Effects of Chronic Alcoholic Disease on M agnocellular and Parvocellular H ypothalam ic Neurons in Men

Effects of Chronic Alcoholic Disease on M agnocellular and Parvocellular H ypothalam ic Neurons in Men

E. V. S iv u k h in a1' 2 A . A . D o lz h ik o v2 lu . E. M o r o z o v3 C . F. J ir ik o w s k i1 V. G r in e v ic h4’5

Abstract

A lthough n u m e ro u s d a ta sh o w in g sev ere m o rp h o lo g ical im p a ir­

m e n t o f m a g n o c e llu la r an d p a rv o c e llu la r h y p o th a la m ic n e u ro n s d u e to ch ro n ic alcoholic c o n s u m p tio n h av e b e e n g a th e re d from a n im al e x p e rim e n ts, on ly o n e stu d y (H ard in g e t al., 1996) w as p e rfo rm e d on p o st m o rtem h u m a n b rain . This s tu d y sh o w e d a re ­ d u ctio n in th e n u m b e r o f v a so p re ssin (V P )-im m u n o reactiv e n e u ­ ro n s in th e su p ra o p tic (SON) a n d p a ra v e n tric u la r (PVN) nuclei, b u t did n o t p ro v id e an y d a ta re g a rd in g th e effect o f c h ro n ic alco ­ hol in ta k e on h u m a n p a rv o c e llu la r n e u ro n s. In o rd e r to assess w h e th e r th e c h a n g e s o b serv ed in th e a n im a l m o d el a lso o ccu r in h u m a n s a n d p ro v id e a s tru c tu ra l b asis for th e re s u lts o f clinical te s ts, w e p e rfo rm e d im m u n o h isto c h e m ic a l a n d m o rp h o m e tric an aly sis o f m a g n o c e llu la r (VP an d oxytocin, OT) a n d p a rv o c e llu ­ lar (c o rtic o tro p in -re le a sin g h o rm o n e , CRH) n e u ro n s in p o s t-m o r­

te m b rain s o f p a tie n ts afflicted w ith ch ro n ic alco h o lic d isease.

W e an aly z ed 2 6 -m a le alco h o lics a n d 22 a g e -m a tc h e d co n tro ls

d iv id ed in to tw o age g ro u p s - “y o u n g ” (< 4 0 yr) an d “o ld ” (> 4 0 yr). H y p o th alam ic sectio n s w e re sta in e d for OT, VP, an d CRH.

The a n aly sis rev ealed : 1) d e c re a s e in V P -im m u n o reac tiv ity in th e SON a n d PVN as w ell as O T -im m u n o reactiv ity in th e SON in alco h o lic p a tie n ts ; 2) in c re a se in O T -im m u n o reactiv ity in th e PVN; 3) in c re a se in C R H -im m u n o reactiv ity in p arv o cellu lar n e u ­ ro n s in th e PVN. F u rth e rm o re , th e p ro p o rtio n o f cells c o n ta in in g CRH a n d VP w a s in c re a se d in alcoholics. T hese findings in d ic a te th a t c h ro n ic alco h o l c o n s u m p tio n d o es in d e e d im p a ir th e m or­

p h o lo g y o f m a g n o c e llu la r n e u ro n s. The e n h a n c e m e n t o f CRH-im- m u n o re a c tiv ity a n d in c re a se d c o -p ro d u c tio n o f CRH a n d VP in p a rv o c e llu la r n e u ro n s m a y be d u e to a d eclin e in g lu co co rtico id p ro d u c tio n , im p lie d by th e h y p o p lasic im p a irm e n t o f a d re n a l co rte x w e o b serv ed in alcoholics d u rin g th e co u rse o f th is study.

Key words

V aso p ressin - oxy to cin • c o rtic o tro p h in -re le a s in g h o rm o n e • h u ­ m a n • alco h o lic d ise a se

Introduction M aterials and M ethods C hronic alcohol e x p o su re p ro fo u n d ly m o d ifies th e m o rp h o lo g y

a n d fu n ctio n o f c e rta in b rain s tru c tu r e s [ 1 ], in c lu d in g h y p o th a la ­ m ic n e u ro e n d o c rin e c e n te rs. D ue o f th e sid e effects o f alcohol in­

ta k e - d e h y d ra tio n a n d ch a n g e in s e ru m o sm o la rity - m any stu d ie s focus o n th e h y p o th a la m ic n e u ro h o rm o n e v aso p ressin (VP), w h ic h is sy n th e s iz e d in m a g n o c e llu la r h y p o th a la m ic n u ­ clei, su p ra o p tic (SON) an d p a ra v e n tric u la r (PVN) n uclei, a n d re ­ leased in to p e rip h e ra l b lo o d c irc u itry fro m th e p o s te rio r p itu i­

ta ry lobe. A n u m b e r o f stu d ie s in ro d e n ts d e m o n s tra te d th e in h i­

b itio n o f VP s y n th e sis by c h ro n ic alco h o l in ta k e [2 ,3 ]. In th e ra t m odel, p ro lo n g e d e th a n o l e x p o su re lead s to a selectiv e loss o f V P-ergic n e u ro n s in th e SON [4] a n d PVN [5], a n d an e le v a tio n in th e VP mRNA levels in th e re m a in in g m a g n o c e llu la r n e u ro n s [4,6]. In th e o n ly stu d y o n th e effects o f ch ro n ic alco h o l a b u se in h u m a n s, H ard in g a n d co lleag u es [7] d e m o n s tra te d a c o n sid e r­

able loss o f V P -im m u n o re a c tiv e n e u ro n s in m a g n o c e llu la r nuclei o f alcoholics. A fter p ro lo n g ed alcohol intake, p la sm a levels o f VP re m a in e d u n c h a n g e d in b o th h u m a n s [ 8 - 1 0 ] an d ro d e n ts [4], b u t in h u m a n s, VP re sp o n se to an o sm o tic c h a lle n g e w as re d u ced [8]. The effects o f alco h o l on th e seco n d p o p u la tio n o f m a g n o c e l­

lu la r n e u ro n s, w h ic h p ro d u c e o x y to cin (OT), are m u ch less know n. No c h a n g e s in OT mRNA h ave b een re p o rte d in ra ts s u b ­ je c te d to p ro lo n g ed e th a n o l e x p o su re [5]. In c o n tra st, p la s m a lev­

els o f OT are e le v a te d in h u m a n alcoholics [10]. P o st-m o rte m m orp h o lo g ical a n aly sis, how ever, w as n o t p e rfo rm ed .

E thanol also has a n effect o n p a rv o c e llu la r h y p o th a la m ic n e u ­ rons. Of p a rtic u la r in te re s t a re th o s e th a t p ro d u c e c o rtic o tro p in - re leasin g h o rm o n e (CRH) [11,12]. As a c e n tra l n e u ro h o rm o n e of th e h y p o th a la m ic -p itu ita ry -a d re n a l (HPA) axis, CRH trig g ers a d re n o c o rtic o tro p ic h o rm o n e (ACTH) a n d g lu co co rtico id se c re ­ tion in re s p o n se to a v a rie ty o f a c u te a n d c h ro n ic stim u li [13].

CRH is c o -p ro d u c e d w ith VP, w h ic h p o te n tia te s effects o f CRH on p itu ita ry c o rtic o tro p h s, e sp ecially u n d e r c h ro n ic s tre ss p a ra ­ digm s [ 1 4 - 1 6 ]. P rolonged alcohol in ta k e in ra ts b lu n ts th e ex­

p ressio n o f b o th CRH a n d VP mRNAs in th e p a rv o c e llu la r PVN [17]. In actively d rin k in g m en , lo w e r basal p la s m a levels o f stre ss h o rm o n e s w ere d e te c te d w h e n c o m p a re d to n o n -alco h o lics, w h ile alcoholics sh o w e d a b lu n te d ACTH re s p o n se in th e CRH stim u la tio n te s t [18]. H ow ever, th e re is no e v id e n c e th a t e th a n o l in d u ces d e a th o r d a m a g e in CRH n e u ro n s [17].

In o rd e r to p rovide a s tru c tu ra l b asis for th e re s u lts o f clinical te s ts a n d to c o m p a re a n im a l m o d e ls w ith c h an g es in h u m a n s, w e p e rfo rm ed a m o rp h o lo g ical an aly sis o f m a g n o c e llu la r (VP an d OT) an d p a rv o c e llu la r (CRH) n e u ro n s in p o s t-m o rte m b rain s o f p a tie n ts afflicted w ith ch ro n ic alcoholic d isease. W e stu d ie d VP- a n d O T -im m u n o reactiv ity in m a g n o c e llu la r n e u ro n s from th e h y p o th a la m ic nuclei a n d in th e p o s te rio r p itu ita ry . To c h a ra c ­ te riz e th e activ ity o f th e HPA axis in alcoholics, w e a n a ly z e d th e c o n te n t o f C R H -im m u n o reactiv e p ro d u c t a n d its c o -lo calizatio n w ith VP in th e h y p o th a la m ic PVN, as w ell as th e m o rp h o lo g y o f th e a d re n a l gland.

Tissue collection

Brains fro m p a tie n ts th a t h ad given th e ir in fo rm ed c o n se n t for b rain a u to p s y a n d th e u se o f b rain tissu e an d m edical files for re­

se a rc h p u rp o s e s w e re o b ta in e d a t a u to p s y from th e D e p a rtm e n t o f Pathology, E m ergency H ospital, Kursk, Russia. The stu d y w as a p p ro v e d by th e Local Ethics C o m m itte e o f th e K ursk S tate M ed­

ical U n iv ersity Council. B rains from 26 m a le p a tie n ts th a t had d ied o f alcoholic d is e a se (alco h o lic e n c e p h a lo p a th y ) ran g in g in ag e fro m 25 to 6 4 y e a rs (41.6 ± 2.2) w e re o b ta in e d a t au to p sy . In all cases, alco h o lic d is e a se w as v erified by several pathological crite ria - liver fa t d e g e n e ra tio n , h e p a to c irrh o sis, alcoholic c ard i­

o m y o p a th y a n d e n c e p h a lo p a th y (fibrosis o f pia m ater, a tro p h ic ch a n g e s o f a b ra in m a tte r, in ju ries o f c e re b ra l blood vessels w ith an in crease in th e ir p e n e tra tio n an d d ia p e d e sis; [19,20]). Pa­

tie n ts d ie d o f b ra in e d e m a , a c u te h e a rt failu re o r re sp ira to ry fail­

ure. R etro sp ectiv e an aly sis o f th e p a tie n ts ’ d rin k in g h isto ry re ­ veale d a n alco h o lic b in g e o f th re e w eek s to six m o n th s in d u ra ­ tion, w h ic h e n d e d in a d m issio n to th e h o sp ital on e to th re e days p rio r to d e a th .

The co n tro l g ro u p c o n ta in e d sa m p le s o f bra in s from 22 m ale in­

d iv id u als (m e a n age: 5 3 .4 ± 3 .9 ) w ith o u t alcoholic p a th o lo g y or p rim a ry n eu ro lo g ical d iseases; th e s e p a tie n ts h ad d ied o f n o n -a l­

co h o l-re la te d ca u se s - a c u te b ra in stro k e (6), a c u te o r re c u rre n t m y o card ial in farctio n (5), d is e a se s o f th e re s p ira to ry sy stem (3), a c u te su rg ical p a th o lo g y (2), a n d o th e rs (6). P o stm o rte m tim e in all cases w as 4 - 1 2 h o u rs. T he co n tro l a n d th e alcoholic p a tie n ts w e re d iv id ed in to tw o g ro u p s: y o u n g e r th a n 4 0 yrs (30 ± 2.8 an d 32 ± 1.3 c o rre sp o n d in g ly ) te r m e d th e “y o u n g " g ro u p an d o ld er th a n 4 0 yrs ( 5 9 .4 ± 2 .7 a n d 4 9 .9 ± 1 .9 co rre sp o n d in g ly ) te rm e d th e “o ld ” g ro u p .

H y p o th alam i w e re d is se c te d from th e o p tic ch iasm to th e p o ste ­ rio r su rfa c e o f th e m a m m illa ry b o d ies a n d fixed by im m ersio n in 4% p a ra fo rm a ld e h y d e (PFA) in p h o s p h a te -b u ffe re d salin e (PBS, pH 7.4) a t ro o m te m p e ra tu re for a b o u t tw o m o n th s. The p itu i- ta rie s w e re d isse c te d an d fixed sep arately . In so m e cases (n = 5, y o u n g alco h o lic g ro u p ; n = 3, old alcoholic g ro u p ; n = 8, age- m a tc h e d c o n tro ls) th e left an d rig h t a d re n a l g lan d s w ere co llect­

ed from each a u to p s y an d fixed in 4% sa lin e -b u ffe re d PFA for a b o u t tw o m o n th s. All tis s u e sa m p le s w e re d e h y d ra te d in graded e th a n o l, e m b e d d e d in p araffin a n d c u t in to serial 6 urn sections.

H y p o th alam i w e re se c tio n e d serially in coro n al p la n e from pre- c h ia s m a tic p a rt to m a m m illa ry bodies. O ne series o f sectio n s w as s ta in e d w ith h e m a to x y lin an d eo sin fo re v a lu a tio n o f ch a n g ­ es in h y p o th a la m ic n e u ro n s in d u ced by ch ro n ic alcohol c o n ­ su m p tio n . C o n secu tiv e u n s ta in e d se c tio n s w e re u sed for fu rth e r im m u n o h isto c h e m ic a l stain in g . P itu ita rie s w e re sectio n ed in h o riz o n ta l p la n e fro m th e to p to b o tto m . Sections o f th e m id d le p a rt o f th e p itu ita ry c o n ta in in g th e p o s te rio r lobe w e re selected for im m u n o h isto c h e m is try . A drenal g la n d s w e re se c tio n e d in fro n tal plan e. S ections c o n ta in in g w e ll-a p p e a re d m e d u lla w ere s ta in e d w ith h e m a to x y lin an d eo sin for an aly sis o f m o rp h o lo g i­

cal c h a n g e s in alco h o lic p a tie n ts .

H em atoxylin and Eosin Stain in g

Sections of h y p o th a l a m i a n d ad re n a l g la n d s w e r e deparaffinized in xylene a n d re h y d r a te d t h r o u g h a g ra d e d e th a n o l series. The sections w e re th a n i m m e r s e d in Ehrlich's h em a t o x y li n for 10 min, dif ferentia ted in 70% e th a n o l and rinse d in distilled water.

After that, the sectio ns w e r e c o u n te r s ta in e d in alcoholic eosin solution c o n ta in in g 1 % eo sin for 3 min. Following w a s h in g u n d e r ru n n i n g w ate r, sections w e r e d e h y d ra t e d , cle are d in xy le n e and cover-slipped.

Im m u noh istoch em istry

Consecutive sectio ns of pitu ita rie s a n d h y p o th a l a m i c o n ta in in g th e d orsola te ral p a r t of th e SON a n d PVN fr om each s u b ject w e re stain ed w ith a n ti s e r a to VP, OT, o r CRH. After deparaffin iza- tion a nd rehydration, slides w e r e p re i n c u b a t e d for a n h o u r a t ro om t e m p e r a t u r e (RT) in 1% Triton X 1 0 0 - p h o s p h a te buffered saline c o n ta in in g 5% n o rm a l g o a t s e r u m (PBS-NGS). O vernight in cubation w ith o n e of t h e p ri m a ry r a b b i t polyclonal a n tib o d ie s - anti-VP or anti-OT (C hem icon In tern atio n al, CA; 1 :1 ,0 0 0 ) , or anti-CRH (Peninsula Laboratories Inc., Division o f Bachem , CA, USA; 1 : 1 ,0 0 0 ) in w o rk in g solution (1 % NGS, 0.3% Triton on PBS, pH 7.4) w as p e rf o rm e d in h u m i d c h a m b e r s a t 4 C. After that, sec­

tions w e r e w a s h e d in PBS for an h o u r a n d in c u b a t e d in w o rk in g solution w ith biotinylated g o a t a n ti - r a b b it IgG (V ector Elite kit, Vector Laboratories, Inc., Burlingam e, CA; 1 : 5 0 0 ) for tw o hours at RT. After w a s h in g in PBS for a n hour, ABC c o m p le x (V ector La­

boratories, CA) in PBS w as ap p li e d (1 h, RT). Finally, after w a s h in g in PBS, th e reaction w as v is ualized w i t h freshly p re p a r e d glu- c o s e - o x id a s e - d ia m in o b e n z id i n e (GOD-DAB) as d e sc r ib e d by Za- borsky a n d H e im e r [21 ]. After r in sin g in distilled water, sectio ns w e re d e h y d ra t e d t h r o u g h a sc e n d i n g c o n c e n t r a ti o n s of eth anol, cleared in xyle ne a nd m o u n t e d w i t h e m b e d d i n g m e d i u m (OTC, G ermany). For co ntrol in c u b a t io n s w e u s e d e i t h e r n o rm a l rab b it serum , d ilu ted 1 :1,0 00 in PBS-Triton in s te a d of t h e p ri m a ry a n t i ­ bodies or anti-OT (anti-VP or anti-CRH), p r e a b s o r b e d w it h s y n ­ th etic OT (VP or CRH) (Sigma).

Double i m m u n o s ta i n in g for CRH a n d VP w as p e rf o r m e d as p re ­ viously describ ed [22]. Briefly, sections w e r e in c u b a t e d w it h th e rabbit polyclonal anti-CRH o v e rn i g h t a t 4 C. After that, bio ­ tinylated g o at a n ti - r a b b it IgG w a s appli ed follo w ed by avidin- ho rs erad is h p eroxidase c o m p l e x (V ector Laboratories, 1 :1 0 0 ) . Im m u n o p re c ip it a te s w e re vis ualized by DAB (Sigma). Next, sec­

tions w e re tr e a te d for 15 m i n in 0.1 M glycine bu ff er a t pH 3.4 in o rd e r to re m o v e t h e i m m u n o c o m p le x e s . S ubsequently , sectio ns w ere i n c u b a t e d o v e rn ig h t in th e ra b b i t polyclonal anti-VP and d evelo pe d by Cy3 -lab el ed g o at a n ti - r a b b it Fab f r a g m e n t (Diano- va) at 1 :2 0 0 . After w ashin g, sections w e r e m o u n t e d o n to glass slides in th e m i x tu r e of ve ro nal-g lycero l (2 :8). W e c o u n t e d th e total n u m b e r of CRH-positive n e u r o n s a n d th e n u m b e r of CRH n e u ro n s in th e V P - im m u n o p r o d u c t fr om t h e PVN. T h en th e p e r­

c en ta g e of single -labele d a n d d o u b le - la b e l e d CRH-positive cells w as calculated. Sections w e r e e x a m i n e d w it h an O ly m p u s BX50 m icroscope. An O lym pus DP10 digital c a m e r a w ith DP-Soft 3.0 softw are w as used for m i c rophotography.

Q uantification o f im m u n o reactivity in neurons o f h ypo th alam ic nuclei

Areas a n d s p e c t r u m of th e optical d e n s i ty of DAB-immunoreac- tive p ro d u c t s in t h e n e u r o n s a n d p o s t e rio r p itu ita ry lobe w ere defined in im m u n o c y t o c h e m i c a ll y s tain ed VP, OT, or CRH sec­

tions w ith th e ImageTool (U niversity of Texas Health Science Center, USA) a n d ImageJ (NIH) softw are packages. Four sections of each s a m p le t h a t w e r e s tain ed e it h e r w ith VP, OT or CRH a n ti­

bodie s w e r e p ro c e s se d for s e m iq u a n t it a tiv e analysis. Correlation o f m e a s u r e d are a (in pixels) a n d avera ge d e n sity of th e gray level w e r e calc u lated (in a r b i tr a r y units, a.u.) for an e s t im a ti o n of the relative optical d e n s i ty of i m m u n o r e a c ti v e pro ducts. On t h e sec­

tions s ta in e d for VP, OT or CRH, a reas o f th e cell s o m a ta a nd th e nuclei of th e n e u r o n s w e r e also m e a s u r e d using ImageTool. After that, a b s o l u te a n d relative (in %) are a s of im m u n o r e a c ti v e p ro ­ d u c ts w e r e calc ulated.

Statistics

The d is tin c tio n s in th e m e a n all m e a s u r e d p a r a m e te r s b e tw e e n t h e subjects as a w h o le a n d a m o n g th e dif ferent ag e gro ups w e r e te s te d using MS Excel 7.0. f-test w a s used for statistical a- nalysis; p < 0 . 0 5 w a s c o n sid e re d significant. Data are p re s e n te d as t h e m e a n ± SE of valu es as in dicated.

Results

Effects o f ch ron ic alcoh olic d isease on m agn o cellu lar neurons o f th e SON and PVN

Histological e x a m i n a ti o n o f t h e SON a n d PVN revealed pyknosis, cytokar yoly sis (Fig. 1 a) an d p ericellula r e d e m a (Fig. 1 b) in a frac­

tion of m a g n o c e llu la r n e urons .

After i m m u n o h i s t o c h e m ic a l staining for OT a nd VP, sim ilar cellu­

lar d is tri b u ti o n w it h i n th e SON and PVN w as o b se r v e d in both control and alcoholic g roups. The largest p a rt of t h e SON - the dor­

solate ra l ( p r e c h ia sm a tic ) p a r t - c o n ta in e d p re d o m i n a n t l y VP-po­

sitive n e u r o n s (F ig.l c); o nly fe w OT-positive n e u r o n s w e r e pres­

e n t in th e cap of this p a r t (Fig. 1 e). In th e PVN, t h e p ro p o r tio n of OT a n d V P -im m u n o re a c tiv e ne u r o n s w as relatively eq ual (Fig. 1 g, i).

In controls, a cle ar re d u c t io n of VP- and OT- im m u n o r e a c ti v it y in t h e SON a nd PVN w a s fo u n d in pa ti e n ts in th e old g ro u p (Table 1).

Analysis of th e SON in t h e y o u n g alcoholic g ro u p revealed no c h a n g e s in OT a nd VP i m m u n o r e a c ti v it y (Table 1). However, p ro ­ found re d u c t io n of OT a nd VP-positive profiles w as observ ed in t h e SON of t h e old g ro u p (Fig.l d, f; Table 1). In th e PVN from al­

coholics, V P -i m m u n o p r o d u c t w as significantly re d u c e d in both ag e g ro u p s (Fig. 1 h; Table 1). In contrast, p ro n o u n c e d increase in OT i m m u n o r e a c ti v it y w a s observ ed in e ld e r p a ti e n ts (Fig.l j; Ta­

ble 1).

Effects o f ch ron ic alcoh olic d isease on th e p osterior pituitary Relatively eq ual a n d m o d e r a t e d is trib u tio n of VP a n d OT i m m u n o - p ro d u c t s w a s o b s e r v e d in controls (Fig. 2 a , c), w ith no difference in th e y o u n g a n d old g ro u p s in optic im m u n o r e a c ti v it y density (Fig.2 e). In alcoholics, irregula r d is tri b u ti o n of VP signal w ith

“l u m p - l ik e ” a c c u m u la ti o n s in te r m i n a ls occu rred in bo th gro ups (Fig. 2b ). Genera l re d u c t io n of optic d e n s i ty of V P -im m u n o reac - tivity w as found in t h e y o u n g g ro u p (Fig. 2 e). In spite of this, OT

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f ” ’ * ^ <*■ % A n e u ro n w ith p e ric e llu la r e d e m a (a rro w ) is

lo c a te d n e a r n o rm a l-s h a p e d n e u ro n s in th e

» * • * * • « W *" X . » sam e n u cle us. S ta in in g w ith h e m a to x y lin -e o -

sin. Scale bars = 50 p m (a, b). c - f T he SON o f c o n tro l (C, E) a n d a lc o h o lic (D, F) p a tie n ts . Th e VP (D) an d OT (F) signals are u b iq u ito u s ­ ly de cre a se d in th e 4 7 -y e a r-o ld a lc o h o lic pa- : * ^ v t ie n t c o m p a re d t o th e 5 1 -y e a r c o n tro l, g - j

^ m A x ’ Th e PVN o f a 5 1 -y e a r o ld c o n tro l (g, i) and a

t m f f s * m . ' •% ,r B r i . '■<* 4 7 -y e a r o ld a lc o h o lic ( h , j ) p a tie n ts . W hereas

^ » *>J* . * •- I S " " ' * '‘1 • V P -im m u n o s ta in in g in th e a lc o h o lic (h) is

- V " X * ■ "*** ' - \ • "* % j ’ w e a k e r th a n in th e c o n tro l (g), O T -p o sitive

— «*» ^ “ ■ } : ' r , • " p cells are h e a v ily s ta in e d in th e a lc o h o lic (j).

• * * '* ' — •' f * - V Im m u n o h is to c h e m ic a l s ta in in g w ith an ti-V P

’j g . '■ , f ,, 4 . J . . ■' * t. ‘ o r a n ti-O T a n tib o d ie s , d e v e lo p e d by G O D -

0

: )AB m e th o d . Scale b a rs = lO O ^ m ( c - j ) .

(t

■»

V v y ; •

e -■ f . P s ,r .. < . v

r- - m « ;< k ;t

1 ( v J ; -f ^ ^ # - • />

■ *■„ » , -:= • ^ - * 'if * ^

4

9 “ • ' : f • -=J£ . • ' > « ■ '

. - i . " I » * ' r ’ » # !: v ’ ' • • ^ ' ■*

/ ■ • ; y - ■ ■< .

I * , - ' ' ■ ' * ’ T ’ " ‘

» f r . ^ . . , • * - . ' • -

c o n te n t re m a in e d u n c h a n g e d (Fig. 2e), b u t “co ars e-lik e” d i s t r i b u ­ tion of O T-im m unoreactiv ity w as o ften ob se r v e d (Fig. 2d).

CRH neurons o f the PVN in alcoh olics

Small a nd m e d i u m - s iz e d CRH-positive n e u r o n s w e r e located along th e third v entr ic le in t h e PVN fr om non-alcoholi c p a ti e n ts (Fig. 3a ). The in te n sity of CRH-staining varied fr om p o o r to very strong; w eakly an d m o d e r a te l y s tain ed cells pre vaile d (Fig. 3b).

In controls, th e n u m b e r of CRH-positive n e u r o n s an d t h e in t e n s i­

ty of th e signal w e r e clearly in creased in e ld e r p a ti e n ts (Table 1).

In alcoholics, th e p o p u la t io n o f CRH-positive n e u r o n s in t h e PVN was m u c h larger in b o th a g e - m a t c h e d g ro u p s (Fig. 3 c). At th e

s a m e tim e, CRH-positive perik ary a a nd pro cesses w e re heavily st a in e d (Fig.3 d ) a n d c ellula r profiles w e r e e nlarged (Table 1).

W e p e rf o rm e d d o u b l e - i m m u n o s t a i n i n g to ev a lu a te co-expres- sion of CRH a n d VP in th e s a m e p arvocellular n e urons . In c o n ­ trols, V P -im m u n o re a c tiv ity w a s found in 11.2 ± 1.74% CRH-posi- tive n e u r o n s fr om y o u n g p a ti e n ts (Fig. 3 e , f) and in 13.21 ±1.65% CRH-positive n e u r o n s in th e old gro up. In alco­

holics, t h e pro p o r tio n of d o u b le - la b e l e d n e u r o n s w as sign ificant­

ly in creased in b o th ag e g roups; 18.38 ± 3.54% in y o u n g p atien ts ( p < 0 . 0 5 ; F ig .3g , h) a n d to 19.4 ±2.41% in e ld e r p atien ts (p < 0.05).

Table 1 R elative areas (in %) o f v a so p re ssin (VP )-, o x y to c in (O T )- an d c o r tic o tro p in -r e le a s in g h o rm o n e (C RH)- im m u n o r e a c tiv e p ro d u c ts in n e u ro n s o f t h e s u p ra o p tic (S O N ) an d p a ra v e n tric u la r (P V N ) n u cle i in a lc o h o lic s

Nucleus/neurohormone Age < 40 yr

Control group Alcoholics

Age > 40 yr

Control group Alcoholics

SON

VP 81.54 ± 0 .6 9 81.55 ± 0 .4 5 78.52 ± 0 .5 4 b 77.93 ± 0 .4 5 b

OT 77.46 + 0.61 76.54 + 0.65 75.09 ± 0 .7 2 b 73.37 ± 0 .7 6 a’ b

PVN

VP 8 2 .9 9 ± 0 .6 7 80.54 ± 0 .6 4 a 81.13 ± 0.61b 78.27 ± 0 .6 7 a' b

o r 78.66 ± 0 .7 6 79.18 + 0.56 74.14 ± 0 .8 7 b 76.31 ± 0 .6 8 a' b

CRH 66.28 ±0.41 73.85 ± 0 .4 1 a 68.31 ± 0 .3 7 b 69.23 ± 0 .3 2 a' b

a p < 0.05 in the same age g ro u p ;b p < 0.05 between different age groups.

Fig. 2 VP- an d O T -im m u n o re a c tiv ity in th e p o s te rio r p itu it a r y in a lco h o lic s , a - d The p o s te rio r p itu ita r y o f a 4 2 -y e a r o ld c o n tro l

(a, c) and a 4 7 -y e a r o ld a lc o h o lic (b, d) p a ­ tie n ts . R e la tiv e ly eq ual and m o d e ra te d is tr i­

b u tio n o f VP- (a) a n d OT- (c) im m u n o r e a c tiv ­ it y is o b s e rv e d in th e c o n tro l p a tie n ts . In th e a lc o h o lic p a tie n t, VP- (b) and OT- (d) im m u ­ n o re a c tiv ity is d is tr ib u te d irre g u la rly , w ith an a c c u m u la tio n in “ c o a rs e -g ra in ” o r “ lu m p ­ lik e ” (b, a rro w s ) axonal te rm in a ls . Im m u n o - h is to c h e m ic a l s ta in in g w ith a n ti-V P and a n ti-O T a n tib o d ie s , d e v e lo p e d by G O D -D A B m e th o d . Scale b a r fo r all panels = 1 0 0 p m . e

M e an o f re la tiv e o p tic a l d e n s ity (O D ) o f im m u n o re a c tiv e p ro d u c ts in p o s te rio r p itu i- ta rie s o f c o n tro l and a lc o h o lic p a tie n ts . S ig­

n ific a n t re d u c tio n o f OD fo r VP (P < 0 .0 5 ) oc­

c u rre d in th e e ld e r a lc o h o lic g ro u p .

- MJ00

3

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>.

i CO 6200

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c 5N00

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e

Effects o f chron ic alcoh olic d isease on th e ad ren al gland In alcoholic patients , a d r a m a ti c re d u c t io n in t h e w i d t h of a d r e ­ nal co rtex w ith a lt e ra t io n in z o n a ti o n w a s ob se r v e d (Fig. 4c). De­

tailed ex a m i n a ti o n revealed pyknosis (Fig. 4d , e) and vacuola ted d ystro p h y (F ig .4 f) of co rtic ocytes in z o n a fasciculata a n d zona glo m eru losa .

D iscussion

In a g r e e m e n t w ith H ardin g a n d co lleagues [7], w h o re porte d n e u ro n a l d e a t h in th e SON a n d PVN, w e fo und signs of neuro nal d e g e n e r a t io n - pyknosis, cytokaryolysis, a nd pericellula r ed e m a - in th o s e b ra in regions. N euronal loss o f VP a n d OT-ergic n e u ­ ro ns w i t h c o m p e n s a t o r y u p - r e g u la t io n of VP a nd OT mRNAs ex­

pres sion in surviv in g n e u r o n s w a s also re p o r te d in anim al m od-

< 40 Vi's old > 411 yrs old < 4 0 \rs old > 4 0 yrs old

VP OT

. . Fig. 3 C R H -im m u n o re a c tiv ity in p a rv o c e llu ­ la r n e u ro n s o f th e PVN in c o n tro l and a lc o h o l­

ic p a tie n ts , a, b C R H -p o sitiv e n e u ro n s in a 4 2 -y e a r-o ld c o n tro l p a tie n t, a t s m a ll (a) and

0 b ig (b) m a g n ific a tio n s , c, d In te n s iv e ly

) f t s ta in e d C R H -p o s itive n e u ro n s and th e ir p ro -

s ' - .. ,, / C? . y cesses in th e PVN o f a 4 7 -y e a r-o ld a lc o h o lic

. \ p a tie n t, a t s m a ll (c) an d b ig (d) m a g n ific a -

# tio n s . Im m u n o h is to c h e m ic a l s ta in in g w ith ' _ > 1f p an ti-C R H a n tib o d ie s , d e v e lo p e d by C O D -

t DAB m e th o d . Scale bars = 1 0 0 |im (a, c),

5 0 ) im (b, d). e , f D o u b le im m u n o s ta in in g fo r CRH (e ) an d VP (f) in th e h y p o th a la m ic

V ^ m M i, PVN o f th e 4 0 -y e a r-o ld c o n tro l. A rro w s in d i­

c a te s in g u la r cells c o n ta in in g b o th CRH- and V P -im m u n o re a c tiv itie s . S in g le -la b e lle d ne u-

j b ro n s are in d ic a te d by a rro w h e a d s , g, h A n in-

_ .< cre ased n u m b e r o f cells s h o w e d co lo c a liz e d CRH- (g) an d VP- (h) im m u n o s ta in in g in th e

s ⁵

s ta in in g w ith an ti-C R H a n tib o d ie s d e v e lo p e d

j > b y C O D -D A B m e th o d a n d w ith a n ti-V P a n ti-

j » ' b o d ie s v is u a lise d w ith flu o re s c e n t (CY3) sec-

^ J L o n d a ry a n tib o d ie s . Scale b a r = 50 (.im (e-h ).

■ 7

V

f '

? ’ ’ * ' / ' %

r *.«*3

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9 .

V. '■

els [4]. W e o b serv ed s u b s ta n tia l d e c re a se in VP im m u n o re a c tiv ­ ity in n e u ro n s o f th e h y p o th a la m ic nu clei an d a c c u m u la tio n of V P -im m u n o p ro d u c t in “c o a rs e -g ra in e d ” a n d “lu m p -lik e ” s tru c ­ tu re s in th e p o s te rio r p itu ita ry . T h ese o b se rv a tio n s, su g g e stin g in h ib itio n o f VP sy n th e sis a n d im p a ir m e n t o f VP release, a re in line w ith th e b lu n te d re s p o n se o f VP to o sm o tic c h allen g es in a l­

coholic p a tie n ts [8]. S im ilar c h a n g e s h ave b e e n fo u n d in OT-ergic p o p u la tio n o f th e SON. H ow ever, c le a r e le v a tio n o f O T -im m u n o ­ reactiv ity w as fo u n d in th e PVN, e sp ecially in e ld e r p a tie n ts . Ac­

tiv atio n o f OT sy n th e sis in th e PVN m ay c o n trib u te to ab n o rm a l

elev a tio n o f p la sm a OT in alco h o lics d u rin g p erio d s o f a b stin e n c e la stin g 4 to 28 d ays [10]. The a b n o rm a l in crease in OT m ay be d u e to alco h o l w ith d ra w a l, as OT is ty p ically released in resp o n se to a c u te s tre s s p a ra d ig m s [ 2 3 - 2 5 ] . H ow ever, it is u n lik ely th a t th e OT re b o u n d w o u ld la st a w h o le m o n th . E nhanced OT p ro d u c tio n m ay c o n tr ib u te to c o m p e n s a tio n o f w a te r loss [2 6 ,2 7 ] an d d e v e l­

o p m e n t o f alcohol to le ra n c e [28].

A ccording to clinical stu d ie s, ch ro n ic alcohol c o n su m p tio n s u p ­ p re s se s c o rtiso l p la sm a levels [18], w h ic h is c o n s is te n t w ith th e

Fig. 4 M o rp h o lo g y o f th e a d rena l gla n d o fa 5 2 -y e a r-o ld c o n tro l and a 4 8 -y e a r-o ld a lc o ­ h o lic . a The a d re n a l g la n d o f a c o n tro l pa­

tie n t. C = c o rte x ; g = zona g lo m e ru lo s a ; f = zona fa s c ic u la ta ; r = zona re tic u la ris ; M = m e d u lla , b M a g n ifie d fr a g m e n t o f a. c - f Pa- th o m o r p h o lo g ic a l cha nges in th e adrenal c o r te x in th e a lc o h o lic : A re d u c tio n o f th e w id th (< 1 m m ) o f a d rena l c o rte x (c), an a l­

te r a tio n in th e z o n a tio n o f th e a d rena l c o rte x (d), p y k n o tic (a rro w s ) and v a c u o la r changes in c o r tic o c y te s in zona g lo m e ru lo s a and zon a fa s c ic u la ta (e), a v a c u o la te d d y s tro p h y (a rro w s ) o f c o rtic o c y te s in zona fa sc ic u la ta (f). S ta in in g w ith h e m a to x y lin -e o s in . Scale bars = 5 0 0 |am (a, c), 100 urn (b, d), 5 0 |im

e, f).

ad ren al cortex hypopla sia o b se r v e d by us a nd re p o r te d by o th e r groups in h u m a n s a n d an im a l m o d e l s [ 2 9 - 3 1 ] . Interestingly, w h ile prolonged e th a n o l intake lead s to d e c re a s e in CRH mRNA levels in parvocellu lar n e u r o n s in rats [ 17,32], w e o b se r v e d a p ro ­ found increase in CRH im m u n o r e a c ti v it y in parv o cellu lar n e u ­ rons fr om th e PVN o f alcoholic pa tie n ts . This increase m a y be ex­

plained by several m e c h a n is m s . First, low levels of circula tin g glu cocorticoids m a y to nically u p r e g u l a te CRH e xpression; sec­

ond, e th a n o l w ith d r a w a l (th e p a ti e n ts w e r e a d m i t t e d to ho sp ita l after lo n g - te rm drinkin g) m a y e x ag g erate CRH p r o d u c t io n and release [ 3 3 - 3 5 ] ; a n d th ird, d e p re s s io n ( 2 5 - 5 0 % o f actively drinkin g alcoholics are d e p r e s s e d [36]) m a y activate CRH s y n ­ thes is [ 3 7 - 4 0 ] .

Co-expres sion of CRH a nd VP in parvocellular n e u ro n s of th e PVN is w e ll - d o c u m e n t e d in ro d e n t s ] 13] a n d re p o r te d in h u m a n s [41,42]. Chronic alcohol c o n s u m p t i o n a n d alcohol w ith d r a w a l do n o t affect VP mRNA levels in parvocellu lar n e u r o n s in ro d e n t s [17]. In alcoholic patients, w e found a n increase in t h e n u m b e r of dou b le-lab eled cells in both , t h e y o u n g a n d th e old g ro ups. Upre- g ulation of VP ex p res sio n usually occurs in s itu a tio n s of insuffi­

cient feed-back of glucocorticoid signaling, su ch as a ft e r a d r e n a ­ le ctom y or prolonged stress [ 1 4 - 1 6 ] . Hyp opla sia of ad re n a l cor­

tex in chronic alcoholics o b se r v e d in o u r s t u d y sugges ts t h a t si­

lencing of n egative glucocorticoid fe ed-back signaling in chro nic

alcohol c o n s u m p ti o n can lead to tonic activation of VP e x p re s ­ sion in CRH-ergic n e urons.

The c h a n g e s o b se r v e d in m a g n o c e llu la r n e u r o n s w e re a ge-de- p e n d e n t. In th e c ontr ol group, VP a n d OT c o n t e n t in t h e SON a n d PVN w as ub iq u ito u s ly lo w e r in old p a ti e n ts (> 4 0 years).

Consistently, d e c re a s e in VP a nd OT c o n t e n t in b o th nuclei (ex­

ce p t OT in t h e PVN) w as m u c h m o r e drastic in alcoholics in th e old g ro u p th a n in y o u n g e r patients. W e ob serv ed an elev ation in CRH i m m u n o r e a c ti v it y in t h e PVN of t h e control old g ro u p in a g r e e m e n t w i t h re p o r te d a ctivation of CRH e xpressio n during ageing in ro d e n t s a n d h u m a n s [43,44]). However, elev ation of CRH i m m u n o r e a c ti v it y w a s m o r e pro f o u n d in t h e y o u n g alcohol­

ic patients .

In conclusio n, t h e analy sis o f m a g n o c e llu l a r h y p o th a la m ic nuclei in alcoholic p a ti e n ts revealed a dec re a s e in VP c o n t e n t in SON an d th e PVN, a n d OT c o n t e n t in th e SON; all th r e e findings w ere m o r e p ro n o u n c e d in t h e e ld e r p a ti e n ts ( > 4 0 years). In spite of this, c o n t e n t of OT w as elev a ted in th e PVN of th e old group. We fo u n d an increase in CRH i m m u n o r e a c ti v it y in individual cells in p a rv o cellu lar n e u r o n s as w ell as an increase in t h e n u m b e r of CRH-positive n e u r o n s co -ex p r essin g VP. These ch a n g e s are likely induced by insufficien t levels of p la s m a glucocortico id s d u e to hy p o p las tic i m p a i r m e n t s of th e a d re n a l cortex.

A cknow ledgem en ts

W e th a n k Dr. Pavel O sten for c o m m e n ts on m a n u s c rip t. W e th a n k Ju lia Kuhl for carefu lly re a d in g a n d c o rre c tin g th e m a n u ­ script. E. S. w as a re c ip ie n t o f a DAAD fello w sh ip .

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