Study On The Modulatory Mechanism Of The Glymphatic Pathway Based On In Vivo Imaging Technology Of The Second Near-infrared Imaging Window

Part One: Establishment of second near-infrared in vivo biological imaging mouse model for studying lymph transport in the central nervous system through the intact skullBackground and objective: The discovery of the glymphatic pathway and meningeal lymphatic vessels in the central nervous system(CNS)has overturned the traditional concept that the CNS lacks lymphatic system and has the privilege of "immune immunity".These innovative studies benefit from the progress of biological imaging technology.However,the current mainstream related imaging technology is challenging to consider the high-resolution imaging effect and the macro,wide field of view imaging requirements.The second Near-infrared(NIR-?)imaging technology has a high temporal and spatial resolution.We want to establish a model for in vivo imaging of CNS lymph transport in mice based on the NIR-? biological imaging technology.Methods: Firstly,we prepared four NIR-? fluorescent probes with different physical and chemical properties and characterized their optical properties.Through the self-built NIR-? wide-field imaging system,we explored the potential application value of these fluorescent probes in the study of CNS lymph transport and screened the ideal probes.Furthermore,we characterized the lymphatic transport pathway and related factors in the skull cavity.Results: Quantum dot nanospheres will stay in the brain cistern for a long time.Molecular probes with poor biological stability were significantly retained in the injection site and brain cistern.Using a compound probe(BSA@780)with both biosafety and visual stability,we successfully established the in vivo NIR-? imaging model to study the lymph transport of CNS.Under physiological conditions,the fluorescent probe injected through the cisterna magna eventually flows out of the skull cavity through the peripheral lymphatic vessels.The faster injection rate will increase the probability of the fluorescent probe flowing back to the ventricular system.When intracranial pressure increases,a large number of fluorescent probes are drained through the superficial venous sinus of the cerebral cortex.The fluorescent probe injected through the lateral ventricle was first drained to the subarachnoid space at the level of the lumbar.The rapid wheezing and respiratory arrest of mice will make the fluorescent probes quickly gather around the arteries.Conclusion: NIR-? in vivo imaging model has high temporal and spatial resolution.It can non-invasively collect dynamic images of deep tissue of mouse skull with high sensitivity and high signal-to-back ratio.It will provide more accurate biological information for the study of lymph transport in CNS and has high application value.Part Two: Effect of core body temperature on the glymphatic pathway and its related mechanismBackground and objective: Current studies have shown that the glymphatic pathway plays a crucial role in metabolite clearance,solute transport,and immune monitoring of the central nervous system.The characterization of the regulation mechanism of CNS lymph transport will bring many new targets for treating conditions.Natural sleep enhances the glymphatic pathway transport and metabolic waste clearance.A variety of sleep-related physiological factors has also been confirmed to regulate the glymphatic pathway transport.The changes in core body temperature are intrinsically related to previously studied physiological factors.However,there is a lack of crucial research on whether the core body temperature is involved in the glymphatic pathway transport.Using our NIR-? in vivo imaging model to study the lymph transport of CNS,we want to characterize whether the change of core body temperature can modulate the carrier of the glymphatic pathway.Methods: Using two anesthetic agents with different effects on the transport of the glymphatic pathway: isoflurane and dexmedetomidine,we formulated two anesthetic strategies for mice: Isoflurane alone,isoflurane and dexmedetomidine combined.The transport path and flow rate of the fluorescent probe were monitored when the core body temperature of mice was maintained at three different stages(hypothermia intervention: 31 ± 0.5 ?;normal body temperature: 37 ± 0.5 ?;continuous hyperthermia: 39.5 ± 0.5 ?).Combined with the realtime changes of respiratory rhythm and heart rate in mice,we summarized the internal mechanism of the change of core body temperature on the regulation of glymphatic pathway.Results: At normal body temperature,dexmedetomidine intervention significantly reversed the inhibitory effect of isoflurane on the glymphatic pathway.Hypothermia significantly reduced the heart rate of isofluraneanesthetized mice but could not reverse the inhibitory effect of isoflurane anesthesia on the glymphatic pathway.Dexmedetomidine reduced the respiratory rate and significantly enhanced the glymphatic pathway of hypothermic mice.The number of fluorescent probes injected into the cranial cavity of hyperthermic mice was significantly reduced in both groups.More probes entering the spinal canal of isoflurane anesthetized and hyperthermic mice.Decompression through cranial fenestration can reverse the inhibitory effect of hyperthermia on glymphatic pathway in mice under combined anesthesia.The heart rate of hyperthermia mice under combined anesthesia is significantly lower than that isoflurane anesthesia.Conclusion: The change of heart rate alone is not enough to regulate the transport of the glymphatic pathway.Respiratory rhythm and heart rate jointly regulate the glymphatic pathway.The increase of respiratory rate and amplitude will lead to the acceleration of fluid flow rate in the skull cavity and inhibit the glymphatic pathway.Low heart rate,low blood pressure,and high cerebrovascular compliance are effective parameters to enhance the glymphatic pathway.