To determine whether released bFGF was playing a role in cell
recovery, a neutralizing polyclonal antiserum against bFGF was added to
BREs that were shocked and recovered, and cell number and viability
were determined 48 hours later. In these studies, the number of BREs
was 43% lower at 48 hours than control cells (
P <
0.05; ANOVA;
Fig. 3 ). In contrast, BREs that were shocked and recovered and then cultured
for 2 days in media with bFGF added (2.5 ng/ml) had a trend toward
increased cell number compared with that of antibody-exposed cells
(1.31 ± 0.36 × 10
5 versus 1.06 ± 0.09 × 10
5). However, the variability in
cell numbers led this increase to be insignificant
(Fig. 3) .
Cells that were shocked and recovered in the presence or absence of
bFGF antibody, exogenous bFGF, or fresh media were examined at 3 and 6
hours for cells undergoing apoptosis. The overall ANOVA was significant
(
P < 0.0001; see
Table 3 ). The percentage of TUNEL-positive cells at 6 hours after shock,
recovery, and removal of media was higher than control, untreated cells
(13.4% ± 3.3% versus 0.6% ± 0.5%,
P < 0.001
Tukey). However, the percentage of TUNEL-positive cells at 3 hours
after shock, recovery, and removal of media was not significantly
higher than control, untreated cells (5.6% ± 2.6% versus 0.2% ±
0.4%,
P = NS). The percentage of TUNEL-positive cells
after treatment was significantly higher after 6 hours than after 3
hours (
P < 0.01). Cells shocked and recovered in the
presence of bFGF antibody were also significantly more TUNEL-positive
at both 3 hours (12.7% ± 3.5%) and 6 hours (13.0% ± 6.0%)
compared with controls (
P < 0.001). When media, and
thus released bFGF, were not removed after shock and recovery, the
percentage of TUNEL-positive cells was lower than when media were
removed after shock and recovery. For example, at 6 hours after injury
and removal of media, there were 13.4% TUNEL-positive cells versus
4.9% when media were not removed (
P < 0.01). However,
the difference at 3 hours was not significant (5.6% versus 4.8%). The
addition of fresh media and bFGF led to a slight but not significant
reduction in the number of TUNEL-positive cells at 3 hours (6.2% ±
5.6%) and 6 hours (6.8% ± 7.3%) compared with cells shocked and
recovered with removal of media (see
Table 3 ).