66
Brain Research, 562 (1991) 66-70 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939117065F
BRES 17065
Hypertension and hyperglycemia in experimental stroke Andrew P. Slivka Department of Neurology, Ohio State University, Columbus, OH 43210 (U.S.A.) (Accepted 28 May 1991) Key words: Hypertension; Hyperglycemia; Experimental stroke
Both diabetes mellitus and hypertension are risk factors for stroke and also influence prognosis following stroke. Experimentally, hyperglycemia augments cortical infarct size in stroke models where collateral circulation exists, and infarct size in hypertensive rats is larger than in normotensive strains. Whether the deleterious effect of hyperglycemia is altered in the setting of hypertension has not previously been studied experimentally. The effect of hyperglycemia on infarct size in spontaneously hypertensive rats was examined in this study. Focal neocortical cerebral ischemia was induced by tandem right common carotid and middle cerebral artery occlusion. Preischemic hyperglycemia had no influence on infarct volume whether the duration of postischemic hyperglycemia was transient or prolonged. Although hyperglycemia increases infarct size in cortical stroke models where collateral circulation is available, this study demonstrates the effect can be modified by the presence of underlying hypertension. INTRODUCTION
H y p e r t e n s i o n is a risk factor for a t h e r o t h r o m b o t i c cerebral infarction 9"25'3°'32. M o r e o v e r , chronic b l o o d pressure elevations occurring prior to a stroke have been correlated with increased 30 day mortality following stroke 43. Experimentally, infarct size is greater in spontaneously hypertensive rats ( S H R ) subjected to focal cerebral ischemia than in normotensive rat strains 6A7'39. D i a b e t e s is also a risk factor for cerebral infarction 31, and influences prognosis following stroke. Early and late mortality as well as disability are increased in diabetics after stroke 3'2°'4°'42. H y p e r g l y c e m i a in the absence of diabetes has also been associated with increased early mortality, p o o r functional recovery, lesion size on CT scan, and cerebral e d e m a 5'8'33'34'36"40'42"46, although these findings have not been confirmed in all studies 1'2'12'21 . W h e t h e r the presence of hypertension may alter the influence of hyperglycemia on stroke morbidity and mortality and thus partially explain these conflicting results has not b e e n adequately addressed. This issue has only been indirectly examined. G o o d k i n r e p o r t e d that hypertension has a greater adverse effect on overall mortality among diabetics than among non-diabetics particularly for young patients 24. Fuller et al. found that most of the increased risk of death from stroke seen in patients with diabetes or glucose intolerance could not be readily explained on the basis of the presence of cardiovascular risk factors such as hypertension 2°.
Studies examining the influence of hyperglycemia in experimental focal infarction have also r e p o r t e d conflicting results. O n e reason for this is that the effect of hyperglycemia in focal stroke m a y be variable and m a y reflect the stroke m o d e l used, species differences, and the degree, timing, and duration of hyperglycemia in relation to the onset of focal cerebral ischemia. In cortical stroke models in which collateral circulation exists, however, most studies have found that preischemic hyperglycemia increases infarct volume 7'14"15'17"41. W h e t h e r the deleterious effect of hyperglycemia is altered in the setting of hypertension has not previously been studied experimentally. The p u r p o s e of this study was to examine the effect of preischemic hyperglycemia and transient or p r o l o n g e d postischemic hyperglycemia on infarct size in S H R using a focal stroke m o d e l which produces p r e d o m i n a n t l y neocortical infarction. MATERIALS AND METHODS Experimental design Two separate experiments were completed in this study. In each experiment male SHR weighing 260-350 g were used. SHR of this weight and age range exhibit significant medial and intimal changes in both peripheral and cerebral arterial vessels 26'35 as well as differences in cerebral blood flow when compared to age-matched normotensive rat strains4'~°. In Experiment 1, 13 SHR were injected intraperitoneally (ip) with 50% dextrose (1 ml/100 rag) and 12 SHR were injected ip with 0.9% NaCI (1 ml/100 mg) 0.5 h prior to middle cerebral artery (MCA) occlusion. In Experiment 2, 12 SHR were injected with 100 mg/kg streptozotocin ip. The streptozotocin was prepared fresh (100 mg in 1 ml 0.9% NaCl, pH 4.5). Streptozotocin-injected SHR were then subjected to focal cerebral
Correspondence: Dr. A. Slivka, Department of Neurology, Ohio State University, 1654 Upham Drive, Columbus, OH 43210, U.S.A.
67 isehemia 2 days later along with 11 SHR controls.
Focal ischemic surgery Animals in both experiments were fasted for 24 h prior to surgery. Haiothane (1.5-2.0%) was mixed with oxygen and nitrogen and delivered through a nose cone using a flow regulator. The tail artery was cannulated with a polyethylene catheter (PE-50) to monitor blood pressure and obtain blood samples for physiological variables. The right common carotid artery (CCA) was exposed through a midline neck incision and occluded with no. 4-0 surgical silk. The right middle cerebral artery (MCA) was exposed through a 2 mm burr hole drilled under a continuous normal saline drip 2-3 mm rostral to the fusion of the zygomatic arch with the squamosal bone. Using a MM 3 mieromanipulator (Narishige Instruments, Tokyo), a hook formed of 20 gauge silver wire was positioned under the MCA. The MCA was then lifted 0.5-1 mm above the cortical surface and cauterized. Body temperature was maintained at 37 °C throughout the procedure with a heat lamp connected to a rectal thermister. Immediately after CCA/MCA occlusion, all wounds were sutured and the animals were allowed to recover from the anesthesia. Surgery for each experiment was completed by a single investigator over a 2-3 week period on rats delivered from a single shipment (Harlan Sprague Dawley Inc., Indianapolis, IN). Physiologic variables Arterial blood pressure was monitored throughout the surgical procedure and then checked 4-6 h after surgery when animals had recovered from anesthesia. Arterial blood p~O2, paCO2, pH, and hematoerit were measured just after tail artery cannulation. Arterial blood gases were repeated again prior to MCA occlusion, and 4-6 h after C C A I M C A occlusion. Hematocrit was measured again just prior to decapitation. The concentration of halothane used during the surgical procedure was such that the mean arterial pressure was always above 90 mm Hg in SHR. This blood pressure is the
lower limit of cerebral autoregulation in SHR 19. The p , O 2 was maintained above 80 mm Hg by adjusting the oxygen concentration. In Experiment 1, where hyperglycemia was produced by ip dextrose injection, glucose was measured just prior to MCA occlusion and then 1-2 h, 4-6 h, and 24 h after MCA occlusion. In Experiment 2 in which diabetes was produced with streptozotocin, glucose was measured just prior to MCA occlusion and 24 h after MCA occlusion.
Brain histology Animals were anesthetized with halothane and decapitated 24 h after CCA/MCA occlusion. The brains were rapidly removed from the cranium and frozen in freon over dry ice. Coronal sections, 20 /~m thick, were cut at 500/zm intervals, fixed in 90% ethanol, and stained with hematoxylin and eosin. Each brain section was magnified using a photographic lens and the infarcted area traced onto paper. Each drawing was then retraced onto a digitizing tablet interfaced to an IBM Personal Computer (Video Image Analysis System, Ted Pelco Company, Redding, CA) which computes infarct areas for each section. To calculate total infarct volume, the infarcted area of sequential sections was summed and multiplied by the thickness between sections. Image analysis for each experiment was done by a technician who was blinded to the treatment groups. Intraobserver reliability using this method on two separate occasions was excellent (product moment coefficient of correlation, r = 0.98, n = 12). Statistical analyses Mean infarct volumes with standard deviations were computed for control and hyperglycemic groups in both experiments. Results were analyzed using a 2-tailed Student's t-test. Power analysis was done for both experiments to estimate the reduction of infarct volume that each experiment would be expected to detect 11.
TABLE I
Psysiological variables Mean _+ standard deviation.
Experiment 1
Preocclusion Mean Art. Pres. (mm Hg) Art. pH PaO2 (ram Hg) paCO2 (ram Hg) Glucose (mg/dl) Hematocrit 1-2 h postocclusion Glucose 3-6 h postocclusion Mean Art. Pres. (mm Hg) Art. pH p , O 2 (ram Hg) p,CO2 (mm Hg) Glucose (mg/dl) 24 h postocclusion Hematocrit Glucose (mg/dl)
Experiment 2
Control (n = 12)
Dextrose (n = 13)
Control (n = 11)
Streptozotocin (n = 12)
124 -+ 16 7.33 -+ 0.03 136 _+ 19 44_+3 127 _+ 10 50_+ 1
106 -+ 18 7.30 _+ 0.04 138 -+ 21 42 _+ 5 4 0 0 + 139 53 _+ 2
130 -+ 21 7.36 -+ 0.03 129 -+ 18 40_+6 158 _+ 36 48_+1
113 -+ 14 7.26 -+ 0.06 124 -+ 26 40_+5 476 _+ 45 50_+1
101 -+ 11
387 + 170
183 -+ 14 7.41 -+ 0.03 104-+6 41+3 115 -+ 8
158 -+ 22 7.33 - 0.05 104 _+ 4 42-+ 3 217 -+ 67
199 _+ 18 7.42 -+ 0.03 101 -+ 14 38_+ 3
158 7.28 106 35
47_+ 2 127 - 20
48_+3 119 -+ 20
49_+2 152 -+ 17
-+ 22 -+ 0.07 -+ 13 -+ 5
49_+3 491 -+ 132
68 TABLE II
ume of 16% or greater in Experiment
Infarct volume
in Experiment Infarct volume (mm3)
DISCUSSION
Normolglycemic
Hyperglycemic
(Dextrose)
141 k 18 (n = 12)
129 k 37 (n = 13)
Experiment 2 (Streptozotocin)
214 l?r 36 (n = 11)
213 -t 38 (n = 12)
Experiment 1
Prior studies examining the influence of hyperglycemia in permanent focal cerebral ischemic models have produced mixed results. Some studies have found an increase in infarct size7*14V15V’7,41 while others have reported
no difference3’,
differences
or a protective
effect22’4”. These
may reflect the focal stroke model employed
or the animal species used as well as the duration and timing of hyperglycemia in relation to the onset of isch-
RESULTS
emia. To explain Physiologic occlusion,
1 and 21% greater
2 would have been detected.
data are presented
all anesthetized
in Table I. Before MCA
rats in both experiments
de-
veloped a mild acidosis and blood pressure depression that was more pronounced in the hyperglycemic groups. Arterial pH and blood pressure measured 4-6 h post MCA occlusion improved in hyperglycemic SHR compared to preocclusion values but remained lower than values in normoglycemic SHR, which normalized. Hematocrit was unchanged over 24 h in all groups. Four animals in Experiment 2 injected with streptozotocin did not develop hyperglycemia. The glucose values of each of these animals was in the range of noninjected normoglycemic controls when measured prior to MCA occlusion and 24 h post MCA occlusion. Since the purpose of this study was to determine the effect of hyperglycemia on infarct size, these streptozotocin-injected animals without hyperglycemia were included in the normoglycemic groups for purposes of analysis. The hyperglycemia produced by streptozotocin injection (Experiment 2) was maintained over 24 h. Glucose values of hyperglycemic animals did not overlap with those of controls when measured preocclusion or 24 h post MCA occlusion. Dextrose injections produced hyperglycemia (Experiment 1) which began to normalize by 4-6 h post MCA occlusion and were in normoglycemic ranges within 24 h post MCA occlusion. No glucose values of dextrose injected animals overlapped with normoglycemic values pre MCA occlusion or l-2 h post MCA occlusion. Some overlap did exist by 4-6 h post MCA occlusion. Infarct volumes from both experiments are presented in Table II. Hyperglycemia produced by dextrose or streptozotocin injection in SHR did not significantly alter infarct size compared to normoglycemic controls (T (19) = 1.04, P = 0.3, Experiment 1; T (21) = -0.08, P = 0.9, Experiment 2). With a power of 80% (B = 0.2), an a of 0.05, and using the number of animals, mean infarct volumes and standard deviations for the control groups of experiments 1 and 2, a change in infarct vol-
the apparent
conflicting
results of hy-
perglycemia following permanent focal cerebral ischemia, Prado et al. postulated that infarcted regions with available collateral circulation such as the cerebral cortex in neocortical infarct models are vulnerable to the deleterious effects of hyperglycemia whereas regions of non-anastamosing (end-arterial) vascular supply such as the striatum in striatal (sub-cortical) infarct models are not41. In support of Prado’s hypothesis, almost all investigators using cortical stroke models with existing collateral supply have reported that preischemic hyperglycemia increases infarct size7~‘4~‘“~‘7~41.Only Zasslow et al. did not find increased infarct size with preischemic hyperglycemia. In that study, however, the infarcted area was measured on only one coronal brain section4s. This method of evaluating infarct size may not be truly representative of total infarct volume. Using the same MCA occlusion model in cats as Zasslow, DeCourten-Myers determined the infarcted area in 6 sequential brain sections and found increased infarct volume with preischemit hyperglycemia”. In contrast to the studies referenced above that demonstrate hyperglycemia increases infarct size in cortical stroke models where collateral circulation exists, in this study no difference in infarct volume was demonstrated between hyperglycemic and normoglycemic hypertensive rats. One possible explanation for the lack of a hyperglycemic effect in SHR may relate to cerebral blood flow. Several investigators have reported decreased cerebral blood flow with acute hyperglycemia’6,23 even in the setting of ischemia”‘. Since cerebral blood flow is depressed to a greater extent in SHR than normotensive rat strains following cerebral ischemia “,ls, acute glucose elevations may not further depress cerebral blood flow in SHR as suggested by Ibayashi et al. who found no difference in parietal or thalamic cerebral blood flow during or after 3 h of bilateral CCA occlusion in hyperglycemic and normoglycemic SHR”. Another possible explanation for the results may relate to the available collateral supply in SHR. Hodde et al. produced brain
69 vessel casts in S H R and normotensive Wistar rats by intra-arterial injection of a low viscosity resin 27. They found at least 3 times more intra-arterial anastomes b e t w e e n the M C A and anterior cerebral or posterior cerebral arteries in Wistar rats than SHR. Since less collaterals are available to iscbemic tissue in S H R than normotensive rat strains, C C A / M C A occlusion in S H R may produce an end-arterial ischemia. If this is the case, the results would be consistent with Prado's hypothesis since others have reported that hyperglycemia is not associated with increased infarct size in normotensive rat strains in models that produce end-arterial infarction T M . The effect of preischemic and transient postischemic hyperglycemia (dextrose injection) on infarct size was no different than that of preischemic and prolonged postischemic hyperglycemia (streptozotocin injection) in this study. Jernigan et al. also found that the effect of hyperglycemia on mortality and morbidity scores in rats following unilateral C C A occlusion and 15 min of hypoxia was the same regardless of whether hyperglycemia was produced by ip glucose injection or by diabetes from an alloxan injection given prior to the hypoxic ischemic insult 29. These results are consistent with work which demonstrates that maximal infarction develops within hours after M C A occlusion 13'45'47 and conform to reports that hyperglycemia induced after M C A occlusion does not influence infarct size 14'38. These studies suggest that the critical time during which hyperglycemia may influ-
REFERENCES 1 Adams, H.P., Olinger, C.P., Biller, J., Brott, T. and Barson, W., Usefullness of admission blood glucose in predicting outcome of acute cerebral infarction, Stroke, 18 (1988) 297. 2 Adams, H.P., Olinger, C.P., Marler, J.R., Biller, J., Brott, T.G., Barson, W.G. and Banwart, K., Comparison of admission serum glucose concentration with neurologic outcome in acute cerebral infarction. A study in patients given naloxone, Stroke, 19 (1988) 455-458. 3 Asplund, K., Hagg, E., Helmers, C., Lithner, F., Strand, T. and Wester, P., The natural history of stroke in diabetic patients, Acta Med. Scand., 207 (1980) 417-424. 4 Barry, D.I., Strandgaard, S., Graham, D.I., Braendstrup, O., Svendsen, U.G., Vorstrup, S., Hemmingsen, R. and Bolwig, T.G., Cerebral blood flow in rats with renal and spontaneous hypertension: resetting of the lower limit of autoregulation, J. Cereb. Blood Flow Metab., 2 (1981) 347-353. 5 Berger, L. and Hakim, A.M., The association of hyperglycemia with cerebral edema in stroke, Stroke, 17 (1986) 865-871. 6 Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J. and Pulsinelli, W., Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries, J. Cereb. Blood Flow Metab., 8 (1988) 474-485. 7 Brint, S., Kraig, R., Kiessling, M. and Pulsinelli, W., Hyperglycemia augments infarct size in focal experimental brain ischemia, Ann. Neurol., 18 (1985) 127. 8 Candelise, L., Landi, G., Orazio, E.N. and Boccardi, E., Prognostic significance of hyperglycemia in acute stroke, Arch. Neurol., 42 (1985) 661-663.
ence infarct size is at the onset of ischemia and during the initial postischemic period. This study also reinforces an important aspect of experimental design for studies using focal stroke models. The variability in infarct size between the control groups seen in this study has been reported previously 6'44. Since the explanation for this variability is unclear and therefore unpredictable, concurrent controls from the same shipment of rats should be used for each experiment. In summary, the influence of hyperglycemia on experimental focal infarction appears complex. Yet in cortical stroke models in which collateral circulation exists, studies report that preischemic hyperglycemia increases infarct volume. Even under these conditions, however, this study demonstrates that the effect of hyperglycemia on infarct size is modified in the setting of long standing hypertension. These results may have clinical implications, and suggest that future clinical studies examining the influence of hyperglycemia on stroke morbidity and mortality should include an analysis of hypertension as a potential modifying factor.
Acknowledgements. The author is grateful to Debbie Gray for her excellent technical assistance, to Drs. John Kissel and Steven Huber for their critical review of the manuscript, and to Bobbie Swank for typing the manuscript. This project was supported in part by a Bremer Foundation Grant from The Ohio State University.
9 Chapman, J.M., Reeder, L.G., Borun, E.R. and Coulson, A.H., Epidemiology of vascular lesions affecting the central nervous system: the occurrence of strokes in a sample population under observation for cardiovascular disease, Am. J. Public Health, 56 (1966) 191-201. 10 Choki, J., Yamaguchi, T., Takeya, Y., Morotomi, Y. and Omae, T., Effect of carotid artery ligation on regional cerebral blood flow in normotensive and spontaneously hypertensive rats, Stroke, 8 (1977) 374-379. 11 Cohen, J., Statistical Power Analysis for the Behavioral Sciences. Academic Press, London, 1977. 12 Cox, N.H. and Lorains, J.W., The prognostic value of blood glucose and glycosylated hemoglobin estimation in patients with stroke, Postgrad. Med. J., 62 (1986) 7-10. 13 Crowell, R.M., Olsson, Y., Klatzo, I. and Ommaya, A., Temporary occlusion of the middle cerebral artery in the monkey. Clinical and pathological observations, Stroke, 1 (1970) 439448. • 14 DeCourten-Myers, G.M., Myers, R.E. and Schoolfield, L., Effects of serum glucose concentration at and following middle cerebral artery occlusion in infarct size in cats, Neurology, 37 (suppl. 2) (1987) 130. 15 DeCourten-Myers, G., Myers, R.E. and Schoolfield, L., Hyperglycemia enlarges infarct size in cerebrovaseular occlusion in cats, Stroke, 19 (1988) 623--630. 16 Duckrow, R.B., Beard, D.C. and Brennan, R.W., Regional cerebral blood flow decreases during hyperglycemia, Ann. Neurol., 17 (1985) 267-272. 17 Duverger, D. and MacKenzie, E.T., The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose, concentration, and
70 age, J. Cereb. Blood Flow Metab., 8 (1988) 449-461. 18 Fujishima, M., lshitsuka, T., Nakatomi, Y., Takami, K. and Omae, T., Changes in local cerebral blood flow following bilateral carotid occlusion in spontaneously hypertensive and normotensive rats, Stroke, 12 (1981) 874-876. 19 Fijishima, M. and Omae, T., Lower limit of cerebral autoregulation in normotensive and spontaneously hypertensive rats, Experientia, 32 (1976) 1019-1021. 20 Fuller, J,H., Shipley, M.J., Rose, G., Jarrett, R.J. and Keen, H., Mortality from coronary heart disease and stroke in relation to degree of glycaemia: the Whitehall study, Br. Med. J., 287 (1983) 867-870. 2l Fullerton, K.J., MacKenzie, G. and Stout, R.W., Prognostic indices in stroke, Q. J. Med., 66 (1988) 147-162. 22 Ginsberg, M.D., Prado, R., Dietrich, W.D., Busto, R. and Watson, B.D., Hyperglycemia reduces the extent of cerebral infarction in rats, Stroke, 18 (1987) 570-574. 23 Ginsberg, M.D., Welsh, F.A. and Budd, W.W., Deleterious effect of glucose pretreatment on recovery from diffuse cerebral ischemia in the cat, Stroke, 11 (1980) 347-354. 24 Goodkin, G., Mortality factors in diabetes. A 20 year mortality study, J. Occup. Med., 17 (1976) 716-721. 25 Gordon, T. and Kannel, W.B., Predisposition to atherosclerosis in the head, heart, and legs. The Framingham study, JAMA, 221 (1972) 661-666. 26 Harper, S.L. and Bohlen, G., Microvascular adaptation in the cerebral cortex of adult spontaneously hypertensive rats, Hypertension, 6 (1984) 408-419. 27 Hodde, K.C., Nordborg, C. and Johansson, B.B., Pial artery casts of normotensive and hypertensive rats, Beitr. Electronenmikroskop. Direktabb. Oberfl., 17 (1984)209-214. 28 Ibayashi, S., Fujishima, M., Sadoshima, S., Yoshida, E, Shirokawa, O., Ogata, J. and Omae, T., Cerebral blood flow and tissue metabolism in experimental cerebral isehemia of spontaneously hypertensive rats with hyper-, normo-, and hypoglycemia, Stroke, 17 (1986) 261-266. 29 Jernigan, J., Evan, O.B. and Kirshner, H.S., Hyperglycemia and diabetes improve outcome in a rat model of anoxia, Neurology, 34 (Suppl. 1) (1984) 262. 30 Kannel, W.B., Role of blood pressure in cardiovascular morbidity and mortality, Prog. Cardiovasc. Dis., 17 (1974) 5-24. 31 Kannel W.B. and McGee, D.L., Diabetes and cardiovascular disease: the Framingham study, JAMA, 241 (1979) 2035-2038. 32 Kannel, W.B., Wolf, P.A., Verter, J. and McNamara, P.M., Epidemiologic assessment of the role of blood pressure in stroke. The Franingham study, JAMA, 214 (1970) 301-310. 33 Kushner, M., Nencini, P., Reivich, M., Rango, M., Jamieson, D., Fazekas, F., Zimmerman, R., Chaivluk, J., Alavi, A. and Alves, W., Relation of hyperglycemia early in ischemie brain infarction to cerebral anatomy, metabolism and clinical outcome, Ann. Neurol., 28 (1990) 129-135.
34 Levy, D.E., Pulsinelli, W.A., Scherer, P.B. and Plum, E, Effect of admission glucose level on recovery from acute stroke, Ann. Neurol., 18 (1985) 122. 35 Limas, C., Westrum, B. and Limas, C.J., The evolution of vascular changes in the spontaneously hypertensive rat, Am. Z, PathoL, 98 (1980) 357-384. 36 Mohr, J.P., Rubenstein, L.V., Tatemichi, T.K., Nichols, ET., Caplan, L.R., Hier, D.B., Kase, C.S., Prince, T.R. and Wolf, P.A., Blood sugar and acute stroke: the NINCDS Pilot Stroke Data Bank, Stroke, 16 (1985) 143. 37 Nakai, H., Yamamoto, L., Diksic, M., Worsley, K.J. and Takara, E., Triple-tracer autoradiography demonstrates effects of hyperglycemia on cerebral blood flow, pH, and glucose utilization in cerebral ischemia in rats, Stroke, 19 (1988) 764-772. 38 Nedergaard, M. and Diemer, N.H., Focal ischemia of the rat brain, with special reference to the influence of plasma glucose concentration, Acta Neuropathol., 73 (1987) 131-137. 39 Ogata, J., Fujishima, M., Morotomi, Y. and Omae, T., Cerebral infarction following bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats. A pathological study, Stroke, 7 (1976) 54-60. 40 Oppenheimer, S.M., Hoffbrand, B.I., Oswald, G.A. and Yudkin, J.S.~ Diabetes meilitus and early mortality from stroke, Br. Med. J., 291 (1985) 1014-1015. 41 Prado, R., Ginsberg, M.D., Dietrich, W.D. and Watson, B.D., Hyperglycemia increases the infarct size in collaterally perfused but not end-arterial vascular territories, J. Cereb. Blood Flow Metab., 8 (1988) 186-192. 42 Pulsinelli, W.A., Levy, D.E., Sigsbee. B., Scherer, P. and Plum, E, Increased damage after ischemic stroke in patients with hyperglycemia with or without established diabetes mellitus, Am. J. Med., 74 (1983) 540-544. 43 Rabkin, S.W., Mathewson, F.A.L. and Tate, R.B., The relation of blood pressure to stroke prognosis, Ann. Int. Med., 89 (1978) 15-20. 44 Slivka, A., Sibersweig, D. and Pulsinelli, W., Carnitine treatment for stroke in rats, Stroke, 21 (1990) 808-811. 45 Weinstein, P.R., Anderson, G.G. and Telles, D.A., Neurological deficit and cerebral infarction after temporary middle cerebral artery occlusion in unanesthetized cats, Stroke, 17 (1986) 318-327. 46 Woo, E., Chan, Y.W., Yu, Y.L. and Huang, C.Y., Admission glucose level in relation to mortality and morbidity outcome in 252 stroke patients, Stroke, 19 (1988) 185-191. 47 Xue, D., Slivka, A. and Buchan, A.M., The time course for infarction in a rat model of transient focal ischemia, Stroke, 21 (1990) 166. 48 Zasslow, M.A., Pearl, R.G.. Shuer, L.M., Steinberg, G.K., Lieberson, R.E. and Larson, C.P., Hyperglycemia decreases acute neuronal ischemic changes after middle cerebral artery occlusion in cats, Stroke, 20 (1989) 519-523.
Brain Research, 562 (1991) 66-70 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939117065F
BRES 17065
Hypertension and hyperglycemia in experimental stroke Andrew P. Slivka Department of Neurology, Ohio State University, Columbus, OH 43210 (U.S.A.) (Accepted 28 May 1991) Key words: Hypertension; Hyperglycemia; Experimental stroke
Both diabetes mellitus and hypertension are risk factors for stroke and also influence prognosis following stroke. Experimentally, hyperglycemia augments cortical infarct size in stroke models where collateral circulation exists, and infarct size in hypertensive rats is larger than in normotensive strains. Whether the deleterious effect of hyperglycemia is altered in the setting of hypertension has not previously been studied experimentally. The effect of hyperglycemia on infarct size in spontaneously hypertensive rats was examined in this study. Focal neocortical cerebral ischemia was induced by tandem right common carotid and middle cerebral artery occlusion. Preischemic hyperglycemia had no influence on infarct volume whether the duration of postischemic hyperglycemia was transient or prolonged. Although hyperglycemia increases infarct size in cortical stroke models where collateral circulation is available, this study demonstrates the effect can be modified by the presence of underlying hypertension. INTRODUCTION
H y p e r t e n s i o n is a risk factor for a t h e r o t h r o m b o t i c cerebral infarction 9"25'3°'32. M o r e o v e r , chronic b l o o d pressure elevations occurring prior to a stroke have been correlated with increased 30 day mortality following stroke 43. Experimentally, infarct size is greater in spontaneously hypertensive rats ( S H R ) subjected to focal cerebral ischemia than in normotensive rat strains 6A7'39. D i a b e t e s is also a risk factor for cerebral infarction 31, and influences prognosis following stroke. Early and late mortality as well as disability are increased in diabetics after stroke 3'2°'4°'42. H y p e r g l y c e m i a in the absence of diabetes has also been associated with increased early mortality, p o o r functional recovery, lesion size on CT scan, and cerebral e d e m a 5'8'33'34'36"40'42"46, although these findings have not been confirmed in all studies 1'2'12'21 . W h e t h e r the presence of hypertension may alter the influence of hyperglycemia on stroke morbidity and mortality and thus partially explain these conflicting results has not b e e n adequately addressed. This issue has only been indirectly examined. G o o d k i n r e p o r t e d that hypertension has a greater adverse effect on overall mortality among diabetics than among non-diabetics particularly for young patients 24. Fuller et al. found that most of the increased risk of death from stroke seen in patients with diabetes or glucose intolerance could not be readily explained on the basis of the presence of cardiovascular risk factors such as hypertension 2°.
Studies examining the influence of hyperglycemia in experimental focal infarction have also r e p o r t e d conflicting results. O n e reason for this is that the effect of hyperglycemia in focal stroke m a y be variable and m a y reflect the stroke m o d e l used, species differences, and the degree, timing, and duration of hyperglycemia in relation to the onset of focal cerebral ischemia. In cortical stroke models in which collateral circulation exists, however, most studies have found that preischemic hyperglycemia increases infarct volume 7'14"15'17"41. W h e t h e r the deleterious effect of hyperglycemia is altered in the setting of hypertension has not previously been studied experimentally. The p u r p o s e of this study was to examine the effect of preischemic hyperglycemia and transient or p r o l o n g e d postischemic hyperglycemia on infarct size in S H R using a focal stroke m o d e l which produces p r e d o m i n a n t l y neocortical infarction. MATERIALS AND METHODS Experimental design Two separate experiments were completed in this study. In each experiment male SHR weighing 260-350 g were used. SHR of this weight and age range exhibit significant medial and intimal changes in both peripheral and cerebral arterial vessels 26'35 as well as differences in cerebral blood flow when compared to age-matched normotensive rat strains4'~°. In Experiment 1, 13 SHR were injected intraperitoneally (ip) with 50% dextrose (1 ml/100 rag) and 12 SHR were injected ip with 0.9% NaCI (1 ml/100 mg) 0.5 h prior to middle cerebral artery (MCA) occlusion. In Experiment 2, 12 SHR were injected with 100 mg/kg streptozotocin ip. The streptozotocin was prepared fresh (100 mg in 1 ml 0.9% NaCl, pH 4.5). Streptozotocin-injected SHR were then subjected to focal cerebral
Correspondence: Dr. A. Slivka, Department of Neurology, Ohio State University, 1654 Upham Drive, Columbus, OH 43210, U.S.A.
67 isehemia 2 days later along with 11 SHR controls.
Focal ischemic surgery Animals in both experiments were fasted for 24 h prior to surgery. Haiothane (1.5-2.0%) was mixed with oxygen and nitrogen and delivered through a nose cone using a flow regulator. The tail artery was cannulated with a polyethylene catheter (PE-50) to monitor blood pressure and obtain blood samples for physiological variables. The right common carotid artery (CCA) was exposed through a midline neck incision and occluded with no. 4-0 surgical silk. The right middle cerebral artery (MCA) was exposed through a 2 mm burr hole drilled under a continuous normal saline drip 2-3 mm rostral to the fusion of the zygomatic arch with the squamosal bone. Using a MM 3 mieromanipulator (Narishige Instruments, Tokyo), a hook formed of 20 gauge silver wire was positioned under the MCA. The MCA was then lifted 0.5-1 mm above the cortical surface and cauterized. Body temperature was maintained at 37 °C throughout the procedure with a heat lamp connected to a rectal thermister. Immediately after CCA/MCA occlusion, all wounds were sutured and the animals were allowed to recover from the anesthesia. Surgery for each experiment was completed by a single investigator over a 2-3 week period on rats delivered from a single shipment (Harlan Sprague Dawley Inc., Indianapolis, IN). Physiologic variables Arterial blood pressure was monitored throughout the surgical procedure and then checked 4-6 h after surgery when animals had recovered from anesthesia. Arterial blood p~O2, paCO2, pH, and hematoerit were measured just after tail artery cannulation. Arterial blood gases were repeated again prior to MCA occlusion, and 4-6 h after C C A I M C A occlusion. Hematocrit was measured again just prior to decapitation. The concentration of halothane used during the surgical procedure was such that the mean arterial pressure was always above 90 mm Hg in SHR. This blood pressure is the
lower limit of cerebral autoregulation in SHR 19. The p , O 2 was maintained above 80 mm Hg by adjusting the oxygen concentration. In Experiment 1, where hyperglycemia was produced by ip dextrose injection, glucose was measured just prior to MCA occlusion and then 1-2 h, 4-6 h, and 24 h after MCA occlusion. In Experiment 2 in which diabetes was produced with streptozotocin, glucose was measured just prior to MCA occlusion and 24 h after MCA occlusion.
Brain histology Animals were anesthetized with halothane and decapitated 24 h after CCA/MCA occlusion. The brains were rapidly removed from the cranium and frozen in freon over dry ice. Coronal sections, 20 /~m thick, were cut at 500/zm intervals, fixed in 90% ethanol, and stained with hematoxylin and eosin. Each brain section was magnified using a photographic lens and the infarcted area traced onto paper. Each drawing was then retraced onto a digitizing tablet interfaced to an IBM Personal Computer (Video Image Analysis System, Ted Pelco Company, Redding, CA) which computes infarct areas for each section. To calculate total infarct volume, the infarcted area of sequential sections was summed and multiplied by the thickness between sections. Image analysis for each experiment was done by a technician who was blinded to the treatment groups. Intraobserver reliability using this method on two separate occasions was excellent (product moment coefficient of correlation, r = 0.98, n = 12). Statistical analyses Mean infarct volumes with standard deviations were computed for control and hyperglycemic groups in both experiments. Results were analyzed using a 2-tailed Student's t-test. Power analysis was done for both experiments to estimate the reduction of infarct volume that each experiment would be expected to detect 11.
TABLE I
Psysiological variables Mean _+ standard deviation.
Experiment 1
Preocclusion Mean Art. Pres. (mm Hg) Art. pH PaO2 (ram Hg) paCO2 (ram Hg) Glucose (mg/dl) Hematocrit 1-2 h postocclusion Glucose 3-6 h postocclusion Mean Art. Pres. (mm Hg) Art. pH p , O 2 (ram Hg) p,CO2 (mm Hg) Glucose (mg/dl) 24 h postocclusion Hematocrit Glucose (mg/dl)
Experiment 2
Control (n = 12)
Dextrose (n = 13)
Control (n = 11)
Streptozotocin (n = 12)
124 -+ 16 7.33 -+ 0.03 136 _+ 19 44_+3 127 _+ 10 50_+ 1
106 -+ 18 7.30 _+ 0.04 138 -+ 21 42 _+ 5 4 0 0 + 139 53 _+ 2
130 -+ 21 7.36 -+ 0.03 129 -+ 18 40_+6 158 _+ 36 48_+1
113 -+ 14 7.26 -+ 0.06 124 -+ 26 40_+5 476 _+ 45 50_+1
101 -+ 11
387 + 170
183 -+ 14 7.41 -+ 0.03 104-+6 41+3 115 -+ 8
158 -+ 22 7.33 - 0.05 104 _+ 4 42-+ 3 217 -+ 67
199 _+ 18 7.42 -+ 0.03 101 -+ 14 38_+ 3
158 7.28 106 35
47_+ 2 127 - 20
48_+3 119 -+ 20
49_+2 152 -+ 17
-+ 22 -+ 0.07 -+ 13 -+ 5
49_+3 491 -+ 132
68 TABLE II
ume of 16% or greater in Experiment
Infarct volume
in Experiment Infarct volume (mm3)
DISCUSSION
Normolglycemic
Hyperglycemic
(Dextrose)
141 k 18 (n = 12)
129 k 37 (n = 13)
Experiment 2 (Streptozotocin)
214 l?r 36 (n = 11)
213 -t 38 (n = 12)
Experiment 1
Prior studies examining the influence of hyperglycemia in permanent focal cerebral ischemic models have produced mixed results. Some studies have found an increase in infarct size7*14V15V’7,41 while others have reported
no difference3’,
differences
or a protective
effect22’4”. These
may reflect the focal stroke model employed
or the animal species used as well as the duration and timing of hyperglycemia in relation to the onset of isch-
RESULTS
emia. To explain Physiologic occlusion,
1 and 21% greater
2 would have been detected.
data are presented
all anesthetized
in Table I. Before MCA
rats in both experiments
de-
veloped a mild acidosis and blood pressure depression that was more pronounced in the hyperglycemic groups. Arterial pH and blood pressure measured 4-6 h post MCA occlusion improved in hyperglycemic SHR compared to preocclusion values but remained lower than values in normoglycemic SHR, which normalized. Hematocrit was unchanged over 24 h in all groups. Four animals in Experiment 2 injected with streptozotocin did not develop hyperglycemia. The glucose values of each of these animals was in the range of noninjected normoglycemic controls when measured prior to MCA occlusion and 24 h post MCA occlusion. Since the purpose of this study was to determine the effect of hyperglycemia on infarct size, these streptozotocin-injected animals without hyperglycemia were included in the normoglycemic groups for purposes of analysis. The hyperglycemia produced by streptozotocin injection (Experiment 2) was maintained over 24 h. Glucose values of hyperglycemic animals did not overlap with those of controls when measured preocclusion or 24 h post MCA occlusion. Dextrose injections produced hyperglycemia (Experiment 1) which began to normalize by 4-6 h post MCA occlusion and were in normoglycemic ranges within 24 h post MCA occlusion. No glucose values of dextrose injected animals overlapped with normoglycemic values pre MCA occlusion or l-2 h post MCA occlusion. Some overlap did exist by 4-6 h post MCA occlusion. Infarct volumes from both experiments are presented in Table II. Hyperglycemia produced by dextrose or streptozotocin injection in SHR did not significantly alter infarct size compared to normoglycemic controls (T (19) = 1.04, P = 0.3, Experiment 1; T (21) = -0.08, P = 0.9, Experiment 2). With a power of 80% (B = 0.2), an a of 0.05, and using the number of animals, mean infarct volumes and standard deviations for the control groups of experiments 1 and 2, a change in infarct vol-
the apparent
conflicting
results of hy-
perglycemia following permanent focal cerebral ischemia, Prado et al. postulated that infarcted regions with available collateral circulation such as the cerebral cortex in neocortical infarct models are vulnerable to the deleterious effects of hyperglycemia whereas regions of non-anastamosing (end-arterial) vascular supply such as the striatum in striatal (sub-cortical) infarct models are not41. In support of Prado’s hypothesis, almost all investigators using cortical stroke models with existing collateral supply have reported that preischemic hyperglycemia increases infarct size7~‘4~‘“~‘7~41.Only Zasslow et al. did not find increased infarct size with preischemic hyperglycemia. In that study, however, the infarcted area was measured on only one coronal brain section4s. This method of evaluating infarct size may not be truly representative of total infarct volume. Using the same MCA occlusion model in cats as Zasslow, DeCourten-Myers determined the infarcted area in 6 sequential brain sections and found increased infarct volume with preischemit hyperglycemia”. In contrast to the studies referenced above that demonstrate hyperglycemia increases infarct size in cortical stroke models where collateral circulation exists, in this study no difference in infarct volume was demonstrated between hyperglycemic and normoglycemic hypertensive rats. One possible explanation for the lack of a hyperglycemic effect in SHR may relate to cerebral blood flow. Several investigators have reported decreased cerebral blood flow with acute hyperglycemia’6,23 even in the setting of ischemia”‘. Since cerebral blood flow is depressed to a greater extent in SHR than normotensive rat strains following cerebral ischemia “,ls, acute glucose elevations may not further depress cerebral blood flow in SHR as suggested by Ibayashi et al. who found no difference in parietal or thalamic cerebral blood flow during or after 3 h of bilateral CCA occlusion in hyperglycemic and normoglycemic SHR”. Another possible explanation for the results may relate to the available collateral supply in SHR. Hodde et al. produced brain
69 vessel casts in S H R and normotensive Wistar rats by intra-arterial injection of a low viscosity resin 27. They found at least 3 times more intra-arterial anastomes b e t w e e n the M C A and anterior cerebral or posterior cerebral arteries in Wistar rats than SHR. Since less collaterals are available to iscbemic tissue in S H R than normotensive rat strains, C C A / M C A occlusion in S H R may produce an end-arterial ischemia. If this is the case, the results would be consistent with Prado's hypothesis since others have reported that hyperglycemia is not associated with increased infarct size in normotensive rat strains in models that produce end-arterial infarction T M . The effect of preischemic and transient postischemic hyperglycemia (dextrose injection) on infarct size was no different than that of preischemic and prolonged postischemic hyperglycemia (streptozotocin injection) in this study. Jernigan et al. also found that the effect of hyperglycemia on mortality and morbidity scores in rats following unilateral C C A occlusion and 15 min of hypoxia was the same regardless of whether hyperglycemia was produced by ip glucose injection or by diabetes from an alloxan injection given prior to the hypoxic ischemic insult 29. These results are consistent with work which demonstrates that maximal infarction develops within hours after M C A occlusion 13'45'47 and conform to reports that hyperglycemia induced after M C A occlusion does not influence infarct size 14'38. These studies suggest that the critical time during which hyperglycemia may influ-
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