TgCRND8 transgenic mice (Chishti et al., 2001
) express a double mutant form of APP695 (K670N/M671L + V717F) driven by the human prion protein promoter, are on a mixed background (C57XC3H/C57), develop Aβ-associated pathology, and exhibit defects in memory as early as 3 months-of-age (provided by A. Chishti and D. Westaway, University of Toronto, Canada). Tg6799 mice (Jackson Laboratory) are double transgenic for APP/Presenilin 1 and co-express five early onset familial AD mutations and rapidly accumulate Aβ42 by 2 months-of-age (Oakley et al., 2006
). Mice heterozygous for the fibrinogen Aα chain (fbg
+/-) (Suh et al., 1995
) were crossed with TgCRND8 and used for CAA determination and behavioral experiments. Non-transgenic (WT) littermates were used as controls in all experiments. Plasminogen-deficient mice (plg
-/-) (Bugge et al., 1995
; Ploplis et al., 1995
), backcrossed onto a C57/BL6 background, and age-matched WT C57/BL6 mice (Charles River) were used in this study. Genotypes were double-checked by taking a tail tissue sample the day of sacrifice. Both genders were used in all experiments, and the proportion of females to males was kept constant when comparing groups. Mice were maintained in The Rockefeller University's Comparative Biosciences Center and treated in accordance with IACUC-approved protocols.
A 500 nl solution containing 500 ng of fluorescently labelled human fibrinogen (Alexa Fluor 488-fibrinogen, Molecular Probes) was stereotactically injected into the right hemisphere (posterior -2.0 mm, lateral 1.8 mm, depth 1.2 mm (Franklin and Paxinos, 2008
)) of 6-month and 9-week-old (pre-depositing) TgCRND8 and WT mice. As control, the same amount of tetramethylrhodamine-BSA (Molecular Probes) was injected in another set of 6-month-old mice. Mice were perfused with a saline/heparin solution after 24 hr, and 20 μm-thick coronal brain sections from the injected hemisphere were fixed in ethanol, washed, and mounted with Vectashield containing DAPI (Vector Labs, Burlingame, CA). Ten sections/mouse (n=3-4 mice/group) were examined for the fluorescent compounds using an inverted LSM 510 laser scanning confocal microscope (Zeiss) equipped with a motorized stage. To quantify the area where fluorescence was present, a tile-scan (4×4) was obtained using a Plan-NeoFluor 25×/0.80 objective and thresholded using Image J (NIH). The results are normalized to the area obtained in WT mice.
Clot formation/degradation and turbidity experiments
Fibrin clots were formed by mixing purified human fibrinogen (10 μM; Calbiochem) or citrated normal human plasma (New York Blood Center, centrifuged at 10,000 × g for 15 min to obtain platelet-deficient plasma), with human thrombin (1 U/ml; Sigma) in the presence of Aβ42 peptide (100 nM - 10 μM; Anaspec) or vehicle (PBS). CaCl2 was adjusted to 5 mM. Absorbance was measured for 10 min at 450 nm. Clots were also formed in the presence of the amyloid peptides calcitonin and amylin (5 μM; Anaspec). Human calcitonin was reconstituted in dH2O, and human amylin (1-37) in DMSO. To allow comparison among all three amyloid peptides, clots formed in the presence of Aβ42, amylin, or calcitonin were controlled for the final concentration of vehicle present. For formation/degradation curves, clots were formed under the same conditions with tPA (14 nM; Genentech) and purified human plasminogen (100 nM; Sigma). Amyloid solutions were shaken for 24-48 hr at room temperature before use.
Confocal image analysis and lysis front retreat rates
Clots were formed as described above on a glass-bottomed dish with Alexa Fluor 488-fibrinogen (50 μg/ml; Molecular Probes) in the presence or absence of 500 nM Aβ42. Some clots contained Congo Red (10 mM; Sigma). For lysis experiments, tPA was injected into the center of the pre-formed clot (20 min after mixing), and time-lapse image stacks were recorded at 15 sec intervals for 5 min as the lysis front retreated from the center. Initial and final images were overlayed, and the distance between lysis fronts was divided by the 5 min collection period (n=3-4 random lysis fronts in 4 separate experiments). Images were obtained with an inverted Axiovert 200 microscope, acquired with LSM 510 v. 3.2 confocal software (Carl Zeiss, Mannheim, Germany), and analyzed with MetaMorph software (Universal Imaging).
Fibrin clots were formed from purified fibrinogen on glass coverslips. After 20 min, clots were washed with sodium cacodylate buffer, fixed with 2% glutaraldehyde, dehydrated, critical point dried, and sputter-coated with gold palladium. Images were obtained using a LEO 1550 scanning electron microscope.
Six-month-old TgCRND8 mice were saline/heparin-perfused, and 20 μm coronal brain sections were prepared, ethanol-fixed, and stained with FITC-conjugated fibrin(ogen) antibody (Dako). Tissue was counterstained for 30 min at room temperature with 0.2 % Congo Red (Sigma) in 70% isopropanol to detect CAA. Immunofluorescence images were acquired using an inverted Zeiss Axiovert 200 microscope.
Human brain immunohistochemistry
Human post-mortem brain tissue was provided by the Harvard Brain Tissue Resource Center and the Washington University AD Research Center. Paraffin sections (7 μm) from the frontal cortex of 5 control (52-90 years-of-age) and 9 AD (77-91 years-of-age) cases were deparaffinized, treated with proteinase K (Dako), and immersed in methanol/H2O2 to inactivate endogenous peroxidases. Immunohistochemistry was carried out using a rabbit polyclonal anti-fibrin(ogen) antibody (Dako) and the Tyramide Signal Amplification™ system (Perkin Elmer) according to manufacturer's instructions. Sections were developed using diaminobenzidine, co-stained for 30 min with 1% Thioflavin S (Sigma) in 70% ethanol, dehydrated, and mounted. Twenty to 25 fields per section were acquired using an inverted Zeiss Axiovert 200 microscope, and vessels > 20 μm that contained fibrin(ogen) were quantified.
Ancrod treatment and CAA determination
Tg6799, TgCRND8, and their WT littermates were treated with ancrod as described (Paul et al., 2007
). To calculate total amyloidosis, 3-month-old TgCRND8 and 4.5-month-old Tg6799 mice implanted with pumps delivering saline or ancrod, and 7-11 month-old TgCRND8-fbg
+/- mice were used. Mice were saline/heparin-perfused, and 20 μm coronal brain cryostat sections were fixed with ethanol and stained for 30 min with 0.5% Thioflavin S (Sigma) in 70% ethanol. Pictures of all the areas with CAA were acquired, thresholded using Image J, and the total CAA area per section was calculated. The average of 2-4 different sections from 4-7 mice per group was determined and plotted relative to the corresponding control group.
Intravital imaging of thrombosis
To observe blood circulation and to induce thrombosis, a cranial window was prepared over the parietal cortex of 6-month and 9-week-old TgCRND8 mice and WT littermates following the surgical procedure described (Mostany and Portera-Cailliau, 2008
) with some modifications (see Supplemental Experimental Procedures
). Two different methods were used to induce clot formation:
Topical application of FeCl3
Increasing concentrations of FeCl3
(2.5%-20%) were added directly to the brain surface with an interval of ~15 min, and thrombosis was recorded using realtime video acquisition with a video camera fitted to an upright Zeiss Axiovert 200 epi-fluorescence microscope (Metavue software). The whole procedure lasted approximately 60 min per mouse (n=2-4 mice/group and up to 10 vessels > 20 μm were thrombosed per animal). After treatment with 10% FeCl3
, some mice were topically administered with recombinant tPA (140 nM; Genentech) to activate fibrinolysis. After imaging, mice were perfused and brains were processed for in situ
zymography as described (Melchor et al., 2003
). Clot formation and dissolution were observed using time-stamped image stacks. Clot size was traced by hand using Metamorph software to calculate the area of the dark zone representing the clot,.
was provoked and imaged using a Fluoview 1000MPE two-photon laser scanning fluorescence microscope (Olympus) equipped with a SpectraPhysics MaiTai DeepSee laser (with a tuneable range of 690-1040 nm) and a 25×/1.05 NA objective. To identify CAA-positive vessels, Methoxy-X04 (Klunk et al., 2002
) (Neuroptix Corporation) was administered via tail vein injection 1 hr prior to imaging (3 mg/kg dissolved as described (Garcia-Alloza et al., 2009
)). The procedure to induce localized laser thrombosis was adjusted from the one described (Nishimura et al., 2006
). See Supplemental Experimental Procedures
for details. We targeted 3-4 vessels/mouse with a diameter greater than 20 μm (n=5-6 at 6 months-of-age; n=2 at 9 weeks-of-age TgCRND8 and WT). A 2×-zoom Z-stack of the area where the clot was formed was acquired for vessel occlusion quantification using Image J (diameter of the clot vs
diameter of the vessel in all the planes).
Morris water maze
TgCRND8 and WT mice infused with saline or ancrod (n=7, 3 to 5-month-old), TgCRND8-fbg
+/- mice and their littermates (n=5-10, ~4-month-old), and plg
-/- and C57/BL6 mice (n=5-6, 3-month-old) were tested in the Morris water maze to evaluate cognitive function (Chishti et al., 2001
) with some minor modifications (see Supplemental Experimental Procedures
for details). Spatial memory was measured by quantifying the percent time spent in the target quadrant and the number of platform crossings during the probe trial test. The experiment was recorded and analyzed using Ethovision video tracking system (Noldus).
TgCRND8-fbg+/- mice and their littermates (n=8-16, 7-11-month-old) were tested in the Y-maze. Experiments were performed in a sound-attenuated room under soft illumination, and visual clues were placed on walls of the testing room. Each trial consisted of two 5 min periods, separated by a 2 min inter-trial interval in which the mouse was placed in its home cage. During the first 5 min period, one of the three arms was blocked by an opaque plexiglass insert; this arm acts as the novel arm in the subsequent 5 min testing period. The entire experiment was recorded, and the first 2 min of the testing period were analyzed using Ethovision (Noldus). Time spent in the novel arm was averaged and compared between groups.
Aβ42 and fibrinogen ELISA
To calculate the total Aβ42 brain content, tissue was weighed and homogenized in 5 M guanidine HCl/50 mM Tris-HCl pH 8 buffer, agitated for 4 hr at room temperature, and centrifuged for 20 min at 16,000 × g to extract total Aβ (Chishti et al., 2001
). Aβ42 concentration was determined by the BetaMark™ x-42 ELISA kit according to manufacturer's instructions (Covance). Tail prick blood samples were taken before, during, and after pump implantation in mice treated with ancrod, and the decrease in fibrinogen levels in plasma was measured by ELISA (GenWay Biotech).
All numerical values presented in graphs are mean ± SEM. Statistical significance was determined using two-tailed t-test analysis comparing control to experimental groups.