Screening AD model rats using the Morris water maze test
In recording adaptive swimming, we found that the sham-operated rats always swam freely, and the composited Aβ-treated rats always swam around the pool perimeter (Fig. 2a). Over the 4 days of testing model rats in the Morris water maze, the time to find the hidden platform (latency) progressively declined in all animals. When the screening ratio (SR), which was based on the latency to find the hidden platform on day 4 for composited Aβ-treated and sham-operated rats, was more than 0.2, this animal was considered as a successful model rat. The percentage of successful model rats was 94.7% (Fig. 2b).
Effect of SBF on rat memory acquisition in the Morris water maze test
The positioning navigation trial was used to evaluate rat memory acquisition on day 1 and 2 of the Morris water maze test. During the 2 days memory acquisition trial, the latency to find the hidden platform progressively declined in all rats. However, as shown in Fig. 3, the latency of the composited Aβ-treated group was 540% and 454% [F (1, 6) = 187.37, P < 0.01] greater than that of the sham-operated group on days 1 and 2, respectively. The prolonged latency of the composited Aβ-treated group was significantly shortened by treatment with SBF at doses of 35 mg/kg [F (1, 6) = 5.71, P < 0.05], 70 mg/kg [F (1, 6) = 17.51, P < 0.01], and 140 mg/kg [F (1, 6) = 79.67, P < 0.01].
Effect of SBF on rat memory retention in the Morris water maze test
The probe trial was used to evaluate rat memory retention on day 3 of the Morris water maze test. As shown in Fig. 4a and b, the time that composited Aβ-treated rats swam in the target quadrant (Q1) decreased by 32.14% within 60 s compared with sham control rats [F (1, 6) = 7.16, P < 0.05]. The reduced swimming time of the composited Aβ-treated group was differently attenuated by 3 doses of SBF, which increased swimming time 4.63% in response to 35 mg/kg SBF, 8.40% in response to 70 mg/kg SBF, and 25.26% in response to 140 mg/kg SBF [F (1, 6) = 3.82, P < 0.05].
Effect of SBF on rat memory re-learning in the Morris water maze test
The reversal trial was used to evaluate rat memory re-learning on days 4, 5, and 6 of the Morris water maze test. Figure 3 shows that the composited Aβ-treated rats took 113, 521, and 652% longer to find the hidden platform than the sham control rats [F (1, 6) = 26.55, P < 0.01]. It is interesting that on days 4, 5, and 6 of the Morris water maze test, the 3 doses of SBF differentially shortened the longer latencies, which decreased 20.43, 31.24, and 55.53% in response to 35 mg/kg SBF [F (1, 6) = 7.23, P < 0.05], 51.77, 50.11, and 61.08% in response to 70 mg/kg SBF [F (1, 6) = 17.51, P < 0.01], and 74.04, 61.5, and 80.51% in response to 140 mg/kg SBF [F (1, 6) = 79.67, P < 0.01].
Effect of SBF on rat swimming speed in the Morris water maze test
The visible platform trial was used to evaluate rat swimming speed on day 7 of training in the Morris water maze test. The times spent finding the visible platform for rats in each group were not significantly different [F (4, 30) = 0.79, P > 0.05]. Therefore, individual differences in rat swimming speed could be excluded, which indicated that motivation and motor skills were essentially intact.
Effect of SBF on rat neuronal injuries induced by composited Aβ-treatment
Three rats from each group were decapitated 60 min after the last administration of SBF or saline on day 38 of drug treatment. In several composited Aβ-treated rats, visual inspection revealed a yellow surface, and a thin or collapsed cerebral cortex. Optical microscopy of HE stained brains from the composited Aβ-treated group showed marked pathological changes in neurons of the hippocampus and cerebral cortex, such as neurofibrillary degeneration, neuronophagia, nuclear pyknosis, and nuclear margination (Fig. 5aB1, B2), as compared with the sham-operated group (Fig. 5aA1, A2, A3). In addition, neurons in part of the cerebral cortex of composited Aβ-treated rats showed typical colliquative necrosis, which was characterized by disrupted cell membranes, fragmented nuclei, and extensive infiltration of inflammatory cells in the necrotic region (Fig. 5aB3). However, in composited Aβ-treated rats that had been treated with SBF for 38 d, neuronal injuries in the hippocampus and cerebral cortex were markedly attenuated in a dose-dependent manner (Fig. 5aC1–E1, C2–E2, C3–E3).
In addition to pathological changes, the number of neurons was significantly reduced in the brains of composited Aβ-treated rats, as compared with those of the sham-operated group. The neuron count was 63.86 ± 4.35% (P < 0.01) lower than that of the sham-operated group in 0.125 mm sections of the hippocampal CA1 area and 55.46 ± 5.48% (P < 0.01) lower in 0.0352 mm2 sections of the cerebral cortex (Fig. 6a). It is noteworthy that the decreased neuron count in composited Aβ-treated rats was dramatically reversed by treatment with SBF for 38 days. The number of neurons was increased 18.98% by 35 mg/kg (P < 0.05), 47.36% by 70 mg/kg (P < 0.01), and by 140 mg/kg 106.81% (P < 0.01) in the hippocampus CA1 subfield and 14.24% by 35 mg/kg (P < 0.05), 59.33% by 70 mg/kg (P < 0.01), and 85.63% by 140 mg/kg (P < 0.01) in the cerebral cortex subfield (Fig. 6a).
The ultrastructure of neurons was examined with electron microscopy. Compared with the sham-operated group (Fig. 6cA), neurons in the composited Aβ-treated group were severely damaged, showing mitochondrial swelling and cristae fragmentation, increased mitochondrial electron density, dilation of the rough endoplasmic reticulum, depolymerization of polyribosomes and polymicrotubules, smaller postsynaptic density (PSD), production of secondary lysosomes, and a large number of lipofuscin deposits in the cytoplasm. The nuclear membrane appeared rough and sunken, euchromatin was condensed and denatured, myelin sheath layers were loose or attenuated, and internal axons and fibers were degenerated (Fig. 6cB). However, 140 mg/kg SBF administered for 38 days dramatically attenuated these neuronal pathological changes induced by composited Aβ, and damage to neuronal subcellular structure was reduced (Fig. 6bC).