Biology Reference
In-Depth Information
frame (Kopf Instruments, Tujunga, CA). A burr hole was made, and a 30-gauge
needle was inserted through the burr hole into the striatum (0.1 mm posterior,
4.0 mm ventral, and 2.0 mm lateral to the bregma). ICH was induced by the
administration of collagenase type IV (0.5 µL saline containing 0.078 U, Sigma)
over a period of 5 min. After remaining in place for another 3 min, the needle
was gently removed.
brain transplantation
F3 and F3.Akt1 hNSCs were dissociated into single cells by a brief trypsin treat-
ment and suspended in PBS at 4×10
7
cells/0.1 ml and kept on ice until trans-
planted. Randomly selected ICH mice of one week after ICH surgery received
2 µL (2×10
5
cells) of F3.Akt1 cell suspension (n = 9), F3 cell suspension (n = 9)
and killed F3 cell suspension (n = 10). F3 cells in glass tube were killed by placing
the tube in boiling water for 1 min, injected slowly for 5 minutes into overlying
cortex of the hemorrhage lesion (0.1 mm posterior, 2.0 mm ventral, and 2.0 mm
lateral to the bregma). In another control group, 2 µL of PBS was injected into the
ICH mice (n = 10). Immunosuppressant was not used in any of the animals. In
order to identify the migration potential to the lesion site of grafted cells, F3.Akt1
hNSCs were infected with an adenovirus vector encoding LacZ gene (pAV.LacZ)
in vitro at 100 MOI (PU/cell) for 24 hr before transplantation (n = 2).
behavioral testing
Behavioral testing was performed weekly with the rotarod (Harvard Instrument)
by 2 individuals blinded to mice treatment status [16], [17]. In the rotarod test,
mice were placed on the rotarod cylinder, and the time the animals remained
on the rotarod was recorded. The speed was slowly increased from 10 to 40 rpm
within a period of 2 min. The trial was ended if the animal fell of the rungs or
gripped the device and spun around for 2 consecutive revolutions. The animals
were trained for 3 days before ICH operation. The maximum duration (in sec-
onds) on the device was recorded with 3 rotarod measurements 1 day before ICH
induction. Motor test data are presented as percentages of the maximal duration
compared with the internal baseline control (before ICH). The modified limb-
placing test is a version of a test previously described [16], [17]. The test consists
of 2 limb-placing tasks that assess the sensorimotor integration of the forelimb
and the hind limb by checking responses to tactile and proprioceptive stimula-
tion. First, the mouse is suspended 10 cm over a table, and the stretch of the fore-
limbs toward the table is observed and evaluated: normal stretch, 0 points; flexion
with a delay (2 sec) and/or incomplete, 1 point; abnormal flexion, 2 point. Next,
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