| Objective:Alzheimer disease(AD) is a chronic, insidious onset and irreversible neurodegenerative disorder mainly occurred in old person, characterized by progressive deterioration of cognitive and mental function, including learning and memory. According to the World Alzheimer Report 2014, the worldwide number of dementia cases currently estimated 44 million, and the patients are nearly 6 million in our country. In addition, with the increase of aging population, the incidence of AD and the number of AD patients will continue to be increased. Therefore, the early diagnosis and treatment of AD have important clinical significance. Nowadays, the popular method of AD diagnosis in clinical work is the combination of clinical symptoms and medial temporal lobe atrophy visual scale evaluation detected by Magnetic Resonance Imaging(MRI). However, the brain atrophy only appeared in advanced AD patients. There is no effective method of early detection and diagnosis of AD patients before the appearance of clinical symptoms and changes of brain morphology until now.The prominent pathological features of AD include the high density of senile plaques, the neurofibrillary tangles(NFTs) and the loss of neurons. As we all known, the main constituent of senile plaque is amyloid β-peptides(Aβ), which consisting of 39-43 amino acids. A lot of research, including genetic studies and evidences from animal models, all support the primary role of Aβ peptide in the cascade of events which leads to the form of neurofibrillary tangles and the neuronal death in AD brain. On the other hand, the apolipoprotein E4(APOE4) allele, which is located on chromosome 19, is a major risk factor of AD. At the same time, apo E4 protein, which translated from APOE4 gene, hasshown strong neurotoxicity, and is related to the occurrence and development of AD. In addition, not only the Aβ and apo E4 itself has neurotoxic effects, there is also the synergistic effect between Aβ and apo E4, which could further aggravate the development of AD.Magnetic resonance imaging(MRI) is a method of medical imageology with high resolution in soft tissues, which is widely used in the research and diagnosis of neurological diseases. Morris water maze(MWM) is one of the most widely used tasks in behavioral neuroscience for studying the psychological processes and neural mechanisms of spatial learning and memory. Hippocampal long-term potentiation(LTP), as a popular electrophysiological model, has been widely used for the research of cellular basis of learning and memory and AD. Some research found that Aβ1-40 could directly cause the expansion of lateral ventricles, the change of the concentration of metabolites detected by 1H-Proton Magnetic Resonance Spectroscopy(1H-MRS) imaging, the impairment of the spatial learning and memory and depression of the induction and maintenance of LTP in rats. Other research also showed that the abilities of spatial learning and memory and working memory were damaged, the hippocampal LTP was depressed in targeted replacement mice expressing human APOE4 gene. However, there are very few reports about the feature of imageology, the effect of spatial learning and memory and hippocampal synaptic plasticity in AD model with the interaction of Aβ and apo E4. Especially, there is lack of research of MRI, behavioral, electrophysiological and morphological test at the same animal model, which could clarify the AD-related alteration from different aspects and finally confirm the AD by pathological test.In the present study, by using MRI, Morris water maze behavioral tests and field potential recording, combined with morphological and western blot techniques, we measured the T2 values and the metabolite concentration of 1H-MRS, detected the changes of spatial learning and memory abilities and synaptic plasticity, and observed the pathological characteristics in three AD models at different time point before and after i.c.v. injection of different drugs. In addition, the correlation between MRI indicators and changes of behavior, electrophysiology and morphology was also investigated. By usingdifferent techniques to detect the AD relative changes in the same animal from different aspects, we hope to contribute to the further understanding of the relationship of different changes observed during the occurrence and development of AD, and to provide new evidences and methods to early diagnosis of AD.Methods:The intracerebroventricular(i.c.v.) injection of Aβ1-40, apo E4 or Aβ1-40 plus apo E4 induced AD rat model rats were used as the research subjects. 60 rats were randomly divided into four groups: control group, Aβ1-40 group, Apo E4 group and co-injection group, with 15 rats in each group. The magnetic resonance imaging(MRI) was done in each rat before and 2 or 4 weeks after i.c.v. injection of different drugs by using multiple echo sequence and 1H-Proton Magnetic Resonance Spectroscopy(1H-MRS). The changes of hippocampal T2 values and metabolite concentrations measured by 1H-MRS was observed. When the second magnetic resonance imaging test finished, the place navigation test, probe trial test and visible platform test were done in rats of 4 different groups. The indicators observed include the mean escape latency and swimming distance for searching the underwater platform, the percentages of total time and distance elapsed in the target quadrant, the mean swimming speed and the escape latency to find visible platform. When the Morris water maze test finished, field excitatory postsynaptic potentials(f EPSPs), paired-pulse facilitation(PPF) and long term potentiation(LTP) in hippocampal CA1 area were recorded immediately by using in vivo field potential recording techniques. When the third magnetic resonance imaging test was finished, HE staining and immunohistochemistry were done in rats randomly selected from four groups to observe the morphological changes of the brain and the deposition of Aβ in the hippocampus and cortex. In addition, western blot techniques were used to measure the content of phosphorylated tau protein in the hippocampus in rats randomly selected from four groups.Results:By doing MRI of multiple echo sequence and 1H-MRS in each rat before and 2 or 4weeks after i.c.v. injection of different drugs, we found that:(1) The characteristics of hippocampal T2 values in three different AD models were different. Before and 2 or 4 weeks after drug administration, the T2 values of control group and Apo E4 group were 80.46±3.06 ms and 80.64±2.27 ms, 81.92±3.48 ms and 81.20±1.67 ms, 80.72±3.47 ms and81.98±3.10 ms, respectively. The hippocampal T2 values in apo E4 group was similar to control at every time point before and after drug application. In addition, in Apo E4 group, the T2 values was not obvious different at any time point before and after drug injection. In Aβ1-40 group, the T2 values were 81.70±3.10 ms, 80.59±3.22 ms and 91.05±3.10 ms before and 2 or 4 weeks after drug application. There was no difference of T2 value between control and Aβ1-40 group before and 2 weeks after drug administration. However, 4 weeks after drug application, the T2 value in Aβ1-40 group significantly higher than control group(P <0.05). Meanwhile, the T2 values at 4 weeks after Aβ1-40 application was significantly higher(P <0.05) than before and 2 weeks after drug administration. In co-injected of Aβ1-40 and apo E4 group, before and 2 or 4 weeks after drug application, the T2 values were 80.86±2.74 ms, 81.71±3.55 ms and 95.95±1.25 ms, respectively. 2 weeks after i.c.v. injection, there was no difference of T2 value between three AD models and control group. However, 4 weeks after drug administration, the T2 value of co-injection group was not only significantly higher than control group, but also higher than Aβ1-40 or Apo E4 group(P <0.05). Meanwhile, 4 weeks after co-injection of Aβ1-40 and apo E4, the T2 value was significantly higher(P <0.05) than before and 2 weeks after drug administration.(2) The hippocampal NAA/Cr+Cr1 was decreased in three different AD models, but the level of decrease was different at three AD model. Before drug application, the NAA/Cr+Cr1 in control group, Aβ1-40 group, Apo E4 group and co-injection group were 1.19±0.06, 1.15±0.12, 1.16±0.05 and 1.16±0.07, respectively. 2 weeks after drug administration, the NAA/Cr+Cr1 in Aβ1-40 group, Apo E4 group and co-injection group were 0.74±0.13, 0.92±0.17 and 0.33±0.12 respectively, which were decreased than 1.16±0.04 in control group(P<0.05). 4 weeks after i.c.v. injection of drugs, the NAA/Cr+Cr1 were 0.44±0.04, 0.73±0.04 and 0.18±0.02 in three different AD models, which were decreased than 1.20±0.10 in control group(P<0.05). In addition, at differenttime point after drug application, the decrease of NAA/Cr+Cr1 in co-injection group was highest and in apo E4 group was lowest. On the other hand, the NAA/Cr+Cr1 decreased more at 4 weeks after drug injection than 2 weeks after drug injection, and decreased more at 2 weeks after drug injection than before drug injection in all AD models we used(P<0.05).(3) The hippocampal Cho/Cr+Cr1 was decreased in all three AD models, but the characteristics of decrease were different. At control group, the hippocampal Cho/Cr+Cr1 were 1.30±0.13, 1.27±0.08 and 1.28±0.05 before, 2 and 4 weeks after drug administration. 2 or 4 weeks after i.c.v. injection of different drugs, the Cho/Cr+Cr1 were 1.01±0.31 and 0.81±0.16, 0.95±0.25 and 0.84±0.09, 0.96±0.28 and 0.85±0.23 in Aβ1-40, Apo E4 and co-injection group, respectively, which were significantly decreased than control group at the same time point(P<0.05), but there was no difference between the three AD models. In addition, compared to the time point before drug application, the hippocampal Cho/Cr+Cr1 was significantly decreased at 2 and 4 weeks after drug injection in each AD model. Meanwhile, the Cho/Cr+Cr1 in Aβ1-40 group was significantly decreased at 4 weeks than 2 weeks after drug application(P <0.05). However, there was no difference of Cho/Cr+Cr1 between 2 and 4 weeks after drug administration in apo E4 alone and co-injection group.(4) The change of hippocampal NAA/Cho showed different characteristics in three AD models. 2 weeks after drug administration, the NAA/Cho in co-injection group was 0.38±0.21, which was significantly lower than the 0.91±0.08 in control group(P <0.05). However, the NAA/Cho of Aβ1-40 or apo E4 alone group were 0.81±0.34 and 1.02±0.33 respectively, which was no significant difference to control group. 4 weeks after drug application, the NAA/Cho of Aβ1-40 or co-injection group were 0.58±0.21 and 0.24±0.14 respectively, which was obviously lower than the 0.94±0.07 in control group(P <0.05), and the NAA/Cho decreased more in co-injection group. On the other hand, 2 weeks after drug injection, the NAA/Cho in co-injection group was significantly decreased compared to the 0.91±0.21 before drug application, and the level of decrease was similar in 2 and 4 weeks after drug injection. In Aβ1-40 group, the NAA/Cho begin to decrease 4 weeks after drug injection and there was no difference between apo E4 and control group at any time point.When the second MRI test finished, the rats were done Morris water maze test. Wefound that:(1) The spatial learning and memory ability was significantly impaired in rats intracerebroventricular(i.c.v.) injected of Aβ1-40 or apo E4 alone, but there was not any significant difference in the extent of damage between those two AD models. On the first day of training in place navigation test, there was no difference between Aβ1-40 or apo E4 injected alone group and the control group. But on the training day 2-5, the mean escape latency and swimming distance of Aβ1-40 group were 35.7±1.8 s and 832.4±55.4 cm, 29.4±1.6 s and 554.3±54.9 cm, 23.8±1.4 s and 459.0±39.0 cm, 23.1±1.3 s and 405.1±45.7 cm, respectively; the mean escape latency and swimming distance of Apo E4 group were 35.1±2.0 s and 826.3±70.4 cm, 28.6±2.1 s and 557.3±53.7 cm, 24.0±2.7 s and 456.1±33.5 cm, 23.4±1.9 s and 404.6±45.8 cm, respectively. Compared to the 26.4±3.8 s and 609.0±67.1 cm, 21.3±3.5 s and 360.2±37.4 cm, 16.1±2.8 s and 267.4±32.3 cm, 14.8±2.5 s and 260.3±34.2 cm in control group, the mean escape latency and swimming distance were significantly larger(P <0.05) on Aβ1-40 or apo E4 injected alone group. In the probe trial test, the percentages of total time and distance elapsed in the target quadrant in Aβ1-40 or Apo E4 group were 34.6±1.9% and 34.1±2.4%, 34.4±1.6% and 34.0±2.2% respectively, which were significantly lower than the 46.3±3.8% and 46.1±3.5% in control group(P <0.01). However, the impairment of spatial learning and memory ability was similar in Aβ1-40 or apo E4 injected alone group.(2) The spatial learning and memory ability was also significantly impaired in the combined i.c.v. injection of Aβ1-40 and apo E4 rats, and the effect of impairment was stronger than Aβ1-40 or apo E4 injected alone group. On the training day 1, there was no difference between control and three different AD models. On the training day 2, the mean escape latency and swimming distance in co-injection group were 41.1±2.5 s and 905.4±80.8 cm, which was significantly larger than control group(P <0.05). However, there was no difference between Aβ1-40 or apo E4 alone group and co-injection group. On training day 3-5, the spatial learning ability in co-administered rats was impaired more severely compared to Aβ1-40 or apo E4 alone group, and the mean escape latency and swimming distance of co-injection group were 36.0±1.1 s and 782.1±71.7 cm, 30.7±1.8 s and 621.7±39.3 cm, 29.8±1.7 s and 588.8±34.1 cm, respectively. In the probe trial test, the percentages of total time and distance elapsed in the target quadrantin co-injection group were 27.3±1.8% and 26.7±1.3%, which were not only significantly lower than control group, but also significantly lower than Aβ1-40 or apo E4 alone group(P <0.01).(3) The visible platform tests showed that all drugs did not affect the vision and motor ability of rats(P>0.05). During all training days, the mean swimming speeds and the escape latency to find visible platform in control group, Aβ1-40 group, Apo E4 group and co-injection group were 19.2±1.6 cm/s and 12.6±0.9 s, 19.2±1.7 cm/s and 12.3±0.8 s, 19.4±1.9 cm/s and 12.7±0.8 s, 19.2±1.8 cm/s and 12.3±0.9 s, respectively. There was not any significant difference in all four groups.The field potentials in hippocampus of rats were recorded when the Morris water maze test finished. The results showed that:(1) Hippocampal LTP was significantly depressed in rats intracerebroventricular(i.c.v.) injected of Aβ1-40 or apo E4 alone, but the depressive effect was similar in those two groups. The amplitudes of f EPSPs was stable in each group before giving high-frequency stimulation(HFS) and the baseline synaptic transmission was not affected. The amplitude of f EPSPs in control group significantly increased to 177.8±4.9% immediately after HFS and the amplitude remained at 149.3±4.1% 60 min after HFS as compared to that measured before HFS and set arbitrarily as 100%. In Aβ1-40 or apo E4 alone group, at 0 min, 30 min and 60 min after giving HFS, the averaged amplitude of f EPSPs decreased to 147.9±5.8% and 144.4±6.1%, 123.4±4.1% and 122.8±4.4%, 117.7±3.8% and 122.4±3.3%, respectively. Compared to the control group, the induction and maintance of LTP were significantly depressed(P <0.01), but the depressive effect was similar in those two AD models.(2) The hippocampal LTP was severely impaired in combined i.c.v. injection of Aβ1-40 and apo E4 group, and the depressive effect was stronger than Aβ1-40 or apo E4 alone group. The baseline synaptic transmission was not affected in co-injection group, but compared to control group, the amplitude of f EPSPs was obviously depressed at different time after HFS(P <0.01). At 0 min, 30 min and 60 min after giving HFS, the averaged amplitude of f EPSPs were 146.5±4.1%, 113.6±3.2% and 105.0±3.8%, respectively. Meanwhile, the amplitude of f EPSPs in co-injection group was similar to Aβ1-40 or apo E4 administered alone group 0 min and 30 min after HFS. However, 60 min after HFS, the LTP was significantlydepressed in co-injection group compared to Aβ1-40(P <0.05) or apo E4(P <0.01) alone group.(3) The paired-pulse facilitation(PPF) was not affected in three different AD models. In control group, Aβ1-40 group, Apo E4 group and co-injection group, the PPF were 194.5±6.1%, 196.8±7.5%, 193.1±6.8% and 194.9±5.4%, respectively. There was no difference between those 4 groups.When the third MRI test finished, HE staining, immunohistochemistry and western blot were done. The results showed that:(1) There were significant morphological changes in hippocampus and cortex, but the degree of changes was different in three AD models rats. Control group showed normal multilayer neuron which closely aligned and arranged in neat rows, with distinct edges, clear nucleus. Apo E4 group only showed mild disorder of hippocampal CA1 multilayer neurons and the gap slightly increased in some cells. In Aβ1-40 group and co-injection group, pyramidal neurons arranged in disorder and the gap expanded, the number of pyramidal neurons reduced, the shape of neuron was irregular and the size of neurons were different. Especially, the morphological chage was more obvious in co-administration group than in Aβ1-40 group. Meanwhile, the reactive proliferation of lymphocytes and microglia in cortex were observed in three AD models, but the inflammatory reaction was more obvious in co-administration group than in other two AD models. At the same time, the malacia was seen in the cortex of Aβ1-40 alone and co-injection group.(2) The different levels of Aβ-positive cells were found in the hippocampus and cortex of three AD models. There was not any Aβ-positive cells in control group. Scattered Aβ-positive cells was found in Apo E4 group. A lot of Aβ-positive cells was found in Aβ1-40 alone and co-injection group, but the maximum number of Aβ-positive cells was in co- injection group.(3) Different AD models exhibited different increasing levels of p Tau(S396). The p Tau(S396)/Tau in Aβ1-40 or apo E4 alone group were 0.969±0.038 and 1.048±0.041 respectively, which were significantly increased(P <0.01) than the 0.606±0.047 in control group, but there was no significant difference between those two AD models. In the co-injection group, the p Tau(S396)/Tau was 1.275±0.051, which was not only significantly higher than control(P <0.01), but also significantly higher than Aβ1-40 or apo E4 alone group(P <0.01).Conclusions:(1) The T2 value was no significant difference between three AD models and control group at 2 weeks after drug administration, which indicate that T2 value was not a reliable method of early diagnosis of AD. 4 weeks after drug application, the hippocampal T2 value elevated in all three AD models and the T2 value in co-injection group elevated more than Aβ1-40 or apo E4 alone group, which indicate that the elevation of T2 value may present the occurrence of AD, and the level of T2 value may be related to the severity of AD.(2) There was significant difference of NAA/Cr+Cr1 between three AD models and control group at different time point after drug administration, which indicate that the change of NAA concentration occurred at the early stage of AD and related to the severity of AD.(3) The spatial learning and memory ability was significantly impaired in all three AD models, and the impairment was more serious in co-administration group than in Aβ1-40 or apo E4 alone group. Considering the changes of NAA/Cr+Cr1 occurred at the early stage of AD and the NAA concentrations reduced to different levels in different AD models, we propose that the reduction of NAA/Cr+Cr1 may be more sensitive than behavioral tests.(4) LTP was significantly depressed in three different AD models, but the impairment of LTP was more seriously in Aβ1-40 and apo E4 co-injected group. Considering the change of NAA/Cr+Cr1 was occurred 2 weeks after i.c.v. injection of drugs and continually decreased 4 weeks after drug injection, and the decreasing level was also different in three AD models, we propose that NAA/Cr+Cr1 has high sensitivity for AD diagnosis.(5) There were typical pathological changes, including Aβ deposition and tau protein phosphorylation, in three AD models, but the pathological changes were more obvious in co-injection group than in Aβ1-40 or apo E4 alone group. The severity of pathological changes was consistent with the changes observed in magnetic resonance imaging, behavioral and electrophysiological experiments in three AD models. |
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