Disparate Recovery of Resting and Stimulated Oxidative Metabolism Following Transient Ischemia

Transcription

1 Disparate Recvery f Resting and Stimulated Oxidative Metablism Fllwing Transient Ischemia 677 R. B. DUCKROW, M.D., J. S. LAMANNA, PH.D., AND M. ROSENTHAL, PH.D. SUMMARY T assess tbe residual effects f transient cerebral ischemia n mitchndria! xidative metablic functin, changes in the reductin/xidatin state f cytchrme a,a, and relative lcal Md vlume were measured in situ frm tbe expsed cerebral surface f rat brain befre and after 10 minutes f cartid artery ligatin. During the ischemlc interval, cytchrme a,a» became reduced and electrcrtical activity was ablished. During the first 20 minutes f reperfusin cytchrme a,a, was hyperxldized beynd baseline with eventual recvery t the riginal steady state. Electrcrtical activity returned mre slwly. Increased energy demand induced by electrical stimulatin f the crtex prduced transient xidatin f cytcbrme a,a%. The amplitude f this xidative respnse was decreased during the first 30 minutes f reperfusin. During tbe first 2 burs f reperfusin the time required fr re-reductin f the xidative respnse was lengthened despite tbe recvery f baseline mitchndria! redx state. These data demnstrate residual metablic dysfunctin after transient ischemia nt apparent under "resting" cnditins but evident when the system is required t perfrm additinal "wrk." We speculate this metablic dysfunctin culd be due t relative substrate limitatin. Strke, Vl 12, N 5, 1981 DURING CEREBRAL ISCHEMIA, neurnal integrity is cmprmised by energy failure due t lack f metablic substrates and xygen. The brain is particularly sensitive t ischemic damage because its energy reserve is very lw. Yet, suppressin f integrated synaptic functin and electrcrtical activity ccurs well befre majr depletin f energy stres. 1 - * Ischemic induced defects f integrated electrcrtical activity can exist after metablic functin returns t nrmal. 1 The dissciatin between neurnal functin and cellular energy stres implies the presence f cntrls that decrease energy demand when energy cnservatin is disrupted. This means that restratin f the baseline metablic state is insufficient fr reestablishment f electrphysilgical activity after transient ischemia, suggesting that the metablic capability t respnd t increased energy demand als is required. This questin may be f critical imprtance in understanding the prcesses that reduce cellular energy demand when the energy supply is threatened by ischemic insult as well as the limitatins placed n the energy cnservatin system by residual defects that exist during reperfusin. T apprach this questin, metablic activity, as indicated by shifts in the reductin/xidatin rati f cytchrme c xidase (cytchrme a,a t ), was recrded in viv by reflectin spectrphtmetry befre and after transient ischemia prduced by the methd f Pulsinelli. 4 Such measurements prvided a direct indicatin f xidative metablic functin since cytchrme a,a t is the final reactant with mlecular xygen in mitchndria and its redx state has been shwn t signal xygen sufficiency and respiratry chain turnver." 1 ' Studies were perfrmed during the "steady state" and during the "active" state prvked Frm the University f Miami Schl f Medicine, Department f Neurlgy, 1501 NW 9th Ave., Miami, FL Reprints: Dr. Duckrw, University f Miami Schl f Medicine, Dcpt. f Neurlgy (D4-5), Bx , Miami, FL by electrical stimulatin f the crtical surface. The latter allwed assessment f the effects f transient ischemia n dynamic changes that ccur when mitchndria respnd t increased energy demand. We cnclude that transient ischemia prduces residual crtical metablic dysfunctin during reperfusin and that this effect cntinues well beynd the time f apparent recvery f the baseline mitchndrial redx state. Electrcrtical activity remains abnrmal until after this residual dysfunctin reslves. We speculate that this residual dysfunctin results frm alteratins in the ability f substrate t enter the respiratry chain. A preliminary reprt f this wrk has been presented. 7 Methds Male Wistar rats with a mean weight f 362 g (range g) were anesthetized with sdium pentbarbital (55 mg/kg i.p.). Plyethylene cannulae were placed in the trachea, a femral artery and a femral vein. Systemic bld pressure was mnitred with the femral artery catheter which was als used fr cllectin f arterial bld samples. The cartid arteries were expsed, the cartid sheaths dissected, and silk ligatures were placed abut the arteries. Silastic tubing split lengthwise was used as a prtective cuff abut each artery t prevent direct cntact by the ligature. The ends f each cartid ligature were threaded thrugh separate lengths f plyethelyene tubing t frm snares. Reversible cclusin was btained by tightening the snares and fixing them with surgical clamps. The resiliency f the silastic cuff assured each snare wuld pen when clamps were remved. Tubcurare hydrchlride (10 mg/kg) was given in a divided dse, 5 mg/kg intraperitnealy and 5 mg/kg subcutaneusly. Artificial ventilatin was then prvided with 30% xygen, 70% nitrgen. Bld samples (150^1) frm the arterial catheter were prcessed with a Radimeter-Cpenhagen PMH Mk2

2 678 STROKE VOL 12, N 5, SEPTEMBER-OCTOBER 1981 acid/base gas analyzer. Arterial bld gas and ph values were maintained within physilgical limits by adjustment f the respiratry minute vlume. Supplemental dses f pentbarbital (0.15 mg/kg) were given at hurly intervals thrugh the femral venus cannula. Bdy temperature was mnitred with a rectal thermistr and maintained at 37 C with a heating pad. The rat was psitined in a head hlder and a midline scalp incisin was extended t the level f the upper thracic vertebrae. The paraspinus musculature was bluntly dissected frm the lamina f the secnd cervical vertebra t reveal the alar framen. The scalp was retracted, and a 4 mm by 6 mm cranitmy was fashined in the right parietal bne by gradually thinning the calvarium with a lw speed dental drill until the bne flap culd be remved with fine frceps. Hemstasis was achieved with the aid f Gelfam (Upjhn) gauze. The dura was left intact. Cytchrme a,a t reductin-xidatin shifts were mnitred by dual-wavelength reflectin spectrphtmetry.* Light frm 2 mnchrmatrs was alternately presented t the ends f 2 ptical fiber bundles whse fibers were randmly cmbined int a single bundle. The light frm this bundle was directed at the surface f the expsed dura. The "sample" wavelength was chsen at the peak f absrptin f reduced cytchrme a,a a (605 nm). A wavelength (590 nm) "equibestic" t the "sample" wavelength fr changes in Hb/HbOi was used as a reference. Scattered light returning frm the crtical surface was cllected with a micrscpe bjective and detected by a phtmultiplier tube hused in the micrscpe barrel. The wrking distance f the bjective was 33 mm and the ptical field was 3.2 mm in diameter. The difference between the light reflected during the perid f illuminatin at 605 nm and the perid f illuminatin at 590 nm ( nm) was cnsidered an index f the relative amunt f reduced cytchrme a,a t. A feedback regulatin circuit was used t maintain the utput f the phtmultiplier cnstant during the reference light illuminatin perid. This was dne by varying the high vltage supply t the phtmultiplier, thus varying its gain. This feedback cntrl vltage has been shwn t be a useful index f lcal bld vlume. 8 A DC-cupled silver-silver chlride wire electrde was placed in the center f the ptical field t recrd the crtical ptential, and a pair f stainless steel stimulating electrdes, separated by 0.8 mm, were psitined 0.9 mm frm the recrding electrde with the aid f an cular reticle. A disk reference electrde was placed under the scalp n the skull midline rstral t the cranitmy. Changes in crtical redx state were prvked by trains f 0.5 ms electrical pulses at 20 Hz lasting 2 secnds. Stimulating vltages ranged frm 7 t 30 vlts and were applied in a gradually increasing fashin t avid prvking spreading crtical depressin. Such stimulatin resulted in cytchrme a,a s xidatins tgether with increases in lcal bld vlume and negative steady ptential (SP) shifts. Eight t 14 such stimuli were given ver a 10 minute perid at selected times thrughut the experiment. After a cntrl series f stimuli were given, the vertebral arteries were electrcagulated in the manner f Pulsinelli 4 by the insertin f a cautery prbe int each f the alar framen. The animal was allwed 60 t 90 minutes t recver befre a secnd stimulus series was perfrmed. Predminance f cartid circulatin t the crtex made ischemic injury due t vertebral cagulatin unlikely. An estimate f the reducible cytchrme a,a % pl size was then determined by allwing the animal t respire 100% nitrgen fr 30 secnds. Recvery withut sequellae was expected after this shrt perid f hypxia. Thirty minutes later the cartid ligatures were tightened fr 10 minutes. Crtical stimulatin was perfrmed 15, 30, 60, 120, and 180 minutes after release f the cartid ligatures. The estimate f reducible cytchrme a.at was again determined at the end f the recvery perid and cmpared t the final level f reduced cytchrme a,a s btained during terminal 100% nitrgen respiratin. Besides pl size estimates, these brief nitrgen induced reductin perids prvided an index f cmparisn fr ischemia induced reductin. The experimental prtcl was arranged t insure that at least 30 minutes elapsed between any supplemental dse f pentbarbital and any stimulus series since phenbarbital alters the xidative respnse t stimulatin.' N supplements were given frm 30 minutes befre cartid cclusin until after the stimulus series 60 minutes after clamp release. Analysis f the respnse f cytchrme a,a t and relative bld vlume t brain stimulatin invlved quantificatin f the evked transients. The abslute magnitude f this xidatin (Pmax), the time frm stimulus nset t attainment f this peak (tpmax), and the time frm the peak t the half recvery pint t the riginal baseline (tl/2 ff) were measured. Derived quantities included the rati f peak bld vlume increase t peak cytchrme a,a t xidatin (the BV:CYT rati) and the slpe f the least squares regressin relating peak cytchrme a,a, xidatin t the crrespnding SP shift. If the range f SP shift amplitude btained by crtical stimulatin was less than ne millivlt, the least squares regressin was nt included in the data cnsidered. Amplitudes f the ptical signals were based n percentages f the full scale signal. Fr cytchrme a,a t, zer was the signal recrded when n "sample" light was available t tissue illuminated by full "reference" light. Full scale was defined when equal "sample" and "reference" signal were recrded under "resting" (unstimulated) cnditins. The bld vlume signal was expressed as a percent f the high vltage initially applied t the phtmultiplier tube. Results Data were cllected frm 14 rats which had bilateral vertebral artery cagulatin fllwed by a 10 minute cclusin f bth cartid arteries. Arterial

3 METABOLIC DYSFUNCTION AFTER Tl/Duckrw et al. 679 bld gas values were maintained within nrmal limits (i.e., Pa, 100 mm Hg, Pac, 35 t 40 mm Hg). Arterial bld samples taken prir t cartid cclusin gave grup means f ph 7.39 ± 0.01 (±SEM), Pc, 38 ± 2 mm Hg, and P, 119 ± 4 mm Hg. The mean systemic arterial bld pressure prir t cartid cclusin was 135 ± 4 mm Hg. Ischemic Interral In all rats, cclusin f bth cartid arteries was fllwed by immediate systemic hypertensin (mean pressure 195 ± 5 mm Hg). In 5 animals this hypertensive respnse was maintained thrughut the entire ischemic interval. In 9 animals the hypertensin reslved, usually beginning at the 6th r 7th minute, and systemic bld pressure returned t a mean f 112 ± 10 mm Hg just prir t release f the ligatures. Cytchrme a,a» quickly became mre reduced after cartid cclusin. Between 15 and 30 secnds after cclusin an inflectin ccurred in the data recrd. This pint was termed the "first reductin plateau." After this pint, cytchrme a,a % either cntinued t becme mre reduced (7 rats) r a brief perid f rexidatin (7 rats) ccurred. Figure 1 shws an example f transient rexidatin f cytchrme a.ai fllwing the first reductin plateau during cartid cclusin. T prvide a mre quantitative index f cytchrme a,a t reductin during cartid cclusin, cmparisns were made with the level f reductin recrded during 30 secnds f respiratin with 100% nitrgen and with 590 ">n (BL. VOL. Increase nm (CYT. a,a 3 xidatin^) S.A.P. (mmhg) ' the cmplete reductin btained during terminal nitrgen respiratin. In 10 f 14 rats the level f cytchrme a,a% reductin prir t release f cartid clamps was higher than the first reductin plateau and 98 ± 4% f the mmentary reductin level achieved by 30 secnds f nitrgen respiratin given prir t cartid cclusin. In 4 f 14 rats the level f reductin prir t release f cartid ligatures was equal t r lwer than the first reductin plateau and 61 ± 14% f the 30 secnd reductin level. There was n crrelatin between the ccurrence f sustained r transient systemic hypertensin during cartid cclusin and the level f cytchrme a,a% reductin prir t cartid ligature release (chi-square crrected: p = 0.92). Fr cmparisn, the mmentary reductin level achieved by 30 secnds f nitrgen respiratin given at the end f the experiment was 86 ± 7% f the "cmplete" reductin level that ccurred during terminal nitrgen respiratin. Electrtrcrtical activity was ablished within 30 secnds f cartid cclusin in all rats. In 3 sme lw vltage activity returned after 2 t 4 minutes and disappeared again after lasting frm 1 t 4 minutes. Tw f these were frm the grup f 4 animals with the lwer degree f cytchrme a,a t reductin prir t reperfusin. Electrcrtical activity remained iselectric thrughut the perid f cartid cclusin in all ther rats. Electrical seizure activity, defined as spikewave discharges r bursts f high amplitude sharp waves, was nt seen during the cclusin r reperfusin perid. 3%f.s. 10%f.a. 10 mln cclusin FIGURE 1. Changes ccurring during and fllwing 10 minutes f crtical ischemia prduced by vertebral cagulatin fllwed by reversible cartid ligatin in the rat. During ischemia the amunt f reduced cytchrme a,a, is increased. After an initial inflectin in the ptical recrd, the amunt f reduced cytchrme a,a, cntinues t increase until cartid ligature release. The early reperfusin perid is characterized by hyperxidatin f cytchrme a^ beynd cntrl levels with eventual return t the resting state. Lcal bld vlume is indicated by crtical light absrptin at 590 nm. S.A.P. signifies systemic arterial bld pressure.

4 680 STROKE VOL 12, N 5, SEPTEMBER-OCTOBER 1981 Crtical absrptin at 590 nm ("bld vlume") shwed a biphasic respnse during cartid cclusin. During the first 30 secnds, absrptin at 590 nm decreased, but then returned t a level abve its starting pint. In 6 rats it remained at this elevated level, in 8 it gradually decreased t a pint at r belw the initial level by the time f ligature release. Perid f Reperfusin The systemic bld pressure decreased immediately upn release f cartid ligatures. In the 5 animals with elevated bld pressure prir t ligature release, it returned t near the precclusin level. If the bld pressure had nt been elevated at the time f ligature release (9 rats), it drpped int a hyptensivc range (69 ± 5 mm Hg). In all animals bld pressure returned t the precclusin range within 15 minutes f reperfusin. The level f reduced cytchrme a,a t als drpped after reperfusin. In mst rats (12 f 14) this xidative respnse was precipitus. In all, the level f reductin fell belw the preischemic baseline. This hyperxidized state f cytchrme a,a t was transient, reaching its peak at 7 ± 1 minutes. The cytchrme redx state was restred t the pre-ischemic baseline in 16 ± 3 minutes. Crtical absrptin at 590 nm increased upn reperfusin. Electrcrtical activity returned gradually during reperfusin. Within 15 minutes sme lw vltage slw and ccasinal sharp activity were seen. At 60 minutes faster frequencies had returned while the vltage remained depressed. At 180 minutes the activity was nearly nrmal t visual inspectin with sme intermittent slwing remaining. This pattern is seen infigure2. 15' 30' J1 SEC Respnse t Crtical Stimulatin T test the ability f the metablic system t respnd t increased energy demand after transient ischemia, trains f electrical pulses f varying vltage were applied t the crtical surface. Such stimulatin prduced a graded respnse in the magnitude f the negative steady ptential shift. Figure 3 demnstrates the xidatin f cytchrme a,a% (CYT Pmax) and transient increase f bld vlume (Bl. Vl. Pmax) that als accmpany such stimulatin. The magnitudes f these changes were crrelated with the magnitudes f the SP shifts (filled circles, fig. 4). The intercept f the cytchrme a,a% regressin line was psitive but nt significantly different frm zer (2 tailed /-test, p = 0.44). The time t peak cytchrme a,a a xidatin (tpmax) and half-recvery (tl/2 ff) were nt strngly crrelated with the magnitude f the xidatin (filled circles, fig. 5). A similar situatin existed fr the times t peak bld vlume nset and half-recvery. Stimulus series were perfrmed befre and after vertebral artery cagulatin prir t cartid cclusin. As seen in the table, vertebral cagulatin did nt prduce a significant change in any f the parameters used t describe the transient metablic and bld vlume respnse t crtical stimulatin. Effect f Cartid Occlusin Of 14 rats subjected t transient cartid cclusin, 11 shwed decreased peak xidatin f cytchrme a,a t prduced by crtical stimulatin after release f the cartid ligatures. Of these, 8 shwed recvery f cytchrme a,a t xidative transient amplitude within the 3 hur reperfusin perid. Three rats shwed sup- 740 FIGURE 2. Electrcrtical activity befre (C), during (Dj, and at indicated times after cartid artery cclusin. 60' 1 / V"! \j 120' 180'

5 METABOLIC DYSFUNCTION AFTER Tl/Duckrw et al. 681 Cntrl 15 min 0 3*1 3mV 180 min FIGURE 3. Respnses f lcal crtical bld vlume (upper traces), cytchrme a,a t redx state (middle traces), and steady ptential (lwer traces) t electrical stimulatin. Respnses befre cartid ligatin (cntrl) and 3 hurs after ligature release (180 min) are similar. In the early reperfusin perid (15 min) the amplitude f the cytchrme a,a, respnse (middle trace) is decreased despite a larger SP shift. The time t half recvery is prlnged. This lengthening f recvery time is shwn mre clearly by nrmalizing the respnse amplitude f the cytchrme a,a> respnses as shwn at the bttm right (slid line = cntrl, dtted line = 15 min f reperfusin, dashed line = 180 min f reperfusin). The bld vlume traces have been scaled t their respective cytchrme a,a, respnses. pressin f peak evked xidatin withut recvery after 3 hurs f rcperfusin. Three rats did nt shw suppressin f evked cytchrme a,a s xidatin at any time, and these were in the previusly mentined grup f 4 having a level f reduced cytchrme a,a % at the end f the cartid cclusin interval less than the "first reductin plateau."» 4 I as as SP Shift (mv> J 0.4 a. O > i 02 m 0.1 SP Shift The reducible cytchrme a,a,, determined by 30 secnds f nitrgen respiratin at the end f the experiment, averaged 91 ± 6% f the reducible cytchrme a,a t determined by the same methd prir t cartid ligatin fr the 11 animals that either shwed n suppressin f the transient cytchrme a,a % respnse r shwed suppressin with recvery. In the (mv) FIGURE 4. Relatinships between the magnitude f cytchrme a,ai xidatin, bld vlume increase, and steady ptential shift (SP shift) evked by direct crtical stimulatin f varying intensity. These data pints represent respnses t crtical stimulatin in a single animal. Open circles represent data taken 15 minutes after remval f cartid ligatures. Filled circles are cntrl data, and squares represent data recrded after 180 minutes f reperfusin. CD CYT. fi.fl, Pm«x (%!.«.)

6 682 STROKE VOL 12, N 5, SEPTEMBER-OCTOBER ~ 10 eg Q_ 6 t? x a Q. c Ol 4 Ol -I 4 / <* > CD CYT.,a 3 Pmax ( BL. VOL. Pmax (%f.s.) s " 20 E 15 c 10 Ol ( CYT. a,a 3 Pmax (%t.8.) BL. VOL. Pmax FIGURE 5. Relatinship between the tpmax and tl/2 ff and the amplitude f the vascular r metablic respnses. These data pints represent respnses t crtical stimulatin in a single animal. Open circles represent data taken 15 minutes after remval f cartid ligatures. Filled circles are cntrl data, and squares represent data recrded after 180 minutes f reperfusin. 3 animals that shwed suppressin f the transient cytchrme a,a t respnse withut recvery the reducible cytchrme a,a t at the end f the experiment was 63 ± 11% f the same pl prir t cartid cclusin. The difference between these 2 grups was nt significant (p = 0.06). Data frm ne f the 8 rats that shwed suppressin and recvery f the peak xidative respnse after transient ischemia is snwed in figure 4. The slpe f the linear relatin between peak cytchrme a,a, xidatin and SP shift was decreased 15 minutes after release f the cartid ligatures. The amplitude f the crrespnding bld vlume increase was unchanged. The intercept f the cytchrme a,a t regressin line increased t a value significantly greater than zer (p = 0.03). After 180 minutes f reperfusin these > ffi 10 values were returning t cntrl levels. The grup means fr the relatinship f the peak cytchrme a,a t xidatin t the peak SP shift during the reperfusin perid are shwn in figure 6 (ACYT Pmax/ASP Shift). Suppressin f the respnse was seen 15 and 30 min after reperfusin. The time required fr cytchrme a.a», transiently xidized after stimulatin, t becme re-reduced t half the baseline reductin/xidatin rati (tl/2 ff) was lengthened during the early reperfusin perid. This is shwn fr individual rats infigures3 and 5. The grup means in figure 6 shw this lengthening (dubling) persisted fr 2 hurs after release f the cartid ligatures and returned t the cntrl level after 3 hurs f reperfusin. Mean values f descriptive indices fr metablic TABLE Stimulus Series - Vertebral Cagulatin Prduced N Significant Change in Transient Metablic and Bld Vlume Respnse t Crtical Stimulatin Cntrl After Cagulatin Units CyttPmax CyttMff BL Vl. tpmax Bl VL M ff BV:CYT rati Slpe 6.4 ±0.3 (12) 6.3 ±0.6 (12) 5.1 ±0.3 (12) 4.4 ±0.4 (12) ± (12) 1.24 ± 0.18 (11) 6.8 ±0.3 (14) 6.5 ±0.7 (14) 5.0 ± 0.2 (14) 4.0 ± 0.3 (14) ± (14) 1.04 ± 0.19 (13) secnds (N) secnds secnds secnds %f.s / mv

7 METABOLIC DYSFUNCTION AFTER Tl/Duckrw et al. 683 CYT. fl, 3 tpmix CYT. ( c) BL. VOL. ti/2 n BV : CYT Rati A CYT Pmix ASP Shift n mv 1 ) 0.10 as 126 \ \ 0 25 I FIGURE 6. Grup means f the parameters describing the transient metablic and vascular respnse t crtical stimulatin befre vertebral artery cagulatin (C), after vertebral artery cagulatin (VC), and during reperfusin after 10 minutes f reversible cartid ligatin fr the eight animate that shwed suppressin and recvery f the cytchrme a,a, xidative transient. The hatched area represents the ischemic interval. (* = p < ** = p < nn-paired \-test cmpared t cntrl (C), errr bars represent ± SEM). vc Time after Reperfusin (min) and bld vlume transients acrss the grup f 8 animals prvide a summary f the effects f the cartid clamping and the recvery that ccurred ver the 3 hur bservatin perid (fig. 6). There was a 14% increase in the cytchrme a,a% tpmax at 15 minutes f reperfusin. There was als a 50% increase in the bld vlume tl/2 ff after 15 minutes f reperfusin. There was suppressin f peak cytchrme a,a t xidatin fr a given SP shift t 30% f cntrl value at 15 minutes f reperfusin. This, in large part, accunts fr the near dubling f the BV:CYT rati (refer t Methds) at that time as well. Discussin Vertebral artery cagulatin fllwed by cartid artery ligatin prduced increased reductin f cerebral cytchrme a,a t and suppressin f electrcrtical activity. Reperfusin was characterized by a perid f hyperxidatin f cytchrme a,a t. A perid f cntinuing metablic dysfunctin intervened between reestablishment f the baseline redx state and return f baseline electrcrtical activity. This dysfunctin was characterized by decreased amplitude f the transient xidative respnse f cytchrme a.a s t increased crtical wrk induced by direct crtical stimulatin. The time required fr cytchrme a,a t re-reductin after stimulus induced xidatin was prlnged. This prlngatin suggests relative substrate limitatin was induced by ischemia and became manifest nly when the quiescent brain was stimulated t perfrm additinal metablic "wrk" during the recvery perid. Increased reductin/xidatin ratis f electrn transprt chain cmpnents have been shwn t ccur during ischemia induced by varius methds.*" 11 Such reductive respnses are expected because the respiratry chain cmpnents f mitchndria in vitr becme reduced when xygen is limited. 5 The degree f reductin f respiratry chain cmpnents during ischemia prvides a criterin fr cmpleteness f ischemia when it is cmpared t the presumably cmplete reductin achieved during terminal nitrgen respiratin. 11 Using this criterin, vertebral cagulatin fllwed by reversible cartid ligatin did nt prduce cmplete cerebral ischemia in rat since cytchrme a,a t reductin did nt attain the level recrded after death. Als, cntinuing reductin f cytchrme a,a 3 during vessel cclusin demnstrated that sme xygen remained available. Nevertheless, since electrcrtical activity was suppressed, energy prvisin was disturbed during the ischemic interval. Crtical reflectance at 590 nm was used as a crrectin fr changes in hemglbin vlume and xygenatin as well as nn-specific changes in light absrptin and scattering by the tissue. During the

8 684 STROKE VOL 12, N 5, SEPTEMBER-OCTOBER 1981 metablic respnse t direct crtical stimulatin this crrectin signal is an indicatr f relative crtical bld vlume. 6 This is pssible because crtical stimulatin prduces few nn-specific changes such as alteratins in brain vlume r shifts f cellular water. During cartid ligatin after vertebral cagulatin, the surface f the brain mves and cellular water shifts are expected. Fr this reasn, care must be taken when interpreting changes in the reference signal as changes in relative crtical bld vlume. Reperfusin after a 10 minute ischemia prduced hyperxidatin fllwed by return f the redx state f cytchrme a,a s t the cntrl level. Interpretatin f these findings requires that the ttal pl f cytchrme a,a s remain unchanged, and there is evidence frm liver" and brain" that ischemia might change this pl size. Hwever, thse rats with reversible abnrmalities f metablic respnse t crtical stimulatin shwed n decrease f the labile (reducible) cytchrme a,a t pl at the end f the 3 hur reperfusin interval. This suggests that, in thse rats, transient ischemia had n permanent effect n cytchrme a,a t pl size, and the decreased absrptin f light at 605 nm immediately after ligature release did, in fact, signal hyperxidatin. Such hyperxidatin has been bserved after ischemia prduced in a variety f mdels." " ie The rapid return f the redx state f cytchrme a,a s frm the hyperxidized cnditin t the baseline state was cnsistent with bichemical data shwing rapid return f ther indexes f metablic functin, including adenylate energy charge, phsphcreatine cncentratin, lactate cncentratin and the lactate/pyruvate rati.'- 1T Electrcrtical activity remained abnrmal, hwever, suggesting decreased energy demand persisted. T determine if metablic dysfunctin persisted during this perid, the brain was stimulated t increased activity by the delivery f electrical pulses t the crtical surface. Such stimulatin has been shwn t be assciated with increases in extracellular ptassium and accmpanied by transient xidatin f mitchndrial NADH and cytchrme a,a e t - " 1B analgus t the state-4 t 3 transitin f islated mitchndria. 6 Cytchrme a,a, xidatins prduced by crtical stimulatin during reperfusin were decreased in amplitude and slwed in time curse. These changes culd be explained by 5 pssibilities: 1) uncupling f xidative phsphrylatin," 2) lss f mitchndrial enzymatic pl size, 11 - " 3) lss f Na + K + ATPasc activity, 31 4) relative substrate deficiency," 118> M r 5) increased tissue xygenatin."-" Uncupling f xidative phsphrylatin des nt appear t be a likely explanatin. If uncupling were present, increased ADP availability wuld cause an verall xidatin f cytchrme a,a % and increase the magnitude f xidative respnses t stimulatin. During reperfusin after 10 minutes f ischemia there was hyperxidatin f cytchrme a,a s, hwever, the magnitude f the xidative respnse t stimulatin was suppressed. Als, the time curses f these changes were different (cf. figs. 1 and 6). The functin f neurnal enzyme systems invlved in the regulatin f cellular metablism can be cmprmised by anxic/ischemic insults. This includes decreases in cytchrme a,a s pl size 11 - M and Na + K + ATPase activity." If the cytchrme a,a, pl size was changed by ischemia, decreasing reduced and xidized fractins prprtinately, the magnitude f any transient signal change wuld decrease withut a change in the resting redx rati. This culd explain the decreased magnitude f the evked xidative transient. This is supprted by data frm 3 animals that did nt shw recvery f evked xidative transient magnitude and als shwed a decrease f labile (reducible) cytchrme a,a t. The small size f this grup precludes the establishment f statistical significance t this finding. If the activity f Na + K + ATPase were decreased by the ischemic insult, as has been reprted in ther preparatins," ne wuld see a uabain-like effect. Ouabain has been shwn t slw the transient xidatin f NADH induced by crtical stimulatin withut affecting the re-reductin rate." Cytchrme a,a s wuld be expected t change similarly. The tpmax increased by 14% at 15 minutes f reperfusin in this experiment but was nt therwise significantly different frm cntrl (fig. 6). Substrate deficiency culd result in inapprpriate xidatin f respiratry chain cmpnents due t a "state-2 like" cnditin. Re-reductin f cytchrme a,a, frm the xidized "active" state t the riginal steady state culd result frm increased reducing equivalent availability t the respiratry chain. Likewise, a blck in reducing equivalent availability culd delay re-reductin f xidized electrn transprt enzymes, including cytchrme a,a s. A drp f NADH belw cntrl levels ccurs during reperfusin after transient ischemia in varius preparatin. 11 ' " ie This suggests a substrate blck despite increased 2-dexyglucse phsphrylatin early during reperfusin after transient ischemia." 1 " We attribute delayed re-reductin f the xidative metablic transient t a relative substrate deficiency resulting frm a blck f reducing equivalent flw t the electrn transprt chain. This blck wuld have t exist after the phsphrylatin f glucse and befre the majr surce f NADH, the tri-carbxylic acid cycle. Transient prlngatin f recvery time has been shwn t ccur after intravenus phenbarbital injectin. This prlngatin abates after 30 minutes in cat preparatins.' Because phenbarbital administered as an anesthetic t the rat culd cause an artifactual prlngatin f the recvery time (t 1 /2 ff), dses were given hurly at least 30 minutes befre crtical stimulatin. Cumulative effect f the drug was unlikely since the recvery time (t 1/2 ff) returned t baseline levels at the end f the 3 hur reperfusin perid. Vascular cntrl f tissue xygenatin may be affected by transient ischemia, and ischemic damage t crtical vasregulatin may accunt fr changes in steady state and dynamic metablic activity. Oxygen delivery t crtical tissue beynd immediate metablic

9 METABOLIC DYSFUNCTION AFTER TI/Duckrw et al. 685 needs culd cause hyperxidatin f mitchndrial respiratry enzymes. As the terminal member f the chain, and direct reactant with mlecular xygen, cytchrme a,a t wuld be expected t be sensitive t tissue xygen delivery. It is likely that reflex hyperemia ccurring after transient ischemia, 11 ' " and indicated by increasing bld vlume in these experiments, plays a rle in the hypcrxidatin f cytchrme a,a, seen during the first 15 t 30 minutes. Vascular cntrl f tissue xygenatin during perids f increased neurnal "wrk" may als be affected by transient ischemia. The rati f the peak increase in crtical bld vlume t the peak xidatin f cytchrme a,a» induced by transient crtical stimulatin increased after ischemia (fig. 6). Hwever, this increase was primarily due t decreased evked cytchrme a,a t xidatin (seen infigs.3 and 4). Als, prlngatin f the recvery time f increased bld vlume induced by crtical stimulatin ccurred 15 minutes int the reperfusin perid. Hwever, this indicatin f a pssible increase in tissue xygen delivery, perhaps sufficient t prlng the perid f transient cytchrme a,a> xidatin, returned t cntrl levels 30 minutes int the reperfusin perid, while prlnged xidatin f cytchrme a,a t cntinued fr 2 hurs (fig. 6). These factrs suggest that abnrmalities f metablic respnse t increased crtical "wrk" seen after ischemia cannt be explained cmpletely by changes in tissue xygen delivery. This study demnstrates that spntaneus electrcrtical activity suppressed by transient ischemia will shw residual abnrmalities f frequency and vltage fr as lng as 2 t 3 hurs after reperfusin despite the return f the basal redx state f cytchrme a,a t. The reperfused crtex remains excitable t crtical stimulatin, hwever, a residual lesin f energy metablism becmes manifest when the crtex is called upn t prvide fr this increase in brain "wrk." Transient xidatins f cytchrme a,a t induced by crtical stimulatin have decreased amplitude and prlnged re-reductin times fr up t 2 hurs after a 10 minute perid f ischemia. These data are cnsistent with the presence f a relative blck in reducing equivalent flw t the respiratry chain. After 2 hurs f reperfusin the metablic respnse t transient stimulatin regains its preischemic qualities and near-nrmal electrcrtical activity returns. This suggests cmplete return f electrphysilgic activity after transient ischemia may require the reslutin f metablic defects that are manifest, nt in the steady state balance f reducing equivalent flw, but, in an inability f the mechanism f energy cnservatin t respnd t transient increases in metablic demand. Acknwledgment This wrk was supprted by PHS grants NS14325 and NS14319 and by the American Heart Assciatin f Greater Miami. JCL is the recipient f RCDA NS References 1. SiesjC BK: Ischemia, In Brain Energy Metablism, New Yrk, Jhn Wiley & Sns, 1978, pp SiesjC BK, Plum F: Pathphysilgy f anxic brain damage. In Gaull GE (ed) Bilgy f Cerebral Dysfunctin, New Yrk, Plenum Press, 1973, Vl 1, pp Ljunggren B, Ratchesn RA, SiesjC BK: Cerebral metablic state fllwing cmplete cmpressin ischemia. Brain Res 73: , Pulsinelli WA, Brierley JA: A new mdel f bilateral hemispheric ischemia in the unanesthetized rat. Strke 10: , Chance B, Williams GR: The respiratry chain and xidative phsphrylatin. Adv Enzyml 17: , JCbsis FF, Keizer JH, LaManna JC, Rsenthal M: Reflectance spectrphtmetry f cytchrme a.a, in viv. J Appl Physil 43: , Duckrw RB, LaManna JC, Rsenthal M: Reversible alteratins f xidative metablism in rat cerebral crtex after transient ischemia. Fed Prc 39: 534, LaManna JC, Crdingley G, Rsenthal M: Phenbarbital actins in viv: Effects n extracellular ptassium activity and xidative metablism in cat cerebral crtex. J Pharmacl Exp Ther 200: , Ginsberg MD, Reivich M, Frinak S, Harbig K: Pyridine nucletide redx state and bld flw f the cerebral crtex fllwing middle cerebral artery cclusin in the cat. Strke 7: , LaManna JC, JObsis FF, Austin GM, Schuler W: Changes in brain metablism in the cat in respnse t multiple brief transient ischemic episdes. Exp Neurl 55: , Rsenthal M, Martel D, LaManna JC, JCbsis FF: In situ studies f xidative energy metablism during transient crtical ischemia in cats. Exp Neurl 50: , Sundt TM Jr, Andersn RE: Reduced nictinamide adenine dinucletide flurescence and crtical bld flw in ischemic and nnischemic squirrel mnkey crtex. 1. Animal preparatin, instrumentatin and validity f mdel. Strke 6: , Mittnacht S Jr, Sherman SC, Farber JL: Reversal f ischemic mitchndrial dysfunctin. J Bil Chem 254: , Clendenn NR, Allen N, Kmatsu T, Liss L, Grdn WA, Heimberger K: Bichemical alteratins in the anxic-ischemic lesin f rat brain. Arch Neurl 25: , Kgure K, Bust R, Schwartzman RJ, Scheinberg P: The dissciatin f cerebral bld flw, metablism, and functin in the early stages f develping cerebral infarctin. Ann Neurl 8: , Welsh FA, Ginsberg MD, Rieder W, Budd WW: Diffuse cerebral ischemia in the cat: II. Reginal metablites during severe ischemia and recirculatin. Ann Neurl 3: , NrdstrCm C-H, Rehncrna S, Siesjfl B: Effects f phenbarbital in cerebral ischemia. Part II: Restitutin f cerebral energy state, as well as f glyclytic metablites, citric acid cycle intermediates and assciated amin acids after prnunced incmplete ischemia. Strke 9: , Lewis DV, Schuette WH: NADH flurescence and [K+], changes during hippcampal electrical stimulatin. J Neurphysil 38: , Lthman E, LaManna JC, Crdingley G, Rsenthal M, Smjen G: Respnses f electrical ptential, ptassium levels, and xidative metablic activity f the cerebral necrtex f cats. Brain Res 88: 15-36, Schutz H, Silverstein PR, Vapalahti M, Bruce DA, Mela L, Langfltt TW: Brain mitchndrial functin after ischemia and hypxia. II. Nrmtensive systemic hypxemia. Arch Neurl 29: 417^19, Schwartz JP, Mrsulja BB, Mrsulja BJ, Passnneau JV, Klatz I: Alteratins f cyclic nucletide-related enzymes and ATPase during unilateral ischemia and recirculatin in gerbil cerebral crtex. J Neurchem 27: , Marshall LF, Welsh FA, Durity F, Lunsbury R, Graham DI, Langfltt TW: Experimental cerebral ligemia and ischemia prduced by intracranial hypertensin. Part 3: Brain energy metablism. J Neursurg 43: , Nrdstrflm C-H, Rehncrna S: Pstischemic cerebral bld flw and xygen utilizatin rates in rats anaesthetized with nitrus xide r phenbarbital. Ada Physil Scand 101:

10 686 STROKE VOL 12, N 5, SEPTEMBER-OCTOBER , Sundt TM Jr, Waltz AG: Cerebral ischemia and reactive hyperemia. Studies f crtical bld flw and mictcirculatin befre, during, and after temprary cclusin f middle cerebral artery f squirrel mnkeys. Circ Res 28: , Rsenthal M, La Manna JC: Effect f uabain and phenbarbital n the kinetics f crtical metablic transients assciated with evked ptentials. J Neurchem 24: , Diemer NH, Siemkwicz E: Increased 2-dexyglucse uptake in hippcampus, glbus pallidus and substantia nigra after cerebral ischemia. Acta Neurl Scandinav 61: 56-63, Levy DE, Duffy TE: Cerebral energy metablism during transient ischemia and recvery in the gerbil. J Neurchem 28: 63-70, 1977 A Case fr Cerebral Thrmbangiitis Obliterans JOSE BILLER, M.D., JORGE ASCONAPE, M.D., VENKATA R. CHALLA, M.D., JAMES F. TOOLE, M.D., AND WILLIAM T. MCLEAN, M.D. SUMMARY The existence f cerebral thrmbangiitis bliterans (CTAO) has been cntrrersial. The clinical, labratry and angigraphic features f a yung wman with recurrent thrmbphlebitis, digital gangrene and a bilateral anterir percular syndrme (Fix-Charany-Marie) are reprted. The cerebral angignun demnstrated significant narrwing f frnt-percular branches f bth middle cerebral arteries. Histlgy f small digital muscular arteries rerealed segmental adventitial flbrsis, narrwing r cclusin f lumen and mild lyraphcitic infiltrates; ccasinal reins shwed phlebitis. An etilgic relatinship between cerebral cclusire disease and peripheral thrmbangiitis bliterans (TAO) is suggested. Strke, Vl 12, N 5, 1981 IN 1879, Vn Winiwarter 1 published an accunt f angipathy in a 57-year-ld man with ft gangrene; he named the angipathy endarteritis bliterans. Buerger, 3 in 1908, designated the cnditin thrmbangiitis bliterans (TAO). Spatz, 8 and Spatz and Lindenberg, 4 in 1939, published cmprehensive accunts f the cerebral frm f TAO (CTAO) distinguishing 2 types accrding t distributin f the lesins. There is a cntrversy in recent literature""" n whether TAO and CTAO are distinct clinicpathlgic entities. A yung wman affected by recurrent thrmbphlebitis, digital gangrene and a Fix-Chavany-Marie (bipercular) syndrme* is reprted as representative f the assciatin between cerebral and peripheral TAO. These bservatins buttress the present trend in angilgy t cnsider TAO and CTAO as distinctive but rare entities. Patient Histry MOC, a 33-year-ld black wman, was admitted t the hspital n August 31, 1980, because f sudden inability t talk and swallw. She had been in gd health until 1966 when she had a thrmbphlebitis in her left leg. One year later she develped a similar episde. In 1968 she had a right deep vein thrmbsis. In 1973, at the age f 26, she suddenly had left Frm the Department f Neurlgy and the Department f Pathlgy, (Sectin f Neurpathlgy) (Dr. Challa), Bwman Gray Schl f Medicine f Wake Frest University, 300 S. Hawthrne Rd., Winstn-Salem, NC This wrk was supprted in part by NIH Grant N. NS Reprints: Dr. McLean. hemiparesis. Radinuclide brain scan at the time shwed increased uptake ver the right parietal regin cnsistent with an area f infarctin. Bilateral cartid angigraphy revealed mild segmental narrwing f several f the left perculfrntal branches and an area f "luxury perfusin" ver the right parietal frntal regin. Rutine bld tests and CSF examinatin were nrmal. Electrcardigram and echcardigram were nrmal. Electrencephalgram shwed mild slwing ver the left hemisphere. During the ensuing weeks she gradually recvered frm her hemiparesis. During the next year she had numerus episdes f pain in her fingers and tes aggravated by cld weather and/r water. A selective left brachial angigram revealed multiple cclusins and n pacificatin f the interdigital arteries (fig. 1A). A femral angigram was unremarkable dwn t the level f the ankle but failed t visualize any vessel beynd that area. Due t recurrent episdes f painful digital ischemia, she required a right transmetatarsal amputatin f the left ring, right index finger, left furth te, and left big te. Histlgic examinatin f the amputated digits revealed segmental invlvement f small muscular arteries several f which shwed narrwing r cclusin f lumens (fig. IB) mainly due t intimal hyperplasia and sparse mnnuclear infiltratin. N evidence f atherma r calcificatin was seen in these vessels. Nne f these arteries shwed fresh thrmbses r vasculitis with fibrinid necrsis. Occasinal veins shwed phlebitis. Fcally invlved vessels were surrunded by cncentric layers f increased cnnective tissue. In Nvember, 1979 she had a bilateral lumbar sym-