Invited Symposium: Stroke/Cerebral Vasospasm



Materials & Methods


Discussion & Conclusion



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Middle Cerebral Artery Function After Ischemia, Reperfusion and Thrombolytic Therapy

Contact Person: Marilyn J. Cipolla (cipollam@ohsu.edu)


The cerebral circulation is a unique vascular bed in that the large extracranial vessels and intracranial pial vessels account for approximately half of total cerebrovascular resistance1. In addition to possessing considerable tone, these large arteries are also highly responsive to changes in arterial pressure and in this way contribute to autoregulation of cerebral blood flow (CBF)1,2. This unusually prominent regulatory role of the large cerebral arteries makes them an integral component of overall cerebral hemostasis.

While there are several influences that determine vessel caliber (e.g., endothelial, neurogenic and metabolic), autoregulation of CBF is primarily due to the myogenic behavior of the cerebral vascular smooth muscle (VSM) that constrict in response to elevated pressure and dilate in response to decreased pressure3-5. The innate myogenic behavior of the VSM is also crucial for establishment of an appropriate cerebrovascular resistance (CVR), which serves to protect downstream arterioles and capillaries in the face of changing perfusion pressures and to maintain tissue perfusion when blood pressure falls4,5. Therefore, altered diameter regulation under pathological conditions could have significant downstream effects, including altered resistance and shear stress, that may affect endothelial cell secretions known to influence tone, vascular permeability and coagulation properties of the whole circulation.

The large middle cerebral artery (MCA) is the most commonly affected artery in clinical ischemic stroke, the occlusion of which produces a large infarction6. Due to its involvement in stroke, this artery is frequently targeted for thrombolytic therapy with tissue plasminogen activator (tPA), a promising treatment for ischemic stroke7. The principal risk of therapeutic plasminogen activator delivery, whether given intra-arterially at the site of occlusion or intravenously, is edema formation and intracerebral hemorrhage following recanalization8-12. One possible mechanism of damage resulting from thrombolysis is an effect of the thrombolytic agents themselves on the function of the cerebral vessels. In our own studies, posterior cerebral arteries that were perfused with 100g/mL tPA had altered diameter regulation that resulted in a significantly decreased pressure at which forced dilatation occurred13. Forced dilatation of cerebral vessels in vivo has been shown to result in blood-brain barrier disruption and edema formation due to the loss of upstream vascular resistance, resulting in increased intravascular pressure on the microcirculation14,15.

In the present study, we hypothesized that tPA may have a direct effect on the cerebral vessels themselves that may be promoting damaging cerebrovascular events such as edema and hemorrhagic transformation. Since both edema formation and hemorrhagic transformation have been reported following tPA treatment of acute thrombotic stroke8,16-18, we investigated the effect of tPA exposure on MCAs that were ischemic for 2 hours. In previous studies, we found that under normal, nonischemic conditions, the MCA possesses considerable tone and responds myogenically to changes in intravascular pressure 19. These myogenic responses were fairly well-preserved in arteries that were ischemic for 2 hours and reperfused for 1-2 minutes19. The present study investigated whether the presence of tPA could affect diameter regulation of ischemic arteries in response to pressure (i.e., myogenic reactivity and tone).

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Materials and Methods

Middle Cerebral Artery Occlusion (MCAO) The focal cerebral ischemia model that we used was a modification of the MCAO model described by Zea Longa et. al.20 and described in detail elsewhere19,21. This model is currently used in numerous laboratories for the study of ischemic brain injury and involves inserting a nylon suture through the right internal carotid artery up to the origin of the MCA in order to block blood flow through the MCA and to block collateral blood flow through all branches of the external carotid artery and all extracranial branches of the internal carotid artery. A detailed description is given below.

The rats were anesthetized by inhalation of 1.5% halothane in a 6-4 mixture of nitrous oxide/oxygen, spontaneously inhaled through a mask. The right common carotid artery was exposed, and the external carotid artery and its branches isolated and coagulated. A 4-0 nylon suture coated with silicon was inserted into the internal carotid artery through the external carotid artery stump and advanced to the anterior cerebral artery, thus occluding the MCA. Body temperature was monitored using a rectal probe and maintained at 370.5C with a heating pad.

Preparation of Arterial Segments and Pressurized Arteriograph System The MCA from the occluded (right) side of the brain was carefully dissected, cleared of extraneous connective tissue and placed in the arteriograph chamber. Dissected arteries were mounted on two glass microcannulas suspended above an optical window within the chamber, perfused with PSS and secured with two strands of nylon thread (diameter=10m) on both the proximal and distal cannulas. For these experiments, the distal cannula was closed off so there was no flow through the vessels. The arteriograph system (Living Systems Instrumentation, Burlington, VT) consisted of a 20mL chamber with inlet and outlet ports for suffusion of PSS and drugs from a 50mL reservoir. The PSS was continually recirculated and pumped through a heat exchanger to warm the PSS to 37C before entering the arteriograph chamber and was aerated with a gas mixture of 5%CO2-10%O2-85%N2 to maintain a constant pH of 7.4 0.05.

Transmural pressure was measured and controlled via a servo system that consisted of an in-line pressure transducer, miniature peristaltic pump and controller connected to the proximal cannula. The arteriograph chamber containing the mounted arteries was placed on an inverted microscope with an attached video camera and monitor to allow for viewing and electronic measurement of lumen diameter. Lumen diameter was measured by the video scan line which detected the optical contrast of the vessel walls on the video monitor and generated a voltage ramp within the video dimension analyzer (VDA) which was proportional to diameter22. The output of the VDA and pressure controller was sent to an IBM compatible computer via a serial data acquisition system (DATAQ, Akron, OH) for visualization of dynamic responses of diameter and TMP, similar to a chart recorder.

Experimental Protocol Arteries were dissected from either nonischemic control (n=8) or after 2 hours of ischemia and perfused with 400g/mL tPA. Isolated arteries were equilibrated in the arteriograph chamber for 1 hour at TMP=75mmHg, after which pressure was increased to 125mmHg. Diameter was continuously recorded and measured at each pressure, once stable, approximately 10 minutes. Following diameter measurement, a single concentration of papaverine (0.1mM) was added to the bath to induce full relaxation and diameter recorded. The amount of tone each artery type possessed was then calculated at each pressure.

Drugs and SolutionsThe perfusate and superfusate for all experiments consisted of a bicarbonate-based phosphate buffer (Ringer's PSS), the ionic composition was (mM): NaCl 119.0, NaCHO3 24.0, KCl 4.7, KH2PO4 1.18, MgSO47H2O 1.17, CaCl2 1.6, EDTA 0.026, and glucose 5.5. PSS was made each week and stored without glucose at 4C. Glucose was added to the PSS prior to each experiment. t-PA (Activase) was a generous gift from Genentech, Inc. and was mixed fresh in PSS in the appropriate concentration and perfused through the MCA once mounted in the arteriograph chamber.

Data Calculations and Statistical Analysis Spontaneous arterial tone was calculated as a percent decrease in diameter from the fully relaxed diameter in papaverine at each TMP by the equation: (1-(Diametertone/Diameterpapav))X100%; where Diametertone=diameter of vessels with tone and Diameterpapav= diameter in papaverine. Differences in diameter and % tone between artery types at the two pressures, as well as the slope of diameter vs. pressure was determined using one-way analysis of variance. Differences in the amount of tone at 75 vs. 125mmHg was determined using analysis of variance with repeated measures. Differences were considered significant at p<0.05.

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A summary of inner diameter and percent tone at 75 and 125mmHg for each artery type is shown in Table 1. Both artery types developed a similar amount of spontaneous tone during equilibration at 75mmHg, and had similar diameters. When pressure was increased to 125mmHg, Control arteries responded myogenically and contracted, decreasing diameter and increasing the amount of tone. In contrast, tPA arteries lacked this myogenic response and dilated to the increased pressure, decreasing the amount of tone. In addition, the slope of the pressure vs. diameter curve for Control arteries was negative (-0.060.01), demonstrating a myogenic response, whereas the slope for tPA arteries was positive (0.860.27), demonstrating a lack of myogenicity.

               75mmHg                        125mmHg
       Diameter         %Tone        Diameter      %Tone 
         (m)                         (m)
Control    232           22           229            26              
           8            3             6           2

tPA        228           24           270**          12**
           15           4             10          3


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Discussion and Conclusion

This study demonstrates that the combination of ischemia and tPA exposure significantly affected myogenic responses of MCAs and impaired diameter regulation in response to increased intravascular pressure. Since previous studies showed that this period of ischemia alone had little effect on myogenic reactivity and tone19, it appears that the presence of tPA in ischemic vessels is a confounding factor that may promote edema formation and hemorrhagic transformation by impairing myogenic responses, a major contributor to autoregulation of CBF3-5.

In normotensive adults, CBF is maintained at approximately 50mL per 100g of brain tissue per minute provided mean arterial blood pressure (MAP) is in the range of 60 to 150mmHg23. Above and below this limit, autoregulation is lost and CBF becomes dependent on MAP in a linear fashion24. Significant tissue damage occurs when autoregulatory mechanisms are lost. For example, at pressures above the autoregulatory limit, the myogenic constriction of VSM is overcome by the excessive intravascular pressure and forced dilatation of cerebral vessels occurs25-29. The loss of myogenic tone decreases CVR and increases CBF, a result that produces blood-brain barrier disruption and edema formation due to increased pressure on the microcirculation26,28,30. These processes may also lead to loss of vascular integrity with potential egress of red blood cells31.

The mechanism by which tPA affects VSM and impairs myogenic responses is not clear from this study. tPA is a serine protease with a high specific activity for converting plasminogen to plasmin through cleavage of a single peptide bond32,33. tPA binds and expresses greater enzymatic activity in the presence of fibrin, which serves to localize and enhance fibrinolytic potential at the sites of fibrin formation33. While the arteries that were exposed to tPA in this study did not have a thrombus, our own studies using transmission electron microscopy have shown significant structural damage to the arterial wall of MCAs that were ischemic for 2 hours, including areas of endothelial denudation and disruption of the internal elastic laminae19. It is possible that this ischemic damage exposes other tPA binding sequences that increases proteolytic activity, such as collagen or other extracellular matrix proteins. In addition, ischemia is known to upregulate endothelial adhesion molecules, such as integrins, which tPA may then bind to and increase or alter its binding and activity34. Although tPA has stringent substrate specificity for plasminogen, a study by Ding et. al. demonstrated that small peptides can mimic determinants that mediate specific proteolysis35. Therefore, it is possible that ischemic damage exposes peptides within the vascular wall that tPA can then bind to and increase its proteolytic activity, thereby affecting either structural or functional components important in mediating myogenic responses.

The effect of tPA on myogenic reactivity and tone may be due to proteolytic damage to the endothelium, which could alter production and release of vasoactive substances. While myogenic responses are inherently smooth muscle mediated, the endothelium, through production and release of vasoactive compounds, has been shown to impair myogenic responses in cerebral arteries36. Alternatively, tPA may have a direct effect on the VSM within the ischemic artery. Preliminary studies with tPA-perfused posterior cerebral arteries showed that the response of cerebral arteries in the presence of tPA (e.g., decreased the pressure of forced dilatation) was similar to that of arteries in the presence of the cytochalasin, a compound known to bind to and inhibit VSM actin polymerization13,29. Since tPA has also been shown to directly bind actin on endothelial cells37, it is possible that the decreased myogenicity in the presence of tPA is due to an effect of this compound on the dynamics of the actin cytoskeleton in either endothelial cells or VSM.

In conclusion, this study demonstrates that exogenous exposure of ischemic MCAs to tPA significantly impairs myogenic reactivity and diminishes myogenic tone. This impairment could have significant consequences during or after reperfusion due to a loss of autoregulation of CBF and diminished CVR. Future studies are needed to determine the mechanism of action of tPA on the arterial wall and the effect of longer time periods of ischemia in combination with tPA exposure on MCA structure and function.

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Cipolla, MJ; Lessov, N; Clark, WM; (1998). Middle Cerebral Artery Function After Ischemia, Reperfusion and Thrombolytic Therapy. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/zhang/cipolla0451/index.html
© 1998 Author(s) Hold Copyright