ACUTE PENTOBARBITAL ‘DEPRESSION {N DOGS, WITH A CONSIDERATION OF THE ANTAGONISTK ACTION OF AMPHETAMINE Timsis for i116 Degree of M. S. MECHEGAN STATE COLLEGE Lee Chang-«Churn ”i950 This is to certify that the thesis entitled Acute Pentobarbital Depression in Dogs, with a Consideration of the Antagonistic Action of Amphetamine presented by Cheng Chun Lee has been accepted towards fulfillment of the requirements for _M.:§_n_degree in Mogy 8c Pharmacology pa.e Ma122 1950 ACUTE PENTOBARBITAL DEPRESSION IN DOGS, WITH A CONSIDERATION OF THE ANTAGONISTIC ACTION OF AMPHETAMINE By lee Cheng-Chun A THESIS Submitted to the School of Graduate Studies of Michigan scene College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology and Pharmacology 1950 x Imus ACKNOhLEDGEMENTS ‘ The author wishes to express his sincere appreciation to Dr. B. V. Alfredson, Head of the Department of Physiology and Pharmacology, for his encouragement and helpful guidance throughout the course of this work, for his assistance in the preparation of this manuscript and for his aid in providing facilities in conducting this work; to Dr. L. B. Sholl, Associate Professor in the Department of Animal Pathology, for his interest and for providing facilities in the blood studies; and to Drs. L. F. Wolterink, E. P. Reineke and J. Meites, Professors in the Department of Physiology and Pharmacology, for their interest and valuable suggestions. Thanks are due also to N . J. Monroe, Mr. J. 0. Reed and Mr. W. S. Gunn, for their assistance in the preparation of the experimental animals. ”'52 1,3(17) CONTENTS Pa INTRODUCTIONOOOOOOOOOOO00.0000000.0.0.000.000000000000000. REVIEI’J OF LITERATUREOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. The Minimal Lethal Dose of Pentobarbital Sodium......... Factors Affectxthe Toxicity of Barbiturates............. Pharmacology of Barbiturates with Particular Reference to the Respiration and Blood Pressure................. Pharmacology of Amphetamine with Particular Reference to the Reapiration and Blood Pressure................. 9 Antagonism to Barbiturates.............................. 11 Effects of Barbiturates and Amphetamine on Blood Pieture'00......0.0.0...0..OOOOOOOOOOOOOOOOOO0.0.0.... 11" 'Q kWVhJ P4N MATERIALS AND METHODSoooooocoo.oeeoeo0.0000000000000000... 17 Experimental Animals and Drugs.......................... 17 Experimental Procedurea................................. 17 Procedure. for Blood Studies............................ 19 Analysis of Data........................................ 20 EXPERDIENTAL RESULTSOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0... 21 The Effect of Pentobarbital Sodium on Respiration and BlOOd PressureOOOOOOOOOOOO000......OOOOOOOOOOOOOOOO... 21 The Effects of Amphetamine Sulfate on Respiration and Blood Pressure........................................ 22 Effects on Respiratory Rate and Ventilation......... 22 Effects on Blood Pressure........................... 2h Response to Sciatic Stimulation..................... 25 The Effects of Pentobarbital Sodium and Amphetamine Sulfate on Blood Picture..................3........... 25 Red and White Cell Counts and Hemoglobin Concentra- tionOOOOOOOOOOOOOO00.0.0.0...OOOOOOOOOOOOOOOOOOOOOO0.... 25 Differential White Cell Count....................... 27 DISCUSSIONOOOOOOO00.0.0000...O0...OOOOOOOOOOO0.0.0.0000... 1*? DosageoeoooeoeooeoooooocoocooQ0oOOOOOOOOOOOOOOOOOOOOOOOO 47 Effects of Pentobarbital Sodium and Amphetamine Sulfate on Respiratory Rate and Ventilation................... 48 Effects of Pentobarbital Sodium and Amphetamine Sulfate on Blood Pressure..................................... A9 Effects of Pentobarbital Sodium and Amphetamine Sulfate on Blood Picture...................................... 51 Total Red Cell Count and Hemoglobin Concentration... 51 Total White cell CountOOOOO0.00000000000000000000000 52 Differential White Cell Count....................... 53 SUMMARYOOOOOOOOOOO0000000coooo00ooOOOOOOOOOOOOOOOOOOOOOOOO 55 BIBLIOGRAPHYOOOOOOOOcocoa...o0000coco0.0000000009000000... 57 APPENDIXoooooooooooooso.oooooooooooooooooooooooooooooooeoo 6# Page 1 INTRODUCTION The widespread use of many barbituric acid derivatives for their hypnotic and anesthetic effects has focused in- creasing attention upon drugs and other measures which may counteract the effects of overdosage with these agents. This problem has been particularly acute in the field of veterinary medicine where certain barbiturates, notably pen- tobarbital, are so commonly employed as surgical anesthetics, especially in canine and feline subjects. ' The primary purpose of the present study is an attempt to determine the optimal dose of one of these, phenyliso- propylamine (amphetamine), required to antagonize the res- piratory depressant effect accompanying severe pentobarbital depression in dogs. 'The eXperimental conditions were designed to yield more data as to degree of depression than could be elucidated by simple clinical trials. No reports have been found in the literature dealing with this specific approach. Certain other collateral findings concerning effects on blood pressure and cellular constituents are also presented. Page 2 REVIEW OF LITERATURE The Minimal Lethal Dose of Pentobarbital Sodium: The median lethal dose, LD 50, which according to Traven (1927) was the dosage that killed 50 per cent of a large group of animals, has been adopted generally by investiga- tors to determine the toxicity of drugs. Swanson and Shonle (1931) found that the LD 50 of pento- barbital sodium injected intraperitoneally for albino rats was 120 mg. per kilogram of body weight, 3 out of 5 animals being killed in this instance. Swanson (1932) found that the LD 50 was 10 mg. higher if the pentobarbital sodium was in- jected subcutaneously. In this case, 9 out of 15 rats were killed. Fitch and Tatum (1932) injected rats intraperiton- eally with twelve barbituric acid derivatives. They found that 75 mg. of pentobarbital sodium per kilogram killed 50 per cent of their rats. Based upon experiments with adult male and non-pregnant female albino rats, Barlow, Duncan and Gledhill (1931) reported the minimal lethal dose as 120 mg. per kilogram when pentobarbital sodium was injected sub- cutaneously. This is 10 mg. lower than Swanson (1932) found by the same method of administration. In a report on the Inedian lethal dose of pentobarbital sodium (LD 50), Carmichael (1938) found that the M.L.D. for young rats, 25 to 260 gm. in 'weight and a few weeks to 9 months old, varied from 110 to 120 mg. per kilogram; while for old rats, 150 to 315 gm. and at least 12 months old, the M.L.D. varied from 85 to 95 mg. Page 3 per kilogram, by intraperitoneal injections of fresh aqueous solution. Carmichael and Johnson (1948) injected intraperitoneally young rats less than 2A hours old with pentobarbital sodium and placed them in an incubator immediately to avoid chilling. They found the LD 50 for the nursed rats to be 40 mg. per kilogram while the LD 50 for those that did not nurse was about 30 mg. per kilogram. Carmichael and Posey (1936, 1937) used the median lethal dose, (LD 50),for determining the toxicity of pento- barbital sodium for guinea pigs. They found that the median lethal doses were 57.5 to 60 mg., 52.5 to 55 mg. and #5 to h7.5 mg. per kilogram body weight for the groups of guinea pigs weighing 200 to 399 gm., #00 to 599 gm. and 600 to 799 gm.,respectively.. The pentobarbital sodium was injected intraperitoneally. O Swanson (1932) found the LD 50 of pentobarbital sodium for cats to be 75 mg. per kilogram by intraperitoneal injec- tion. He reported death in h out of 6 of his cats. Fitch and Tatum (1932) administered several barbituric acid derivatives intraperitoneally and orally to rabbits. The median lethal dose of pentobarbital sodium was found to be 65 mg. and 175 mg. per kilogram body weight respectively. The ratio of intraperitoneal to oral toxicity was 1:2.72. Swanson and Shonle (1931) determined the minimal lethal dose of pentobarbital sodium for dogs injected intravenously to be 50 mg. per kilogram, and given orally to be 85 mg. per Page A kilogram. In both of these instances, the M.L.D. was given as one that killed more than 50 per cent of animals; 3 out of 5 animals being killed in the first instance and 5 out of 5 being killed in the second instance. The ratio of intra- venous to oral M.L.D. was 1:1.7. With barbiturates taken with suicidal intent in humans, Kempf (l9h6) indicated that adult patients ingesting less than 50 grains (3.25 gm.) of pentobarbital sodium did not die when simply allowed to sleep in a position that did not inter- fere with their respiration. Factors Affect the Toxicity of Barbiturates: Nicholas and Barrow (1932) found a marked sex difference in the response of rats to isoamylethyl barbituric acid (amytal). The anesthetic dose for adult females was about one-half that for the adult males when given subcutaneously. Maturing male rats fell between adult males and females, and immature males and females gave satisfactory deep anesthesia with adult female dosage. Holck and Kanan (1935) used a ‘ variety of barbiturates to study the sex difference in white rats. They also found that the female white rat was much more sensitive than was the male to isoamylethyl barbituric acid (amytal), pentobarbital sodium, N-methylcyclohexenyl methyl barbiturate (evipan or evipal),’b§tyl-B-broma11yl barbituric acid (pernocton or pernoston), and, not quite as marked, to n-hexylethyl barbiturate (ortal). But no sex difference to N-methylcyclohexenyl methyl barbiturate (evipan) was detected by them in the dog, cat, rabbit, guinea pig, Page 5 white mouse, turtle or frog. This agreed with results re- ported by Fitch and Tatum (1932) upon rabbit sensitivity to barbiturates in general, with Kennedy (1934) in regard to white mice, and with their own study (l93h) upon isoamylethyl barbituric acid (amytal) in the dog and rabbit. Barron (1933) could demonstrate no sex difference with pentobaribtal sodium in rats. This is contrary to what Holch and Kanan (1935) found with the same preparation. Using isoamylethyl barbituric acid (amytal) and employing deep anes- thesia as a criterion, Barron (1933) found the sex difference to appear in rats weighing between 50 and 60 grams. In the immature animals, he found no significant difference in the response between the males and females. He suggested that this sex difference in response to isoamylethyl barbituric acid (amytal) may be due either to a testicular influence, or to the influence of the hypophysis and the suprarenal glands. Castration of the mature males (Barron, 1933) largely removed this sex difference. Castration of the males before maturity, however, did not interfere with the deve10pment of the normal male resistance to isoamylethyl barbituric acid (amytal). He has stated (1933) that, "It is interesting to note that the weight at which the sex difference appears -- between 50 and 60 grams -- is also the weight at which the differential growth relation of both the hypOphysis and the suprarenals first appear between the males and females (Donaldson, 192A). The female rat has the heavier hypophysis and suprarenal glands." Page 6 Moir (1937) affirmed that mature female albino rats were definitely less resistant to pentobarbital sodium than cor- responding males. He also found that very young females were more resistant to pentobarbital sodium than corresponding males, and the slightly older females approximated males in their resistance. As castration did not remove the resistance of the males to pentobarbital sodium entirely, he believed (1937) that the superior resistance of the males might be due to some factor or factors other than the male gonads. Both Holck 92 El. (1942) and Gaylord and his coworker (l9hh) showed that the spayed female rats possessed more resistance to pen- tobarbital sodium than did the normal female rats. The latter group (l94h) attributed the shorter duration of sleep of the spayed rats to their extra subcutaneous fat. I Cameron (1938) found that warmth tended to decrease the duration of sleep following pentobarbital sodium. It was confirmed by Gaylord and his coworker (19AA) that the dura- tion of sleep caused by subcutaneous injection of 30 mg. of pentobarbital sodium per kilogram of body weight decreased with the increase of environmental temperature, and proved by Carmichael and Johnson (1948) in one day old young rats. The influence of age upon the toxicity of barbiturates has been investigated by different groups (Carmichael and Posey, 1936 and 1937; Carmichael, 1938; Moir, 1937). They found (Carmichael and Posey, 1936 and 1937; Carmichael, 1938) that the older the animal, the greater the tolerance to pen- tobarbital sodium. The LD 50lfor one-day-old rats was less than one-third to one-fourth of that for the adult rats Page 7 (Carmichael and Johnson, 19h8). Holck and Kanan (l93h) have determined that in rabbits there was no correlation between body weight and the magni- tude of the fatal dose of isoamylethyl barbituric acid (amytal). They have reported that, by eliminating the ex- tremes in the weight, the LD 50 of individual dogs was not significantly different from that of the whole series. Rats were much more resistant to dilute solutions of isoamylethyl barbituric acid (amytal) (Nicholas and Barron, 1932; Barron, 1933). Intravenous saline injections revived rats fatally poisoned with concentrated solutions of, isoamylethyl barbituric acid (amytal) (Barron, 1933). Swanson and Shonle (1931a) foundthat 10 and 20 per cent of isoamyl- [ethyl barbituric acid (amytal) gave about similar results in dogs when the drug was administered rectally. Animals have shown a marked difference in the magnitude of lethal dosage with different methods ofdggginistration (Fitch and Tatum, 1932; Swanson and Shonle, 1931). These investigators all found that oral administration caused the least toxic effects. In the case of intravenous injection, the toxicity was directly proportional to the speed of the injection (Swanson and Shonle, 1931 and 1931a; Hirshfeld, Hyman and wanger, 1931). Pharmacology of Barbiturates with Particular Reference to the Respiration and Blood Pressure: With oral administration, the hypnotic doses of barbi- turates caused slight slowing of the respiration (Sollmann, Page 8 19h8). Page and Coryllos (1926) found that large doses of isoamylethyl barbituric acid (amytal) depressed the res- piratory center directly, reducing the depth and rate, often with irregularities. Marshall and Posenfeld (1936) stated that barbiturates appeared to depress the central respira- tory mechanisms more than the sino-aortic mechanism. Lehman (1939), analyzing the depressant effects of barbiturates on respiration, supported the view of the previous authors that the barbiturates possessed high central paralytic effects. Even hypnotic doses might paralyze the respiratory center, if they were too rapidly injected intravenously (Sollmann, 19h8). With intravenous injection of the short-acting bar- biturates in cats, Burstein and Rovenstine (1938) demonstra- ted that apparent hyperactivity of laryngeal reflexes was produced, particularly involving adduction of the vocal chords. This was abolished by atropine, and they attributed the stimulation of the adduction reflex to the barbiturates. Following the administration of barbiturates to labora- tory animals under ether anesthesia, a fall in blood pressure has been reported by Gruber and Baskett (1925), and Gruber and Roberts (1926). Page and Coryllos (l926)stated that the intravenous injection of isoamylethyl barbituric acid (amytal) produced a rapid fall in blood pressure in dogs, and that it returned to an approximately normal pressure of 110 to 120 mm. Hg within a minute, 2. ., when anesthesia was induced. Bleckwenn (1930) found that the acute injection of barbiturates Page 9 generally caused a drop in blood pressure in both laboratory animals and in man. This difficulty was largely overcome by a very slow administration. Swanson and Shonle (1931a) con- firmed Bleckwenn's observation that the reduction in the rate of injection could prevent the fall in blood pressure to a very considerable extent. Sollmann (l9h8) stated that with rapid intravenous injection there was a prompt fall of blood pressure, generally with prompt recovery to the normal level. He considered this to be due to the temporary precipitation of the sparingly soluble barbituric acids on contact with the blood. It might therefore be prevented by slow injection of dilute and alkalinized solutions. Gruber and Baskett (1925) have called attention to the fact that toxic doses of sodium phenobarbital caused par- alysis of respiration. They further observed that the heart continued to beat for some seconds, even minutes, after cessa- tion of the respiration. Pharmacology of Amphetamine with Particular Reference to Respiration and Blood Pressure: B-Phenylisopropylamine was first synthesized by Alles (1928), while searching for a substitute for ephedrine, which would be more economical and easier to prepare. Benzedrine is a common trade name for this chemical compound which was given the nonproprietary name "Amphetamine" by the council on Pharmacy and Chemistry of the American Medical Association (Bett, 19w). Detrick _e_t_. 3;. (1937) have discussed its pharmacology. It was partly sympathomimetic, partly Page 10 amusculotropic. Bett (1946) has published a detailed review of the literature on its clinical applications. Analyzing the effects of dl-B-phenylisopropylamine' upon respiration in dogs, Alles (1933) found that there was a short period of apnea or diminished respiratory rate fol- lowed by an increase in rate but not by any increased ampli- tude. Alles and Prinzmetal (1933) showed that six amines, including amphetamine, intravenously injected into pithed dogs or cats, dilated bronchi that exhibited natural or in- duced tone byrilocarpine injection. They also reported some bronchoconstriction in intact guinea pigs, and little, if any, effect in perfused guinea pig lungs. Pedden 35,31. (1935), and Cameron and Tainter (1936) have reported that ampheta- mine was a poor and undependable bronchodilator, but the higher doses (up to 10 mg. per kilogram) were effective (Cameron and Tainter, 1936). By direct microscopic tech- nique on fresh sections of excised lung, Sollmann and Gilbert (1937) found that amphetamine contracted the bronchiolar muscle. Following injections of amphetamine (0.25 to 4 mg. per kilogram) into pentobarbital-anesthetized dogs and cats, and urethaneeanesthetized rabbits, Detrick gg’gl. (1937) found that there was a marked increase in rate and depth of respirations. _He showed that the action was central, but Sollmann (1948) stated that, under acute effects, ampheta- mine caused a moderate increase in respiration from the caro- tid sinus reflex. Different groups (Alles, 1933; Alles and Prinzmetal, Page 11 1933) showed amphetamine to possess pressor actions in dogs and cats. Pedden gt 31. (1935), and Cameron and Tainter (1936) confirmed the pressor effects of amphetamine in dogs. It has been shown in man by Prinzmetal and Bloomberg (1935), and Myerson and Ritvo (1936) that there was an increase in blood pressure of moderate intensity and duration following oral or parenteral administration of amphetamine. Detrick 23,31. (1937) found that the comparative potency of ampheta- mine with respect to epinephrine varied from l/lOO to 1/500. The variability in intensity of the responses was great; as small a dose as 0.25 mg. per kilogram caused a 50 mm. rise in blood pressure in one animal, and 4 mg. in another animal caused but 20 mm. increase in blood pressure. Cats seemed more sensitive than rabbits to the pressor action of ampheta- mine. Sollmann (1948) stated that amphetamine, as observed in dogs, caused a prolonged rise of blood pressure, marked slowing of the heart (chiefly reflex) and increase in cardiac amptitude. He attributed this pressor effect to vasocon- striction. Antagonism to Barbiturates: The physiological antagonism between barbiturates and certain convulsant drugs and more recently dextrose has been the subject of considerable investigation. In the earlier experimental work, several groups called attention to the superior efficacy of picrotoxin as an anti- dote to barbiturate poisoning (Maloney 32.31., 1931; Maloney and.Tatum, 1932; Maloney, 1933). This has been confirmed Page 12 experimentally by many workers (Barlow, 1935; Maloney, 1936a; Koppanyi 32.31” 1936; Marshall 33 31., 1937; Hjort, 1938; Werner and Tatum, 1939). Clinically, it has also been re- ported to be of value in the treatment of barbiturate poison- ing (Murphy 33 31., 1937; Kline 33 31., 1937; Kohn 33 31., 1938; Rovenstine, 1938; Reifenstein, 1940; Anderson, 1941). Metrazol was proved to be also effective as an antidote to barbiturate poisoning (Barlow, 1935; Marshall 33 31., 1937; Hjort 33 31., 1938; Werner and Tatum, 1939). Zipf 33 31. (1937) showed that metrazol was thexnost efficient antagonist to the actions of several barbiturate derivatives while coramine and other analeptics were considerably less effective. Picrotoxin was not included in their list of stimulants. Alfredson (1941) reported that metrazol pro- duced a definite respiratory stimulant effect in dogs with profound pentobarbital sodium anesthesia. Coramine was also recommended experimentally as an antidote (Maloney 23,31., 1931; Maloney and Tatum, 1932; Barlow, 1935; Maloney, 1936; Werner and Tatum, 1939). Other analeptics introduced as antidotes to barbiturate poisoning were ephedrine, strychnine, caffeine, cocaine, brucine, physostigmine, and also calcium gluconate and insulin Maloney, 1933; Barlow, 1935; Hjort 33 31., 1938; Werner and Tatum, 1939). ' It was found (Quastel and Wheatley, 1932) that barbitu- rates depressed the respiration of brain tissue 13,11353, by inhibiting the oxidation of glucose, lactate and pyruvate. Page 13 The oxidation of succinate was not affected. Soskin and Taubenhaus (1943) recommended that sodium succinate could be used as a safe and effective antidote against toxic doses of pentobarbital sodium in rats. It shortened the effective duration of anesthetic doses of both pentobarbital sodium and isoamylethyl barbituric acid (amytal). Glucose, sodium lactate, and sodium malate did not produce effects comparable to succinate. DeBoer (1946) confirmed that both sodium succinate and sucrose lessened the sleeping time in dogs with pentobarbital anesthesia in doses of 30 mg. per kilogram body weight. The length of pentobarbital sodium anesthesia was correlated in general with the degree of diuresis pro- duced by different drugs used and the method of injection. Marshall 33 31. (1937) have contended that the antidotal action of picrotoxin against barbiturate depression of res- piration was due to its decreasing narcosis rather than to specifically stimulating the respiratory center. Krantz 33 31. (1937) considered that its action against barbiturate poison- ing appeared to be due to some special mechanism termed "awakening effect". , Reifenstein and Davidoff (1938) first demonstrated that the depth and duration of'narcosis with soluble isoamylethyl barbituric acid (amytal) in man were effected by amphetamine sulfate, since in every case, narcosis was completely coun- teracted by the injection of 20 mg. to 30 mg. of amphetamine sulfate intravenously, and in five instances by only 10 mg. Myerson (1940) suggested that amphetamine sulfate could be Page 14 used as an antidote in counteracting poisoning by the barbi- turates in humans. Freireich and Landsberg (1946) in Meadowbrook Hospital, Nassau County, New York, used ampheta- mine sulfate in humans with coma from an overdosage of bar- biturates taken with suicidal intent. Among the fourteen patients treated with amphetamine sulfate intravenously, thirteen patients recovered without any ill effect except for some headache. They claimed that the only patient who died had not received a sufficient quantity of amphetamine sulfate because no more of the drug was available. The central nervous system stimulant effects of ampheta- mine were first reported by Alles (1933) in its awakening effect on barbiturate anesthetized animals and in its in- somnia-producing effects in man. Therapeutic use of the cen- tral effects of amphetamine was first made by Prinzmetal and Bloomberg (1935) in the treatment of narcolepsy. They found it was approximately three times as effective as ephedrine. Bailey (1943), utilizing amphetamine sulfate to lower the electrical convulsion threshold in the therapy of mental disorder, found that it exerted its effect centrally rather than by the accompanying peripheral changes, such as the increased blood pressure. Effects of Barbiturates and Amphetamine on the Blood Picture: Such barbiturates as isoamylethyl barbituric acid (amytal), pentobarbital sodium, N-methylcyclohexenyl methyl barbiturate and thiobarbiturate (pentothal sodium) had been studied by many workers as to their effect on blood constituents (Cook and Rose, 1930; Adolph and Gerbasi, 1933; Essex 33 31., 1936; Page 15 Hausner 33,31., 1938; Haury 33 31., 1939; Hahn, 1943; Carr and Essex, 1944). These reports indicate that anesthesia under the above drugs caused a hemodilution resulting in lowered red blood cell count and hemoglobin concentration. They were also in agreement that the spleen was the principal factor in reducing the red cell count in the peripheral blood and that it became engorged under the influence of these drugs. This action was believed to be due to the trap- ping of red blood cells in the sinusoids, as splenectomy . prevented these changes in blood cell count (Essex 33 31., 1936), and in hemoglobin concentration (Carr and Essex, 19hh). Tatum (1939), in his review, stated that, in general, leucocytes were apt to increase in number under barbiturates. Total leucocyte count was found to remain fairly constant in man after daily oral administration of amphetamine sulfate (10 mg.) for 6 to 12 days (Davis and Harris, 1942). Wakim (1946) found in dogs that anesthesia with pentobarbital so- dium produced a reduction in white blood cell count averaging 2.3 thousands (0.9 to 3.9). After splenectomy it caused a‘ reduction of the white cell count averaging 3.8 thousands (0.5 to 9.4). Their blood samples were drawn about a half hour after anesthesia. Amphetamine had been reported to cause a rise of the red blood cell count in rats (Enrich and Krumbhaar, 1937), in the dog (Pinkston andfinkston, 1939; Davis, 1941), and in man (Davis and Harris, 1942). The polycythemia resulting from daily oral administration of amphetamine for a period of one Page 16 to two weeks was explained by assuming that amphetamine re- duced the blood flow to the bone marrow, thus diminishing its oxygen supply, and thereby stimulating erythropoiesis (Davis, 1941; Davis and Harris, 1942). By acute administration, however, it was attributed to the fact that contraction of the spleen induced by amphetamine caused the liberation of the red cells from the spleen pulp into the blood stream (Pinkston and Pinkston, 1939; Palitz, 1939; Cioglia and Frada, 1940). In a study using daily doses varying from 1 mg. to 80 mg. of amphetamine sulfate on Albino rats, Ehrich and Krumbhaar (1937) found that the individual 1eucocyte_counts were not as uniform as the erythrocyte counts, and there were no distinct differences in the animals treated with different doses. In a study concerning the toxicity of amphetamine sulfate, Ehrich and Krumbhaar (1939) found that all dogs receiving 2 mg. per kilogram or more of amphetamine per day orally de- veloped macrocythemia and granulocytosis. No changes were found in the hemoglobin concentration or the number of ret- iculocytes. In general, the larger the dose of amphetamine, the higher the degree and especially the longer the duration of the granulocytosis. No lymphocytosis was found at any time. Page 17 MATERIALS AND METHODS Experimental Animals and Drugs: The dogs used in all the experiments were obtained from a city dog pound. These animals had been kept in individual cages and maintained on a commercial dog food diet*. They were all Mongrels and ranged, with the exception of one animal**, from 5.5 kg. to 16 kg. in body weight. The amphetamine sulfate used was a commercial 5% solu- tion***. The pentobarbital sodium**** employed was in a 4% solution in 10% alcohol, freshly prepared before use. Experimental Procedure: At the beginning of each experiment, the dog was anesthetized'with pentobarbital sodium at the rate of 30 mg. per kilogram body weight administered intravenously by way of the cephalic or the saphenous vein. When anesthesia was complete, the animals were arranged to record respiratory rate, respiratory volume (called ven? tilation all through this experiment) and blood pressure on the smoked surface of a paper belt operated by a kymograph. Blood pressure was recorded by means of a mercury manoé meter connected through the medium of 6% citrate solution in a rubber tube attached to a glass cannula inserted into the *Dickinson's dog nuggets manufactured by the Albert Dickinson Company, Chicago, Illinois. **The exception was dog No. 34 which weighed 21 kg. #**"Amfetasul", Pitman-Moore Company, Indianapolis, Indiana. ****Abbott Laboratories, North Chicago, Illinois. Page 18 carotid artery in the region of the neck. Respiratory rate was obtained by means of a bellowstype stethograph. The values for respiratory volume were obtained by measuring the expired air by means of a wet test gas meter connected through the necessary valve system to a metal cannula in- serted into the trachea. The volume was recorded at each 250 cc. mark by means of an electrically operated signal mag- net. In addition, the sciatic nerve was exposed in the region of the thigh and sectioned. The central end was attached to a shielded stimulating electrode which was connected to a variable voltage stimulating device*. All drugs were injected into the femoral vein through a rubber tube leading from a burette to a glass cannula in- serted into,the vein. As a routine procedure, 5 cc. of physiological saline solution was allowed to flow into the vein immediately following each drug injection. After obtaining a normal record, additional injections of pentobarbital sodium were given at approximately five Ininute' intervalS. The amount injected per dose depended upon ‘the apparent stage of depression of the animal and varied from 3 to 10 mg. per kilogram body weight. The serial injec- tions were continued until the respiratory depression was marked as evidenced by very noticeable decrease in respiratory *Electrodyre stimulator -- the Electrodyre Company, Boston 15, Massachusetts. Page 19 volume. In many instances it was noted at this point that the recovery from the blood pressure depressing effect of the barbiturate injection was poor. After deep anesthesia was attained, 6 of 24 dogs were given amphetamine intravenously at the rate of 20 mg. per kilogram body weight, 6 at the rate of 10 mg., 6 at the rate of 5 mg., and 6 at the rate of 2.5 mg. The recording of the drug effects was then continued for about one hour. The animals were then killed by the injection of saturated mag- nessium sulfate solution. There was no significant difference in the average body weight between the four groups of dogs (Tables 1 to 4). Procedure for Blood Studies: Five samples of blood were drawn from each of the twelve dogs during the course of the eXperiment. The time at which blood samples were drawn was as follows: 1. Before the dog was anesthetized. 2. After the surgery was completed, 133;, thirty to sixty minutes after each experiment was begun. 3. At the deep depression stage, 1131, after the last injection of pentobarbital sodium. 4. At the high peak of the stimulation of amphetamine sulfate,(as indicated by the increased respiratory rates and ventilation, from two to thirty-eight minutes after the am- phetamine injection in these twelve dogs). 5. After the dog was killed by the saturated magnesium' sulfate solution. Page 20 The first blood sample was drawn from the cephalic or saphenous vein, and the others from the femoral vein which was not used for injecting drugs. About 2 cc. of blood was withdrawn each time/Tglaced in a small vial contained 4 mg. of potassium oxalate. Within twelve hours, the red and white cell counts were made by the usual procedure using Hayem solution for the red count and 1% acetic acid for the white cell count. The hemoglobin concentration*was determined at the same time by the Sahli method. The same pipettes and chamber were used throughout the series. A blood smear of each sample was made and stained by Wright's method. The differential white cell count was made later. Blood samples were placed in a refrigerator if storage was necessary prior to counting. Analysis of Data: It will be noted that data from one or two animals have been rejected in some of the tabulations set forth in the section on Experimental Results. In every case, this deci- sion was based upon the Chauvenet criterion as cited by Calvin 33 31. (1949) *As used in this thesis, the term hemoglobin concentration denotes the number of grams of hemoglobin per 100 cc. of blood. Page 21 EXPERIMENTAL RESULTS The Effects of Pentobarbital Sodium on the Respiration and Blood Pressure: The average total dose of pentobarbital sodium required to produce severe depression in dogs was 56.486.6 mg. per kilogram body weight (Tables 1 and 2). The initial anesthetic dose of 30 mg. per kg. is included in this value. Each serial injection of pentobarbital sodium was followed by a further decrease in ventilation and respiratory rate in all of the experimental subjects. After the final injection, the average ventilation in the 23 dogs studied was 6173168 cc. of air per minute (Table 1); and the average respiratory rate was 10.22t4.32 per minute (Table 2). Following each injection of pentobarbital sodium, the blood pressure dropped in all of the subjects. In the early stages of the experiments, this was followed by a prompt re- turn (within two minutes) to the previous normal. As the experiment progressed and as the depression increased, this recovery became less and less evident. The blood pressure dropped from 155.9114.9 mm. Hg after surgery to 103.1922.1 mm. after the final injection of pentobarbital sodium (Table 3). This difference is significant. The minimal electric stimulus in volts required to cause a noticeable respiratory stimulation increased with an increase in the depth of the pentobarbital depression. After surgery, it was 9.838.85 volts and following the final dose of Page 22 pentobarbital sodium, it ranged from 50 to 250 volts (Appendix 4) with an average of 154.5853.3 volts (Table 4). Variations between individual animals was great. Ten dogs died during the period of pentobarbital sodium administration (Appendix 5). In these cases, the average lethal dose was 61.0‘9.0 mg. per kg. body weight. It was observed that the heart continued to beat for one to two min- utes after cesation of respiration (Figure I). The average respiratory rate before paralysis was 10.3883.66 per minute, and the ventilation averaged 5138128 cc. of air per minute. This value does not include dogs No. 22 and No. 25 where the ventilation was 1000 cc. per minute in each case. The blood pressure dropped rapidly to a very low level and remained low in seven of the ten cases. In the three remaining dogs, the blood pressure went up slowly and then fell suddenly when respiration ceased. Before death, the pressure averaged 88.8323.8 mm. Hg. The injection of amphetamine or electric stimulation of the central end of the cut sciatic nerve with as much as 250 volts failed to produce any favorable response in these subjects. The Effects of Amphetamine Sulfate on Respiration and Blood Pressure: Effects on Reppiratory Rate and Ventilation As shown in Table 1, the injection of amphetamine during the pentobar- bital depression caused an increase in ventilation. After the final injection of pentobarbital sodium, the ventilation Page 23 of the four groups ranged from 517 to 675 cc. of air per min- ute with no significant difference between the different dos- age groups. The injection of amphetamine was followed in all cases by a marked increase in ventilation which attained its maximum within two to thirty-eight minutes (Appendix 1). This I increase was from 6758186, 6333133, 5173169 and 6501197 cc. of air per minute to 17753337, 22333628, 22423549 and 27708693 cc. for the four groups treated with amphetamine from the low to the high dose 1evels,respectively. Again there was no significant difference between the four groups of dogs. The average time intervals required to reach the maximum respiratory stimulant effect after amphetamine were 28.339.7, 23.7‘11.0, 22.037.9 and 3.832.7 minutes for the four groups receiving this drug at levels of 2.5, 5.0, 10 and 20 mg. per kg.,respectively (Table 1). There was no significant dif- ference in the time required to reach the point of maximum stimulant effect in the three groups treated with 2.5, 5.0, and 10 mg. per kg. of amphetamine. In the case of the group receiving the drug at the rate of 20 mg. per kg., this inter- val was significantly less. The data on individual animals are presented in Appendix 1. Table 2 shows the effect of pentobarbital and amphetamine on the respiratory rate. The rate did not show the same pat- tern of change in all of the dogs following the administra- tion of amphetamine. It was increased from minima of 10.673 4.86, 10.67t2.38, 8.67i4.03 and ll.00*6.63 per minute during deep pentobarbital depression to maxima of 22.33 9.58, 18.17 Page 24 i3.37, 19.5035.6l and 24.6035.73 per minute after amphetamine in the four groups treated with the drug from the low to the high dose levela respectively. There was also no significant difference in the respiratory rate between the four groups at the stage of maximum pentobarbital depression or amphetamine stimulation. The data on individual animals are presented in Appendix 2. Effects on Blood Pressure Table 3 shows the effects of pentobarbital and amphetamine on the blood pressure. After the surgery, the average blood pressure in each of the four groups was 162.4312.8, 156.039.0, 146.0318.0 and 158.7817.6 mm. Hg. During deep pentobarbital depression, these values were decreased to 117.6tl9.9, 87.7:8.7, 106.0e22.2 and 106.3 ‘26.1 mm.,respectively.i The injection of amphetamine uni- formly caused a marked increase in blood pressure, reaching a maximum within a few minutes after the drug was adminis- tered. The blood pressures at this point were, respectively, 261.8‘9.8, 226.7‘15.3, 217.6‘9.2 and 209.7‘14.9 mm. for the groups on the 2.5, 5.0, 10 and 20 mg. per kg. dose levels of amphetamine. While there was no significant difference be- tween the average blood pressures of the four groups of dogs either immediately after surgery or during deep pentobarbital depression, there was a significant difference following amphetamine between the average for the group on the 2.5 mg. per kg. level and the other three groups. Although the pres- sure dropped slowly, it was still above any previous normal in all subjects at the termination of the experiments one-half Page 25 to one hour following amphetamine administration. The data on individual animals are presented in Appendix 3. Response to Sciatic Stimulation The effect of pento- barbital sodium and amphetamine on the respiratory response to electrical stimulation of the afferent sciatic nerve is set forth in Table 4. This table shows that the stimulation required after amphetamine was comparable to that which was necessary early in the experiment (immediately after surgery) and that deep pentobarbital depression raised the threshold of respiratory response to sciatic stimulation enormously. The average of the voltage required for all the animals was 9.7738.85 at the beginning of the experiment, 154.5353.3 during deep pentobarbital depression, and 21.8818.0 following amphetamine. No significant difference is shown in the response of the dogs in the different dose levels of ampheta- mine. The data on individual animals are presented in Appendix 4. The Effects of Pentobarbital Sodium and Amphetamine Sulfate on the Blood Picture: Blood samples were obtained from twelve of the twenty- four successfully-experimented dogs. The average total dose of pentobarbital sodium which these dogs received was 55.3 17.3 mg. per kg. body weight. Of these twelve dogs, four received 2.5 mg., one received 5.0 mg., one received 10 mg. and six received 20 mg. of amphetamine per kg. body weight. Red and White Blood Cell Counts and Hemoglobin Concentra- tion: The effects of pentobarbital and amphetamine on total Page 26 cell counts and hemoglobin concentration are summarized in Table 5 and Figure VIII. The data on individual animals are presented in Appendix 6. Under pentobarbital sodium anesthesia, only a slight decrease in red blood cell count was observed in six dogs. There was a remarkable increase in three of the remaining dogs in the third blood sample drawn, 113, during the stage of deep pentobarbital depression; and in one dog in the second blood sample, 113., after surgery was finished. The red cell count was increased in seven out of twelve dogs after injection of amphetamine. However, it was in- creased in four of the five remaining dogs in the later period of pentobarbital depression, and a high count was continued in three of them following the injection of amphetamine._ The fourth dog (No. 32) showed some decrease and the remaining dog (No. 28) evidenced no appreciable change in the red cell count (Appendix 6). The hemoglobin concentration was decreased during pento- barbital depression, and increased under amphetamine, but was not correlated exactly with the changes in red blood cell count. 0n the average, as shown in Table 5 and Figure VIII, there was a rise in the red blood cell count from 7.1 mil- lions before anesthesia to 7.4 millions during deep pento- barbital depression, and to 7.9 millions per c.mm. after injection of amphetamine. The hemoglobin concentration de- creased from 120% before anesthesia to 111% during deep Page 27 pentobarbital depression, and then increased to 129% after amphetamine administration. There was a definite and progressive decrease in the total white cell count as the pentobarbital depression in- creased. With the exception of one dog (No. 34), this phe- nomenon was not greatly influenced by amphetamine administra- tion. In the case of the one exception, the count increased early in the experiment and remained high for the balance of the experimental period. After amphetamine injection, the white cell count, in each of the eleven dogs, was only about two-thirds to one-sixth of the value observed before the dog was anesthetized (Appendix 6). As shown in Table 5 and Figure VIII, the reduction of white blood cell count was from an average normal value of 13.5 thousand to 8.2 thousand per c.mm. during deep pento- barbital depression, and to 6.4 thousand after the adminis- tration of amphetamine. No noticeable differences in the total cell counts or hemoglobin concentration were found between the groups of dogs on the different dose levels of amphetamine. Differential White Cell Count: The normal differential white cell counts and the counts after pentobarbital sodium and amphetamine sulfate are summarized in Tables 6 and 7, and Figures IX and X. The data on individual animals are pre- sented in Appendix 7 and 8. As shown in these tables and figures, the polymorpho- nuclear leukocytes progressively decreased both in percentage Page 28 and in actual total count during the entire experimental period. While the percentages of lymphocytes and bands, 1131, young forms of polymorphonuclear leukocytes, increased, their actual total counts decreased. The degree of this change was independent of the dosage level of amphetamine in the case of the polymorphonuclear leukocytes and bands. There were, however, some apparent differences in the case of the lymphocyte counts where the total values were greater in the group receiving 2.5 mg. per kg. than in the group on the 20 mg. per kg. body weight. Page 29 >.Nem.m o.neo.~m o.HHen.mm b.0em.mm poemme mafia umumnma< Hmsfixms homes on A.sasv saws sswoaeuao ,mmoeonnm moneowom aneOmo mamemqmw Nooemmma owaebam mmoemmmm meoqsooa mmaemmo enmemeea mmmemmma madameo poeueo soepoomea Heeacsseopscd newsmpmnme< ecflsmponma< mo coauomnca Hmsfiwms poems puma nmpm< mo psaoa p< hHmpMHmeEEH A.:HB\.oov soapmafipam> .poeuo campuses new uses Hmpoass .eoosaoae so: ea em wont **©.oes.om om o.¢em.~m OH H.heo.oo o.m m.oeu.om m.m m.4em.~m lus\uea ass\uev enemasm finance cams Hanan Imposmad Inmnopdem emoa Hence was mean .mwon cw cowpmawpco> co opmmasm mafiamposmsd use asavom Hepwnnsnopcom mo poommm one .H manna N:.meoo.m em as.me~e.m o ee.e.eo.oH c mo.HeN4.m o A.wxv mwop .93 Atom no “oz Page 30 mm.meoo.am H©.me0m.mH bm.meba.ma mm.memm.mm sconce endswpmnmad Hmawxms mo pesos pa seam.semm.oa- no.0eoo.4N no.0eoo.aa oo.~eb©.ma no.4euo.m oo.©eoo.¢H mm.meb©.oa om.oeoo.ba om.¢eho.oa dofiuoohca Hmpfinumhopcom esfismpmnma< mo nomenoncw seems puma pepm< haepmflvosaH “.mfls\.oov coapmafiunep .mwoo ca ovum encumhwamem no enemasm onwamponma< was asHuom Hepanusnopcmm no poommm one ON OH o.m m.~ isa\see enemas» unwaspondaa Hepapusnopcem .QOHHO flhwflflmpm Uflw “~de HMUOB§§ .ooosaosa cos ea em wont sso.oes.cm o.s.e.ea H.5eo.oo o.oeb.mm 0.4em.mm xua\uei finance euoa annoy was mean .N eHnma Ne.meoo.e ss.memm.m es.~.ao.oa me.a.~e.e x.wx. .ez aeom *m amen “oz Page 31 ¢.Heo.a m.aeo.e N.He~.m o.oe©.m vacuum odes amuezds< Hmswxss noses op x.ceev mafia .popho campuses one sees Hmpoa** .ueusaocw no: one mm was m moms **H.N~ea.moa **0.4Hwa.mma cm.aaen.mom H.0mem.ooa o.naen.mma ow N.me©.uam N.NN+O.©OH c.mflro.©¢H OH m.mH«h.©NN B.meh.hm o.m«o.mmH O.m m.oem.H©N m.oaeo.>aa , w.NHe4.NoH m.N o.e.~.cm em.m.oa.aa e.eeo.oo om.meom.e e.o.a.en os.e.so.oe e.mes.mm ee.aeoo.s avenue Heuflnamnoucem hummusm wa\wev Amxwma A.mxv ecfismpends< Mo compound“ popu< enemasm a: no .93 hcom Hmusms puma seams saws Hanan mo ucfiom u< ae>oa pmezoq unmanned nhmnopnom Awm.asv ehsmmeum uooam econ Haves use mean .nmon ca easemeum voon no enemasm ecwssponda< one season Heueotecoeeoi no possum one .m oases an *m emoc no ”oz Page 32 .uouum uumncmpu mam :mma amuoess .vmusaoca no: cum 4 cam m woos *so.wa«mtam mum.mmem.ama sumw.m«m.w m.mm«m.am m.oo«m.mma ms.m«m.w om , o.o«~.cm em.mu0m.aa o m.Na«©.aa b.0neo.0ma an.oeo.m oa u.meo.mo om.meoo.m *m a.ame~.mm 4.5meo.0ma om.aaea.ma o.m m.meo.ao mm.meoo.oa um n.5aeo.~m w.aaeo.mba oo.mew.o m.m 0.4em.~m mm.aeam.m o ocasmpmnasa amuaphmnoucmm hummusu Amx\mav wa\wsv A.wxv awov no no soauooaaa tapes oeeuaee . season .93 seem no coauomnca hmpm< puma henna ecaampmsaam ampanhmnounmm "oz Auuao>v usa55apm amsacaz euoa ampoe cum mama .umom ca :oapmassauw hqumAamuum eosvoum on emuasvmm unassaum amoahpomam mo spwthpm one so epmmasm esasmpmnasa cam asavom ampannmnopcum mo vacuum 039 .a magma Page 33 mNa oma maa Naa oNa oma aqa «Na 04a moa ooa no 00a Noa oma Oma moa moa paa ammo you seam ooo.moo.s ooo.msa.a ooo.oseus ooo.oaa.a ooo.oeo a ooo.omm.m ooo.o-.e ooo.oao.s ooo.oac.o ooo.osm.o ooo.ome.u ooe.omm.s 00m.~ma.m oos.oma”m ooo.oas o ooo.~om.o .Ea.o hum one u ..~ m N a o.om 0m m AN m N a 0.0a mm m .ON m m a o.m 40 m d m N a m.~ mm *.oz oeaeeuosoea aauaoueoousod eamsmu uooam “.mx\.msv ewmuoo mam wean .umon ca coapmppcmocob :anoamosem vcm meadoo aaeo spasm dam mum ampoa one no epmuasm emwempemms< ecu asueou aeeeetseoesom uo poouum one . eanma ewov Mo “oz Page 34 4ma oma aaa aaa oma ammo hum seam .umov ubamzp on» aam mo mmmno>am wcausu uwon amucmaapmdwm one Bonn amuse vooan use mumoavca uhmnas: mamsmu cooan ease cmo.m ooc.msm.m m o«s.c oom.oem.s e cm~.m co“.mos”s m . oc~.~a oom.mma a N omm.ma cem.saH.s a .as.o hum .sa.o pom *.oz on: can eaasmu eooam Avesnaucoov ucaampmnms< tsetse. .n eanms m.mm aeoaotsooesou emmuoa mam mama ssema umou no “oz Page 35 Table 6. The Effect of Pentobarbital Sodium and Amphetamine Sulfate on the Differential White Cell Count (per cent) in Dogs. No: Drug and Dosage (mg/kg) Blood Cells (per cent) of Pentobar- sample Lympho- dogs bital Amphetamine No.* Polys Bands cytes 4 52 2.5 1 49.3 13.8 32.3 2 38.0 15.8 40.2 3 25.0 15.0 50.3 4 24.8 23.5 44.8 5 13-7 l9;0 59-0 1 64 5.0 1 21.0 27.0 38.0 2 22.0 20.0 47.0 3 36.0 18.0 36.0 4 39.0 10.0 34.0 5 40.0 20.0 24.0 1 55 10.0 1 50.0 10.0 38.0 3 49.0 12.0 35.0 4 , 20.0 7.0 58.0 5 39.0 19.0 40.0 6 56 20.0 1 50.8 16.0 25.8 2 51.5 18.5 24.5 3 46.2 25.2 23.7 4 27.8 29.8 36.3 5 24.0 33.5 34.5 12** 55.3 1 47.8 15.7 30.0 2 42.2 17.4 34.0 3 39.7 20.5 33.1 4 27.1 24.2 40.8 5 24.0 25.6 42.1 *The blood sample numbers indicate the blood drawn from the experimental dogs during five different stages as described under materials and methods. **Average of the twelve dogs. Page 36 Table 7. The Effect of Pentobarbital Sodium and Amphetamine Sulfate on the Total Differential White Cell Count in Dogs. No: Drug and Total Dose Blood Cells (per c. mm.) of sample Lympho- dogs Pentobarbital Amphetamine No.* Polys Bands cytes 4 52 2.5 l 5565 1587 3771 2 4251 1635 4891 3 1728 1318 5463 4 1467 1361 2843 5 717 935 3923 1 64 5.0 1 4022 5171 7277 2 3333 3030 7121 3 2232 1116 2232 4 3939 1010 3434 5 3980 1990 2388 1 55 10-0 g 3375 775 2945 3 2524 618 1803 4 900 315 2610 5 956 466 980 6 56 20.0 1 7598 2425 4065 2 6389 2416 2630 3 3817 2035 2031 4 2056 1986 1934 . 5 1122 1660 1632 12** 55.3 1 6195 2220 4148 2 5099 2137 4134 3 2985 1627 2972 4 1921 1557 2418 5 1286 1323 2407 *The blood sample numbers indicate the blood drawn from the experimental dogs during five different stages as described under materials and methods. **Average of all the twelve dogs. 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OHH oo« aH 00 «.HH 2 mm oHH OOOH Nm 5« «H m «N No 00« m on «.o m «N ONH OOOH «m o« «H E NN om o«o NH 00 «.0H m 0N OOH o«> c o« «.m 2 9H N5 00: NH «0 4H. m 0H mm 00: « m« a S «H o« 004 «H on NH m N. 40 00« HH om 0H m o A.mm.sev A.aHa\.oov A.aHs away Amx\mav A.mxv *xmm .oz oaammeAd aOHpmHHuama open venomnaH saHaom .pz moa aoon haoumaHamom HepHpamnouaem Mo hvom uaaos< Hence gamma whomma hHopmHaesaH .saHvom HmanamQOpamd mo memoa omamH maH>H>aam poz mmoa co mama oaammoam UOOHa use haopwaHamem .« NHaaeQQH Page 69 0HH 000.m 000.0H«.e « 0~H 000.4 000.000.0 s 0HH 00H.e 000.00~.0 m NHH 000.0 000.000.e N 0~H 000.0 000.0m~.e H «.N 04 on mmH 0«~.« 000.000.m « 0~H 0«H.0 000.0m0.w s 00H 00H.« 000.00m.0 m 00 00s.: 000.~HH.0 N mHH 00H.HH 000.0~«.0 H «.N 0« 0m III III III « 04H 000.H 000.0«H.e m «NH 0«0.0 000.00«.0 N mmH 00~.m 000.00H.e H «.N . ~« mm 0HH 0««.0 000.0ao.e « mmH 005.0 000.00e.u H 00H 0«a.0H 000.000.« m 00H 0«m.mm 000.000.0 N ~0H 0«0.«H 000.0«0.0 H «.N 0« am paoo pom .as.o pea .sa.o pea *.oz eaHsmuanBH Haanamnouaem .oz *nnm on: can eHasmm moa eoon Amx\mav omoa Hmpoe cam maua .mmoa :H aoHpmupaeoaoo aHQOHmoam: ace mpaaoo HHoo epHax use can Hmaoa esp so mumMHam oaHSmponas<. can anHeom anHnnnnopcmm «0 panama use .0 xHegcQQH 'Page 70 NNH NMH ONH NNH «HH NHH HOH mHH OdH HNH OmH mmH OmH HHH NNH OHH OOH OOH 5O OOH NOH ammo pea **Om O: .m OOH. .NH oom. .NH OO«. 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O .Ea.o pea opp « +~ m N H ON « a m N H ON « AN m N H ON « +~ .« N H OH « J m N H O.« *.oz oaHamuonmad oHaemm aoOHm me\msv emoa Hmpoa 0am maaa AvoaaHpaooV .O anaoam< «5 m« m: «« 4O kuHOHmnoaaem 4m Nm Hm mm «m .02 moa Page 71 OHH O«H ONH OMH HmH 4mH OHH ONH OHH OOH OOH no am 9:00 pea **nm 0«0.m 000.0 0«H.« 000.0 005.0 00«.~ 00«.H 005.5 00H.H 0«0.m 000.0 0««.0H 000.0H .Ea.o pea up: .Omma enap HHnmm map aH OooHn mo .00 OOH tea madam 5H mHmaUe &OOH** .mvoapea 0am mHmHamoms nocaa vmnHaommn mm ummmpm oaoammmHa e>Hm maHaaO mmou HmpamsHamdwe map Scam gamut OOOHO map opmoHuaH mamnsaa mHasmm OOOHO ease 000.0«N.0 000.050.5 000.050.0 000.00~.5 000.000.0 000.005.5 000.000.0 000.00m.5 000.000.0 000.0««.5 .000.005.0 000.000.0 000.0m«.0 .Ea.o pea one AaeaaHuaoov « .4 m N H ON «« « ‘N m N H ON m« « d m N H ON O« *.oz eaHampmsas< HmaHnnmnopaem eHasmm aoOHm Amx\mav omoa Hence and maha .O xHaaeaa< mm 5m Om .oz moa Page 72 Appendix 7. The Effect of Pentobarbital Sodium and Amphetamine Sulfate on the Differential White Cell Count (per cent) in Dogs. Blood Dog sample Cells_(pgr cent) No. No.* Polys Bands Lymph. Mono. Baso. Eosino. 27 1 47 8 36 3 2 4 2 32 12 50 2 - 4 3 6 12 66 4 1 l4 4 20 23 46 1 3 7 5 5 6 72 4 5 8 28 1 59 17 20 4 - - 2 46 21 3O 2 - 1 3 -- -- -- - - - 4 13 36 43 3 - 1 5 -- -- -- - - - 29 1 44 21 31 3 - 1 2 26 17 47 5 2 3 3 36 20 35 2 - 7 4 29 4 53 5 - 4 5 2O 18 59 2 - 1 30 1 ' 4g 19 42 1 - 1 2 4 - 3 33 13 28 E .. 2 4 32 31 32 2 - 3 5 16 33 46 - - 5 35 1 21 27 38 - - 14 2 22 20 47 - - 11 3 36 18 36 - - 10 4 39 10 34 - - l7 5 40 20 24 4 - 12 38 1 50 10 38 2 - .. 2 -- -- -- - - - 3 49 12 35 3 1 - 4 2O 7 58 3 2 - 5 39 19 4O \ 2 - - 31 1 48 19 29 3 - 1 2 49 13 34 l - 6 3 41 22 30 1 - 6 g 19 23 52 2 - 4 Page 73 Appendix 7. (continued) Blood Dog sample Cells (per cent) No. No.* Polys Bands Lymph. ’Mono. Baso. Eosino. 32 § 53 11 29 - - 7 3 38 19 37 2 1 3 4 44 21 30 - ' 5 5 -- -- -- - - - 34 1 45 11 28 - - l6 2 47 18 23 2 - 10 3 44 24 19 - - 13 4 29 47 17 1 - 6 5 23 33 30 2 - 12 36 1 56 16 23 2 1 2 2 55 25 17 - - 3 3 54 . 29 15 - - 2 4 15 28 47 1 2 7 5 19 31 4O 2 2 6 37 l 64 10 21 1 - 4 2 55 18 24 3 - 1 3 42 38 19 1 - - 4 18 47 31 l 1 2 5 9 56 31 1 1 ,2 39 % 39 29 25 4 3 - 3 58 19 22 - 1 - 4 42 13 41 2 - 2 5 45 14 37 3 - 1 *The blood sample numbers indicate the blood drawn from the experimental dogs during five different stages as described under materials and methods. Page 74 'Appendix 8. The Effect of Pentobarbital Sodium and Amphetamine Sulfate on the Total Differential White Cell Count in Dogs. Drug and Total Dose Cells (per c.mm.) Pentobarbital Amphetamine Blood Lympho- Dog sodium sulfate sample Polys Bands cytes ' No. (mg/kg) (mg/kg) No.* 27 50 2.5 1 7450 1268 5706 2 7472 2802 11675 i 100 2010 11083 194 2231 44 5 478 573 6876 28 52 2.5 l 4838 1394 1640 2 3703 1691 2415 i '55. 15:. 255. 5 --- --- --- 29 58 2.5 1 6213 2965 4377 2 1222 799 2209 3 1836 1020 1785 4 1784 246 3567 5 1050 945 3098 30 48 2.5 l 3760 720 3360 2 4608 1248 3264 3 2343 923 3550 4 1280 1240 1280 5 624 1287 1794 ' 35 64 5 1 4022 5171 7277 2 3333 3030 7121 3 2232 1116 2232 4 3939 1010 3434 5 3980 1990 2388 38 55 10 5 3875 775 2945 3 2524 618 1803 4 900 315 2610 5 956 466 980 ‘31 48 20 1 7008 2774 4234 2 5243 1391 3638 3 2727 1463 1995 4 798 966 2184 5 --- --- --- Page 75 Appendix 8. Drug and Total Dose Cells (per c.mm.) (continued) Pentobarbital Amphetamine Blood Dog sodium sulfate sample Lympho- No. (mg/kg) (mg/kg) No.* Polys Bands cytes 32 58 2O % 11846 2459 6482 3 4522 2261 4453 5 6116 2919 4170 34 73 20 '1 4455 1089 2772 2 5875 2250 2875 3 5632 3072 2432 4 3509 5687 2057 5 2001 2871 2610 36 50 20 1 11172 3192 4589 2 10203 4638 3154 3 4968 2668 1380 4 458 854 1434 5 779 1271 1640 37 53 20 1 --- -O- --- 2 4235 1386 1848 3 1890 1710 855 4 450 1175 775 5 333 2072 1147 39 55 20 g 3510 2610 , 2250 3 3161 "“1536 1199 4 1008 312 984 5 1373 427 1129 4*The blood sample numbers indicate the blood drawn from the experimental dogs during five different stages as described under materials and methods. MI HI AN STATE UNIV RSITY LIBRARIES 0 IE 3 1293 (53046 2945