motor performance in rats exposed to severe forebrain ischemia

503

Motor Performance in Rats Exposed to SevereForebrain Ischemia: Effect of Fasting and

1,3-Butanediol

David J. Combs and Louis G. D'Alecy

Functional assessment of animals following experimental cerebral ischemia is often difficult due to thepassive nature of many neurologic exams. We attempted to increase the objectivity of motor functionevaluation by adapting quantifiable behavioral tests and actively testing rats' motor capability follow-ing a cerebral ischemic insult. It was hypothesized that active testing would reveal motor deficits whichwere not readily apparent upon casual observation and that such testing would provide a moresensitive means of experimental neurologic assessment. Wistar rats were exposed to reversible severeforebrain ischemia using the four-vessel occlusion technique. Motor function was evaluated using thetotal motor score (sum of scores for screen test, balance beam test, and prehensile-traction test) overthe 48 hours which followed 20 minutes of cerebral ischemia. To manipulate neurologic outcome, ratswere fed or fasted the day prior to ischemia and then pretreated with either 1,3-butanediol or saline.Fasted saline-treated animals demonstrated improved total motor performance compared with fedanimals by 48 hours after ischemia. There was no improvement in motor performance by fasted vs.fed rats from among the butanediol-treated animals. Pretreatment with butanediol resulted in signifi-cantly better total motor performance among fasted rats 24 hours after ischemia; however, by 48hours postischemia, no difference was detectable. This is the first demonstration of motor deficitsproduced by four-vessel occlusion in rats. The motor tests devised appear to be adequately sensitive todetect changes in motor function that are not apparent with passive observation in this model. (Stroke1987;18:503-511)

A TTEMPTS to quantify neurologic recovery have/ \ been made by numerous investigators in a

J. A . wide variety of species.1"7 Subjectivity is aparticular problem in neurologic assessment often dueto the passive nature of the testing. We have attemptedto increase the objectivity of motor function evaluationby adapting quantifiable behavioral tests and activelytesting the motor capabilities of rats exposed to a cere-bral ischemic insult. We hypothesized that active test-ing would reveal motor deficits which were not readilyapparent to casual observation, and that such testingwould provide a more sensitive means of experimentalneurologic assessment.

The four-vessel occlusion (4-VO) model was devel-oped by Pulsinelli and Brierley8 and has been heavilyscrutinized with regard to its histologic, metabolic,and blood flow characteristics.9"16 With the exceptionof the work on learning and memory by Volpe et al,17

little has been published on the functional deficits pro-duced in this model.

To test the sensitivity of the motor tests in differenti-ating between animals of varying levels of impair-ment, it was necessary to attempt modification of the

From the Departments of Physiology and Surgery, The Universi-ty of Michigan, Ann Arbor, Michigan.

Supported in part by the Department of Surgery and the AnnArbor Veterans Administration Hospital.

Address for reprints: Louis G. D'Alecy, 7703 Medical ScienceII, Department of Physiology, The University of Michigan MedicalSchool, Ann Arbor, MI 48109.

Received September 17, 1985; accepted October 20, 1986.

neurologic outcome from cerebral ischemia. To ac-complish this, animals were manipulated as to theirnutritional status prior to the ischemic insult. Fed orfasted rats were pretreated just prior to ischemia withsaline or the ketogenic alcohol 1,3-butanediol (BD).These treatments were chosen because fasting has beenshown to favorably influence the outcome of cerebralischemia18"20 and BD treatment has been shown to havesignificant protective effects in hypoxia and ischemic-hypoxic insults.2122

Materials and MethodsWe adapted the 4-VO model814 using male Wistar

rats (Charles River Breeding Farms) that weighed230-290 g and were maintained in a 12-hour light-darkcycle in quarters on standard rat chow and water. Therats were anesthetized with sodium pentobarbital (65mg/kg i.p.) and supplemented (i.p. or i.v.) as needed.The right and left common carotid arteries were isolat-ed and cardiovascular silk (5-0) was passed throughunderlying musculature and around each carotid ar-tery, carefully excluding the vagus nerve from thesnare. Each snare exited the wound site through a 2.5cm long guide tube (PE-50); the free ends of each snarewere knotted to prevent the guide tube or the snarefrom being displaced. The left jugular vein was cathe-terized, and the cannula exited at the nape of the neck.The tail artery was cannulated with a 5 cm long can-nula (PE-50 tubing) filled with heparinized saline (100U/ml). Elastic surgical tape coated with quinine wasused to close the tail wound and to cover the catheter.

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504 Stroke Vol 18, No 2, March-April 1987

The vertebral arteries were electrocauterized (BantamBovie Unit) through the alar foramen. EEG leads, con-sisting of self-tapping 3/16-in. stainless steel screwssilver-soldered to 30-gauge multistranded stainlesssteel wire (5-7 cm long), were inserted into 3 holes inthe skull and insulated from surrounding tissue withdental acrylic. Immediately after surgery, each rat wasgiven physiologic saline (0.5 ml/100 g i.v.) to providepostsurgical hydration and placed in individual cageswith free access to water throughout the remainder ofthe experiment. Fasted rats received no food until afterthe ischemic insult, whereupon food was provided forthe remainder of the experiment. Fed groups had ac-cess to food at all times. After surgery, all rats werehoused in a 12-hour light-dark cycle facility. The isch-emic insult occurred 22-26.5 hours after the last anes-thetic supplement.

Approximately 30 minutes before 4-VO, rats re-ceived an i.p. injection of either 47 mmol/kg BD (50%v/v BD in water, 8.43 ml/kg) or an equivalent volumeof physiologic saline. Five to 10 minutes before theinduction of cerebral ischemia the tail artery cannulawas connected to a pressure transducer (StathamP23Dc). The cannula was kept patent by flushing withheparinized saline (100 U/ml); the total amount of hep-arin given to any rat never exceeded 75 units. The EEGleads were connected to an amplifier (Grass Model7P511G) and displayed on an oscillograph (GrassModel 7 polygraph).

Induction of 4-VOFor the induction of 4-VO the rat was restrained by

hand, and the carotid arteries were occluded by placingtraction on the snares and securing the snares withbulldog clamps. Once the animal became uncon-scious, a thermistor probe (Yellow Springs Instru-ments Model 402) was inserted 8 cm into the rectum toallow an initial measurement of body temperature ap-proximately 2 minutes into the insult. A heat lamp wasused to maintain body temperature at this value. Thenthe rat's tail was tapped to test the righting response.The righting response was also tested just before theend of the ischemic insult.

Approximately 3 minutes before the end of 4-VO,2% lidocaine was injected into the wound site aroundthe guide tubes. After 20 minutes of carotid arteryocclusion the bulldog clamps were removed as werethe guide tubes and snares. The wound was quicklyreopened and reestablishment of flow confirmed bylooking at and manipulating each carotid artery. Thewound was then closed with wound clips, and the rat'stemperature, EEG, and blood pressure were monitoredfor the 2-hour postischemic period (the carotid arteriesof responsive rats [see "Results"] were not checkeddue to excessive movement). The rat was then returnedto its individual cage and allowed free access to foodand water.

Any rats with unabolished or returning EEG activityduring 4-VO were eliminated from the study as wererats that died of respiratory failure during the ischemicinsult. Any rats that died from seizures were not in-

TaWe 1. Experimental Protocol

Day 1 — Surgery

Pentobarbital anesthesia

Carotid artery snares

Vertebral artery occlusions

EEG leads

Blood vessel cannulas

Day 2 — Four-vessel Occlusion (4-VO)

Preischemic motor tests

Screen test

Balance beam test

Prehensile-traction test

Drug treatment (approx. 30 minutes before 4-VO)

Induction of 4-VO (22-26.5 hours after anesthesia)

Day 3 — 24 Hours after 4-VO

Motor tests

Screen test

Balance beam test


Day 4 — 48 Hours after 4-VO

Motor tests

Screen test

Balance beam test


Carotid check (halothane anesthesia)

eluded in the study. After the 48-hour neurologic as-sessment, the rats were placed under halothane anes-thesia, and their carotid arteries were visually checkedfor patency; a clogged carotid artery resulted in remov-al from the study.

Experimental ProtocolThe experiment took place over 4 days. The events

of each day are listed in Table 1 to indicate the timingof the neurologic tests relative to all other aspects ofthe experiment. To limit the influence of chance orobserver bias each of the motor tests was given twiceon each occasion, and the best performance was cho-sen as the rat's score. A 2-3 minute rest period be-tween each type of motor test was given to minimizeany fatigue factor. All tests were carried out in a quietarea under subdued lighting. The scoring of the motorperformance tests is described in Table 2.

SCREEN TEST. This test is a variation of the inclinedscreen test used for mice and rats and serves as anindicator of general muscle strength.2324 We used a29 x 30 cm screen that could be rotated from 0° (hori-zontal) to 90° (vertical). Grid size of the screen was0.6 x 0.7 cm. The screen was situated such that whenin the vertical position, it was 70 cm above a foamrubber pad (7.5 cm thick). Each rat was placed on thehorizontally positioned screen. The screen was thenrotated to the vertical position, and time on the screenwas recorded to a maximum of 15 seconds.

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Combs and D'Alecy Functional Assessment After Cerebral Ischemia 505

Table 2. Scoring Codes for Individual Motor Tests

Score Screen Balance beam Prehensile-traction

0 Falls off in 0-5 seconds



3 Stays on screen full 15seconds

Unable to maintain grip or balance on woodenbeam

Remains on beam up to 10 seconds

Remains on beam 11-20 seconds

Remains on beam 21-30 seconds

Hangs on 0-2 seconds

Hangs on 3—4 seconds

Hangs 5 seconds; no third limb up to rope

Hangs on 5 seconds; brings rear limb up to rope

All measured times were rounded to the nearest whole second. The best time of 2 trials determined the coded score for all tests during eachtesting session. Scores of 3 were assigned to animals that climbed to the top of the screen, walked to the balance beam supports, or climbed tothe prehensile-traction rope supports.

BALANCE BEAM TEST. This test serves as an indicatorof equilibrium, although body strength becomes themajor factor tested once the rat loses its balance.23 Anunfinished wooden rod (2.5 cm diameter) was posi-tioned horizontally 40 cm above a foam pad (7.5 cmthick). The rod was supported by clamps 70 cm apart.The rat was placed at the center of the wooden rod, andthe time to falling off was recorded to a maximum of30 seconds.

PREHENSILE-TRACTION TEST. This test is adapted fromsimilar tests in mice.2326 The prehensile portion of thetest involves the rat's ability to hang onto a horizontalrope by its forepaws and is a measure of musclestrength. The traction portion involves the animal'sability to bring one of its rear limbs up to the rope anddepends on equilibrium and muscle strength. A 0.5 cmdiameter nylon cord was placed horizontally 70 cmabove a foam rubber pad (7.5 cm thick), A rat's fore-paws were placed on the rope, and the animal wasreleased. The rat was allowed to hang onto the rope forup to 5 seconds. Time to falling was noted as well aswhether or not the rat brought a rear limb up to therope.

TOTAL MOTOR SCORE. This score is the sum of thescores for the screen, balance beam, and prehensile-traction tests, and therefore ranges from 0 to 9.

Treatment GroupsManipulation of the animals' nutritional state and

the drug treatment produced 4 groups. Rats that wereeither fed or fasted before cerebral ischemia receivedeither BD (47 mmol/kg i.p.) or an equivalent volumeof physiologic saline about 30 minutes prior to 4-VO.The resulting experimental groups, therefore, werefasted-saline, fasted-BD, fed-saline, and fed-BD.Since some of the fasted-saline and fed-saline rats re-sponded differently (details given in "Results") to 4-VO, responsive and unresponsive categories were des-ignated within both of these groups and are comparedin "Results."

To control for the effect of surgery, nutritional state,and drug treatment on motor performance, 4 controlgroups were tested: fasted-saline, fasted-BD, fed-sa-line, and fed-BD (n = 3 for each group). These ani-mals received the full surgical procedure. The nextday, they were given a preischemic neurologic exami-nation, treated with an appropriate i.p. injection, andabout 30 minutes later had the guide tubes removed

from the neck wound without occluding the arteries.These rats were then returned to their cages and treatedin the usual manner.

Statistical AnalysisBoth parametric and nonparametric statistical tests

were used and are described in "Results."Responsive and unresponsive rats within both the

fasted-saline and fed-saline groups were compared us-ing Student's t test for all variables except the motorperformance scores (Kruskal-Wallis test for rankdata). Statistical significance was accepted for p<0.05.

Physiologic data such as blood pressure, body tem-perature, and EEG return time were compared amongthe 4 experimental groups using one-way analysis ofvariance (ANOVA). When ANOVA indicated a sig-nificant difference (p <0.05), further analysis wasdone with Student's t test using a Bonferroni correctionfactor for a error protection. In this case, only 4 specif-ic comparisons were made to examine the effects ofdrug and nutritional status: fasted-saline vs. fasted-BD, fed-saline vs. fed-BD to examine the effects ofBD; fasted-saline vs. fed-saline, fasted-BD vs. fed-BDto test the effect of nutritional status. Since no groupwas involved in more than 2 comparisons, a differencewas considered significant at the 5% level ifp < 0.05/2or/j < 0.025.

Motor performance data for all 4 control groupswere compared using the Kruskal-Wallis test forranked data. Since the testing indicated no significantdifferences among these control groups (carotid notoccluded) for any motor performance variable, theywere combined. To test for changes in performanceover 3 days of motor testing, the 24- and 48-hourpostischemic total motor scores of the combined con-trol group were compared to the preischemic perform-ance using the Wilcoxon paired rank test, and a errorprotection was provided by acceptingp < 0.025 for the5% significance level.

Individual groups were compared using the Krus-kal-Wallis test to examine motor performance datafrom experimental groups. Three comparisons weremade for each group: one to test the effect of BD, oneto test the effect of preischemic nutritional status, andone to compare with appropriate operated controls. Toaccount for multiple comparisons, a error protectionwas provided. Since any experimental group was in-

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volved in 3 comparisons, a comparison was consideredsignificant at the 10% level only when p <O.O33.

ResultsSix of the 48 rats exposed to 4-VO (12.5%) failed to

show a fully abolished EEG tracing during cerebralischemia. Four rats (8.3%) died of respiratory failureduring the ischemic insult, while 1 rat (2.1%) died ofseizures between 24 and 48 hours after ischemic in-jury. Two animals were eliminated because they failedto meet the criterion of 2 observably patent carotidarteries at the 48-hour carotid check.

Among the saline-treated rats (fasted or fed) ex-posed to ischemia, 2 subgroups were observed: re-sponsive and unresponsive. Unresponsive rats showedabolished EEG activity for the full duration of theischemic period, no response to tail tap at either thebeginning or end of 4-VO, and no significant move-ment throughout the ischemic period. Responsive rats,in contrast, maintained isoelectric EEG but eithershowed a remnant of the righting response to one of thetail taps or moved significantly during the insult; sig-nificant movement was considered spontaneous right-ing with or without transient running motion, or re-verse rolling (i.e., the rat rolled over in a directionopposite to that expected for righting). BD-treated ratswere all unresponsive. Unresponsive fasted saline-treated rats showed no significant differences in phys-iologic or neurologic parameters when compared withresponsive fasted saline-treated rats, and they werecombined to form the fasted-saline group. A compari-son between unresponsive fed saline-treated (UFS)rats and responsive fed saline-treated (RFS) rats wasalso made. Only one significant difference was found,for blood pressure 10 minutes into the ischemic period.UFS rats had a higher blood pressure (156 mm Hg)than RFS rats (142 mm Hg; p = 0.03, Student's t).

Given the similarity of the 2 groups, the UFS and RFSrats were combined to form the fed-saline group.

Physiologic DataThe physiologic data were analyzed by ANOVA

followed, when appropriate, by multiple Student's ttests using the Bonferroni correction factor for a errorprotection. Four experimental group comparisonswere made for the physiologic data: fasted-saline vs.fasted-BD, fed-saline vs. fed-BD, fasted-saline vs.fed-saline, and fasted-BD vs. fed-BD.

In Figure 1, blood pressure increased markedly dur-ing ischemia and returned to control levels after ische-mia. ANOVA detected no differences among the 4groups at either control or postischemic time points.Ten minutes into ischemia, blood pressure in the fed-saline group was significantly greater than in either thefasted-saline or fed-BD group. ANOVA indicated thatat 20 minutes of ischemia there was a significant dif-ference among the means; however, none of the 4comparisons of interest were significant by Student's ttest.

ANOVA with accompanying Student's t tests re-vealed that fasted-saline rats had a significantly higherbody temperature (38.7 ± 0.1° C) than did fasted-BDrats (37.6 ± 0.2° C; p <0.05, Student's t test). Thesame difference was observed among the fed animals,with fed-saline rats showing a greater body tempera-ture (38.8 ±0.1° C) than fed-BD rats (38.1 ± 0.1° C;p<0.05, Student's t test). No significant differencesexisted between fed and fasted rats within each drugtreatment. No significant differences were found in theEEG return data, with the overall average being 961seconds.

Motor PerformanceNo significant differences were found among the 4

operated control groups when analyzed by the Krus-

BLOOD PRESSURE

BLOOD PRESSURE(mmHg)

ISO

140

130

120

no

IOO

90- 3 0 - 1 5 0 IS 30 45 fcO 75 90

TIME (MINUTES)

105 120

FIGURE 1. Mean blood pressure before( — 20 minutes), during, and after 4-vessel oc-clusion (4-VO, shaded area). SEM bars areshown only for data points during the ischemicinsult (* significantly different from fasted-sa-line orfed-butanediol [BD] group, p <0.05,Student's t test with Bonferroni correction).

-O- FASTED SALINE (N-10)-•- FASTIDBD(W-«>

•O-FED SALINE 01*9)

••- FED JD<N«3>

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Combs and D'AUcy Functional Assessment After Cerebral Ischemia 507

TOTAL MOTOR SCORE - CONTROL RATS (N= 1 2)

90

80

70

60PERCENTAGE 5 0OF HATS IN

GROUT 40

30

20

10

0

90

SO

70

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PERCENTAGE 50OF RATS IN

OROUP 4 0

30

PREISCKEHIA

1 2 3

24 HAS POST

1 2 3 4 5 6 7 8 9TOTAL HOTORSCORI

FIGURE 2. Distribution of total motor scores for the combinedcontrol groups over the course of the experiment.

kal-Wallis test for each motor test variable. These 4groups were therefore combined and tested for changesin performance over the 3 days of testing. There wereno significant differences between preischemia per-formance level and the performance levels at either 24or 48 hours after sham ischemia in any individual per-formance test (screen, balance beam, prehensile-trac^tion tests, and total motor score). The distributionamong total motor scores for all of trie groups is shownin Figure 2. These results indicate that neither thesurgical procedure, the nutritional status prior to ische-mia, nor the drug treatment accounted for the changesin performance observed after ischemia in the experi-mental rats.

Fasted-Saline vs. Fasted-BDThe fasted-BD rats showed no significant differ-

ences in total motor score compared with their operat-ed controls at any time after ischemia. The fasted-saline rats were different at the 10% level from theiroperated controls 24 hours after ischemia (p = 0.01,Kruskal-Walks) but not at 48 hours. While no differ-ence was detected between groups before 4-VO, therewas a difference significant at the 10% level 24 hours

after ischemia (Figure 3). The fasted-saline rat distri-bution was considerably further down the coded scoreaxis than the fasted-BD group distribution, indicatingbetter performance by the fasted-BD group. This dif-ference, however, was gone by 48 hours.

Fed-Saline vs. Fed-BDOverall motor performance in fed-BD rats was re-

duced below that of their operated controls at 24 hoursafter 4-VO (p = 0.02, Kruskal-Wallis) but becamestatistically equivalent by 48 hours. However, totalmotor performance among fed-saline rats remainedsignificantly below that of their operated controlsthroughout the postischemia phase of the experiment(p = 0.01 at both 24 and 48 hours postischemia,Kruskal-Wallis). There was no significant differencebetween the 2 groups (Figure 4). It does appear that thefed-BD rats showed greater improvement by 48 hourspostischemia; however, the p value of 0.09 does notallow the improvement to be considered significant atthe 10% level.

TOTAL MOTOR SCORE - FASTED RATS

PERCENTAGEOF RATS INGROUP

PERCENTAGEOr RATS INGROUP

PERECENTAGEOr RATS INGROUP

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p-O.OCH*

In n1 2 3 4 5 6 7 9

46 HRS POST

n • In0 1 2 3 4 5 6 7 8 9

TOTAL HOTORSCORI

| O TASTED SALINT(N-ll) B TASTED BD (N-6)|

FIGURE 3. Distribution of total motor score for fasted-salinevs. fasted-butanediol (BD) rats over the course of the experi-ment (p value shown for between-group comparison, * signifi-cant at the 10% level).

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TOTAL MOTOR SCORE - FED RATS

70

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PERCENTAGE 4 0OF RATS IN

CROUP M

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GROUP 30

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I iI 2 3

24 HRS POST

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p>0 09

TOTAL HOT OR SCORE

| D FEP SALINE ( N ' U ) H rEDBD(H-6) |

FIGURE 4. Distribution of total motor score for fed-saline vs.fed-butanediol (BD) rats over the course of the experiment (pvalue shown for between-group comparison).

Fasted-Saline vs. Fed-SalineOverall motor performance of the fed-saline group

was significantly depressed below that of their controlsthroughout the 48-hour recovery phase. The differencebetween fasted-saline and their controls was signifi-cant at 24 hours but not at 48 hours after 4-VO. Per-formance fell similarly in the 2 groups 24 hours after 4-VO (Figure 5). By 48 hours after the insult, however,the fasted-saline rats performed significantly betterthan the fed-saline animals.

Fasted-BD vs. Fed-BDOnly the fed-BD rats were significantly worse in

total motor score than their respective controls (p =0.02, Kruskal-Wallis) 24 hours after 4-VO. Neithergroup was statistically distinguishable from their con-trols at 48 hours postischemia. The overall motor per-formance of these 2 groups is illustrated in Figure 6.Although the motor performance of fasted-BD ratsappears to be better than that of fed-BD rats 24 hoursafter 4-VO, this difference was not significant.

DiscussionInterpretation of Changes in Total Motor Score

We have attempted to increase the objectivity ofmotor function evaluation by adapting quantifiable be-havioral tests and actively testing the abilities of the ratbefore and after cerebral ischemia. One advantage ofactive testing over passive observation may be thatdeficits which are not necessarily obvious on simpleobservation are uncovered. With the exception of thework on learning and memory by Volpe et al,17 littlehas been done on the neurologic deficits produced bythis model, perhaps because the deficits seemed rela-tively mild or nonexistent. Indeed, Pulsinelli et al27

described fasted saline-treated rats 24 hours after 20minutes of 4-VO as "No animal pretreated with salineconvulsed, and all walked well with no signs of neuro-logic dysfunction at 24 hours." While fasted saline-treated rats in our study also looked normal on casualobservation, active testing indicated that motor per-formance was seriously impaired 24 hours after 4-VO,and that by 48 hours postischemia there was still atrend indicating that performance was below control

TOTAL MOTOR SCORE - SALINE RATS

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PERCENTAGE 4 0

OF ANIMALSIN GROUP 5 0

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OF RATS INGROUP JO

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0R0U? S O

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2 4 HRS POST

_Q m n.0 1 2 3 4 5 6 7 8 9

48 HRS POST

p-0.01

l n Q i0 1 2 3 4 5 6 7 8 9

TOTAL MOTOR SCORE

| D TASTED SALIWE(N-ll) D rED SALINI <N'l 2) |

FIGURE 5. Distribution of total motor score for fasted-saline vs.fed-saline rats over the course of the experiment (p value shownfor between-group comparison, * significant at 10% level).

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TOTAL MOTOR SCORE - BD RATS

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50PERCENTAGE « 0

OF RATS INGROUP 3 °

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GROUP 30

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i48HRSPOST

a10 1 2 J 4 5 6 7 8 9

TOTAL MOTOR SCORE

|E2 TASTED BP(N-6) S rEDBD<N-6) |

FIGURE 6. Distribution of total motor score for fasted-butane-diol (BD) vs. fed-BD rats over the course of the experiment (pvalue shown for between-group comparison).

levels. The fasted-BD group was the only one that didnot fall significantly below its respective control intotal motor score 24 hours after ischemia. Experimen-tal groups manifested varying degrees of improvementby 48 hours postischemia. The fact that total motorscore could discern both deteriorations and improve-ments in motor performance suggests that this param-eter is more sensitive than casual observation. Al-though the scores probably reflect decreased motorcapability, motivational considerations cannot beeliminated as a factor contributing to rat performance.

Total motor score followed a distinct pattern aftercerebral ischemia with a sharp drop after 4-VO, fol-lowed by improvement by 48 hours after the ischemicinsult. Whether the drop in performance representsactual cerebral ischemic damage or a diaschisis-likefunctional depression is not known. Recovery mayrepresent a dual-phase process involving reemergenceof function from depressed areas or recovery throughneural reorganization. The rapidity of recovery makesreorganization unlikely,28 and this tends to favor thehypothesis that improved motor performance repre-sents reemergence of functionally depressed neurons.

The rapid onset of improvement is particularly surpris-ing in view of a study which indicated that histologi-cally evaluated neuronal damage progresses between24 and 72 hours in rats exposed to 30 minutes of 4-VO." Clearly correlative studies of neuronal changeswith motor recovery over more extended periodswould address many of these questions. It is possiblethat a shorter time course of motor recovery will befound to indicate a smaller lesion.

The 4-VO ModelAn observation, which to our knowledge is unique,

deals with the criteria used to define an acceptable 4-VO preparation. According to Pulsinelli et al,29 theonly animals included for analysis are those that "be-come unresponsive and completely lose their rightingreflex for the duration of carotid artery occlusion;"they tested the righting reflex just after carotid occlu-sion and just before the release of the occlusion. Thereflex was tested using the tail or hind paw flick whilethe animals were on their right and left sides. Thesecriteria seem rigid, except that the meaning of com-plete loss of righting reflex is not clear. In anotherstudy from the same laboratory, loss of the rightingreflex referred to the loss of the "ability to arise from arecumbent position,"30 thus allowing some range ofmovement as long as the rat does not arise from recum-bency. In the data reported here, tail tap and EEGhelped define classification and rejection criteria. Al-though all rats in the study maintained isoelectric EEGduring the insult, saline-treated rats fell into responsiveand unresponsive categories (see "Results"). The factthat responsive rats maintained isoelectric EEG im-plies that cortical blood flow must have been below thethreshold for failure of electrical neuronal function31

(functional threshold). Since the righting reflex doessurvive low decerebration,28 we deduce that subcorti-cal structures regained enough blood flow for rightingfunction to be maintained in the responsive animals.How great the disparity between forebrain blood flowin responsive vs. unresponsive groups cannot be di-rectly addressed. However, the difference was rela-tively minor in terms of the neurologic endpointsmeasured.

The BD-treated rats showed no such dispersion intoresponsive and unresponsive groups. A possible expla-nation for this is that cerebral function may have beendepressed by the intoxicating effect of BD and couldhave resulted in rats that had relatively higher cerebralblood flow but remained unresponsive nonetheless. Itcould be argued that BD may have helped rats "pass"the inclusion criterion of isoelectric EEG by these verysame depressive effects, thereby allowing rats withexceptionally high forebrain blood flows to remain inthe study. This is unlikely since there is evidence thatBD decreases the functional threshold for isoelectricEEG.22

Effects of Fasting and BD on Motor PerformanceFasting has been shown to be protective in cerebral

ischemia in terms of neurologic,18 metabolic,19 and

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cerebral blood flow20 indices. In this experiment, fast-ing was beneficial to neurologic recovery as well (Fig-ure 5). Physiologic considerations do not account forthe improved motor recovery, for neither cerebral per-fusion pressure nor body temperature favored the fast-ed-saline vs. fed-saline rats. Whether or not the 2groups would maintain their difference over more ex-tended periods was not addressed in this study.

As can be seen from Figure 3, fasted rats given BDhad better motor performance when compared withfasted rats given saline. This improvement was rela-tively mild in that by 48 hours postischemia, there wasno longer a significant difference between the fasted-BD and the fasted-saline group. While these results arenot as dramatic as the >500% increases in hypoxictolerance we observed earlier in hypoxic mouse stud-ies,21 it suggests that BD does, at least transiently,favorably alter the effects of cerebral injury. The fast-ed-BD rats did not benefit from higher cerebral perfu-sion pressure, but body temperature changes may havebenefited them in that their body temperature was sig-nificantly lower than that of their saline-treated coun-terparts. Hypothermia protects the central nervoussystem from hypoxia, hypoxia-ischemia, and isch-emia.32"35 Although no studies have demonstrated thata temperature difference of only 1.1° C is protective tothe brain during ischemia, a recent study on spinal cordischemia suggests that such changes in body tempera-ture can be beneficial to recovery of function in thecentral nervous system.33 As a result, we cannot ruleout the possibility that lower body temperature ac-counted for the improvement in motor performanceprovided by BD. It is also possible that the sedativeeffects produced by BD contributed to its effective-ness; however, the protection conferred by BD in thehypoxic survival model in mice21 could not be fullyexplained by its sedative effects since similar doses ofBD and ethanol, an alcohol with similar central ner-vous system effects,3* did not produce the same in-creases in hypoxic survival time.37 The ability of BD toinduce ketosis may have also contributed to its abilityto alter cerebral ischemic outcome.1'2122

In summary: The motor tests designed for these ex-periments appear to be more sensitive than passiveobservation in detecting changes in the level of motordeficits in rats after 20 minutes of 4-VO. This moresensitive testing was able to detect both increases anddecreases in motor function that were not apparent oninspection. Fasting significantly improved perform-ance among saline-treated rats, supporting the obser-vations of other investigators. Fasted rats given BDalso had an improved time course of recovery in thatthe return of function occurred earlier than in the sa-line-treated animals.

AcknowledgmentsWe would like to express our gratitude to Jan Dyk-

stra and Karla Barton for their excellent technical helpand to Daniel Reuland and Eugene Weng for theirassistance. We are also in the debt of Dr. Gerald Zelen-ock for his support and advice during this project.

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KEY WORDS • cerebral ischemia • motor deficit • fast-ing • 1,3-butanediol • rat

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D J Combs and L G D'Alecy1,3-butanediol.

Motor performance in rats exposed to severe forebrain ischemia: effect of fasting and

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