Author + information
- Received October 7, 2011
- Revision received January 17, 2012
- Accepted January 26, 2012
- Published online November 1, 2012.
- John W. Chen, MD, PhD⁎,†,
- Jose-Luiz Figueiredo, MD⁎,
- Gregory R. Wojtkiewicz, MS⁎,
- Cory Siegel, MD⁎,
- Yoshiko Iwamoto, BS⁎,
- Dong-Eog Kim, MD‡,
- Marc W. Nolte, PhD‡,
- Gerhard Dickneite, PhD‡,
- Ralph Weissleder, MD, PhD⁎ and
- Matthias Nahrendorf, MD, PhD⁎,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Matthias Nahrendorf, Center for Systems Biology, 185 Cambridge Street, Boston, Massachusetts 02114
Objectives The purpose of this study was use molecular imaging targeting coagulation pathway and inflammation to better understand the pathophysiology of silent brain ischemia (SBI) and monitor the effects of factor XIIa inhibition.
Background SBI can be observed in patients who undergo invasive vascular procedures. Unlike acute stroke, the diffuse nature of SBI and its less tangible clinical symptoms make this disease difficult to diagnose and treat.
Methods We induced SBI in mice by intra-arterial injection of fluorescently labeled microbeads or fractionated clot into the carotid artery. After SBI induction, diffusion-weighted magnetic resonance imaging was performed to confirm the presence of microinfarcts in asymptomatic mice. Molecular imaging targeting the downstream factor XIII activity (single-photon emission computed tomography/computed tomography) at 3 h and myeloperoxidase activity (magnetic resonance imaging) on day 3 after SBI induction were performed, without and with the intravenous administration of a recombinant selective factor XIIa inhibitor derived from the hematophagous insect Triatoma infestans (rHA-Infestin-4). Statistical comparisons between 2 groups were evaluated by the Student t test or Mann-Whitney U test.
Results In SBI-induced mice, we found abnormal activation of the coagulation cascade (factor XIII activity) and increased inflammation (myeloperoxidase activity) close to where emboli lodge in the brain. rHA-Infestin-4 administration significantly reduced ischemic damage (53% to 85% reduction of infarct volume, p < 0.05) and pathological coagulation (35% to 39% reduction of factor XIII activity, p < 0.05) without increasing hemorrhagic frequency. Myeloperoxidase activity, when normalized to the infarct volume, did not significantly change with rHA-Infestin-4 treatment, suggesting that this treatment does not further decrease inflammation other than that resulting from the reduction in infarct volume.
Conclusions Focal intracerebral clotting and inflammatory activity are part of the pathophysiology underlying SBI. Inhibiting factor XIIa with rHA-Infestin-4 may present a safe and effective treatment to decrease the morbidity of SBI.
Behavioral changes, neuropsychological deficits, and aggravated vascular dementia can be observed in patients after cardiovascular surgery (e.g., coronary bypass surgery, valve replacement, carotid endarterectomy) and in patients undergoing vascular interventions (1). With the advent of sensitive imaging modalities such as diffusion-weighted magnetic resonance imaging (MRI), there has been an increasing awareness of injury to the brain in patients without major symptoms. These lesions, called silent brain ischemia (SBI), are thought to be caused by microembolism from debris, air, fat, or fragmented thrombi (2,3). The overall incidence in the general patient population is 2% to 3%, with a prevalence increasing with age of up to 20% in the elderly (3,4). Vascular procedures, such as angiography, carotid endarterectomy and stenting, and heart surgery can increase the risk for SBI substantially (1,3). The presence of SBI has also been found to be associated with dementia and increased risk of stroke (4). Currently, no effective therapy exists for treating this condition.
In this work, we reasoned that coagulation factor XII (FXII) can serve as a potential therapeutic target in SBI. FXII is a serine protease that initiates the intrinsic coagulation cascade by activation of factors XI to XIa (5) and also has immunomodulatory functions (6). It binds an Fc receptor on monocytes, increases the release of interleukin-1 and -6 from monocytes and macrophages and stimulates neutrophils (7–9). FXII also activates chemerin, a chemoattractant that binds a G protein–coupled receptor on dendritic cells and macrophages, directing an immune response to the site of injury (10). Additionally, FXII activates prekallikrein and thus bradykinin formation, which in turn is a potent inflammatory mediator that induces vasodilation and vascular leakage (11). FXII-deficient mice have decreased plasma bradykinin levels, suggesting that FXII may ameliorate a potentially harmful inflammatory response that can cause further damage (12,13). It has been suggested that FXII improves the strength and size of a developing thrombus (11). Interestingly, deficiency or inhibition of FXII protects against arterial thrombus formation and reduces cerebral injury in mouse models of stroke without increasing the risk of bleeding (14,15). In humans, hereditary deficiency of FXII is not associated with abnormal bleeding diathesis (16). That a FXII-driven pathway is required for pathological thrombus formation but is not involved in hemostasis suggests that FXII inhibition could be a promising and safe therapeutic strategy (17,18) for SBI.
Infestins are a class of serine protease inhibitors derived from the midgut of the hematophagous insect, Triatoma infestans, a major vector for the parasite Trypanosoma cruzi, known to cause Chagas disease (19). The insect uses these inhibitors to prevent coagulation of ingested blood. The Infestin gene encodes 4 domains that express proteins inhibiting different factors in the coagulation pathway (19), including Infestin-4, a strong inhibitor of activated coagulation factor XII (FXIIa). In this study, we found that rHA-Infestin-4 (recombinant Infestin-4, fused to human albumin for prolongation of blood half-life) decreased the amount of ischemic injury in both a thromboembolic and a microbead embolic model that mimics SBI caused by other materials such as air and fat. We used 2 targeted molecular imaging approaches to examine treatment effects on the coagulation cascade by imaging activity of the downstream clotting factor XIIIa and to study brain inflammation by MRI of myeloperoxidase (MPO) activity. We found that in murine SBI, microembolism caused clotting and inflammation in the brain and that rHA-Infestin-4 reduced microinfarctions via anticoagulatory effects without changing the inflammatory response.
All experiments were performed in adult Balb/c mice obtained from Charles River Laboratories, Inc. (Wilmington, Massachusetts). Thirty-six mice were used to optimize the animal model with respect to surgical access and the type and amount of injected materials. The institutional review board at Massachusetts General Hospital approved all animal experiments. Mice were anesthetized in an isoflurane chamber with 2% isoflurane by inhalation with 2 l/min of supplemental oxygen and transferred to a warm heating pad in a supine position while under isoflurane.
Most likely, SBI in patients is caused by a shower of minuscule emboli that lodge in the small vessels in the brain and result in diffuse ischemic injury, whereas large-scale blood flow is not interrupted. Therefore, to leave the internal carotid artery intact, we chose to retrogradely inject embolic material into the external carotid artery. Thromboemboli and microbeads were injected 1-sided through the external carotid artery into the internal carotid artery while the common carotid artery was temporarily occluded. This forced the emboli into the arterial system of the brain without compromising perfusion. Ethicon 8-0 mononylon sutures (Johnson & Johnson, Brussels, Belgium) were used for all arterial ligations. A temporary ligation was made on the common and internal carotid arteries to stop the blood flow during catheter placement. The distal suture around the external carotid artery was permanently tied off, and a second suture was placed around the artery to secure the catheter. The external carotid artery was opened 5 mm distal to the carotid bifurcation. We then inserted a modified intramedic polyethylene PE10 catheter (inner diameters of 0.28 and 0.61 mm, Becton, Dickinson and Company, Sparks, Maryland) retrogradely into the external carotid artery, with the tip of the catheter facing the bifurcation (Fig. 1). The ligation on the internal carotid artery was then released, allowing for flow of injected material into the internal carotid artery. Flashback of arterial blood into the catheter was observed, and then either microbeads or thromboemboli were injected. In a control group, we only ligated the external carotid artery, which did not lead to ischemic injury of the brain. After injection of emboli, the catheter was removed, and the external carotid artery was ligated to stop bleeding. The ligatures at the internal and common carotid arteries were removed to re-establish arterial flow, and the wound was closed with 7-0 nylon sutures.
After optimization experiments, we found that intracarotid injection of 500 microbeads of 43-μm diameter resulted in reproducible, clinically silent microlesions. A syringe loaded with 500 Fluoresbrite Plain YG 45 Microspheres (excitation/emission: 441 nm/486 nm, Polysciences, Inc., Warrington, Pennsylvania) was attached to the catheter to inject the beads (Fig. 1).
Fresh mouse blood (500 μl) was withdrawn from donor mice via cardiac puncture and immediately added to sodium citrate solution for initial anticoagulation (final concentration, 0.32%). After centrifugation at 1,500 rpm for 10 min to remove cells, 20 μmol CaCl2 and 10 units of high-activity human thrombin were added to the plasma to induce clotting (molecular weight, 37,000, 100 U/ml, specific activity 2,800 NIH U/mg protein, Calbiochem, EMD Chemicals, Inc., Darmstadt, Germany). To label the thromboemboli, 2 nmol of Genhance 680 (Perkin Elmer, Massachusetts) was added. The material was stored at 4°C for up to 72 h. Before use, the clot was washed with normal saline solution and treated with a tissue homogenizer to fractionate the clot into small particles ∼10 μm in diameter (Fig. 1). Optimization experiments determined that a volume of 10 μl of this preparation did not cause overt neurological deficits.
These emboli sizes were chosen to better account for the variability of different types of injury that may occur in the clinical setting. The human middle cerebral artery is ∼4 mm in diameter, with large emboli considered to be those >2 mm in size. Mouse middle cerebral artery is ∼80 microns in diameter. Therefore, we chose 2 types of emboli that can be considered small (10 microns) and relatively large (43 microns, just >50% of the diameter of mouse middle cerebral artery).
After recovery from anesthesia, mice were assessed for signs of stroke using the Benderson scale (20) after SBI induction. We also observed the animals for dyskinesia, lethargy, grip, limb weakness, eyelid droop, gait disturbance, circling, and rolling. Observation of any of these symptoms was considered to be an indicator of stroke, whereas lack of these symptoms and a Benderson score of 0 (normal) indicated that no overt stroke had occurred, consistent with the possibility of SBI. If no stroke symptoms were present, the mice were used for this study; otherwise, they were excluded. A total of 4 mice were excluded after thromboembolism and 7 mice after microbead injection.
After optimization of the animal model, we proceeded to study 4 cohorts (n = 5 to 9 per group): mice with induction of SBI by thromboemboli or microbeads (untreated controls), and 2 additional groups in which mice were treated with rHA-Infestin-4 (CSL Behring GmbH) at a dose of 200 mg/kg via intravenous tail vein injection. The mice received their first injection immediately after SBI induction. Single-photon emission computed tomography (SPECT)/computed tomography (CT) of FXIII activity was done 3 h after SBI. The cohorts that were imaged 3 days after SBI by MRI received daily injections until and including day 3 after injury. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was assessed 3 days after SBI to measure the microinfarct burden.
MRI to assess infarct volume and MPO activity
MRI was performed using a Bruker (Billerica, Massachusetts) 4.7-T scanner with a RARE T1-weighted (T1w) sequence (repetition time [TR] 1,500 ms; echo time [TE] 8 ms; averages 8; matrix 192 × 192 × 22; voxel size 0.133 × 0.13 mm × 0.5 mm); and a RARE T2-weighted (T2w) sequence with the same geometry (TR 5,622 ms; TE 20 ms; averages 4). To evaluate the model, we also used diffusion-weighted imaging after SBI induction. An echo planar imaging diffusion-weighted sequence with 6 diffusion directions was used (TR 4,800 ms; TE 32 ms; matrix 128 × 128 × 22; voxel size 0.195 × 0.195 × 0.5 mm).
MPO is an abundant enzyme secreted by many inflammatory myeloid cells such as neutrophils, Ly-6Chigh monocytes, subsets of activated macrophages, and microglia during inflammation and thus is a suitable imaging biomarker for the local inflammatory response. Mice were scanned before and 90 min after intravenous administration of 0.3 mmol/kg of MPO-gadolinium contrast agent (MPO-Gd [bis-5-hydroxytryptamidediethylenetriaminepentaacetatic acid-gadolinium]) (21–23) 3 days after SBI induction. MPO-Gd is a gadolinium-encaging chelator (diethylenetriaminepentaacetate) derivatized with 2 serotonin moieties and can react with other MPO-Gd molecules to form oligomers in tissue with high MPO activity, but remain as single molecules in the absence of MPO activity (21,24). MPO-Gd causes highly MPO-specific tissue enhancement (22,23). After imaging, mice were killed, perfused with normal saline solution, and the brains were harvested and sliced for fluorescence imaging on a mesoscopic fluorescent imager (OV-100, Olympus Corp., Center Valley, Pennsylvania) to verify that emboli were present.
Amira (Visage Imaging, San Diego, California) and Matlab (MathWorks, Natick, Massachusetts) software was used for data analysis. T2w images were segmented by region-based thresholding for volume computation and 3-dimensional visualization of lesions. Regions of interest were identified on each image slice of the 90-min post-injection T1w scans. The brain ventricles were excluded by manual segmentation of T2w images before automated segmentation of MPO-Gd positive areas on T1w scans. MPO-Gd–positive voxels were quantitated using a Matlab procedure that counted voxels with a signal intensity that was 3 SDs above the average signal in normal brain tissue. To determine whether Infestin has additional effects on inflammation, the MPO-Gd–positive volume was normalized to the T2 volume to account for change in infarct size. In addition, we quantified the contrast-to-noise ratio, which reflects the degree of inflammation in tissue (22,23). This approach was validated previously, including imaging in MPO−/− mice (22).
SPECT-CT to assess clotting cascade activation
Because cross-linking of fibrin by FXIII is considered the final step in the formation of a clot, it is a useful measure of overall clotting activity. We performed in vivo SPECT-CT imaging using the FXIII-specific molecular probe FXIII-indium (25,26) during the acute stage after SBI induction. This imaging agent consists of an indium 111–labeled peptide that is recognized by FXIII as specific substrate and is then incorporated into the forming clot. Previous work carefully characterized the specificity of the imaging agent using a scrambled peptide sequence (27) and FXIII−/− mice (26). Four groups of mice underwent SBI induction and treatment 1 h before injection of approximately 1 mCi of a factor XIII substrate peptide labeled with indium 111 (actual amount injected was 731 to 1,273 μCi). SPECT-CT was performed using the Gamma Medica-Ideas (Northridge, California) XSPECT small animal imaging system. We used a cone beam (50 kVp, 500 mA) x-ray tube with a solid-state CMOS detector over 256 projections. These projections were reconstructed using the modified Feldkamp reconstruction algorithm. The SPECT scan used dual gamma cameras with 1-mm medium energy pinhole collimators through 64 projections (32 projections from each camera) at 90 s/projection. The SPECT images were reconstructed using the ordered subsets expectation maximization algorithm and fused to the CT images for accurate anatomic colocalization of molecular information.
Animals were killed immediately after SPECT-CT imaging. The brain was excised and tissue analyzed with a Wallac Wizard 1480 gamma well counter (Perkin-Elmer, Waltham, MA). Afterward, the brain was sliced into 2-mm thick sections and imaged by fluorescence reflectance imaging as described in the following to verify the presence of fluorescent emboli. Afterward, the tissue slices were placed on phosphorimager plates overnight for autoradiography and analyzed on a Molecular Dynamics Typhoon phosphor imager plate reader (Sunnyvale, California).
For SPECT-CT data analysis, the entire brain and skeletal muscle were manually segmented from the CT images using Amira software to calculate the target-to-background ratio, with muscle activity serving as background. Data were normalized for the mass of the brain and the injected dose of each animal. Autoradiography and gamma counting data were decay corrected to the time of killing of the mice. Three-dimensional visualizations of the SPECT-CT data were rendered using Osirix software (Geneva, Switzerland).
Ex vivo imaging and pathology
After the mice were killed, the brains were cut into 2-mm thick coronal sections using a mouse brain slicer (Zivic Instruments, Pittsburgh, Pennsylvania) and imaged with an OV-100 small animal imaging system (Olympus Corp.), a hybrid of a planar reflectance fluorescence imaging system and a high-power microscope. The brain sections were imaged using the green fluorescent protein channel (excitation, 400 nm; emission, 508 nm) for the microbeads and the near-infrared channel for the fluorescent thromboemboli (excitation 680 nm) with as high as 16× magnification. White-light images were also acquired to assess potential hemorrhage caused by injury.
To assess microinfarct burden 3 days after SBI, brain slices were produced and placed in a 1% TTC in phosphate-buffered saline solution for 30 min at 37° C. The brain sections were then washed 3 times with phosphate-buffered saline solution for 1 min each and then imaged using a digital camera (Olympus FE-280) to assess TTC staining. Viable brain tissue stained red with TTC, whereas infarcted regions did not stain.
For further histological analysis, slices of brain tissue were embedded in O.C.T. compound (Sakura Finetek, Torrance, California), and serial 5-μm frozen sections were cut. The avidin-biotin peroxidase method was used for immunohistochemistry, and tissue sections were incubated with factor XIIIA: C-20 (Santa Cruz Biotechnology, Inc., Santa Cruz, California) followed by a biotinylated anti–goat IgG secondary antibody (Vector Laboratories, Inc., Burlingame, California). The reaction was visualized with a 3-amino-9-ethylcarbazole substrate (DakoCytomation, Carpinteria, California), and all sections were counterstained with Harris hematoxylin solution. The slides were digitized automatically at ×400 magnification, and images were captured using NanoZoomer 2.0-HT (Hamamatsu Photonics K.K., Shizuoka, Japan).
Results are expressed as mean ± SEM. Statistical comparisons of the 2 groups were evaluated by the Student t test or Mann-Whitney U test (if the variances were significantly different in the 2 groups, assessed by the F test). A value of p < 0.05 was considered to indicate statistical significance.
Mouse model of SBI
We found that in 5 control mice, ligation of the external carotid artery and placement of the catheter but without injection of thrombogenic material did not cause ischemic injury. When these mice were imaged by SPECT-CT or MRI, we did not find injury sequelae. Next, we chose 2 different materials to mimic microembolism, accounting for the heterogeneous nature of SBI. Fluorescent microbeads 43 μm in diameter, as well as fractionated blood clots that were labeled with fluorochromes (∼10 μm in size), resulted in variable symptoms and degree of tissue damage, depending on the amount of material injected. In optimization experiments, we adjusted the quantity of embolic material to avoid neurological deficits that are the hallmark of stroke, which we frequently found if >1,000 microbeads or >30 μl of thromboembolic material were injected. Specifically, 500 microbeads and 10 μl of thromboembolic material rarely caused strokelike symptoms, and we chose these quantities for all further experiments. If stroke symptoms occurred nevertheless, animals were excluded from the study.
Both embolic materials were fluorescent; thus, we could observe how they produced occlusions in small cerebral vessels (Figs. 1A, 1B, and 1C) and microinfarctions in the brain. Fluorescent labeling of emboli also allowed us to locate them in the target tissue; thus, fluorescence imaging served as quality control for the procedure in all subsequent experiments. Most emboli and subsequent molecular imaging signal was located in the ipsilateral hemisphere; however, some spillover, likely through anastomoses provided by the circle of Willis, was observed in the contralateral hemisphere. Diffusion-weighted MRI showed the typical subtle changes that are described in human patients with SBI (Fig. 1D).
rHA-Infestin-4 administration reduces tissue damage in SBI
To assess the amount of brain tissue damage caused by emboli, we computed the percentage of the brain that is infarcted for each model from MRI (Figs. 2A and 2B). We found both SBI models resulted in infarct volumes that were, on average, <4% of the brain volume, which is substantially less than the average infarct volume in human stroke patients, which averages ∼9% (28). Interestingly, the microbead model generated slightly larger average infarct volume compared with the thromboemboli model (3.5 ± 1.3% vs. 1.1 ± 0.4%, p = 0.09). To corroborate the imaging data, we also performed TTC staining of slices harvested from mice 3 days after embolization. Both types of microembolization produced small infarcts (Figs. 2C and 2D) that occupied <5% of the brain by area, similar to the volume results from MRI. Again, we found that injection of microbeads produced slightly more tissue damage compared with the thromboemboli (4.9 ± 0.9% vs. 2.1 ± 0.5%, p = 0.06).
When mice were treated with rHA-Infestin-4, significantly decreased microinfarction was detected by MRI for both materials (microbeads model 53% reduction; thromboemboli model 85%) (Figs. 2A and 2B). TTC staining further confirmed these findings (microbead model 54% reduction; thromboemboli model 66% reduction) (Figs. 2C and 2D).
We found evidence of microhemorrhages in 6 of 8 mice (75%) injected with microbeads and in 5 of 9 mice (56%) injected with thromboemboli (Fig. 3). After rHA-Infestin-4 administration, the occurrence of microhemorrhage did not increase. In fact, treatment reduced the occurrence of microhemorrhages from 75% to 67% in the bead model and from 55% to 12% in the clot model (p = NS and 0.07, respectively) (Fig. 3).
rHA-Infestin-4 treatment decreases clotting secondary to SBI
To assess the effect of rHA-Infestin-4 on coagulation, we studied the activity of FXIII, which is downstream from and affected by FXII and responsible for cross-linking fibrin clots. In mice with SBI, we found diffuse SPECT signal in the ipsilateral brain hemisphere, indicating that the injected microemboli induced intravascular coagulation once they lodged in the brain. The location of the SPECT signal was corroborated by ex vivo autoradiography, which also showed that activity colocalized with microbeads and thromboemboli on fluorescence reflectance images (Figs. 4 and 5).⇓⇓
There was a significant reduction in the amount of FXIII activity after rHA-Infestin-4 administration in SBI caused by both injection of thromboemboli (Fig. 4) and microbeads (Fig. 5). This was corroborated by ex vivo autoradiography exposure of brain sections (middle panels of Figs. 4 and 5). The overall degree of factor XIII activity reduction is visualized on the 3-dimensional SPECT-CT images (Figs. 4 and 5, left panels). Immunohistochemical staining for FXIII confirmed in vivo imaging results (Fig. 6).
rHA-Infestin-4 does not alter MPO activity in the brain after SBI
To assess MPO activity in vivo, we performed MRI 3 days after SBI using the molecular imaging agent MPO-Gd. In a cohort of normal mice, no enhancement was found after injection of MPO-Gd (data not shown). In mice with SBI, we found diffuse enhancement after injection of MPO-Gd, colocalizing with the embolic material on ex vivo fluorescence images (Figs. 7 and 8).⇓⇓ Injection of microbeads induced more MPO activity compared with thromboemboli (contrast-to-noise ratio: microbeads 36 ± 1; thromboemboli 23 ± 2; p < 0.05) and larger lesions (number of MPO-Gd–positive voxels in the brain: microbeads 3,162 ± 1,435; thromboemboli 548 ± 207; p = 0.05).
In both models, there was a trend toward a decrease in MPO-Gd–positive volume after rHA-Infestin-4 treatment. Interestingly, after accounting for infarct volume, we found that the normalized MPO-positive volume did not change after treatment (Figs. 7 and 8). The average contrast-to-noise ratio of MPO-positive lesions was also unchanged (Figs. 7 and 8).
Due to the absence of severe clinical signs and symptoms, SBI is a complication of medical procedures that is difficult to diagnose and therefore often overlooked. Increased use of diffusion-weighted MRI has provided evidence that the incidence of SBI increases with age and vascular procedures. There is no clinical treatment available. We used a mouse model that reflects the heterogeneous pathophysiology of SBI by using microthrombi and microbead embolism to study the sequelae of SBI with respect to intravascular activation of the coagulation system and the inflammatory response. Regardless of the material for the microemboli, in the absence of stroke-like symptoms, we found activation of the clotting cascade in the vicinity of the microemboli, measured by SPECT-CT imaging FXIII activity, a clotting factor that is involved in the final step of the coagulation cascade. We further detected increased inflammatory activity, reported by MPO MRI. Furthermore, we report that rHA-Infestin-4, a recombinant factor XIIa inhibitor fused to human albumin for prolongation of its half-life, significantly reduced both FXIII activity and the burden of microinfarction. These findings suggest rHA-Infestin-4 as a potential treatment in patients with a diagnosis of SBI.
Clinicians frequently encounter neuropsychological symptoms in patients who underwent major cardiovascular interventions. Because conventional brain imaging techniques showed no anatomic correlates, these “subtle” changes in alertness, orientation, mood, and character were often dismissed as difficulty to adjust to the hospital environment or advanced age. The advent of imaging techniques that have very high sensitivity, such as DWI MRI, has brought to light that an astounding number of patients have post-procedural impairment of diffusion in their brains (1,3). Improving our understanding of SBI biology, we found that in mice after microembolization, regardless of the material of the emboli, there is considerable pathology, even in the absence of stroke symptoms. Specifically, FXIII activity was increased 3 h after embolization, indicating increased coagulation at the site where embolic material lodged. Three days later, these brain areas showed significant inflammation as a response to the tissue injury. We speculate that the arrest of blood flow in the smallest vessels in the brain after fragmented clot or beads lodged in them caused secondary clotting and ischemic death of small volumes of brain tissue and in some cases microhemorrhages. Secondary intravascular clotting may thus serve as a therapeutic target, as seen in the mouse cohorts treated with rHA-Infestin-4. The described mouse model can be used for future studies of SBI, especially to test therapeutic approaches to prevent it. The use of fluorescent embolic material is advantageous because it can serve as a quality control for the procedure and informs on where the injected materials lodged, which is otherwise difficult to determine. Commercially available fluorescent microbeads proved particularly suitable because the amount of injury can be standardized due to the homogeneous size of beads and the ease of dosing.
We characterizedSBI in mice using 2 molecular imaging approaches that are well positioned to detect subtle changes early in the course of disease. MPO activity was imaged by MRI on day 3, when the inflammatory activity peaks in stroke (23). The increased MPO activity suggests that a response occurs that parallels stroke, specifically, the recruitment of innate immune cells to the site of injury. At this point, it is unclear whether subsequent inflammation exacerbates ischemia of brain cells and whether it also could be targeted therapeutically. In the current study, rHA-Infestin-4 did not change the normalized MPO-positive volume after accounting for infarct size, suggesting that this treatment did not have additional effects on inflammation other than from the decrease in infarct size. In addition, MPO signal on day 3 after SBI was not changed by treatment. Thus, the MPO-Gd–positive areas seen on imaging may represent breakthrough injured areas that escaped rHA-Infestin-4 treatment. As such, these areas did not demonstrate any change in MPO activity.
As early as 3 h after induction of injury in our murine models, there was significant SPECT signal, indicating heightened FXIII activity. Given the lack of overt symptoms in SBI, recombinant tissue plasminogen activator is likely not suitable for SBI. Even in stroke, patients with mild symptoms are not treated with thrombolytic therapy due to the substantial risk of hemorrhagic complications (29). Conversely, similar to previous studies on stroke (14,17), FXII inhibition did not increase frequency of hemorrhage in SBI, pointing to a favorable profile of unwanted side effects. In general, anticoagulatory therapy carries an increased risk of bleeding, and this would reduce enthusiasm for a therapy targeting SBI, which is not a life-threatening condition. However, this was not the case for rHA-Infestin-4. This finding is in line with the symptoms of hereditary FXII deficiency, which do not include inadequate bleeding. Altogether, the data suggest that FXII inhibition could serve as prophylactic therapy in high-risk cases and may be safe in patients who undergo procedures involving arterial catheterization or vascular surgery. These hypotheses will have to be tested in future studies, especially because therapy was started 1 h after embolization in our study.
Limitations of the model include the observed microhemorrhages, which have not been reported in clinical cases of SBI. Future studies should also focus on long-term sequelae and explore whether these are comparable to the clinical situation.
Our findings in mice demonstrate that in SBI, both coagulation and inflammatory pathways are activated, regardless of whether the cause is a migrating thromboembolus or other material. The coagulation abnormality and, more important, the amount of ischemic injury resulting from microembolism were effectively reduced by factor XIIa inhibition, pointing to a novel treatment option to decrease the morbidity of SBI. The molecular imaging technologies used here allowed not only a better understanding of the biology of SBI, but may also have translational value to determine the severity of SBI in patients. The study further indicates that these molecular imaging strategies could be a sensitive tool to monitor therapeutic efficiency.
The authors thank Dr. Thomas Weimer (CSL Behring GmbH) for providing rHA-Infestin-4.
For an expanded Methods section, please see the online version of this article.
Selective Factor XIIa Inhibition Attenuates Silent Brain Ischemia
This work was supported by a grant from CSL Behring GmbH, and by grants from the NIH (grants R01HL095629 and R01HL096576 to Dr. Nahrendorf, R24-CA92782 to Dr. Weissleder, and R01-NS070835 and R01NS072167 to Dr. Chen). Dr. Chen has received a research grant from Pfizer (unrelated to this project). Dr. Nolte holds employee shares of CSL Limited. Dr. Nahrendorf has received grant support from CSL Behring. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Chen, Figueiredo, and Wojtkiewicz contributed equally to this work.
- Abbreviations and Acronyms
- computed tomography
- factor XII
- factor XIII
- bis-5-hydroxytryptamide-diethylenetriaminepentaacetic acid-gadolinium
- magnetic resonance imaging
- silent brain ischemia
- single-photon emission computed tomography
- 2,3,5-triphenyltetrazolium chloride
- Received October 7, 2011.
- Revision received January 17, 2012.
- Accepted January 26, 2012.
- American College of Cardiology Foundation
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