2.3 Results and discussion
2.3.3 Increasing Aβ accumulation, apoptotic signal, and oxidative stress
in the CA1 regions of hyperglycemic mice injected with oligomer Aβ
1–40At first, we confirmed that the infusion site in each mouse was exactly in the CA1 region of the hippocampus by cresyl violet staining of mouse brains (Fig. 5). Immunohistochemical analyses showed that Aβ depositions were identified in both of the hyperglycemic and
normoglycemic mice at the CA1 region after infusion with oligomer Aβ1–40 (Figs. 6A-1 and A-2). However, the extent of the Aβ1–40
accumulation was more evident in the hyperglycemic mice than in the normoglycemic mice (p < 0.001; Fig. 6B). No positive staining of Aβ accumulation was observed in vehicle-treated mice (Figs. 6A-3 and A-4).
Therefore, we suggested that acute oligomer Aβ1–40 might result in increased accumulation of Aβ1–40 around CA1 region in hyperglycemic mice compared to normoglycemic mice. Because a common feature of AD pathology is neuron loss, we used caspase-3 immunostaining to detect the presence of apoptosis in mice with impaired spatial reference memory. Although caspase-3 immunopositive reactions were identified in the CA1 regions of both hyperglycemic and normoglycemic mice treated with oligomer Aβ1–40, the apoptotic CA1 region in hyperglycemic mice was greater than that in normoglycemic mice (p < 0.001; Figs. 7A-1, A-2, and B). Apoptotic signals in the dentate gyrus subregion of hippocampus were also observed in the hyperglycemic mice treated with oligomer Aβ1–40 or vehicle (Figs. 7A-1 and A-3). No signal, however, was observed in the normalglycemic mice infused with vehicle even though there was some cell loss at the infusion site (Fig. 7A-4). Furthermore, in examining the correlation between apoptosis and oxidative stress, we found MnSOD
immunopositive reaction in the CA1 region of hyperglycemic and
normoglycemic mice injected with oligomer Aβ1–40 (Figs. 8A and B). The oxidative stress signal in the CA1 region is correlated to the result of the Aβ1–40 accumulation and caspase-3 staining. No signal was identified in the dentate gyrus subregion of the hyperglycemic mice (Figs. 8A-1 and A-3), although apoptotic signal was previously observed in this region.
There was no staining observed in CA1 region of hyperglycemic or normoglycemic mice injected with vehicle (Figs. 8A-3 and A-4). Our findings from the serial immunostaining for Aβ1–40, caspase-3, and MnSOD suggest that the increasing Aβ1–40 accumulation induced more apoptotic CA1 neuron in hyperglycemic mice than that in the
normoglycemic mice. Previous evidence also indicates that around 70%
of neurons in the CA1 region of the hippocampus die during the progression of AD [78]. Therefore, we propose that the interaction
between the hyperglycemia and oligomer Aβ1–40 enhanced the damage of the CA1 subregion over the “safe threshold”. In addition, we found the oxidative stress of the CA1 region was also more evident in
hyperglycemic mice than in normoglycemic mice. Therefore, these data suggested that acute intrahippocampal CA1 administration of oligomer Aβ1–40 induced more severe damage in hyperglycemic mice than in normoglycemic mice through increasing Aβ accumulation, oxidative stress, and apoptotic neuron. One previous study also suggests a positive feedback loop between the oxidative damage and glucose metabolic dysfunction [79]. A previous dose response experiment doses ranging from 0.01 to 5 μM confirmed the selective vulnerability of CA1 to soluble oligomeric Aβ [80]. Evidence showed that brain tissues from AD
patients have more nerve cells with activated caspase-3 than do those from people who died of other causes [81]. Su et al. (2001) reported that neurons with caspase-3 are found in brains of AD mice or cultured nerve cells [82]. Aβ, in ways that are inhibited by free radical antioxidants like vitamin E, causes brain cell protein oxidation, lipid peroxidation, reactive oxygen species formation, and other oxidative stress responses,
suggesting that this peptide is a source of oxidative stress in brain [83].
Furthermore, we also found cell apoptosis in the dentate gyrus of hyperglycemic mice injected with oligomer Aβ1–40 or vehicle. These results are consistent with previous pathological studies in humans and animals showing hyperglycemia preferentially induces neuronal death in the CA1, subiculum, and dentate gyrus of the hippocampus, as well as in the superficial layers of the cortex, or in the striatum [84]. We did not, however, observe any MnSOD immunoreactivity in the dentate gyrus or CA1 region of these mice. A study of 3- and 12-month old rats showing nerve conduction deficits after STZ treatment revealed no changes in antioxidant enzymes except for increased catalase in 12-month-old rats [85]. Therefore, we suspect that the apoptosis in the dentate gyrus was induced by STZ treatment through another signaling pathway instead of from oxidative stress in CA1 region arising from accumulation of Aβ.
The anatomical and behavioral data from our study suggest that hyperglycemia itself induced the apoptosis in the dentate gyrus of the hippocampus, but had no effect on spatial learning and memory. However, hyperglycemia group with oligomer Aβ1–40 treated mice induced more accumulation of Aβ1–40, oxidative stress, and apoptosis in the
hippocampal CA1 neurons. Many studies have suggested that lesions in
the CA1 and dentate gyrus of the hippocampus induce the impairment of spatial learning and memory. The hippocampus has been well known to play a critical role in certain types of learning, including spatial learning [86-88]. Specific cells within the hippocampus become selectively activated when an animal is replaced in particular locations within its environment [89]. Within the hippocampus, cells of the dentate gyrus serve to restrict or amplify signals that originate in extra-hippocampal sites and propagate into the hippocampus proper [90]. The CA1 region of hippocampus plays significant roles in associational memories [91,92].
Therefore, hyperglycemia in oligomer Aβ1–40-treated mice induced the impairment of spatial learning and memory. In summary, our results not only provide the animal model to evaluate in detail the behavioral and neuroanatomical effects of the interaction between abnormal glucose metabolism and Aβ1–40 in vivo but also provide experimental support for the epidemiological literature indicating that the amyloid accumulation and metabolic dysfunction may interact to exacerbate the pathogenesis of AD.
Figure 3. Hyperglycemia of C57BL/6J male mice induced by STZ treatment. (A) Body weights examined before (day 1) and after STZ injection (days 10, 15, and 23) and (B) Blood glucose levels examined before (day 1) and after STZ injection (days 10, 15, and 23). Reduced body weight and significantly increased blood glucose revealed the effectiveness of hyperglycemic induction by STZ. Error bars indicate standard error of the means. *p < 0.05.
Figure 4. Effect of Aβ1-40 peptide on performance of water maze task. The 4 training days were scheduled on days 17–20. The 3 testing trials were carried out on day 21. (A) Hyperglycemic mice injected with oligomer Aβ1-40 showed significant impairment in the spatial reference memory compared to mice treated with other treatments. (B) The escape latency of oligomer-treated mice in the water maze during the 4 training days. The oligomer Aβ1-40 injected hyperglycemic mice showed a slower learning ability than the normoglycemic mice (p < 0.05). (C) The escape latency of hyperglycemic mice in the water maze during the 4 training days. The oligomer Aβ1-40 injected hyperglycemic mice showed a slower learning ability than the monomer or vehicle-treated hyperglycemic mice. (D) Swim velocity of hyperglycemic mice in the water maze during the 4 training days. No significant difference was identified among the 3 groups of mice with different Aβ treatments. (E) The performance of mice during probe trial conducted on day 23. The hyperglycemic mice with oligomer Aβ1-40 injection spent less time in the target quadrant than in the other 3 quadrants. These results show that hyperglycemia accelerated the impairment of the spatial reference learning and memory with oligomer Aβ1-40 treatment. Double-distilled water was used as vehicle treatment in the study. Error bars indicate standard error of the means. *p < 0.05.
Figure 5. The Aβ1-40 injection sites confirmed by cresyl violet staining. (A) Oligomer Aβ1-40 injection into hyperglycemic mice. (B) Oligomer Aβ1-40 injection into
normoglycemic mice. (C) Vehicle injection into hyperglycemic mice. (D) Vehicle injection into normoglycemic mice. Cresyl violet staining reveals that injection sites were in the CA1 region of the hippocampus. Arrows indicate the injection site. Scale bar＝100 μm.
Figure 6. Immunohistochemistry of Aβ1-40 deposition in the CA1 region of
hippocampus. (A) The Aβ1-40 immunoreactive granules by DAB staining in the CA1 region of the hippocampus of the mice. (1) Oligomer Aβ1-40 injection into
hyperglycemic mice, (2) oligomer Aβ1-40 injection into normoglycemic mice, (3) vehicle injection into hyperglycemic mice, (4) vehicle injection into normoglycemic mice. Scale bar ＝ 100 μm. (B) The percentage of staining area of the Aβ1-40
deposition in the CA1 region. Compared to the normoglycemic mice treated with oligomer Aβ1-40 (n = 3), there was a significantly increased area of Aβ1-40 deposition in the CA1 of hyperglycemic mice treated with oligomer Aβ1-40. *p < 0.001.
Figure 7. Results of caspase-3 signal showing apoptosis in the subregions of the mouse hippocampus. (A) Caspase-3 immunoreactivity in the hippocampus of the mice. (1) Oligomer Aβ1-40 injection into hyperglycemic mice, (2) oligomer Aβ1-40
injection into normoglycemic mice, (3) vehicle injection into hyperglycemic mice, (4) vehicle injection into normoglycemic mice. Caspase-3 positive signals are observed in the CA1 and the dentate gyrus (DG) subregions of the hippocampus in the
hyperglycemic mice. Normoglycemia show the caspase-3 positive signals only in the CA1 subregion of the hippocampus with oligomer Aβ1-40-treated mice. Scale bar ＝ 100 μm. (B) The percentage of relative area in the CA1 of the mice. Compared to the normoglycemia group treated with oligomer Aβ1-40 (n = 3), hyperglycemia group treated with oligomer Aβ1-40 (n = 3) showed a significant cell loss in CA1 region. *p
< 0.001.
Figure 8. Results of MnSOD staining showing oxidative stress occurred in mouse brains. (A) MnSOD immuno reactivity in the CA1 region of the hippocampus. (1) oligomer Aβ1-40 injection into hyperglycemic mice, (2) oligomer Aβ1-40 injection into normoglycemic mice, (3) vehicle injection into hyperglycemic mice, (4) vehicle injection into normoglycemic mice. Positive oxidative stress signal is observed in the CA1 subregion of the hippocampus only in oligomer Aβ1-40 treated hyperglycemic or normoglycemic mice. Scale bar＝ 100 μm. (B) The percentage of relative area of the normoglycemia and hyperglycemia-treated oligomer Aβ1-40 mice in the CA1 region.
Compared to the normoglycemia group-treated oligomer Aβ1-40 (n = 3), hyperglycemia-group treated with oligomer Aβ1-40 (n = 3) show a significant oxidative stress in CA1 region. *p < 0.001.