ti it

SEN ILE PLAQUE FORMATIO N AND SEVERECARDIOV ASCULARDISEAS E

BY

CARLOS F. SONE IRA RU:I Z

A th e si s subm i tted tothe Schoo l of Graduate Studie s inpar t i alfu l f ilnl"!n t of th e

re quiremen ts for the degreeof Ma ster of Sci e nce

Faculty ofMedicin e Memo r ial Un iversi t y of Newfou n d lan d

19 94

St.John' s Newfo undl a nd

1+1

~~f"!a,io ~cw-AOnlariol

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ABSTRACT

Senileplaques are considered by many to be the most consis t entneuropathologic feat u r e of Alzheimer'sdisease,but theyare abo prese n t inthebrains of non-demented, elderly subj ects. Recent re po r t s (Spa r ks ec al, 19 9 0 , 19931 sU9gest that no n - deme nted pa t i e nts wi t h cr itical coro nary artery disease show a higher prev a lenc e of sen ileplaqu e s tha n no n- demented subj e c t s withou t heart dis ea s e . The present st udy analyzes 40 aut opsy brains which were di v i de d into three groups acc ordi ng to the cHnic o-pat holog i c al findi ngs : an Alzhe imer ' s disease group [n ..12), a severe cardIovaecuLar di sease group (n_17) and a control gr\lup (n_ll). The brain are asex ami ne dwe r e the midd l efrontalgyrus.the superiorand inferio r wate rshedareas, the hippocampa l formation with the trans entorhinalcortex,theprimaryvisualcortex, the head of the caudate nucleus and the anterio rlobe of the cerebellum.

Consecutive sections wer e stained with cresyl viol et, the modifie d Bielscbowsky method and immunohistochemistry for amy l o i d bet a -pro t ein. Senile plaques and neu rof i b r i llary tangles were counted in threeand sixmi c r o s c opic fields at XIOO and X200magn ification ,respectively,and the mean ve Iue a of the counts were ca l c u l a t e d and used for statistical analysis. Patients with severe ca r di o v a s c ula r diseas e (critical coro n ary arterydisease and/o r hy per tension )showe d

H

a hi g he r pr e val e nc e of senileplaques tha n the non-demented controls. In the cardrcveecurardisease group nenile pla q ue counce we r e signific antly lar g e r in the in f e r i o r watershed area" dentate gyrus , subiculum and transentiorhi ne L cortex.

Conl;rol and cardiovascular di sea s e patien ts showed no diff '2r en ce regardi ng the pre val e nce and number of neu r o f i brilla r y ta ng l e s . In the sa mple exa mine d , cardiovascular disease patients occup ied an in t e r me dia te position in the spectrumof senile pla que formatio nbetween Al z he i me r ' s disease and non-heart disease patients. These resu ltssuggest that ther emight be a cardiovascularcomponent in the genesisof senile plaques and thatstudyof pat i en t s wi t h severecardiovascula r disease may help to clarit y the originand evol utionofthese les i o ns.

iii

This thesi s is ded i ca t ed tothe memor yof mygr a ndmot he r

ACKNOWLEDGEMENTS

It is my great pleasure to express my gratitude to my supervisor, Dr.Thomas M.Scott, for giving me the opportunity to learn in his laboratory and for making thiswork possible through his support, orien tat ion and many fruitful discussions. Thanks are also due the other members of my super viso ry committee, Dr. David Haegert and Dr. Alan Goodridge ,for theirva l u a b l e suggestions along the cours eof this study.

My appreciation is extended to Linda Chafe fo r her friendlyhelp in many aspectsof this workand to Geraldine Kufflick for her assistance in the preparation of the manuscript.I am also especiallygrateful to Ed Evelly and to the staff of the Laboratory of Pathology of the Hea l th Sciences Centrefor their technicalas s i sta n c e.

I extend my sincere thanks to my co-workers in the Division of Biological Sciences of the Canadian Me mo r i al Chiropractic college for their understanding and support during the final stages of thiswo r k.

Fina lly, I am especially indebtedto mywife ,Ma r i el a , for givingme strength and unlimited love in spite of th e circumstance s that havekeptus physica l l y ap ar t.

CONTENTS

page ABSTRACT . ... ....••.• .••••••. ...• •.. ii AKNOWLEOOEMENTS •.• • • ..• •• •• ..• • . • •.. . .•... . . ....• LIST OF TABLES .••. ••. •.• .• .•..••.•..•••. .. .. ... viii LISTOF FIGURES .•• . ... . . ... ... ...••. .. •.. .•. • • ix LISTOF ABBREVI ATIONS •.... .... .. . .• •. • •... .•.. ... xi Chapter 1 INTRODUCTION ....•• . ....••... . .. .

1.1l De me nt i " an d Alzheimer's di s ease .•.••.

1.2l Ne ur op a t holog yof Alzheime r ' s disease.

1.2 . 1 ) General features .

1.2.2 1 Seni le plaque s . ... . . . 14 1.2.3) Neurofibrillarytangles... . . . .. 2S 1. 3 ) Neuropsychopathology of cardiovascular

diseases .. • • • • . • . • • •... • • ..• . • •••• • • • • . ••. 31 Chap t e r 2 MATERIALS AND METHODS•.•..•.•.. . . ....• 40 2.1} Descri pt i on ofthe sample... .. . ... 40 2.2) Neu ropathologica l ae e eeeeenc •. • ... . . 43 2.2 . 11 In i t ia.lprocedure s .... .. .. ... 43 2.2 . 2 1 Stai n i ngme t h od s ... .. .. .. . . .. . 46 2.2 .31 Quan t if icati o n of SPandNFT . . . 52 2.3) Statistical ana lysis

Chapte r3 RESULTS •••. . . ..

vi

53 55

Chapter 4 DI SCUSS ION.•... . . 4.1) Brainwe igh t

pa g e 89 89 4. 2) Severecv eandSP formation .... 90 -1.3) Qualitativeaspectsand stainingofSP 95 4. 3 . 1)Pr i mar y vis ua l cortex 99

4.3.2)Caudatenucleus 100

4.3.3)Cerebellum....••.. 10 3

'1.4)Hippocampal formation •...•... 108 4.5) Severecve end SP: Pathogenet i c

relationsh i p 113

4.5.1) Blood-brainbar rierimpairment. ... 114 4.5.2) Dene rva tingmicroangiopathy 119 4.5.3)Cerebralhypoper fusrLcn ... 12 3

4.6) Summaryand Conc lusion s .. 12 9

REFERENCES•...• ... •• ....••... .•... .•...

vii

131

LIS TOF TABLES

Table 2.1:Desc ript ive charac ter isticsof the

sample , .

Table2.2:Description of the subgroupsof CVD

patie nt s . . .

viii

page

41

42

LI STOF FIGURES

page

Figure3.1: Comparisonof brainwei ghts 56 Figure 3.2: SP in neocorticalareas ;.. sa

Fi g u r e 3.3: SP inmed ial temporal lobe 59

Fig ure3.4: NFT in medial tempora l lob e 60

Figure 3.5: <;lPin neocorticalareas(CCAD) 62 Figure 3.6: SP inmedialtempora l lob e (CCAD) 63 Figure 3.7: NFT in medial tempora l Lobe (CCAD)... 64 Figure3.8: SPin neocortical areas (HT) 65 Figure 3.9: SPin medial temporal lobe (HT) 67 Figure 3.10:NFT in medial temporallobe (HT) 68 Figure 3.11: Diff u s e p Laquea inthe molecularlayer

of the subiculumof a CVD patient 70

Figure 3.12: Cl a s s i c plaques in the pyramidal cel l la y e r of the subi culumof an AD patient ... 71 Figure 3.1 ~ : Bielschowsky-stained SPinth e molecular

layer of the dentategyrus of an AD patient... . ... ... . .... . .. ... 73 Figure 3. 14 : Anti-amyloidimmt:.noh istochemistryshowing

the same SP (a n in Figure 3.13) inthe molecular layer ofthe dentate gyrus of an AD patient.. . ... . . 74 Figure 3.15:SP in the inferior watershed area of a

CVD patient 75

FigureJ.16 : SP in the molecular layer of the dentate

gyrus of an AD patient 76

Figure 3.17:se in the molecularlayer of the dentate gyrusof a CVD patient ,.... . ... .. 77

ix

page

Fi g ur e 3.18:SP inthe pyramidal cell layerof the

subiculum of an AD patient 78

Fi gure 3.19: SP in the pyram idalcell layer of the

subiculumof a CVD patient 79

Fi g u r e 3.20: SP in the transen to rhinalcortexofan

AD patient 81

Figure3.21:SP inthetransentorhinal cortex ofa

CVD patient 82

Figure3.22:SP inthe primaryvisual cortexof an

ADpatient 83

Figu r e 3.23 :SP inthehead of the caudatenucleus of an AD patient .... .. ... ... . . . 85 Fi g ure 3.24: Diffuse plaques in the molecular layer

of the cerebellarcorte xofan AD

patient. 86

Fi g u r e 3.25 : To r p ed o-lik e swe llingof the axon of a Pu r k i n j e cel lin the cer eb ellumof an

ADpati e n t 87

Fi gu re 3.26:Amyloidangiopat hyof meninge al and pa r e n c hyma l bloodvessels of the

cerebellumof an AD pati ent 88

LISTOF ABBREVIAT:IONS

AD: Alzheimer'sdisease BBB: Blood-brai n ba r ri e r

CAl: CAl regionof the hippocampus prop er CCAD: Critical coronar y arterydisease CVO: Cardiovascular df seeee DG: Dentat egyrus HT:Ar t e ri al hypeaten sio n IW/\: Inferiorwatershedarea MFG: Middle frontalgyrus NF'T: Ne ur o f i bril l a rytang l es SF:Senile pl a qu e s

Sub: Subiculumof thehippoca mpal format ion SWA: Supe r i o r waters hed area

TEC: Tr an s e n t orh ina l cortex vcrpr imary visualcorte x

xi

INTRODUCTION

1. 1) Dementia and Alz h eimer's disease

Dementia is a clinical syndrome with many different causes. According to the American Psychiatric Association jl98?) , the essential feature of dementia is impairment in short- and long-term memory, associated with impairment in abstract thinkingand jUdgement, other disturbances of higher cortical function, or personalitychange, in tho absence of gross clouding of conciousness. The disturbance is enoughto interferesignificantlywith wo rk or usual social activities or r-e LazLonehips withothers.

In the last decade, developments in br a i n ima g i ng, preciseps y c h o l o g i c a l teat e , immunohistological techniques, and molecularbiologyhave greatly increased ourknowledge of many aspects of the dementingsyndrome, and in particular of Alz h e i me r ' s disease (AD). This explosion in investigationand knowledge hasbee ndriven not only by advancing techniques, butal s oby the recognitionof the impactthat the increasing lifespan of the populationha s ha d onsocial and fina ncia l structuresof the economically advancedcountries.

Allwe s t e r n nationshav e seen grea t increases inbo t h the number and the proportion of elderly individuals in their pop u la t i ons.As moreand morepeop lesurvive int o oldag e ,th e

incidence of dementing illnesses, and part i cularlythat of AD, increases , laying a great erburdenon hospital se r v i c e s.Th e s e demographi c ch a ng e s and th e i r consequences are profoundly in flue nc i n g changes in the patternsof health ca r e as wellas priorities inbiomedicalresearch.

De me n ti aca n occurat any age. It is,however, uncommon below60 years butincreasessharplyinprevalence afterth i s eqe.The most frequently quoted rates suggesttha t around5\

of th e populat i on above 65 and up to 25\ of those ov e r 80 ye a r s are demented (To mli ns o n, 199 2 ).Henderson and Kay(1 9 84) summarized a group of impo rta nt surveys onth e prevalence of dementia. The actualpercentage ofaffected people in five surveys between 1956 and 19 75 was widely variable, but all re vea l e d steep rises abov e 75 years and in all the surveys t.oere wassome evidence of dementi ainmo r eth an 20\of people above 85 years. Amore re c ent report (Evans et a L,, 1989), ba s e d on a study of noninstitutionalizedindividualsin East Boston, showed clinically diagno sed probable AD in 3% of subjects aged 6Sto 74, 18 .7 \of subjects aged 75 to 84, and an ast onishing 47.2\ of SUbjects ov e r 85 years.

In the age group over 60 years, there is a very wide varietyofcau s e s of deme n t i a . These include cerebrovascular ch a n g e s (mUl t i- infarct dementia l; central nervous system infections (te r t i a r y neurosyphil is, tubercu lous and fungal meningitis, vi ralencephalitis, human immunodeficiency vi rus-

related disorders, Creut zfeldt-Jakobdisease}; brain trauma (e s pe c i a l l y chronic subdural hematoma ) ; toxic-metabolic disturbances (pe r n i c i ous anemia, folic-acid deficiency, hypothyroidism); normal·pre ssure hydrocephalus; neurologic diseases(Huntington'schorea,multiplesclerosis ,Parkinson's disease); and postanoxic orposthYP031ycemic sta tes.However, despi t ethesemany causes,the most common con d ition pr od uc ing demen t i a is AD (American Psych i a t ric As s oc i a tio n, 1987 ; Roo and Price, 1993 ; Tomlinson,1992 ) .

In an autop sy study of demen t ed pat ients Tomlin s o n , Bless ed and Roth (1970)obse r vedthat about sot of the cases showed fe a t u r e s of ADin a pureform. Inaround 15\ extensive evide nceof cerebral softening was the only majorpa thologic~l lesion fo un d , and in about10\ the twodegenerative processes were combined. In a further 15\ there were Alzheimer-like chang es,with andwi t hout some cerebralsoftenings.but their quant itativegradingwasno t high enoughfor a firm diagnosis. In about 4t other specific ebnorma Lf t.Iea such as pick's di...eas e or tumors were found, but no adequate morp ho logical basis fordementia was detec ted in the remaining 6\ .

A later st ud y re port ing on a larger group of demente d patien ts (Jellinger, 1976) als o found th a t dementia of Alzheimertype accoun t ed for just over hal f the cases, ~'hlle ne a r l y a quar t er belongedtothe vascular type , andthe two were combined in 13\;. Ce r eb r a l tumors , Pick 's disea s e and

other rarer conditions accounted for the remaining 12%.

These studies found that in 10 to 1S% of the cases the le s i o ns of AD and cerebral infarctionoccurred toge the rto an extent that may justify the diagnos isof dementia due to mixed se ni l e and vascularchanges.

The terminology rela ted to hD is now more or le s s consistent, al t ho ug h th ere are still contradict i ons about separatingpresenile fro m seni leAD(Rot h, 1965).The former is largelygen eticallydeterminedand associated with severe neuropat hologi cal changeswhichdifferentiate it comp let e l y from anything seenin age-matched con t r o l s ; by comparison, the la t t e r may oc c u r with little or no evidence of genetic determinat ionand may present changes which are closer to thos e fo und in the normal aged brain.

Some authorspr e f f,r to usethe term"Alzheimer's disease"

for the preseni leformth at matchesbetter theca s eor i gina l l y described by Al oiaAlzheimerin 1907,while theterms "senile dementia"or"seni l e dementia ofthe Alzheimertype" have been commonly used to refer to the senile form. However, the proposed separation of AD and senile dementia has never wen totallyaccepted, and the majority opinion now places these two disorders in one category(To ml i n s o n , 1992) . We are going to accept this general crit e r i o n, and the term "Alzheimer's disease" will be used throughou t this work to refer to both the presenileand senileforms.

Clinically,AD is characterizedby anins i d i o u s onset and a progressiveanddeteriorating course. In the ea rlystages, memory impairment may bethe only apparentcogn itivedeficit . Ther emay alsobe subtlepersonalitycha n g e s, su c h as apathy, lack of spontaneity, and qui et withdrawal fr om social interact ions. pe o p l e usually remain neat and wel l - g roome d , and , asi de from an occasio nal ir r i t a b l e ou t b u rst, are cooperative and be ha v e in a socia l l y approp r i at e way. Dis t u r b a n ceof sp eech functions can be also an early sympt om (e.g., an omi a , echola lia ,di f f icu ltyincomp r ehen d ingwr itt e n or or a l speech ) . I f the pati e n t has insight into the dete rior ation,he maybe come depressed,and depressionoccurs inabout 25\ of the patients.withprogressionof the disease, va r i o u s cognitivedisturhances become qu ite apparent, and behavior and personali tyaremor eobvious l y affected . By the latestage, the personmay becomple t ely mut eand inattentive. At this point the patient is tot a llyinc a p ab l e of caringfor himself and many are partlyor completely incontinent. In many,but notall patients,thereis extremewa s t i ng.Terminal br on chopneumonia is the commonestpostmort em findi ng t.owhic h dea t h is immedia te l y att r i bu t a b le (Ame ri can Psycia t ric Association, 1987:Katzman , 1989; To ml inson, 1992).

Women are affected moreof ten than men, theproporti on be ingabout 2:1 (Ar o n s o n et al., 1990; Rocca, Amadu cc i and Schoenberg, 19B6 ; Tomlinson, 1992). The duration of the

illness is variable , but it is considered to be 5 years average. Occasional cases survive onl y a few months, but survival as long as 21 years has been re c o r ded. It has been observed thatprogress ionof the dise asein presi nile-onset ca s e s is fa s t e r than in senile-ons et ca ses (Se l tz er an d Sherwing, 198 3 ).

Afield that has attractedincreasing interest is that of po t e nt i al risk factors for AD. So far, the most convincin g associationwith AD, beyond increasingage,is the prese nceof a family history of dementia (Henderson, 1990; Mohset al., 1987). A re c e n t st udy (Me n de z et; aI., 1992) showedsimilar resul ts, the AD patients ha d more first-degree de men ted relatives than the non-Andementiagroup or thenormalelderly cases, but surprisingly, inthe group of ADpatients witha familyhistory of dementiathe diseasestartedata la ter ag e than in those without an aff e c ted relative, a findingthat contradic tsthe more tradi t ional vi ewwh i c h considersfamily historyof dementia more closely re l a t ed to early -onset AD than to Lace-cneeeAD(Br e itn e r et al., 198 8 ) .

Two othe r fa c t o r s seem to be al s o associ ated wi t h incre ased risk for AD: he ad trauma and Down ' s syndrome (Ho r vat h etaL, , 1989), althou gh the va lid i t y of the form e r has be e n que s tio ne d bysome studies (Aronson et al., 19 9 0i Me ndez et aL,, 19 92) . The presence of Alzhe imer~ type histological le sions inall Down'ssyndrome pa t i e nt swhodie

over the age of 30 years (Ma l a mud, 1912; Wi sniewsk i. Wisniews kiand Wen, 198 5 1 ha s suggested that the pathological proces sin Down's syndromefro m early middlel ife is similar to tha toccurring inA'Jseveraldecadesla t e r . Thisprobab le linkhas given additionalsup portto the ger.etichypot hesesof ADand has become stronge r af terdiscovering that the gene cod ing for the precursormol eculeof beta -amyloidpr o t e i n ia located on chromosome21 (Gol dga beret al., 1981).

At the same time, st udies wi th mo no zyg ot i ctwtnu have sho wntha t concordance for ADis le sa tha n60\,indi cati ng tha t othe r factors, proba bly envi rome ntal (e.g. , tox i c, infec tious),playanimp o r tantroleinthepa thogene~isof the di s e ase (Horvathet al., 1989;Katzman, 198~, 19 8 9).

1.2) Neuropathologyof AlzheiJIIsr'edieeaeft

1.2 . 1 )Ge ne ral feature s

Gross ly , the brain of pat ients with ADis us uall y strikingly atroph ic, par ticularly in pr e s enil e cases, a'.'eragingabout 1000 g.This results in an abnormallylarg e sub a r a c hno i d sp a ce, with pool s of cerebra-spi n al fluid sepa r a ting the arac hn oid from the ret r ac t ed co rtex, and enl a r gementofth e vent ricle s.The corticalatrop hyis us ual l y

symmetricaland although to some degree generalized, tends to affec tparticU lar ly the frontalandte mpor al lobes (Ame ri c a n Academy of Neurology, 1980; Tomlinson , 1992). Some studies, however, have shown posterior parietal atrophy td be most ma r k e d (Brun andEng l u nd , 1981; de la Monte,1989), a result that correlates with ~reports of reduced metabolic activi tyand blood flow in the temporoparietal reg i o nof AD patients (Burnset aL;,1989;Eberlinget a L. , 1992;Friedland et al. ,1987; Jobstet al .• 19 92; Prohovniket al.,1988).The degreeof shr i n k ag e , ingeneral ,isfar from constant and may occ a sion a lly beabs e n t, particularlyinold age,even though widespread histological evidenceof AD is found (To mlins o n , 1992).

AccordingtoCo r s e l li s (197 6) , the average reduction in volume of the cerebral hemispheresin elderly women withAD is about16%. Hubbardand Anderson(l98 1) observedtha t inolder AD patients (more th a n 80years) ce r e b r alat r o phy was less marked and largely affectedthe temporal lobe s by comparison with the youngergroupin which the gyral atrophy was more diffuse and significantly greater.

Amo r e recentand verydetailedwork(de 10. Monte,1989) showed reduc t i o nincross-sectionalarea (nto 19%") atfive different le v el s of th e brains of AD patients. The lo s s of cer e b r a l cortex ranged from 13\ to 24% and that of white matter from3%to 19';'.Ventricular enlargement,greatest in

the occ ipitalslices, wa s pre sentinall theAlz he i me r cases.

The author concluded that in AD the cerebral hemispheres undergo a degree of collapse or contraction along wi t h the progressive atrophy, thisobservationbeing sup portedbythe fact that the relati v e dilatation of theventricula r system was considerablyless thanwo ul d be expectedon the basis of tissue loss.

There is still some doubt about whether the cortical atrophyin AD involves narro win gof the cortical ribbon,or shorteni ngof cortica l le ng t h oracombination of both.Many older textbooks stressed the marked na rrowi ng of the cortica l ribbonas an important factor leading to cortica l at r op hy , but Tomli nson(1992)ment i o ned that his personal observationshav e never confirmed tha t this isobv i o us, exc e p t in the me dial te mp o r a l cortex . Furtherm ore . two quantitative studies (Duyckaerts et al. , 198 5; Na j l e r a h i m and Bowen, 1988 ) have conc ludedthat reduction incortical le ng t h is a majorfactor in cortica l atrophy.

Mi cros c opi cally , AD is characte ri zed byth e pr e s en c e of senile pl aques (SP) and neurons conta i n i ng neu rofi bri l lary tang les (NFT) throughout most az-aae of the corte x and in many subcort ical nuclei. In the hi ppocampus , numer o us pyr ami da l cells are affect ed by gra nul ova c uo Jar degenera t i o n , andmany Hira no bod i e s arepre s en t . Loss of neuro ns, pa r t icula rlylar ge ne ur o n s, is severe insomepart s

10 of the cortex and hippocampus and in so me deep nuclei, especially the basal nucleus of Me y n e r t and the locus coeruleus. Deposition of amyloid within th e wall of blood vesse ls (congophilic or amyloid angiopathy) is seen in the majorityof cases ofAD. Adeqz-ee of leuko-araiosis is also frequently present (Ameri ca n Ac a demy of Ne ur ology , ⁱaeo,

Horvath et al., 1989; Katzman, 1969; Koo and Pric e. 1993;

Tomlinson, 1992).

In order to make clearer the general picture of the histological changes that characte ri ze AD, the pa r t i c u l ar pat ho l o g i c a l alterotions will be described briefly in this se c t i on , Amoredetailed description ofSP and NFTfo l l ows this section.

~ Senileplaques:

Senile plaque is the term most frequently us ed to name the le s i ons originally de s c ri b e d by Blog and Ma r ine s c o in 18 92. They are spherical areas of alteredneuropilof up to 200 micrometers in diamete r that typically consist of a cen tra lcoreof extr a ce llu la r amyloid-likematerialsurrounded byswol len nerve processes (dy s trop h i c neurite s). ttRe acti ve ceLka'' (astrocy tes, macrophages or microglial cells) are al way s includedin the plaque are a in variablenumbers.SP are dif fi cult to see in sections stained with hema toxylin and eosinor cresyl vi ole t . butea s ily demo ns t rat ed withsilve r tec hn i q ue s (Bielscho wsk y, Badian, von Braunmuh l), Congo red ,

11 thioflavi n S or immu nohistoc hemistry fo r amyloidbe t a -protein (Amer!::-an Aca d e my of Neuro logy, 198 0 ; Koo and Pr i c e, 19 93;

To mlins o n,1992).

. Neurofibrillarytangl e s :

Thes e le s ion s were described for the first:. time by Al z heimer in 1907. In light microscopic pr e para t i on s the tangl e appea r s as a thi c k en i ng and tortuos ity of fib r ils within the ne u r on a l cytoplasm. As wi t h gp, the s e lesions ar e not easy toseeinsect ion s stainedwith hematoxylinand eosin or cresylviole t. Theyare moat read ilyseeninla r g e neur o ns us i ng silver tec h n ique s, thioflavinS or antibodi es rai s e d ag a i n s t dif f erG: n t:. componen ts of paired helica l filaments (Amer i c anAcademy of Neuro logy, 1980; Koo and Pr i ce, 19 93 ; Tomlinson, 19 92).

-Gr anulovac uo lardegeneration ;

This change, original l yde scribedby Simchowicz in 1911, is largely confined tothepyrami da l cellsof the hippoc a mpus andconsists atone ormore va cuoles, 3 to 5 mic romet e rs in diameter, in the cytoplasm of the s e neuron s . Ea c h vacuol e contains a single, cen t r a l granule tha t me a sures 1 to 2 micrometers. cl us te rs of vacuoles may cau s e the neuron to bulgeand displ <'1ce thenu cle us to an ec centriclocation.The ch an g e is easilyseen with hematoxylinand eosinan din many silver imp regn at i o ns whe n the gra nule is intenselysta ined (Amer i c a n Academy of Neurology , 198 0 ; Tomlins on , lS92).

"

Accor ding to Tomlin30n and Ki t chen e r (19 72). gra nulovacuo lar deg e n era t ionis rar e l yfoundbefor etheageof 65, but afte r that it occurs increasingl y oftenin non-dement e d patient s , and bythe ninth decadeit can be identifiedin3 ou t of 4 cas es,although no moretha n 9':ofthe cells of the Sommer' s sector areaffecte d. Th i scontras t e d mar kedlywith theseries of demen t ed pa t ients in whi ch over 20\ of ce l l s in the Somme r ' s sector were commonlyinvo l ved . The fact that so me antibodies that reactwit hNFTalso immunostainedthe gr an u les (Dick so n et al., 1987) has sugg e s t ed that gr a nulo v a cuo la r degenerati on possibly repr e s en ts autop hagy of proteins of cy t o s ke l etal origin or pho sph o rylated ep itope s of such prot eins. and ther efo r e ha s some rel at i on shipwi th othe r man i f es t ationsofabnormal cy t os k e l e t a l proteins whic h oc cur in olde r in divid u a l s,ADandother neurodegenera tive di so r d e rs of laterlife.

- Hira nobodies:

Hir a no bodi escan rea dil ybe iden tified inhe ma toxy lin and eosi n st ai ne d sections. They were fi r st ident ified by Hirano and co-wo r k e rs in 1966 and tend to be brightly eosinophilicbodies, ovoi d inshape,and10 to 30 mi c rome ters in le n g th by B mi crometers They occur in intellectually normal ol d people and in AD pati ents , and alt h oug h ther e is a con s i d e r ab l e overla p be t we en them, the latt e r groupissigni f i c an t l ymoreinvo l ved.Hiranobodi e s are

1J most oft enfoundamongand usuallyadjacenttothehi ppocampal pyram ic:.al ce ll s. Ultrastructur ally ,they consistof paral l e l fila ment s (60 to 100om in le n g t h) wh i c h al ternatewi thlo nge r she e t - likematerial.Th e irlocat i o n 1nrelationtoneuron s is deb a t able, but wi th el ectr o n microscopy they oft e n see mto indent,rathe r tha ntooccupy thepr ikaryo n (Tomlins on, 19 9 2 ). Th e sizeofthefila mentsin Hiran o bodiesand thei r pos i t ive immu no re ac tions to actin and ac tin -a s soc ia ted proteins (Ga l l owa y, Peryand Gambet t i , 198 7 ) suggests tha t th e yresult froman ab normal configurati.onof microfil ame n ts·:

- Leuk o-ara i o sis :

Leuk o-ara i o sisis thetermapplied to rare fac t io nof the whitema tte r,largelype riven tri cula rindistri but i o nand well seenoncompute r ized tomogr aphy . It has been describe din both deme n t e d and non-dementedpatients'.Brunand Eng lund (19 8 6) id en tified the le sion in 60\"of ca.ses of AD ina post mort e m study, and a similar figu re {5S\} was repor t e d by Ahar on· Per et z, Cumming s andHi ll{l98 8}in aco mp ut eri zedtomography st ud y. Mi c r osc opic a lly,thelesi onischa ra c ter i2edbypar ti a l loss of mye li n,axonsandoli gode ndrogliacel lsbut with only sl i g h t as t rocytic glio sis and macrop hage in filtra tion

(To ml i n s o n,1992 ).

14 1. 2.2 ) Sen ilepla qu e s

The most'striking featureofthe cer e bralcortexin AD is the presence of SP wh i c h ma y occur in vas t numb e r s . Th e pr e s en c e of numerous neocortica l SP, alone, is consid e re d by ma ny authorstobeenou g h tomak e thepathologica l di a g n o s i s of AD,andwel l recog nized hi s t ol og i cal criteria are based on quantitat iveor semiquanti tativeassessmen t ofpl a q ue densit y and its correl ationwiththe age ofth e patient IKachaturian, 1985;Mi r r a , Hart andTe r r y , 19 93 ) , Others suggest that the diagnosis can be made only when nume r o us SP and NFTare presentinthe neocortex(To ml ins on, 1982 );however , Terryan d co-workers (1 9 B7) found tha t 30%of case sabove74 ye ars had no tanglesin the neo c orte x , and cons ide redtha.t patien t swi t h and without tan gles we r e not different on clinica l or neu rochemica l grounds.

Several va r i et ies of plaques have bee n describe d dependingon the predominance of on e or the otherof their constitutingel e me nt s .The featu re s ofthe socalle d "cl as s ic "

plaque have alread y been descr ibed . "Primiti ve" plaques consistofa small numbe r of distend ed neu r i t eswith eithe r no central amyloid fibr il s or on ly a sma ll number. The thir d typ e, th e Mburntout" plaque, consi s ts of a dens e foc us of amy loid-like ma te ri al wi t h veryfewor no de tect able ao norma f neuri t es at its pe riphery (Prob s t et aL, , 19B7; Terry and

15 Wisniewski, 1970). Electron microscopy has identified some of the swollen neurites as axonal terminals, but accordLng to recent immunocytochemical observations, many abnormal dendrites also seem tote r mi n a t e in SP (Tomlinson, 1992).

Another form of amyloid depositionalso occurs in normal individuals and in patients with AD. These lesions have received different names (diffus e plaques, pre amyloid deposits, senile plaque-like structures, very primitive plaques) and can be demonstrated using the modified Bielschowski stain, the periodic acid-methenamine silver stain , or by immunostainingwith antibodies raised against beta-amyloidprotein(the main componentof amyloidfibrils of SP and congophilic angiopathy).They are see nas ill-defined an d more or less circular areas of fine fibrillary, amorphous or granu larmate r Laj, with no swollenneurites and occasional morphologicallynormal neurons (Probst et al., 1987;Ya mag uch i et al., 1988a, 1988b).Probst et al. (1987) also re port edthe presence of a central cell (probably mi c r og lia l ) in 15 diffus e plaq ues rec on s t r u c t e d by meansof seri a l sections and suggested that this cell is an essential component of this typeof plaqueandmi gh t have a role in its pathogenesis.

Diffuse plaques, wh i ch are bel ieved to represent a precursorto SP,occur in the cerebral corte xin greatnumbe r in AD, but the ycan alsobefound in r~ g i o n swhe r e clas sic pl aques are few, such as the brain stem, basa l ga ng l ia and

16 cerebel lum (Cole et a1. . 1993 ; Joachim, Morris andSe l k o e , 19891Man n et aL, , 19 90 ; Suenagaet al., 1990; Yama zaki et al., 1992).

Ele c tro n microscopy ha s failedto demonstra t ethat this diffuse l y stainingmaterialconsists of recogni zable amyl o i d fi la me n t s.Joachim, Mor r i sand Selkoe (1989)didno t find any ultrastructural abn ormalit ythat could be cor rela t ed withthe modified Bi e l s c hows ky stained and beta-amyloid protein re a c t i v e lesions observed on lightmicroscopy.A morerecent study of cerebella r diffuse plaques (Ya maza k i et a1. , 1992) showedthat although immunoelectron microscopyde mo ns t r a t e d many immunoreactive substances in the plaque s, routi ne ele c tro n microscopy showedonlyve r y sma llamountsofamyloi d fibrilsin a few diffu s e plaques.

SP may be found in an increasingproportio n of peopl e from midd l eage onwards.particu larlyinthe anteromed ial part of the temporal lo b e including the amygdaloid nucleus. Tomlinso n, Bl essedand Roth (19 6 8 ) and Mann , TuckerandYate s (1987 )ident i f ied Sil in 50% of individualsin their 60s andin 82\ of indi v i dua l s in th eir 70s. Using anti - beta-arnyloi d an t i s e r u m in sections of the fr o n t a l lob e , t)gomori et al.

(1969) fou ndno plaque s in ind ivi d ua l s underSO ye arsof age, but 40\ of thosein their60s,44% of thos e inth ei r70s , and 78\of those in their aos showe d SP. These lower figurescould be attr i b ut a b leto us ing only se ct ionsof the fron ta l lobe,

17 because the first appearance of SP in many cases occurs in isolated areasof the bra in,especiallyinthe temporal lobe.

Mo r e recently, Arria g a d a , Marzloff and Hyman (1992 ) studied the brains of 25 non-dementedind i v i d ual s (48 to103 ye a r s , mean age 71.8 years) and found SP in 11 of them, but the degreeof SP pathology didnot correlate with age.

I t is important tomen Lion th a t plaqueco u n t s equal to thoseto u nd in AD can occurin intellectually wellpreserved old people.Katzmanet al. (1968 ) studied the brains of 137 previous re s i de n t s of a nursing facility (10 8 demen ted, 29 non-demen ted) and found that one-t h i rd of the non-demented subjects (me a nag e 66.7 ye ars ) had pathological featur es of mild AD. TheseBubj~ctsshowedplaque counts thatwe r e aotrof th o s e of demented patients wi th AD. A higher figure was reported by Crystal et al. (1988 ). They found plaque counce greaterthan 30 SP/mm2 in the frontal cortex of 6 out of 9 ncn-demenced subjects (two-thirds),althoughtheir samplewa s sma llerthan that ofKa t zma n et a L,

Ina study of 20 Frenc hcentenarians (15no n - d eme n t ed and 5 demen te d) beta-amyloid deposits were found in the pa rah ippocam p al andsuperiortemporal gyriofallth e cases, wha t ev e r the cl inical cr at.e , and the density of the le s i o n s wa s not correlated wit h the severity of th e mental dete rioration (Delaere et al., 1993). Ano the r re c e n t paper (Gi a nna kop ou l o s et aL,, 1993) reported on a series of 31

18 elde rly pa t i e n t s (7 wit h AD and 24 with no or very mild cogni tiveimpairment) ag e d from 96 to 102ye a r s . They studied the hippocampus, entorhinal cortex, superio r fr o nt a l and inferiortemporalgyri. ann observedtha t the densitiesof SP we r e comparable in both groups {demented andno n-de me n t e d ) , withno direct correlationbetweenthe number of SP and th e clinical ma n ife s t a t i o n of AD.

In advancedcases of AD, no area of the cerebralcortex may betotally spared, althoughthe concentrationsof plaques in the medial temporal cortexand the amygdaloidcomplexoften appear to be greater than elsewhe re, and the pri marymotor and somaticsensory corticalareas are scarcelyaf f ected . SPare also foun d wit h i n the majorityof subcorticalgraystructures, particular ly in the walls of th e thir d ven tr i cle , the mamillarybodies and th etegmentumof the midb ra in (Arno l det aL,, 1991 ; Esiri, Pearson and Powell,1986; Pe a r s o n et al., 1985; Toml inson, 1982,1992). Diffu s e pla q u e s or pre amyloid de p o s i t s are more wide l y distributed than classic pla qu e s throughout the brain. They can be found in the cortex, ne os t r i a t u m, globus pallidus, thalamic nu clei , br a i n stem, cerebe llar cortex , andupper spinal cor d (8ugb n i eta1., 19 8 9 ) .

Plaquescan occur in al l la y e r s ofthe cere b ral cortex, butwi t h conventiona l8i els c ho wskystaining they are commone st in the secondand thirdcortica l laye rs (Duyckae r ts et al. ,

19 1986; Pearson et al., 19 8 5l . Using Ca mpbell ' s silver technique, Braak, Braak and Kalus(1 98 9) studied the occipi tal is o co r t e x in 18 ADpatients and ob s e r v e d also a laminar distribution of SP with layers II and III being the most affected. They also pointed out that the cor t e xco v e ri n gthe depth of the sulcige:nerall yshows aLa t-qe r numberofplaques than thecortex spreading over thecr e s t of thegy ri. Bea ch and McGeer (19 92 ) analyzed the distrib u t ion of SP in the primary visualco r t e x (ar e a 17 of Brodmann !. They also found a la mi na r di str ibu t i on of the lesio n s with a si g n i f i c a n t aggregationof plaques at the interface of laye r s rvc and V, and prominent diffuse deposits within Layer-a I an d IVe. In additionto theseobservations , it has be e n reportedthat in cortical areaswhereth e accumulati onofbet a - a myloidprotei n is relative lysp a r s e , the depo si ts tend to showa col u mnar arrangement , which might be related to the columnar organizationof th e cereb ralcortex (Akiy~maet al., 1993 ).

Amy l o i d fibrils in plaques and congophilicangiopathyare comp o s e d of a 4.-Kd protein ref erred to as the beta-amyl oid protein.Ot h erco n st i t u e n t s ident if iedwithinthe amyloi d cu r e in c lud e alphal·antichymotrypsin ,sulfated glycoaaminoglycans, comp lement factors, and aluminosi lica tes. The beta-amyloid protein consists of 4.2 to 4.3 amino acid residues, with a hydrophobic region of 11 ree xduee (po s iti on s 29 to 42) that rendersth e peptideinsoluble. Molecular cl o n i n g studies have

20 determined that thebe t a-amy loidpeptide is a t runcet.ed form of a much larger pro t ei n called amyloid pr e cu r s o r protein (APP).Thearran gement.of ami noacids in the APPsugg e s t sthat it has onemembrane-spanning seg men t, a large extrace l lula r amino-terminal region. and a shorter intracellula r car boxy terminus.The beta-amyloidpeptideencompasses14 amino acids ofthe putativetransme mbranedomainand28 amino acidsof the adjacent extr ace l Iukar domain of APP (Kanget aL. , 1987; KeD and Price.1993; Selkoe, 1991; Tomlinson, 1992).

At least four speciesof APPmessengerribonucleic acid s (mRNA), encoding 563, 695, 751, and 770 ami n o ac i d s respectively, have been ide ntified. The APP-75 1 an d APP- 7 70 mo l e c u l e s are~dentical toAPP- 6 9 5 exceptthatthey contain a domaintha t shareshomology wit h the so-calledKunitztype of protease inhibitors, wh i ch could playafundamental ro l e in the generatio l1of beta -amyloidprotein (Ponteet al., 19 8 8 ; Ta n z i et al. , 1988; Tan zi , St George-H yslop and Gusella , 1989). APP- 6 9 S mRNA is expresse dpredominantl yin thene r vous system, wherea s APP-751 andAPP-77 0 t.r-arrac z-Lpte are expre s sed both in braintissue an d systemicor gans. Curre nt lylit tle: is known ab ou t the shortestAPP transcript (APP·S 6 3), which lacks the membranedomain (xoo and Pric e , 1993 ).

Recentre p o r t s have determinedthatincul turesystems a natur al clea v ag eoftheAPPmoleculeoccursnearthemi dd l eof th ebeta -amyloid domai n ,wh i c h suggests tha t normal process ing

21 of APP prec lude s the fo rma ti o n of an intac t amylo i doge nic fragment.Asa result .amylo idoge nesi spresumabl yrequi r es the absenceofthis normal cleavageeven t andmi g htbethere s ul t of an aberrant pr o c e ssing of the APP molecule (Gandy and Gr e e n g a r d , 19 9 2; Koo and Price, 1993; Mat t s on etal. , 19931 Se l ko e, 1991 ) .

The physiologi crolesof APPare not known , and neither is the part played by the beta·amyloid protein in the pathogenesisof the disease. Atrophic or autocrine property of APPhas be e nsuggested. This hypo thesi s isin accord with the discovery that the APP with the Kun i tz domain is a spe c if ic cellsec re t e d proteaseinhi bitor (pro t easene x ! n Ill.

capableof fo nn ! ng co mplexes wit h epidermal grow;:h eeeeee- bindingprote i n andthe gammasubunit of nervegrowt hfactor. In addit.ion, protease ne xin II is a potent. inhi bitor of Chymotryps i n , a fac t that could he-..e some rele va nc e , con s i d e r i ng thatpre v i o u swor-kshavesugg e s t e d the inv olv ement of a chymot rypsi n-lik e proceeee in the de po sit i on of bet .a- amy l o i d prot e in (Oltersdorf et a L., 1999 ; Va n Norstrand et al., 1989) .

Matts o n and co-workers (1 9 9 3 ) have suggested thatAPPis involv ed in ca Icrumho me o sta si s. According to the m, normal enzymatic proc esDing of APP results in the libe r at i o n of secretedforms of APP which presumably bind to and activa te specific cell sur f a c e receptors on target cella. This

22

act ivationwould cause a reduction in intracellularcalc ium le v e l s and wou l dprotect against aberrant rises that can occur when neur-one are subjected to adverse conditions (e.g., ischemia, trauma, excitotoxins). The abnormal processing of APP, then, would have two major consequences for neurons. First, the normal int r a c e l l u l a r calcium-stabilizing and neuroprotective functionofAPPis compromised.Second,beta- amyloid pept ide aggregates and further destabilizes calcium homeostas is.

Recently, Nishimoto et 811. (l9 93 ) proposed a novel possibility: APP is a neuronal receptor coupled to Go(a major GTP-binding protein in the brain) and abnormal APP-Go signalling is involved in the pathogenesis of AD. They suggested that after the proteolytic release of the beta- amyloid fragment, the residual C-t e r mi n a l portionof APP may remaininthe cell membrane and could injure neurons through constitutive stimulation of a cell signalling pathway involvingGo.

~ studies with dissociated hippocampal neurons have shown that beta-amyloidfragments seem to be neurotrophic to immature neurons but neurotoxic to mature neurons (Yankner, Duffy and Kirschner, 1990). Malouf (1 9 92 ) observed deqenez-at.Lve changes after the application of the whole beta- amyloid prote into cultured neurons, but fa':'ledto demonstrate any neurotoxicity using beta 1-28 or beta 25-35 fragments.

2'

However, Pike et al. (1993) reported neurodegenerationinduced by different beta-amyloid fragments. but their most significant result that to x i c i ty was associated specificallywiththose peptides that exhibi ted significant aggregation. Another recent ~ study (Carpenter, Crutcher and Kater, 1993)analyzedthe effectof SP on living neurons using cryostatse c t i o ns fr o m the ce r e bra l cortex of Alzheimer patients as a substratum for cultured rat hippocampal neurons. The authors conclude d th at SP ca n have deleterious ef fe ct s on neurona l organiza t ion in...!il, affecting the amount, complexi ty anddirection of neuri te outgrowthas well as cell survival .

The results of .in...Y. studies have been more contradictory. Various groups have observed no significant mo r p h o l o g i c a l difference after becavamy Lc Ld fragment injections, as comparedto vehicle control injectionsin rat or rhesus monkey brains(Ga me s et al.,1992; Podlisnyet aL,. , 1992; Stein-Behrenset al., 1992). On the other hand, there are reports of significant toxicityof beta-amyloid and the beta 25 - 3 5 fra g~ent intracereb ra lly injected in the same species (Emr e et al., 19 92 ; Kowal l et a L. , 1992).

Methodological variations could account for some of the differences, but in general it seems that the simple intraparenchy mal inj e c ti o n of beta-amyloid is not a good model for the study of Alzheimer-relatedne ur o t o x i c i ty .

24 Th eor i ginof the beta-amyl oidpeptidethat ac cumulates in SP and in the wall ofbl ood vesse lsis co ntrovers ial.Two prevailing hypotheses, no t mu tua lly excLue Lve, argue for neuronalandva sc ula r ori gins . Evidencefor a neuronal origin ha s been given by the topographicdistributi on of SP within th e gray matter; the neurona l loc al i za t io n of API' mRNA, incl u d i ng hig h le vel s of APP-69 5 ; the transport of API' in axons; the loca l i zat i on of N- and C- t e rmi nus of API' in neurites surrounding amyloid deposits ; and the finding of be ta - a myl o i d depositsad j acent to neurites inplaques (Koo and Price, 1993).

Evidencefor a vascularori gin in clude s the proximity of bloo dvessels to SP;deposits of amyl oi d inme n inge a l vessels outs ide the brai nparenchyma; thepres e nce of full -lengthAPP molecules in pla t ele t s,' the presence of serum components (e. g.,comp l eme nt facto r s )inamyl o i d;the possibleincreased permeability of the blood-brainbarrier in aging; and the presence of beta-amyloid immunoreactivity in the skin of individualswi thAD(Koo and Price, 1993 ).

These two hypotheses; however, do not exclude the possibilitythat thedeposition of amyloidin brain parenchyma and in wall of bl ood vessels may be the result of two independe ntprocesses that arise from differentcompartments of the brain (Ro z e mulle r et al., 1993 ) .

25 1.2.3 ) Neurofibrill a rytangles

Altho u g h NFTare a ma j o r histopathological hallmarkof AD, othe r conditions exhibit va ri ab l e degre es of NFT formation. These include normal agIng. aged individu a lswith Do wn' s syn drome. de ment ia p\lg i l i s tic a. th e Pa r k i nsonism- dementi acarr.plex ofGua m, poste n c e p h aliti c Parkinsoni sm ,and sub a c ut e sc l e r os ingpanence phaLftLa (Wisniewskiet. al .• 1979).

Withsilver stai n the y appe a r as fibri llary structures withinper i k a r y a, butthei r con fig u r a t i on may vary according to the sit e of the tangle, the type of neu r onaffe c t ed , and the stag e of its developmen t (Banche ret. al.• 1989;Vickers , Del acour t e and Mor r ison, 1992). In most cells with NFT the nucleuscan still be identi f i ed , but sometimes, pres uma bl yin the final stages. the cell ou t li ne disappears an d only the distortedfi b r i l sre mai n intheneuropil.giving ri s e to what have bee n called -ghost tangle s " (Koo and Pri ce, 199 3 ; Tomlin s o n, 1992).

Recently. a new type of les ion has bee n de s cr ibed in re lation to -g ho st tangles- (tang l e ·associa t e d neurit i c cl u s t er) , in which abnormal neurites formdense aggregates , each centered byan extracellular (g hos t) tiingleinstead of by an amyloid deposi t (Mun oz and Wang, 199 21 . Tangl e-associated neuritic cl us t ers were ab undantin thehippo camp us of all AD ca ses analyz ed by these autho r s (n_ 14 ) , an d a few cou ld be

26 found inth e nucleus hasalisof Meynert and occasional l yin thene oco r t e x . Similar lesions werere po r t e d byIkeda et al. (1992) in the hippocampus of AD patients und e r the term

"de gene rative ne ur i t e - be a r i ng ghost tangles ", but they were found onl y in 13 out of 30 patients (43\).

Electron microscopy has revealed that the NFT of AD consists of dense bundles of long, unb r a n c h i n g filaments th at measure approximately 20 om across, with a regular constriction to 10 om occurring every 80 nm. Wi s n i e ws ki , Na rangand Te r r y (1976) suggestedthat NF'Twere constitutedby paired filame n t wo u nd in a double helix (paired helical filaments), a view that has beenla r g e l y accepted since then.

However, although the great majority of ta ngles found in normalagingand inADconsistpredominantly of pa i red he li cal filame nt s (PHF), some aut ho r s have described also straight fila ments (Shibayama and Kitoh, 1978;Yagaohita et a L,, 19 81) and straighttubules (Gibson, Stones and Tomlinson, 1976) in NFT.

NFT occur more in the anterior fr on tal and tempo ral cortex than in the posterior partsof the hemisphe res,and the pr e - and post-central gyri and the pri ma ry sensoryareasof the cortex ar e usually little involved. Some ca ceacxtce of cells are particularly resistant and Pur k i n j e ce lls, Betz cells, and large motor neurons appe a r to esc ape almost en t irel y (Arnol d et a L. , 1991; Pearsonet a1., 1985).

27 Innormal old age andinADthe heavies t densi t yof NFT is found in the anterome d i al tempora l cortex, inc ludingthe unc a l co rtex, the cort. i comedial pa r t of the amy gd a l oi d nuc l e u s . the hippocampus and the adjacen t pa r ah i ppocampa l gyrus.Tomli n s on. Bles s e dand Roth (1968) andTomlinsonand Kitchener (197 2 )obs e rved tan gl e formationinthe s e area s in about 5'\"of norma l peo ple dyi ng in thei r 40s ; the figure increasedto50\"for peopledying intheir70s, and by the 90s all cases showe d some af fe cted neuron s. Although the r e is ag r e e me nt abo uttheincreaseintangle formatio n th a t occ urs in non-dame n t c d indiv i dua l s th ro u gho u t mi d dle and late life, qua ntitat i v e estim a tions have de mo nstrat e d a flignif i c u n t differen ce inthe number of affe cte d hippocampa l pyra mi d a l cel lsinADcompar e dwithintell e c tuallynormal per sonsofthe same age (Ball,1977; Tomli nson ,Blessedand Roth, 1970).N~'"

are alsoab un d a nt inneocor tica lareasinADpa tien ts,but in a signif icant proportion of older cas e s the y can be very scarc eor no tpre sent atal l (Braa kand Br aa k ,19 9 0a;Terry et al., 1987).

Neuronsin the subco r t i c a l re g i o ns canbe also affe c t e d in AD. NFT are common and sometImee nume r ous in the basal nucleusofMe y ne r t,the periventriculartissues along the wall of the III ventricle, the septalnuc lei , the periaqueduc tal gr a y matte r of the midbrain, the raphe nuclei and the locus coerul eu s (xccan dPrice, 1993;Toml ins on, 19 92 ) .

28 The insol ubi lityof PHF ha s frustatednumerous attempts at purification ,and identification01:thechemicalcomponent s has been difficult. Nevertheles,re c e n t evidence suggeststhat NF"I' re pr e eent sites of cy t o a kelet a l disruption.

Immun o c y t o che mi cal studieshaveid e nt i fi ed several cellular constituents within NFT, including microtubule-associated proteins (MAP2 and tau l, A6e (a 68-kd prote in, probably a hig hly modified fo rm of tau, enriched in AD brains an d cerecro-eptneIfluidandorig i n a ll y identified withth e ALZ-SO antibody), neurofilaments (pa r t i c u l a r l y phosphorylated ep.itopent, and ubiquitin (a protein impl icated in the no rr- lysosomalbreakdownofshortli v ed and abnormal proteins) (Cor k et al., 1986; Grundke-Iqbal et; al., 1986; Ko s i k et al.,1984; Ko s ik , Joachimand Selkoe, 1986; Lee et al., 1991; Perry et al., 19B7; WolozinandDa v i e s , 198 7) .

PHF are also presentin dystrophicneuritesof SP and in

"neu r o p i l t.hreads", which thickened and twisted argyrophiliccell processes dispersed in the neuropil betwee n SP and NFT. Braak and Braak (1988) and Ihara (198el have pr odu ce d evidence that neuropil threads are th e res u l t of massivedendri tic sproutingof cort ical neuronsinvolved in NFT formation, and Duyckaerts et al. (198 9 ) reported a correlation between thenumberof these ab norm al fibe rs and the severit yof de mentia.

Id e nti fi c a t i o n of neurons forming NFT prior to actu a l

29 accumu lat ion of PHFhasnot been succes sfu l. Tab atonet al. (1988) sugg e ste d tha t themonoclonal antibo dy ALZ-S O ma y well ectecc suc hneurons, since it immunostai nsneuronsbothwit h and wi t ho u t ta ngle s ,

Inthe norma l br ai n , ta uis mostabun dan t in axon e where i t is no t ph o s ph o r yl a t ed, although low levels of pho s phory l a t e d tauareal so present in the cellbodiesand dendrites . Ac cu mu l at i on of la r g er quantities ofphos phoryl a ted tau insomatodendrit iccompartments, as detectedby ant i - tau antibodies.is one ofthe earliestidentified abnor malitiesin AD andoc c ursprior to the form ationof PHF(Bancher at al., 1989). Phosphorylate d tau is les a effe c t i v e inmicrotubu le asse mb l ing ; as a result, tau may not properlyassoci ate with tubul in and fre e ta u may co -e a ee mm e into PHF (Montejo de Gar c in i et al., 1988 ).

Anoth e r pr o babl e marker is MAPS , an early fo r m of mdcrot.u buk e - u a s ocLate d proteins that is normally expr e ssed dur i n g th e deve lopmental growthof ne urons . Aberra n t MAPS expr ess i o nhas beenob serve din thehippocampusof AD patien ts (Ge d d es, Lun d gren and Kim , 1991l, but ill co ntras t to ta u a Lt ez-ataona , wh i c hare observedpr ima r i lyin neu rons wit h a pred i l e c t i o n for ADpathology, MAPSimmu n ore ac t i vi t yhas bee n obs erv e d in both vuln erable and non -v ulne ra b l e hi p po campal neu rons. It is th e r eforeuncertain whe the r thealterations in MAPSimmunorea c ti v ity prece d eoroccu r in response to the AD

'0

neuropathology.

Despite con sid e r a b l e progess made duringthe last decade in understanding the molecular pathology of NFT and the amyloid fibrils of SP , the problem of their relationship remainsunsolved.

Masters et al. (1 9 85) proposedth a t amyloidfibrils in SP and inblood vessels were related to intraneuronal PHF and that all three fibrous deposi ts were composed of the same prot ein, bu t Selkoe et al. (1 986) observed that the amyloid core of SP was no t immunosta ine d using polyclonal and monoclonal antibodies that label PHF, including anti-tau an t i b o d i e s .

For many years, a number of diseases have been ch ar ac t eri z e d by NFT formationinth e absenceof plaques. The Parkinsonism-dementiasyndromeof Guam, dementiapugilistica, subacutesclerosing panencephalitis,progressivesupranuclear palsy, and pos tencephalitic Parkinsonism are well known examples. It has been recentlyshown, however, that at least in some cases of progressive supranuclear palsy and dementia pugilist ica, diffue:~ plaques ca n be readily demonstrated (Rober ts, Allsop and Bruton, 1990; Tan et a L,, 1988). A reverse situation is observed in about 30%of ADpatients aged more tha n 74, in whom absenceof neocorticaJ tanglesin the presence of numerous SP haa been reported (Te r r y et; al., 198 7).

31 Lig ht and electron immunocytochemical analyses of ~he br ai n sof pat i e nt s withsubacu te scle rosing pane nc e phalit ie (SSPE). AD and age-matched co ntro l s (Tabaton et al .• 19 8 9 ) revealedtha t abnormal neurites LaSSPE we r e identicalto the widespread ab no rm al neurit es of AD, but they occur in the absence of amyloid deposi t s . Tt-.e y als o observed tha t the dist r i bu t i onof the ne u ri t icalt e r a t i on correla t ed with that ofNFTinbot hcondit ions , but no t lrIiththat of SP in AD. The autho rssuggest ed that theirre s ul t s arecons i s t e ntwith the hypothesistha t in AD the wi de sp rea dalter a t.i on of neurona l proce csee isnot secon dary tothe depositionofamylo id .

The report s of Probs t et al. (1988, 19a~)hav e showna different i al patter n of immunore ac t i v i t y of SP neurites depe nd i ng on the presence or ab s e n c e of NFT inthe neocortex.

tn pa tients withNFT, the ne uritesof SP wer epos itive for anti-PHF an dan t i -ta u antibodies,but in AD pat ients wit h only neocort ica l SP , the neurite s weredevoidof immuno reactive ma t e r i a l. The s efindings indi c atethatPHF and ta upolyp e pti de are not essential compone n t s of SP, suggesting tha t the de v elopmen t of SPin the neocort.exmay occur in d epe n d e nt l yof PHF pat ho logyof neoc orticalneurons.

32 1.3) Neuropsychopathologyof ca rdi ovasculardisease s

The relationship between cardiovascular diseases and their pa t h o l o g i ca1effects on the centr alnervou s system is very complex, and Borne areas of this field hav e not been studied extensively enough. The most fr e q u e n t neurological complication of cardiac disease is the cardiogenic bra in embolism, that; arises when embolic material from the heart occludes a brain artery producing stroke. Some studies (Cerebral Embolism Ta s k Force, 1986; Foulkea et;a1., 198 8 ) have shownthat about 15 % (range 6%to 23%) of all is c hemi c strokes are cardioembolic.

The listof ca rdiacdisorders capable of producingbrain emboliis extensive (Helgason and Sherman, 19 8 9 ) , but atrial fibrillation with or without associated ischemic or hype r-t.ensLve heazt; disease, acute and chronic ischemic heart disease, and valvular heart disease (both rhe uma t i c and prosthetic) are the most frequent types of heart diseases causing cardiogenicstrokes.

Res e arc hinthis ~'ieldhasbeenvery ac t iv e , partly due to the de va s t a ti n g effects that strokes have on pat ients' live.However, apartfr o m their emboliceffects, lessisknown about thecerebral res po n s e to alteredcardiac rhyt hm, car di ac isch emi a, cardiac failure, and hype rte ns ion , whi ch mayle ad to subt lepsyc ho l og i ca l changesandfocal neu r o l o g i c a l def icits.

3J Kurosawa and co-worke rs (l9S3) studieda group of 163 patient swithacutemyocard i alinfarction who wer e admi t t e d to a Coronary Care Unit during a periodof two years. andfou nd that about 64\ of all the patients showed some mental disorde rs, wh i c h we r e conside red to be mild or mod era t e in most of the caeea. An x i o ua and depressi ve st a t e s we re the ddeordera mo st fr-equenn kv found , wi t h 42 and 30 af f e ct ed pa ti e n t s, res p ec t ively . Theyal s o observed tha t about 26\ of the subjec t s were in need of psych o t r op i c med ica tion . and abo u t lot showed di s o r i en ta tion or cloudy co ncfou eaeee, ruetbermc r e,it has beenrep ort e dtha t depressi on iscommonly manife s t ed inpatie nts wi th seriouscard i a c disea s es an d has be e n no t ed in many cases to predate the diag nos i s of the cardiacabnormality (Treschet al..1985) .

Another paper (Reichet al.•1983). reporting ona series of six long-term survivors of cardiac arrest.showedthat this groupof patientsmay suffe r frommildcerebr a l dys f un c t i o n , manifested primar i l yby pers on a lity cha ng e s and beha vio ra l symptoms that can be mistaken fo r emotiona l respons e s to illne s s (dep ression in mo s t of the cases). The autho rs sugge s t e d that the presence of chronici t y, dysinhibit ion, apathy, and disturbances of judgement and insight are ind i cat ive of cerebra l dys f unc tio n.

In the las t few yea rs, centra l sys t e m comp l i c a tionsobservedincoro nary ar te r y byp assgraft (CABG)

34 surgery have attracted the at t e nt ion of various research groupe,mainlybecause CABG is the most frequentlyperformed cardiac surgery in North America and due to the fact that central system complications (s t r o ke and en c e p h a l opathy)ac c ou n t for the major adv ers e sequelaeof this procedure (Brillman,1993).

In a re t ros p e c t i v e studyof 1669 patients whosurvived thi s kind of surgery, Coff eyet ei. (l983) found a central nervous sys t emco mplica tion rate of3.at, includingaltered mental state , str oke, and siezures, with a mo r t a l i t y of approximate ly 30% in patients withneurologicalcomplications.

Ala t e r pr o s pe c t i ve analysis (Sha w et aL,, 1985) showed a complication rate as high as 61\ in the earlypostoperative period. In addit ion to altered me n t a l state, seiz ures and stroke , the authors also reported the presence of opht halmologicabnor malities, pdmitive reflexes,psychoses, depression , and some peripheral disorders, such as brachial plexopathy.

A later study of the same group (shawet al., 1987) compa redthene ur ologi c al complicationsin patientsunde r going CABG surgerywi t h agro up of matchedpatients whound erw e n t eurqc z-yfor periphera l vascu lardisease no t ass oc i ated with bypass.Theauthorsfound that the patientswho underwentCABG surgeryhada complication rate more than twi ce as hig h as thatof pa t ientswho ha dperipheralvascu l arar te r i a lsurge r y,

3'

implicating bypass as a significant.factor in the development of neurological complications.

Itseemsalso that a substant ialnumberofpa t i e n t sha v e mi no r cognitive impairmentafterCABGprocedures. 'ccordingt.o Newmanan dco-workers (1990), these abnor ma lit iesmay occurin up to75%of patientseight days postoperatively, andmaybe present in about cne-unrrd of patients even one year after surgery. psychiatric disturbances are also commonfollo wing CABGsurgery. These include agitation. del us i ons, paranoia, nightmares, sleep disturbances, depression and anxiety, most of which are associated with cognitive deficits (Hel ler and Kornfeld, 1986).

Over the years some reports have also suggested that.

hyp e r t e n s i v e pati en ts show a poorer per forma ncein various cogni t ive ,perceptual, andpsyc homo t o r tasks whe n comparedto normotensivecontrols, althoughthe defic its aresubtleand require sensitive ps yc h ological tests for detection (Battersb y et al., 1993; Bolleret a l,, 1977; Fra n c e s chi et a L,, 1982 ; Go l d ma n et aL., 1974; Miller et al., 1984). Some studiesha v e indicated tha t thedisease process itself is responsi bl efo r these behaviora l defic its(Franceschi.:!t al., 1982 ; Mil l e ret a.l . , 1984), wh il e others haveshown impaired fu nctioningwi t h antihypertensive drugs (Lasser et al., 19 8 9 ; Lichter, RichardsonandWyke , 1989).

Th e s e behav ioral chang es are accomp anie d and probably

36 relatedto a variety of morphologicalbrainchanges, bu t only in the last deca d e, with the help of more accurate quantitative methods, it has been possible to demonstrate them.

Many stud ieshave beendoneonspontane ouslyhypert ensi ve rats (SHR),compar ingthemwithag e - and s.ex-mac ched Wistar - Kyoto (WKY) rats, the normotens ive control strain for SHR.

Lehr, Browning and Myers (1980) were the first to report quant i tat ive differences in brain size between SHR and WKY rats. They studi e d a-mo nth-old animalsand found that th e thickness of the pons andoverlyingcerebellumwas greater in SHR than WRY rats, but that the lengthofthepons, midbrain and diencephalonwas les s in SHR thanWKY rats. Nelson and Boulant (1981) observed that in az-week-o Ld male SHR total brain weight and volu me were about11\' less than in control ani mals.

The stud ies conducted by Ritter and Dinh (1 986) and Ritteret al. (19 8 8 ) showed that the ventxdc le a of SHR we r e enlarged from 8 to S6 weeks of age, but this ventricul ar dilatationwas notdiminishedappreciablyby maintaininglower arterial blood pr e s s u r e by captopxil administration.

Fur t he r more,theyreportedthatthe cerebralven triclecrOBS- sect ional area wa s no r mal in spr ague-Dawl e y rats made hypertensive byre mov i ng oneki d n ey and partia llyclip pingthe renal artery on the othe r side. They suggested that

37 ventricular dilatation in hypertem::ion may be genetica lly linkedtotheelevat ionof blood pressure but not drivenby it,

Bendel andEilam (1992) assessed ventr icularvolumesJ.n

~inSHRand WKY rats usingmagneti cresonance ima g i n g. At 2.5 monthsof age, the ventricu larvolume swereid e n ti c alin the twostrains.By3to5 mo nths,me a n ventricularvolume was significa ntlylarge:.- in SHRtha n WKY rats (37.5 mm3 VB 28 mma t, andby 6toemon thsthe differ e nc ewasevengr e a te r(58 mm3 va 33 mmar•

Amo r e recent report (Ta jlma et al., 1993 ) based on a morphometric study of 6 to 7 -month- oldSHR and WKY rata showed tha t ventricular volume was twofold greater in SHR than control rats. The volumes oftheen t irebr a inandof allthe gray matter struc tures studied (s ub st antia nig ra. periaqueductalgray matter, inferior and super ior col l iculi, caudate-putamen, and thalamus), and the thickness of the frontal and temporal cor tex were 11% to 2St le s s in SHR.

Neuronal frequency was similarin the two rat strains, but having smaller brains, SHR ha d fewer neurons per brain structure than control rats.

In humans, the resultsreported so far have shown subtle but definitealterations that might accountfor some ofthe cognitive and beha viora l changes previouslydescribed, and seem to correspond withthe dataob t a i ne d from animal models.

38 A recent .i.n...tiY study (Sale r n o et al. , 1992 ) of 18 ca r efu lly selected long-te rm hypertensive patients and 17 con trol subj e cts, using magnetic resonance ima g i ng, showed eba - the hype rtensi ve patien ts had signifi cantly larger volumes of the right and left latera l ventr icles and a significantly smal l e r lef t hemispherevolume (n o r malize d to intracranial vo lume) .

The wo rkcarried ou t by a gr oupofrese a r c he r s at the Universi ty of Kentucky Medi cal Cente r (Sp arks et al.. 19 90) br oughtabou t a previ ou s ly unrecog ni zed rel at i onshi p betwe en 'cr it i c a l coro naryarte rydisease (eeAD) and thepresenceof SP in the brain. They st u d i e d th e brains (f r o n t a l pole, hippocampus and parahippocampal gyrus) of 20 non-demented subjects dy i ng as a resul t of CCAD or having CCAD as an incidenta l finding at autopsy. 16 non-demen ted, no n- he a r t di sease subjects , and 17 individuals with clinical manifestations and pat h o l og i c a l co nfi rmat ion of AD. Their resultsindicatedthat SP fo r ma t io n wasabu ndantinthe brains of patients with CCAD compared to the brai n s of control subjects . Furthermore, there was a significantly greater prevalenceof SP in theCCAD group:15 out of 20 CCADpatients had SP. but they were onlypresent in 2 out of 16 control

Amorerecentreport of the same group (Sp arks et al., 1993) corroborated these results us ing a largersample (1 04

as CCAD subjects and 91 controls). In addition to having a significant lygreater prev alenceofSPthannon-be art disease subjects, CCAD pat ients also ha d a gr e a t e r pre valence of neuropi l thr e a d s andall of the mshowedALZ-SO immunoreactive neurons in the parahippocampal gyrus. The author s sugg este d th at the sequence of plaque formation is initiatedby the presence ofALZ-SOimmunoreactive neuronsfol l owed in orderby neuropil threads, amyloid beta-protein deposition and format ionof diffuse SP.

Based on the results of Sparks and co-wor kers (1990, 19 93) , thepresent study has intended to corroborate their observations expanding the analysis to other brain are a s , in c l ud i n g subcortical structures, and co n s i d e r i n g not only pa t i e n t s withCCAD • but also hypertensivesubj ect s.

The two m&inobjectives of thiswo r k ar e : 1) To determineif the brains of patients with severe

cardiovascula r diseases(CCAD and/or hypert ension) show anysignof SPformat ion.

2) IfSP are prese n t , to knowhow theirdistribution comp ar e s withthatof ADpat ient s .

MATERIALSANDMETHODS

2.1) Descript ionof th esample

In order to acomplish th e objectives previously mentioned, the brains of 40 patients,obtained at. post -mortem examination, were studied. This sample was divided into 3 groups,accord ingto thecli nico -p a t hol og i cal findingsof the individual cases: a cardiovasculardi s e ase (CVD) group, an Alzheimer's disease (ADI group, and a control group. Al l brains wereobt a i ned from the Department of Pathologyof the Memorial universityof Newfou ndland,St.John's,Newf o undland. The CVD group inc l ude d 17 pa tients (13 males and 4 females , mean age: 70 years), the AD group comp ris ed 12 patients (4 malesandB females,mean age:80 yearu! , and the controlgroupconsistedof 11pat ients (6 males and 5fe male s , mean age: 68 year-a]. All ind ividua lswere60years of age or older.The CVD andcontrolgroups we r e similarregarding age , butAD pat ients were significant l yol der than patients of th e other two groups. Ta b l e 2.1 surr,rnarizes the descriptive cha r acte r isticsof thesample .

Wi t h the purpose of st udying the eff e c t s of diffe r ent cardiovascular problems, th e CVD group was further divided into two subgroups: pa t i ents wi thcritical corona ry artery dise ase(CCAD).and hyperten sive (HTIpatient s. Thefo r mer

41 TABLE 2.1 ;DESCRI PTIVECHARACTER IS TICSOFTHE SAMPLE

eve AD Control

17 12 11

males

"

fe ma l e s

age 70 .3 .1- 6. 9 80+/- 5. 9 68.4 .1 - 8.0

consistedof 12 patients,withan averageageof 71years, and the latterinclude d 11 patient swith a mean age of 69 years.

The descriptive dat acorresponding to the two subgroups are summarized in Table 2.2. six out of 17 patientsof the CVO group had bo t h , CCAD and HT. and thus we r e included in ooth subgroups.The remaining 11 cases comprisedsix CCADpatients andfi v eHTpa t i e nt s.

The criteria to co nformeach group wereas follows : Al zheime r's diseasegroup:

1) Pa t i ent swi t h a clinical diagnosisof dementia.

2) Pathological confirma tion of AD according to Kachaturi a n's crit eria (Kac ha t u r l a n , 198 5).

42 TABLE 2.2: DESCRIPTION OF THE SUBGROUPS OF CVD PATIENTS

males

females

age

CCA D

12

10

71.6+/-7.2

HT

11

69.6 +/- 6.9

Cardia-v asc ular diseasegroup:

1)No n -dementedpatie n t s .

2) Normal neur opathological examination (no tumor, hemo rrhage, in f a r c t , trauma, or evi de nc e of a degenerative disease l .

3) cHoic o -pathologicaldiagnos i sof CCAD and/or HT.

4) CCADwas defined as more than75%of oc clu s i o n ofthe right coronar y artery, the main trunk of the left coronary artery, the anteriorin t erve nt r i c u l a r artery or the circumf lex arte ry . Onlypatien ts in whom CCAD was th e direct ca u s e of deathorcneof the principaldiagnose swe r e included.IfCCAD was an incidental finding at autopsy, the case was not