During development, neurons become assembled into functional networks by growing out axons and dendrites (collectively neurite outgrowth) that connect synaptically to other neurons. Strikingly, neurons retain their capacity for growth and synapse formation in the adult brain. Only those neurons that manage to establish adequate contacts with neighboring cells escape cell death. Stimulation and acceleration of neurite outgrowth is thus important for restoring neuronal function after injury or for neurodegenerative diseases. A promising strategy for drugs targeted against neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease is to promote the regeneration of neurons. Thus, new therapies are focusing on identifying molecules that affect neurite outgrowth. For this purpose, QPS Neuropharmacology provides cell culture models to assess the neurotrophic effects of your developmental compounds.
Neurite Outgrowth Solution 1
Primary rat (mouse) hippocampal or cortex neurons are treated with compounds for 48 h on DIV1. Cells are subjected to indirect immunofluorescence analyis using anti-beta-III tubulin antibody. The following parameters using image based analysis (Axio.Imager Z1 microscope (20x), 10 image deep z-stacks, collapsed to 2D (ImageProPlus), 12 images per slide from 12 predefined fields, Image enhancement, filtering) are determined of approximately 200 cells per experimental group.
- number of neurites
- total length of neurites
- length of the longest neurite
Figure: Effects of Compound X and reference item (RI) on neurite outgrowth of primary rat hippocampal neurons.
Neurite Outgrowth Solution 2
Adult rat dorsal root ganglion (DRG) explants are cultured under serum free conditions in the presence or abesence of neurotrophic factors for 5 days. Explants are are subjected to indirect immunofluorescence analyis using anti-beta-III tubulin antibody. Parameters such as the neuritic halo (the total area specifically occupied by neurites) are analyzed by using a software-supported automatic quantification method.
Figure: Neuritic halo of adult dorsal root ganglion explants upon treatment with NGF.
Neurite Outgrowth Solution 3
Microfluidic chambers (MFCs) can provide unique insights into the axonal compartment independent of the soma. We thus offer a model of axonal injury and re-growth in MFCs using dissociated dorsal root ganglion (DRG) neurons from adult wild type mice. Already 3 days after seeding neurons into the somal side of the MFC, axons start to cross the microgrooves. After 5 days in culture, axons have completely crossed the grooves and built a beautiful network on the axonal side (Fig 1 A). Neurons can then be axotomized by fast removal of the medium from the axonal compartment (Fig. 1 B).
Developmental compounds can be selectively applied to either axonal or somal side alone, by using a hydrostatic pressure difference between the somal and the axonal chamber.
The effect of NGF application to the axonal side in comparison to vehicle-treated cells was quantitatively analyzed, showing a significant increase of branching and axonal length, while no impact on the number of crossing axons could be observed (Fig.2).
Figure 1. Representative images of DRG neurons from adult wild type mice in microfluidic chambers. DRG neurons were cultured until DIV5 and fixed before (A) and after (B) mechanical axotomy. Tubulin III (TubIII) labeling to visualize neurons and their extensions and DAPI to counterstain for nuclei. Crossing axons on the axonal side before axotomy (A) and removal of axons by axotomy (B). Scale Bar 1,000 µm.
Figure 2. Effect of NGF treatment on axonal re-growth 24 h after axotomy. The following marker were assessed using image-based quantification of total number of crossing axons (A), the number of branch points per crossing axon (B) and the total length of all axons (C); n = 4 wells per group. Unpaired t-test; *p<0.05; **p<0.01. VC: vehicle control; NGF: nerve growth factor.