[Neuroscience] Re: Pertussis toxin?

Bill via neur-sci%40net.bio.net (by connelly.bill from gmail.com)
Tue Jul 31 04:12:21 EST 2007


> I reckon the real problem is how to keep a very small volume (e.g., 2
> ml) properly oxygenated for several hours, so that slices don't become
> ischemic while you incubate them in PTX?

Yeah, I realized this almost as soon as I posted. I was thinking that
a 10mL Bijou or centrifuge tube with a fine bubbler made out of some
1.5mm tubing might work too.

> How about using a small 1-2 ml airtight sealable vial? Fill all the
> way to the lid with PRE-oxygenated slosh, containing PTX, plunk in the
> slice, then screw on the airtight cap. No deadspace, no loss of
> oxygen.
>
> Could check this method easily to see if slices in control ACSF are
> still viable after a couple hours of this storage. Worth a try at
> least, eh?

Yeah, that's a good thought. I know my supervisor would like that
idea, he is always trying to get us to entrust our oxygenation needs
to air tight vials. I know it makes perfect scientific sense, but
somehow the idea that aCSF would stay oxygenated over that length of
time just strikes me as wrong.

> On the other hand, I've had a long standing suspicion that sitting in
> even a large oxygenated holding chamber for several hours is WORSE
> than being on the rig under continous perfusion. The evidence being
> that the first slice you put on the rig always seems to be the
> healthiest one. I.e., even after a few hours, when you go to get a new
> slice fresh from the holding chamber, it never looks as good as the
> one you JUST removed from the rig! My hypothesis (i.e.,
> unsubstantiated fantasy) is that the slices are constantly releasing
> "bad stuff" (they are in the process of slowly dying, after all), and
> sitting around in this same "bad stuff" leads to "bad things". Whereas
> the first slice on the rig has had its "bad stuff" continuously
> removed by the rig perfusion.

I've noticed the same thing too. I've wondered the same thing, i.e.
the "bad stuff" theory; but I've also always read that the ultimate
holding chamber solution is actually an interface chamber. I have
definitely read a paper that showed that slices kept in a submersion
chamber undergo more oedema than slices kept in an interface chamber.

> Do you have any experience with perfusing the holding chamber itself?

As a matter of fact, yes. I knew a Japanese physiologist who would
constantly perfuse his 100-200mL holding chamber with carbogenated
aCSF at about 2mL/min. His chamber was made from a 200mL beaker, so
the overflow would just spill out the spout, down the outside and down
a pan into the sink.

> BTW, if any of this works, definitely let me know!


> Matt
>
> PS - Sorry for responding so consistently to your highly varied
> questions. I'm actually not THAT much of a know-it-all. But yours are
> among the only questions in this newsgroup anymore that I even
> understand, let alone can contribute to.

I honestly appreciate the feedback. Yeah, it's a great shame this
group doesn't get more traffic, it's all "How could the aliens control
your brain ions from outer space?" or "Because the TD E/L =increases=
the Iranians must never -discount- the topography of the 'nervous
system' [AoK Ap8]".



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