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Calbindin-1 Expression in the Hippocampus following Neonatal Hypoxia-Ischemia and Therapeutic Hypothermia and Deficits in Spatial Memory - PubMed

  • ️Tue Jan 01 2019

Calbindin-1 Expression in the Hippocampus following Neonatal Hypoxia-Ischemia and Therapeutic Hypothermia and Deficits in Spatial Memory

Janasha Goffigan-Holmes et al. Dev Neurosci. 2019.

Abstract

Hippocampal injury following neonatal hypoxia-ischemia (HI) leads to memory impairments despite therapeutic hypothermia (TH). In the hippocampus, the expression of calbindin-1 (Calb1), a Ca2+-buffering protein, increases during postnatal development and decreases with aging and neurodegenerative disorders. Since persistent Ca2+ dysregulation after HI may lead to ongoing injury, persistent changes in hippocampal expression of Calb1 may contribute to memory impairments after neonatal HI. We hypothesized that, despite TH, neonatal HI persistently decreases Calb1 expression in the hippocampus, a change associated with memory deficits in the mouse. We induced cerebral HI in C57BL6 mice at postnatal day 10 (P10) with right carotid ligation and 45 min of hypoxia (FiO2 = 0.08), followed by normothermia (36°C, NT) or TH (31°C) for 4 h with anesthesia-shams as controls. Nissl staining and glial fibrillary acidic protein (GFAP) immunohistochemistry (IHC) were used to grade brain injury and astrogliosis at P11, P18, and P40 prior to the assessment of Calb1 expression by IHC. The subset of mice followed to P40 also performed a memory behavior task (Y-maze) at P22-P26. Nonparametric statistics stratified by sex were applied. In both anterior and posterior coronal brain sections, hippocampal Calb1 expression doubled between P11 and P40 due to an increase in the cornus ammonis (CA) field (Kruskal-Wallis [KW] p < 0.001) and not the dentate gyrus (DG). Neonatal HI produced delayed (P18) and late (P40) deficits in the expression of Calb1 exclusively in the CA field (KW p = 0.02) in posterior brain sections. TH did not attenuate Calb1 deficits after HI. Thirty days after HI injury (at P40), GFAP scores in the hippocampus (p < 0.001, r = -0.47) and CA field (p < 0.001, r = -0.39) of posterior brain sections inversely correlated with their respective Calb1 expression. Both sexes demonstrated deficits in Y-maze testing, including approximately 40% lower spontaneous alterations performance and twice as much total impairment compared to sham mice (KW p < 0.001), but it was only in females that these deficits correlated with the Calb1 expression in the hippocampal CA field (p < 0.05) of the posterior sections. Hippocampal atrophy after neonatal HI also correlated with worse deficits in Y-maze testing, but it did not predict Calb1 deficits. Neonatal HI produces a long-lasting Calb1 deficit in the hippocampal CA field during development, which is not mitigated by TH. Late Calb1 deficit after HI may be the result of persistent astrogliosis and can lead to memory impairment, particularly in female mice.

Keywords: Astrogliosis; Calbindin; Memory; Neonatal brain injury.

© 2019 S. Karger AG, Basel.

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Conflict of interest statement

DISCLOSURE STATEMENT

We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Figures

Figure 1.
Figure 1.. Developmental changes in Calb1 expression in the hippocampus.

Representative Calb1 IHC of hippocampal sections in anterior (A1 to A3) and posterior (B1 to B3) coronal sections at P11 (A1 and B1), P18 (A2 and B2), and P40 (A3 and B3). Bar, 1000 μm. Two photomicrographs (4X) of the hippocampus in the posterior coronal section (i and ii), were used to compose the final figure (B1 to B3). Box and whiskers plot represent mean optical densities (OD) adjusted for background for Calb1 immunoreactivity in anterior and posterior coronal brain sections for the whole hippocampus (A4 and B4, respectively), the CA field (A5 and B5, respectively), and DG (A6 and B6, respectively). Boxes are limited by the 75th and 25th percentiles (interquartile range, IQR) and whiskers are limited by the last data point within 1.5 times the IQR from the median (continuous line inside the box). Analysis by Kruskal-Wallis ANOVA with Dunn-Bonferroni correction; *, adjusted p value <0.05. CA, cornus ammonis; DG, dentate gyrus.

Figure 2.
Figure 2.. Impaired hippocampal expression of Calbi in posterior coronal sections following HI and lack of protection by TH at P18.

Box and whiskers plot represent mean optical densities (OD) adjusted for background for Calb1 immunoreactivity in whole hippocampus (A1 and B1), CA field (A2 and B2), and DG (A3 and B3) in anterior and posterior, respectively. Boxes are limited by the 75th and 25th percentiles (interquartile range, IQR) and whiskers are limited by the last data point within 1.5 times the IQR from the median (continuous line inside the box). Analysis by Kruskal-Wallis ANOVA with Dunn-Bonferroni correction; *, adjusted p-value <0.05. Representative Calb1 IHC section hippocampus within posterior coronal brain sections of sham (B4), NT (B4’), and TH (B4”). Bar, 1000 μm. Two photomicrographs (4X) of the hippocampus (i and ii) within the posterior coronal section were used to compose the final figure (B4). CA, cornus ammonis; DG, Dentate gyrus; NT, normothermia; TH, therapeutic hypothermia.

Figure 3.
Figure 3.. Persistent Calbi deficits in the hippocampus in posterior coronal sections following HI linked to persistent astrogliosis at P40.

Box and whiskers plot represent mean optical densities (OD) adjusted for background for Calb1 immunoreactivity in whole hippocampus (A1), CA field (A2), and DG (A3) in anterior and posterior coronal sections. Boxes are limited by the 75th and 25th percentiles (interquartile range, IQR) and whiskers are limited by the last data point within 1.5 times the IQR from the median (continuous line inside the box). Analysis by Kruskal-Wallis ANOVA with Dunn-Bonferroni correction; *, adjusted p-value <0.05. Representative Calb1 IHC hippocampal section in posterior brain sections of sham (A4), NT (A4’), and TH (A4”). Bar, 1000 μm. Two photomicrographs (4X) of the hippocampus (i and ii) within posterior coronal sections were used to compose final figure (A4). Calb1 expression in the whole hippocampus (B1), CA field (B2), and DG (B3) of posterior coronal sections were correlated with their respective GFAP score. Spearman correlations were applied. Continuous line represents the predicted linear regression and discontinuous lines represent the projection to the 95% confidence interval. p-value < 0.05 were considered significant. CA, cornus ammonis; DG, Dentate gyrus; GFAP, glial fibrillary acidic protein; HIP, hippocampus; NT, normothermia; Post, posterior; TH, therapeutic hypothermia.

Figure 4.
Figure 4.. Temporal evolution of GFAP immunoreactivity between P11 to P40 in sham and HI-injure mice treated with NT or TH.

Representative GFAP IHC of posterior brain sections containing hippocampi of sham, normothermia (NT), and therapeutic hypothermia (TH). Bar, 1000 μm. Two photomicrographs (4X) of the hippocampus (i and ii) within the posterior coronal section, were used to compose final figure. White arrows, areas of columnar injury or cellular loss within the CA pyramidal cell layer (Py). White arrow heads, glial scars with the CA Py. Black arrow heads, area of diffuse astrogliosis with the CA Py.

Figure 5.
Figure 5.. Memory deficits in Y-maze task correlated with Calb1 deficits in the hippocampus of posterior coronal sections following HI at P40.

Box and whiskers plot represent percentage of spontaneous alternation performance (SAP, A), impairment (sum of alternate arm returns and same arm returns, B) and time spend in new arm during Y-maze phase 2 (C), stratified by sex. Boxes are limited by the 75th and 25th percentiles (interquartile range, IQR) and whiskers are limited by the last data point within 1.5 times the IQR from the median (continuous line inside the box). Analysis by Kruskal-Wallis ANOVA with Dunn-Bonferroni correction; *, adjusted p-value <0.05. Calb1 expression in the whole hippocampus (A1, B1 and C1) and CA field (A2, B2 and C2) in posterior coronal sections were correlated with % SAP (A1 and A2), % impairment (B1 and B2), and % time in new arm (C1 and C2) stratified by sex (i, males; ii, females). Spearman correlations were applied. Continuous line represents the predicted linear regression and discontinuous lines represent the projection to the 95% confidence interval. p-value < 0.05 were considered significant. CA, cornus ammonis; NS, nonsignificant; NT, normothermia; TH, therapeutic hypothermia.

Figure 6.
Figure 6.. Hippocampal atrophy at P40 resulting from neonatal HI at P10 does not correlate with Calb1 deficits.

Box and whiskers plot (A) represent the residual hippocampal volumes in mm3 for of sham, normothermia (NT), and therapeutic hypothermia (TH). Boxes are limited by the 75th and 25th percentiles (interquartile range, IQR) and whiskers are limited by the last data point within 1.5 times the IQR from the median (continuous line inside the box). Analysis by Kruskal-Wallis ANOVA with Dunn-Bonferroni correction; *, adjusted p-value <0.05. Calb1 expression in the whole hippocampus were correlated with residual hippocampal volumes in males and female mice (B). Spearman correlations were applied. Continuous line represents the predicted linear regression and discontinuous lines represent the projection to the 95% confidence interval. p-value < 0.05 were considered significant.

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