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Brain Slices, THC and PGE2.

Doktor DynaSoar targeting at OMCL.mil
Sun Jan 4 17:21:59 EST 2004


On Sun, 04 Jan 2004 08:36:06 GMT, BilZ0r
<BilZ0r at TAKETHISOUThotmail.com> wrote:

} Anything immuno/cytokine related is always a bit out of my depth, so I 
} was wondering if anything had anything to add to this hypothesis.
} 
} I've been interested in in vitro THC-induced neurotoxicity since: Chan et 
} al., J Neurosci. 1998 Jul 15;18(14):5322-32. He shows that THC induced 
} pronouced apoptosis in hippocampal slices, and that this was PGE2 
} mediated.
} 
} Now whether THC induces neurotoxicity in whole animals is a point of much 
} contension. Its been shown that THC induces PGE2 release in animals, and 
} that PGE2 mediates behavioural changes due to THC, as well as the fact 
} that inhibiting eicosinoid synthesis inhibits some behavioural changes 
} normally seen in THC treated -HUMANS-.
} 
} So, although it looks likely that THC increases the level of PGE2 in 
} human brains, what if the trauma that a brain slice recieves, causes the 
} production of heaps of PGE2, and then THC just pushes it over the edge as 
} far as PGE2 production goes?
} 
} So, basically my question is, do you think brain slices have a much 
} higher PGE2 concentration or COX activity?
} 
} But then, THC is also toxic to cultured neurons. Do you think cells in 
} culture have a higher PGE2 concentration or COX activity?
} 
} Thanks for any input.

With regards to "what if the trauma that a brain slice recieves,
causes the  production of heaps of PGE2, and then THC just pushes it
over the edge as far as PGE2 production goes?"

No slicing was performed. The samples were cultured on plates, hence
no trauma due to slicing. Below is their method, from the paper.

"Primary neuron cultures and measurement of cell death. Primary
hippocampal neurons were established from postnatal day one Sprague
Dawley rat pups and maintained in defined medium with minimal
supplements. Pups were killed by decapitation and the hippocampi were
digested in 5 ml of 10 U/ml papain at 37°C for 20 min. After two
rinses, the tissue was triturated (two rounds, 10-15 pipetting each)
in dissociation medium (27 mM K2S04, 1 mM kynurenic acid, 15 mM MgCl2,
74 mM Na2SO4, 18 mM glucose, 225 µM CaCl2, 0.0012% phenol red, and 2
mM HEPES, pH 7.4) using a 5 ml disposable plastic pipette.
Ninety-six-well plates were coated with poly-D-lysine (66 µg/ml).
Cells were plated into the inner 60 wells of the 96-well plate (the
peripheral wells were filled with water to act as humidity barrier) at
5 × 104 cells/cm2 and maintained in 200-250 µl of Neurobasal medium
(Life Technologies, Gaithersburg, MD) in the presence of N-2
supplement (Bottenstein and Sato, 1979) with 10 U/ml penicillin, 10
µg/ml streptomycin, and 0.5 µg/ml glutamine for 10-16 d before use.
Because Neurobasal medium and N-2 did not maintain neuron viability
for extended periods, the media was modified. These modifications
included adding 0.1% chicken ovalbumin (Sigma, St. Louis, MO) and
increasing sodium selenite to at least 300 nM. The amount of selenite
added (up to micromolar) was determined empirically for each batch of
N-2. Primary neurons cultured under these conditions survived 4-5
weeks. Serum-free Neurobasal medium with supplements has been used
extensively for sustaining the survival of cultured CNS neurons
(Brewer et al., 1993; Otsuki et al., 1994; Takeshima et al., 1994;
Morrison et al., 1996; Xiang et al., 1996). However, supplements with
very high contents of antioxidants, such as B27, were not suitable for
our studies (see Results). Neurons maintained in the presence of serum
experience serum withdrawal-induced cell death at the time of THC
treatment. All cannabinoid dilutions were performed in siliconized
tubes. Absolute ethanol was used as carrier or vehicle for THC.
Controls and vehicle-treated controls were obtained from cells treated
with comparable concentration of ethanol as THC-treated samples. MTT
assays were performed as previously described (Hansen et al., 1989).
At the end of the incubation, 25 µl of MTT (5 mg/ml) was added to the
cells in 100 µl of growth media. The plates were incubated for 2 hr at
37°C in a CO2 incubator and solubilized overnight in 9% SDS and 22%
DMF before determination of absorbance at 570 nm. SR141716A was
provided by Research Biochemicals (Natick, MA) as part of the Chemical
Synthesis Program of the National Institutes of Mental Health,
Contract N01MH30003."

So, because the treatment you suggest as the cause is not performed,
this does not support your suggestion. On the other hand, for the same
reasons, it does not negate it. 




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