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Capillary patency after transient middle cerebral artery occlusion of 2 h duration
Neuroscience Letters 253 (1998) 191–194
Capillary patency after transient middle cerebral artery occlusion of 2 h duration Ping-An Li a ,*, Johannes Vogel b, Maj-lis Smith c, Qing-Ping He a, Wolfgang Kuschinsky b, Bo K. Siesjo¨ a a
Center for the Study of Neurological Diseases, The Neuroscience Institute, Queen’s Medical Center, 1356 Lusitana Street, UH Tower 8th floor, Honolulu, HI 96813, USA b Department of Physiology, University of Heidelberg, D-69120 Heidelberg, Germany c Laboratory for Experimental Brain Research, University of Lund, Lund, Sweden Received 12 June 1998; received in revised form 28 July 1998; accepted 4 August 1998
Abstract Reperfusion after transient focal ischemia of 2 h duration is followed by secondary bioenergetic failure after 4 h of reperfusion. The objective of the present study was to explore whether or not this secondary deterioration is due to secondary microcirculatory compromise. Normal fasted rats were subjected to 2 h of MCA occlusion and allowed reperfusion for 2, 4, 6 and 8 h. At predetermined reperfusion times, rats were injected with Evans blue and decapitated. Capillary patency was determined using a fluorescent double-staining technique. No capillary perfusion deficits were detected in the ischemic neocortical penumbra, neocortical focus or striatal focus. We concluded that the secondary deterioration of bioenergetic state is not due to microcirculatory compromise. Since hyperglycemic animals show pan-necrotic lesions, a hyperglycemic group was added at 8 h of reperfusion to test if the adverse effect of hyperglycemia on ischemic damage is related to capillary compromise. The results showed that, in hyperglycemic rats, capillary perfusion in the striatal focus was compromised after 8 h of recirculation following 2 h of MCA occlusion. It is concluded that when normoglycemic rats are subjected to 2 h of MCA occlusion, capillary patency is not affected during the first 4–6 h of reflow. At 8 h of reflow, though, particularly in hyperglycemic rats, microcirculation is compromised in the caudoputamenal focus, probably reflecting infarction. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Capillary patency; Focal ischemia; Microcirculation; Fluorescent double staining; Blood flow; Brain
Reperfusion after focal ischemia of 2 h duration is followed by partial recovery of tissue bioenergetic state, but a secondary deterioration is observed after 4 h of reperfusion [2]. The question arises whether or not this is due to secondary microcirculatory compromise. The previous data on local blood flow suggest that blood flow is not compromised in the first 1–6 h of reperfusion, following 1–2 h of MCA occlusion [7,11], i.e. in the period in which the bioenergetic state deteriorates. However, a recent study reports that the amelioration of tissue damage by PBN is related to an improved reflow during early reperfusion [10]. In that
* Corresponding author. Tel.: +1 808 5377923; fax: +1 808 5377899; e-mail: [email protected]
study, PBN was given just before and 30 min after induction of focal ischemia of 90 min duration, and blood flow was assessed by laser Doppler flow techniques, and by NMR imaging. The results obtained with the latter technique demonstrated that, in untreated animals, flow index in the first 1–2 h of reperfusion was ,50% of control. In the above mentioned studies local blood flow was measured by the autoradiographic technique which should permit an assessment of the adequacy of restoration of microcirculation. However, the question remains whether compromised perfusion could have existed at the level of individual capillaries. To provide an answer to this question, we assessed capillary patency after 2, 4, 6 and 8 h of reperfusion, following 2 h of MCA occlusion. Since normoglycemic animals did not show any evidence of capillary compromise, we added a hyperglycemic group with 8 h of
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00643- 0
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P.-A. Li et al. / Neuroscience Letters 253 (1998) 191–194
reperfusion to testify whether preischemic hyperglycemia which is known to shorten maturation of damage will influence the capillary perfusion. Male fasted Wistar rats (Møllegaard Breeding Center, Copenhagen, Denmark), weighing 310–340 g, were used for the experiments. Operative procedure and induction of transient 2 h MCA occlusion were conducted as described previously [5,11]. Blood pressure, blood gases, arterial pH, and body temperature were maintained close to physiological range. Animals without neurologic deficit were excluded for analysis. The body temperature was controlled between 37 and 38.5°C by air cooling during the reperfusion period. Capillary patency was determined using a double-staining technique [3,5,6]. The results are given as the percent ratio between perfused and existing capillaries. Animals were decapitated at predetermined recirculation periods of 2, 4, 6 and 8 h (n = 8 each normoglycemic Fig. 2. Percentage of capillary perfusion in hyperglycemic animals subjected to 2 h of MCA occlusion with 8 h of recirculation. Perfusion deficit could be detected in the ipsilateral striatal focus. *P , 0.01 vs. contralateral striatal focus.
Fig. 1. Percentage of capillary perfusion in normoglycemic animals subjected to 2 h of MCA occlusion with 2, 4, 6 and 8 h of recirculation. (A) Neocortical penumbra, (B) neocortical focus, (C) striatal focus. Data are collected from eight animals in each time point. Bars denote mean values and circles individual rats. No statistical significant differences could be found using either ANOVA plus post Scheffe’s test for chronological data or unpaired t-test for ipsi- and contralateral comparison.
group). Hyperglycemic rats (8 h reperfusion only, n = 7) were i.v. infused with 25% of glucose solution for 30 min (2.5 ml/h) before MCA occlusion to raise the plasma glucose concentration to ~20 mM. Normoglycemic rats were infused the same amount of Krebs-Hanseleit solution and the preischemic plasma glucose concentration was 5–6 mM. ANOVA followed by Scheffe’s F-test and two-tail unpaired t-test was employed for statistical analyses. The results of capillary patency in normoglycemic animals are summarized in Fig. 1. Non-ischemic contralateral hemisphere showed close to 100 percent capillary perfusion in the neocortical penumbra (Fig. 1A), neocortical focus (Fig. 1B), and striatal focus (Fig. 1C). In the ischemic ipsilateral side all animals showed close to 90–100% capillary perfusion except one rat after 2 h and two rats after 8 h of reperfusion had 40–60% of reperfusion in the striatal focus. There were no significant differences in capillary patency between ipsi- and contralateral hemispheres. The results of capillary patency in the hyperglycemic animals are given in Fig. 2. Capillary perfusion in the contralateral hemisphere was not influenced by glucose infusion. Thus, 100% of capillary perfusion was detected in all the three regions studied. However, in the ipsilateral hemisphere, compromised capillary perfusion was detectable in the striatal focus. Thus, five hyperglycemic rats showed compromised capillary perfusion in the striatal focus. Sets of representative microphotographs showing capillary patency in striatal focus after 8 h of recirculation in both normo- and hyperglycemic rats are given in Fig. 3. The present results thus demonstrate that capillary patency remains close to normal during the first 6 h of reperfusion, following 2 h of transient focal ischemia in normoglycemic rats. These results, and those previously obtained with autoradiographic [11] and laser-Doppler tech-
P.-A. Li et al. / Neuroscience Letters 253 (1998) 191–194
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Fig. 3. Microphotographs showing microvascular patency, determined by fluorescent double staining technique, in striatal focus of rat brain at 8 h reperfusion after 2 h focal ischemia subjected to normoglycemia (A,B) and hyperglycemia (C,D). Left (A,C), morphologically detectable capillaries marked by fibronectin antibody (coupled to fluorescein isothiocyanate) stain. Right (B,D), perfused capillaries labeled by intravascular Evans blue. Circulation time of Evans blue: 10 s. Evans blue perfused virtually all (95%) of the morphologically existing capillaries in the normoglycemic rat but not in the hyperglycemic one. Scale bar, 50 mm.
niques for assessing local CBF and erythrocyte velocity [8], as well as, those obtained with tissue PO2 electrodes [8], demonstrate that deterioration of tissue bioenergetic state after 4 h of recirculation is not likely due to microcirculatory dysfunction. Focused on in vitro results reflecting the performance of mitochondria, we favor the view that secondary deterioration of cellular bioenergetic state is due to mitochondrial dysfunction, rather than a microcirculatory compromise. Our results are at variance with those reported by Schulz et al. [10]. However, their data on laser-Doppler flow are similar to ours [8], apart from the fact that they found a higher blood flow at one time of reperfusion (30 min). Possibly, this result could be explained by the fact that PBNinjected animals had an increased blood pressure. Thus, the major difference in results relates to the NMR data of Schulz et al. [10]. The data suggest that blood flow is decreased well below 50% of control during the first hour
of reflow, and that PBN corrects this ‘microcirculatory deficiency’, bringing flow rates close to control. There are two major problems with this conclusion. First, several other laboratories, using a variety of techniques, have failed to find such a microcirculatory deficiency. Second, since PBN was given in a pretreatment paradigm, it appears likely that the MRI results reflect the improvement of tissue bioenergetic state rather than an improvement of blood flow. However, this issue remains to be settled. Our previous data have shown that preischemic hyperglycemia, which is known to exaggerate brain damage following both global/forebrain and focal ischemia, did not exert its detrimental effect on capillary perfusion after a short period (10 min) of forebrain ischemia [6]. However, when ischemic duration was prolonged to 20–30 min, a narrowing of capillary lumina [9], a large fraction of capillaries with formed elements [4], and an increased leakage of tracer through the blood–brain barrier [1] were found after reper-
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P.-A. Li et al. / Neuroscience Letters 253 (1998) 191–194
fusion in hyperglycemic animals. Similar with forebrain ischemia, focal ischemia of short duration (30 min) did not influence capillary perfusion [5]; however, as the present study shows, when ischemic duration was increased to 2 h microcirculatory compromise occurred in hyperglycemic animals. Therefore, the ischemic duration appears to be an important factor. It remains to be explored why the added effect of hyperglycemia on capillary perfusion could be observed after long duration of ischemia but not after a short period of ischemia. Probably, even if a short period of ischemia could lead to some upregulation of adhesion molecules, and to sticking of polymophornuclear leukocytes, the adverse effects of such events are limited. When ischemic period is prolonged long enough, such adverse effects are more pronounced and could be easily detected. The present study was supported by the Swedish Medical Research Council, the Juvenile Diabetes Foundation International, the US Public Health Service via the NIH, and the Deutsche Forschungsgemeinschaft. [1] Dietrich, W.D., Alonso, O. and Busto, R., Moderate hyperglycemia worsens acute blood-brain barrier injury after forebrain ischemia in rats, Stroke, 24 (1993) 111–116. [2] Folbergrova´, J., Zhao, Q., Katsura, K. and Siesjo¨, B.K., N-Tertbutyl-a-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia, Proc. Natl. Acad. Sci. USA, 92 (1995) 5057–5061. [3] Go¨bel, U., Theilen, H. and Kuschinsky, W., Congruence of total and perfused capillary network in rat brains, Circ. Res., 66 (1990) 271–281.
[4] Kalimo, H., Rehncrona, S., So¨derfeldt, B., Olsson, Y. and Siesjo¨, B.K., Brain lactic acidosis and ischemic cell damage. 2. Histopathology, J. Cereb. Blood Flow Metab., 1 (1981) 313–327. [5] Li, P.-A., Gisselsson, L., Keuker, J., Vogel, J., Smith, M.-L., Kuschinsky, W. and Siesjo¨, B.K., Hyperglycemia-exaggerated ischemic brain damage following 30 min of middle cerebral artery occlusion is not due to capillary obstruction, Brain Res., (1998) in press. [6] Li, P.-A., Vogel, J., He, Q.-P., Smith, M.-L., Kuschinsky, W. and Siesjo¨, B.K., Preischemic hyperglycemia leads to rapidly developing brain damage with no change in capillary patency, Brain Res., 782 (1998) 175–183. [7] Nagasawa, H. and Kogure, K., Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion, Stroke, 20 (1989) 1037–1043. [8] Nakai, A., Kuroda, S., Kritia´n, T. and Siesjo¨, B.K., The immunosuppressant drug FK506 ameliorates secondary mitochondrial dysfunction following transient focal cerebral ischemia in the rat, Neurobiol. Dis., 4 (1997) 288–300. [9] Palja¨rvi, L., Rehncrona, S., So¨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. [10] Schulz, J.B., Panahian, N., Chen, Y.I., Beal, M.F., Moskowitz, M.A. and Rosen, B.R., Facilitation of postischemic reperfusion with a-PBN: assessment using NMR and Doppler flow techniques, Am. J. Physiol., 272 (4 Pt 2 ) (1997) H1986– H1995. [11] Tsuchidate, R., He, Q.P., Smith, M.-L. and Siesjo¨, B.K., Regional cerebral blood flow during and following 2 hour of middle cerebral artery occlusion in the rat, J. Cereb. Blood Flow Metab., 17 (1997) 1066–1073.
Capillary patency after transient middle cerebral artery occlusion of 2 h duration Ping-An Li a ,*, Johannes Vogel b, Maj-lis Smith c, Qing-Ping He a, Wolfgang Kuschinsky b, Bo K. Siesjo¨ a a
Center for the Study of Neurological Diseases, The Neuroscience Institute, Queen’s Medical Center, 1356 Lusitana Street, UH Tower 8th floor, Honolulu, HI 96813, USA b Department of Physiology, University of Heidelberg, D-69120 Heidelberg, Germany c Laboratory for Experimental Brain Research, University of Lund, Lund, Sweden Received 12 June 1998; received in revised form 28 July 1998; accepted 4 August 1998
Abstract Reperfusion after transient focal ischemia of 2 h duration is followed by secondary bioenergetic failure after 4 h of reperfusion. The objective of the present study was to explore whether or not this secondary deterioration is due to secondary microcirculatory compromise. Normal fasted rats were subjected to 2 h of MCA occlusion and allowed reperfusion for 2, 4, 6 and 8 h. At predetermined reperfusion times, rats were injected with Evans blue and decapitated. Capillary patency was determined using a fluorescent double-staining technique. No capillary perfusion deficits were detected in the ischemic neocortical penumbra, neocortical focus or striatal focus. We concluded that the secondary deterioration of bioenergetic state is not due to microcirculatory compromise. Since hyperglycemic animals show pan-necrotic lesions, a hyperglycemic group was added at 8 h of reperfusion to test if the adverse effect of hyperglycemia on ischemic damage is related to capillary compromise. The results showed that, in hyperglycemic rats, capillary perfusion in the striatal focus was compromised after 8 h of recirculation following 2 h of MCA occlusion. It is concluded that when normoglycemic rats are subjected to 2 h of MCA occlusion, capillary patency is not affected during the first 4–6 h of reflow. At 8 h of reflow, though, particularly in hyperglycemic rats, microcirculation is compromised in the caudoputamenal focus, probably reflecting infarction. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Capillary patency; Focal ischemia; Microcirculation; Fluorescent double staining; Blood flow; Brain
Reperfusion after focal ischemia of 2 h duration is followed by partial recovery of tissue bioenergetic state, but a secondary deterioration is observed after 4 h of reperfusion [2]. The question arises whether or not this is due to secondary microcirculatory compromise. The previous data on local blood flow suggest that blood flow is not compromised in the first 1–6 h of reperfusion, following 1–2 h of MCA occlusion [7,11], i.e. in the period in which the bioenergetic state deteriorates. However, a recent study reports that the amelioration of tissue damage by PBN is related to an improved reflow during early reperfusion [10]. In that
* Corresponding author. Tel.: +1 808 5377923; fax: +1 808 5377899; e-mail: [email protected]
study, PBN was given just before and 30 min after induction of focal ischemia of 90 min duration, and blood flow was assessed by laser Doppler flow techniques, and by NMR imaging. The results obtained with the latter technique demonstrated that, in untreated animals, flow index in the first 1–2 h of reperfusion was ,50% of control. In the above mentioned studies local blood flow was measured by the autoradiographic technique which should permit an assessment of the adequacy of restoration of microcirculation. However, the question remains whether compromised perfusion could have existed at the level of individual capillaries. To provide an answer to this question, we assessed capillary patency after 2, 4, 6 and 8 h of reperfusion, following 2 h of MCA occlusion. Since normoglycemic animals did not show any evidence of capillary compromise, we added a hyperglycemic group with 8 h of
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00643- 0
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P.-A. Li et al. / Neuroscience Letters 253 (1998) 191–194
reperfusion to testify whether preischemic hyperglycemia which is known to shorten maturation of damage will influence the capillary perfusion. Male fasted Wistar rats (Møllegaard Breeding Center, Copenhagen, Denmark), weighing 310–340 g, were used for the experiments. Operative procedure and induction of transient 2 h MCA occlusion were conducted as described previously [5,11]. Blood pressure, blood gases, arterial pH, and body temperature were maintained close to physiological range. Animals without neurologic deficit were excluded for analysis. The body temperature was controlled between 37 and 38.5°C by air cooling during the reperfusion period. Capillary patency was determined using a double-staining technique [3,5,6]. The results are given as the percent ratio between perfused and existing capillaries. Animals were decapitated at predetermined recirculation periods of 2, 4, 6 and 8 h (n = 8 each normoglycemic Fig. 2. Percentage of capillary perfusion in hyperglycemic animals subjected to 2 h of MCA occlusion with 8 h of recirculation. Perfusion deficit could be detected in the ipsilateral striatal focus. *P , 0.01 vs. contralateral striatal focus.
Fig. 1. Percentage of capillary perfusion in normoglycemic animals subjected to 2 h of MCA occlusion with 2, 4, 6 and 8 h of recirculation. (A) Neocortical penumbra, (B) neocortical focus, (C) striatal focus. Data are collected from eight animals in each time point. Bars denote mean values and circles individual rats. No statistical significant differences could be found using either ANOVA plus post Scheffe’s test for chronological data or unpaired t-test for ipsi- and contralateral comparison.
group). Hyperglycemic rats (8 h reperfusion only, n = 7) were i.v. infused with 25% of glucose solution for 30 min (2.5 ml/h) before MCA occlusion to raise the plasma glucose concentration to ~20 mM. Normoglycemic rats were infused the same amount of Krebs-Hanseleit solution and the preischemic plasma glucose concentration was 5–6 mM. ANOVA followed by Scheffe’s F-test and two-tail unpaired t-test was employed for statistical analyses. The results of capillary patency in normoglycemic animals are summarized in Fig. 1. Non-ischemic contralateral hemisphere showed close to 100 percent capillary perfusion in the neocortical penumbra (Fig. 1A), neocortical focus (Fig. 1B), and striatal focus (Fig. 1C). In the ischemic ipsilateral side all animals showed close to 90–100% capillary perfusion except one rat after 2 h and two rats after 8 h of reperfusion had 40–60% of reperfusion in the striatal focus. There were no significant differences in capillary patency between ipsi- and contralateral hemispheres. The results of capillary patency in the hyperglycemic animals are given in Fig. 2. Capillary perfusion in the contralateral hemisphere was not influenced by glucose infusion. Thus, 100% of capillary perfusion was detected in all the three regions studied. However, in the ipsilateral hemisphere, compromised capillary perfusion was detectable in the striatal focus. Thus, five hyperglycemic rats showed compromised capillary perfusion in the striatal focus. Sets of representative microphotographs showing capillary patency in striatal focus after 8 h of recirculation in both normo- and hyperglycemic rats are given in Fig. 3. The present results thus demonstrate that capillary patency remains close to normal during the first 6 h of reperfusion, following 2 h of transient focal ischemia in normoglycemic rats. These results, and those previously obtained with autoradiographic [11] and laser-Doppler tech-
P.-A. Li et al. / Neuroscience Letters 253 (1998) 191–194
193
Fig. 3. Microphotographs showing microvascular patency, determined by fluorescent double staining technique, in striatal focus of rat brain at 8 h reperfusion after 2 h focal ischemia subjected to normoglycemia (A,B) and hyperglycemia (C,D). Left (A,C), morphologically detectable capillaries marked by fibronectin antibody (coupled to fluorescein isothiocyanate) stain. Right (B,D), perfused capillaries labeled by intravascular Evans blue. Circulation time of Evans blue: 10 s. Evans blue perfused virtually all (95%) of the morphologically existing capillaries in the normoglycemic rat but not in the hyperglycemic one. Scale bar, 50 mm.
niques for assessing local CBF and erythrocyte velocity [8], as well as, those obtained with tissue PO2 electrodes [8], demonstrate that deterioration of tissue bioenergetic state after 4 h of recirculation is not likely due to microcirculatory dysfunction. Focused on in vitro results reflecting the performance of mitochondria, we favor the view that secondary deterioration of cellular bioenergetic state is due to mitochondrial dysfunction, rather than a microcirculatory compromise. Our results are at variance with those reported by Schulz et al. [10]. However, their data on laser-Doppler flow are similar to ours [8], apart from the fact that they found a higher blood flow at one time of reperfusion (30 min). Possibly, this result could be explained by the fact that PBNinjected animals had an increased blood pressure. Thus, the major difference in results relates to the NMR data of Schulz et al. [10]. The data suggest that blood flow is decreased well below 50% of control during the first hour
of reflow, and that PBN corrects this ‘microcirculatory deficiency’, bringing flow rates close to control. There are two major problems with this conclusion. First, several other laboratories, using a variety of techniques, have failed to find such a microcirculatory deficiency. Second, since PBN was given in a pretreatment paradigm, it appears likely that the MRI results reflect the improvement of tissue bioenergetic state rather than an improvement of blood flow. However, this issue remains to be settled. Our previous data have shown that preischemic hyperglycemia, which is known to exaggerate brain damage following both global/forebrain and focal ischemia, did not exert its detrimental effect on capillary perfusion after a short period (10 min) of forebrain ischemia [6]. However, when ischemic duration was prolonged to 20–30 min, a narrowing of capillary lumina [9], a large fraction of capillaries with formed elements [4], and an increased leakage of tracer through the blood–brain barrier [1] were found after reper-
194
P.-A. Li et al. / Neuroscience Letters 253 (1998) 191–194
fusion in hyperglycemic animals. Similar with forebrain ischemia, focal ischemia of short duration (30 min) did not influence capillary perfusion [5]; however, as the present study shows, when ischemic duration was increased to 2 h microcirculatory compromise occurred in hyperglycemic animals. Therefore, the ischemic duration appears to be an important factor. It remains to be explored why the added effect of hyperglycemia on capillary perfusion could be observed after long duration of ischemia but not after a short period of ischemia. Probably, even if a short period of ischemia could lead to some upregulation of adhesion molecules, and to sticking of polymophornuclear leukocytes, the adverse effects of such events are limited. When ischemic period is prolonged long enough, such adverse effects are more pronounced and could be easily detected. The present study was supported by the Swedish Medical Research Council, the Juvenile Diabetes Foundation International, the US Public Health Service via the NIH, and the Deutsche Forschungsgemeinschaft. [1] Dietrich, W.D., Alonso, O. and Busto, R., Moderate hyperglycemia worsens acute blood-brain barrier injury after forebrain ischemia in rats, Stroke, 24 (1993) 111–116. [2] Folbergrova´, J., Zhao, Q., Katsura, K. and Siesjo¨, B.K., N-Tertbutyl-a-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia, Proc. Natl. Acad. Sci. USA, 92 (1995) 5057–5061. [3] Go¨bel, U., Theilen, H. and Kuschinsky, W., Congruence of total and perfused capillary network in rat brains, Circ. Res., 66 (1990) 271–281.
[4] Kalimo, H., Rehncrona, S., So¨derfeldt, B., Olsson, Y. and Siesjo¨, B.K., Brain lactic acidosis and ischemic cell damage. 2. Histopathology, J. Cereb. Blood Flow Metab., 1 (1981) 313–327. [5] Li, P.-A., Gisselsson, L., Keuker, J., Vogel, J., Smith, M.-L., Kuschinsky, W. and Siesjo¨, B.K., Hyperglycemia-exaggerated ischemic brain damage following 30 min of middle cerebral artery occlusion is not due to capillary obstruction, Brain Res., (1998) in press. [6] Li, P.-A., Vogel, J., He, Q.-P., Smith, M.-L., Kuschinsky, W. and Siesjo¨, B.K., Preischemic hyperglycemia leads to rapidly developing brain damage with no change in capillary patency, Brain Res., 782 (1998) 175–183. [7] Nagasawa, H. and Kogure, K., Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion, Stroke, 20 (1989) 1037–1043. [8] Nakai, A., Kuroda, S., Kritia´n, T. and Siesjo¨, B.K., The immunosuppressant drug FK506 ameliorates secondary mitochondrial dysfunction following transient focal cerebral ischemia in the rat, Neurobiol. Dis., 4 (1997) 288–300. [9] Palja¨rvi, L., Rehncrona, S., So¨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. [10] Schulz, J.B., Panahian, N., Chen, Y.I., Beal, M.F., Moskowitz, M.A. and Rosen, B.R., Facilitation of postischemic reperfusion with a-PBN: assessment using NMR and Doppler flow techniques, Am. J. Physiol., 272 (4 Pt 2 ) (1997) H1986– H1995. [11] Tsuchidate, R., He, Q.P., Smith, M.-L. and Siesjo¨, B.K., Regional cerebral blood flow during and following 2 hour of middle cerebral artery occlusion in the rat, J. Cereb. Blood Flow Metab., 17 (1997) 1066–1073.