Resorufin analogs preferentially bind cerebrovascular amyloid: potential use as imaging ligands for cerebral amyloid angiopathy

Abstract

BackgroundCerebral amyloid angiopathy (CAA) is characterized by deposition of fibrillar amyloid β (Aβ) within cerebral vessels. It is commonly seen in the elderly and almost universally present in patients with Alzheimer's Disease (AD). In both patient populations, CAA is an independent risk factor for lobar hemorrhage, ischemic stroke, and dementia. To date, definitive diagnosis of CAA requires obtaining pathological tissues via brain biopsy (which is rarely clinically indicated) or at autopsy. Though amyloid tracers labeled with positron-emitting radioligands such as [11C]PIB have shown promise for non-invasive amyloid imaging in AD patients, to date they have been unable to clarify whether the observed amyloid load represents neuritic plaques versus CAA due in large part to the low resolution of PET imaging and the almost equal affinity of these tracers for both vascular and parenchymal amyloid. Therefore, the development of a precise and specific non-invasive technique for diagnosing CAA in live patients is desired.ResultsWe found that the phenoxazine derivative resorufin preferentially bound cerebrovascular amyloid deposits over neuritic plaques in the aged Tg2576 transgenic mouse model of AD/CAA, whereas the congophilic amyloid dye methoxy-X34 bound both cerebrovascular amyloid deposits and neuritic plaques. Similarly, resorufin-positive staining was predominantly noted in fibrillar Aβ-laden vessels in postmortem AD brain tissues. Fluorescent labeling and multi-photon microscopy further revealed that both resorufin- and methoxy-X34-positive staining is colocalized to the vascular smooth muscle (VSMC) layer of vessel segments that have severe disruption of VSMC arrangement, a characteristic feature of CAA. Resorufin also selectively visualized vascular amyloid deposits in live Tg2576 mice when administered topically, though not systemically. Resorufin derivatives with chemical modification at the 7-OH position of resorufin also displayed a marked preferential binding affinity for CAA, but with enhanced lipid solubility that indicates their use as a non-invasive imaging tracer for CAA is feasible.ConclusionsTo our knowledge, resorufin analogs are the fist class of amyloid dye that can discriminate between cerebrovascular and neuritic forms of amyloid. This unique binding selectivity suggests that this class of dye has great potential as a CAA-specific amyloid tracer that will permit non-invasive detection and quantification of CAA in live patients.

Contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged Tg2576 mice

“…To explore the potential mechanisms by which anti-ROS therapy reduces CV deficits in aged Tg2576 mice, we visualized CAA and neuritic plaque loads using the congophilic fibrillar amyloid dyes methoxy-X04 (for in vivo imaging of CAA and neuritic plaques) (40), methoxy-X34 (for in situ staining of CAA and neuritic plaques) (40), and resorufin (for in situ staining of CAA alone) (41). Similar to our previous report (13), substantial deposition of CAA and neuritic plaques was noted in 15-mo-old, vehicle-treated Tg2576 mice where CAA deposits had progressed to encompass almost the entire leptomeningeal arteriolar system without interruption ( Fig.…”

Cerebral Amyloid Angiopathy Increases Susceptibility to Infarction After Focal Cerebral Ischemia in Tg2576 Mice

“…In a subset of aged Tg2576 mice (N=4), CAA and neuritic plaque load was quantified post mortem as per our established protocol. 27 Briefly, mice were sacrificed 72 hours post-MCAO, followed by brain extraction, fixation (4% paraformaldehyde), equilibration (30% sucrose), and coronal sectioning (50μm). Brain sections were stained with 1μM resorufin (which selectively stains CAA) and 2μM methoxy-X34 (which stains both CAA and neuritic plaques).…”

Molecular Neuroimaging in Vascular Cognitive Impairment

“…43 Resorufin (a phenoxazine derivative) and multidentate 18 F-polypegylated styrylpyridines showed preferential binding of cerebrovascular Aβ deposits over neuritic plaques in vitro but they have not been shown to cross blood-brain barrier in vivo. 44,45 More recently, 99m…”