Acid-Induced Death in Neurons and Glia
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The Journal of Neuroscience, August 1991, 1 I(9): 2499-2497 Acid-induced Death in Neurons and Glia Maiken Nedergaard, Steven A. Goldman, Smita Desai, and William A. Pulsinelli Department of Neurology and Neuroscience, Raymond and Beverly Sackler Foundation, Cornell University Medical College, New York, New York 10021 Lactic acidosis has been proposed to be one factor pro- glucoseand glycogenconcentrations ofthe affected tissue(Smith moting cell death following cerebral ischemia. We have pre- et al., 1986). Local accumulation oflactic acid to cytotoxic levels viously demonstrated that cultured neurons and glia are killed may play a causalrole in the genesisof brain infarction following by relatively brief (10 min) exposure to acidic solutions of cerebral ischemia (Meyer and Yamaguchi, 1977; Siemkowitz pH ~5 (Goldman et al., 1989). In the present series of ex- and Hansen, 1978; Pulsinelli et al., 1982; Nedergaard, 1987). periments, we investigated the relationship between changes Several authors have addresseddirectly the issue of acid- in intracellular pH (pH,) and cellular viability. pH, was mea- induced cell death. Cultured neurons and astrocytes die follow- sured using fluorescent pH probes and was manipulated by ing relatively brief exposures to extracellular pH (pH,) below changing extracellular pH (pH,). Homeostatic mechanisms 5.3 (Norenberg et al., 1987; Goldman et al., 1989). In viva, regulating pH, in neurons and glia were quickly overwhelmed: exposure of rat parietal cortex to lactic acid below pH, 5.3 for neither neurons nor glial cells were able to maintain baseline 20 min causedfrank brain infarction (Kraig et al., 1987). How- pH,when incubated at pH, below 8.8. Neuronal and glial death ever, such extremesof tissuepH may not be encounteredduring was a function of both the degree and the duration of intra- in viva global or focal ischemia, unlessthe animals are exposed cellular acidification, such that the LD,, following timed ex- to marked degreesof preischemic hyperglycemia (Kraig and posure to HCI increased from pH, 3.5 for lo-min acid incu- Chesler, 1990). Rather, tissue pH may be moderately lowered bations to pH, 5.9 for 2-hr exposures and pH, 8.5 for 8-hr for many hours in those areas of focal ischemia that ultimately exposures. necrose(Kobatake et al., 1984; Nedergaard et al., 1990). This Replacement of HCI with lactic acid raised the LD, to pH, article examines the possibility that the prolongation of acid 4.5 for lo-min acid exposures, but did not change the LD, exposure does in fact lower the threshold for acid-induced cell for longer exposures: pH, measurements concurrent with ex- death, thereby providing a mechanism by which brain tissue tracellular acidification suggested that the greater cytotox- might infarct at pH levels within the range achieved in vivo icity of lactic acid relative to that of HCI was caused by the during ischemic acidosis. more rapid intracellular acidification associated with lactic To test this hypothesis, neurons and glial cells were loaded acid. The onset of death after exposure to moderately acidic with pH-sensitive dyes and exposed to extracellular acid for solutions was delayed in some cells, such that death of the periods ranging from 10 min to several hours. Neuronal and entire cell population became evident only 48 hr after acid glial intracellular pH (pH,) was determined by microspectro- exposure. During this latency period, cellular viability indices fluorometry and was correlated with cell viability, as assessed and ATP levels fell in parallel. The course of death could be by trypan blue exclusion. The results indicate that the cellular experimentally manipulated: hypothermia initiated after the LD,, for H+ ions is an inverse function of the duration of acid acid exposure attenuated the degree of cell death, indicating exposure. that processes occurring during this postinjury period were responsible for demise. Thus, a delayed process of cell death Materials and Methods can be initiated in vitro by an initial insult other than gluta- Tissue dissociation and culture. Mixed forebraincultures were derived mate exposure, and this process can occur in glia as well from 16-d-gestationrat embryosand preparedas previously described as in neurons. (Goldmanet al., 1989);lo6 cellswere plated in eachof 24 wellsin poly- L-lysinetreated plates (Falcon). For pH, measurements,2 x IO6cells Cerebralhypoxia-&hernia induceslactic acid formation through wereplated on poly+lysinecoated25mm roundcoverslips maintained in 35-mmComing dishes. The cultureswere kept at 37°Cin 5% CO, the accentuation of anaerobic glycolysis. The magnitude of this humidifiedair. Cultureswere exposed to acid after 8-10d in vitro (DIV), lactic acid accumulation dependslargely upon the preischemic unlessotherwise stated. Culture medium. The culture mediumconsisted of 20% fetal calf serumand 80% Dulbecco’smodified Eagle’s medium, supplemented Received Nov. 29, 1990; revised Mar. 26, 1991; accepted Mar. 27, 1991. with 4 mM L-glutamine,100 PM nonessentialamino acids,8 mg/ml We thank Dr. Martin Lesser for his assistance with the statistical analysis of n-glucose,5 &ml insulin, 20 U/ml penicillin-G, 20 &ml strepto- these data. This research was supported in part by Kobmand i Odense Johann and Hanne Weimann, f. Seedor@ legat, by NIH Grant NS-03346, by National mycin, and 50 rig/ml amphotericin.Half-volume media changes were Institute of Neural Disorders and Stroke Grant NS-013 16, and by grants from performedevery third day. the G. Harold and Leila Y. Mathers Charitable Foundation and from Mr. and Acid exposure. Followingan 8-10-d incubationperiod, test cultures Mrs. Fowler Merle-Smith. S.A.G. is a Cornell Scholar in Biomedical Science. wereexposed to HEPES-bufferedRinger’s solution (HBS), titrated with Correspondence should be addressed to Dr. M. Nedergaard, Department of HCI to a pH within the rangeof 2.2-7.3.Each culture was washed three Neurology and Neuroscience, Cornell University Medical College, 1300 York timesin the appropriateacidified HBS, then exposedto the testsolutions Avenue, New York, NY 1002 1. from 10 to 360 min in a CO,-freeincubator at 37°C.The test solutions Copyright 0 1991 Society for Neuroscience 0270-6474/91/l 12489-09$03.00/O were changedevery 2 hr during thoseacid exposuresexceeding 120 2490 Nedergaard et al. * Acid-induced Death in Culture min. Following acid exposure, each culture was washed once in fresh and excess medium was aspirated; complete solution exchange was medium and left for 24 hr in the incubator before viability counting, accomplished in less than 5 sec. All measurements of pH, were made unless otherwise indicated. The HEPES-buffered solution was composed in HBS. Intracellular pH calibration standards for individual cells were of 150 mM NaC1, 1 mM MgSO,, 1 mM K,HPO,, 20 mM HEPES (N-2- established by exposing the cultures, after measurement, to a potassium hydroxyethylpiperazine-N-2-ethenesulfonic acid), 10 mM glucose, and Ringer’s solution containing 10 PM of the ionophore nigericin. Nigericin 1.5 rnr+r CaCl,. In some experiments, 20 mM sodium L-lactate was sub- in combination with high potassium equalizes extracellular andintra- stituted for 20 mM NaCl. Solutions containing CO, were bubbled with cellular DH (Chaillet and Boron. 1985). The potassium Rineer’s solution 5% CO,/95% air for 30 min before titration with HCl. Extracellular pH employed consisted of 70 mM’KC1, ‘10 rn& glucose, 1 rn& CaCl,, 2.5 was determined with an electronic pH meter (Beckman Instruments, mM K,HPO,, 10 mM HEPES, 1 mM MgSO,, and 90 mM sucrose. model 55) in combination with a Microelectrodes MI-410 pH probe. Fluorescence from individual neurons and glia, as well as from small Both were reliable to 0.01 pH unit. All pH measurements were made cellular aggregations, was monitored by a microspectrofluorometer. at 37°C. Measurements were obtained from a IO-pm-diameter field with an ad- Hypothermia.The effect of lowered incubation temperatures upon justable field aperture placed in front of the photomultiplier tube. In all acid-induced injury was examined by lowering the ambient incubation measurements of neurons, we focused upon the cell body. As a result chamber temperature to 32°C after acid exposure at 37°C. After the of poor dye uptake among glia, relative to their neuronal counterparts exposure, one set of cultures was kept in this incubator for 4 hr and (Connor et al., 1987), as well as to the thin, well-spread topography of then transferred to an incubator at 37°C. cultured astrocytes, the contamination of neuronal signal by glial counts Viability criteria and index. Loss of membrane integrity, as deter- was low. Also, we attempted to measure neurons positioned in areas mined by trypan blue inclusion, was used as a marker of cell death free of underlying glia. Monochromatic excitation was accomplished (Phillips, 1969; Choi et al., 1987; Goldman et al., 1989). A total of496 using a Photon Technology Delta Scan, in which a xenon arc beam is cultures were exposed to acidified solutions, spanning a wide range of split by a rotating chopper into two beams, each of which passes through pH, and exposure duration, as noted above. After appropriate postex- a narrow-range monochromotor (4-nm band width), which permits pre- posure survival times, the cells, both adherent and detached, were count- cise wavelength selection. In this study, dual excitation at 495 nm (pH ed by hemocytometer, following 15 min incubation in trypan blue as sensitive) and 440 nm (pH insensitive) was used for both BCECF and previously described (Goldman et al., 1989). Neurons and glia were CDCF, while DCF was excited at 487 nm (pH sensitive) and 450 nm pooled for this determination, as no effort was made to assess their (pH insensitive). The Delta Scan was attached to a Leitz Diavert mi- differential sensitivities to acidosis. croscope equipped with a Nikon 40 x 1.3-NA Fluor objective.