Biology Reference
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Fig. 2
Brain injury. Panel (
a
) shows the isolated common carotid artery (
arrow
). Panel (
b
) shows a brain with
severe gross injury 48 h after ligation.
Arrows
outline the injured region. Panels (
c
) and (
d
) show TTC staining
of injured brain from an anterior section (
c
), and posterior (
d
)
1. Place pups in the preheated hypoxia chamber (heated to 36°C)
(see Note 4).
2. Turn on the 8% oxygen tank at 15 L/min for 5 min to purge
the tank. Decrease the flow to 5 L/min for duration of hypoxic
event (30 min to 3.5 h, depending on severity of brain injury
desired) (see Note 5).
3. When hypoxia exposure is completed and animals are stable,
return pups to the dams.
3.2.3
Hypoxia Exposure
Weight gain, growth, timing, and cause of mortality and morbidity
should be recorded. All brains should be scored for gross injury at
the time of sacrifice. Gross brain injury is visually apparent 24 h
after injury (Fig.
2b
), and can be evaluated using an ordinal scale
modified from Vannucci (
61
) as shown in Table
1
. If animals are
not perfusion-fixed, tetrazolium chloride (TTC) staining, which is
incorporated into actively respiring tissues, can be used to clearly
demarcate areas of non-vital tissue (Fig.
2c, d
). The intensity of the
color is proportional to the rate of respiration in the tissues, so liv-
ing tissue stains red, while infarcted areas appear white.
3.3 Evaluation
of Injury
3.3.1 Gross Evaluation
of Injury
3.3.2 Triphenyltetrazolium
Chloride Staining
When TTC diffuses into
actively respiring
tissues it accepts elec-
trons from the mitochondrial electron transport chain and the stain
is reduced to the pink compound formazan. The accumulation of
this pink compound stains actively metabolizing tissues red, and
the intensity of the red is proportional to the rate of respiration in
those tissues. This method can therefore be used to distinguish
between live (stain, red) and infarcted (no stain, white) areas on
2 mm thick brain sections.
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