6/1/2023 0 Comments Essential anatomy 3 freeBriefly, preheated (37 ☌) Enzyme mix 1 was added to each sample, and incubated under continuous rotation (100 rpm) for 15 min. The supernatant was carefully removed, and the pellet enzymatically dissociated using an Adult Brain Dissociation Kit (130-107-677, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Suspensions were transferred to 15 mL tubes and ice-cold HBSS was added to a final volume of 4 mL, then centrifuged at 300× g for 5 min at 4 ☌. Brains were mechanically dissociated with sterile scissors in HBSS and further disrupted by pipetting up and down with 1 mL pipette tips until a homogenous suspension of ~1 mm 3 pieces was achieved. Brains were dissected out, the cerebellum and olfactory bulb removed and the rest suspended in ice-cold HBSS (#14175129, Gibco, Carlsbad, CA, USA). WT ( n = 8, including 6 male and 2 female) and Aqp9 −/− ( n = 8, including 6 male and 2 female) adult mice were deeply anesthetized and transcardially perfused with heparin saline (0.05% heparin (5000 IU/mL) in 0.9% NaCl sterile saline) solution 20 mL/min for 4 min. These antibody-based studies need to be complemented by other methodological approaches to get a more accurate picture of AQP9 localization in the brain. In a more recent study, the expression of AQP9 in the mouse brain, and in particular substantia nigra, was confirmed using Aqp9 knock-out mice as controls. Later and more elaborate analyses based on high-resolution immunogold cytochemistry revealed AQP9 in astrocytic plasma membranes and mitochondria in the rat brain. Early immunocytochemical analyses identified AQP9 in tanycytes, astrocytes, and dopaminergic neurons. More than 20 years have elapsed since AQP9 was identified in the brain, yet there are still significant voids in our knowledge as to the localization and function of this aquaporin in brain tissue. The present analysis provides novel insight into the role of AQP9 in the brain and opens new avenues for research in the field of neuroinflammation and chronic neurodegenerative disease.Īquaporin-9 (AQP9) is a member of the aquaglyceroporin subfamily, with permeability to a wide range of noncharged solutes such as glycerol, urea, monocarboxylates, purines, and pyrimidines in addition to water. Further, in isolated cell subsets, validated by flow cytometry we demonstrated that Aqp9 transcripts are expressed in microglial cells, albeit at lower concentrations than in astrocytes. After intrastriatal injections of MPP +, the increase in transcript levels of proinflammatory genes was less pronounced in AQP9 −/− mice compared with wild-type controls. This toxin induces a strong inflammatory response in brain. ![]() Our results show that targeted deletion of Aqp9 significantly suppressed the inflammatory response to the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP +). We also explored whether Aqp9 is expressed in microglial cells, which would be supportive of this hypothesis. ![]() ![]() In this study, we hypothesized that AQP9 plays a proinflammatory role in the brain, analogous to its role in the periphery. In peripheral tissues, AQP9 is expressed in leukocytes where it is involved in systemic inflammation processes. Yet its precise localization and function in brain tissue remain unresolved. More than 20 years have passed since the first demonstration of Aquaporin-9 (AQP9) in the brain.
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