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Vascular smooth muscle and red cell sodium and potassium in haemorrhagic shock measured by lithium substitution… Day, Brian 1978

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VASCULAR SMOOTH MUSCLE AND RED CELL SODIUM AND POTASSIUM IN HAEMORRHAGIC SHOCK MEASURED BY LITHIUM SUBSTITUTION ANALYSIS by M.B.,Ch.B., University of Manchester, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANATOMY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA BRIAN DAY MASTER OF SCIENCE in Brian Day, 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Anatomy. The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date March 30, 1978 i ABSTRACT A new method of measuring i n t r a c e l l u l a r Na and K using L i substitu-tion was applied to a study of vascular smooth muscle and red c e l l Na changes i n haemorrhagic shock. A rat haemorrhagic shock model was used. Controlled haemorrhage was allowed with a syringe reservoir and the a r t e r i a l blood pressure was maintained,at 30 mm Hg. In a p i l o t study, using 20 rats, the plasma Na and plasma K were monitored. A f a l l i n plasma Na and a r i s e i n plasma K were observed. Both returned towards normal following retransfusion and recovery for one hour. In vascular smooth muscle, s i g n i f i c a n t changes i n both c e l l Na and K occurred following a 2 hour period of haemorrhagic shock. The vascular smooth muscle c e l l Na i n control animals was 27.0+1.5 mEq/kg dry weight and 42.7+1.4 mEq/kg dry weight i n the shocked animals (P<0.001). The c e l l K was 127.8+6.0 i n the control animals and 74.7+4.2 i n the shocked animals. In red c e l l studies, s i g n i f i c a n t increases in red c e l l Na were found. The red c e l l Na in controls was 7.09i0.29 mEq/litre c e l l s , whilst in the shocked animals the red c e l l Na was 8.26+0.33 mEq/litre c e l l s (P<0.025). This was associated with a small but not s t a t i s t i c a l l y s i g n i f i c a n t f a l l i n red c e l l K. In both sets of experiments, the plasma Na and K were monitored and similar changes to those of the p i l o t study were found. Following retransfusion and recovery for 1 hour in the vascular tissue study and 2 hours in the red c e l l study, no s i g n i f i c a n t recovery of c e l l u l a r Na or K occurred. The results of these studies are consistent with a s i g n i f i c a n t impairment of c e l l membrane function i n haemorrhagic shock. The importance of both normal vascular responses and red c e l l function following severe haemorrhage i s obvious. The fact that both may be impaired may have important implications i n r e l a t i o n to the treatment and prognosis of haemorrhagic shock. i i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1 REVIEW OF LITERATURE 3 ORIGIN OF THE TERM "SHOCK" 3 EARLY CONCEPTS OF PATHOPHYSIOLOGY . 3 MODERN CONCEPTS OF THE PATHOPHYSIOLOGY OF SHOCK . . . 5 FUNCTIONAL ABNORMALITIES OF ORGANS IN SHOCK . . . . 6 CELL ABNORMALITIES IN SHOCK 7 PHYSIOLOGY OF SODIUM AND POTASSIUM DISTRIBUTION . . . 8 SODIUM, POTASSIUM AND WATER DISTRIBUTION IN SHOCK . . 10 LITHIUM SUBSTITUTION ANALYSIS OF Na AND K IN VASCULAR SMOOTH MUSLCE IN THE RAT 14 LITHIUM SUBSTITUTION ANALYSIS OF RED CELL Na AND K 15 PURPOSE OF STUDY 16 MATERIALS AND METHODS 17 HAEMORRHAGIC SHOCK MODEL 17 « PLASMA SODIUM AND POTASSIUM MEASUREMENTS 18 CELL SODIUM AND POTASSIUM IN VASCULAR SMOOTH MUSCLE . . 18 RED CELL SODIUM AND POTASSIUM 19 i v Page RESULTS 21 PILOT STUDY 21 CELL SODIUM AND POTASSIUM IN VASCULAR SMOOTH MUSCLE . . . 21 RED CELL SODIUM AND POTASSIUM 22 DISCUSSION 23 THE EXPERIMENTAL SHOCK MODEL 23 METHODS OF CELL SODIUM AND POTASSIUM MEASUREMENT . . . 23 EARLY STUDIES OF SODIUM AND POTASSIUM IN SHOCK . . . . 24 CELL SODIUM AND POTASSIUM CHANGES IN HAEMORRHAGIC SHOCK . . 25 THERAPEUTIC IMPLICATIONS OF CELL SODIUM AND POTASSIUM CHANGES IN SHOCK 28 SUMMARY AND CONCLUSIONS 32 TABLES I - IV 33 FIGURES 1 - 6 37 BIBLIOGRAPHY 43 V LIST OF TABLES Page I THE COMPOSITIONS OF PHYSIOLOGICAL SALT SOLUTIONS . . . 33 I I I PLASMA Na + AND K + CHANGES IN HAEMORRHAGIC SHOCK . . . 34 I I I INTRACELLULAR AND PLASMA Na AND K MEASUREMENTS IN VASCULAR SMOOTH MUSCLE 35 IV RED CELL AND PLASMA Na AND K IN HAEMORRHAGIC SHOCK . . 36 v i LIST OF FIGURES Page 1 DISTRIBUTION OF ELECTROLYTES IN BODY FLUID COMPARTMENTS. . 37 2 REPLACEMENT OF EXTRACELLULAR Na BY L i AFTER INCUBATION IN LiPSS AT 2°C 38 3 RAT HAEMORRHAGIC SHOCK MODEL 39 4 RAT TAIL ARTERY EXCISION 40 5 EXTRACELLULAR (PLASMA) Na AND K (RESULTS) 41 6 CELL Na AND K IN VASCULAR SMOOTH MUSCLE (RESULTS) . . . 42 ACKNOWLEDGEMENTS I wish to express my sincere gratitude to my sponsor Dr. S.M. Friedman, who i n i t i a t e d and sustained my interest i n this subject, for his experienced advice and help with every stage of the project. I would also l i k e to thank Dr. K.S. Morton, who allowed me to spend a year i n the Department of Anatomy as a part of my orthopaedic training. I would f i n a l l y l i k e to thank Miyoshi Nakashima, who taught me many of the p r a c t i c a l methods I used in this project and, with the s k i l l e d help of Roseanne Mclndoe and Gisela Spieckermann, performed many of the d i f f i c u l t technical procedures used. 1 INTRODUCTION A major source of controversy concerning the subject of shock has centred around an acceptable d e f i n i t i o n . The reason for this d i f f i c u l t y has been a lack of understanding of the nature of this syndrome. Messmer^ defined shock as follows: Shock i s a syndrome characterized by an acute reduction i n the n u t r i t i v e blood supply to the v i t a l tissues, associated with a disproportion between oxygen supply and demand and inadequate elimination of acid metabolites from the tissue. As a result of this haemodynamic disturbance, functional and structural changes take place i n the organs affected. g The c l a s s i f i c a t i o n of Blalock has become widely accepted. He divided shock into four categories: 1. Haematologic (oligaemic), 2. Neurogenic, 3. Vasogenic, 4. Cardiogenic, The neurogenic and vasogenic types of shock can be correlated with regard to a change i n resistance of the vessels as the primary cause; the cardiogenic type involves f a i l u r e of the heart as a pump, and the haematologic type results from a decreased blood volume due to a loss of f l u i d from any of the f l u i d compartments of the body. The common denominator of the above four categories i s a state of reduced flow to the v i t a l (and non v i t a l ) organs of the body. This work of Blalock's was a milestone i n the understanding of hypovolaemic shock. He ended many years of f u t i l e attempts by experimental workers to find a toxic agent to explain the c l i n i c a l syndrome of hypovolaemic shock. The four categories of shock may be brought under one d e f i n i t i o n at a c e l l u l a r l e v e l , and shock may be defined as a f a i l u r e of c e l l metabolism caused by poor c a p i l l a r y perfusion. With this d e f i n i t i o n i n 2 mind, those interested i n the underlying mechanisms of haemorrhagic shock have studied the abnormalities of c e l l function i n this syndrome in great d e t a i l . They have used many different methods of study and have looked at both morphological and biochemical changes occurring at the c e l l u l a r l e v e l . 3 REVIEW OF LITERATURE ORIGIN OF THE TERM "SHOCK" The word shock as described previously is used to define a c l i n i c a l syndrome. The exact origin of the term as used to define a state occurring after some gross injury or insult to the body is not known. According to Wiggers,^ Le Dran^ in 1743 was the f i r s t to use the word "choc", but he used i t to refer to a state of collision. The change to its present day c l i n i c a l meaning probably occurred insidiously over many years. By the mid nineteenth century, the word was widely used in a context similar to its present usage. Although obvious and severe trauma was recognised as a cause of shock i n i t i a l l y , i t was not u n t i l later that 43 i t was realised by Latta that the onset of shock could follow other severe illnesses such as cholera. It became widely believed that a toxic agent was responsible for both traumatic and non-traumatic shock. Subsequently, the term shock became overused and was applied to a wide variety of unrelated c l i n i c a l states, including syncope, hysterical illness, and often any severe generalised illness. In 1862, Sir James 53 Paget cautioned against the excessive use of the term and slowly the term evolved to its present day meaning. EARLY CONCEPTS OF PATHOPHYSIOLOGY Early concepts of the aetiology of shock centred around neurogenic 34 or cardiogenic mechanisms. In 1850, Hall described spinal shock following cord transection and further confused attempts to define shock. In the late nineteenth century, many investigators were studying the 4 physiology of c i r c u l a t i o n and gradually there evolved an understanding 9 of the effects of hypovolaemia. Blum, in 1876, concluded that r e f l e x vagal action resulting i n impaired cardiac function was responsible for the state of shock. C r U e ^ is credited with the f i r s t s i g n i f i c a n t experimental work on the subject of shock. He concluded that f a i l u r e of a r t e r i a l pressure was the primary cause of shock and that cardiac, respiratory, and neurological failu r e s were related to this primary abnormality. About this time, many workers began studying the various types of shock syndrome, both c l i n i c a l l y and experimentally. In 1910, Henderson^"* wrote: Venous pressure i s , so to speak, the fulcrum of the circulation...shock, as surgeons use the word is due to f a i l u r e of the fulcrum. Because of the diminished venous supply, the heart i s not adequately distended and f i l l e d during diastole. Hence the picture of a ' f a i l i n g heart 1 is revealed by the pulse. For the same reason, a r t e r i a l pressure ultimately sinks i n spite of intense a c t i v i t y (not because of failur e ) i n the vasomotor system and in spite of contraction (not because of relaxation) of the a r t e r i o l e s . His clear understanding of the role of poor venous return i n the pathophysiology of shock is obvious from his writings. Over the next twenty years or so, including the period embracing World War I, a great deal of effo r t went into the search for a humoral agent, which might be the major factor i n the causation of shock. Some considered that 61 histamine was such an agent. Swingle performed experimental studies on adrenalectomised animals and concluded that cir c u l a t o r y f a i l u r e i n shock was related to changes i n c e l l u l a r permeability with s h i f t s i n water and electrolytes and reduction i n circulatory volume. 5 MODERN CONCEPTS OF THE PATHOPHYSIOLOGY OF SHOCK 47 Recently, Messmer has reviewed the pathophysiology of shock. He has stressed that an inadequate supply of nutrients to the tissues and an a l t e r a t i o n of transcapillary gradients for water soluble metabolites and ions occurs i n shock. These metabolites accumulate i n the extra-c a p i l l a r y space. The manifestations of shock i n various organs have been studied i n d e t a i l . P a r t i c u l a r attention has been focused on the abnormalities i n cerebral, pulmonary, renal, and gastro-intestinal systems. The pathological changes which occur i n shock are widespread and ultimately a l l organs are involved, but to different degrees depending on the l o c a l supply of tissue nutrients. The cardiac output i s distributed i n a disproportionate way i n both low and high cardiac output shock. Acute low cardiac output, with a decreased venous return,. as occurs i n hypovolaemia, causes a re f l e x sympathetico-adrenal reaction, mediated largely through baroreceptors i n the aorta and carotid sinus. The blood flow i s p a r t i c u l a r l y decreased i n regions with a high concentra-tion of alpha receptors including skin, kidneys, and splanchnic bed. The coronary and cerebral c i r c u l a t i o n s , which lack alpha receptors ,are spared at the expense of these other systems. Thus the cardiac output i s redistributed and the a r t e r i a l blood pressure i s s t a b i l i s e d , at least temporarily. In high output shock, as occurs i n severe sepsis or severe trauma without f l u i d loss, peripheral arteriovenous shunting occurs and although the cardiac output i s raised, there i s a functional decrease i n blood flow and supply of nutrients. In both types of shock an increase in blood v i s c o s i t y may occur with erythrocyte aggregations i n the post c a p i l l a r y venules. This results i n further impairment of the microcircu-l a t i o n . 6 FUNCTIONAL ABNORMALITIES OF ORGANS IN SHOCK Decreased blood flow and metabolic depression of various organs i n post traumatic shock was studied i n d e t a i l during the Korean war. A f a l l i n blood Na and a r i s e i n K was shown. The peripheral eosinophil count f a l l s due to adrenal cortex a c t i v i t y . An early r i s e i n serum b i l i r u b i n and increased bromsulphein retention are indicators of l i v e r function disturbance. Muscle metabolism is increased with an increase i n urinary creatine. Evidence of i n s u l i n resistance i s indicated by a diabetic type glucose tolerance curve and a flattened i n s u l i n tolerance curve. Decreased glomerular f i l t r a t i o n and urine output with an increased blood urea nitrogen occur early and both gastric and salivary secretions are decreased. Decreased coronary blood flow may contribute to the f a l l i n cardiac output due to myocardial depression. The entity of "shock lung"^^ is observed with severe multiple trauma and with severe sepsis. Whilst a great deal is now known about this syndrome i t s exact aetiology remains unknown. Low blood flow i t s e l f is not thought to be a major primary factor i n producinggthis syndrome. There is a complex endocrine response i n response to most types of shock. The adrenal glucocorticoids and mineralocorticoids, the adrenal medullary catechol amines, angiotensin, a n t i d i u r e t i c hormone are a l l secreted i n increased amount i n shock. The metabolic responses are widespread and non-specific. Essen-t i a l l y , the impaired supply of nutrients, including oxygen, and the accumulation of metabolites due to the poor microcirculation result i n widespread abnormalities. Because of the hypoxia, energy r i c h phosphate synthesis i s decreased and anaerobic metabolism occurs. 7 CELL ABNORMALITIES IN SHOCK C e l l metabolic products, including H +ions, lactate, and ketones accumulate. The increased ADH and aldosterone cause further o l i g u r i a and Na retention with K loss occurs. Many of the lo c a l tissue changes cannot be detected i n the peripheral blood because of the microcircula-tory disturbance. These l o c a l changes include c e l l organelle (lysosome 19, 12 and mitochondria ) and c e l l membrane damage. These l o c a l reactions are, i n themselves, damaging and the l o c a l release of lysosomal enzymes combined with a poor supply of nutrients and hypoxia result i n further damage. A vicious c i r c l e occurs and a continuous and increasing deterioration of the microcirculation occurs leading to further c e l l damage and ultimately to c e l l disruption. The metabolic and biochemical changes i n the c e l l s of different organs during shock have been studied by many different techniques. Such studies have added considerably to our knowledge of the patho-physiology of shock. C e l l function studies i n shock have included those concerned with 19 7- 55 44 morphology, lysosomal enzymes, c y c l i c AMP, Nucleic acid synthesis, ATPase activity,-* and cation transport changes. ^  The tissues used i n these studies include l i v e r , kidney, skeletal muscle, ' q - > J Z 5 J / j ° 10,15 , „ 17,18,39 . . 21,60 T myocardium, red c e l l s , connective tissue. In this study we are concerned with changes i n c e l l u l a r Na and K which occur i n haemorrhagic shock. The basic physiology of Na and K d i s t r i b u t i o n and current l i t e r a t u r e on c e l l Na and K changes i n shock w i l l therefore be reviewed. 8 PHYSIOLOGY OF SODIUM AND POTASSIUM DISTRIBUTION The approximate d i s t r i b u t i o n of electrolytes i n the various body compartments are shown i n figure 1. The values given for the c e l l e lectrolyte concentrations vary s l i g h t l y i n different tissues. I t is seen that Na and CI are mainly e x t r a c e l l u l a r , whereas K and PO^ are mainly i n t r a c e l l u l a r . I t is also notable that the i n t e r s t i t i a l f l u i d has a very low protein content compared with the c e l l and plasma concen-trations. The barriers which separate these compartments are one of the main reasons for the different compositions seen. The forces responsible for water and electrolyte movement across these barriers ( i . e . the c a p i l l a r y wall and the c e l l membrane) are dif f u s i o n , solvent drag, active transport, exocytosis and endocytosis. The particular roles of each of these forces are described i n standard texts. The c e l l membrane separates the,cellular and i n t e r s t i t i a l f l u i d and is responsible for the different concentrations of ions and water i n the two phases. The inside of a c e l l has a negative potential r e l a t i v e to the exterior. This resting membrane potential varies i n different tissues, from -lOmV to -lOOmV. The membrane permeability i s a very important barrier mechanism. Thus, c e l l membranes are r e l a t i v e l y impermeable to large organic anions, including proteins. They are quite freely permeable to K + and C l ~ , but less so to Na +. Thus, K + permea-b i l i t y i s 50 to 100 times the Na + permeability, whether or not membranes possess pores which determine these differences i n permeability is un-known. In any case, membranes behave as i f they have pores of about 0.7nm diameter. The difference between Na and K may be related to the fact that when hydrated the Na + ion is larger than the K + ion. Forces 9 acting across c e l l membranes on various ions can be examined and analysed. The tendency of Cl to diffuse along i t s concentration gradient into c e l l s is balanced by the e l e c t r i c a l gradient caused by the negative i n t r a c e l l u l a r charge. The membrane potential at which equilibrium occurs is called the equilibrium potential and can be calculated from the Nernst equation. Na has a special status with regard to di f f u s i o n across membranes. With the system as described, Na has both e l e c t r i c a l and concentration gradients tending to cause Na to enter c e l l s . However, the c e l l Na concentration remains low due to active transport of Na out of the c e l l and K into the c e l l . This is achieved by means of a Na-K pump mechanism, located i n the c e l l membrane and deriving i t s energy source from ATP. Na and K transport are coupled i n a variable r a t i o . The Na-K pump a c t i v i t y i s proportional to the Na concentration inside c e l l s . The pump may be electrogenic i f i t extrudes more Na molecules than i t takes i n K, i.e. by increasing the 'coupling r a t i o 1 . The pump is inhibited by cardiac glycosides and by metabolic poisons which prevent ATP formation. The pump i s also very temperature sensitive and Na-K pump a c t i v i t y is inhibited by low temperatures. The above knowledge is applied i n experimental studies of the nature of the pump mechanism. Na-K ATPase, a large lipoprotein with a molecular weight of 670,000, i s the enzyme which hydrolyses ATP to ADP i n order to produce the energy required to drive the pump. This enzyme requires Mg for a c t i v i t y . The negative membrane potential described above depends on the nature of the membrane and the d i s t r i b u t i o n of ions across the membrane. The Na-K pump i s also related to c e l l volume maintenance. I f active Na transport is inhibited i t w i l l diffuse into the c e l l and water would follow against the osmootic gradient. The evidence for the above pump mechanism has been reviewed by Glynn. J" The role of ATP as an energy source has been suspected for many years, but proof was lacking u n t i l experiments on giant squid nerve fibres which involved the i n t r a c e l l u l a r i n j e c t i o n of ATP were performed. Further proof was obtained by experiments using red blood c e l l s . SODIUM, POTASSIUM AND WATER DISTRIBUTION IN SHOCK F l e a r ^ studied patients undergoing major surgery and performed Rectus Abdominis biopsies at various stages throughout the procedure. He measured the tissue levels of Na, K, and water and concluded that muscle as a tissue gained water, sodium and CI and lo s t K during the 33 procedure. Hagberg performed measurements on tissue f l u i d and plasma electrolytes i n dogs subjected to haemorrhagic shock and found greatly increased levels of tissue f l u i d K with s l i g h t increases i n plasma K and he suggested loss of i n t r a c e l l u l a r K to i n t e r s t i t i a l f l u i d as the l i k e l y cause. The same worker performed i n vivo muscle biopsies of single isolated muscle fibres and using X-Ray fluorescence microanalysis 32 measured the i n t r a c e l l u l a r K levels. He found a 26 per cent f a l l i n i n t r a c e l l u l a r K using this technique and concluded that this loss was induced by some c e l l membrane damage occurring i n haemorrhagic shock. He did comment, however, that the c e l l membrane was traumatised during i s o l a t i o n of the single muscle f i b r e . Data based on indirect methods using CI as an extr a c e l l u l a r tag have 23 been c r i t i c i s e d because of the tendency of CI to enter the c e l l . 3 8 Johnson and Tucker used Na2S0^ as an ext r a c e l l u l a r tag and took biopsies from the Rectus Abdominis muscle of dogs subjected to haemorr-11 hagic shock. They found evidence of a r i s e in water, K and Na i n plasma measurements and a f a l l in i n t r a c e l l u l a r Na and a r i s e i n i n t r a c e l l u l a r K. These results were inexplicable even by the authors themselves. They therefore performed more experiments using deltoid biopsies and obtained further equivocal results. They concluded that t h e i r extra-c e l l u l a r tagewas unreliable i n shock. Shires et. a l . ^ 9 used ultramicroelectrodes to monitor c e l l membrane function in shock. Using baboons, subjected to severe haemorrhage, they monitored the transmembrane potential difference i n skeletal muscle c e l l s impaled with Ling electrodes. They also analysed the f l u i d and electro-l y t e content of muscle samples. They found increased levels of i n t r a -c e l l u l a r Na i n association with sustained muscle membrane depolarisation. They noted that these changes were reversed on resuscitation of the animal. S i m i l a r l y , with prolonged and severe haemorrhagic shock i n dogs they found that the resting membrane potential of muscle decreased from -90mV to -60mV. With this change i n potential difference and according to the Cl space, i t was calculatedffrom the Nernst equation that Na had entered the c e l l and K had l e f t . The i n t e r s t i t i a l space around the muscle was analysed with a micropipette and high K levels upxto 18 mEq/1 52 were found. Trunkey, i n simi l a r studies using baboons, came to similar conclusions and showed a 40 per cent reduction i n amplitude of the action potential and a prolongation of both the depolarisation and repolarisa-tio n times. Following resuscitation, amplitude and depolarisation time recovered, but the repolarisation time remained prolonged for up to 10 days, indicating that some impairment of c e l l membrane function may persist for this long period. Coleman^ investigated the electrolyte changes i n cardiac muscle of dogs during haemorrhagic shock. He measured the t o t a l Na and K levels and concluded that there was no s i g n i f i c a n t loss of i n t r a c e l l u l a r K. Brand^ performed similar studies and found sim i l a r results. Based on these studies, they concluded that a f a i l u r e of the transport mechanism for Na and K i n heart muscle with a consequent d e f i c i t i n myocardial energy metabolism was not a major factor in the pathogenesis of i r r e v e r s i b l e shock. 2 1 Essiet and S'fcahl studied water and electrolyte changes i n skeletal muscle, connective tissue and kidney i n rats subjected to haemorrhagic shock and surgical trauma. They observed a balanced movement of water, Na and K i n roughly normal proportions into r e l a t i v e l y a c e l l u l a r connec-tive tissue, but i n the more c e l l u l a r renal andmmuscle tissue examined they found that Na levels were increased s i g n i f i c a n t l y more than water and K. They postulated an Na-K pump f a i l u r e as the mechanism involved. Studies of changes i n red c e l l cations during shock have been 1 8 carried out i n both monkeys and humans. Curreri observed elevated c e l l Na levels in patients suffering from burn shock and also showed that the red c e l l Na level returned to normal levels as the patient's 39 c l i n i c a l condition improved. Johnson and Bagget measured red c e l l Na and K i n monkeys subjected to varying degrees of haemorrhagic shock. They found that i n prolonged haemorrhagic shock there was a movement of Na into red c e l l s and K outwards. Cunningham^ carried out similar studies on the red c e l l s of humans using normal volunteer controls and patients i n varying degrees of shock. They found also that i n severe or prolonged shock, there was a s i g n i f i c a n t r i s e in c e l l Na. Liver c e l l function has been widely studied by workers i n t h i s f i e l d . Sayeed and colleagues"^ have investigated i n d e t a i l the a b i l i t y of the 13 l i v e r to support active ion transport and maintain normal c e l l volume i n haemorrhagic shock. Using a rat experimental model, they showed that i n s l i c e s of l i v e r tissue there was a 100 per cent increase i n Na i n late shock and also a s i g n i f i c a n t loss of K. The same workers have demon-strated in other experiments that l i v e r , muscle and kidney ATP concentra-tions are decreased i n shock. Based on this they have used ATP-MgCl^ 13 for the treatment of experimental shock. In addition to metabolic studies, Baue described the morphological o / : changes i n l i v e r tissue i n haemorrhagic shock. Holden also studied the morphological changes i n greater d e t a i l . Sayeed has studied active Na and K transport and ATP levels i n the lung i n rats subjected to haemorrhagic shock. He found that the energy dependent transport of Na and K i n the lung was not altered. He postulated that direct u t i l i s a t i o n of atmospheric oxygen i n the lung might allow maintenance of c e l l u l a r energy levels during the low flow state which occurs. The same worker had previously shown that mitochon-d r i a l function i n the lung was not deficient i n haemorrhagic shock. 49 M i l l e r studied the metabolic changes in the brain i n shocked rats and showed only very minor changes. He suggested that selective compen-satory cardiovascular adjustments were responsible for preserving normality i n this tissue. 14 LITHIUM SUBSTITUTION ANALYSIS OF Na AND K IN VASCULAR SMOOTH MUSCLE IN THE RAT Although the vasogenic type of shock was one of the major categories as c l a s s i f i e d by Blalock and despite the importance of the vascular homeostatic mechanisms i n shock, very l i t t l e i s known about the changes which occur in vascular tissue during shock. The role of Na i n the regulation of vascular smooth muscle tension and the importance of reciprocal movements of Na + and JC1" i n association with acute vasocon-26 s t r i c t i o n have been discussed by Friedman and Friedman. The c e l l Na and K changes i n vascular smooth muscle have not been previously studied during haemorrhagic shock. U n t i l recently, there was no simple method available for the measurement of c e l l Na and K i n vascular smooth muscle. Whilst indirect methods, based on the use of ex t r a c e l l u l a r space markers have been used, the accuracy of such methods depends on the r e l i a b i l i t y 40 of ex t r a c e l l u l a r f l u i d volume measurements. In studying vascular smooth muscle c e l l Na and K there are special problems related to the paracellular matrix. This matrix binds ions within i t s network and also l i m i t s the r e l i a b i l i t y of e x t r a c e l l u l a r space markers, which are unable to gain access to this space. Friedman et a l . have recently studied this problem and have devised a method based on lithium iOJK'). •> subs t i t u t i o n , which has been used to measure vascular smooth muscle c e l l Na and K i n both normal and hypertensive states. Lithium was chosen because of i t s monovalent nature and because the a f f i n i t y of chondroitin sulphate (which forms the major part of the polyanionic gel of blood vessels) i s greater for L i than for Na throughout the physiological range. The p r i n c i p l e of this method is based on the fact that L i does not replace c e l l Na or K at 2°C, but exchanges readily with ex t r a c e l l u l a r Na at a l l temperatures. In his studies, Friedman showed that there was a d i f f u s i o n barrier preventing free exchange between Na and L i and that c e l l disruption removed the barrier and that the exchange was metabolically regulated. He showed that on incubating an artery i n a physiological saline solution, in which Na i s replaced by L i at 2°C for 30 minutes, the e x t r a c e l l u l a r Na was replaced by L i , leaving the c e l l Na unaffected (Figure 2). LITHIUM SUBSTITUTION ANALYSIS OF RED CELL Na AND K 29 In other studies Friedman et a l . have measured red c e l l Na and K using L i substitution analysis. They showed that L i was unable to cross the red c e l l membrane in either d i r e c t i o n at low temperatures and used the method to study the red c e l l Na and K changes i n hypertensive rats. Other workers have used MgC^ to wash out e x t r a c e l l u l a r Na i n order 17 18 39 to measure the red c e l l Na concentration i n haemorrhagic shock. ' ' However, the use of MgC^ for this purpose is undesirable, because i t s 3 different ionic strength may affect the result and because of i t s known 13 effects on ATP metabolism. I t has been shown that Mg i n vivo and i n 31 v i t r o i n h i b i t s the deamination and dephosphorylation of ATP. I t has 2+ also been shown that bivalent ions, including Mg play an important role in the structural i n t e g r i t y of the c e l l membrane. For these reasons the L i substitution method offers theoretical advantages for red c e l l s Na and K measurements. PURPOSE OF STUDY The purpose of this study was as follows: In a p i l o t study, to v e r i f y the v a l i d i t y of a rat haemorrhagic shock model and to measure the plasma sodium (Na) and potassium (K) changes occurring i n haemorrhagic shock. To apply a new ion exchange method, based on lithium (Li) substitu-tion, to the measurement of vascular smooth muscle and red c e l l Na and K i n haemorrhagic shock. To add to our understanding of the changes i n c e l l function which occur i n haemorrhagic shock. 17 MATERIALS AND METHODS HAEMORRHAGIC SHOCK MODEL (Figure 3) Adult male albino rats weighing 300 to 400 G were anaesthetised with an intraperitoneal i n j e c t i o n of 4-5mg sodium pentobarbital per 100 gm body weight. (The solution contained 15mg Na pentobarbital per ml.) After anaesthesia was obtained (10-15min) the rat was placed in the supine position and the hind legs were restrained loosely using a rubber band. Rectal temperature was monitored during the experiment. The forelegs were not restrained. A 2cm i n c i s i o n was made i n each groin and both femoral ar.terji'eswere exposed and dissected free. Both arteries were cannulated with polyethylene tubing (P.E.50). The cannulae were ti e d i n place with fine black s i l k . The d i s t a l end of one tube was attached to a 3-way stopcock and a p l a s t i c syringe was attached to permit bleeding into the syringe and the taking of blood samples. The other tube was used to monitor a r t e r i a l blood pressure v i a a transducer and polygraph recorder. After cannulation, 250 units of heparin were given i n t r a -a r t e r i a l l y through one of the cannulae. An i n i t i a l blood sample (0.1ml) was obtained and bleeding was i n i t i a t e d by allowing controlled haemorrhage into the syringe u n t i l the mean a r t e r i a l blood pressure was reduced to 30-35mm Hg. This degree of hypotension usually occurred after the removal of 8-9ml of blood i n 2-3 minutes. The blood pressure was then maintained at 30mm Hg for the desired length of the experiment by the removal or addition of small amounts of blood as necessary. Following the defined period of hypovolaemia, the animal was retransfused at 1ml per minute. Rectal temperature and syringe volume were recorded at 5 minute intervals. The body temperature was kept above 35 C by the use of a warming lamp when necessary. For each animal, the average bleeding volume was recorded and the estimated blood volume was calculated from the body weight. A l l animals were resuscitated at the end of the procedure and were returned to the cage, where they were allowed food and water ad l i b as soon as they were awake. I f an animal survived more than 48 hours i t was considered a survivor. The overall mortality for a 1 hour period of hypovolaemia at 30,235mm Hg was 20 per cent. PLASMA SODIUM AND POTASSIUM MEASUREMENTS A l l plasma Na and K measurements were made on 0.1ml samples of blood taken at intervals throughout the experiment. A glass electrode method, as devised by Friedman e_t aX_' was used for estimating the Na and K levels. This method uses cation sensitive glass electrodes and is not only fast and accurate, but can be used with very small blood samples. As a p i l o t study asseries of 20 rats were subjected to a 1 hour period of haemorrhagic shock, following which each animal was retrans-fused and allowed to recover for 1 hour. A pre-shock blood sample was taken and further samples were taken at 10 min, 30 min, 1 hour and f i n a l l y 1 hour after retransfusion. CELL SODIUM AND POTASSIUM IN VASCULAR SMOOTH MUSCLE A series of 30 rats was used i n this experiment. Ten paired rats were bled to an a r t e r i a l pressure of 30mm Hg as described above. This pressure was maintained for a 2 hour period. One of the pair was then retransfused and allowed to recover for 1 hour. Ten anaesthetised, but unshocked rats were used as controls. The glass electrode method described previously was used to monitor the plasma Na and K levels at 30 min intervals. With the rat i n the supine position, the rat t a i l artery was exposed by i n c i s i n g skin and fascia through a longitudinal ventral i n c i s i o n . The entire length of the artery was exposed by gentle dissection and the c o l l a t e r a l vessels were divided (Figure 4). The artery was transected at the base of the t a i l and at the d i s t a l end. The artery was then divided into equal halves, placed between 2 f i l t e r papers and gently pressed to remove blood from the lumen. The arteries were placed i n a physiological s a l t solution i n which Na had been replaced by WdtJpxac.t. «C 2°C, for 45 minutes. The artery was then blotted dry between 2 f i l t e r papers and weighed. I t was processed by dessication, defatting with ether and was re-weighed prior to extraction for 7 days with 0.75 N i t r i c acid. Cation measurements were made using atomic absorption ion analysis. The composition of the physiological solutions used for incubation are shown i n Table I. RED CELL SODIUM AND POTASSIUM In this experiment, 11 rats were subjected to haemorrhagic shock for a 2 hour period. Eleven control animals were anaesthetised and their femoral arteries were cannulated, but they were not bled. These controls were alternated with the test animals i n pairs. Again the plasma Na and K were monitored during the experiment. Blood samples of 2.0ml were taken from each of the experimental 20 animals at the end of the 2 hour period of haemorrhagic shock. Following t h i s , the shocked animals were retransfused with the reservoir blood and allowed to recover for 2 hours and a 2.0ml blood sample was again taken. Each sample was washed twice i n normal Kreb's solution at 2.0°C. The c e l l s were allowed to stand, with occasional s t i r r i n g , for 30 minutes. The suspended c e l l s were then centrifuged at 1550 rpm for 5 minutes, and 0.1ml samples of precipitated c e l l s were transferred to 5ml of d i s t i l l e d water to which 5ml of 10 per cent t r i c h l o r a c e t i c acid were added to precipitate the proteins. The Na and K were then measured by atomic absorption spectrophotometry. Sim i l a r l y , red c e l l Na and K measurements were made on 2.0ml samples from the controls. 21 RESULTS PILOT STUDY After one hour of hypotension at 30mm Hg, there was a small decrease i n plasma Na, from a mean pre-operative vale of 14L+1.0 mEq/litre to 138il.OmmEq/litre (Table I I ) . At the same time, plasma K rose from 4.7io.l mEq/litre to 6.0io.2 mEq/litre. Following retransfusion and recovery for 1 hour, the plasma Na returned to i t s pre-shock le v e l of 141±0.3 mEq/litre, and the plasma K f e l l to 5.2±0.2 mEq/litre. These results are summarised i n figure 5. The overall mortality of this degree of shock was 20 per cent within the 48 hour period following retransfu-s ion. CELL SODIUM AND POTASSIUM IN VASCULAR SMOOTH MUSCLE In vascular smooth muscle, s i g n i f i c a n t changes i n c e l l Na and K occurred following the 2 hour period of haemorrhagic shock (Table I I I ) . The mean c e l l Na i n the control animals was 27.0+1.5 mEq/kg dry weight whilst i n shocked animals the c e l l Na was 42.7+1.4 mEq/kg dry weight (p< 0.001). C e l l K was 127.8±6.0 mEq/kg dry weight i n controls and 74.7±4.2 mEq/kg dry weight i n the shocked animals (p<0.001). On retransfusion and recovery for 1 hour, c e l l Na was 43.1+2.0 mEq/kg dry weight and c e l l K was 81.9+4.3 mEq/kg dry weight. I t was not possible to correlate the degree of c e l l Na or K changes with the overall mortality, which was 25 per cent. Figure 6 is a histogram which summarises the findings i n this experiment. 22 RED CELL SODIUM AND POTASSIUM The results of red c e l l Na and K changes are summarised i n Table IV. Red c e l l Na i n control animals was 7.09+0.29 mEq/litre c e l l s and i n the shocked animals c e l l Na was 8.26±0.33 mEq/litre c e l l s (p<0.025). Red c e l l K i n control animals was 118.67+2.08 and i n the shocked animals i t was 115.87+2.11. Following retransfusion and recovery for 2 hours there was no s i g n i f i c a n t changes i n either red c e l l Na (8.22+0.28) or in red c e l l K (115.43±2.06). In both the vascular smooth muscle and red c e l l experiments there were s i g n i f i c a n t plasma Na and k changes, as summarised i n Tables I I I and IV. These findings simply confirmed those of the p i l o t study. I t was interesting that i n both experiments there was a persisting abnor-mality of c e l l Na changes after retransfusion, whilst the plasma Na and K both returned towards normal. 23 DISCUSSION THE EXPERIMENTAL SHOCK MODEL Haemorrhagic shock has been widely studied in the experimental ,66 laboratory, with the use of a canine model subjected to the Wiggers 4 procedure or a modification of his model. Bacalzo et a l . , i n experiments using male albino, rats, devised a standardised rat model., They analysed several c r i t i c a l factors, such as the role of bleeding volumes, haemody-namic levels and duration of hypotension. They concluded that the preferred model for haemorrhagic shock i n rats was based on allowing bleeding into an adjustable reservoir to induce sustained hypotension. 11 The rat haemorrhagic shock model was also studied by Butcher et a l . , who summarised i t s advantages as follows: (1) The reproducability of experi-ments was greater i n rats of a pure inbred s t r a i n than i n mixed pedigree dogs available i n most laboratories. (2) The time required to perform si m i l a r experiments was less i n the rat. (3) Experiments i n a given number of rats were less expensive than i n the same number of dogs. For these reasons, the rat model was used i n the present study, and the procedure used was similar to that of Bacalzo and Butcher. METHODS OF CELL SODIUM AND POTASSIUM MEASUREMENT The methods used for the measurement of c e l l Na and K i n this study 'ar.eebased on the observation that the entry of L i into c e l l s is blocked at low temperatures. In vascular tissue there i s an extensive paracellular matrix surrounding the vascular smooth muscle c e l l s . Previous methods of measuring c e l l Na and K i n this t i s s u e have been based on the use of e x t r a c e l l u l a r markers, which are u n r e l i a b l e because of t h e i r l i m i t e d access to t h i s p a r a c e l l u l a r space. L i , a monovalent ion, has a higher a f f i n i t y than Na for chondroitin sulphate, which i s a major component of the polyanionic gel of the p a r a c e l l u l a r matrix. I t was therefore chosen by Friedman as the basis of the method he devised for measuring c e l l Na 27 28 and K i n vascular smooth muscle. '>^° He showed that L i does not enter c e l l s at low temperatures and this allows the e x t r a c e l l u l a r Na to be r e a d i l y exchanged for L i at 2°C, leaving the i n t r a c e l l u l a r Na i n t a c t . C e l l Na and K could then be estimated simply as the r e s i d u a l a f t e r incu-bation i n a p h y s i o l o g i c a l s a l i n e s o l u t i o n i n which Na had been replaced by L i . Friedman has used t h i s method to measure c e l l Na i n vascular smooth muscle i n normal and i n hypertensive states. 29 The method has also been modified to measure red c e l l Na and K. In these studies i t was shown that L i does not cross the red c e l l membrane in e i t h e r d i r e c t i o n at low temperatures and i n applying t h i s method i t was shown that there was increased permeability to both L i and Na i n hypertensive r a t s . EARLY STUDIES OF SODIUM AND POTASSIUM IN SHOCK Early experimental workers had shown that the blood K increased i n a v a r i e t y of c l i n i c a l conditions, including haemorrhage, traumatic shock, severe g a s t r o i n t e s t i n a l disorders and following i n t r a p e r i t o n e a l injections 66 67 of glucose. Zwemer and Scudder believed that K released by c e l l i njury was the elusive toxic agent responsible for shock. However, experiments i n which the p h y s i o l o g i c a l e f f e c t s of intravenous K i n j e c t i o n were studied failed to substantiate the theory. Large doses simply caused arhythmias and not a shock like state. It was also noted that such arhythmias were not a feature of c l i n i c a l shock. 66 Wiggers, in his monograph on shock, stated: "... a marked degree of salt depletion can induce a l l the signs of shock independently of trauma or haemorrhage, and lesser degrees may aid the production of shock from trauma and other causes." 22 In 1940, Fenn showed that K le f t muscle cells after haemorrhage 54 and returned after blood or saline was transfused. Price also noted that the plasma K increased during terminal shock and also noted a slight 14 increase in plasma Na and CI. Clarke and Cleghorn were also able to demonstrate a rise in K in shock, but did not find any change in Na or CI. They also noted a f a l l in K in the liver of rats subjected to severe 48 haemorrhage. Miller found increased K levels in both skeletal muscle and in serum after scalding and haemorrhage and believed that this was due to loss of muscle protoplasm without an equivalent loss in K. CELL SODIUM AND POTASSIUM CHANGES IN HAEMORRHAGIC SHOCK Tissues used in studies of c e l l electrolyte changes in shock have • T A A i - 57 , . , 21 , , ^  . , 21,24,32,37,62 ,. included l i v e r , kidney, skeletal muscle, ' ' ' ' cardiac muscle, 15 r e ( j c e \ i s } 17,18,39 a n ( j connective tissue. ^1> 60 Although no one has studied smooth muscle Na and K changes in shock, studies on skeletal muscle have shown consistently that there is a gain in Na and a 24 loss of K in haemorrhagic shock. Flear, in c l i n i c a l studies of patients undergoing major surgical procedures, and in experimental studies in which he performed microanalysis of single muscle fibres, has noted an increase 26 i n Na concentration i n muscle. In association with these findings he found a 26 per cent f a l l i n c e l l K i n the isolated muscle fi b r e micro-21 32 62 analysis.. Many workers have reported si m i l a r findings, ' ' the 3 8 exception being Johnson and Tucker who used Na2S0^ as an ex t r a c e l l u l a r marker and studied the plasma Na and the c e l l Na and K i n the rectus abdominis muscle of dogs subjected to haemorrhagic shock. They found an increased Na and K i n plasma and a decreased c e l l Na with an increased c e l l K. They were unable to explain these results, however, and concluded that t h e i r e x t r a c e l l u l a r tag was not r e l i a b l e i n shock. Coleman and Glaviano''""' and Brand^ were unable to find any change i n c e l l K i n cardiac muscle during haemorrhagic shock. They concluded that a f a i l u r e of the Na and K transport mechanisms with a consequent f a i l u r e of myocardial effi c i e n c y was not a major factor i n the pathogenesis of haemorrhagic shock. The studies of c e l l Na and K changes i n vascular smooth muscle performed i n the present study have shown that these c e l l s gain Na and lose K i n haemorrhagic shock. This fact may have important implications with regard to the pathogenesis and treatment of haemorrhagic shock. The importance of Na and K movements i n the regulation of vascular 26 tension have been reviewed by Friedman and Friedman. In view of the role of Na and K i n the regulation of vascular responses i t i s l i k e l y that a s i g n i f i c a n t change i n the transmembrane d i s t r i b u t i o n of these ions, as shown i n the present study, might result in impairment of the usual vascular homeostatic mechanisms which occur following severe haemorrhage. Alterations i n red c e l l Na concentration has been shown to occur i n 63 a number of different c l i n i c a l conditions. Welt et a l . reported that red c e l l Na was raised i n patients with uraemia. The same worker noted 27 64 39 similar changes i n cancer and severe burns. Johnson and Baggett studied red c e l l f l u i d and electrolyte changes during haemorrhagic shock i n the monkey. In these studies, they measured red c e l l Na and K after washing the c e l l s i n isotonic MgCl^- They found a small r i s e i n c e l l Na and a f a l l i n c e l l K. They also found a s i g n i f i c a n t r i s e i n plasma K and a small, but s t a t i s t i c a l l y i n s i g n i f i c a n t r i s e i n plasma Na. 17 Cunningham e_t al_. carried out studies on patients i n varying degrees of shock. They noted a marked increase i n red c e l l Na i n severe shock. They used a similar method to that of Johnson and Baggett and found that red c e l l Na i n severely shocked patients was 17.0+5.8 mEq/litre c e l l s , whilst i n controls i t was only 7.5+2.1 mEq/litre c e l l s . They noted that the magnitude of c e l l Na increase was a function of the severity and duration of shock and could be well correlated with changes i n the c l i n i c a l course when sequential measurements were made. In the present study we have confirmed that the red c e l l Na does r i s e i n haemorrhagic shock. The L i method of red c e l l Na and K measure-ment used i n this study has two potential advantages over MgC^ washing of red c e l l s . F i r s t , MgCl2 has the disadvantage that i t s different ionic 3 strength may affect the results. Second, i t may influence the Na-K 31 pump through i t s known effects on ATP. Green and Stoner have shown that both i n vivo and in v i t r o Mg i n h i b i t s the deamination and dephosphorylation of ATP. MgCl2 has been used i n studies concerned with the therapy of 13 haemorrhagic shock because of these effects on ATP metabolism. The fact that the red c e l l membrane appears unable to maintain a normal c e l l Na concentration may have important implications i n r e l a t i o n to red c e l l function i n shock. The f a l l i n plasma Na and the r i s e i n plasma K noted i n the present 28 study were expected in view of the c e l l changes found. The r i s e i n plasma K has been a consistent finding i n many studies, but there have been 38 39 54 studies where an increased plasma Na was found. ' ' On retransfusing the animals i t was noted that after a 2 hour period of recovery, the plasma Na and K recover, whilst the c e l l Na and K do not. This was the case i n both the vascular tissue and red c e l l studies. Thus, the fact that the plasma Na and K have recovered does not necessarily mean that the c e l l membrane dysfunction has recovered. These findings are consis-41 tent with those of Kenney and Randall, who described long term changes in plasma volume and c e l l u l a r electrolytes following haemorrhagic shock i n dogs. Thus, the impairment i n c e l l membrane function responsible for the c e l l Na and K changes i n shock appears to persist for some time after retransfusion, even though this may not be reflected by the plasma Na and K levels. THERAPEUTIC IMPLICATIONS OF CELL SODIUM AND POTASSIUM CHANGES IN SHOCK The apparently widespread changes i n c e l l u l a r and ex t r a c e l l u l a r Na and K in haemorrhagic shock have possible therapeutic implications. Some 2 early workers, such as Amberson, regarded the effects of isotonic saline infusions as harmful. Others disagreed and supported the use of saline solutions i n shock.''" More recently, experimental studies in rats have confirmed that increased survival i s achieved when electrolyte solutions (such as normal saline or Ringer's lactate) are used to supplement blood transfusions.''"''"'"'^ These studies have shown that i n severely shocked rats survival i s doubled when blood transfusion i s supplemented by 5 8 Ringer's lactate infusions. Shires has been one of the strongest 29 advocates of the use of balanced s a l t solutions i n the treatment of shock. Using tagged Na2SC>4 he noted a decrease i n ext r a c e l l u l a r f l u i d space following severe haemorrhage, which was not reversed by the replacement of shed blood alone. He concluded that there was a functional d e f i c i t i n the ext r a c e l l u l a r f l u i d compartment with haemorrhage and that el e c t r o l y t e was required with blood for proper treatment. The advantages of saline solutions either alone or i n combination with other infusions i n the management of shock might be explained by changes in transmembrane d i s t r i b u t i o n of Na and K which occur i n shock. As Na enters c e l l s and K leaves, a vicious c i r c l e is set up, whereby the c e l l membrane and the Na-K pump might suffer further damage. Attempts to maintain the normal ratios of ions across c e l l membranes by the infusion of appropriate electrolyte solutions seems a l o g i c a l step to take in an ef f o r t to stop this vicious c i r c l e . The arguments i n favour of electrolyte solutions for early resuscita-52 t i o n have been discussed i n d e t a i l by Moss. He quoted the following 42 paragraph from W. Arbuthnot Lane, written i n 1891, and thought to be the f i r s t documented use of s a l t solution i n the treatment of haemorrhage: I t came to my knowledge but a few weeks ago that our l a t e lamented colleague, Dr. Wooldridge, had, shortly before his death, been making experiments on transfusion i n animals, by means of which he was able to show that the usually accepted views as to the i n u t i l i t y of introducing saline solutions into the circ u l a t o r y system to replace blood l o s t i n severe haemorrhage were absolutely false. He found that by the in j e c t i o n of a s u f f i c i e n t l y large quantity of saline solution into the vessels of a dog which had lost enough blood to result i n death he was able at once to restore the animal to a c t i v i t y and health. He thereby showed that after an animal had sustained a loss of blood s u f f i c i e n t to terminate i t s l i f e , there was l e f t i n the blood vascular system enough haemoglobin to sustain l i f e , i f only enough f l u i d be added to keep i t in c i r c u l a t i o n . Moss further reviewed the known c l i n i c a l experience and t r i a l s using saline solutions and provided evidence that there was an increased requirement for sodium during shock. He based this need partly on evi-dence for the contraction of the i n t e r s t i t i a l space and the adsorption of Na and water on i n t e r s t i t i a l collagen during shock. He noted that the use of saline was not associated with the development of; pulmonary oedema. He also described the t r i a l s carried out by the United States Navy unit i n Da Nang, which showed that massive infusions of saline (12 l i t r e s ) i n addition to blood replacement i n massively injured patients resulted in improved survival. The other therapeutic implications of the changes i n c e l l Na and K found relate to the use of more s p e c i f i c agents such as membrane s t a b i l i s i n g drugs (e.g. steroids) and Na-K pump stimulators (ATP-MgC^) • The use of high dose steroid therapy i n shock i s at present undergoing extensive c l i n i c a l investigation. Based on studies which have shown depleted ATP levels i n shock"^ and on the theory that the changes i n c e l l Na and K found i n shock may be due to impairment of the Na-K pump, attempts have been made to treat shock with infusions of ATP-MgC^ i n 13 addition to volume replacement. Chaudry et a l . have shown that this treatment i s beneficial i n experimental studies and have shown that there i s improved survival in rats treated with such infusions. The above discussion of the therapeutic implications of the c e l l Na and K changes relates to the generalised change i n c e l l Na and K found in haemorrhagic shock. In r e l a t i o n to the present study, in which the changes in vascular smooth muscle and red c e l l Na and K have been studied, there are some more sp e c i f i c implications which have been mentioned previously. Thus, the possible impairment of vascular r e a c t i v i t y resulting from the changes i n vascular smooth muscle and the impaired red c e l l function which may resul t from the red c e l l Na changes may s i g n i f i c a n t l y affect the course and prognosis of haemorrhagic shock. 32 SUMMARY AND CONCLUSIONS The effects of haemorrhagic shock on the changes i n c e l l u l a r and extr a c e l l u l a r (plasma) Na and K were studied i n the rat. Shock was induced by allowing controlled haemorrhage into a syringe reservoir, u n t i l the a r t e r i a l pressure was 30mm Hg, and this pressure was maintained for a defined period by the removal or addition of blood as necessary. C e l l Na and K changes i n vascular smooth muscle and i n red c e l l s were studied by an L i substitution method. This method is based on the fact that L i does not enter c e l l s at low temperatures. The plasma Na and K were measured by a glass electrode method. After a 2 hour period of haemorrhagic shock, there was a s i g n i f i c a n t r i s e i n c e l l u l a r Na i n both vascular smooth muscle and i n red c e l l s . There was a corresponding f a l l i n c e l l K i n vascular smooth muscle, but only a small f a l l i n red c e l l K. Plasma Na f e l l and plasma K rose during haemorrhagic shock. By i n t e r f e r i n g with the normal vascular homeostatic mechanisms which occur following severe haemorrhage, and by impairment of red c e l l function, the disturbed c e l l membrane function demonstrated i n these studies may have important implications with regard to the treatment and prognosis of haemorrhagic shock. 33 TABLE I The Compositions of Physiological Salt Solutions (mM), Aerated with 95% 0 2, 5% C0 2 (ph 7.4±0.1 at 37°C) Solution Na K Ca Mg Cl HCOo HPO, L i COq Gluco se Norm 141.2 5 1.7 1.2 123.4 25 1.2 - - - 11 (PSS) L i Subst. - 5 1.7 1.2 123.4 - 1.2 141.2 25 11 (LiPSS) 34 TABLE I I Plasma Na"1" and K + Changes i n Haemorrhagic Shock A l l values expressed i n mEq per l i t r e ± S.E. of the mean TIME (min) Na K Pre-Shock 141±1.0 4.7+0.1 lfl!0 140.4±0.7 5.4±0.2 30 139.0+0.6 5.2+0.2 60 138±1.0 6.0±0.2 120 (recovery) 141+30.3 5.2+0.2 TABLE I I I I n t r a c e l l u l a r and Plasma Na and K Measurements in Vascular Smooth Muscle Control (10) Shock (10) Shock Plus Recovery (10) C e l l Na 27.0+1.5 42.7±1.4 43.1+2.0 C e l l K 127.8+6.0 74.7±4.2 81.9±4.3 Plasma Na 141.0+1.0 137.0±1.0 140.0+0.4 Plasma K 4.8+0.1 6.1+J0.2 5.3+0.2 (Cell Na and K values i n mEq/kg dry weight, Plasma Na and K i n mEq/1 + S.E. of the mean.) 36 TABLE IV Red C e l l and Plasma Na and K in Haemorrhagic Shock Control (11) Shock (11) Recovery (11) Red C e l l Na (mEq/1 c e l l s ) 7.09±0 .29 8.26+0.33* 8.22±0. 28 n s Red C e l l K (mEq/1 c e l l s ) 118.67±2 .08 115.87+2.ll n s 115.43±2. 06 n s Plasma Na (mEq/litre) 140.5 ±1 .1 136.0 ±1.2* 141.2 ±0. 8** Plasma K (mEq/litre) 4.6 ±0 .1 5.9 ±0.1** 4.7 ±0. Values are mean ± S.E. *p 0.025 **p 0.005 ns = not s i g n i f i c a n t 37 Blood plasma Interstitial fluid Cell fluid Fig. 1 Note that the Na and CI are mainly e x t r a c e l l u l a r , whereas K and PO^ are mainly i n t r a c e l l u l a r . The barriers separating these compartments are responsible for the different compositions. (From Leaf and Newburghi Significance of Body Fluids i n C l i n i c a l Medicine, 2nd Ed. Thomas, 1955). 38 o Fig. 2 Diagramatic representation of L i replacing Na i n the e x t r a c e l l u l a r space, including the paracellular matrix, following incubation i n LiPSS at 2°c. .39 Fig. 3 Rat Haemorrhagic Shock Model. Both femoral arteries are cannulated, one being connected to a transducer and polygraph recorder to monitor the a r t e r i a l blood pressure and the other to a syringe to allow bleeding. V E N T R A L T A I L A R T E R Y E X C I S I O N Fig. 4 The entice length of the rat t a i l artery i s exposed by i n c i s i n g skin and f a s c i a , and after dividing c o l l a t e r a l vessels i t i s transected at i t s base and d i s t a l end. 41 Fig. 5 Extracellular (Plasma) Na and K Changes. The s l i g h t f a l l i n plasma Na coincides with a marked, r i s e i n plasma K. Following retransfusion and recovery for one hour, both tend to return towards the control l e v e l s . 42 Fig. 6 I n t r a c e l l u l a r Na and K Changes i n Vascular Smooth Muscle. There i s a sig n i f i c a n t r i s e i n c e l l Na i n the shocked animals, with no si g n i f i c a n t recovery after retransfusion and recovery for one hour. There i s a corresponding f a l l i n c e l l K, which also f a i l s to recover within that period. 43 BIBLIOGRAPHY 1. A l l e n , F.M. Salt solutions in shock. Am. J. Surg. 38:155, 1939. 2. Amberson, W.R. Blood substitutes - review. B i o l . Rev. 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