Neurotrophic effects of brain-derived neurotrophic factor and vascular endothelial growth factor in major pelvic ganglia of young and aged rats

Abstract

Objective: To investigate the neurotrophic effect of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in cultured major pelvic ganglia (MPG) derived from young and aged rats.

Materials and methods: The dorsocaudal region of the MPG was isolated from 12 6-month-old male rats and 12 24-month-old male rats. The MPGs were treated with BDNF, VEGF, or both, at 0, 12.5, 25, 50, 100 and 150 ng/mL to determine the effective concentration for 50% activity (EC(50)) and optimum dosage for promoting neurite growth. Neurite outgrowth from treated MPGs was measured by microscopy. NADPH diaphorase and tyrosine hydroxylase (TH) staining was used to characterize neurites.

Results: Both BDNF and VEGF promoted neurite sprouting from MPG. Neurite growth was more robust in MPGs derived from young rats (6 months) than from aged rats (24 months). The EC(50) for BDNF, VEGF and combined treatment were 10.6, 11.9 and 52 ng/mL in young rats, and 11.3, 12 and 0.75 ng/mL in old rats, respectively. The optimum dosage of both factors for promoting MPG neurite growth in all groups was 25-50 ng/mL. VEGF appeared to favour NADPH diaphorase-positive neurites, whereas BDNF favoured TH-positive neurites.

Conclusion: BDNF and VEGF promote neurite growth from cultured MPG; combined treatment produced the most robust neurite outgrowth. Neurite growth from MPGs derived from aged rats was not as robust as it was from MPGs from younger rats. Further studies on the effect of neurotrophins after cavernous nerve injury are warranted.

FIG. 1

Neurites sprouting from MPG after stimulation with BDNF, VEGF and VEGF+BDNF at 48 h. The MPG were embedded in RGFM and cultured in vitro. Neurotrophic factors at 0, 12.5, 25, 50, 100 and 150 ng/mL were added to the culture solution. Neurites were longer at all dosages of neurotrophic factors than were control neurites. The number in the upper left hand corner is the number of neurites in the given image, and the arrow indicates the longest neurite in each field. ×100.

FIG. 2

Difference of neurites from young and old MPG at 48 h; the MPG from 6-month-old ‘young’ rats (top panel) and 24-month-old ‘aged rats’ (bottom panel) were treated with 25 ng/mL BDNF (A,D), VEGF (B,E) and VEGF+BDNF (C,F) for 48 h. The combined factors (C,F) induced longer neurites than either factor alone. Neurite outgrowth was more robust in the young group. The number in the upper left hand corner is the number of neurites in the given image, and the arrow indicates the longest neurite in each field. ×100.

FIG. 3

Neurite length from MPG at 48 h; MPGs from young and old rats were treated with BDNF, VEGF, or BDNF+VEGF. The mean (sd) length of the neurites (12 for each point) is shown. BDNF- and VEGF-treated MPGs had longer neurites than control MPGs at most concentrations (#P < 0.05 vs control MPG within the same age group). Compared to young MPG, the aged MPG produced significantly shorter neurites at any dose (*P < 0.05 for young vs aged MPG when comparing similar concentrations of neurotrophic factors).

FIG. 4

Neurite length from MPGs at 72 hl MPGs from young and old rats were treated with BDNF, VEGF or BDNF+VEGF. The mean (sd) length of the neurites (12 for each point) is shown. The neurites were longer by ≈100 μm on average than at 48 h. BDNF- and VEGF-treated neurites tended to be significantly longer than control neurites at most doses (#P < 0.05 vs control MPG within the same age group). Neurites in the aged group tended to be shorter than in the young group at 72 h, but the difference was statistically significant only in the groups treated with the lowest concentrations (12.5 and 25 ng/mL) of VEGF alone (*P < 0.05 for young vs aged MPG when comparing similar concentrations of neurotrophic factors).

FIG. 5

NADPH diaphorase and TH staining of neurites at 72 h; NADPH diaphorase (A) and TH (B) immunostaining was used to characterize neurites. Neurites positive for NADPH diaphorase were stained blue and neurites positive for TH were stained brown. (C). VEGF with or without BDNF led to more NADPH diaphorase-positive neurites than with BDNF alone (a, *P < 0.05 vs MPG treated with BDNF only). BDNF with or without VEGF led to more TH-positive neurites than MPG treated with VEGF only (b, #P < 0.05 vs VEGF only). There was no significant difference in TH and NAPDH diaphorase positivity between young and aged rats in any group (P > 0.05).