Effects of ethanol on brain lactate in experimental traumatic brain injury with hemorrhagic shock

Brain Research 837 Ž1999. 1–7 www.elsevier.comrlocaterbres

Research report

Effects of ethanol on brain lactate in experimental traumatic brain injury with hemorrhagic shock Brian J. Zink b

a, )

, Carol H. Schultz a , Xu Wang a , Michelle Mertz a , Susan A. Stern a , A. Lorris Betz

b

a UniÕersity of Michigan, Section of Emergency Medicine and the Emergency Medicine Research Laboratory, Ann Arbor, MI 48109-0303, USA UniÕersity of Michigan, Department of Surgery, Section of Neurosurgery, and Departments of Pediatrics and Neurology, Ann Arbor, MI 48109-0303, USA

Accepted 18 May 1999

Abstract ObjectiÕe: Previous studies of traumatic brain injury ŽTBI. and hemorrhagic shock ŽHS. models, have shown cardiorespiratory depression in ethanol-treated animals. This study investigated the effects of ethanol ŽET. on brain lactate concentrations and acidosis in a TBIrHS model. Methods: Anesthetized swine were instrumented and subjected to injury ŽINJ. consisting of fluid percussion TBI of 3 atm with concurrent 30 mlrkg graded hemorrhage over 30 min. Three groups were studied: Sham, INJ and INJrET. ET was given preinjury as a 2-grkg i.v. bolus over 30 min, and an infusion of 0.4 g kgy1 hy1. Cardiorespiratory and cerebral physiologic data were monitored continuously for 150 min postinjury. Cerebral and renal blood flow was measured with colored microspheres. Brains were frozen in situ with liquid nitrogen. Lactate was measured with an enzymatic method. Results: ET levels at injury were 219 " 24 mgrdl. The INJrET group had increased mortality, impaired ventilation, and reduced renal blood flow. Brain Žcortical. lactate levels were significantly higher and cerebral venous lactate concentrations were increased in the INJrET group during the postinjury period. Cerebral venous glucose was significantly higher in the INJrET group, and cerebral venous pH was significantly lower. Conclusion: In this TBIrHS model, ethanol-induced increases in lactate concentrations in brain tissue and cerebral venous blood are associated with respiratory depression and reduced organ blood flow. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Ethanol; Brain injury; Lactate; Shock

1. Introduction Ethanol intoxication increases the risk of traumatic brain injury ŽTBI.. Up to 50% of TBI patients have a blood ethanol concentration over 0.1% at the time of injury w9,17,23,40,41,47x. Some clinical and laboratory investigations suggest that acute ethanol intoxication worsens outcome from TBI w1,11,12,17,27,29,41,42x. The effects of ethanol on TBI appear to be active in the first hour postinjury w47x. We previously found that ethanol intoxication caused respiratory depression, and decreased cerebral perfusion pressure ŽCPP. and regional cerebral blood flow ŽrCBF. in a porcine fluid percussion brain injury model w48,49x. We also noted increased blood lactate levels, and decreased blood pH in ethanol-treated animals. The addi) Corresponding author. University of Michigan, Section of Emergency Medicine, TC B1354, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-0303. Fax: q1-734-936-9414; E-mail: [email protected]

tion of hemorrhage to TBI magnifies ethanol effects w50,51x. Previous animal studies have shown that brain and cerebrospinal fluid ŽCSF. lactate levels increase following fluid percussion and cortical impact brain injury w16,21,22,25,30,33,45x. In humans with severe TBI, CSF lactate levels are increased, while CSF and brain tissue pH are decreased w5–7,10,28,30,34x. Acute ethanol intoxication, without injury, is also associated with increased blood and brain lactate levels in humans and animals w19,24,32,35x. However, chronic ethanol administration did not significantly increase brain lactate levels in a rat fluid percussion model w36x. Hemorrhagic shock is a major factor in secondary brain injury following TBI, and up to one-half of TBI patients have concurrent injuries that may lead to hemorrhagic shock w2–4,8,18,31,43x. Hemorrhagic shock increases systemic lactic acidosis, and this may be exacerbated by ethanol w14,15,20,39,46x. The effects of acute ethanol intoxication on brain lactate levels following

0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 6 4 6 - 7

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B.J. Zink et al.r Brain Research 837 (1999) 1–7

TBI and hemorrhagic shock have not been previously reported. Therefore, we evaluated brain lactate levels in a porcine model of combined fluid percussion brain injury and hemorrhagic shock, with the hypothesis that the physiological effects of ethanol in this model are associated with increases in brain lactate concentration.

Yorkshire swine weighing 18 to 23 kg were sedated with intramuscular ketamine Ž20 mgrkg., anesthetized with nosecone isoflurane Ž2%., endotracheally intubated, and maintained on isoflurane Ž1.15%. with an FIO 2 of 28. Vascular access was obtained via cutdown for catheter placement in both femoral arteries, one femoral vein, and the right carotid artery for blood sampling, hemorrhage, and pulmonary artery and systemic arterial pressure monitoring. A catheter was inserted via the femoral artery into the left ventricle of the heart for injection of dye-labeled microspheres to determine regional blood flow measurements. A 16-mm craniotomy was made with a drill in the right parietal area, adjacent to the sagittal suture, and anterior to the coronal suture. A T-shaped bolt was screwed into the craniotomy site until it abutted the intact dura. The bolt was connected to the fluid percussion device ŽStevenson Machine, Cincinnati, OH. and a high pressure transducer to measure injury, as previously described w50,51x. A second craniotomy was performed in the left posterior parietal region, 3 mm anterior and 5 mm lateral to the bregma. A neonatal intraventricular catheter ŽPhoenix Biomedical, Valley Forge, PA. was placed in the left lateral ventricle and connected to an intracranial pressure ŽICP. transducer. A brain temperature probe ŽCole-Parmer, Niles, IL. was placed adjacent to the intraventricular catheter. A third, T-shaped craniotomy was made just anterior to the inion over the midline, and a 4-F fiberoptic oximetric catheter ŽAbbott, North Chicago, IL. was placed in the sagittal sinus for cerebral oxygen saturation monitoring and blood sampling. The craniotomy sites were sealed with dental cement.

tion and brain injury. Cardiac output ŽCO. was measured by thermodilution technique ŽAmerican Edwards Cardiac Output Computer, Irvin, CA.. Ventilatory parameters, including minute ventilation Ž VE ., tidal volume Ž VT ., ventilatory frequency Ž VF ., and end-tidal carbon dioxide concentration ŽETCO 2 ., were measured continuously ŽDatex Capnomac Ultima, Datex Instrumentarium, Helsinki, Finland.. Hypercapnic ventilatory response was determined by infusing 6% CO 2 into the ventilatory circuit while maintaining all other gas concentrations at the standard levels. Ventilatory and physiologic responses to hypercapnia were recorded before and after 5 min of 6% CO 2 inhalation. The hypercapnic response is the slope of the line created by calculating the change in minute ventilation divided by the change in end-tidal CO 2 concentration Ž DV ErDETCO 2 .. Arterial and venous blood was sampled at baseline Žafter instrumentation., preinjury Žafter ET or NS fluid bolus., and 15, 45, 75, 120, and 150 min postinjury. Analysis included systemic arterial and cerebral venous blood gases, hematocrit, hemoglobin, and serum sodium, potassium, calcium, glucose and cerebral venous lactate ŽGem Premier Blood Gas and Chemistry Analyzer, Mallinckrodt Sensor Systems, Ann Arbor, MI; Kodak Ektachem DT 60II and DTSC II Multichemistry Analyzer, Rochester, NY.. Serum ethanol levels, obtained preinjury and at 120 min postinjury Žor at death if animals expired prior to 120 min postinjury. were measured with an NADralcohol dehydrogenase colorimetric assay ŽSigma Diagnostics, St. Louis, MO. and read on a UV spectrophotometer ŽHewlett Packard, 8452A, Diode-Array, Waldbronn, Germany.. Cerebral and renal blood flow determinations were made at baseline, and 15, 45, and 150 min postinjury using dye-labeled microspheres ŽDye-Trak, Triton Technologies, San Diego, CA. with a reference sample methodology as has been previously described w50,51x. At the completion of the monitoring period, while the animals remained anesthetized, the brain was frozen in situ for 30 min with liquid nitrogen with the method described by Wagner and Myers w44x. The animals were then decapitated and heads immersed in liquid nitrogen, and then stored at y708C. Heads were sectioned with a band saw, and 0.5 g tissue samples were taken from right cerebral cortex, cerebellum, and medulla. Brain tissue was mixed with 8% perchloric acid, buffered to a pH of 7 with 3.25 molar KOH, and lactate was measured with a colorimetric, peroxidase-based enzymatic assay ŽSigma..

2.2. Measurements

2.3. InterÕentions

A computerized physiologic data acquisition system ŽBiopac, Santa Barbara, CA. was used for continuously monitoring systolic and diastolic arterial blood pressure, mean arterial pressure ŽMAP., ICP, CPP ŽCPP s MAPy ICP., pulmonary artery pressures, end tidal CO 2 concentra-

Three experimental groups were studied. Animals in the sham group had all surgical procedures performed, but did not undergo injury. An injury group ŽINJ., was subjected to fluid-percussion brain injury, as previously described, with a target injury level of 3.0 atm w50,51x. Concurrent

2. Methods This investigation was approved by the University of Michigan Committee on Use and Care of Animals. Animal care standards were in compliance with the ‘‘Guide for the Care and Use of Laboratory Animals’’. 2.1. Instrumentation

B.J. Zink et al.r Brain Research 837 (1999) 1–7

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Table 1 Preinjury characteristics — values are means with standard deviations in parentheses. Measurements were obtained at the preinjury timepoint. p Values are reported for ANOVA. CV s cerebral venous blood measurement Parameter

Sham

INJ

INJrET

p

Number in group Weight Žkg. Brain injury Žatm. Brain temp. Ž8C. CPP ŽmmHg. ICP ŽmmHg. Cardiac output Žlrmin. Lactate cv Žmmolrl. Glucose cv Žmgrdl. pH cv Žunits. Oxygen sat. cv Ž%.

9 19.7 Ž1.3. 0 37.0 Ž0.3. 87 Ž16. 6 Ž3. 3.7 Ž0.4. 1.27 Ž0.26. 100 Ž15. 7.37 Ž0.39. 76 Ž5.2.

13 20.5 Ž1.8. 2.85 Ž0.5. 37.0 Ž0.4. 81 Ž16. 7 Ž4. 3.8 Ž0.7. 1.44 Ž0.42. 98 Ž13. 7.36 Ž0.40. 76 Ž5.4.

13 20.5 Ž1.5. 3.05 Ž0.5. 37.0 Ž0.6. 78 Ž16. 10 Ž4. 3.6 Ž0.7. 2.20 Ž0.95. 124 Ž17. 7.35 Ž0.02. 80 Ž5.4.

0.47 0.43 0.94 0.47 0.048 0.79 0.016 - 0.001 0.64 0.10

with brain injury, INJ animals were subjected to a 30 mlrkg graded hemorrhage over 30 min using a computerized roller pump ŽMasterflex, Cole-Parmer Instrument, Chicago, IL.. To simulate the dynamics of acute hemorrhage, the hemorrhage rate was decreased exponentially over the 30-min span. In the third group ŽINJrET., ethanol was administered 1 h prior to injury as an intravenous bolus of 2 grkg of a 20% solution over 30 min, followed by a 0.4-g kgy1 hy1 maintenance intravenous infusion for the duration of the experiment. Intravenous loading and infusion of ethanol was performed rather than gastric loading, as was done in previous studies, in order to reduce variance in ethanol levels, and to produce levels that were consistently in the 200–300 mgrdl range. Sham and INJ group animals received equivalent volumes of normal saline. Animals that became apneic in the postinjury period were ventilated with a volume-cycled ventilator to maintain PaO 2 at 90– 120 Torr, and PaCO 2 at 40–50 Torr. Animals were monitored for 150 min postinjury, without fluid resuscitation, and were euthanized with pentobarbital 30 mgrkg i.v. following brain freezing. In animals that were judged to have impending death, brain freezing was initiated when MAP fell below 30 mmHg, CPP was below 20 mmHg, and the heart rate was slowing with widening of the arterial pulse wave. In our experience with the model, these physiological changes indicate that death will occur within 15 min.

group made statistical comparisons beyond the 120 min postinjury mark less meaningful. A p value of - 0.05 was considered to be a statistically significant result.

3. Results The three groups were well matched, with no significant differences in weight, magnitude of injury, MAP, CPP, or CO ŽTable 1.. The mean ethanol level was 219 " 24 mgrdl at the time of injury, and rose to 263 " 35 mgrdl by 120 min postinjury. Animals in the INJrET group had significantly higher ICP, cerebral venous lactate, and cerebral venous glucose levels at the preinjury timepoint. However, cerebral venous pH was not significantly lower. All other preinjury metabolic parameters were similar between groups ŽTable 1..

2.4. Data analysis A power analysis Ž b s 0.91, a s 0.05. predicted that 10 animals per group would be required to detect a difference in brain lactate of 3 mmolrg assuming a standard deviation of 2 mmolrg. Group means and standard deviations were calculated for all experimental measurements, and compared using analysis of variance ŽANOVA. with Tukey post hoc test. Repeated measures ANOVA or a two-sample t-test was used to compare the two injury groups. The premature death of four animals in the INJrET

Fig. 1. Brain lactate concentrations in three brain regions. p Values are ANOVA comparisons for the three groups.

B.J. Zink et al.r Brain Research 837 (1999) 1–7

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Table 2 Postinjury metabolic parameters — values are means with standard deviations in parentheses. ANOVAs p value for comparison of all three groups at timepoint; rm ANOVAs p value for comparison between INJ and INJrET groups at preinjury, and postinjury timepoints using repeated measures ANOVA Parameter Lactate cv Žmmolrl. Glucose cv Žmgrdl. pH cv Žunits. A-v pH difference Hemoglobin cv Žgrdl. PaCO 2 ŽTorr. HCO 3 cv ŽTorr. Oxygen sat. cv Ž%.

45 min 120 min 45 min 120 min 45 min 120 min 45 min 120 min 45 min 120 min 45 min 120 min 45 min 120 min 45 min 120 min

Sham

INJ

INJrET

ANOVA

rm ANOVA

1.40 Ž0.23. 1.53 Ž0.56. 95 Ž13. 91 Ž33. 7.37 Ž0.03. 7.35 Ž0.06. 0.050 Ž0.035. 0.077 Ž0.058. 10.3 Ž0.80. 10.6 Ž0.88. 43 Ž5. 44 Ž8. 33 Ž2.0. 33 Ž3.3. 77 Ž5. 79 Ž8.

2.79 Ž0.80. 3.88 Ž2.57. 117 Ž25. 117 Ž34. 7.30 Ž0.06. 7.29 Ž0.10. 0.083 Ž0.105. 0.102 Ž0.069. 9.37 Ž1.16. 9.30 Ž1.37. 40 Ž5. 38 Ž7. 28 Ž2.6. 28 Ž3.8. 61 Ž15. 60 Ž18.

4.50 Ž1.43. 5.19 Ž3.06. 148 Ž49. 143 Ž47. 7.19 Ž0.10. 7.17 Ž0.10. 0.180 Ž0.094. 0.139 Ž0.059. 9.64 Ž1.00. 9.44 Ž1.39. 46 Ž11. 48 Ž13. 28 Ž3.9. 27 Ž4.5. 54 Ž15. 62 Ž15.

- 0.001 0.009 0.004 0.027 - 0.001 0.001 0.004 0.127 0.139 0.058 0.124 0.041 0.002 0.007 0.002 0.062

0.056

Four of 13 animals in the INJrET group died prior to the termination of the experiment at 75, 80, 80, and 116 min postinjury. One animal in the INJ group died prematurely at 145 min postinjury. In all animals that died, apnea requiring mechanical ventilation occurred prior to circulatory collapse and death ŽTable 3.. Animals in the INJrET group had significantly higher brain tissue lactate levels in the right cerebral cortex. Brain lactate levels were also higher in the INJrET group in the cerebellum, and medulla, but not to a significant degree ŽFig. 1.. If the four animals in the INJrET group and one animal in the INJ group that died prematurely were excluded, mean cortical lactate levels were 4.09 " 3.98 vs. 3.14 " 2.54 mmolrg, respectively Ž p s 0.567, two-sample t-test.. For all three groups, mean brain tissue lactate levels were higher in the cerebellum and medulla than in the cerebral cortex. A significant increase in cerebral venous lactate concentration was seen in the INJrET group in the postinjury

0.006 0.012 0.014 0.377 0.048 0.973 0.929

period ŽTable 2.. Cerebral venous glucose was significantly higher in the postinjury period in the INJrET group, and cerebral venous pH was significantly lower, while cerebral venous bicarbonate concentrations were not significantly different. Even when animals that died prematurely were excluded, cerebral venous lactate was higher in the INJrET group Ž p s 0.066, repeated measures ANOVA comparing INJ and INJrET group.. Arterial PCO 2 levels were significantly higher in the INJrET group in the postinjury period. The arterialrcerebral venous blood pH difference was significantly greater in the INJrET group in the postinjury period. Cerebral venous oxygen saturation decreased significantly in the injury groups, but was not significantly different between the INJ and INJrET groups in the postinjury period ŽTable 2.. Physiological changes were noted in the injury groups, including significant decreases in MAP, CPP, and CO ŽTable 3.. Mean values were lower in the INJrET group compared with the INJ group, but not to a significant

Table 3 Postinjury physiological parameters — values are means with standard deviations in parentheses. ANOVAs p value for comparison of all three groups at timepoint; rm ANOVAs p value for comparison between INJ and INJrET groups at preinjury, and postinjury timepoints using repeated measures ANOVA Parameter

Sham

INJ

INJrET

ANOVA

Survival time Žmin. CPP ŽmmHg.

150 Ž0. 92 Ž15. 86 Ž16. 3.22 Ž0.39. 2.97 Ž0.59. 67 Ž22. 74 Ž30. 265 Ž116. 360 Ž73.

149.6 Ž1.4. 57 Ž8. 57 Ž12. 1.96 Ž0.28. 2.01 Ž0.36. 48 Ž15. 74 Ž20. 189 Ž92. 232 Ž44.

130.8 Ž31.4. 43 Ž18. 52 Ž17. 1.57 Ž0.53. 1.86 Ž0.70. 42 Ž19. 59 Ž35. 105 Ž81. 131 Ž91.

0.029 - 0.001 - 0.001 - 0.001 - 0.001 0.370 0.658 0.002 0.001

Cardiac output Ž1rmin. CBF-R cortex Žml 100 gy1 min. RBF-R kidney Žml 100 gy1 min.

45 min 120 min 45 min 120 min 45 min 150 min 45 min 150 min

rm ANOVA 0.353 0.234 0.826 0.339

B.J. Zink et al.r Brain Research 837 (1999) 1–7

Fig. 2. Minute ventilation. ps 0.016, ANOVA, comparing all three groups; ps 0.05, repeated measures ANOVA comparing INJ vs. INJrET for the entire experimental period.

degree. ICP increased to approximately 15 mmHg in the early postinjury period, but returned to baseline values by 45 min postinjury, and was not significantly different between injury groups. Therefore, the primary effect in lowering of CPP was the reduction in MAP. Cerebral blood flow ŽCBF. was significantly reduced in the injury groups at 15 min postinjury, but returned to near baseline levels by 45 min postinjury. The degree of CBF lowering with injury was not different between the cerebral cortex and medulla. Renal blood flow ŽRBF. was more compromised by injury than was CBF ŽTable 3.. When expressed as a percent change from baseline values, RBF, but not CBF, was significantly lower at 45 min postinjury in the INJrET group compared with the INJ group Ž p s 0.009, two-sample t-test for right kidney sample.. Ventilation was significantly impaired in the INJrET group in the postinjury period when compared with the INJ group. Minute ventilation was decreased to a greater extent than the hypercapnic response ŽFig. 2.. The primary effect in lowered minute ventilation was a reduction in ventilatory frequency. A resultant increase in arterial PaCO 2 to near 50 Torr was noted, but PaO 2 remained above 120 Torr in the INJrET group ŽTable 2..

4. Discussion In this porcine model, which was designed to simulate the common scenario in human trauma victims of acute ethanol intoxication, TBI, and hemorrhagic shock, significant elevations in brain lactate, cerebral venous lactate and

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glucose, and a reduction in cerebral venous pH were found in ethanol-treated animals. The degree of ethanol intoxication in this model Žmean postinjury levels of 220–260 mgrdl. is similar to human studies of TBI, where mean ethanol levels greater than 200 mgrdl are commonly reported w9,23,47x. The biochemical alterations observed in this experiment were associated with detrimental physiological changes that have been previously characterized in this model w50,51x. Accentuated brain acidosis may play a role in the increased mortality, impaired ventilation, and decreased blood flow observed in ethanol-treated animals in this study. Human studies of patients with severe TBI have found that elevated lactate concentrations in both CSF and microdialysate fluid are correlated with worse neurological outcome and increased mortality w5–7,10,28,30,34,37x. Some clinical studies suggest that factors that contribute to secondary brain injury, such as hemorrhagic hypotension, may be associated with elevated brain lactate levels w7,30x. A number of previous studies in animal models of TBI have found that brain lactate increases within 30 min of injury w21,22,25,33,45x. Only one other published study has examined ethanol effects on brain lactate following TBI. Prasad et al. w36x found that lateral fluid percussion injury produced a three-fold increase in brain lactate concentration within 5 min. However, animals that had 6 weeks of chronic ethanol administration did not have higher brain tissue lactate concentrations following TBI than animals fed a control diet. This study differed from ours in that it used a less severe TBI Žmean 1.8 atm. without the addition of hemorrhage, excluded animals that experienced apnea, and had acute, superimposed on chronic ethanol intoxication, with mean levels at the time of injury of 121 mgrdl. A number of mechanisms have been proposed for the increase in brain lactate following TBI. Some studies suggest that elevated lactate levels in CSF and brain tissue may be secondary to an increase in systemic lactate production w22,28,38x. An increase in systemic blood lactic acidosis has been observed following TBI, even in the absence of circulatory shock or hypoperfusion. Since the blood brain barrier is permeable to lactate, systemic lactic acidosis may contribute to brain lactic acidosis. Another theory is that decreased cerebral perfusion and CBF following TBI causes cerebral ischemia, and this leads to elevated brain lactate concentrations w38x. However, one clinical study w10x found no correlation between decreased CBF and elevated CSF lactate levels, and another w6x found that higher lactate levels were associated with higher CBF. The most recent proposed mechanism for brain lactate elevation following TBI centers around mitochondrial dysfunction and disordered cellular energy metabolism in the early postinjury period. Work by Hovda et al. w21x and Kawamata et al. w25x using microdialysis in rats suggests that altered transmembrane ionic gradients Žrelated to glutamate release. increase energy demands following TBI

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and that subsequent hyperglycolysis results in lactate accumulation. Ethanol, however, is known to inhibit NMDAmediated cytotoxicity, which could theoretically reduce hyperglycolysis w26x. Kelly et al. w26x using a rodent cortical contusion model, found that ethanol intoxication increased early death, presumably due to cardiorespiratory compromise, but improved neurological outcome in those animals that survived. The authors propose that the neuroprotective effect of ethanol seen in this study is due to ethanol-induced blockade of NMDA receptor-mediated excitotoxicity. Blood glucose concentration is another factor that may influence lactate accumulation. In a rat model of cerebral tissue hypoxemia, higher blood glucose levels were associated with increased brain lactate concentrations w13x. Since blood glucose levels were increased following intravenous ethanol loading in our model, this may also be a factor in elevated brain lactate. Ethanol intoxication without injury leads to increased blood lactate levels w24,32x. The effects on brain lactate accumulation have not been established. The approximate doubling of blood lactate levels that was seen following ethanol-loading and prior to injury in our study has also been reported in healthy humans with low levels of ethanol intoxication w24x. The mechanism for ethanol-induced lactate acidosis is thought to be a metabolic consequence of the build-up of NADH during ethanol oxidation that stimulates pyruvate dehydrogenase to form lactate from pyruvate w19,35x. Therefore, a number of factors Žethanol, TBI, hemorrhage, glucose, ischemia, increased glycolysis. could contribute to increased brain lactate in this model. Our data do not provide definitive answers to which factors are most important, and are limited by the lack of preinjury brain tissue lactate levels. In a previous study that compared ethanol effects in TBI with and without hemorrhagic shock, we found that blood lactate levels were significantly higher when hemorrhage was added to the TBI model, and ethanol intoxication magnified this effect w51x. It is likely that systemic hypoperfusion and tissue ischemia from hemorrhage and TBI contributes to elevated brain lactate levels in our model. However, in the current study, the evidence is not strong that brain ischemia was worse in ethanoltreated animals. Although the INJrET group had lower CPP, CO, and CBF, the mean CBF in all tested regions was well above the level that is commonly used to denote ischemia Ž20–30 ml 100 gy1 miny1 .. The reduction in blood flow to the kidney was proportionately greater than to the brain, suggesting that cerebral autoregulation of blood flow remained intact in this model. We did not acquire data to calculate an arterial–cerebral venous lactate difference, but the increase arterial–cerebral venous pH difference in the INJrET group suggests that brain acidosis from elevated brain lactate was more pronounced in these animals. Therefore, the source of increased brain lactate in ethanol-treated animals in this model may be

partly due to systemic lactic acidosis, but it appears that the brain is also producing, or accumulating lactate, resulting in increased levels. Other effects, such as mitochondrial dysfunction and hyperglycolysis may be active at the cellular level, but were not assessed in our model. A number of limitations are present in this study. We did not include a group that received ethanol without injury. Also, higher mean ethanol levels that rose throughout the experiment were seen in this experiment, compared to our previous experiments which used gastric ethanol loading. However, intravenous ethanol infusion did not produce greater cerebral venous lactate elevation or worse physiological effects when compared with these previous studies w50,51x. The early deaths in the INJrET group are a confounding factor in assessing brain lactate. It is unclear whether the higher degree of brain lactate concentration found in these animals was due to the worsened hemodynamics and cerebral dysfunction that resulted in premature death, or if it was due to sampling earlier in the postinjury period. The results of this study demonstrate that ethanol may increase blood and brain lactate concentration following TBI and hemorrhagic shock, and that this metabolic effect is associated with detrimental physiological changes. Further investigation will determine if attenuating ethanol-induced elevations in brain lactate will improve the physiological response to brain injury.

Acknowledgements This study was supported by the National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, a5 K08-AA00184-02A2.

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