Fibroblast Growth Factor-2 Induced by Enriched Environment Enhances Angiogenesis and Motor Function in Chronic Hypoxic-Ischemic Brain Injury

<div><p>This study aimed to investigate the effects of enriched environment (EE) on promoting angiogenesis and neurobehavioral function in an animal model of chronic hypoxic-ischemic (HI) brain injury. HI brain damage was induced in seven day-old CD-1® mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At six weeks of age, the mice were randomly assigned to either EE or standard cages (SC) for two months. Rotarod, forelimb-use asymmetry, and grip strength tests were performed to evaluate neurobehavioral function. In order to identify angiogenic growth factors regulated by EE, an array-based multiplex ELISA assay was used to measure the expression in frontal cortex, striatum, and cerebellum. Among the growth factors, the expression of fibroblast growth factor-2 (FGF-2) was confirmed using western blotting. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and α-smooth muscle actin (α-SMA) were also evaluated using immunohistochemistry. As a result, mice exposed to EE showed significant improvements in rotarod and ladder walking performances compared to SC controls. The level of FGF-2 was significantly higher in the frontal cortex of EE mice at 8 weeks after treatment in multiplex ELISA and western blot. On the other hand, FGF-2 in the striatum significantly increased at 2 weeks after exposure to EE earlier than in the frontal cortex. Expression of activin A was similarly upregulated as FGF-2 expression pattern. Particularly, all animals treated with FGF-2 neutralizing antibody abolished the beneficial effect of EE on motor performance relative to mice not given anti-FGF-2. Immunohistochemistry showed that densities of α-SMA⁺ and PECAM-1⁺ cells in frontal cortex, striatum, and hippocampus were significantly increased following EE, suggesting the histological findings exhibit a similar pattern to the upregulation of FGF-2 in the brain. In conclusion, EE enhances endogenous angiogenesis and neurobehavioral functions mediated by upregulation of FGF-2 in chronic hypoxic-ischemic brain injury.</p></div

Environmental enrichment also enhanced endogenous angiogenesis in the hippocampus.

<p>(A, B) After exposure to EE, the capillary count and densities of PECAM-1⁺ cells were quantified using the MetaMorph Imaging System. Scale bar 150 μm. (A) Capillary count was increased in the dentate gyrus of the hippocampus at 8 weeks following the exposure to EE. (B) In particular, the densities of PECAM-1 in the EE mice were significantly higher than those in the SC controls at 8 weeks after treatment (^*<i>p</i><0.05, n = 5 each). HIE: hypoxic-ischemic encephalopathy, EE: enriched environment, SC: standard cages.</p

Western blot analysis also confirmed activin A upregulation in the brain.

<p>(A–C) Analysis of activin A level in the striatum, cerebellum, and frontal cortex using western blotting. (A) Activin A was significantly upregulated in the striatum at 2 weeks after exposure to EE (^*<i>p</i><0.05, n = 3 each). (B) The activin A in the cerebellum is also highly expressed at 2 weeks after EE treatment (n = 3 each). (C) On the other hand, activin A significantly increased in the striatum at 8 weeks after exposure to EE (^*<i>p</i><0.05, n = each). HIE: hypoxic-ischemic encephalopathy, EE: enriched environment, SC: standard cages.</p

Environmental enrichment enhanced endogenous angiogenesis in the frontal cortex and striatum.

<p>(A–H, K–R) Two and 8 weeks after exposure to EE, the densities of α-SMA⁺ cells and PECAM-1⁺ cells were quantified using the MetaMorph Imaging System. Scale bar 50 μm. (I) The density of α-SMA⁺ cells was significantly higher in frontal cortex of EE mice than SC controls 8 weeks after treatment (^*<i>p</i><0.05, n = 5 each). (J) The HIE-EE mice also showed an increase in PECAM-1⁺ angiogenesis 2 weeks and 8 weeks after treatment (^*<i>p</i><0.05, n = 5 each). (S, T) The densities of α-SMA⁺ cells (S) and PECAM-1⁺ cells (T) were higher in the striatum of EE mice than SC controls at 2 weeks after treatment (^*<i>p</i><0.05, ^†<i>p</i><0.1, n = 5 each). (T) The densities of PECAM-1⁺ cells were also significantly increased at 8 weeks following the EE treatment (^*<i>p</i><0.05, n = 5 each). HIE: hypoxic-ischemic encephalopathy, EE: enriched environment, SC: standard cages.</p

Infusion of FGF-2 neutralizing antibody reversed functional recovery.

<p>(A, B) To confirm the relationship between EE-induced FGF-2 upregulation and behavioral benefits, subgroups of EE and SC mice with HI brain injury were randomly assigned to either SC/anti-FGF-2 (n = 3) or EE/anti-FGF-2 (n = 4). (A) All animals treated with anti-FGF-2 abolished the beneficial effect on motor performance relative to EE mice not given anti-FGF-2 at 8 weeks after treatment (^*<i>p</i><0.05). (B). Mice treated with EE/anti-FGF-2 also showed the decreased grip strength whereas grip strength of EE mice not given anti-FGF-2 was significantly increased compared to the other groups (^*<i>p</i><0.05).</p