Journal of Cerebral Blood Flow and Metabolism 20:21-27 © 2000 The International Society for Cerebral Blood Flow and Metabolism Published by Lippincott Williams & Wilkins, Inc., Philadelphia

Magnetic Resonance Imaging Indexes of Therapeutic Efficacy

of Recombinant Tissue Plasminogen Activator Treatment of Rat

at 1 and 4 Hours After Embolic Stroke

*:j:Quan Jiang, *Rui Lan Zhang, *Zheng Gang Zhang, *:j:Jarnes R. Ewing, *Ping Jiang,

tGeorge W. Divine, *:j:Robert A. Knight, and *:j:Michael Chopp

Departments of *Neurology and tBiostatistics and Research Epidemiology, Henry Ford Health Sciences Center. Detroit, and

:f:Department of Physics, Oakland University, Rochester, Michigan, U.S.A.

Summary: With use of magnetic resonance imaging (MRI), the effects of early and delayed treatment of embolic stroke in rat with recombinant tissue plasminogen activator (rt-PA) were investigated, Rats with embolic stroke were treated with rt-PA at 1 (n = 9) or 4 (n = 7) hours after stroke onset or were untreated (n = 15). Diffusion-weighted imaging, perfusion­weighted imaging, and T2-weighted imaging were performed before and after embolization from 1 hour to 7 days. No sig­nificant differences were detected in the relati ve areas with low cerebral blood flow (CBF), apparent diffusion coefficient of water (ADCw), and T2 between the 4-hour treated group and the untreated group. Significant decreases in the average rela­tive areas with low CBF were detected in the I-hour treated

Cerebral artery thromboembolism is a major cause of human cerebral infarction. Studies using recombinant tis­sue plasminogen activator (rt-PA) have demonstrated a significant improvement of neurological outcome in pa­tients with ischemic stroke, although the risk of hemor­rhagic transformation increased in patients receiving rt­PA treatment (NINDS, 1995). Diagnostic tools to predict and characterize the efficacy and safety of thrombolysis will greatly improve the management of stroke patients. Diffusion-weighted imaging (DWI), perfusion-weighted imaging, and T2-weighted magnetic resonance imaging (MRI) have been used to characterize the efficacy of

Received April 21, 1999; final revision received August 16, 1999; accepted August 17, 1999.

Supported by National Institute of Neurological Disorders and Stroke grants POI NS23393, ROI NS33627, and ROI NS38292.

Address correspondence and reprint requests to Dr. Michael Chopp, PhD, Neurology Department, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202, U.S.A.

Abbreviations used: ADCw, apparent diffusion coefficient of water; CBF, cerebral blood flow; DWI, diffusion-weighted imaging; MRI, magnetic resonance imaging; rt-PA, recombinant tissue plasminogen activator.

21

group from 4 to 48 hours after embolization as compared with the untreated group. The increase in T2 in the I-hour treated group was significantly lower than in the untreated and 4-hour treated groups. A significant increase in ADCw was detected in the I-hour treated group at 3 and 24 hours after embolization as compared with the untreated and 4-hour treated groups. Sec­ondary embolization was detected by both MRI and laser scan­ning confocal microscopy. The data suggest that MRI can de­tect the efficacy of rt-PA treatment and secondary ischemic damage. Key Words: Diffusion-Perfusion-Magnetic reso­nance imaging-ThromboJysis-Therapeutic window­Cerebral ischemia.

acute rt-P A intervention after experimental stroke (Rother et aI., 1996; Jiang et ai., 1998). The values for cerebral blood flow (CBF) and apparent diffusion coef­ficient of water (ADCw) obtained 1 hour after injection of rt -P A correlate with histological outcome in the tissue, and the beneficial effect of early rt-PA treatment of em­bolic stroke is reflected in an increase in CBF and ADCw (Jiang et aI., 1998).

Therapeutic intervention with rt -PA is an important breakthrough in the treatment of acute ischemic stroke (NINDS, 1995). However, the majority of stroke patients currently do not present within the required 3-hour time window for rt-PA intervention and so cannot be treated (Barsan et ai. , 1993). Therefore, it is important to inves­tigate the physiological and biophysical effects of throm­bolytic therapy at various durations of ischemia with the ultimate goal of extending the therapeutic window of rt-PA treatment. Previous studies have demonstrated that treatment with rt-PA alone improves neurological defi­cits and reduces mean infarct volume if it is administered at 1 and 2 hours but not at 4 hours after onset of embolic stroke (Zhang et aI. , 1999a). However, employing a

22 Q. JIANG ET AL.

combination of adhesion molecule antibody and rt -P A treatment at 4 hours after stroke reduces infarct volume. In the present study, we investigated the physiological and biophysical effects of early (l hour) and delayed (4 hours) rt-PA intervention by means of MRI and histopa­thology in a rat model of embolic stroke.

MATERIALS AND METHODS

All experimental procedures were approved by the Care of Experimental Animals Committee of Henry Ford Hospital.

Three groups of male Wi star rats weighing 300 to 450 g (n = 3 1 ) subjected to embolic stroke were investigated: untreated animals (n = 1 5), early-treated animals in which rt-PA was administered I hour after embolization (n = 9), and delay­treated animals in which rt-PA was administered 4 hours after embolization (n = 7). The method to produce embolic stroke and rt-PA treatment in this study was identical to that previ­ously described (Zhang et aI., 1 997; Jiang et aI., 1998). In brief, an aged white clot (prepared 24 hours before ischemia) was slowly injected into the internal carotid artery to block the middle cerebral artery and to cause ischemic stroke (Zhang et aI., 1 997).

Magnetic resonance imaging measurements were performed using a 7 T, 20-cm-bore superconducting magnet (Magnex Sci­entific, Abingdon, U.K.) interfaced to a console (SMIS, Surrey, England). A 1 2-cm-bore actively shielded gradient coil set, capable of producing magnetic field gradients up to 20 G/cm, and a birdcage radiofrequency coil were used. Stereotaxic ear bars were used to minimize movement during the imaging pro­cedure. During MRI measurements, anesthesia was maintained using a gas mixture of N 20 (69%), O2 (30%), and halothane (0.75 to 1 %). Rectal temperature was kept constant using a feedback-controlled water bath. A modified fast low angle shot (FLASH) imaging sequence was employed for reproducible positioning of the animal in the magnet at each MRI session (Knight et aI., 1 99 1 ; Jiang et aI., 1 -(93). The MRI measurements were performed before ischemia, repeatedly for 4 to 5 hours (for untreated and I-hour treated groups) or 7 hours (for 4-hour treated group) after onset of embolization, and at 24, 48, and 1 68 hours after onset of embolization (for all three groups). Methods to set up and perform DWI, perfusion-weighted im­aging, and T2-weighted imaging measurements as well as the analysis of MRI data employed in this study are identical to those previously described (Jiang et aI., 1 998).

Laser scanning confocal microscopy was used to measure cerebral plasma perfusion volume and to identify distribution of clot materials in an additional two animals (one untreated and one treated at I hour) using a double-labeled plasma flow method (Zhang et aI., I 999c). The embolus was labeled by mixing Evans blue (2% solution in saline, 0.2 mL/ 100 g of body wt; Sigma Chemicals, St. Louis, MO, U.S.A.) with blood. Animals were administered (intravenously) I mL of fluorescein isothiocyanate/dextran (50 mg/mL, 2 x 106 molecular weight; Sigma) immediately after obtaining the last MR image at 4 to 5 hours after embolization, and fluorescein isothiocyanatel dextran was allowed to circulate for 1 minute. The animals were decapitated, and the brains were rapidly removed and placed in 4% paraformaldehyde for 24 hours. Vibratome coro­nal sections ( 1 50 �m) were analyzed with a Bio-Rad MRC 1 024 laser scanning confocal microscope mounted onto a Zeiss microscope (Bio-Rad, Cambridge, MA, U.S.A.). A lOx objec­tive or a 40x oil immersion objective was used for data acqui­sition. Green (fluorescein isothiocyanate/dextran) and red

J Cereb Blood Flow Metab. Vol. 20, No. I, 2000

(Evans blue) fluorochromes on the sections were excited by the laser beam at 488 and 568 nm, respectively, and emissions were acquired with photomultiplier tubes through 522- and 605-nm emission filters, respectively. Histological processing and measurements of brain tissue employed in the current study were identical to those previously described (Jiang et aI., 1 998).

Statistics Observations are summarized as means ± SE. The data

analysis was carried out using paired t tests for ti me effects and by two-sample t tests for rt-PA effects. As the effect of rt-PA treatment of thromboembolic stroke is most evident at 24 and 48 hours (Zhang et aI., 1 997; Jiang et aI., 1 998) and we have the largest sample size at 24 hours, multiple-comparison issues with respect to rt-PA effects were addressed by considering the 24-hour comparison as the primary test of an rt-PA effect. Unadjusted P values for the secondary tests at other times are also presented.

For tests of time effects (changes from baseline for CBF and ADCw and for normal versus ischemic hemisphere for T2), Bonferroni-adjusted P values were used. There were eight com­parisons for CBF and T2 and seven for ADCw' For these tests, P values of 0.05/8 = 0.0065 and 0.0517 = 0.0071 , respec­tively, were considered significant.

RESULTS

All physiological parameters after embolization were within the normal range. Figure 1 shows temporal pro­files of the relative areas with low CBF (relative area containing CBF below 50 mLilOO g/min to the area of the ipsilateral hemisphere within the reference coronal section) in the ipsilateral hemisphere before and after embolization. As a result of embolization, the relative

LL III o

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C 1 2 3 4 5 6 7 8 24 48 Time (hours)

168

FIG. 1. Average relative area with low cerebral blood flow (CBF; relative area containing CBF below 50 mU100 g/min to the area of the ipsilateral hemisphere) in the ipsilateral hemisphere of treated and nontreated animals plotted as a function of time after embolization. As a result of embolization, the relative areas with low CBF in the ipsilateral hemisphere increased (P < 0.01) from preischemic control values in all three groups. The decrease in the mean values of the relative areas with low CBF was greater in the 1-hour treated group than in the untreated group (4 hours, P = 0.008; 5 hours, P = 0.006; 24 hours, P < 0.001; 48 hours, P = 0.025) and 4-hour treated groups (24 hours, P < 0.026; 48 hours, P = 0.048) after embolization. No statistically significant differences were detected in the areas with low CBF between untreated and 4-hour treated groups.

MRI INDEXES OF EFFICACY OF rt-PA TREATMENT 23

FIG. 2. Diffusion-weighted images (b = 800) of coronal sections without (A) and with 1-hour (8) and 4-hour (C) recombinant tissue plasminogen activator treatment show the evolution of the diffusion-weighted hyperintensity (left-hand side of image), obtained before and 1 to 48 hours after embolization. The hematoxylin and eosin-stained 6.0-�m-thick coronal sections of untreated (A, 168 h_histology), Hour treated (8, 168 h_histology), and 4-hour treated (C, 168 h_histology) animals subjected to diffusion-weighted imaging were obtained 1 week after embolization. The damaged areas in the 1-hour treated animal were redistributed from ipsilateral caudate putamen and insular cortex during acute ischemia to smaller areas in the cortex at chronic time pOints (24 and 48 hours) compared with the larger damaged area in the untreated and 4-hour treated animals that encompassed the caudate putamen and the parietal and insular cortical areas.

FIG. 3. A representative rat killed at 4 hours after 1-hour recombinant tissue plasminogen activator treat­ment investigated by laser scanning confocal microscopy and histology. (A) A segment of Evans blue­stained clot is lodged downstream of the origin of the middle cerebral artery (blue). (8) A coronal section shows an Evans blue-stained clot (arrow). Also shown (C and 0) are images obtained from laser scan­ning confocal microscopy of the co-ronal section in 8. (C) The xy pro- C jection through the stack of 20 opti-cal sections (5-�m-thick section) is shown. The clot appears red (ar-row) and the perfused cerebral vas­culature green (an absence of vas-cular perfusion was evident around the clot). (0) A high-magnification image from C (arrowhead) shows fragments of the parent clot (red) blocking cerebral plasma perfusion (green).

B

J Cereb Blood Flow Metab. Vol. 20, No. 1, 2000

24 Q. JIANG ET AL.

areas with low CBF in the ipsilateral hemisphere in­creased (P < 0.01) from preischemic control values in all three groups. In the untreated and 4-hour treated groups, relative areas with low CBF in the ischemic hemisphere remained large up to 24 hours. No statistically significant differences were detected in the relative areas with low CBF between the untreated and 4-hour treated groups, although the relative areas with low CBF in the 4-hour treated group decreased slightly compared with the un­treated group. The areas with low CBF in the I-hour treated group gradually decreased after administration of rt-PA. Significant differences in the relative areas with low CBF were detected between the I-hour treated and the untreated and 4-hour treated groups at 4 hours (with untreated, P = 0.008), 5 hours (with untreated, P = 0.006),24 hours (with untreated, P < 0.001; with 4-hour treated, P = 0.006), and 48 hours (with untreated, P = 0.025; with 4-hour treated, P = 0.048) after embolization.

Figure 2 presents DWI results (b = 800 s/mm2) ob­tained from 1 to 48 hours after injection of the embolus. A hyperintensity is apparent in the DWI of the ipsilateral hemisphere in all three animals after embolization. In the untreated and 4-hour treated animals, a DWI hyperinten­sity is apparent in the ipsilateral caudate putamen and insular cortex after the onset of ischemia; image contrast and the area of hyperintensity increased as ischemia pro­gressed and were maximal at 24 to 48 hours. In the I-hour treated animals, changes of DWI hyperintensity were variable after injection of rt-P A. One I-hour treated animal exhibited a pattern and temporal profile of DWI hyperintensity similar to that for the control animals, whereas three I-hour treated animals exhibited an early disappearance of DWI hyperintensity (within 24 hours). The remaining I-hour treated animals exhibited a slower increase in image contrast and area of hyperintensity than the control untreated animals. In the I-hour treated animals, four of nine animals had redistributed lesion locations from the ipsilateral caudate putamen and insu­lar cortex during acute ischemia to smaller areas in the cortex at chronic time points (24 and 48 hours). In the 4-hour treated animals, two of seven animals exhibited a reduction of hyperintense areas after rt -P A treatment, and the remaining animals exhibited a profile of DWI hyperintensity similar to that of the untreated animals.

Figure 2 shows the corresponding hematoxylin and eosin-stained coronal sections labeled as 168 h_histol­ogy. The damaged area in the hematoxylin and eosin­stained section from untreated and treated animals cor­responds closely with the hyperintense area in the DWI obtained 24 hours after embolization. The representative untreated animal exhibited a lesion area incorporating putamen and parietal and insular cortex, whereas the rep­resentative I-hour treated animal exhibited a small lesion area localized to the parietal cortex. The I-hour rt-PA­treated animals exhibited smaller normalized areas (P =

J Cereb Blood Flow Metab. Vol. 20. No.1, 2000

0.04) and volumes (P = 0.01) of ischemic damage (34 ± 9%, percent of the lesion area to the area of the contra­lateral hemisphere within the reference coronal section; 20 ± 11 %, percent of the lesion volume to the volume of the contralateral hemisphere) than those in the untreated animals (65 ± 20%, percent of the lesion area to the area of the contralateral hemisphere within the reference co­ronal section; 43 ± 14%, percent of the lesion volume to the volume of the contralateral hemisphere). The 4-hour rt-PA-treated animals exhibited intermediate areas (63 ± 7%, relative percent of the lesion area) and volumes (36 ± 8%, relative percent of the lesion volume) of ischemic damage.

As illustrated in Fig. 2b, redistribution of lesioned ar­eas was detected by DWI in 44% of I-hour rt-PA-treated animals. The newly appearing lesions may be caused by incompletely dissolved clots moving downstream to oc­clude small vessels. To confirm this, laser scanning con­focal microscopy was performed to identify the frag­ments of original clot and cerebral plasma perfusion vol­ume. Figure 3 shows the results from a rat killed at 4 hours after I-hour rt-PA treatment. A segment of Evans blue-stained clot lodged downstream of the origin of the middle cerebral artery (Fig. 3A, blue). A coronal section shows the Evans blue-stained clot (Fig. 3B, arrow). Fig­ures 3C and D comprises images obtained from laser scanning confocal microscopy of the coronal section in Fig. 3B. Figure 3C shows the xy projection through the stack of 20 optical sections (5-f.Lm-thick section). The clot appears red (arrow) and the perfused cerebral vas­culature green (an absence of vascular perfusion was evident around the clot). Figure 3D is a high-magni­fication image from Fig. 3C (arrowhead) and shows frag­ments of the parent clot (red) blocking cerebral plasma perfusion (green).

Figure 4 presents the temporal profiles of normalized ADCw (ADCw values for ischemic damaged tissue nor­malized to preischemic control tissue) for a region of interest localized to the center of the DWI abnormality in the ipsilateral hemisphere in all three groups. After em­bolization, the ADCw values in the ischemic striatum significantly (P < 0.001) decreased from preischernic values in all three groups. In the untreated and 4-hour treated groups, ADCw in the ischemic striatum remained below preischemic ADCw values up to 24 hours. In the 4-hour rt-PA-treated group, however, ADCw transiently increased after rt-PA injection and then returned to low levels as in the untreated group at 24 hours after middle cerebral artery occlusion. There were no significant dif­ferences in the relative ADCw between the 4-hour treated group and the untreated group. In the I-hour treated group, ADCw gradually increased after injection of rt­PA. Higher mean ADCw values were present from 1 to 24 hours after administration of rt-PA in the I-hour treated group compared with the untreated and 4-hour

MRI INDEXES OF EFFICACY OF rt-PA TREATMENT 25

1.6

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FIG. 4. Relative apparent diffusion coefficient of water (ADCw) in the lesion under untreated and recombinant tissuse plasminogen activator-treated conditions plotted as a function of time after embolization. The relative ADCw values in the ischemic area sig­nificantly (P < 0.001) decreased from preischemic values after embolization in all three groups. Significant differences were de­tected after embolization between the 1-hour treated and un­treated (3 hours, P = 0.039; 4 hours, P = 0.017; 24 hours, P < 0.023) and 4-hour treated (3 hours, P < 0.001; 24 hours, P < 0.025) groups. No statistically significant differences were de­tected in the relative ADCw between untreated and 4-hour treated groups.

treated groups. Statistically significant differences were detected in normalized ADCw values between the I-hour treated with untreated and 4-hour treated groups at 3 hours (with untreated, P = 0.039; with 4-hour treated, P

< 0.001), 4 hours (with untreated, P = 0.017), and 24 hours (with untreated, P = 0.023; with 4-hour treated, P

< 0.025) after embolization. Figure 5 shows the postembolic temporal profiles of

.1T2 in the lesion (the same region of interest as in ADCw measurements) in the rt-PA-treated and untreated groups. A small increase in Ll T2 was detected within 4 hours; .1 T2 was maximized at 24 hours in the untreated group (P < 0.001) and 4-hour rt-PA-treated group (P < 0.004) and at 48 hours in the I-hour treated group (P = 0.006) and then declined at 1 week after middle cerebral artery occlusion. However, LlT2 at 1 week remained elevated relative to baseline in all three groups. The I-hour rt-PA­treated group had a lower Ll T2 than the control and 4-hour treated groups at 24 hours (untreated group, P =

0.003; 4-hour treated group, P = 0.020) and 48 hours (4-hour treated group, P = 0.009) after embolization. There were no significant differences in Ll T2 between untreated and 4-hour rt-PA-treated groups.

DISCUSSION

In this study, we used MRI to investigate 1- and 4-hour rt-PA intervention after embolic stroke. Our data suggest that delayed rt-PA treatment (4 hours) does not significantly alter the MRI parameters of ADCw• CBF, and T2 or reduce ischemic tissue damage as compared

with those in control untreated animals. However, early rt-PA treatment (I-hour) reduces ischemic tissue dam­age, as reflected in a rapid increase in ADCw, a reduction of areas with low CBF, and a reduction of the increase in T2.

Our data indicate that tissue perfusion is not signifi­cantly increased when rt-PA treatment is instituted at 4 hours after embolic stroke. We have not performed MRI angiography, and we do not know the status of the clot after thrombolysis. We also cannot exclude the possibil­ity that the clot was effectively lysed but that microvas­cular plugging occurred downstream of the clot from emboli, from leukocyte adhesion, or from fibrin deposi­tion (del Zoppo et aI., 1991). In addition, erythrocyte stasis, reduction of local blood perfusion pressure, in­creased blood viscosity in vessels, and narrowed lumina due to perivascular glial swelling (Ames et aI., 1968; Ginsberg and Myers, 1972; del Zoppo et aI., 1991) may also account for the hypoperfusion after 4-hour rt-PA treatment. The cause of low tissue perfusion after 4 hours of rt -P A treatment requires further investigation.

The indexes of MRI measurements of the efficacy of rt-PA intervention in the animal study may have poten­tial for the management of human stroke. Our data indi­cate that rapid recanalization of an embolic ally occluded brain artery leads to a reduced lesion. In recent study of rt-PA treatment in patients, Heiss et aI. (1998) demon­strated that the percentage of CBF changes measured using e50]H20 positron emission tomography predicted the degree of clinical improvement achieved within 3

24 ---------------c7.,0------------------, _ , - treated at 1 h -.to- nontreated #.

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-4 C 1 2 3 4 5 6 7 24 48 168

Time (hours)

FIG. 5. The temporal profiles of � T2 (the difference of T2 values in homologous regions of the ipsilateral and contralateral hemi­spheres) in the lesion before and after embolization in all three treated and untreated groups. A small increase in � T2 was de­tected within 4 hours, and �T2 was maximized at 24 hours in untreated (P < 0.001) and 4-hour recombinant tissue plasmino­gen activator (rt-PA)-treated (P < 0.004) groups and at 48 hours in the 1-hour treated group (P < 0.01) and then declined at 1 week after embolization. The 1-hour rt-PA-treated group had a lower � T2 than the untreated and 4-hour treated groups at 24 hours (untreated group, P= 0.003; 4-hour treated group, P= 0.020) and 48 hours (4-hour treated group, P = 0.009) after embolization. There were no significant differences in �T2 between untreated and 4-hour rt-PA-treated groups.

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26 Q. JIANG ET AL.

weeks when patients were treated within the 3-hour therapeutic window of rt-PA. Studies by many investi­gators (Sundt and WaItt, 1971; Pulsinelli et aI., 1982; Garcia et aI., 1993) using a variety of animal models have demonstrated a critical period of 2 to 7 hours after ischemia in which reversible cell damage transforms to irreversible damage. The time to salvage tissue is related to the degree and duration of CBF reduction, the specific tissue, and the animal species. The efficacy of reperfu­sion in reducing cell damage strongly depends on the rapidity of reperfusion, as survival of ischemic tissue decreases exponentially with time (Astrup et aI., 1981; Jones et aI., 1981; Heiss and Rosner, 1983). Thus, in our model of embolic stroke in the rat, early restoration of CBF may be a good indicator of the therapeutic efficacy of rt-PA intervention.

The duration of ischemia and residual CBF are re­flected in the evolution of changes of other MRI­measurable biophysical parameters such as ADCw and T2. Busza et ai. (1992) have reported that the decrease in ADCw is related to a threshold of CBF. However, ADCw is a function of a number of variables (Cooper et aI., 1970; Tanner, 1978; LeBihan et aI., 1989; Moseley et aI., 1990; Benveniste et aI., 1992; Helpern et aI., 1992), not only CBF. Benveniste et ai. (1992) demonstrated that ADCw declines following direct microdialysis treatment of rat brain with ouabain, a known inhibitor of Na+,K+­ATPase, which acts without changing CBF. Therefore, in the acute ischemic phase, the decline in ADCw may be related to energy failure. Mintorovitch et ai. (1994) dem­onstrated a significant decline in Na+,K+-ATPase activity at 30 and 60 minutes after middle cerebral artery occlu­sion in the rat. Rapid restoration of CBF induces an early recovery of ADCw, which may indicate the efficacy of rt-PA intervention, as has been demonstrated after cere­bral venous embolization and rt-PA treatment of rat (Rother et aI., 1996). T2 changes in cerebral ischemic tissue occur much later than changes in ADCw and are related to tissue edema (Knight et aI., 1994; Jiang et aI., 1997). Previous studies have demonstrated that early re­canalization reduces infarction and edema (Overgaard, 1994). Therefore, reduction of the increase in T2 with I-hour rt -P A treatment reflects the therapeutic efficacy of the thrombolytic intervention.

The efficacy of thrombolysis depends on not only the arterial recanalization but also the secondary effects of thrombolysis such as redistribution of clot material. In this study, our data demonstrate that MRI and laser scan­ning confocal microscopy can detect the secondary isch­emic damage caused by secondary embolization. Sec­ondary ischemic events have long been recognized (Dinsmore et aI., 1986), and progressing ischemia ex­tends to previously spared cerebral regions (Haley et aI., 1988). However, it is difficult to identify and monitor the processes that cause secondary ischemia in the patient.

J Cereb Blood Flow Metab, Vol. 20, No. 1, 2000

Early diagnosis of secondary ischemia is of clinical in­terest. Our study demonstrates that MRI and laser scan­ning confocal microscopy techniques and the new model of embolic stroke may be useful to investigate secondary ischemic damage.

Extending the time window of thrombolytic interven­tion would greatly benefit the management of stroke pa­tients. A potentially efficacious treatment strategy to ex­tend the window for thrombolysis would be to employ a combination thrombolytic and neuroprotective therapy (Bowes et aI., 1995). A combination of rt-PA and anti­leukocyte adhesion molecule antibody (anti-CDI8, anti­ICAM-l) treatment after 4-hour embolic stroke in rat significantly reduces the infarct volume as compared with rt-PA-treated animals (Zhang et aI., 1999a,b). In the present study, a reduction in the mean relative areas with low CBF «50 mL/lOO g/min) and a transiently elevated mean relative ADCw were detected after rt-PA treatment in the 4-hour rt-PA-treated group compared with the un­treated group, although no improvement in the ischemic outcome was present. We speculate that the CBF and ADCw profiles may provide an index to the window of opportunity for using combination therapy after acute stroke.

In summary, our data indicate that select MRI mea­surements can distinguish between thrombolytic therapy with rt-PA performed within (1 hour) and outside (4 hours) of the therapeutic window. Early changes in tissue perfusion and ADCw values identify later therapeutic benefit. Secondary ischemic events likely caused by em­boli derived from the lysed clot may contribute to the ischemic lesion.

Acknowledgments: The authors thank Upul Senaratne and Jonathan Prostak for data analysis and Denice Bliesath for manuscript preparation.

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J Cereb Blood Flow Metab, Vol. 20. No.1, 2000