E .E. Otukonyong et al. Brain Research 887 2000 70 –79
73
and coverslipped using Eukitt reagent O. Kindler, MA-2091, Japan. The stored images were analyzed for
Feiburg, Germany and examined under a light micro- cell count and intensity by selecting appropriate menus
scope. NADPH-d-positive cells were counted per section. such as slice density under the ‘option’ menu at 256 gray
The counting and staining intensity quantification was levels pixel, the ‘tools’ menu to map out the area to be
done by using computer software NIH image version 1.58. counted and the ‘lut’ menu to set the intensity threshold
Some cells were counted manually at 320 objective using an NIH Image, version 1.58 software with a mini-
magnification. mum particle size setting of 10 pixels and standard
threshold. The cells were counted regardless of the intensi- 2.5. Experiment 4
ty of staining since the weaker intensity observed in some sections may possibly have resulted from fixation. Some
In this experiment, we investigated the effect of fasting cells were counted manually at 320 objective magnifica-
treatment and leptin repletion on ovulation. This was to tion. The intensity values which represent the product of
know whether leptin treatment was effective in reversing signals that take both the numbers and the intensity of
the starvation-induced inhibition of estradiol secretion, and staining into account are presented in arbitrary units.
which by implication and extension infers that probably the secretion of LH ovulatory hormone may have
2.8. Statistical analysis occurred in the afternoon of proestrus. The treatment
procedure and the leptin dosing is as described in Experi- At least six sections were counted for each animal and
ment 1. Ovariectomy was performed under light ethyl five sections with the highest numbers of NADPH-d-
ether in the morning of estrus. The excised ovary and the positive cells were used for statistical analysis. Data are
isolated oviduct were covered with drops of saline 0.9 presented
as means6standard
error of
the mean
and coverslipped. The oviduct portion of the ovary was means6S.E.M.. NADPH-d-positive cells, staining in-
examined under a light microscope for an extruded ovum. tensity, estrogen and leptin data were analyzed using the
Mann–Whitney U-test. Incidence of ovulation was ana- 2.6. Experiment 5
lyzed by Fisher’s exact probability test. Comparison between groups for body weight change, food intake and
This experiment was designed to investigate the effect water intake was by Student’s t-test.
of leptin treatment on dependable variables such as food intake, water intake and body weight change. This was to
be used as a strong independent measure of ascertaining
3. Results
that the dose of leptin used in this experiment was at least adequate to alter the neural processes associated with
3.1. Effect of fasting on body weight change and plasma fasting-induced inhibition of the estradiol secretion. Food
leptin levels intake, water intake and body weight change were investi-
gated after food was returned to the animals. Pre-weighed Four days starvation caused a 20 fall in body weight
food and water were given to the animals fasted and given whilst ad libitum feeding over the same period caused a
vehicle control or leptin. Food and water were weighed 5 increase in body weight. Significant P ,0.01 change
to the nearest 0.1 g on an electronic balance Mode in body weight occurred in the fasted animals compared
RE-Zero, A D Co. Ltd., Japan. The difference from the with the fed control Fig. 1. The total body weight loss in
previous measurement was recorded as the intake for the fasted vehicle-treated animals was about 45 g 23967.3
period. Food intake and water intake were quantified 30 initial, 19467.0 g final, whilst in the fed rats body weight
min, 1, 4, 8, 12 and 24 h after recommenced feeding. The increased by about 11.5 g 22965.0 initial, 24068.2 g
change in body weight was also quantified. final. The change in body weight following fasting and
leptin treatment did not differ significantly from that in the 2.7. Quantification of NADPH-d-positive cells and
fasted vehicle-treated
ones leptin;
24065.1 initial,
staining intensity 19064.0 g final, Vehicle; 23969.3 initial, 19469.0 g final,
i.e., 50 vs. 45 g, P ,0.06, suggesting that leptin treatment NADPH-d staining intensity and cell counts were quan-
did not result in a further decrease in body weight. Plasma tified using Macintosh II X Apple computer Inc., CA,
leptin levels decreased significantly P ,0.01, Mann–Whit- USA equipped with a quick capture frame media grabber
ney test in the fasted vehicle-treated rats at all time-points 1.7 Data translation, MA, USA, and Nikon Japan
compared with the fed control. Compared with the fasted camera-microscope Diaphot-TMD EF2 set up. With this
vehicle-treated rats, leptin treatment significantly P ,0.01, ensemble, the images of the sections were transferred to a
Mann–Whitney test increased leptin levels in the fasted magnetic optic MO disc MA-M128, MAC. The images
leptin-treated rats Table 1. Thus, leptin treatment pre- were then analyzed using a power Macintosh 9600 360
vented the fall in plasma leptin in postfasted animals PC connected to a Flex Scan computer Model E 66 T,
without producing supraphysiological levels. This shows
74 E
25.763 intensity, 261 cells, 11.461 intensity in the fasted vehicle-treated rats and 762 cells, 34.563 intensity in the
fasted leptin-treated ones Fig. 2.
3.3. Effect of fasting and leptin repletion on the plasma estrogen levels
Plasma estrogen levels decreased significantly P , 0.001, Mann–Whitney U-test in the fasted vehicle-treated
rats at 10.00, 14.00, 18.00 and 22.00 h compared with the fed control. Compared with the fasted vehicle-treated rats,
leptin repletion significantly P ,0.001, Mann–Whitney U-test increased estrogen levels at 10.00, 14.00, 18.00,
and 22.00 h Fig. 3. Leptin may have acted at the pituitary–ovarian axis levels to bring about the increase in
estrogen secretion [42].
Fig. 1. Body weight change in fed, fasted vehicle-treated and fasted leptin-treated female rats. Values are presented as means6S.E.M. n 55
3.4. Effect of fasting and leptin repletion on ovulation
group. P ,0.01 fasted vehicle-treated; fasted leptin-treated vs. fed control Student’s t-test.
Ovulation incidence decreased significantly P ,0.05 in the fasted vehicle-treated rats compared with the fed
that the fall of leptin with fasting is sufficient to produce control as 1 out of 5 rats ovulated 20 vs. 100. In the
these observed changes and signifies the ‘physiological’ fasted leptin-treated rats, the incidence of ovulation in-
nature of the experiment. creased significantly P ,0.05 compared with the fasted
vehicle-treated rats as 4 out of 5 rats ovulated 80 vs. 20 3.2. Effect of fasting and leptin repletion on NADPH-d
Table 2. reactivity
3.5. Effect of leptin repletion after refeeding on In the VMH, NOS-positive cells number and staining
dependable variables intensity increased significantly P ,0.001, Mann–Whitney
U-test in the fed and fasted leptin-treated rats compared Food intake, water intake and body weight change were
with the fasted vehicle-treated ones. NOS-positive cells significantly P ,0.05, Student’s t-test inhibited in the
number and staining intensity in the VMH of fed rats was fasted leptin-treated rats compared with the fasted vehicle-
7661 cells, 97.562 intensity, 963 cells, 38.364 intensity treated rats Fig. 4. The reduction in food intake and
in the fasted-vehicle treated rats, and 7464 cells, 95.263 water intake may have accounted for most of the weight
intensity in the fasted leptin-treated ones. Fewer NOS- loss observed in the leptin-treated animals. It is also
positive cells were generally expressed in the ARC area. possible that an increase in the thermogenesis induced by
Compared with fasted vehicle-treated rats, the ARC NOS- ob protein leptin administration may have contributed in
positive cells and staining intensity in the fed and fasted part to the negative energy balance and resultant weight
leptin-treated rats increased significantly P ,0.05, Mann– loss. Another factor could be a reduction in hypothalamic
Whitney U-test. In the ARC, NOS-positive cells number neuropeptide Y levels [23,48] in the leptin-treated rats
and the intensity of staining in the fed rats were 661 cells, which resulted in inhibition of fasting-induced hyperphagia
[20].
Table 1 Effect of fasting and leptin repletion on the plasma leptin levels during
a
4-day fast in female rats
4. Discussion
