Effect of neurotrophin-3 on SOD and MDA in rats after acute spinal cord injury

Journal o f Medical Colleges o f PLA 2007;22(1)

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Effect of neurotrophin-3 on SOD and MDA in rats after acute spinal cord injury G U O Shu-zhang ($$#$),REN Xian-jun" ({3%%), JIANG T a o

Department o f Orthopaedics

(B 8$), O U Y A N G

Zhong

<@FB,$)

Xinqiao Hospital, Third Militury Medical University, Chongqing 400037,

China 0bjective:To investigate the effect of neurotrophin-3 on the expressions of SOD and MDA in

[Abstract]

the injured spinal cord of rats. Methods: Totally 105 SD rats were randomly divided i n t o 3 groups ( n =

35) : sham group. control group and experimental group. Animal model of acute spinal cord was inflicted with Allen's method by a thin plastic tube situated in subarachnoid space below the injury level for perfusion. Rats in experimental group received 20 pl NT-3 (200 ng) from the tube at 0 , 4 , 8 , 1 2 , 24 h and 3 ,

7 d after injury, and those in control group got the equal volume of normal saline at the same time points. T h e animals in sham group only received opening vertebral plate and putting tube in subarachnoid space. T h e rats were sacrificed at 4 , 8 , 1 2 , 24 h , and 3 , 7 , 14 d postinjury ( n = 5 ) . And the levels of superoxide dismutase (SOD) and malondialdehyde ( M D A ) in blood were observed with colorimetric method. Results : T h e serum level of SOD reduced obviously and the level of MDA raised obviously in rats after the injury, and the activity of SOD reached the lowest o n day 3 and the concentration of MDA reached peak at the 7 d.

In the experimental group, the SOD level was obviously higher (P
of free radical and lipid peroxidation is attenuated. [Key words]

neurotrophin-3; spinal cord injury ; superoxide dismutase ; malondialdehyde

T h e greatest jeopardize of spinal cord injury

rats of both sexes, weighing 220-270 g , were equal-

was functional impairment resulted from nerve in-

ly and randomly divided into 3 groups. Experimen-

jury. U p t o n o w , there were not effective methods

tal group ( N T - 3 g r o u p ) : 20 pl N T - 3 (200 n g ) was

for primary injury. T h u s preventing secondary in-

injected shortly after the injury and 4, 8 , 1 2 , 24 h ,

jury was more important. T h e experiment detected

and 3 , 7 d post-operation viu the subarachnoid

the level of superoxide dismutase (SOD) and malon-

space tube. Control group: 20 pl saline was injected

dialdehyde ( M D A ) in blood by colorimetric methods

a t the same time points. Sham group: only received

and studied the mechanism of promotion for the re-

opening vertebral plate and putting tube in sub-

covery of motor function.

arachnoid space.

MATERIALS AND METHODS

Establishment of animal model

Animals were

anesthetized by abdominal injection of 20% Ure-

SOD kit and MDA kit were pur-

t h a n e ( 1 000 mg/kg). T, spinous process was taken

chased from Jiancheng Bio-engineering research in-

a s center and a dorsal median incision of 5 cm was

stitute (Nanjing, China). N T - 3 was bought from

performed t o expose

Cytolab Corporation. SD rats were provided by the

T, whole vertebral lamina were bitten off t o expose

Materials

Animal Center of Daping Hospital of our university. Spectrophotometer type 752 and thermostatic waterbath trunk were used.

Animal grouping

One hundred and five SD

T, to

T,,, spinous process and

a round area of 3 mm in diameter as the spinal cord injury area. T h e right vertebral lamina of T," was bitten off t o expose dura mater of spinal cord as putting the subarachnoid space tube area. As the center of the spinal posterior middle vessels, T8

*

:Corresponding author. Tel: 86-23-68774081

spinal cord injury were prepared by adding 6 g X 5

Journal o f Medirul Colleges of PLA 2007;22(1)

29

Allen

h , and 3 , 7 and 14 d after operation, and the super-

method. Then a small hole on the dura mater near

natants were obtained by centrifugation. The level

to the posterior middle vessels was acquired. Cere-

of SOD and MDA in the supernatant was deter-

brospinal fluid flowed out, and a thin plastic tube

mined

was gently inserted 3 mm deep toward the injury

group, 20 p1 NT-3 (ZOO ng) was injected shortly af-

(MDA 1 chromometry , with spectrophotometer at 550 nm (SOD) and 532 nm (MDA) wave length. Statistical analysis Data were analyzed by SPSSlO. 0 and expressed with mean+SD. t test was

ter the injury and 4 , 8 , 1 2 , 24 h t 3 and 7 d after op-

used to detect difference among groups. P
eration via the subarachnoid space tube. Control

was

group: 35 spinal cord injury animal models were

P
prepared, and the same volume saline (20 pl) was

cant.

cm impaction

followed

the technique

of

site into the subarachnoid space. The tube was fixed to the inter-vertebral soft tissues and skin. In NT-3

injected at the same time. Sham group : 35 rats were

with

xanthinoxidase

considered

( S O D ) and TBA

statistically

significant,

and

RESULTS

only received opening vertebral plate and putting

Praxiological score

tube in subarachnoid space.

BBB score showed the re-

BBB score"]

covery of motor function had very statistically sig-

was applied to observe the recovery of motor func-

nificant difference between experimental group and

Assessment of motor function

tion in the rats. T o assess the motor function after

control group (P
spinal cord injury, the animals were assessed at the

perimental group was obviously superior to control

time points of 24 h , 3 , 7 and 1 4 d after operation.

group. The result indicated NT-3 obviously pro-

SOD and MDA detection

The blood was taken

moted motor function recovery of rats after spinal

from cor dextrum at the time points of 4 , 8, 1 2 , 24

cord injury.

Tab 1 BBB score in rats after acute spinal cord injury T i m e after injury

Groups

24 h 0.2210.43 0. 70f 0. 4Vd 1 9 . 7 0 f 0 . 95

Control NT-3 Sham "P<0. 05, bP
715

control group;

3d 3. ZOf 2 . 3 9 8. 01 f2. 05bd 20.601k0.52

7d 9 . 3 0 k l . 49 14. 3 2 1 2 . llbd 20.9OkO. 32

14 d 12.80+0.92 18. 40+1. 08bd 21. O O f O . 00

dP
Serum levels of SOD and MDA

T h e level of

SOD was notably descended in both experimental group and control group 4 h after operation, then

DISCUSSION Spinal cord injury (SCI) is one of the most de-

reached the lowest point at post injury 3 d. After

bilitating and costly injuries that anyone can suffer.

this, the level gradually elevated. Till post injury

As there is no cure, it is arguable that primary pre-

14 d , there was no significant difference between

vention should receive substantially greater empha-

experiment group and sham group. The level of

sis. Upon initial impact the vertebral fracture caus-

MDA was ascended in both experimental group and

es a local, segmental-limited damage of the spinal

control group at post operation 4 h after operation,

cord (primary damage). As consequences of rupture

then reached the peak at post injury 7 d. After this,

or contusion of axons, hemorrhage, ischemia and

the level was gradually lowered. The result showed

edema develop. The damage considerably expands

that NT-3 significantly inhibited the abnormal ex-

during the first weeks due to further destruction of

pression of MDA and elevated the activity of SOD

neuronal and glial cells (secondary damage)[*]. The

(Tab 2 and T a b 3 ) .

secondary damage is not only

propagated

by

Journal of Medical Colleges of PLA 2007 ;22( 1 )

30

Tab 2 SOD level in rats after acute spinal cord injury Groups Control NT-3 Sham

Time after injury

4 h 120.20f15.74 151. 7 3 k 6 . 63"d 199.71f8.55

'PP
Tab 3 Groups

Control NT-3 sham

71s

8h 12 h 24 h 3d 86.79k13.48 7 8 . 0 2 f 2 2 . 18 62.49f13.92 44.14f14.58 141. 8 1 f 1 2 . 71bd 105. 9 2 f 1 2 . 27"d 88. 47k16. 7Vd 6 6 . 0 5 f 9 . 95"d 198.40110. 52 192. 6 6 1 1 8 . 71 192.10*31.52 182.18f12.53

7d 14 d 99.43f13.26 135.33k16. 98 173. 1 4 f 2 0 . 58bd 211. 4 9 k 2 1 . 89' 208.14f19.72 227.82+12.24

control group; dP
MDA level in rats after acute spinal cord injury Time after injury 4h 2. 9 0 k 0 . 13 2. 0 5 k 0 . 4fjbc

8h 3.24f0.18 2. 4 8 1 0 . 36"

12 h 3.50f0.78 2. 7 7 f 0 . 27bd

4 . 0 l f 0 . 84 3. 2 5 f 0 . 24bd

24 h

1.49f0.18

1.83f0.25

2. 18fO. 15

1. 30+0. 19

3d 4.3410.34 3. 611tO. 22hd 1 . 4 2 f O . 36

7d 4 . 6 9 f 0 . 42 3. 5 4 f 0 . 61bd 1.41f0.31

14 d 3. 3 8 1 0 . 31 2. 9 2 1 0 . 22bd 1.36+0.52

'PP(0. 05, bP
systemic effects but also by lesional-parenchymal

result showed that N T - 3 can significantly inhibit

phenomena that influence each other reciprocally,

free radical and lipid peroxidation, mitigate the sec-

like excitotoxicity , inflammation, edema formation

ondary damage t o spinal cord vessel and nerve cell,

and lipid peroxidation/radical formation, and so on.

which was significant for recovering motor function

Lipid peroxidation/radical formation played a

of SCI, mitigating the damage of nerve cell and pro-

key role in the secondary spinal cord injury. SOD

moting nerve regeneration. We concluded that ex-

reflected the production of radical and MDA reflect-

ternal N T - 3 can mitigate secondary injury of spinal

ed the degree of lipid peroxidationC3'. SOD can resist

cord in vivo.

oxygen free radical, promote the regulating function

In our research, we found that there was a time

of H,O, density, protect tissue and cell by catalyze

interval difference between the lowest point level of

the 0' dismutation reaction. It has proved"'

the level of SOD was descended and the level of

SOD and the peak point level of M D A , which hinted increasing the activity of SOD couldn't completely

MDA was ascended after SCI, which hinted the

interrupt the lipid peroxidation.

lipid peroxidation was reinforced. So it is necessary

should take measures in many ways to prevent lipid

t o inhibit and remove free radical a t early period of

peroxidation and others factors of secondary SCI,

SCI. ErtenCslfound increasing the activity of SOD has obviously protective effect on SCI.

co-promote the recovery of spinal cord injury.

that

In our s t u d y , we observed that the level of

SOD notably descended both experimental group and control gro.up 4 h after SCI, then reached the

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Spinal Cord,

(Received 2006-10-08; revised 2006-1 1-25) (Editor GUO Jian-xiu)