Manganese induces cell swelling in cultured astrocytes.

Manganese in excess is neurotoxic and causes a CNS disorder that resembles Parkinson's disease (manganism). Manganese highly accumulates in astrocytes, which renders these cells more vulnerable to its toxicity. Consistent with this vulnerability, manganese has been shown to cause histopathological changes in astrocytes (Alzheimer type II change), generates oxidative stress and bring about mitochondrial dysfunction, including the induction of the mitochondrial permeability transition (mPT) in astrocytes. In addition to manganism, increased brain levels of manganese have been found in hepatic encephalopathy, a chronic neurological condition associated with liver dysfunction, wherein Alzheimer type II astrocytic changes are also observed. As low-grade brain edema, possibly secondary to astrocyte swelling, has been reported in hepatic encephalopathy, we hypothesized that manganese may contribute to such edema. We therefore exposed cultured astrocytes to manganese (Mn(3+)) acetate (25 and 50microM) for different time periods and examined for changes in cell volume. Manganese dose-dependently induced astrocyte swelling; such swelling was first observed at 12h (28%), which further increased (54%) at later time points (24-48h). Pretreatment of astrocyte cultures with antioxidants, including vitamin E, the spin trapping agent PBN, and the iron-chelating agent desferroximine, as well as the nitric oxide synthase inhibitor l-NAME, all significantly blocked (50-80%) astrocyte swelling caused by manganese, suggesting that oxidative/nitrosative stress is involved in the mechanism of such swelling. Cyclosporin A, an inhibitor of mPT also blocked (90%) manganese-induced astrocyte swelling. The data indicate that manganese exposure results in astrocyte swelling and such swelling, at least in part, may be caused by oxidative stress and/or mPT. Astrocyte swelling by manganese may represent an important aspect of manganese neurotoxicity, and may be a factor in low-grade brain edema associated with chronic hepatic encephalopathy.

Immunohistochemical detection of inducible nitric oxide synthase, nitrotyrosine and manganese superoxide dismutase following hyperglycemic focal cerebral ischemia.

We have characterized the temporal changes in iNOS, MnSOD and nitrotyrosine immune reactivity in a rat model of permanent middle cerebral artery occlusion under acute hyperglycemic or normoglycemic conditions followed by either 3- or 24-h recovery. We found that the macroscopic labeling pattern for all three antibodies colocalized with the ischemic core and penumbra which was determined by cresyl violet histological evaluation in adjacent sections. Hyperglycemia induced prior to ischemia resulted in earlier infarction which correlated with increased immunoreactivity for iNOS, MnSOD and nitrotyrosine. In the penumbral region of the frontal cortex, labeling of specific cell structures was largely limited to cortical neurons near the corpus callosum and was apparent earlier in the hyperglycemic rats. Increased polymorphonuclear leukocyte adhesion in blood vessels was observed at 24 h in the hyperglycemic group. At both of the recovery times studied, we observed only minor vascular staining for nitrotyrosine and none for iNOS. Our results are consistent with hyperglycemia resulting in an early and concomitant increase in both superoxide and nitric oxide production which can lead to peroxynitrite formation that then nitrates tyrosine residues. It would appear that hyperglycemic ischemia contributes to the early induction of key enzymes involved in nitric oxide bioavailability.

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy.

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.

Thiamine deficiency results in metabolic acidosis and energy failure in cerebellar granule cells: an in vitro model for the study of cell death mechanisms in Wernicke's encephalopathy.

Thiamine deficiency (TD) in both humans and experimental animals results in severe compromise of mitochondrial function and leads to selective neuronal cell death in diencephalic and cerebellar structures. To examine further the influence of TD on neuronal survival in relation to metabolic changes, primary cultures of rat cerebellar granule cells were exposed to thiamine-deficient medium for up to 7 days in the absence or presence of the central thiamine antagonist pyrithiamine (Py). Exposure of cells for 7 days to thiamine-deficient medium alone resulted in no detectable cell death. On the other hand, 50 microM Py treatment led to reductions of thiamine phosphate esters, decreased activities of the thiamine-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase, a twofold increase in lactate release (P < 0.001), a lowering of pH, and significant (58%, P < 0.001) cell death. DNA fragmentation studies did not reveal evidence of apoptotic cell death. Addition of 50 microM alpha-tocopherol (vitamin E) or 100 microM of butylated hydroxyanisole (BHA) to Py-treated cells resulted in significant neuroprotection. On the other hand, addition of 10 microM MK-801, an NMDA receptor antagonist, was not neuroprotective. These results suggest that reactive oxygen species (ROS) play a major role in thiamine deficiency-induced neuronal cell death. Insofar as this experimental model recapitulates the metabolic and mitochondrial changes characteristic of thiamine deficiency in the intact animal, it might be useful in the elucidation of mechanisms involved in the neuronal cell death cascade resulting from thiamine deficiency.

Effects of ammonia on glutamate transporter (GLAST) protein and mRNA in cultured rat cortical astrocytes.

Ammonia is a neurotoxic substance which accumulates in brain in liver failure and it has been suggested that ammonia plays a key role in contributing to the astrocytic dysfunction characteristic of hepatic encephalopathy. In particular, the effects of ammonia may be responsible for the reduced astrocytic uptake of neuronally-released glutamate and high extracellular glutamate levels consistently seen in experimental models of hepatic encephalopathy. To further address this issue, [(3)H]-D-aspartate uptake was examined in primary rat cortical astrocyte cultures exposed to 5 mM ammonium chloride for a period of 7 days. In addition, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot studies were performed to examine the mRNA and protein expression respectively of the glutamate transporter GLAST in ammonia-treated cells. Studies revealed a 57% (p<0.05) decrease in [(3)H]-D-aspartate uptake and a concomitant significant decrease in GLAST transporter protein (43%, p<0.05) and mRNA (32%, p<0.05) expression. The reduced capacity of astrocytes to reuptake glutamate following ammonia exposure may result in compromised neuron-astrocyte trafficking of glutamate and could thus contribute to the pathogenesis of the cerebral dysfunction characteristic of hyperammonemic syndromes such as hepatic encephalopathy.

Hepatic encephalopathy: An update of pathophysiologic mechanisms.

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that occurs in both acute and chronic liver failure. Although the precise pathophysiologic mechanisms responsible for HE are not completely understood, a deficit in neurotransmission rather than a primary deficit in cerebral energy metabolism appears to be involved. The neural cell most vulnerable to liver failure is the astrocyte. In acute liver failure, the astrocyte undergoes swelling resulting in increased intracranial pressure; in chronic liver failure, the astrocyte undergoes characteristic changes known as Alzheimer type II astrocytosis. In portal-systemic encephalopathy resulting from chronic liver failure, astrocytes manifest altered expression of several key proteins and enzymes including monoamine oxidase B, glutamine synthetase, and the so-called peripheral-type benzodiazepine receptors. In addition, expression of some neuronal proteins such as monoamine oxidase A and neuronal nitric oxide synthase are modified. In acute liver failure, expression of the astrocytic glutamate transporter GLT-1 is reduced, leading to increased extracellular concentrations of glutamate. Many of these changes have been attributed to a toxic effect of ammonia and/or manganese, two substances that are normally removed by the hepatobiliary route and that in liver failure accumulate in the brain. Manganese deposition in the globus pallidus in chronic liver failure results in signal hyperintensity on T1-weighted Magnetic Resonance Imaging and may be responsible for the extrapyramidal symptoms characteristic of portal-systemic encephalopathy. Other neurotransmitter systems implicated in the pathogenesis of hepatic encephalopathy include the serotonin system, where a synaptic deficit has been suggested, as well as the catecholaminergic and opioid systems. Further elucidation of the precise nature of these alterations could result in the design of novel pharmacotherapies for the prevention and treatment of hepatic encephalopathy.

Increased "peripheral-type" benzodiazepine receptor sites and mRNA in thalamus of thiamine-deficient rats.

"Peripheral-type" benzodiazepine receptors (PTBRs) are highly expressed on the outer mitochondrial membrane of several types of glial cells. In order to further elucidate the nature of the early glial cell changes in thiamine deficiency, PTBR sites and PTBR mRNA were measured in thalamus, a brain structure which is particularly vulnerable to thiamine deficiency, of thiamine-deficient rats at presymptomatic and symptomatic stages of deficiency. PTBR sites were measured using an in vitro binding technique and the selective radio ligand [3H]-PK11195. PTBR gene expression was measured by RT-PCR using oligonucleotide primers based upon the published sequence of the cloned rat PTBR. Microglial and astrocytic changes in thalamus due to thiamine deficiency were assessed using immunohistochemistry and antibodies to specific microglial (ED-1) and astrocytic (GFAP) proteins respectively. Significant increases of [3H]-PK11195 binding sites and concomitantly increased PTBR mRNA were observed in thalamus at the symptomatic stage of thiamine deficiency, coincident with severe neuronal cell loss and increased GFAP-immunolabelling (indicative of reactive gliosis). Positron Emission Tomography using 11C-PK11195 could provide a novel approach to the diagnosis and assessment of the extent of thalamic damage due to thiamine deficiency in humans with Wernicke's Encephalopathy.

Chronic exposure of rat primary astrocyte cultures to manganese results in increased binding sites for the 'peripheral-type' benzodiazepine receptor ligand 3H-PK 11195.

Alterations of 'peripheral-type' benzodiazepine receptors (PTBRs) in brain are a feature of hepatic encephalopathy (HE). Although ammonia toxicity has been implicated in the disorder, recent findings suggest an accumulation of manganese in the brains of cirrhotic patients dying in hepatic coma. In this study, we examined the expression of PTBRs as well as the binding of the selective PTBR ligand 3H-PK 11195 in cultured astrocytes following chronic exposure to manganese. When astrocytes were exposed to 100 microM manganese for 1 week, a 57% increase in Bmax for 3H-PK 11195 binding was detected (P < 0.01) with no change in the Kd value. However, an examination by RT-PCR of the expression of the isoquinoline-binding moiety of the PTBR complex in these cells revealed no change in PTBR mRNA levels following manganese treatment. These findings suggest that manganese up-regulates 3H-PK 11195 binding sites by a process which does not involve a change in transcription. In view of the proposed role of astrocytic PTBRs in 'neurosteroid' synthesis, manganese-induced increases of PTBRs could contribute to the pathogenesis of HE.

Increased expression of glyceraldehyde-3-phosphate dehydrogenase in cultured astrocytes following exposure to manganese.

Manganism is a disorder characterized by hyperintensities in basal ganglia structures on magnetic resonance imaging which may be the consequence of manganese deposition in these areas. Since manganese is taken up avidly into astrocytes and is known to interfere with cerebral energy metabolism, we studied the effect of this metal on the expression and activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in primary cultures of astrocytes. Treatment with 100 microM manganese for 7 days increased both the Vmax and Km values for GAPDH which was not reproducible with other divalent metals. Using RT-PCR, increased GAPDH expression was detected in cells exposed to manganese compared with controls. No changes in cytochrome oxidase activity or ATP levels were observed, and lactate production was unaffected, in manganese-treated cells. These findings provide evidence of a possible role for GAPDH in the mediation of the effects of manganese on central nervous system function.

Alcohol-thiamine interactions: an update on the pathogenesis of Wernicke encephalopathy.

Wernicke encephalopathy is a neurological disorder commonly observed in chronic alcohol abuse, in patients with AIDS, and in other conditions of compromised nutritional status. The underlying cause of the disorder is thiamine deficiency. The present review highlights data focusing on alcohol-thiamine interactions and their relationship to the pathogenesis of Wernicke encephalopathy. Recent findings on the effects of alcohol on thiamine absorption and storage and on thiamine phosphorylation to the enzyme co-factor form (thiamine diphosphate) are discussed with regard to the postulated "biochemical lesion" of Wernicke encephalopathy. Also discussed are new findings on the molecular genetics of the thiamine-dependent enzyme transketolase in patients with Wernicke encephalopathy. A discussion of the hypotheses regarding the mechanisms underlying the phenomenon of selective neuronal cell death observed in this disorder including cerebral energy deficit, focal lactic acidosis, glutamate excitotoxicity, increased expression of immediate-early genes, free radicals and perturbations of the blood-brain barrier are presented. Finally, the possible role of thiamine deficiency in alcoholic peripheral neuropathy is reviewed.

Mechanisms of neuronal cell death in Wernicke's encephalopathy.

Wernicke's Encephalopathy (WE) is a serious neurological disorder resulting from thiamine deficiency, encountered in chronic alcoholics and in patients with grossly impaired nutritional status. Neuropathologic studies as well as Magnetic Resonance Imaging reveal selective diencephalic and brainstem lesions in patients with WE. The last decade has witnessed major advances in the understanding of pathophysiologic mechanisms linking thiamine deficiency to the selective brain lesions characteristic of WE. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase, a rate-limiting tricarboxylic acid cycle enzyme are significantly reduced in autopsied brain tissue from patients with WE and from rats treated with the central thiamine antagonist, pyrithiamine. In the animal studies, evidence suggests that such enzyme deficits result in focal lactic acidosis, cerebral energy impairment and depolarization resulting from increased release of glutamate in vulnerable brain structures. It has been proposed that this depolarization may result in N-Methyl-D-Aspartate receptor-mediated excitotoxicity as well as increased expression of immediate early genes such as c-fos and c-jun resulting in apoptotic cell death. Other mechanisms involved in thiamine deficiency-induced cell loss may involve free radicals and alterations of the blood-brain barrier. Additional studies are still required to identify the site of the initial cellular insult and to explain the predilection of diencephalic and brainstem structures due to thiamine deficiency.

Regional activation of L-type voltage-sensitive calcium channels in experimental thiamine deficiency.

During pyrithiamine-induced thiamine deficiency (PTD), specific regions of the brain develop histological damage. The basis of this selective vulnerability is unknown but the mechanism may involve a glutamate-mediated excitotoxic process in affected structures, leading to alterations in membrane potential and disturbances in calcium homeostasis. In this study, we have examined the volume of distribution of [3H]nimodipine, an L-type voltage-sensitive calcium channel (VSCC) antagonist, in the brain of the PTD rat. An increase in specific binding of [3H]nimodipine was detected only in the posterior thalamus at the symptomatic stage, immediately following the loss of righting reflexes (P < 0.0001). There was also an increase in nonspecific binding in the medial geniculate and inferior colliculi. Replenishment with thiamine at the symptomatic stage returned [3H]nimodipine binding to normal levels. These findings provide evidence that depolarization and activation of L-type VSCCs occur in the posterior thalamus and may contribute to the appearance of histological lesions in this structure during experimental thiamine deficiency.

Immediate-early gene expression in the brain of the thiamine-deficient rat.

Pyrithiamine-induced thiamine deficiency (PTD) in the rat is associated with neuronal loss in the thalamus and inferior colliculus. Recently, we were able to demonstrate the occurrence of apoptosis in the thalamus of these animals. Given that immediate-early genes (IEGs) participate in signal transduction pathways that mediate programmed cell death, the present study utilized in situ hybridization and immunohistochemistry to examine the expression of four IEGs (c-fos, c-jun, fos-B, and NGFI-A) during the progression of PTD. Elevated c-fos mRNA levels were initially observed in the posterior medial thalamus on d 12 of the deficiency. At the acute symptomatic stage (characterized by a loss of righting reflex on d 16-17), the posterior-medial thalamus exhibited increased mRNA for all genes examined, whereas the inferior colliculus demonstrated mRNA induction for c-fos, c-jun, and NGFI-A. Immunohistochemical analysis revealed that elevations of IEG mRNA associated with the acute symptomatic stage were consistently translated into protein in the thalamus. In contrast, whereas elevated Fos- and Jun-like immunoreactivity were detected in the inferior colliculus at this stage, NGFI-A-like immunoreactivity declined significantly below basal levels, suggesting a translational block. These results are consistent with our recent findings of apoptotic cell death, and indicate that differential patterns of IEG expression occur in the thalamus and inferior colliculus during PTD, which may contribute to the pathogenesis of this disorder.

Ammonia and manganese increase arginine uptake in cultured astrocytes.

Recent work has suggested a possible role for nitric oxide (NO) in the development of hepatic encephalopathy (HE). In this study, we examined the effect of ammonia and manganese, factors implicated in the pathogenesis of HE, on the transport of arginine (a precursor of NO) into primary cultures of astrocytes. Treatment with 5 mM ammonia for 1-4 days produced a maximal (53%) increase in L-arginine uptake at 3 days when compared to untreated cells. Kinetic analysis following 4-day treatment with 5 mM ammonia revealed an 82% increase in the Vmax and a 61% increase in the Km value. Similar analysis with 100 microM manganese showed a 101% increase in Vmax and a 131% increase in the Km value. These results suggest that both manganese and ammonia alter L-arginine uptake by modifying the transporter for arginine. A decrease of 32% in the non-saturable component of L-arginine transport was also observed following treatment with ammonia. When cultures were treated separately with 5 mM ammonia and 100 microM manganese for 2 days, the uptake of L-arginine increased by 41% and 57%, respectively. Combined exposure led to no further increase in uptake. Our results suggest that ammonia and manganese may contribute to the pathogenesis of HE by influencing arginine transport and thus possibly NO synthesis in astrocytes.

Manganese decreases glutamate uptake in cultured astrocytes.

Recent data have shown an accumulation of manganese in the basal ganglia in patients with chronic hepatic encephalopathy (HE). Astrocytes and ammonia are critically involved in the pathogenesis of HE, and we have recently demonstrated that ammonia decreases glutamate uptake in cultured astrocytes. Since failure by astrocytes to take up glutamate may represent an important pathogenetic mechanism in HE, we, therefore, examined the effect of manganese on glutamate transport in these cells. Treatment of cultured astrocytes with 100 microM manganese for 2 days resulted in a 54% decrease in the uptake of D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed a 28% decline in Vmax, with no change in the K(m). Treatment of cultures with 5 mM NH4 Cl inhibited D-aspartate uptake by 21%, and a combination of 5 mM NH4Cl with 100 microM manganese produced an additive effect on uptake inhibition. These results suggest a pathogenetic role for manganese in HE, possibly involving glutamate transport.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) decreases glutamate uptake in cultured astrocytes.

The deleterious effect of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopaminergic neurons of the substantia nigra is well established. In addition, increased glutamatergic drive to basal ganglia output nuclei is considered a likely contributor to the pathogenesis of Parkinson's disease. One possibility for the increased excitatory tone may be related to an impairment in glutamate uptake. As astrocytes possess efficient transport mechanisms for both MPTP and glutamate, we have examined the effect of this agent on D-aspartate uptake into these cells. Treatment of cultures with 50 microM MPTP for 24 h decreased uptake by 39%. Kinetic analysis revealed that this effect was due to a 35% decrease in Vmax with no change in the Km. Treatment with deprenyl, a monoamine oxidase B inhibitor, produced a complete reversal of MPTP-induced uptake inhibition, but was ineffective following exposure of cells to the MPTP metabolite, 1-methyl-4-phenylpyridinium (MPP+). Removal of MPTP from cultures resulted in a complete restoration of glutamate uptake after 24 h. These results show that MPTP reversibly compromises glutamate uptake in cultured astrocytes, which is dependent on the conversion of MPTP to MPP+. Such findings suggest that the glutamate transporter in astrocytes plays an important role in MPTP-induced neurotoxicity and possibly in parkinsonism.

Cerebral vulnerability is associated with selective increase in extracellular glutamate concentration in experimental thiamine deficiency.

Microdialysis in the awake, freely moving rat was used to determine the effect of pyrithiamine-induced thiamine deficiency on the levels of amino acids in the brain. Studies were carried out on (a) presymptomatic animals immediately before the development of behavioral changes and (b) acute symptomatic animals within 6 h following loss of righting reflexes. This latter stage precedes the appearance of histological lesions. The results were compared with pair-fed controls. Dialysis probes were implanted in one vulnerable structure [ventral posterior medial thalamus (VPMT)] and one nonvulnerable area [frontal parietal cortex (FPC)] on the contralateral side. In VPMT of acute symptomatic animals, the glutamate concentration was significantly increased (3.37 +/- 0.64 microM; p < 0.005) compared with control values (0.93 +/- 0.09 microM), whereas in FPC no change in glutamate content was evident. These results suggest that glutamate plays a significant role in the development of central thiamine deficiency lesions. The absence of any increase in glutamate levels in the nonvulnerable FPC suggests that a glutamate-mediated excitotoxic mechanism may be responsible for the selective cerebral vulnerability in thiamine deficiency.

The transport of leucine and aminocyclopentanecarboxylate across the intact, energy-depleted rat blood-brain barrier.

The transport across the blood-brain barrier of the large neutral amino acid leucine and the nonmetabolised aminocyclopentanecarboxylate (ACPC), of similar molecular size, was studied in the perfused, energy-depleted rat brain. It was found that when both leucine and ACPC were perfused for periods of up to 10 min their accumulation in the brain increased in a linear fashion. The ratio of perfusate radioactivity per milliliter and tissue radioactivity per gram (Rt/Rp) rose to above unity for both leucine and ACPC, indicating continued uptake against a concentration gradient of the radiolabel within the CNS. When the effect of increasing the concentration of the amino acid upon its influx into the brain was studied, it was found that under these conditions the kinetics of transport for both leucine and ACPC were of a similar order of magnitude to those reported previously in vivo. The values for the Michaelis constant for transport (Km), maximum rate of transport (Vmax), and the constant for the apparently linear, nonsaturable component (Kd) for leucine into the cerebrum were 84.5 +/- 29.0 microM, 45.5 +/- 1.5 nmole/min/g, and 2.62 +/- 0.15 microliters/min/g, respectively, and for ACPC 381 +/- 64 microM, 54.0 +/- 1.5 nmole/min/g and 0.35 +/- 0.10 microliter/min/g, respectively. Comparing this data with previously reported values it is suggested that the transport of leucine into the central nervous system from a perfusate or bolus where no other competing amino acids are present, is flow dependent. Furthermore, ACPC enters the brain almost entirely by a carrier-mediated process, with little or no nonsaturable influx despite a similar oil/water partition coefficient as leucine.

The effect of a low pH saline perfusate upon the integrity of the energy-depleted rat blood-brain barrier.

The effect of a low pH perfusate upon the integrity of the rat blood-brain barrier was studied using an in situ supravital brain perfusion technique in which high-energy phosphates are depleted. Control animals were perfused for 10 min with a Ringer's salt solution containing the metabolic inhibitor 2,4-dinitrophenol (DNP) and adjusted to a pH of 7.4. In two separate experimental groups the perfusate, consisting of either the same medium as the controls or with additional buffering from Tris maleate, was switched after 5 min at a pH of 7.4, to a medium adjusted to pH 5.5 with lactic acid. Following a total perfusion time of 10 min, the integrity of the blood-brain barrier was assessed using the small molecular weight tracer [14C]mannitol. The cerebral perfusate flow rates (CPFR) after 10 min of perfusion were also determined in the three groups by perfusing for 40 s with [14C]iodoantipyrine. In each group, mannitol was excluded from the tissue of the brain to the same degree as has been previously reported in vivo, indicating an intact blood-brain barrier. There was also no significant pH-dependent change in CPFR. Ultrastructural examination of animals that had been perfusion fixed following in situ perfusion revealed no obvious differences between the cerebral endothelium of the control and low pH perfused animals. These results demonstrate that in the absence of energy-producing metabolism a perfusate pH of 5.5 is insufficient to disrupt the blood-brain barrier.