Effects Of Testosterone Treatment On Spatial Learning And Memory Deficits Induced By Aβ1-42Oligomers Combined With Gonadectomy

Alzheimer’s disease (AD), a neurodegenerative disorder resulting inprogressive memory loss and neuronal death, is characterized by intracellularneurofibrillary tangles (NFT), extracellular senile plaques (SP) composed ofsoluble β-amyloid peptide (Aβ) and synaptic neuronal loss. The accumulationof soluble Aβ oligomers in the brain plays a critical role in the degeneration ofneurons during the pathogenesis of AD. Pathogenesis of AD include:oxidative stress, inflammation, cholinergic doctrine, calcium homeostasis, Tauprotein abnormalities modified, apolipoprotein E genotype mutation, as wellas Aβ injury doctrine. As an incidence initiating factors of AD, Aβ has beenwidely recognized.Aβ is a product that type I transmembrane protein amyloid precursorprotein (APP) is sheared sequentially by β-secretase enzyme (BACE-1) andγ-secretase enzyme (γ-secretase), according to the length of the fragments ofits amino acid, APP can be divided into Aβ25-35, Aβ1-40and Aβ1-42.Aβ1-42is the longest fragment and most toxic, which plays the strongest rolein the neurodegenerative process.At present, various types of transgenic mice, senescence acceleratedmouse (SAM) and animal models induced by intrahippocampal orintracerebroventricular injection of Aβ are used for AD study. Althoughtransgenic mice can mimic the neuropathological features of AD, which haveshowed no or very little senile plaque, limited cell death and varied learningcognitive dysfunction. Many studies in animals have established linksbetween soluble Aβ oligomers and cognitive performance. Whether injectionsof soluble Aβ1-42oligomers can induce reliable cognitive deficits in animals is still controversial, as the symptoms of AD are likely caused by interactionsof β-amyloid with various co-factors.Not only AD patients have Aβ deposition in brains, but accompany by thereduction of serum sex hormone level. The reduction of the serum testosterone(T) level is the age-related risk factors of male patients with AD. Anincreasing number of reports in human males support the positive effect ofphysiological levels of androgens on spatial learning and memory, althoughsome studies have found no or even a negative relationship between T andspatial ability.Beneficial actions of androgens support the hypothesis that agerelatedandrogen depletion may increase the risk of developing AD. As previouslydiscussed, several studies have identified androgens as endogenous regulatorsof Aβ. The mechanism (s) by which androgens regulate Aβ is not known, butpresumably involves one or more of three general pathways; direct actionsthrough androgen receptors (ARs)-dependent pathways, indirect actionsthrough estrogen pathways via T aromatization to estradiol, indirect actionsthrough gonadotropin pathways via T modulation of thehypothalamic-pituitary-gonadal axis.The high density of the ARs in fundamental centers of learning andmemory in brain, such as the hippocampus, suggests that there must be somerelationship between ARs and cognitive aspects of the brain. In mostmammals, the hippocampus has a well-documented role in spatial memoryacquisition. The effects of T on behavioral performances may be due to localaromatization of T to estradiol or direct interaction with ARs located in thehippocampus.It is well known that cognitive performance is closely related to androgenlevels, but the mechanism that androgen affects cognitive performance isunclear. The abnormal synaptic changes may be a core part of thepathogenesis of AD. Sex hormones have a closely relationship to theregulation of synaptic plasticity. Elevated estrogen levels can induce newsynapses and dendrites in learning and memory-related brain regions. In recent years, the effect and mechanism of androgen on regulation of synapticplasticity have begun to pay more attention.Hippocampus is involved in the spatial learning and memory access,reproduction and consolidation in early period, and the earliest lesion regionof AD. Therefore, this study is conducted to determine whether solubleAβ1-42oligomers injection combined with gonadectomy (Aβ+GDX) canimpair spatial learning and memory in rats as assessed by the MWM test, andwhether Aβ1-42and gonadectomy have a synergistic effect on impairment ofspatial learning and memory. The serum T levels in all groups are analyzed, tocorrelate the serum T level with spatial ability and investigate the protectiveeffects and potential mechanism of T on cognitive performance, and thepotential involvement of ARs is assessed using the antagonist flutamide (F). Inaddition, by detecting the expression levels of synaptophysin (SYN) andPSD-95, and to explore possible signaling pathway by detecting theexpression levels of BDNF, CaMK II (p-CaMK II) and CREB (p-CREB), toexplore whether T affect the spatial learning and memory by changing thesynaptic plasticity, to provide theoretical and experimental basis for androgenfor improvement in cognitive performance.Part One The rat model of spatial learning and memory deficitsinduced by Aβ1-42oligomers in combination with gonadectomyObjectives: To explore whether soluble Aβ1-42oligomers injectioncombined with gonadectomy (Aβ+GDX) could impair spatial learning andmemory in rats, and whether Aβ1-42and gonadectomy have a synergisticeffect on impairment of spatial learning and memory.Methods:Experiment1Forty rats were randomly divided into four groups (n=10): GI(gonadally-intact), SGI (sham-gonadally intact), vehicle (cannulae injectedwith sterile water) and Aβ (cannulae injected with soluble Aβ1-42oligomers).Two weeks after the injection of soluble Aβ1-42oligomers, the MWM testwas administered to analyze the effect of soluble Aβ1-42oligomers on spatial learning and memory in rats.Experiment2Thirty rats were randomly divided into three groups (n=10): intact, sham(sham-gonadectomized) and GDX (gonadectomized). Two weeks after thegonadectomy, the MWM test was administered to analyze the effect of thegonadectomy on spatial learning and memory in rats.Experiment3This experiment consisted of five groups (n=10per group): control, Aβ,GDX, S-Aβ+GDX (Animals underwent an identical cannulation except thatthey received no injections, and experienced the same gonadectomizedoperation except the testes were exposed but not ligated or removed) and Aβ+GDX (injection of soluble Aβ1-42oligomers combined with gonadectomy).Two weeks after all procedures, the MWM test was administered to evaluatethe effect of the Aβ1-42injection in combination with gonadectomy on spatiallearning and memory in rats.SurgeryRats were anesthetized by intraperitoneal (i.p.) injection of2%pentobarbital sodium and then placed in a stereotaxic instrument. Bilateralguide cannulae were implanted in the left and right hippocampal CA1regionsand attached to the skull surface using dental cement and jeweler’s screws.Stereotaxic coordinates based on Paxinos and Watson’s atlas of the rat brainwere: anterior-posterior (AP),3.8mm from bregma; medial-lateral (ML),±2.5mm from the midline; and dorsal-ventral (DV),2.9mm from the skullsurface.After7days, CA1region injections were administered through the guidecannulae using injection needles connected by polyethylene tubing to5μlHamilton microsyringes. Either5μl of soluble Aβ1-42oligomers (2μg/μl) or5μl of sterile water (vehicle) was stereotaxically injected bilaterally into theCA1regions. Sham group animals underwent an identical operation exceptthat they received no injections.MWM test Each animal received4trials per day for5consecutive days. For each trial,the time and path length required for the rat to find the hidden platform wasrecorded. On day6, the platform was removed, and the rat was allowed toswim freely for120s as in the probe trial. The time spent in the targetquadrant and the number of platform crossings were measured.Blood sampling and tissues preparationOn the day following the final day of the MWM test, animals wereanesthetized, and0.5ml of blood was drawn by cardiac puncture into aheparinized syringe. The sample centrifuged at5000×g for20min at4°C.The supernatant was then transferred to a clean microcentrifuge tube andstored at80°C until assayed.Following blood samples, rats were sacrificed by decapitation, and brainswere removed. The brain tissues were fixation in4%paraformaldehyde,dehydration in a series of ethanol, embedded in wax and sectioned into5μMthick sections. Tissue sections were then deparaffinized, dehydrated, andstained with Cresyl violet, followed by differentiation, dehydration andclearance before they were mounted. The number of intact pyramidal cells(per1mm linear length of the same section of CA1hippocampus) was thencounted by three experimenters and analyzed.Results:1Effects of soluble Aβ1-42oligomers on performance in the MWM testby adult male Sprague-Dawley ratsGroup differences were found among all groups (escape latency: P <0.001;path length: P <0.01); however, no significant differences were observedamong the vehicle, GI and SGI groups. Analyses of escape latencies withineach day revealed a significant effect of soluble Aβ1-42injection on day4(P<0.05) and day5(P <0.001), the Aβ group had significantly longer escapelatencies than the GI group, no significant differences were found among anyof the other groups. Analyses of path lengths within each day revealed asignificant effect of soluble Aβ1-42injection on day5(P <0.05), the Aβgroup had significantly longer path length than the GI group (P <0.01), no significant differences were found among any of the other groups. Nosignificant differences were found in mean swim speed among all groups.During the probe trails, there were significant differences between groupsin the amount of time spent in the target quadrant (P <0.05). The Aβ groupspent a significantly smaller percentage of time in the target quadrant than didthe GI group (P <0.05), no significant differences were observed among thevehicle, GI and SGI groups. No significant differences were found in theserum T level among all groups.Nissl staining results showed that no significant differences were found inthe number of intact pyramidal cells between the GI, SGI and Vehicle rats.Compared to the GI group, the number of intact pyramidal cells of the Aβ rats(55.6%) was significantly reduced (P <0.001).2Effects of gonadectomy on performance in the MWM test by adult maleSprague-Dawley ratsGroup differences were found among the intact, sham and GDX group rats(escape latency: P <0.01; path length: P <0.05); however, no significantdifferences in behavioral performances were observed between the intact andsham groups. Analyses of escape latencies within each day revealed asignificant effect of gonadectomy on day4(P <0.05) and day5(P <0.001),the GDX group had significantly longer escape latencies than the intact group.Analyses of path lengths within each day revealed a significant effect ofgonadectomy on day4(P <0.05) and day5(P <0.01), the GDX group hadsignificantly longer path lengths than the intact group. No significantdifferences were found in mean swim speed among all groups.During the probe trails, significant differences were found between groupsin the amount of time spent in the target quadrant (P <0.05), the GDX groupspent a smaller percentage of time in the target quadrant than did the intactgroup. Significant differences were found in the serum T level between theintact, sham and GDX groups (P <0.001), there was a significant decrease inthe serum level in the GDX group.No significant differences were found in the number of intact pyramidal cells between the intact and sham rats. Compared to the intact group, thenumber of intact pyramidal cells in the GDX group rats (47.0%) wassignificantly reduced (P <0.001).3Effects of injecting soluble Aβ1-42oligomers in combination withgonadectomy on performance in the MWM test by adult maleSprague-Dawley ratsGroup differences were found among all groups (escape latency: P <0.001;path length: P <0.001). Analyses of escape latencies within each day revealeda significant effect of soluble Aβ1-42injection in combination withgonadectomy on day3(P <0.05), day4(P <0.001) and day5(P <0.001),the Aβ+GDX group had significantly longer escape latencies than the controlgroup. Analyses of path lengths within each day revealed a significant effectof soluble Aβ1-42injection in combination with gonadectomy on day4(P <0.05) and day5(P <0.001), the Aβ+GDX group had significantly longerpath lengths than the control group. No significant differences were found inmean swim speed among all groups.During the probe trails, there were significant differences between groupsin the amount of time spent in the target quadrant (P <0.05). The Aβ+GDXgroup (P <0.05) spent a significantly smaller percentage of time in the targetquadrant than did other groups. Significant differences were dound in theserum T level among all groups (P <0.001).Nissl staining results showed there were no differences in the number ofintact pyramidal cells in the Control and S-Aβ+GDX rats. The number ofintact pyramidal cells in the Aβ+GDX rats was the least, the number of cellsdecreased by82.8%compared to the Control group, significant differenceswere found compared to other groups (all P <0.001).Conclusions:1Soluble Aβ1-42oligomers injection had an effect on theimpairment of spatial learning and memory in rats.2Gonadectomy impairedthe spatial learning and memory in rats.3Aβ1-42injection and GDX eachimpaired cognitive performance and that the combination of these treatmentswas additive, leading to even greater impairment. Part Two The protective effect of testosterone on spatial learning andmemory impairment induced by Aβ1-42oligomers combined withgonadectomy.Objectives: The serum testosterone (T) levels in all groups were analyzed,to correlate the serum T level with spatial ability and investigate the protectiveeffects and potential mechanism of T on cognitive performance, and thepotential involvement of ARs was assessed using the antagonist flutamide (F).Methods:Experiment1Fifty-six Aβ+GDX rats were randomly divided into seven groups (n=8),all receiving subcutaneous (s.c.) injections of0.75mg T dissolved in0.1ml ofsesame oil, and then blood samples were collected at0,6,12,24,48,72or96h. This experiment was used to confirm the peak time of serum T levels afteradministration.Experiment2Twelve days after soluble Aβ1-42injection combined with gonadectomy,fifty-six Aβ+GDX rats were randomly divided into seven groups (n=8), andreceived s.c. injections of either only0.1ml of sesame oil (0mg group) or0.25,0.50,0.75,1.00,1.50or2.00mg of T dissolved in0.1ml of sesame oilbeginning2days before the start of the MWM test and then throughout6daysof testing (8days of injections total). This experiment was used to determinethe optimal T dose for administration in Aβ+GDX rats.Experiment3Twelve days after soluble Aβ1-42injection combined with gonadectomy,thirty-two Aβ+GDX rats were randomly divided into four groups (n=8):C-Aβ+GDX (Aβ+GDX rats received no injections), T-Aβ+GDX (Aβ+GDX rats with T), F-Aβ+GDX (Aβ+GDX rats with F), F+T-Aβ+GDX(Aβ+GDX rats with F and T). T-Aβ+GDX rats and F+T-Aβ+GDX ratswere s.c. injected1h before testing daily with0.75mg T dissolved in0.1mlof sesame oil for8days beginning2days before the MWM test and thenthroughout6days of testing. F-Aβ+GDX rats and F+T-Aβ+GDX rats were pre-injected with5μg F dissolved in0.5μl of DMSO into each side ofthe hippocampus. All F injections were given1h before testing dailybeginning2days before the start of the MWM test and then throughout6daysof testing (8days of injections total).Results:1Serum T levels in Aβ+GDX rats at different times after TadministrationSerum T levels in all groups sampled at different time points up to96hafter administration of T were significantly higher than that of the0h group,and they differed significantly among the groups (P <0.001). The48h grouphad the highest serum T level among all the groups.2Effects of different T doses on performance in the MWM test by Aβ+GDX ratsDifferences in performance in the MWM test were found among all groupsadministered different T doses (escape latency: P <0.001; path length: P <0.01). Analyses of escape latencies within each day revealed a significanteffect of T on day4(P <0.01) and day5(P <0.001), the0.75mg T grouphad significantly shorter escape latencies than the0mg T group. Analyses ofpath lengths within each day revealed a significant effect of T on day4(P <0.05) and day5(P <0.001), the0.75mg T group had significantly shorterpath lengths than the0mg T group. No significant differences were found inmean swim speed among all groups.During the probe trails, significant differences were found between groupsin the amount of time spent in the target quadrant (P <0.05). The0.75mg Tgroup spent a significantly greater percentage of time in the target quadrantthan did the0mg group (P <0.05). Significant differences were found in theserum T level among all groups, administered different doses of T can lead tothe serum T level increasing, the serum T level was dependent on theadministered T dose (P <0.001).The number of intact pyramidal cells gradually increased from the0.25mggroup to the1.00mg group; the number of pyramidal cells in the0.75mg group rats was the most, increased by162.6%compared to the0mg group (P<0.001).3Effects of F on performance in the MWM test by Aβ+GDX ratsDifferences in performance in the MWM test were found among all groups(escape latency: P <0.001; path length: P <0.05), however, no significantdifferences were found in cognitive performances among the C-Aβ+GDX,F-Aβ+GDX and F+T-Aβ+GDX groups. Analyses of escape latencieswithin each day revealed a significant effect of T on day5(P <0.001). TheT-Aβ+GDX group had significantly shorter escape latency than the C-Aβ+GDX group (P <0.01). Analyses of path lengths within each day revealed asignificant effect of T on day5(P <0.05). The T-Aβ+GDX group hadsignificantly shorter path length than the C-Aβ+GDX group (P <0.01).There were no significant differences in mean swim speed among all groups.During the probe trails, significant differences were found between groupsin the amount of time spent in the target quadrant (P <0.05). The T-Aβ+GDX group spent a significantly larger percentage of time in the targetquadrant than did the C-Aβ+GDX group (P <0.05). We found no significantdifferences among the C-Aβ+GDX, F-Aβ+GDX and F+T-Aβ+GDXgroups. Significant differences were found in the serum T level among allgroups, administered0.75mg T can lead to the serum T level increasing, Fhad no effect on the serum T level (P <0.001).The number of intact pyramidal cells in the T-Aβ+GDX group rats wasthe most, the T-Aβ+GDX group had significant differences (P <0.001)compared to the C-Aβ+GDX group. No significant differences were found inthe number of pyramidal cells among the F-Aβ+GDX, F+T-Aβ+GDX andC-Aβ+GDX rats.Conclusions:1The spatial learning and memory is closely related to theserum T level in Aβ+GDX rats.2Moderate dose of T can improve spatiallearning and memory damage in Aβ+GDX rats.3The effect of T onbehavioral performances was partly mediated via ARs in Aβ+GDX rats. Part Three The mechanism of testosterone treatment on spatiallearning and memory deficits induced by Aβ1-42oligomers combinedwith gonadectomy.Objectives: By detecting the expression levels of SYN, PSD-95, BDNF,CaMK II (p-CaMK II) and CREB (p-CREB) to explore possible signalingpathway, to explore whether T affect the spatial learning and memory bychanging the synaptic plasticity, to provide theoretical and experimental basisfor androgen for improvement in spatial learning and memory.Methods: We used Western blot and RT-PCR to explore the effect of T onthe expression levels of SYN, PSD-95, BDNF, CaMKII (p-CaMKII) andCREB (p-CREB) in different group.Results:1The expression levels of SYN and PSD-95in different groupWestern blot results showed that the expression levels of SYN andPSD-95are relatively high in the Control group and S-Aβ+GDX group, therewere no significant differences between two groups; compared to the Controlgroup, the expression levels of SYN and PSD-95were significantly decreasedin the Aβ, GDX and Aβ+GDX groups (P <0.001), the most significantdecline was found in the Aβ+GDX group (P <0.001).RT-PCR results are basically same as Western blot results, there were nosignificant differences in the β-actin mRNA among all groups. The expressionlevels of SYN mRNA and PSD-95mRNA are relatively high in the Controlgroup and S-Aβ+GDX group, there were no significant differences betweentwo groups; compared to the Control group, the expression levels of SYNmRNA and PSD-95mRNA were significantly decreased in the Aβ, GDX andAβ+GDX groups (P <0.001), the most significant decline was found in theAβ+GDX group (P <0.001).2Effects of T on the expression levels of SYN and PSD-95Western blot showed that there were no significant differences in theexpression levels of SYN and PSD-95among the C-Aβ+GDX, F-Aβ+GDXand F+T-Aβ+GDX groups. The expression levels of SYN (P <0.001) and PSD-95(P <0.01) in T-Aβ+GDX group were significantly increasedcompared with the C-Aβ+GDX group. RT-PCR results were basically sameas Western blot results, there were no significant differences in the β-actinmRNA among all groups. The expression levels of SYN mRNA (P <0.01)and PSD-95mRNA (P <0.001) in T-Aβ+GDX group were significantlyincreased compared with the C-Aβ+GDX group. There were no significantdifferences in the expression levels of SYN mRNA and PSD-95mRNAamong the C-Aβ+GDX, F-Aβ+GDX and F+T-Aβ+GDX groups.3Effects of T on the expression levels of CaMK II (p-CaMK II)Western blot showed that there were no significant differences in theexpression level of p-CaMK II among the C-Aβ+GDX, F-Aβ+GDX and F+T-Aβ+GDX groups. The expression level of p-CaMK II in T-Aβ+GDXgroup was significantly increased compared with the C-Aβ+GDX group (P<0.05). We found no significant differences in the expression level of CaMKII among all groups.4Effects of T on the expression levels of CREB (p-CREB)Western blot showed there were no significant differences in theexpression level of CREB among all groups; there were no significantdifferences in the expression level of p-CREB among the C-Aβ+GDX, F-Aβ+GDX and F+T-Aβ+GDX groups; a significant difference was found inexpression level of p-CREB between the T-Aβ+GDX group and C-Aβ+GDX group (P <0.05).5Effects of T on the expression levels of BDNFWestern blot showed that no significant differences were found in theexpression level of BDNF among the C-Aβ+GDX, F-Aβ+GDX and F+T-Aβ+GDX groups. The expression level of BDNF in the T-Aβ+GDXgroup was significantly increased compared with the C-Aβ+GDX group (P<0.001). RT-PCR results were basically same as Western blot results, therewere no significant differences in β-actin mRNA among all groups. Theexpression level of BDNF mRNA in the T-Aβ+GDX group was significantlyincreased compared with the C-Aβ+GDX group (P <0.01), there were no significant differences among the C-Aβ+GDX, F-Aβ+GDX and F+T-Aβ+GDX groups.Conclusion：1The effect of soluble Aβ1-42oligomers injection combinedwith gonadectomy on cognitive dificits may be related to the change ofsynaptic plasticity.2Testosterone improved the spatial learning and memoryin Aβ+GDX rats by Ca2+-CaM/CaMK II-p-CREB signal transductionpathway, thereby increased BDNF, SYN and PSD-95expression, enhancedthe regulatory role on synaptic plasticity.