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(Stroke. 1995;26:1035-1038.)
© 1995 American Heart Association, Inc.

Articles

Preischemic But Not Postischemic Zinc Protoporphyrin Treatment Reduces Infarct Size and Edema Accumulation After Temporary Focal Cerebral Ischemia in Rats

Chitoshi Kadoya, MD; Edward F. Domino, MD; Guo-Yuan Yang, MD; Joseph D. Stern, MD A. Lorris Betz, MD

From the Departments of Pharmacology (C.K., E.F.D.), Surgery (Neurosurgery) (G.-Y.Y., J.D.S., A.L.B.), Pediatrics (A.L.B.), and Neurology (A.L.B.), University of Michigan, Ann Arbor.

Correspondence to Dr Edward F. Domino, Department of Pharmacology, A220E MSRB III, University of Michigan, Ann Arbor, MI 48109-0630.

	Abstract

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Background and Purpose Zinc protoporphyrin (ZnPP) has multiple actions. It is an interleukin-1 antagonist as well as a hemeoxygenase inhibitor. Interleukin-1 is produced in ischemic brain and probably contributes to ischemic injury, although the role of heme oxygenase during ischemia is unknown. Whether ZnPP treatment is more effective before or after ischemia, as well as whether it is more protective in permanent or temporary cerebral ischemia, is also unknown. Therefore, we investigated the effect of ZnPP on infarction size and edema in a rodent model of temporary and permanent focal cerebral ischemia.

Methods Two groups of adult male Sprague-Dawley rats were pretreated with either 50 mg/kg ZnPP IP or saline and subjected to permanent middle cerebral artery occlusion 30 minutes later. Four additional groups of animals were subjected to 2 hours of temporary middle cerebral artery occlusion followed by 22 hours of reperfusion. Two of these groups were pretreated 30 minutes before middle cerebral artery occlusion with either 50 mg/kg ZnPP IP or saline. The other groups received ZnPP at either 2 or 4 hours after middle cerebral artery occlusion. Regional cerebral blood flow in the ischemic cortex was monitored with laser Doppler flowmetry. Cerebral infarct size and brain water were measured 24 hours after the onset of either form of ischemia.

Results Regional cerebral blood flow after occlusion was approximately 13% to 20% of baseline after either permanent or temporary ischemia. ZnPP had no effect on regional cerebral blood flow, infarct size, or edema formation in permanent ischemia. In contrast, pretreatment significantly reduced infarct size (17.2±6.6% in controls versus 6.2±2.9% in pretreated rats) and edema formation (center zone, 4.00±0.71% water in controls versus 1.18±0.26% water in pretreated rats) in the model of temporary ischemia, but treatment after occlusion had no effect.

Conclusions ZnPP treatment protected the brain when administered early in the temporary ischemia model.

Key Words: cerebral ischemia, focal • interleukin-1, receptor antagonist • neuronal protection • rats

	Introduction

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Zinc protoporphyrin (ZnPP) has anti-inflammatory properties.¹ It acts as an interleukin-1 (IL-1) antagonist as well as an inhibitor of heme oxygenase and other heme-dependent enzymes.² Although the role of heme-dependent enzymes in brain ischemia is not well known, the effects of IL-1 are well described.^{3 4 5 6} Yamasaki et al³ reported that intraventricular administration of recombinant human IL-1 increased brain edema formation after 1 hour of ischemia and 24 hours of reperfusion in rats, whereas the IL-1 antagonist ZnPP decreased ischemic brain edema. Additional data are needed to determine whether ZnPP is more effective before or after ischemia, as well as in temporary or permanent cerebral ischemia.

The experiments described here were designed to determine whether ZnPP, given before or after ischemia, alters infarction volume or edema formation resulting from either temporary or permanent middle cerebral artery occlusion (MCAO).

	Materials and Methods

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This study was approved by the Committee on Animal Research at the University of Michigan. A total of 74 adult male Sprague-Dawley rats (Charles River Laboratories), weighing between 230 to 300 g, were each initially anesthetized with 3% halothane in a mixture of 0.1 L/min O₂ and 0.7 L/min air. Subsequently, anesthesia was maintained with 0.9% to 1.0% halothane. A PE-50 catheter was introduced into the femoral artery to allow continuous monitoring of arterial blood pressure. Arterial blood samples were obtained for analysis of blood gases and pH with a 170 pH/blood gas analyzer (Corning Medical) and of blood glucose with a Glucometer II (Miles Laboratories). Blood gases were analyzed twice during each experiment. Blood pressure was maintained above 90 mm Hg by adjusting the halothane concentration. The right temporal muscle temperature was monitored with a thermometer (YSI model 73A, Yellow Springs Instrument Co) and maintained at 37°C with a heating pad. Body temperature was monitored with a digital dual-channel thermometer (Fischer Scientific).

The skull was exposed through a midline incision, and two burr holes approximately 1.5 mm in diameter were made 1 mm posterior and 5 mm lateral to the bregma. The laser Doppler flow probe was placed on the dura away from the cortical vessels. Cerebral blood flow (CBF) was measured using a laser Doppler flowmeter (Laserflo BRM², Vasamedics Inc) before and 30 minutes after occlusion to confirm induction and maintenance of ischemia. The CBF data were expressed as a percentage of baseline CBF (milliliters per 100 grams per minute). If CBF increased over 35 mL/100 g per minute during the occlusion, the animal was excluded from the study.

The MCA was occluded using a modification of the method of Longa et al.⁷ The right common carotid artery was exposed through a midline incision and visualized with an operating microscope. The branches of the external carotid artery, including the occipital terminal lingual and maxillary arteries, were isolated and coagulated. The internal carotid artery was then isolated, and its extracranial branch, the pterygopalatine artery, was ligated with a 5-0 silk suture near its origin. A 5-cm length of 3-0 nylon suture with a slightly enlarged and rounded tip was introduced into the transected lumen of the external carotid artery and gently advanced from the external into the internal carotid artery until resistance was felt to passage of the suture. The distance from the tip of the suture to the bifurcation of the right common carotid artery in the brain was about 19.5 to 20.5 mm. The 3-0 nylon sutures were presoaked in heparin solution (1000 U/mL). In temporary ischemia, 8 U (0.4 mL) heparin was injected intra-arterially 10 minutes before reperfusion.

In the permanent occlusion experiments, animals were killed by decapitation 24 hours after MCAO, and the brains were quickly removed. In the temporary occlusion experiments, animals were subjected to a 2-hour period of MCAO. Reperfusion of the MCAO was accomplished by withdrawal of the suture and ligation of the external carotid artery. Each animal was killed 22 hours after reperfusion.

There were six experimental groups, each consisting of six or seven rats randomly assigned to each group. Animals in all groups were operated on, and the various procedures were carried out as described above. Both the infarction area and brain edema were measured. The experimental groups were as follows: (1) permanent ischemia with control 0.9% NaCl treatment: animals received 0.9% NaCl 0.5 mL/kg IP 30 minutes before the onset of ischemia; (2) permanent ischemia with ZnPP treatment: animals received ZnPP in a dose of 50 mg/kg (0.5 mL/kg) IP 30 minutes before the onset of ischemia; (3) temporary ischemia with 0.9% NaCl treatment: animals received 0.9% NaCl 0.5 mL/kg IP 30 minutes before the onset of ischemia; (4) temporary ischemia with ZnPP treatment: animals received ZnPP in a dose of 50 mg/kg (0.5 mL/kg) IP 30 minutes before the onset of ischemia; (5) temporary ischemia with ZnPP treatment: animals received ZnPP in a dose of 50 mg/kg (0.5 mL/kg) IP 2 hours after the onset of ischemia; and (6) temporary ischemia with ZnPP treatment: the animals received ZnPP in a dose of 50 mg/kg (0.5 mL/kg) IP 4 hours after the onset of ischemia.

Each rat brain was sliced in 2-mm coronal sections for measurement of stroke area. Coronal slices were incubated for 20 minutes in a solution of 2% triphenyltetrazolium chloride (TTC, Sigma Chemical Co) in 25 mmol/L potassium phosphate–buffered saline (Sigma). After incubation, all of the samples were fixed in buffered formaldehyde. The slices were then photographed, and the total cross-sectional area of the infarcted tissue was measured in square millimeters using computer-assisted planimetry (Sigmascan, Jandel Scientific). Infarct area was expressed as a percentage of the total ischemic hemisphere area. The total size of the cerebral stroke was calculated as the sum of the infarct areas from the frontal pole through all six slices.

For brain water measurements, samples were removed from flattened cortical mantels using 7-mm and 10-mm cork borers to obtain tissue samples from the center, intermediate, and outer zones of the ischemic cerebral cortex and from corresponding areas of the contralateral nonischemic cortex as described by Betz and Coester⁸ and Martz et al.⁹ Brain samples were placed in preweighed crucibles and reweighed to obtain the wet weight. A Mettler AE100 balance (Mettler Instrument Corp) was used. The brain samples were then dried for 48 hours at 100°C to determine their dry weight. The percentage of water in the samples was calculated as the difference between wet and dry weights divided by the wet weight and multiplied by 100. Values are shown as the water content in the ischemic cortex.

Protoporphyrin IX Zinc (II) was purchased from the Aldrich Chemical Company Inc. It was converted to ZnPP disodium salt with 0.1 N NaOH and dissolved in 25 mmol/L potassium phosphate–buffered saline.

Physiological data are expressed as the mean, and other values are expressed as mean±SE. Statistical evaluation was performed using Student's unpaired t test in permanent ischemia and one-way ANOVA (Dunnett's one-tailed) in temporary ischemia for comparison of sham-operated and experimental groups; Student's paired t test was used for comparison of paired data obtained in each animal in permanent and temporary ischemia. Differences that achieved a value of P<.05 were considered significant.

	Results

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Levels of arterial blood gases, pH, mean arterial pressure, plasma glucose, and hematocrit and body and temporal muscle temperatures were similar between the corresponding control and ZnPP-treated groups before and after the ischemic period. CBF data for control and ZnPP-treated groups with permanent and temporary ischemia are summarized in the Table. The regional CBF in the ischemic cortex after occlusion was approximately 13% to 20% of baseline in both the permanent and temporary ischemic experiments. There were no significant differences among either permanent or temporary ischemic groups.

View this table: [in this window] [in a new window]   Table 1. Effect of Zinc Protoporphyrin on Percent Regional Cerebral Blood Flow in Ischemic Core During Permanent and Temporary Ischemia

The mean percent cerebral infarct sizes for groups 1 and 2 (permanent ischemia) are illustrated in Fig 1. ZnPP treatment did not affect infarct size resulting from permanent MCAO (64.7±4.8% for 0.9% NaCl rats versus 58.5±7.0% for ZnPP-treated rats). The effect of ZnPP on ischemic brain edema was measured in the center, intermediate, and outer zones of the infarct after 24 hours of permanent occlusion (Fig 2). Brain edema accumulation was greatest in the center and least in the outer zone. There were no significant differences among the 0.9% NaCl and ZnPP-treated groups.

View larger version (31K): [in this window] [in a new window]   Figure 1. Graphs show effect of zinc protoporphyrin (ZnPP) on infarct area (top) and total size (bottom) in permanent ischemia. The animals were treated with either 0.9% NaCl or ZnPP 30 minutes before middle cerebral artery occlusion. After 24 hours of ischemia, the percentage of cross-sectional area of the right hemisphere that did not stain with triphenyltetrazolium chloride was measured for each of six brain slices of each animal. Values are mean±SE for six animals.

View larger version (41K): [in this window] [in a new window]   Figure 2. Bar graph shows effect of zinc protoporphyrin (ZnPP) on brain edema in rats subjected to permanent ischemia. The animals were treated with either 0.9% NaCl or ZnPP 30 minutes before middle cerebral artery occlusion. After 24 hours of ischemia, brain samples were taken from the center, intermediate, and outer zones of the infarct in the ischemic cortex. Values shown are the mean±SE of the water content in the ischemic cortex (n=6).

In contrast, in groups 3 to 6 (temporary ischemia), total infarct size was significantly smaller in the group treated with ZnPP before ischemia (17.2±6.6% in controls versus 6.2±2.9% in pretreated rats, P<.05, Fig 3). Brain edema accumulation was smaller in the group treated with ZnPP before ischemia (center, 83.3±0.7% water in controls versus 80.3±0.3% water in pretreated rats, P<.05; intermediate zone, 81.1±0.7% water in controls versus 79.2±0.1% water in pretreated rats; Fig 4). Although the stroke size (Fig 3) and brain edema formation (Fig 4) were lower in animals treated at the time of reperfusion (ZnPP+2 hours) than in controls, these differences did not reach statistical significance. An additional 2-hour delay in therapy (ZnPP+4 hours) had no effect on stroke or edema.

View larger version (25K): [in this window] [in a new window]   Figure 3. Graphs show effect of zinc protoporphyrin (ZnPP) on infarct area (top) and total size (bottom) in temporary ischemia. The percentage of the cross-sectional area of the right hemisphere that did not stain with triphenyltetrazolium chloride is shown for each of six slices in 0.9% NaCl-treated and ZnPP-treated rats. *P<.05 compared with 0.9% NaCl pretreatment (n=6 to 7).

View larger version (47K): [in this window] [in a new window]   Figure 4. Bar graph shows effect of zinc protoporphyrin (ZnPP) on brain edema in rats subjected to temporary ischemia. The animals were treated with 0.9% NaCl or ZnPP 30 minutes before or ZnPP 2 hours or 4 hours after middle cerebral artery occlusion. After 22 hours of reperfusion, brain samples were taken from the center, intermediate, and outer zones of the infarct in the ischemic cortex. Values are shown as the water content of mean±SE of the ischemic cortex. **P<.01, *P<.05 compared with 0.9% NaCl pretreatment (n=6 to 7).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present findings indicate that pretreatment with ZnPP results in a reduction in cerebral infarct size, brain edema, sodium accumulation, and potassium loss after 2 hours of MCAO followed by 22 hours of reperfusion. When ZnPP was administered 2 hours after MCAO, it tended to reduce ischemic brain damage, although the results were not statistically significant. When ZnPP was administered 4 hours after MCAO, no protection against brain ischemia was detected. Yamasaki et al³ reported that brain edema formation was significantly reduced with intraventricular ZnPP treatment immediately after reperfusion following 1 hour of ischemia. The present data indicate only partial protection with systemic ZnPP treatment after 2 hours of ischemia. Thus, ZnPP must be present in the brain in a very early postischemic stage to provide any benefit.

The present study sheds no light on the mechanism of action of ZnPP, although its major effect has been suggested to be due to IL-1 antagonism.³ When ZnPP is given intraperitoneally, it must be absorbed into the systemic circulation and then cross the blood-brain barrier. In transient ischemia, reperfusion would improve the concentration of the drug in postischemic brain areas, whereas with permanent ischemia the drug would be delivered only to brain areas with good collateral circulation. The failure of pretreatment to attenuate ischemic brain damage in permanent ischemia may be related to reduced delivery of the drug to ischemic tissue or the delayed appearance of IL-1. On the other hand, it is possible that IL-1 is most important in reperfusion.

The mechanism of action of ZnPP protection may be mediated by inhibition of IL-1 receptors or heme oxygenase inhibition. The role of heme oxygenase inhibition in ischemia is not known, whereas that of IL-1 is clearly implicated in ischemia.^{6 10 11 12 13}

In summary, ZnPP reduces brain infarct volume and edema accumulation after temporary but not permanent MCAO. ZnPP has at least two possible major mechanisms of action. Further research is needed to determine whether the major mechanism of ZnPP in preventing ischemic injury is mediated by the inhibition of IL-1 and/or heme oxygenases.

	Acknowledgments

 
This study received major support from the Psychopharmacology Research Fund (361024) and partial support from a grant from the National Institutes of Health (NS-23870).

Received February 3, 1994; revision received November 22, 1994; accepted January 31, 1995.

	References

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