(316e) Efflux of Cerebrospinal Fluid through Cervical Lymph Vessels Is Reduced in Aged Mice

Introduction: Incidences of Alzheimer’s disease (AD) and other dementias increase with age and are linked to the reduced clearance of the toxic waste products (such as amyloid-β (Aβ)) accumulating in the brain ^[1]. Cerebrospinal fluid (CSF) entering and leaving the brain is integral to this clearance. CSF is propelled into the brain along perivascular spaces, mixes with the brain’s waste-laden interstitial fluid (ISF) and then drains these toxins predominantly via the cervical lymph vessels of the neck. This transport of fluid deteriorates with age, presumably because lymphatic function is compromised ^[1]. However, the dominant physiological mechanisms that drive cervical lymphatic flow (or fail in aging) remain unclear as most studies assess fluid transport using isolated lymph vessels. These conditions do not accurately represent physiological transport, where a network of vessels undergoes coordinated pulsations to transport fluid. In this talk, we present measurements of fluid flow in the cervical lymph vessels of mice in vivo. We use these first-of-their-kind measurements to explore the failure mechanisms impacting fluid clearance in mouse models of aging.

Methods: Intravital two-photon microscopy was used to visualize and quantify CSF flow in the cervical lymph vessels of both young (2-month) and aged mice (18 – 22-month, n>5). The mice were anaesthetized using ketamine/xylazine, and the cervical lymph vessels were surgically exposed. We simultaneously visualized vessel diameter fluctuations and the motion of 1 µm fluorescent microspheres (injected in the cisterna magna) flowing in the vessels. Detailed velocity measurements from particle motion and correlations to the changes in vessel diameter were performed using custom particle tracking and vessel wall detection algorithms in MATLAB ^[2]. All experiments were approved and conducted in accordance with the relevant guidelines and regulations stipulated by the University Committee on Animal Resources of the University of Rochester Medical Center, certified by the Association and Accreditation of Laboratory Animal Care.

Results: Particle tracking revealed significantly impaired clearance in the cervical lymph vessels of aged mice. The reduced speeds and lower volumes cleared were strongly correlated to the decrease in the contraction frequency (intrinsic rate) of the lymphatic vessels – the dominant driving force of fluid transport. Additionally, valve function is compromised in aged vessels, leading to stronger retrograde flows that reduce the net fluid volumes cleared.

Discussion: The techniques and results presented here allow for the first in vivo investigation of fluid dynamics and vessel function in the cervical lymph vessels – a dominant exit pathway draining CSF from the brain. With simultaneous particle tracking and vessel contraction measurements, we present mechanistic evidence that the severely reduced volumes of CSF cleared through these vessels is strongly correlated to the decrease in vessel function with age. Our measurements bolster the hypothesis that the cervical lymph vessels are a bottleneck in the aged brain’s fluid transport and restoring their function could be a powerful therapeutic target.

Acknowledgements: This work was supported by the NIH/National Institute on Aging (grant RF1AG057575).

References:[1] Nedergaard, M., et al., Science. 2020, 370(6512);[2] Kelley, D.H., et al., Am. J. Phys. 2011, 69, 267