Dynamic changes of beta-amyloid protein deposition in hippocampus of female ovariectomized rats

BACKGROUND： To evaluate and summarize the effects of cerebral perfusion and vascular reserve on the treatment of SICAS. Recently, research on β-amyloid protein has focused on the regulatory effects of estrogen or phytoestrogen on its deposition. However, there have been only a few reports on dynamic changes of β -amyloid protein deposition in hippocampus of ovariectomized rats. OBJECTIVE： To measure β -amyloid protein deposition in the hippocampal formation of ovariectomized rats by using immunohistochemistry; to observe time-dependent dynamic changes. DESIGN： Randomized controlled animal study. SETTING： Third Xiangya Hospital of Central South University. MATERIALS： The experiment was carried out in the Central Laboratory of the Third Xiangya Hospital of Central South University from November 2005 to December 2006. Fifty healthy female Sprague Dawley （SD） rats, weighing （293 ± 10） g, were provided by the Animal Laboratory of Xiangya Medical College, Central South University. All rats had neither a childbearing history nor hepatic or renal disease, or skeletal deformity. β-amyloid protein immunohistochemical kit was provided by Wuhan Boster Company. The experiment was in accordance with animal ethics standards. METHODS： All rats were randomly divided into five groups, including normal control group （n = 10）, sham operation group （n = 10）, and ovariectomized group （n = 30）. After anesthesia in the ovariectomized group, the bilateral ovaries were separated and resected. The same volume of fat was resected in the sham operation group. Rats from the normal control group, however, did not receive any surgical treatments. Rats in the normal control group and sham operation group were sacrificed by anesthesia 7 weeks after surgery. Every ten rats from the ovariectomized group was respectively sacrificed at 7, 15, and 30 weeks after surgery. lmmunohistochemistry was used to detect β-amyloid protein deposition in hippocampal sections. Cell counting and gray value measurements served to record the dynamic changes in β-amyloid protein deposition. MAIN OUTCOME MEASURES： （1） Morphological changes. （2） Positive cell counts from β -amyloid protein stainings and gray value measurements. RESULTS： All 50 rats were involved in the final analysis. （1） Morphological changes. β -amyloid-positive cells were detected in the hippocampus of all rats. Biebrich scarlet stained neurites with a swollen cytoplasm. A few β -amyloid-positive cells were observed in all groups 7 weeks after surgery, and plasma and neurites were slightly stained. By 15 weeks after surgery, a number of β -amyloid-positive cells were observed in the ovariectomized group, and plasma and neurites were also slightly stained. By 30 weeks after surgery, however, many β-amyloid-positive cells were observed in the ovariectomized group. These cells were partially aggregated and darkly stained. （2） Positive cell counts and gray value of β-amyloid protein in hippocampus. At 7 weeks after surgery, cell counts and gray value measurements were not significantly different in the ovariectomized group compared to the sham operation group and normal control group （P 〉 0.05）. Cell counts and gray value measurements were higher in the ovariectomized group by 15 weeks compared to those by 7 weeks in the normal control group, sham operation group and ovariectomized group （P 〈 0.05）. At 30 weeks after surgery, cell counts and gray value measurements were higher in the ovariectomized group compared to the normal control group. In addition, there were significant differences between sham operation group and ovariectomized group, at 7 and 15 weeks after operation （P 〈 0.05-0.01 ）. Cell counts and gray value measurements increased in all groups over time. CONCLUSION： Extended estrogen deficiency in rats can increase β-amyloid protein deposition in the hippocampus and the deposition increases over time.

Beta-amyloid precursor protein cleavage enzyme-1 expression in adult rat retinal neurons in the early period after lead exposure

Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein （Aβ）,and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 （BACE-1） in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer＇s disease.Adult rats were intraocularly injected with different doses of lead acetate （10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L）.The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer＇s disease.

Impact of Sub-chronic Aluminium-maltolate Exposure on Catabolism of Amyloid Precursor Protein in Rats

Objective To investigate the impact of sub-chronic Aluminium-maltolate [Al（mal）s] exposure on the catabolism of amyloid precursor protein （APP） in rats. Methods Forty adult male Sprague-Dawley （SD） rats were randomly divided into five groups： the control group, the maltolate group （7.56 mg/kg BW）, and the Al（mal）s groups （0.27, 0.54, and 1.08 mg/kg BW, respectively）. Control rats were administered with 0.9% normal saline through intraperitoneal （i.p.） injection. Maltolate and Al（mal）s were administered to the rats also through i.p. injections. Administration was conducted daily for two months. Rat neural behavior was examined using open field tests （OFT）. And the protein expressions and their mRNAs transcription related with APP catabolism were studied using enzyme-linked immunosorbent assay （ELISA） and real-time polymerase chain reaction （RT-PCR）. Results The expressions of APP, 13-site APP cleaving enzyme 1 （BACEI） and presenilin-1 （PSi） proteins and their mRNAs transcription increased gradually with the increase of Al（mal）3 doses （P〈0.05）. The enzyme activity of BACEI in the 0.54 and 1.08 mg/kg Al（mal）s groups increased significantly （P〈0.05）. The expression of 8-amyloid protein （AS） 1-40 gradually decreased while the protein expression of A81-42 increased gradually with the increase of Al（mal）s doses （P〈0.05）. Conclusion Result from our study suggested that one of the possible mechanisms that Al（mal）s can cause neurotoxicity is that Al（mal）s can increase the generation of A81-42 by facilitating the expressions of APP, β-, and γ-secretase.

Comparative study of histopathology changes between the PS1/APP double transgenic mouse model and Aβ_(1-40)-injected rat model of Alzheimer disease

Objective To identify the genetype of the PS1/APP double transgenie mouse model, then to analyse the histopathological changes in the brain and compare the differences between the transgenie mice models and Aβ1-40-injeeted rats models of Alzheimer disease. Methods The modified congo red staining, Nissl＇s staining and immunohistology staining was used to observe the Aβ deposits, activation of astrocyte respectively. Results ①The PS1/APP transgenic mouse extensively displayed Aβ deposits in the cortex and hippocampal structures, and GFAP positive cells were aggregated in mass and surrounded the congo red-positive plaque. ②The Aβ1-40-intrahippocmnpal-injeeted rat model showed the Aβ plaque deposits in the dentate gyrus of the hippocampus, with the astrocyte surrounded. The neurons loss was significant in the injection point and pin hole of injection with Nissl＇s staining methods. GFAP-positive cells increased significantly compared with the uninjected lateral of the hippocampus. Conclusion Although Aβ1-40-injected rat models could simulate some characteristic pathological features of human Alzheimer diseases, Aβ deposits and neurons loss in partial hippocampal, it would not simulate the progressive degenenration in the brain of AD. The double transgenie PS1/APP mice could simulate the specific pathogenesis and progressive changes of AD, mainly is Aβ deposits and the spongiocyte response , while no neurons loss were observed in this model.

Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of p-amyloid protein (P-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP1-40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP1-40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP1-40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K+] solution was rather weak. In control animals the percentage of increase of ACh-release during behavioral performance was 57%, while in β-AP1-40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP1-40 -treated rats. The results show that the injection of β-AP1-40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.

Protective Effects and Mechanism of Total Coptis Alkaloids on Aβ_(25-35) Induced Learning and Memory Dysfunction in Rats

Objective： To observe the effect of total coptis alkaloids （TCA） on β -amyloid peptide （A β 25-35） induced learning and memory dysfunction in rats, and to explore its mechanism. Methods： Forty male Wistar rats were randomly divided into four groups： the control group, the model group, the TCA low dose （60 mg/kg） group and the TCA high dose （120 mg/kg） group, 10 in each. A β 25-35 （5 μl, 2 μg/μl） was injected into bilateral hippocampi of each rat to induce learning and memory dysfunction. TCA were administered through intragavage for consecutive 15 days. Morris Water Maze test was used to assess the impairment of learning and memory; concentration of malondialdehyde （MDA） in cerebral cortex was determined by thiobarbituric acid reactive substance to indicate the level of lipid peroxidation in brain tissues; activity of manganese-superoxide dismutase （Mn-SOD） in cerebral cortex was determined by xanthine-oxidase to indicate the activity of the enzyme; and NF-κB protein expression in cerebral cortex was measured by SP immunohistochemistry. Results： （1） Morris Water Maze test showed that, during the 4 consecutive days of acquisition trials, the rats in the model group took longer latency and searching distance than those in the control group （P〈0.01）, which could be shortened by high dose TCA （P〈0.05）; during the spatial probe trial on the fifth day, the rats in the model group took shorter searching time and distance on the previous flat area than those in the control group （P〈0.01）, which could be prolonged after TCA treatment （for low dose group, P〈0.05; for high dose group, P〈0.01）. （2） Analysis of cerebral cortical tissues showed that, compared with the control group, MDA level got significantly increased and Mn-SOD activity decreased in the model group （both P〈0.01）. After having been treated with TCA, the MDA level got significantly decreased （P〈0.05 and P〈0.01 respectively for low and high dose group）, while relative increase of Mn-SOD activity only appeared in high dose group （P〈0.05）. （3） Immunohistochemistry analysis showed the protein expression of NF- κB got significantly increased after modeling, while high dose TCA can significantly inhibit it. Conclusion： TCA could improve A β 25-35 induced dysfunction of learning and memory in rats, and its protective mechanism is associated with its actions in decreasing MDA level, increasing Mn-SOD activity and inhibiting the expression of NF-κB in cerebral cortex.