$45915 1.1359114? Michigan grate 1 University l fl This is to certify that the thesis entitled "Protective Mechanisms of Intestinal Luminal Glucose—Oleic Acid Instillation in Hemhorragic Shock" presented by I Joshua M. Halper has been accepted towards fulfillment of the requirements for M.S. . Ph 1 1 degree 1n ys O ogy eff/‘17 M/éw Major professor February 13, 1985 I)ate 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES m RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. i .__-_ _- WWISMSOFMESPINALHl‘flNALm-OLEIC ACIDINSTIIIATIQ‘IINHEIDRRHAGICSI-m Joshua Park Halper A THESIS Suhnitted to Michigan State University in partial fulfillment of the requirements forthedegreeof MOFSCIM Department of Physiology 1984 PIUI'EITI'IVE MECHANISdS OF INTESTML IUMINAL GLUCOSE-OLEIC ACID INSTILIATIQJ IN HEIDRRHAGIC SHIXZK By Joshua Mark Halper Beneficial effects of perfusing solution containing 150 um glucose and 40 nM oleic acid (G+O.A.) through the intestinal luren of dogs during severe herorrhagic shock were determined in anesthetized dogs. Aortic pressure was lowered to 35 man for 3 hrs followed by reinfusion of all shed blood. All dogs were treated with a duodenal instillation of either G+O.A. or nomal saline (N/S). Perfusion of own. increased survival rate (67%) in dogs. Blood glucose (as) decreased to 20-40 mg% in N/S dogs and died within 8 hours. G+O.A. dogs absorbed 70% of perfused glucose and maintained normal BG for 26—41 hrs. Intestinal volume absorption was same for both treatment groups. Intestinal biopsies showed nucosal sloughing, congestion and hamrrhage in N/S dogs, while GI-O.A. dogs had normal intact mucosal surface. The activities of cardiodepressants were unaltered in all sanples for GI-O.A. dogs but increased in all sanples of N/S dogs. DEDIQTION Tomyfamilyandfriendswhogavenethestrength andperseverancetocarplete this work. ii I would like to thank Dr. William Frantz for serving on my calmittee. Special thanks to Dr. Robert Pittman for his valuable assistance and support. I especially would like to thank Dr. (thing—Chung Chou for his guidance and wisdan during my study of physiology, research training, and in carpletion of this thesis. I also would like to thank Denise Ingold Wilcox for her assistance and technical expertise. iii IJiSt Of Tables 0 0 I O O O O O O O O O O O O O O O O O O O 0 List Of Figures 0 O O O O O O O O O O O I O I O O O O O O 0 List of Abbreviations . . . . ...... . ........ I. TABIEOFWS Literature Review A. B. C. D. E. Introduction......... ..... General Circulatory Sequence in Response to Hmmage O O O O I O O O O O O O O O O O CardiacOutputandtheHeartinShock . . . . . Total Peripheral Resistance inShock . . . . . . SynpathoadrenalRespmsesinShock....... RenalRespcnseinShock............ Response of Intestinal and Splanchnic CirculationinShcck............. InterstitialFluidNbvementinShock. . . . . . IntestinalFluidAbsorptiminshock. . . . . . GlucoseandbbtabolisninShock . . . . . . . . ToxicFactorsinShock............. IntestinalPathologyinShock iv 10 14 18 20 26 27 TABLE OF WIS (Continued) II. murals m W O O O O O O O O O O O O A. B. C. D. E. Protocol for Hanorrhagic Hypctension . . . AbsorptionandCardiodepressantSeries.. Histopatholcgy....... ..... .. III . Results A. B. C. D. SurvivalSeries............. Iv. Dime“ I O O O O O O O O O O O O O O O O O V. SmmaryandConclusimsW... VI. ListofReferenoes.............. Page 30 30 33 33 35 36 38 45 .58 59 63 77 78 Table 1 . Table 2. Table 3 . Table 4 . LISTOF'EBIES Page Nurber Carposition of the glucose—oleic acidsolution............... 32 Fluid volune and glucose absorption in thedogstreatedwithGi-O.A.amiN/S.... 52 Time-course of plasma protein concen- tration (g/100 ml) in the own. andN/S-treateddogs............ 56 Tine-course of pH and blood gases (nuflg)1ntheG+O.A. andN/S- treateddogs.............. 57 vi LIST OF FIGURES Figure l. Synpathoadrenal responses in cardio- vascular changes associated with hypotension and circulatory shock. Alpha adrenoceptors associated with arteries and veins cause vasoconstriction , label with a (+) . adrenoceprtors associated with the causes increased myocardial ccntractility and heart rate: (+) . Beta adrenoceptors associated with arteries cguse vasodilatation; (-l . "Dopaminergic" receptors associated with mesenteric and renal arteries cause vasodilatatiom(-)............. 5 Figure 2 . Schenatic representation of the patho- physiological mechanism responsible for the developrent of mucosal lesions of the small intestine in a hypotensivestate.............. .16 Figure 3. Tine-course of mean arterial blood pressure (upper panelland glucose concentration (lower panel) in dogs treated with G+O.A. (salid line) and \mtreateddogs(dashedline)........ 40 Figure 4. Tine-course of changes in arterial blood pressure and glucose concentration in seven dogs treated with normal saline instillation through the intestinal lumen. Arrow indicates the initiation oftheinstillation.n=7. ......... 44 Figure 5. Tine-course of changes in henatocrit and heart rate during the experiment in dogs treated with G+O.A. (solid line) and N/S (dashed line) in the survival series. n=9 for G+O.A., and n=7 for N/Sgrcup 4'7 LIST OF FIGURES (Continued) Page Figure 6. Time-course of blood vclmne and mean arterial blood pressure in dogs treated with Gd-O.A. (solid line) and normal saline (dashed line) before, during, and after hencrrhagic hypo- tension for 3 hours. Arrow denotes the beginning of the treatments. 'Ihe nunber in parenthesis indicates the voltme of fluid absorbed fran the intestine in ml/kg. n=8 for each treatment . Asterisks indicate values significantly different from the corresponding values of the normal salinetreateddog.............. 50 Figure 7 . Time-course of changes in henatocrit andheartrateduringtheexperiment in dogs treated with (30$. (solid line) andN/S (dashed line) in the 'on series.. n=8 for G+O.A. (GroupA) andn=8foerng1p (GroubB).................. 55 viii LIST OF FIGURES (Continued) Figure 8 . Representative paper chrcmatogram showing 1) plasma fran blood reservoir, 2) plasma f ran pre-herorrhage feroral arterial sample (2A), 3) plasma from pre-helorrhage portal venous sample (3V) , 4) plasma fran post-heronhage fetoral arterial sample (4A), 4) plasna frun post-tarorrhage portal venous sanple (5V), and 6) serine standard. Rsvaluesareshcwnandeachspotis given a letter designate in order of * increasing migration frm the origin. Darkest spots are the serine standard (C), followed by large dense lines (D), thedcttedspots (A), andopen faint spots (B, C, E andF). Double arrows indicate location of cardiodepressants and single arrow denotes the serine standard. The density of the lines in spot D represents the intensity of the spot, hence no lines inside the spot indicates less intense spot. ‘IheRsvaluereferstothemigration distanceofanyspotonthechrana— tographinrelationshiptothe distancethattheserinestandard hasmigrated................ 61 C.O. WUA. Gch H O R. MABP “3F N/S LISP OF ABBREVIATIODB adenosine triphosphate cardiac output Glucose-Oleic acid solution, bubbled with 95% oxygen - 5% carbon dioxide gas arterial glucose concentration henatocrit heart rate mean arterial blood pressure myocardial depressant factor Normal saline, bubbled with 95% oxygen and 5% carbon dioxide gas W10)! Chou, Kvietys, Post and Sit (1978) showed that instillation of chyrre through the lumen of the small intestirne markedly increased the local intestinal blood flow. Glucose (Chcal, Burns, Hsien and Dabney, 1972) and oleic acid (Gnu, Hsien, Yu, Kvietys, Yu, Pittman arnd Dabney, 1976) are the major cantributing element of the physiological chyue that produces the hyperennic state. The nutrient-induced intestinalhypereniaisconfinedinthenucosallayerandnotinthe nuscularis layer of the gastrointestinal tract (Chou et al., 1976). 'nnerationale forthepresent studywas thatperfusionof glucoseand oleic acid would increase local blood flow and oxygen delivery, especially tonthe nucosal tissues, and would sumly energy subStrates totl‘neischennicandhypcxicnucosaduringhercrrhagic shock. The purpose of this study was to elucidate the possible mechan- isns of the beneficial effects of the glucose-oleic acid solution in prevention of irreversible hencrrhagic shock. Specifically we deter- mined whether the treatment would prevent hypoglycemia, maintained blood volume by intestinal fluid absorption , prevent intestinal nucosal danage and cardiodepressant release. I. LITERATURE REVIEW A. Introduction A basic feature of hennorrhagic shock is that blood flow through thevessels ofthemicrocirculatim issoinpairedastoresultin cellulardamage. Aconterporaryquestianiswhethertheetiologyof shock is due to poor cardiac performance and/or peripheral insuffic- iency. Shock my exacerbate beyond the limits of control if hemrrhage is not corrected (Wiggers, 1950) . 'Ihe trarnsition fran reversible to irreversible status has been suggested by Crowell and Guyton (1961) tobeduetoanacutecardiacfailure. Ithasbeenproposedthatthe ”circulatory weak spot" in irreversible shock is the heart itself. B. GeneralCirculatorySequenceinResponsetoHenorrhage 'n'nerearemmerouswaystoecplainthephasesofhetorrhagic shock. A sinplified approach used by Guytcrn (1976) is based'ou the eventual outcate. With mild hemrrhage (less than 10-20% of total blood volume) the body will carpensate, preventing deterioration of the circulation. This form of shock is called nonprogressive or carpensated shock. The first line of defense is mediated ttmngh the baroreceptor reflexes. The reduction inneanarterialbloodpressure (MABP) andpulsepressureduringhenorrhageresultsindiminished stitmlationofthebaroreceptors locatedinthecarotidsinusesand aorticarch. 'n'nediminisl'nedingnlsedecreasesstinulationofthe vasarotorcenterandresultsinanincreasesynpatheticdrivetothe heart andblood vessels (Berna arnd Levy, 1977). By further reduction in MABP below 50—60 mag, Neil (1962) dermstrated an increased sympathetic drive via the chempreceptor. reflex. This reflex is also responsible for peripheral vasoccnstriction, an increase in force and rateofrespirationandanincreaseinvemsretum. However, the chaloreceptor reflex is not responsible for an increase in a cardiac synpathetic response (Downing and Siegel, 1963) . If the herorrhage goes beyond 20% of one's total blood volume, the state of shock will worsen. Beyond a critical set point, shock becomes progressive. The events themselves will becane a vicious cycle until canplete deterioration of the circulation occurs. Hypotension or hypcvolenia causes activatian of cardiovascular reflexes which result in a general increase in syupathoadrenal activ- ity as sham schematically in figure 1. ‘lhis figure also illustrates the deleterious positive feedback loop associated with circulatory shock. Hypcvolenia results in sympathetic discharge stixmlating release of epinephrine and norepinephrine fran the adrenal medulla, arnd increased release of norepinephrine at syrrpathetic neuroeffector junctions of the heart, kidney and blood vessels. Release of these catecholenines cause increased myocardial ccntractility, heart rate, and peripheral vasoconstriction. Figure 1 also shows the splanchnic vascconstriction and resulting visceral hypoxia and ischenia leading to breakdown of lysosanal metbranes. The result is release of auto- lytic lysosanal enzyms into the circulation , formation of endogenous vasoactive substances, and possibly formation and release of a specific toxic factor(s) like myocardial depressant factor (NF) (Mans and Parker, 1979) . Figure 1. Synpathoadrenal responses in cardiovascular changes associated with hypotension and circulatory shock. Alpha adrenoceptors associated with arteries and veins cause vasoconstriction, label with a (+). Beta1 adrenoceptors associated with the heart causes increased myocardial contractility and heart rate; (+). Beta2 adrenoceptors associated with arteries cause vasodilatation; (-) . "Dopaminergic'I receptors associated with mesenteric and renal arteries cause vasodilatation; (-) . Figural Adrenal <£H [(1% Medullo u: 9 £4." ARKMDC] 9 Reninémg e - M +‘EJRenflod Vessels Reflexes * T o 'W:cerol Blood Vessels ' - ‘ Peripheral; Blood Vessels I A ° Hypolenslon .. * CNS‘ Stagnant Hypoxia Lysosoml Rupture Vaeoactive Autecoide MD! 0 . alpha odren‘oceplors a a beta odrenoceplors o -doponninergi_c receptors C. Cardiac Ougnut and the Heart in Shock The fmndamental physiological defect in shock is a reduced cardiac output (C.O.) which may result fronn decreased myocardial efficiency or fron reduced circulatory filling. Investigators have differing opinions of the relative importance of cardiac failure during the earlier stages of hypotension. Herorrhage reduces the rate of coronary blood flow and therefore tendstodecreaseventricular function. Theconsequentreductionin C.O.duetohetorrhage leadstoafurtherdeclineinarterial pressure, a classical exanple of a positive feedback mechanism (Berne and Levy, 1977). The myocardial ischennia results fronn inadequate coronary perfusion during severe hypotension (Carlson, Selinger, Utley and Hoffman, 1976; Hackel, Ratliff and Mikat, 1974). Studies by Kleinman, Krause and Hess (1979) and Jones, anith, DuPont and Williams (1978) provided evidence for decreased coronary blood flow, subendocardial ischenia, necrosis, hemrrhage, and increased coronary vascular resistance in henorrhagic shock. 'lhey detonstrated by tracer technique that regions of the left ventricle were totally unperfused in late henorrhagic shock. To prove cardiac hypofunction, Crowell and Guyton (1962) used cardiac output curves (plotting atrial pressure against the resulting cardiac output) to show progressive changes during the course of irreversible herorrhagic shock. AlthoughC.O. isausefulindexofmyocardialfunction,Rothe and Selkurt (1964) used stroke work or minute work as a more inclusive factor for evaluating cardiac function. Rothe and Selkurt showed an increase in heart rate following hennorrhage, however, at the last hour of hypotension, but prior to transfusion, there was a significant decrease in heart rate over the maxinun hemrrhage (bleed out) period. Myocardial dearession is a factor in the developnent of irrever- sible hemorrhagic shock but the dearession is not observed until at least 20% of the henorrhage blood volume had spontaneously returned fronn the hemorrhage reservoir to the animal (Rothe, 1966). Early declinesincardiacoltpltareduetotheprogressivereductionin cardiac filling. the noderate herorrhagic hypotension and the cardiac/peripheral vascular damage could be corrected by fluid transfusions (Rothe and Selkurt, 1964). D. Total Peripheral Resistance in Shock Hemorrhagic shock generally has a greater effect on C.O. than arterial pressure. With a small blood loss (10% blood volme) the arterial pressure is maintained, while C.O. usually decreases. mny investigatorsusingacaninenodelhavefonmdanincreaseintotal peripheral resistance (TPR) after severe henorrhage (Bathe, Love and Selkurt, 1963). With prolonged hemorrhagic hypotension, TPR gener- ally decreases toward the prehenorrhage control levels. This gradual decline of TPR can be due to 1) a release of vascdilator materials into the circulation fronn ischemic organls), 2) the local accnmu- 1ation of metabolites that cause vasodilation, 3) a decrease of synpathetic vasoconstrictor inpulses, 4) a reduction of circulating catecholamines, and/or 5) the refractoriness of blood vessels to catecholannines and possibly acidosis, which reduces vascular reactiv- ity (Chien, 1967). Another question arises concerning central nervous systen depression. One possible explanation for the decline in TPR may stern frondamagetothenervepatlmwvmethertheoriginbecentralor peripheral, still remains unclear. Hinshaw (1971) has shown a suppression of autoregulation and spontaneous vasorotion, and Zweifach (1965) docnmented a diminished activity of the vascular smoothnuscleduringandaftertl'neherorrhagic insult. Duringthe laterstagesofhenorrhagicshock, theterminalvascularbedbehaves more as a passive structure and no longer exhibits active adjustments to circulatory disturbances (Crowely and Tnm'p, 1982) . E. Syupathoadrenal Responses in Shock With a significant reduction in blood volute, cardiovascular reflexes are activated and result in a generalized increase in synpatmadrenalactivity. missynpathoadrenalresponsecausesthe release of a large amount of activating catecholamines . Epinephrine and rnorepinephrine are released from the adrenal medulla. Norepin— ephrine is also released from adrenergic nerve terminals innervating blood vessels and the heart. 'Ihese endogenous synpathetic nediators areactiveinthecotpensatorycirculatorychangesseenduringthe various stages of shock (Adams and Parker, 1979) . Freednan arnd Miller (1941) , with infusions of epinephrine at low concentrations (3.4-16.4 ug/kg) produced shock and death in dogs. Poole and Watts (1959) showed that Freedman and Miller's lowest infusion rate produced epinephrine arterial blood levels of 39 ug/l. This levelproducedshockanddeathinnornovolenicanimals. In 1964, Watts and Westfall were unable to detect catecholamines in a series of eight dogs whose arterial pressure was experimentally lowered ' from their prehemrrhagic control levels to approximately 80 mag (in30angstepreduction). Onlyafterfurtherreductionin pressure were significant levels observed. Watts (1956) docnmented a 5-10-fold increase in rnorepinephrine and a 50-100-fold increase in epinephrine during henorrhagic shock in the anesthetized dog. Richardson (1965) and other investigators have verified Watts' observations arnd also have show significant increases in arterial rnorepinephrine in the dog during shock. The use of steroids will alleviate sore effects of catechola- mine. High levels of cortisol and corticosterone, are produced in excessduringshockevenwhenhypotension is severe, prolonged, arnd whenbloodflowtotheadrenaloortetiscorpronised. largedbsesof glucocortiooids have been shown to exert a beneficial influence in ecperinentalshockonlywhenadninisteredwithinthefirst few minutes of the onset of shock and in the proper dosage (Griffiths, 1972) . Generally, the beneficial effects of steroids may be related to vasodilation , increased production of adenosine triphosphate (ATP), increased conversion of lactic acid to glycogen, and stabil- ization of lysosonal nerbranes . F. Renal Response in Shock With decreased perfusion pressure, blood supply to renal vascular beds will be tenporarily maintained by the autoregulatory control. If the hemrrhagic hypotension persists, renal blood 10 flow is diminished and urine formation ceases when systenic arterial pressure falls to 50 mnflg (Bell, 1972). If blood pressure is, not brought under control, an accorpanying rise in renal sympathetic rerveactivitycausesthesecretionofreninfronthejmctaglotenllar apparatus. Renin converts angiotensinogen to angiotensin I. A cowertingenzynefomdinthelmngsandothertissueconvertsthis octapeptide into a very potent vasoconstrictor, angiotensin II, both fomnsof angiotensinaidindirectlyinwaterconservationbythe kidney. AngiotensinIIhasalsobeenshomtostinulatetheadrenal cortetinreleasingaldosteronewhichactstoconservebodysodiun arnd neintain blood volure (Guyton, 1976) . Hypotension has been shown by Logan, Jose, Eisner, Lilienfield and Slotkoff (1971) to cause an intrarenal redistribution of blood flow to the inner cortical nephrons. Several factors may play a role in the redistribution of intrarenal blood flow. The following‘alter- ations inrenalhenodynannicsmayberelatedtohetorrhage: increased hmoral release of rnorepinephrirne, increased huncral release of angiotensin, enhanced adrenergic stimllation, and diminished renal perfusion pressure. The intrarenal infusion of either norepinephrine or angiotensin, as well as renal nerve stimulation, all increased renal resistance, but did not alter cortical distribution of blood flow (Rector, Stein, Bay, Osgood and Cerris, 1972). G. Response of Intestinal and Splanchnic Circulation in Shock Considerableinteresthasbeenfocusedontheintestineasa target organ in shock. Lillihei (1957) introduced the original concept of an "intestinal factor” in irreversible hemrrhagic shock. 11 The theorywhich inplicatesthe intestineas atargetorganwasbased on two unrelated findings: 1) cardiac depression occurred late in the shock state even in the absence of coronary insufficiency, pointing to possible production of circulatory cardiotocic factor(s) . Second- ly, marked splanchnic hypoperfusion is a proninent feature of early shock and, if prevented, results in an inproved survival (lefer, 1978). During the beginning stages of hypovolenia , autoregulation is a proru'nent feature in the splanchnic as well as the hepatic renal, demal, and skeletal nuscles (Brdzmann, mndermcd, McCoy, Price and Jacobson, 1970). As the hypotension continues, the autoregulatory mechanism wanes and flow is significantly reduced. The coronary and cerebralvascularbedsaremthanperedbythecentralvasoconstric- tion. The fraction of C.O. perfusing coronary and cerebral vascular beds is actually increased during shock (Roding and Schenk,’1970). RodingandSchenkobservedin92%oftheirev «3.2m finnnH uO I ~\._.|H/HI H. n.—.nn..._\)1 H 3 1x.” ...nnn.\\\\H.n H .— .F\ \\ If \\\ 9+0. >. e e #0 l 9 \\r _ . _ _ . . ..m o a u o a new I ._. MOO .l .—. \\HVIlllw—HIIITA— 1“ 0. +0.). \ \ .... H \.n\..F .— Inflow/7% d .Vm .lo\\ I67 \h 20:33. H I m)...zm H 30 V. 9 “he _ r _ _ . 0 .d N 0 5 ...—Zn 2.3. 48 (Figure 6). Tie control value of MABP for N/S treated dogs (n=8) (GroupB) wasl43 r 10.1mngahdwasreducedtc42 r 2.0mmHgdurirg tle initial hererrhage period. Figure 6 also shows tle lnenerrhage blood volune (ml/kg) recorded from tle calibrated blood reservoir during tle hypotensive period. Over tle 2’: hours after tle initiation of hemorrhage, tle bleeding volumes of tle two groups were net significantly different. However, duringtlelast30minutesofhypotensionthebleedingvoluneintle N/S group was significantly less than that in tle G+O.A. gronp. 'Ile maximum sled blood volunnes were 47.7 i 3.8 ml/kg at 115 holrs for tle G+O.A. gronp, annd 48.5 i 3.0 ml/kg at 1 horr for tle N/S grolp. Duringthelast30mirmtes ofhypotension, theshedblcodvolmewas 42.9 r 3.4 ml/kg for tle G+O.A. grolp, whereas that for tle N/S gronp was 29.3 i: 3.3 ml/kg. This indicates that tle N/S treated dog requiredspontareousuptakeof 40.0% oftlesledblood fromtle reservoir during the last 30 minutes of hypotension to maintain blood pressurearound37 imeflg. Athirdgronpofdogs (n=4) (GroupC) wlewerealso treatedwithan instillation ofN/Shadaspontareous uptake of only 4.4% from the reservoir. Tle maximum shed blood volune for this gronp was 46.6 i 4.3 ml/kg at 215 holrs after initi- ation of henorrhage annd decreased to 44.6 r 4.0 m1/kg during tle last 30 minutes of lnypotension. Tle reason for tle differeee between thisandtleaboveN/Sgroups (GroupB)wasduetointestinal absorption of fluid as will be described below. After3hoursofhypotension, allrenainingsledbloodwas returned. As slncwrn in Figure 6, tle MABP for G+O.A. gronp returned Figure 6 . Time-conrse of blood volume and mean arterial blood pressure in dogs treated with G+O.A. (solid line) and normal saline (dashed line) before, during, and after henerrhagic hypo- tensionfor3ho1rs. Arrowdenotestlebeginningoftle treatments. Tle nunber in parentlesis indicates tle volume of fluid absorbed from tle intestine in ml/kg. n=8 for eacln treatment. Asterisks indicate values significantly differ- ent from tle correspoding values of tle rermal saline treated dog. 49 50 BLOOD VOLUME MEAN ARTERIAL BLOOD \I no .9 .K V .. m as .8. (m. H .3 m m .8 E R 8 U S . S 8 F. R P .3 3.95m . * \H/// 4 H2111 zomzbne/ . mEzm ,. so. Nee , H P jam 3% o)- b Zoom is 33% 51 tc109t4.7mngupm‘b1ocdtransfusicnandmcreasedtollors.4 mmHgat4leursardfell slightlytolo3 15.8mmHgat5hcurs. nempcttheN/Sgroupnormpb)mthectherhandremrmdto9s 18.2mmfigandfe11to78 18.3-.ard 43.8 12.7at4and5hours, respectively. TheMABP of tle thirdgroup (N/S treated,-Gro1pC) rebinedto100t8.9mmflgardincreasedt010318.3mmHgat4hours butfellonlyte98i3.2mmflgat5hours. Intestinal fluid volume and glucose (when applicable) absorption werenneasmnedinallthreegronpsmentioedaboveandareecpressed inTable 2. Therewasne significant differeee in tlevoluneof fluidabsorbedinGronpsAardB,butt1evoluneabsorbedbyGroupB ardAwassignnificantlylessthanth‘atbyGroan. IntleG-IO.A.gron(GronpA),intestinalglucoseabsorptionwas reamed. 'lle G+O.A. solution contained 27 mg/ml (150' ml!) of glucose:atotalof945mg/kgofglnlcosewasinstilledatarateof 81 rug/min. Trnus, 70.8% oftleinfusedglucosewasabsorbed. Arterialglucosecoeentrationwasmeasuredinallthreegronps. 'neGch forGronpA (G+O.A.)was9l.218.0mg%before henorrhage. At IkhoJrscf hypotension, tleGlChincreasedtol96.8 137.5ngn andfellt0105.3i:22.4mg%attleedof5hours. Control value forGroan (N/S treated)was105.515.0mng%ardinncreasedt0204.3 r22.6mg%atlhourofhypotension. Duringtlehypotennsionand after reinnfusion of shedblood,G1CA fell progressivelyto53.3r9.8 mg%attleedof5hours. GlCAforGroan (N/Streated)was100r '10.4mg%before hennorrhageard increasedatl’nleursofhypotension 52 Table 2: Fluid Volune and Glucose Absorption in 'l‘re Dogs Treated With G+O.A arnd N/S. Volune Volume Glucose Infused Absorbed Absorbed (ml/kg) (ml/kg) (mg/kg) Gronp A 35 22.2 i 4.0 669.5 r 123.3 G+O.A. (n=8) Group B 35 20.2 i 1.8 -— N/s ' (n = 8) Group G 35 32.7 r 0.5"r -- N/S (n=4) * p<0.05 relative to Grolp A and B. 53 to 220.0 1: 17.3 mg%. G1CA fell to 77.2 r 11.3 mg% at 5 hours of enperinrents. T‘ledata forhematocritandleartrateareshowninFigure 7. TlechangesinhenatocritintleWgronpsshowninFigureSwere net significantly different. However, Grolp C (not represented) did show sore lnenedilution during tle last hour of hypotension. The heretocrit in Group C irncreased to 50.0 r 1.7% at 30 mninutes of hypotension,whichwassimilartotleotlerowogrolpsrepresentedin Figure 7. Over tle next 235 holrs of hypotension, tle henatocrit decreasedto43.5::2.9%,wlereastheotleroegronpsslewedan irncrease at tle end of hypotension. Tlere was re significant differ- eeeinleartrateamogthethreegroupsofdogs. The data for plasma protein concentration are shown in Table 3 for six dogs. Plasma protein concentration did net signnificantly changefroncontrolvaluesduringorafterhenerrhagichypotension. Furtlermore, tlevalues fortleGnO.A. ardN/Sgronpswerernot significantly changed. T'learterialpaardbloodgases (POZardPCDZ) weremeasuredin seven dogs. Table4 shows tleresults fromtwo treatmentgronps (G+O.A. and N/S). T‘le control pH was between 7.44 - 7.45 for tle twp gronps. T'repHdroppedto7.40 intheGiO.A. treatedgroup (n=4) and to 7.39 for the N/S treated gro1p(n=3) at tle beginning of hypo- tension. nnne pH was 7.26 and 7.19 for tle G.+.O.A. and N/S treated grolps, respectively, by tle last lnolr of hypotension. Two hours afterreinfusingtleremainingsledblood, pHincreasedto7.35and 7.31 for tle G+O.A. and MS treated groups, respectively; Neitler Figure 7. Time-course of changes in hematocrit and leart rate during the experiment in dogs treated with G+O.A. (solid line) and N/S (dasled line) in tle absorption series. n=8 for G+O.A. (GroupA) andn=8 for N/S grow (Grow B). 54 55 HEMATOCRIT (96) HEART RATE (beats/min) ...n 4.. n1\\ 0 mm 22.. ... (hint)... w\._. \\MX‘H‘F/H\ mo: mun...“ ... n+9? \ \\P 3.. x e s u. 3.. . on 9 A\— . . . P _ l.m O n N w .0 Num 1 ...: . s .IH/sls e O O '/ “‘00,. o\nm. o\ I mac n . ._. 25 .111" “W. . . _ . . Jm o u N w s we?!“ 4 ...:sn 2.2; 56 Table 3. Time-Conrse of Plasma Protein Concentration (9/100 ml) in tle G+O.A. and N/S Treated Dogs. Time (loirs) Treatnnennt -.5 0a 1ID 2 3c 4 G+O.A. 5.9r0.6 5,210.3 5.1ro.4 4.9r0.3 4.9r0.4 5.9r0.3 (n=3) mls 5.7r0.3 5.5105 5,310.4 5.2ro.4 5.92043 6.81:0.3 (n=3) ; Beginrning of hypotension. c Instillation begun. End of hypotension. 57 .edlo £5 confines... B 933.8 heave .. .o 9n» B gflflos 3.on a .ccancooconn_oo one o .cscon_ccaunasaoncH .sowncouooan mo oficcwoomm N m.~ao.om m.ens.n~ sm.cae.~m m.esn.em o.~ae.a~ n.oam.em m.ono.mm coo m.~nc.nn c.~ae.~m o.swm.ma a.aas.ea n.oan.~a o.ono.~a ~.Hno.nn sauce enc.casm.s smoc.onn~.s amc.caae.a «No.cam~.a seo.oaom.a ~o.caam.n Ho.onae.n area m e on N be co m... .nuscs. mans. inns. unseen aneuczp o.onm.n~ o.das.n~ sm.HaH.o~ H.Hae.n~ n.~a~.n~ o.mn~.on ~.~as.sm N8o e.mae.~e ~.~ns.ea e.cen.Hs n.onm.ca m.maa.as n.na~.en e.mam.ns Nod «Ho.onmm.s amc.oaam.s wee.cno~.s ano.oas~.s «mc.onem.a mo.onoe.s ~.onme.a an m a on N be co m.) 3.305 meme $05 $.05 .88 caused. m? o8 .466 can 5 Acres. romeo 8ch can so no «his .e wanna 58 growreturredtocontrolvalues atShonrsbuttheN/S treatedgrow showedalowerpHattlelastholrofobservation. OveralltlepH between tle two treatment grows is significantly different (F value = 7.06) . Tle partial pressure of arterial oxygen (P02) was 78.2 mmHg for GOO.A. and 85.0 mmHg for N/S treated grows for tle pre-henorrhage period (-.5 honrs). Tle arterial PO2 continued to increase during tle 3 hours of hypotension and fell slightly by 5 lnours. T're arterial PO2 remained elevated but fell to 92.4 mnnflg at 5 hours for tle GnO.A. treated grow. Arterial PO2 of tle N/S treated grow returned within control values to 88.4 mun-lg at tle same time period. Overall tle arterial PO2 between the two treatment grows is significantly different (F value = 5.21) . Tle control partial pressure of arterial carbon dioxide (P002) was 37.4 mmHg and 33.6 for tle G+O.A. and N/S treated grows, respec- tively. In both grows, tle arterial PCO2 decreased during tle hypotension. All animals were taken off from tle respiration during entire hypotensive period. Rapid deep breaths were reted in all animals after tle first honr of hypotension, thus contributing to the lowered arterial sz observed during latter holrs of hypotension. 'I’re arterial PCO2 was 28.3 mmHg for tle G+O.A. grow and slightly in- creased to 30.0 mmHg for tle N/S treated grow at 5 holrs. There was asignificantchangebytheBrdhonrofhypotensionovertinneO withineachgrow. 59 C. Cardiodepressants: Results from two experiments are slewn schematically in Figure 8. In both grows of dogs, blood samples were obtained from the blood reservoir (#1), femoral artery (#A) and portal vein (#V) before and after henorrhage. In tle N/S treated dogs, little or re cardio- depressants were present in tle blood reservoir (#1) or those obtained before herorrhage (#2A and 3V) . Tle deproteinized sannples takenfromtleN/Sgrowsslewednospotsintlepre—henorrhage samples. Afterhenorrhage, spotswere fond inA, B, C, DarndE (see leged of Figure 8 for explanation). Tle large dense lines of spot D represents tle cardiodepressants (donble arrows) that have a migra- tion distaree of approximately Rs = 1.7 to that of tle serine standard (deeted by single arrow). In the G+O.A. treated dogs, tlere were some cardiodepressants (indicated by less dense lines, letter D) present in deproteinized plasma sanrples taken before henorrhage (#2A and 3V: pre-herorrhage) . No cardiodepressants were present in tle samples taken from tle blood reservoir (#1). Several otler spots appear in pro—hemorrhage sanmples of glucose-oleic acid treated grow at B, C and F. Hemorrhage, however, did net increase tle amonnt of cardiodepressants in spot D (re lines) deeted by tle dolble arrows. Other spots of letters B, C and F were smaller in tle post-henorrhage samples. Spot A is present in samples from tle blood reservoir and post-henorrhage sannples from both G+O.A. and tle N/S treated grows. Figure 8. Representative paper chromatogram slewing: 1) plasma from blood reservoir, 2) plasma from pre=henorrhage femoral arterial sannple (2A), 3) plasnna from pro-hemorrhage portal veeus sample (3V), 4) plasma from post-lnenorrhage fenoral arterial sample (4A), 5) plasma from post-lnenorrhage portal veeus sample (5"), and 6) serine standard. Rs values are slewnandeachspotisgivenaletterdesignateinorderof increasing migration from the origin.* Darkest spots are the serine standard (C), followed by large dense lines (D), tledottedspots (A), andcpenfaintspots (B, C,EandF).' Donble arrows indicate location of cardiodepressants and single ‘arrow deetes tle serine standard. Tle density of tlelinesinspotDrepresentstleintensityoftlespot, heeerelinesinsidetlespotindicates lessintensespot. * 'ne Rs value refers to tle migration distance of any spot ontlechromatographinrelationshiptotledistancethat tle serine standard has migrated. 60 61 E u. 2.3 urooo Nb van- rue—owning“ w< A.) m< m mnazn 0 ®. 0 § 2033).. mgr—Zn 163 I 1 MSOafl—trn n v.00 DO 3 amino urooo Nb vanuznzoaaxgn w< ab m< a «Sin Pm a 3m @- 102...: ngoaarhfin a + 9 b 0000 UB®© -fl ODD 000 p O N.m Nb Pm '9 £5» 0 Q 62 D. Histmathclogy: Tissue sanples were collected for determination of any morpho- logical changes (gross and histological) that may have occurred during the experimental procedure in both G+O.A. (n=3) and N/S (n=3) treated groups. According to Chin _e_t al. (described in methods) , the three dogs who received N/S at one hour after herorrhage showed histological damage in the range between grades two to four. Macro— scopically, a cannon finding for dogs treated with N/S was uucosal sloughing, congestion and herorrhage. In contrast, the three dogs treated with the G+O.A. solution only received a grading between zero to one upon histological observation. Macroscopic appearance showed a normal intact mucosal surface with no evidence of congestion but petechiaewere fourdintheduodenmmandileuninonedog. IV. DISCIJSSIQI Dogs subjected to hsnorrhagic hypotension may succunb to a state of irreversibility if the shock is of sufficient degree and duration (Wiggers and Werle, 1942; Walcott, 1945). There are two communly used methods to experimentally produce henorrhagic shock. 'I'ne first islcnownastl'nesinglewithdrawalorthenonreservoirmetl'nod (Walcott, 1945) . 'L’ne nonreservoir method consists of the renoval of apredetenninnedquantityofbloodandtheimmediaterehnrnofafixed percentage of blood collected. 'Ihe amount returned can be predeter- minedtoproducel‘nsnorrhagicshockofvaryingdegreesfromtle mildest symptoms to 100% fatalities. 'Ihe secod method is the graded hemorrhage method described by Wiggers and Werle (1942) . The removal of blood at definite time intervals or prrportionately smaller volunes of blood renoved until the mean arterial blood pressure falls to a ”shock level“. A modified version of Wiggers henorrhage method wasemplcyedinthisstudy. UsingthemethoddescribedbyLamsonard Deturk (1945) areeervoirmsconnectedtoafenoralarterycatheter forbleeding. Agradedhsnorrhageof 35mmflgfor3hourswasshown inourlaboratorytoproduce irreversibleshockarddeathinalldogs (Senko, 1980). Brohnenn et a1. (1970) has demonstrated that autoregulatory mechanisms will sustain blood flow teporarily to all vascular beds inthebeginning stagesofhsnorrhage. Asthehsnorrhagichypotension progresses, only essential vascular beds (e.g., brain and heart) will receive sufficient blood flow. The intestinal vascular bed * 63 64 among others suffer extensively in severe hemorrhagic hypotension. Roding and Schenk (1970) reported that the mesenteric fraction of the cardiac ontput is significantly reduced during a sustainned honor- rhage. Marked splanchnic hypoperfusion is a prominent feature of prolonged henorrhagic hypotension produced by graded henorrhage (Reynell, Marks, Chidsey, 1955; Roding and Schenk, 1970). 'Ihe splanchnic hypoperfusion nno longer maintains adequate oxygenation of the highly metabolic mucosa. As a result, the ischemic splannchnic region liberates a number of tonic factors (Haglund and mndgren, 1972; Lefer 1978). At autopsy, mucosal congestion and necrosis of the small and large bowel are conmon findings (Wiggers, 1950; Chiu et al., 1970; Haglurd et al., 1975). These toxic factors contribute to the vicious deteriorating cycle observed during circulatory shock, ultimately leading to death. Lillehei (1957) reduced the mortality rate by 80% in experimentally-induced hemorrhagic shock through maintenance of intestinal perfusion pressure. Our rationale was to maintain local intestinal mucosal blood flow and metabolism durinng the hsmhagic hypotension by duodenal instillation of a glucose-oleic acid solution. The solution has been shown to inncrease blood flow and metabolism (Chou et al., 1972, 1976, 1978). As a control, normal saline was administered one hour after initiation of hanorrhage . The rate and volume of administration was standardized for all dogs to be 3 ml/min and 35 ml/kg body weight, respectively. The rate of volume infusion approximates normal gastricsnptyingandtheamnmtofvoluneinfusedwasfonndndtto disted the intestine after conpletion of infusion. 65 Prolonged hypovolemia produces ischemic and hypoxic coditions of the intestine as shown by Selkurt and Brecher (1956); Haglund and Lundgrunn (1974) . Adequate perfusion should prevent irreversible damage from occurring. Carl Wiggers (1950) described the classical response of arterial blood pressure after the return of hemorrhaged (shed) blood. Mean arterial blood pressure returns within prelnerorrhage levels after retransfusion and progressively falls over the next several hours. Tl'etime intervalbeweenretransfusionanddeath isknownasnormo— volemic shock (Ehrlich, Kroner, and Watkins, 1969). The MABP of the untreated group (Figure 3) and the individual blood pressures treated with normal saline (Figure 4) illustrate Wiggers' original descrip- tion of the progressive stages of irreversible shock. Prolonged survival for 26 to 41 hours after reinfusion of all shed blood occurred in dogs treated with a duodenal instillation of tle glucose-oleic acid solution. We were able to demonstrate a 67% survivability in tle dogs treated with G+O.A. Dogs untreated and treated with a duodenal instillation of rnormal saline showed 100% mortality within 3 to 6 hours after reinfusion of shed blood. The protective effects of an intraluminal glucose solution was shown by Chiu, Scott and Gurd (1970) to maintain the integrity of the intestinal mucosa . Hypertonic glucose not only acts as an energy substrate but also as an osnotic force (Moffat et a1. , 1968) . In our study, instillation of the glucose-oleic acid solution improved the maintenance of the arterial glucose concentration. Maintenance of 66 blood glucose levels within the nnormal range throughout the hypo- volemic and normovolemic periods coincides closely with improved survival of the animal (Figure 3). Hift and Strawitz (1961) have demonstrated a tanporal relationship between blood glucose changes and morphologic damage in lepatic mitochondria. ‘Ihe tolerance to hemorrhage was also shom by Drucker et al. (1958) to be related to maintenance of blood glucose concentration. Survival was signifi- cantly prolonged in dogs who received an intravenous infusion of hypertonic glucose. Treatment was instituted when tolerance to hypcvolemiabegantodeconpensatebytl'eneedtoreinfuseblood from tle hemorrhage reservoir (Moffat et al., 1968). Initial stores of glucose may only last approximately four hours, forcing tl'e body to utilize alternate energy sources during normovolsmic shock. Tie predonninately anaerobic metabolism during severe hemorrhagic hypotension significantly reduces the glycogen stores early in shock while only producing stall quantities of much needed ATP. This lack of glucose forces glucocorticoid—stimulated gluooeogeesis to produce amino acids, fatty acids, and glycerol (Schumer and Erve, 1975). Wiggers (1950) and McCormick et al. (1969) have observed the hyperglycemic state early in honorrhage . Sympathoadrenal mechanism is prinnarily responsible for the hepatic glucose mobilization (Jorley and Watts, 1964; Adams and Parker, 1979). Blood glucose was maintained for all dogs who received tle glucose-oleic acid (Figure 3). The arterial blood pressure coincides well with the maintenance of blood glucose. Blood glucoseremrnstonearcontrolvaluesasdoesthearterialblood 67 pressure after retransfusion. Tte instillation of glucose-oleic acid solution maintains blood glucose throughout the hypcvolemic and normovolemic periods. In contrast, treatment with normal saline, although possibly acting as a tenporary plasna expander, did little to maintain arterial glucose concentrations and survival in this group of dogs who received the instillation of normal saline at one honr of hypotension (Figure 4) . 'ne close correlation between arterial blood pressure and arterial glucose concentration represents the need for maintenance of blood glucose levels for prolonging survival. Alldogsinthisgrouptreatedwithnormal salirewere hypoglycemic prior to death. Tl'e fall in arterial glucose concentration below normal levels reduces tle pump capacity for transport of gluooeogenic substrates to the cells. This, conbired with a lack of oxygen, causes an increase in lactic acid, producing intracellular acidosis and an extracellular acidemia (Schmer and Erve, 1975). Markov, Oglethorpe, Yomg.and Helkens (1981) showed that glycolysis is interrupted during severe metabolic acidosis. Ptosplofructokinase is a mnultivalent enzyme that catalyzes the rate-limiting reaction of fructose-6- phosphate plosphorylation. Tl'e intracellular acidosis seen during stock decreases tle catalytic action of phosptofructokinase, reducing tleamountofATPproduced fromthennbdem-Meyertofpathway. 'Ii'e plasma pH of tie dogs treated with tie glucose-oleic acid solution fell significantly during tle period of hypotension from time 0 through 5th hour. The fall in pH was also significant within tle N/S treated group during tl'e hypotension and tie two hours after. 68 Mukov's group (1981) usinng a similar hemorrhage protocol of 3S mmflg for31'onrsshowedsinmilarreductioninarterialpfi. All dogs showed hyperventilation indicating signs of respiratory stress throughout the henorrhagic hypotension. The arterial PO2 (Table 4) for both groups were increased due to the persistent hyperventilation. Overall there was a significant difference in pH andPOzbutrotfoerzbeuveenttemotreamentgronps. Itis reasonabletoassnmne thatthedogsintl'enormalsalinegroupwere sufferinng from a metabolic acidosis. 'Ihe instillation of the glucose-oleic acid solution starting at He first hour of hypotension possibly contributed to the better respiratory conpensation during tle 3rd, 4th and 5th hours after initiation of herorrhage. Through tl'e maintenanoe of blood glucose levels , the glucose-oleic acid solution prevented the severity of a metabolic acidosis by making available adequate levels of substrate for oxidative plospl'orylation. Moffat et a1. (1968) , by administering hypertonic glucose durinng the physiological deconpensation of slnock , has reported better respira- tory values for tlose animals as conpared to otters receiving hyper- tonic sucrose. Conpensatory mechanisms were considered to be operating maxi- mally one hour after initiation of hemorrhage. The influence of fluid treatment at this time, to support tle compensatory mechanisms, would supplement interstitial fluid shifts and aid in restoring fluid loss by hemorrhage (Miller and Dale, 1978) . Previous investigators (Van Lieve et al., 1938; Goldberg and Fine, 1945; Miles at al., 1968) have indicated that intestinal fluids do not participate in plasma 69 volune restoration after hanorrhage in the dog. These investigators agreeinpartwithMiller andDale inthat normal salineabsorption ismaintainedandenhanncedtosonedegreeduringshock. Millerand Dale used a hemorrhage procedure that approximated 35% of measured blood volnne. This hsnorrhage volure was sufficiently below tle IDso blood volume of 42 to 43% (Swan, 1965). The hemorrhage insult used by Miller and Dale was of sufficient magnitude to activate conpensa— tory mechanisms innvolved in plasma volume restoration, but not to such a degree as to cause 100% mortality. Van Lieve and associates used 3.2% of body weight to approximate hanorrhage volume and Goldberg and Fine used a systolic pressure of 50 mmHg for several roars to induce stock. Results from oir study showed no significant difference in volume absorption between tle glucose-oleic acid (Grow A) and normal saline (Grow B) treatment grows (Table 2). Item was a third gronw of dogs (Grow C) wlo also received an instillation of normal saline. Trese animals not only survived the henorrhage insult but stoned significant absorption of fluids over the glucose-oleic grow (Grow A) and treir normal saline comterparts; Grow B. Our findings follow ttose of Miller and Dale's in that a sizeable amount of fluid was absorbed in all treatment grows. This suggests that placerent of oxygenated fluid (i.e., normal saline) will assist in the continmnalmaintenanceoftl'eabsorptivecapacityintl'esnallbmel duringseverehypotension. Grodeidnotsuccumbtotleheror- rhagicsl'ockandwasabletocontinueandenhanceabsorptionof normal saline. 70 Several possible explanations exist for tie larger or equivalent fluidabsorptionintlesalinegrowsasconparedtoglucoseoleic acid grow. First, a limited mount of work (eergy) is required for absorption of normal saline thann for the absorption of glucose. Secodly, a depletion of salt (i.e., chlorides) thronglout the tissues oftrebodyduetohenorrhage. Whensodiumchloridewas placedintheintestinetl'erewasahigherdiffusiongradient, and salt passed into tle bloodstream more readily. 'Ihirdly, severity of thehsnorrhageprotocolmaybeafactorinabsorptionofnomal saline. Jenkins et al. (1961) and Ehrlich et a1. (1969) ford that corpensatorymechanisnebeganntodeteriorateBhonrsafterahsnor— rhage. After henorrhage exceeds 40% of blood volune, tle ecperi- mnental annimal becones unnresponsive to trerapeutic maneuver (i.e., administration of normal. saline) (Ehrlich et al., 1969) . Jenkins et a1. (1961) also sl'nooed that plasna dilution in hanorrhaged dogs is well correlated quantitatively with the degree of blood pressure experimentally reduced. 'l‘legrowofdogswloreceivedtl'eduodenalinstillationofthe glucose-oleic acid solution absorbed 71% of the glucose administered. In on study, not only did glucose improve survival, but it enhannced and maintained arterial glucose levels, prevented tle severe decom- pensationphaseseeninGrowBandmaintainedbetterrespiratory conpensation. Glucose also contributes directly to tle metabolic maintenance of the myocardium and extracellular volune (Opie, 1970) . Alargeamonmtofexperimnentaldatahasrevolvedabontwl'etier glucose is "good” and/or free fatty acids (FFA) are "bad" for tle 71 ischemic myocardium. A number of changes acconpanny myocardial ischemia: depressed insulin response, augmented catecholomines, corticosteroids and growth lormone. Also, inncreased glycogen levels occur and are responsible for a variety of metabolic alterations seen during stock. All these edocrinne changes are responsible for the relative glucose intolerannce and inncreased FFA levels (Koes, 1975). Tl'e "glucose hypotlesis" discussed by Opie (1970) postulates that glucose is "good" for the heart based upon tle following predictions. Glucose availability enhannces the rate of anaerobic glycolysis, reverses ion losses, alters impaired menbrane electrophysiology, effects extracellular volune , decreases plasma FFA concentrations, and alters plasma osnolarity. Tne FFA are supposedly "tonic" to the isotonic leart (e.g. during severe henorrhagic hypotension) in that anhythnuas are more likely to occur and contractility is depressed. However, Wildenthal (1971) ewlains the well-oxygenated myocardium shifts from FFA as tle preferred substrate to glucose dnn'ing ischemia or anoxia and depeds nwon tle glycolytic patlmy for energy production. The plasma protein concentration (ppc) for do two treatment grows fell from control values in He initial stages of hemorrhage (Table 3). Tie fall in ppc continued for tle first 2 honrs during hypotension. Haddy, Overbeck and Daugnerty, Jr. (1968) have reported that an inncrease in tle precapillary resistance will predominate early in henorrhage, favoring absorption of excellular fluid. Tie reverse istruelateinhsnorrhage, filterationpredoninatesdueto prOportionately higter post-capillary resistance. Also, the increase 72 in capillary permeability may be oe explanation for tle loss of fluid late in stock, both before and after reinfusion of the shed blood. The gradual change observed in ppc for tie glucose-oleic acid treated grow correlates with the change in henorrhaged blood volune. This correlation is also ecsnplified in tle normal saline treated grow where tl'e ppc falls gradually during tl'e first two hours of hypotension and tlen inncreases above control for the 3rd honr. Although no significant differeee was evident between tle two treatment grows for ppc. The "spontaneons uptake" of blood from the blood reservoir represents one of tie earliest signs of cardiovascular deconpensation (Hollenberg, Waters, 'Ioevs, Davies and Nickerson, 1970). A spontan- eonsnwtakeof40%oftlesredbloodfromtnebloodresenroirwas observedinthenormalsalinetreatedgrow (Figure6,GrowB), while tle glucose-oleic acid treated group (Grow A) spontaneously returnedonly10% fortresametimeperiod. Tleanmomtoftleremrn ofbloodfrumtlereservoirtomaintainthehypotensivepressurewas show to be significantly different between grows A and B (Figure 6). Bathe (1970) has suggested that, at 40% uptake of the maximum shed blood, there is significant depression of myocardial funnction. Simnilarresponsemightoccurinonrnormalsalinetreatedgrow (GrowB) andtheresponsemighthavecontributedtotrefall inMABP during the normovolemic period and eventually lead to death of all dogsinthisgrow. Tlethirdgrowofdogs,GrowC,whilealso receiving tle normal saline instillation, returned only 4% of the hemorrhaged blood volune. Grow C absorbed significantly larger 73 amounts of fluid (Table 2) as opposed to Grow B, but the maximal amount of henorrhaged blood (measured in He reservoir) for Grow C occurredlhonrandBO mins laterthanthatofGroupB. IngrowC, normal saline was absorbed in significantly larger quantity and may haveactedasatenporaryplasmaenpandertomaintaincirculating blood volume late in tl'e hypotensive period. Rothe and Selkurt (1964) suggest that an inncrease in hetatocrit isduetoadischargeoferythrocytesfromtl'espleen. Furtlerloss of fluid from tle vascular system may continue to concentrate the red blood cells and plasna proteins. Results from onr study indicate an inncrease of henatocrit for both treatment grows (Figure 5 and 7). Marty and Zweifach (1971) , using spleectomized dogs, reported a drop in hematocrit of 25 to 40% from control values during tle first hour of hypotension, with little change after tle first honr. Our henato- critdatawassimilartofindingsbyRotleandSelertwtorepbrteda significant inncrease in henatocrit ratios by the time tle blood pressure was bronght to 35 mmflg. The henatocrit increased at tle beginningofhypotensionbutdroppedsnddenly, retainingbelcwthe initial inncrease unntil the secod hour of hypotension. Coleman, Kallal, Feiger and Glaviano (1975) showed an 8% increase in hereto- crit 2 hours after reinfusion of shed blood. Significant increases were evident for both saline treated grows series. Tie highest heart rate recorded (Figure 7) coincides with the poinnt of maximal hemorrhaged blood volume (Figure 6) . Bod, Manning and Peissner (1979) also showed a maximal heart rate of 217 beats/min and a peak sled blood volume of 47 ml/kg at 1’5 honrs after henorrhage 74 was initiated. Rotle and Selkurt (1964) showed a significant decreaseinteartratebebweenperiodsofmaximalbloodvclnmeand timejustpriortotransfusion,snggestingsonedegreeofcentral nervoussystemdmression. Ourdatashcweimilartredstothatof RotheandSelkurt,eccepttredifferencebetweenthesetwoperiods are not signnificant. Rather than using a fixed time interval for hypotension, Ratio and Selkurt maintained the hypotensive pressure until there was 30% nwtake over the maximal blood volume. 'l‘hepaperchromatographictechniquedescribedbyYamadaand Pettit (1977) offers a simple and highly specific assay for cardio- depressannts. Only 200 ul of deproteinized plasna sample is required, eliminating the need for volume replacenent. Serial sampling of several milliliters is required for analysis in papillary muscle preparations and other bioassays . Tie paper chromatographic tech- nique is cleann, reproducible, requiring snall sample size, and particularly useful in serial studies innvolving stall animals. 'necardiodepressantsinonrstndywerefomdtoappearintte post—renorrhage samplestakenfromttenormalsalinetreateddog (Figure 8). Cardiodepressants were located by conparison of tie mnigration distanoe or Rs value to a serine standard (#6) . This spot, designatedDinonrstudy, shonldappearatamigrationdistance between Rs 1.6-1.8 times tie distance that serine had migrated. This spot correspods to simnilar data presented by Barenrolz et a1. (1973) , Yamada and Pettit (1977): Regal and Hilewitz (1978). Yamada and Pettit fond that a large elution volune (85-105 ml plasma) preparedandrenovedfromapaperchronatographhavingamigration 75 distance between Rs 1.6-1.8 contained tle higiest depressant activity in papillary muscle preparation. This slate contained 42 units of higher depressannt activity compared to a spot eluted with a migration distance of Rs 2.0-2.4. Sane cardiodepressants were present in the pre-henorrhage sannwle taken from tl‘e glucose-oleic acid treated dog, probably due to surgi— calmanipulation. However,henorrhagedidnotincreasetleamonntof cardiodepfissantsinarterialnorportalvemsblood. SpotAmay representttelargeamomntofcirculatingcatecholominespresentin thesl'ockplasnaonly fromsamplestakenfromtl'ebloodreservoir (#1) and samples takenafterhenorrhage (4Aand 5V). Haglind, Haglund, Lundgren, Romanmns and Scherstern (1980) have correlated tle amount of intestinal mucosal damage and mortality with thedegreeofgradeddecreasesinblood flcwtotheintestines. The degree of mucosal damage correlates significantly with mortality. Using Chiu et a1. (1970) schene for morpl'ologic characterization of tie intestinalmucosa (see Material andMetl'ods), Haglind and assoc— iates showed a high mortality rate in animals with histological damagebetweengradesB-S. 'n'eanimalswithgradesbetweenO-Zhada low mortality rate. Our study shoved the instillation of normal salineoffers littletonoprotectionofthemucosaduringhenor— rhage. In 3 dogs, tle histological damage was between grades 2-4. Chiu et al. reported tl'e placenent of sodium chloride (0.9% and 1.8%) solutions into intestinal sacs of dogs. Tie histological annalysis of these sacs after 60 minutes of ischemia showed sone protection, receiving a grade between 2-3. Although the experimental design in 76 Chiu's study differs from tle modified Wiggers' protocol, tle analysis of mucosal damage correlates fairly well. Tl'e 3 dogs in on study wl'o received tle instillation of the glucose-oleic acid solution were given a grading between 0-1. ‘lhe protective effects of glucose aloe has been shown by Chiu, Scott and Gurd (1970) and Chiu, McArdle, Brown, Scott and Gurd (1970) to offer conplete protection. Macroscopic daservation reveals a normal mucosa with an occasional spot of petechiae after treatment with tle glucose-oleic acid solution. Mucosal slonghing and henorrhage was present thronghout the snall intestinal tract of dose dogs wlo received tl'e instilla- tion of normal saline. V. SW AN) MUSIONS ‘Iheuseofaherorrhageprotocol that induces totalbcdyhemor- rhage demands replacement of lost fluids tenporarily until whole blood is made available and, most important, provisions of a readily utilizable eergy substrate (such as glucose) as suggested by Opie (1970) . 'lhe instillation of a solution containing 150 mM glucose, 40 mM oleic acid and bubbled with 95% oxygen-5% carbon dioxide gas into the inntestinnal lunen of dogs subjected to irreversible hemorrhagic shock had tle following beeficial effects: A. Tie glucose-oleic acid solution prevented irreversible henorrhagic shock and increased survival rate (67%) for 26-41 hrs. B. ReducedttemdforspontaneonsnwtakeofshedbloOdfrom tle henorrhage reservoir to maintain tle low pressure at 37 mmHg thronghout the 3 hr hypotensive state. i C. Maintained arterial glucose levels between 87-127 min after reinfusion of all shed blood and throughout normovolemic stage of stock. D. 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