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 1
Jul 22, 2024
Abstract:
Alzheimer's disease (AD) yog tus cwj pwm los ntawm kev tsis txaus ntawm kev kawm thiab kev nco. Lub pathological feature ntawm AD yog qhov kev hloov pauv ntawm tus lej thiab qhov loj ntawm synapses, axon ntev, dendriticcomplexity, thiab dendritic tus txha nraub qaum hauv hippocampus thiab prefrontal cortex.
Alzheimer's disease yog kab mob neurodegenerative uas feem ntau cuam tshuam rau cov neeg mob kev nco, kev muaj peev xwm xav, thiab muaj peev xwm ua neej nyob txhua hnub. Txawm li cas los xij, txawm hais tias lub cim xeeb ntawm cov neeg mob Alzheimer's muaj kev cuam tshuam, peb tseem tuaj yeem nkag siab thiab pab lawv los ntawm ntau qhov kev xav.
Ua ntej, txawm hais tias lub cim xeeb ntawm cov neeg mob Alzheimer's tus kab mob yuav maj mam poob qis, lawv lub peev xwm hnov mob thiab kev xav muaj peev xwm tsis cuam tshuam. Yog li ntawd, peb tuaj yeem pab lawv los ntawm kev saib xyuas lawv cov kev xav tau ntawm lub cev thiab saib xyuas lawv cov kev xav tau kev xav.
Qhov thib ob, kev nco ploj ntawm cov neeg mob Alzheimer's tus kab mob tuaj yeem kho tau los ntawm kev cob qhia thiab txhim kho kev mloog. Piv txwv li, cov neeg mob tuaj yeem tawm dag zog lawv lub hlwb thiab txhim kho lawv lub cim xeeb los ntawm kev nyeem phau ntawv thiab koom nrog kev ua suab paj nruag lossis kev ua si. Ntxiv mus, yog cov neeg hauv tsev neeg thiab cov zej zog tuaj yeem muab kev txhawb nqa thiab kev pab rau cov neeg mob, cov txiaj ntsig ntawm cov dej num no yuav tseem ceeb dua.
Thaum kawg, peb yuav tsum txhawb kom zej zog saib xyuas thiab nkag siab ntau ntxiv rau cov neeg mob Alzheimer's. Cov neeg mob xav tau peb lub tuam txhab thiab kev txhawb nqa, tsis yog cia siab rau kev nco. Tsuas yog thaum ib puag ncig kev sib raug zoo puv nrog kev hlub thiab kev saib xyuas tus neeg mob tuaj yeem kho thiab tswj tau zoo dua.
Nyob rau hauv luv luv, kev sib raug zoo ntawm Alzheimer's kab mob thiab nco yog heev nyuaj, tab sis peb yuav tsum nquag pab cov neeg mob, txhim kho lawv lub neej zoo, thiab txhawb cov zej zog kom xyuam xim rau thiab nkag siab txog lawv qhov teeb meem. Kuv vam tias peb txhua tus tuaj yeem tsim kom muaj lub neej zoo dua thiab muaj kev sib haum xeeb 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 tias nws tuaj yeem tswj hwm qhov sib npaug ntawm cov neurotransmitters, xws li nce qib ntawm acetylcholine thiab kev loj hlob, uas tseem ceeb heev rau kev nco thiab kev kawm. Tsis tas li ntawd, Cistanche tseem tuaj yeem txhim kho cov ntshav khiav thiab txhawb nqa cov pa oxygen, uas tuaj yeem ua kom lub hlwb tau txais cov khoom noj txaus thiab lub zog, yog li txhim kho lub hlwb tseem ceeb thiab kev ua siab ntev.
Nyem Paub txhawm rau txhim kho kev txawj ntse
Treadmill ce tuaj yeem txhim kho synaptic plasticity hauv nas lossis nas qauv ntawm kev mob stroke, ischemia, thiab dementia. Txoj kev tshawb no tsom los tshuaj xyuas cov teebmeem ntawm treadmill ce rau kev kawm thiab kev nco, thiab structuralsynaptic plasticity hauv 3 × Tg-AD nas, tus qauv nas ntawm AD.
Ntawm no, peb qhia tau hais tias 12 lub lis piam ntawm treadmillexercise pib nyob rau hauv peb-hli cov nas txhim kho spatial ua hauj lwm nco nyob rau hauv 6-hlis-laus-Tg-AD nas, thaum uas tsis yog-kev xyaum rau-hli-hnub nyoog 3 × Tg-AD nas pom impaired spatial ua haujlwm nco.
Txhawm rau tshawb xyuas cov txheej txheem muaj peev xwm rau kev tawm dag zog kev tawm dag zog-vim kev txhim kho ntawm kev kawm thiab kev nco, peb tau tshuaj xyuas cov txheej txheem synaptic plasticity hauv hippocampus thiab prefrontal cortex ntawm rau lub hlis 3 × Tg-AD nas uas tau ua tiav 12 lub lis piam ntawm kev tawm dag zog treadmill.
Peb pom tias treadmill ce ua rau muaj kev nce hauv cov lej synapse, cov qauv ntawm cov qauv synaptic, kev qhia ntawm synaptophysin (Syn, presynaptic marker), qhov ntev axon, dendritic complexity, thiab tus naj npawb ntawm dendritic spines hauv 3 × Tg-AD nas thiab rov qab los. cov kev txwv no rau qib zoo sib xws ntawm cov tsis-Tg tswj cov nas tsis muaj kev tawm dag zog treadmill. Tsis tas li ntawd, kev tawm dag zog treadmill kuj tau txhim kho cov kev txwv no hauv cov nas uas tsis yog Tg tswj.
Txhim kho cov txheej txheem synaptic plasticity tuaj yeem sawv cev rau lub peev xwm los ntawm qhov kev tawm dag zog treadmill tiv thaiv kev poob qis hauv kev kawm spatial thiab nco thiab synapse poob hauv 3 × Tg-AD nas.
Keywords: kev ua si; nco; synaptic; dendritic qaum; structural synaptic plasticity; 3 × Tg-AD nas.
1. Taw qhia
Alzheimer's disease (AD) yog ib qho kab mob neurodegenerative uas feem ntau tshwm sim los ntawm kev nco tsis zoo thiab kev paub tsis meej [1]. Nyuaj hauv kev nco qab cov ntaub ntawv kawm tshiab, lossis kev nco tsis ntev, yog ib qho tsos mob ntxov ntawm AD [2].
Raws li tus kab mob loj hlob zuj zus, AD cov neeg mob maj mam poob lub cim xeeb mus ntev, uas cuam tshuam rau lub neej zoo. AD pathology yog tus cwj pwm los ntawm kev sib sau ntawm amyloid- (A) plaques thiab tau neurofibrillary tangles nyob rau hauv hippocampus thiab prefrontal cortex (PFC), ob qho tib si yog cov qauv tseem ceeb hauv kev kawm thiab kev nco [3-6].
Cov kev tshawb fawb yav dhau los qhia tias A thiab tau tuaj yeem ua rau tsis nco qab los ntawm kev cuam tshuam synaptic plasticity hauv hippocampus thiab prefrontal cortex [7,8].
Qhov no synaptic plasticity suav nrog kev ua haujlwm-raws li kev hloov pauv hauv kev ua tau zoo thiab kho dua tshiab, axonal sprouting thiab dendriticremodeling, thiab dendritic spine dynamics.
Hauv kev tshawb fawb tsiaj, soluble A -oligomers ntawm nanomolar concentrations, rho tawm ncaj qha los ntawm cerebral cortex ntawm AD cov neeg mob los yog tsim los ntawm hluavtaws peptide, cuam tshuam lub cim xeeb ntawm kev kawm tus cwj pwm bypotently inhibiting lub sij hawm ntev potentiation (LTP), txhim khu kev nyuaj siab mus ntev. (LTD), txo qis dendritic spine ceev ntawm hippocampus [9–11].
Lub caij no, nws tau pom tias kev kawm spatial thiab kev nco tsis zoo yog tshwm sim los ntawm kev ua haujlwm tsis zoo ntawm ob tus neeg mob thiab cov qauv hloov pauv hloov pauv ntawm AD, xws li AppNL-GF [12,13], Tg2576 [14], APP/PS1 [15], 5 × FAD [16], and 3 × Tg-AD [17].
Qhov txo qis synaptic function feem ntau pom nyob rau hauv cov nas me ua ntej txoj kev loj hlob ntawm cov quav hniav, uas qhia tau hais tias A oligomers ua rau synaptic deficits ua ntej plaque tsim [18]. Lub caij no, kev tshawb fawb siv suav tomography (CT) thiab magnetic resonance imaging (MRI) qhia tau hais tias lub hippocampal thiab prefrontal cortex ntawm cov neeg uas muaj Alzheimer's tus kab mob ntsws loj zuj zus tuaj [19,20].
AD muaj ntau zuj zus thoob plaws ntiaj teb no, thiab tseem tsis tau muaj kev kho mob tuaj yeem kho lossis qeeb ntawm qhov kev loj hlob ntawm tus kab mob no. Cov ntaub ntawv pov thawj qhia tias kev tawm dag zog lub cev txo qis amyloid lub nra txhim kho synaptic plasticity thiab kev kawm thiab kev nco tsis txaus hauv cov qauv nas ntawm AD thiab ncua kev nce qib ntawm cov kab mob pathological. hauv hlwb [21–28].
Kev tawm dag zog lub cev tuaj yeem ua los ntawm ntau yam txheej txheem hauv cov qauv kev sim sib txawv. Aerobicexercise tuaj yeem ua rau cov ntshav ntws hauv hlwb, txhim kho kev siv cov pa oxygen, thiab txhawb kev nthuav tawm ntawm kev loj hlob yam tseem ceeb uas txhawb nqa synaptic plasticity thiab neurogenesis thiab tuaj yeem ua rau muaj txiaj ntsig zoo rau lub hlwb ua haujlwm [29-31].
Cov txheej txheem koom nrog suav nrog kev nce qib ntawm cov noob qhia ntawm cov hom phiaj molecular cuam tshuam nrog kev kawm thiab kev nco, synaptic plasticity, thiab neuronal ciaj sia [30].
Tsis tas li ntawd, kev tawm dag zog aerobic tuaj yeem txhim kho kev kawm thiab kev nco tsis txaus los ntawm kev thim rov qab synapse poob hauv hippocampus thiab cortex hauv cov laus nas, tus qauv nas ntawm vascular dementia, thiab nruab nrab cerebralartery occlusion / reperfusion (MCAO / R) qauv [31-33].
Txoj kev tshawb no yuav tshuaj xyuas qhov cuam tshuam ntawm 12 lub lis piam ntawm kev tawm dag zog ntawm spatial kev kawm thiab kev nco siv aneight-arm radial maze hauv rau-hli 3 × Tg-AD nas.
Tom qab ntawd, cov txheej txheem muaj peev xwm koom nrog raug tshawb xyuas. Lub hippocampus thiab prefrontal cortex paub tias yog cov cheeb tsam tseem ceeb rau kev kawm spatial thiab nco [34].
Nws ntseeg tau tias synaptic plasticity nyob rau hauv lub hippocampus thiab prefrontal cortex yog lub hauv paus ntawm cellular ntawm kev kawm thiab kev nco, uas nyob ntawm lub zog ntawm synaptic kev twb kev txuas [35].
Cov kev tshawb fawb yav dhau los tau pom tias qhov loj dua synaptic active cheeb tsam muaj txiaj ntsig zoo dua ntawm kev zoo siab postsynapticneurons thiab shortening ntawm thaj chaw nquag tuaj yeem cuam tshuam txog kev ua haujlwm tsis zoo ntawm kev sib kis ntawm synaptic [36].
Cov neurotransmitter diffuses thoob plaws lub synaptic cleft thiab khi rau receptor proteins ntawm daim nyias nyias ntawm lub postsynaptic neuron, ua rau ionicchannels nyob rau hauv lub postsynaptic membrane yog qhib los yog kaw.
Qhov zoo tshaj plaws shortening ntawm synaptic cleft raws thiab thickening ntawm postsynaptic ntom (PSD) tuaj yeem txhim kho kev sib kis tau zoo [37–39]. Tsis tas li ntawd, axon, dendritic, thiab dendritic spines tsim lub hauv paus ntawm cov txheej txheem synaptic plasticity.
Nyob rau hauv txoj kev tshawb no, peb tau txiav txim siab seb 12 lub lis piam ntawm treadmill ce pretreatment hloov A -induced impairment nyob rau hauv structural synaptic plasticity nyob rau hauv lub hippocampus thiab prefrontal cortex insix-hli-laus 3 × Tg-AD nas.
Peb pom tias 12 lub lis piam ntawm kev tawm dag zog kev tawm dag zog tiv thaiv kev poob qis hauv kev kawm spatial thiab nco hauv rau-hli-hnub 3 × Tg-AD nas thiab treadmillexercise ua rau txhim kho cov txheej txheem synaptic plasticity los ntawm kev nce cov lej synapse thiab cov qauv tsis sib xws, kev qhia ntawm synaptophysin (Syn ), qhov ntev axon, dendritic complexity, thiab tus lej dendritic qaum. Cov txheej txheem no tuaj yeem ua rau cov treadmill tawm dag zog ua kom txhim kho kev kawm thiab kev nco.
2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Tsiaj txhu
Triple-transgenic Alzheimer's disease (3 × Tg-AD) txiv neej nas (harboring PS1M146Vknockin, APPswe, thiab TauP301L transgenes) tau txais los ntawm Jackson Laboratory.Age-matched C57BL / 6J txiv neej nas tau ua hauj lwm raws li non-transgenic (Non-Tg) tswj.
Txoj kev tshawb no tau ua tiav ntawm 3-hli-hnub 3 × Tg-AD (n=20) thiab nas tsis-Tg (n=20). Txhua tus tsiaj tau txais kev tso cai nkag mus rau zaub mov thiab dej hauv chav uas tswj tau qhov kub thiab txias (23 ± 1 ◦C) thiab av noo (40-60%), nrog rau 12 h lub teeb-tsaus voj voog.
Cov nas tau tuav txhua hnub rau 3-6 hnub ua ntej kev sim coj cwj pwm. Txhua tus tsiaj tu thiab siv tau los ntawm cov txheej txheem pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Beijing SportUniversity (2015015).
2.2. Treadmill Exercise Protocol
Thaum muaj hnub nyoog 3 lub hlis, cov nas tau muab faib ua plaub pawg (n=10 txhua): Cov pab pawg uas tsis yog-Tg, pawg tsis-Tg, 3 × Tg-AD pawg tswj, thiab 3 × Tg-AD kev tawm dag zog .
Tag nrho cov nas siv tau raug tso cai hloov mus rau treadmill khiav rau 30 minon 3 hnub sib law liag (thawj hnub ntawm 5 m / min; hnub thib ob thiab thib peb ntawm 10 m / min).
Tom qab ntawd, cov nas no tau raug ua raws li txoj cai kev tawm dag zog ntawm qhov ceev ntawm 12 m / min ntawm 0◦ nqes hav rau 10 min, tom qab ntawd 15 m / min rau 50 min. Nyob rau hauv tag nrho, nas raug cob qhia rau 1 teev ib hnub twg, 5 hnub hauv ib lub lis piam, rau tag nrho 12 lub lis piam.
Raws li peb qhov kev ntsuam xyuas los ntawm ib qho chaw nyob ib leeg-lanetreadmill txuas nrog Oxymax / Comprehensive Laboratory Animal Monitoring System, cov txheej txheem kev cob qhia mus sij hawm ntev no tswj kev siv zog ntawm 50% mus rau 65% ntawm VO2max.
Cov nas nyob rau hauv cov tswj tsis-Tg thiab 3 × Tg-AD tswj pawg tau tso rau ntawm lub treadmill, tsis tau khiav, rau tib lub sij hawm raws li cov pab pawg. Peb thawj zaug ua qhov kev xeem yim-armradial maze rau txhua tus nas (10 nas hauv txhua pab pawg, hnub nyoog 6 hli).
Tom ntej no, peb tshuaj xyuas cov kab mob histological txawv txav los ntawm Golgi staining (3 nas nyob rau hauv txhua pab pawg), Western blot (3 micein txhua pab pawg), thiab electron microscopy (4 nas nyob rau hauv txhua pab pawg) (Daim duab 1A).
Daim duab 1. Treadmill kev tawm dag zog tiv thaiv kev poob qis hauv spatial kev kawm thiab kev nco hauv 3 × Tg-AD nas.(A) ncua sij hawm ntawm treadmill kev tawm dag zog lossis kev tswj tsis tau, kev ntsuas tus cwj pwm, thiab kev kuaj mob histological. (B)
Qhov feem pua ntawm kev ua haujlwm nco tsis raug rau hnub 5 thiab hnub 6 tau nce ntau hauv 3 × Tg-ADmice piv rau pawg tswj tsis-Tg (B; ** p < 0.01, n=10 nas).
Qhov nce hauv feem pua ntawm kev ua haujlwm nco tsis raug yog tiv thaiv los ntawm treadmill ce pretreatments (B; ## p < {{0}}}.01,n=10 nas). (C) Tsis muaj qhov cuam tshuam loj rau qhov feem pua ntawm kev siv lub cim xeeb yuam kev ntawm kev tswj tsis-Tg, tsis yog-Tg ce, 3 × Tg-AD tswj, thiab 3 × Tg-AD ce nas (C; p > 0.05,n {{ 11}} nas).
2.3. Yim-Arm Radial Maze Test
Lub yim-caj npab radial maze muaj ib lub hauv paus platform nrog ib txoj kab uas hla ntawm 18 cmand yim caj npab uas txuas los ntawm lub hauv paus platform, txhua txhua 30 cm ntev, 7 cm dav, thiab 15 cm siab, nrog ib tug sib npaug angular nrug ntawm ib sab caj npab. Lub yim-caj npab radialmaze tau pleev xim xiav.
Qhov kawg ntawm txhua caj npab tuav lub khob zaub mov uas yog 3 cm nyob rau hauv lub cheeb thiab 1 cm tob. Ib lub qhov rooj tau nyob nruab nrab ntawm txhua sab caj npab thiab qhov nruab nrab ntawm txoj kab. Lub koob yees duab yees duab nyob saum toj no tau txuas nrog lub khoos phis tawj los sau cov kev txav ntawm cov tsiaj.
Qhov kev tshawb fawb tau nce siab 35 cm saum toj no theem hauv pem teb hauv chav dimly lit nrog ntau qhov kev tshawb fawb ntxiv. Tag nrho cov txheej txheem sim siv chocolate ua zaub mov. Cov ntaub ntawv tau raug tshuaj xyuas nrog ethology video tsom xam software (JLBehv-8ARMM, ShanghaiJiliang Company, Shanghai, Suav).
Txhawm rau txhim kho kev txhawb siab rau cov qhob noom xim kasfes hauv txoj cai radial maze raws tu qauv, nas tau txwv tsis pub noj zaub mov rau ib lub lim tiam ntxiv kom ncav cuag 80-85% ntawm lub cev qhov hnyav thiab khaws cia raws li cov xwm txheej no thaum kawm tag nrho.
Txoj haujlwm radial maze tau muab faib rau hauv kev hloov kho thiab kev yuav khoom. Nyob rau ob hnub sib law liag ntawm kev hloov kho, txhua tus nas tau muab tso rau hauv lub hauv paus pib platform.
Tom qab 10 s, txhua lub qhov rooj tau qhib, thiab tus nas raug tso cai tshawb nrhiav thiab noj zaub mov (0.08 gchocolate) hauv txhua caj npab ntawm qhov tshawb nrhiav rau 10 feeb. Lub rooj sib tham nrhiav tau muaj ib qho kev sim ua ib zaug ib hnub rau 10 hnub sib law liag.
Plaub ntawm caj npab (1, 3, 5, thiab 7) tau baited nrog chocolate khoom plig. Cov nas tau muab tso rau hauv nruab nrab ntawm qhov kev tshawb nrhiav, thiab tom qab 10 s, txhua qhov rooj tau qhib. Cov kev ua ub no ntawm nas tau kaw cia li thiab tom qab ntawd txheeb xyuas qhov kev soj ntsuam video.
Ib sab caj npab nkag tau suav thaum tag nrho plaub lub paws ntawm tus nas hla qhov pib ntawm caj npab. Qhov kev sim txuas ntxiv mus txog thaum tus nas nkag mus rau tag nrho plaub lub caj npab.
Kev nkag mus rau hauv ib qho uas tsis yog-baitedarm tau suav tias yog kev siv lub cim xeeb yuam kev thiab rov nkag mus rau hauv lub caj npab uas tau mus xyuas yav dhau los tau suav tias yog qhov ua haujlwm nco ua yuam kev.
Hauv tabulating qhov kev ua tau zoo, qhov feem pua ntawm kev ua haujlwm nco yuam kev (lub sij hawm luv luv) tau txhais tias yog ib qho kev rov ua dua ntawm tag nrho cov kev nkag, thiab feem pua ntawm kev siv lub cim xeeb yuam kev (ntev-termmemory) raws li feem pua ntawm kev nkag mus rau hauv ib lub caj npab thiab cov uas tsis tau noj. bait thaum nkag baited caj npab tawm ntawm tag nrho cov nkag [40,41].
2.4. Golgi Staining
Rau qhov kom muaj nuj nqis ntawm morphological hloov pauv, hippocampus thiab prefrontalcortex tau stained nrog Golgi staining kit (FD Rapid Golgi Stain Kit PK-401, FD NeuroTechnologies, Columbia, MD, USA) ua raws li cov chaw tsim khoom cov lus qhia.
Hauv luv luv, lub hlwb raug muab rho tawm thiab muab tso rau hauv jars uas muaj 20 mL ntawm Golgi-Cox tov rau 17 hnub nyob rau hauv qhov tsaus ntuj ua ntej txiav rau hauv 60 µm slices siv lub cryostat (Leica, Nussloch, Lub teb chaws Yelemees).
Cov seem tau muab tso rau hauv cov tshuaj staining rau 10 feeb, lub cev qhuav dej hauv ascendingethanol series (50%, 70%, 95%, thiab 100%), thiab tshem tawm hauv xylene ob zaug. Thaum kawg, slideswere cover-slipped nrog Permount.
Pyramidal hlwb los ntawm CA1 cheeb tsam ntawm lub hippocampus los yog txheej V ntawm lub prefrontal cortex tau kos siv lub tshuab microscope ntawm ib tug magnification ntawm 200 × thiab 1000 ×.
Neurons raug xaiv yog tias lawv tsis pom tseeb (los ntawm vasculature, glial hlwb, los yog lwm yam neurons) thiab tshwm sim tag nrho stained thiab zoo nyob rau hauv seem.
Qhov ntev ntawm lub axon yog ntsuas los ntawm manually taug qab ib tug neurite los ntawm ciam teb ntawm lub soma mus rau lub ntsis ntawm lub axon [42]. Dendritic morphology tau soj ntsuam los ntawm Sholl tsom xam.
Lub pob tshab gridwith equidistant (10 µm) concentric rings tau nyob nruab nrab ntawm cov ntoo dendritic tracings. Tom qab ntawd tus lej ntawm lub nplhaib sib tshuam tau siv los kwv yees tag nrho qhov ntev dendritic thiab dendriticarborization [43–45].
Thaum kawg, peb tau sau 9-13 ntu hauv CA1 hippocampus thiab layerV prefrontal cortex los ntawm 3 nas nyob rau hauv txhua pawg sim. Txhawm rau xam tus txha nraub qaum, qhov ntev ntawm dendrite (tsawg kawg ntau dua lossis sib npaug ntawm 10 µm ntev, 2nd-oder dendrites) tau taug qab (ntawm 1000 ×). Qhov ntev ntawm txhua ceg dendritic raug xam, thiab tus naj npawb ntawm spines raws qhov ntev suav (kom yield spines / 10 µm) [43].
The length of dendritic filopodia is normally >3m og ib<10 µm [46]. The morphology of spines is commonly categorized into three types: thin, mushroom, and stubby [47].
Cov nqaj qaum raug suav tias yog nyias yog tias qhov ntev ntau dua lub caj dab txoj kab uas hla thiab lub taub hau thiab caj dab zoo sib xws. Spines raug cais raws li nceb yog tias txoj kab uas hla ntawm lub taub hau yog ntau dua li txoj kab uas hla ntawm lub caj dab.Spines raug suav tias yog stubby yog tias qhov ntev thiab dav sib npaug.
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