Figure 1.
LBPs were administered each day by the intragastric (i.g.) route for 2 weeks, when SCO administration was commenced by a subcutaneous (s.c.) osmotic minipump. Administration of LBP and SCO continued for a further 4 weeks. Behavior tests were performed immediately 1 day after SCO/LBP treatment. Some of animals were sacrificed immediately for biochemical analysis after drug treatment. Twenty-four hours after behavioral testing the rest of animals were sacrificed for immunohistochemical analysis.
Figure 2.
LBPs protect against SCO-induced impairments of working memory.
(A) Diagram of the novel object recognition (NOR) task. Left (training phase), animals are exposed to two identical objects (a1 and a2). Right (test phase), animal are exposed to two different objects, a familiar (a1) object from the training phase and a new object (b) not seen before. (B) The total exploration time in two phases was recorded. Control (vehicle/saline) rats spent more time on exploring the novel object whereas vehicle/SCO animals explored the novel and familiar objects equally. LBP treatment (LBP/SCO group) restored the preference for the novel objects. (C) Discrimination indices in the test phase were calculated as the percentage of time spent exploring the novel object over the total time spent exploring both objects. The dashed line indicates the 50% (chance) level. (D) The object location recognition (OLR) task. Left (training phase), animals are exposed to two identical objects (c1 and c2). Right (test phase), animals are exposed to two same objects. One object (c1) is at the same location, but the other (c2) is relocated versus the training phase. (E) The total time exploring the objects in familiar and novel locations was recorded. Control rats spent more time exploring the relocated object whereas rats treated with SCO (vehicle/SCO group) showed no preference for the object at the new location. LBP treatment (LPB/SCO group) restored the preference for the novel location. (F) Discrimination indices in the test phase were calculated as the percentage of time spent exploring the new location over the total time spent exploring both locations. The dashed line indicates the 50% chance level. Values are means ± SEM (Vehicle/saline, n = 12; vehicle/SCO, n = 10; LBP/SCO, n = 11); *,P<0.05; **,P<0.01.
Figure 3.
LBP treatment reverses the SCO-induced increase in latency time in the Morris water maze.
(A) The apparatus for Morris water maze. The black circle in the northeast quadrant represents the location of the hidden platform. Animals were introduced into the southwest quadrant. (B) The latency time, swim distance and swim speed to find the submerged platform were recorded over six consecutive days. The animals of three groups have the similar swim speed, but SCO treatment (vehicle/SCO group) increased the latency and swim distance to find the hidden platform whereas LBP administration (LBP/SCO group) restored latency and distance to the levels of the vehicle/saline control group. (E) The percentage of time spent in 4 quadrants during the probe trial. The animals in control and LBP/SCO groups spent more time in the target quadrant while SCO treatment decreased time in target quadrant. Values are means ± SEM (Vehicle/saline, n = 12; vehicle/SCO, n = 10; LBP/SCO, n = 11); *,P<0.05 versus the vehicle/SCO group.
Figure 4.
Effect of SCO/LBP treatments on cell proliferation in the hippocampal dentate gyrus (DG).
(A) Representative immunofluorescence staining for Ki67, a marker of cell proliferation. Sections were subjected to Ki67 antibody after SCO or/and LBP treatment. Arrows indicate Ki67-positive cells in hippocampus. The dashed line is the location of subgranular layer in DG. (B) Quantification of Ki67-positive cells in the DG in A. SCO treatment significantly decreased cell proliferation compared to the vehicle/saline group. The LBP/SCO group showed a significantly higher proportion of Ki67-positive cells than the SCO group. Values are means ± SEM (n = 6 animals per groups); *,P<0.05; **,P<0.01; scale bars, 50 µm.
Figure 5.
LBP treatment increases neuroblast differentiation in the hippocampal DG.
(A) DCX immunostaining showed the newborn neurons in the subgranular zone of the DG. DCX-immunoreactive neuroblasts (green) were abundant in the DG in the vehicle/saline group. SCO treatment markedly decreased the number of DCX-positive cells. In the LBP/SCO group, the number of DCX-positive cells was restored. Scale bars, 200 µm. (B) Quantification of DCX-positive cells in DG of the three groups in A. (C) The representative images of Calretinin immunostaining. The length of dendrites is markedly injured by SCO compared with control and LBP treatment groups. Scale bars, 100 µm. (D) Quantification of number of Calretinin positive cells in DG in A. Values are means ± SEM (n = 6 animals per groups); **,P<0.01.
Figure 6.
LBP protects the processes of newborn neurons in DG of hippocampus.
(A) Representative images of doublecortin (DCX)-positive neuroblasts in the subgranular zone of DG. The lower panels are the enlargement of the frames in the upper panels. The arrows indicates the tertiary neurites of DCX positive neurons in DG. In control vehicle/saline and LBP/SCO groups DCX-immunoreactive neuroblasts have well-developed processes extending to the molecular layer of the DG. SCO treatment (vehicle/SCO group) led to significant reduction of tertiary dendrites. (B) Quantification of number of the DCX-immunoreactive cells with tertiary dendrites in the three groups. Values are means ± SEM (n = 6 animals per groups); ***,P<0.001; scale bars, 50 µm.
Figure 7.
Anti-oxidative Effects of LBP in hippocampus.
The SOD (A), GPX (B) specific activities, GSH (C) and MDA (D) levels were determined by using the rat hippocampus homogenates. Values are means ± SEM (n = 4 animals per group), *,P<0.05 and **,P<0.01 vs. SCO alone-treated group.
Figure 8.
Effects of LBP on the levels of AChE in the hippocampus of the vehicle, scopolamine (SCO), SCO+LBP groups (n = 4 animals per group).
The AChE substrate acetylthiocholine in the kits is incubated with hippocampal homogenates. Quantification of the thiocholine produced from the hydrolysis of acetylthiocholine reflects the AChE activities. Values are means ± SEM; *,P<0.05 vs. SCO alone-treated group.
Figure 9.
LBP did not alter the expressions of BDNF and IGF-1.
(A) The hippocampus homogenates were separated in SDS page and bloted with BDNF, IGF-1 and GAPDH antibodies. GAPDH is the internal standard. (B) Quantification of BDNF in A indicates SCO-decreased BDNF was not reversed by LBP treatment. (C) Quantification of IGF-1 indicates both of SCO and LBP did not influence the level of IGF-1 in hippocampus. Values are means ± SEM (n = 4 in each group). **,P<0.01.
Figure 10.
Effects of LBP on the apoptosis in hippocampus.
(A) The hippocampus homogenates were separated in SDS page and bloted with Bcl2 and Bax antibodies. Bcl2 level decreases after SCO administration and is reversed by LBP treatment, while Bax shows opposite pattern after SCO or LBP treatment. GAPDH is the internal standard. (B) Quantification of the ratio Bax/Bcl2 in A shows that SCO alone increases the ratio of Bax/Bcl2 which is reversed by LBP treatment. Values are means ± SEM (n = 4 in each group). **,P<0.01 and ***,P<0.001.