Brain Research, 561 (1991) 106-119 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-89931911503.50 ADONIS 000689939117037E

106

BRES17~7

Dynamic changes in local cerebral glucose utilization following cerebral concussion in rats: evidence of a hyper- and subsequent hypometabolic state Atsuo Yoshino, David A. Hovda, Tatsuro Kawamata*, Yoichi Katayama* and Donald P. Becker Division of Neurosurgery, UCLA School of Medicine, University of California at Los Angeles, Los Angeles, CA 90024-6901 (U.S.A.) (Accepted 21 May 1991) Key words: Concussion; Metabolism; Glycolysis; Hypermetabolism; Hypometabolism; Autoradiography; Excitatory amino acid; Ion flux

Following cerebral concussion, in which there is no evidence of direct morphological damage, cells are exposed to an increase in extracellular potassium as well as an accumulation of calcium. This concussion-induced ionic flux most likely alters the cellular energy demands thereby modifying metabolic processes. To investigate the metabolic changes after cerebral concussion, local cerebral metabolic rates for glucose (1CMRglc) utilizing [t4C]2-deoxy-D-glucose were studied in rats (n = 98; 250-300 g) immediately, 30 min, 6 h, 1, 2, 3, 5 and 10 days following a unilateral frontoparietal fluid percussion (F-P) injury (3.7-4.3 atm). Compared to sham controls, animals exhibited bilateral hypermetabolism immediately following brain injury. However, this effect was more pronounced in structures ipsilateral to the site of F-P and was especially marked for the cerebral cortex (46.6-30.1% higher than control) and hippocampus (90.1-84.4% higher than control). By 30 min post-trauma many ipsilateral regions still showed evidence of hypermetabolism, although their 1CMRglc had subsided. Beginning as early as 6 h following injury many regions within the ipsilateral cortex and hippocampus went into a state of metabolic depression (16.433.7% of control) which lasted for as long as 5 days. These results indicate that, although not mechanically damaged from the insult, cells exposed to concussive injury dramatically alter their metabolic functioning. This period of post-concussive metabolic dysfunction may delineate a period of time, following injury, during which cells are functionally compromised. INTRODUCTION Cerebral concussion is defined as a closed head injury characterized clinically by an immediate and transient impairment of neural function such as loss of consciousness, and equilibrium as well as amnesia in the absence of overt morphologic brain damage 4'5'36. Historically, there have been two main theories regarding the mechanisms responsible for the i m p a i r m e n t of neural function following concussive brain injury. O n e invokes the concept of neural depression 1° and the o t h e r neural excitation sS. The localization of dysfunction following concussion has been classically attributed to a depression of the cerebral cortex 3s, although, recent work 19 indicates that neurological symptoms may develop via an injury-induced activation of brainstem regions. A t the cellular level, cerebral concussion results in the transient increase of extracellular potassium (K÷) 11'27 and glutamate 13'24"27'37 as well as an accumulation of cal-

cium (Ca2+) 8'16'26"44. O f

the mechanisms p r o p o s e d for

this injury-induced ionic shift, neural firing and excitatory amino acid ( E A A ) stimulation of ligand-gated ionic channels a p p e a r to be the most plausible 27. This ionic flux, particularly that of K +, exhibit a profile similar to that seen for spreading depression 9'32'33'36'46. In general, ionic fluxes have been associated with an increase in glucose utilization 49 adhering to a p r o p o s e d concept of energy compartmentalization where glucose appears to be the fuel of choice to provide the necessary energy to maintain ionic m e m b r a n e balance 1'2. Therefore, given the dramatic ionic shifts seen following concussive brain injury, m a r k e d alterations in glucose metabolism would be expected. A few studies have addressed this question of glucose utilization following concussive brain injury. In the cat, glucose metabolism was m e a s u r e d one hour following a fluid percussion (F-P) brain injury using [14C]2-deoxy-Dglucose (2-DG) 49. The results indicated an increase in

* Present address: Department of Neurological Surgery, Nihon University, School of Medicine, 30-10yaguehi-Kamimachi Itabash-ku Tokyo 173, Japan. Correspondence: D.A. Hovda, Division of Neurosurgery, UCLA School of Medicine, CHS 74-140, University of California at Los Angeles, Los Angeles, CA 90024-6901, U.S.A. Fax: (1) (213) 206 3732.

107 g l u c o s e utilization p r i m a r i l y w i t h i n r e g i o n s o f t h e brains t e m (dorsal p o n t i n e t e g m e n t u m ) w i t h o t h e r structures s h o w i n g a m o d e r a t e d e p r e s s i o n 19'2°. H o w e v e r , in a m o r e r e c e n t study m e a s u r i n g a r t e r i a l - v e n o u s d i f f e r e n c e s foll o w i n g a F - P b r a i n injury, glucose utilization i n c r e a s e d by 243% as early as 5 m i n a f t e r t h e insult a n d t h e n gradually r e t u r n e d to n o r m a l 2 h p o s t - t r a u m a 2. In t h e rat, the effect o f 2 - D G u p t a k e has b e e n m e a s u r e d at 10 and 20 m i n f o l l o w i n g a F - P b r a i n injury 45. T h e results f r o m this study i n d i c a t e d t h a t 20 m i n f o l l o w i n g injury the upt a k e o f 2 - D G was n o t d i f f e r e n t f r o m c o n t r o l s , h o w e v e r , at 10 m i n p o s t - i n j u r y t h e u p t a k e was h i g h e r in c o r t e x and l o w e r in subcortical structures. O t h e r s h a v e also r e p o r t e d an i n c r e a s e in glucose utilization f o l l o w i n g a F - P b r a i n injury 5°. H o w e v e r , in this case t h e e n h a n c e d 2 - D G was r e s t r i c t e d to t h e c o r t e x a n d s e e n in o n l y a few animals (2 o u t o f 6) at 2 h p o s t - t r a u m a . T h e a b o v e studies diff e r e d b o t h in the species s t u d i e d a n d t h e d e g r e e o f in-

brief, after completion of the surgery, the animals were removed from the stereotaxic frame and loosely restrained with continuing anesthesia. The injury screw was connected to the saline-filled reservoir with rigid polyethylene tubing (i.d. 2 mm; 20 o n in length). A strain-gage transducer (Statham PA85-100) between the injury cylinder and injury screw measured the amplitude (3.7-4.5 atm) and duration (21-23 ms) of the fluid-pulse wave. After the animal was attached to the tubing, the anesthesia was terminated, and following a 60 s recovery period a transient pressure flnid-pulse was delivered to the epidural space. While transient apnea was typically induced at this injury level, systemic circulatory collapse was not observed. If apnea persisted for more than 10 s, respiration was mechanically supported with a mixture of oxygen and room air during the period of the apnea. If respiratory support was required for longer than 45 s the animal was excluded from the study. As control experiments, sham-injuries were conducted with procedures identical to those for the actual injury except for the administration of the flnid-pulse wave. With the exception of the acute experiments (immediately and 30 min following injury, see below), when animals recovered from unconsciousness (judged as when the animal exhibited a toe-pinch withdrawal reflex), inhalation of anesthetic gases was reinstated and the scalp sutured. All animals were monitored in a recovery cage for 6 h after surgery and, when stable, were returned to their home cage.

j u r y w i t h m e a s u r e m e n t s b e i n g r e s t r i c t e d to o n l y a few t i m e p o i n t s f o l l o w i n g injury. C o n s e q u e n t l y , a c o m p r e h e n s i v e study o f glucose utilization f o l l o w i n g t h e t i m e c o u r s e o f r e c o v e r y a f t e r a c o n c u s s i v e b r a i n injury rem a i n s to be c o n d u c t e d . Such a c o m p r e h e n s i v e study is critical since cells m a y b e in d i f f e r e n t states o f m e t a b o l i c r e c o v e r y d e p e n d i n g o n t h e t i m e p o s t - i n j u r y and s u b s e q u e n t l y w o u l d r e s p o n d differentially to p h a r m a c o l o g i c a l o r surgical i n t e r v e n t i o n d e p e n d i n g h o w s o o n f o l l o w i n g injury t h e y w e r e i m p l e m e n t e d . I n o r d e r to m e a s u r e m e t a b o l i c c h a n g e s o v e r the c o u r s e o f r e c o v e r y w e d e s i g n e d t h e f o l l o w i n g study to c a l c u l a t e t h e local c e r e b r a l m e t a b o l i c rates for glucose (1CMRglc; # m o l / 1 0 0 g / m i n ) at d i f f e r e n t t i m e s f o l l o w i n g a l a t e r a l F - P b r a i n injury in t h e rat. MATERIALS AND METHODS

Subjects Young male Sprague-Dawley rats (n = 98; 250-300 g) were studied immediately (n = 5), 30 rain (n = 5), 6 h (n = 4), 1 (n = 5), 2 (n = 4), 3 (n = 4), 5 (n = 5) and 10 days (n = 5) following a lateral F-B brain injury. At each time point sham-surgeries (n = 4) were conducted with procedures identical to those for the actual injury, except for the administration of the F-P.

Surgical preparations Following induction of general anesthesia (33% oxygen, 66% nitrous oxide and enflurane (1.5-2.0 ml/min)) the animals were placed in a stereotaxic frame and the scalp was sagittally incised. The rectal temperature was kept between 37.0 and 38.0 °C with a thermostatically controlled heating pad and all surgical wounds were infiltrated with local anesthesia (1% xylocaine). For administration of the F-P injury (see below) a craniotomy was made and a rigid plastic hollow screw (o.d. 6.5 mm; i.d. 4.5 nun) was secured over the exposed dura using cyanoaerylate and dental acrylic. The injury screw was positioned 1.0 mm posterior to bregma and 6.0 mm lateral (left) of the midline.

Procedures for injury induction The F-P injury has been previously described in detail 12"34. In

Local cerebral glucose utilization ICMRglc was determined by the 2-DG method as originally described by Sokoloff et al.49 with cannulation of the femoral artery and vein being conducted under general anesthesia. To assure that the cannulas remained in place for the 2-DG experiment animals were restrained on a cardboard plank using adhesive tape. For animals studied immediately following trauma 2-DG (150 #Ci/kg) was slowly administered (i.v.) over a period of 30 s beginning 30 s prior to the initiation of the F-P pulse. Anesthesia was not reinstated following injury in either the 'immediate' or the '30 rain' animals. In animals studied at 6 h, 1, 2, 3, 5 and 10 days following injury, cannulation was performed on the day when the animal was to be studied. These animals were allowed to recover from anesthesia for at least 3 h before injection of 2-DG. For all animals, timed arterial samples were collected throughout the 45 min experiment. Blood samples were immediately placed on ice, centrifuged, and the plasma assayed for t4C activity and glucose concentration. Forty-five minutes following the 2-DG injection a lethal dose of sodium pentobarbital was administered (100 mg/kg, i.v.). The brain was immediately removed, frozen in pulverized dry ice, coated with an embedding matrix, and stored at -80 °C until sectioning. Coronal (20 #m) frozen sections were processed for thionin histology with adjacent sections being processed for autoradiography together with calibrated 14C methaerylate standards. For quantification of the autoradiography optical density was measured in 25 regions of the brain using a computerized image analysis system (JAVA: Jandel Scientific). ICMRgic was calculated using the equation originally described by Sokoloff et al. 49.

Physiological parameters In order to determine the physiological state of the animals at the time of the study a final blood sample was taken just prior to euthanasia. Blood gas (pH POE, pCO2) measurements were made with a gas analyzer (1304 pI-I/BIood Gas Analyzer, Instrumentation Laboratory) and were found to be within the normal range. To evaluate the state of the animals during the administration of the F-P pulse, 6 rats were catheterized via the femoral artery as described above. Systemic arterial pressure was recorded continuously before and after brain injury. In addition, arterial blood gases (pH, pOE,pCO2) were analyzed at regular intervals throughout the experiment and were found to be within normal range. To determine if the F-P injury disrupted the blood-brain barrier permeability 6 additional rats were injected with Evans blue (25 mg/2.5% aqueous solution; i.v.) 30 s before injury (n = 3) and at 5 h following injury (n = 3). One hour following Evans blue injec-

108 tion, the animals were anesthetized and transcardially perfused with 0.9% saline, followed by 10% buffered formalin. The brains were removed and histological sections were evaluated. Data analysis

ICMRgic data were compared for each time point after-injury

IMMEDIATE

between experimental (injured) and control (sham-operated) groups utilizing a one way analysis of variance with post hoe comparisons using appropriate contrasts in a least squares design. For physiological variables an analysis of variance was also employed with comparisons requiring a P value of 0.05 to be considered statistically significant.

DAY I

~!i~Pi~ii¸ J~~:

Fig, 1.

(For legend,

see next page.)

109 RESULTS

Acute neurological evaluation The F-P injury resulted in 10 deaths, and only 4 ani-

DAY 5

mals had to be excluded from the study due to respiratory complications. For the remaining injured animals, the severity of the insult was quite uniform across all animals as defined by the degree of apnea and the pe-

DAY 10

Fig. 1. (Continued.) Coronal 2-DG autoradiographs through the caudate/putamen, dorsal hippocampus, brainstem and cerebellum processed immediately, 1, 5 and 10 days following lateral F-P injury or sham. Note the hypermetabolism evident immediately following injury, primarily in the side (left) ipsilateral to the F-E This hypermetabollsm gave way to metabolic depression by 6 h which was sustained for as long as 5 days.

110 TABLE I Mean (-+ S.E.M.) local cerebral metabolic rate for glucose (amolllO0 g/min) in selected structures of the cerebral cortex

I, ipsilateral to site of injury; C, eontralateral to site of injury; Imm., immediately following injury. Frontal cortex Injured

Parietal cortex Sham

Injured

Temporal cortex Sham

Injured

Occipital cortex Sham

Injured

Sham

lmm.

I 114.8-+7.88** C 90.8-+7.41

78.3-+4.16 111.0-+5.13"* 76.9-+3.08 85.5-+5.88

74.9-+3.06 128.9-+6.19"* 74.7-+2.96 106.2+_7.59

89.7---7.60 121.9+_5.80** 90.1-+7.13 110.5-+6.74

93.7+_5.94 92.5-+5.47

30 min

I C

89.5-+4.60** 78.7-+2.75

76.3-+0.66 77.3-+0.60

82.2-+5.86 73.9-+6.42

73.7-+1.41 75.7-+2.55

99.5+_10.04 88.6-+8.25

89.5-+2.64 107.6-+3.22"* 89.0-+2.82 95.1-+2.74

97.8-+1.39 96.3-+1.07

6h

I C

75.3-+5.85** 82.0-+2.34

97.0-+6.14 95.9-+2.76

78.4-+3.58 83.4-+2.48

93.8-+8.35 92.2+_7.08

61.4+2.66 ** 73.3-+2.59**

92.6-+6.51 94.4-+3.69

78.1-+4.24"* 89.8-+3.37

100.3---0.60 96.6-+0.98

Day l

I C

73.2-+5.07** 97.1-+1.46

96.3-+4.95 94.9-+4.81

71.0-+4.51"* 92.2-+2.49

94.0-+2.69 94.2+_2.09

72.3-+3.79** 90.9-+3.96

94.5-+5.00 93.7-+4.83

70.3-+4.98** 96.9-+3.50

99.6-+2.11 97.5+_2.46

Day 2

I C

75.2-+5.35* 97.3-+3.56

97.6-+8.15 96.2-+7.97

72.1-+0.93"* 91.0-+3.48

94.6-+5.59 93.2-+6.47

62.4---5.00** 95.8-+7.81

95.3-+5.61 95.5-+5.73

70.1-+3.68** 99.0-+5.92

96.9-+5.01 98.0-+4.91

Day 3

I C

79.2-+5.62** 99.3-+3.74

97.6-+2.30 95.9-+2.64

78.5-+2.13"* 92.1-+3.00

92.9-+1.61 92.9-+2.38

77.1-+2.68" 95.9+_5.68

95.7-+4.94 94.5-+4.26

76.9-+6.67* 96.3-+7.51

95.0-+1.94 95.9-+1.64

Day 5

I C

88.6-+4.65* 99.4-+2.49

95.6-+2.53 95.3-+2.85

80.2-+6.07 93.9-+5.31

92.3-+2.51 92.4+3.85

69.7-+4.66* 93.4+_7.91

91.5-+3.34 92.4-+2.95

85.9-+5.61 98.0+_6.15

97.1+_1.64 94.4-+3.71

Day 10

I C

95.4-+3.75 94.6-+3.52

96.3-+2.90 98.4-+3.75

94.6-+1.54 93.6-+1.40

95.3+_4.35 93.7-+3.38

91.2-+5.03 91.6-+5.07

92.1+_4.56 93.9-+4.71

99.5-+3.49 97.7-+3.49

96.7+_4.89 98.4-+4.04

*P < 0.05; **P < 0.01.

riod of unconsciousness. The m e a n a p n e a time p e r group ranged from 15.4 to 27.4 s showing little variability between time points (F7,36 = 0.648, P < 0.713). H o w e v e r , the p e r i o d of unconsciousness was m o r e variable, with the mean time ranging from 83.0 to 178.0 s (F7.36 = 1.106, P < 0.386). The variability seen in the unconsciousness m e a s u r e m e n t most likely reflects the fact that, unlike apnea, it cannot be continuously monitored. Therefore, some variability is due simply to the different times after injury when the e x p e r i m e n t e r tested for unconsciousness.

regards to the b l o o d - b r a i n b a r r i e r studied at 1 and 6 h following injury there was no evidence of extravasation of Evans blue albumin in any of the animals. Finally, histological evaluation of all brains u n d e r light microscopy did not indicate any trauma-induced morphological damage. The cerebral cortex directly u n d e r n e a t h the injury cap was u n r e m a r k a b l e although there a p p e a r e d to be a slight indentation at the site of the percussion. Underlying white m a t t e r was clear and there was no evidence of hemorrhageing, or necrotic tissue formation.

Physiological and pathophysiological variables The results of the b l o o d gas m e a s u r e m e n t s indicated

Measurement o f lCMRglc The autoradiographs exhibited very g o o d differentiation between structures allowing for accurate measure-

that for both the injury and the control groups p H , p O E and p C O 2 were all within n o r m a l limits. The mean and standard error p H for the injury group was 7.47 _+ 0.01 with controls exhibiting a m e a n value of 7.43 -+ 0.02 (FIA 3 = 3.446, P < 0.881). F o r the p O E the injured group showed a m e a n -+ standard e r r o r of 100.6 _+ 6.7 with controls exhibiting a value of 107.9 _+ 4.7 (F1,13 = 0.679, P < 0.426). Finally, the p C O 2 for the injured group showed a m e a n -+ s t a n d a r d e r r o r of 29.5 - 1.7 with controls having a value of 36.1 -+ 1.7 (Ft.13 = 6.49, P < 0.026). In the animals m e a s u r e d for systemic blood pressure following injury there was a transient (43.3 -+ 3.4 s) increase from 102 -+ 3 (baseline) to 161 -+ 6 m m H g . With

ments of selected regions (see Fig. 1 and Tables I - V I ) . F o r the s h a m - o p e r a t e d animals there was no evidence of metabolic asymmetry at any time following the surgery. F u r t h e r m o r e , these animals did not exhibit any differences in 1CMRglc for any of the structures m e a s u r e d across time beginning at 6 h post-sham operation. However, for the i m m e d i a t e and 30 min post-surgery time points, ICMRglc for all structures m e a s u r e d in the sham control animals were lower c o m p a r e d to the later time points. This was particularly evident for the frontal and parietal cortex where rates were reduced by as much as 21.1% ( P < 0.01) and 21.4% ( P < 0.01) c o m p a r e d to the later time points respectively. This metabolic depression was most likely due to the effects of anesthesia.

111 The injured animals showed a marked difference in 1CMRglc for many structures at different times follow-

ing trauma. In general these changes could be described as an increase in 1CRMglc immediately after injury re-

CONTRALATERAL

IPSILATERAL

Frontal Cortex 140 -

140 '~

120 . ~

~

120-

0..

loo -

100 -

80"

80

u

60"

60

E

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40

z

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30rain 6h Day1

D.~yS

De;y10

Parietal 140 "~m

120

~0

loo

, I ) ,

3()min 6h Dayt

DayS

Day10

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Cortex 140 -

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.o

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20

60

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40

40-

T Z

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~" 0

2o

•

#l,

3()min 6h Day1

Day5

DaylO

20

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30min 6h Day1

Day5

Day10

Occipital Cortex 140

140

"E

120

120

o

100

O m ¢m

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--~Z

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30min 6h Day1 Day5 Time after Injury

.

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v

I. = ,

a,--.- Injury s Sham

30rain 6h Dayt Day5 Time after Injury

D~ylO

Fig. 2. Mean (-+ S.E.M.) local cerebral metabolic rates for glucose (1CMRglc) within selected regions of the cerebral cortex following injury. Note that immediately following injury the entire cerebral cortex ipsilateral to the site of percussion exhibits a significant increase in glucose metabolism and that, beginning at 6 h after trauma, these same regions go into a state of metabolic depression which does not completely alleviate until 10 days post-injury. *P < 0.05; **P < 0.01.

112 turning toward control values by 6 h and then a subsequent decrease in ICMRglc which spontaneously recov-

ered over the course of 10 days. These changes were seen primarily within the hemisphere ipsilateral to the

CONTRALATERAL

IPSlLATERAL Dorsal Hippocampus

E

120

120 -

80

80 -

60

60 -

40

40 •

20

I 30min 6h Day1

DayS

Day10

20

30min 6h Day1

Stratum Lacunosum-moleculare Dorsal Hippocampus

"~

120

120 -

100

100,

Day5

Day10

Day5

O,;yl0

of

0. 80-

60-

60

~

°. o m

E

40"

40

O 2O

,

I

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30min 6h Day1

Days

'DAY10

20

,II,

3C)min 6h Day1

Ventral Hippocampus

=D

120 - •"

120 -

100-

100 '.

o

80=L

60

60-

40

40-

m

rJ 20

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30rain 6h Day1 Day5 Time after Injury

Day10

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, m 30rain dh l . ~ vDay1 Day5 Time =tier Injury

u DaylO

Fig. 3. Mean (-+ S.E.M.) local cerebral metabolic rates for glucose (ICMRglc) within selected CA1 regions of the hippocampus following injury. Note that like the cerebral cortex in Fig. 2, the hippocampus ipsilateral to the percussion exhibited a significant increase in metabolism immediately following injury with this increase still being present at 30 min post-trauma. As depicted, the subsequent metabolic depression lasted for only 2 days. *P < 0.05; **P < 0.01.

113 TABLE II

Mean (-+ S.E.M.) local cerebral metabolic rate for glucose (izmolllO0 g/min) in selected structures of the CA1 region of the hippocampus I, ipsilateral to site of injury; C, contralateral to site of injury; Imm., immediately following injury.

Lacunosum-moleculare ventral

Lacunosum-moleculare dorsal Ventral

Dorsal Injured

Sham

Injured

Sham

Injured

Sham

Injured

Sham

Imm.

I 90.1-+8.78"* C 55.4-+3.09

47.4-+3.55 47.9-+3.38

108.4+11.33"* 75.8-+9.12

74.0-+3.91 73.6+4.03

106.6-+7.53"* 66.0-+3.43

58.0-+2.51 57.7-+2.62

106.9+6.37 ** 76.1-+6.62

75.9-+3.97 75.7-+4.23

30 min

I 61.0-+6.98" C 47.3-+8.31

51.2-+3.57 50.0+-3.88

94.5-+4.86** 71.3-+8.13

73.1---1.88 73.4-+2.07

97.5-+9.20** 76.8-+6.63

58.7-+5.01 58.8-+5.16

108.2-+9.23" 85.6-+7.48

78.5-+4.69 78.7+-5.03

6h

I 39.1-+4.58" C 41.7-+3.72

50.6-+2.98 51.6-+1.81

60.8-+3.24** 60.9-+2.59**

76.6-+0.58 74.3-+1.78

58.4---4.32* 59.8-+1.60

71.1-+4.44 71.9-+5.09

73.6-+4.64 75.0-+7.87

78.6-+2.77 78.8-+2.62

Day 1

I 44.3-+5.42 C 49.5+6.28

52.7-+1.82 51.0-+2.47

65.3-+6.61 74.5-+7.23

77.0-+3.73 76.0-+4.18

58.4-+3.23* 70.0-+7.00

72.2-+1.85 71.8-+1.90

65.4-+6.13 78.2+-9.85

78.7+5.47 79.1-+5.43

Day 2

I 39.4-+9.30 C 47.6-+8.47

47.3-+6.28 49.8-+6.74

63.8-+5.95 74.7-+2.16

74.5-+4.35 74.5-+4.16

68.8-+5.14 72.4-+6.58

72.4-+8.21 71.5-+8.67

73.9-+4.81 79.7-+6.94

81.8-+7.21 82.2-+6.72

Day 3

I 45.0-+3.08 C 49.5-+1.80

47.8-+1.46 48.1-+1.03

70.8-+3.64 73.8---2.81

75.0---3.01 73.4-+4.45

68.9-+4.14 73.1-+2.33

74.6-+4.58 74.7-+4.59

73.2-+5.48 83.6-+4.89

82.5---6.01 82.8-+6.05

Day 5

I 42.6-+4.27 C 47.7-+5.21

48.3-+4.05 48.9-+3.08

71.4-+3.19 73.8-+4.47

77.6-+3.81 77.4-+4.34

72.3-+3.97 72.3-+2.63

71.3-+4.30 71.8-+4.18

83.2-+2.45 82.9-+1.36

82.4-+3.82 80.4-+2.97

Day 10

I 50.5-+1.47 C 50.5-+2.07

51.6-+4.13 51.3-+2.64

72.1-+4.85 73.8-+5.21

74.3-+3.19 73.4-+3.06

74.1-+3.44 74.3-+2.66

73.4-+2.64 71.4-+3.00

80.8-+3.87 80.1-+4.22

82.7-+4.10 81.6-+3.27

*P < 0.05; **P < 0.01.

site of the F-P injury, however, there were small changes in ICMRglc in the contralateral side. The structures most

affected were the cerebral cortex and the hippocampus (Fig. 1).

TABLE III

Mean (-+ S.E.M.) local cerebral metabolic rate for glucose (l~molllO0 g/min) in selected structures of the thalamus I, ipsilateral to site of injury; C, contralateral to site of injury; Imm., immediately following injury.

Anterior thalamus

Medial dorsalis

Ventral thalamic nucleus Lateral geniculate nucleus Medial geniculate nucleus

Injured

Sham

Injured

Injured

Sham

Injured

Sham

Injured

Sham

I C

59.1---5.81 67.2-+2.39

67.1---4.60 67.7-+3.80

75.6-+10.23 73.1-+4.03 75.9-+9.01 72.2-+5.04

43.5-+8.00 57.2-+4.26

61.9-+5.97 60.6-+6.87

54.6-+9.99 58.4-+8.11

62.0-+1.12 61.8---1.04

79.9-+8.03 83.3-+8.39

72.3-+4.96 71.4-+5.06

30 rain I C

65.5-+5.36 67.6-+3.59

64.8-+4.20 66.2-+4.08

75.5-+6.99 76.0-+6.26

76.8-+3.24 75.9-+3.76

64.7---7.79 64.0_-.4.90

64.6-+4.33 63.8-+4.17

58.3-+8.11 58.0-+6.48

68.8-+4.95 68.3---5.70

70.6-+7.19 70.5-+6.40

71.5-+3.02 71.3-+3.20

6h

56.2-+3.12 ** 72.6-+3.92 67.0-+2.05 73.9-+3.69

73.6-+3.79 73.6-+4.16

77.3+6.30 76.5-+7.02

56.5-+6.74 * 71.9-+2.97 63.0-+2.25 72.7-+3.27

47.5-+5.01"* 70.7-+2.23 59.1-+3.61" 71.9-+2.60

48.7-+4.66** 88.9_+3.35 57.6-+1.74" 93.2---2.14

Day 1 I C

65.4-+4.51 75.2-+5.92

73.1-+4.24 70.7-+5.68

68.6-+4.92 78.5-+6.23

78.0-+3.95 77.6-+4.24

51.5-+6.07" 72.4-+4.98 72.0-+4.27 70.3-+6.21

52.0-+3.26* 71.3-+7.39

70.3-+4.78 68.5-+3.73

82.5---4.08 94.0---4.37

89.7-+7.33 91.2-+7.68

Day 2 I C

77.8-+2.80 75.7-+1.87

77.2-+5.43 76.5-+5.55

68.3-+5.76 75.3-+6.51

78.9-+5.87 78.3+-4.98

56.4-+2.17" 75.6-+6.97 69.6-+4.99 74.9-+7.22

48.7-+1.82"* 69.2-+3.62 71.8-+2.76 69.3-+4.79

81.4---7.89 88.3-+8.08

90.0-+6.61 90.3-+5.31

Day 3 I C

77.7--+6.19 79.4-+6.26

80.9-+3.55 76.7---5.17

74.4-+4.44 78.8+4.28

75.5-+2.41 73.5-+1.99

69.5-+1.73 73.0-+2.77

68.3---6.70 71.0-+5.61

60.3-+4.22* 70.5-+2.26

70.3-+1.83 70.5-+2.48

83.7-+2.79 90.6-+4.83

90.4-+5.46 87.6-+3.65

Day 5 I C

73.2-+5.84 75.7-+6.30

75.2-+7.00 77.8-+5.00

73.9-+7.82 73.9-+7.72

75.1-+4.87 75.4-+3.88

66.2-+5.33 69.3-+6.73

72.4-+5.04 73.6-+6.21

67.9---6.11 72.9-+5.98

72.9-+2.68 72.6---1.17

83.2-+3.43 92.3-+3.75

86.1+-2.21 90.0-+3.24

Day 10I C

75.0-+3.42 80.5-+4.02

80.4---3.05 76.8-+4.99

75.6-+7.89 76.4-+7.81

77.7-+3.99 77.1-+3.00

70.7-+5.39 70.4_+6.40

71.6-+7.72 72.9-+8.93

71.0-+6.99 72.7-+6.51

71.9-+1.40 72.3-+1.16

85.6-+3.31 90.8+--2.26

91.9-+7.61 92.4-+7.64

Imm.

I C

*P < 0.05; **P < 0.01.

Sham

114 TABLE IV

Mean (-+ S.E.M.) local cerebral metabolic rate for glucose (l~molllO0 mg/min) in selected structures of the brainstem I, ipsilateral to site of injury; C, contralaterai to site of injury; Imm., immediately following injury.

Substantia nigra reticulata

Superiorcolliculus

Red nucleus

Dorsal raphe

Injured

Sham

Injured

Sham

Injured

Sham

Injured

Sham

Imm.

I 50.0-+7.21 C 51.7-+7.14

45.6-+1.48 45.9-+1.75

83.5-+8.70* 76.5-+6.46

62.2-+4.53 62.2-+4.59

57.8---7.54 64.6-+8.88

54.9-+2.92 54.9-+3.16

57.8-+6.86 57.2-+7.04

43.8-+4.37 43.4-+4.23

30 min

I 50.7-+2.93 C 48.5-+2.74

45.4-+0.91 45.5-+0.93

65.5-+4.38 63.7-+4.28

63.8-+3.13 62.9-+3.46

52.0-+0.95 53.8-+1.54

53.5-+3.73 53.4-+3.38

49.3-+5.71 49.5-+5.75

48.7-+2.87 48.4-+3.03

6h

I 52.7-+2.13 C 52.0-+1.95

54.6-+1.59 53.7-+3.13

58.6-+3.93 57.5-+3.25

67.6-+3.79 68.0-+4.21

62.8-+3.37 60.3-+2.39

58.9-+4.24 61.0-+4.00

46.6-+5.26 46.4-+5.31

50.3-+0.21 50.2_+0.13

Day 1

I 48.5-+4.01 C 53.5-+5.17

55.1-+4.00 54.9-+3.31

64.7-+2.14 68.9-+1.79

67.4-+4.35 65.5-+4.05

58.6-+1.90 63.2-+2.15

59.1-+2.45 59.7-+2.17

53.4-+4.45 53.4-+4.34

51.3-+1.29 50.6-+1.24

Day 2

I 52.1-+3.72 C 54.0-+2.74

54.1_+4.53 63.2-+8.07 53.2-+4.31 69.4-+4.46

69.0-+4.03 68.0-+3.54

53.0-+6.89 57.2-+5.12

59.3-+4.57 59.9-+4.85

49.9-+9.48 50.1-+9.43

49.7-+1.28 50.3-+1.34

Day 3

I 51.6+3.21 C 52.4-+3.44

54.7-+3.12 53.4-+2.97

65.1-+4.59 65.4-+5.05

67.6-+1.42 68.1---1.45

55.7-+3.54 57.7-+3.32

60.8-+1.86 60.1-+2.35

51.0-+3.91 50.8-+3.90

49.1-+0.93 49.3-+0.77

Day 5

I 51.9-+7.07 C 51.9-+7.47

56.0-+2.50 55.5-+3.03

62.2-+2.47 65.2-+2.55

66.8-+3.52 67.1-+4.38

58.8-+2.60 59.1-+2.38

61.3-+3.64 61.1-+3.34

50.9-+6.33 50.4-+6.37

50.0-+1.24 50.0-+1.40

Day 10

I 55.2-+3.77 C 54.0-+3.68

52.7-+6.00 51.5-+6.76

65.6-+5.76 66.3-+5.78

67.4-+4.57 69.5-+3.92

58.8-+4.85 59.0-+4.64

57.5+3.82 58.1+3.95

48.3-+1.62 47.6-+1.59

49.5-+4.38 49.6-+4.43

*P < 0.05; **P < 0.01.

Cerebral cortex (layers 3-5). Immediately following the F-P injury all regions of the cerebral cortex ipsilateral to the concussion site exhibited a significant increase in ICMRglc (P < 0.01). Thirty minutes after F-P only the ipsilateral frontal and occipital cortex still showed a significant increase in 1CMRglc (P < 0.01) compared to controls (Fig. 2 and Table I). At 6 h following injury the ipsilateral cerebral cortex began to show signs of a metabolic depression. This depression primary affected the cortex ipsilateral to the injury involving all areas measured and lasted for as long as 5 days returning to control values 10 days after injury. The rates for glucose metabolism of the cerebral cortex contralateral to the site of trauma were also affected following the F-P injury, however, not to the same degree as the ipsilateral side. Immediately following the injury the regions within the contralateral cerebral cortex showed a slight increase in ICMRglc compared to controls of between 14.5% and 19.5%, but this was not statistically significant. By 30 min post-injury these regions had returned to control levels. Just like the ipsilateral cortex, the contralateral cerebral cortex showed a reduction in 1CMRglc at 6 h posttrauma. This was particularly evident for the temporal cortex which showed a significant reduction of ICMRglc of 22.4% (P < 0.01) compared to sham controls. By 24 h after the injury all regions of the contralateral cerebral

TABLE V

Mean (-+ S.E.M.) local cerebral metabolic rate for glucose (Itmol/lO0 g/min) in selected structures of the cerebellum I, ipsilateral to site of injury; C, contralateral to site of injury; Imm., immediately following injury.

Cerebellar cortex (Crus I)

Deep cerebellar nuclei (Lateral)

Injured

Injured

Sham

Sham

Imm.

I 41.9-+4.46 C 45.2-+5.68

39.1+3.80 39.3-+3.75

73.8-+5.90 77.6-+9.49

74.1-+3.01 73.4-+1.62

30 min

I 41.3---4.94 C 40.0-+2.75

42.2-+2.19 43.8-+2.42

82.1-+4.43 79.3---2.83

79.9-+2.20 79.6---1.75

6 h

I 42.9---4.96 C 41.3---3.48

50.3-+5.34 50.6-+5.31

71.7-+3.66 70.6+3.04

81.7-+3.40 81.1-+3.71

Day 1

I 49.7-+2.16 C 49.2-+3.48

51.9-+2.61 51.6-+1.43

80.9-+6.29 81.8-+5.74

78.4---5.19 81.1-+4.69

Day 2

I 50.5-+4.92 C 47.9-+5.85

47.9-+1.77 47.2---1.53

80.2-+6.24 81.7-+6.00

79.6-+3.57 80.9-+3.40

Day 3

I 52.6±4.19 C 50.0-+3.01

49.1---7.81 79.2-+5.62 48.0-+8.82 79.8-+6.58

82.5---7.67 82.2-+5.26

Day 5

I 50.3-+0.90 C 48.0-+1.57"

51.9-+0.19 52.4-+1.01

79.5---4.72 77.6-+6.33

83.9-+2.39 82.2-+3.10

49.9-+7.69 51.7-+8.08

81.1-+5.87 81.6-+5.52

81.0-+8.18 77.5-+9.69

Day 10 I

50.3-+1.98 C 50.2-+3.64

*P < 0.05; **P < 0.01.

115 TABLE VI Mean (+- S.E.M.) local cerebral metabolic rate for glucose (l~molllO0 g/rain) in selected miscellaneous structures

I, ipsilateral to site of injury; C, contralateral to site of injury; Imm., immediately following injury; CC-Splen., corpus callosum splenium. Caudate/putamen

Septal nucleus

Globus paUidus

Ventromedial hypothalamus Amygdala

Injured

Injured

Sham

Injured

Sham

Injured

Sham

Injured

C

68.2+--11.27 83.1-+3.70 83.4-+7.98 82.8-+3.63

39.7---9.47 42.8-+9.29

41.9-+2.85 42.4-+3.21

37.3-+7.28 31.4-+4.71

34.7-+3.32 35.1-+3.23

41.7-+5.00 43.1-+5.06

47.2-+3.24 46.9-+3.69

C

81.1-+4.81 82.8-+5.51 83.1-+5.51 82.6-+5.70

49.1-+8.37 47.2-+8.48

45.7-+4.29 46.3-+4.50

38.9-+3.21 37.0-+3.63

37.9-+3.71 37.1-+3.67

49.0-+1.97 49.9-+1.96

C

95.8-+7.14 86.3-+2.66 100.4-+7.80 89.1-+1.28

47.7-+4.75 50.1-+5.14

48.7-+5.68 48.0-+5.55

35.1-+2.15 40.0-+3.14

40.2-+3.77 40.1-+2.89

C

79.0-+3.06 87.1-+5.26 88.2-+2.90 87.9-+3.89

47.0-+1.18 49.1-+1.36

49.8-+5.07 49.0-+4.50

33.1-+3.21 39.8-+1.08

C

77.9-+5.83 89.7-+3.41 86.3-+6.87 88.3-+4.47

42.9-+9.10 47.3-+8.24

46.2-+2.80 46.5-+2.72

C

79.0-+3.70 88.2-+1.84 85.3-+4.35 87.0-+1.30

49.9-+2.05 50,6-+1.09

C

87.6-+6.28 87.1-+7.81 87.9-+4.39 84.9-+7.74

C

84.0-+3.59 87.8-+2.79 86.3-+3.66 86.4-+4.46

lmm. 30 rain 6h Day 1 Day 2 Day 3 Day 5 Day 10

Sham

CC-Splen. Sham

Injured

Sham

81.3-+7.86 76.7-+2.43 83.0-+10.12 75.9-+2.83

32.6-+4.95* 32.5-+4.92*

20.0-+0.99 20.2-+0.75

48.0-+4.88 47.9-+4.90

77.6-+7.67 80.7-+6.98

76.5-+4.66 74.8-+5,96

22.2-+3.24 23.0-+1.43 22.0-+3.23 23.2-+1.42

47.0-+4.06 47.3-+4.15

54.6-+4.29 53.5-+5.31

71.1-+4.37 72.1-+4.28

78.3-+3.12 79.2-+3.28

26.8-+3.25 26.7-+2.29 27.0-+3.32 26.8-+2.04

39.2-+3.46 40.6-+3.94

51.1-+3.21 54.6-+4.18

54.5-+2.64 55.7-+2.49

69.7-+2.66 82.0-+3.67

78.8-+2.41 78.1-+2.32

25.8-+1.38 28.0-+2.66 26.3-+1.40 28.0-+2.57

38.8-+2.54 43.0-+4.00

41.4-+2.89 40.8-+2.54

56.6-+2.86 55.5-+3.36

53.4-+1.50 53.5-+1.79

60.0-+4.10"* 80.1-+3.05 81.8-+4.32 80.3-+3.17

28.2-+9.32 25.6-+5.87 27.2-+9.52 25.0-+5.38

50.1-+2.87 47.3-+4.09

37.4-+2.34 40.8-+2.50

43.6-+2.12 43,1-+1.71

57.7-+3.01 60.0-+3.96

55.9-+4.23 55.9-+3.88

73.2-+1.12 79.8-+3.88

81.5-+4.89 79.5-+3.91

26.9-+1.51 26.3-+1.86 27.0-+1.49 25.6-+1.72

46.9-+2.88 47.4-+3.15

50.2-+2.84 49.2-+2.91

40.8-+3.91 43.6-+4.65

43.4-+2.94 44.0-+2.16

55.8-+2.49 56.9-+2.54

58.2-+3.48 58.0-+3.08

79.5-+5.45 82.0-+5.74

83.0-+3.12 83.6-+3.61

26.1-+2.02 27.2-+1.30 26.3-+2.04 27.8-+1.02

47.7-+4.39 47.9-+4.07

48.6-+5.49 49.6-+5.32

39.8-+3.18 41.3-+3.06

42.1-+3.26 42.2-+3.82

57.8-+6.91 58.7-+6.42

58.0-+7.56 58.6-+8.19

85.4-+6.20 85.9-+6.28

83.4-+1.35 83.6-+1.13

25.2-+1.44 24.4-+2.89 25.0-+1.49 24.4-+3,01

*P < 0.05; **P < 0.01.

cortex returned to sham control levels and remained at these levels throughout the remaining time points studied. Hippocampus. Like the cerebral cortex, all areas of the ipsilateral hippocampus exhibited a marked increase in ICMRglc during the first few minutes following trauma. However, these increases were much more dramatic within the CA1 stratum pyramidale of the dorsal and the ventral hippocampus. Analysis of the stratum lacunosum-moleculare of the dorsal and the ventral CA1 hippocampus indicated that they also increased their rates of metabolism exhibiting levels that were 46.5% (P < 0.01) and 40.8% (P < 0.01) higher than controls respectively. By 30 min post-injury this increase in 1CMRglc had subsided to some degree in the CA1 stratum pyramidale of the dorsal hippocampus and in the stratum lacunosum-moleculare of the ventral CA1 hippocampus. In contrast the CA1 stratum pyramidale of the ventral hippocampus and the stratum lacunosum-moleculare of the dorsal CA1 hippocampus remained significantly higher than controls (Fig. 3 and Table II). As the time post-injury increased these same regions of the ipsilateral hippocampus, like the cerebral cortex, went into a state of metabolic depression. At 6 h postinjury, this was particularly evident for the stratum lacunosum-moleculare of the dorsal CA1 hippocampus. The remaining regions of the ipsilateral hippocampus were not as depressed. At 1 day following injury the ipsilateral hippocampus began to spontaneously recover with its ICMRglc showing only a slight depression of metabolism ranging from

a mean of 15.2% to 19.1% lower than controls. This residual depression lasted for as long as 3 days recovering by the 5th day after trauma. The hippocampus contralateral to the site of injury showed a slight but non-significant increase in ICMRglc compared to controls at the immediate and 30 min time points. It also exhibited some later signs of metabolic depression but this was restricted to the 6th hour point and only the stratum lacunosum-moleculare of the dorsal CA1 hippocampus reached a level of statistical significance (P < 0.01). By 1 day post-injury the contralateral hippocampus was indistinguishable from controls and remained unremarkable for the remaining time points. Thalamus. Neither the ipsilateral or contralateral thalamic nuclei showed any evidence of an increase in 1CMRglc following F-P injury. In contrast, many regions of the thalamus showed a marked depression of ICMRglc beginning at 6 h post-trauma with this effect being especially evident in the thalamic nuclei ipsilateral to the site of injury. At the 6th hour time point these affected regions and their corresponding percent of metabolic depression compared to controls included the anterior thalamus 22.6% (P < 0.01), ventral thalamic nuclei 21.4% (P < 0.05), lateral geniculate nucleus 32.8% (P < 0.01), and the medial geniculate nucleus 45.2% (P < 0.01). Many of these regions remained depressed over the next 2 days with the lateral geniculate nucleus not returning to control values until 5 days post-injury. For the contralateral thalamus, the above regions also exhibited a reduction in ICMRglc beginning at 6 h post-

116 injury although not to the same degree. Furthermore, the contralateral thalamus was not different from controis by 1 day post-injury and remained unchanged for the remaining time periods studied (Table III). Brainstem. Within the brainstem only the superior colliculus showed any evidence of hypermetabolism following the F-P injury. This was restricted to the period immediately following the injury and was more apparent in the ipsilateral compared to the contralateral colliculus. For this structure the ipsilateral superior colliculus showed an increase in ICMRglc of 34.2% (P < 0.05) with the contralateral colliculus exhibiting an increase of 23.0% (P < 0.2). However, by 30 min post-injury this hypermetabolic state had subsided and this structure remained at control levels for the remaining periods studied (Table IV). Cerebellum. Within the cerebellum both the cerebellar cortex (Crus I) and the deep cerebellar nuclei (lateral) were measured. For both of these regions there was no evidence of a hypermetabolic state following F-P injury. However, 5 days post-injury the contralateral cerebellar cortex exhibited a metabolic depression which was 91.6% of control (P < 0.05). This depression spontaneously alleviated by 10 days. This crossed cerebellar metabolic diaschisis within Crus I was the only remarkable finding seen within the cerebellum with the remaining regions staying at control levels across all post-injury time points (Table V). Miscellaneous structures. Of the remaining areas studied only the caudate/putamen and the amygdala showed changes following injury. For the caudate/putamen, there was a slight reduction (19.9%) immediately following injury. This effect was restricted to the side ipsilateral to the injury and did not reach statistical significance. There were no other changes in this structure throughout the extent of study. Although the amygdala did not show any evidence of an increase of metabolism in response to the brain injury it did exhibit a depression of ICMRglc beginning at 6 h after surgery. This depression progressed gradually reaching its greatest extent at 2 days showing a reduction of ICMRglc by 25.1% (P < 0.01). Thereafter, it slowly recovered, and by 10 days, reached control levels (Table VI). DISCUSSION

Calculation of glucose utilization following F-P injury The use of the 2-DG method with its standard equation for calculation of ICMRglc soon following an insult to the brain may, depending on the injury, violate some of the basic steady-state assumptions required in order to obtain valid results 17'35. Factors which can affect this validity include alterations in blood-brain barrier (BBB)

permeability, direct morphological damage to the region of interest and dramatic reductions in cerebral blood flow (CBF) to ischemic levels. In the F-P model of cerebral concussion (as used in the current study) many observations indicate that the basic steady-state assumptions are most likely met. First, as described in the current study, there is no evidence of disruption of the BBB as determined by the lack of extravasation of Evans blue albumin. However, using a similar injury devise, others have reported a breakdown of the BBB following a F-P injury as measured with Evans blue albumin 34 and immunocytochemical detection 43. The difference between these reports and the current study most likely reflects the fact that, unlike in other studies where the rat is positioned in direct contact with the injury device, our method of injury involves the interconnection of a 20 cm rigid polyethylene tube between the rat and the injury devise which may dampen the F-P pulse. Given that Evans blue albumin is a gross measure for BBB integrity we cannot exclude the possibility that the animals in the current study did experience some degree of injury-induce BBB breakdown. However, several lines of evidence would suggest that this may not explain the metabolic results. First, using either Evans blue or immunocytochemical detection, the vascular disruption reported following F-P was localized to regions close to the site of injury 34'43. However, the increase in ICMRglc immediately following F-P seen in the current study was not restricted to just one region of the cerebral cortex or the hippocampus. Second, in a recent study 57 we have reported that when the CA3 region of the hippocampus is removed (via kainic acid) the increase in ICMRglc typically seen within the CA1 region is prevented with 1CMRglc remaining at control levels. Therefore, any change in BBB permeability following injury was not enough to mask the lack of an injury-induced change in 1CMRgic within CA1 following removal of its excitatory amino acid input. Finally, even within regions of BBB breakdown following F-P in cats, measurements of 1CMRglc indicated that the mechanisms of glucose transport are not grossly disturbed 35. Another factor which could affect ICMRglc following brain injury is morphological damage to regions of interest. However, as reported in the current study there was no evidence of morphological damage. Finally, there is the question of CBF, which if it reaches ischemic conditions (