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 elsewhere^19,21. 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 37±0.5°C 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=10µm) 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 37°C 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 400µg/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 4°C. 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-(Diameter_tone/Diameter_papav))X100%; where Diameter_tone=diameter of vessels with tone and Diameter_papav= 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.

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 (-0.06±0.01), demonstrating a myogenic response, whereas the slope for tPA arteries was positive (0.86±0.27), demonstrating a lack of myogenicity.

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

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

**p<0.01 

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 CBF^3-5.

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 occurs^25-29. 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 microcirculation^26,28,30. 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 bond^32,33. 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 polymerization^13,29. 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.

1. Heistad DD, Kontos HA (1979): In "Handbook of Physiology: The Cardiovascular System III" (RM Berne and N Sperelakis, Eds.), Ch. 5, pp. 137-182. American Physiological Society, Bethesda, MD.
2. Heistad DD, Marcus ML, Abboud FM (1978): Role of large arteries in regulation of cerebral blood flow in dogs. J Clin Invest 62:761-8.
3. Johansson B (1989): Myogenic tone and reactivity: definitions based on muscle physiology. J Hyperten 7:(Suppl 4):S5-S8.
4. Mellander S (1989): Functional aspects of myogenic vascular control. J Hyperten 7:(Suppl 4):S21-S30.
5. Osol G (1995): Mechanotransduction by vascular smooth muscle. J Vasc Res 32:275-292.
6. Mohr JP, Gauthier JC, Hier DB (1992): Middle cerebral artery disease. In: "Stroke: Pathophysiology, Diagnosis, and Management" (HMJ Barnett, BM Stein, JP Mohr, FM Yatsu, eds.), 2nd ed., pp.361-417. Churchill Livingstone, NY.
7. del Zoppo GJ (1994). Antithrombotic therapy in cerebrovascular disease. In Thrombosis and Hemorrhage. (J. Loscalzo and AI Schafer, Eds.) pp.1225-52. Blackwell Scientific, Boston.
8. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group (1995): Tissue plasminogen activator for acute ischemic stroke. NE J Med 333:1581-7.
9. Sasaki O, Takeuchi S, Koike T, Koisumi T, Tanaka R (1995): Fibrinolytic therapy for acute embolic stroke: intravenous, intracarotid, and intra-arterial local approaches. Neurosurg 36:246-53.
10. Hacke W, Kaste M, Fieschi C, et. al. (1995): Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS). J Am Med Assoc 274:1017-25.
11. Brott T (1991): Thrombolytic therapy for stroke. Cerebrovasc Brain Metab Rev 3:91-113.
12. Pessin MS, del Zoppo GJ, Estol CJ (1990): Thrombolytic agents in the treatment of stroke. Clin Neuropharmacol 13:271-89.
13. Cipolla MJ and Porter J (1998): The effect of tissue plasminogen activator on cerebral artery vasoreactivity. Stroke29(1):325.
14. Lassen NA, Agnoli A (1972): Upper limit of autoregulation of cerebral blood flow: On the pathogenesis of hypertensive encephalopathy. Scan J Clin Lab Invest 30:113-115.
15. Johansson B, Li C-L, Olsson Y, Klatzo I (1970): Effect of acute arterial hypertension on the blood-brain barrier to protein tracers. Acta Neuropath 16:117-124.
16. Koudstaal PJ, Stibbe J, Vermeulen M (1988): Fatal ischaemic brain oedema after early thrombolysis with tissue plasminogen activator. Br Med J 297:1571-4.
17. Brott TG, Haley, EC, Levy DE, et. al (1992): Urgent therapy for stroke: Part I. Pilot study of tissue plasminogen activator administered within 90 minutes. Stroke 23:632-40.
18. Haley CE, Levy DE, Brott TG, et al (1992):Urgent therapy for stroke: Part II. Pilot study of tissue plasminogen activator administered within 91-180 minutes from onset. Stroke 23:641-5.
19. Cipolla MJ, McCall AL, Lessov N, Porter JP (1997): Reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats. Stroke 28:176-80.
20. Zea Longa E, Weinstein PR, Carlson S and Cummins R (1989): Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84-91.
21. Clark WM, Lessov N, Lauten JD, Hazel K (1997): Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat. J Mol Neurosci 9:1-6.
22. Wiederhielm CA: Continuous recording of arteriolar dimensions with a television microscope. J Appl Physiol 18:1041-1042,1963.
23. Phillips SJ, Whisnant JP (1992): Hypertension and the brain. Arch Intern Med 152:938-945.
24. Heistad DD, Kontos HA (1979): In "Handbook of Physiology: The Cardiovascular System III" (RM Berne and N Sperelakis, Eds.), Ch. 5, pp. 137-182. American Physiological Society, Bethesda, MD.
25. Lassen NA, Agnoli A (1972): Upper limit of autoregulation of cerebral blood flow: On the pathogenesis of hypertensive encephalopathy. Scan J Clin Lab Invest 30:113-115.
26. Johansson B, Li C-L, Olsson Y, Klatzo I (1970): Effect of acute arterial hypertension on the blood-brain barrier to protein tracers. Acta Neuropath 16:117-124.
27. Kontos HA, Wei EP, Navari RM, Levasseur JE, Rosenblum WI, Patterson JL, Jr. (1978): Responses of cerebral arteries and arterioles to acute hypotension and hypertension. Am J Physiol 234:H371-H383.
28. Kontos HA, Wei EP, Dietrich WE, Navari RM, Povlishock JT, Ghatak NR, Ellis EF, Patterson JL, Jr.(1981): Mechanism of cerebral arteriolar abnormalities after acute hypertension. Am J Physiol 240:H511-H527.
29. Cipolla MJ and Osol G (1998): Vascular smooth muscle actin cytoskeleton in cerebral artery forced dilatation. Stroke 29(6):1223-8.
30. Tamaki K, Sadoshima S, Baumbach GL, Iadecola C, Reis DJ, Heistad DD (1984): Evidence that disruption of the blood-brain barrier precedes reduction in cerebral blood flow in hypertensive encephalopathy. Hypertension 6(Suppl I):I75-I81.
31. Batjer HH (Ed.) (1992): In "Spontaneous Intracerebral hemorrhage," Vol. 3. Saunders, Philadelphia.
32. Francis CW (1995): The fibrinolytic system: normal physiology and pathophysiology. In: Thrombolytic therapy for peripheral vascular disease. (AJ Comerota, Ed). Ch. 3, pp. 25-40, JB Lippincott Co, Philadelphia.
33. Madison EL, Coombs GS, Corey DR (1995): Substrate specificity of tissue-type plasminogen activator. J Biol Chem 270:7558-62.
34. Okada Y, Copeland BR, Hamann GF, Koziol JA, del Zoppo GJ (1996): Integrin alphavbeta3 is expressed in selected microvessels after focal cerebral ischemia. Am J Pathol 149:37-44.
35. Ding L, Coombs GS, Strandberg L, Navre M, Corey DR, Madison E (1995): Origins of the specificity of tissue-type plasminogen activator. Proc Nat'l Acad Sci USA 92:7627-31.
36. Cipolla M, Porter JM, Osol G (1997): High glucose concentrations dilate cerebral arteries and diminish myogenic tone through an endothelial mechanism. Stroke 28:405-11.
37. Dudani AK, Ganz PR (1996): Endothelial cell surface actin serves as a binding site for plasminogen, tissue plasminogen activator and lipoprotein. Br J Haemat 95(1):168-78.