Superoxide dismutase reduces permeability and edema induced by hypertension in rats XIU-ME1 ZHANG AND EARL F. ELLIS of Pharmacology and Toxicology, Medical College of Virginia, Department Virginia Commonwealth University, Richmond, Virginia 23298-0613

ZHANG, XIU-MEI, ANDEARLF. ELLIS. Superoxide dismutase reduces permeability and edema induced by hypertension in rats. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H497-H503, 1990.-These studies determined whether superoxide dismutase (SOD), an oxygen free-radical scavenger, affects brain and lung vascular protein extravasation and water content after acute hypertension. Hypertensive vascular injury was induced in rats by bolus injection of norepinephrine. Vascular permeability was assessed with lz51-labeled serum albumin and water content determined by wet and dry weight measurement. Pretreatment with SOD prevented or reduced the increase in brain water content and brain and lung protein extravasation caused by hypertension, whereas inactivated SOD had no effect. SOD also reduced mortality caused by acute hypertension. Treatment 30 min after hypertension with SOD or polyethylene glycol-conjugated SOD reduced edema caused by hypertension. In some instances SOD reduced tissue water content and permeability to below normal control levels found in animals without hypertension. These studies show that oxygen radicals contribute to increases in permeability and water content after hypertensive injury and also suggest that oxygen radicals may contribute to regulation of vascular permeability and water content in normal animals.

oxygen radicals; tion; blood-brain

free-radical scavengers; barrier; norepinephrine;

cerebral microcirculabrain; lung

ACUTEDRUG-INDUCEDHYPERTENSION isknowntocause abnormalities in the cerebral circulation. These abnormalities include increased cerebral vascular permeability to dye and proteins (9, 12), a loss or “breakthrough” of autoregulation, and forced dilation of the arterial vasculature, which imparts a “sausage-string” appearance (19, 28). Additionally, we and our colleagues have shown that after acute hypertension induced by pressor agents or experimental fluid percussion brain injury, abnormalities of cerebral arterioles are caused by oxygen radicals formed during increased arachidonic acid metabolism (15, 16, 33). The abnormalities include sustained arteriolar dilation, decreased cerebral arteriolar reactivity to hypocapnia, pial arteriolar endothelial lesions, and altered reactivity to vasoactive substances. These abnormalities are known to be caused by cyclooxygenasedependent free radicals, since they can be prevented by cyclooxygenase inhibitors and oxygen radical scavengers

(16, 33). In the past several years investigators have become interested in the endothelium and its generation of relaxing factors. These endothelium-derived relaxing fac0363-6135/90

$1.50 Copyright

tors (EDRFs) are formed by the endothelium in response to various vasoactive agents and are short lived (6). Recently, it has been shown that EDRFs are destroyed by oxygen radicals (22, 25-27, 32). This implies that a condition that increases the formation of oxygen radicals may alter vascular reactivity by decreasing or eliminating the action of EDRFs (3). Conversely, free-radical scavengers may protect against destruction of EDRFs by free radicals. We recently tested the effect of the free-radical scavenger superoxide dismutase (SOD) on the blood pressure and cerebral blood flow response to acute hypertension induced by increasing bolus injections of norepinephrine in rats (5, unpublished observations). We found that SOD pretreatment reduced the pressor and blood flow response to given submaximal pressor doses of norepinephrine and thus shifted the dose-response curves for these parameters to the right. Additionally, we found that the mortality associated with administering 10 pg/kg norepinephrine was eliminated by pretreatment with SOD. Eight out of eight rats pretreated with SOD survived, whereas only three out of eight (37%) of the untreated rats survived. The mechanism by which SOD reduced mortality was uncertain. The purpose of the present investigation was to begin to address the possible mechanisms by which SOD protects against the effects of acute hypertensive injury. Because acute hypertension is known to increase bloodbrain barrier permeability, we examined the effect of SOD on brain water content and cerebrovascular permeability to ?-labeled bovine serum albumin (1251-BSA). Also, acute hypertension, whether induced by experimental fluid percussion brain injury or by pressor agents, often produces a bloody exudate in the respiratory tract. Therefore, we also examined the effect of SOD on lung water and vascular protein extravasation. Both pre- and posthypertension administration of SOD were examined. The results show that administration of SOD before hypertensive injury reduces both edema and permeability, whereas administration of SOD after injury reduces edema formation. Additionally, our data suggest that oxygen radicals may affect permeability and water content in normal animals. MATERIALS

AND

METHODS

General preparation and induction of hypertension. These experiments were conducted in 64 Sprague-Dawley male rats (300-400 g) anesthetized with Inactin (140

0 1990 the American

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Society

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mg/kg ip). After tracheotomy, the rats were ventilated with room air and the respirator rate and volume adjusted such that the end-expiratory COa was controlled at a level of -30 mmHg. The temperature of all animals was maintained at 37OC. Mean arterial blood pressure was measured with a pressure transducer connected to a cannula introduced into the aorta through the left femoral artery. The right femoral artery and vein were cannulated for withdrawal of arterial blood samples and infusion of superoxide dismutase, respectively. Blood gases and pH were analyzed to ensure adequate and consistent ventilation. Hypertension was produced by intravenous administration of increasing bolus doses of norepinephrine. Based on the results of our previous studies (5, unpublished observations), we used three successive doses of norepinephrine: 1, 3, and 10 pg/kg. The multiple norepinephrine doses were used simply to try to ensure vascular damage. Blood pressure was recorded during the maximum response to each dose of norepinephrine and also at 1, 2, and 5 min after each dose of norepinephrine. Ten micrograms per kilogram norepinephrine was chosen as the maximum dose, since our previous studies (5) showed that with this dose both control and SOD-treated animals responded with the same maximum degree of blood pressure increase. Control and SOD-treated animals were examined in a random order. Determination of permeability and water content. Vascular permeability to plasma protein was evaluated as we have done previously (7) by counting the extravascular radioactivity in the tissue after administration of 30 &i/kg 12”1-BSA (New England Nuclear, Boston, MA). The l”“I-BSA was administered through the femoral vein 10 min before the administration of norepinephrine in the studies where SOD was given before hypertension or at 30 min after the 10 pg/kg dose of norepinephrine when SOD was given after hypertension. At the end of the experiment, the thoracic cavity was opened and a blood sample was obtained from the right ventricle by puncturing the ventricle with a lo-p1 measuring pipette. This volume of blood was then counted for radioactivity, and all tissue radioactivity was normalized to the number of counts per milliliter of blood. To clear the brain vasculature of intravascular 1251-BSA, the animal was perfused via the left ventricle with saline at a perfusion pressure not exceeding 100 mmHg. The vena cava was severed during this perfusion process so that perfusate returning from the brain could exit the vasculature. After the brain was perfused, the lungs were also cleared by a similar process, wherein saline was infused through the right ventricle, but the perfusion pressure did not exceed 50 mmHg. Next, both hemispheres of the brain and two pieces of lung were placed in tared test tubes and their weight determined to the nearest 0.01 mg on a Sartorius 2474 analytical balance. The test tubes were then placed in an oven at 130°C for at least 16 h until the brain and lung samples reached a constant weight. The samples were covered while returning to room temperature to prevent the accumulation of moisture, and the dry weight was then determined. The radioactivity of the blood and dried brain and lung was

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counted by a Beckman gamma scintillator. The index for permeability of the brain vasculature and the extravasation of protein in the lung was calculated by the following formula: (counts/min of 1251per g dry wt tissue divided by counts/min of 12”1per ml blood) x 100. It should be noted that although extravasated albumin can be used as an index of vascular permeability in the brain, lung interstitial albumin does not necessarily reflect only vascular permeability. Changes in lung interstitial albumin can also be altered by changed in vascular hydrostatic pressure or changes in capillary surface area. Therefore, we have referred to changes in lung 12’1labeled protein content as “protein extravasation” and not permeability. Brain and lung percent water content was calculated by the formula: [(wet wt - dry wt)/wet wt] x 100. Administration of superoxide dismutase. Rats pretreated with SOD were given a 0.5-ml bolus dose of 24,000 U/kg followed by a constant infusion of 1,600 U/kg per minute in a volume of 33 pl/min. SOD (3,400 U/mg) was from bovine erythrocytes and was obtained from Sigma (St. Louis, MO), and the vehicle was saline. Therefore, the animals received an initial loading dose plus a continuous supplement throughout the norepinephrine and postnorepinephrine injection period. Control animals received saline administered in a similar manner. For animals receiving SOD after the hypertensive challenge, the procedure was as follows. Thirty minutes after the 10 ,ug/kg dose of norepinephrine 1251-BSA was administered, and 5 min later native SOD was given as a bolus injection and then as an infusion, as described above for pretreatment with SOD. Another group of animals received polyethylene glycol-conjugated SOD (PEG-SOD) instead of native SOD (3,18). The rationale for the administration of PEG-SOD was that PEG-SOD is stable for long periods of time and thus a one-time bolus injection could be administered to the animals. The dose of PEG-SOD was 2,000 U/kg. In both posttreatment groups, the animals were killed 1 h after administration or initiation of the SOD therapy. In some experiments, inactivated SOD was tested to be certain that SOD effects were not merely a consequence of administration of protein. SOD was inactivated by heating it in a pressure cooker for 8 h. This length of time was necessary because SOD is an extremely resistant enzyme, and shorter heating periods did not produce 100% inactivation of SOD activity, as determined by a cytochrome c reductase assay for SOD activity (21). Experimental groups: protocols. There were five groups of animals in the first series of experiments where the effects of the administration of SOD before hypertension were examined. The first group was the sham control group, which had all surgical procedures performed, but no norepinephrine or SOD was administered. This control group did, however, receive equivalent volumes of the saline vehicle. The second group of animals was given norepinephrine but did not receive SOD. The third group of animals received SOD before the administration of norepinephrine. The fourth group of animals received heat-inactivated SOD before the administration of nor-

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epinephrine, and a fifth group of animals received only SOD, without norepinephrine, to determine the effects of SOD alone on brain and lung water content. The protocol for the animals pretreated with SOD was as follows. First, stable blood pressures were obtained for at least 10 min, the bolus SOD was given, and the SOD infusion was started. Ten minutes after the bolus SOD the intravenous dose of 1251-BSA was administered. In those animals receiving norepinephrine, the first dose of norepinephrine was given 10 min after the administration of the 1251-BSA. The subsequent two doses (3 and 10 pg/kg) were given at lo-min intervals after the first dose of norepinephrine. Within 10 min after the 10 pg/ kg dose of norepinephrine, the animals were perfused and the tissues were weighed as described above. Therefore, in the experiments where pretreatment with SOD was one of the modalities tested, the total time elapsed from the first dose of norepinephrine to the end of the experiment was -30 min. In the experiments where SOD was given after hypertensive injury, the protocol was as follows. First, a stable base-line blood pressure was obtained, and then the 1, 3, and 10 lug/kg doses of norepinephrine were administered at lo-min intervals. In those animals surviving the 10 pg/kg dose of norepinephrine, 1251-BSAwas administered 30 min after the 10 pg/kg dose of norepinephrine. Native SOD, PEG-SOD, or saline vehicle was given 5 min after 1251-BSA. The animals were then monitored for one additional hour, and they were then perfused and tissue weights and permeability were determined. Therefore, in these experiments the total elapsed time from the first dose of norepinephrine to the end of the experiment was -2 h. Statistical analysis of the various treatments was performed by analysis of variance followed by Fisher’s least significant difference test. In some instances it was necessary to perform a log transformation of the data because the distribution of the data was not normal. P 5 0.05 was considered to be statistically significant.

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1. Blood pressure data and survival rates in superoxide dismutase pretreatment studies

TABLE

MABP, Group

n Control

Control NE SOD+NE SOD iSOD + NE

8 5 5 5 5

mmHg Survival Rate

NE-induced maximum

106t8 102t6 108t6 112t5 105t8

8of8 1 of5 5of5 5of5 3of5

184t6 185Ik3 184t5

Blood pressure data and survival rates in superoxide dismutase (SOD) pretreated studies. Values are means t SE; n, no. of rats. NE, norepinephrine-induced hypertension; MABP, mean arterial blood pressure; SOD, superoxide dismutase pretreatment; iSOD, inactivated superoxide dismutase pretreatment. 12

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1. Effect of superoxide dismutase pretreatment on cerebrovascular permeability and lung protein extravasation after norepinephrineinduced hypertension. Values are means t SE; see text for calculations. NE, norepinephrine-induced hypertension; SOD, superoxide dismutase pretreatment; iSOD, inactivated superoxide dismutase pretreatment. Because of abnormal distribution of lung protein extravasation values, log transformation of the data was performed before analysis of variante. *p ZZ 0.05 vs. control. **P 5 0.05 vs. NE and control. FIG.

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ti 5 .i

The control blood gases and pH of all groups of rats were examined by analysis of variance, and no significant differences were found. The average Pace,, Pao,, and pH for all groups were 32 t 1 mmHg, 101 t 2 mmHg, and 7.46 t 0.01, respectively. SOD administered before hypertension. As shown in Table 1, control blood pressures for all four groups of animals were the same. Also, in those groups of animals receiving norepinephrine, the maximum blood pressure responses to 10 pg/kg norepinephrine were similar. Additionally, we found that administration of SOD or 1251BSA had no effect on resting blood pressure (data not shown). Table 1 also shows the number of animals in each group that survived the 10 pg/kg dose of norepinephrine. As in our previous work (5, unpublished observations), we found that norepinephrine was lethal in animals that were not given SOD. However, all animals receiving SOD before norepinephrine survived. Fisher’s exact test was used to analyze mortality in the three groups receiving

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2. Effect of superoxide dismutase pretreatment on brain ana lung water content after norepinephrine-induced hypertension. Values are means t SE. NE, norepinephrine-induced hypertension; SOD, superoxide dismutase pretreatment; iSOD, inactivated superoxide dismutase pretreatment. P 5 0.05 vs. control. FIG.

norepinephrine. Of these three groups, the only groups that were significantly different from each other were the norepinephrine and the norepinephrine plus SOD groups (P = 0.048). Figure 1 shows that norepinephrine increased brain permeability by -100% (P < 0.05). However, animals pretreated with SOD were protected from changes in permeability, whereas animals pretreated with inactivated SOD displayed the same increase in permeability as those animals receiving only norepinephrine. Figure 2

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shows the percent brain water in these same groups of animals plus an additional group of animals to which only SOD and not norepinephrine or lz51-BSA was administered. It can be easily seen that norepinephrine administration in the absence of SOD caused a statistically significant (P < 0.05) increase in brain water. Animals receiving SOD before norepinephrine were protected from increases in brain water and, in fact, actually have statistically significantly decreased levels of brain water when compared with the control animals (P < 0.05). Giving animals only SOD induced a percent water content that was on the average lessthan that in controls but was not statistically significantly decreased compared with controls. Again, as with the brain permeability response, those animals that received inactivated SOD were not protected from the damaging effects of acute hypertension. Figure 1 also shows that animals subjected to the hypertensive regime have a greatly increased lung protein extravasation. As indicated by the standard error of the norepinephrine-treated group, the variability in lung protein was large, and this required a log transformation of the data for statistical evaluation purposes. Animals receiving SOD before the norepinephrine had lung protein extravasation ratios that were closer to control than to the norepinephrine-treated animals. Animals pretreated with inactivated SOD had average ?-protein ratios that were higher than those of control animals but were not on the average as high as those that received only norepinephrine. Again, however, variability in the protein extravasation response in the group receiving inactivated SOD was apparent. Figure 2 shows the effect of hypertension and SOD on lung water content. Unlike the preceding results, norepinephrine did not affect lung water content in this experimental paradigm. Treatment with SOD before giving norepinephrine produced a tendency toward a decreased lung water content that was not statistically significant. Treatment of animals with only SOD, however, produced a statistically significant decrease in lung water (P < 0.05), suggesting that superoxide free radicals may be important modulators of lung water content even in normal animals. Effects of SOD posttreatment. Table 2 shows that there TABLE

2. Blood pressure data and survival rates

Group

n Control

NE SOD+NE PEG-SOD + NE

6 6 6

93t6 94t6 93t4

NEinduced maximum

183t3 186k3 186&3

mmHg Before SOD or vehicle

85t4 83t6 73t9

60 min suffer

Survival Rate

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were no significant differences between the groups with respect to control blood pressure, the maximum blood pressure effect of 10 pg/kg norepinephrine, or blood pressure immediately before SOD administration or at death. It might be noted, however, that the average blood pressure was slightly decreased at 30 min after hypertension, which is immediately before SOD administration. However, mean pressures were similar to control at 60 min after either SOD or vehicle just before death. Table 2 survival data demonstrates that more than 6 rats had to be used to obtain the data shown in each group in Table 2. Analysis by Fisher’s exact test showed that mortality was the same in all three groups that received norepinephrine only or norepinephrine and then SOD after hypertension. Combining these three groups shows that the average survival was 50% and was significantly different from animals receiving norepinephrine and pretreatment with SOD (Table l), at P = 0.05. Interestingly, the 50% survival rate in the animals without SOD pretreatment is similar to the 60% survival rate in animals receiving inactivated SOD before norepinephrine (Table I). Figure 3 shows the brain permeability results obtained when SOD or PEG-SOD was administered at 30 min after the hypertensive insults. Comparison of these results with Fig. 1 shows that permeability in the group receiving only norepinephrine was not significantly increased when 12”1-BSA is given after the acute hypertensive insult. These results indicate that the change in permeability is associated more with the actual acute hypertensive challenge and occurs as a direct and immediate result of hypertension. Figure 3 also shows that the average brain permeability in the SOD or PEG-SOD group was lower but not significantly different from the untreated norepinephrine group. However, when the animals receiving SOD either in the native or conjugated form are combined (mean permeability t SE, 3.52 t 0.46) and compared with the 8

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Blood pressure data and survival rates in superoxide dismutase (SOD) posttreatment studies. Values are means t SE; n, no. of rats. MABP, mean arterial blood pressure; NE, norepinephrine-induced hypertension; SOD, superoxide dismutase given 30 min after norepinephrine; PEG-SOD, polyethylene glycol-conjugated superoxide dismutase given 30 min after norepinephrine. Blood pressure data are given only for those animals that survived acute hypertensive insult and posthypertension study period.

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3. Effect of superoxide dismutase posttreatment on cerebrovascular permeability and lung protein extravasation after norepinephrine-induced hypertension. Values are means t SE; see text for calculations. NE, norepinephrine-induced hypertension; SOD, superoxide dismutase treatment at 30 min after hypertension; PEG-SOD, polyethylene glycol-conjugated SOD treatment at 30 min after hypertension. Individual SOD and PEG-SOD groups are not different from norepinephrine-only group when using Fisher’s least significant difference test. However, when animals that received SOD, in either the native or the conjugated form, are combined and compared with rats receiving only norepinephrine using a two-tailed unpaired Student’s t test, animals receiving SOD are significantly different from those not receiving SOD; "P 5 0.05. FIG.

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norepinephrine group using a two-tailed unpaired Student’s t test, a P value of 0.05 is derived. Under this statistical analysis the presence of the superoxide dismutase molecule causes a slight but significant decrease in permeability, suggest)ing that free-radical mechanisms may play a role in the tonic control of vascular permeability. Figure 4 shows that brain water content after acute hypertension was increased even more than in the studies where SOD was given before injury (Fig. 2). However, it should be remembered that a longer period of time has elapsed between norepinephrine administration and termination of the experiment in the groups receiving posthypertension therapy. Figure 4 also shows that both SOD and PEG-SOD reduced the brain edema compared with the group receiving only norepinephrine. In this respect PEG-SOD appeared to be slightly more efficacious than unconjugated SOD in reducing brain water content. It should be noted that the brain water content found in the PEG-SOD group is essentially identical to that found in normal sham control animals not challenged with norepinephrine-induced hypertension (Fig. 2) . Comparison of Figs. 3 and 1 shows that there was no large increase in lung 1251-protein extravasation in the norepinephrine-treated group when the tracer was given 30 min after norepinephrine challenge. This again implies that the increase in extravasation is transient and associated more directly in time with the actual hypertensive challenge. Figure 4 shows that the lung water content in the animals receiving only norepinephrine was elevated above control (Fig. 2). Because lung water was not significantly increased in the short-term experiments where SOD was given before injury and the animals were killed soon after the hypertensive challenge, these posttreatment results suggest that a longer period of time after hypertensive challenge is needed before increases in lung 81.5

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FIG. 4. Effect of superoxide dismutase posttreatment on brain and lung water content after norepinephrine-induced hypertension. Values are means t SE. NE, norepinephrine-induced hypertension; SOD, superoxide dismutase treatment 30 min after hypertension; PEG-SOD, polyethylene glycol-conjugated SOD treatment 30 min after hypertension. *P 5 0.05 vs. norepinephrine group. Note that brain water content of SOD- and especially PEG-SOD-treated groups is similar to brain water content of normal control animals without hypertensive injury (Fig. 2). Also, note that lung water content in animals receiving only norepinephrine is greater than lung water content in control and norepinephrine groups in SOD pretreatment study (Fig. 2). This implies that the longer experimental protocol in posttreatment studies allows time for expression of an increased water content in hypertension-injured animals.

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water occur. Again, as with brain water content, treatment with SOD or PEG-SOD after the hypertensive challenge lowered lung water content, implying a role for oxygen radicals in the edema response to acute hypertension that lasts beyond the actual hypertensive insult. DISCUSSION

Our results show that SOD but not inactivated SOD pretreatment prevents increases in brain vascular permeability and lung protein extravasation that are associated with acute hypertension. These results imply that oxygen radicals are important in the mediation of these changes. Although previous studies have shown that oxygen radical scavengers prevent permeability and water changes induced by neural injury such as that produced by cold lesions (1, 4, lo), there are no studies, to our knowledge, examining how systemic administration of superoxide dismutase or polyethylene glycol-conjugated SOD can affect the brain’s response to acute hypertension. The mechanism by which norepinephrine increases cerebral vascular permeability likely involves alterations in permeability in the venous side of the microcirculation, since recent studies have shown increased venous permeability induced by acute hypertension (2, 20). The cellular and molecular mechanisms by which free radicals increase permeability are uncertain; however, free radicals have been shown to affect a number of enzyme systems, including, for example, Na+-K+-adenosinetriphosphatase (8, 14). The mechanism by which the production of oxygen radicals is triggered is likely associated with cyclooxygenase-dependent metabolism of arachidonic acid. This is true since previous studies from our institution have shown that after acute hypertension cyclooxygenasedependent free radicals are formed with concomitant effects on the arterioles (16). These abnormalities produced by acute hypertension include sustained arteriolar dilation, endothelial lesions, and reduced arteriolar responsiveness to hypocapnia. The arteriolar abnormalities have also been shown to be associated with experimental concussive brain trauma, which produces a transient hypertensive response after the actual traumatic insult (33). Additionally, the work of Johannson (11) supports a role for cyclooxygenase-dependent free-radical alteration of the blood-brain barrier, since she showed that the increased cerebrovascular permeability produced by epinephrine can be reduced by pretreatment with the cyclooxygenase inhibitor indomethacin. Previous studies show that with acute drug-induced hypertension there is a transient opening of the bloodbrain barrier (13, 24). Our results with administration of 12?-BSA after hypertension confirm that the barrier opening is transient, since cerebrovascular permeability was not as dramatically affected when the tracer was given at 30 min after the norepinephrine-induced hypertensive injury. The transient nature of the permeability response is supported by the work of Nag (23). She found that the transient increased permeability of cerebral vessels during acute hypertension is associated with a similar transient loss of the terminal sialic acid groups on the luminal plasma membrane of permeable cerebral

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vessels. However, since most studies of permeability use a limited number of different-sized molecular tracers, we cannot rule out that in our study or other studies there may be continuing permeability to various yet unexamined molecules in the period after acute hypertension. Our data (Fig. 3) also suggest that free radicals may modulate cerebrovascular permeability in normal animals, since posttreatment with SOD reduced the average permeability to 1251-BSA to slightly below control. This implies that control permeability may be influenced by some tonic level of free-radical production. However, until now little has been done with free-radical scavengers in normal animals. Most studies in the past have concentrated on examining the effects of free-radical scavengers in injured or otherwise challenged animals. Other investigators have also examined the effect of free-radical generating systems on cerebrovascular permeability. Wahl et al. (30) and Wei et al. (31) found that topical administration of arachidonic acid on the brain surface, which is known to produce free radicals (30), increased permeability. Wei et al. (31) also showed that the free-radical scavengers SOD plus catalase were able to reduce the permeability increases, as measured 1251-labeled serum albumin extravasation. However, by when Unterberg et al. (29) examined the effect of topical brain application of the oxygen radical-generating system hypoxanthine plus xanthine oxidase, they found that visually observed permeability to fluorescein was increased in one of three experiments while no extravasation of fluorescein isothioscyanate (FITC)-dextran was observed microscopically in two experiments. While some of the above studies support a role for free radicals in increasing cerebrovascular permeability, it is difficult to compare the results, since widely different experimental approaches were taken. It may be especially difficult to compare studies with topical applications of radicalgenerating systems with our approach, which relies on permeability increases induced by endogenous mechanisms in response to hypertension. In addition, our studies do not pinpoint the precise brain areas in which the permeability increases occur. SOD reduced water content of the brain when given either before or after hypertension. The increase in water induced by hypertension may be related to the increased permeability to solute molecules during hypertension, which in turn bring water after them. Whereas increased brain permeability to 1251-BSA was transient, it should be noted from Figs. 2 and 4 that water content tended to increase with time after the hypertensive insult. Thus the rat brain tissue at -1.5 h after the hypertensive insult had a greater water content than that of animals that were examined within minutes of the hypertensive challenge. However, in both the pretreatment and posttreatment experiments, SOD reduced brain water content to control levels. This implies that some continuous ongoing free-radical-producing event is responsible for producing brain edema. This effect of SOD is due to its free-radical-scavenging effect and not to an osmotic effect of this protein molecule, since inactivated SOD had no effect on brain water content (Fig. 2). With respect to lung water, we were somewhat sur-

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prised that in the SOD pretreatment series of animals hypertension did not increase lung water, given that a watery and bloody exudate often emanated from the trachea after hypertension. However, in the longer term experiments where SOD was given posthypertension, lung water was increased. In these instances, SOD or PEG-SOD given at 30 min after the hypertensive challenge was able to reverse the lung water increases seen in animals treated only with norepinephrine. Another interesting finding with respect to lung water content (Fig. 2) was our observation that SOD, in the absence of hypertension, significantly reduced lung water content. Also, in animals pretreated with SOD and given the hypertensive challenge, there was a similar but statistically insignificant tendency for decreased lung water. These results, like the brain water results, suggest that chronic free-radical production may influence water content. Polyethylene glycol-conjugated SOD, which became available to us in the latter part of our studies, has a much longer half-life and enters endothelial cells and maintains activity much longer than unconjugated SOD (3). For this reason, PEG-SOD can be given once and will have a long-lasting effect, thus eliminating the need for continuous intravenous supplementary infusion. This longer half-life may make PEG-SOD a superior therapeutic agent. Our results show that the one-time bolus injection of PEG-SOD was as effective as the much larger dose of SOD given as a bolus and followed by continuous infusion. With respect to mortality, the current studies clearly show that animals pretreated with SOD survive the hypertensive challenge better. This is in agreement with our previous studies (5, unpublished observations), which showed that eight out of eight animals pretreated with SOD survived, whereas only three out of eight control animals lived. Although pretreatment with SOD prevented the increased brain permeability, brain water, and lung protein extravasation in response to the hypertensive challenge, it seems unlikely to us that these effects are what reduced mortality. This impression is based on the fact that the magnitude of the brain permeability and edema response was not large enough to be life threatening. Additionally, there was no increase in water content in the lungs of untreated animals in the pretreatment study (Fig. 2). We are therefore uncertain of the mechanism by which SOD decreased mortality. We would note, however, that Levasseur et al. (17) have recently found that pretreatment of rats with SOD greatly reduced mortality after fluid percussion-induced experimental brain injury. Interestingly, this model of brain trauma produced transient hypertension reminiscent of that produced by injection of norepinephrine. In summary, these studies demonstrate that acute hypertension induces alterations in vascular permeability and tissue water content that are free-radical dependent. Previous studies showing the cyclooxygenase-dependency of this free-radical generation imply that arachidonic acid metabolism is important in initiating this response. Additionally, our results show that oxygen

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and water

content

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in

17 August

1989; accepted

in final

15. 16.

We thank Sallie Holt, Richard Police, Lyn Rice, and Marcia Heizer for assistance. Our research is supported by a grant-in-aid from the American Heart Association, National Institute of Neurological Disorders and Stroke Grants NS-27214, NS-23432, and NS-12587, and National Heart, Lung, and Blood Institute Grant HL-41788. E. Ellis is an Established Investigator of the American Heart Association and the recipient of a Javits Neuroscience Investigator Award. X.-M. Zhang is a Visiting Scholar supported in part by the People’s Republic of China. Address for reprint requests: E. F. Ellis, Box 613, MCV Station, Richmond, VA 23298. Received

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Superoxide dismutase reduces permeability and edema induced by hypertension in rats.

These studies determined whether superoxide dismutase (SOD), an oxygen free-radical scavenger, affects brain and lung vascular protein extravasation a...
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