Methods to study glymphatic system in the rodent brain during physiological and pathological processes

Abstract: Over the past decade, our understanding of brain-waste clearance underwent a revolution after the discovery of the so-called glymphatic system. The glia-lymphatic (abbreviated, glymphatic) system was first described in 2012 as a paravascular macrosystem involved in the distribution of nutrients and clearance of metabolic waste from the brain parenchyma. The main actor of the system is the cerebrospinal fluid (CSF), which from the subarachnoid space (SAS) flow into the perivascular space (PVS) of brain penetrating arteries, and from there into the brain parenchyma, facilitated by aquaporin-4 (AQP4) water channels on astrocitic endfeet surrounding blood vessels.Intriguingly, once in the parenchyma, the CSF mixes with the interstitial fluid (ISF), releasing nutrients and collecting metabolic wastes and toxic proteins, including Aβ and Tau, two proteins involved in the pathogenesis of Alzheimer´s disease (AD).Importantly, recent studies have shown that the glymphatic system, and therefore its function in “cleaning” the brain from toxic wastes and proteins, is impaired during aging. Age is the main risk factor for developing neurodegenerative disease caused by accumulation of toxic proteins, e.g. AD or Parkinson´s diseases (PD). Therefore, the understanding of how glymphatic system functions is of utmost importance.Because of the novelty of this field, it is needed to find consensus on the techniques and methodology to study glymphatic system, as well as interpretation of the results.The aim of this thesis was then to describe methodologies useful for glymphatic studies in rodents, and to apply these methods to study CSF movement in the brain both in physiological and pathological settings (i.e., hypothermia and PD).Specifically, in Paper I we described the CM injection method to study glymphatic function in the rodents´ brain. In Paper II, we quantitatively compare the efficiency of different lectins and staining methods to label vasculature in rodents, as an important step for AQP4 polarisation studies. In Paper III, we investigate how AQP4 is affected byantisense oligonucleotides (ASO) targeting Aqp4 mRNA. In Paper IV, we investigated glymphatic system function in two different mouse models of PD, and test whether glymphatic system is involved in the clearance of α-syn (a protein involved in PD pathogenesis) from the brain parenchyma. Finally, in Paper V we explored glymphaticfunction and AQP4 in hypothermia, a condition often related to anaesthesia.Overall, this thesis helped the field of brain fluid dynamics investigation by providing a description of the techniques that can be used in pre-clinical research to investigate glymphatic function on a macroscopic level and dissect the microscopic players of the system. The methods described in this thesis can be adapted to the investigation of glymphatic function in different physiological and pathological settings, as well as different preclinical models. Advancing pre-clinical research with reproducible and standardised methods is fundamental for following translational applications.