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Experimental Neurology 161, 115–126 (2000)doi:10.1006/exnr.1999.7250, available online at http://www.idealibrary.com on

Neuronal Subclass-Selective Loss of Pyruvate DehydrogenaseImmunoreactivity Following Canine Cardiac Arrest and Resuscitation

Yolanda E. Bogaert,*,1 Kwan-Fu Rex Sheu,†,2 Patrick R. Hof,‡ Abraham M. Brown,† John P. Blass,†Robert E. Rosenthal,* and Gary Fiskum*,3

*Departments of Biochemistry and Molecular Biology and Emergency Medicine, George Washington University School of Medicine,Washington, DC 20031; †Burke Medical Research Institute, Cornell University Medical College, White Plains, New York 10605; and ‡KastorNeurobiology of Aging Laboratories and Fishberg Research Center for Neurobiology, and Departments of Geriatrics and Adult Development,

and Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029

Received December 28, 1998; accepted September 16, 1999

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Chronic impairment of aerobic energy metabolismccompanies global cerebral ischemia and reperfusionnd likely contributes to delayed neuronal cell death.eperfusion-dependent inhibition of pyruvate dehy-rogenase complex (PDHC) enzyme activity has beenescribed and proposed to be at least partially respon-ible for this metabolic abnormality. This study testedhe hypothesis that global cerebral ischemia and reper-usion results in the loss of pyruvate dehydrogenasemmunoreactivity and that such loss is associated withelective neuronal vulnerability to transient ischemia.ollowing 10 min canine cardiac arrest, resuscitation,nd 2 or 24 h of restoration of spontaneous circulation,rains were either perfusion fixed for immunohisto-hemical analyses or biopsy samples were removed forestern immunoblot analyses of PDHC immunoreactiv-

ty. A significant decrease in immunoreactivity wasbserved in frontal cortex homogenates from both 2nd 24 h reperfused animals compared to samples fromonischemic control animals. These results were sup-orted by confocal microscopic immunohistochemicaleterminations of pyruvate dehydrogenase immunore-ctivity in the neuronal cell bodies located withinifferent layers of the frontal cortex. Loss of immunore-ctivity was greatest for pyramidal neurons located inayer V compared to neurons in layers IIIc/IV, whichorrelates with a greater vulnerability of layer V neu-ons to delayed death caused by transient global cere-ral ischemia. r 2000 Academic Press

Key Words: cardiac arrest; cerebral ischemia; lasercanning microscopy; mitochondria; neocortex; reper-usion.

1 Present address: University of Colorado Health Science Center,enver, CO 80262.2 Deceased.3 Present address: Department of Anesthesiology, University of

aaryland, Baltimore School of Medicine, Baltimore, MD 21201.

115

INTRODUCTION

Delayed neuronal death following transient globalerebral ischemia and reperfusion occurs by both necro-is and apoptosis. Mitochondrial dysfunction has beentrongly implicated in mediating both forms of neuro-al death (1, 2, 25). Alterations in mitochondrial oxida-ive phosphorylation, Ca21 transport, free radical gen-ration, release of apoptotic factors, and metabolicnzymes contribute to oxidative stress, cellular Ca21

verload, activation of cell death proteases, and meta-olic failure (8).Using a clinically relevant canine model of cardiac

rrest and resuscitation, we have demonstrated ahronic exacerbation of anaerobic cerebral energy me-abolism that is related to neurological outcome (33).lthough studies on ischemic alterations of mitochon-rial oxidative energy metabolism have focused onlterations in electron transport chain activities (1, 9,1), observations indicating a postischemic hyperoxida-ion of pyridine nucleotides and electron transporthain components have suggested that a deficiency inhe generation of reducing power to be used by thelectron transport chain may be just as important27, 30). Consistent with these observations are thendings by several laboratories that reperfusion causessignificant decrease in the maximal activity of brain

yruvate dehydrogenase complex (PDHC), which cata-yzes the critical oxidation/reduction reaction that linkslycolysis to the tricarboxylic acid cycle (5, 11, 18,6–48).Abnormally low levels of PDHC have also been

ssociated with chronic neurodegenerative disordersncluding Alzheimer’s disease (35), Huntington’s dis-ase (43), Wernicke–Korsakoff syndrome (20), and aariety of hereditary ataxias (37). Although a sufficienteduction in PDHC activity could lead to metabolicailure, oxidative stress, and cell death, no direct cause

nd effect relationships have been established. One

0014-4886/00 $35.00Copyright r 2000 by Academic Press

All rights of reproduction in any form reserved.

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116 BOGAERT ET AL.

pproach that would aid in establishing this relation-hip would be to compare the pattern of inhibition ofDHC among neuronal subclasses with the selectiveulnerability of neuronal subtypes to acute or delayedeath.The mechanism responsible for reduced PDHC activ-

ty in animal models and many human disorders is, athis juncture, unknown. However, in vitro studies haveuggested that inhibition could be due to either oxida-ion of critical sulfhydryl groups (45) or metal-catalyzedite-specific oxidation of any one of a number of suscep-ible amino acids (5). Site-specific protein oxidation haseen observed in some but not all models of cerebralschemia and reperfusion (10, 19, 21, 26), in Alzhei-

er’s disease (42), during normal aging, and in variousodels of neurotoxicity, such as paraquat toxicity,agnesium deficiency, and chronic exposure to alcohol

4). Oxidative damage to proteins is also promoted byonditions known to exist following cerebral ischemia,.g., low pH (12), as well as high Ca21 in the case ofDHC (5). Evidence suggests that this mechanism of

nhibition is responsible for the reduction in brainlutamine synthetase activity following global isch-mia (26). Studies also indicate that site-specific oxida-ion of this and many other proteins results in anncreased rate of proteolytic degradation, as detectedy a decrease in immunoreactivity (44). The presenttudy was conducted to determine if the immunoreactiv-ty of PDHC is reduced in the cerebral cortex followingardiac arrest and resuscitation and to determine if theattern of reduction matches the relative vulnerabilityf cortical neuronal subclasses to delayed death.

MATERIALS AND METHODS

lobal Cerebral Ischemia and Reperfusion

All experiments were performed in accordance withhe guidelines of the Institutional Animal Use and Careommittee of the George Washington University. Aanine model of cardiac arrest and resuscitation as alinically relevant model for global cerebral ischemiand reperfusion has been described (32, 33). Adultemale beagles weighing 10–15 kg were initially anes-hetized with thiamylal sodium and prolonged anesthe-ia was induced by infusion of a-chloralose (75 mg/kg).nimals were endotracheally intubated and ventilatedith room air prior to induction of cardiac arrest.uscle paralysis was maintained with i.v. pancuro-

ium bromide and antibiotic prophylaxis was adminis-ered with ceftriaxone. Resuscitative drugs were admin-stered via a venous catheter advanced to the level ofhe right atrium. Arterial pressure was continuouslyonitored through a femoral arterial catheter. Pulse,CG, and rectal temperature were also continuouslyonitored and the temperature maintained at between

7 and 39°C by lights and heating blankets. 0

A thoracotomy was performed on all animals, includ-ng nonarrested controls. Ventricular fibrillation car-iac arrest was induced with a train of electricalurrent applied directly to the epicardium of the rightentricle following incision and reflection of the pericar-ium. Artificial respiration was discontinued at thenset of fibrillation. Following 10 min of cardiac arrest,nimals were either sacrificed or cardiopulmonary re-uscitation (CPR) was initiated to allow for reperfusioneriods of either 2 or 24 h. Resuscitation was initiatedy open chest cardiac massage, administration of epi-ephrine and sodium bicarbonate, and ventilation with00% O2. Open chest CPR was continued for 3 min at aate of 50/min followed by internal defibrillation. Arte-ial blood gas samples were measured prior to arrest, 2in following defibrillation, and frequently thereafter.he ventilator was adjusted following the first bloodas determination and thereafter to maintain arterialO2 between 80 and 100 mm Hg and pCO2 between 25nd 35 mm Hg. Artificial ventilation was maintainedor 22 h, at which time dogs were weaned from con-rolled ventilation. Animals were maintained underonstant intensive care and were presumed to beubject to postoperative pain, which was controlledsing a constant infusion of morphine sulfate at 1 mg/hhroughout the experiments.

issue Acquisition

Biopsy samples for immunoblot analyses were ob-ained from the frontal cortex of chloralose-anesthe-ized animals following a craniotomy and immediatelyrior to euthanasia with 0.25 ml/kg Euthanasia-6Veterinary Labs, Lenexa, KS). These samples weremmediately immersed in liquid nitrogen and stored at75°C. For immunohistochemistry, dogs were perfused

ranscardially with ice-cold 1% paraformaldehyde inhosphate-buffered saline (PBS, pH 7.4) for 1 minollowed by cold 4% paraformaldehyde in PBS for 10–15in at a flow rate of 300 ml/min as previously described

15–17). After perfusion, the brains were immediatelyemoved from the skull.

mmunoblot Measurements

Polyclonal antibodies against bovine PDHC wereaised as described previously (34). Antibodies specificor PDHC components were raised against the respec-ive bovine proteins that were excised from SDS–olyacrylamide gels. Samples of frozen frontal cortexapproximately 0.2 g wet wt) that had been stored at75°C were weighed and homogenized with a Brink-an Polytron homogenizer in a medium previously

escribed for use in enzyme activity measurements (5).he ice-cold medium (9 ml/g sample) contained 50 mMotassium phosphate buffer (pH 7.8), 1 mM EDTA, and

.1% (wt/vol) Triton X-100. The protein concentration of

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117PYRUVATE DEHYDROGENASE IMMUNOREACTIVITY AFTER ISCHEMIA

he homogenate was determined by a modified Biuretrocedure (41). Brain proteins were resolved on 10%DS–polyacrylamide gels (1 mm thick) by electrophore-is and then electrotransferred to nitrocellulose mem-ranes for 3–4 h at 5 V/cm in a Bio-Rad Transblot Cellith plate electrodes (Bio-Rad Laboratories, Hercules,A). These conditions were optimized for detection of

he aE1p and E2p components of the PDHC. Thelectrotransfer of aE1p was variable, and under theseonditions, bE1p passed through the membrane andas detected poorly. For Western blotting analysis, theembranes were preincubated with a blocking buffer

ontaining 150 mM NaCl, 50 mM Tris–HCl (pH 7.6)nd 0.05% Tween 20 (TBST), and 2% bovine serumlbumin (Sigma, St. Louis, MO) and then incubated forh with the anti-PDHC antibody diluted 1:1200 in thelocking buffer. After three rinses with TBST, theembranes were probed for 2 h with the 125I-labeled

oat anti-rabbit IgG secondary antibodies (Amershamife Science, Arlington Heights, IL) diluted in TBST atconcentration of 3 µCi/100 cm2 of membrane. The

lots were rinsed six times with TBST and the boundadioactive antibody was detected with a Phosphorim-ger (Molecular Dynamics, Sunnyvale, CA). The abun-ance of aE1p and E2p for each sample was quantifiedy integration of the total phosphoimager signal forhat band. A standard box was used for all bands, whichere completely enclosed by this box. Backgroundalues were measured in an adjacent region of the sameane using the standard box and were subtracted fromhe integrated band intensity. Variations in labelingnd transfer efficiency between blots were corrected byormalization of sample signals to a standard sample

ncluded on each blot.

mmunohistochemistry

Perfusion fixed brains were blocked into coronalections that included the frontal cortex area 8a in theyrus proreus and postfixed in 4% paraformaldehydeor an additional 6 h (15–17). Following postfixation,oronal blocks to be used in immunofluorescence stud-es were transferred to a PBS buffer and sectioned on aibratome at 20 µm within 2 days of fixation. All otherlocks were frozen on dry ice after cryoprotection in araded series of PBS-buffered sucrose solutions andectioned on a cryostat at 40 µm. All sections from eachlock were kept in anatomical series and every 10thection was processed for immunohistochemistry. Theemaining sections were stored in serial order at 220°Cn a cryoprotection solution consisting of glycerol, ethyl-ne glycol, distilled H2O, and PBS (3:3:3:1 volumeatio). The sections were incubated for 48 h at 4°C withhe primary antibodies in PBS containing 0.3% Triton-100 and 0.5 mg/ml bovine serum albumin. Following

ncubation, the sections were processed by the avidin–

iotin method using a Vectastain ABC kit (Vector w

aboratories, Burlingame, CA) and 3,38-diaminobenzi-ine as a chromogen and intensified in osmium tetrox-de. For double labeling fluorescence studies, sectionsere incubated simultaneously for 2 h with relevant

econdary antibodies conjugated to Texas-Red (TR) oro fluorescein isothiocyanate (FITC). A fully character-zed, specific rabbit polyclonal antibody to PDHC wassed at a working dilution of 1:1000 (24, 35, 36). Aouse monoclonal antibody to the microtubule-associ-

ted protein (MAP2) was used to visualize the neuronalnd dendritic organization of the dog neocortex (Sigma,t. Louis, MO; working dilution: 1:3000). A paralleleries of sections was stained with cresyl violet tolarify cytoarchitecture.Sections double labeled for PDHC and MAP2 were

nalyzed using a Zeiss 410 laser scanning confocalicroscope (LSM, Zeiss, Oberkochen, Germany). This

ystem includes a Zeiss Axiovert inverted microscope,n Ar/Kr laser, a PC computer and optical disk storageevice. Optical densities of labeling were assesseduantitatively according to the paradigm developed byazzaley et al. (13). Sections were initially screenednder epifluorescence using a 103 Zeiss Plan-Neofluarbjective and filters selective for visualizing FITC or TRo select areas of interest. The area selected was theniewed through a Zeiss 633/1.25 NA Plan-Neofluarbjective for analysis. Images were digitized on theomputer display as sections were scanned with anr/Kr laser, whose wavelength was specific to thexcitation of the fluorescent marker (i.e., 488 nm forITC and 568 nm for TR), reflected to the specimen byn FT488/568 dichroic mirror and a 90% neutral den-ity filter attenuation. A contrast/brightness settingas determined for each marker, yielding high-resolu-

ion images for both bright and dim zones of theections without regions where pixel intensity reachedaturation. Images were subsequently enlarged, yield-ng a final magnification of 1893, to clearly delineateell boundaries. Several serial optical planes of sectionithin a neuron were analyzed for level of immunolabel-

ng to determine an average intensity/area for a giveneuron. This is especially important in the case of largeyramidal neurons where some of the fluorescence mayie out of focus on a single optical plane of section. Theoma of the neuron in layers III/IVc and V were thenraced and their ratio of fluorescence intensity/areaas recorded. Since PDHC is exclusively present initochondria, the unstained nuclei were chosen to

etermine the amount of background staining whichas subtracted from the intensity of the staining in theerikaryon. The intensity/area of the nucleus wasetermined in order to ensure that the level of back-round staining was comparable among sections. Sub-raction of background fluorescence was used to estab-ish a pixel intensity threshold below which a pixel

ould not contribute to the average pixel intensity (or

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rea) of the field, thus delineating precisely the specificeuronal area to be quantified (13). Twenty to 30eurons per layer and per section were analyzed usingt least five sections per animal. Comparative datanalysis used one-wayANOVAand the Duncan’s methodor multiple pairwise comparisons.

RESULTS

anine Cerebral Cortex Pyruvate DehydrogenaseImmunoreactivity

Previous experiments suggested that the reperfusion-ependent reduction in canine cerebral cortex PDHCctivity could be due to site-specific protein oxidation5). As this form of oxidative injury has been shown toark proteins for proteolytic degradation (44), we

ested the hypothesis that PDHC immunoreactivity iseduced following cerebral ischemia and reperfusionue to cardiac arrest and resuscitation. Figure 1Aescribes a representative PDHC immunoblot, usingifferent amounts of frontal cortex homogenate proteino establish the relationship between levels of proteinnd immunoreactivities as quantified with phosphorim-ger signals obtained from the blots. The antibodiesaised against bovine PDHC recognized canine brain

FIG. 1. Western blot analysis of PDHC proteins in canine frontalntibodies and quantified with a phosphorimager. (A) Immunoblot sanging from 2 to 15 µg. (B) The relationship of phosphoimager sigomogenate protein analyzed in blots such as that shown in A. Line

espectively.

DHC proteins and the electrophoretic mobilities of theanine proteins were similar to those observed previ-usly for bovine proteins (34). The identities of the E2p72 kDa), aE1p (41 kDa), and bE1p (34 kDa) proteinsere also verified with antibodies specific for each of

he individual proteins (data not shown). The phospho-imager signals for both E2P and aE1p were linearlyelated to the quantity of homogenate protein within ateast the range of 2–11 µg protein (Fig. 1B). The signalor bE1p was relatively weak and therefore no attemptsere made to quantify this signal.Comparisons of PDHC immunoblots for frontal cor-

ex homogenates obtained from nonischemic controlnimals (C), animals following 10 min cardiac arrest-nduced ischemia, and animals that were reperfusedor 2 or 24 h following 10 min ischemia are described inig. 2. Figure 2B shows a representative immunoblotnd Fig. 2A presents the average levels of aE1p and2p immunoreactivity (6SEM, n 5 4) for the experi-ental groups normalized to levels found in the nonisch-

mic control tissue. Although there appears to be alight reduction in both aE1p and E2P immunoreactiv-ty following ischemia alone, this was not statisticallyignificant. There was, however, a significant reductionf approximately 45% in aE1p reactivity and a 30–35%

tex. Immunoreactivities were developed with 125I-labeled secondarying aE1p and E2p at loaded levels of cortical homogenate protein

s for aE1p and E2p (in arbitrary units) compared to the amount ofregression analysis shows r2 5 0.974 and 0.998 for aE1p and E2p,

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119PYRUVATE DEHYDROGENASE IMMUNOREACTIVITY AFTER ISCHEMIA

eduction in E2p reactivity following ischemia plusither 2 or 24 h reperfusion (P , 0.05).

ytochemical Localization of PDHC Immunoreactivitywithin the Frontal Cortex of Control, Ischemic, andReperfused Brains

Given the finding that immunoreactivity of PDHCroteins aE1p and E2p is reduced in homogenates ofanine frontal cortex following 10 min ischemia plus asittle as 2 h reperfusion, cell-selective distribution ofDHC was measured throughout layers of the frontalortex that are known to exhibit variable sensitivity toostischemic neuronal death. The dorsal anterior pre-rontal cortex, corresponding to area 8a on the gyrusroreus, was characterized by a dense Nissl-stainingayer II that contained high numbers of small pyrami-al neurons and a layer III with relatively larger andparser pyramidal neurons (Fig. 3A). The distinctionetween the deep portion of layer III (IIIc) and layer IVas difficult to establish on Nissl-stained materials due

o the rather poor definition of layer IV. Layer IVppeared as a thin zone predominantly composed ofelatively small granular neurons intermingled with

FIG. 2. Western blot determination of PDHC aE1p and E2p immschemic (ISC), and 2 or 24 h reperfused animals (2 h, 24 h). (A) Mnimals per experimental group. *, Statistically significant differencontrol sample as a standard of reference.

ayer IIIC that was characterized by the occurrence of i

yramidal neurons. Layer V was easily distinguishednd had much larger pyramidal neurons than layerIIc. Layer VI was much more heterogeneous andontained small pyramidal and fusiform neurons (Fig.A; 15).In control animals, strong PDHC immunoreactivityas observed in the neurons of layer III and predomi-antly in large pyramidal neurons of layer V, whereasmaller neurons tended to be less intensely labeledFigs. 3B and 4A). MAP2 immunoreactivity revealedhe neuronal characteristics of the different corticalayers with a prominent staining of large pyramidaleurons in layers IIIc and V. The small pyramidaleurons in layer II appeared as a dense band of MAP2

mmunoreactivity, and the layer VI polymorphic neu-ons were intensely stained. Also, the dendritic arbor ofll neuron types was remarkably labeled by the anti-AP2 antibody and the presence of dendritic bundlesas observed throughout the cortical thickness (Fig.C).Pyruvate dehydrogenase is exclusively localized toitochondria, which are present to varying extents in

ll brain cells. Mitochondria are predominantly located

reactivities in canine frontal cortex samples from control (C), 10 minimmunoreactivity normalized to control samples (6SEM) for four

rom Control (P , 0.05). (B) Representative immunoblot including a

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120 BOGAERT ET AL.

odies but can also be found to a lesser extent in axons.rightfield microscopy examination of DAB-treatedaterials revealed a global decrease in staining inten-

ity at 2 and 24 h reperfusion compared to controlnimals and dogs with 10 min ischemia (Fig. 4). It isorth noting that after 10 min ischemia, no major

hanges were observed in the staining intensity com-ared to that seen under the control condition and that,n particular, the large layer IIIc and layer V pyramidaleurons exhibited a morphology comparable to that inontrol animals (Figs. 4A and 4B). A gradual decreasen intensity of PDHC immunolabeling was observed atand 24 h reperfusion, although discerning differences

n staining intensity among neuronal subtypes is diffi-ult at low magnification. At higher magnification (Fig.), it is clear that 2 h reperfusion resulted in a relativelyelective loss of immunostaining in large pyramidaleurons within layer V vs layer IIIc/IV and that themall neurons appeared to be less affected.Laser scanning confocal microscopy analysis at even

igher magnification showed that immunofluorescenttaining of PDHC in layer V of the frontal cortexisplays a punctate or thread-like pattern within bothhe cell soma and neuropil that is consistent with thenown distribution of mitochondria (Fig. 6A). Althoughittle, if any, change in layer V PDHC immunostaining

FIG. 3. Cytoarchitecture of area 8a in the frontal cortex of a nonyramidal cells in layers III and V. Layer IV is poorly differentiated anncubated with a polyclonal antibody to PDHC, staining is observedeurons are less intensely stained. (C) MAP2 immunoreactivity is obyramidal cells. Cortical layers are indicated by Roman numerals. Sc

as apparent following 10 min ischemia alone (Fig. r

B), there was a striking reduction in the immunostain-ng of both the pyramidal cell soma and surroundingeuropil in layer V following either 2 or 24 h reperfu-ion (Figs. 6C and 6D). The reduction in immunostain-ng following 2 h reperfusion cannot be explained byidespread necrosis. Although approximately 30% of

ayer V pyramidal neurons appear necrotic at 24 heperfusion (6), essentially no evidence of cell death isresent within any cortical layers at 2 h reperfusion inhis model or other models of transient global cerebralschemia. Moreover, robust MAP2 immunostaining ofell bodies and dendritic processes was present through-ut the cortical layers at 2 h reperfusion (Fig. 6E). Inontrast to the reduction in PDHC immunostainingbserved in the large pyramidal cells of layer V follow-ng 2 to 24 h reperfusion, immunostaining of theelatively small neurons present within layer IIIc andn the thin layer IV was preserved following 2 heperfusion (see Fig. 6F, which shows layer IIIc/IV in aouble-labeled section using TR to stain PDHC andITC to stain MAP2; primary colocalization was in theeuron soma, verifying the neuronal enrichment ofDHC).A quantitative assessment of PDHC immunofluo-

esce was conducted using laser scanning confocalicroscopy in order to provide further evidence for a

emic dog. (A) Nissl stain shows a dense layer II and the presence ofayer VI contains a polymorphic population of neurons. (B) In sections

arily in medium to large neurons in layers III and V, while smallerved in most neuronal types and reveals the dendritic arborization ofbar (on C) 5 100 µm.

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121PYRUVATE DEHYDROGENASE IMMUNOREACTIVITY AFTER ISCHEMIA

eduction in PDHC immunoreactivity. The results sum-arized in Table 1 represent data collected from two

nimals per group, using five sections per animal andeasuring 20–30 neurons per cortical layer and sec-

FIG. 4. Low magnification photomontages of area 8a in the dog frty in a control animal (A), following 10 min ischemia alone (B), andntensity in the 24-h reperfusion animal compared to that seen in thifferences in PDHC staining intensity and the involvement of particlearly visible at higher magnification. Cortical layers are indicated b

ion. Although no reduction in the immunofluorescence i

er unit area of layer V neuronal cell bodies wasvident following ischemia alone, a significant, approxi-ately 50% reduction was apparent following 2 h

eperfusion (P , 0.05). In contrast, no reduction in

al cortex showing the laminar distribution of PDHC immunoreactiv-ter 2 h (C) and 24 h (D) reperfusion. Note the decrease in stainingther conditions. However, at this level of resolution, subtle laminarr neuronal populations are not reliably detectable, whereas they areoman numerals. Scale bar (on D) 5 160 µm.

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euronal cell bodies located in layers IIIc–IV followingither 10 min ischemia or 2 h reperfusion. In order toontrol for possible differences in staining of differentpecimens, the ratio of immunofluorescence for cells inayer V compared to cells in layers IIIc/IV withinndividual sections were obtained and are summarizedn Table 1. No reduction in the ratio of fluorescenceetween the two areas was observed following ischemialone. However, a significant reduction was observedollowing 2 h reperfusion (P , 0.05).

DISCUSSION

A reperfusion-dependent decrease in brain pyruvateehydrogenase activity has been reported for severalifferent models of cerebral ischemia (5, 11, 18, 46, 47).sing the canine cardiac arrest model, we observed arofound, 40–70% inhibition of PDHC activity in ca-ine frontal cortex following 30 min to 24 h reperfusion5). The results of the present study using the sameodel indicate that the reduction in activity in brain

issue homogenates is at least qualitatively related tohe reduction in PDHC immunoreactivity (aE1p and2p) following both 2 and 24 h reperfusion. Ten min-tes ischemia alone resulted in no loss of immunoreac-

FIG. 5. PDHC immunoreactivity in layer IIIc/IV (top row) and inschemia (middle column), and following 2 h of reperfusion (right colun all conditions except in layer V of the 2-h-reperfusion animal. Inarrows). Compare with Table 1. Scale bar 5 75 µm.

ivity, which is consistent with the lack of an effect of r

schemia alone on PDHC maximal enzyme activity (5).he reperfusion-dependent loss of PDHC immunoreac-

ivity is in contrast to the finding of Zaidan et al. (48),ho used a rat four-vessel occlusion model of global

erebral ischemia and observed no loss of PDHC immu-oreactivity. However, the rat model differs from theanine model in several respects. In the rat model,lthough PDHC activity is reduced in the relativelyulnerable dorsolateral striatum, PDHC activity ap-ears unchanged in the cerebral cortex (48). The extentf inhibition of PDHC activity in the rat striatum is alsoonsiderably less than the inhibition we have observedn the canine cortex (5). Also, the spatial and temporalattern of neuronal injury in cardiac arrest comparedo arterial occlusion models of global cerebral ischemias somewhat different (29).

The mechanism by which PDHC is inhibited duringeperfusion may provide a further explanation for thencoupling between PDHC enzyme activity and immu-oreactivity. We have previously postulated that reper-

usion-dependent inhibition of PDHC activity may beue to site-specific protein oxidation (5). Others haveuggested that inactivation is specifically due to sulfhy-ryl oxidation (45). Site-specific protein oxidation doesot necessarily result in an immediate loss of immuno-

er V (bottom row) of a control dog (left column), following 10 min of). Note the presence of large, intensely stained neurons in both layerss case large layer V neurons exhibit a considerably lighter staining

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123PYRUVATE DEHYDROGENASE IMMUNOREACTIVITY AFTER ISCHEMIA

eins for proteasomal degradation (44). It is likely thathe susceptibility to degradation is dependent upon thextent of protein oxidation. Therefore, although a givenxidative insult may be sufficient to inactivate annzyme, it may not be sufficient to result in proteolyticegradation. This possibility could help explain whyhe reduction in PDHC aE1p and particularly E2pmmunoreactivity is not as great as the reduction in PDHctivity that we have previously reported (5). The fact thate observed a greater loss of PDHC activity in a modelhere site-specific protein oxidation is known to exist (21)

han was observed by Zaidan et al. (48) could also explainhe difference in the effects of ischemia/reperfusion on

FIG. 6. Confocal imaging of PDHC (A–D and F) and MAP2 (E, F)DHC immunoreactivity in control (A) and 10-min-ischemic (B) tissueuropil appears reduced in both 2-h (C)- and 24-h (D)-reperfused ani

n layers IIIc/IV (E, F) and in layer V (not shown) following ischemia aeuron (green) in layers IIIc/IV following 2 h reperfusion. Scale bar in

DHC immunoreactivity observed in these two systems. a

The finding that PDHC immunoreactivity was al-ered during cerebral ischemia and reperfusion allowedor comparisons between the levels of PDHC in neuronsresent in different layers of the frontal cortex thatxhibit different vulnerabilities to ischemic cell death.ypically, relatively large neurons in the middle ofortical layer III and throughout layer V exhibit thereatest vulnerability to delayed death, with neuronsn layers I, IV, and VI being more resistant to injury28). The relative susceptibility of different neuronalubclasses to ischemic injury is multifactorial androbably dependent on the degree to which these cellsndergo and withstand Ca21 overload, oxidative stress,

the frontal cortex of control and experimental dogs. Cortical layer Vppears similar, while immunofluorescence in both the cell soma and

ls. Robust somatic and dendritic MAP2 immunostaining was present2 h reperfusion. (F) Robust PDHC staining (red) in a MAP2-stainingD and F 5 12 µm and in E 5 30 µm.

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nd metabolic failure (8, 40). We decided to compare

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he pattern of PDHC levels in layers IIIc/IV comparedo that in layer V to test the hypothesis that a reductionn the level of this important enzyme in cerebral energy

etabolism is a prelethal marker of susceptibility toelayed death. The neuronal somata of these particularayers were chosen due to the relative ease of identifica-ion and demarcation and due to the fact that signifi-antly greater delayed neuronal death is apparent inayer V than in layer IV (i.e., our layer IIIc/IV) in bothat and canine models of global cerebral ischemia (28,9, and P. Hof, unpublished observations).The finding that the level of PDHC in either neuronal

ubclass was unchanged following ischemia alone isonsistent with the results of the western immunoblots,nd consistent with the lack of inhibition of PDHCctivity reported previously (5). The observation thatDHC immunoreactivity in cortical layer V neuronalell bodies was reduced by approximately 50% follow-ng 2 h reperfusion is consistent with both the Westernmmunoblot measurements and with enzyme activity

easurements performed on frontal cortex homoge-ates. Although PDHC immunoreactivity was reducedollowing 2 h reperfusion in the neuropil as well as inhe cell bodies within cortical layer V, there was noeduction in PDHC staining intensity in the cell bodiesocated within layers IIIc/IV. This finding was verifiedy independently measuring the ratio of immunofluores-ence per unit area for cell soma in layer V versusayers IIIc/IV, since the ratio of fluorescence for the tworeas within individual sections should be unaffectedy variations in staining between samples. The observa-ion that there was a significant decrease in this ratioollowing 2 h reperfusion but not following 10 minschemia alone is consistent with the immunofluores-ence measurements performed on individual neurons

TABLE 1

Quantitative Laser Scanning Confocal Analysis of PDHCImmunoreactivity in Canine Neocortex Following

Ischemia/Reperfusion

Animal group Layer VLayerIIIc/IV

RatioV:IIIc/IV

ontrol 107.2 6 6.8 79.4 6 2.8 1.35 6 0.040 Min ischemia 106.0 6 16.0 78.5 6 3.5 1.34 6 0.15H reperfusion 55.4 6 3.4* 82.5 6 4.5 0.68 6 0.08*

Note. Results represent the means 6 SEM of immunofluorescencentensity per unit area as described under Materials and Methods.ata were obtained using two animals per group and five frontal

ortex sections per animal. Immunofluorescence was measured forhe cell bodies of 20–30 neurons per section, yielding 200–300easurements per group. The ratio of fluorescence for layers V:IIIc/IVas determined for individual sections, yielding 10 measurementser group.* Significantly different from the control group (P , 0.05).

resent in different sections from different perfusion T

xed specimens. It is therefore concluded that 10 minardiac arrest and 2 h reperfusion results in a reductionn PDHC immunoreactivity that is relatively specificor large pyramidal neurons present in layer V com-ared to the smaller neurons present in layers IIIc/IV ofhe canine neocortex.

The selective, prelethal loss of PDHC in the corticalayer V pyramidal cells is consistent with the relativeulnerability of these neurons in this and other modelsf global cerebral ischemia and reperfusion (28, 39).lthough these results do not establish a cause-and-ffect relationship between the loss of PDHC andeuronal death, they do support the hypothesis thathe reduction in PDHC is a sensitive molecular markerf irreversible neuronal injury. If, as we and othersave postulated, the reperfusion-dependent loss ofDHC activity is due to one or more forms of oxidative

njury (5, 45), the reduction in PDHC levels may serves a marker of differential sensitivity to oxidativetress. It should therefore be determined if the loss ofDHC is related to histochemical indicators of oxida-

ive stress, e.g., products of protein oxidation and lipideroxidation products, and whether such relationshipsre evident within different neuronal subclasses.In addition to the possible relationship between

DHC levels and oxidative stress, it is likely that someells lose sufficient PDHC activity to severely impairhe production of reduced pyridine nucleotides thatould limit production of ATP by oxidative phosphoryla-ion. A shift in metabolism toward anaerobic glycolysisaused by impaired PDHC activity would promotehronic lactic acidosis and alter other metabolic path-ays that are dependent on normal tricarboxylic acid

ycle activity, such as neurotransmitter biosynthesis.ny or all of these sequelae could easily contribute to

he pathogenesis of ischemic cell death. In addition tohe correlative results described in the present study,upport for the involvement of altered PDHC activity inschemic neurodegeneration comes from animal experi-

ents demonstrating neuroprotection by agents thatither stimulate PDHC activity, such as dichloroac-tate (7, 18), or metabolic substrates that bypass theDHC step to generate acetylCoA, such as 1,3-utanediol (22, 14) and acetyl-L-carnitine (3, 23, 33, 38).t remains to be determined to what extent PDHCevels and PDHC activity must be reduced to actuallyrecipitate or potentiate neuronal death and whetherharmacological agents directed at counteracting PDHCnhibition are therapeutically effective in human isch-mic brain disorders.

ACKNOWLEDGMENTS

We thank A. P. Leonard for expert technical assistance. This workas supported by NIH Grants NS34152 and AG09014 and by Sigma

au, S.p.A.

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125PYRUVATE DEHYDROGENASE IMMUNOREACTIVITY AFTER ISCHEMIA

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