Background and Purpose The potential of thrombolytic agents to improve outcome after ischemic stroke could be negated if recanalization of an occluded artery exacerbates cerebral edema. We examined whether infarctions associated with reperfusion have more edema than those without reperfusion and whether the time course for the development of cerebral edema varied with and without reperfusion.
Methods Infarct volumes were measured 24 hours after permanent and 1, 2, and 3 hours of temporary right middle cerebral artery (MCA) occlusion in spontaneously hypertensive rats. Hemispheric volume, water, sodium, and potassium were measured 3, 6, 12, 24, 36, and 48 hours after permanent and 3 hours of temporary MCA occlusion and also determined 24 hours after permanent and 2 and 3 hours of temporary MCA occlusion.
Results Minimal tissue damage occurred after 1 hour of temporary ischemia. Infarct sizes were similar after permanent and 3 hours of temporary MCA occlusion and significantly greater than after 2 hours of temporary ischemia. Hemispheric volume, water, and sodium from the infarcted right hemisphere were significantly greater than those from the left hemisphere beginning 6 hours after MCA occlusion and continuing for 48 hours, with a peak at 24 hours. Right hemispheric water measured 24 hours after 2 hours of temporary ischemia was significantly less than after permanent or 3 hours of temporary ischemia.
Conclusions This study demonstrates that cerebral edema after focal stroke is related to infarct size and is independent of reperfusion status. The results suggest that exacerbation of cerebral edema will not occur after thrombolytic treatment or spontaneous recanalization of occluded cerebral vessels.
Cerebral edema associated with transtentorial herniation is a major cause of early mortality after stroke.1 Recanalization of an occluded artery occurring spontaneously or after thrombolytic therapy might be expected to exacerbate cerebral edema by reestablishing blood flow to ischemic brain where the blood-brain barrier is disrupted. In 1988 Koudstaal et al2 described two patients with disabling middle cerebral artery (MCA) distribution strokes who were treated with tissue plasminogen activator within 3.5 hours after onset of symptoms. Both patients subsequently deteriorated and died from transtentorial herniation despite autopsy evidence of reperfusion in one of the patients. Autopsy was refused in the other patient. Since then several reports have found that therapeutic or spontaneous reperfusion of an occluded vessel is not associated with increased mass effect on CT, defined as sulcul or ventricular effacement, lateral displacement of the midline structures, or amount of hypodensity.3 4 5 One study demonstrated that infarct size was the major determinant of swelling on CT scan.3 Because CT scans provide only an indirect measurement of edema, we studied the development of cerebral edema in a focal stroke model in rats to address two issues: (1) whether the time course for development of cerebral edema varies between rats with and without reperfusion and (2) whether infarctions associated with reperfusion have more edema than those without reperfusion.
Materials and Methods
Five separate experiments were completed in this study. The type of ischemic insult and outcome measures for each experiment are outlined in Table 1⇓.
Male spontaneously hypertensive rats (SHR) weighing 230 to 360 g were fasted for 24 hours before surgery. Halothane (1.5% to 2.0%), mixed with oxygen and nitrogen, was delivered through a nose cone with the use of a flow regulator. The tail artery was cannulated with a polyethylene catheter to monitor blood pressure and to obtain blood samples for the assessment of physiological variables. Permanent focal neocortical ischemia was produced by occluding the right common carotid artery (CCA) with 4-0 silk and cauterizing the right MCA.6 Temporary focal cerebral ischemia was produced by permanently occluding the right CCA with 4-0 silk and using a No. 1 microclip on the right MCA (Codman).7 Flow interruption of the MCA was observed after clip placement. Sham procedures were performed as previously described.8 Body temperature was maintained at 37°C throughout the procedure with a heat lamp connected to a rectal thermistor. Surgery for each experiment was completed by a single investigator during a 2- to 3-week period on rats delivered from a single shipment (Harlan Sprague-Dawley, Inc, Indianapolis, Ind). The method of MCA occlusion and euthanasia to be described was approved by the Institutional Laboratory of Animal Care and Use Committee at Ohio State University.
Animals subjected to transient ischemia were reanesthetized at various time points after the onset of ischemia. After verification of MCA occlusion, the clip was removed, and blood flow in the MCA was visually verified. Wounds were reclosed, and the rats were returned to their cages. Sham-operated rats and rats with permanent MCA occlusion were reanesthetized 3 hours after MCA manipulation or occlusion. The MCA and surrounding cortex were visualized to simulate the conditions that occurred in rats with transient MCA occlusion. Wounds were resutured, and the rats were allowed to recover from anesthesia.
Arterial blood pressure was monitored throughout the surgical procedure and then checked 4 to 6 hours after surgery when the animals had recovered from anesthesia. Arterial blood Pao2, Paco2, pH, glucose, and hematocrit were measured before MCA occlusion. Arterial blood gases and temperature were measured again 3 to 6 hours after CCA/MCA occlusion. Hematocrit was determined just before decapitation. During the surgical procedure mean arterial blood pressure was maintained above 90 mm Hg.
Animals from experiment 1 were anesthetized with halothane and decapitated 24 hours after CCA/MCA occlusion. The brains were rapidly removed from the cranium and frozen in isopentane over dry ice. Coronal sections 20 μm thick were cut at 500-μm intervals, fixed in 90% ethanol, and stained with hematoxylin and eosin. Each brain section was magnified by means of a photographic lens, and the infarcted area was traced onto paper. Each drawing was then retraced onto a digitizing tablet interfaced to an IBM personal computer (Sigma Plot, Jaudeo), which computed infarct areas for each section. We calculated total infarct volume by summing the infarcted areas of sequential sections and multiplying by the thickness between sections. Image analysis for each experiment was done by a technician blinded to the study groups.
Brains used for edema analysis were quickly removed from the cranium and dissected free of the olfactory bulbs and cerebellum. The brain was then sagittally sectioned at the midline. Each hemisphere was weighed on a digital analytic balance (Mettler Instrument Corp) to obtain wet weight. Right and left hemispheres were then separately suspended on a wire through the occipital lobe in a preweighed beaker of distilled water. The weight of the beaker and water was then subtracted from the weight of the beaker, water, and suspended hemisphere. This difference represented the weight of water displaced by the brain. Since the specific gravity of distilled water is 1.0, the weight of displaced water equaled the volume of displaced water and was equivalent to hemispheric volume.9 Each hemisphere was placed in an oven at 90°C to 100°C for 48 hours and then reweighed to obtain dry weight. Tissue water content was expressed as percentage of wet tissue weight (wet weight−dry weight/wet weight×100).
The dehydrated hemispheres were crushed and then digested in 2 mL of 35% perchloric acid at 180°C for 2 hours. Samples were centrifuged, and the supernatant was decanted. The residue was washed with 5 mL of water and centrifuged. The supernatants from the two centrifugations were brought to a total volume of 25 mL. Aliquots of 1 mL were diluted to 20 mL with deionized water, and potassium and sodium were then measured by atomic absorption spectroscopy (Varian Techtron PPY Limited). Potassium and sodium values were expressed as milligrams per gram dry weight.
Mean infarct volumes and SDs were computed for each of the groups in experiment 1, and results were analyzed with the use of a one-way ANOVA and Newman-Keuls test. The Student’s t test was used to compare brain volume, water, sodium, and potassium of the right hemisphere to the left hemisphere for all five experiments as well as mean right hemispheric volume, water, sodium, and potassium of the two groups in experiment 5. To characterize temporal changes in experiments 2 and 3, brain volume, water, sodium, and potassium of the right hemisphere for each of the time points was analyzed with the use of a one-way ANOVA and Newman-Keuls multiple comparisons test. In experiment 4 mean right hemispheric volume, water, sodium, and potassium of the three groups were analyzed with the use of a one-way ANOVA and Newman-Keuls post hoc test.
In all five experiments no significant differences were found between any of the groups for blood pressure, arterial blood gases, glucose, hematocrit, or temperature (data not shown).
Infarct volumes after temporary and permanent focal ischemia are presented in Table 2⇓. Three hours of transient ischemia resulted in infarct volumes comparable to those seen after permanent ischemia. One hour of temporary ischemia caused less damage than permanent and 2 or 3 hours of temporary ischemia (P<.01), and 2 hours of temporary ischemia produced significantly smaller infarcts than permanent or 3 hours of temporary ischemia (P<.05).
Because infarct sizes after permanent and 3 hours of temporary MCA occlusion were similar, the time course for development of cerebral edema was examined in rats after these two types of insults. This allowed control for infarct volume so that the variable being studied was reperfusion status. The volume of the infarcted right hemisphere was significantly greater than the left hemisphere from 6 to 48 hours after permanent MCA occlusion (P<.05, Fig 1⇓), and volume in the right hemisphere was greater at 24 and 36 hours compared with 3 and 12 hours (P<.05). Right hemispheric volume was greater than left hemispheric volume from 12 to 48 hours after 3 hours of temporary ischemia (P<.05, Fig 1⇓), and right hemispheric volume was greater at 36 hours than at 6 hours (P<.05). Water content in the right hemisphere was greater than that in the left hemisphere from 6 to 48 hours after both permanent and 3 hours of temporary ischemia (P<.05, Fig 2⇓). Water content in the right hemisphere was greater at 24 and 36 hours than at 6, 12, and 48 hours after permanent ischemia but only at 6 and 12 hours after temporary ischemia (P<.05). Right hemispheric sodium was greater and potassium less than left hemispheric levels from 3 to 48 hours after permanent and temporary ischemia (P<.05, Figs 3⇓ and 4⇓), except for the 6-hour sodium level in temporary ischemia. Right hemispheric sodium at 24 hours was greater than at 3, 6, and 12 hours after permanent ischemia but only at 12 hours after temporary ischemia (P<.05, Fig 3⇓). Right hemispheric potassium at 24 hours was less than at 3, 6, 12, and 48 hours after permanent ischemia, and right hemispheric potassium at 6 hours was less than at 48 hours after temporary ischemia (P<.05, Fig 4⇓).
Since hemispheric water peaked at 24 hours after permanent and 3 hours of temporary ischemia, cerebral edema was measured 24 hours after temporary and permanent focal ischemia to determine whether reperfusion increases cerebral edema. Right hemispheric water content was significantly less after 2 hours of temporary ischemia than after permanent or 3 hours of temporary ischemia (P<.05, Table 3⇓). No difference between right and left hemispheric volume or water was seen in sham-operated SHR, although right hemispheric water after permanent and 2 or 3 hours of temporary ischemia was significantly greater than after sham operation (P<.05). A trend for higher water content in the infarcted hemisphere after 3 hours of temporary MCA occlusion compared with permanent MCA occlusion was seen initially (Table 3⇓). Since a major aim of this study was to determine whether reperfusion increases edema formation, we repeated this experiment to confirm or refute this trend (experiment 5). No difference in hemispheric water content after permanent and 3 hours of temporary MCA occlusion was seen (Table 4⇓).
The effect of reperfusion on the time course and extent of edema formation has not previously been adequately examined. Clinical studies suggest that mass effect on CT scan after ischemic stroke is independent of reperfusion status; however, edema is difficult to distinguish from infarcted tissue on CT. In addition, mass effect is only an indirect measurement of edema and is seen only with large infarctions. Furthermore, performing a sufficient number of serial CT scans to determine the time course of edema formation in patients is not practical. Using a focal ischemic model in the rat, we demonstrated a similar time course for development of cerebral edema after permanent and 3 hours of temporary MCA occlusion. Furthermore, cerebral edema seems to be related to the size of infarction rather than to the occurrence of reperfusion. Hemispheric water content and infarct size after 2 hours of temporary ischemia was less than after permanent or 3 hours of temporary ischemia. No difference in edema or infarct volume was seen between permanent and 3 hours of temporary MCA occlusion.
Cerebral edema formation after focal cerebral ischemia has been measured in specific brain regions rather than the entire hemisphere in several studies.10 11 12 13 14 A gradient in the extent of edema was seen that was greatest in the most ischemic regions, less but increased in the periphery of the infarct, and minimally changed in noninfarcted regions.10 12 13 Because this gradient of edema formation is well described and because we felt that mass effect seen on CT would be more reflective of changes in hemispheric water than regional brain water, we measured hemispheric water in this study.
This study confirms findings from others that cerebral ischemia in rats sustained for greater than 1 to 2 hours results in infarction that is maximal if ischemia persists for 3 hours or more.7 15 The close correlation between increased sodium and water content after focal cerebral ischemia seen in our study has also been reported by others.10 11 13 14 16
Our results confirm those of other studies that found that cerebral edema peaks 1 to 3 days after permanent MCA occlusion.10 11 12 14 16 Data on the time course of cerebral edema formation after temporary ischemia are limited to one study by Ito et al,17 in which hemispheric water, sodium, and potassium in gerbils were measured after only brief periods of temporary ischemia. After 30 minutes of temporary CCA occlusion, brain water increased for 8 hours and then slowly decreased over the next 64 hours. Brain water increased to a peak at 72 hours and then fell to baseline levels at 1 week after 1 hour of temporary CCA occlusion. In the study of Ito et al, hemispheric water content after permanent CCA occlusion was measured at various times up to 20 hours. Hemispheric water after 30 minutes and 1 hour of temporary CCA occlusion was less than that after permanent CCA occlusion for each of the time points measured between 3 and 20 hours, and the peak levels of water content seen in both the temporary ischemia groups were less than the maximal value seen after permanent ischemia at 20 hours. Small or no infarction occurred with 1 hour or less of temporary MCA occlusion, and thus little edema formation would be expected after this type of insult. We did not measure edema formation after brief periods of temporary focal cerebral ischemia because we were interested in whether reperfusion influenced edema formation while controlling for infarct size. We therefore measured edema after permanent and 3 hours of temporary ischemia and found that the time course of changes in hemispheric water, sodium, and potassium was similar after these different ischemic insults.
Prior work comparing cerebral edema after temporary and permanent focal cerebral ischemia is limited and was not designed to answer issues addressed in this study.17 18 19 20 Brief periods (30 to 60 minutes) of temporary focal ischemia result in a small increase or no increase in brain water compared with either permanent ischemia or longer periods of temporary focal ischemia. Yang et al18 demonstrated increased water in 2-mm-thick hemispheric sections 24 hours after 1 hour and 2 hours of MCA occlusion but not after 30 minutes of MCA occlusion in rats. Although we did not measure edema after temporary MCA occlusion of less than 2 hours, we did demonstrate that infarct size and brain water were less after 2 hours of MCA occlusion compared with permanent or 3 hours of temporary ischemia. Ito et al17 found that hemispheric water was greater in gerbils after 3 hours of temporary CCA occlusion and 3 hours of reperfusion than after 6 hours of permanent CCA occlusion and also greater after 6 hours of temporary CCA occlusion followed by 3 hours of reperfusion compared with 9 hours of permanent CCA occlusion. The time of peak edema formation after permanent and 3 hours or 6 hours of temporary CCA occlusion was not determined in the study of Ito et al. We found that cerebral edema peaked 24 hours after permanent and 3 hours of temporary ischemia. At that time no difference in water, sodium, and potassium in the infarcted right hemisphere was seen between these groups. Furthermore, in contrast to the study of Ito et al, no difference was seen in hemispheric water after 3 hours of temporary MCA occlusion followed by 3 hours of reperfusion and 6 hours of permanent MCA occlusion (79.8±0.6% versus 79.8±0.6%, Fig 2⇑).
In conclusion, this study demonstrates that cerebral edema after focal stroke is related to infarct size, independent of reperfusion status. By demonstrating that cerebral edema is related to infarct size rather than the presence or absence of reperfusion, this study, along with clinical observations, suggests that exacerbation of cerebral edema will not occur after thrombolytic therapy or spontaneous recanalization of occluded cerebral vessels.
This study was supported in part by the William H. Davis Scholarship for Medical Research. The authors are grateful to Debbie Gray for technical assistance, to Dr John T. Kissel for critical review of the manuscript, and to Hancy Hodges and Cindy Murphy for typing the manuscript.
Reprint requests to A. Slivka, MD, Department of Neurology, Ohio State University, 1654 Upham Dr, Columbus, OH 43210.
- Received June 9, 1994.
- Revision received December 21, 1994.
- Accepted March 3, 1995.
- Copyright © 1995 by American Heart Association
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