Induction of tolerance to ischemia: Alterations in second-messenger systems in the gerbil hippocampus

Brain Research Bulletin,Vol. 29, pp. 559-565, 1992

0361-9230/92 $5.00 + .OO

Copyright0 1992 Pergamon Press Ltd.

Printed in the USA. All rights reserved.

Induction of Tolerance to Ischemia: Alterations in Second-Messenger Systems in the Gerbil Hippocampus HIROYUKI

KATO,’

TSUTOMU

ARAKI,

KENSHI

MURASE

AND

KYUYA

KOGURE

Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, l-l Seiryo-machi, Aoba-ku, Sendai 980, Japan Received

16 December

199 1; Accepted

12 April

1992

KATO, H., T. ARAKI, K. MURASE AND K. KDGURE. Induction of tolerance to ischemia: Alterations in second-messenger systems in thegerbil hippocampus. BRAIN RES BULL 29(5) 559-565, 1992.-Preconditioning the brain with sublethal ischemia protects against neuronal damage following subsequent ischemic insult. Using [3H]inositol 1,4,5-triphosphate (IP,), [3H]phorbol 12,13dibutyrate (PDBu), [3H]fotskolin, [‘HIcyclic adenosine monophosphate (CAMP) and [3H]rolipmm, we performed quantitative autoradiography to determine postischemic alterations in second-messenger systems in the gerbil hippocampus following preconditioning the brain with sublethal ischemia. At 7 days of reperfusion, no alterations were observed in brains subjected to 2 min of forebrain ischemia which produced no neuronal damage. However, 3-min ischemia caused a 75% reduction in [)H]IP, binding (p i 0.01 vs. control) and l5-25% reductions in [‘Hlforskolin (p < 0.01 vs. control), [‘HIcAMP (p < 0.05 vs. control), and [3H]rolipram (p < 0.01 vs. control) binding in the CA1 subfield coincident with histopathological CAI pyramidal cell destruction, but no significant alterations in [3H]PDBu binding. Preconditioning the brain with 2 min of ischemia followed by 4 days of reperfusion prevented both histopathological cell death and the reductions in binding following subsequent 3 min of ischemia. Interestingly, [‘H]IP3 and [‘Hlrolipram binding in CA I showed a transient reduction, by 30% and 20% (both p < 0.01 vs. control), respectively,in the early reperfusion period. This downregulation of the IP3system may play a role in the protection against cell death. Cerebral ischemia Second messengers

Hippocampus Gerbil Inositol triphosphate

Preconditioning

WE have recently reported that preconditioning the brain with sublethal &hernia alters selective neuronal vulnerability showing characteristic postischemic time course (16). One h after 2 min of forebrain ischemia in Mongolian gerbils, the neuronal vulnerability is increased. As a result, neuronal damage following subsequent ischemic insult induced after a l-h interval becomes extensive as compared to damage at shorter and longer intervals or after single &hernia (2,l I, 12,16,24,34).By contrast, tolerance is observed when secondary &hernia is rendered after a I- to 7-day interval, and the brain is protected against neuronal damage (16,21,22). The tolerance is not acquired following sham operation and I min of ischemia (I 6,182 I ,22). We have investigated the mechanisms of the increased vulnerability, and reported, as possible causative factors, postischemic hypopetfusion which is maximal 1 h after 2 min of ischemia (l4), impairment of protein synthesis which is depressed for several hours after 2 min of ischemia (l), alterations in receptor sensitivity and second messenger systems ( 15, I7), and the excitotoxic mechanism ( 13).

Tolerance

Autoradiography

However, the mechanism of the induction of tolerance is not known and remains to be elucidated. Extracellular signals to neurons are transmitted by neurotransmitters which activate specific receptors on the neuronal surface, followed by the responses of receptor-linked secondmessenger systems (38). Calcium influx via voltage-operated and receptor-operated calcium channels also operates as a second messenger in the brain, especially during pathologic conditions such as ischemia (28). There are two major second-messenger systems in the brain, the adenylate cyclase and phosphoinositide systems. Recent experimental evidence indicates that marked alterations in the intracellular signal transduction take place early after ischemic insult (26,27). The purpose of this study was, therefore, to reveal the alterations in the second-messenger systems in the gerbil hippocampus following &hernia induced 4 days after preconditioning with sublethal ischemia, in which case the tolerance is observed. To examine the phosphoinositide system, we used [3H]inositol l,4,5-triphosphate (IP,) (to label IP3

’ Requests for reprints should be addressed to Hiroyuki Kato, M.D.

559

KATO

receptors) and [3H]phorbol 12,13-dibutyrate (to label protein kinase C). To examine the adenylate cyclase system, we used [3H]forskohn (to label adenylate cyclase), [3H]cyclic adenosine monophosphate (CAMP) (to label particulate CAMP-dependent protein kinase), and [3H]rohpram (to label CAMP selective phosphodiesterase). METHOD

Induction oflschemia A total of 40 male Mongolian gerbils (Seiwa Experimental Animals, Fukuoka, Japan), aged I3- 14 weeks and weighing 6892 g, were used. They were allowed free access to food and water before and after surgery. They were maintained under a standardized light and dark cycle (light 800 to 2000), and the surgeries were done during the daytime. Anesthesia was induced with 2% halothane in a mixture of 30% oxygen and 70% nitrous oxide. A midline cervical skin incision was made and bilateral common carotid arteries were gently exposed. The arteries were then occluded with aneurysm clips. Anesthesia was discontinued when the clips were in place. Occlusion and reperfusion of the carotid arteries were verified by visual observation. Body and cranial temperature during surgery and ischemia was maintained at 37.0-37.8”C. Postischemic body temperature was also monitored for 2 h to confirm the presence of postischemic hyperthermia and the absence of hypothermia. Body temperature of all animals subjected to ischemia increased above 38°C after ischemia, as reported previously (16). Animals were subjected to single 2 and 3 min of ischemia, and to 3 min of ischemia induced 4 days after preconditioning with 2 min of ischemia. Animals subjected to single ischemia were decapitated at 7 days of survival. Animals subjected to double ischemia were decapitated at 1 h, 1 day, and 7 days. Six normal animals served as controls. The brains were quickly removed and frozen in powdered dry ice. Coronal frozen sections, I5 pm in thickness, were cut in a cryostat and thaw-mounted onto gelatin-coated cover slips. and stored at -80°C until assay.

ET AL.

NaCl and 1 mM CaCl, for 60 min at room temperature. The sections were then washed twice in the buffer for 2 min at 4°C. Nonspecific binding was determined using 1 rMPDBu (Sigma). [“H]Forskolin Forskolin binding sites were localized autoradiographically as described previously (27,36). Sections were incubated with 10 nM [3H]forskolin (20 Ci/mmol, New England Nuclear) in the buffer containing 50 mMTris-HCl (pH 7.7), 100 mM NaCl and 5 mM MgClz for IO min at room temperature. The sections were then washed twice in the buffer for 2 min and rinsed briefly in distilled water at 4°C. Nonspecific binding was determined using 10 puM forskolin (Sigma). [3H]Cyclic .4denosine

Monophospha~e

[3H]Cyclic adenosine monophosphate (CAMP) autoradiography was performed as described previously (8). Brain sections were preincubated in the Krebs-HEPES buffer (118 mM NaCl, 5 mM KCI, 2.5 mM CaCl,, 1.18 mM KH2P04, 1.18 mM MgSO,, I I mM glucose. and 25 mM HEPES; pH 7.4) for 20 min at 22°C. Then the sections were incubated with 10 nM [3H]cAMP (5 I Ci/mmol. Amersham) and 1 mM IBMX in the same buffer for 90 min at 22°C. The sections were then washed in the buffer for I min at 4”C, and in distilled water rapidly at 4°C. Nonspecific binding was determined using 10 PM CAMP (Sigma).

[3H]Rolipram autoradiography was performed as described previously (19). Brain sections were incubated with 5 nM [3H]rolipram (31.32 Ci/mmol, kindly donated by Meiji Seika Co. Ltd., Tokyo, Japan) in the I50 mM phosphate buffer (pH 7.4) containing 2 mM MgClz and 100 PM dithiotheitol for 60 min at 4°C. The sections were then washed twice in the buffer for 30 sat 4°C. and in distilled water rapidly at 4°C. Nonspecific binding was determined using I wA4rolipram (kindly donated by Meiji Seika Co. Ltd.).

HisloputholoK? Adjacent sections were stained with cresyl violet and used for histopathology. Neuronal damage to the hippocampus was semiquantitatively graded using a O-3 rating system with 0 = normal, I = a few neurons damaged, 2 = many neurons damaged. and 3 = majority of neurons damaged. The average of values of both hemispheres was considered. [‘H]lno.sitol

1.4,5-7iiphosphute

The method for the autoradiographic

visualization

of inositol

1.4,5-triphosphate(IP,) binding using [3H]IP3 has been described previously

(27,37). Sections were incubated with 10 ti [3H]IP3 New England Nuclear) in the buffer containing 20 mM Tris-HCl (pH 7.7), 20 mM NaCI, 100 mM KCI, 1 mM EDTA and I mg/ml bovine serum albumin for 10 min at 4’C. The sections were then washed twice in the buffer for 2 min at 4°C. Nonspecific binding was determined using 10 palm IP3 (Sigma, St. Louis, MO).

( 17.0 Ci/mmol,

[‘H]Phorhol

12, I_?-Dibutyrate

[3H]Phorbol 12,13-dibutyrate (PDBu) autoradiography was performed as described previously (26,35). Brain sections were incubated with 2.5 nM [3H]PDBu (20 Ci/mmol, New England Nuclear) in a solution of 50 mA4 Tris HCI (pH 7.7). 100 mM

The sections were dried under a stream of cold air and apposed to Hyperfilm-3H (Amersham) for 2-6 weeks. The optical density of the regions of interest was measured using a computerassisted image analyzer system (IBAS image analyzer system, Zeiss). The relation between optical density and radioactivity was determined using a third order polynomial function with reference to tritium standards ([3H]microscale, Amersham) exposed along with the tissue sections. Optical densities of the brain regions measured were in the range in which the radioactivity of the [3H]microscale showed a near-linear relation. Postischemic alteration of the beta quenching level in the hippocampus is minimal (25). In the present study, the animals that were killed after ischemia showed no significant alteration in nonspecific binding levels compared to control animals (data not shown). Therefore, we made no correction in the beta quenching level after ischemia. Stulistics Each group contained 6-7 animals. Binding assays were performed in duplicate. Values were expressed as means f SD. Statistical significance was analysed using the Kruskal-Walhs nonparametric analysis of variance and the Williams-Wilcoxon rank sum test.

ISCHEMIC

TOLERANCE

AND

SECOND

561

MESSENGERS

TABLE [‘H]IPI

BINDING

(fmol/mg

1

TISSUE) IN THE HIPPOCAMPUS FOLLOWING 3-MIN ISCHEMIA INDUCED 4 DAYS AFTER

2- AND 3-MIN ISCHEMIA 2-MIN ISCHEMIA

AND FOLLOWING

2-Min + 3-Min Ischemia Control

CA I subfield CA3 subfield Dentate gyrus Mean

f SD, n =

143 + 17.9

51 + 11.6 91 f 13.4

2-Min Ischemia I Days

3-Min Ischemia 7 Days

lh

I Day

I Days

134 f 14.4

36 f 14.2t

95 IL 26.61_

100 k 13.0t

126 f 14.7

39 + 5.3 49 f 7.8T

42 f 9.0 67 + 18.9*

57 f 20.6 87 Ifr 15.1

[3H]Forskolin Binding

RESULTS

Following 2-min ischemia, there were no alterations in [3H]forskolin binding in the hippocampus. Three-min ischemia caused a 19% reduction (p < 0.0 1 vs. control) in the CA 1 subfield by 7 days (Fig. l), but otherwise unchanged. Following secondary ischemia, [3H]forskolin binding was comparable to control levels at any time of reperfusion (Table 3).

Two-minute ischemia produced no neuronal damage (the damage score, 0 + 0). However, 3-min ischemia destroyed exclusively CA 1 pyramidal cells of the hippocampus (2.3 + 0.8, p < 0.01 vs. control). By contrast, preconditioning with 2-min ischemia prevented the CA 1 neuronal damage at any reperfusion period (0 +- 0 each). CA3 pyramidal cells and dentate granule cells showed no visible damage at any time.

[3H]cAMf’ Binding

[-‘H]IP, Binding

Following 2-min ischemia, there were no alterations in [3H]cAMP binding in the hippocampus. Three-min ischemia caused a reduction in the CA1 subfield by 7 days (p < 0.05 vs. control), and a narrow band of high density of [3H]cAMP binding in the stratum pyramidale of the CA 1 subfield disappeared (Fig. 1). Following secondary ischemia, [3H]cAMP binding was comparable to control levels at any time of reperfusion (Table 4).

We found no alterations in [3H]IPJ binding 7 days after 2min ischemia. However, 3-min ischemia caused a 75% reduction in [3H]IPX binding in the CA1 subfield (p < 0.01 vs. control) and a 46% reduction in the dentate gyrus (p < 0.0 1 vs. control) (Fig. 1). Following secondary ischemia, [3H]IP3 binding was reduced by 25-35% in CA1 (p < 0.01 vs. control) and dentate gyrus (p < 0.05 vs. control) at 1 h and 1 day of reperfusion, but recovered to control levels by 7 days (Table I).

[3H]Roliprum Binding Two-min ischemia caused no alterations in [3H]rolipram binding in the hippocampus. Following 3-min ischemia, the binding in stratum pyramidale of CA1 decreased by 16% (p < 0.01 vs. control) (Fig. 1). Following secondary ischemia, transient I l-20% reductions in [3H]rolipram binding was observed in the strata pyramidale (p < 0.01 control) and radiatum (p < 0.05 vs. control) of CA1 at I day of reperfusion (Table 5).

[3H]PDBu Binding We did not observe statistically significant alterations in [3H]PDBu binding in the hippocampus (Table 2). But in half of the animals subjected to 3 min of ischemia, conspicuous decrease in binding in the strata pyramidale and lacnosum-moleculare of CA 1 was observed (Fig. 1).

TABLE BINDING

(fmol/mg

2

TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3-MIN ISCHEMIA 3-MIN ISCHEMIA INDUCED 4 DAYS AFTER 2-MIN ISCHEMIA 2-Min Ischemia 7 Days

Control

CA

46 f 13.9 80 f 9.4

6-7, *p < 0.05, tp < 0.01 compared to control.

Histopathology

[‘H]PDBu

39 + 7.2 65 + 8.1*

3-Min Ischemia 7 Days

AND FOLLOWING

2-Min + 3-Min Ischemia

I Day

Ih

7 Days

I subfield

Stratum oriens Stratum pyramidale Stratum radiatum Stratum lac-mol CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum

moleculare

Mean i. SD, n = 6-7.

901 1060 880 735

871 1074 902 771

t 80.9 + 101.7 + 118.1 k 152.2

842 925 879 662

f 113.6 f 77.7 f 76.7 + 93.0

888 1068 898 854

f f + f

192.1 180.1 179.4 209.1

850 1031 868 754

+ 94.2 XL113.1 -t 94.1 k 75.2

830 1010 780 653

f f f f

87.5 95.2 87.7 78.0

f f + f

126.1 102.6 108.4 100.6

840 + 835 f

93.0 70.6

792 f 79.6 843 + 126.0

807 f 101.3 851 f 72.4

853 f 153.8 891 + 177.4

791 f 831 *

90.1 71.1

701 f 47.4 710 f 61.4

765 f 102.6

831 f 132.2

879 +

883 k 157.1

806 f 101.5

712 f 64.3

73.9

562

KATO

ET AL.

FIG. 1.Representative autoradiographs of [‘H]inositol 1,4,5_triphosphate (IP>). [3H]phorhol 12,13-dibutyrate (PDBu), [3H]forskolin (Fors), [3H]cyclic adenosine monophosphate (CAMP) and [-‘H]rolipram (Roli) binding in the gerbil hippocampus of control gerbil brains (a, d, g. j, m). and in gerbils killed 7 days after 3 min of ischemia (b, e. h, k. n). and 7 days after 3 min of ischemia induced 4 days after pretreatment with 2 min of ischemia (double ischemia; c, f. i. I. 0). The binding in CA1 of all ligands, especially [3H]IP, binding, is reduced following 3 min of ischemia. However. the reductions are prevented by preconditioning with sublethal ischemia (double ischemia).

DISCUSSION The present study showed that 2-min forebrain ischemia in gerbils caused no alterations in the binding of second-messenger systems in the hippocampus, where no histopathological neuronal damage was observed. By contrast, 3-min ischemia, which produced moderate to severe destruction of CAI pyramidal cells, caused a marked (75%) reduction in [‘H]IP3 binding and 15-25s reductions in [3H]forskolin. [‘HIcAMP and [‘Hlrolipram binding in the CA1 subfield. In a half of the animals subjected to 3 min of ischemia, conspicuous decrease in [3H]PDBu binding was seen (Fig. 1), but the change was not statistically significant. The striking reduction in [3H]IP3 binding following ischemia and the relative preservation of [3H]PDBu and [3H]forskolin binding are consistent with previous reports (17). Postischemic alterations in [3H]cAMP and [3H]rolipram binding have not been reported. The different rates of reductions among the five second-messenger system ligands may be explained by the different localizations of the binding sites in the CA1 subfield because 3-min ischemia produces selective CA 1 pyramidal cell destruction without causing damage to presynaptic sites, interneurons, and glia (20). Previous reports indicate that [3H]IP3

binding sites are exclusively located on postsynaptic CA1 pyramidal cells, but that [‘H]PDBu and [3H]forskoIin binding sites may also be located on cells other than postsynaptic pyramidal cells such as presynaptic sites. interneurons, and glia (3,9,17.27). Following secondary ischemia, we observed transient reductions in [3H]IP3 and [3H]rolipram binding. [‘H]IP3 binding was decreased by 30% in the CA I subfield at 1 h and I day of reperfusion, and [3H]rolipram binding was also decreased by 20% in the stratum pyramidale of CA 1 at 1 day. However, the [3H]IP, and [‘Hlrolipram binding recovered to control levels by 7 days. t3H]PDBu, [3H]forskoIin, and [3H]cAMP binding remained at control levels throughout the reperfusion period after secondary ischemia. In our previous study, [‘H]IP3 binding following 2 min of ischemia in gerbils decreased by 30% at 6 h, 1 day. and 4 days of reperfusion. but was not statistically different from control at I month (17). In the present study, [3H]IP3 binding was not decreased at 7 days. Taken together, these results suggest a transient downregulation of IP3 system following 2 min of ischemia. Following secondary ischemia. the [3H]IP3 binding was reduced to 70% ofcontrol at I h and I day of reperfusion. This continuous

ISCHEMIC TOLERANCE

AND SECOND MESSENGERS

563

TABLE 3 [‘HIFORSKOLIN

BINDING

(fmol/mg TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3-MIN 3-MIN ISCHEMIA INDUCED 4 DAYS AFTER 2-MIN ISCHEMIA

2-Min + 2-Min lschemia

Control CA 1 subfield CA3 subfield Dentate hilus Dentate gyrus

54 * 162 + 224 k 109 f

5.1 18.3 10.2 4.2

7 Days 59 f 5.1 159 f 24.9 219 f 20.2 112f II.9

3-Min lschemia 7 Days 44 f 161 + 229 + ll6k

5.3* 13.4 16.2 5.7

3-Min Ischemia

I

Ih 54 f 162 + 221 + 105 f

AND FOLLOWING

ISCHEMIA

6.9 11.6 19.4 7.1

Day

51 + 162 f 223 f I10 f

4.5 14.2 11.8 5.7

7 Days

55 f 160 f 223 + Ill f

6.8 22.3 18.4 11.8

Binding in the CA3 subfield was measured in the stratum lucidum. Mean f SD, n = 6-7, *p < 0.01 compared to control

of [‘H]IP3 binding is likely to ameliorate ischemit neuronal damage following secondary ischemic insult (9,lO) because stimulation of IP3 receptors mobilizes calcium from intracellular calcium stores (4,7,3 1) and because elevated intracellular calcium concentrations are believed to lead to cell death (28). The excess stimulation with IP3 (5,39) and calcium (29) during and after ischemia (9) is the likely reason to cause the downregulation. Therefore, the reductions in [3H]IP3 binding may play a role in the protection against neuronal damage following preconditioning with sublethal ischemia. However, the downregulation of IP3 system cannot solely explain the tolerance phenomenon because the tolerance persists for 1 to 7 days after preconditioning while the downregulation of IP3 system was observed from 6 h to 4 days. [3H]Rolipram binding also transiently decreased at 1 day of reperfusion. However, the role of the CAMP selective phosphodiesterase, which [3H]rolipram labels ( 19) in ischemic neuronal damage is not known at present, as it is not the role of the adenylate cyclase system. Therefore, the role of CAMP selective phosphodiesterase in the protection following preconditioning is obscure. A behavioral study shows that the preconditioning the gerbil brain with sublethal ischemia prevents CA I neuronal destruction following secondary 5 min of ischemia, but that the result of passive avoidance tests performed 4 days after secondary ischemia were impaired (33). However, the present study showed that the second-messenger systems, especially the IP, system, exhibit transient dysfunctions after secondary ischemia, which may be one of the reasons for the disturbed learning

downregulation

ability.

The mechanisms of the induction of tolerance is not fully understood at present. Previous studies suggest a role of a protective protein such as heat shock/stress proteins and superoxide dismutase (21,32). In fact, pretreatment with hyperthermia protects the brain against subsequent ischemic insult (6,23). Furthermore, a brief period of ischemia induces the synthesis of heat shock proteins (21,30). However, the temporal pattern of heat shock protein-70 staining does not correlate well with the pattern of induced tolerance. Kirino et al. (2 1) reported that the tolerance was observed 1 day after 2-min ischemia in gerbils, but there was only minimum staining in the CA1 subfield at that time. Therefore, the induction of tolerance may not simply be explained by stress proteins. Taken together with the present result, the tolerance may be acquired through multifarious postischemic events. In conclusion, we showed postischemic alterations in the second-messenger systems using [3H]IP3, [3H]PDBu, [3H]forskolin, [3H]cAMP and [3H]rolipram binding following 2 and 3 min of ischemia and following 3 min of ischemia induced 4 days after preconditioning with 2 min of ischemia. Protection of the brain against ischemic brain damage by preconditioning with sublethal ischemia was accompanied by preservations of the second-messenger systems although [3H]IP3 and [3H]rolipram binding showed transient reductions in the early reperfusion period. This downregulation of the second-messenger systems, especially the IP3 system, may be one of the mechanisms of tolerance because ischemia-induced stimulation of IP3 receptors leads to intracellular calcium mobilization which is believed to be related to cell death.

TABLE 4 [‘HICAMP

BINDING

(fmol/mg

TISSUE) IN THE 3-MIN ISCHEMIA

HIPPOCAMPUS FOLLOWING 2- AND 3-MIN ISCHEMIA INDUCED 4 DAYS AFTER 2-MIN ISCHEMIA

AND FOLLOWING

2-Min + 3-Min Ischemia Control

CA 1 subfield CA3 subfield Dentate hilus Dentate gyrus

160 f 429 f 380 f 344 k

29.4 29.4 32.5 56.4

2-Min Ischemia 7 Days

161 + 434 + 384 + 344 t

35.2 50.7 45.8 49.7

3-Min lschemia 1 Days

122 f 427 f 368 + 305 f

9.6* 31.8 30.1 49.3

I Day

Ih

181 f 449 f 382 f 345 f

42.0 38.7 18.1 26.8

150 + 436 f 391 f 319 f

37.8 28.6 31.7 38.6

7 Days

I56 417 350 297

k + * k

24.3 41.9 43.3 43.1

Binding in the CA 1 and CA3 subfields was measured in the stratum pyramidale. Binding in the dentate gyrus was measured in the granular layer. Mean f SD, n = 6-7, *p < 0.05, compared to control.

564

KATO ET AL. TABLE [‘HIROLIPRAM

BINDING

5

(fmol/mg TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3-MIN 3-MIN ISCHEMIA INDUCED 4 DAYS AFTER 2.MIN ISCHEMIA

ISCHEMIA

AND FOLLOWING

?-Min + 3-Min lschemia Control

CA 1 subfield Stratum oriens Stratum pyramidale Stratum radiatum Stratum lac-mol CA3 subfield Average Dentate gyrus Stratum moleculare

94 89 97 65

& * + +

4.8 5.0 6.8 2.9

?-Min lschemia I Days

107 100 113 68

3-Min Ischemla I Days

81 75 90 57

? 13.2 -t 8.9 * IO.1 + 4.1

i4 r +

9.9 4.8t 6.0 1.6

Ih

105 82 102 63

I Day

+ 22. I i- Il.3 -t ‘4.0 i: 6.6

72 !Z 4.3

76 Z!Z 3.2

73 i 3. I

73 r

65 t 4.8

70 +

66 + 3.0

63 + 6.3

Mean + SD, n = 6-7. *p < 0.05, tP < 0.01 compared

3.3

5.

7 Days

85 t 6.9 71 + 4.6t 86 ? 9.0* 58 t 5.4

89 88 92 67

67 +- 4.9

68 _t 6.1

65 ? 3.9

6X *

i I+ &

11.4 8.9 12.2 2.x

4.2

to control.

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24.

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