Assessment of Blood–Brain Barrier Breakdown - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 385

2.2. Materials
and Instruments

1. Anesthesia: a mixture of ketamine (100 mg/ml, 0.08 ml/100 g,
Ketaset ® ) and xylazine (20 mg/ml, 0.06 ml/100 g, VMD).
Some reports suggest that isofl urane may be a potent BBB
opener and thus should probably be avoided ( 29 ).
2. Artifi cial cerebrospinal fl uid (ACSF): 129 mM NaCl, 21 mM
NaHCO 3 , 1.25 mM NaH 2 PO 4 , 1.8 mM MgSO 4 , 1.6 mM
CaCl 2 , 3 mM KCl, and 10 mM glucose. The ACSF is equili-
brated with a gas mixture comprising 95% O 2 and 5% CO 2 (pH
7.4). All Chemicals are purchased from Sigma-Aldrich.
3. Elevated K + solution: For experiments with elevated K + (e.g.,
to imitate some aspects of red blood cells hemolysis following
subarachnoid hemorrhage), KCl was added to the ACSF in
exchange with NaCl to maintain osmolarity within the physi-
ological range.
4. Sodium fl uorescein (MW = 376 Da, 1 mg/ml, Novartis), in
saline.
5. FITC-Albumin (MW = 66 kDa, 2 mg/ml, Sigma-Aldrich), in
saline.
6. Bone cement (UNIFAST Trad, GC AMERICA INC).

2.2.1. Solutions

1. A stereotactic frame
2. A fl orescence stereomicroscope (Zeiss, SteReO Lumar V12)
3. Heating pad for monitoring body temperature
4. A driller (DREMEL)
5. Perfusion pump or an automatic injector

2.2.2. Instruments

Surgical Procedure

1. EMCCD camera (Andor Technology, DL-658 M-TIL)
2. Computer monitor

Imaging

1. Adult SD rats (weighing 200-300 g) are deeply anesthetized by
intraperitoneal (IP) injection of ketamine and xylazine mixture.
2. The tail vein is catheterized with a sterile canula (BD Neofl on,
24 G).
3. The animal is placed in a stereotactic frame under the micro-
scope and body temperature is continuously monitored and
maintained at 37.0 ± 0.5°C.
4. A bone window is drilled over the motor-somatosensory cortex
(4 mm caudal, 2 mm frontal, 5 mm lateral to bregma) of one
hemisphere.
5. An infl ow and outfl ow tubes are inserted under the bone
cement rim (created around the bone window), allow continu-
ously superfusion of the brain cortex with ACSF.
6. The dura is carefully opened.

2.2.3. Methods

Animal Preparation