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Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction
Brain Research, 408 (1987) 79-85
79
Elsevier BRE 12477
Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction M. Nedergaard Institute of Neuropathology, Universityof Copenhagen, Copenhagen (Denmark) (Accepted 26 August 1986)
Key words: Hyperglycemia; Focal ischemia; Infarction; Middle cerebral artery; Lactacidosis; Rat
To study whether transient ischemia is influenced by hyperglycemia, the middle cerebral artery was occluded for 5, 10 and 15 min in normo- and hyperglycemic rats. Five-minute ischemia induced minor lesions in both groups. After 10-min ischemia a significant greater infarct volume was found in hyperglycemia compared with normoglycemia (29 + 9 mm 3 vs 4 + 4 mm 3, P < 0.001). Fifteen-minute artery occlusion induced even more damage in both hyper- and normoglycemia (63 + 20 mm 3 vs 13 + 12 mm 3, P < 0.006). The lateral part of striatum was infarcted in all hyperglycemic animals exposed to 10 or 15 min of ischemia. In the same area selective neuronal injury occurred in 6 out of 9 uormoglycemic animals. The findings show that hyperglycemia increases brain damage during transient ischemia by conversion of selective neuronal injury into cerebral infarction
INTRODUCTION Diabetic patients have an increased incidence of cerebrovascular disease and the stroke o u t c o m e seems worse 16. The p o o r e r o u t c o m e in diabetic patients has been a t t r i b u t e d to the complicating conditions of diabetes, namely micro- and m a c r o a n g i o p a thy, arterial h y p e r t e n s i o n and diabetic c a r d i o m y o p a thy. E x p e r i m e n t a l global ischemia, however, has amply evidenced e n h a n c e d brain d a m a g e in hyperglycemia indicating a d e t r i m e n t a l effect of hyperglycemia p e r se 15. The e n h a n c e d brain d a m a g e is p r o b a b l y caused by increased lactate production in the hyperglycemic condition during ischemia 15. Nevertheless, the volume of infarction is not enlarged by hyperglycemia in e x p e r i m e n t a l focal ischemia induced by permanent occlusion of the middle cerebral artery ( M C A ) tl. This discrepancy m a y either reflect a pathophysiological difference between global and focal ischemia or b e t w e e n transient and p e r m a n e n t ischemia.
To evaluate this question infarction volume was studied in transient focal ischemia following M C A occlusion for 5, 10 and 15 min in n o r m o - en hyperglycemicrats MATERIALS AND METHODS
Animals Thirty-two male Wistar rats (S.P.F. Strain, Panums-avlslaboratorium, C o p e n h a g e n ) , weighing 3 3 0 350 g were used for the e x p e r i m e n t . In half of the animals hyperglycemia was induced 2 days before operation by a single i n t r a p e r i t o n e a l injection of streptozotozin (60 mg/kg). Before the o p e r a t i o n , the normoglycemic animals were starved overnight with free access to water. The animals were anesthetized with 3% halothane, intubated and mechanically ventilated with 0 . 5 - 1 . 0 % halothane in a 2:1 NEO:O z mixture, by a respirator ( H a r v a r d r o d e n t respirator). The tail artery was cannulated for continuous recording of mean arterial b l o o d pressure and monitoring o f p C O 2
Correspondence: M. Nedergaard, Institute of Neuropathology, University of Copenhagen, 11-Frederik V's Vej, Copenhagen 2100DK, Denmark. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
80 and pO 2 during the operation. Animals with a blood pressure of less than 90 mm Hg throughout the experiment were discarded. The p C O 2 was kept between 35 and 42 mm Hg, and pO2 within the range of 110-180 mm Hg. A constant rectal temperature of 37 °C was maintained with a heating lamp. In all animals the right MCA was exposed through a subtem-
loomm3 ~ 90 hyperglycemia oo [7777 norrnoglycemia 70 6o 5o 40
poral craniectomy 2°. a needle (6-0) was placed under the main trunk of the artery proximal to the origin of the lateral lenticulostriate branch. The needle was drawn until arrest of perfusion (controlled by microscopy) for 5, 10 or 15 min according to Table 1. In sham-operated animals the MCA was exposed, the
3o I I r r ~ [Ic~Iz~~-P~°°°' 20 lo ~ _ ~ o 5 10 t5 rain of ischemia Fig. 1. Diagram of volume of infarction in the 6 experimental groups. Mean + S.E.M. Levels of significance were tested by comparinghyperglycemicto normoglycemia.
needle placed under the artery for 10 min and then removed. After the operation, the tail artery catheter was removed, and ventilation continued (N20:O 2 mixture) until spontaneous respiration allowed for extubation,
T 9<0.006 _L q |
/
paraffin. For every 200pm interval three 10-pro thick coronal sections were stained with Hematoxylin-Eosin (HE), Acid fuchsin/Cresyi violet or Kliiver-Barrera.
Histologicprocedure At sacrifice the animals were reanesthetized with halothane and perfusion-fixed through the ascending aorta with Lillies phosphate-buffered formaldehyde for 20 min at 120 mm Hg with a roller pump (Dick, Copenhagen). The brains were stored for 4 days in the fixative, dehydrated in alcohol and embedded in
J!!
Infarct volume
Quantificationof brain damage Infarction was evident after 4 days as tissue with decreased stainability. An example is shown in Fig. 2. The slightly stained area of infarction was detected
....
Fig. 2. Coronal section from a hyperglycemic animal exposed to 10-min ischemia. The area of infarction shows decreased stainability. The infarct is localized to the lateral part of the neostriatum. A very sharp transition between the infarct and normal tissue is evident. Hematoxylin-Eosin stain.
81 by videodensitometry on a computerized image analyzer (Leitz TAS) in all the HE-stained sections. The volume of infarction was calculated by the formula: V- A x d L2 Where V is the infarct volume, A the sum of infarcted area in the sections measured, d the distance between the serial sections (200/~m) and L the linear magnification. The shrinkage was determined by comparing the area of 3 sections in each animal to freeze-dried sections at the same level in control animals. The infarct volume was corrected for the volume shrinkage of 16 _+ 3% (mean _+ S.E.M.).
RESULTS All animals survived in the hyperglycemic and the normoglycemic groups. The rats were clumsy, but walked and drank immediately after the operation. None of the animals had apparent neurological deficit. Five-minute artery occlusion induced minimal lesions in the cortex situated directly below the craniectomy in both normo- and hyperglycemic animals (Fig. 1). After 10-rain MCA occlusion all hyperglycemic rats had sharply demarcated infarcts in the lateral part of the striate body (Fig. 2). One normoglycemic rat had a minor striate infarct and in two out of 4 rats sporadic neuronal injury was evident in the upper
Fig. 3. Neostriatum in a normoglycemic animal exposed to 10-min ischemia. The lateral part of the striatum demonstrates sporadic neuronal injury seen as a dense gradient of infiltrating macrophages. Damage is worse superiorly. Acid fuchsin/Cresyl violet stain. x 180.
82
Fig. 4. The upper part of neostriatum after 10-min ischemia in a normoglycemic animal. Sporadic neurons are shrunken with pyknotic nuclei (arrows). Macrophages infiltrates the tissue. Acid fuchsin/Cresyl violet stain. × 450.
Fig. 5. Minor infarct in the lateral part of neostriatum in a normoglycemic animal exposed to 15-rain ischemia. The transition between infarcted and normal tissue is gradual. Hematoxylin-Eosin stain. × 180.
83
part of the striatum (Figs. 3 and 4). In the hyperglycemia duced complete
DISCUSSION
g r o u p , 15 m i n o f i s c h e m i a i n -
major
i n f a r c t i o n in t h e s t r i a t e b o d y
The present study indicates that transient ischemia
( T a b l e I). I n 4 a n i m a l s p a r t s o f t h e a d j a c e n t n e o c o r -
is a s s o c i a t e d w i t h a h i g h e r r i s k o f p e r m a n e n t
rex was infarcted as well. One normoglycemic
i n f a r c t i o n in h y p e r g l y c e m i c
rat had
a minor
i n f a r c t in s t r i a t u m
but the lesion was not
sharply
demarcated.
transition
farcted and normal
The
tissue was gradual
out of 5 normoglycemic
animals
between
in-
( F i g . 5). I n 4
sporadic
neuronal
It is c l e a r l y d o c u m e n t e d
that hyperglycemia
e t al. o b s e r v e d t h a t t h e p r e i s c h e m i c significantly affected
nutrional state of
the recovery
i n j u r y w a s f o u n d in t h e u p p e r p a r t o f t h e s t r i a t e b o d y ,
i n g i s c h e m i a o r h y p o x i a 9'1°. S i e m k o w i c z
whereas
s h o w e d t h a t a f t e r 10 m i n o f c o m p l e t e
the histological
appearance
of neocortical
all h y p e r g l y c e m i c
In 3 out of 6 sham-operated
animals minor cortical
adver-
sely affects the recovery after global ischemia. Myers monkeys
structures was inconspicuous,
cerebral
animals.
moglycemic
rats died within
rats survived
l e s i o n s w e r e f o u n d in t h e c o r t e x s i t u a t e d d i r e c t l y b e -
covery
low the craniectomy,
cits 19. A l s o t h e m e t a b o l i c
or with minor
brain ischemia
12 h , w h i l e n o r -
with either
persisting
follow-
and Hansen
complete
neurological
and neurophysiological
redeftre-
TABLE I
Histopathological details on the animals SNI, selective neuronal injury in the striate body. Student's t-test.
Hyperglycemia Infarct volume (mm 3) Shamoperated No. 1 No. 2 No. 3
.
SN1
. 2 3
Normoglycemia
.
Striate infarct
Cortical infarct
-
-
.
-
Plasma Infarct glucose volume (mmol/liter) (ram3)
22 24 23
. .
. . 5
2+2 5 min No. No. No. No.
1 2 3 4
.
.
3 11 22
. -
. +
-
24 27 29 20
18 24 34 38
19 46 63 87 103 63 + 20
. .
-
+ + + +
+
25 20 27 22
-
-
+ + + + +
+ + + +
21 24 25 20 28
Plasma glucose (mmol/liter)
8.3 6.7 9.8
n.s.
. . 3 9
. . -
. . -
1 1 3 9
2 10 10 10 10 13 + 12
-
5.9 6.3 9.5 8.2
n.s.
+ + -
+
4+4
-
Cortical infarct
. .
3+4
29+9 15 min No. 1 No. 2 No. 3 No. 4 No. 5
. .
Striate infarct
2+3
9+10 10 min No. 1 No. 2 No. 3 No. 4
SN1
-
7.3 8.8 5.7 9.2
P < 0.001
+ + + +
+
P < 0.006
8.1 6.6 7.8 6.1 9.5
84 covery are depressed in hyperglycemic rats after global ischemia 15. Histopathological, marked clumping of nuclear chromatin and cell sap was evident after 30 min of incomplete ischemia in hyperglycemic animals, whereas the changes were strikingly discrete in normoglycemic animals 7. The findings indicate that acidosis in association with energy failure exaggerates the damage. However, lactic acidosis did not significantly impair the postischemic recovery of mitochondrial function evaluated in vivo -~. Acidosis alone probably does not induces irreversible damage. Thus, rats exposed to severe hypercapnia develop no deterioration of brain energy state and even extreme hypercapnia-altered ultrastructure to such a moderate degree only, that irreversible cell damage is unlikely TM. Adequate reperfusion is of critical importance for recovery following global ischemia. Severe lactacidosis impairs postischemic circulation. Glucose given prior to 30-min incomplete global ischemia in cats caused a virtually complete impediment of perfusion, Kg~gstr0m et al. found that fasted rats had a better flow than fed animals after 15 min of incomplete global ischemia s. A morphometric study on cortical capillaries evidenced that the lumen narrowed as a result of endothelial swelling in hyperglycemic animals exposed to severe incomplete ischemia for 30 min 13. in normoglycemia, only minimal vascular changes were seen. It seems likely that endothelial swelling hampers reperfusion after ischemia accompanied by high levels of lactate, In this study, the injury after 5-min ischemia was minimal in both normo- and hyperglycemic rats, whereas 10-min artery occlusion induced infarction in the striate body in all hyperglycemic animals and in one normoglycemic rat. After 15-min ischemia part of the neocortex was also infarcted in hyperglycemia but only one of the normoglycemic rats had an infarct in the striatum. In normoglycemia, selective neuronal injury in the striate body occurred after 10-min
REFERENCES 1 Folbergrova, J., Ponten, U. and Siesj0, B.K., Patterns of changes in brain carbohydrate metabolites, amino acids and organic phosphates at increased carbon dioxide tensions, J. Neurochem., 22 (1974) 1115-1125. 2 Garcia, M.J., McNamara, P.M., Gordon, T. and Kannell,
ischemia and became more pronounced after 15-rain artery occlusion. Thus, in hyperglycemic animals the selective neuronal injury was converted into infarction. The volume of infarction is not increased by acute hyperglycemia in experimental stroke induced by permanent artery occlusion. Probably this reflects that the area of dense ischemia (with lactacidosis) is equal in size in normo- en hyperglycemia I1. If lactate production in this area is above the threshold for infarction in both groups the size of infarction is not influenced by hyperglycemia. In contrast, permanent occlusion of the middle cerebral artery in rats with chronic streptozotocin diabetes (4 months duration) lead to an increased volume of infarction (P < 0.046) 12. Jakobsen et al. found a 36% reduction in length of capillary network in neocortical tissue of rats with 1 year's diabetes °. The capillary loss was out of proportion to the 16% loss of neurons. The increased volume of infarction in chronic diabetic rats might, therefore, be a result of decreased collateral blood flow to the ischemic area. Since lactacidosis is not a feature of hypoglycemia it is noteworthy that the area of infarction is minor and the tissue structure better preserved if the animals are rendered hypoglycemic before permanent occlusion of the MCA 12. The significance of preischemic hyperglycemia was emphasized in this study. The poorer outcome after stroke in diabetic patients is probably related to both the complications of diabetes and to the detrimental effects of increased lactacidosis. Schoenberg et al. found that diabetes mellitus provided a major risk factor for complete stroke but not for an increased incidence of transient ischemic attack 17. This indicates that transient ischemic attacks in patients with raised blood glucose might result in complete infarction due to impaired reperfusion of the ischemic area.
U.H., Morbidity and mortality in diabetes in the Framingham population, Diabetes, 23 (1974) 105-111. 3 Ginsberg, M.D., Welsh, F.A. and Budd, W.W., Deleterious effect of glucose pretreatment on recovery from diffuse cerebral ischemia in the cat. Local cerebral blood flow and glucose utilization, Stroke, 11 (1980) 347-354. 4 Hall, T.C., Miller, A.K.H. and Corsellis, J.A.N., Varia-
85 tions in the human purkinje cell population according to age and sex, Neuropathol. Appl. Neurobiol., 1 (1975) 267. 5 Hillered, L., Smith, M.L. and Siesj¢, B.K., Lactid acidosis and recovery of mitochondrial function following forebrain ischemia in the rat, J. Cereb. Blood Flow Metab., 6, in press, 6 Jakobsen, J., Sidenius, P., Gundersen, H.J. and Osterby, R., Quantitative changes of cerebral neocortical structure in long-term streptozotocin diabetic rats, Diabetes, in press. 7 Kalimo, H., Rehncrona, S., Soderfeldt, B., Olsson, Y. and Siesj~, B.K., Brain lactic acidosis and ischemic cell damage. 2. Histopathology, J. Cereb. Blood Flow Metab., 1 (1981) 313-327. 8 K~gstrom, E., Smith, M.L. and Siesj~, B.K., Recirculation in the rat brain following incomplete ischemia, J. Cereb. Blood Flow Metab., 3 (1983) 183-192. 9 Myers, R.E., A unitary theory of causation of anoxic and hypoxic brain pathology. In S. Fahn, J. Davis and L. Rowland (Eds.), Cerebral hypoxia and its consequences, Advances in Neurology, Raven, New York, 1979, pp. 195-213. 10 Myers, R.E. and Yamaguchi, S., Nervous system effects of cardiac arrest in monkeys, Arch. Neurol., 34 (1977) 65-74. 11 Nedergaard, M. and Diemer, N.H., Focal ischemia of the rat brain. With special reference to the influence of plasma glucose concentration, Acta Neuropathol., in press, 12 Nedergaard, M. and Diemer, N.H., Influence of hyperglycemia on ischemic brain damage after middle cerebral artery (MCA) occlusion in the rat, J. Cereb. Blood Flow Metab., Suppl. 5, 11 (1985)231-232. 13 Paljfirvi, L., Rehncrona, S., S~derfeldt, B., Olsson, Y. and
Kalimo, H., Brain lactic acidosis and ischemic cell damage: quantitative ultrastructural changes in capillaries of rat cerebral cortex, Acta Neuropathol., 60 (1983) 232-240. 14 Paljfirvi, L., S~derfeldt, B., Kalimo, H., Olsson, Y. and Siesj~a, B.K., The brain in extreme respiratory acidosis. An ultrastructural study in the rat, Acta Neuropathol., 58 (1982) 87-94. 15 Plum, F., What causes infarction in ischemic brain? The Robert Wartenberg Lecture, Neurology, 33 (1983) 222-233. 16 Pulsinelli, W.A., Levy, D.E., Sigsbee, B. and Plum, F., Increased damage after stroke in patients with hyperglycemia with or without established diabetes mellitus, Am. J. Med., 74 (1983) 540-544. 17 Schoenberg, B.S., Schoenberg, D.G., Pritchard, D.A., Lillinfeld, A.M. and Whisnant, J.P., Differential risk factors for complete stroke and transient ischemic attacks: study of vascular disease (hypertension, cardiac disease, peripheral vascular disease) and diabetes mellitus, Trans. Am. Neurol. Ass., 105(1980) 165-167. 18 Schr~der, K.F., Hopf, A., Lange, H. and Th~rner, G., Morphometrisch-statische strukturanalysen des striatum, pallidum und nucleus subthalamicus beim menschen. 1. Striatum, J. Hirnforsch., 16 (1975) 333. 19 Siemkowicz, E. and Hansen, A., Clinical restitution following cerebral ischemia in hypo-, normo- and hyperglycemic rats, Acta Neurol. Scand., 58 (1978) 1-8. 20 Tamura, A., Graham, D.I., McCulloch, J. and Teasdala, G.M., Description of technique and early neuropathoiogical consequences following middle cerebral artery occlusion, J. Cereb. Blood Flow Metab., 1 (1981) 53-60.
79
Elsevier BRE 12477
Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction M. Nedergaard Institute of Neuropathology, Universityof Copenhagen, Copenhagen (Denmark) (Accepted 26 August 1986)
Key words: Hyperglycemia; Focal ischemia; Infarction; Middle cerebral artery; Lactacidosis; Rat
To study whether transient ischemia is influenced by hyperglycemia, the middle cerebral artery was occluded for 5, 10 and 15 min in normo- and hyperglycemic rats. Five-minute ischemia induced minor lesions in both groups. After 10-min ischemia a significant greater infarct volume was found in hyperglycemia compared with normoglycemia (29 + 9 mm 3 vs 4 + 4 mm 3, P < 0.001). Fifteen-minute artery occlusion induced even more damage in both hyper- and normoglycemia (63 + 20 mm 3 vs 13 + 12 mm 3, P < 0.006). The lateral part of striatum was infarcted in all hyperglycemic animals exposed to 10 or 15 min of ischemia. In the same area selective neuronal injury occurred in 6 out of 9 uormoglycemic animals. The findings show that hyperglycemia increases brain damage during transient ischemia by conversion of selective neuronal injury into cerebral infarction
INTRODUCTION Diabetic patients have an increased incidence of cerebrovascular disease and the stroke o u t c o m e seems worse 16. The p o o r e r o u t c o m e in diabetic patients has been a t t r i b u t e d to the complicating conditions of diabetes, namely micro- and m a c r o a n g i o p a thy, arterial h y p e r t e n s i o n and diabetic c a r d i o m y o p a thy. E x p e r i m e n t a l global ischemia, however, has amply evidenced e n h a n c e d brain d a m a g e in hyperglycemia indicating a d e t r i m e n t a l effect of hyperglycemia p e r se 15. The e n h a n c e d brain d a m a g e is p r o b a b l y caused by increased lactate production in the hyperglycemic condition during ischemia 15. Nevertheless, the volume of infarction is not enlarged by hyperglycemia in e x p e r i m e n t a l focal ischemia induced by permanent occlusion of the middle cerebral artery ( M C A ) tl. This discrepancy m a y either reflect a pathophysiological difference between global and focal ischemia or b e t w e e n transient and p e r m a n e n t ischemia.
To evaluate this question infarction volume was studied in transient focal ischemia following M C A occlusion for 5, 10 and 15 min in n o r m o - en hyperglycemicrats MATERIALS AND METHODS
Animals Thirty-two male Wistar rats (S.P.F. Strain, Panums-avlslaboratorium, C o p e n h a g e n ) , weighing 3 3 0 350 g were used for the e x p e r i m e n t . In half of the animals hyperglycemia was induced 2 days before operation by a single i n t r a p e r i t o n e a l injection of streptozotozin (60 mg/kg). Before the o p e r a t i o n , the normoglycemic animals were starved overnight with free access to water. The animals were anesthetized with 3% halothane, intubated and mechanically ventilated with 0 . 5 - 1 . 0 % halothane in a 2:1 NEO:O z mixture, by a respirator ( H a r v a r d r o d e n t respirator). The tail artery was cannulated for continuous recording of mean arterial b l o o d pressure and monitoring o f p C O 2
Correspondence: M. Nedergaard, Institute of Neuropathology, University of Copenhagen, 11-Frederik V's Vej, Copenhagen 2100DK, Denmark. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
80 and pO 2 during the operation. Animals with a blood pressure of less than 90 mm Hg throughout the experiment were discarded. The p C O 2 was kept between 35 and 42 mm Hg, and pO2 within the range of 110-180 mm Hg. A constant rectal temperature of 37 °C was maintained with a heating lamp. In all animals the right MCA was exposed through a subtem-
loomm3 ~ 90 hyperglycemia oo [7777 norrnoglycemia 70 6o 5o 40
poral craniectomy 2°. a needle (6-0) was placed under the main trunk of the artery proximal to the origin of the lateral lenticulostriate branch. The needle was drawn until arrest of perfusion (controlled by microscopy) for 5, 10 or 15 min according to Table 1. In sham-operated animals the MCA was exposed, the
3o I I r r ~ [Ic~Iz~~-P~°°°' 20 lo ~ _ ~ o 5 10 t5 rain of ischemia Fig. 1. Diagram of volume of infarction in the 6 experimental groups. Mean + S.E.M. Levels of significance were tested by comparinghyperglycemicto normoglycemia.
needle placed under the artery for 10 min and then removed. After the operation, the tail artery catheter was removed, and ventilation continued (N20:O 2 mixture) until spontaneous respiration allowed for extubation,
T 9<0.006 _L q |
/
paraffin. For every 200pm interval three 10-pro thick coronal sections were stained with Hematoxylin-Eosin (HE), Acid fuchsin/Cresyi violet or Kliiver-Barrera.
Histologicprocedure At sacrifice the animals were reanesthetized with halothane and perfusion-fixed through the ascending aorta with Lillies phosphate-buffered formaldehyde for 20 min at 120 mm Hg with a roller pump (Dick, Copenhagen). The brains were stored for 4 days in the fixative, dehydrated in alcohol and embedded in
J!!
Infarct volume
Quantificationof brain damage Infarction was evident after 4 days as tissue with decreased stainability. An example is shown in Fig. 2. The slightly stained area of infarction was detected
....
Fig. 2. Coronal section from a hyperglycemic animal exposed to 10-min ischemia. The area of infarction shows decreased stainability. The infarct is localized to the lateral part of the neostriatum. A very sharp transition between the infarct and normal tissue is evident. Hematoxylin-Eosin stain.
81 by videodensitometry on a computerized image analyzer (Leitz TAS) in all the HE-stained sections. The volume of infarction was calculated by the formula: V- A x d L2 Where V is the infarct volume, A the sum of infarcted area in the sections measured, d the distance between the serial sections (200/~m) and L the linear magnification. The shrinkage was determined by comparing the area of 3 sections in each animal to freeze-dried sections at the same level in control animals. The infarct volume was corrected for the volume shrinkage of 16 _+ 3% (mean _+ S.E.M.).
RESULTS All animals survived in the hyperglycemic and the normoglycemic groups. The rats were clumsy, but walked and drank immediately after the operation. None of the animals had apparent neurological deficit. Five-minute artery occlusion induced minimal lesions in the cortex situated directly below the craniectomy in both normo- and hyperglycemic animals (Fig. 1). After 10-rain MCA occlusion all hyperglycemic rats had sharply demarcated infarcts in the lateral part of the striate body (Fig. 2). One normoglycemic rat had a minor striate infarct and in two out of 4 rats sporadic neuronal injury was evident in the upper
Fig. 3. Neostriatum in a normoglycemic animal exposed to 10-min ischemia. The lateral part of the striatum demonstrates sporadic neuronal injury seen as a dense gradient of infiltrating macrophages. Damage is worse superiorly. Acid fuchsin/Cresyl violet stain. x 180.
82
Fig. 4. The upper part of neostriatum after 10-min ischemia in a normoglycemic animal. Sporadic neurons are shrunken with pyknotic nuclei (arrows). Macrophages infiltrates the tissue. Acid fuchsin/Cresyl violet stain. × 450.
Fig. 5. Minor infarct in the lateral part of neostriatum in a normoglycemic animal exposed to 15-rain ischemia. The transition between infarcted and normal tissue is gradual. Hematoxylin-Eosin stain. × 180.
83
part of the striatum (Figs. 3 and 4). In the hyperglycemia duced complete
DISCUSSION
g r o u p , 15 m i n o f i s c h e m i a i n -
major
i n f a r c t i o n in t h e s t r i a t e b o d y
The present study indicates that transient ischemia
( T a b l e I). I n 4 a n i m a l s p a r t s o f t h e a d j a c e n t n e o c o r -
is a s s o c i a t e d w i t h a h i g h e r r i s k o f p e r m a n e n t
rex was infarcted as well. One normoglycemic
i n f a r c t i o n in h y p e r g l y c e m i c
rat had
a minor
i n f a r c t in s t r i a t u m
but the lesion was not
sharply
demarcated.
transition
farcted and normal
The
tissue was gradual
out of 5 normoglycemic
animals
between
in-
( F i g . 5). I n 4
sporadic
neuronal
It is c l e a r l y d o c u m e n t e d
that hyperglycemia
e t al. o b s e r v e d t h a t t h e p r e i s c h e m i c significantly affected
nutrional state of
the recovery
i n j u r y w a s f o u n d in t h e u p p e r p a r t o f t h e s t r i a t e b o d y ,
i n g i s c h e m i a o r h y p o x i a 9'1°. S i e m k o w i c z
whereas
s h o w e d t h a t a f t e r 10 m i n o f c o m p l e t e
the histological
appearance
of neocortical
all h y p e r g l y c e m i c
In 3 out of 6 sham-operated
animals minor cortical
adver-
sely affects the recovery after global ischemia. Myers monkeys
structures was inconspicuous,
cerebral
animals.
moglycemic
rats died within
rats survived
l e s i o n s w e r e f o u n d in t h e c o r t e x s i t u a t e d d i r e c t l y b e -
covery
low the craniectomy,
cits 19. A l s o t h e m e t a b o l i c
or with minor
brain ischemia
12 h , w h i l e n o r -
with either
persisting
follow-
and Hansen
complete
neurological
and neurophysiological
redeftre-
TABLE I
Histopathological details on the animals SNI, selective neuronal injury in the striate body. Student's t-test.
Hyperglycemia Infarct volume (mm 3) Shamoperated No. 1 No. 2 No. 3
.
SN1
. 2 3
Normoglycemia
.
Striate infarct
Cortical infarct
-
-
.
-
Plasma Infarct glucose volume (mmol/liter) (ram3)
22 24 23
. .
. . 5
2+2 5 min No. No. No. No.
1 2 3 4
.
.
3 11 22
. -
. +
-
24 27 29 20
18 24 34 38
19 46 63 87 103 63 + 20
. .
-
+ + + +
+
25 20 27 22
-
-
+ + + + +
+ + + +
21 24 25 20 28
Plasma glucose (mmol/liter)
8.3 6.7 9.8
n.s.
. . 3 9
. . -
. . -
1 1 3 9
2 10 10 10 10 13 + 12
-
5.9 6.3 9.5 8.2
n.s.
+ + -
+
4+4
-
Cortical infarct
. .
3+4
29+9 15 min No. 1 No. 2 No. 3 No. 4 No. 5
. .
Striate infarct
2+3
9+10 10 min No. 1 No. 2 No. 3 No. 4
SN1
-
7.3 8.8 5.7 9.2
P < 0.001
+ + + +
+
P < 0.006
8.1 6.6 7.8 6.1 9.5
84 covery are depressed in hyperglycemic rats after global ischemia 15. Histopathological, marked clumping of nuclear chromatin and cell sap was evident after 30 min of incomplete ischemia in hyperglycemic animals, whereas the changes were strikingly discrete in normoglycemic animals 7. The findings indicate that acidosis in association with energy failure exaggerates the damage. However, lactic acidosis did not significantly impair the postischemic recovery of mitochondrial function evaluated in vivo -~. Acidosis alone probably does not induces irreversible damage. Thus, rats exposed to severe hypercapnia develop no deterioration of brain energy state and even extreme hypercapnia-altered ultrastructure to such a moderate degree only, that irreversible cell damage is unlikely TM. Adequate reperfusion is of critical importance for recovery following global ischemia. Severe lactacidosis impairs postischemic circulation. Glucose given prior to 30-min incomplete global ischemia in cats caused a virtually complete impediment of perfusion, Kg~gstr0m et al. found that fasted rats had a better flow than fed animals after 15 min of incomplete global ischemia s. A morphometric study on cortical capillaries evidenced that the lumen narrowed as a result of endothelial swelling in hyperglycemic animals exposed to severe incomplete ischemia for 30 min 13. in normoglycemia, only minimal vascular changes were seen. It seems likely that endothelial swelling hampers reperfusion after ischemia accompanied by high levels of lactate, In this study, the injury after 5-min ischemia was minimal in both normo- and hyperglycemic rats, whereas 10-min artery occlusion induced infarction in the striate body in all hyperglycemic animals and in one normoglycemic rat. After 15-min ischemia part of the neocortex was also infarcted in hyperglycemia but only one of the normoglycemic rats had an infarct in the striatum. In normoglycemia, selective neuronal injury in the striate body occurred after 10-min
REFERENCES 1 Folbergrova, J., Ponten, U. and Siesj0, B.K., Patterns of changes in brain carbohydrate metabolites, amino acids and organic phosphates at increased carbon dioxide tensions, J. Neurochem., 22 (1974) 1115-1125. 2 Garcia, M.J., McNamara, P.M., Gordon, T. and Kannell,
ischemia and became more pronounced after 15-rain artery occlusion. Thus, in hyperglycemic animals the selective neuronal injury was converted into infarction. The volume of infarction is not increased by acute hyperglycemia in experimental stroke induced by permanent artery occlusion. Probably this reflects that the area of dense ischemia (with lactacidosis) is equal in size in normo- en hyperglycemia I1. If lactate production in this area is above the threshold for infarction in both groups the size of infarction is not influenced by hyperglycemia. In contrast, permanent occlusion of the middle cerebral artery in rats with chronic streptozotocin diabetes (4 months duration) lead to an increased volume of infarction (P < 0.046) 12. Jakobsen et al. found a 36% reduction in length of capillary network in neocortical tissue of rats with 1 year's diabetes °. The capillary loss was out of proportion to the 16% loss of neurons. The increased volume of infarction in chronic diabetic rats might, therefore, be a result of decreased collateral blood flow to the ischemic area. Since lactacidosis is not a feature of hypoglycemia it is noteworthy that the area of infarction is minor and the tissue structure better preserved if the animals are rendered hypoglycemic before permanent occlusion of the MCA 12. The significance of preischemic hyperglycemia was emphasized in this study. The poorer outcome after stroke in diabetic patients is probably related to both the complications of diabetes and to the detrimental effects of increased lactacidosis. Schoenberg et al. found that diabetes mellitus provided a major risk factor for complete stroke but not for an increased incidence of transient ischemic attack 17. This indicates that transient ischemic attacks in patients with raised blood glucose might result in complete infarction due to impaired reperfusion of the ischemic area.
U.H., Morbidity and mortality in diabetes in the Framingham population, Diabetes, 23 (1974) 105-111. 3 Ginsberg, M.D., Welsh, F.A. and Budd, W.W., Deleterious effect of glucose pretreatment on recovery from diffuse cerebral ischemia in the cat. Local cerebral blood flow and glucose utilization, Stroke, 11 (1980) 347-354. 4 Hall, T.C., Miller, A.K.H. and Corsellis, J.A.N., Varia-
85 tions in the human purkinje cell population according to age and sex, Neuropathol. Appl. Neurobiol., 1 (1975) 267. 5 Hillered, L., Smith, M.L. and Siesj¢, B.K., Lactid acidosis and recovery of mitochondrial function following forebrain ischemia in the rat, J. Cereb. Blood Flow Metab., 6, in press, 6 Jakobsen, J., Sidenius, P., Gundersen, H.J. and Osterby, R., Quantitative changes of cerebral neocortical structure in long-term streptozotocin diabetic rats, Diabetes, in press. 7 Kalimo, H., Rehncrona, S., Soderfeldt, B., Olsson, Y. and Siesj~, B.K., Brain lactic acidosis and ischemic cell damage. 2. Histopathology, J. Cereb. Blood Flow Metab., 1 (1981) 313-327. 8 K~gstrom, E., Smith, M.L. and Siesj~, B.K., Recirculation in the rat brain following incomplete ischemia, J. Cereb. Blood Flow Metab., 3 (1983) 183-192. 9 Myers, R.E., A unitary theory of causation of anoxic and hypoxic brain pathology. In S. Fahn, J. Davis and L. Rowland (Eds.), Cerebral hypoxia and its consequences, Advances in Neurology, Raven, New York, 1979, pp. 195-213. 10 Myers, R.E. and Yamaguchi, S., Nervous system effects of cardiac arrest in monkeys, Arch. Neurol., 34 (1977) 65-74. 11 Nedergaard, M. and Diemer, N.H., Focal ischemia of the rat brain. With special reference to the influence of plasma glucose concentration, Acta Neuropathol., in press, 12 Nedergaard, M. and Diemer, N.H., Influence of hyperglycemia on ischemic brain damage after middle cerebral artery (MCA) occlusion in the rat, J. Cereb. Blood Flow Metab., Suppl. 5, 11 (1985)231-232. 13 Paljfirvi, L., Rehncrona, S., S~derfeldt, B., Olsson, Y. and
Kalimo, H., Brain lactic acidosis and ischemic cell damage: quantitative ultrastructural changes in capillaries of rat cerebral cortex, Acta Neuropathol., 60 (1983) 232-240. 14 Paljfirvi, L., S~derfeldt, B., Kalimo, H., Olsson, Y. and Siesj~a, B.K., The brain in extreme respiratory acidosis. An ultrastructural study in the rat, Acta Neuropathol., 58 (1982) 87-94. 15 Plum, F., What causes infarction in ischemic brain? The Robert Wartenberg Lecture, Neurology, 33 (1983) 222-233. 16 Pulsinelli, W.A., Levy, D.E., Sigsbee, B. and Plum, F., Increased damage after stroke in patients with hyperglycemia with or without established diabetes mellitus, Am. J. Med., 74 (1983) 540-544. 17 Schoenberg, B.S., Schoenberg, D.G., Pritchard, D.A., Lillinfeld, A.M. and Whisnant, J.P., Differential risk factors for complete stroke and transient ischemic attacks: study of vascular disease (hypertension, cardiac disease, peripheral vascular disease) and diabetes mellitus, Trans. Am. Neurol. Ass., 105(1980) 165-167. 18 Schr~der, K.F., Hopf, A., Lange, H. and Th~rner, G., Morphometrisch-statische strukturanalysen des striatum, pallidum und nucleus subthalamicus beim menschen. 1. Striatum, J. Hirnforsch., 16 (1975) 333. 19 Siemkowicz, E. and Hansen, A., Clinical restitution following cerebral ischemia in hypo-, normo- and hyperglycemic rats, Acta Neurol. Scand., 58 (1978) 1-8. 20 Tamura, A., Graham, D.I., McCulloch, J. and Teasdala, G.M., Description of technique and early neuropathoiogical consequences following middle cerebral artery occlusion, J. Cereb. Blood Flow Metab., 1 (1981) 53-60.