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HUCB-Derived MSCs in Canine Cerebral Ischemia 3557

Fig. 2. Pre- and postembolization arteriogram of middle cerebral
arteries taken from basilar artery (ventral view). A: Angiography prior
to middle cerebral artery occlusion. B: Angiography after middle cer-
ebral artery occlusion; arrows in A indicate patent middle cerebral ar-
tery prior to the thromboembolism. The arrow in B indicates the
same middle cerebral artery. Arteriogram in B shows sluggish contrast
enhancement and partial filling of the left embolized middle cerebral
artery compared with the right intact middle cerebral artery. Fig. 3. Transverse MR images 1 day after cerebral ischemia. A: T2-
weighted MR image. B: FLAIR MR image. C: T1-weighted MR
and coworkers (2006) used T2-weighted MR images to mea- image. D: Contrast-enhanced T1-weighted image. There is a well-
sure the volume of the ischemic lesion; however, in the pres- defined hypersignal area in middle cerebral artery territory zone and
ent study, the lesion area was measured from FLAIR MR in the thalamus in T2-weighted and FLAIR MR images (within
images in imaging software (MRIcro; C. Rorden, University circle). On FLAIR MR images, the appearance of the lesion is simi-
of South Carolina, Columbia, SC). For each slice, higher in- lar to that of the lesions on T2-weighted MR images. However, the
tensity lesions in the FLAIR images (signal intensity 1.25 FLAIR MR image suppresses the signal from cerebrospinal fluid, so
better visualization of periventricular lesion is obtained. Hyposignal
times higher than the counterpart in the contralateral brain
intensity is seen in the same area in the T1-weighted MR image.
lesion) were marked as ischemic lesion areas, and the infarct Slight enhancement is seen in the contrast-enhanced T1-weighted
volumes were calculated taking slice thickness (4 mm/slice) MR image. Midline shift from the mass effect caused by brain edema
into account. To avoid overestimation of the infarct volume, is also seen.
the corrected infarct volume (CIV) was measured as follows:
CIV 5 [RT – (LT – LI)] 3 d, where RT is the area of the
right hemisphere in square millimeters, LT is the area of the into ethylenediaminetetraacetic acid (EDTA) tubes, and one
left hemisphere in square millimeters, LI is the infarct area in was sent for a complete blood cell count (CBC).
square millimeters, and d is the thickness of the slice (4 mm;
Neumann-Haefelin et al., 2000; Kurozumi et al., 2004; 2,3,5-Triphenyl Tetrazolium Chloride (TTC) Staining
Honma et al., 2006). The relative infarction lesion area was Immediately after euthanasia, the brains were removed
expressed as the percentage of the right hemisphere volume. and brain slices were prepared that included the cortex and
With this equation, the effect of edema formation or tissue basal ganglia area. The brain slices were then immersed in a
shrinkage on the estimation of infarct size is minimized (Lin 2% solution of TTC in normal saline at 378C for 20 min (Liu
et al., 1993). The lesion volume at 1 day, 1 week, and 2 et al., 2006). TTC stains normal gray areas of the brain deep
weeks after cerebral ischemia modeling was measured, and the red but does not stain infarcted tissue (Bederson et al., 1986).
change in lesion volume after cerebral ischemia was compared After TTC staining, the brain slices were fixed in 10% phos-
between the HUCBC group and the control group. The phate-buffered neutral formalin and photographed.
lesion volume on day 1 was considered to be 100%, and grad-
ual changes at weeks 1 and 2 were compared with the day 1
Post-Mortem and Histopathological Examination
lesion volume.
All experimental dogs were euthanized at 4 weeks after
the cerebral ischemic induction through deep anesthesia with
an intravenous injection of propofol and an overdose injection
Blood Sample Collection of potassium chloride. After sacrifice, the brains were removed
Blood samples were taken prior to the modeling; on the and evaluated for gross lesions. Brain slices, including the cor-
day of the modeling at 1, 3, 6, and 12 hr; and 1, 2, 3, 5, 7, tex, basal ganglia, and thalamus, were placed in 10% phos-
10, 14, 21, and 28 days after modeling through venipuncture phate-buffered neutral formalin. After at least 48 hr of immer-
of the jugular vein. A total of 5–10 ml blood was collected sion-fixation, fixed tissues were processed and embedded in

Journal of Neuroscience Research