A massive increase of extracellular glutamate levels, resulting from an increased release and/or a decreased uptake, occurs during hypoxic-ischemic events with consequent neuronal death by excitotoxicity.

In this example, we have investigated the neuroprotective effect of nerve growth factor (NGF) and Riluzole on the mature neurite network in primary cultures of rat cortical neurons after glutamate intoxication. Riluzole, a glutamate antagonist receptor and nerve growth factor (NGF) were used as reference compound.

Biological model :
Cortical neurons of rat embryo.

Culture conditions :
Rat cortical neurons were cultured following a modified protocol initially described in Singer at al., 1999.

Treatement :
The conditions tested in this study were:

• Control medium.
• Control medium with glutamate.
• Riluzole at 3 concentrations with glutamate.
• NGF with glutamate.
  

Six wells per experimental condition were carried out. At the end of the intoxication, cultures were washed and placed in fresh culture medium without glutamate.

Immunohistochemistry :
Cortical neurons were labeled by a mouse monoclonal anti neurofilament (NF) antibody. This antibody labels the neurite network of mature and viable neurons in culture. This antibody was chosen because glutamate intoxication induces toxicity in mature neurons which specifically express neurofilaments and receptors to glutamate. The quantity of labelled neurofilaments was proportional to the quantity of mature functional neurons.
Astrocytes were labeled by a rabbit polyclonal anti glial fibrillary acidic protein (GFAP). GFAP is an intermediate-filament (IF) protein that is highly specific of astroglial cells.

After incubation, the cells were washed in PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG to reveal NF antibody and with Alexia Fluor 568 goat anti-rabbit (to reveal GFAP).
The nuclei of the cells were labeled by a fluorescent marker (Hoechst dye solution).

Analysis :
Analysis of the neurite length of neurons was carried out using In Cell Analyzer 1000. The results were expressed in number of cell bodies per picture. Statistical analysis was performed with Student unpaired t-test.

3.1. Effect of NGF and Riluzole on the total neurite length of mature neurons after glutamate intoxication.

The results are shown in table 1 and figure 1.

After glutamate intoxication, the density of the neurite network was decreased compared to the control medium without intoxication (16%).

In this condition, NGF had a protective effect on the neurite network (48 % compared to the control medium without glutamate intoxication). This protection was significant (p<0.001 %) compared to control medium after glutamate intoxication.

Riluzole had a dose dependant protective effect on the neurite network (respectively 57 %, 47 % and 23 % compared to the control medium without glutamate intoxication). These protections were significant (p<0.001 %) compared to control medium after glutamate intoxication.

Photograph 1: effects of the control medium and NGF and Riluzole on neurons network (Green: neurons labeled with neurofilament antibody. Red: astrocytes labeled with GFAP antibody. Blue: nuclei stained with Hoechst dye).

3.2. Conclusion

The NFG had a neuroprotective effect against glutamate intoxication, and Riluzole, an antagonist of glutamate receptor had a dose-dependant neuroprotective effect on the neurite network of mature neurons. The analysis of neurofilament length for neuronal cell viability allows the quantification of the mature neurons which express the glutamate receptors.

This model of glutamate intoxication is adapted to test several compounds on the neurons survival.

All the last Pharmaceutical news