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NEUROPATHOLOGY OF OVINE CERO I D - L I POFUSCI NOS I S
A t h esis presented i n part ial fu lfi lment
the requi remen t s for
(30%) of
the degree of
Mas t e r of P h i l osophy in Ve t e r i nary Pathology at
Massey Un iversity
Ak i n o r i Sh imada
1987
ii
ABSTRACT
The purposes of t h i s study we re to describe the
neuropathol ogy of ovine ceroi d - l i pofuscinosi s ,
to c ompare
find ings with those of the related ent i t ies in humans and other
domest i c animals , and t o pr ovi d e morphological informat i on that
might h e l p eluci date the pathogenesis of these d iseases .
An
establ ished flock of South Hamps h i r e sheep carrying the ceroid­
l i pofuscinosis gene have made it possible to perform a
l ongitu d i nal study on the central ne rvous system of affected
sheep of vari ous ages incl uding foetuses .
The most s t r i k i ng gross pat hological change of affected
sheep was brain atropy .
At t e rm i nal d isease,
the brain we ights of
affected sheep were 55% of those of normal sheep .
Atrophy
affected mainly the ce rebrum .
Sudan black and luxol fast blue posi t i ve autof luorescent
neuronal pigment granu l e s were d e t e cted by l i ghtmic roscopy as
early as the m i d stage of foetal devel opment ,
examined .
the ear l i est stage
Postnatal l y t h e re we r e t opograph i cal differences in the
quantity of accumulated l i popigments in neurones of var i ous
areas .
S i mi lar l y ,
there we re age r e lated topograph i cal
d ifferences in secondary d egene rative changes .
Neuronal
l oss was
most seve r e in the par i e ta l l obe cortex showing an ini t ial
l aminar d i st r i but i on .
Th is pat t e rn was we l l demonstrated by a
concomi tant astrocytosis .
In add i t i on to the compl e x e l e c t ron dense cytosomes simi l a r
t o those reported in t h e h uman syndromes ,
there w e r e l ess compl e x
cytosomes o f sma l l e r s i z e i n affe cted foetal brains . T h e lat t e r
we r e c l early bounded by a t r i l am i nar membrane and contained
who rls o r l oose stacks of t r i l aminar membranes resembl ing those
of the l im i t ing membranes .
I n some e l e ctronmi c rographs t h e re was
a sugges t i on of con t i nu i t y between the surrounding membrane and
iii
the internal membrane s ,
but th i s wa s not d e f i n i t e l y demon s tra t e d .
Th i s i s prov i s i ona l ly interpreted a s being due
interna l i z a t ion of
to an
surrounding l imi t ing membrane rather than a
re c y c l ing of membrane .
S ome of the s e sma l l cyt o s omes a l s o showe d
comp l e x mul t i l a me l l a r pro f i l e s s imi l ar to those of large compl e x
cyto some s .
The s e l atter a ppeared to be formed b y c oa l e s cence o f
sma l ler comp l e x one s .
There thus appeared to be a sequence o f
change s i n t h e d ev e l opment o f storage cytosome s .
Th i s s tudy reve a l e d that the ovine d i s e a s e h a s not only many
neuropa thologi c a l findings in c ommon with ana l ogous human
d i s e a s e s , but a l s o s ome pa tho l o g i c a l fea ture s wh ich have not b e e n
reported i n af f e c t e d humans o r anima l s .
l i pofu s c i no s i s
Ovine cero id­
is thus a useful an imal mod e l for the s tudy of t h e
human cero i d - l i pofus c i n o s e s .
iv
ACKNOWLEDGEMENTS
Th is study was pe rformed using the faci l i t i es of t h e
Department of Vete r i nary Pat h o l ogy and P u b l i c Health at Massey
Unive rsi ty.
I wou l d l ik e to t hank P r ofessor B. W . Mank t e l ow ,
Head of the D e pa r tment ,
for mak ing these ava i l able. I am
parti c ularly grateful t o Professor R . D . Jo l ly , my supe rviso r ,
for providing me w i t h t h e oppo rtun i ty to unde rtake t h i s pro je c t
and h i s continuous h e l p and encouragement throughout t h e stud y .
Advice on t h e inte rpretat i on of l i ght and e l ect ron
m i c r oscopy was also pr ovided by Dr. W . J.
at Taronga Z o o ,
Mart inus ,
Hartley ,
patho l ogist
Austral ia. D r. D.N. Palme r and Mr. R . D.
who are membe rs of the team work ing on ov ine c e r o i d ­
l i pofuscinosis in t h i s l aboratory ,
have a l so provided h e l p in
my unde rstand ing of b i o chemi cal aspects of the d isease.
The i r
encou rageme nt was great ly appr e c i ated.
I also wish to acknowl edge the h e l p of Mr. M.J. B i rt l e s of
the Department of Physi o l ogy and Anatomy in the preparat i on of
t issues for the immuno cytochemi cal study ,
Dr. A.S. Dav i e s of
the Department of Physi o l ogy and Anatomy in the study of ovine
neu roanatomy , and Mrs .
L.R.
D i ckson of t h e Department of
Veterinary C l i n i ca l S c i ence and fifth year B . V. S c . students i n
t h e caesarean sect i ons wh i ch provided foetuses for t h e study .
Special thanks a r e also due to Mr .
R .J.
D . H. Hopcroft and M r .
Bennett of t h e D e partment of S c ientific and Industrial
Research wh o gave advi c e on t h e preparat i on of t issues for
e l ect ron m i c r oscopy and printed t h e e l e c t r onmi c r ograph s , and
Mr. T . G .
Law and t h e staff at t h e Central Photographi c Uni t who
printed the l i g h tm i c r o g raphs .
I would l i k e t o t hank Mrs .
P.M.
S lack and Mrs .
P.M.
Davey
for t h e i r g r eat h e l p in the preparat i on of tissues for l i ght
and f l uorescent m i c r os c opy , Mr .
F.K.
Sharpe for his assistance
i n po s t -mortem room ,
a nd Mrs .
A.A.
Scott f or her a s s i s ta n c e i n
the u s e o f word pro ce s s i n g program and i n printing the f in a l
copy .
F i na l ly I would a l s o l i k e to e x pre s s thank f u l n e s s to my
f ami ly i n Japan f o r t h e ir con t i nuous encouragement ,
wi f e ,
Naok o ,
a nd my s on ,
Kentaro ,
a nd my
f or their patience and h e l p .
.V
TABLE OF CONTENTS
page
ABSTRACT
ii
ACKNOWLEDGEMENTS
iv
V
TABLES OF CONTENTS
L IST OF F I G URES
vii
L IST OF TABLES
xiv
1
INTRODUCTI ON
CHAPTER
I:
Review - the n e u r o n a l ceroidl i pofusci noses .
2
Gross pathol ogy .
23
CHAPTER I I I :
Light rn i c roscopy .
26
CHAPTER
IV :
E l e ctron rni c roscopy.
41
C HAPTER
V:
Gen e r a l d iscussi o n .
49
CHAPTER
II :
B I B L I OGRAPHY
53
vii
LIST OF FIGURE S
F o l l owing
page
Figure
2. 1
Brain weig h t s of
sheep with c e roid­
lipo f u scinos i s and age matched norma l
24
cont r o l s r e l ative to e a ch o t h e r and age .
2.2
Late r a l view of b rains f rom a 2 5 months o l d
a f f e c t e d s h e ep and f rom a 27 months o l d norm a l
s h e ep .
T h e greate st d e gr e e o f at rophy invol ve s
t h e c e rebrum .
Note marked thinning o f t h e gyri
24
o f t h e c e r e b r um o f the a f f e cted anima l .
2.3
Transv e r s e s e c t i ons of the ce rebrum f rom a
2 5 months o l d a f f e cted sheep and f rom a
month s o l d normal
o f t h e c e rebrum ,
c o rtex,
sheep.
27
Note reduced si z e
marked thinni ng o f the
seve r e l y at tenua ted corpu s cal l o s um
and s l ight enla rgement of the lateral
vent ric l e in the a f f e cted s h e ep. M i l d
a t r ophy o f t h e d i encepha l on and moderate
r e d u c t i on in si z e o f t h e subcortic a l wh i t e
matte r a re a l s o noted in the a f fe cted
24
sheep .
2. 4
Transve r s e s e c t i on of t h e ce rebrum of a 2 5
month s o l d a f fe cted s h e ep.
d e s c r i bed in F i g.
demon s t ra t e d.
Change s
2 . 3 a r e we l l
Note mye l in l o s s in the
s ub c o rt i ca l white m a t te r .
3.1
28
Vent r a l h o rn ce l l s of t h e ce rvica l spina l
c ord of a 23 month s o l d a f f e cted sheep .
Note c l u s t e r s o f a u t o f luore s cent g ranu l e s
in t h e neurona l pe rika rya .
3. 2 a
28
Vent r a l h o rn c e l l o f a 23 months old
a f f e cted sheep .
Lipopigment granu l e s
a c c umul ating i n t h e cytop l a sm stain b l a ck
with Sudan b l a ck B .
3 . 2b
28
Ven t r a l h o rn c e l l o f a 2 3 months o l d
a f f e cted sheep .
Lipopigment granu l e s
f i l ling t h e cytopl a sm s tain b l ue with l uxol
28
f a s t b l ue .
3 . 2c
Ven t r a l h o rn c e l l o f a 23 months o l d
a f f ected s h eep .
Lipopigment g ranu l e s
f i l l ing t h e cytopl a sm stain r e d with PAS .
28
viii
Figur e
Following
page
3 . 3a
Ventral h o rn cells o f a f ull t e rm foetus
a f f e c ted with ovine c e r oid-li po f u scinosis .
Nume r o u s fine lipopigment g r anule s
d i f f usely located in the c y topla sm.
3 . 3b
29
Ventral horn cell o f a 5 mont h s old
a f f e cted lamb .
Note clu s t e r s o f lipopigment
g r anule s of rela tively la r g e r s i z e in the
neu r onal pe rik a rya w i th i n c r e a s e d intensity
of
the s taining with luxol f a s t blue .
They
h ave a pe rinucl e a r location .
3. 3c
29
Vent ral h orn c ell of a 18 mont h s old
a f f e cted lamb .
The l i popi gment granule s a r e
l a r g e r a n d st ain m o r e intensely than those
i n F i g s . 3. 3a & 3. 3b and h ave a pe ri nuclear
29
loc a t i on.
3. 4
Ce rebellar cortex f rom an o bligate
h omozygous a f f e cted mid t e rm f oetus.
Dis crete S udan black s t a i ning inclu s i ons
a r e already pre sent in P u r k inje cells.
3.5
29
C e rebral cortic e s of a f ull te rm a f fected
f oe t u s
( b) ,
( a) ,
a 5 months old a f f e cted lamb
a 18 month s old a f f e c ted lamb
a 23 months old a f f e cted s h eep
( d).
( c) and
The
amount of Suda nophilic d e po s i t s in ne urone s
a s well a s in t h e c e rebral cortex incre a s e s
w i t h advancing age .
Large clu sters o f
Sudanoph i lic d e posit s w i t h va riation in t h e
s taining a n d a neuronal lo s s a r e noted by
te rminal d i s e a se.
3.6
29
Ventral horn c ells o f a 18 months old
a f f e c t e d lamb .
L i po p i gment g r anule s
a c c umulat e in the neu ronal pe rikarya ,
extending into axon hillock and even into
t h e proximal part of t h e axon .
3.7
29
Cerebral cortex of a 23 mont h s old a f fected
sheep ,
s howing neu ronal los s and pronounced
gliosis .
Depo sit m a t e r i a l i s mainly in
macrophage s .
29
ix
Following
Figure
page
3. 8
A t ransve r s e s e c t i ons of t h e ce rebral
cortex of a normal 5 mont h s old lamb
( a) i s
compa r e d with t h o s e o f a 5 month s old
and a 18 month s old
( b)
( c) a f f e c t e d lambs.
At
5 month s t h e r e i s a lready a evi dence o f
a t rophy w i t h pa r t i c ular lo s s o f neurone s in
t h e m i ddle area o f t h e i s o cortex . Th i s i s
exa cerbated i n t h e 18 mont h s old animal
with a gene r a l lo s s of ne u rone s in other
areas a s well.
Note t h e f o cal a rrangements
of rema ining neurone s in t h e s upe r f i c i a l
a re a o f t h e i s o cortex o f t h e 1 8 months old
animal.
3.9
30
Cerebral cortex of a 23 mont h s old a f fected
s h eep ,
showing ma rked neu r onal lo ss and
promi nent gli o s i s.
a r rangement of
Note t h e f ocal
rema ining neu rone s in the
s u pe r f i cial area o f t h e i s ocortex and a
clu s t e r of l i p i d laden cells interpreted a s
mac rophages.
3.10
31
A small glial nodule a s soci ated with a
small f ocal a r r angement o f remaining
neurones in the supe r f i c i al area of the
c erebral c o r t e x f rom a 23 months old
·31
a f fected sheep.
3.11
A coronal s e c t i on of t h e pa ri etal lobe o f
t h e c e rebrum i n a 5 month s old normal lamb
( a) i s compa r e d w i t h s im i la r secti ons of a
5 month s
( b) and a 18 mont h s old ( c)
a f f e cted lamb s .
The immunocytochemi cal
s ta i n for glial f i b r i lla ry a c i dic protein
d emon s t rat e s a s trocyto s i s in the mi ddle
a rea of
band .
the i socortex wi th a clear - c ut d a rk
T h i s o c c u r s f i r s t in the dor s a l gyri
( b) extending into t h e lateral gyr i with
inc r e a s ed a g e
( c) .
Concom i t ant with t hi s i s
a t r ophy o f t h e s ame a r ea s .
As trocyto s i s o f
t h e wh i te m a t ter i s a l s o prominent.
3 . 12
31
M e d i um powe r magni f i ca t i on of the c e rebral
cor tex f rom t h e s ame s e c t i on of 5 mont h s
old a f f ec t e d lamb a s s hown in Fig. 3 . 11 .
It
conf i rms t h a t t h e d a r k band interpreted a s
a n a st r ocyt o s i s d o e s c o ns i s t of cellular
and f i brous component s .
3 . 13
31
High power magni f i ca t i on o f the dark band
as s hown in F i g s . 3 . 11 & 3 . 1 2 . Note
hyperpla s t i c and hype r troph i c a s trocyt e s
wi t h prominent f i br i llary proce s s e s .
31
X
Fol l owing
Figur e
page
3 . 14
C e r e b r a l c o r t e x o f a 23 month s old a f f e c t e d
s heep .
Hyper t rophic a s t r o c y t e s with bi z a r r e
n u c l ei contain lipopigment granu l e s i n t h e
cytop l a sm .
3 . 15
31
Higher power magnification o f a
pe riva s c u l a r c u f f o f lipid l a den
m a c rophage s in the c e r e b r a l c ortex o f a 23
month s old a f f e c t e d s h e e p .
Note
considerable h e t e r ogeneity in the staining
of the g r unu l a r d e po sit s in t h e cytop l a sm .
3 . 16
I nterna l c a p s u l e o f a
31
23 mont h s old
a f fected s h e e p . Many va c u o l a r space s are
dif f u s e l y seen in t h e white matter .
3. 1 7
Higher power magni fication of one part o f
Fig . 3. 16.
A phagocyte w i t h c e l l debris in
its cytopl a sm is p r e s ent in a vacuo l a r
space .
3 . 18
32
32
Note many mye l in ovoid s .
S ubcortic a l white mat te r s o f a 5 month s o l d
a f f e c t e d l amb a n d o f an a ge -matched normal
l amb .
Note the inc re a sed fibri l lary
a st r ocytic proce s s e s in t h e a f f ected
32
anima l .
3. 19
S ubcortic a l white matte r s o f a 23 months
o l d a f f e c t e d sheep and of an age matched
norm a l sheep .
Note a marked l o s s of myelin
in the a f f e c t ed anima l .
4.1
32
Cereb r a l cortic a l neu rone f rom a mid t e rm
ovine f oetus a f f e cted with c e roid­
l ipo f u s cino s i s .
Note pre sence of e l e ct r on
dense r e sidu a l bodie s up t o 0 . 7 pm in
diamet e r in the cytop l a sm .
4.2
P a r t o f a c e rebr a l c o rt ic a l neu rone o f a 7
month s o l d a f f e c t e d l amb .
This cytosome
a ppe a r s to b e f o rmed by c o a l e s cence o f the
sma l l e r den s e bodie s .
4 .3
42
Mye l inated a xon in t h e c e re b r a l cortex of a
2 2 month s o l d a f f ec t e d s h e e p contains a
storage cyto s ome and a mitochondria .
4.4
42
42
Astro cyte in the c e r e b r a l c or t ex o f a 7
month s o l d a f f e c t ed l amb contains s torage
cyt o s om e s and thick bund l e s o f fibril s in a
hypertrophic proce s s .
I t i s adjacent to t h e
neurone t h a t is int e r p r e t e d a s showing
signs of d egene r ation .
42
xi
Following
Figur e
page
4.5
Dis c rete r e sidual bodie s within a membrane
bounded c y t o s ome in a m a c r ophage in the
cerebral c ortex of a 7 month s o l d affected
lamb .
4.6
Blood ve s sel in t h e c e r ebral cortex of a 2 2
month s old affe c ted s he ep .
Note e l e c tron
42
dense bodie s in t h e endothelia l cells .
4.7
42
Ele ctron dense body within a cerebr a l
cortical neurone from a full t e rm ovine
foetu s with cer oid-lipofus cinosis .
Note a
43
s u r r o unding trilamina r membrane.
4. 8
Part of a storage cyto s ome within a
ce rebral cortic a l neu r one from an affected
full t e rm ovine foetu s ,
showing fingerprint
and c urvilinear profi l e s .
4. 9
S torage cyto some within a ce rebral cortic a l
neurone from a
sheep ,
2 2 months o l d affected
sh owing a "tubular"
st ructur e s and
43
an ele c t r on- l u c ent va c u o l e .
4 . 10
43
Part of a s torage c yt o s ome within an
a s t rocyte in t h e c e r e b r a l cortex of a 2 2
mont h s old affected s h eep,
c r y sta lloid pat tern .
showing a
Note g l i a l fibri l s on
t h e l eft .
4. 11
43
Part of a cerebral c o r tical neurone from a
full t e rm ovine foetus with c eroid­
lipofuscinosis .
Note e l ect r on dense body ,
s howing t h r e e -layered lame llae with
approxima tely 6 . 5 nm in thickne ss and
s ta c k s of multilamel l a r a r rays.
On the
43
bottom i s a mit o c hondria .
4 . 12
P a r t of a multilam e l l a r cytosome within a
cerebral cortical neu r one from a full t e rm
ovine foet u s with c e r o id -lipofuscinosis .
It
s how s five -laye r e d lamellar structures with
a periodicity of a pproximately 12 nm and
s ta c k s of multilamellar a r ray s .
4 . 13
43
P a r t of a cerebral c o r tical neurone from a
full t e rm ovine foetu s with ce roid­
lipofus cinosi s ,
s howing a variety of
abnormal pigment c yto s ome s le ss than 1 . 2 �
in diame t e r in t h e cytopla sm .
The s e
c ont r a s t with a n d a re generally smaller
than mit o c h ondria .
43
xii
Figu r e
Fol l owing
page
4 . 14
Pigment cytosom e s up t o 0 . 8 pm in diameter
within a cereb r a l c ortica l neurone f rom a
f u l l t e rm ovine f oe t u s with ce roid­
lipo f u s cinosis ,
showing a di s c rete
s u rrounding membrane .
Note a granu l a r
matrix and mul ti l ame l l a r c omponents within
t h e s e cytosome s .
4 . 15
43
P a r t of a neurite in t h e c e r e b r a l cortex o f
a f u l l t e rm ovine f o e t u s with ceroid­
lipo f u s cinosis ,
showing a m u l ti lame l l a r
cytosome . i n a neurite o n the cent e r .
4 . 16
43
P a r t of a c e r e b r a l cortic a l neurone f rom a
f u l l t e rm ovine f o e t u s with ce roid­
lipo f u s cinosis ,
showing l ame l l a r cyto s ome s
l e s s than 0. 8 pm in diame t e r .
Note whor l s
and l o o s e s t a c k s o f t r i l aminar membranes .
4 . 17
44
High er power magnification of one part of
the l ame l l a r c y t o s ome with who r l s in Fig.
4. 16 .
Arrow poin t s to t h e
site at which
continuity between the s u r rounding membrane
and the interna l membrane is s uggested .
4. 18
44
P a r t of a c e r e b r a l c o r tica l neurone f rom a
f u l l t e rm ovine f o e t u s with ce roid­
lipo f u s cinosis ,
showing a variety of
inc l u sion bodie s .
Mu l t i-l ayered and five ­
l ayered l amel l a e a re s een in the cytosome s .
The long a r row indica t e s the site at which
a f ive - l ayered l ame l l a e appe a r s to be
f o rmed by the f u s ion of two three-l ayered
l ame l la e .
4 . 19
44
P a r t o f a cerebr a l c o r tica l neurone f rom a
f u l l t e rm ovine f oe t u s with ceroid­
lipo f u s cinosi s ,
showing a cytosome on the
l owe r right , in which lame l l a r a r rays
a ppear t o change t o a fine granu l a r
profile .
Note m u l tive sic u l a r body ,
swo l len
r ough endopla smic r e t i c ul um and part o f a
mitochondria .
4 . 20
44
M a c r ophage in the c e r e b r a l cortex f rom a 9
months o l d a f f e cted l amb ,
containing
s torage cytosome s o f simil a r s t ru cture to
t h o s e in neurone s , amorphous e l e c tron dense
materia l , membranous bodie s and degene rated
mye lin in the cytopl a sm .
44
xiii
Figur e
Fol lowing
page
4 . 21
A st rocyte in t h e c e r e b r a l cortex f rom a 2 2
months o l d a f f e c te d sheep ,
showing nu c l e u s
o f bi z a r r e s h a p e a n d a hype rtrophic proce s s
w i t h i n c r e a s e d numb e r o f f i br i l s and
storage cyto s ome s .
Note dense network o f
a st r ocytic f i b r o u s proce s s e s in the
a djacent neuropi le.
4. 22
44
P a r t o f a c e r e br a l c o rt i c a l neurone f rom a
norm a l f u l l t e rm ovine f o e t u s ,
showing
r e sidual bodi e s c omposed o f a f i ne granu l a r
mat r i x and an e l e c t r on-l u cent vacuole.
44
xiv
L I S T OF TABLES
Fo l l owing
Tab l e
l.I
page
C l in i c a l c l a s s i f i ca t i on of t h e neuronal
cero i d - l i po f u s c i n os e s .
l.II
T i s su e s valuab l e
5
in morph o l og i c a l d i a gno s i s
in t h e neurona l c e ro i d - l i po f u s cinose s .
14
2 . II I
L i s t o f a f f e c ted s h e e p u s e d in t h i s s tudy .
23
2. IV
L i s t o f norma l c ontrol sheep u s e d in t h i s
study .
3.I
Evolut i on of h i s t olog i cal changes accord ing
to age in bra ins
of
s heep a f f e c ted wi t h
ceroid-l i po f u s c i n o s i s .
3. I I
23
29
Rela t i ve d i s t ri b u t i on and s eve r i ty o f
l e s i ons in t h e s elected a r e a s of the
cent ral nervous s y s tem o f 1 2 to 25 mont h s
old s h eep a f f e c t e d wi t h c e r o i d­
30
lipo f u s c i nos i s .
3.I I I
D i s t r ibut i on and evolut i on of Walle r i an
type degene r a t i on in brains of
sheep
a f f e c ted with c e r oi d-l i po f u s cino s i s .
3 . IV
30
Aut o f l uore s cence and h i s t o chemi cal
rea c t i ons o f ne u r ona l pigment granu l e s i n
sheep a f f e cted w i th c e r o i d - l i po f u s c i no s i s .
3.V
32
Autofluore s cence and h i s to c h emi cal
r e a c t i ons of neu rona l p i gment granu l e s
( li po f u s c in) i n norma l
sheep .
33