Visualization of Clot Lysis in a Rat Embolic Stroke Model
Application to Comparative Lytic Efficacy
Background and Purpose—The purpose of this study was to develop a novel MRI method for imaging clot lysis in a rat embolic stroke model and to compare tissue plasminogen activator (tPA)-based clot lysis with and without recombinant Annexin-2 (rA2).
Methods—In experiment 1 we used in vitro optimization of clot visualization using multiple MRI contrast agents in concentrations ranging from 5 to 50 μL in 250 μL blood. In experiment 2, we used in vivo characterization of the time course of clot lysis using the clot developed in the previous experiment. Diffusion, perfusion, angiography, and T1-weighted MRI for clot imaging were conducted before and during treatment with vehicle (n=6), tPA (n=8), or rA2 plus tPA (n=8) at multiple time points. Brains were removed for ex vivo clot localization.
Results—Clots created with 25 μL Magnevist were the most stable and provided the highest contrast-to-noise ratio. In the vehicle group, clot length as assessed by T1-weighted imaging correlated with histology (r=0.93). Clot length and cerebral blood flow-derived ischemic lesion volume were significantly smaller than vehicle at 15 minutes after treatment initiation in the rA2 plus tPA group, whereas in the tPA group no significant reduction from vehicle was observed until 30 minutes after treatment initiation. The rA2 plus tPA group had a significantly shorter clot length than the tPA group at 60 and 90 minutes after treatment initiation and significantly smaller cerebral blood flow deficit than the tPA group at 90 minutes after treatment initiation.
Conclusions—We introduce a novel MRI-based clot imaging method for in vivo monitoring of clot lysis. Lytic efficacy of tPA was enhanced by rA2.
Previous studies in experimental stroke models demonstrated that tissue plasminogen activator (tPA) mediated recanalization and subsequent reperfusion leads to reduced final infarct volumes.1,–,3 Currently available MRI methods to determine arterial patency such as time of flight angiography in small animals are sensitive to a number of parameters, including flow, blood longitudinal relaxation time, and sequence parameters.4 Signal intensity is based on a combination of these parameters and may make quantitative analysis of thrombolysis difficult. This might explain why a relationship between recanalization and reperfusion has not been clearly established.5 Direct clot imaging has been proposed, but these techniques typically require a multimodality approach or targeted contrast agents not readily available to investigators.6,–,8 Although in vitro assays are available to test the effects of thrombolytics on blood clots, these systems cannot sufficiently mimic the in vivo environment.9 Direct in vivo MRI-based visualization of the embolus will allow for near real-time quantification of clot dissolution and will provide unique information regarding lytic efficacy of thrombolytic agents.
In this study, we present a novel MRI methodology for clot visualization in an experimental stroke model using a commercially available contrast agent. Further goals of this study were to characterize clot lysis dynamics during thrombolytic therapy and to evaluate the lytic efficacy of tPA with or without recombinant Annexin-2 (rA2), a compound thought to enhance the lytic action of tPA.1
Materials and Methods
This study consisted of 2 experiments. The first (in vitro) experiment involved designing a robust method for modifying standard clots for visualization on MRI and histology. The second (in vivo) experiment characterized the temporal evolution of clot lysis mediated by tPA versus tPA combined with rA2 (rA2 plus tPA) utilizing MRI.
In Vitro Experiment: Experiment 1
Standard clots10 were modified by mixing 250 μL of blood with 4 different contrast agents at volumes ranging from 0 to 50 μL to determine optimal contrast agent type and volume for MRI visualization. The contrast agents used were Magnevist (Berlex Laboratories, Montville, NJ), Combidex (Combidex Threading Tools bv; Nieuwe Waterwegstraat 5, Schiedam, Holland), Omniscan (GE Health Care, Princeton, NJ), and Eovist (Bayer Healthcare Pharmaceuticals, Wayne, NJ). An animal surgeon with experience in embolic stroke modeling inspected the quality of the clots. At the contrast volumes used, Magnevist-infused clots showed consistent stability, whereas the use of the other contrast agents resulted in fragile and unstable clots. Magnevist therefore was used for the in vitro and in vivo experiments.
For in vitro quantification of contrast intensity, Magnevist clots were placed in saline and imaged using the T1-weighted imaging (T1WI) sequence described in the MRI section. The resulting signal intensity of the clot and surrounding saline were measured. The clot enhancement ratio was calculated by dividing the clot signal intensity by the saline signal intensity. Subsequent histological visualization was made possible by incubating the clot in 2% Evans Blue for 24 hours at 4°C.
In Vivo Experiment: Experiment 2
All procedures used in this study were approved by University of Massachusetts Medical School Institutional Animal Care Use Committee. Spontaneously breathing male Wistar rats (n=22; Taconic Farms, Hudson, NY) weighing 318±27 grams were anesthetized with isoflurane (5% for induction, 2% for surgery, 1.2% for maintenance) in room air. PE-50 polyethylene tubing was inserted into the femoral artery for monitoring of mean arterial blood pressure, blood gases, electrolytes, and glucose. Additional tubing was inserted into the femoral vein to allow for intravenous (intravenous) infusion of tPA, rA2 plus tPA, or vehicle. Body temperature was monitored continuously with a rectal probe and maintained at 37.0°C±0.5°C with a thermostatically controlled heating pad. Embolic stroke (ES) was induced in all 22 animals as previously described11 with the modified clot reported in experiment 1. The animals were randomly assigned to either vehicle (n=6), tPA (n=8), or rA2 plus tPA (n=8) treatment. The intravenous infusion of thrombolytics or vehicle was administered at 90 minutes after ES with a 10% bolus and the remainder was infused over 1 hour. The vehicle group received 1 mL of saline, whereas the tPA groups received 10 mg/kg tPA dissolved in either 1 mL of saline or rA2 solution (5 mg/kg). Histidine-tagged rA2 was produced in Escherichia coli from a bacterial expression vector containing full-length human annexin-2 cDNA according to the method previously described.12 Purity of rA2 was confirmed by SDS-PAGE followed by Coomassie blue staining, and its identity was verified by Western blot analysis. The rA2 was finally eluted and kept in elution buffer (20 mmol/L sodium phosphate, 0.5 mol/L NaCl, 500 mmol/L imidazole; pH 7.4). Imaging was performed from 30 to 180 minutes and animals were electively euthanized at 3.5 hours after ES to remove their brains. Brains were imaged using an optical scanner and Evans Blue-stained clots were identified and their lengths were measured.
MRI experiments were performed on a Bruker system utilizing a 4.7-T/400-mm horizontal magnet equipped with Paravision software (Billerica, MA) and a 20-G/cm gradient insert (insert diameter=120 mm, 120 μs rise time). A surface coil (inner diameter=23 mm) was used for brain imaging and an actively decoupled neck coil was used for perfusion labeling.13 T1WI and perfusion-weighted imaging (PWI) were performed at 60, 90, 105, 120, 135, 150, and 180 minutes after ES. Diffusion-weighted imaging (DWI) was performed at 30, 90, and 160 minutes after ES. Time-of-flight MRA was performed at 60 and 150 minutes after ES. PWI, DWI, and MRA were performed as previously described in detail.11 Two contiguous slices with in-plane visualization of the bifurcation of the middle cerebral artery (MCA), anterior cerebral artery (ACA), and internal carotid artery (ICA) were recorded sequentially using a T1WI-centric ordered turbo spin-echo with an echo train length of 4, an echo time of 3.2 ms, a recovery time of 290 ms, a field of view of 25.6×25.6 mm, a slice thickness of 750 μm, and a matrix size of 256×128. Saturation slabs 1-cm-thick were placed posterior and anterior to the circle of Willis to reduce flow-induced imaging artifacts.
Analysis of MRI Data
A maximum intensity projection image was created from the 2 T1WI slices acquired and analyzed using the Medical Image Processing, Analysis, and a Visualization software package (NIH [mipav.cit.nih.gov]). To identify the clot on the maximum intensity projection of the T1WI, ≥30% signal intensity within the ICA, MCA, and ACA compared to the surrounding brain parenchyma was chosen. An investigator (R.P.W.) blinded to treatment assignment outlined the clots using the software's tracing tool. The length of the clot in each vessel was summed and the total clot length during and after treatment was reported as a percentage of the initial clot length.
DWI, PWI and MRA were analyzed blinded to treatment assignment using QuickVol II (http://www.quickvol.com/).14 Quantitative apparent diffusion coefficient (ADC) and cerebral blood flow (CBF) maps were created and their corresponding threshold-derived lesion volumes were calculated as described previously.13,14 The thresholds used to define respective abnormal ADC and CBF regions were reduced to 0.53×10−3 mm2/s and 0.3 mL/g/min, respectively, as previously validated.13 Diffusion and perfusion lesion volumes are expressed as percentages of the initial volumes at 30 (DWI) and 60 (PWI) minutes, respectively.
We introduce a metric to summarize the evolution of the clot length and perfusion deficit volume defined as the α distance, which is expressed by: where t and i represent the treatment time point and initial value, respectively. Equation 1 was normalized to pretreatment values of clot length and perfusion deficit volume.
For comparative purposes, MRA images were visually analyzed by a blinded investigator (K.M.S.) and were scored on a 3-point scale as follows: 1, complete MCA occlusion; 2, partial MCA reperfusion; and 3, complete reperfusion of the MCA. MCA was selected for analysis to reveal an overall picture of vessel recanalization.
Analysis of Evans Blue Clot Histology
The entire surface of all the rat brains was scanned with a high-resolution (1600 dpi) scanner. The Evans Blue clot was identified and measured by summing the length of all clot fragments. Total clot length was assessed and correlated with MRI to validate T1WI-derived clot length and to determine the lytic efficacy of each treatment.
Data are presented as mean±SD. Statistical comparisons were performed using 1-way ANOVA with post hoc least significant difference test for multiple comparisons. Correlation analysis was performed using Pearson 2-tailed test (version 15.0; SPSS, Chicago, IL). P<0.05 was considered significant.
In Vitro Clot Experiments
Figure 1 shows a Magnevist-enhanced clot suspended in saline. The signal enhancement ratio of the clot was calculated for concentrations ranging from 0 μL to 50 μL of Magnevist. Signal enhancement was undetectable in clots with the lowest contrast agent volumes of ≤5 μL. The Table displays the signal enhancement ratios of the clot at various Magnevist volumes. The 25- and 30-μL Magnevist volume provided significantly higher signal enhancement ratios relative to all other concentrations used, with insignificant differences between these 2 concentrations. We opted to use 25 μL Magnevist in all further experiments to obtain the highest clot signal enhancement ratio while utilizing the lowest contrast agent volume.
In Vivo Clot Experiments
The Magnevist/Evans Blue-modified clots were visible on MRI and histology. They produced consistent arterial occlusion and subsequent ischemic changes as assessed by T1WI, PWI, DWI, and MRA in all animals. When exposed to tPA, the clot slowly dissolved over the course of 90 minutes after treatment initiation. In both the vehicle and the tPA group, T1WI results matched well with Evans Blue stained clot found in the MCA, ACA, and ICA postmortem (Figure 2). T1WI-derived 180-minute clot length correlated significantly with the clot length assessed on histology (r=0.93; P<0.05).
Initial clot localization was determined in all animals before treatment was initiated. Clot localization was as follows: MCA+ICA+ACA (n=13 [59%]), ICA+ACA (n=4 [18%]), ACA+MCA (n=3 [14%]), and ICA+MCA (n=2 [9%]), respectively. Mean arterial blood pressure, blood gases, electrolytes, and glucose were not significantly different between groups, and values remained within physiological limits throughout the study (data not shown).
Clot length and CBF lesion volumes were significantly smaller than vehicle at 15 minutes after treatment initiation in the rA2 plus tPA group, whereas in the tPA group no significant reduction from vehicle was observed until 30 minutes after treatment initiation. Beginning at 60 minutes after treatment, clot length in the rA2 plus tPA group was significantly smaller than in the tPA group. From 90 minutes after treatment initiation, CBF lesion volume in the rA2 plus tPA group was significantly smaller than in the tPA group (Figure 3). The absolute CBF volumes pretreatment were 311±65 in the vehicle group, 319±40 in the tPA group, and 327±32 in the rA2 plus tPA. There were no initial statistical differences between these groups (P=0.801).
Plotting CBF lesion volume versus clot length gives the clot lysis profile for each animal over time. In the vehicle group, no clot lysis was observed as indicated by consistent CBF lesion volumes and clot lengths over time (Figure 4A–C). After thrombolysis, all animals demonstrated a reduction in CBF lesion volumes and clot lengths, with rA2 plus tPA group animals displaying the most complete clot lysis and reperfusion. The α distances of the thrombolysis groups were significantly shorter than those of the vehicle group at all time points after treatment initiation. Combining rA2 with tPA improved clot dissolution and CBF restoration as indicated by a significantly shorter α distance in the rA2 plus tPA group compared to the tPA alone group at 15, 60, and 90 minutes after treatment initiation (Figure 4D).
There was a nonsignificant reduction in the ADC lesion volumes between the 2 treatment groups and the vehicle group at 150 minutes after ES (Figure 5).
MRA showed that the treatment groups had significantly better recanalization of the MCA than the vehicle group, but no difference between the treatment groups was found (data not shown).
This study describes a novel approach for MR visualization of clot and clot lysis in a rat embolic stroke model by adding Magnevist, a widely available MR contrast agent, to the blood before clotting.
The utility of this approach is to directly compare the efficacy of thrombolytic agents on speed and completeness of clot lysis. Previous studies indirectly assessed thrombolytic efficacy by comparing DWI/PWI-derived lesion volumes or final infarct volume on histology.2,4,15 However, directly visualizing clot integrity is especially important at late therapeutic time points at which the “no or low reflow phenomenon” may occur.16 Complete recanalization can potentially occur with a small or nonexistent response of the CBF or DWI lesion volume. Information about clot lysis enhances the overall understanding of the results of treatment and more completely answers questions about the mechanisms of success or failure of an investigational stroke treatment. Further, this methodology allows for more complete characterization of the ES model. Our results show that a wide variety of clot configurations occur in the cerebral vasculature, which may explain some of the variability seen in this model.10
Compared to established methods used in the preclinical setting to determine vessel patency after treatment, the new clot imaging technique offers a number of advantages. It does not require the injection of a contrast agent, which is critical to digital subtraction angiography and some MRA techniques. Vessel visualization on noncontrast MRA techniques, such as time of flight, are dependent on a combination of pulse sequence parameters, vessel blood flow velocity, and blood flow patterns.4 The new clot imaging technique allows for high-quality visualization of the clot using standard T1WI. It is conceivable that with the use of targeted contrast agents, direct clot visualization may become a novel diagnostic technique in patients.8
Clot length was used to determine clot lysis in vivo. This metric directly defines the temporal efficacy of different thrombolytic agents independent of surrogate markers such as perfusion deficit. Together with perfusion and diffusion imaging, imaging of clot lysis allows for a more thorough characterization of the efficacy of thrombolytics in an ES model.
To show the utility of this method, we compared the effects of vehicle, tPA alone, and rA2 plus tPA treatment. The rA2 plus tPA treatment more reliably produced thrombolysis relative to tPA alone. Our results show that half of all tPA group animals had some clot remaining in the arteries, whereas no animals in the rA2 plus tPA group had clot remaining. After completion of treatment, the more consistent action of rA2 plus tPA resulted in statistically significant smaller perfusion lesion volume after treatment as compared to tPA alone. Further, rA2 plus tPA resulted in an earlier statistically significant difference in CBF deficit volume versus vehicle compared to tPA alone. These findings are supported by the α distance analysis, which shows an initial stronger response to rA2 plus tPA relative to tPA alone. Further, at late time points during and after treatment, rA2 plus tPA showed a shorter α distance than tPA alone or vehicle. This observation provides further evidence that the addition of rA2 to standard tPA treatment results in enhanced thrombolysis in this ES model.
The rA2 is thought to operate as a cell-surface receptor for both plasminogen (the inactive precursor of plasmin) and its activator, tPA.17,18 The rA2–tPA–plasminogen triple complex is more effective than tPA alone in converting plasminogen into plasmin.19,20 Our previous study confirmed the enhanced plasmin generation by rA2 plus tPA in vitro and demonstrated rA2 plus tPA improved neurological outcomes in a rat focal embolic stroke model.1 It is possible that rA2 plus tPA generates more plasmin at the clot site because it has been demonstrated that the plasmin precursor, plasminogen, binds to the endothelial cell surface and is enriched in the clot.20,21 Therefore, rA2 plus tPA may locally bind plasminogen and consequently amplify plasmin generation in the vicinity of the clot, resulting in more effective fibrinolysis.
Although we report faster and more complete clot lysis and reperfusion, there was no statistically significant difference in the final ADC lesion volume between groups. We hypothesize that this finding was attributable to the late initiation of treatment in this experiment. Previous findings in the rat suture model of stroke showed that after 95 minutes of ischemia, recovery of the ADC lesion was nonsignificant.22 Future experiments should investigate the relationship between lysis efficacy and ADC lesion resolution.
There was an observed difference between the results from MRA characterization of clot lysis and the clot imaging technique in regard to the efficacy of tPA compared to rA2 plus tPA. Clot lysis may result in partial recanalization as well as blood vessel damage, which can cause changes in blood flow velocity and profile.23 The inherent limitations of MRA to detect vessel patency under these conditions may reduce the sensitivity of this technique to visualize recanalization and may explain this result.4
The clot imaging protocol was limited because it is only sensitive to clot localized in the bifurcation of the ICA, ACA, and MCA. Sections of the MCA distal to the M1 segment were not visible. At the current resolution, larger imaging field of views would require lengthier scans and therefore would impede the ability to perform other scans and limit the temporal scanning during the acute stroke phase and at multiple time points during treatment.
To summarize, in this study we successfully developed a methodology that enables us to visualize clots using a contrast reagent Magnevist in a focal rat embolic stroke model. We used this new method to characterize clot lysis dynamics during thrombolytic therapy. We also documented that tPA plus rA2 yields faster and more consistent thrombolysis than tPA alone. This new methodology provides a novel approach to preclinically evaluate investigational thrombolytic agents.
Sources of Funding
This study was supported by institutional grants. X.W. was supported by a NIH grant.
The tPA was a kind gift from Genentec (R01-NS065998 to X.W.).
- Received September 8, 2010.
- Accepted November 22, 2010.
- © 2011 American Heart Association, Inc.
- Hilger T,
- Niessen F,
- Diedenhofen M,
- Hossmann KA,
- Hoehn M
- Gralla J,
- Schroth G,
- Remonda L,
- Fleischmann A,
- Fandino J,
- Slotboom J,
- Brekenfeld C
- Henninger N,
- Sicard KM,
- Schmidt KF,
- Bardutzky J,
- Fisher M
- Ishii H,
- Yoshida M,
- Hiraoka M,
- Hajjar KA,
- Tanaka A,
- Yasukochi Y,
- Numano F
- Schmidt KF,
- Ziu M,
- Schmidt NO,
- Vaghasia P,
- Cargioli TG,
- Doshi S,
- Albert MS,
- Black PM,
- Carroll RS,
- Sun Y
- Sakharov DV,
- Rijken DC
- Bardutzky J,
- Shen Q,
- Henninger N,
- Schwab S,
- Duong T,
- Fisher M