Example Report
Behavioral and Morphological Evaluati on of
Neuroprotection by Three Company Compounds in
MPTP-receiving Mice
(DATE)
T h i s s tu d y w a s c on d u c t e d u n d e r t h e t e r m s o f a S e r v i ce s
A g r e e me n t b e t we e n N e u r o D e t e c t i v e I n c . a n d C o m p a n y , d a t e d
2
1.
E x e c u t i v e Su m m a r y
Purpose:
Design:
T h i s s tu d y ( 1 ) e x am i n e d t h e e f f e c ts o f 3 d i f f e re n t
compounds supplied by Company on behavior in an
M P T P m o u s e m o de l o f P a r k i n s o n ’s d i s e a s e ; a n d ( 2 )
c o m p a r e d t h e a b i l i t y o f t h e s a me t h re e c o m p o u n d s t o
p r e s e r v e d o p a m i n e f u n c t i o n i n th e n i g r o - s t r i a t a l
pathway of the same animals used in the behavioral
study.
I n t h e b e h a v i o r a l s t u d y , f i v e g r o u ps o f a d u l t m a l e
C 5 7 B 1 / B 6 m i ce r e ce i v e d i m p l a n ts o f o s m o t i c
minipumps containing a 14-day supply of either vehicle
o r o n e o f t h r e e c om p o u n d s s u p p l i e d b y C o m p a n y
(designated 1, 2, and 3). Three days later the animals
r e c e i v e d t w o b e h av i o r a l a s s a y s o f s tr i a t a l f u n c t i o n
(Open Field test, Tail Hanging). Immediately following
t h e s e t e s ts t h e a n i m a l s r e ce i v e d s y s te m i c i n j e c t i o n s o f
a t o x i n k n o w n t o k i l l d o p a m i n e n e u r on s i n t h e
s u b s t a n t i a n i g r a (M P T P ) . T h e s a m e b e h a v i o r t e s t s
were then administered 10-12 days post-MPTP (i.e.,
w h i l e th e p u m ps we r e s t i l l a c t i v e ) a n d t h e n a g a i n 3 0 4 0 d a y s p o s t - M P T P ( w h e n t h e p u m ps w e re i n a c t i v e ) .
T h e s e t e s ts ( O p e n F i e l d , T a i l H a n gi n g ) w e r e a l s o
a d m i n i s te r e d a f o u r t h t i m e , 5 0 - 5 5 d a y s p o s t - M P T P . A
g r o u p o f u n o p e r a te d , u n t r e a t e d ( n o rm a l ) m i c e s e r v e d
a s c o n t r o l s . T h e re w e re 1 0 m i ce i n e a c h g r o u p .
I n t h e s t u dy o f d o p a m i n e f u n c t i o n , th e b r a i n s o f t h e
s a m e a n i m a l s t e s t e d b e h a v i o r a l l y w e r e r e m ov e d f o r
histological examination. The brains were sectioned
a n d s t a i n e d w i t h a t y r o s i n e h y d r ox y l a s e
i m m u n oh i s t o c h e mi c a l ( T H ) s t a i n . T h e s t a i n e d s e c t i on s
w e r e m o u n t e d o n m i c r o s c o p e s l i de s a n d t h e c e l l s
s t a i n e d f o r TH i n th e s u b s t a n t i a n i g ra ( S N ) a n d v e n t r a l
t e g m e n t a l a r e a ( V T A ) w e r e c o u n te d . N u m b e rs o f T H p o s i t i v e n e u r o n s i n t h e s e t w o s t r u c tu r e s a r e a m e a s u re
of the number of functioning dopaminergic neurons.
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Results:





I n t h e m o s t c o m mo n l y u s e d be h a v i or a l
m e a s u re i n t h i s mo d e l ( l o c o m o t o r a c t i v i t y ) ,
both 1 and 2 but not 3 tended to attenuate
M P T P - i n d u c e d h y pe r a c t i v i t y , w i t h 1 ’ s
i m p r o v e me n t re a c h i n g s t a t i s t i c a l s i gn i f i c a n ce
w h i l e i t w a s s t i l l b e i n g d e l i v e re d ( 1 0 D a y s
post-MPTP).
I n t h e b e h a v i o r a l m e a s u re m o s t s e n s i t i v e t o
l o s s o f n i g r o - s t r i a ta l d o p a m i n e ( u n s u p p o r t e d
r e a r i n g , i .e . t h e ab i l i t y t o b a l a n c e on t h e
h i n d l i m b s w i t h ou t f o r e l i m b s u p p o r t ) , 1 b u t n o t
2 o r 3 s i g n i f i c a n t l y r e d u ce d t h e ch ro n i c M P T P i n d u ce d i m p a i r me n t i n t h i s b a l a n c i n g t a s k ( 6 0
D a y s p o s t - M P T P ) . S i n ce t h i s r e s u l t o c c u r r e d a
m o n t h a f t e r t h e d ru g c e a s e d be i n g de l i v e re d ,
it implies a long-lasting protective effect.
1 b u t n o t 2 o r 3 s i g n i f i c a n t l y a t t e n u at e d M P T P i n d u ce d i m p a i r me n t o f f o r e l i m b e x t e n s i on
w h i l e th e c o m p o u n d w a s b e i n g a d m i n i s t e re d
( 1 0 D a y s p o s t - M PT P ) . A t 3 0 D a y s an d 6 0 D a y s
post-MPTP, mice that had received either of
t h e t h r e e c o m p o u n d s t e n de d t o b e l e s s
i m p a i r e d , t h o u g h th i s ch r o n i c i m p r o v e m e n t
r e a c h e d s t a t i s t i c a l s i g n i f i c a n ce o n l y w i t h 1 a t
3 0 D a y s p o s t - M P TP .
T h e r e we r e m o re T H - p o s i t i v e n e u r on s i n t h e
s u b s t a n t i a n i g r a (S N ) o f a n i m a l s t r e a t e d w i t h
1 , 2 , o r 3 t h a n i n v e h i c l e t re a t e d a n i m a l s . T h e
n u m b e r s o f t h e s e d o p a m i n e r g i c n e u ro n s w e re
n o t d i f f e re n t a m o n g t h e t h r e e t e s t c om p o u n d s .
A l l g r o u p s of a n i ma l s w i t h M P T P l e s i o n s h a d
s i g n i f i c a n t l y f e we r T H - p o s i t i v e S N n e u r o n s
t h a n u n l e s i o n e d co n t r o l s .
O n l y a n i m a l s r e ce i v i n g 1 h a d s i g n i f i ca n t l y
m o r e T H - p o s i t i v e n e u r on s i n t h e v e n t r a l
t e g m e n t a l a r e a ( V T A ) , c o m p a r e d t o l e s i on e d
v e h i c l e a n i m a l s . A l l g r o u p s of l e s i on e d
a n i m a l s h a d s i g n i f i c a n t l y f e we r T H - po s i t i v e
n e u r on s i n t h e i r V T A c o m p a r e d t o u n l e s i o n e d
controls.
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2.
3.
3.1.
I nt r o d uc t io n
M i c e t h a t r e c e i v e s y s te m i c i n j e c t i on s o f t h e p y ri d i n e
t o x i n 1 - me t h y l - 4 - ph e n y l - 1 ,2 , 3 ,6 - t e t ra h y d r o p y r i d i n e ( MP T P )
s e l e c t i v e l y l o s e s i g n i f i c a n t n u m b e rs o f d o p a m i n e r g i c
n e u r on s i n t w o m i d b r a i n s t r u c t u re s , t h e s u bs t a n t i a n i gr a
( S N ) a n d t h e v e n t ra l t e g m e n t a l a r e a ( V T A ) . L o s s o f
d o p a m i n e c e l l s i n S N m i mi c s t h e c l i n i c a l c o n d i t i o n i n
P a r k i n s on ’ s d i s e as e , a n d l e a d s t o m ot o r d y s f u n c t i o n . T h e
d o p a m i n e r g i c l os s i n m o u s e V T A i s o f u n k n o wn r e l e v an ce t o
P a r k i n s on ’ s d i s e as e , b u t m a y c o n t r i bu t e t o t h e c o g n i t i v e
d e f i c i ts o f P a r k i n s o n ’ s d i s e a s e , b e c au s e of t h e s e n e u r o n s ’
projections to the frontal cortex.
I n t h i s s t u d y w e te s t e d t h e a b i l i t y of t w o C o m p a n y
compounds, 1 and 2 as well as a comparison compound, 3,
t o p r e v e n t t h e l o s s o f d o p a m i n e r gi c n e u r on s i n t h i s M PT P
m o u s e m o d e l o f Pa r k i n s on ’ s d i s e as e , a s w e l l a s t o p r e v e n t
t h e m o t o r d e f i ci t s t h a t o c c u r i n t h i s m o d e l .
Methods and Experimental Design
Design
F i f t y a d u l t m a l e C5 7 B l 6 mi c e we r e ra n d o m l y d i v i de d
i n t o 5 g r o u p s . Al l w e r e t e s t e d i n t h e O p e n - F i e l d a n d T a i l H a n g i n g t e s t s o f s t r i a t a l f u n c t i o n . T h e O pe n F i e l d t e s t i s
t h e m o s t f re q u e n t l y u s e d b e h a v i o r a l a s s a y i n t h e M P T P
mouse model, and is thought to be sensitive to loss of
dopaminergic input to the striatum from the substantia
nigra. The Tail Hang test is known to be sensitive to direct
striatal damage.
F o u r o f t h e 5 g r o u p s o f m i c e r e ce i v e d a n i n f u s i o n o f 1
o f t h e 3 t e s t c o m p o u n d s o r a n i n f u s i o n o f a v e h i cl e
s u b s t a n ce c o n t a i n i n g n o c o m p o u n d . T h e i n f u s i o n s w e re
a d m i n i s te r e d v i a s u b c u t a n e o u s l y i m pl a n t e d o s m o t i c m i n i p u m p s o v e r a 1 4 da y p e r i o d ( A l z e t # 1 0 0 2 ; 0 . 2 5 u l /h r f l o w
r a t e ) . T h r e e d a y s f o l l o w i n g i m p l a n t a t i o n o f t h e p u m ps t h e
m i c e w e r e g i v e n th e O pe n F i e l d a n d T a i l H a n g t e s t s , as w a s
t h e g r o u p o f u n o p e r a t e d ( c o n t r o l ) mi c e . I m m e di a t e l y
f o l l o w i n g t h e b e h av i o r a l t e s ts , t h e pu m p - c o n t a i n i n g mi c e
MPTP Mouse Example Report-NeuroDetective- 2008
5
r e c e i v e d a 4 0 m g /k g i n j e c t i on o f M PT P s u b c u t a n e ou s l y . A l l
g r o u p s w e re t h e n g i v e n th e O pe n F i e l d a n d T a i l H a n g t e s t s ,
a t 1 0 - 1 2 d a y s a n d 3 0 - 4 0 d a y s p os t - M P T P . T h e O p e n F i e l d
a n d T a i l H a n g t e s ts w e re a l s o g i v e n a g a i n a t 5 0 - 5 5 d ay s
post-MPTP.
For the Open Field test, total distance traveled and
t o t a l n u m b e r o f v e r t i c a l m o v e me n t s (i . e . r e a rs ) w e r e
r e c o r d e d , e x c e p t th a t o n l y n u m b e r of r e a r s w a s re c o r de d a t
5 0 -5 5 d a y s p os t - M P T P . T h e n u m be r o f u n s u p p o r t e d r e a r s
( i . e . , u s i n g t h e h i n d l i m bs o n l y wi t h n o s u p p o r t f r o m th e
f o r e l i m b s a l o n g t h e w a l l s o f t h e t e s t e n c l os u r e ) i s t h e m o s t
r e l i a b l e m e a s u re o f d o p a m i n e n e u r on l o s s i n t h i s t e s t , a n d
i s m o s t e v i de n t a t l o n g p o s t - l e s i o n t i m e s .
F o r t h e T a i l H a n g t e s t , t h e d e g re e to w h i c h t h e
f o r e l i m b s a n d h i n dl i m b s we r e e x te n de d w h e n t h e a n i ma l
w a s s u s p e n de d b y t h e t a i l w a s r a t e d o n a 0 - 3 s c a l e . T h e
r a t e r w a s b l i n d t o t h e e x p e ri m e n t a l c o n d i t i o n s .
3.2.
Subjects
T h e m i ce w e re C 5 7 B 1 /B 6 t y p e a n d w e r e o b t a i n e d f r om
J a c k s o n L a b o r a t o r i e s . T h e y w e re 8 -1 2 w e e k s o l d u p o n
a r r i v a l a t t h e N e u ro D e t e c t i v e a n i m a l c o l o n y . T h e y w e re
allowed to acclimatize for 10-14 days prior to any
e x pe r i me n t a l m a n i p u l a t i o n s . A l l m i ce w e r e h ou s e d i n gr o u p s
o f 3 - 4 p e r c a g e a n d w e r e p r o v i de d wi t h a d - l i b i t u m a c c e s s t o
f o o d a n d w a t e r . T h e y we r e m a i n t a i n e d o n a 1 2 h r : 1 2 h r
l i g h t / d a r k c y c l e . T h e a n i m a l s w e re ra n d o m l y a s s i g n e d t o
f i v e g r o u p s ( 3 g r ou p s r e ce i v e d M P T P p l u s o n e o f t h re e
c o m p o u n d s , a 4 t h g r o u p r e c e i v e d M PT P p l u s t h e v e h i c l e , a n d
the 5th group were controls and did not receive either a
compound or MPTP treatment).
T h e r e we r e i n i t i a l l y 1 0 mi c e i n e a ch g r o u p . F o l l o w i n g
M P T P i n j e c t i o n s 9 m i c e d i e d : 3 f r o m t h e V e h i cl e g r o u p , 1
from the 1 group, 2 from the 2 group, and 3 from the 3
g r o u p . A C h i - S q u a r e a n a l y s i s s h o we d n o s i gn i f i c a n t
d i f f e r e n ce i n t h e m o r t a l i t y r a t e s f o r t h e d i f f e r e n t g r o u p s o f
lesioned animals.
T h r o u g h t h e c ou r s e o f b e h a v i o r a l t e s t i n g p o s t M P T P
i n j e c t i on , 1 m o u s e f r o m t h e V e h i c l e g r o u p a n d 1 m o u s e
f r o m t h e C o n t r o l gr o u p d i e d . D u ri n g h i s t o l o g i c a l p r o c e s s i n g
t h e t i s s u e f r o m s om e b r a i n s d i d n o t r e s p o n d s u f f i c i e n t l y t o
MPTP Mouse Example Report-NeuroDetective- 2008
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t h e s t a i n i n g t o p e rm i t a c c u r a t e c e l l co u n t i n g . T h u s , t h e
final set of neuron counting data was from 6 Vehicle, 8 1, 8
2 , 6 3 , a n d 7 C o n tr o l b r a i n s .
3.3.
Procedures
A f t e r t h e m i c e h a d b e e n a c c l i m a t e d t h e y we r e
r a n d o m l y a s s i g n e d t o o n e o f t h e 5 g ro u p s ( s e e a b o v e ) .
E a c h m o u s e w a s t h e n g i v e n a n i n i t i a l O p e n - Fi e l d t e s t a n d a
T a i l H a n g i n g t e s t ( d e s c r i be d b e l o w ) . F o l l o w i n g t h e s e te s t s
e a c h m o u s e i n 4 of t h e g r o u p s w as i m p l a n t e d w i t h a mi n i o s m o t i c p u m p . T h e i m p l a n t s we r e pe r f o r me d u n de r
i s o f l u r a n e a n e s t h e t i c . T h e m i n i - p u mp s c o n t a i n e d o n e o f
three compounds: 2 (3 mg/kg/day in DMSO vehicle), 1 (3
m g / k g / d a y i n D MS O v e h i c l e ) , o r 3 ( 1 m g / k g / d a y ) ; o r v e h i cl e
o n l y . T h e i n f u s i o n s w e re g i v e n a t t h e r a t e o f 0 .2 5 u l / h r f o r
14 days. Three days following implantation of the pumps,
a n d f o l l o w i n g b e h a v i o r a l t e s t i n g (s e e D e s i g n ) , t h e m i ce
r e c e i v e d a s u b c u t a n e o u s i n je c t i o n o f 4 0 m g / k g M P T P . T e n t w e l v e d a y s f o l l o wi n g M P T P i n je c t i o n s e a c h m o u s e w as
tested again in the Open-Field and Tail Hanging tests (see
b e l o w ) . T h e s e t e s t s w a s r e p e a t e d a t 3 0 - 4 0 d a y s p os t MPTP, and again at 50-55 days post MPTP injections.
BEHAVIORAL TESTING:
(1) Open-Field test
E a c h a n i m a l i s p l ac e d i n a c l e a r p l as t i c o p e n - f i e l d b o x
( 3 6 c m x 3 6 c m ) w i th t w o r o w s o f p h o t o - b e a m s m o u n t e d o n
t h e s i de s t o d e t e c t a n d d i s t i n g u i s h be t w e e n h o r i z o n t a l
m o v e me n t s ( f r o m w h i c h d i s t a n c e t r a v e l e d i s c a l cu l a t e d) a n d
v e r t i c a l m o v e me n ts , i . e . re a r s ( D i g i s c a n A c t i v i t y Me t e r ,
O m n i te c h E l e c t r o n i c s ) . A m b i e n t c o n d i t i o n s i n c l u de l o w
n o i s e a n d d i m l i gh t i n g . T h e a n i m a l ’s m o v e me n t s w i t h i n t h e
f i r s t 5 m i n u t e s of b e i n g p l a c e d i n t h i s b o x a r e c a l c u l a t e d
f r o m a u t o m a t e d r e c o r d i n g s o f t h e c ro s s i n g o f t h e s e
photobeams (“beam-breaks”). On the last of the four Open
F i e l d t e s t s , o n l y re a r s w e re c o u n te d, a n d t h e s e we r e
d i v i d e d i n t o “ s u p po r t e d ” a n d “ u n s u pp o r t e d ” r e a r s . I n
s u p p o r t e d r e a rs , t h e m o u s e p l a ce s at l e a s t o n e o f i ts
f o r e l i m b s u p on t h e s i de w a l l o f t h e b o x w h i l e a r e a r i s
MPTP Mouse Example Report-NeuroDetective- 2008
7
r e c o r d e d . I n u n s u p p o r t e d r e a r s , t h e r e i s n o p l a c e me n t o f
t h e f o r e l i m bs o n t h e w a l l a n d t h e mo u s e i s b a l a n c e d s o l e l y
o n i t s h i n d l i m bs wh i l e a r e a r i s r e c o rd e d . U s e o f t h e
f o r e l i m b s i s d e t e rm i n e d f r o m v i de o re c o r d i n gs o f t h e te s t .
( 2 ) T a i l - H a n g i ng
T h e m i ce a r e h u n g b y t h e i r t a i l s 3 t i m e s e a c h , f o r
a p p r o x i m a t e l y 1 0 s e c e a c h t i m e . E a ch m o u s e i s h u n g b y t h e
b a s e o f t h e t a i l a bo u t 3 0 c m a b o v e th e s u r f a c e of a t a b l e
until the mouse turns either to the left or to the right. A
left turn is given a score of 0, a right turn receives a score
of 1.
W h i l e h a n g i n g d u ri n g t h e t u rn s t e s t , f o r e l i m b
p l a c e m e n t i s a n a l y z e d . T h e l i m bs a r e s c o re d o n a 4 - p o i n t
s c a l e . L i m b s w h i ch a r e p r o p e r l y e x t e n d e d o u t a n d a b o v e
t h e h e a d a r e g i v e n a s c o r e o f 0 . L i m b s w h i ch a r e c l as p e d
or held against the body are given a score of 3. Scores of
1 o r 2 a r e a s s i g n e d f o r r e l a t i v e s t a g e s b e t we e n th e t wo
e x t re m e s .
T h e h i n d - l i m bs a r e a l s o a n a l y z e d w h i l e h a n g i n g i n t h e
t u r n s t e s t . A s c a l e o f 0 t o 3 i s u s e d a s p e r t h e f o re l i mb
rating test.
F o r a l l b e h a v i o r a l t e s t s , t h e r e s u l ts o n e a c h m e as u re
w e r e f i r s t a n a l y ze d u s i n g a r e pe a t e d m e a s u re s A N O V A
f o l l o w e d b y t e s t s o f t h e b e t w e e n g ro u p s e f f e c t u s i n g
F i s h e r ’s H S D m e th o d .
NEURON COUNTING:
B e t w e e n 5 5 a n d 6 0 d a y s p o s t M P T P i n j e c t i on s , t h e
m i c e w e r e s a c r i f i ce d b y a n o v e r d os e o f S o d i u m N e m b u t a l .
T h e i r b r a i n s w e re p e r f u s e d w i t h ph os p h a t e b u f f e re d s al i n e
f o l l o w e d b y L a n a ’ s f i x a t i v e ( p a r a f o r m a l d e h y de a n d p i cr i c
a c i d ) . T h e b r a i n s w e r e re m o v e d a n d p l a c e d i n L a n a ’s
fixative for 7 – 10 days. The brains were then cut
c o r o n a l l y a t 5 0 µ m u s i n g a v i b r a t o m e , a n d t h e s e c t i on s
stained with an antibody to tyrosine hydroxylase (TH), the
r a t e - l i m i t i n g e n z y m e i n d o p a m i n e s y n t h e s i s wh i c h i s a l s o
MPTP Mouse Example Report-NeuroDetective- 2008
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h i g h l y c o n c e n t r a t e d i n t h e ce l l b o d y a n d i t s p r o c e s s e s . T h e
s p e c i f i c p r o t o c o l w a s a c c o r d i n g t o t h e V e c t a s t a i n PK - 6 1 0 2
M o u s e I gG s t a i n i n g k i t s a n d V e c t o r S K - 4 1 0 0 D AB k i t (S i g m a A l d r i c h ) . O n c e s t a i n e d a n d c o v e r - s l i p p e d t h e t i s s u e wa s
e x a m i n e d u n d e r a m i c r o s c o p e a t 1 0 0 X m a g n i f i c a t i o n . Tw o
t i s s u e s l i ce s (– 2 .9 m m a n d – 3 .6 m m p o s t e r i o r t o B r e g ma )
were selected for cell counting in the SN and the VTA.
These sections are at the midpoint of the rostral half and
the midpoint of the caudal half of SN respectively. Each THl a b e l e d ce l l t h a t w a s c l e a r l y v i s i b l e a n d h a d b e t w e e n 2 a n d
6 n e u r i t e s w a s c on s i de r e d a n e u r o n a n d c o u n t e d . A n
o v e r a l l a v e r a g e c ou n t f o r e a c h a n i ma l w a s c a l c u l a t e d f r o m
t h e f o u r s e c t i on s (r o s t r a l a n d c a u d a l , l e f t a n d r i g h t ) . A n
average was obtained for the four sections. Separate
a n a l y s e s w e r e p e rf o r m e d o n t h e n e u r o n c o u n t s f r o m S N R
a n d V T A . T h e s e co u n t s w e re a n a l y ze d u s i n g a r e p e a t e d
m e a s u re s A N O V A f o l l o w e d b y t e s t s of t h e b e t w e e n g r ou p s
e f f e c t u s i n g F i s h e r’ s L S D m e t h o d ( p ’ s < . 0 5 f o r s t a t i s t i ca l
s i g n i f i c a n ce ) .
4.
Results
4.1. Open Field test
Prior to MPTP lesioning there was no significant
d i f f e r e n ce a m o n g t h e 5 g r o u ps i n t h e o p e n - f i e l d a c t i v i t y
t e s t i n d i s t a n c e t ra v e l e d o r n u m b e r o f r e a r s , a l t h o u gh t h e 3
group tended to be the least active and Company compound
g r o u p s t h e m os t a c t i v e i n o v e r a l l l o co m o t o r a c t i v i t y ( p . A 1 ) .
T h e s e t e n de n c i e s c o u l d r e f l e c t s l i g h t d i f f e re n ce s i n o v e r a l l
r e s p o n s i v e n e s s t o t h e d i f f e re n t d r u gs a f t e r 3 d a y s , a b s e n t
any lesion effect.
L o c o m o t i o n : F o l l o w i n g M P T P l e s i o n i n g , t h e v e h i c l e g ro u p
w a s s i g n i f i c a n t l y m o r e a c t i v e t h a n co n t r o l s a t 1 0 d a y s p o s t M P T P ( p < 0 . 0 2 ) ( p p . A 5 , A 6 ) , a n d t h i s e f f e c t p e rs i s t e d a t 3 0
d a y s p o s t - M P T P ( p < 0 . 0 2 ) ( p p . A 7 , A8 ) . O v e r a l l A N O V A ,
first test: F(4,36) = 3.51, p<.02; second test: F(4,34) =
2 . 8 4 , p < . 0 4 . 3 t re a t m e n t d i d n o t s h o w t h e s l i g h t e s t
s u g g e s t i o n o f a l t e r i n g t h e M P T P - i n du c e d g r e a t e r a c t i v i t y a t
either post-lesion time point. However Company 196923
MPTP Mouse Example Report-NeuroDetective- 2008
9
d i d s i g n i f i c a n t l y r e d u c e a c t i v i t y c o m pa r e d t o v e h i cl e
( p < 0 . 0 0 8 ) a t 1 0 d a y s p o s t - M P T P ( p p . A 5 , A 6 ) , t o t h e po i n t
o f n o t b e i n g d i f f e re n t f r o m c o n t r o l , a n d 2 s h o we d a s l i g h t
t r e n d i n t h e s a me d i r e c t i o n ( p = 0 . 1 7 ). N e i t h e r C o m p a n y
c o m p o u n d w a s s i gn i f i c a n t l y d i f f e re n t f r o m v e h i c l e a t 3 0
d a y s p o s t - M P T P , t h o u g h 2 te n d e d i n t h a t d i r e c t i o n ( p = 0 . 1 3 )
and in fact was not significantly different from normal (pp.
A7, A8).
T o t a l R e a r i n g : M P T P t e n d e d t o r e du c e t h e t o t a l n u mb e r o f
t i m e s t h e m i ce m ad e v e r t i c a l re a r i n g m o v e me n t s a t 1 0 d a y s
p o s t - l e s i o n , b u t t h i s d i f f e r e n ce ( V e h i c l e v s . C o n t r o l ) j u s t
m i s s e d s t a t i s t i c a l s i g n i f i c a n ce ( p < 0 .0 8 ) ( p p . A 9 , A1 0 ) .
O v e r a l l A N O V A : F (4 , 3 6 ) = 4 .7 6 , p < . 0 0 4 . A t 3 0 d a y s p o s t l e s i o n t h e r e w a s n o s u g g e s t i o n o f a n M P T P e f f e c t o n ov e r a l l
r e a r i n g ( p . A 1 1 ) . B o t h C o m p a n y c o m p o u n d s we r e n o t
s i g n i f i c a n t l y d i f f e re n t f r o m V e h i c l e a t e i t h e r p o s t - l e s i on
t i m e , a l t h o u g h a t 1 0 d a y s p o s t - M P T P m i c e re c e i v i n g e i t h e r
C o m p a n y c o m p o u n d d i d h a v e s i g n i f i ca n t l y r e d u ce d o v e ra l l
rearing compared to Control (p<0.0009 for 1 and p<0.004
f o r 2 ) , p p . A 9 , A 1 0 . S i n ce t h e r e w as n o s i gn i f i c a n t MP T P
e f f e c t , t h e s e re s u l t s a r e d i f f i c u l t t o i n t e r p r e t .
U n s u p p o r t e d R e a r i n g : A t 5 0 - 6 0 d a y s p o s t - M P T P , w h i ch i s
4 0 -5 0 d a y s a f te r t h e c o m p o u n ds c e as e d b e i n g d e l i v e r e d ,
m i c e t h a t h a d r e c e i v e d 2 m a d e s i gn i f i c a n t l y m o r e
u n s u p p o r t e d r e a rs t h a n m i c e t h a t h a d r e c e i v e d V e h i c l e
( p < 0 . 0 5 ) , w h i ch i n t u r n m a d e s i gn i f i c a n t l y f e we r
u n s u p p o r t e d r e a rs t h a n n o r m a l s ( p < 0 . 0 0 0 1 ) ( p . A 1 3 ) .
O v e r a l l A N O V A : F (4 , 3 4 ) = 7 .7 5 , p < . 0 0 0 3 . T h a t i s , 2
s i g n i f i c a n t l y a t t e n u a t e d t h e M P T P - i n d u c e d r e du c t i o n i n
unsupported rearing.
T h e r e w a s n o s i gn i f i c a n t e f f e c t o f e i t h e r M P T P o r a n y
treatment on supported rearing (p. A14).
4 . 2 . T a i l - H a n g i ng
N o s i g n i f i c a n t g r ou p d i f f e r e n c e s w e re o b s e rv e d i n t h e t a i l
h a n g t e s t p r i o r t o M P T P t r e a t m e n t , a n d n o n e we r e o bs e r v e d
post-MPTP in the hindlimbs. However MPTP did
significantly impair forelimb extension at all three postl e s i o n t i me p o i n t s : p < 0 . 0 0 0 1 a t 1 0 , 3 0 a n d 6 0 d a y s ( p. A 1 7 ,
MPTP Mouse Example Report-NeuroDetective- 2008
10
p p . A 1 9 a n d A 2 0 , a n d p . A 2 3 r e s p e ct i v e l y ) . T h u s t h e re w a s
n o r e c o v e r y o f t h i s i m p a i r me n t o v e r t i m e . O v e r a l l AN O V As :
F ( 4 , 3 5 ) = 1 3 . 8 , p < . 0 0 0 1 (1 0 d a y s ), F ( 4 , 3 4 ) = 1 6 . 0 , p <
. 0 0 0 1 (3 0 d a y s ) , an d F ( 4 , 3 5 ) = 1 6 . 0 , p < . 0 0 0 1 (6 0 d ay s ) .
2 s i gn i f i c a n t l y re d u c e d t h i s i m p a i r me n t w h i l e t h e c o mp o u n d
was being administered (10 Days post-MPTP), p<0.05 (p.
A 1 7 ) , w h i l e 1 a n d 3 s h o we d n o s u c h t r e n d . H o w e v e r a l l
t h r e e c o m p o u n d s te n d e d t o r e du c e th e i m p a i r me n t w h e n
m e a s u re d p o s t - d o s i n g , a t e n de n c y th a t a c h i e v e d s t a t i s t i c a l
s i g n i f i c a n ce f o r 1 a t 3 0 D a y s p o s t - MP T P ( p < 0 . 0 5 ; p p . A 1 9 ,
A 2 0 ) . I t s h ou l d be n o t e d t h a t t h e m e a s u re u s e d i n t h i s t e s t
i s a t r u n c a t e d s c a l e ( 4 p o i n ts ) a n d t h e re f o r e re a l b u t p a r t i a l
effects can be difficult to detect with smaller sample sizes.
4 . 4 N e u r o n C o un t s
Substantia nigra. The graph below shows the average
count of tyrosine hydroxylase (TH) positive neurons in the
S N f o r e a c h g r o u p.
70
Number of cells
60
50
40
30
20
Vehicle
1
2
3
Control
O n a v e r a g e , g r ou p s t h a t r e c e i v e d s y s t e m i c M P T P
i n j e c t i on s h a d 5 8 % f e we r T H n e u r o n s t h a n u n l e s i on e d
c o n t r o l m i c e . H o w e v e r l e s i o n e d m i c e t h a t r e c e i v e d on e o f
t h e 3 t e s t c o m p o u n d s s h o w e d s i g n i f i c a n t l y l e s s o f a l os s ,
a v e r a g i n g a 4 2 % re d u c t i o n i n T H l a be l e d n e u r o n s . O v e r a l l
MPTP Mouse Example Report-NeuroDetective- 2008
11
A N O V A : F ( 4 , 2 9 ) = 2 4 .6 5 2 , p < 0 . 0 0 0 1 . P o s t - h o c F i s h e r ’s
tests showed that each test compound produced significant
s p a r i n g o f T H p o s i t i v e n e u r on s , c o mp a r e d t o V e h i cl e
a n i m a l s , b u t t h a t t h e t e s t c o m p o u n ds w e re n o t d i f f e r e n t
from each other in this effect (see table below).
Diff.
Vehicle vs. 3
Vehicle vs. 1
Vehicle vs.
2
Vehicle vs.
Control
3 vs. 1
3 vs. 2
3 vs. Control
1 vs. 2
1 vs. Control
2 vs. Control
P-value
7.950
10.229
11.317
.0411*
.0064*
.0029*
34.783
.0001*
2.279
3.367
26.833
1.088
25.554
23.467
.5176
.3413
.0001*
.7381
.0001*
.0001*
Ventral Tegmental Area. The following graph shows the average number
of TH labeled neurons found in the VTA for each group.
Number of cells
70
60
50
40
30
20
Vehicle
1
2
3
Control
M P T P i n j e c t i o n s r e s u l t e d i n a n a v e r a g e o f 2 8 % f e w e r TH
n e u r on s i n t h e V TA ( V e h i c l e v s . C on t r o l ) , a d i f f e r e n c e t h a t
MPTP Mouse Example Report-NeuroDetective- 2008
12
w a s s i g n i f i c a n t ( s e e b e l o w ) . T h e t e s t c o m p o u n d s r e du c e d
t h i s l os s b y o n l y ab o u t 1 0 % , o n a v e ra g e . S t a t i s t i c a l a n a l y s i s
o f t h e s e d a t a u s i n g r e p e a t e d m e a s u re s AN O V A s h o w e d a n
overall significant effect (F(4,29) = 6.278, p < 0.0010), but
p o s t - h o c F i s h e r ’s t e s t s s h o w e d t h a t o n l y 2 t re a t m e n t l e d t o
significantly less loss of TH neurons compared to vehicle
( s e e t a b l e be l o w ) .
Diff.
Vehicle vs. 3
Vehicle vs. 1
Vehicle vs.
2
Vehicle vs.
Control
3 vs. 1
3 vs. 2
3 vs. Control
1 vs. 2
1 vs. Control
2 vs. Control
P-value
4.283
6.083
8.371
.2783
.1042
.0283*
18.383
.0001*
1.800
4.087
14.100
2.288
12.300
10.012
.6233
.2689
.0011*
.5010
.0020*
.0099*
MPTP Mouse Example Report-NeuroDetective- 2008