Biology Reference
In-Depth Information
Using this differential staining of the cells and nuclei and a confocal
microscope capable of exciting and capturing fluorophores at three
different wavelengths, we can determine the number of apoptotic
cells in control conditions and in presence of a neurotoxic insult
(
33
). We have used two chemical inducers of neurodegeneration:
N
-methyl-
D
-aspartate (NMDA) and SNOC (s-nitrosocystein).
Both result in oxidative stress in neurons and induce neuronal
apoptosis by free oxygen radicals, similar to events in ischemic
brain (
34-36
). The determination of neuronal apoptosis requires
that at least several criteria for neuronal degeneration are simulta-
neously detectable in the exposed cells (
37
). Meeting these require-
ments helps us to distinguish apoptosis from necrosis, which is an
irreversible event and will not respond to neuroprotective treat-
ment. In a third model of neuronal injury cerebrocortical cells
were incubated with HIV/gp120, an envelope protein of the HIV
virus known to induce HIV-associated neurocognitive disorders in
transgenic mice and humans (
38
). Cerebrocortical cultures 17-21
days in culture are incubated with HIV/gp120 (50 mM) for 24 h
and apoptotic neurons displaying NeuN and TUNEL staining
counted.
4
Notes
1. It is important to notice that expression of a functional EPO-R
in brain tissue was disputed recently and the specificity of anti-
bodies used questioned by Sinclair and colleagues (
39
).
However, in a response focusing on neuroprotective effect of
EPO (
10
) we challenged that conclusion.
2. Glass coverslips are critical. We found that this type of glass
used to produce the coverslips results in superior adherence of
neuronal cells.
3. This step is determining for the quality of the primary neu-
ronal cultures. All meninges have to be removed diligently
(and rapidly) to minimize overgrowth of cultures with
fibroblasts. Furthermore, it is crucial that this procedure is car-
ried out in petri dishes filled with Hanks Medium that is equili-
brated at RT and completely cover the brains. Successful isola-
tion will yield in square cortex sections measuring 1 × 1 mm.
4. Quality and suitability of the fetal calf serum varies from batch
to batch. It is highly recommended to test several batches from
one or more manufacturers and to determine the one that
results in the best outcome. Once identified, our laboratories
buy a large quantity of the most suitable batch of fetal calf
serum and store at −80°C.
5. Proper trituration will determine the final number and viability
of neurons in primary cultures. Triturating with not perfectly
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