Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors

Abstract

In postnatal tissues, angiogenesis occurs in nontumoral conditions on appropriate stimuli. In the nervous tissue, hypoxia, neural graft, increased neural function, and synaptic activity are associated with neoangiogenesis. We have investigated the occurrence of neoangiogenesis in the superior cervical ganglia (scg) of newborn rats treated for 8--21 days with 6-hydroxy-dopamine (6-OHDA), nerve growth factor (NGF), or 6-OHDA + NGF. The two latter treatments induced a significant increase in scg size. However, the increase after combined treatment far exceeded that of NGF alone. Similarly, histological and histochemical analysis revealed neuronal hypertrophy and endothelial cell hyperplasia associated with stromal hypertrophy (as described by laminin immunostaining) and increased vascular bed (as revealed by platelet/endothelial cell adhesion molecule-1 immunostaining) in 6-OHDA + NGF-treated pups. NGF, either alone or associated with 6-OHDA, also induced a significant up-regulation of NADPH diaphorase, neuronal nitric oxide synthase, and vascular endothelial growth factor expression in scg neurons. The present investigation suggests that the increase of scg size induced by NGF and 6-OHDA + NGF is associated with neoangiogenesis, and that the induction of vasoactive and angiogenic factors in neurons represents a further and previously undisclosed effect of NGF.

Figure 1

(A) Whole-mount preparation of superior cervical ganglia of littermate rats (26 days) treated with NGF and 6-OHDA, NGF, 6-OHDA, and saline. 6-OHDA administration induces a well known degeneration of the sympathetic neurons, which, on the contrary, is prevented by NGF treatment. scg are larger in NGF-treated pups than in control pups; however, growth induced by double treatment is impressive. Toluidine-blue micrographs show general morphology in control (B), NGF (C), and 6-OHDA + NGF- (D) treated pups. High-magnification micrographs show intragangliar blood vessels in controls (E), NGF-treated (F and G), and 6-OHDA + NGF-treated (H–M) pups. In the latter group, within the vessel and capillary wall, high cellularity can be observed, and buds of newly formed vessels are clearly detectable (arrow, H). (Bars = B–D, 200 μm; E–M, 100 μm.)

Figure 2

Histochemical composition of scg after treatment. The figure shows laminin, p75, and PECAM-1 IR in scg of controls (Left) and pups treated for 8 days with 6-OHDA associated with NGF (Right). The graph shows the quantitative histochemical composition. Data are expressed as area covered by the immunoreactive signal inside a standard sampling window (proportional area). As also seen in micrographs, in the 6-OHDA-treated group laminin IR occupies the largest proportional area because of the very low presence of neuronal component. In double treatment preparations, a true increase of the stromal component can be measured. p75 IR can be detected intra- and extracellularly: the percentage related to its proportional area increases in NGF and 6-OHDA + NGF-treated pups. PECAM-1 IR depicts nets of newly formed vessels in double-treated pups. An overall increase of the percent of area covered by PECAM-1 IR can be measured. Statistical evaluation: ANOVA and Dunnett's test; *, P < 0.05. [Bars = 100 (laminin also for p75) and 50 (PECAM-1) μm.]

Figure 3

VEGF IR and NADPH–diaphorase staining in neurons in scg of controls and newborn rats treated with NGF (1 and 8 days) and 6-OHDA + NGF (8 days) and density evaluation. Eight days of NGF treatment induces a significant increase in VEGF expression; a greater increase occurs after concomitant 6-OHDA and NGF administration. Single NGF administration strongly up-regulates NADPH–diaphorase staining, but the increase induced by 8 days of combined 6-OHDA and NGF treatment far exceeds that of NGF single administration. Statistical analysis: ANOVA and Dunnett's test; *, P < 0.05. (Bar = 100 μm.)