Localized conditional induction of brain arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia

Background Longitudinal mouse models of brain arteriovenous malformations (AVMs) are crucial for developing novel therapeutics and pathobiological mechanism discovery underlying brain AVM progression and rupture. The sustainability of existing mouse models is limited by ubiquitous Cre activation, which is associated with lethal hemorrhages resulting from AVM formation in visceral organs. To overcome this condition, we developed a novel experimental mouse model of hereditary hemorrhagic telangiectasia (HHT) with CreER-mediated specific, localized induction of brain AVMs. Methods Hydroxytamoxifen (4-OHT) was stereotactically delivered into the striatum, parietal cortex, or cerebellum of R26CreER; Alk12f/2f (Alk1-iKO) littermates. Mice were evaluated for vascular malformations with latex dye perfusion and 3D time-of-flight magnetic resonance angiography (MRA). Immunofluorescence and Prussian blue staining were performed for vascular lesion characterization. Results Our model produced two types of brain vascular malformations, including nidal AVMs (88%, 38/43) and arteriovenous fistulas (12%, 5/43), with an overall frequency of 73% (43/59). By performing stereotaxic injection of 4-OHT targeting different brain regions, Alk1-iKO mice developed vascular malformations in the striatum (73%, 22/30), in the parietal cortex (76%, 13/17), and in the cerebellum (67%, 8/12). Identical application of the stereotaxic injection protocol in reporter mice confirmed localized Cre activity near the injection site. The 4-week mortality was 3% (2/61). Seven mice were studied longitudinally for a mean (SD; range) duration of 7.2 (3; 2.3−9.5) months and demonstrated nidal stability on sequential MRA. The brain AVMs displayed microhemorrhages and diffuse immune cell invasion. Conclusions We present the first HHT mouse model of brain AVMs that produces localized AVMs in the brain. The mouse lesions closely resemble the human lesions for complex nidal angioarchitecture, arteriovenous shunts, microhemorrhages, and inflammation. The model’s longitudinal robustness is a powerful discovery resource to advance our pathomechanistic understanding of brain AVMs and identify novel therapeutic targets. Supplementary information The online version contains supplementary material available at 10.1007/s10456-023-09881-w.

Company, Reno, NV, USA, model number: 80008).Supplemental Table 1 shows the stereotaxic coordinates used to deliver 4-OHT into the striatum, parietal cortex, and cerebellum of neonatal mice on postnatal day 1 and were established based on previous reports. 3,4 he exclusion criteria were immediate periprocedural adverse events, any procedure-related complications compromising the normal mouse behavior, or a loss of body weight >20%.

Systemic Injection of Tamoxifen
Tamoxifen (Sigma Aldrich, catalog number: T5648) was dissolved in corn oil at 25 mg/mL and was administered intragastrically to neonatal mice (50 mg/kg body weight) and intraperitoneally to adult mice (200 mg/kg body weight).

Magnetic Resonance Imaging
Mice were anesthetized with isoflurane in oxygen and nitrous oxide at 3% for induction and 1-2% for maintenance to maintain a respiratory rate between 80 and 100 breaths per minute measured with a pillow sensor under the abdomen.Mice were settled on a circulating warm water blanket to maintain normal body temperature.Magnetic resonance imaging (MRI) and 3D time-of-flight magnetic resonance angiography (MRA) were performed using a 7 Tesla smallanimal, 30-cm horizontal-bore magnet and a BioSpec Avance III spectrometer (Bruker Corp., Billerica, MA, USA) with a 116-mm high-power gradient set (600 mT/m) and a 30-mm wholebody mouse quadrature coil.Fast spin-echo scout images of the brain were acquired in three
Images were captured with the Nikon Eclipse Ti2 Confocal Microscope (Nikon Corp., Minato City, Tokyo, Japan).

Prussian Blue Staining
Prussian blue staining was performed using the Iron Stain Kit (Abcam, Cambridge, UK, catalog number: ab150674) according to the manufacturer's instructions.Briefly, mouse brain sections were equilibrated in distilled water and incubated with potassium ferrocyanide and hydrochloric acid (1:1) for 5 minutes.The sections were mounted with Prolong Gold Antifade Reagent (Thermo Fisher Scientific, catalog number: P36930) and imaged with the MZ8 stereomicroscope (Leica Microsystems).