Treadmill Exercise Tiv Thaiv Kev poob qis hauv Kev Kawm Spatial Thiab Nco hauv 3 × Tg-AD nas Los ntawm Kev Txhim Kho Ntawm Cov Qauv Synaptic Plasticity Ntawm Hippocampus Thiab Prefrontal Cortex Part 2

2.5. Western Blot

Cov nas raug tshuaj loog los ntawm isoflurane inhalation thiab decapitated. Cov ntaub so ntswg ntawm lub hippocampus los yog prefrontal cortex tau lysed nyob rau hauv txias RIPA lysis buffer (Thermo ScientificPierce, Waltham, MA, USA) supplemented nrog protease thiab phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA).

Lysis buffers tso cai rau cov kws tshawb fawb hauv cov chaw sim tshuaj lom neeg kom tswj tau qhov kev sim zoo dua, yog li kev kawm zoo dua ntawm kev loj hlob thiab kev hloov ntawm cov hlwb, uas ua lub luag haujlwm tseem ceeb hauv kev kawm thiab kev tshawb fawb kho mob.

Ntxiv nrog rau kev pab rau kev tshawb fawb, lysis buffers kuj tseem muaj txiaj ntsig zoo rau kev txhim kho tib neeg kev nco. Raws li kev tshawb fawb tshiab, cov khoom siv tshuaj lom neeg nplua nuj hauv lysis buffers tuaj yeem pab txhawb kev loj hlob thiab kev loj hlob ntawm cov paj hlwb, yog li txhim kho lub hlwb kev nco.

Ib txoj kev tshawb nrhiav pom tias qee yam khoom hauv lysis buffers tuaj yeem txhawb kev sib kis ntawm qee cov paj hlwb, yog li txhim kho lub hlwb kev kawm thiab kev nco muaj peev xwm. Tsis tas li ntawd, cov khoom no tuaj yeem txhawb kev loj hlob ntawm cov paj hlwb thiab pab lub hlwb kom hloov mus rau qhov chaw tshiab thiab kev kawm tshiab. Qhov tshwm sim ntawm cov teebmeem no yog tias tib neeg tuaj yeem nkag siab cov kev paub tshiab zoo dua thiab nco ntsoov nws ntev dua.

Tau kawg, lysis buffers lawv tus kheej tsis tuaj yeem daws txhua yam teeb meem nco. Cov kws tshawb fawb tseem xav tau kev tshawb fawb ntxiv txog kev siv cov kev tshawb pom no los txhim kho tib neeg kev nco. Txawm li cas los xij, qhov tseem ceeb ntawm qhov kev tshawb pom no twb paub meej heev: lysis buffers tuaj yeem muab qhov pib zoo rau kev txhim kho tib neeg lub cim xeeb thiab muab ntau txoj hauv kev rau kev tshawb fawb yav tom ntej. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche tuaj yeem txhim kho kev nco zoo vim Cistanche yog cov khoom siv tshuaj suav tshuaj suav nrog ntau yam tshwj xeeb, ib qho ntawm kev txhim kho kev nco. Cov nyhuv ntawm Cistanche los ntawm ntau yam khoom xyaw uas nws muaj, suav nrog tannic acid, polysaccharides, flavonoid glycosides, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb lub hlwb kev noj qab haus huv ntau txoj hauv kev.

Nyem Paub Txoj Kev Txhim Kho Kev Nco

Tag nrho cov protein tau ntsuas los ntawm kev ntsuas BCA (Thermo Scientific Pierce, USA). Cov qauv tau ua kom sov ntawm 100 ◦C rau 15 feeb. Rau Westernblot, sib npaug ntawm cov protein tau thauj khoom (20 ug / zoo) mus rau 12% gel. Tom qab electrophoresis thiab hloov mus rau PVDF daim nyias nyias (Millipore, IPVH00010, Burlington, MA, USA), lawv tau incubated rau 1 h nyob rau hauv chav tsev kub nrog ib tug blocking tsis (5% BSA tsis).

ThePVDF membranes tau incubated ib hmos nrog cov thawj antibody (GAPDH, 1:10,000,Abcam, Boston, MA, USA; PSD95, 1:500, Abcam, USA; Syn, 1:500, Abcam, USA) .

Tom qab ntawd lawv tau ntxuav nrog TBST tsis pub peb zaug ntawm 5 feeb ib ntus thiab incubated rau 1 h nrog HRP-conjugated tshis los tiv thaiv luav los yog HRP-conjugated tshis anti-nas secondaryantibody (1:15,000, Proteintech Group, Rosemont , IL, USA), ua raws li TBST ntxuav ntawm 5 min intervals.

Cov qib protein tau kuaj pom nrog chemiluminescent reagents (ThermoScientific Pierce, Waltham, MA, USA) thiab cov qhab raug ntsuas siv Image J software.

2.6. Electron Microscopy

Cov nas tau muab tshuaj loog nrog chloral hydrate thiab perfused transcardially nrog PBS, ua raws li 4% paraformaldehyde thiab 2.5% glutaraldehyde tov hauv 0.1 Mphosphate tsis rau 20 min.

Lub paj hlwb raug tshem tawm thiab muab tso rau hauv tib lub fixative hmo ntuj. Lub hippocampus thiab prefrontal cortex tau stained nrog 1% osmium tetroxide, thiab tom qab ntawd lub cev qhuav dej hauv cov qib ntawm acetone.

Cov ntaub so ntswg raug txiav rau hauv cov ntu ultrathin, stained nrog uranyl acetate thiab lead citrate, thiab tshuaj xyuas nrog Hitachi H-7100 electron microscope. Cov duab ntawm random txoj hauj lwm los ntawm cov qauv no tau coj ntawm × 30, 000 magnification (22 µm2).

Synapses nrog puag ncig vesicles, asymmetric synapses, thiab ib qho loj synaptic sib cuag tau suav tias yog excitatory synapses, thaum presynapticterminals nrog pleomorphic flattened vesicles thiab symmetric synapses tau suav tias yog inhibitory synapses [48].

Peb tau txheeb xyuas tus naj npawb ntawm cov kev xav tau zoo tshaj plaws (22 µm2 / txhua daim duab teb, Daim duab 2) hauv hippocampus thiab prefrontal cortex ntawm txhua tus nas, thiab tom qab ntawd nruab nrab ntawm cov synapses ntawm rau daim duab teb los ntawm tib pab pawg (6 daim duab teb los ntawm 4 nas. hauv txhua pab pawg).

Qhov ntev ntawm lub synaptic active cheeb tsam, qhov dav ntawm lub synaptic cleft, curvature ntawm lub synaptic interface, thiab thickness ntawm postsynaptic density ntawm 12-15 randomly xaiv synapses tau quantified thiab muab piv los ntawm 4 nas pergroup (Daim duab 3).

Qhov ntev ntawm thaj chaw nquag thiab qhov tuab ntawm postsynaptic ntom ntom tau ntsuas raws li Güldner [49]. Synaptic cleft tau txhais tias yog thaj tsam ci ntsa iab tshaj plaws nyob nruab nrab ntawm cov kab mob ua ntej thiab postsynaptic [50].

Synaptic curvature tau txiav txim siab siv cov mis: R=a/2 + b2/8a, qhov twg b yog kab koom nrog ob qhov kawg ntawm postsynapticthickening thiab a yog qhov kev ncua deb ntawm postsynaptic membrane rau b [51 ,52](saib daim duab 3A).

Daim duab 2. Treadmill kev tawm dag zog ua kom cov lej synapse ntawm hippocampus thiab prefrontal cortex in3 × Tg-AD nas. (A) Cov neeg sawv cev electron microscope imaging ntawm hippocampus thiab prefrontalcortex nyob rau hauv non-Tg tswj, tsis-Tg ce, 3 × Tg-AD tswj, thiab 3 × Tg-AD ce nas.

Cov xub xiav xiav cim cov synapses. Lub thawv liab sawv cev rau qhov loj synapse. Ib qho kev nthuav dav, siab-magnification pom ntawm synapses nyob rau hauv lub prefrontal cortex yog pom nyob rau hauv lub liab square box nyob rau hauv qab. (B, C) Cov lej synapse ntawm hippocampus (B) thiab prefrontal cortex (C) tau poob qis hauv 3 × Tg-AD tswj pawg piv rau pawg tswj tsis-Tg (*** p < 0 . 9}} duab seem).

Treadmill exercisepretreatment nce tus lej synapse ntawm hippocampus (B) thiab prefrontal cortex (C) innon-Tg nas (** p < 0.01, n=6 duab seem). Txhua cov ntaub ntawv tau muab los ntawm 4 nas.

Daim duab 3. Treadmill qoj ib ce txhim kho synaptic structural parameters ntawm hippocampus thiab prefrontal cortex nyob rau hauv 3 × Tg-AD nas. (A) Kev ntsuas tus sawv cev ntawm cov qauv synaptic.

Cov mis mos R=a/2 + b2/8a, qhov twg b yog txoj kab txuas nrog ob qhov kawg ntawm postsynaptic thickening thiab a yog qhov kev ncua deb ntawm postsynaptic membrane rau b, tau siv los txiav txim siab curvature.

(B, C) Qhov ntev ntawm lub synaptic active cheeb tsam ntawm hippocampus (B) thiab prefrontalcortex (C) tau txo qis hauv 3 × Tg-AD tswj pawg piv rau pawg tswj tsis-Tg (*** p < {{ 3}}. {18}} 001, n=12–15). (D, E) Qhov dav ntawm thesynaptic cleft ntawm hippocampus (D) thiab prefrontal cortex (E) tau nce ntau hauv pawg tswj hwm 3 × Tg-AD piv rau pawg tswj tsis-Tg (*** p < {{ 30}}. * p < 0.001, n=12–15 synapses) thiab prefrontal cortex (E; ** p < 0.01, n=12–15). (F, G) Lub synapticcurvature ntawm hippocampus (F; *** p < 0.001, n=12–15), thiab prefrontal cortex (G; ** p < 0.01, n=12–15 ) tau txo qis hauv pawg tswj hwm 3 × Tg-AD piv rau pawg tswj tsis-Tg, thiab qhov kev txo qis no tau thaiv los ntawm kev tawm dag zog treadmill pretreatment ob qho tib si hauv hippocampus(F; ** p < 0.01, n=12- 15) thiab prefrontal cortex (G; *** p < 0.001, n=12–15). (H, I) Lub thickness ntawm postsynaptic ceev ntawm hippocampus (H) thiab prefrontal cortex (I) tau txo qis hauv pawg 3 × Tg-AD tswj piv rau pawg tswj tsis-Tg (*** p < 0.001, n {{ 50}}–15), thiab qhov txo qis no tau thaiv los ntawm kev tawm dag zog treadmill pretreatment ob leeg hauv hippocampus (H; ** p < 0.01,n=12–15) thiab prefrontal cortex (I; *** p < 0.001, n=12–15).

Txhua cov ntaub ntawv teev muaj 12 thiab 15 synapses los ntawm 4 nas nyob rau hauv txhua pab pawg.

2.7. Kev txheeb cais

Cov ntaub ntawv tsom xam tau qhov muag tsis pom ntawm genotypes thiab keeb kwm kev kho mob ntawm cov nas. Cov ntaub ntawv raug nthuav tawm raws li qhov nruab nrab ± SEM. Cov ntaub ntawv teev tseg tau muab piv nrog ob-txoj kev ANOVA ua raws li Tukey's post hoc tsom xam. Kev soj ntsuam tom qab-hoc tau ua tsuas yog thaum ANOVA yieldeda cov txiaj ntsig tseem ceeb lossis kev cuam tshuam tseem ceeb ntawm ob yam. Cov txiaj ntsig tau suav tias yog qhov tseem ceeb ntawm p < 0.05.

3. Cov txiaj ntsig

3.1. Treadmill Exercise Tiv Thaiv Kev poob qis hauv Kev Kawm Spatial thiab Nco hauv 3 × Tg-AD nas

Peb thawj zaug nrhiav kev txiav txim siab seb puas muaj hnub nyoog rau lub hlis 3 × Tg-AD nas tau nthuav tawm spatiallearning thiab kev nco tsis zoo thiab seb treadmill ce pretreatment tiv thaiv qhov poob ntawm kev kawm spatial thiab nco hauv rau-hli-hnub 3 × Tg-AD nas.

Non-Tg controlmice thiab 3 × Tg-AD nas tau txais 12 lub lis piam ntawm kev tawm dag zog treadmill lossis kev tswj tsis tau kev tawm dag zog pib thaum muaj hnub nyoog peb hlis (2 × 2 factorial tsim: genotype vs. exercise).

Tom qab 12-kev cob qhia lub limtiam, qhov kev xeem yim-caj npab radial maze tau siv los tshawb xyuas qhov kev kawm paub thiab nco txog nas. Ob lub cim xeeb ua haujlwm (lub peev xwm nco tau rau lub sijhawm luv luv) thiab kev siv lub cim xeeb (nco rau cov ntaub ntawv uas nyob tas li lub sijhawm) raug ntsuas (qhov txawv ntawm kev ua haujlwm thiab kev siv lub cim xeeb tau piav qhia hauv Tshooj 2, Daim duab 1A).

Ob txoj kev ANOVA tau qhia tias genotypeand treadmill ce muaj qhov cuam tshuam rau feem pua ​​​​ntawm kev ua haujlwm nco yuam kev hnub 5 (genotype: F1, 39=8.6, p=0.006; treadmill ce: F1, {{ 8}}.5, p=0.024; genotype × treadmill ce sib cuam tshuam: F1, 39=4.2, p=0.047 thiab hnub 6 (genotype: F1 , 39=6.1, p=0.019; treadmill ce: F1, 39=6.4, p=0.016; }}.6, p=0.039; Daim duab 1B) ntawm kev yuav khoom.

Tukey's post hoc tests qhia tias feem pua ​​​​ntawm kev ua haujlwm tsis ua haujlwm nco tau nce ntau hauv pawg 3 × Tg-ADcontrol piv rau pawg tswj tsis-Tg (ob hnub 5 thiab hnub 6: p < 0.01; Daim duab 1B ).

Qhov nce hauv feem pua ​​​​ntawm kev ua haujlwm nco tsis raug yog tiv thaiv bytreadmill ce pretreatments (ob hnub 5 thiab hnub 6: p < 0.01; Daim duab 1B). Txawm li cas los xij, ob txoj kev ANOVA pom tias genotype thiab treadmill ce tsis muaj qhov cuam tshuam rau feem pua ​​​​ntawm kev siv lub cim xeeb yuam kev (piv txwv li, hnub 10, genotype: F1, 39=0.2, p=0.6; treadmill kev tawm dag zog: F1,39=0.04, p=0.8; tag nrho 10 hnub ntawm kev yuav khoom.

Ua ke, cov txiaj ntsig no qhia tias 6-hli-xyoo-laus 3 × Tg-AD nas tau nthuav tawm kev ua haujlwm tsis zoo ntawm lub cim xeeb tab sis tsis nco qab, thiab kev tawm dag zog treadmill tiv thaiv kev poob qis hauv spatial ua haujlwm nco hauv 3 × Tg-AD nas.

3.2. Treadmill Exercise Nce Synapse Numbers thiab Txhim Kho Synaptic Cov Qauv Tsis Zoo ntawm Hippocampus thiab Prefrontal Cortex hauv 3 × Tg-AD nas

Txhawm rau tshawb xyuas seb treadmill kev tawm dag zog los txo qis hauv kev kawm spatial thiab nco tau cuam tshuam nrog cov qauv synaptic plasticity, peb suav cov lej synapse thiab synaptic structural parameters ntawm hippocampus thiab prefrontal cortex uas yog qhov tseem ceeb rau kev xa cov ntaub ntawv ntsig txog kev kawm thiab kev nco (Daim duab 2A).

Ob txoj kev ANOVA qhia tias genotype thiab treadmill ce muaj qhov cuam tshuam loj rau cov lej synapse ob leeg hauv hippocampus (genotype: F1,23=59.3, p < 0.0{{9} }1; treadmillexercise: F1,23=51.{{20}}, p < 0.001; p=0.027; Daim duab 2B) thiab prefrontal cortex (genotype: F1, 23=48.6, p < 0.001; treadmillexercise: F1, 23=59.8, p < 0.001; genotype × treadmill qoj ib ce: F1, 23=8.5, p=0.009; Tukey's post hoc tests qhia tias hippocampus thiab prefrontal cortex synapse tus lej tau txo qis hauv 3 × Tg-AD tswj pawg piv rau pawg tswj tsis-Tg (p <0.001; Daim duab 2B, C).

Treadmill exercisepretreatment thaiv qhov txo qis ntawm cov lej synapse ob leeg hauv hippocampus thiab prefrontal cortex hauv 3 × Tg-AD nas (p < 0.001; Daim duab 2B, C).

Lub caij no, treadmill exercise tau nce cov lej synapse hauv hippocampus thiab prefrontal cortex hauv cov nas uas tsis yog Tg (p < 0.01; Daim duab 2B, C).

Txhawm rau ntsuas qhov ua tau zoo ntawm kev sib kis ntawm synaptic, peb tau ntsuas thiab ntsuas qhov ntsuas qhov ntsuas ntawm qhov ntsuas hluav taws xob los ntawm electron microscopy (EM), suav nrog qhov ntev ntawm thaj chaw synaptic active, qhov dav ntawm lub synaptic cleft, synaptic curvature, thiab qhov tuab ntawm postsynaptic ntom ntom hauv lub hippocampus thiab prefrontal cortex (Daim duab 3A).

Cov kev tshawb fawb yav dhau los tau pom tias qhov loj dua synaptic active cheeb tsam yog qhov ua tau zoo ntawm kev zoo siab postsynaptic neurons, thiab qhov shortening ntawm thaj chaw nquag tuaj yeem cuam tshuam txog kev ua haujlwm tsis zoo ntawm kev sib kis ntawm synaptic [36].

Ob txoj kev ANOVA tau qhia tias genotype thiab treadmill ce muaj qhov cuam tshuam loj rau qhov ntev ntawm thaj chaw synapticactive ob qho tib si hauv hippocampus (genotype: F1,53=10.6, p=0.0{{ 20}}2; treadmill ce: F1,53=5.0, p=0.03; {14}}.004; Daim duab 3B) thiab prefrontal cortex (genotype: F1, 56=17.4, p < 0.001; treadmill ce: F1, 56=5.0, p=0. 03; genotype × treadmill qoj ib ce: F1, 56=6.8, p=0.012;

Tukey's posthoc xeem tau qhia tias qhov ntev ntawm cov synaptic active cheeb tsam ntawm hippocampus thiab prefrontal cortex tau txo qis hauv 3 × Tg-AD tswj pawg piv rau pawg tswj tsis-Tg (p < 0.001; Daim duab 3B, C).

Treadmill ce pretreatment txhim kho qhov ntev ntawm lub synaptic active cheeb tsam ob leeg nyob rau hauv lub hippocampus (p < 0.001; Daim duab 3B) thiab prefrontal cortex (p=0.001; Daim duab 3C) nyob rau hauv 3 × Tg-AD nas .Lub synaptic cleft yog ib tug ~ 20 nm nqaim qhov chaw nruab nrab ntawm lub axon davhlau ya nyob twg ntawm lub presynaptic neuron thiab daim nyias nyias ntawm postsynaptic neuron.

Qhov zoo tshaj plaws shortening ntawm thesynaptic cleft tej zaum yuav muaj ib tug adaptive muaj nuj nqi ntawm optimizing synaptic lub zog los ntawm kev txhim kho qhov zoo concentration ntawm tso tawm neurotransmitters thiab txo qhov zoo cleftresistance [37,38].

Ob txoj kev ANOVA tau pom tias genotype thiab treadmill ce muaj qhov cuam tshuam rau qhov dav ntawm cov synaptic cleft ob qho tib si hauv hippocampus (genotype: F1,55=21.5, p < 0.0{ {24}}1; treadmill ce: F1, 55=15.1, p < 0.001; thiab prefrontal cortex (genotype: F1, 53=10.6, p=0.002; treadmill ce: F1, 53=5.0, p=0.03; genotype × treadmill kev sib cuam tshuam: F1, 53=9.2, p=0.004;

Tukey's post hoc tests qhia tias qhov dav ntawm cov synaptic cleft ntawm hippocampus thiab prefrontal cortex tau nce ntau hauv pawg 3 × Tg-AD tswj piv rau pawg tswj tsis-Tg (p < 0.001; Daim duab 3D, E).

Treadmill ce pretreatment txo qhov dav ntawm cov synaptic cleft ob leeg hauv hippocampus (p < 0.001; Daim duab 3D) thiab prefrontal cortex (p=0.001; Daim duab 3E) in3 × Tg-AD nas.

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