W. A. THOMAS, R. A. FLORENTIN, S. C, NAM, J. M. REINER AND K. T. LEE 
865 
tions from the 37-hour stock and cholesterol 
diet-fed swine. The results showed that, in both 
groups, most of the labeled cells had the DNA 
content of diploid cells. This supports the find- 
ing that most labeled cells had divided shortly 
after synthesizing DNA and very few remained 
in G2. No significant difference was found be- 
tween the stock swine and cholesterol swine up 
to thi"^ ^jeriod. By 42 hours, however, the choles- 
terol swine showed the beginning of a second 
peak of labeled mitoses. Although this could 
represent a second division of the labeled cells, 
another possibility is that it is due to post-pulse 
labeling. In in vivo situations, the availability 
time of ^H-thymidine is generally no longer 
than 30-60 minutes, so that this can be con- 
sidered a pulsing period. Labeling of cells at 
some time after this period can be referred to 
as post-pulse labeling. Post-pulse labeling is 
presumably due to re-utilization of label sal- 
vaged from dead cells in the intestine and 
elsewhere.^'^ 
In order to find out whether post-pulse label- 
ing occurred, another experiment was carried 
out (Figure 3) . A segment (No. 2) of the right 
carotid artery was surgically exposed and 
wrapped with parafilm and vascular clamps 
were applied at two points to isolate the seg- 
ment from the general circulation during the 
period of pulsing with ^H-thymidine. This tech- 
nique was tested, and we found that the seg- 
ment was completely isolated from the general 
circulation and that it was still capable of syn- 
thesizing DNA after a two-hour period. After 
the segment of right carotid artery was iso- 
lated, the swine were given ^H-thymidine intra- 
venously. Segments No. 1 and No. 3 were not 
isolated. Two hours later, long after completion 
of the pulsing period, the clamps were released 
so that any labeling of cells in the previously 
isolated segment would have to be due to post- 
pulse labeling. The swine were killed at 5 hours, 
2 days, and 7 days. No post-pulse labeling was 
seen at 5 hours. However, at 2 days and 7 days 
there was considerable post-pulse labeling. 
Grain counts of the post-pulse labeled cells 
showed that most of them had less than 9 
grains. In contrast, the non-isolated segments 
had heavily labeled cells. 
Since most of the post-pulse labeled cells had 
RIGHT CAROTID 
ARTERY. 
CLAMPS 
APPLIED 
2 HOURS 
LEFT CAROTID 
ARTERY 
ARCH OF 
- AORTA 
Figure 3. — Schematic drawing of carotid arteries illus- 
trating where vascular clamps are applied and where 
sections are taken. 
less than 9 grains, we reexamined the percent- 
ages of labeled mitoses using 9 grains as the 
"cut-off" point (Figure 4) . When this was done, 
the second peak of labeled mitoses virtually dis- 
appeared. This suggests that it was due to 
post-pulse labeling and not to a second division 
100- 
80- 
60- 
40- 
20- 
0- 
LABELED MITOSES c a 9 GRAINS 
TOTAL NUMBER OF MITOSES 
X 100 
STOCK 
CHOLESTEROL — o-o— 
17 22 27 32 37 42 47 
TIME IN HOURS 
Figure 4. — Graphic presentation of percentages of la- 
beled mitoses data using 9 grains as the "cut off" 
point since most of the post-pulse labeled cells had 
less than 9 grains. The second wave of labeled mi- 
toses (Figure 2) virtually disappeared, indicating 
that it was due to post-pulse labeling and not to a 
second division of the labeled cells. 
