were plotted against those obtained from Ce-l4l. There was a highly significant (P