Further Evidence of Neuroprotective Effects of Recombinant Human Erythropoietin and Growth Hormone in Hypoxic Brain Injury in Neonatal Mice

Abstract

Experimental in vivo data have recently shown complementary neuroprotective actions of rhEPO and growth hormone (rhGH) in a neonatal murine model of hypoxic brain injury. Here, we hypothesized that rhGH and rhEPO mediate stabilization of the blood−brain barrier (BBB) and regenerative vascular effects in hypoxic injury to the developing brain. Using an established model of neonatal hypoxia, neonatal mice (P7) were treated i.p. with rhGH (4000 µg/kg) or rhEPO (5000 IU/kg) 0/12/24 h after hypoxic exposure. After a regeneration period of 48 h or 7 d, cerebral mRNA expression of Vegf-A, its receptors and co-receptors, and selected tight junction proteins were determined using qRT-PCR and ELISA. Vessel structures were assessed by Pecam-1 and occludin (Ocln) IHC. While Vegf-A expression increased significantly with rhGH treatment (p < 0.01), expression of the Vegfr and TEK receptor tyrosine kinase (Tie-2) system remained unchanged. RhEPO increased Vegf-A (p < 0.05) and Angpt-2 (p < 0.05) expression. While hypoxia reduced the mean vessel area in the parietal cortex compared to controls (p < 0.05), rhGH and rhEPO prevented this reduction after 48 h of regeneration. Hypoxia significantly reduced the Ocln+ fraction of cortical vascular endothelial cells. Ocln signal intensity increased in the cortex in response to rhGH (p < 0.05) and in the cortex and hippocampus in response to rhEPO (p < 0.05). Our data indicate that rhGH and rhEPO have protective effects on hypoxia-induced BBB disruption and regenerative vascular effects during the post-hypoxic period in the developing brain.

Keywords: VEGF-A; angiogenesis; angiopoietins; blood–brain barrier; hypoxia; hypoxic brain injury; neurovascular unit; occludin; tight junction proteins; vasculogenesis.

Figure 1

VEGF-A gene (A,B) and protein (C,D) expression and Angpt-2 gene expression (E,F) in normoxic and hypoxic developing mouse brains after a regeneration period of 48 h (A,C) or 7 d (B,D). *, p < 0.05; **, p < 0.01.

Figure 2

Vascular development in normoxic and hypoxic developing mouse brains with and without rhGH and rhEPO treatment. The vessel area in the parietal cortex (A,B) and hippocampus (C,D) was quantified by Pecam-1 IHC after a regeneration period of 48 h (A,C) and 7 d (B,D). Data are presented as mean ± SEM. *, p < 0.05.

Figure 3

Gene expression of Ocln relative to PBGD in normoxic and hypoxic brains of neonatal mice with and without rhGH and rhEPO treatment after a regeneration period of 48 h (A) or 7 d (B). *, p < 0.05.

Figure 4

Quantification of OCLN⁺ area by immunofluorescence analysis of OCLN and PECAM-1 protein in normoxic and hypoxic developing mouse brains with and without rhGH and rhEPO treatment after a regeneration period of 48 h (A,C) and 7 d (B,D) in the parietal cortex (A,B) and hippocampus (C,D). Data are presented as mean ± SEM. *, p < 0.05.

Figure 5

Representative photomicrographs of PECAM-1 (green) and OCLN (red) protein co-staining of vascular endothelial cells in hypoxic developing mouse brains (B,D,F,H) compared to normoxic controls (A,C,E,F) after a 48 h regeneration period in NT (A,B), VT (C,D), rhGH- (E,F), and rhEPO-treated brains (G,H) in the parietal cortex. Blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain.

Figure 6

Protein levels of (A) IL-2, (B) IL-6, and (C) IL-10 in normoxic and hypoxic mouse brains after a 48 h regeneration period. *, p < 0.05; **, p < 0.01; ***, p < 0.001.