istological Aspects of Sepsis-Induced Brain Changes | a 7 oainpraiee ae oe ary a stress is vortance for determining the outcome of sepsis PF 2 Baboon Model™ shly reactive aldehyde 4-hydroxynonenal (HNE) is one of the most imp. K. Zankovie, N. ZaRKovic, G. SCHLAG, H. Rept, and G, WaEc recently (38), specific monoctonal n (oF peptide) conjugate(s) that allow morphological analysis of the tissue fcate that HNE and related aldehydes are ~ ina Introduction Encephalopathy is one of the organ dysfunctions seen in critically il patients with fe organ dysfunction syndrome (MODS) secondary to sepsis (defined ponse to infection) | calor central nervous system (CNS) status is usually included in mul- organ dysfunction scores part on the Glasgow Coma Scare (see olume). Its often dificult to evaluate the CNS status i abnormal amino-acid patterns may also account for brain alterations (e.g, reutrotransmitter) [20] ‘Studies on the pathophysiology of sepsis due to the specific permeability of the brain-blood barrier, unique inflammatory responses (engagement of the glial cells), and the specific anatomy and histology of different brain regions ight into the role of oxidative stress in the able supplement to: nonhuman primate model of sepsis [32], which involves pathophysiology of brain damage caused by sepsis involving one of two strain .chave performed an immunohistochemical analysis ofthe distibut a the brain of the septic baboons and investigated whether the morphological | defined brain damage was also reflected in plasma markers of brain damage, eg, neuron specific enolase (NSE). NSE i the neuronal form of the glycolytic enzyme 2- phospho: ‘damage (6), in serum asa marker of cerebral injury after head traur jacob disease [32], and hypoxic brain very similar to humans, offers many advantages as. addition to our previous studies in which we have primati 1], and mediator systems (e.. spproack to include the brain. sons for brain dysfunction are numero: iple brain microabscesses (see Young, this volume) or the action of , phospholipase A) {9], Creutzfeld- smage {3}, and a marker of tumors rophage system, which induces the inflammatory euroendocrine properties [35 her source of ROS perfusion asa result of sepsis-induced microcirculatory mismatch i yy, ROS produced by the inflammatory cells and the endothelial es phy ole a ieee Gefense agaist germs [284 Experimental Procedure lecules), generating highly reactive aldehydes, which are considere: Hes), generating highly reactive aldehyds se ‘Male animals of the species Papio tha weight of about 20 kg were sxic messengers” of free radicals [11,41]. Aldehydes produced in this way are very 4 damage tissue [10], meaning that various organs are damaged simulta sly by infective agents as well as by the toxic products of sepsis. The permeabi investigated. Fasting animals were sedated with 6-8mg/kg ketamine hydrochloride (Ketalar, Parke Davis Co., Ann Arbor, MI), placed in the supine position. Following intubation, the tube was connected (o an oxygen blender via a T-piece. The FiO, was fained with pentobarbital at a dosage of Ts paper is dedicated to Prof, Hermann Esterbauer. We lst a fantastic colleague and kept within 239% + 28 Anesthesia was n wonder fiend 2-dmgikg per hour.Following exposure of the right femoral vein, an introducer for a heparinized sn-Ganz catheter (7F) was positioned in the pulmonary artery. A catheter was nto the right brachial artery for blood withdrawal and for pressure moni 1d of the acute study catheters were ced in a subcutaneous pouch; the wound was closed with serted into the right brachial vein for infusion (anesthesia, medicatio the start of E. coli infusion), Baboons were infused wit the course of the experiment. In Is were administered addition: .ous fluid, depending on the measured pulmonary wedge pressure assessed 48h following recovery. h measurement and for infusion therapy after the acute phase of the loride (Ketalar) 6-8mg/kg, sterile conditior lines or to the infusion pump. The wound was reclosed after the procedure and the animals were her kept without anesthesia, Blood samples were drawn in ethylenediaminetetra 1m tubes (Greiner, Kremsmiinster, Austria) centri Total blood 1890 (Coulte nercial is a double antibody assay. NSE in the sample competes with a fixed inding sites of the specific antibodies. Bound and free NSI d by the addition of a second antibody immunoadsorbent followed by cen- nd decanting, The radioa is, hemoglobin, and hematocrit were determined by a co -ctronics Inc., Hialeah, FL, USA). NSE was measured using a c: ia coli Preparation in a fermenter using Tryptone soy broth for 150min at 37°C with n, aeration, and pH control to maximize living cell counts and to minimize ipopolysaccharide (LPS) levels as previously described [32] Brain Fixation and Morphological Analysis: consecutive paraffin section as modified by Greevie ‘was removed by autopsy within 1h after death and fixed in 10% formalin. Upon fixation, the brains were grossly inspected and cut by consecutive coronal sect slabs of about Sim thickness, After detailed examination, the material was photo- graphed, The samples of brain tissue fixed in 10% buffered formalin were dehydrated in graded ethanol, and embedded in paraffin, aralin blocks of different sizes, corresponding to the sizes ofthe brain slabs, ‘microtome into sections of Sum and swith B-cells ofa BALBe mouse immunized with HNE modified keyhole specific for the HNE. i Immunohistochemical Analysis For the immunohistochemical detection of HNE adducts, the immunoperoxidase tech ‘ed, with secondary rabbit-anti-mouse antibodies (Dako, Denmark). Nonspecific binding was prevented by the use of normal, nonimmunized rabbit serum while endogenous peroxidase reactions were prevented by H,0, treatment of sections using a 1% BSA solution as scavenger for washing the s ‘The affinity of the FINE 1g4 antibody was determined by testing the serial dilutions jody on Hela cells fixed in 4% buffered paraformaldehyde that had been treated by HNE in vitro for 1 concentrations of the aldehyde ranging between Ol and 100M. The specificity of HNE 1g4 monoclonal antibody was previously verified by adsorption with its primary antigen (HNE-histidine epitope) in the form of an HNE-BSA conjugate (100 1M HINE/25mgBSA/ml saline solution). ‘The HNE-BSA conjugate was washed of the excess of free aldehyde by filtering fon the Amicon membrane ultrafiltration system using an 0.5kDa membra (Amicon, Ireland) under pressure of nitrogen, with the sample being constantly rabydrochloride (DAB, Dako, Denmark) and nickel chi ). Contrast staining was done using 19 acridine orange solu- tion (Sigma, USA) and 196 Safranin-O-stock solution (Sigma, USA). Positive immunostaining thus produced a dark gray-to-black color, with contrast of a light yellow-to-orange st Results and Discussion Experimental sepsis caused the death of the experimental animals within 2 days, irrespective of the strain of E. coli used to induce sepsi fluence the inflammatory response to the bacteria within the blood vessels, asi; | 3] z presented in Tabl blood vessels ofthe contro septic baboons injected 2] og z with SNS E.coli contained mainly lymphocytes (including plasma cells), neuteop let gE g tnd macrophages. The blood vesels ofthe brains of animals injected With $5 coll J [Epo Feds contained mainly neutrophils and monocytes, while lymphocytes were only found 2 ‘occasionally The V-R and subarachnoidal spaces were infiltrated by the leukocytes in | & the SNS E. coli injected control animals. It can therefore be assumed that the strain of | |g g bacteria plays a role in the induction of the systemic inflammatory response and i & = consequences on the brain, However as could be expected, there were no prominent glee & inflammatory cells found in the brain tissue itself, except in the brain of one baboos 2| 22 3 in which the phenomenon of neuronophagia was manifested As can be seen from 3| #22 g “Table 2, very strong immunchistochemical staining on HINE was observed for almost | |e] 333 e all in the subarachnoidal space and the bra 1).In | [Sls is] e ‘ presence ofthe aldehyde was less obvious i the gray matter of the ba | E and could not be even detected for some animals, Interestingly, the animals in which g there was no positive testing for HNE in gray matter were those who lived for a | 22 g relatively short | |elegee & “The difference in HINE content observed for the white and gray matter of septic 3) 2285 & baboons was even more obvious if the positive HINE testing stemmed from a je/2/2537 2 2 [8 in the gray and white matter respectively (Table 3, Fig. 2). Numerous astroglial ce ee |S |eeee 2.2. / 8 rhe sea ear peeren he cine see epe inl le z detected forthe same glial cells in the white matter. While the strain of E, cli used to Va! 2 induced sepsis did not seem to be of importance concerning astroglial “oxidative gi eie Q burst” in white matter, it seemed to be of relevance for the HNE positivity of astro- ele) E g cytes in gray matter. Thus, weak HNE positivity was seen for the astrocytes in the gray E/ Els 2 matter of 3 of S baboons infected with SNS E, col but only in 1 ofS baboons infected 2\2|2 z with $8 B. co Al 2 lea, The fc of expernentalspg onthe presence ofthe iid peroxidation rot E/E |egese | treatment Position of the blood vessels 2/g |aawaa S88] 2 cbarachnoidal White matter Gray matter 2|f |88822a9e2|§ 2 nies a ai S 8 Seal - He + eae 568 SE. coll pene o + Els Boe sr 40 SNS Eccl 2 4 + z| 225 | SNS Eeal = ? 7 g\igs | egeseeanar| FFp & 16 SNS Ecol o + = eee gee & 8 SSE cal + “ + z ETE reese + ? + Z gee on a a + “14 age ieee + + : Alec eos euy eel ede n non-sensitive; 8S, serum sensitive; Immaunohistochemial positivity to HAE: + { ‘ery strong Hh moderate:# weak negativebecause oftheir role in the generalized stress reaction, ine system function (which has to be stable for recovery Fig. 5. ress to be possible) and inflammatory response {7,34,35]- Due to the uid (400)Particular observations on the presence ofthe lipis peroxidation product 4-hydrosynonenal (HNE) in the bran tissue of septs abowns ‘Animal Survival Experimenta Ependim Subepenitnsl — Hemolytic Edema fyi no. a) chotiosd plexus’ while matter” content of 55 8 SNS coli an 7 + aa + + 56 148 SNS & coli He é s + 37 40 SNS E col we + a 39 7 SNS E colt + ” + # + 6 16 SNS E coli 4 - = 7 z 66 8 88 E.coli + 7 - 7 + + o 33 SSE coli 4 4 + at ' 7 ® 2 SSE coli 4 7 a . a 3 28 SSE coll o + “ 7 e = HINE postvity-+4, more than ten HNE-postive cells per high magnification (+400) field (HME); ++, five to ten HNE-positivecells per HM + less than five HNE-pastive cells per HME; ~, no HNE-positive cells, Nimoderate 0 strong intensity ofthe HNE immunopositive staining, ~, no FINE positivity. g ee 5 aghh ; Pe : z 2 3 2 es a2 aa Pr] eg as jo vuseyd wosyrediso> ato) pur gso uF fuo> ado, st pe P8598 1 sisdoe Buuunp paonpoad (924s 30 seer Sunsozoqur ue poqeanan ‘aBewep GND Jo 199seU 24) “(G Fig) BSN JO SIs z g = gk : fee Fe Ae 2 3 ze B38 = a Zs ge é Fo g28we presence of HNE and was much the other brain regions, fe neurons were found in the midl than in not depend on the cellular inflam- brain damage induced by sepsis could be he blood-brain barrier and on the release of the (such as HNE) from the blood vessels, Rea lamage can be seen in the plasma enolase values, which results of this study can be summarized as follows: Herero ee onlenicuaia aie ‘ular edema fluid of the brain and the hemolytic content of the blood ‘Acknowledgments, We thank James Davies for his siipport performing the animal adducts. experiments and Eva Paul for enolase measurements. in the brain membranes and blood vessels,na AM, Clark RL, Goodwin FK (1978) Measurement 1d now neuronal specific enolase (NNE) isoenzymes skey and human nervous tisse. | Neurochem 33:319-329 ‘Morell F, Raya A, Roma J, Aldasoro M, Vila J, Luch S, Romero FJ peroxidation product, induces relaxation of he ‘Metab 14:593-696 imeno M, Belova Ny Kamat A, Rettori V (1995) duce the pattern of pituitary hormone sect 5-395 dV (1993) Nitric oxide synthesis clfecis on brain blood flow. A R (1994) Nitric oxide and References: 1998) Response ofthe micr eds) Pathophysiology of shock, sepsis, few York, pp 230-256 ber DE. (1994) Vat epsis and organ fsilure-nitrc oxide. Fo ger Berlin Heidelberg New York, pp 243-258 hi (1994) Blochervical markers of cerebral ischaemia, In: Sebel PS, Fitch 2s, arteries. 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In Schlag G, Red H (eds) Pathophysiology of shock, sepsis snd organ failure. Springer Be berg New York, pp 908-984 : 38 Waeg G, Dimsity G, Esterbauer I (1996) Monoclonal antibodies for dete hydroxynonensl proteins. Free Radic Biol Med 25:149-159 39, Wakayama ¥, Shi Sagawa game ¥ (1987) High ne , Thompson Ry (1980) Human 14-3-2 protein: enolase level in cerebeoxp inthe early stage of Creutfeld-Jakob ribysion in human tissue. Biochim Biophys Acta Wochenschr 65:798-80 40, Waterfall AH, Singh G, Fry JR, Marsden CA (1995) Detection ofthe lipid peroxidation B, CadenasE, Bovers A (1986) The relation of free radical production product malondyaldehyde in dhe rat brain fn vivo, Newrosct Let 200:69-72 Te npr 41, ZarkovieN, thie 2,Jurin M, Schau RJ, Publ H, Esterbauer H (1993) mulation of HeLa cell 1973) Besin’ mone-amines in human hepatic encephalopathy. Acta {growth by physiological concentrations of 4-hydroxynonenal, Cell Biochem Funct 11.279 opathol (Bee) 48:53-68 286 at hagel |, Consentino F, Vanhoutte PM (1993) Endothelium-dependent con- 2 ‘ah H, Jurin M, Esterbaver H (1994) Mutual dependence of growth fractions to oxygen-derived ftee radicals in the canine basilar artery. Am J Physiol | modifying effects of 4-hydroxynonenal and fetal calf serum in vitro. Free Radic Biol Med 257:H1859-H858 163877-8841, Schour RJ, Esterbauer H (1991) Biological Stes H (ed) Oxidative stzess. Academic London, pp 337-369, Discussion long. Did you have ike to make in regard ‘oxide synthase or nitric oxide and possil th damage that you observed? lar rene ry cellular reaction analysis and in these cases with L ensity ofthis reaction in brain tissue a i hat would you conclude from that? That there was less cellular interaction with F nitric oxide synthase? jon is that itis too early to draw conclusions, because it is an ongoing ‘we have only first preliminary results. hat is your cusrent interpretation of the data? Are the pathological changes in the ie to intoxication from endotoxin, or other bacterial produtcts, or due to blood. erat progressing ischemia? y right that it might be mainly due to blood low changes by ind of local ischemic reperfusion, in addition to the inflamma- ved out, there is a lot going on on the vessel wall and it might be that real sin the blood-brain barrier are decisive. Bolton: Very interesting results. The thing that I was struck by was the amount of cerebral edema. Clinical cal studies that we have observed in humans, we ddo nat see cerebral eclema in septic encephalopathy. We have, however, had at cone patient, where cerebs death of the patient. 1 bi cases from around the world, It is almost like a Reye syndrome, but affecting adults, Cultures have been negat know they were in ours, The patient had all the systemic signs of sepsis. But in general, { do not think cerebral edema in humans has been observed in septic encephalopathy. st we have a big bacterial challenge, so we infuse 10° and the blood stream. Iti not comparable with humans, more toxicity wwe have a level of about 10° where there are about 10° bacteria, So we have mucl jon. It scems somewhat reminiscent, Do you see any meningeal involve- is model; is the bacterial challenge great enough that you actually see K. Zarkovic: Maybe this isan effect of endotoxi thas HNE, which damage and maybe of “second toxie messengers” for free 1¢ blood-brain barrier. Schlag: But our baboons, if they de, they die of adrenal bleedings, so they are really compa- rable with the Waterhus-Fridre very interesting, degeee comparable to children with meningococei sepsis. Kochanel [think it would be very interesting also to see what model te and nitrates were in your Schlag: ‘We measured it. We see an increase, Kochanck: Again, thinking about this with regard to Dr. Traber’s presentation, there have bet several reports particularly of an association between elevations of nitrite and nitrates in pediatric septic shock and the development of multiple organ failure. Many of these children have overwhelming infections and we obviously goto the wall for them “try anything you can”, massive doses of pressors, etc. Have you modeled the situa- tion, where you produce what would be an overwhelming insult with # need for‘ical care intervention, in sheep, i. producing & situation where the sheep are so sick they need to be intubated and require inotropic support. [ wonder if you have jing septic shock tes and nitrates of those animals. We have had them to ‘of having to put them on oxygen, these animals are not included in the groups we presented. We have had to give inotropic support, we have taken therm to Recommendation CB. Bourow, D.S. PRouGH, CL. SpRUNG, and G.B. YOUNG ‘At present, the Glasgow Comaa Score (GCS) can be used to assess brain dysfunction in ic patients. The rationale for its continued use in septic patients ist 1. The demonstrated correlation of scores with outcome Problems with the GCS are found wit Assessment in sedated patients Intubated patients . Patients whose eyes are swollen shut |. GCS design: Fragmentation into subcategories rather than comprising single ordinal scale. Other scoring systems, eg, the Reaction Level Scale-85, that do not have these s should be compared with GCS in large comparative studies. Electro: , eg, continuous or serial EEG, is a valuable supplement especially in the evaluation of cerebral cortical function in