Dear Isabelle,

We used such procedure for astroctytes while studying newly expressed channels under OGD conditions. If it is interesting to you? We do not use slices but astrocytic culture, OGD protocol which is easily can be applied for slices.  You may use our procedure to remove oxygen from the CO2 incubator using an anaerobic chamber to put inside the incubator. So, the method could be applicable for organotypic brain slices.

To simulate ischemia in vitro, cultured astrocytes were subjected to anoxia and hypoglycemia.  Astrocytes were plated in 60×15 mm petri dishes for Western blot experiments or 24 well plates for glutamate clearance assays and allowed to grow to confluence. When confluent, anoxia was achieved by removing growth medium from the cultures, rinsing gently by immersion in a bicarbonate-buffered balanced salt solution (BBSS) that contained (in mM): NaCl 127, KCl 3, NaHCO3 19.5, NaH2PO4 1.5, MgSO4 1.5, D-glucose 25, and CaCl2 1, and placing the cultures

at 37°C in fresh BBSS previously gassed for 5 min with 5% CO2 and 95% N2. (anoxia buffer; pH 7.4). Hypoxia and hypoglycemia (simulated ischemia) were achieved by decreasing the concentration of D-glucose in these solutions (25% or 10% of normal glucose). The cultures were then placed in a chamber (Billups-Rothenberg, Del Mar, CA) flooded with 5% CO2 and 95% N2 and incubated at 37°C for 24 hrs. Control astrocytes were placed in BBSS (pH 7.4) under normoxic conditions.

Please see attached our PDF for details of results.

Cordially, Serguei.

Thank you so much Serguei for your help and for sharing your paper!

Could you let me know which chamber from Billups-Rothenberg you recommend to use? Is it the modular incubator chamber or the hypoxia chamber? My organotypic brain slices are either cultivated in 6 well plates (one slice/well) or on MEAs  (I put 3 MEAs in 150 mm diameter petri dish).

Kind regards,

Isabelle

you could try this protocol if it fufill your request.

Meyer et al., The Journal of Neuroscience, June 11, 2014 • 34(24):8259 – 8267

Slice pharmacology and oxygen and glucose deprivation. Acutely prepared coronal slices and or oxygen and glucose deprivation (OGD) treatment for ex vivo studies were conducted as described previously (Nishi et al., 1997; Sahin et al., 2007, 2008). Briefly, WT and Cdk5 CKO mouse brains were placed in ice-cold oxygenated Kreb’s bicarbonate buffer bubbled with 95% O2 and 5% CO2. Coronal slices (350 m) were made and striatal or cortical slices (2 mm from bregma) were microdissected and incubated at 30°C. For OGD, slices were equilibrated in Kreb’s buffer for 30 min, as before, followed by exchange with buffer in which glucose has

been replaced by 10 mM sucrose and bubbled with 100% N2. For slice treatments with the Cdk5 inhibitor indolinone A (Indo A), incubation periods were 1 h at the concentrations indicated. Calpeptin and calpain inhibitor 3 treatments were with 20 M for 1 h. SFK81297 treatment was at 1 M for 5 min. Control untreated slices were incubated for equal amounts of time, and treated with vehicle-containing buffer. Following these treatments, slices were snap frozen in dry ice and stored at 80°C

until processed for quantitative immunoblot analysis.

http://www.jneurosci.org/content/34/24/8259.short

Dear Isabelle,

the type of the chamber plays a secondary role, while the principle is how long OGD should be (10-30 minutes) for reproducible results. In the article that Wen Lu suggested (Meyer et al., 2014), authors used proper OGD, but the finding of Cdk5 conversion (p32 to p25) and why at low calcium they obtained neuroprotection is not well understood. Why?

First, since Cdk5-biochemistry is strongly linked to endogenous polyamines (He et al., 2016, J.Neurosci. 36910:3079-3091), the GLIAL store of polyamines in the brain (Laube and  Veh, 1997) and retina (Skatchkov et al., 2000) are ignored. However polyamines may play great role in neuroprotection.

Second, when authors used zero-calcium media (Fig. 2C) they showed strong neuroprotection  but the interpretation of data is not satisfying (not complete). In fact, it is known that low calcium triggers opening of glial Cx43 gap junction (Benedikt et al., 2012) and hemichannels in glia (Contreras et al., 2002; 2003) and therefore possible release of polyamines from glia to neurons via Cx-hemichannels.

Then, since polyamines have neuroprotective effect accordingly growing literature on polyamine related protection (rev. Skatchkov et al., North Am.Ps2014) and recent evidence is justified by Noro et al., (Cell Death Dis. 2015 Apr 16;6:e1720), polyamines stored in glia may be extremely important.

Third, since brain slices are completely removed from blood circulation there is no more polyamine fueling from blood to brain(!) and the only sources of endogenous polyamines are the glial cells. However, during few hours of incubation without polyamines, the endogenous polyamines can be released, washed and wasted. THEREFORE a supplement of polyamines  should be always in media. Most of the people are still ignoring the fact of polyamine supplement. 

Importance of polyamines can not be more underestimated. On the other hand, if polyamines are oxidized they are converted to aldehydes and reactive radicals vulnerable for cells. So, to prevent oxidation there are two ways: (1) using low molecular weight polyamines (agmatine, putrescine, spermidine) and/or  (2) to depress polyamine oxidases.

I attached some key articles and may give you more. So, hope that more clear view of organotypic strategy is highlighted.

Very cordially, Serguei. 

Dear Wen,

Thank you so much for your answer. You mention a protocol for fresh slices that could also help me to design the experiment.

Thanks!

Isabelle

Dear Serguei, again thank you very much for providing such detailed information that will be very very useful for developing my assay from organotypics slices. I will keep you in touch for sharing my first results. With my kind regards, Isabelle