Journal: Annals of Clinical and Translational Neurology

Article Title: Calcium-sensing receptor (CaSR) as a novel target for ischemic neuroprotection

doi: 10.1002/acn3.118

Figure Lengend Snippet: Generation and basal phenotype of Hipp CaSR −/− mice with CaSR KO targeted to hippocampus. (A) The Hipp CaSR −/− mice were made by breeding floxed-CaSR mice, which carried loxP sequences flanking the exon 7 of Casr genes, with CAMPKIIa-Cre mice expressing bacteriophage P1 Cre recombinase under the control of the CAMPKIIa gene promoter. (B) PCR analyses of genomic DNAs from hippocampi of control ( Casr wt/wt , Casr wt/flox , and Casr flox/flox mice without Cre expression), heterozygous Hipp CaSR +/− (Het), and homozygous Hipp CaSR −/− (Hom) mice were performed with specific primers to confirm the excision of exon 7 as indicated by the presence of a 284-bp cDNA band (ΔExon7) in the Het and Hom KO mice. (C) Immunohistological detection of CaSR in the CA1, CA3, and DG of the hippocampi from 6 months old Hipp CaSR −/− and control (Cont) mice shows that CaSR expression, depicted by brown DAB staining, in different regions of hippocampus in Cont mice is abrogated in the same regions of the KO mice. Bar: 20 μ m. (D) Vasculature patterns and sizes in Cont and Hipp CaSR −/− mice were examined after infusion with MICROFIL™ contrasting reagent (in red) (Flow Tech Inc., MA). Quantification scores for the sizes of left and right posterior communicating (PCOM) arteries shown in the right panel indicate comparable vascular sizes in the Cont and Hipp CaSR −/− mice. N = 4–6 mice. (E) Neurobehaviors were assessed by MWM tests on 4 months old male mice as described in Materials and Methods. Latency, distance traveled, and swimming speed during visible-platform (day 1–2) and hidden-platform (day 3–5) sections are not different between Hipp CaSR −/− (KO) and Cont littermates, indicating comparable ability of spatial learning. Nor did the % time spent in target zone (Zone 3), where the platform was set previously, differ in the final probe trial between the Cont and KO mice, suggesting comparable memory retention. Mean ± SE; n = 7 mice per group. CaSR, Ca 2+ -sensing receptor; KO, knockout; CA, cornu ammonis; DG, dentate gyrus; DAB, 3,3′-diaminobenzidine; MWM, Morris water maze.

Article Snippet: Mice were anesthetized with a cocktail of ketamine/acepromazine, heparinized, and sequentially perfused via the left ventricle with 25 mL of PBS, 4% PFA, and a colored (red) contrasting agent MICROFIL® (Flow Tech Inc., Carver, MA, USA).

Techniques: Expressing, Control, Staining, Knock-Out