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well, and cells were incubated for 10 min after which the amount of ATP was
quantified through a luciferase reaction. The luminescence intensity was determined
using a luminometer with lysates in a standard opaque-walled multi-well plate. The
ATP content was determined by running an internal standard and expressed as a
percentage of untreated cells (control).
23.2.2
In Vivo Study
23.2.2.1
Transient Focal Middle Cerebral Artery Occlusion (MCAO)
Male adult Sprague-Dawley rats (weighing 260-300 g, Harlem Chicago, IL)
were given access to food and water ad libitum. Before surgery, rats were fasted
overnight with free access to water prior to surgery, and the following day they
were weighed and anesthetized by IP injection with ketamine hydrochloride
(80 mg/kg body weight IP; Putney) and xylazine hydrochloride (20 mg/kg body
weight IP; Vedco) (McCollum et al.
2010
). During the experiment, core tempera-
ture was maintained at 37°C by a thermostatically controlled heating pad regu-
lated via a rectal temperature probe (CMA 450). Local cerebral blood flow (LCBF)
was monitored in the cerebral cortex of left hemisphere in the supply territory of
the middle cerebral artery (MCA) by laser Doppler flowmeter (LDF) (Perimed
Inc., OH, USA). Transient focal cerebral ischemia of the middle cerebral artery
(MCA) for 2 h was induced by the suture occlusion technique (Longa et al.
1989
;
Sun et al.
2011
). Briefly, the left common carotid artery and the left external
carotid artery were exposed through a midline neck incision. A 4-0 monofilament
nylon suture coated with silicon (Doccol Co., NM, USA) was inserted through an
arteriectomy in the external carotid artery, gently advanced into the internal
carotid artery, and positioned approximately 17 mm from the carotid bifurcation.
LCBF was monitored continuously during the MCAO surgery. With the use of
this technique, the tip of the suture occludes the origins of the MCA, the proximal
anterior cerebral artery, and the posterior communicating artery. Reperfusion was
accomplished by withdrawing the filament 2 h after MCAO (Longa et al.
1989
;
Sun et al.
2011
) .
23.2.2.2
Rat Treatment Schedules
After surgery, animals were allowed to recover from the anesthesia and given
food and water ad libitum. Fifteen rats were randomly assigned as controls
(MCAO rats which received only the vehicle, saline 0.9%), experimental
(MCAO rats which received taurine, 40 mg/kg), and sham-operated (received
the same surgical procedure without insertion of the silicon filament). Taurine
was delivered subcutaneously to the experimental group 24 h after the reperfu-
sion for 4 days.
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