C-type natriuretic peptide functions as an innate neuroprotectant in neonatal hypoxic-ischemic brain injury in mouse via natriuretic peptide receptor 2

Abstract

Neonatal hypoxia-ischemia (HI) is the most common cause of brain injury in neonates, which leads to high neonatal mortality and severe neurological morbidity in later life (Vannucci, 2000; Volpe, 2001). Yet the molecular mechanisms of neuronal death and brain damage induced by neonatal HI remain largely elusive. Herein, using both in vivo and in vitro models, we determine an endogenous neuroprotectant role of c-type natriuretic peptide (CNP) in preserving neuronal survival after HI brain injury in mouse pups. Postnatal day 7 (P7) mouse pups with CNP deficiency (Nppc^lbab/lbab) exhibit increased brain infarct size and worsened long-term locomotor function after neonatal HI compared with wildtype control (Nppc^+/+). In isolated primary cortical neurons, recombinant CNP dose-dependently protects primary neurons from oxygen-glucose deprivation (OGD) insult. This neuroprotective effect appears to be mediated through its cognate natriuretic peptide receptor 2 (NPR2), in that antagonization of NPR2, but not NPR3, exacerbates neuronal death and counteracts the protective effect of CNP on primary neurons exposed to OGD insult. Immunoblot and confocal microscopy demonstrate the abundant expression of NPR2 in neurons of the neonatal brain and in isolated primary cortical neurons as well. Moreover, similar to CNP deficiency, administration of NPR2 antagonist P19 via intracerebroventricular injection prior to HI results in exacerbated neuronal death and brain injury after HI. Altogether, the present study indicates that CNP and its cognate receptor NPR2 mainly expressed in neurons represent an innate neuroprotective mechanism in neonatal HI brain injury.

Keywords: C-type natriuretic peptide; Natriuretic peptide receptor 2; Neonatal hypoxic-ischemic brain injury; Neuronal death; Oxygen-glucose deprivation.

Fig. 1.

CNP deficiency (Nppc^lbab/lbab) increases the vulnerability of the neonatal brain to HI insult. (A) CNP gene mutated or wildtype (Nppc^+/+) mouse pups at postnatal day 7 (P7) were subjected to HI treatment (20 min of hypoxia, 8% O₂), and brain infarct size was detected 48 h after HI. n = 8 pups/group. B) Rotarod test for locomotor function evaluation performed one month after HI. Data are expressed as mean ± SEM. n = 9–10 pups/group, *, p < 0.05 vs. Nppc^+/+. ns, no significant difference. Two-tailed Student’s t-test.

Fig. 2.

Optimization of OGD treatment on primary neuron death. Primary cortical neurons were exposed to OGD treatment for 0.5, 1.0 or 1.5 h followed by reoxygenation for 24 h. Cells cultured in normal condition were used as control (Normal). Neuronal damage was determined by assessment of LDH release (A) and cell viability assay (B) at the end of reoxygenation. (B) Representative images of live (green) and dead (red) cells and quantification of the percentage of dead cells over total cells following the exposure of 1.0 or 1.5 h of OGD. Scale bar, 20 μm. Data are expressed as mean ± SEM. n = 3 independent experiments. *, p < 0.05 vs. Normal. ns, no significant difference. Two-tailed Student’s t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Recombinant CNP protects primary cortical neurons from OGD insult. Primary cortical neurons were incubated with 0, 5, 25, 100 or 500 nM of recombinant CNP for 6 h, and then exposed to OGD treatment for 1.0 h followed by reoxygenation for 24 h. LDH release (A), cell viability (B), and TUNEL staining (C) were performed at the end of reoxygenation. (B) Representative images of live (green) and dead (red) cells and quantification of the percentage of dead cells over total cells with recombinant CNP treatment (100 nM) after OGD/reoxygenation. Scale bar, 40 μm. (C) Representative images of TUNEL (red) staining and quantification of the TUNEL-positive cells over total cells with recombinant CNP treatment (100 nM) after OGD/reoxygenation. Data are expressed as mean ± SEM. n = 3 independent experiments. *, p < 0.05 vs. Normal. #, p < 0.05 vs. OGD. ANOVA following by Newman-Keuls post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

The neuroprotective effect of recombinant CNP is mediated by NPR2, but not NPR3. (A and B) Primary cortical neurons were incubated with 100 nM of recombinant CNP, NPR2 antagonist P19, or NPR3 antagonist AP811 alone, or combination of CNP with either P19 or AP811 for 6 h, and then exposed to OGD for 1.0 h followed by reoxygenation for 24 h. LDH release (A) and cell viability (B) were detected at the end of OGD/reoxygenation. (C and D) Primary cortical neurons were incubated with 0, 5, 25, 100 or 500 nm of ANP or BNP, and then exposed to OGD for 1.0 h followed by reoxygenation for 24 h. LDH release (C), and cell viability (D) were detected at the end of OGD/reoxygenation. Data are expressed as mean ± SEM. n = 4 independent experiments. *, p < 0.05 vs. Normal. #, p < 0.05 vs. Ctrl. &, p < 0.05 vs. CNP. ns, no significant difference. ANOVA following by Newman-Keuls post hoc test.

Fig. 5.

NPR2 is abundantly expressed in neuron cells. (A) the levels of NPR2 protein in cerebrovascular endothelial cells and neurons isolated from the cortices of mouse pups were detected by western blotting. (B) Representative confocal images of the colocalization of NPR2 (red) and neurons (NeuN, red) in the cerebral cortex (upper panel) and hippocampus (lower panel) in brain slices. Left, low magnification images; Right, high magnification images, scale bar, 20 μm. Bar graph shows the ratio of NPR2-positive neurons in total NPR2-positive cell in cortex and hippocampus. (C) Representative confocal images of the colocalization of NPR2 (green) and primary cortical neurons (MAP2, red). Left, low magnification images; Right, high magnification images, scale bar, 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.

NPR2 antagonist administration increases brain infarct size and neuronal death after neonatal HI. NPR2 antagonist P19 was administered into the ipsilateral hemisphere via i.c.v. injection prior to HI, then HI insult was conducted. (A) Representative images of TTC staining and quantification of brain infarct size 48 h after HI. Data are expressed as mean ± SEM. n = 8 pups/group. *, p < 0.05 vs. vehicle. Two-tailed Student’s t-test. (B) Representative confocal images of the colocalization of TUNEL (red) with neurons (NeuN, green) in the cortex of the ipsilateral hemisphere of the brain 48 h after HI. Scale bar, 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)