Project Grant F30AG101787

GAMMA-LAMININ IN MICROVASCULAR FUNCTION AND BRAIN WASTE CLEARANCE - PROJECT SUMMARY CEREBRAL SMALL VESSEL DISEASE (CSVD) IS A PROGRESSIVE, AGE-RELATED NEUROLOGICAL CONDITION THAT AFFECTS 11 MILLION AMERICANS AND CONTRIBUTES TO 20% OF STROKES AND 45% OF DEMENTIA CASES.2, 57 CRITICALLY, THERE ARE NO FDA- APPROVED THERAPIES THAT MECHANISTICALLY TARGET CSVD.1, 3 A MARKER OF CSVD ON MAGNETIC RESONANCE IMAGING (MRI) IS THE PRESENCE OF ENLARGED PERIVASCULAR SPACES (PVS).1 THE PVS FUNCTIONS AS A KEY ENTRY ROUTE OF CEREBRAL SPINAL FLUID (CSF) INTO THE BRAIN PARENCHYMA. CSF MOVEMENT WITHIN THE PVS IS THOUGHT TO BE DRIVEN BY VASOMOTOR OSCILLATIONS OF PENETRATING ARTERIOLES AND NEUROVASCULAR COUPLING (NVC).4-8 FURTHER, PVS ENLARGEMENT AS SEEN ON MRI IN CSVD HAS BEEN THEORIZED AS A MARKER OF ALTERED BRAIN WASTE CLEARANCE BASED ON PRECLINICAL STUDIES IN MICE.4 RECENT GENETIC AND NEUROPATHOLOGICAL CLINICAL STUDIES HAVE ASSOCIATED G-LAMININ, A VASCULAR BASEMENT MEMBRANE PROTEIN, WITH CSVD. HOWEVER, IT REMAINS UNCLEAR HOW ALTERED G-LAMININ EXPRESSION AFFECTS CEREBROVASCULAR FUNCTION AND BRAIN WASTE CLEARANCE. THESE QUESTIONS ARE DIFFICULT TO ANSWER IN HUMANS, BUT RIGOROUS STUDIES CAN BE PERFORMED IN MOUSE MODELS WITH G-LAMININ DEFICIENCY. HERE, I AM PROPOSING TO UTILIZE IN VIVO TWO-PHOTON IMAGING AND THINNED-SKULL CRANIAL WINDOWS IN MICE TO TEST THE MECHANISTIC ROLE OF G-LAMININ IN CSVD PATHOPHYSIOLOGY. ALTOGETHER, I HYPOTHESIZE THAT G-LAMININ DEFICIENCY WILL LEAD TO ARTERIOLAR DYSFUNCTION, WITH CORRESPONDING PVS BURDEN AND DECREASED PARENCHYMAL WASTE MOVEMENT. SPECIFIC AIM 1 PROPOSES TO TEST THE ROLE OF G-LAMININ IN ARTERIOLAR VASOMOTION AND NVC, USING TWO-PHOTON IMAGING THROUGH THINNED-SKULL CRANIAL WINDOWS IN MICE LACKING G-LAMININ FROM MURAL CELLS ("PKO"). SPECIFIC AIM 2 PROPOSES TO TEST THE ROLE OF G-LAMININ IN PVS BURDEN (AIM 2A) AND BRAIN WASTE CLEARANCE (AIM 2B). IN AIM 2A, I WILL USE A NOVEL APPROACH FOR PVS VISUALIZATION THAT LABELS SURROUNDING PERIVASCULAR CELLS TO CREATE A NEGATIVE SPACE WHERE THE PVS IS EXPECTED. THIS CONTRASTS WITH THE TRADITIONAL APPROACH OF A CISTERNA MAGNA DYE INJECTION WHICH CAN INTRODUCE ECTOPIC PRESSURE AND A DIFFUSION GRADIENT, POTENTIALLY ALTERING PVS ANATOMY AND COMPLICATING INTERPRETATION. USING THIS NOVEL APPROACH IN CONJUNCTION WITH TWO-PHOTON IMAGING THROUGH THINNED-SKULL CRANIAL WINDOWS, I WILL COMPARE HOW PVS AREA FLUCTUATES WITH VESSEL MOTION IN WT AND PKO MICE. I WILL ALSO ANALYZE IF PVS METRICS DIFFER ACROSS VESSEL TYPE (PENETRATING ARTERIOLES VS ASCENDING VENULES) AND SIZE. IN AIM 2B, I WILL IMPLANT A CHRONIC OSMOTIC PUMP THAT WILL DELIVER A CAGED TWO-PHOTON ACTIVATABLE FLUORESCENT DYE TO THE PARENCHYMA.40, 41 TWO-PHOTON UNCAGING WILL BE PERFORMED TO TRACK DIFFUSION AND DIRECTIONAL MOVEMENT OF UNCAGED DYE AS A METRIC OF BRAIN WASTE CLEARANCE IN VIVO. THIS RESEARCH DIRECTLY SUPPORTS THE MISSION OF NIH AND NIA BY ADVANCING OUR UNDERSTANDING OF AGE-RELATED VASCULAR CONTRIBUTIONS TO COGNITIVE IMPAIRMENT AND DEMENTIA. LONG TERM, THIS MECHANISTIC PROPOSAL AIMS TO REVEAL EARLY CSVD PATHOPHYSIOLOGY TO INFORM FUTURE NEUROPATHOLOGICAL AND CLINICAL IMAGING STUDIES IN PATIENTS WITH CSVD.