Brain Damage Secondary to Hemorrhagic Traumatic Shock in Baboons* G, SeutaG, K. ZARKOVIC, H. ReDL, N. ZARKOVIC, and G. Ware Introduction Trauma and hemorrhage in experimental models should closely mimic human polytraume to create a valid besis for drawing definitive conclusions zbout the pathomechenisms and pathophysiology of polytrauma. Pi" Te baboon is the only large animal (eg. dog, pig, sheep) that is able to maintain spontaneous respiration under general anesthesia in the supine position for hours normal gas exchange parsmeters, It is therefore ideelly suited for she stady of posttraumatic events in a setting similer to that of human tau baboon offers @ multitude of other similarities 10 humans, eg, its eroce-rezctiity ‘with several homan antibodies. In order to simulate the condition of treumatic shock w polytreums in the baboon and the resulting organ damage correspo boman condition, the animals have so be exposed to severe shock for a cert Itis well knoven [1,4.22,23) that decreasing the cerebral blood fiow 1 about ermal or below will resule in the development of ischemic brain edema v: increased weter accumulation in the brein perenchyma. n addition, 3 ofirzeversible tissue damage such as infarction may be possible, Such brein infarction cen occur as disseminated hemorshagic infarction and be of importance for the ‘outcome of the experiment. Animels thus affected cennat be used for the eveluetion se trauma, and fra mediators may provoke a ge: fiammetory reaction, especially in vital organs such as the lung, liver, kidney, and g ion we refer to 2s : eck.” ‘Most medistors released in traxmatic shock originate from the other humoral systems (coagulation, Sbbrinclysis, kalikre! tems, in particvler from activated polymorphonuclear nevtrophil (PMN), such as the sutrophil elestese, a marker of Ph tion. Many of these relea! in plasma and tissu ight on the pethomech plement and ini) ond cellular sys nd and in part supported by was supported by ication Minis of cence4 G.Sehlageta if y _NoMvaDousd ——Saan —‘evrnatic Shock in Beboons 5 ned several in vitro To cope with the problem of species differences we perfor tigations to es for exazrple, whether there are dtferences in neutrophil exygen injeal release between humans and baboon (unpublished data). Reactive oxygen ROS) released by phorbol myzistic acid (PMA) and endotoxin in baboons is ble to that in humens, These preliminary data convinced us to employ the {ahton as 2 model to stody troumatic shock. This model was designed to produce species ( compara veer insights into the pathomechanisms involved in treumms and to provide 2 highly penful tool {0 investigate the efficacy of pharmacologic interventions in traumatic shock. Proteinase release is accompanied by 1 of toxic onygen radicals, both of shih RECSURETGT UeRDE Damagerdering shock peTewary To WARWEH ischera and oxide {7.8:16-19a73- The Tseue-damaging eect of toxic ae cals aT en in lipid peroxidation products [e.g., formation of conju- fated dienes, malondialdehyde, loss ofa E, SH groups of elbumin ete). Verioos etpephysclogicel conditions (induced nd mediated by diferent biochemical peth- Fey) generate ROS and thus indace lipid peroxidation (Fig. 2). Highly reactive echydes are also produced as a consequence of lipid peroxidation (10,11,28). Be- Gause of their toxicity, they are considered “secondary toxic messengers” of free icals which cause secondary tissue demage [11,242]. One of the most important troduets of pid peroxidetion is highly reactive aldehyde 4-hydroxyaenenal (NE) cli). There ate data indicating thet HNE end related aldehydes are involved in 120S-induced demage to various organs, particulary the brain, efter different patho- Togical ev However, cescades) of events leading to oxidative Gamage in the brain under diferent petholosical conditions such as stroke, trauma, tnd sepsis can not be analyzed from a morphological point of view. The rease the deta incicating involvement of ROS and their secondary toxie messe diferent diseases were obtained by biochemsical analysis of che activity of enzymes involved in ROS production or detoxification, or from the presence of HNE or related aldehydes in serum, CSF or the tttue homogenates [10,12,28]. Consequently, mor~ phological evaluation of the tissue and cellulr distribution of the mediators of oxida- ‘stress that would show probable differences between the different types of cells or ris of orgens involved in pathological eve 7 recently cific mo: in (Or pep side) conjugate(s) that allow bution of the aldehyde. The result abteined using a bodies forthe immu- nohistochemicel analysis of HNE di experimental hemorzhegic shock will be presented. HNE monoclonal a in of baboons exposed 10 Materials and Methods Animals male beboons (Pap enimals had been ceged at the nonhuman primete unit of Biocon Reseerch Lid Leborztories in Pretoria, South Afric irus) weighing between 20 and 23kg we6G Schlag et a linical condition of the beboons had been checked prior to their entry into quaran- tine. At the begining of the study all the animals had been found 10 be iseases. Anesthesia ‘The baboons were fasted for 12h before each experiment and had water ad I The animals were sedated and immobilized on the morning of the experiment with intramuscular Ketamine hydrochloride [8-20mg/xg body weight (BW)]. ‘The animals were anesthetized with intravenous pentobarbital sodiem edminis- tered via an electroencephalogrem (EEC) controlled closed loop feedback system as previously described [20,11]. The animals received berween 1-3mg pentobarbitel/a per kilogrem BW. The baboons were sponteneously ventilated throughout the experiment with @ positive end expiratory pressure (PEEP) of 0-2mm Hg to Keep the alveoli open with a controlled end expiratory CO, measurement. For this purpose the animals were intubated (tube no. 8-8) and connected to a servoventiletor C900 with a continuous positive airway pressure (CPAP) device for spontaneous bresthing. The FiO, (inspiretory O, concentration) was adjusted to about 25% + 2, : The administration of pentobarbital was automatically the end expiratory CO, exceeded 55mmHg scomtinved whenever Instrumentation Following exposure of the right femoral vein, en introducer with a heperinized TF Swan-Ganz catheter wes posi 1 leced in a pulmonary artery branch to measure cardiac output (CO) and pulmonary wedge pressure (PWP), 1 I pressure (RAP), and also to sample mixed venous blood. A triple lumen cesheter was inserted into the leh brachial vein for infusions, anesthe- .nd blood sampling. The arterial blood pressure was monitored via I artery, andin addition the line served for blood alysis for bacterial blood cultures. Ipsilateral to the Swan-Ganz catheter a Jarger catheter (14-16F) wes placed in the femoral blood removal during the hemorzhege period. To determine the hourly only, a catheter was placed in with an ultre-red lamp to mai Ganz catheter) at no less tha: increased to more than 37.5°C. sia, medication, sampling for g2s he experiment were warmed via the Swan sn the temperature fore (mon ned off w C. The lamp was essesuanca amin diustiviinasitivesciniiaie/stessibbudanieitainiiaiitenuttaiharsiasinGrerinertat taco reumatc shee) ‘The experimental protocol was reviewed en y the Institutional Animal Care and Use Committee of Biocon Research Laboratories, Pretoria, South Afri ‘Afier the instrumentation a period of 60min was given for stabilization of the animals. “Traumatic shock was produced first by a simulation of trauma (soft sissue injury, fractures) with complement activation, Recent clinical studies have shown that complement activation occurs very early in treumetic shock (12-14). For ethics] yeasons, fractures and soft tissue trauma ate only justifiable in acute shock models ichere the animals are killed at the end of the experiment. In our subchronic model the animals recover from anesthesia and shock and may survive. Complement activa tion was achieved by the administration of cobra venom factor (10 U/kg) at the beginning of the experiment and 1h after the start of reperfusion (5U/Kg). ‘A ttel of 30min after the bolus application ef cobra venom factor the hemor- shage was activated. Blood was withdrawn trom nonheparinized animels into blood ‘bags containing 70m CPDA (sodium citrate, sodium dihyérophosphate, adenine) ‘The amount of blood removed was varied until the mean arterial pressure (MAP) Qos Sms ne cerdiae output was reduced by ssure dropped below 35 mmHg, the animals received & bolus of Ringer sol {20-30ml). In the absence of response, shed blood (uptake of blood) was rein‘used The time point of shed blood reinfusion and the overall quantity of supplied blood was documented. The overall blood loss (includi was defined as the otal quantity of removed blood, Usually zbou ¢ total Bleod volume vas remove (total bl00d volume is approxima the BW), The start of complement activation and the subsequent Blood removal take 10 more then 3h (time-limited ) (Fig. 1). After the hypotension period (low flow stage), the reperfusion of the shed blood with the seme smount of Ringer solution was started, which pulmonary pressure (PAP) must not exceed the threshold value of 23mm Hg. At the same time, cardige cv!put was monitored se in addition to the hou! measurement and should not exces bas Afer t triple lumen eatherer in the left brachial Yela and the Swen Genz aving the introducer in place. Both the are trogucer were filled with diluted heparin solution @ ets pose The sabeuteneoys pow conditions. Then the wounds \ si the experiment for the h 60% of UilOml) and placed in a n opened under sterile ng the subchronic period nis, a Sican-Ganz catheter wes jl line) were reconnected 10 removed end e procedures w measurements end blood samplings durin (24, 22, 48, 72h) fe repested for je phase of the experiment the subchroBrain Dam ary to Hemorvhagie Trewmatic Shock in Beboons 19 edi ‘ye see similar effets actually in the liver where the injury iselso very focal. schleg: Fjust want to show you 2 few slides. You see, here we measured the neuron specific Nave in the serum of these beboons and we see an increase at about 24) the green Tine is a group of baboons in which we used an antiselectin for treatment, and this ad a significant survival compared to the control group, the red line, One EEpcon showed really severe brain damage which we showed in the histology. We see ae eevere increase of the neuronie specific enolase s0 that it would be a nice predic~ tor bot mostly itis too late because we do this retrospectively. So the points, whet we seeiooking for is to have « predictor to say when it is enough hypotension and we Jove to start with the reperfusion, otherwise you get this severe damage. So we are Tpoking for a coatinuovs measurement of cerebral blood flow and also of P02 in the group h brein tissue.aa ne ee ey eee (18 -G. Schlag eta they are actuzlly opersted there is brain ischemia for some time, Some of these e patients actually show some similerities to what you have shown in your study. There is clinical importance o our study and, knowing this, I think the substances should be tested te protect such brain damage. Vour model is ideal for that because you could actually give i preoperatively, tying to avoid brain damage. Kocheneke L have one other comment releted to that. There is one other correlation to your study, Peter Safar in his research with the US Army and the US Navy has frequently discussed how the families of the survivors of sustained hemorthagic shock often complain that these people are just not the same person after the episode. In particu- Jan, their memory is often disturbed. Related to this, 1 was intrigued to see the hippocampal injurys it would be what you would expect to see if hemorrhagic shock ‘were actually producing brain injury. Young I wes interested in your thoughts cbout where the presumed free radicals are pro. duced, To follow up on Dr. Baethmann’s question, I wes wondering if you did find damage with in other organs, or do you think there wes @ disruption of blood-brain barrier and activated free radical production intrinsically within the brain rather than in the vascular system? @ sang Thave to give this a) nto Dr. Zarkovie: Could yo N. Zerkovic: ‘The pattern of oxidative or free radicel-induced damage during ischemic.reperfusion injury is quite general. Whether this happened in these baboons | really do not know wwe anelyzed s0 far, However, one could assume that it probably happened oughout d Redl ‘We have a lot of systemic evidence where we heve done ex peroxidation paremeters, events znd in addition activa adical action from previous baboon studies monitoring of plasma antioxidants and lipid cf oxygen radicals are ischemie-reperfusion Young So you think it p a systemic thing that causes free radical damage to th brain? @ Red Twould believe it is both local ischemia-reperfusion and, in addition, what you have seen around these hemorrhage areas when the phagocytes come in. N. Zarkovic: One could in fect assu activated, because ther make 2 locel focal dem coll en wherever there i erfusion kind of damage ed on the general principle.ane Se a ane Basin Damage Secondary wo HemontagicTrsmate Sheen abe Discussion Beethmennt ips faras traumatic-hemorthagic shock without any primary involvement of the brain Seconcerned, I think there are few data available of whet is actual! brain unless systemic hypotension is so severe that cerebral blood ‘tow is affected, ‘hen one may expect ischemic damage of the brain in the selectively vulnerable areas. My question is concerned with the specific effects of activating the complement iyatem, oF what Would occur in the brain then without inducing hemorrhegic shock i sddition? In other words, whet is more damaging for the brain: complement vation or hemorrhagic hypovolemia’ Schlag Wwe did these same studies in hemorrhagic shock without complement activation and ‘cave exactly the same results, First we thought the complement activation hes some Jimels with hemorrhagic shock jnflvence on this event, and therefore we did five and in two animals we also found Baethmenn: Did blood pressu: ce Srovasculer ev! Schlag Yes, Tthink so. Because in the future we will meas wwe are not able to do this, ‘mam Hg. But as you knows the baboon is quite resis ss daing his studies in pigs with hemorrhagic shock. We also did studies in pigs they ere even more resistant b¢ ‘emain with 3mm Hg for 3-£h without showing any change in the basic seis. The baboon is really very similar to humans, Baethmena: Altogether the changes you are ol hypovolemia are quite si date 1960s (or early 1 thagie published in the co surviving periods of severe arterial aber : e Jement sctiv want to meke a comment sion toy ur study in the ¢Brain Damage Seco jemorthagic Traumatic Sbcck in Babons Finally it should be mentioned that while HNE could contribute to the develop- ment ofthe tissue damage, it might on the other hand also be ithe damaged tissve since HNE can also ect as reguletor of cellular Of importance in this regerd would be not only the content of HNE within the tissue tot also the neture of the local tissue end the humoral, blood-originating factors present at the site of damage which would be releted to the histological and anatomi- Gal features ofthe brain. Conclusions ss end the resultant secondary toxic messenger of free redicals, HNE, ‘mining the brain damage induced by 1. Oxidative: play a rele in the cescede of events det hemorshagic shock. 2, HNE present in the brain tissue seems o be of vascular origin and could play azole in the fanction of the blood-brain barrier and the zegulation ofthe cerebral blood fiow 3, Asa result, HNE could be of relevance for determining the outcome ofthe hemor- gic shock, or at least its consequences for the brain References 1. Beethmana A, KempskiO (1951) The brain shack. Secondary dimurbence of eerbr feneen. Chee 1052058-2088 2 Bar § Sig 6 ao 7M, Rel 0939) Imehene icone in hemornaie she Dt Sho: 7 fons. In: Schisg G, Reel sth Wig Beilin Heidelberg New Yorks pp :D (1989) Central nervous system i ons of oxygen radical formation and lipid IB], Meldrum BS( ing from profound arterial hrpcientien. correlees, Brain Res 15.6 ‘ey JB, Excell 8] (1968) The effects of profound systemic hypaen rhesus. Physiological and pathological observations. Brain induced aheretiont WW (€Gs) Host defense berg New ¥ Seakine eysfunetion in trauma, shack and sepsis Springer, 80 & Cole G, Cowl Wa, (1887) Long ter Cyrotosies Trs8-7885 Ww (1867) tb Se:1€84~168) 1514 G, Schlag etal Big. & Strong immune GHNE) (<0) these territories wha2 G Schlager al zones between regions 2p also induce prSchlag et ‘Table 1. Histological evaluation ofthe brain damage induced by hemorthage in baboon ‘Type of eels or tissue Brain strueturelregion Immunohistochemical positsty to BNE Inflammatory cells ~ Blood vessels! ‘Moderate to strong monocyes and peutrophilst Blood vessels (the wll) Meni Moderate Subarchnoidal space Moderate White mater Weak Grey matier Weak Astrocytes White matter Raretand moderate Gray matier Negative Neurons Often’ and scrong. Often’ and strong, Cersbalum Often‘ and moderate Ependimel cells Plexus chorioideus Oftent and strong White maner Subependimal region strong! Pio-glil border strong! Astrocytes around the Blood-brain barzier Often’ and strong ‘blood vessels Infarction, necrosis, Cerebellom . Negative, both in neurons Only monocytes and nevizophils were notice. Mnflammatory cele were seen only within the Blood vessels, notin she brain tissue "Positivity of reaction depending on the intensity of immunohistachemics) staining end the ipeidence of positive celle cn high megnifcstion felé microscopy "Between one end two HNE-poshive cel ‘More then ten HNE-postive cells ‘Difuase and intensive immunesta shock model in the baboon and were responsible for our wish to further examine these clinical casualties on a neuropethologicel basis. On the coronal section of tissue was reduced in size due to the evident brain edema. On the horizontal section through the brain stem and cerebellum, acute hemorthi ction with perifocal edema wes noticed in the rostral pert of the left sphere (Fig, 2), Accordi the tissue damage, the infarction occurred only few hours before only a few inlemmatory cells, monacjzes, end neutrophil §n and around the blood vessels of the affecied segion. Interestingly, neurons and glial cells in the damaged tissue did aot show a presence of HNE, indicating that the oxidative lipid peroxidation and generation of BNE did not 1 inferction (Table 1). However, moderately the surrounding cerebeller white metier and in the astrocytes near the necrotic tissue (Fig. 3). On the other hand, soft membzenes, and suberachno‘dal and cerebral Blood vessels showed marked immunopositive reac if the e brain, ventricul es were noticedResults and Discussion tis well known thet circulatory failure and hem: tant ex tors which stron ties [25]. In our pelytrauma baboon model in connecti hypotension developed, causing a blood pres re for 2h, Brierley etal. [4] demonstrated jtaphan, shed blood or combi the cerebral perfusion pressure in 4 monkey seas seduced £0 below 25 mmHg and kept at this level eriods of time (up to 58min). Brain damage did not occur when the cer sion pressure re mained above 25mmHg. Brain damage wes else de; onset of hypotension: the fester the hypotension was achieved, the more se brain damage, In our model the fell in blood pressure a 40min). Arterial byp 0-50 mg Hg wes after the onset of blood withérawsl. The hypotension period lasted 90-150min. In cases apnoea was observed, which twas man led ventilation, ‘were on CP: fer 2 short while of profound arter 40mm Bg mean aortic pres: 2d slowly (30- pressure causing he pesk Jevel of fn er complete retransfusion of th blood fow. Apnoes was ob Ringer solution. Clinic nevrol8 Gu Schlag eta Special Postmortem of the Brain Brain Fheation and Morphological Analysis The brain was exemined by subseriel consecutive paraffin section as modified by Greevie [15]. The brain was removed by autopsy within Ih after death end fixed in 10% formalin, Upon fixation, the brein was grossly inspected and cut by consecutive coronal section into slabs ebout Smmn thick. After detailed examination, the materiel was photographed. The samples of brain tissue fixed in 10% buffered formalin were dehydrated in graded ethanol and embedded in paraifin, Perafin blocks of different sizes, corresponding to the sizes of the brain slabs, were cut subserially by the lerge Tetrener-Jung microtome into 5-im sections and used for histological and immunohistechemical analysis. Monocional Antibodies Against HNE Monoclonal antibodies to detect HNE-modified proteins were obtained ftom the culture medium of the clone "HNE 1s" which was derived ftom a fosion of Sp2-4 ryelome cells with B-cells of e BALBc mouse immunized with HNE-modified keyhole limpet hemocyanine (28). The ansibocy is specific for the HNE-istidine epitope in HNE-protein (peptide) conjugetes, HNE-lysine ané HNE-cysteine give 5 and 49% reactivity with HNE lg Immunohistochemical Analysis For immunohistochemical detection of HNE adducts, the im dase tech- nique was used, with secondary rebbit-anti-mouse antibodies (Deko, Denmark) Nonspecific binding was prevented serum while endogenous peroxidase reaction was abolished by H.0, treatment of the sections sing 19 bovine serum albumin (BSA) solution es scavenger for the slide washing (three times). Affinity for HNE 1g4 antibody was determined by testing the serial dilution of the antibody on the 4% buffered dehyde fixed HeLa cells treated by # 0 for Lh with a range of alde centretions (0.1-100 4M). ‘The specificity of HNE 1g4 monoclonal antibody was previously verified by adsorp- tion with its primary antigen (HNE-histidine epitope) in the form of a HNE-BSA conjugate (100 18f HNE/25 mg BSA/ml saline solution). 7 ‘washed, removing excess free aldehy¢ stirring of th ‘was stained by immunohistochemicelly positive inobe jerahydrachloride (DAB, reaction to } Dako, Denmark) end nickel chloride ( done using 19% Acridine orange sclution (Sigma, USA) and 1% Sefranin-O-stock solution (Sigma, USA). Pos ing thus produced a derk gray-to-black color with contrast ight yellow