Journal of Alzheimer’s Disease31(2012)865–878
DOI10.3233/JAD-2012-120211
IOS Press
865 The Load of Amyloid-?Oligomers
is Decreased in the Cerebrospinal Fluid
of Alzheimer’s Disease Patients
Giulia M.Sancesario a,Maria T.Cencioni b,Zaira Esposito a,Giovanna Borsellino b,Marzia Nuccetelli a, Alessandro Martorana a,b,Luca Battistini b,Roberto Sorge a,Gianfranco Spalletta b,Davide Ferrazzoli a, Giorgio Bernardi a,b,Sergio Bernardini a and Giuseppe Sancesario a,b,?
a Tor Vergata General Hospital,Faculty of Medicine and Surgery,The University of Rome Tor Vergata,Rome,Italy
b Santa Lucia Foundation,Rome,Italy
Accepted19May2012
Abstract.Amyloid-?(A?)oligomers are heterogeneous and instable compounds of variable molecular weight.Flow cytometry and?uorescence resonance energy transfer(FRET)-based methods allow the simultaneous detection of A?oligomers with low and high molecular weight in their native form.We evaluated whether an estimate of different species of A?oligomers in the cerebrospinal?uid(CSF)with or without dilution with RIPA buffer could be more useful in the diagnosis of Alzheimer’s disease (AD)than the measurement of A?42monomers,total tau(t-tau),and phosphorylated tau(p-tau).Increased t-tau(p<0.01)and p-tau(p<0.01),and decreased A?42(p<0.01),were detected in the CSF of patients with AD(n=46),compared to patients with other dementia(OD)(n=35)or with other neurological disorders(OND)(n=56).In native CSF(n=137),the levels of A?oligomers were lower(p<0.05)in AD than in OD and OND patients;in addition,the ratio A?oligomers/p-tau was lower in AD than in OD(p<0.01)and OND(p<0.05)patients,yielding a sensitivity of75%and a speci?city of64%.However, in CSF diluted with RIPA(n=30),A?oligomers appeared higher(p<0.05)in AD than in OND patients,suggesting they become partially disaggregated and more easily detectable after RIPA.In conclusion,FRET analysis in native CSF is essential to correctly determine the composition of A?oligomers.In this experimental setting,the simultaneous estimate of low and high molecular weight A?oligomers is as useful as the other biomarkers in the diagnosis of AD.The low amount of A?oligomers detected in native CSF of AD may be inversely related to their levels in the brain,as occurs for A?monomers,representing a biomarker for the amyloid pathogenic cascade.
Keywords:A?42,Alzheimer’s disease,amyloid-?,biomarkers,cerebrospinal?uid,?ow cytometry,?uorescence resonance energy transfer,FRET,oligomers
INTRODUCTION
The amyloid-?peptides1-40and1-42(A?40,A?42) are physiological derivatives of the amyloid-?protein precursor,a normal constituent of neuronal and glial membranes.A?oligomers are soluble aggregates of a ?Correspondence to:Prof.Giuseppe Sancesario,Department of Systems Medicine,1Montpellier Street–00133Rome, Italy.Tel.:+39620903013;Fax:+39620903118;E-mail: sancesario@med.uniroma2.it.variable number of A?monomers and constitute the precursors of insoluble large?brils that deposit in amy-loid plaques in the brain,representing,together with neuro?brillary tangles,the histological hallmarks of Alzheimer’s disease(AD).A?42,and to a minor extent A?40,oligomers have been regarded in recent years not just as the intermediate to A??brils,but as a pri-mary pathological factor,affecting synapses’function and neuronal viability,and secondarily rising an amy-loidogenic cascade to the large insoluble A??brils,and the formation of amyloid plaques and neuro?brillary
ISSN1387-2877/12/$27.50?2012–IOS Press and the authors.All rights reserved
866G.M.Sancesario et al./AβOligomers in CSF
tangles[1–3].A soluble pool of A?40and A?42aggre-gates together with the large insoluble A??brils can be extracted by analytical ultracentrifugation of tissue homogenates from the cerebral cortex of AD affected brains,demonstrating that soluble oligomeric A?species are intrinsic to the brain AD pathology[4–6]. The proposed role of A?oligomers in the brain pathology has stimulated an extensive search for their presence in the cerebrospinal?uid(CSF),since,unlike the insoluble?brils deposited in brain tissue,solu-ble oligomers can diffuse into the CSF.If detected in sensitive assays,they would constitute,in essence,a useful biomarker for the amyloid pathogenic cascade. An increase of A?oligomers in the CSF of AD patients would be expected as a corollary of their increased occurrence and neurotoxic role in the brain tissue. The detection of single A?aggregates in the CSF was?rst observed using?uorescence correla-tion spectroscopy,a very sensitive biophysical method, demonstrating the occurrence of a faster A?self assembly in the CSF of AD affected patients[7]. Although several methods have been recently devel-oped attempting to detect diffusible A?oligomers in CSF,their detection in the CSF has not been an easy task to be pursued for diagnostic purposes in the setting of common biomedical research institutions[8].San-tos and co-workers[9]published a very sensitive and speci?c method for the detection of different species of A?oligomers in the CSF of non-demented neurologi-cal patients,based on a?uorescence resonance energy transfer(FRET)setup,and subsequent detection by ?ow cytometry.
We used FRET and?ow cytometric analysis and unexpectedly found that levels of A?oligomers are lower in the CSF of probable AD than in control patients in contrast to the results recently reported by Santos et al.[10].These?ndings raise the possibility that the detectability of A?oligomers with the FRET cytometric analysis could be dependent on different pre-analytical and analytical factors.We sought to val-idate the quantitative reproducibility of storage and detection procedures of oligomers by FRET cytometric analysis,and?nally we evaluated whether an estimate of oligomer A?species in their native form in the CSF could be more useful in the diagnosis of AD than A?monomers and tau.
PATIENTS AND METHODS
One hundred and thirty seven patients were included in this study after admission to the Neurological Centre of the Tor Vergata General Hospital between2009 and2011because of cognitive or motor impairments. Exclusion criteria were acute trauma of the head and of the spinal cord,chronic subdural hematoma, hydrocephalus,brain tumors and paraneoplastic neu-rological syndrome,acute and chronic infections of the CNS,vitamin de?ciency,hypothyroidism,liver and kidney insuf?ciency,or https://www.doczj.com/doc/ce15895167.html,rmed written consent was obtained from each patient or relatives for routine clinical and instrumental investigations. The study was conducted under the guidelines of Hip-pocrates deontology and following approval of the local Ethical Committee.
Clinical evaluation
Medical history,thorough clinical and neurolog-ical investigation,Mini-Mental State Examination (MMSE),complete blood screening,electrocardio-gram,electroencephalogram,and magnetic resonance imaging of the brain were performed on all patients.
A more comprehensive neuropsychological and psy-chiatric examination was performed on patients with cognitive de?cit,using the standardized Mental Dete-rioration Battery[11]and the Hamilton Rating scale [12].Moreover,CSF was obtained as part of routine clinical evaluation;A?42,total tau(t-tau),and phos-phorylated tau(p-tau)were measured for differential diagnosis of dementia[13,14].
Patients were?rst divided in two groups:patients with different degrees of cognitive impairment (demented,MMSE=18.2±6.1)and patients without cognitive impairment(non demented, MMSE=27.8±1.2).The non demented group (n=56;40women,16men)included patients with heterogeneous neurological diseases:arterial hypertension and chronic ischemic encephalopathy (n=9),Parkinson’s disease without cognitive impair-ment(n=13),cerebellar atrophy(n=3),familial tremor(n=3),late onset epilepsy(n=5),spondilo-genetic mielopathy(n=4),optic papillitis(n=1), Sjogren syndrome(n=1),peripheral neuropathies (n=7),pseudotumor cerebri(n=2),narcolepsy(1), depressive pseudo-dementia(n=6),and psycho-somatic syndrome(n=1).Demented patients were further classi?ed,according to clinical criteria,as affected by probable AD(n=46;27women,19 men;MMSE=20.1±6.5)and patients affected by other dementias(n=35;14women,21men; MMSE=15.5±3.8).The latter group consisted of patients affected by vascular dementia(n=15), frontotemporal dementia(n=1),Creutzfeldt-Jacob
G.M.Sancesario et al./AβOligomers in CSF867
disease(n=1),Parkinson’s disease with dementia (n=14),progressive supranuclear palsy(n=1), amyotrophic lateral sclerosis with dementia(n=1), and chronic depressive state(n=2).The diagnosis of probable AD was made according to the DSM IV and NINCDS-ADRA guidelines[15].Diagnosis of frontotemporal dementia was suggested accord-ing to neuropsychological de?cits and behavioral symptoms[16].Diagnosis of probable vascular dementia included patients according to NINDS-AIREN criteria[17].Clinical and neuropsychological examination was repeated six months later in each patient to check the clinical course and accuracy of the diagnosis.
CSF withdrawal
Lumbar punctures were performed following stan-dard procedures.Brie?y,the skin in the lumbar area was cleansed with povidone-iodine solution10%, and local anesthesia was induced with ethyl chlo-ride.A20-gauge needle was inserted at the L3/L4 or L4/L5interspaces while the patients were at bed rest in the lateral decubitus or in the sitting position. 6–8ml were routinely drained in three consecutive polypropylene tubes without preservative,and imme-diately carried to the chemistry department.Blood samples were taken at the same time to evaluate blood-brain albumin ratio and blood-brain barrier integrity. Polypropylene tubes(Eppendorf Safe-Lock tubes) and polypropylene pipette tips(Gilson-DIAMOND Tips)were used in the collection and storage of CSF [18].
One CSF sample from each patient was used for routine chemical analysis and microscopic observa-tion.Patients with blood contaminated CSF samples containing>500erythrocytes/?l were excluded from this study.CSF samples were centrifuged(2000rpm) at4?C for10min,divided in aliquots of300?l,and stored at–80?C within2h after withdrawal.Within1 to3months after freezing,the samples were thawed just once and immediately handled for the detection of A?oligomers and for the measurements of A?42, t-tau,and p-tau.
Aβ42,t-tau and p-tau measurements
The levels of A?42,t-tau,and p-tau,classical biomarkers in CSF,were determined according to pre-viously published standard procedures[19,20],using commercially available sandwich enzyme-linked immunosorbent assays(ELISA)(Innotest?-Amyloid 1-42,Innotest h-T-tau,Innotest PhosphoT-tau–Inno-genetics).
Preparation of Aβaggregates
We obtained aggregates from A?monomers using Dahlgren’s modi?cation of Lambert’s protocol[21, 22].Recombinant A?42and synthetic A?40were purchased respectively from rPeptide(Athens,GA) and American Peptide(Sunnyvale,CA).Both A?40 and A?42monomers(1mg)were dissolved in anhy-drous dimethyl sulfoxide to5mM,and sonicated for 10min.Thereafter,the monomeric A?solutions were diluted to100?M either in cold HAM’s-F12cul-ture medium(Euroclone)and incubated over night at 4?C for oligomer formation,or in HCl10mM and incubated at37?C overnight for?bril formation.All samples were then stored at?20?C.
Analysis of synthetic Aβaggregates
Frozen oligomer and?bril preparations were thawed just once,and used to con?rm the formation of A?aggregates by SDS-PAGE analysis without heat denat-uration followed by silver staining.Monomers and aggregated forms of A?40and A?42,corresponding to an original content of1?g of monomers for each sample,were separated in16%SDS-PAGE tris-tricine buffer(5ml Acrylamide Bis/tri33%,0.88ml Glyc-erol,3.3ml Tris-HCl3M pH8.45,1ml SDS1%, 100?l APS10%,10?l TEMED).Polypeptide SDS PAGE molecular weight standards(Bio-Rad)were included as reference.After run,gels were?xed in 40%methanol and10%acetic acid over night,and then were silver stained(Bio-Rad Silver Stain kit).Densit-ometric analysis of scanned blots was performed using the NIH ImageJ version l.29program(NIH,Bethesda, MD,USA).
Fluorescence labeling of antibodies
The monoclonal A?antibodies4G8(Chemicon) and6E10(Covance),recognizing different epitopes of the A?peptide(17-24and1-16,respectively), were labeled respectively with the?uorescent chro-mophores Alexa Fluor488and Alexa Fluor594using the Monoclonal Antibody Labeling Kit(Invitrogen), following manufacturer’s instructions.
868G.M.Sancesario et al./AβOligomers in CSF
FRET and?ow cytometric analysis
Extensive FRET analysis was carried out in frozen samples of native CSF(n=137)and of synthetic oligomer and?bril preparations;frozen samples were thawed just once and directly incubated with 4G8-Alexa Fluor488(2nM)and6E10-Alexa Fluor 594.Moreover,some CSF samples(n=15of patients with AD,and n=15with other neurological disor-ders(OND))were studied in duplicate to evaluate the interference of dilution and detergents on detec-tion of oligomers.To this purpose,two CSF aliquots of each patient were studied:the undiluted one (de?ned as native)was directly incubated with?uo-rescent antibodies,according to our protocol,whereas the other was preliminary diluted(1:1)with RIPA buffer which contains both non ionic(0.5%NP-40and0.25%deoxycholate)and ionic detergents (SDS0.05%)according to Santos et al.[9,10]. Furthermore,in a separate and adjunctive group of patients(n=5)FRET analysis was carried out in duplicate in fresh native CSF samples incubated with?uorescent antibodies within2h after lum-bar puncture,as well as after one freezing-thawing cycle.
Equal volumes(300?l)of native or diluted CSF, of synthetic A?40and A?42monomers,and of A?aggregate preparations(corresponding to an original content of0.1?g of monomers for each sample)were incubated for90min at4?C with4G8-Alexa Fluor 488(2nM)and6E10-Alexa Fluor594(8nM),accord-ing to Santos et al.[9].CSF samples and standard in vitro A?aggregates were analyzed for monomer and oligomer content with a FACS Calibur cytome-ter(BD biosciences).FRET analysis was acquired from samples incubated either with the?uorescent chromophores Alexa Fluor488and Alexa Fluor594 (blank),or with the?uorescent antibody4G8-Alexa Fluor488only or with6E10-Alexa Fluor594,and ?nally with both?uorescent antibodies.FRET involves the energy transfer between two?uorescent chro-mophores,from the excited donor molecule,Alexa Fluor488,to the acceptor molecule,Alexa Fluor594. The FRET can occur only if the distance between the two chromophore-carrying antibodies is less than 10nm,that is the distance between the donor and acceptor probe at which the energy transfer is(on average)50%ef?cient(F¨o rster distance).According to Santos et al.[9],oligomer particles were gated in logarithmic forward/side scatter dot plot(FSC ver-sus SSC).The green or red?uorescence respectively of the dyes Alexa Fluor488and Alexa Fluor594were detected in logarithmic scale by the correspond-ing FL1and FL3photomultipliers through530/30 or670LP bandpass?lter,respectively.To avoid dif-ference in analysis,known number of Count Bright absolute counting beads(Invitrogen)were added to all sample and gated in logarithmic FL4/SSC dot. Incubation of synthetic Aβ42oligomers in human CSF
To evaluate the stability of A?42oligomers in human CSF,known amounts of synthetic A?42oligomers were incubated in CSF of control(n=5)and AD patients(n=5).Brie?y,synthetic A?42oligomer preparation,corresponding to an original content of 0.5?g monomers,was diluted to25?M in native CSF 300?l of AD and control patients,and incubated at 37?for5h.SDS PAGE was used to determine the frequency distribution of synthetic A?42monomers and oligomers:samples of control and probable AD patients were run in duplicate in16%tris-tricine SDS-PAGE at room temperature.After the run,the gels were alternatively?xed in40%methanol and10% acetic acid for silver staining(Bio-Rad),or were trans-ferred onto nitrocellulose membrane in Tris glycine 20%methanol for western blot.Thereafter,nitrocellu-lose membranes were boiled3min in distilled H2O, blocked in3%BSA,and probed with the mono-clonal anti A?antibody6E10(Covance)at a1:2,000 dilution in PBS at4?C overnight.Bound antibodies were detected by incubation for2h at room tem-perature with an HRP-conjugated secondary antibody (DAKO)1:10,000in PBS.HRP reaction products were detected using the ECL system(Perkin Elmer), followed by ChemiDoc acquisition(Bio-Rad).Densit-ometric analysis of scanned blots was performed using the NIH ImageJ version l.29program(NIH,Bethesda, MD,USA).
Statistical analysis
All data analysis was performed using the Statistical Package for the Social Sciences Windows,version17.0 (SPSS,Chicago,Illinois,USA).Descriptive statistics consisted in the mean±SD for variables with Gaus-sian distributions after con?rmation with histograms and the Kolgomorov-Smirnov test,or median(min-max)for parameter categorical(non–parameters).The homogeneity of the variance was evaluated with Lev-ene’s test.One-way analysis of variance(ANOV A) and test for multiple comparison(Bonferroni test) was used to evaluate signi?cant differences among
G.M.Sancesario et al./A βOligomers in CSF 869
groups.Receiver operating characteristic (ROC)curve analysis determined cut-off of sensibility and speci-?city of the data.Statistical signi?cance was set at p value <0.05.RESULTS
Synthetic A βoligomers
Silver stained SDS polyacrylamide gels were used to distinguish aggregated species of synthetic A ?40and A ?42after applying the oligomer-or ?bril-forming conditions.Analysis of the A ?40gel showed a large band of monomers (～3.5kDa)and an unspe-ci?c (～9kDa)band equally detectable under different experimental conditions (Fig.1A).Analysis of the A ?42gel showed an heterogeneous complex of monomers and oligomers of different sizes depend-ing on the experimental conditions (Fig.1B):oligomer preparations contain monomer,tetramer,and pentamer bands of ～14–17kDa,corresponding to low molecular weight (LMW)oligomers;?bril preparations contain monomer and tetramer bands,and large aggregates of 27and ～50–100kDa,which correspond to high molecular weight (HMW)oligomers.The presence of ?brils in the ?bril preparation was not,however,detected by SDS-PAGE and immunoblotting:?brils instead could be visualized by electron microscopy and atomic force microscopy according to previous works [21,22].Although it is not possible to equate structural A ?42oligomer species observed in vitro and in CSF,it is worth noting that A ?42?bril prepara-tion contains A ?42monomers,and LMW as well as HMW oligomers,corresponding at least in part to the heterogeneous molecular size of the oligomer mixture detected in CSF
[23].
Fig.1.SDS-PAGE and silver staining of synthetic A ?40(A),and A ?42(B),corresponding to an original content of 1?g of monomers,in monomeric (lane 1),?bril (lane 2),or oligomeric preparations (lane 3).M =molecular weight markers.
FRET and Flow cytometric analysis of in vitro formed A βaggregates
The particles positive only for 4G8-Alexa Fluor 488can be detected by the FL1photomultiplier through a 530/30bandpass ?lter,while the particles posi-tive for both 4G8-Alexa 488and 6E10-Alexa 594can be detected by FL3photomultiplier through the 670LP bandpass ?lter,and can be identi?ed as cor-responding to FRET events indicative of oligomer particles (Fig.2).FRET analysis readily distinguishes A ?monomer and A ?aggregate preparations.Sam-ples containing synthetic A ?42monomers did not show detectable FRET signal after incubation with both monoclonal 4G8and 6E10anti-A ?antibodies,labeled respectively with Alexa Fluor 488or Alexa Fluor 594(data not shown).Moreover,no FRET signals were detected in samples containing A ?42oligomer preparation after incubation either with both chromophores Alexa Fluor 488and Alexa Fluor 594not bound to anti-A ?antibodies (Fig.2A),or after incubation with just one labeled antibody,i.e.,4G8-Alexa Fluor 488or 6E10-Alexa Fluor 594separately (Fig.2B,C,E).On the contrary,after incubation with both anti-A ?antibodies 4G8-Alexa Fluor 488and 6E10-Alexa Fluor 594,the samples of oligomer and ?bril preparations displayed highly speci?c FRET signals in the FL3photomultiplier (670LP),indica-tive of single A ?aggregates and of their numbers in the sample (Fig.2D,F).FRET signal intensity was stronger for ?bril preparation,containing both LMW and HMW oligomers (Fig.2F),compared to oligomer preparation containing LMW oligomers only (Fig.2D).However,it is not presently clear whether ?brils in ?bril preparation remain in solution and are ef?ciently detected by FRET signals.Anyway,?bril preparations were more stable than oligomer prepa-rations and were used to assess the sensitivity of the assay.As shown by Santos et al.[9],FRET signals are dose-dependent and linear for the concentrations of monomers used for A ?42aggregates,ranging between 10pM to 2.0nM (Fig.3).Although estimation of the A ?oligomer size is possible due to FRET signal intensity of each single particle [24],characteriza-tion of the molecular size of the detected particles cannot be easily inferred among a multiplicity of FRET signals.Moreover,a reliable internal standard for the detection of A ?oligomer is presently not avail-able,since both natural and synthetic A ?oligomer mixtures are heterogeneous,composed by variable species of different molecular sizes and conforma-tions.Therefore,our FRET analysis is not indicative
870G.M.Sancesario et al./AβOligomers in CSF
Fig.2.Flow cytometric detection of A?42oligomers prepared in vitro(original content of0.1?g of monomers).Fluorescent signals are represented as black spots.The FRET signals were detected by the FL3photomultiplier(670LP bandpass?lter)and represented toward the middle of each graph if the acceptor Alexa Fluor594was excited by a very close donor Alexa Fluor488,whose signals were instead only detected by the FL1photomultiplier(530/30bandpass?lter)and represented on the left of each graph.A–D)Samples of A?42oligomer preparations after:A)?uorescent chromophores Alexa Fluor488and Alexa Fluor594,or B)4G8-Alexa Fluor488,or C)6E10-Alexa Fluor594labeled antibodies were added separately,or D)4G8-Alexa Fluor488and6E10-Alexa Fluor594labeled antibodies were added in combination.E, F)Samples of A?42?bril preparations after the6E10-Alexa Fluor594acceptor antibody was added alone(E),or in combination with the 4G8-Alexa Fluor488labeled antibody(F).The numbers reported on the corners of each?gure represent the percentage of?uorescent signals detected in each channel respective of the total of the?uorescent signals detected on the whole sample.
G.M.Sancesario et al./AβOligomers in CSF
871
Fig.3.Sensitivity of the detection of A?42oligomers in?bril prepa-ration by?ow cytometric analysis.A?42oligomers were prepared in vitr o using?bril preparation as described in methods,and progres-sively diluted from2nmol to10pmol(original content of0.1?g of monomers).All data points represent mean values(±standard error) of measurements performed in triplicate.
of the molecular size of the detected oligomers in the samples.
FRET and?ow cytometric analysis of native CSF Human CSF incubated with both chromophores Alexa Fluor488and Alexa Fluor594did not emit?uo-rescent signals(Fig.4A).Incubation of CSF with one labeled antibody plus a cromophore,i.e.,4G8-Alexa Fluor488plus Alexa Fluor594or with6E10-Alexa Fluor594plus Alexa Fluor488,displayed?uorescence signals in the FL1photomultiplier only,as expected (Fig.4B,C).Incubation of CSF with both labeled antibodies4G8-Alexa Fluor488and with6E10-Alexa Fluor594showed?uorescence signals in the FL3pho-tomultiplier,detecting the energy transfer of FRET events between contiguous donor and acceptor probes, and demonstrating the presence of A?oligomers in human CSF(Fig.4D).
The quantitative evaluation of FRET signals in CSF samples from control and AD patients’groups pre-sented a large variation in the amount of detected oligomers(Fig.5).In order to avoid differences in the measurements due to uncontrolled variations in vol-ume of the CSF samples and of other undetermined variables,FRET events in each sample were normal-ized to the amounts of Count Bright absolute counting beads detected in the same sample.In restricting the analysis to the relationship between the A?FRET events relative to the counting beads,we unexpectedly found that the levels of A?oligomers were lower in the CSF of probable AD patients than in OND and other dementia(OD)patients(p<0.05)(Fig.6B). Relationship between Aβoligomers and other biomarkers of AD
Low levels of CSF A?oligomers were detected in AD patients in the early as well as in the late stage of the cognitive impairment assessed with MMSE scores(data not shown),even though the number of AD patients in the different severity stages in our study is insuf?cient to clarify whether CSF oligomers could be related to the disease progression.To vali-date our results with other investigated biomarkers for AD,we also measured the levels of monomeric A?42, t-tau,and p-tau in this set of samples as detected by ELISA.Consistent with previous reports,increased t-tau(p<0.01)and p-tau(p<0.01),and decreased A?42(p<0.01and p<0.05)levels were detected in the CSF of patients with probable AD,compared to OND and to OD patients,respectively(Fig.6A,C,E). The values of A?42,t-tau,and p-tau detected in our lab were within the con?dence interval of the average±2 SD in the external quality control program for CSF biomarkers of the Alzheimer’s Association[25]. Thereafter,we studied whether any relationship exists between A?oligomers detected by FRET and the other biomarkers of AD detected by ELISA. According to Santos et al.[9]and Fukumoto et al.
[23],the ratio between A?oligomers and monomeric A?42in CSF was not signi?cant(data not shown). Conversely,the ratio of A?oligomers/t-tau was signi?cantly lower in the CSF of patients with probable AD compared to the OND control group (p<0.01)but not to the OD group(Fig.6D);the ratio of A?oligomers/p-tau was signi?cantly lower in AD patients compared to OND control(p<0.05) as well as to OD patients(p<0.01)(Fig.6F).We next performed ROC curve analysis to assess the ability of the ratio A?oligomers/t-tau or A?oligomers/p-tau to discriminate the patients with probable AD from the other two groups.The cutoff level of0.40for the ratio A?oligomers/t-tau yielded a sensitivity of78% and a speci?city of71%,while a cutoff level of2.50 for the ratio A?oligomers/p-tau yielded a sensitivity of75%but a speci?city of64%.Such values were comparable to sensitivity and speci?city yielded by the other biomarkers alone or in combination in our study:A?monomer sensitivity78%,speci?city65%; t-tau sensitivity80%,speci?city62%;p-tau sensitivity 80%,speci?city63%;A?monomers/t-tau sensitivity
872G.M.Sancesario et al./A βOligomers in
CSF
Fig.4.Representative ?ow cytometric detection of A ?42native oligomers in CSF samples.A)CSF with ?uorescent chromophores Alexa Fluor 488and Alexa Fluor 594.B–D)CSF after 4G8-Alexa Fluor 488(B),or 6E10-Alexa Fluor 594(C)labeled antibodies were added separately,or (D)in combination.Signi?cant FRET signals were detected by the FL3photomultiplier (670LP bandpass ?lter)only in D.
78%,speci?city 71%;A ?monomers/p-tau sensitivity 80%,speci?city 62%.
In?uence of CSF sample preparation on A βoligomers
To examine whether the RIPA buffer,which was used in the original protocol of Santos et al.[9,10],could affect the detection of A ?oligomers,we also conducted FRET analysis in duplicate in native CSF and in CSF diluted with RIPA buffer,using CSF sam-ples obtained from 30individuals (15patients with AD,and 15with OND).Taking into account the dilu-tion of CSF with RIPA (1:1)compared to native CSF,there was no correspondence between the two ana-lytical procedures since most of the data set of CSF diluted with RIPA lie above the identity line in a scatter diagram of pooled data from AD and control patients (Fig.7A)(p <0.01);indeed,the aliquots of CSF diluted with RIPA showed a concentration of A ?oligomers signi?cantly higher than the aliquots of native CSF from the same individuals examined at the same time (Fig.7B).Moreover,the in?uence of CSF prepara-tions was comparatively evaluated in the AD and OND group.In native CSF,the levels of A ?oligomers were not signi?cantly different between the two groups due to the large standard deviation and to the small sample size of this subset of patients (Fig.7C).Conversely,the levels of A ?oligomers in the CSF aliquots diluted with RIPA were signi?cantly higher for AD compared to OND patients (p <0.05):RIPA buffer increases the overall amount of A ?oligomers and reduces the large
G.M.Sancesario et al./AβOligomers in CSF
873
Fig.5.Absolute(abs)levels of A?oligomers detected by?ow cyto-metric analysis in the CSF of different group of patients.Values, expressed as FRET events,are represented as standard deviation with mean and C.I.95%.AD,Alzheimer’s disease;OD,other dementia; OND,other neurological disorders.NS=non signi?cant. variability in their concentration,compared to values detected in native CSF(Fig.7C).However,the values of CSF oligomers detected with and without RIPA were not related to the values of MMSE(data not shown). In contrast to the above results,similar amount of A?oligomers were detected for?ve CSF samples studied before and after one freezing/thawing cycle(data not shown),according to Santos et al.[9].
In?uence of CSF on Aβoligomers
We then addressed the possibility that the CSF from control and AD patients could differentially affect solubility of A?oligomers,explaining their different concentrations in the two groups of patients.There-fore,we incubated equal amounts of synthetic A?42 oligomer preparations diluted to25?M in human CSF of OND control and AD patients,correspond-ing to an original content of0.5?g monomers for each sample,and used SDS-PAGE to examine stability of native aggregated species in the different CSF micro-environment.
Silver stained SDS-PAGE of human CSF demon-strated that the pattern of CSF protein bands was similar in OND and probable AD patients(Fig.8A, lane2,5).It is worth noting that silver stained SDS-PAGE as well as the monoclonal anti A?antibody 6E10and western blot analysis failed to detect respec-tively the presence of the endogenous monomers (Fig.8A,lane2,5),or of the endogenous aggregated species of A?in CSF of OND and AD patients (Fig.8B,lane2,5).When synthetic A?42oligomer preparations were analyzed alone or after incubation for5h in human CSF,the silver stained SDS-PAGE gel detect the presence of the A?42monomers but not of the A?42oligomers(Fig.8A,lane1,3,6).However, under the same experimental conditions western blot analysis demonstrated that synthetic A?42oligomer preparations can be detected as a mixture of monomers and LMW oligomers of different molecular weight even if diluted to25?M in CSF(Fig.8B,lane1,3,6), showing the advantage of this technique compared to the silver stained SDS-PAGE gel.Densitometric anal-ysis of scanned blots demonstrated that the amount of monomers and of the different bands of oligomers were comparable after incubation of synthetic A?42 oligomer preparations in the CSF of OND and AD patients(data not shown).
DISCUSSION
Our and previous searches of A?42oligomers in CSF have been implicitly based on the working hypothesis that the level of A?aggregates would be higher in body ?uids of AD than in control patients,according to the theory that it is the soluble A?42oligomers which pro-voke neurodegeneration,rather than plaques deposited in the brain.However,such a hypothesis implies that A?oligomers could paradoxically be more diffusible than A?monomers from the brain tissue to the CSF, contradicting all previous studies clearly demonstrat-ing that CSF A?levels in AD correlate inversely with the A?load in the brain.
Aβoligomers in the CSF:A controversial research topic
All previous studies on oligomers of A?in CSF have reported increased levels in patients with AD, even though different techniques have been used [7,23,26,27].Although very sensitive methods have been used in these studies,their results raise some concerns about the nature of the parameters effectively detected.Pitschke et al.[7]investigated more properly the aggregability of synthetic A?in the CSF rather than the levels of oligomers per se. Georganopoulou et al.[26]demonstrated that amyloid-beta derived diffusible ligands(ADDLs)(molecular weights between17and42kDa)are elevated sig-ni?cantly in AD patients compared to age-matched controls.Moreover,Fukumoto et al.[23]detected ele-vated levels of HMW A?oligomers of45-to90-KDa
874G.M.Sancesario et al./A βOligomers in
CSF
Fig.6.A,C,E)CSF levels of A ?42,t-tau,and p-tau detected by ELISA in control patients affected by neurological diseases without cognitive impairment (OND)(n =56),and in patients affected by probable Alzheimer’s disease (AD)(n =46),or by other dementias (OD)(n =35).The samples from each patient were assayed in vitro in duplicate.Values are expressed as pg/ml.B)Levels of A ?oligomers detected by ?ow cytometric analysis in the CSF of different group of patients.Values of oligomer FRET signals were normalized (norm)to the amounts of Count Bright absolute counting beads in each sample,and expressed as relative FRET events.D,F)Ratio of CSF A ?oligomers,detected by ?ow cytometric analysis,to CSF levels of t-tau (D)and p-tau (F)detected by ELISA.Values are represented as standard deviation with mean and C.I.95%.One-way ANOV A followed by Bonferroni test for multiple comparisons.NS =non signi?cant,?p <0.05,??p <0.01.
in the CSF,accounting however for a very small amount (<1%)of the total oligomer mixture in the CSF of AD patients,which is instead predominantly composed of LMW oligomers [23].In addition,Gao et al.[27]using a synthetic ligand,surprisingly found only an enrichment of A ?40oligomers,but not of A ?42oligomers in the CSF of AD patients.Actually,such studies advantageously detected one speci?c species of aggregates,the HMW oligomers,but they likely missed the LMW oligomers,the predominant fractions of the total oligomer mixture in the CSF as demon-strated by the study of Fukumoto et al.[23].
A recent paper casts doubt as to whether the increased levels of A ?oligomers in the CSF of AD reported by Georganopoulou et al.and Fukumoto et al.[23,26]are true or due to the interference of the
G.M.Sancesario et al./AβOligomers in CSF875
heterophilic antibodies in the detection of A?oligomer
with ELISAs[28].Anyway,our study shows lower
levels of A?oligomers in AD patients and leads to
opposite results to those of Santos et al.[10],although
both studies use FRET-based method.We have demon-
strated that the different conclusions between the two
studies depend on preparation of CSF samples with and
without RIPA,which determines a signi?cant increase
in the number of A?oligomers detected in the CSF
of AD in comparison to control patients.RIPA,which
contains both ionic and non-ionic detergents,can dis-
aggregate HMW oligomers,increase the number of
smaller oligomers and stabilize A?dimers[23,29].
Fig.8.A)Non denaturing SDS-PAGE and silver staining of syn-
thetic A?42oligomer preparation(0.5?g),of CSF,and of synthetic
A?42oligomer preparation(0.5?g)incubated in https://www.doczj.com/doc/ce15895167.html,ne1,A?42
oligomer preparation;lane2,CSF from a control patient;lane3,
A?42oligomer preparation in the CSF of a control patient;M,molec-
ular weight markers;lane5,CSF of an AD patient;lane6,A?42
oligomer preparation in the CSF of the AD patient.B)Western blot
of synthetic A?42oligomer preparation(0.5?g)alone or after incu-
bation in CSF,separated by SDS-PAGE and probed with monoclonal
https://www.doczj.com/doc/ce15895167.html,ne1,A?42oligomer preparation;lane2,CSF
of the control patient;lane3,A?42oligomer preparation in the CSF
of the control patient;M,undetected run of the molecular weight
markers;lane5,CSF of the AD patient;lane6,A?42oligomer prepa-
ration in the CSF of the AD patient.A?oligomers were prepared as
described in the methods’section.
Fig.7.Levels of A?oligomers detected by FRET analysis in the
native CSF and in CSF diluted with RIPA buffer(1:1)from AD
(n=15)and OND control patients(n=15).A)Scatter diagram of
pooled data from AD and control patients(n=30).The45?identity
line(in case x=y)is used as a reference comparing the correspon-
dence between the two sets of data from CSF with and without RIPA:
the corresponding data points are not equal to each other,since most
of the scatters of CSF diluted with RIPA lie above the identity line
(p<0.01).B)Diagram of A?oligomers detected in native CSF com-
pared with values of the same samples of CSF diluted with RIPA
(1:1).Differences in dependence of CSF preparation were consid-
ered in AD and OND patients pooled together(n=30).Data are
represented as error bars with mean and C.I.95%.C)Comparison
of A?oligomers detected by FRET analysis in patients affected
by probable AD(n=15)and in OND patients(n=15)either in
native or diluted CSF from the same subjects.Data are represented
as error bars with mean and C.I.95%.One-way ANOV A followed
by Bonferroni test for multiple comparisons.?p<0.05.
876G.M.Sancesario et al./AβOligomers in CSF
On the basis of this?nding,we emphasize that FRET analysis without RIPA,as performed in our protocol, is essential to correctly determine the composition of A?oligomers in native CSF.It is noteworthy,indeed, that in native CSF the A?dimers were more frequently selected from CSF of people cognitively normal than in CSF from patients with AD[30].The effect of RIPA could be otherwise relevant since in increasing the number of LMW oligomers,it may indirectly sug-gest that the native CSF of AD patients contains more HMW oligomers than the control group. Advantages of FRET-?ow cytometric analysis
of Aβoligomers
In comparison to the methods described above, FRET signals can allow an estimate of different species of the LMW and HMW A?oligomers in their native state in CSF.It is not clear however whether FRET signals can also involve the detection of A??brils, but this point could not be relevant in this context because?brils have not been detected in the CSF[23]. In addition,the FRET analysis should receive little or no interference from the endogenous CSF heterophilic antibodies,which instead can give false positive results in the detection of A?oligomer with ELISAs[28]. Indeed,unlike A?oligomers labeled speci?cally with multiple donors and acceptors,the heterophilic anti-bodies contain only two antigen binding sites which can be un-speci?cally labeled either by two donors, two acceptors,and just occasionally by one donor and one acceptor,giving no or very weak FRET signals. Therefore,FRET-?ow cytometric analysis can give an opportunity to assay speci?cally A?aggregates of dif-ferent sizes,although there is no evidence proving that all species of A?oligomers,arising from full-length as well as from truncated A?peptide[31],are indeed detected in the CSF by this method.
FRET-?ow cytometric analysis of Aβoligomers Using such setup,our study demonstrates that the global levels of A?oligomers are lower in the CSF of probable AD than in control patients;however,they do not exclude that minor fractions of oligomeric species could be increased in the CSF.In our opinion,indeed, our data do not represent outcomes completely oppo-site to previous results,rather they could suggest that the increase of HMW oligomers[23],representing a very small amount(<1%)of the oligomer mixture, might well coexist with and could not overcome the decrease of global A?oligomers we observed in the CSF of probable AD patients.It is conceivable,instead, that the multimerization of HMW oligomers could proceed at the expense of LMW A?oligomers and monomers in CSF decreasing their global number. This hypothesis is supported by our previous work demonstrating that a higher proportion of monomers of A?42can be detected by ELISA in CSF of AD compared to control patients by increasing temper-ature to37?C prior the analysis,suggesting that A?42aggregation could be reversible[20].Similar results were obtained analyzing A?42in the CSF under both denaturing and non-denaturing conditions with respectively SDS-PAGE-western blot or ELISA [32].
Whether increased HMW A?in CSF mirrors their content in the brain tissue or whether their formation might further increase by spontaneous seeded multi-merization at the expense of monomers and LMW oligomers[7]in the CSF should be further investi-gated.We demonstrated that the CSF composition of AD and control patients does not affect the solubility and stability of synthetic A?aggregates,at least under our experimental conditions when they were incubated at37?C for5h.These data may suggest that the content of total A?aggregates in the CSF is likely dependent on their diffusion from the brain tissue,so that if entrapped in the brain in AD patients they can diffuse less in the CSF.
Our study is in accordance with a recent study on the relationship between A?peptides and oligomers in the brain and CSF of aged canines,which naturally accumulate human-type A?within diffuse plaque, exhibiting patterns similar to A?deposited in the human brain[33].This study clearly demonstrates that the amount of soluble oligomers in CSF was inversely related to the brain extractable A?,suggesting that as the total amount of brain A?increases,the amount of soluble oligomers in the CSF decreases.By analogy,in updating the“sink hypothesis”of AD,we could sug-gest that the count of both monomeric and oligomeric A?in the CSF is decreased because aggregated A?is deposited in plaques in the brain.
Diagnostic power of FRET-?ow cytometric analysis of Aβoligomers
The?nding of low levels of A?oligomers in the CSF of AD patients appears to be related to the high levels of t-tau and p-tau which represent CSF biomark-ers of neuronal damage.Indeed,the ratios of A?oligomers to t-tau and p-tau are signi?cantly differ-ent in AD patients,and the latter can differentiate AD
G.M.Sancesario et al./AβOligomers in CSF877
from control as well as from other forms of dementia. However,the AUC values in the ROC analysis indi-cated that the levels of A?oligomers and their ratio to t-tau and p-tau did not display better discrimina-tion as a diagnostic biomarker for AD than the level of monomeric A?42alone.This lack of better diag-nostic values of A?oligomers could be dependent on the fact that both A?monomers and oligomers in CSF mirror an inverse interplay with the A?in the brain.
Some technical considerations should also be rele-vant to sensitivity and speci?city of the test.To fully evaluate the usefulness of A?oligomers as a marker of AD,the patients should be followed longitudi-nally to increase the accuracy of the clinical diagnosis. Moreover,the study does not contain any healthy con-trols,due to the ethical and practical dif?culties in ?nding volunteer healthy old subjects on which to per-form a lumbar puncture.This represents a weakness of the study,limiting the comparison of our results between patients affected by AD and age-matched patients affected by other neurological disorders with or without dementia.Moreover,FRET-?ow cytometric analysis is likely to be in?uenced by multiple fac-tors such as CSF sample preparation,the amount of incubation time before?uorescence measurement,the binding of A?aggregates to other proteins in the CSF, the possibility that binding of both antibodies was partly unspeci?c,and other unknown variables[9]. To account for the variability of the assay,indeed,the FRET events in each sample should be normalized to the amounts of counting beads detected in the same sample.The in?uence of multiple factors in CSF prepa-ration should be further standardized,before FRET analysis of A?oligomers can be used for diagnostic purposes.
In conclusion,FRET analysis in native CSF is essen-tial to correctly determine the composition of A?oligomers.Under such experimental setting,our data demonstrate that the load of A?oligomers is lower in CSF of patients with AD than in the control patients, suggesting that it may be inversely related to their content in the brain,as occurs for A?monomers.To reconcile our report with other opposite results[7,23, 26],we hypothesize that the multimerization of HMW oligomers proceeds at the expense of LMW oligomers and A?monomers,decreasing their number in the CSF.FRET analysis of the CSF A?oligomers is as useful as the other biomarkers in the diagnosis of AD; in addition,the low level of A?oligomers in CSF of AD patients can represent a biomarker for the amyloid pathogenic cascade.ACKNOWLEDGMENTS
We thank Graziano Bonelli for excellent techni-cal assistance in the preparation of the manuscript. The work was supported by Italian Ministry of Health grants RF08.32,and RC10.
Authors’disclosures available online(https://www.doczj.com/doc/ce15895167.html,/disclosures/view.php?id=1370). REFERENCES
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阿尔茨海默病诊断进展 卫生部北京医院彭丹涛 一、临床诊断标准 （一） NINCDS — ADRDA 临床诊断标准 1.I 很可能（ probable) 诊断标准： 临床检查和神经心理检查认为痴呆；两种及以上认知领域缺陷；记忆和其它认知障碍进行性加重；无意识障碍； 40-90 岁起病，常在 65 岁以后，且非其它全身系统性疾病及脑部疾病所致认知障碍。 2.II 支持很可能（ probable) 诊断标准： 特殊认知功能的进行性衰退（如：失语、失用、失认）；日常生活能力损害及行为改变；家族中有类似病人，尤其有神经病理证实者；实验室检查结果：腰穿脑脊液压力正常；脑电图正常或无特异性改变，如慢波增加； CT 检查证实有脑萎缩，且随病程进行性加重。这是1984 年制订的标准。被称为 AD 病人诊断的“金”标准。其诊断准确率达 80 ％一 100 ％，敏感性达 81 ％一 88 ％，特异性达 90 ％。在应用中发现，该诊断标准在应用中存在局限性， 2007 年对其进行了重新修订。 （二）修订 NINCDS — ADRDA 诊断标准 1. 可能为 AD : A+B 、 C 、 D 或 E 中至少一个核心症状： A. 早期、显著的情景记忆障碍，包括以下特点： 逐渐出现的进行性的记忆功能下降，超过 6 个月；客观检查发现显著的情景记忆损害，主要为回忆障碍，在提示或再认试验中不能显著改善或恢复正常；情景记忆障碍可在起病或病程中单独出现，或与其它认知改变一起出现。
2. 支持特征： B. 存在内颞叶萎缩 MRI 定性或定量测量发现海马结构、内嗅皮层、杏仁核体积缩小（参考同年龄人群的常模）。 C. 脑脊液生物标记异常 A β 1-42 降低、总 tau(t-tau) 或磷酸化 tau(p-tau) 增高，或三者同时存在。 D. PET 的特殊表现：双侧颞叶糖代谢减低；其它有效的配体，如 FDDNP 预见 AD 病理的改变。 E. 直系亲属中有已证实的常染色体显性遗传突变导致的 AD 。 该诊断标准出台后，对临床和科研有很大的帮助。 二、欧盟针对 AD 的诊治的建议（一） 2010 年，欧盟针对 AD 的诊治提出了建议： 应由知情者补充临床病史（ A ），知情者问卷，如有可能应使用（最佳实践建议）。 认知功能下降导致的生活功能独立性受损在痴呆的诊断标准中是一项关键内容，需在诊断评价中进行测试（ A ）；对所有痴呆患者都应进行神经系统和躯体检查（最佳实践建议）；对所有患者都应进行认知功能评估（ A ），疑似或极早期的 AD 患者应进行定量神经心理学测试（ B ）；认知功能评估应包括主要认知域（尤其是延迟回忆）及一般认知评估（ A ）。对于中度记忆障碍者，线索回忆比自由回忆更为合适（ B ）；所有患者都应进行 BPSD 评估（ A ），适当的评分量表从知情者处获取信息（最佳实践建议），对 AD 患者伴随疾病的评估。 （一）记忆 建议采用加州言语学习测验 (CVLT) Buschke 自由回忆和线索选择性提醒回忆测验——可鉴定是否为早期 AD 。 Rey 听觉言语学习试验（ The Rey Auditory Verbal Learning Test ， RAVLT ) 可以区分出 AD 患者和非痴呆患者，或将 AD 和其他类型的痴呆患者予以
病因成谜 毫无来由的大脑萎缩，只能观察到脑部细胞内多处形成B淀粉的沉淀，使脑部失去原有的功能，逐渐坏死，并且不再新生。这种现象主要发生在大脑皮质和”海马回区域”(与学习、记忆关系极为密切的前脑的一部份)，因此，老年痴呆症患者最明显的症状是：记忆力丧失；语言、判断、推理和方向感等认知功能也逐渐降低；接着出现妄想、幻觉、游走、忧郁等异常行为；意识时而清醒，时而模糊。在人体正常的衰老过程中，大脑中会产生一种叫作B淀粉样蛋白(amyloid beta)的物质，这种蛋白被认为是发生阿尔茨海默症的关键。 阿尔茨海默病的7大危险信号为： 1一次又一次地重复问相同的问题。 2一字又一字，一遍又一遍的重复叙述相同的事情。 3以前很容易和经常做的事，像做饭，缝补或玩牌，现在却忘记了。 4丧失了支付和如何管理自己财务的能力。 5在很熟悉的环境中迷路走丢，或家中的物品方错位置。 6经常忘记洗澡或长期穿同一件衣服，却强调已经洗过澡或说他的衣服是很干净的 轻度症状：阿尔茨海默病起病缓慢，通常以健忘为首发症状。如果你怀疑周围的亲人可能出现了轻度的阿尔茨海默病的临床表现，还需要观察是否有以下变化： ?记忆丧失和语言表达能力的改变 ?获取新知识的能力下降 ?完成日常生活活动的时间延长 ?情绪和人格改变，像抑郁或焦虑 ?饮食安排或按时服药出现困难 ?完成简单的算术计算、理财、持支票簿或牌出现障碍
?对所熟悉的地方位置感到迷惑 ?判断能力下降，以至于做出错误的决断 提示：即使出现上述症状的几种，患者任然可能完成绝大多数的活动甚至可以驾车。此时仍然需要看医生 中度阿尔茨海默病，大脑内的病理变化更加严重，并且向其他控制语言、认知、感觉和思维的中枢蔓延，症状变得更加显著，可以出现行为问题（此阶段约有大于65%的患者仍然不能诊断出，直到进展为中至重度）。 阿尔茨海默病中度症状： ?健忘加重，发展到忘记过去的事情像曾从事过的职业和朋友的名字 ?集中精力专注的时间缩短 ?组织思绪的能力障碍 ?认知和思维障碍（像判断能力下降，礼节的遗忘） ?语言障碍，包括表达、朗读、理解和书写方面的障碍 ?获取新知识或处理意外情况时能力下降 ?反复重复相同的语言，动作或问题 ?出现朋友和家人认知困难 ?赖散、易怒、焦虑和妄想，尤其是发生在下午或晚上时 ?幻觉、妄想、多疑或偏执 ?不注意仪表和个人卫生 ?情绪控制能力下降（像餐桌上不讲礼貌、在不恰当的时间或地点不穿衣服、 说脏话） ?象在使用淋浴和厕所或做记录时出现困难 重度症状：在阿尔茨海默病的最后阶段，脑内神经元出现广泛的损害，所有的
血管性痴呆诊疗指南 在我国，血管性痴呆是仅次于阿尔茨海默病的第二位引起痴呆的原因。血管性痴呆的危险因素包括高血压、高龄、糖尿病、既往卒中史、卒中病灶的大小和部位等。缺血性和出血性卒中引起的脑损伤均可导致痴呆和认知功能障碍。血管性痴呆的特点包括：出现记忆障碍和至少两个其他认知领域，包括定向、注意、语言词汇功能、视空间能力、计算、执行功能、习惯动作、抽象、判断能力的障碍，且这些障碍足以引起日常生活能力和社会功能的缺损。 尽管卒中可以增加痴呆的危险性，但目前就脑血管病与痴呆的关系之间如何确定，还非常模糊。卒中的次数、卒中的部位、梗死脑组织的总容量到底要达到多大的量，方能引起痴呆，目前尚无定论。因此，在某些情况下，很难确认卒中是否是痴呆的唯一病因。总之，通过影像学检查未发现脑血管病的证据，说明痴呆患者没有血管病基础；反之，发现了血管病变并不能说这就是痴呆的病因，尤其是在另一个引起痴呆的病因，如阿尔茨海默病与脑血管病并存的情况下。 【临床表现】 血管性痴呆可以表现为不同的临床亚型。 1.皮层综合征特点是临床反复出现脑血栓形成或栓塞，伴有明显的局灶感觉和运动缺损症状和体征，失语更为严重，认知障碍的起病急骤。
2.皮层下综合征伴有深部白质病变的皮层下综合征的特点是有假性球麻痹的体征，锥体束征。抑郁或情绪不稳，额叶释放症状。记忆障碍相对较轻。定向力障碍，对新鲜事物反应减少，兴趣减少，判断事物间关系的能力下降，在交谈时难于将焦点从一个话题转移到另一个话题，注意力不集中。刻板语言和动作。 3.常染色体显性皮层下白质脑病的遗传性脑动脉病（CA-DASIL）所致的血管性痴呆一般在30～50岁起病，与19号染色体上的notch3基因突变有关。由于皮层下多发梗死导致腔隙状态，突出的临床特点是假性球麻痹，伴有情感障碍和尿失禁，双侧锥体束征。步态平衡障碍、步距小，病史中有偏头痛史。家族史常阳性。 【辅助检查】 头颅MRI检查可以发现皮层下多发腔隙性梗死灶。此外，在皮层下动脉硬化性白质脑病（Binswanger’s病）的患者，还可以在更广泛的皮层下白质发现脱髄鞘病变。MRI的敏感性比CT高。 其他实验室检查包括除外外源性栓塞、红细胞增多症、血小板增多症、中枢神经系统血管炎、脑膜血管梅毒所致的多发性脑梗死，这在较年轻的患者、尤其是没有高血压病的患者尤其重要。 【诊断】
2010年欧洲神经病学联盟阿尔茨海默病诊疗指南 一、概述 (一)目的 2008年成立工作组的目的是修改前一版欧洲神经病学联盟(EFNS)的 阿尔茨海默病(AD)诊疗指南。前一版指南采用了第4版诊断和统计手册(DSMⅣ)及美国国立神经病学、语言障碍和脑卒中研究所一阿尔茨海默病及相关疾病学会(NINCD-ADRDA)对痴呆综合征和AI)的诊断标准。本次修改的指南特别强调疾病生物标记物依据，如磁共振成像(MRI)、18F一脱氧核糖葡萄糖正电子扫描(PET)和脑脊液(CSF)检测等技术所获取的进一步证据，以提高临床诊断的可信度。此外，特别注意了近期在疾病认知和行为方面的AD临床试验结果。由于AD是该指南所关注的焦点，非AD型痴呆，如血管性痴呆(VaD)、额颢叶变性(FTLD)、帕金森病痴呆(PDD)、路易体痴呆(D1，B)、皮质基底节变性(CBD)、进行性核上性麻痹(PSP)、皮质一纹状体一脊髓变性痴呆(CJD)及其 他痴呆将另述。本指南足指导临床工作的理想标准，但并不一定适用于所有情况，还应该考虑每个患者的临床表现和可用资源。该指南未讨论经济学效益，因为各国差异较大，不同国家会得出不同的结论。 (二)背景 痴呆患者在65岁及以上人群中占5．4％，患病率随年龄增长而不断升高，其中以AD为主。欧洲痴呆研究协会与欧洲AI)协会合作研究发现，目前在欧洲有845万人患有AD。痴呆给社会造成了严重的经济负担，整个欧洲每年为此花费约1410亿欧元，其中56％为非正式护理花费。
每年痴呆患者的花费为21 000欧元，因病致残约为350／10万人，相比之下，糖尿病为247／10万人。随着人类寿命的延长，预计未来30年痴呆患者数将成倍增长。早发型AD(年龄<65岁)值得特别关注，因为这种情况遗传易感性强。具有不同的临床和认知表现，与晚发型病例比较。其病程进展更快。另外患者可能仍处在劳动和生育年龄。因此对早发型AD需要特殊处理。患者在临床AD之前常先经过一个轻度认知功能障碍(MCI)时期。此时一个或多个认知域出现主观和客观障碍，但仍能保持日常活动(ADL)能力。专家组决定不对MCI综合征进行详细总结，因为MCI的疾病分类学及其与AD的关系尚在讨论中。 (三)检索及评价策略 本指南的循证依据来自于考科蓝图书馆(Cochrane Library)资料库、荟萃分析和系统综述及通过联机医学文献分析和检索系统(Medline)数据库检索到的2009年5月之前期fu中发表的原始科学论文。专家组根据科学可靠性水平评价证据(证据分级I、Ⅱ、Ⅲ和Ⅳ)，并采用EFNS 手册中的定义依据可靠性水平划分推荐等级(A、B或C)。对于尚无证据的重要临床问题，则根据经验和专家工作组的共识给出最佳实践建议。 二、临床诊断：病史、实验室检查、神经系统检查和躯体检查患者和知情人提供的病史应该集中在受损的认知领域、疾病进展过程、日常生活能力的损害及任何相关的非认知症状。既往病史、伴随疾病、家族史和教育史是病史的重点。神经系统检查和全身体检对于区分AD 与其他原发性退行性和继发性痴呆及伴随疾病尤为重要。虽然目前尚
泥炭沉积的类脂化合物（正构烷烃、脂肪醇、脂肪酸、甾酮、三萜类化合物和类异戊二烯、直链酯类等）、纤维素中C，H，O 同位素，以及泥炭腐殖化度和孢粉、生物化石等都是恢复古环境的良好指标。虽然泥炭的这些气候代用指标能够反演古环境的相对干湿、冷暖，但并不能定量地给出温度值的大小。 1、GDGTs（甘油二烷基甘油四醚脂） 研究较多的GDGTs化合物主要包括类异戊二烯类（GDGT-0~GDGT-4）和支链类（I~III）两大类，类异戊二烯GDGTs被认为是古菌细胞质膜中所特有，是古菌存在的生物标志化合物。 与该指标的相关内容： （1）CBT：环化指数（the Cyclisation ratio of Branched Tetraethers） （2）MBT：甲基化指数（the Methylation index of Branched Tetraethers （3）研究发现支链GDGTs 结构中甲基个数（MBT指数）主要受当地年平均大气温度（MAAT）影响，其次受环境pH影响；支链GDGTs结构中环戊烷个数（CBT指数）主要受环境pH控制。 （4）环化指数（CBT）/甲基化指数（MBT）是近年来根据支链四醚膜类脂（GDGTs）提出的定量化重建土壤pH和陆地年平均大气温度（MAAT）的生物标志物指标。 （5）Weijers等人提出的MBT/CBT 指标在近海、湖泊沉积中都得到了较好应用，并依此将MBT/CBT 指标应用到泥炭沉积中，讨论了指标在泥炭沉积中的适用性和应用潜力。文章发表在2007年的《Geochimica et Cosmochimica Acta》上。 （6）许云平等利用GDGTs来重建全新世渤海湾有机碳的来源及沉积能量（2010年国家自然科学基金项目）。由GDGTS衍生出的指标BIT比值可用作湖相、河口、滨浅海环境沉积物中判识有机质来源的重要指标。 （7）高效液相色谱-质谱仪（HPLC-MS）进行GDGTs分析（当前存在的主要问题）。 2、脱-A-三萜烯系列化合物（属脂肪族） 脱-A-三萜类是地质体中重要的生物标志化合物，已在石油和各种沉积物中多有报道，认为是高等植物三萜类经光化学和/或微生物氧化使得A环丢失的降解产物。该系列化合物在沉积物中的出现一方面说明被子植物的输入，另一方面显示A环的丢失是高等植物五环三萜类较为普遍的转换途径。 与该指标的相关内容 （1）可反映气候的干湿、温度高低以及沼泽水位的高低； （2）研究发现，该指标在泥炭中的积累与沼泽发育期生物群落结构组成差异密不可分；（3）脱-A-三萜烯变化序列与植被群落结构演替具有相关性（可以与孢粉、植物大化石的结果相互验证） （4）GC-MS分析采用惠普6890气相色谱与HP5973质谱联用仪
The Lancet：阿尔茨海默病最新国际诊断标准 2014-05-31 20:32来源：丁香园作者：辛夷籽 字体大小： 过去8年内，国际工作组（IWG）及美国国家老龄问题研究所---阿尔茨海默病协会，推出了阿尔茨海默病(AD)的诊断标准；通过更好的定义临床表型以及将生物标志物整合进诊断进程中，从而全面覆盖疾病各个时期（从无症状到最严重痴呆阶段）。 这些标准最重要的实际应用是允许在疾病前驱期便开展更早的预防手段，并且有利于临床前期AD二级预防的研究。通过这些标准的研究应用，最终将会发展出一项常规领域能够真正探查AD存在的通用标准。 考虑到现有的IGW研究诊断标准的优缺点，近日The Lancet杂志刊登了一篇文章，提出新的先进性建议，从而对之前诊断框架进行改善。基于这些提炼，AD的诊断得以简化，只需通过AD临床表型（典型/非典型）联合同AD病理相一致的病理生理生物标志物即可。 另外文章建议疾病的下游标志物，如MRI容积、脱氧葡萄糖PET可更好的用于检测及监控疾病过程。本文分别详细阐述了典型AD、非典型AD、混合型AD以及AD的临床前阶段的特异诊断标准。具体如下： 一.典型AD的IWG-2 诊断标准（任何时期的A加B两方面） A:特异临床表型：存在早期及显著情景记忆障碍（孤立或与暗示痴呆综合症或轻度认知障碍相关的其他认知、行为改变），包括下述特征： 1.患者或知情者诉有超过6个月的，逐步进展的记忆能力下降； 2.海马类型遗忘综合症的客观证据，基于AD特异检测方法---通过线索回忆测试等发现情景记忆能力显著下降。（在疾病中度及重度痴呆阶段海马遗忘综合症可能难于鉴定，体内AD病理证据中足以存在痴呆综合症的相关特点）； B：体内AD病理改变的证据（下述之一） 1.脑脊液中Aβ1–42水平的下降以及T-tau或P-tau蛋白水平的上升； 2.淀粉样PET成像，示踪剂滞留增加； 3.AD常染色体显性突变的存在（常携有PSEN1、PSEN2、APP突变）；
呆病（阿尔茨海默病）中医诊疗方案 一、诊断 （一）疾病诊断 1.中医诊断标准 参照《实用中医内科学》（第2版）（王永炎、严世芸主编，上海科学技术出版社，2009年）。 （1）记忆障碍，包括短期记忆障碍（如间隔5分钟后不能复述3个词或3件物品名称）和长期记忆障碍（如不能回忆本人的经历或一些常识）； （2）认知损害，包括失语（如找词困难和命名困难）、失用（如观念运动性失用及运动性失用）、失认（如视觉和触觉性失认）、执行功能（如抽象思维、推理、判断损害）等一项或一项以上损害； （3）上述两类认知功能障碍明显影响了职业和社交活动，或与个人以往相比明显减退。 （4）起病隐匿，发展缓慢，渐进加重，病程一般较长。但也有少数病例为突然起病，或波动样、阶梯样进展，常有中风、眩晕、脑外伤等病史。 神经心理学检查、日常生活能力量表、MRI或脑脊液检查等有助于痴呆临床诊断。 2.西医诊断标准 参照中国痴呆临床实践指南工作组《中国痴呆诊疗指南》的中文版阿尔茨海默病临床诊断标准（田金洲主编，人民卫生出版社，2012年）： （1）记忆或认知功能损害逐渐出现6个月以上，且进行性恶化。 （2）神经心理学测试证实存在显著的情节记忆损害。 （3）精神状态检查或神经心理学测评提供认知功能损害的客观证据。 （4）工作或日常生活能力受损。 （5）神经影像学证据：海马体积缩小或内侧颞叶萎缩。 （6）除外其他病因：1）认知损害发生或加重在明确的卒中后3个月内，或存在多发梗塞或严重白质高信号等血管性痴呆的典型特征；2）或具有波动性认知损害、形象生动的视幻觉及自发的帕金森综合征等路易体痴呆的核心特征；3）或具有行为变异和额叶和/或前颞叶明显萎缩等额颞叶痴呆的突出特征；4）或其他可逆原因如激素或代谢异常如甲状腺功能减退或叶酸/维生素B12缺乏；5）或谵妄或其他精神及情感疾病，如精神分裂症、抑郁症。
生物标志物 科技名词定义 中文名称：生物标志物 英文名称：biomarker 定义：用于监测和评价能够导致生物有机体的生物化学和生理学改变的化学污染物。 所属学科：海洋科技（一级学科）；海洋科学（二级学科）；环境海洋学（三级学科） 本内容由全国科学技术名词审定委员会审定公布 生物标志物：在亚个体和个体水平上既可以测定污染物暴露水平，也可以测定污染物效应的生理和生化指标。 对于疾病研究，生物标志物一般是指可供客观测定和评价的一个普通生理或病理或治疗过程中的某种特征性的生化指标，通过对它的测定可以获知机体当前所处的生物学过程中的进程。检查一种疾病特异性的生物标志物，对于疾病的鉴定、早期诊断及预防、治疗过程中的监控可能起到帮助作用。寻找和发现有价值的生物标志物已经成为目前研究的一个重要热点。 自1994年蛋白质组概念提出，定量蛋白质组学已经成为蛋白质组学研究的热点和中心。定量蛋白质组学便是检测正常与疾病状态下组织全部表达蛋白质在量上的差别。 定量蛋白质组学中的蛋白质定量技术也成为发现生物标志物的重要途径。 生物标志物是生物体受到严重损害之前，在不同生物学水平（分子、细胞、个体等）上因受环境污染物影响而异常化的信号指标。它可以对严重毒性伤害提供早期警报。 这种信号指标可以是细胞分子结构和功能的变化、可以是某一生化代谢过程的变化或生成异常的代谢产物或其含量，可以是某一生理活动或某一生理活性物质的异常表现，可以是个体表现出的异常现象，可以是种群或群落的异常变化，可以是生态系统的异常变化。 生物标志物分类 从功能上一般分为: 接触（暴露）生物标志物 （biomarker of exposure）; 效应生物标志物
倍,经χ2检验,差异均有显著性;二项分布拟合与Edward检验均显示,扬中胃癌的发病存在明显的家庭聚集性,符合多基因遗传方式;先证者家庭成员发生胃癌的危险性显著高于均衡可比的对照家庭成员,核心家系成员间患病率的差异,可能与胃癌遗传易感性和家庭内环境因素暴露的差异有关[5,6]。 分析胃癌家族史在家庭聚集性中的作用,结果显示(资料未列出):先证者家系有胃癌家族史的比例为28134%(761/2685),对照家系胃癌家族史的比例为2170%(69/2557),两者差异有极显著性,χ2 =64612,P=01001;同样,胃癌病例有家族史的比例为41175%(291/697),也显著高于非胃癌对照家族史的比例11186%(539/4545),表明遗传易感性因素在胃癌发生中有重要地位。 同时,也应该看到,以肿瘤发病率为观察研究的终点指标,对遗传易感性作用相对较弱的散发性肿瘤而言,敏感性较低,出现一些难于解释的阴性结果,需要借助分子遗传学、分子生物学技术,准确判断肿瘤早期生物学表型与遗传易感性(基因型)之间的关系。根据国内外现有流行病学资料:胃癌是在多种环境和遗传因素长时间、多步骤、交互作用下的结果[2,7],无论是外源性致癌物,或是机体产生的内源性致癌物,都要通过宿主遗传易感性因素(研究比较成熟的是各种代谢酶基因多态性)的作用,才能最终导致癌变,因此,有必要采用分子流行病学方法,进一步阐明在致癌物代谢的各条通路中,易感基因及其多态性所起的作用[8212],我们已经利用在扬中胃癌高发区获得的环境暴露与基因多态性资料,对此进行了探讨。有关结果将另文报道。 参考文献 1李茂森,耿昌友,朱阳春,等.扬中市1991～1995年恶性肿瘤发病及死亡情况调查研究1肿瘤,1997,17:47724781 2C orrea P1Human gastric carcinogenesis:a multistep and multifactorial process2first American cancer s ociety award lecture on cancer epidemiology and prevention1Cancer Res,1992,52:6735267401 3Perera FP1Environment and cancer:who are susceptible?Science, 1997,278:1068210731 4S tadtlander CT,W aterbor JW1M olecular epidemiology,pathogenesis and prevention of gastric cancer1Carcinogenesis,1999,20:2195222081 5Nagase H,Ogino K,Y oshida I,et al1Family history2related risk of gastric cancer in Japan:a hospital2based case2control study1Jpn J Cancer Res,1996,87:1025210281 6La Vacchia C,Negri E,Franceschi S,et al1Family history and the risk of stomach and colorectal cancer1Cancer,1992,70:502551 7T oy oshima H,Hayashi S,Hashim oto S,et al1Familial aggregation and covariation of diseases in a Japanese rural community:com paris on of stomach cancer with other diseases1Ann E pidemiol,1997,7:44624511 8K ato S,Onda M,M atsukura N,et al1G enetic polym orphisms of the cancer related gene and Helicobacter pylori in fection in Japanese gastric cancer patients1An age and gender matched case2control study1Cancer, 1996,77:1654216611 9K ato S,Onda M,M atsukura N,et al1Helicobacter pylori in fection and genetic polym orphisms for cancer2related genes in gastric carcinogenesis1 Biomed Pharmacother,1997,51:14521491 10Ng EK,Sung JJ,Ling TK,et al1Helicobacter pylori and the null genotype of glutathione2S2trans ferase2mu in patients with gastric adenocarcinoma1Cancer,1998,82:26822731 11National Institute of Environmental Health Science.Research on environment2related disease1Environmental G enome Project119981 Available from:http://w w w1niehs1nih1g ov/envgenom1 12沈靖.人类基因组计划与肿瘤预防研究面临的机遇.肿瘤,2000, 20:682721 (收稿日期:2000202220) (本文编辑:邵隽一) ?名词小词典? 生物标志物(biological marker) 能够反映致病因素或毒物从暴露到效应过程各个环节性质的特异性生物分子,如DNA、蛋白质、酶、脂质、糖类等。生物标志物的确定和检测是流行病学研究中的关键问题,因为这种确定和检测可被用来进行病因探讨、危险因素的评价、致病因子致病机理的研究、人群易感性评估、疾病流行规律的掌握、疾病防治措施的研究和评估等。 生物标志物大致上可分为两大类,一类是根据表型和基因型的特点分为表型生物标志物和基因型生物标志物,前者包括蛋白质、多肽、脂质、糖类和其他在血清和体液中可检测到的特异性分子,后者主要包括基因类型及突变型、DNA加合物、DNA多态性等;另一类是根据致病因子作用机体的过程,可划分为暴露生物标志物、作用生物标志物、效应生物标志物等。 随着分子生物学理论和技术的深入发展,研究生物标志物的技术手段日趋先进、完善。现可用先进的核酸研究技术、蛋白质研究技术、酶学研究技术、免疫学研究技术等检测和研究生物标志物。 (方福德100005北京市中国医学科学院基础医学研究所) (收稿日期:2000209219) (本文编辑:邵隽一) ? 6 3 ?中华预防医学杂志2001年1月第35卷第1期 Chin J Prev M ed,January2001,V ol35,N o. 1
阿尔茨海默病最新国际诊断标准 过去8年内，国际工作组（IWG）及美国国家老龄问题研究所---阿尔茨海默病协会，推出了阿尔茨海默病(AD)的诊断标准；通过更好的定义临床表型以及将生物标志物整合进诊断进程中，从而全面覆盖疾病各个时期（从无症状到最严重痴呆阶段）。 这些标准最重要的实际应用是允许在疾病前驱期便开展更早的预防手段，并且有利于临床前期AD二级预防的研究。通过这些标准的研究应用，最终将会发展出一项常规领域能够真正探查AD存在的通用标准。 考虑到现有的IGW研究诊断标准的优缺点，近日The Lancet杂志刊登了一篇文章，提出新的先进性建议，从而对之前诊断框架进行改善。基于这些提炼，AD的诊断得以简化，只需通过AD临床表型（典型/非典型）联合同AD病理相一致的病理生理生物标志物即可。 另外文章建议疾病的下游标志物，如MRI容积、脱氧葡萄糖PET可更好的用于检测及监控疾病过程。本文分别详细阐述了典型AD、非典型AD、混合型AD以及AD的临床前阶段的特异诊断标准。具体如下： 一.典型AD的IWG-2 诊断标准（任何时期的A加B两方面） A:特异临床表型：存在早期及显著情景记忆障碍（孤立或与暗示痴呆综合症或轻度认知障碍相关的其他认知、行为改变），包括下述特征： 1.患者或知情者诉有超过6个月的，逐步进展的记忆能力下降； 2.海马类型遗忘综合症的客观证据，基于AD特异检测方法---通过线索回忆测试等发现情景记忆能力显著下降。（在疾病中度及重度痴呆阶段海马遗忘综合症可能难于鉴定，体内AD病理证据中足以存在痴呆综合症的相关特点）； B：体内AD病理改变的证据（下述之一） 1.脑脊液中Aβ1–42水平的下降以及T-tau或P-tau蛋白水平的上升； 2.淀粉样PET成像，示踪剂滞留增加； 3.AD常染色体显性突变的存在（常携有PSEN1、PSEN2、APP突变）； 典型AD排除标准（补充检查：如血检、脑MRI以排除其它导致认知紊乱或痴呆的疾病，或伴发病症） 1.病史：a.突然发病 b.早期出现下述症状：步态障碍、癫痫、行为改变； 2.临床特征：a.局灶性神经特征 b.早期锥体外系体征 c.早期幻觉 d.认知波动；
老年痴呆(阿尔茨海默病)测试表
病因成谜 毫无来由的大脑萎缩，只能观察到脑部细胞内多处形成β淀粉的沉淀，使脑部失去原有的功能，逐渐坏死，并且不再新生。这种现象主要发生在大脑皮质和”海马回区域”（与学习、记忆关系极为密切的前脑的一部份），因此，老年痴呆症患者最明显的症状是：记忆力丧失；语言、判断、推理和方向感等认知功能也逐渐降低；接着出现妄想、幻觉、游走、忧郁等异常行为；意识时而清醒，时而模糊。在人体正常的衰老过程中，大脑中会产生一种叫作β淀粉样蛋白（amyloid beta）的物质，这种蛋白被认为是发生阿尔茨海默症的关键。 阿尔茨海默病的7大危险信号为: 1 一次又一次地重复问相同的问题。 2 一字又一字，一遍又一遍的重复叙述相同的事情。 3 以前很容易和经常做的事，像做饭，缝补或玩牌，现在却忘记了。 4丧失了支付和如何管理自己财务的能力。 5 在很熟悉的环境中迷路走丢，或家中的物品方错位置。 6 经常忘记洗澡或长期穿同一件衣服，却强调已经洗过澡或说他的衣服是很干净的
阿尔茨海默病中度症状： ●记忆障碍-健忘加重，发展到忘记过去的事情像曾从事过的职业和朋友的名字 ●集中精力专注的时间缩短 ●组织思绪的能力障碍 ●认知和思维障碍(像判断能力下降，礼节的遗忘) ●语言障碍，包括表达、朗读、理解和书写方面的障碍 ●获取新知识或处理意外情况时能力下降 ●反复重复相同的语言，动作或问题 ●出现朋友和家人认知困难 ●懒散、易怒、焦虑和妄想，尤其是发生在下午或晚上时 ●幻觉、妄想、多疑或偏执 ●不注意仪表和个人卫生 ●情绪控制能力下降(像餐桌上不讲礼貌、在不恰当的时间或地点不穿衣服、说脏话) ●像在使用淋浴和厕所或做记录时出现困难 重度症状：在阿尔茨海默病的最后阶段，脑内神经元出现广泛的损害，所有的自我感觉消失。在这一阶段，需要全天护理检测，
阿尔茨海默病 集团企业公司编码：（LL3698-KKI1269-TM2483-LUI12689-ITT289-
阿尔茨海默病 阿尔茨海默病（AD）是一种起病隐匿的进行性发展的神经系统退行性疾病。临床上以、失语、失用、失认、视空间技能损害、执行功能障碍以及人格和行为改变等全面性表现为特征，病因迄今未明。65岁以前发病者，称早老性痴呆；65岁以后发病者称老年性痴呆。 基本信息 别????称早老性痴呆，老年性痴呆，老年前期痴呆 英文名称Alzheimerdisease，AD 就诊科室神经内科多发群体70岁以上 常见症状记忆障碍，失语，失用，失认，视空间技能损害，执行功能障，人格、行为改变等 传染性无 阿尔茨海默病病因 该病可能是一组异质性疾病，在多种因素（包括生物和社会心理因素）的作用下才发病。从目前研究来看，该病的可能因素和假说多达30余种，如家族史、女性、头部外伤、低教育水平、甲状腺病、母育龄过高或过低、病毒感染等。下列因素与该病发病有关： 1.家族史 绝大部分的流行病学研究都提示，家族史是该病的危险因素。某些患者的家属成员中患同样疾病者高于一般人群，此外还发现先天愚型患病危险性增加。进一步的遗传学研究证实，该病可能是常染色体显性基因所致。最近通过基因定位研究，发现脑内淀粉样蛋白的病理基因位于第21对染色体。可见与遗传有关是比较肯定的。 先天愚型（DS）有该病类似病理改变，DS如活到成人发生该病几率约为100%，已知DS致病基因位于21号染色体，乃引起对该病遗传学研究极大兴趣。但该病遗传学研究难度大，多数研究者发现患者家庭成员患该病危险率比一般人群约高3~4倍。St.George-Hyslop等（1989）复习了该病家系研究资料，发现家庭成员患该病的危险，父母为14.4%；同胞为3.8%～13.9%。用寿命统计分析，FAD一级亲属患该病的危险率高达50%，而对照组仅10%，这些资料支持部分发病早的FAD，是一组与年龄相关的显性常染色体显性遗传；文献有一篇仅女性患病家系，因甚罕见可排除X-连锁遗传，而多数散发病例可能是遗传易感性和环境因素相互作用的结果。 与AD有关的遗传学位点，目前已知的至少有以下4个：早发型AD基因座分别位于2l、14、1号染色体。相应的可能致病基因为APP、S182和STM-2基因。迟发型AD基因座位于19号染色体，可能致病基因为载脂蛋白E（APOE）基因。 2.一些躯体疾病
阿尔茨海默病临床路径2016 一、阿尔茨海默病临床路径标准 （一）适用对象。 第一诊断为阿尔茨海默病 (ICD-10: G30.904)。 （二）诊断依据。 根据《中国痴呆与认知障碍诊治指南》(中华医学会神经病学分会痴呆与认知障碍学组、中国阿尔茨海默病协会编写，中华医学杂志，2011年) 、《美国国立老化研究所与阿尔茨海默病协会诊断指南写作组对阿尔茨海默病诊断指南的推荐和介绍》（中华神经科杂志，2012年）和国际工作组标准-2（Advancing research diagnostic criteria for Alzheimer’s disease: the IWG-2 criteria Lancet Neurol，2014年），符合痴呆的诊断标准：慢性隐袭起病，数月或数年，认知功能障碍导致工作能力或日常生活功能受到影响。排除其它疾病导致的痴呆。影像学可能见到内侧、底部、外侧颞叶、顶叶的萎缩。 1.一项或一项以上的认知功能障碍： （1）工作能力或日常生活功能受到影响。 （2）比以往的功能和执行力水平有所下降。 （3）无法用谵妄或主要精神障碍解释。 （4）通过联合以下两者来检测和诊断患者的认知损害：①来自患者和知情人的病史采集；②客观的认知评价——简单的精神状态检查或神经心理学测验。当常规的病史和简易精神状态检查（MMSE）结
果不足以形成确凿的诊断时．应进行全面的神经心理学测验。 （5）包括以下至少一个和／或两个领域以上的认知或行为损害：①学习并记住新信息的能力受损。症状包括：重复问题或谈话，乱放个人财物．忘记重要事件或约会，在一个熟悉的路线上迷路等。②推理能力和处理复杂任务的能力受损，判断力差。症状包括：理解力差，无法管理财务，决策制定能力差，无法规划复杂或连续的活动。③视空间功能受损。症状包括：不能识别面孔或常见物品，尽管视力很好仍不能通过直接观察找到物品，不能操作简单的工具，穿衣定向障碍等。④语言功能受损(说、读、写)。症状包括：说话时找词困难、犹豫不决，有语音或语义错语、拼写或书写错误。⑤人格、行为或举动改变。症状包括：异常的情绪波动如激动不安、动机缺乏、主观努力、淡漠、失去动力、回避社交．对以往活动的兴趣减低、失去同理心、强迫的或强迫观念行为、同社会相悖的行为等。神经系统检查没有其他异常发现。同时要符合阿尔茨海默病的特征性认知功能障碍：比如记忆障碍是最突出的异常，可以有语言问题、视空间问题、推理判断问题。同时要鉴别路易体痴呆以及额颞叶痴呆的特征性表现。 2.辅助检查：对所有首次就诊的患者进行辅助检查有助于揭示认知障碍的病因或发现伴随疾病，基因检测有助于提高诊断级别。 （三）治疗方案及药物的选择依据。 根据《中国痴呆与认知障碍诊治指南》 (中华医学会神经病学分会痴呆与认知障碍学组、中国阿尔茨海默病协会编写，中华医学杂志，2011年) 制定治疗方案。目前无特效治疗，采用综合治疗方案，药物治疗和护理照顾结合，药物治疗主要如下：
广东化工 2010年第4期· 150 · https://www.doczj.com/doc/ce15895167.html, 第37卷总第204期 生物标志物监测环境污染研究新进展 姜元臻 (中山市环境监测站，广东中山 528400) [摘 要]生物标志物在环境污染监测方面的应用日益重要，文章侧重于对生物标志物在此方面的应用进行全面阐述，包括：生物标志物的定义及分类，生物标志物的特征及优势，生物标志物在检测环境污染的应用，最后还提出了生物标志物在环境监测方向的展望。 [关键词]生物标志物；环境污染；生物监测 [中图分类号]O65 [文献标识码]A [文章编号]1007-1865(2010)04-0150-03 New Advances of Study on Monitoring Environmental Pollution by Biomarkers Jiang Yuanzhen (Zhongshan Environmental Monitoring Station, Zhongshan 528400, China) Abstract: Biomarkers is becoming more and more important in the application of environmental monitoring. The article focased on a comprehensive exposition of biomarker application in this regard, which included definition and classification of biomarker, characteristics and advantages of biomarker, biomarker’s application in the detection of environmental pollution, finally made an outlook of biomarker in the direction of environmental monitoring. Keywords: biomarker；environmental pollution；biomonitoring 1 生物标志物概述 1.1 生物标志物的定义 目前，中国的环境监测工作还主要是针对环境中化学成分的存在量进行检测。物理化学监测虽然能清楚地知道环境中各化学成分的具体含量及其变化，但却不能直接反应环境对生物所造成的毒害作用。另外，由于环境中的许多污染物含量很低，相互混合，体系复杂，仅用化学因子监测的手段往往不能够全面的反映环境的污染状况。在环保观念日益增强的今天，社会对环境评价的全面性和准确性的要求也日益增高，这就要求建立一个综合的、多手段的、多参数的环境监测体系以实现快速、高效、准确地对环境状况作出全面的评价。而生物监测正好补充了理化监测的不足。 生物标志物是生物体受到严重损害之前，在分子、细胞、个体或种群水平上因受环境污染物影响而产生异常变化的信号指标。一种标志物应能敏感有效地反映出生物体发生严重损伤之前的生物变化，并能准确评估生物体所处的污染状态及其潜在危害，为环境污染提供早期警报。随着分子生物学理论和技术的迅速发展，生物标志物(biomaker)的研究作为一个崭新的领域逐渐引起了国内外共同关注[1]。1987年美国国家科学院首先将生物标志物定义为由生物体或样品可测出由外来化合物导致的细胞学或生物化学组份或过程、以及结构或功能的变化[2]。Benson和DiGiulo[3]认为生物标志物是在生物个体所测得的生物化学、生理学或病理学反应，而这些生物学反应能给出环境污染物的暴露，或由暴露所引起的亚致死效应资料。 生物指示物(Bioindicators)自上世纪70年代污染生态学中出现并一直沿用至今。最初只是将耐污的生物物种称为指示生物(Indicator species或Bioindicator)，随着污染生态学的野外研究和实验室毒性试验研究，逐渐将生物指示物的应用范围扩大至污染生态学的不同生物学组织层次，小至分子水平，大至生态系统结构与功能，包括发生在分子、生物化学、生理、病理组织、生物个体、种群、群落和生态系统等不同生物学组织水平上的生物学效应，从生物学的角度为环境质量的监测和评价提供依据。简单地讲，生物标志物就是可衡量环境污染物的暴露及效应的生物反应。一个理想的生物标志物应具备化学特异性，能够微量鉴定、试验费用低廉、检验快速，与环境样品中污染物有量的相关性等。寻找理想的生物标志物一直是环境监侧、环境毒理学及环境医学领域研究的重要内容。 1.2 生物标志物的分类和各种类型的生物标志物 从功能上看，生物标志物一般可分为三类[4]，即暴露生物标志物(Biomarkers of exposure)，反应或毒性效应生物标志物(Biomarkers of responser or toxic effect)，易感性生物标志(Biomarkers of susceptibility)。 1.2.1 暴露生物标志物 暴露生物标志物指示机体经化学品的暴露，即污染物引起的物体的反应，如指示对重金属暴露的金属硫蛋白(MTs)，但此类标志物不能指示污染物的毒性效应，有助于研究生物对化学分析方法很难检测到的的环境中的不稳定化合物的暴露。暴露生物标志物一般依靠测定体液和组织中特定化学物质或者其代谢物，或者与生物分子相互作用形成的产物。 1.2.2 反应或毒性效应生物标志物 效应标志物是指在一定的环境暴露作用下，生物体产生相应的可测定的生理生化变化或其它病理方面的改变，即指示污染物对生物体健康状况的损害效应，如指示DNA损伤的DNA 加合物(DNA-adducts)，它可能是生物机体中某一内源性成分或测定机体功能容量，产生疾病或障碍的改变等。确定化学物质的生物学效应的生物标志物很多，从最简单的标志物如监测体重变化至复杂的标志物如采用免疫化学技术测定特定同功酶[5]。酶活性抑制持久，因此，可作为重要的效应生物标志物。如血细胞数和血细胞损伤的检测可提供各种资料，出现姊妹染色单体交换指示染色体潜在损伤，可由环氧乙烯暴露引起；缺乏特有淋巴细胞指示免疫抑制，可由二恶英(TCDD)等化学物质引起。HSP70家族是序列最保守并且对污染物的应激反应最为显著的一类应激蛋白。沈骅等[6]以鲫鱼为实验动物，Cu，EDAT-Cu，Zn，Pb，Cd，染料橙(HC Orange 1)及两种金属同时进行长期低浓度暴露，在不同浓度下，应激蛋白HSP70被不同程度地诱导，并有明显的剂量效应关系。研究发现，在低于国家渔业水质标准的浓度下，HSP70仍然有显著的诱导表达，说明水体中污染物在低于现行渔业水质标准的浓度下，长期暴露仍然会对鱼类产生一定的损伤。HSP70比传统的生长、繁殖等生物指标更为敏感。 1.2.3 易感性生物标志物 易感性标志物是指当生物体暴露于某种特定的外源化合物时，由于其先天遗传性或后天获得性缺陷而反映出其反应能 [收稿日期] 2009-07-31 [作者简介]姜元臻(1982-)，男，山东人，硕士，主要从事环境监测方面的工作。