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Fig. 6
The impact site in the right parietal cortex 2 weeks after mild TBI, showing typical small contusion in the Epo
treated animals (
a
) compared to the much more extensive cortical contusion in animals in the saline group (
b
)
the brain is elevated in front with a spatula, disconnected from
the cranial nerves at the base and collected into a bottle with 4%
paraformaldehyde. The specimen is later transferred into 30%
sucrose solution (after 24-48 h) and preserved at 4°C. The
specimens are further processed to study the contusion volumes,
assess the cell counts in hippocampus and for immunohis-
tochemical studies (see Note 14).
2. Preparation of 100 ml of 4% Paraformaldehyde (procedure
performed in the hood): (a) Heat 50 ml distilled water in a
beaker on a hot stir plate to 60-65°C; (b) Measure 4 g of para-
formaldehyde and add it to the water; (c) Add few drops (1-4,
one by one) of 1 N NaOH till the solution is clear. A pH strip
(not pH meter) can be used to check the pH, (d) Cool the
solution and then add 50 ml of 0.2 M buffer, and (e) Store the
solution at 4°C (see Notes 15 and 16).
3. Preparation of 0.2 M buffer: To prepare 2 l of 0.2 M buffer,
add 12.5 g of Sodium Phosphate Monobasic/Monohydrate
and 44.2 g of Sodium Phosphate Dibasic anhydrous to 2 l of
ddH
2
O (see Note 16).
4. Preparation of 30% sucrose: To prepare 100 ml of 30% sucrose,
add 30 g of sucrose to PBS and stir it. Store the solution at 4°C
(see Note 16).
3.5
Typical Study
A study (
3
) was conducted in Long Evans rats to evaluate the time
window for neuroprotection with Epo after CCI. Recombinant
Erythropoietin (rhEpo) was administered (5,000 U/kg, subcuta-
neous) at various times points after TBI (1, 3, 6, 9, or 12 h). The
rats were randomly assigned to one of the Epo groups or to the
saline group. Typical examples of the impact site in Epo treated
and saline treated animals are shown in Figs.
6a, b
, respectively.
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