The Effects of Nerve Growth Factor on Adult and Aged Dorsal Root Ganglion Neurons Maintained in Primary Culture

Jiang, Zhi-Gang (1994) The Effects of Nerve Growth Factor on Adult and Aged Dorsal Root Ganglion Neurons Maintained in Primary Culture. PhD thesis, University of Glasgow.

Abstract

Nerve growth factor (NGF) is, a prototype neurotrophic factor, well documented for its biochemical character, distribution and functions in embryonic and neonatal animals. However, only very tentative evidence exists for its effects on mature neurons, and results are controversial. Studying NGF by using mature neurons should be a significant and attractive field, not only for its clinical value in nerve regeneration by adults, but also for understanding the mechanisms of aging. Studies in vitro present a comparatively simple environment for analysis of the effective factors. Dorsal root ganglia (DRGs), containing an enormous heterogeneity of sensory neuronal types, are a useful model in understanding the nervous system. Hence, in the present study, mouse DRG neurons were maintained in vitro supplemented with NGF V5 cultures without NGF or with anti-NGF present, to examine any NGF effects on survival, morphological phenotype, neuropeptide expressions and neurite outgrowth (equivalent to regeneration in vitro). Comparison of adult (6 months) and aged (2 years) animals was made throughout the present study. In the survival study, a range of NGF (25-200ng/ml) was added to adult neurons in co-cultures, and neuronal survival monitored for 14 days in vitro (div). 100ng/ml NGF effectively maintained survival, compared with cultures without exogenous NGF. Aged as well as adult neurons were cultured [with non-neuronal cells (NNCs) present] without exogenous NGF or with 100ng/ml NGF added for up to 29div. NGF enhanced the survival of both adult and aged neurons (P<0.005 by ANOVA). To exclude possible endogenous NGF from NNCs, or any mediation of an NGF effect in co-culture by NNCs , a neuron- enriched culture system was also used. In these neuronal loss was avoided during cell preparation and well dispersed neurons were obtained. 100ng/ml NGF and/or 1:100 anti-NGF were re-examined in the modified neuron-enriched cultures for 9div and an enhanced survival of both adult and aged neuron persisted, although this effect was lower than in co-cultures. In general, about 20% of DRG neurons were NGF-dependent for their survival in adult and aged cultures. By size analysis of over 6500 neurons, intermediate-sized neurons (24-33mum in diameter) were predominant for both adult and aged in co-culture in the presence of NGF. The immunocytochemistiy (ICC) study using the avidin-biotin- peroxidase complex (ABC) method revealed that higher proportions of SP- immunoreactive (ir), CGRP-ir and NPY-ir aged, as well as adult, neurons were present in enriched cultures supplemented with 10 or 100ng/ml NGF than in cultures without NGF added (P<0.05-0.01 by ANOVA and t-tests), whereas SOM-ir adult and aged neurons showed little difference in cultures without or with NGF added. By counting at the peak day (9div) of SP expression, enhancement of the SP-ir subset in the presence of vs absence of NGF was similar for adult or aged neurons; the proportion of the SP-ir subset was not reduced following aging. Hence the SP-ir subset, together with the subsets of CGRP-ir and NPY-ir, were considered as NGF-dependent survival neurons in adult, and even aged mouse DRGs. Enhancement of the SP-ir proportion was more rapid in aged neuronal cultures supplemented with NGF than in cultures without exogenous NGF. Also, the staining intensities for CGRP and NPY were greater in both adult and aged cultures with NGF added than those in cultures without NGF. In addition to maintaining survival, NGF affected the mature neuronal phenotypes. Scanning election microscopy demonstrated that in the presence of NGF, abundant microvilli projections were distributed on the neuronal surface; in contrast, neurons were deformed and lost their smooth appearance, if 1:100 anti-NGF was present in enriched cultures. In the study of neurite geometry in enriched culture, major neurite length (maximal extension of a neurite), entire neurite length (lengths of major neurite plus all its branches), total neurite length (lengths of all neurite outgrowth from each neuron), soma size, neurite number (for each neuron) and branch number (per neurite) were selected as parameters. Tracing was carried out blindly at 1, 3, 6 and 9div by computer image analysis and a digitizing tablet. Neurites and neurons from at least 5 mice for each adult or aged cultures were measured directly from culture or from photomicrograph montages and data were statistically tested by ANOVA. The results demonstrated that NGF enhanced neurite outgrowth (P<0.0005) at 6 and 9div for both adult and aged neurons Total neurite lengths of adult and aged neurons were distinct in the presence of NGF; neurite length was predominantly enhanced by adult neurons, whereas increased branch lengths were evident for aged neurons. Neurites predominantly extended from intermediate- and large-sized neurons. In summary, aged neurons showed comparatively lower abilities for survival and functional peptide expression compared to adult neurons in vitro, but continued to be responsive to exogenous NGF. In addition, reconstruction of neurites by aged and adult neurons in vitro was demonstrated in the present study.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Robert A Smith
• Keywords: Neurosciences
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-74760
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 16:37
• Last Modified: 27 Sep 2019 16:37
• URI: https://theses.gla.ac.uk/id/eprint/74760