Background and Purpose We previously reported that the endothelium-dependent dilation of pial arterioles by either topical acetylcholine (ACh) or bradykinin (BK) was markedly inhibited after 10 minutes of near total ischemia after bilateral carotid occlusion. The present study tests the responses after 10 minutes of reperfusion and investigates the effect of either oxygen or oxygen radical scavengers on the results.
Methods Mice were subjected to bilateral carotid ligation or sham ligation. Pial arteriolar diameters were monitored by an image-splitting technique at a craniotomy site. In separate studies, the responses to topically suffused ACh, BK, or sodium nitroprusside (SNP) were tested before ischemia. After 10 minutes of ischemia and 10 minutes of reperfusion, the response was assessed again. Sham-operated mice were observed in each study. Cerebral blood flow was continuously monitored with a laser-Doppler technique. Additional separate studies were conducted as follows: presence of superoxide dismutase plus catalase during ischemia and reperfusion, or increase in the inspired oxygen (arterial oxygen) and oxygen in suffusate.
Results The response to ACh was significantly impaired after 10 minutes of reperfusion. The responses to BK and SNP were unaffected. Radical scavengers failed to influence the impaired response to ACh. Elevations of arterial and suffusate oxygen levels to over 300 mm Hg failed to prevent the impairment.
Conclusions After 10 minutes of reperfusion, a selective impairment of the response to ACh remains. The response to another endothelium-dependent dilator, BK, recovered, and the response to endothelium-independent SNP was unaffected. Because neither radical scavengers nor oxygen altered the outcome with respect to ACh, I suggest that neither radical generation nor hypoxia accounts for the selective impairment of dilation by ACh. Rather, I hypothesize that reduced shear during ischemia diminishes the ability of the endothelium to synthesize and/or release the endothelium-derived relaxing factor for ACh. I hypothesize further that this impaired release or synthesis persists throughout the 10-minute period of reperfusion.
There have been surprisingly few publications concerning the effect of ischemia or of ischemia/reperfusion on the vasomotor properties of brain blood vessels.1 2 3 4 5 6 Recently, I7 reported that in mice, 10 minutes of profound ischemia produced a selective diminution in the endothelium-dependent relaxation of pial arterioles normally produced by three endothelium-dependent dilators, each with its own distinct endothelium-derived mediator.8 9 10 11 Thus, the responses to ACh, BK, and calcium ionophore A-23187 were all inhibited. The dilation produced by SNP, an endothelium-independent dilator, was not affected by 10 minutes of ischemia. The work presented below uses the same murine model of ischemia to investigate the effect of ischemia/reperfusion on the responses to ACh, BK, and SNP. I show that the response to ACh remains selectively depressed. Moreover, the depression is not affected by scavengers of superoxide, H2O2, or singlet oxygen or by supplying the vessels with very large amounts of oxygen.
Materials and Methods
All procedures were approved by the institution's animal care and use committee.
Male ICR mice (Harlan-Sprague-Dawley; weight, 27 to 35 g) were used. They were anesthetized with urethane, and the pial surface was exposed as previously reported.12 13 Briefly, a tracheostomy and craniotomy were performed, and the dura was stripped from the craniotomy site to expose the pial vessels beneath the transparent arachnoid. The mouse was maintained at 37°C with a heated mattress, and the pial surface was continuously suffused with mock cerebrospinal fluid (Elliott's solution14 ) at 37°C. The pH of the suffusate was adjusted by bubbling CO2 through it and was always between 7.34 and 7.35, differing slightly within this range from mouse to mouse but always remaining constant for any individual mouse. All drugs were dissolved in the Elliott's solution. All drug solutions, when suffused, had the same pH as the Elliott's solution.
In each mouse, a pial arteriole was selected for continuous monitoring with the use of incident light, microscope, television camera, and monitor. An image-splitting system was used to measure diameter.15 Such systems are capable of measuring changes <0.5 μm in size, as explained by Dyson.16 In our laboratory, an object 10 μm wide was measured on 20 consecutive occasions with a standard error of 0.2 μm. With 10 μm as a standard, a 5-μm distance was measured and deviated by only 1% (0.05 μm) from the predicted size.
The only criterion for selection of the arteriole to be monitored was an ID between 25 and 45 μm with a straight segment of at least 100 μm long.
Each mouse had a ligature of 4-0 suture material loosely placed around each common carotid artery. The artery was narrowed by pulling on both ends of the ligature. In these studies, both arteries were ligated in this way except in sham-operated mice, in which the ligatures were not tightened.
A laser-Doppler flowmeter and probe17 18 19 were used to continuously monitor flow over the cerebral hemisphere. This method provides a very accurate record of flow expressed in arbitrary units.17 18 19 The probe was placed over the craniotomy at a position adjacent to the monitored vessel.
All drugs were obtained from Sigma Chemical Co: ACh chloride, BK diacetate, SNP, SOD, catalase (thymol free), and histidine dihydrochloride. Only one dilator was tested per mouse. Cumulative dose-response curves were obtained with the use of two ascending doses. Each was applied for 2 minutes (a plateau was reached) before ligation of the carotids. After washout of the dilators, the carotids were ligated. After 10 minutes of ischemia followed by 10 minutes of reperfusion, the vessels were tested again with the dilator.
Systemic BPs were continuously monitored through a femoral artery catheter. In some studies, expired CO2 was continuously monitored in arbitrary units using a Columbus Instruments microcapnometer. The purpose of this measurement was to document that the changes in vascular responsiveness were unrelated to coincidental changes in arterial CO2.
In all studies, the mice breathed spontaneously. In one set of studies, we attempted to prevent the adverse effects of ischemia/reperfusion by supplying large amounts of oxygen throughout the experiment. From the onset of the experiment, the inspired mixture was enriched with oxygen, and the suffusate of Elliott's solution was equilibrated with 100% oxygen. Although the craniotomy was open, the rapid suffusion rate (2 mL/min) over a shallow, small (approximately 4×4×0.5 mm) craniotomy site limited the loss of oxygen to the atmosphere. Samples of suffusate obtained from the far edge of the craniotomy site showed that the partial pressure of oxygen was always >250 mm Hg.
In another set of experiments, we tested the hypothesis that superoxide and/or H2O2 were generated during ischemia or reperfusion and were responsible for the selective impairment of the response to ACh. In these experiments, the suffusate contained 60 U/mL SOD and 46 U/mL catalase, amounts that scavenge significant amounts of superoxide and H2O2.9 10 The scavengers were present from the onset of ischemia through the end of the experiment.
Throughout the studies shown, time control experiments were performed comparing initial responses with responses at 25 minutes later. In these studies, the ligatures were present but were never tightened. The passage of time had no effect on the responses.
Two-way ANOVA was used to analyze the data. Major treatments were before versus after ischemia/reperfusion. Minor treatments were the two doses of the dilator being tested. All values are expressed as mean±SD.
Fig 1⇓ shows the marked impairment of the dose-dependent dilation to ACh at the end of 10 minutes of reperfusion. In this experiment, bilateral ligation of the carotids reduced flow to 26±9% (mean±SD) of control level (initial flow, 143±82 flow units). At the end of reperfusion, flow was 83±36% of control (preischemic) value. Expired mean CO2 and BP levels were the same during the preischemic and reperfusion periods in which ACh was tested.
Acetylcholine With Oxygen
Fig 2⇓ shows that the response to ACh was severely impaired despite the presence of high levels of oxygen during ischemia and during reperfusion. The oxygen partial pressure in the suffusate was 380±30 mm Hg and was 397±165 mm Hg in arterial blood. During ischemia, flow was reduced to 6±2% of its initial level (120±21 flow units). During reperfusion, at the time of the second test of ACh, mean flow was not only restored but was higher than its preischemic level (124±36% of initial value). Expired CO2 was unchanged during the experiment, but BP was reduced to 78±10% of initial value (P<.05).
Acetylcholine With Radical Scavengers
The impairment of the response to ACh was unaffected by SOD+catalase (Fig 3⇓). The flow was reduced to 8±1% of base during ischemia and was restored to 76±37% of base during reperfusion. Initial flow was 120±31 flow units. Expired CO2 was unchanged during the course of the study. BP during reperfusion was 81±25% of preischemic levels (P<.05). These negative results indicate that neither superoxide nor H2O2 played a role in the impairment of response to ACh. To rule out singlet oxygen, we used 10−3 mol/L histidine. Only two mice and two controls were examined because, again, the scavenger had absolutely no protective action against the effect of ischemia. Mean responses to the ACh were dilations of 5% and 13% before and 3% and 7% after ischemia in the pressure of histidine. The no-histidine controls gave responses of 6% and 16% before and 4% and 7% after ischemia.
Bradykinin and SNP
Fig 4⇓ shows that neither the responses to BK nor to SNP were affected by ischemia/reperfusion. In the study of BK, flow during ischemia was reduced to 7±2% of control and restored to 98±49% (initial value, 145±40 flow units). BP was unchanged in either study. Expired CO2 was constant in the BK study but increased 29±15% (P<.01) during reperfusion in the SNP study.
The new findings are (1) the response of pial arterioles to ACh is selectively and severely impaired after 10 minutes of ischemia and 10 minutes of reperfusion; (2) the responses to another endothelium-dependent dilator, BK, and to SNP, an endothelium-independent dilator, were unaffected; and (3) neither enrichment of the local environment with oxygen nor the scavenging of oxygen-centered radicals or H2O2 prevented ischemia/reperfusion from impairing the response to ACh.
The impairment of the response to ACh was similar to that previously reported during ischemia in this model.7 Thus, the effect lasts for at least 10 minutes of reperfusion. The effect of impairment after reperfusion was essentially as great as that reported after 10 minutes of ischemia without reperfusion.7 In the latter study, bilateral ligation of the carotids produced an approximately 90% reduction in blood flow. This degree of reduction was at first not achieved during the present investigation. Thus, the impaired response after reperfusion and shown in Fig 1⇑ was found when flow during ischemia had been reduced by only about 75%. The failure to achieve greater flow reduction was due to improper use of the ligatures. When this was corrected, flow reductions in the successive experiments reported here (eg, Figs 2 and 3⇑⇑) again approximated 90%. The data shown in Fig 1⇑ indicate that transient reductions of flow need not approach 100% to produce profound impairment of subsequent responses to ACh.
Was the impaired response to ACh caused by a lack of oxygen during ischemia and/or during reperfusion? The answer appears to be no, as shown in Fig 2⇑. The response after reperfusion was still suppressed even though the suffusate contained oxygen at levels >250 mm Hg during the period of ischemia and reperfusion and even though arterial Po2 was ≥300.
An alternate hypothesis suggests that radicals formed either during incomplete ischemia or during reperfusion account for the impaired response to ACh. This hypothesis may be considered the opposite of the hypoxia hypothesis, since oxygen is required for radical formation. In any event, the scavengers failed to prevent the impairment of the response to ACh. The present study shows that this is so at the end of 10 minutes of reperfusion. The earlier study7 showed an identical failure after 10 minutes of ischemia without reperfusion. Thus, radicals do not seem to account for impairment of the response to ACh either during ischemia or reperfusion in this model. Of course, we cannot rule out the possibility that radicals were responsible for the selective impairment of the response to ACh but were inaccessible to the scavengers. However, this is unlikely because scavengers similarly applied do affect pial arteriolar responses.4 9 10
In this model, the amount of reperfusion varied greatly from mouse to mouse, as did the change in BP during the experiment. We were unable to relate this variability in reperfusion to changes in or to absolute levels of BP, initial flow, or expired CO2. Nor could we relate the degree of reperfusion to the degree of initial ischemia.
Many models of ischemia/reperfusion actually show transient hyperemia during reperfusion. This only occurred in some of the mice observed here, and a mean increase in blood flow over preischemic values was found in only one of the subsets of experiments reported here. At first, one may consider that absence of hyperemia limits the ability to extrapolate conclusions from our model to the other models. However, in all of our experiments, whether hyperemia occurred or not, there was a profound depression of the response to ACh. Therefore, I believe that the selective impairment of the response to ACh is unrelated to factors that produce hyperemia during reperfusion.
If the selective impairment of the response to ACh is due neither to a lack of oxygen nor to generation of radicals, what is its cause? It is not due to inactivation of guanylate cyclase, the enzyme whose activity underlies the response to ACh, because the response to SNP was not impaired even though that response is also dependent on guanylate cyclase.
I suggest that the persistent inhibition of the response to ACh may reflect a persistent diminution in the shear-dependent release of the EDRF22 for ACh (EDRFACh). I have previously presented data compatible with the hypothesis that brief, large increases in shear can lead to increased release of EDRFACh for up to 20 minutes.23 It is possible that the release of EDRFACh by ACh is impaired for at least 10 minutes after a prolonged episode of drastically decreased shear such as occurs during 10 minutes of profound ischemia.
There is one fact that contradicts my hypothesis. If a drop in shear is responsible for a loss of the response to ACh, how can we explain the presence of response to ACh in vitro in preparations without shear? I cannot explain this paradox. However, the in vitro response of cerebral arterioles occurs under artificial conditions and after excision of the vessel. These conditions and procedures might alter the production or release of EDRFACh and negate the effect of reduced shear.
It is interesting that unlike the response to ACh, the response to BK recovered during 10 minutes of reperfusion even though it, like the response to ACh, is abolished after 10 minutes of ischemia alone.7 This is consistent with several lines of data showing that in the surface arterioles of the brain, the EDRF for BK is not the same as that for ACh and is not a product of the action of nitric oxide synthase.8 9 10
It is possible that recovery of the response to ACh would have occurred if the period of reperfusion were prolonged. This would still indicate a difference between the mechanism responsible for the loss of response to ACh and that of the more transient loss of response to BK.
Previous reports in piglets and cats5 6 also describe selective impairment of some dilating responses after ischemia/reperfusion. However, piglets cannot be compared with mice and cats because piglets do not normally display endothelium-dependent dilation to ACh. The cats, like the mice described here, showed impairment of the response to ACh with an intact response to SNP. In the cat studies,6 BK was not tested nor was the effect on the inhibition of oxygen or of radical scavengers. In cats, the selective inhibition of the response to ACh persisted for 60 minutes of reperfusion. However, at that time, cerebral blood flow was still low in most cats. Our study shows, at least in mice, that the response to ACh will be depressed after at least 10 minutes of reperfusion even in mice with hyperemia at that time.
Selected Abbreviations and Acronyms
|EDRF||=||endothelium-dependent relaxing factor|
This study was supported by grant HL-35935 from the National Heart, Lung, and Blood Institute.
Reprint requests to William I. Rosenblum, Neuropathology, MCV/VCU, Box 980017, Richmond, VA 23298-0017.
Review of this article was directed by Guest Editor Michael S. Wolin, MD.
- Received October 4, 1996.
- Accepted November 13, 1996.
- Copyright © 1997 by American Heart Association
Rosenblum WI. Contractile responses of pial arterioles in gerbils with unilateral carotid ligation. Stroke. 1981;12:83-85.
Mayhan WG, Amundsen SM, Faraci FM, Heistad DD. Responses of cerebral arteries after ischemia and reperfusion in cats. Am J Physiol. 1988;255:H879-H889.
Nelson CW, Wei EP, Povlishock JT, Kontos HA, Moskowitz MA. Oxygen radicals in cerebral ischemia. Am J Physiol. 1992;263:H1356-H1362.
Leffler CW, Beasley DG, Busija DW. Cerebral ischemia alters cerebral microvascular reactivity in newborn pigs. Am J Physiol. 1989;257:H266-H271.
Clavier N, Krisch JR, Hurn PD, Traystman RJ. Effect of postischemic hypoperfusion on vasodilatory mechanisms in cats. Am J Physiol. 1994;267:H2012-H2018.
Rosenblum WI, Wormley B. Selective depression of endothelium-dependent dilations during cerebral ischemia. Stroke. 1995;26:1877-1882.
Rosenblum WI, Nelson GH, Shimizu T. l-Arginine suffusion restores response to acetylcholine in brain arterioles with damaged endothelium. Am J Physiol. 1992;262:H961-H964.
Rosenblum WI. Hydroxyl radical mediates the endothelium-dependent relaxation produced by bradykinin in mouse cerebral arterioles. Circ Res. 1987;61:601-603.
Kontos HA, Wei EP, Kukreja RC, Ellis EF, Hess ML. Differences in endothelium-dependent cerebral vasodilation by bradykinin and acetylcholine. Am J Physiol. 1990;258:H1261-H1266.
Rosenblum WI, Nelson GH. Endothelium dependence of dilation of pial arterioles in mouse brain by calcium ionophore. Stroke. 1988;9:1379-1382.
Rosenblum WI, Nelson GH, Povlishock JT. Laser-induced endothelial damage inhibits endothelium dependent relaxation in the cerebral microcirculation of the mouse. Circ Res. 1987;60:169-176.
Rosenblum WI, Zweifach BW. Cerebral microcirculation in the mouse brain. Arch Neurol. 1963;9:414-423.
Baez S. Recording of microvascular dimensions with an image splitter television microscope. J Appl Physiol. 1966;21:299-301.
Dyson J. Precise measurement by image splitting. J Opt Soc Am. 1960;50:754-757.
Haberl RL, Heizer ML, Marmarou A, Ellis EF. Laser-Doppler assessment of brain microcirculation: effect of systemic alterations. Am J Physiol. 1989;256:H1247-H1254.
Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 2nd ed. Oxford, UK: Clarendon Publishers; 1989:69.
Kukreja RC, Loesser KE, Kearns AA, Naseem SA, Hess MJ. Protective effects of histidine during ischemia reperfusion in isolated perfused rat hearts. Am J Physiol. 1993;264:H1370-H1381.
Murata S, Rosenblum WI, Shimizu T, Nelson GH. Delayed platelet adhesion/aggregation at sites of endothelial injury in mouse cerebral arterioles after transient elevations of blood pressure and shear. Stroke. 1995;26:650-654.
With use of nitroblue tetrazolium reduction as an index of superoxide anion generation, it has been previously observed that oxygen free radicals are generated after cerebral ischemia/reperfusion in the newborn pig and adult cat.1R 2R Moreover, it has been well documented that oxygen radicals inactivate the EDRF from ACh.3R 4R ACh-induced dilation is reversed to constriction after cerebral ischemia/reperfusion.2R Because the pretreatment with oxygen radical scavengers partially restores ACh dilation after the above injury, it has been suggested that the production of such radicals contributes to altered cerebral responsiveness to ACh after ischemia/reperfusion.2R
Results of the present study show that a protocol of 10 minutes of ischemia plus 10 minutes of reperfusion results in an impairment of mouse pial arteriolar dilation to ACh, while responses to BK and SNP were unaffected. Provocative new data suggest that this impairment does not originate from hypoxia, extracellular reactive oxygen species, or singlet oxygen, since it was observed to occur in the presence of hyperoxia, SOD and catalase, or histidine, respectively. While reasons for differences between the present and previous observations are uncertain, several possibilities should be considered. First, SOD and catalase are rather large substances that would have somewhat restricted access to intracellular compartments where free radicals are formed. Therefore, the response to ACh may be inhibited by a source of superoxide or peroxide inaccessible to these scavengers. Second, the lack of effect of a hyperoxic suffusion on the impairment of the relaxation to ACh by ischemia/reperfusion could be due to the accumulation of a tissue metabolite formed during ischemia. Although these and other explanations will need to be considered in future studies, the findings of the present study may have important implications for the maintenance of cerebral perfusion after cerebrovascular trauma.
Selected Abbreviations and Acronyms
|EDRF||=||endothelium-dependent relaxing factor|
Armstead WM, Mirro R, Busija DW, Leffler CW. Postischemic generation of superoxide anion by newborn pig brain. Am J Physiol.. 1989;255:H401-H403.
Nelson CW, Wei EP, Povlishock JT, Kontos HA, Moskowitz MA. Oxygen radicals in cerebral ischemia. Am J Physiol.. 1992;263:H1356-H1362.
Kontos HA, Wei EP, Povlishock JT, Kukreja RC, Hess ML. Inhibition by arachidonate of cerebral arteriolar dilation from acetylcholine. Am J Physiol.. 1989;256:H665-H671.