acidosis induces necrosis and apoptosis of cultured hippocampal neurons
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Experimental Neurology 162, 1–12 (2000)doi:10.1006/exnr.2000.7226, available online at http://www.idealibrary.com on
Acidosis Induces Necrosis and Apoptosis of CulturedHippocampal Neurons
Ding Ding,*,1 Shaye I. Moskowitz,†,1 Rong Li,† Sean B. Lee,‡,2 Mariano Esteban,‡,3
Kevin Tomaselli,§ Jane Chan,¶ and Peter J. Bergold*,†*Department of Pharmacology and Physiology, ‡Department of Biochemistry, and ¶Department of Neurology, †Program in Neural and
Behavioral Science, State University of New York-Downstate Medical Center, Brooklyn, New York 11203; §IDUN Pharmaceuticals,11085 North Torrey Pines Road, Suite 300, La Jolla, California 92037
Received June 7, 1999; accepted August 23, 1999
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Acidosis, hypoxia, and hypoglycemia rapidly andransiently appear after reduction of cerebral bloodow. Acidosis also accompanies head trauma and sub-rachnoid hemorrhage. These insults result in ne-rotic and apoptotic loss of neurons. We previouslyemonstrated that transient acidification of intracellu-
ar pH from 7.3 to 6.5 induces delayed neuronal loss inultured hippocampal slices (49). We now report thatcidosis induced both necrotic and apoptotic loss ofeurons. Necrosis and apoptosis were distinguishedemporally and pharmacologically. Necrosis appearedapidly and was dose dependent with the duration ofhe acidosis treatment. Apoptosis was delayed withaximal number of apoptotic cells seen with a 30-min
cidosis treatment.Apoptotic neuronal loss was accom-anied by DNA fragmentation and was blocked by
nhibitors of protein and RNAsynthesis, ectopic expres-ion of the anti-apoptotic gene bcl-2, or an inhibitor ofaspases, proteases known to be activated during apop-osis. Necrotic neuronal loss was unaffected by thesereatments. Hypothermia, a treatment known to attenu-te neuronal loss following a variety of insults, blockedoth acidosis-induced necrosis and apoptosis. Theseesults indicate that acidosis is neurotoxic in vitro anduggest that acidosis contributes to both necrotic andpoptotic neuronal loss in vivo. r 2000 Academic Press
Key Words: acidosis; neurodegeneration; gene expres-ion; cerebral ischemia; trauma; bcl-2.
INTRODUCTION
Acidosis, hypoxia, and hypoglycemia are immediateetabolic consequences of reduced blood flow to the
1 An equal contribution was made by the first two authors.2 Present address: Department of Genetics, Massachusetts Gen-
ral Hospital, Boston, MA 02114.3 Present address: National Laboratory of Biotechnology, Campo
dniversita Automica, Canto Blanco 280049, Madrid, Spain.
1
rain (22, 47). These insults, individually or in combina-ion, induce neuronal loss as a result of two distincteath mechanisms—necrosis and apoptosis. In animalodels of ischemia, neuronal loss within 24 h is pre-
ominantly necrotic, while more delayed neuronal deaths apoptotic (12, 37, 60). Acidosis also rapidly appearsollowing trauma or subarachnoid hemorrhage (10, 48).
Neuronal loss is the consequence of necrosis orpoptosis. Necrosis results from collapse of cellularetabolism and loss of ion homeostasis. It requires
either energy nor new gene expression and is accompa-ied by cell lysis, release of intracellular contents, andctivation of inflammatory responses (1, 41). Apoptosis,n contrast, is a regulated alteration of cellular metabo-ism that culminates in ‘‘cell suicide’’ or programmedell death (1, 41). During apoptosis, there is extensiveuclease and protease activity, resulting in a character-
stic condensation of chromatin and release of monond oligonucleosomes into the cytoplasm without cellysis (1, 41).
The contribution of hypoxia, hypoglycemia and acido-is to necrotic or apoptotic neuronal loss is poorlynderstood. In disassociated neuronal cultures or cul-ured hippocampal slices, necrosis is induced by com-ined hypoxia-hypoglycemia, or by hypoglycemia alone11, 43, 51, 57, 58). Activation of NMDA receptorsnderlies this neuronal loss (15, 26, 43, 53, 54, 57, 58).cidosis was protective against hypoxia-hypoglycemiand glutamate toxicity. This is consistent with inactiva-ion of NMDA receptors at pH , 6.8 (2, 11, 15, 26,2–60). In addition, acidosis was not toxic to disassoci-ted neurons unless exposed to pH , 6.0 (17, 42). TheH of the brain rarely acidifies to this extent duringtroke or other neurodegenerative diseases (37, 48).hese results suggested that acidosis protects againstxcitotoxicity in vitro and may be protective in vivo55). Studies using disassociated embryonic neurons,owever, may not adequately model many forms ofeuronal damage. Ischemic injury is highly age depen-
ent: when compared to adults, the neonatal brain is0014-4886/00 $35.00Copyright r 2000 by Academic Press
All rights of reproduction in any form reserved.
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2 DING ET AL.
xtremely resistant (61). The toxic effect of acidosisay only be apparent in older neurons. Although a
irect effect of acidosis has not been examined in vivo,cidosis toxicity has been examined indirectly. Hyper-lycemia preceeding ischemia results in more severecidosis (pH 5.8–6.2) than ischemia alone. Hypercapniareceeding ischemia also results in more severe acido-is. Neuronal injury was more severe in rats madeyperglycemic or hypercapnic before ischemia (24, 27,8, 29, 34, 50). Increased acidosis is thought to underliehe more extensive ischemic injury seen in diabeticatients (34). These studies, albeit quite suggestive, doot provide direct evidence for a role for acidosis ineurodegeneration.Acidosis toxicity has been studied using cultured
ippocampal slices. Isolated from 9- to 25-day-old rats,ippocampal slice cultures retain much of the tissuerchitecture, neuronal density, and synaptic connec-ions of the hippocampus (7, 14). These features of sliceultures may be critical for the expression of acidosisoxicity. Disassociated neuronal cultures that lack theseeatures do not display acidosis-induced neuronal loss17, 42, 55). In contrast, following transient acidifica-ion of intracellular pH (pHi) from 7.3 to 6.6 inducedeuronal loss in cultured slices (49). In this study, weemonstrate that mild acidification (pH 6.6) induces anarly necrosis and a more delayed apoptosis.
MATERIALS AND METHODS
Hippocampal slice cultures. Hippocampal slicesere isolated from 9- to 25-day-old Sprague–Dawley
ats and cultured in vitro (7). Transverse slices (400m) were cut using a McIllwain tissue chopper (Brink-an, Westbury, NY) and cultured on Millicell-CMembranes (Millipore, Bedford, MA). Slice culturesere maintained at 35°C in 5% CO2 atmosphere in 50%agle’s basal media, 25% Earle’s balanced salt solution,5% horse serum, 25 mM Hepes, pH 7.3, 1 mM gluta-ine, and 6.5 mg/ml glucose. All media componentsere from GIBCO/BRL (Grand Island, NY). Unless
ndicated, all other chemicals were from Sigma (St.ouis, MO). Cultures obtained from postnatal days 9-o 11-day-old rats were treated at 4 days in vitro with00 nM 5-flourodeoxyuridine, 100 nM uridine, and 100M cytosine arabinoside for 24 h.Induction of acidosis in cultured slices. After 2eeks in vitro, slice cultures were examined and the0–80% of all cultures retaining intact granule cell andyramidal cell layers were included. Cultures werelaced in Hank’s balanced salt solution (30 ml, HBSS)ontaining 4.0 mM NaHCO3 and vigorously bubbled at7°C with 95% O2, 5% CO2 that was sterilized through
0.2-µm syringe filter. This treatment induced ancidification of extracellular and intracellular pH of
lice cultures to 6.6 (49). Temperature during the tcidosis treatment was monitored with a thermocouplerobe (YSI 401, Cole-Palmer, Niles, IL). Control cul-ures were similarly treated with modified HBSS con-aining 25 mM NaHCO3 NaCl was reduced to 116 mMaCl to maintain osmolarity. Treatment of slice cul-
ures with modified HBSS does not result in intracellu-ar or extracellular acidosis (49).
Measurement of pHi. Slice cultures were loaded forh at 35°C with BCECF-AM (10 µM, 28,78-Bis(carboxy-
thyl)-4 or 5-carboxyfluorescein, diacetoxymethyl ester,olecular Probes, Eugene, OR) in Earle’s balanced salt
olution containing 20 mM glucose. pH determinationsere done on cultures receiving a 15-min acidosis
reatment since BCECF leaked from the culture by 30ins (J.C. and P.J.B., unpublished result). Culturesere excised from Millicell-CM filter inserts and placedn coverslips. Fluorescent video images were obtainedsing a fluorescence ratio imaging system (Photonechnology International, Princeton, NJ). Images wereaptured on a Novicon intensified video camera, using a80-nm-long pass filter and alternating excitation be-ween 440 and 500 nm. Fluorescence ratios were con-erted to pH values using a standard curve obtainedrom slice cultures treated with the ionophores nigeri-in (10 µM) and monesin (10 µM) in: (in mM) KCl 70,epes 20, sucrose 90, CaCl2 1, MgSO4 1, Na2HPO4 2.5,
lucose 10, buffered to pH 6.20, 6.60, 6.73, 7.20, and.38. No perfusion was used in this study; as a resultlices from postnatal day 10 were used due to theiresistance to hypoxic damage (49). pHi did not changeuring the 15-min BCECF recordings.Propidium iodide and TUNEL assays of slice cul-
ures. Cultures were stained for 30 min with 0.5%ropidium iodide in HBSS containing 20 mM Hepes,H 7.3, and 25 mM glucose at 35°C. The number ofpifluorescent propidium iodide nuclei in the CA3 andA1 pyramidal cell layers were counted by two indepen-ent observers. Propidium iodide fluorescence withinhe pyramidal cell layer accurately assays neuronaloss in hippocampal slices cultures (57). Cell countseflect the number of dying cells at the time of assay. Tonsure that propidium iodide staining is similar torypan blue exclusion, slice cultures were made acidicor 30 min and neuronal loss assayed at 8 h withropidium iodide followed by assay of the same cultureith trypan blue. No significant difference was ob-
erved between the two assays (propidium iodide,71 6 85; trypan blue, 163 6 29 n 5 4; P . 0.1, Stu-ent’s t test), thus demonstrating the validity of theropidium iodide to assay neuronal loss in slice cul-ures. Trypan blue-stained cells in the pyramidal cellayer had the spindle-shaped morphology of pyramidaleurons. A major source of variability in these studiesesulted from the slice culture preparation. Variabilityrom individual dissections was less than among mul-
iple dissections. Culture variability did not prevent
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3ACIDOSIS-INDUCED NECROSIS AND APOPTOSIS
tatistical significance to be obtained in all experi-ents. TUNEL staining was performed en bloc in slice
ultures using the Apotag kit (Oncor, Gaithersburg,D), according to the manufacturer’s instructions.
imilar en bloc immunohistochemistry was performedith anti-Bcl-2 antisera. En bloc staining was requiredue to the difficulty in resectioning 250-µm slice cul-ures to slices less than 50 µm. As a result, the image inig. 2 is from a culture many cells thick.Inhibition of RNA and protein synthesis. Slice cul-
ures were treated with the translation inhibitor aniso-ycin (10 µM) for 2.5 h. Thirty minutes into the
nisomycin treatment, the cultures were labeled for 2 hith L-[35S]methionine (20 µCi, .1000 Ci/mmol, Du-ont NEN, Boston, MA). Cultures were quick frozen onry ice, and solubilized in 10 mM Tris–HCl, pH 8.0,.5% sodium dodecyl sulfate. L-[35S]methionine incorpo-ation into protein was determined by precipitationith 25% trichloroacetic acid (21). Inhibition of protein
ynthesis was determined as the percentage differenceetween anisomycin-treated cultures and mock-treatedultures. To determine the reversibility of anisomycin,ultures were treated with anisomycin (10 µM) for 2.5. The drug was removed and the cultures labeled with-[35S]methionine for 30 min. Recovery of protein syn-hesis was determined as the percentage differencerom mock-treated cultures. Slice cultures were treatedith the irreversible transcription inhibitor actinomy-
in D (0.5 µg/ml). After 30 min, cultures were labeledor 2 h with 5-[3H]uridine (20 µCi, .20 Ci/mmol,upont NEN, Boston, MA). The cultures were quick
rozen on dry ice and RNA isolated using Microfastrack kit (Invitrogen, San Diego, CA) with the omissionf oligo-dT chromatography. RNA was precipitatedsing trichoroacetic acid. Inhibition of RNA synthesisas determined as the percent difference betweenctinomycin D-treated and mock-treated cultures.Transfer and expression of bcl-2 using vaccinia virus
ectors. A cDNA encoding human bcl-2 (provided by S.orsmeyer, Washington U., St. Louis, MO) was excisedith EcoRI. The EcoRI ends were converted to blunt
nds using the Klenow fragment of DNA polymerase Ind cloned into SmaI site of the inducible vacciniairus expression vector pPR35 (46). Bcl-2 was cloned inoth orientations with respect to the virus late pro-oter p4b. Recombinant vaccinia virus expressing bcl-2
n the sense (WR-bcl-2) or the antisense (WR-bcl-2anti)rientation were isolated and titered (33). To determinef WR-bcl-2 directs regulated expression of bcl-2 pro-ein, 1 3 105 HeLa cells were infected with 5 3 105
laque forming units of WR-bcl-2. Protein was ex-racted after 24 h and analyzed by immunoblot analysissing antisera directed against human bcl-2 proteinSanta Cruz Biotech, Santa Cruz, CA). To determine if
R-bcl-2 directs regulated expression of bcl-2 protein5
n slice cultures, 2 3 10 plaque forming units in a dolume of 2 µl was applied to the pyamidal and granuleell layers of the cultures using a microdrop technique.
pulled glass micropipette was positioned with aicromanipulator over the center of a neuronal layer
Narishige, Japan). A 200-nl drop was formed on thend of the micropipette and lowered on the neuronalayer. This procedure was continued 8–10 times untilll neuronal layers had been repeatedly transduced.ultures were maintained for 24 h in a 35°C incubator
n the presence or absence of isopropylthiogalactosideIPTG, 3 mM). The cultures were fixed with 4% parafor-aldehyde, 0.5% glutaraldehyde, and processed for
mmunohistochemistry (8) using antisera against hu-an bcl-2 (1:100 dilution). Immunocytochemistry was
one en bloc since cultures could not be resectioned. Asresult the immunocytochemistry in Fig. 5 is from a
ulture many cells thick. To determine if WR-bcl-2revented apoptosis, cultures were transduced withR-bcl-2 or control vectors and treated with IPTG.fter 3 h, cultures received a 15- or 30-min acidosis
reatment. Sixteen hours following acidosis neuronaloss was assayed by propidium iodide. To determine if
R-bcl-2 prevented necrosis following acidosis, vac-inia vectors were transduced and the cultures treatedith IPTG. Eleven hours following transduction, slice
ultures received a 30-min acidosis treatment. Eightours following the acidosis treatment, neuronal lossas determined using the propidium iodide assay. Thisnsured comparable times of bcl-2 accumulation hadccurred prior to assay of neuronal loss in the assay ofpoptosis or necrosis.
RESULTS
cidosis Induces a Dose-Dependent Loss of SliceCulture Neurons
An acidosis treatment of thirty minutes induced apecific loss of neurons that was seen 8 and 16 h later49). To analyze if acidosis-induced neuronal loss de-ended on the duration of acidosis, slice cultures re-eived acidosis treatment of 15, 30, 45, and 60 min andeuronal loss assayed 8 and 16 h later (Fig. 1). Neuro-al loss was assayed by counting propidium iodide-tained nuclei. Neuronal loss was observed at 16 h, butot 8 h following a 15-min acidosis treatment. Addi-ional loss was observed at both 8 and 16 h following a0-min acidosis treatment. Nuclei were uniformlytained at 8 h, but at 16 h staining was more punctate,uggesting the nuclear fragmentation that occurs dur-ng apoptosis. Further increasing the duration of thecidosis treatment resulted in greater neuronal loss ath and less at 16 h. This suggests that neuronal loss 8following acidosis is dose-dependent. Loss at 16 h isaximal following a 30-min acidosis treatment and
ecreases with longer acidosis treatments. Control
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r mock acidosis does not result in significant neuronaloss at 8 or 16 h.
cidosis Induces Apoptosis and Necrosis of SliceCulture Neurons
Different durations of acidosis induced maximal neu-onal loss at 8 or 16 h following acidosis. This suggestedhat neuronal loss at different times utilized differentechanisms. We tested if the hypothesis that the early
euronal loss was necrotic and the later loss was apoptotic.The TUNEL assay is one method to distinguish
poptosis from necrosis. TUNEL alone is not sufficientince the method labels both late stage necrotic cells asell as those undergoing apoptosis (1). There is exten-
ive fragmentation of nuclear DNA during apoptosishat are labeled in situ using TUNEL. Cultures re-eived a 30-min acidosis treatment, maintained for andditional 8 or 16 h and stained using TUNEL. At 8 h,UNEL staining was not observed despite propidium
odide stained nuclei in the pyramidal cell layer (Fig. 2).t 16 h, both TUNEL staining and propidium iodidetaining were observed (n 5 4). TUNEL-stained cellsere found predominantly in the primary neuronal
ayers, suggesting DNAfragmentation occurred primar-ly in neurons. Both TUNEL and propidium iodidetaining were observed sixteen hours following a 15-in acidosis treatment (n 5 3, not shown). Neither
ntracellular acidification nor propidium iodide-stainedells were observed in cultures treated in modifiedBSS, suggesting that intracellular acidification was
FIG. 1. Acidosis induces a dose-dependent neuronal loss in cultucidosis treatment or were mock treated and returned to the incubollowing acidosis. Neuronal loss was assayed by counting propidiumean of four to six determinations 6 SEM. The amount of neuronal
F 5 69.783, ANOVA; **P . 0.001, Bonferoni posttest). The amount ocidosis treatment (F 5 21.389, ANOVA; *P . 0.01, Bonferoni posttes
esponsible for DNA fragmentation (n 5 4, not shown). b
Necessary Role for New Gene Expression inAcidosis-Dependent Apoptosis
Protein synthesis inhibitors attenuate neuronal lossollowing ischemia (18, 35, 44). Protein or RNA synthe-is inhibitors also prevent apoptotic neuronal lossnduced by reactive oxygen species or removal of tro-hic factors (36, 45). Necrotic neuronal loss, in con-rast, has no requirement for production new protein orNA (35). Inhibitors of RNA and protein synthesis weresed to test for a necessary role of new gene expression
or neuronal loss following acidosis and to furtheristinguish necrosis from apoptosis. The protein synthe-is inhibitor anisomycin was used since it effectivelynd reversibly inhibited synthesis of protein in sliceultures. Anisomycin (10 µM) treated cultures had.2 6 1.2% (n 5 4) of [35S]methionine incorporationnto slice culture protein as compared to mock treatedultures. This inhibition was reversible, [35S]methio-ine incorporation was 118.8 6 3.9% (n 5 4) of control0 min after removal of the drug. Anisomycin waspplied 30 min prior to a 30-min acidosis treatment andashed out 2.5 h later (Fig. 3). Neuronal loss at 8 h wasnaffected, while neuronal loss at 16 h was signifi-antly attenuated. Anisomycin was also effective ineducing neuronal loss at 16 h following a 15-mincidosis treatment (Fig. 3). These data suggest thatrotein synthesis was required for neuronal loss at6 h, but not at 8 h following acidosis.The observations that a 3-h anisomycin treatment
hippocampal slices. Slice cultures received a 15-, 30-, 45-, or 60-minr. The cultures were stained with propidium iodide at 8 and 16 hide-stained nuclei counted in the pyramidal cell layer. Values are thes at 8 h significantly increased as the duration of acidosis increaseduronal loss at 16 h did not significantly change except after a 60-min
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is are reversible suggest that synthesis of new proteinor apoptosis occurs within 3 h following acidosis. Thisindow for new protein synthesis was further tested by
reating cultures with 15 min of acidosis. The culturesere returned to the incubator for 3 h and then treatedith anisomycin for 3 h. Neuronal loss at 16 h did not
ignificantly differ between the anisomycin-treatedroup (267 6 28, n 5 4) and the mock-treated group318 6 79, n 5 4; P . 0.05, Student’s t test). The abilityf anisomycin to block neuronal loss during the first 3 hollowing the acidosis treatment, but not the subse-uent 3 h suggests the need for new protein synthesisuring the initial 3 h following acidosis.Attenuation of apoptosis by anisomycin may not be
ue to inhibition of translation. Inhibition of transla-ion alters the redox state of the cell by accumulation ofethionine and stimulation of glutathione synthesis
45). Alteration of the redox state of the cell mayttenuate acidosis induced neuronal loss since neuro-al loss was blocked by removal of oxygen, suggesting aole for reactive oxygen species (49). To address theseossibilities, we used actinomycin D, an irreversiblenhibitor of RNA synthesis. By sparing protein synthe-is, actinomycin D inhibits new gene expression with-ut elevating amino acid levels. We first tested ifctinomycin D blocks RNA synthesis. 5-[3H]uridinencorporation was 10.7 6 3.9% (n 5 3) in actinomycin D0.5 µg/ml)-treated cultures as compared to controls,uggesting effective inhibition of transcription. Actino-ycin D treated cultures had 92.6 6 8.1% (n 5 3) of
35S]methionine incorporation as compared to mock-reated controls, suggesting that protein synthesis
FIG. 3. Apoptosis, but not necrosis, following acidosis requires nith anisomycin (15 µM) or actinomycin D (0.5 µg/ml). 30 min later th
odide-positive nuclei were counted in the pyramidal cell layer 8 or 16ignificant difference from the corresponding mock treatment (ANOV
as uneffected. Actinomycin D (0.5 µg/ml) was applied u
0 min prior to a 30-min acidosis treatment. At 8 h,euronal loss was observed, but at 16 h neuronal lossas attenuated (Fig. 2). Actinomycin D (0.5 µg/ml) waslso effective at reducing neuronal loss at 16 h following15-min acidosis treatment (Fig. 3). These data sug-
est that transcription and translation were needed fornduction of neuronal loss at 16 h following acidosis. Inontrast, neuronal loss at 8 h showed no requirementor either transcription or translation.
Experiments using anisomycin suggested a need forrotein synthesis during the three hours followingcidosis. Actinomycin D (0.5 µg/ml) was added 3 hollowing a 15-min acidosis treatment to test for aimilar need for RNA synthesis. No significant differ-nce in the number of propidium iodide-stained nucleias observed in the pyramidal cell layer in an actinomy-
in plus acidosis-treated group (327 6 122, n 5 4) asompared to an acidosis alone group (391 6 79; n 5 4;
. 0.5, Student’s t test). These data suggest a need forew transcription for apoptotic neuronal loss within 3 h
ollowing acidosis.
cl-2 Expression Blocks Apoptosis, But Not NecrosisFollowing Acidosis
Ectopic bcl-2 expression suppresses apoptosis in-uced by a wide variety of agents and provides andditional means to distinguish apoptosis from necro-is (25, 31, 40). A human bcl-2 cDNA was transferrednto slice culture using a recombinant vaccinia virusector WR-bcl-2. In WR-bcl-2, the bcl-2 cDNA was
gene expression. Slice cultures were either mock-treated or treatedltures received a 15- or 30-min (right) acidosis treatment. Propidiumllowing acidosis. Values are the mean 1 SEM. An asterisk indicates a
5 7.0504, P , 0.01; Boneferroni posttest).
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7ACIDOSIS-INDUCED NECROSIS AND APOPTOSIS
een modified by placing lac operator sites between theaccinia promoter and the bcl-2 cDNA. WR-bcl-2 alsoxpresses Escherichia coli lac repressor (lacI) to permitnducible expression of bcl-2. Inducible bcl-2 expressionas tested by transducing WR-bcl-2 to HeLa cells. Two
ontrol vectors were used to control for effects of theiral vector other than bcl-2 expression. WR-bcl-2antiontained bcl-2 in an antisense orientation and WR-lacirected the expression of E. Coli b-galactosidase. Werst examined if WR-bcl-2 directs inducible expressionf human bcl-2 in cultured slices. Vaccinia virus vectorsere applied to slice cultures using a microdrop method
hat resulted in infection of greater than 70% of sliceulture cells. Both neurons and glia were infected.ultures were maintained for 24 h, either in the pres-nce or absence of IPTG, and then immunostained withnti-bcl-2 sera (Fig. 4). A large increase in bcl-2 immu-oreactivity was observed in IPTG-stained cultures asompared to untreated cultures, suggesting that expres-ion of bcl-2 was induced by the IPTG treatmentn 5 8). The increase in bcl-2 immunoreactivity wasbserved in soma of cells in the granule and pyramidalell layers. These data suggest that WR-bcl-2 directednducible expression of bcl-2 in cultured slice neuronsnd nonneurons. To further verify that WR-bcl-2xpressed bcl-2 protein, HeLa cells were transducedith WR-bcl-2, WR-bcl-2anti, or WR-lac, and humancl-2 expression was assayed by immunoblot assaysing antisera specific for human bcl-2. A Mr 25,000rotein immunoreactive protein was observed in WR-cl-2-infected cultures treated with the lack inducerPTG that was absent when IPTG was omitted (bcl-2ef ) (Fig. 4). The Mr 25,000 immunoreactive proteinas not observed in cultures transduced with WR-lacr WR-bcl-2anti and treated with IPTG. These datauggest that WR-bcl-2 directs inducible expression ofuman bcl-2 in HeLa cells. To demonstrate a protectiveffect of bcl-2 expression against acidosis induced neu-onal loss, slices were transduced with WR-bcl-2, WR-cl-2anti, or WR-lac in the presence or absence of IPTG.hree hours following transduction, cultures received a0-min acidosis treatment and neuronal loss was as-ayed 16 h later (Fig. 4). The number of propidiumodide stained cells in the pyramidal cell layer wasignificantly reduced in the WR-bcl-2 plus IPTG-reated cultures than WR-bcl-2-transduced cultures inhe absence of IPTG. This suggests that expression ofcl-2 was responsible for the attenuation of neuronaloss 16 h following acidosis. A similar IPTG-dependentrotection was observed in WR-bcl-2-transduced cul-ures following a 15-min acidosis treatment. No signifi-ant protection was observed in cultures transducedith WR-lac or WR-bcl-2anti, further suggesting thatrotection against acidosis toxicity resulted from in-reased bcl-2 expression. These data provide additional
vidence that neuronal loss 16 h following acidosis is pue to apoptosis. The protection of bcl-2 against apopto-is is likely due to neuronal expression of bcl-2. Sinceoth neurons and glia were infected, the possibilityxists that glial expression of bcl-2 also participated inrotection against neuronal loss.While bcl-2 is thought to protect against apoptosis, it
s believed to be ineffective against necrosis (25, 31, 40).e hypothesized that neuronal loss 8 h following
cidosis would be unaffected by increased bcl-2 expres-ion. A protocol was designed to ensure that humancl-2 protein levels at 8 h following acidosis would beimilar to levels that provided protection at 16 h. Sliceultures were transduced with WR-bcl-2 in the pres-nce or absence of IPTG and incubated for 11 h.ultures received a 30-min acidosis treatment andeuronal loss assayed 8 h later. Similar neuronal lossas observed in the absence or presence of IPTG,
uggesting that bcl-2 did not protect against neuronaloss at 8 h (Fig. 4). The absence of protection againsteuronal loss at 8 h provides additional evidence thatcidosis kills neurons by two mechanisms—an earlyecrosis and a late apoptosis.
Necessary Role for Caspases inAcidosis-Dependent Apoptosis
Caspase action likely mediates all forms of apoptosis1). A need for caspase activity for neuronal loss follow-ng acidosis was tested using zVAD-fmk (N-benzyloxy-arbonyl-Val-Ala-Asp-fluoromethylketone), a cell perme-ble, irreversible caspase inhibitor. zVAD-fmk inhibitedaspases three to four times more effectively than thenrelated cysteine proteases, calpain I and cathepsin, suggesting that zVAD-fmk was a specific caspase
nhibitor (4, 39). zVAD-fmk was applied at differentoncentrations to slice cultures 30 min before and 3, 6,nd 9 h in series following a 30-min acidosis treatment.elayed neuronal loss was assayed using propidium
odide 8 and 16 h later (Fig. 5). While zVAD-fmk at 10nd 5 µM significantly blocked acidosis-induced neuro-al loss at 16 h, 1 µM did not protect. These datauggest that a need for protease activation in acidosis-ependent neuronal loss at sixteen hours. zVAD-fmk (5M) also protected against neuronal loss 16 h following15-min acidosis treatment. In contrast, zVAD-fmk
5 µM) had no effect on neuronal loss 8 h following a0-min acidosis treatment. These data suggest theeed for protease activation for neuronal loss observedt 16 h, but not at 8 h. A selective need for caspase activityrovides additional evidence that neuronal loss at eightours is necrosis and neuronal loss at 16 h is apoptosis.
ypothermia Blocks Both Necrosis and Apoptosis
Hypothermia strongly protects against ischemic brainamage (5, 16). It has not yet known if hypothermia
rotects against necrosis or apoptosis following isch-
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9ACIDOSIS-INDUCED NECROSIS AND APOPTOSIS
mia, although there have been suggestions of a protec-ive effect against apoptosis (13, 32). We thereforeested if hypothermia protected against necrosis orpoptosis following acidosis (Fig. 6). Cultured sliceseceived a 15-min acidosis treatment at 37, 35, or 32°C.mmediately after the acidosis treatment, slices wereeturned to 35°C, and neuronal loss was assayed 16 hater. Neuronal loss at 16 h was attenuated at 35°C asompared to 37°C and completely suppressed at 32°C. Aimilar suppression of neuronal loss was observed at 16
FIG. 5. The caspase inhibitor zVAD inhibits apoptosis, but not0-min acidosis treatment. The cultures were maintained at 35°C fyramidal cell layer. Values are the mean plus SEM. A single asterisk15-min acidosis treatment (Student’s t test, P , 0.005). A double a
eceiving a 15-min acidosis treatment (F 5 12.579, ANOVA; P , 0.05
FIG. 6. Both apoptosis and necrosis are temperature dependent.2°C. The cultures were returned to the incubator and maintained atyramidal cell layer. Values are the mean plus SEM. A significant diffe7°C (F 5 12.579, ANOVA; *P , 0.05; **P , 0.01; Bonferroni). A sign
reatment at 37°C (F 5 17.76, ANOVA; ***P , 0.01; Bonferroni post testfollowing a 30-min acidosis treatment. These datauggest that hypothermia protected against apoptosis.o test if hypothermia protected against necrosis, cul-ured slices received a 30-min acidosis treatment at 37r 32°C and propidium iodide staining was assayed 8 hater. Neuronal loss was also strongly suppressed at2°C, suggesting that hypothermia attenuated necrosiss well. Hypothermia has been reported to attenuateschemic damage by preventing acidosis (32). We there-ore examined pHi after a 15-min acidosis treatment at
rosis induced by transient acidosis. Slice cultures received a 15- oror 16 h and propidium iodide positive nuclei were counted in the
icates a significant difference in neuronal loss from cultures receivingrisk indicates a significant difference in neuronal loss from culturesnferroni post test).
ce cultures received a 15- or 30-min acidosis treatment at 37, 35, orC for 8 or 16 h. Propidium iodide-positive nuclei were counted in thece was observed in cultures receiving a 15-min acidosis treatment atnt difference was observed from cultures receiving a 30-min acidosis
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7 or 32°C. pHi acidified to a similar extent at 32°C6.49 6 0.03, n 5 6) and 37°C (6.49 6 0.06, n 5 6). Theresence of acidosis during hypothermia suggests thathe protective effect of hypothermia is subsequent tohe induction of acidosis.
DISCUSSION
cidosis Induces Both Necrosis and Apoptosis inCultured Hippocampal Cultures
The central finding of this study is that brief andransient acidification of pHi to 6.6 induced neuronaloss by two mechanisms—necrosis and apoptosis. In allxperiments, propidium iodide staining was centeredn the pyramidal cell layer that is highly enriched ineurons and absent in other regions of the culture thatere predominantly glial. Since propidium iodide stains
he nuclei of any dying cell, the possibility exists thatcidosis is toxic to astrocytes, oligodendrocytes, oricroglia as well. Even though prolonged acidosis
illed over 1400 cells, a individual slice culture containoughly 105 cells in the slice culture remained viable.he number of neurons lost even in the most extremecidic conditions in this study is likely less than 20%.The preferred way to delineate apoptosis from necro-
is is to use multiple criteria to distinguish betweenhese two death mechanisms (1). Our hypothesis washat neuronal loss at eight hours is necrotic and atixteen hours it is apoptotic. Apoptotic neuronal lossas induced by treatments as brief as 15 min. Nuclei ofpoptotic neurons stained strongly with TUNEL (Fig. 2).demonstration of nucleosome-sized DNA fragments
y gel electrophoresis was not feasible due to insuffi-ient numbers of apoptotic cells, even when slice cul-ure DNA was hybridized to 32P-labeled probes (P.B.nd H. J. Federoff, unpublished results). Apoptoticeuronal loss was also reduced by inhibition of tran-cription, translation or caspases (Figs. 3 and 5), asell as ectopic bcl-2 expression (Fig. 4). Apoptoticeuronal loss was maximally induced by a thirtyinute acidosis treatment. These multiple lines of
vidence strongly suggest that the neuronal loss in-uced at 16 h was apoptotic.In contrast to apoptotic loss, neuronal loss at eight
ours was not induced with a 15-min acidosis treat-ent (Fig. 1) and was not stained by TUNEL (Fig. 2).ight-hour neuronal loss was not blocked by inhibitionf transcription, translation, or caspases (Figs. 3 and), nor ectopic bcl-2 expression (Fig. 4). Necrosis has aery different dose–response to acidosis than apoptosis:s the duration of acidosis increased, necrotic neuronaloss at 8 h increased, while apoptotic death at 16 hecreased. These data strongly suggest that the acido-is could induce either necrosis or apoptosis, dependingpon the duration of the acidosis treatment.
A 15-min acidosis treatment induced predominantly tpoptosis, suggesting that apoptosis is more readilynduced than necrosis in cultured slices. Necrotic celloss increased as the duration of acidosis was in-reased. Like acidosis, exposure to NMDA or genera-ors of reactive oxygen species (ROS) induced necrosist higher doses than apoptosis (3, 9). Apoptosis alsoccurred more slowly. These data suggest that the rapidnduction of necrosis overcomes the cell before apopto-is is completed.Apoptosis following acidosis needed new gene expres-
ion in the first 3 h (Fig. 3). Apoptosis following cerebralschemia shows a similar need for gene expressionhile necrosis does not (35). It is not yet known whenene expression must be altered following ischemia forpoptosis to occur. Experiments using inhibitors ofrotein and RNA synthesis suggest that alterations inene expression for apoptosis following acidosis muste very rapid—within 3 h following acidosis (Fig. 3).It is not yet known how mild and transient acidosis
esults in both necrosis and apoptosis. Neurons canndergo apoptosis or necrosis following exposure toOS, glutamate or glutamate agonists (3, 9). In con-
rast, other apoptotic stimuli such as loss of trophicupport results only in apoptosis. Acidosis readilynduces reversible synaptic depression in hippocampallice cultures and no long-term change in slice culturexcitability. These results suggest that excitotoxic con-entrations of glutamate are not released (Bergold andiang, manuscript in preparation). We believe thatecrosis and apoptosis following acidosis depends uponeneration of ROS (20, 35). Both necrosis and apoptosisollowing acidosis are blocked by hypoxia (49) as well as
variety of antioxidants (Bergold, unpublished data),uggesting a necessary role for ROS in acidosis-inducedeuronal loss.
oes Acidosis Contribute to Neurodegenerative Injury?
Acidosis is a ubiquitous feature of cerebral ischemia,rauma and hemorrhage. The role of acidosis in brainnjury, however, has been controversial (10, 47, 48).cidosis has been proposed to have either a toxic orrotective role (47, 48, 55). During focal and globalschemia, pH of the brain rapidly acidifies to pH 6.2–6.86, 19, 23, 27, 38, 43). The degree of acidification iseterogenous; areas of greatest acidosis occur in therain regions that are most susceptible to ischemicnjury (56). Trauma also acidifies the brain to pH 6.548). Subarachnoid haemorrhage induces prolongedcidosis of 6.5 that lasts up to 1 h (10). Acidosis ofimilar duration and extent in slice cultures inducesoth apoptosis and necrosis to slice culture neurons.hese data suggest a neurotoxic role for acidosis.lthough this study emphasizes a toxic role for acido-is, substantial evidence exists to suggest a neuroprotec-
ive role as well (55). Acidosis inactivates NMDA
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11ACIDOSIS-INDUCED NECROSIS AND APOPTOSIS
eceptors and reduces calcium influx (30). Both ofhese effects are potentially neuroprotective.
The studies of acidosis in slice cultures may providen explanation to the apparent paradox of acidosiseing protective or harmful. Acidosis toxicity requiresxygen and prolonged exposure to low pHi (Fig. 1) (49).epending upon the ischemia model utilized and isch-mia duration, pH either returns to normal (23) oremains acidic (6, 38, 56). During trauma and subarach-oid hemorrhage, there is prolonged acidosis in theresence of oxygen (10, 48). Short exposure to low pHin the absence of oxygen may be neuroprotective.uring brain injury, the toxic or protective conse-uences of acidosis will depend upon amount of oxygen,he extent of acidosis and the amount of time requiredo return to physiological pH.
ACKNOWLEDGMENTS
We thank Drs. Howard Federoff, Ira Kass, and Todd Sacktor forritically reading this manuscript, Dr. Ilham Muslimov for assistanceith the photography, and Mr. Taosheng Liu for help with experi-ents at the early stage of this study. This work is supported byD31300 (to P.J.B.).
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