Flumazenil does not affect the increase in rat ... - Springer Link

Metabolic Brain Disease, Vol. 11, No. 4, 1996

Flumazenil Does Not Affect the Increase in Rat Hippocampal Extracellular Glutamate Concentration Produced During Thioacetamide-Induced Hepatic Encephalopathy Philip McArdlei, Donald H . Penning2A, Franklin Dexter2 , and James D . Reynolds2 Received: 1 March 1996; Accepted: 16 June 1996

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that often occurs as a consequence of acute or chronic liver failure. Previous reports have suggested that alterations in amino acid neurotransmission, particularly glutamate, may play an important role in the pathogenesis of HE . The objectives of the present study were to test the hypothesis that extracellular glutamate concentration is increased during HE, and to determine if flumazenil, a benzodiazepine antagonist, alters the extracellular concentration of glutamate during HE. The experimental approach involved using microdialysis probes to measure rat hippocampal extracellular glutamate concentration. HE was brought about as a result of thioacetamide-induced liver failure . Thioacetamide produced behavioral and metabolic effects, such as somnolence, hyperventilation and hyperammonemia, consistent with stage three HE . Comparison with saline-treated rats demonstrated that HE was associated with a significant increase (p=0 .010) in extracellular hippocampal glutamate concentration . Administration of flumazenil caused a transient increase in arousal level, but did not affect the increase in glutamate concentration (p=0,93) . These results corroborate the theory that glutamate neurotransmission is altered during HE and suggest that the flumazenil arousal of HE rats is not mediated by a change in extracellular glutamate concentration . Keywords : Flumazenil; glutamate; hepatic encephalopathy ; hippocampus ; microdialysis

l Present Address : Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama 2 Department of Anesthesia, University of Iowa, Hospitals and Clinics, Iowa City, IA 3 To whom correspondence should be addressed at Department of Anesthesia, Duke University Medical Center Durham, NC 27707 ; Tel. : (919) 681-3345, Fax : (919) 681-8357

329 0885-7490/96/1200-0329$09 .50/0 0 1996 Plenum Publishing Cor

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INTRODUCTION Hepatic encephalopathy (HE) is a neuropsychiatric disorder which often occurs as a consequence of acute or chronic liver failure (Mousseau and Butterworth, 1994) . In cases of acute failure, death can occur in a matter of days as a result of extensive brain edema and increased intracranial pressure . Currently, HE and accompanying cerebral edema is the leading cause of death in people diagnosed with fulminant hepatic failure (Hawker, 1993) . Patients with HE exhibit a deterioration in brain activity progressing from mild stupor and loss of muscle tone to coma . Impairment of normal liver function is associated with an increase in blood and brain ammonia concentration . This increase can impact negatively on normal neuronal function ; alterations in neurotransmission have been proposed to account, at least in part, for the loss of brain function produced by HE (Record, 1991) . The glutamate neuronal system is very sensitive to the changes in ammonia concentration that can result from liver injury (Rao et al., 1992) . Enzymes that are important in the regulation of glutamate function can be affected by changes in ammonia concentration (Fonnum and Hassel, 1995) . Furthermore, neuronal synaptic enzymes involved in glutamate metabolism may be affected differently compared to non-synaptic enzymes . Hyperammonemia and acute HE stimulate cerebral cortical synaptic mitochondrial glutamate dehydrogenase activity in the direction of glutamate oxidation . In contrast, cortical non-synaptic mitochondrial activity is increased in the direction of glutamate formation (Faff-Michalak and Albrecht, 1993) . Ammonia may also alter glutamate uptake (Rao et al., 1992) . Acute HE, induced by thioacetamide, decreases glutamate uptake in rat cerebrum synaptosomes (Oppong et al., 1995), but chronic HE has no affect on rat cerebrocortical glutamate uptake (Maddison et al., 1996) . These apposing results suggest that acute and chronic HE may have different effects on glutamate uptake or that species differences exist ; the same authors have reported that glutamate uptake is increased in cerebral cortical synaptosomes prepared from dogs diagnosed with congenital chronic HE (Maddison et al., 1995) . Studies in both humans (Lavoie et al., 1987) and animals (Zimmermann et al., 1989 ; Bosman et al, 1990 ; Swain et al ., 1992) have reported that total brain glutamate concentration, which includes both metabolic and neurotransmitter pools, is decreased with HE . However, such changes may not reflect what is happening to the neurotransmitter pool of glutamate (an estimated 1 to 5% of total glutamate in the CNS) and the variant effect of HE on synaptic enzymes involved in glutamate metabolism (Faff-Michalak and Albrecht, 1993) . As well, in some studies glutamate concentration was quantitated in terms of brain tissue wet weight which may be misleading due to the increase in tissue water content and subsequent cerebral edema associated with HE . Other researchers have used microdialysis to determine the extracellular concentration of glutamate in vivo during experimentally-induced HE with conflicting results . Glutamate concentration in the frontal cortex of portacaval-shunted rats did not change even when plasma ammonia concentration was increased by administration of ammonium acetate to produce the hyperammonemia associated with HE (Rao et al., 1995) . In contrast, rabbit cerebral cortical glutamate concentration was increased by acute ischemic liver failure and by acute hyperammonemia

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(de Knegt et al., 1994) . It is debatable if these experimental conditions accurately mimic HE . Rats that recover from portacaval shunting exhibit few overt signs of the neurologic impairment associated with HE (Rao et al., 1995), while hyperammonemia alone, produced by ammonia infusion, may not reflect all the deleterious effects that liver failure has on brain function (Hilgier et al., 1991) . The first aim of the present study was to test the hypothesis that hippocampal extracellular glutamate concentration, determined by microdialysis, is increased during HE . We chose to look at the hippocampus because of its rich glutamate innervation and its important role in higher cortical functions . Liver failure was induced by thioacetamide, a hepatotoxin that produces a course of neurologic symptoms and metabolic effects which resemble HE (Zimmermann et at., 1989). Inhibitory neurotransmitter systems may also be altered during HE . Clinical studies indicate that there may be an increase in the level of endogenous benzodiazepines in cases of fulminant hepatic failure (Basile et al ., 1991) . Such an increase would be expected to enhance gamma-aminobutyric acid (GABA) inhibition in the CNS . The GABA neuronal system plays an important role in regulating glutamate release (Traub and Miles, 1991) such that perturbations in one system would affect the other . While the exact role of the GABA neurotransmitter system in the pathogenesis of HE still needs to be resolved (Jones et al., 1994), attention has focused on the ability of benzodiazepine antagonists to reverse the somnolence associated with HE . One such antagonist is flumazenil (Jones et al., 1994 ; Howard and Seifert, 1993) . Preliminary studies have reported flumazenil administration significantly improved reaction time of patients with latent HE (Gooday et al., 1995) . The interrelationship between the glutamate and GABA systems suggest that part of flumazenil's action could alter glutamate release. In this regard, it is interesting to note that flumazenil has also been used in the treatment of epilepsy (Scollo-Lavizzari, 1984 ; Sharief et al, 1993), another neurologic condition associated with increased glutamate concentration . At this point, the effect of flumazenil on hippocampal glutamate concentration during HE is not known . Therefore, in the second part of this study, we tested the hypothesis that flumazenil alters the extracellular concentration of glutamate in the hippocampus during thioacetamide-induced liver failure .

MATERIALS and METHODS Chemicals, solutions and supplies All chemicals were at least reagent-grade quality and were purchased from a variety of commercial suppliers . Flumazenil (Romazicon®) was generously donated by Dr . Jerry Sepinwall of Hoffman-LaRoche (Nutley, NJ) . HPLC-grade methanol and HPLC-grade sodium acetate were used to prepare the mobile phase for the quantitation of glutamate . o-Phthalaldehyde/B-mercaptoethanol complete reagent solution (pH 10 .4) for the glutamate analysis was from Sigma Chemical Company (St . Louis, MO) . Reagent solutions were prepared using deionized water, obtained from a Nanopure® water-purification system . Microdialysis probes (CMA/20, 400 .tm ID and 500 .pm OD, with a 2-mm dialysis

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membrane tip), guide cannulas and FEP inlet/outlet tubing (120 gm ID and 680 µin OD) were supplied by CMA Microdialysis (Acton, MA), and the polyethylene (PE) tubing for the arterial and venous catheters was from Becton Dickinson (Parsippany, NJ) . Experimental animal preparation The surgical and experimental protocols were approved by the University of Iowa Animal Care and Use Committee . Male Sprague-Dawley strain rats (Harlan Incorporated, Indianapolis, IN), weighing 300-325 g, were used for all experiments . Rats were individually housed in Thoren® cages with a 12-h light/dark cycle and were allowed ad libitum access to food and water. On the day of instrumentation, each rat was anesthetized for surgery with an ip injection of sodium thiopental (50 mg/kg ; Pentothal®, Abbott Laboratories, North Chicago, IL) . A small incision was made in the right groin and catheters (PE 50) were inserted into the right femoral artery and vein . The opening was sutured and then the head was secured in a Stoelting stereotaxic frame (Wood Dale, IL) for probe implantation. Following a midline scalp incision, the skull was exposed using a periosteal elevator . A guide cannula was stereotaxically placed into the left hippocampus through a burr hole, drilled 5 mm posterior/4 min lateral to bregma (Paxinos and Watson, 1986) . The microdialysis probe was then inserted into the guide cannula ; final probe depth was 5 mm below the dura . (Immediately prior to implantation, the microdialysis probe was perfused with filtersterilized, degassed artificial cerebral spinal fluid (ACSF) until the outlet tubing was filled . The ends of the inlet and outlet tubing were then heat-sealed) . The probe assembly was secured to the skull with dental acrylic (L .D . Caulk Co ., Milford, DE) and the scalp incision was sutured shut over the hardened cement . The microdialysis inlet/outlet tubing and the arterial and venous catheters were tunneled subcutaneously and exteriorized through a small incision in the back of the neck . Each rat was monitored until it regained consciousness and exhibited normal exploratory behavior . All animals were allowed three days to recover from surgery before inducing 1'ver failure . Hepatic encephalopathy and hippocampal glutamate concentration Liver failure and subsequent HE were induced with thioacetamide. Rats in the HE group (n=10 with 8 survivors) were dosed twice by gavage with 300 mg/kg thioacetamide (5% w/v in normal saline) with a 24-h period between doses . Control animals (n=7) were dosed with an equivalent volume of saline (approximately 2 ml) over a similar time interval . Blood glucose levels were determined daily for all animals . For the saline control rats glucose concentration was 107±19 mg/dl . Thioacetamide-dosed rats with a blood glucose concentration < 80 mg/dl received 8 ml of 5% dextrose/0.3% potassium chloride in normal saline, given subcutaneously every 12 h, to combat hypoglycemia and as prophylaxis against dehydration and renal failure (Zimmermann et al., 1989) . Neurologic impairment was continually assessed as the thioacetamide-dosed rats passed through the different stages of HE : Stage One, initial lethargy ; Stage Two, ataxia ; Stage Three, loss of motor and stimulatory reflexes ; and Stage Four, coma (Bures, 1976) . All of the thioacetamide-treated rats reached stage three, approximately 24 h after the last dose . The presence of stage three


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HE in the thioacetamide-treated rats and the absence of neurologic impairment in the salinetreated control rats was confirmed by independent observers having no knowledge of the treatment regimen that was used . The dialysis experiment was started 24 h after the last dose of thioacetamide or saline . An arterial blood sample was taken and then the inlet tubing for the microdialysis probe was unsealed, attached to an infusion pump (Harvard Model 22, Natick, MA), and perfused with ACSF pumped at a rate of 2 pl/min . After a probe equilibration period of 30 min, consecutive dialysate samples were collected at 15-min intervals . After four samples had been collected, each animal was euthanized with 2 ml of saturated potassium chloride solution . Two more dialysate samples were collected as a positive control to demonstrate that the microdialysis probe could detect the increase in extracellular glutamate concentration produced by massive neuronal depolarization and death (i .e. that the probe was functional) . Prior to conducting this study, a criteria was set that any experiment that did not record at least a one-fold postmortem increase in dialysate glutamate concentration was to be excluded from further analysis . After completion of the experiment, probe placement was verified by injection of india ink into the inlet tubing with sufficient force to rupture the microdialysis membrane . Each rat brain was excised, partially dissected, and then visually inspected to confirm that the probe was located in the left hippocampus . Livers from several rats that received thioacetamide were isolated for gross examination . Flumazenil and extracellular glutamate concentration in hepatic encephalopathy Thioacetamide was used to induce HE in 12 rats, three days after surgical instrumentation and probe implantation . Similar to the previous experiment, all rats exhibited stage three HE, 24 h after the second dose of thioacetamide . One group of rats (n=5) was randomly assigned to receive 10 mg/kg flumazenil . The remaining rats (n=7) made up the control group assigned to receive vehicle (i ml of 0.15% Tween 80 v/v in normal saline) . The microdialysis tubing was connected to the infusion pump, the probe was equilibrated for 30 min, and then consecutive dialysate samples were collected at 5-min intervals . The collection period was shortened to 5 min to account for the expected short duration of HE reversal by flumazenil . The previous experiment demonstrated that a 5-min dialysate sample (10 pl volume) contained enough glutamate to be accurately quantitated by the HPLC method . Nine dialysate samples were collected and then each animal received an intravenous injection of flumazenil or vehicle . Each rat was closely observed to determine if there was any change in the level of arousal . A further .nine samples were collected and then each animal was euthanized with potassium chloride and four post mortem dialysate samples, at 5 min intervals, were collected . As before, the probe was injected/ruptured with india ink ; subsequent visual inspection verified placement in the left hippocampus . Quantitation of dialysate glutamate concentration The glutamate concentration in each dialysate fraction was measured in a single determinant by an HPLC procedure that was a modification of a previously described

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method (Reynolds and Brien, 1992) . This method involved precolumn derivitization of glutamate with o-phthalaldehyde/B-mercaptoethanol to form a thio-substituted isoindole derivative, which was separated by isocratic reverse-phase HPLC and quantitated by fluorescence detection . Each dialysate sample was diluted with nanopure water to a final sample volume of 150 p.1 . A 100 µl aliquot of derivitizing solution was reacted with 100 µl of sample and allowed to react for 2 min before injecting the reaction mixture onto an octadecylsilane reverse-phase column (Supelcosil LC-18®, 4 .6 mm ID x 150 mm length, 5 µm particle size ; Supelco, Bellefonte, PA) . The mobile phase, comprised of 33 .7 mM aqueous sodium acetate/methanol (67 .3/32 .7, v/v) at pH 7 .5, was pumped at a flow rate of 2 .0 ml/min . The within-day coefficient of variation of the assay did not exceed 4% ; the recovery of glutamate was >92% ; and the lower limit of quantitative sensitivity was 9 pmol glutamate/ml . Determination of blood metabolic parameters An arterial blood sample was taken to determine ammonia and glucose concentrations, and blood gas status just prior to collecting microdialysis samples . Due to an equipment failure, we were not able to quantitate blood gas status of the saline-gavaged rats from the first series of experiments . The saline metabolic data was obtained from rats that were instrumented with femoral catheters and microdialysis probes and gavaged with saline, but these rats were not used in the glutamate analysis . Arterial plasma ammonia concentration was quantitated with an ammonia assay kit (Sigma), and arterial blood glucose concentration was quantitated using an Accu-Chek®IIm blood glucose monitor (Boehringer Mannheim Diagnostics, Indianapolis IN) . Arterial blood Po t , Pco2 , and pH were determined using a blood-gas analyzer (Instrumentation Laboratory, Lexington, MA) . Data presentation and statistical analysis Blood metabolic data are presented as group means ± SD . To preserve power, these data were not statistically analyzed . Different statistical approaches were used to analyze the glutamate results from the two experiments . For the first study, a median value of glutamate dialysate concentration was - calculated for each saline control and thioacetamidetreated rat . The one-sided Wilcoxon-Mann-Whitney test was used to determine if thioacetamide-induced HE increased extracellular hippocampal glutamate concentration . A distribution-free method was chosen because of the small concentration of glutamate in the control dialysate samples relative to the HE rats ; these data were not normally distributed even after logarithmic transformation . In the second study, analysis of covariance was used to test if flumazenil affected the extracellular glutamate concentration during thioacetamideinduced HE . Baseline glutamate concentration was used as a covariate to account for any variations in probe glutamate recovery among animals . All of the standard regression diagnostics were satisfied at the p >0 .05 level .


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RESULTS Behavioral and blood metabolic data In the first series of experiments, two of the thioacetamide rats died before the microdialysis protocol was initiated . None of the saline-treated animals died . No rats died in the flumazenil part of the study . The progression of thioacetamide-gavaged rats through the stages of HE was similar to previously described reports (Zimmermann et al., 1989), and all these rats exhibited stage three HE at the start of the dialysis experiment, approximately 24 h after the last dose of thioacetamide. Values for arterial blood Pot, Pco2 for arterial ammonia concentrations, following saline or thioacetamide gavage are given in Table I . In the thioacetamide group, arterial pH was decreased, while Pot and ammonia concentration were increased compared to the saline-treated rats . Livers from rats that received thioacetamide were swollen and hemorrhagic . Gross examination revealed areas of necrosis widely scattered throughout the lobules indicative of severe liver injury . Table I . Rat arterial blood gas and metabolic data

Treatment Saline Thioacetamide

pH 7 .45 ± 0 .06 7 .28 ± 0 .08

86±9 127 ± 21

Pco2

Ammonia

40±4 43 ± 13

25 .8 ± 4.5 245 ± 82

Data are expressed as group means ± SD . Values for the thioacetamide-treated rats (n=13) were obtained from blood samples taken immediately prior to perfusing the microdialysis probe . Values for the saline control group (n=7) were obtained following two days of saline gavage. Units for the Pot and Pco2 data are mm Hg ; ammonia values are expressed as pmol/l .

Hippocampal extracellular glutamate concentration Representative rat hippocampal glutamate concentration plots following gavage with saline or thioacetamide are presented in Figure 1 . For saline, the glutamate concentration remained reasonably stable during collection of the four dialysate fractions . In contrast, thioacetamide-treated rats exhibited an elevation in extracellular glutamate efflux that occurred over a wide concentration range . Also depicted in each plot is the post-mortem increase in glutamate concentration after euthanization with potassium chloride . All saline and thioacetamide-treated rats in this part of the study exhibited at least a one-fold postmortem increase in glutamate, indicative of correct probe function . Figure 2 presents the median glutamate dialysate concentration for each rat that received either saline or thioacetamide . As a group, the median dialysate concentration in rats that received saline (n=7) was 169 ng/ml compared to 1314 ng/ml for the thioacetamide-treated rats (n=8) . Thioacetamide-induced liver failure resulted in a significant increase in hippocampal glutamate efflux (p=0 .010) .

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Flumazenil injection produced a transient biphasic increase in the arousal level of all thioacetamide-treated rats . The initial phase, which lasted for 2 to 3 min, was characterized by rapid head and limb movements along with sniffing/exploratory activity . This was followed by a second phase in which movement and the incidence of exploratory behavior gradually decreased . Somnolence returned approximately 10 min after injection of flumazenil . Vehicle injection had no effect on arousal level . Representative hippocampal glutamate concentration plots of thioacetamide-gavaged rats before and after iv injection of flumazenil or vehicle are presented in Figure 3A . One of the vehicle-treated rats did not exhibit a post-mortem increase in glutamate efflux and was excluded from analysis . Comparison of glutamate efflux levels between the two groups, before and after injection, demonstrated that flumazenil had no effect on hippocampal glutamate efflux during thioacetamide-induced liver failure (p=0.934; Figure 3B) . Saline 2000 1900, l

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Figure 3. Graph A : Representative rat hippocampal dialysate glutamate concentration-plots (24 h after two days of gavage with thioacetamide) before and after ip injection of vehicle or flumazenil . Graph B : Mean (± SD) glutamate dialysate concentrations, corrected for baseline, following ip injection of vehicle or flumazenil . There was no difference in glutamate concentration between the two groups (p=0 .934) .

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DISCUSSION Glutamate is the major transmitter in the CNS . Alterations excitatory am o acid neurotransmission have been proposed to contribute to the depression of brain function associated with HE . With this in mind, the objective of the first part of the study was to determine if extracellular hippocampal glutamate concentration is increased during thioacetamide-induced liver failure and subsequent HE in the rat . Administration of thioacetamide produced behavioral and metabolic changes similar to those previously reported . One day after the second dose of thioacetamide, rats were comatose with a loss of motor and stimulatory reflexes, indicative of stage three HE . The decrease in pH may be due to an increase in serum lactate concentration (Hawker, 1993) . The observed increase in ammonia concentration (Table 1) is consistent with the hyperammonemia that occurs during liver failure . The hyperammonernia may also be responsible for the observed somnolence . Both in vitro (Lombardi et al., 1994) and in vivo (Szerb and Redondo, 1993) experiments have demonstrated that elevations in ammonia concentration can decrease excitatory synaptic transmission in the hippocampus and other cortical regions . Post mortem liver examination revealed extensive areas of necrosis, indicative of liver injury following thioacetamide exposure. Extracellular glutamate concentration was increased (p=0.010) in the rat hippocampus following thioacetamide-induced HE compared with saline-gavaged rats . Within the thioacetamide group there was a large variation in observed glutamate concentration (Figure 2), with amounts ranging from 200 to almost 7000 ng/ml . The reason for this variability is unclear as all thioacetamide-treated rats had similar blood gases and metabolic measurements, and they exhibited a similar degree of somnolence, identified as stage three HE . It is unlikely that this increase in glutamate concentration is due to a direct hippocampal effect of thioacetamide as it is rapidly metabolized by the liver . In rats, the plasma concentration of thioacetamide is reduced by 95%, 24 h after its administration (Porter et al., 1979) . In one study, thioacetamide was quantitated in rat brain homogenate by NMR spectral analysis, following a total dose of 1800 mg thioacetamide/kg body weight (Peeling et al., 1993). However, analysis of post-mortem brain homogenates can not determine if thioacetamide is in the actual brain tissue or if it is simply present in the cerebral blood supply . In fact, the latter possibility is more likely . The levels of thioacetamide (0 .05 to 1 mM ; 4 to 75 µg/ml) reported were similar to the plasma concentrations predicted by extrapolating the pharmacokinetic data obtained by Porter et al. (1979) . Definitive in vivo and in vitro studies to determine if thioacetamide is present in the CNS and what effect, if any, it may have on neurotransmitter release have not been conducted . Liver failure can produce a wide spectrum of metabolic effects . As such, the increase in hippocampal glutamate concentration may be due to several factors . HE can impair highaffinity glutamate uptake (Oppong et al ., 1995) leading to an accumulation of glutamate in the synaptic cleft. As well, HE can alter the synthesis and catabolism of glutamate . Brain glutamate metabolism is regulated by several different enzymes and by a complex interrelationship between neurons and astrocytes (Yudkoff et al., 1993), which may be


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sensitive to HE-induced changes such as hyperammonemia (Albrecht and Faff, 1994) . The observed increase in extracellular glutamate concentration is in contrast to other studies that have reported a decrease in total glutamate concentration in various brain regions, including the hippocampus, with HE produced by thioacetamide (Peeling et al., 1993 ; Zimmermann et al ., 1989) . These observations, combined with the results of the present study, indicate that the metabolic and neuronal/neurotransmitter pools of glutamate are affected differently during HE . The increase in extracellular glutamate concentration suggests that glutamate neurotransmission is enhanced during HE . An expected consequence of increased glutamate neurotransmission would be down-regulation of glutamate receptor populations . Indeed, several studies report decreased glutamate binding, specifically decreased NMDA subtype binding, in various brain regions, during HE and/or hyperammonemia (for a review see Rao et al., 1992) . Another consequence could be neuronal damage . Glutamate, when present in the synapse at high concentration, can cause excitotoxic injury in the hippocampus leading to neuronal cell death . While postmortem verification of microdialysis probe function and location precluded histologic analysis in the present study, a previous report demonstrated that thioacetamide-induced liver failure does produce neuronal injury to the hippocampus and other brain regions (Peeling et al., 1993) . The pattern of damage resembled the distribution of neuronal injury that can result from an episode of focal ischemia, which also appears to be mediated by increased glutamate concentration (Schousboe and Frandsen, 1995) . The neuronal injury produced by thioacetamide-induced liver failure may result from increased extracellular glutamate concentration but it has caused some authors to question the use of thioacetamide as a model of HE because the resultant neurologic injury is more severe than that typically seen in some clinical situations (Peeling et al., 1993) . The most common neuropathologic finding in human HE is the transformation of astrocytes to Alzheimer type II glia with little neuronal damage (Norenberg, 1987) . In fact, with humans, if the underlying disorder is corrected reasonably early, full neurologic recovery from HE can be expected (Morris and Ferrendelli, 1990) . However, if HE persists, irreversible changes to the cerebrum can occur leading to a condition called hepatocerebral degeneration . Hepatocerebral degeneration is characterized by necrotic lesions and severe neuronal cell loss (Morris and Ferrendelli, 1990 ; Asao and Oji, 1968), reminiscent of both ischemic brain injury and the histologic damage produced by thioacetamide . In the clinical setting, manifestations of HE can blend imperceptibly into those of hepatocerebral degeneration suggesting a common underlying mechanism or mechanisms (Morris and Ferrendelli, 1990) . As such, thioacetamide-induced liver failure in the rat may represent an acceleration of the clinical progression from "simple" HE to hepatocerebral degeneration . Benzodiazepine antagonists, such as flumazenil, have been reported to reverse the somnolence associated with HE (Jones et al., 1994) . The objective in the second part of the study was to determine the effect of flumazenil on extracellular glutamate concentration during thioacetamide-induced HE. Similar to the first part of the study, hippocampal extracellular glutamate concentration was elevated following thioacetamide exposure . Attempts to determine the concentration of GABA in the dialysate fractions were unsuccessful as the amounts were too low to be reliably quantitated by the HPLC method

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(