ABSTRACT ENDOTOXIC SHOCK IN THE HORSE By Kenneth E. Gertsen In this study 4 horses were used to study the cardiovascular effects of intravenous injection of an endotoxin of E. aoli. Clinical signs of endotoxic shock were noted. The horses all exhibited similar signs within 1 hour of the injection. They included depression, cold, clammy extremities, decreased capillary perfusion time, and decreased blood pressure. Hemoconcentration and acidosis were noted in the blood of each horse. Three of the horses died within 14 hours of the injection of the endotoxin. One mare survived 3 weeks before being euthanatized. Necropsy examinations were performed on all of the horses. Hyper­ emia and numerous petechial and ecchymotic hemorrhages were noted grossly throughout the body. Histopathologic examination confirmed the lesions of hyperemia and hemorrhages noted on gross examination. ENDOTOXIC SHOCK IN THE HORSE s' vi3 Kenneth E. Gertsen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Surgery and Medicine 1970 ProQuest Number: 10008723 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest, ProQuest 10008723 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 G>-Q5 / O f t-2071 ACKNOWLEDGEMENTS The author wishes to thank his graduate degree committee members, Dr. F. H. Oberst, Dr. G. H. Conner, and Dr. H. Kitchen, for their guidance in the planning and writing of this thesis. Appreciation is also expressed to those who helped along the way, including Dr. R. W. Van Pelt, Dr. W. F. Riley, Jr., Dr. P. J. Tillotson, and Dr. J. D. Krehbiel. Thanks are extended to Miss Judy Lee and Mrs. Pat Rosenberger for their assistance wi t h the laboratory samples, Miss Dixie Middleton for the help with the illustrations, and Mrs. D. Boerma, Mrs. T. Wolcott, Miss D. Stanton, and Mrs. C. Janssen for their help with the manuscript. Lastly, sincere gratitude is expressed by the author to his wife, Diana, for the continuing encouragement to complete this work. ii TABLE OF CONTENTS Page INTRODUCTION ........................................................... 1 REVIEW OF LITERATURE .................................................. 2 Description of the D i s e a s e ...................................... 2 S h o c k ........................................................... 4 E t i o l o g y ......................................................... 4 En d o t o x i n s ...................................................... 5 Septic (Endotoxic) 7 Shock in M a n ............. Therapy in M a n .................................................. Adrenergic therapy .................................... Corticosteroid therapy ................................ 8 9 12 Digitalis th e r apy...................................... 13 Antibiotic therapy 13 .................................... Miscellaneous therapy..................................... 13 Shock Therapy in the H o r s e ....................................... 14 MATERIALS AND M E T H O D S ..................................................... 16 R E S U L T S .....................................................................19 Horse #5773 .................................................... 19 Horse #6659 .................................................... 20 Horse #6035 .................................................... 20 Horse # 6 1 6 2 ....................................................... 21 Monitored Results ............................................. 22 Blood p H .................................................. 22 iii Page Blood p C 0 2 ................................................ 22 Oxygen s a t u r a t i on ..........................................22 Central venous p r e s s u r e ................................... 33 Arterial blood p r e s s u r e ................................ 33 Respiratory r a t e ......................................... 33 Body t e m p e r a t u r e ......................................... 33 P l a t e l e t s ...................................................41 H e m o g r a m s .................................................. 41 Serum p r o t e i n .............................................. 41 Blood urea nitrogen (BUN).................................. 41 Serum Na, K, and C l ....................................... 50 N e c r o p s y ......................................................... 50 B a c t e r i o l o g y .................................................... 52 D I S C U S S I O N ............................................................. 54 S U M M A R Y .................................................................. 62 B I B L I O G R A P H Y ...................... 65 iv LIST OF TABLES Table 1 Page Central venous pressure from preinjection time to 48 hours postinjection of endotoxin (mm H 2 O * ............... . 34 2 Pulse rate (min.)/respiratory rate (min.)/temperature (F°) per minute from preinjection time to 48 hours postinjection of e n d o t oxin....................................... 40 3 Serum sodium (in Eq./L.), potassium (in Eq./L.) and chloride (in Eq./L.) from preinjection time to 48 hours postinjection of e n d o t oxin....................................... 48 4 Total protein (gm/100 ml.)/BUN (mg./lOO ml.) from preinjection time to 48 hours postinjection of e n d o t o x i n ......................................................... 49 v LIST OF FIGURES Page Blood pH of horse #5773 from preinjection to 12 hours postinjection of endotoxin....................... 23 Blood pH of horse #6035 from preinjection to 14 hours postinjection of endotoxin....................... 24 Blood pH of horse #6659 from preinjection to 13 hours postinjection of endotoxin....................... 25 Blood pH of horse #6162 from preinjection to 48 hours postinjection of endotoxin....................... 26 Venous blood pC02 of all 4 horses from preinjection to death of horse or 48 hours postinjection ......... 27 Blood pCC>2 of horse #6659 from preinjection to 12 hours postinjection of endotoxin....................... 28 Blood pCC>2 of horse #6162 from preinjection to 24 hours postinjection of endotoxin............. . . . . 29 Blood pCC>2 of horse #6035 from preinjection to 12 hours postinjection of endotoxin ....................... 30 Blood CO 2 of horse #5773 from preinjection to 12 hours postinjection of e ndotoxin...................... 31 Venous oxygen saturation of horses #6659, #6035, and #5773 from preinjection to death of the horse 32 . . Arterial oxygen saturation of all 4 horses from preinjection to death of horse or 40 hours post­ injection. Note the drop in saturation of all horses except #6162, who survived the 48-hour test period ............................................. 35 Arterial blood pressure of horse #6659 from pre­ injection to 13 hours postinjection of endotoxin. 36 . . Arterial blood pressure of horse #5073 from pre­ injection to 12 hours postinjection. Note rise of pressure prior to death of animal while he was s t r u g g l i n g ............................................... vi 37 Figure Page 14 Arterial blood pressure of horse #6035 from pre­ injection to 12 hours postinjection of endot o x i n .............. 38 15 Arterial blood pressure of horse #6162 from pre­ injection to 48 hours postinjection. Note mitral increase followed by a slight drop and then a gradual rise. This horse survived the 48-hour trial period . . . . 39 Platelet count of death of horse or 42 16 17 all 4 horses from preinjection to 48 hours postinjection of endotoxin . . . Packed cell volume of to death of animal or period. Horse #6162, period, leveled out 2 all 4 horses from preinjection the end of the 48-hour trial which survived the 48-hour trial hours postinjection .................. 43 18 White blood cell count and differential of horse #6659 from preinjection to 12 hours postinjection of e n d o t o x i n ....................................................... 44 19 White blood cell count and differential of horse #5773 from preinjection to 12 hours postinjection of e n d o t o x i n ....................................................... 45 20 White blood cell count and differential of horse #6162 from preinjection to 48 hours postinjection of e n d o t o x i n......................... . . ..................... 46 21 White blood cell count and differential of horse #6035 from preinjection to 11 hours postinjection of e n d o t o x i n ........................... 47 Lung of horse following endotoxin injection. Note marked congestion and influx of cells ....................... 51 Adrenal gland of horse following endotoxin injection. Hemorrhage and congestion seen in zona glomerulosa. . . . . 51 22 23 24 Kidney of horse following endotoxin injection. The glomerulus is showing congestion................................ 53 25 Liver of horse following injection of endotoxin. Note marked congestion of sinusoids.................................. 53 26 Pin k mucous membranes of normal h o r s e .......................... 55 27 Mucous membranes of horse #6659, 5 minutes after injec­ tion of endotoxin. The somewhat blanched appearance is due to the potent sympathomimetic activity of the injected e n d o t o x i n ................................................ 55 28 Mucous membrane of horse #6659, 12 hours after injec­ tion of endotoxin. Note the congested appearance and the cyanotic gum margins outlining the incisor teeth. . . . vii 56 Figure 29 30 31 Page Arterial blood pressure before endotoxin injection and 7 minutes after injection in horse # 6 0 3 5 ................ 58 On horse #6035, the arterial blood pressure seen at 2 hours after endotoxin injection. Blood pressure began to stabilize at half the preinjection level . . . . . 58 Arterial blood pressure of horse #6035 at 7 hours post­ injection about the same seen 2 hours postinjection. At 11 hours the pressure decreases further just before horse's death .................................................. 58 32 Dual trace recorder (A) for recording blood pressures is on left; Statham transducer (B) is on IV stand next to water manometer used to measure central venous pressure ( C ) .............................................. 63 33 Arrangement of dual trace monitor (A), Statham trans­ ducer (B) and water manometer (C) next to horse in stocks prior to endotoxin injection ......................... 34 63 Radiometer gas monitor (A) and American Optical Micro-Oximeter (B)................................................ 64 viii INTRODUCTION In recent reports seen in veterinary literature (Carrol et at. , 1965; Coffman and Bracken, 1968; Roberts, 1965) endotoxic shock in the horse has been discussed. Only one report (Carrol et a t . , 1965) was concerned with the production of experimental endotoxic shock; the other two discussed clinical cases diagnosed as endotoxic shock (Coffman and Bracken, 1968; Roberts, 1965). It has been suggested that endotoxins from various gram-negative bacteria play important roles in the colitis "X" syndrome. This research was conducted to establish dosages, cardiovascular and clinical response of the horse to intravenous injection of endo- toxin. The clinical responses seen were compared to signs seen in colitis "X". With the data obtained, it should be possible to use this model of endotoxic shock to further study the syndrome, as well as to develop therapeutic techniques for use in animals in clinical shock. 1 REVIEW OF LITERATURE Description of the Disease Various writers have recognized the colitis "X” syndrome by the following clinical signs: culatory collapse, acute onset, profuse diarrhea, peripheral cir rapid dehydration, and hemoconcentration (Bryans, 1963; Olson, 1966; Pickrell, 1968; Rooney et 1963; Rooney, 1965). Most affected horses collapse, are unable to rise (Pickrell, 1968), become dyspneic (Pickrell, 1968; Rooney, 1965), and develop initial temperature rises which later drop to normal or subnormal (Olson, 1966; Rooney, 1963; Bryans, 1963). w ithin 24 to 48 hours The disease usually terminates in death (Rooney, 1963; Rooney, 1965). In the early stages of the syndrome the hemoconcentration is accompanied by leukopenia that is predominantly neutropenia (Bryans, 1963; Rooney, 1963). Leukocytosis may b e seen later (Rooney, 1963). In most cases the blood urea nitrogen level is elevated to over 60 mg./ 100 ml. There is loss of serum sodium, potassium and bicarbonate (Olson 1966). In the usual history the affected horse has suffered from recent illness (Rooney, 1965) 1966; Rooney, 1963). or stressing conditions (Bryans, 1963; Olson, It has been suggested that the hemorrhagic colitis syndrome, which followed transportation by rail, reported in years past was the same or similar type disease (Olson, 1963; Rooney, 1963). The disease occurred in horses of all ages and was usually sporadic (Bryans, 1963; Rooney, 1963; Rooney, 1965). 3 The most striking necropsy lesions were in the cecum and the colon (Rooney, 1963; Rooney, 1965). mucosae wer e hyperemic The serosal surfaces were cyanotic and the (Rooney, 1965). Edema and hemorrhages occurred in the submucosa (Bryans, 1963; Rooney, 1963; Rooney, 1965). The liver and spleen were engorged and the adrenal glands had cortical hemorrhages (Rooney, 1963; Rooney, 1965). The lungs were emphysematous and the heart might be either normal or flabby (Bryans, 1963; Rooney, 1963). No sig­ nificant lesions were found in the stomach, small intestine, pancreas, urogenital tract or central nervous system (Rooney, 1963). Microscopic examination of the submucosa of the colon and cecum revealed dilatation of the venules and varying degrees of constriction of the arterioles (Bryans, 1963; Rooney, 1963). Necrosis with sloughing of the mucosal epithelium was seen when tissues had not undergone post­ mortem changes before examination (Rooney, 1963). The lymph nodes drain­ ing the cecum and colon were hyperemic with the lymphocytes undergoing necrosis (Bryans, 1963; Rooney, 1963). Rooney (1965) reported micro­ scopic evidences of other disease processes including respiratory infec­ tion, metritis, or myocarditis. Acute salmonellosis is one of several diseases which resembles the colitis "X" syndrome (Olson, 1962; Rooney, 1965). In both diseases affected animals have been stressed, have high BUN levels, and have elevated temperatures (Lowe, 1969; Olson, 1966). In salmonellosis the pulse is full and strong with no sign of peripheral vascular collapse. Bacteriological examination of the feces may help confirm the diagnosis of salmonellosis (Olson, 1966). Viral arteritis is another similar sporadic disease to consider in differential diagnosis. Epizootic outbreaks are occasional. The onset is acute wit h temperature rise, panleukopenia, increased packed cell 4 volume and increased blood urea nitrogen. Some affected horses exhibit signs of respiratory disease, but others show signs of colic and diarrhea. Pregnant mares usually abort; necropsy lesions of the aborted foals are nonspecific. The mortality rate is low (Olson, 1966). Clinically acute thromboembolic colic and torsions of abdominal viscera resemble colitis "X". Thorough clinical and necropsy examinations are essential to confirm diagnoses (Rooney, 1965) . Shock Shock has been defined as the syndrome resulting from many circum­ stances in which there is impairment of the effective circulating blood volume (Collins et al.» 1964b). The theory of perfusion defect of tissues and vital organs in shock is widely accepted (Anderson et al. , 1967; Dietzman and Lillehei, Spink, 1962). 1969; Shires and Carreco, 1966; Sodeman, 1967; Hardaway (1968) discussed the normal phenomenon of blood flowing through the capillary and its ability to furnish nutrients to and carry metabolites from individual cells. He defined shock as an inadequate capillary perfusion resulting from the impairment of blood flow through the capillary. Spink (1962) agreed that in shock the micro­ circulation is the final common denominator — unit, the capillary. the cell and its supporting Weil and Shubin (1967) presented a classification based on etiology which includes: hypovolemia, cardiac failure, hyper­ sensitivity, neurogenic impediment of blood flow, endocrine failure, and bacteremic (endotoxic) shock. The purpose of the classification of shock is to aid clinicians in an organized approach to diagnosis and treatment. Etiology Attempts to discover the etiological agent of colitis "Xn have not bee n conclusive (Bryans, 1963). It has been suggested that endotoxins 5 from various gram-negative bacteria play important roles in the syndrome (Bryans, 1963; Coffman and Bracken, 1968; Rooney et at., 1963). These endotoxins have been injected intravenously and have produced signs simi­ lar to colitis "X" (Rooney et at., 1963). Carrol et at. (1965) injected an endotoxin prepared from a culture of Aevdbactev aevogenes intraperitoneally and noted clinical signs resembling colitis "Xu . The horse collapsed in 1 hour and died 8 hours after the injection. Endotoxins Endotoxins are lipopolysaccharides bonded to proteins in the cell wal l of gram-negative bacteria (Carrol et at. , 1965; Gilbert, 1960; Kwaan and Weil, 1969; Lillehei et at. , 1967). In endotoxic shock the lipopolysaccharide has been released from the cell wall of the gramnegative bacteria (Lillehei et a t . , 1967; Sukandan and Thai, 1965). Endotoxic shock can be reproduced experimentally by intravenous injec­ tion of the endotoxin of Escherichia coti in dogs (Bell and Schloerb, 1966; Duff et at. , 1965; Evans et at., 1967; Grable et at., 1963; Lillehei and MacLean, 1958; Thomas et at., 1969), in rabbits (Grable et at., 1963), in cats (Granway et at., 1969), and in monkeys at. , 1968). (Nies et When viable E. ooti, which was isolated from a case of pyelonephritis, was injected intravenously the same circulatory responses were produced in dogs and in monkeys as had been obtained when purified endotoxin was injected (Thomas et at., 1969). Unlike exotoxins, endo­ toxins produce the same general symptoms regardless of the animal injected or the bacterium that produced the toxin (Brande, 1964; Carroll et at., 1965; Fine, 1961; Weil and Spink, 1957). Experimentally the acute signs usually include high fever, decreased blood pressure, diarrhea, muscle pains, and hyperpnea (Brande, 1964; 6 We i l and Spink, 1957). Endotoxins of gram-negative bacteria are said to cause their lethal effects through their potent sympathomimetic activity (Lillehei and MacLean, 1958). vasoconstriction The basic hemodynamic alteration is severe (Spink, 1962; Fine, 1968), which results in increased peripheral vascular resistance (Duff et al., 1965; Lillehei et al. , 1967). The vasoconstriction of the hepatic veins causes pooling in the abdominal viscera with less blood available to the active circulation (Lillehei et a l . , 1967; Weil and Spink, 1957). There is also loss of plasma due to increased hydrostatic pressure in the congested capillaries (Lillehei et a l . , 1967). The pooling of blood and congestion in the viscera causes decreased venous return to the heart lowering both cardiac pressure and cardiac output 1960). The decreased blood flow to the kidneys causes marked decrease in urine output and resulting renal failure 1962) . (Gilbert, (Kwaan and Weil, 1969; Spink, The decreased blood flow to the viscera decreases motility and produces visceral ischemia (Fine, 1968; Lillehei and MacLean, 1967; Longerbeam et a l . , 1962). Necropsy findings in experimental endotoxic shock are consistent. The principal lesion is hemorrhagic necrosis of the mucosa of the gastro­ intestinal tract, especially the small intestine and colon (Carroll et a l . , 1965; Evans et a l . , 1967; Lillehei and MacLean, 1967). Petechial hemorrhages occur on the serosa of the gastrointestinal tract, the heart and the kidneys (Carroll et al. , 1965). Snell (1969) believes the sig­ nificance of pulmonary edema is minimal, whereas Sukandan (1965) indicates it contributes to cardiac failure. 7 Septic (Endotoxic) Shock in Man Because of the marked similarities, endotoxic shock has been used as a model for study of septic shock in man (Lillehei et a l . , 1958). Shock associated with gram-negative bacterial infections of the blood stream have been suggested to be caused by the liberation of endotoxin from the bacteria (Weil and Spink, 1958). Gram-negative bacteria are the most com­ mon organisms isolated from wound infections, abscesses, urinary tract infections and septicemias in man (Dietzman and Lillehei, 1969). Septic shock is said to be second only to myocardial infarction as a cause of shock in hospitalized patients (MacLean, 1962; Weil and Spink, 1958). Mortality from septic shock is high, ranging from 60% to 80% (Kwaan and Weil, 1969; Weil et al . , 1964). In prolonged shock, accumulated toxic products resulting from poor tissue perfusion and anoxia of the cells from anaerobic metabolism produce lethal effects (Sodeman, 1967). Shock can be produced in severe infections in the abscence of a bacteremia (Ebert and Abernathy, 1961). In one clinical report manipulation of the genitourinary tract and considerable trauma were considered important in producing septic shock (Weil et al. , 1964). In a study of 169 clinical cases, Weil (1964) provided a clear pic­ ture of the clinical aspects of shock caused by gram-negative organisms. Initially there was fever with cold, clammy, cyanotic extremities, plus rapid, shallow breathing, vomiting and diarrhea. leukopenia, especially a neutropenia. Concurrently, there was Serum sodium and chloride levels fell and metabolic acidosis developed, and serum lactate concentration increased (Kwaan and Weil, 1969). and the BUN levels rose. Blood flow to the kidneys decreased, In one clinical study patients that died had elevated serum potassium levels (Blair et al. , 1969). Of the gram-negative bacteria, E. ooli , A. aerogenes , Pseudomonas aeruginosa , and Proteus sp. 8 w ere those most commonly Isolated (Spink, 1962; Weil and Spink, 1964; W ilson et al. , 1967). Therapy in Man No single treatment regimen has been successful in all patients in shock since the clinical manifestations, though similar, are from widely differing causes (Thai and Wilson, 1965). In treating shock it is important to restore the effective circulating volume while correcting the hemodynamic disturbance occurring in the microcirculation of the viscera (Lillehei et al. , 1967). Fluids. Initially fluid therapy is the major consideration. A loss of more than 30% of the initial blood volume is critical, but there are good reserves of red blood cells and hemoglobin from which to draw (Weil and Shubin, 1967). of fluid loss The type of fluid administered depends on the nature (Thai and Wilson, 1965). Hardaway (1967) has stated that adequate fluid volume is the most important concern in therapy in non­ cardiac shock, and that fluid administration should continue until there is elevation of the central venous pressure or the pulmonary artery pres­ sure. Some of the more effective replacement fluids are colloid solutions (Lillehei et al. , 1967), plasma (Lillehei et al. , 1967; Longerbeam et al. , 1962; Artz and Fitts, 1962), low molecular weight dextrans (Lillehei et al. , 1967; Lillehei et al. , 1964; Artz and Fitts, 1962), and balanced electrolyte solutions (Lillehei et al. , 1967; Thai and Wilson, 1965). Measuring central venous pressure has proven to be a valuable guide in monitoring fluid restoration (Anderson et al. , 1967; Lillehei et al. , 1967; Weil, 1969; Jennings, 1967). Fluids can be administered to the point of elevating the central venous pressure. The venous pressure reflects the competence of the myocardium to handle the fluid volume 9 returned to it. A n increase in central venous pressure indicates the heart is loaded to capacity Ad r energic t h e r a p y . (Thai and Wilson, 1965; Weil, 1969). When the body is severely stressed, the overall defense reaction may not be in the best interest of that individual (Veilleux, 1963). Endotoxins cause generalized vasoconstriction, which is not limited to the body surface; the kidneys stop producing urine; the gastrointestinal tract stops functioning; and the retention of metabolic wastes produces acidosis (Fine, 1968). The endotoxins cause their lethal effects b y acting as potent sympathomimetic agents or by sensitizing the animal to levels of endogenous sympathomimetic agents which are ordinarily nontoxic (Lillehei and MacLean, 1958). There has been some controversy over the choice of adrenergic drugs to use in the treatment of shock. Ahlquist receptors in the sympathetic system. (1948) described 2 types of The alpha receptors cause vasocon­ striction, stimulate the ureter and the uterus, and promote intestinal relaxation. lation. The beta receptors cause vasodilation and myocardial stimu­ Of the more commonly used adrenergic drugs, isoproterenol is mainly a bet a stimulator, epinephrine is mainly alpha with some beta activity on the heart. at the alpha receptors Methoxyamine and phenoxybenzamine block activity (Kaiser et oil., 1964; Moran, 1963). Vasopressor drugs such as epinephrine and norepinephrine are given to maintain blood pressure, but the resulting vasoconstriction and tissue anoxia may worsen rather than improve the circulation (Collins et al. , 1964a). Others have suggested that excessive sympathetic nervous system stimulation does more h a r m than good when treating shock (Brau et al. , 1966; Blair et al. , 1969; Collins et al. , 1964a; Hermeck and Thai, 1968; Lillehei et al. , 1969) . In acute disease states where blood volume is 10 reduced, the vasomotor and adrenergic receptors are already under intense stimulation. The alphamimetic drugs will increase the blood pressure but at the expense of decreased blood flow to most organs 1968). (Hermeck and Thai, Studies have shown that epinephrine will slow blood flow in the capillaries through intense venous constriction and may even cause back­ flow from the venous end of the loop (Dennis and Zimmer, 1964). It has been shown that continuous infusion of norepinephrine to maintain blood pressure can cause serious local tissue sloughs (Olgesby and Baugh, 1968). Vasopressor drugs are generally said to have limited use in septic shock (Collins, 1964b; Sodeman, 1967). Recent clinical studies concerning septic shock have recommended the use of vasodilators rather than vasopressors Bradley and Weil, 1967; Collins, (Anderson et al., 1967; 1964b; Hermeck and Thai, 1968; Lillehei et al. , 1964; Nickerson and Gourzes, 1962; Wilson et al. , 1964; Wilson et al. , 1967). In a study using cats the alpha blocking agents proved to be competitive antagonists to adrenaline at the adrenergic receptor sites (Birmingham et al. , 1967). The disadvantage of alpha adrenergic blockage is diversion of blood flow from the heart and brain, but blood pressure can be kept at an adequate level with fluid replacement Harrison, 1969). (Perlroth and Reasonable oxygenation can result during hypotension if the circulating blood volume is adequate (Brau et al. , 1966). Vaso­ dilators must be given after adequate fluid therapy, but if given before fluid therapy, the results can be dangerous Nickerson and Gourzes, (Hardaway et al. , 1967; 1962; Perlroth and Harrison, 1969). Chlorpromazine has been shown to increase peripheral blood flow significantly in dogs, but the effects were only transient (Kilman, M or e dogs pretreated with chlorpromazine at 25 to 59 mg./kg. 1969). survived and had fewer significant lesions on necropsy examination than those receiving 11 2.5 to 15 mg./kg. w h e n both groups were given 7.5 mg./kg. toxin (Lillehei and MacLean, 1958). of E. coli endo­ Rats suffering from massive hemor­ rhagic shock suffered 50% lower mortality in the group pretreated with chlorpromazine (Collins, 1964b). Phenoxybenzamine increased the number of survivors in hemorrhagic shock w h e n dogs wer e pretreated before bleeding. In this study the alpha- lytic agents were of no value when there was failure to respond to replace­ ment transfusions (Jacob et a l . , 1956). Arbulu and Thai (1966) gave phenoxybenzamine to dogs in hemorrhagic shock thereby causing decreased total peripheral resistance with decreased left ventricular work. It was found that myocardial response to loading was enhanced after alpha adrenergic blockage. Phenoxybenzamine had no effect on the metabolic acidosis but did increase capillary perfusion in dogs given endotoxin (Abrams et a t . , 1969). In another study dogs were given E. ooli endotoxin at 3 to 5 mg./kg. body weight followed by phenoxybenzamine. There was increased pulmonary blood flow with falling total vascular resistance. When norepinephrine was given with phenoxybenzamine, there was markedly decreased vascular resistance with increased blood flow (Sukandan and Thai, 1965). Phenoxybenzamine administered at the level .2 mg./kg. to 2.0 mg./kg. body weight produced overall improvement in the clinical response of patients in shock (Wilson et a l . , 1964). The metabolic acidosis follow­ ing the use of phenoxybenzamine was thought to be due to washout from muscle masses, skin, and other organs previously isolated by vasocon­ striction (Hermeck and Thai, 1968). Anderson et al. (1967) felt that volume replacement should be the first and at times the only therapy for patients in shock. After conventional therapy has failed, phenoxybenzamine can be used to improve capillary perfusion. 12 Corticosteroid t h e r a p y . Massive doses of corticosteroids administered over a short period appeared to increase the overall survival rates in persons suffering from severe shock (Blair et a l ., 1969; Melby, 1961) even though there is not adrenal cortical failure in shock (Ebert and Abernathy, 1961). Using a rat heart lung experiment, corticosteroids were shown to increase the left ventricular work index (Sayers and Soloman, 1960). Corticosteroids w ill protect the cell by maintaining membrane integrity and stabilization of lysosomes (Melby, 1964). Endotoxin has been found to increase lysosomal enzymes in plasma after infusion (Nies et al. , 1968; Weisman and Thomas, 1962). In animals treated with cortisone therapy there was definitely decreased release of lysosome enzymes (Weisman and Thomas, 1962). Corticosteroid protection against cellular damage is nonspecific in response to noxious stimuli. The degree of protection is proportional to the concentration of corticosteroid in a given volume of tissue. Short term therapy does not alter protein or carbohydrate metabolism and there is no pituitary or adrenal cortical suppression (Melby, 1961). Cortisol increases myocardial contractibility and reduces peripheral resistance after large doses (Melby, 1964). Lillehei et al. (1964) reported high dosages of corticosteroids act as alpha adrenergic block­ ing agents. In experimental studies, massive doses of glucocorticoids such as methy1-prednisolone (195-30 mg./kg.) and dexamethasone (2-6 mg./kg.) reduced the sympathetic response to endotoxin (Dietzman and Lillehei, 1969) . In 2 separate clinical studies, more patients in gram-negative bactermic shock survived after receiving massive doses of corticosteroids (Blair et al. , 1969; Weil et al. , 1964). Wilson et al. (1967) recommended administering hydrocortisone at levels of 50 mg./kg. body weight to 13 patients in septic shock. Adrenocorticosteroids are recommended by others as part of routine shock therapy in treating septic shock Weil, 1967; Shires and Carreco, 1966; Spink, 1962). (Bradley and Phenoxybenzamine and/or cortisone in massive doses may be combined with fluids for best results (Lillehei et al. , 1967). Digitalis t h e r a p y . in shock. Weil (1969) suggests a "V.I.P." approach to the patient The "V" is for ventilation of the patient, to restore volume, "I" is for infusion and "P" is for the pump-augmenting cardiac function with cardiotonic drugs. Thai and Wilson (1965) and Hardaway et al. (1967) suggested that digitalis be used in face of heart failure and especially if the central venous pressure remains high Shires and Carreco (Lillehei et a l . , 1967). (1966) suggested a maximum therapeutic dosage of digi­ talis be given promptly whe n there is a rise in central venous pressure. Antibiotic t h e r a p y . Large doses of antibiotics are recommended immediately for animals in shock (Lillehei et al. , 1967; Muller et al. , 1965; Spink, 1962; Thai and Wilson, 1965). Shires and Carreco (1966) recommended the use of 10 to 15 million units of penicillin or 2 grams of Chloromycetin intravenously during the first 24 hours, but Lillehei et al. m ended the use of 1 gram of Chloromycetin. (1964) recom­ It should be remembered that shock will persist in spite of sterilization of the blood stream (Weil and Spink, 1958) . Care should be taken when administering antibiotics since the death of many bacteria may release more endotoxin thereby worsening the state of shock (Spink et al., 1948). Miscellaneous shock. therapy. Other forms of therapy have been recommended in Survival of dogs given E. ooli endotoxin was not markedly increased whe n hyperbaric oxygen was given (Evans et a l . , 1964). In another study 14 it was suggested that hyperbaric oxygen used in dogs in hemorrhagic shock might improve survival through better oxygenation of tissues and Ferguson, (Navarro 1968). Hardaway and Johnson (1963) suggested that endotoxic shock is pro­ duced in part b y disseminated intravascular coagulation. In studies using 1.5 ml./kg. body weight of E. ooVi endotoxin in dogs, there were prolonged Lee White clotting times, dramatic falls in platelet counts, and increased prothrombin times. The decline in fibrinogen and platelets was inter­ preted to have resulted from platelet clumps and the intravascular clot­ ting process. Preheparinization did not affect mortality but did prevent the loss of clotting activity. The packed cell volumes did not increase as much in heparinized dogs as in controls (Hardaway and Johnson, 1963; al. > 1967). West Shock therapy in the h o r s e . dehydration; The syndrome of colitis "X" produces severe consequently fluids must be given to correct the resulting fluid-electrolyte imbalance. Horses will dehydrate rapidly with colitis "X" since fluid is not only lost in the feces but there is marked accumu­ lation of fluid within the gastrointestinal tract and fluid intake is reduced (Tasker and Olson, 1964). Sodium, potassium, and bicarbonate are suggested to be the main electrolytes lost in colitis "X"; therefore, it is recommended that balanced electrolytes be administered and that sodium bicarbonate or sodium lactate be added to these solutions 1967b; Tasker and Olson, 1964). (Tasker, Laboratory tests should be conducted during fluid therapy since too much bicarbonate or potassium can harm the patient (Tasker and Olson, 1964). Promazine hydrochloride was used successfully as an alpha adrenergic blocking agent for part of the therapy in a horse exhibiting clinical 15 endotoxic shock. Coffman and Bracken (1968) reported depression follow­ ing promazine administration may not be deleterious because the relief of anxiety may be desirable in any stressed horse. (1968) and Roberts Coffman and Bracken (1965) recommend massive doses of corticosteroids for horses in endotoxic shock. A pharmacological dosage of .5 mg./kg. of body weight of 9-fluoroprednisolone has been recommended and can be repeated every 4 hours for several times with no harmful side effects (Roberts, 1965). Antibiotics are recommended to help prevent septicemia. Oral anti­ biotics are recommended early in the course of the disease, but later during endotoxic shock they should be administered cautiously. If large numbers of organisms die, increased levels of endotoxin are released in the intestine and are available for absorption (Coffman and Bracken, 1968). MATERIALS AND METHODS For this study 4 healthy horses, ranging in age from 6 to 20 years, were used. Two mares were of unknown breeding, one mare was an Appaloosa, and the fourth was a purebred Arabian gelding. All animals appeared to be in good health and had been observed at the veterinary clinic for over 2 months. The right carotid artery of each horse was surgically exteriorized to form a permanent carotid loop (McClymont, 1950). The surgical sites were allowed to heal for at least 2 weeks before experimentation was started. Prior to injection of endotoxin, blood was collected from 3 of the horses for bacteriologic culture. Forty cubic centimeters of blood were withdrawn by aseptic technique into a sterile heparinized syringe from the jugular vein of each horse. Blood from each horse was placed on Tryptose agar slants to which 20 cc. of brain heart infusion broth had bee n added. The samples were incubated for 2 weeks at 37 C. and were observed daily for growth. Complete blood counts as well as platelet counts were performed on blood collected preinjection and at 15 minutes, 30 minutes, hours, 8 hours, 1 hour, 12 hours, 36 hours, and 48 hours postinjection. A Coulter Counter* was used for couting the white blood cells. *Coulter Counter, Model A, Coulter Electronics, Hialeah, Fla. 16 4 17 Blood samples were also obtained from the jugular vein of each horse for determinations of serum sodium (Ganbrino, 1968), (Ganbrino, (Henry, 1968), 1964) and serum chloride serum potassium (Henry, 1964) as well as total protein b l o o d urea nitrogen (Ormsby, 1942; Crocker, 1967). The clots w e r e allowed to retract at 20° C. and the samples were centrifuged* at 5° C. to obtain clear supernatant serum. -70° + 5 ° The samples were stored at C. until analyses were conducted. A polyethylene catheter** was placed in the left jugular vein by first making a venipuncture w ith a 12-gauge needle. The catheter was inserted through this needle into the jugular vein and into the great veins of the thorax or the right atrium. The needle was then removed. The catheter was connected to a water manometer to record central venous pressure. By the use of a 3-way valve, venous blood samples were w i t h ­ drawn through the catheter. A 6-inch, 15-gauge intra-arterial catheter*** was placed in the carotid loop. Direct blood pressure was obtained by connecting the catheter to a pressure transducer^ and recording the data on a monitor A 3-way valve was connected to the blood pressure transducer which allowed for collection of samples of arterial blood for partial pressure determinations of carbon dioxide as well as oxygen saturation and pH. *Model PR-2, International Portable Refrigerated Centrifuge, International Equipment Co., N e edham Heights, Mass. **PE 205 Polyethylene Tubing, Clay Adams, Inc., New York, N.Y. ***Jelco IV Catheter, Jelco Labs, Raritan, N.J. t statham P23AC Pressure Transducer, Statham Laboratories, Inc., Hato Rey, Puerto Rico. t +D u a l Trace Monitor, IR-2T, Electronics for Medicine, Inc., 30 Virginia Road, White Plains, N.Y. 18 Oxygen saturations wer e obtained using an Oximeter.* Oxygen saturations wer e also determined on venous blood and all samples were analyzed within 60 seconds after collection. Partial pressures for carbon dioxide and the pH of venous and arterial bloods were obtained with a blood gas analyzer.** Analyses were made w ithin 60 seconds after collection. Lyophilized E . aol-i endotoxin*** was used in each experiment. It was reconstituted in 10 cc. of sterile saline 1 hour before intravenous injection. *MicroOximeter, American Optical Co., Bedford, Mass. **Radiometer Micro Gas Monitor, Radiometer, Copenhagen, Denmark. ***Lipopolysaccharide E. g o IA 0 1 2 7 :B8, Difco Laboratories, Detroit, Mich. 48210. RESULTS Horse #5773 Weight: 478 kg. Sex: Mare Age: 12 years Horse #5773, an excitable and difficult to handle mare, was given 28 mg. of E. ooZi. endotoxin in 10 cc. of sterile saline resulting in a dosage of .059 mg./kg. body weight. The injection extended out over a 30-second period. Three minutes after injection of the endotoxin, rapid, the mare exhibited labored respirations and leaned heavily against the side of the stocks and tail rope; however, 7 minutes later she regained equilibrium. The mare w ent down 70 minutes postinjection, but she refused to lie quietly. Restlessness consisted mostly of abortive attempts to rise, but on several occasions she was able to stand for short periods. hours postinjection, At 7-1/2 the mare stood for approximately 90 minutes. The horse continued to sway back and forth while standing and held her head low. The gingival mucous membranes were cyanotic and the capillary filling time was 8 seconds. The mare stood at 9-1/2 hours postinjection and collapsed 1 hour later. At this time the mare began to exhibit nystagmus and the pupils were markedly dilated. During the last few minutes of life, she became violent. gled trying to rise, only to collapse in extreme exhaustion. wer e made to restrain the mare during this time. She strug­ No attempts The horse died 12-1/2 hours after the injection and a complete necropsy examination was performed. 19 20 Horse #6659 Weight: 484 kg. Sex: Horse #6659 received 27 Gelding Age: mg. <5f E, q o Z% endotoxin in 10 saline resulting in a dosage of .056 mg./kg, body weight. 6 years cc. of sterile Three minutes after the injection the horse began to sweat profusely and was uneasy. He supported muc h of his weight on tail rope, of the restraining stocks, but wi t h i n 8 minutes he was much steadier. The horse collapsed 90 minutes postinjection. was increased and the movements were labored. The respiratory rate The horse stood for the first time at 4 hours postinjection and remained standing for 45 minutes. The horse got to his feet 9 hours postinjection and remained standing for 30 minutes. He constantly shifted his weight while standing as if his legs had difficulty supporting him. The horse collapsed in extreme exhaustion but continued to make abortive attempts to rise. His death was immediately preceded by violent paddling and thrashing. The horse died 13-1/2 hours postinjection and a complete necropsy examination was then performed. Horse #6035 Weight: 448 kg. Horse #6035 received Sex: 27 Mare Age: mg. of E. aoli endotoxin in 10 saline, resulting in a dosage of .066 mg./kg. body weight. 20 years cc. of sterile The injection extended over a 30-second time period. Two minutes after injection the horse became restless. She leaned heavily against the side of the stocks and nearly fell to the floor. respiratory movements were rapid, labored, and deep. The Beads of perspira­ tion appeared on the neck in 5 minutes, sweating became profuse, and by 10 minutes the animal was completely wet with perspiration. became much steadier 10 minutes following the injection. The mare 21 The mare's status remained stable for the next 90 minutes, at which time she collapsed and was moved to the casting stall. The horse lay quietly for the next 5 hours and then began abortive attempts to rise. There were deep sighing respiratory movements during the time she was in lateral recumbency. The pupils were markedly dilated. The blo o d did not clot normally and bleeding continued after veni­ puncture and formed h e m a t o m a s . The mare died 10-1/2 hours postinjection and a necropsy examination was performed. Horse #6162 Weight: 485 kg. Sex: Mare Age: 20 years Horse #6162 received 19.1 mg. of E. oot'i endotoxin in 10 cc. of sterile saline, resulting in a dosage of .035 mg./kg. body weight. The injection was completed in 15 seconds. Within 5 minutes after the injection there were urticarial plaques over her entire body. There was an initial blanching of the gingival mucous membranes, but the gums were markedly cyanotic by 3 hours postin­ jection. The animal was extremely lethargic and continually shifted weight from limb to limb. After 3 hours the mare's condition began to improve. She drank approximately 2 gallons of water when it was offered at 12 hours post­ injection. She ate a small quantity of hay when it was offered at 24 hours. The mare was led to her stall 48 hours after the initial injection of endotoxin. At that time she began to show clinical symptoms of founder. She reluctantly bore weight on her forefeet and insisted on lying down. The horse otherwise appeared normal clinically and was alert with normal appetite for the next 2-1/2 weeks. The mare began to lose condition 22 rapidly toward the end of the third wee k and had trouble standing. The mare was euthanatized and a complete necropsy examination performed. Monitored Results Blood pH (Figures 1-4). In each of the 4 horses there was a definite drop in the p H of the blood 2 hours after the endotoxin was injected. of the 3 horses that died (#5773, #6659, and #6035), In each there was a signifi­ cant drop in both the arterial and venous blood pH values (P> .05). Initially in each experiment there were essentially no differences between the arterial and venous blood pH levels. In the terminal stages in each there were marked differences between arterial and venous blood pH values at all times Blood pCO? (P> .01). (Figures 5-9). Differences between the arterial and venous blood pC02 values w ere slight and not significant. pC 0 2 definitely dropped and the venous blood pC 0 2 #5773 in which it dropped. The arterial blood rose except in horse There were significant differences between the preinjection arterial and venous blood pC 0 2 just before each horse died (P > levels and those taken .05). Oxygen saturation (Figures 10-11). There were no significant changes in arterial oxygen saturation levels, but venous oxygen saturation levels fell dramatically in a pattern of falling initially, rebounding, and then steadily declining (P > .05). The differences between arterial and venous oxygen saturations in the control sampling were not as great as the dif­ ference seen in the terminal s a m p l e s . In horse #6162 there was essen­ tially no change in the arterial oxygen saturation level, but the venous oxygen saturation level declined slightly. 23 o = Arterial x = Venous 7.45 7.40 7.35 7.30 7.25 7.20 7.15 7.10 7.05 0 min. 15 30 60 2 hr 4 6 8 10 12 hr Time Figure 1. Blood pH of horse #5773 from preinjection to 12 hours postinjection of endotoxin. 24 o = Arterial x = Venous 7.40 7.35 7.30 7.25 7.20 7.15 pH 7.10 7.05 7.00 6.95 6.90 x 6.85 6.80 0 min. 15 30 60 2 hr. 4 Time 6 10 12 14 hr. Figure 2. Blood pH of horse #6035 from preinjection to 14 hours postinjection of endotoxin. 25 o = Arterial x = Venous 7.50 7.45 7.40 7.35 7.30 7.25 7.20 7.15 7.10 7.05 7.00 min 10 30 12 13 hr Time Figure 3. Blood pH of horse #6659 from preinjection to 13 hours postinjection of endotoxin. 7.50 4. Blood JO >< m oo o oo #6162 from CM 00 v£> VC o o 00 •H d m CM o CM m o H m o o o e to 48 hours ■ 3 Figure o = Arterial 26 00 A m O n 00 m CN CN O Venous O 00 blood 5. 4 horses from to death vO CN O >>0 of horse O preinjection or 48 hours iH postinjection. M of all 00 pC02 II o Figure 205773 206162 206035 206659 27 oo 28 . = Arterial o = Venous 55 50 20 10 0 min Time Figure 6. Blood p C ^ of horse #6659 from preinjection to 12 hours postinjection of endotoxin. 29 00 U o QJ 3 O d •<-) d •H ■P (0 o ft CO M 3 x o ,d 00 Sf CN OJ r* e •H E-I O P d o *H 4-1 O a) •i-} d •H CD d ft e o d X, CN vO V4 xi X CN iH vO a) to o u o vO -d O CO CN O C_> ft m X T) O O m o vO O O m .05). Horse #5773 experienced a rise at the time of the terminal sampling, but this was observed while the mare was struggling before death. The catheter was dislodged during the struggling and the horse died before it could be replaced. There were no significant changes in pulse pressures in any of the horses during the experiments. Respiratory rate (Table 2). The respiratory rates were the most variable parameters monitored in the experiment. The changes of the respiratory movements during each trial were dramatic. No panting or shallow breath­ ing was noted, but deep groaning respirations were frequent. Just before death respiratory movements were abdominal in nature with deep s i g h s . Body t e m p e r a t u r e . Body temperatures varied slightly during the trials but rose in only 2 animals (#6659 and #5773). These rises were recorded terminally when the animals were quite excitable and were vigorously attempting to stand. Initially there were temperature drops in 3 horses (#6659, #6035, and #5773). sixth hour (P > In each this fall was significant by the .05), after which the temperature began to rise. 34 Table 1. Central venous pressure from preinjection time to 48 hours postinjection of endotoxin (mm H 2 O) Horse Preinjection #6659 #6162 #6035 #5773 13 2 13 -1 15 minutes 7 1 6 6 30 minutes 9 1 1 6 60 minutes 4 3 3 -4 2 hours 5 -2 15 8 4 hours 1 -1 23 0 5 6 hours -1 8 hours 20 - 5 (died) 10 hours 12 hours 13 hours -4 0 +1 18(died) 24 hours -1 36 hours +5 48 hours + 2 (lived) 2 2 (died) 35 . + o x 100 = = = = #6162 #6035 #6659 #5773 98 96 94 92 90 88 86 84 82 80 78 15 « • . 30 • • 60 2 hr. • 4 » 6 • 8 • • lo 12 • 24 • 36 Time 11. Arterial oxygen saturation of all 4 horses from preindeath of horse or 40 hours postinjection. Note the drop in of al horses except #6162, who survived the 48-hour test • 48 36 . = Systolic pressure o = Diastolic pressure 160 150 140 130 120 110 100 90 80 70 60 50 0 tin. 15 30 60 2 hr. 4 Time 6 10 12 hr. Arterial blood pressure of horse #6659 from preinjection stinjection of endotoxin. 37 . = Systolic pressure o = Diastolic pressure 170 160 150 140 130 120 110 100 90 80 70 60 50 0 r ,n. 15 30 60 2 hr. 4 6 8 10 12 hr. Time Arterial blood pressure of horse #5073 from preinjection re ] rs p istinjection. Note rise of pressure prior to death of anihe as struggling. 38 . = Systolic pressure o = Diastolic pressure 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 0 lin. 15 30 60 2 hr. 4 6 8 10 12 hr. Time Arterial blood pressure of horse #6035 from preinjection tinjection of endotoxin. 3 CU Vi M 3 to St 00 CO CO CO a ) (U Vi M CX O. \ \ o LO o St o CO o CN co St CN •o I CM o o o 00 o rs 00 o ,3 VI CM \0 O o on cn •H 3 B from preinjection to 48 hours postinjection. a gradual rise. This horse survived the 48- Vi .3 \£> *rf CU H s Figure 15. Arterial blood pressure of horse #6162 mitral increase followed by a slight drop and then trial period. aj Note hour Systolic Diastolic 39 40 time to O l VO OV i-'r-. m ov O '1 00 vO r l CM CO st VO LO 00 00 vO vO ooi 00 O'* o OM Ov OV CMI OV OV o CO rH ■ s. o St s tl oo OV "s vO CO CO CM CD 00 CD r—1 Q VOI 'd ' OV Oh CD S t CD CM Q 001 T3 CD H o o rH St CM CM CO O cd vO 00 m CO o vO CM '— VO CO CO o o CM S . CM ml 00 OV 00 rH '— CM OOI CO 001 X st ov 00 ov •H o st CO CM CO Oh -s. .05). .05). All animals suffered hemoconcentration. increases in packed cell volumes significant (P > The (PCV) and hemoglobin concentrations were Blood collected as each animal approached death was quite thick and viscous. It appeared to be coincidental that the ranges of PCVs and death were from 61.0 to 63.5 (number of samples insuf­ ficient for statistical analysis). In each case there was an initial leukopenia, primarily a neutropenia, w ithin 15 minutes after endotoxin injection. in 4 hours (P > .05). Lowest levels were reached In each case numbers of neutrophils tended to increase from the fourth hour postinjection until death. Initially there were insignificant decreases in numbers of circulat­ ing lymphocytes; however, the decreases were not as marked as for the neutrophils. The total white cell counts of 3 horses (#6659, #6035, and #5773) at the time of death were significantly different from the preinjection counts (P > .05). The gradual increases in the white counts as death approached reflected the increases of circulating neutrophils. Serum protein (Table 4). The quantity of total protein in serum and the albumin globulin ratio did not change significantly in any of the horses. Blood urea nitrogen (BUN) (Table 4). Blood urea nitrogen (BUN) levels did not increase markedly in 2 of the cases during the experiments and #6035). (#5773 Horse #6659 had an increase of 11.5 mg./lOO ml. over the 42 #6659 #6162 #6035 #5773 170 160 150 140 130 120 110 cells X 103/mm3 100 50 15 min. 30 60 2 hr. 4 6 8 10 12 24 36 48 hr. Time Figure 16. Platelet count of all 4 horses from preinjection to death of horse or 48 hours postinjection of endotoxin. 43 + . o x 64 = = = = #5773 #6162 #6035 #6659 62 60 58 56 54 52 50 48 46 44 42 40 38 36 x 15 • in. • 30 • • _• 60 2 hr. 4 •. 6 • 8 Time •t 10 12 24 • 36 •1 48 hr. ire 17* Packed cell volume of all 4 horses from preinjection to animal or the end of the 48-hour trial period. Horse #6162, vived the 48-hour trial period, leveled out 2 hours postinjection. 44 . x o + 8000 = = = = WBC Segmented neutrophils Nonsegmented neutrophils Lymphocytes 7000 6000 5000 4000 3000 2000 10000 0 min. 15 30 60 2 hr. 4 Time 6 8 10 12 hr. Figure 18. White blood cell count and differential of horse #6659 from preinjection to 12 hours postinjection of endotoxin. 45 . x o + 7000 = = = = WBC Segmented neutrophils Nonsegmented neutrophils Lymphocytes 6000 5000 4000 3000 2000 1000 12 hr min Time Figure 19. White blood cell count and differential of horse #5773 from preinjection to 12 hours postinjection of endotoxin. 46 . x o + 7000 = = = = WBC Segmented neutrophils Nonsegmented neutrophils Lymphocytes I 6000 5000 4000 3000 2000 1000 0 min. 15 30 60 2 hr. 4 6 Time 8 10 12 24 36 Figure 20. White blood cell count and differential of horse #6162 from preinjection to 48 hours postinjection of endotoxin. 48 47 WBC Segmented neutrophils Nonsegmented neutrophils Lymphocytes 7000 6000 5000 4000 3000 2000 1000 10 min. 12 hr. Time Figure 21. White blood cell count and differential of horse #6035 from preinjection to 11 hours postinjection of endotoxin. m '• • cr •a B lo co o VO =Ste iH u from preinjection time co CM vO rH VO =S= ov Eq./L.) LO VO VO =te CO r-^ O i—1 ov CM • rs Ov CM • rs OV CO • CO O i—i sr • CO o rH r• oo ov oo • sr O rH CM • O. OV CM • rs ov sr « vO OV CO • CM • o o !—1 OV LO • ov OV CM • rs OV 00 • S l­ av CM • rs OV CM « rs ov CM • rH O r—I CM • rH CO • CO • vO 00 CM • rs ov 00 • i—1 OV CO O o r- m OV ov • rH • rH O rH o 1—1 o 1—I o VO • 00 OV VO • 00 Ov CO • CO sr • 00 Ov rH o i —i VO • ST CM * -d - m o OV • St CM • lO vO • ST 00 • ■vd- o • LO LO • m 00 • LO CM vO rH vO =fte o • sr VO • CO CO • CO av « CM CO . CM OV • CM vO • CO LO • CO ov o • sr CO • CO • CM LO • CM sr • CM CO • CO OV • CM ov • CO CO r-^ r>LO 00 CM i—i rs CM rH CO CM i—1 LO CM i—1 sr CM i—1 OV CM i—1 /- s LO • CO X o vO • LLfe C7 sr CO rH 00 CM rH CM CM rH O CO VO CM i—1 CM i—1 O CO rH sT CO s - / CM vO T—1 3 vO =fte VO CM i—1 00 CM i—1 CO CM rH o vO CM i—1 vO CM i—1 CM CM OV LO vO vO =s= sr co i—i LO CO rH CM co i—1 CM CO i—1 rs CM r—1 00 CO I—1 m CO i—i vO CO CO potassium (in Eq./L.) 48 hours postinjection of endotoxin CM • « • LO r>. r>. O • LO X i—t • r-^ vO =»= CO vO vO =!te B rt 0 o •rl -P a 0) •o 3. Table 00 sT QJ CO M O 33 fl •r l a) U Pu I— 1 CO 1—1 B s s LO rH o o CO VO OV OV CM LO r-~ OV CO CM m o CO rH i—1 u d I— 1 rH H rH d 0 o d X .d o X ST vO oo CM rH o CO rH CO o X • • sr CO o CO rH CO K d •K • CO IH d 0 X