Middle aged CAMKII-Cre:Cbsfl/fl mice: a new model for studying perioperative neurocognitive disorders

Figures

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  Fig. 1.. Cbs was successfully deleted in CAMKII-Cre cells. (A, B) Protein expression of CBS in the hippocampus was analyzed by western blotting. GAPDH was used as a loading control (mean ± SD, unpaired t-test, ****P<0.0001, n=3). (C) Relative expression of Cbs mRNA in the hippocampus was analyzed by real-time PCR. Gapdh was used as a loading control (mean ± SD, unpaired t-test, ****P<0.0001, n=3).

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  Fig. 2.. Anesthesia and surgery induced significant cognitive deficits in 8-month-old CAMKII-Cre:Cbsfl/fl mice. (A) Experimental timeline of surgery and behavioral tests. (B) Representative movement tracks in the open-field test. (C) Time spent in the central zone in the open-field test (ns: no significant difference, mean ± SD, One-way ANOVA). WT: n=8, Littermate: n=6, CBS-CKO: n=7. (D) Locomotive ability was evaluated by the average speed in the open-field test (ns: no significant difference, mean ± SD, one-way ANOVA). (E) and (F) show the statistical results of spatial learning (mean ± SEM, two-way ANOVA, **P<0.01, ****P<0.0001) during four consecutive training days and spatial memory (mean ± SD, one-way ANOVA, ****P<0.0001) on the test day of the Barnes maze test. (G) Contextual memory was also evaluated by the ratio of freezing time in the contextual fear conditioning test (mean ± SD, one-way ANOVA, ***P<0.001, ****P<0.0001).

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  Fig. 3.. 8-month-old CAMKII-Cre:Cbsfl/fl mice demonstrated similar cognitive deficits to 18-month-old wild type counterparts after undergoing exploratory laparotomy. (A) Experimental timeline of surgery and behavioral tests. (B) Representative movement tracks in the open-field test. (C) Time spent in the central zone in the open-field test (ns: no significant difference, mean ± SD, unpaired t-test). PND: n=8, CBS-CKO: n=7. (D) Locomotive ability was evaluated by the average speed in the open field test (ns: no significant difference, mean ± SD, unpaired t-test). (E) and (F) show the statistical results of spatial learning (ns: no significant difference, mean ± SEM, two-way ANOVA) during four consecutive days and spatial memory (ns: no significant difference, mean ± SD, unpaired t-test) on the test day of the Barnes maze tests. (G) Contextual memory was also evaluated by the ratio of freezing time in the contextual fear conditioning test (ns: no significant difference, mean ± SD, unpaired t-test).

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  Fig. 4.. Immunofluorescence staining of the CA1 region of the hippocampus two days after isoflurane anesthesia and exploratory laparotomy. (A) Representative confocal microscopic images of Iba1 (red) and DAPI (blue) expression in the CA1 region of wild-type, littermates, and CAMKII-Cre:Cbsfl/fl mice, scale bar, 50 µm. (B) Representative confocal microscopic images of GFAP (red) and DAPI (blue) expression in the CA1 region of wild-type, littermates, and CAMKII-Cre:Cbsfl/fl mice, scale bar, 50 µm. (C) Iba1 expression of CAMKII-Cre:Cbsfl/fl mice in the CA1 region was remarkably increased compared to that of wild-type and littermate mice (mean ± SD, One way ANOVA, ****P<0.0001, n=9 slices from 3 mice, 3 views per slice). (D) GFAP expression of CAMKII-Cre:Cbsfl/fl mice in the CA1 region was remarkably increased compared to that of wild-type and littermate mice (mean ± SD, One way ANOVA, ****P<0.0001, n=9 slices from 3 mice, 3 views per slice).

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  Fig. 5.. Cbs conditional knockout in CAMKII positive neurons did not cause abnormality in the brain morphology. (A) Representative microscopic images of cresyl violet stained neurons in the dentate gyrus (DG) of wild-type, littermates, and CAMKII- Cre:Cbsfl/fl mice, scale bar, left: 100 µm, right: 50 µm. (B) There was no significant difference in the density of hippocampal neurons of wild-type, littermates, and CAMKII-Cre:Cbsfl/fl mice (ns: no significant difference, mean ± SD, One-way ANOVA, n=3). (C) Representative microscopic images of cresyl violet stained neurons in the cortex of wild-type, littermates, and CAMKII- Cre:Cbsfl/fl mice, scale bar, left: 100 µm, right: 50 µm. (D) There was no significant difference in the density of cortical neurons of wild-type, littermates, and CAMKII-Cre:Cbsfl/fl mice (ns: no significant difference, mean ± SD, One-way ANOVA, n=3).