Journal of Cerebral Blood Flow and Metabolism 9:234-242 © 1989 Raven Press, Ltd" New York

The Effect of a Low pH Saline Perfusate Upon the Integrity of the Energy-Depleted Rat Blood-Brain Barrier

J, Greenwood, A. S. Hazell, and P. J. Luthert

Department of Neuropathology, Institute of Psychiatry, DeCrespigny Park, London, United Kingdom

Summary: 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 high­energy 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 blood­brain barrier was assessed using the small molecular weight tracer [14C]mannitol. The cerebral perfusate flow rates (CPFR) after 10 min of perfusion were also deter-

Lactic acidosis has been strongly implicated in the aetiology of brain cell damage in cerebral isch­aemia (Siesj6 198 1, 1985; Plum 1983; Pulsinelli and Petito 1983; Ono et aI., 1986), hypoxia (Myers 1979; Olesen 1986), trauma (Rosner and Becker 1984), and thiamine deficiency (Hakim, 1984). There is considerable evidence that when the intracellular concentration of this anaerobic metabolite rises, it becomes increasingly cytotoxic, such that in cere­bral ischaemia the degree to which the tissue pH drops (Smith et aI., 1986) and the amount of cellular damage and dysfunction can be correlated with the circulating glucose concentration-hyperglycaemic animals suffering greater damage than hypoglycae­mic animals (Myers and Yamaguchi 1977; Pulsinelli

Submitted August 23, 1988; accepted Oct. 17, 1988. Address correspondence and reprint requests to Dr. J. Green­

wood at Department of Neuropathology, Institute of Psychiatry, DeCrespigny Park, London SE5 8AF, United Kingdom.

This work was presented in part as a preliminary communica­tion to the Physiology Society, UK (Greenwood et al., 1986).

Abbreviations used: CPFR, cerebral perfusate flow rate; DNP, 2,4-dinitrophenol; lAP, iodoantipyrine.

234

mined 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 pH­dependent 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. Key Words: Blood-brain barrier-Brain perfusion-Cerebral isch­aemia-Energy-depleted brain-Lactic acid-Perme­ability.

et aI., 1982; Siemkowicz and Hansen 1978; Gins­berg et aI., 1980; Kalimo et aI., 1981a, 1981b; Rehn­crona et aI., 198 1). Indeed, a problem encountered during severe incomplete ischaemia is that residual blood flow may continue to supply the tissue with sufficient glucose to fuel anaerobic glycolysis that will result in a progressive accumulation of lactate (Rehncrona et aI., 1979, 1981; Kalimo et aI., 1981a). In complete ischaemia, lactic acidosis is limited by the pre-ischaemic stores of glucose and glycogen (Ljunggren et aI., 1974a), with the degree of tissue lactate accumulation being important in post ischaemic recovery (Rehncrona et aI., 1981). The extent, however, to which the pH of the extracel­lular fluid has to drop before cell necrosis occurs is considerable, a reduction to below a pH of 5.3 being reported as the threshold for necrotic damage (Kraig et aI., 1987). It has further been shown that following complete ischaemia improved functional recovery can be achieved by the enhanced clear­ance of metabolic waste products from the vascu­lature (Hossmann and Olsson, 1970; Olsson and Hossmann, 197 1).

LOW pH AND BLOOD-BRAIN BARRIER 235

In addition to the generalised tissue damage found in ischaemia, lactic acidosis, and low pH are also thought to contribute specifically to endothelial cell damage and blood-brain barrier disruption (Broman and Lindberg-Broman, 1945; Klatzo, 1983; Paljarvi et aI., 1983; Kuroiwa et aI., 1985; Nagy et aI., 1985), although this has been refuted in a recent study using perfused frog pial venules in which it was found that severe hypoxia and meta­bolic inhibition disrupted the blood-brain barrier, but reduction in pH to 5.1 did not (Olesen 1986; Olesen and Crone 1986). The study of Nagy et aI. ( 1985) is of particular relevance to the current in­vestigation, as he demonstrated barrier opening fol­lowing intracarotid infusion of lactic acid in vivo.

The mechanism of barrier opening under these con­ditions is unclear, although it has been suggested that the increased permeability is either due to cell membrane damage and tight junction disruption (Nagy et aI., 1985) or to the stimulation of vesicular formation, and transfer across, the capillary endo­thelium (Westergaard et aI., 1976), a process that is rare in normal cerebral endothelium. The interpre­tation of vesicular profiles is, however, contentious as their presence does not necessarily indicate that net transfer is occurring and it does not preclude the possibility that they are in continuity with the cell surface membrane (Brightman et aI., 1983 ; Bundgaard et aI., 1979). If, on the other hand, ve­sicular transfer does occur, the processes of endo­and exocytosis are thought to involve the utilization of metabolic energy whilst cytosolic movement might be due to Brownian motion (Cervos-Navarro et aI., 1983). In support of this theory is the sugges­tion that the refractory period observed between two periods of barrier opening following transient ischaemia is due to a temporary depletion of energy supplies, which inhibits the energy-requiring pro­cess of pinocytosis that leads in turn to a reduction in vascular permeability (Kuroiwa et aI., 1985).

Recently, the passive properties of the blood­brain barrier have been studied in an energy­depleted brain (Greenwood et aI., 1985; Luthert et aI., 1987), in which the rat brain is perfused in situ

with a simple nonoxygenated saline perfusate free of glucose. The addition of 2,4, -dinitrophenol (DNP) in the perfusate, as an uncoupler of oxidative phosphorylation, and the absence of added glucose and oxygen accelerates the depletion of high-energy phosphates and allows breakdown of endogenous substrates without subsequent renewal. Mter 5 min of perfusion, ATP and phosphocreatine concentra­tions drop below 10 and 1% of control levels, re­spectively (J. Greenwood et aI., in preparation), a rate of degradation similar to that reported previ­ously following decapitation (Lowry et aI., 1964) or

total ischaemia (Ljunggren et aI., 1974a; 1974b). Under these perfusion conditions, the barrier re­mains intact, and adequate cerebral flow was main­tained for periods of at least 12.5 min (Luthert et aI., 1987). Using this in situ perfusion technique, an at­tempt has been made to determine whether low pH perfusate within the luminal compartment per se disrupts the blood-brain barrier and hence to ascer­tain whether the mechanism of low pH endothelial disruption demonstrated in vivo (Nagy et aI., 1985) may be due to passive processes, such as tight junc­tion disruption and cell membrane damage, or to an energy-requiring process, such as vesicle forma­tion. Vascular permeability and cerebral perfusate flow rates (CPFR) have been used as indices of bar­rier integrity and adequate perfusion, with exami­nation of the microvasculature at the light and elec­tron microscope level.

MATERIALS AND METHODS

Surgery Experiments were carried out on male Wi star rats

weighing 360-460 g that were anaesthetised with pento­barbitone (50--{)0 mg/kg, i.p.). The surface of the skull was exposed, and a burr hole made with a dental drill, at the intersection of lambdoid and sagittal sutures, to the level of the confluence of sinuses, but without penetration of the latter. The thorax was rapidly opened, followed im­mediately by cutting of the right atrium to allow perfusate drainage. The left ventricle was opened to permit cannu­lation of the proximal ascending aorta, the cannula being tied in place. The interval between opening the thorax and successful commencement of perfusion was always less than 30 s. Opening of the confluence of sinuses then followed, allowing further efflux of perfusate, and ligation of the thoracic descending aorta was effected.

Perfusion medium and apparatus The perfusion medium consisted of either a saline Ring­

er's solution (Greenwood et aI., 1985; Luthert et aI., 1987) or a Tris maleate buffer (0.05 M; KCI 4 mM; CaCl2 2.3 mM) adjusted to 300 mOs with sodium chloride. Both contained the metabolic inhibitor DNP (2 mM), and were adjusted to pH 7.4 with 1 M NaOH. For the low pH perfusates, subsequent acidification to pH 5.5 with lactic acid was carried out giving a lactate concentration of be­tween 0.53 to 0.6 1 mM in the Ringer's perfusate and 9.2 1 to 1 1.23 mM with the Tris maleate perfusate. Control animals were perfused for 10 min with the pH 7.4 Ringer­DNP solution. Experimental animals were perfused with either Ringer-DNP or Tris maleate-DNP at a pH of 7.4 for 5 min and then for a further 5 min at pH 5.5. These 10 min pretest perfusions were then followed by the test perfusate which contained, in addition to the above, ei­ther e4C]mannitol (2.2 MBq/j-Lmol) or e4C]iodoantipyrine [(lAP) 2.2 MBq/j-Lmol] for quantitative estimation of blood-brain barrier permeability and CPFR, respectively. Quadruplicate samples of test perfusates were taken and prepared for measurement of radioactivity by liquid scin­tillation counting. The unbound dye Evans blue (MW 96 1) was added to the test perfusate in all cases as a qualitative visual indicator of barrier integrity.

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236 J. GREENWOOD ET AL.

As previously reported (Luthert et aI., 1987), the per­fusion apparatus consisted of two independent, recycling, parallel circuits connected to the aortic cannula via a three-way tap, thereby allowing rapid switching from one solution to another when required. Perfusion medium was pumped at a rate of 25 ml/min via a peristaltic pump through a water jacket, in-line filter, and bubble trap, the jacket being maintained at a temperature sufficient to en­sure the head of the animal remained at approximately 37.4°C throughout the perfusion. The temperature was monitored once every minute during the perfusion by a thermistor probe positioned in the upper oesophagus of the animal. Changes in perfusate pressure during the ex­periments were measured using a pressure transducer (Statham P23AC) connected to a Grass Polygraph Model 5 chart recorder.

Blood-brain barrier permeability studies At the end of the 10 min pretest perfusion the test per­

fusate, containing e4C]mannitol (0.15-0.20 J.LM), was pumped through the brain of each animal (at the same pH as the immediate pretest perfusate) for a period of 2.5 min (Greenwood et aI., 1985; Luthert et aI., 1987). Following this test period, a 20-s high-pressure saline washout was effected, ensuring rapid removal of radiolabel from the cerebral vasculature.

Upon termination of the perfusion, the brains were rap­idly removed and, following brief washing in saline, were frozen in liquid hexane cooled over dry ice. Each brain was subdivided while still frozen into paired forebrain, cerebellum, and brainstem portions. The areas known to lack a blood-brain barrier, i.e., the choroid plexus and pineal gland, were removed from the bulk of tissue prior to it being weighed into scintillation vials. Tissue samples were dissolved in a solution of an organic base (Optisolv, Pharmacia, Milton Keyres, U.K.), with the addition of a toluene-based scintillation mixture neutralized with ace­tic acid, radioactivity was measured in an automatic scin­tillation counter (Tricarb 4430, Packard Instruments). Quench corrections when necessary were carried out us­ing the external standard method. Barrier integrity was assessed quantitatively as a ratio RtlRp (tissue dpm per gram wet weight)/(perfusate dpm per milliliter) for the 2.5-min test perfusion.

Perfusate flow rate studies To measure the cerebral perfusate flow rate the lO-min

pretest perfusion was followed by a 40-s perfusion with the test medium containing [14C]IAP and calculated as described previously (Luthert et aI., 1987). No saline vas­cular washout was carried out on these animals. Subse­quent removal and processing of tissue for scintillation counting was as described above for the permeability studies.

Statistical analysis Statistical comparisons between similar brain regions

in pH 7.4 and the two pH 5.5 treated groups were carried out for CPFR, RtlRp ratios, and water content data utilis­ing an unpaired, two sided Student's t test and taking 95% as the limit of significance.

Change in perfusate pH across the cerebral

vascular bed In a series of animals (n = 6) in which bilateral ligation

of the external carotid and pterygopalatine arteries had

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been effected, serial samples of the perfusate effluent were taken from the breached superior sagittal sinus dur­ing the 10-min perfusion in which the perfusate was changed from a pH of 7.4 to 5.5. The pH of each effluent sample was then measured using a micro-pH electrode (Amagruss pH-C-1080, Flowgen Instruments Ltd., Kent, UK).

Morphological investigations For microscopical examination of capillary morphol­

ogy, animals were perfused with freshly prepared one­half strength Karnovsky's fixative for 15 min at a rate of 12 mllmin following the lO-min pretest perfusions. The head of each animal was removed, cleared of skin and musculature, and stored in the same fixative overnight at 4°C. The brains were then removed and slices (150 J.Lm thick) were prepared using a tissue vibrotome (Vibroslice 752/M, Campden Instruments, London, UK) just rostral to the optic chiasma, at the level of the anterior commis­sure. These were washed in cacodylate buffer and then postfixed in I % osmium tetroxide for 1 h. Slices were resin embedded between Teflon-coated slides. One­micron sections were cut and stained with toluidine blue for examination at the light microscope level. Ultra-thin sections were heavy metal stained and examined on a Hitachi H-600 transmission electron microscope.

RESULTS

Macroscopic examination of each brain for Evans blue leakage showed colouration of pineal gland, choroid plexus, and other circumventricular re­gions. Apart from such areas, the brains appeared uniformly yellow due to the partial lipid solubility of DNP. The pH 5.5 brains were more deeply stained yellow due to the increased solubility of DNP in nervous tissue at low pH (Caldwell, 1957, 1960).

Integrity of the blood-brain barrier, expressed as an RtlRp e4C]mannitol ratio, after either a to-min pretest perfusion of pH 7.4 (n = 8) or a 5-min per­fusion at pH 7.4 followed by 5 min at pH 5.5 (Ringer/DNP, n = 6 and Tris maleate/DNP, n = 4), is shown in Table 1. Also shown for comparison is a similar e4C]mannitol ratio recently obtained in

vivo (Luthert et aI., 1987). There was no significant difference in RtlRp ratios between any of the four groups of animals.

The CPFR for the three brain regions studied is shown in Table 2 for the pH 7.4 and two pH 5.5 sets of animals. No significant difference in perfusate flow rates were obtained between these groups for any of the three regions examined. The different perfusion media had no effect upon reflected perfu­sion pressure which, as in previous studies (Luthert et aI., 1987), reached a final value of approximately 55 mm Hg at the end of each experiment. When the Ringer-DNP medium was used to perfuse the brain, the pH of the brain effluent remained consistently below 7.4, even during the 5-min perfusion with a 7.4 adjusted perfusate. When the perfusate was

LOW pH AND BLOOD-BRAIN BARRIER 237

TABLE 1. Barrier permeability expressed as the ratio of tissue radioactivity to that of the perfusate (RtlRp)

following a 2.5 min perfusion with [14C] mannitol

Rt/Rp (xW)

Cerebrum Cerebellum Brainstem

Ringer's-DNP pH 7.4 (8) 1.0 ± 0.3 2.0 ± 0.6 3.2 ± 1.2

Ringer's-DNP pH 5.5 (6) 1.0 ± 0.2 1.3 ± 0.2 1.2 ± 0.4

Tris-DNP pH 5.5 (4) 1.7 ± 0.4 1.2 ± 0.3 3.1 ± 0.6

In vivoo (6) 1.6 ± 0.4 2.2 ± 0.6 3.8 ± 1.5

The test perfusate was carried out after 10 min of pretest per­fusion of differing pH and buffering capacity as described in the text. For comparison the in vivo Rt/Rp ratios for [14C]mannitol following maintenance of steady levels for 2.5 min within the circulation, are given. The values are means ± SEM with num­ber of animals given in parentheses.

° From Luthert et at. (1987).

switched to pH 5.5, the pH of the effluent failed to drop dramatically, reaching a final pH of no less than 6.5. The pH of the cerebral effluent from the Tris maleate-DNP perfused brains remained at be­tween 7.1 and 7.3 during the first 5 min of a pH 7.4 perfusion. On changing to the low pH the effluent pH rapidly fell to between 5.6 and 5.7 (Fig. 1).

Histological examination did not reveal any ob­vious changes in the appearances of vasculature ex­posed to the different perfusate acidities. Thick (1 flm) sections stained with Toluidine blue showed little or no astrocytic swelling in cortical regions in either the pH 7.4 or the two pH 5.5 groups.

Ultrastructural examination revealed no notice­able difference in the appearance of the capillary endothelium of the three groups. The microvascu­lature was well preserved in both the pH 7.4 (n = 4) and the pH 5.5 (Ringer-DNP, n = 4 and Tris male­ate-DNP, n = 3) groups (Figs. 2-4). Astrocyte foot processes occasionally showed signs of oe­dema particularly in the white matter and around larger vessels (Fig. 5). These alterations were not sufficient to cause any deformation of the capillar­ies, and in no case were partially or totally occluded vessels observed. The vessels in both groups were largely indistinguishable from those fixed without prior saline perfusion. No oedema was seen in the

capillary endothelial cells; there was no increase in vesicular profiles, and no obvious disruption of the tight junctions or luminal membrane was observed.

DISCUSSION

It had recently been shown that the blood-brain barrier can be opened to the high molecular weight tracer horseradish peroxidase when Ringer's, con­taining lactic acid, is perfused in vivo into the ca­rotid circulation of the rat (Nagy et aI., 1985). The present study, however, shows that under condi­tions in which the brain is depleted of high-energy phosphates (J. Greenwood et aI., in preparation) the perfusion of a solution containing lactic acid at a pH of 5.5 for a total of 7.5 min fails to open the blood­brain barrier to either unbound Evans blue or the small tracer e4C]mannitoi (Fig. 2). These findings are consistent with a recent report demonstrating that perfusion of frog pial vessels with a perfusate of pH 5.1 does not disrupt the vasculature (Olesen, 1986), although the same group did find vessel dis­ruption with hypoxia and metabolic inhibition (Ole­sen and Crone, 1986).

The failure of luminal lactic acid and low pH to open the blood-brain barrier in this study may sug­gest that they are not important in ischaemic barrier disruption or that metabolic energy is required in addition. Hyperosmolar barrier opening has been shown to occur in the absence of energy-producing metabolism (Greenwood et aI., 1988), but Hoss­mann and Olsson ( 197 1) demonstrated suppression of barrier opening, with hypertensive and chemical insults, following a period of ischaemia during which energy depletion must have occurred. It is a reasonable supposition, therefore, that there may be more than one way in which the barrier is opened. The morphological correlates of barrier opening are discussed below. If lactic acid is not the cause of ischaemic barrier damage, perfusion of lac­tic acid in vivo must be disrupting the barrier in other ways, perhaps by provoking local vasodilata­tion, loss of autoregulation, and subsequent eleva­tion of cerebral blood flow, a factor known to cause barrier opening (Rapoport, 1976; Kuroiwa et aI., 1985). Recently, however, it has been reported that

TABLE 2. Cerebral perfusate flow rates following the different perfusion protocols

Ringer's-DNP pH 7.4 (4) Ringer's-DNP pH 5.5 (4) Tris-DNP pH 5.5 (4)

Cerebrum

0.81 ± 0.19 0.86 ± 0.06 1.00 ± 0.09

CPFR (ml/min/g)

Cerebellum

1.19 ± 0.08 1.13 ± 0.08 1.19 ± 0.12

Brainstem

1.63 ± 0.22 1.54 ± 0.09 1.11 ± 0.09

Body weight (g)

398 ± 17 373 ± 6 393 ± 2

Values are means ± SEM with numbers of animals in parentheses.

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238 J. GREENWOOD ET AL.

Ringer

Tris maleate

o 4 6 8 10

Perfusion time (min)

FIG. 1. Examples of the perfusate effluent pH sampled from the superior saggital sinus (see text) during a 5-min perfu­sion with a medium at pH 7.4 followed by a further 5-min perfusion at pH 5.5. The open circles represent the effluent pH of the Ringer's-DNP perfusate and the closed circles rep­resent the effluent pH of the Tris-maleate-buffered perfusate.

lactic acid infused into the circulation of conscious piglets does not increase CBF (Laptook, et aI., 1988), although these differences may be due to the route of administration.

The relatively good buffering capacity of the ce­rebral vasculature is demonstrated by the small pH difference across the vascular bed of the pH 5.5 Ringer's perfused animal. Conversely, when Tris maleate buffer is added to the perfusate a dramatic arterio-venous difference in pH is found when the perfusate is switched from a pH of 7.4 to 5.5. What is perhaps more surprising, however, is the obser­vation that the pH of the effluent for both the per­fusates during the pH 7.4 perfusion remains below 7.4. It is possible that this may be due to the efflux of lactic acid from the tissue, although separate ex­periments looking at the efflux of lactic acid from perfused brains do not support this (J. Greenwood et aI., in preparation). Furthermore, if this were so

A

then one would expect a greater drop in the effluent pH of the poorly buffered Ringer's perfusate. It is clear from these experiments that, at least in the absence of energy-yielding metabolism, a pH of 5.5 within the lumen is insufficient to damage the cere­bral endothelium and cause them to become dis­rupted.

The relationship between pH, cerebral oedema, and cerebral blood flow/CPFR is one that requires close examination. It has been shown in the past that the severity of brain oedema is likely to be proportional to the degree of tissue acidosis (Taka­hashi, 1966; Paljarvi et aI., 1983). In the present study, there was no significant increase in the water content of the brain following the low pH perfu­sions, although the values were slightly greater than those of the pH 7.4 perfused animals (Table O. Ex­amination of the state of the microvasculature at both light and electron microscope levels showed limited perivascular swelling of astrocytic foot pro­cesses at either pH 7.4 or the two pH 5.5 groups of animals (Figs. 2-4), although this was more marked in white matter (Fig. 5). These appearances are sim­ilar to those described by Paljarvi et al. ( 1983) and Kalimo et al. ( 198 1b), who demonstrated that the microvasculature and parenchyma remained well preserved if saline was infused prior to the is­chaemic period. In animals in which they infused a glucose-saline mixture, however, there was consid­erable astrocytic and endothelial swelling and greater lactic acidosis. In these high lactic acid preparations the preservation of the tissue was poor, with severe structural changes and vessel oc­clusion.

The buffering capacity of the two perfusates used

B

FIG. 2. Electron micrographs of cerebrum following perfusion with Ringer's-DNP at a pH of 7.4 for 10 min prior to fixation. (A) Parenchyma (x3,500). (8) Capillary (x11,000).

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LOW pH AND BLOOD-BRAIN BARRIER 239

A 8

FIG. 3. Electron micrographs of cerebrum following perfusion with Ringer's-DNP at a pH of 7.4 for 5 min followed by a further 5 min at pH 5.5 prior to fixation. (A) Parenchyma (x4,900). (8) Capillary (x11,000).

in the present study differ considerably, and as a result the concentration of lactic acid required to reduce the pH to 5.5 differed by over an order of magnitude. It was only in the well-buffered Tris maleate perfusate that the concentration of lactate approached that generally found in plasma in is­chaemic states; 9.2 1 to 11.23 f.LmoVml in this study compared to 10 to 15 f.LmoVml during ischaemia in

vivo (Rehncrona et aI., 1981). Indeed, it has previ­ously been found that severe brain cell damage in experimental ischaemia only occurred when the tis­sue lactate rose to a level of 35 f.Lmol/g whilst at a level of 15 f.Lmol/g there was very little morpholog­ical change (Kalimo et aI., 1981a,b; Rehncrona et

A

aI., 198 1). The tissue lactic acid threshold at which morphological abnormalities occur has been put at between 16 and 20 f.Lmol/g. Unfortunately the amount of lactic acid used to acidify the Krebs­Ringer's bicarbonate buffer used by Nagy et ai. ( 1985) in their in vivo opening of the blood-brain barrier is not reported. The higher concentrations of lactic acid required to cause damage are consistent with the recent report by Kraig et ai. ( 1987) and Petito et ai. ( 1987), who reported that necrosis oc­curred only when the extracellular fluid pH dropped to below 5.3. It is also perhaps important to con­sider that the in vivo relationship between lactic acid concentration and pH is complex, for protons

FIG. 4. Electron micrographs of cerebrum following perfusion with Ringer's-DNP buffered with Tris-maleate and adjusted to a pH of 7.4 for 5 min followed by a further 5 min at pH 5.5 prior to fixation. (A) Parenchyma (x4,300). (8) Capillary (x 11 ,000).

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240 J. GREENWOOD ET AL.

FIG. 5. Electron micrograph of cerebral capillary in white matter, following perfusion with Ringer's-DNP at a pH of 7.4 for 10 min prior to fixation. Mild swelling of the perivascular astrocytic foot process can be seen (x4,500).

may be produced from sources other than lactic acid, such as during the anaerobic hydrolysis of ATP (Paschen et aI., 1987). In a recent study in which lactic acid was applied directly to the spinal cord, morphological changes were found both in the parenchyma and blood vessels, the latter showing signs of capillary wall necrosis and leakage of HRP. The experimental conditions in this study were, however, extremely severe, with a pH of 1.8 and lactic acid concentrations of 6.3 M and above; both conditions are highly unlikely to be found in vivo.

A further consideration is the difference between the tissue concentration of lactic acid and the per­fusion medium concentration. In the present study, lactic acid and low pH are introduced via the vas­culature whilst in vivo, during ischaemia, lactate is produced intracellularly and not in the lumen. Al­though it is known that at the blood-brain barrier the monocarboxylic acid carrier for lactate dramat­ically increases its translocation at low pH values (Oldendorf et aI., 1979) it is unclear how much of the perfusate lactic acid will enter the tissue under our experimental conditions. What is known, how­ever, is that during physiological hyperlactic aci­demias in which blood levels of lactic acid can reach as high as 10 mM, it takes many hours for the lac­tate to equilibrate with the CSF (Pardridge, 1983), indicating a very low rate of influx across the blood-brain barrier. Indeed, the concentration of lactic acid present in the brain tissue under these conditions increase by only tenfold from control values (J. Greenwood et aI., in preparation) to lev­els that are believed to be insufficient to cause ul-

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trastructural damage (Plum, 1983). Intracellular, rather than intraluminal, lactate concentration may be of prime significance in the development of cell swelling and damage, a hypothesis consistent with the results of Rehncrona et aI. ( 1981), who found identically raised blood lactate concentrations in both saline- and glucose-infused ischaemic animals, but with a significantly higher tissue concentration in the latter, the group which also showed the se­vere pathological change.

A pump flow rate of 25 mllmin gave CPFR values for the pH 7.4 and both the pH 5.5 groups of per­fused animals that are similar to anaesthetised in

vivo CBF rates (Reid et aI., 1982; Braun et aI., 1985). There was no significant difference between the groups, suggesting that perfusate access to the cerebral vascular bed was similar in all cases. The variation in CPFR between the three brain regions examined in each group (Fig. 3) has been observed previously (Luthert et aI., 1987) and may be due to a loss of regulatory mechanisms that normally con­trol the differential supply to different areas of the brain.

Reported endothelial cell injury following lactic acid-based perfusion (Nagy et aI., 1985), in isch­aemia (Paljarvi et aI., 1983; Chiang et aI., 1968), following intracerebral lactate injections (Petito et aI., 1987), and after direct application of lactic acid to the spinal cord (Balentine and Greene, 1987) was not observed in this energy-depleted preparation. It is clear from these previous studies that only at high tissue lactate concentrations and low pH deteriora­tion of the microvasculature occur, the threshold for damage being reported to be a tissue pH of around 6 (Mabe et aI., 1983); more recently the threshold within the extracellular fluid has been put at 5.3 (Kraig et aI., 1987; Petito et aI., 1987). Of direct relevance to the failure to open the barrier was the lack of an increase in the normal number of micropinocytotic vesicle profiles seen. It has been suggested that microvesicular transport (rarely seen in abundance in normal cerebral endothelium) is an active process and is responsible for the increase in permeability found in ischaemic brain damage (Westergaard, 1977; Kuroiwa et aI., 1985). The fail­ure to open the barrier at low pH in the absence of energy-producing metabolism in this system could tentatively be explained by the inhibition of an ac­tive process of this nature taking place. An alterna­tive explanation would be that under these, or any other, conditions the vasculature is simply resistant to acidity as low as pH 5.5. This does not rule out the possibility that vesicular transport does occur in ischaemic brain, but would imply that it occurs only upon restitution of blood flow and energy status.

LOW pH AND BLOOD-BRAIN BARRIER 241

In conclusion, perfusion with a low pH solution containing lactic acid is insufficient to bring about blood-brain barrier opening in the absence of en­ergy-producing metabolism. This is despite the fact that lactic acidosis has been strongly implicated in barrier disruption in cerebral ischaemia and other pathological conditions. The absence of an increase in vascular permeability may be due to the process being one that requires the presence of metabolic energy or to the likelihood that the endothelium are resistant to external acidity of as low as pH 5.5

Acknowledgment: This work was supported by grants from the Wellcome Trust.

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