N2乙酰半胱氨酸对内毒素增敏的新生大鼠缺氧缺血性脑损伤的治疗作用
http://www.100md.com
2007年4月13日
·Original Article in English·
[ Received Date] 2006 - 02 - 13
[ Foundation Item] Supported by the National Natural Science Foundation of
China (30571972)
[ Biography] NIE Chun - xia ,female ,chief doctor ,postgraduate student ,major
in neonatal brain damage.
Therapy Effect of N- Acetylcysteine on Lipopolysaccharide - Sensitized Neonatal
Rat with Hypoxic - Ischemic Brain Damage
NIE Chun - xia , WANG Xiao - yang , ZHU Chang - lian
(Department of Pediatrics ,the Third Hospital Affiliated to Zhengzhou University ,Zhenghou 450052 ,China)
Abstract : Objective To evaluate the effect of N - acetylcysteine (NAC) on lipopolysaccharide (LPS) - sensitized neonatal rats with hypoxic
- ischemic brain damage (HIBD) and possible mechanism except the antioxidant . Methods With the total number of 98 Wistar pups at postnatal
day 8 of either sex was used in this study. There were 86 pups which were divided into three groups to evaluate the brain injury :vehicle group ( n =
29) ,low dose (25 mg/ kg) ( n = 31) and high dose NAC (200 mg/ kg) ( n = 26) treatment group. The pups were injected with LPS(0. 1 mg/ kg)
intraperitoneally 3 days before hypoxic - ischemic (HI) insult . Multiple dose of NAC (25 mg/ kg or 200 mg/ kg) or vehicle was injected intraperi2
toneally before and after HI. Brain injury was evaluated 7 days after HI. For the Caspase - 3 activity and immunoblotting analysis , the samples were
collected at 24 h after HI treated either with vehicle or high dose NAC ( n = 6 per group) . Results The brain injury volume was significantly re2
duced by high dose NAC (200 mg/ kg) treatment compared with that of vehicle (77 % reduction , P < 0. 001) . The tissue loss was reduced 67 % ( P
< 0. 001) in high dose NAC treated group compared with that of vehicle. However ,there was no significant reduction of brain injury in the low dose
NAC treatment group compared with vehicle group. Caspa2se - 3 like activity measurement showed that the activity decreased 53 % after high dose
NAC treatment ( P < 0. 001) compared with that of vehicle treatment . The immunoblots showed that the active formof Caspase - 3 , 17 kDa band ,
was abolished by the high dose NAC treatment . Conclusions NAC treatment attenuate LPS - sensitized neonatal HI brain injury is dose dependent .
The neuroprotective effect involves Caspase - 3 inhibition.
J Appl Clin Pediatr ,2006 ,21( 6) :378 - 381
Key words : hypoxic - ischemic ;neuroprotection ;N - acetylcysteine ;lipopolysaccharides
N2乙酰半胱氨酸对内毒素增敏的新生大鼠缺氧缺血性脑损伤的治疗作用
聂春霞,王小阳,朱长连
(郑州大学第三附属医院儿科,郑州450052)
[摘要] 目的 探讨N2乙酰半胱氨酸(NAC) 对内毒素(LPS) 增敏的新生大鼠缺氧缺血性脑损伤(HIBD) 的防治效果和作用机
制。方法 8 日龄Wistar 大鼠98 只,性别不拘, 其中86 只随机分为3 组,安慰剂组(29 只) 、小剂量(25 mg/ kg) (31 只) 和大剂量(200
mg/ kg) NAC 治疗组(26 只) 。新生大鼠予腹腔注射LPS(0. 1 mg/ kg) ,3 d 后结扎左侧颈总动脉并吸入7. 7 %的氧气40 min 制备成
LPS 增敏的HIBD 动物模型。在应用LPS 后1 h、缺氧缺血(HI) 前2 h、HI 后0、24 h 腹腔注射NAC(25 mg/ kg 或200 mg/ kg) 或同体积
生理盐水,HI 后7 d 评价脑损伤的程度。余下12 只分别给予大剂量NAC (6 只) 和安慰剂(6 只) 并在HI 后24 h 测定脑组织Caspase2
3 的活性和蛋白印迹。结果 大剂量NAC 治疗组脑梗死体积较安慰剂组减少77 % ( P < 0. 001) , 而组织丢失体积均减少67 % ( P
< 0. 001) 。小剂量NAC 治疗组脑梗死及脑组织丢失体积与对照组相比无明显差别。HI 后24 h 大剂量NAC 治疗组Caspase23 活性
较安慰剂组显著降低(53 % , P < 0. 001) 。结论 NAC 对LPS 增敏的新生大鼠HIBD 的防治作用呈剂量依赖性,其神经保护作用与
抑制Caspase - 3 相关。
实用儿科临床杂志,2006 ,21 (6) :378 - 381
[关键词] 缺氧缺血;脑神经保护;N2乙酰半胱氨酸;脂多糖类
[中图分类号] R722. 1 [文献标识码] A [文章编号] 1003 - 515X(2006) 06 - 0378 - 04
Introduction
Hypoxic - ischemic brain damage (HIBD) is an important cause
of neurodevelopmental impairment and disability despite marked im2
provements in perinatal practice aimed at improving neonatal out2
comes. The causes of the perinatal HIBD are complicated , but as2
phyxia and inflammation seem to be important risk factors. The princi2
pal mechanisms are initiated by energy depletion , followed by activa2
tion of glutamate receptors. The cascade of subsequent events to neu2
ronal death involved accumulation of cytosolic calcium and activation
of a variety of calcium - medita2ted deleterious events , including es2
pecially generation of free radicals[1 ] . A growing body of evidence
supports the beneficial effects of antioxidants[2 ,3 ] . N - acetylcysteine
(NAC ,Sigma ,Germany ,01039) , a precursor of glutathione and a po2
tent antioxidant , has been identified to reduce the oxidative stress and
inflammation , and protect against brain damage in adult animal mod2
els[4 - 8 ] . Previous study has shown that brain injury is related with
brain maturity[9 ] . There is no report to show the effect of NACon im2
mature brain injury. The purpose of this study is to investigate the ef2
fect of NAC on LPS - sensitized hypoxic2ischemic (HI) neonatal brain
injury and possible mechanisms except antioxidant.
Materials and methods
Materials Eight - day - old Wistar rat (from Charles Ri2ver ,
Sulzfeld ,Germany ,weight 15 - 20 g) pups of either sex were injected
with LPS (0. 1 mg/ kg) ( E. coli 055 :B5 ,Sigma Germany) intraperi2
toneally. Unilateral hypoxic - ischemia was induced on postnatal day
11 according to the Rice - Vannucci model and modified[10 ] . Pups
were anesthetized with halothane (5 %for induction , 1. 5 % - 3. 5 %
for maintenance) in a mixture of nitrous oxide and oxygen (1∶1) and
the duration of anesthesia was < 5 min. The left common carotid
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实用儿科临床杂志第21 卷第6 期2006 年3 月 J Appl Clin Pediatr , Vol . 21 No. 6 March 2006
© 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
artery was cut between double ligatures of prolene sutures (6 - 0) .
After the surgical procedure the wounds were infiltrated with a local
anesthesia , and the pups were allowed to recover for 1 h. The pups
were placed in a chamber perfused with a humidified gas mixture (7.
7 % oxygen in nitrogen) for 40 min. The temperature in the incubator
and the temperature of the water used to humidify the gas mixture
were kept at 36 ℃. After hypoxic exposure the pups were returned to
their dams until were sacrificed.
Experimental groups The rat pups were divided into three
groups when evaluated the brain injury :1. vehicle group ( n = 29) ;
2. low dose NAC (25 mg/ kg) treatment group ( n = 31) ;3. high dose
NAC (200 mg/ kg) treatment group ( n = 26) . Multiple dose of
saline or NAC (25 mg/ kg or 200 mg/ kg) were given intraperitoneally
1 h after LPS injection , 2 h before carotid artery ligation , 0 h and 24
h after HI respectively. For the mechanism study , the rat pups were
divided into vehicle and high dose NAC (200 mg/ kg) treatment
groups ( n = 6 per group) . Multiple injections of saline or NAC were
given intraperitoneally as above but not 24 h. Tissue preparation and
MAP2 immunostaining. The pups were deeply anesthetized and perfu2
sion - fixed with 5 %formaldehyde in 0. 1 mol / L PBS at 7 days after
HI. The brains were rapidly removed and immersion - fixed in 5 %
formaldehyde at 4 °C for 24 h. After dehydration with graded ethanol
and xylene , the brains were paraffin - embedded and cut into 6μm
frontal sections. Every 100th section was stained with an antibody a2
gainst microtubule - associated protein 2 (MAP2) . Antigen recovery
was performed by heating the sections in 0. 01 mol/ L boiling sodium
citrate buffer (pH 6. 0) for 10 min. Nonspecific binding was blocked
for 30 min with 4 % horse serum in PBS. Anti - MAP2 (clone HM-
2 , 1∶2000 , Sigma) incuba2ted for 1 h at room temperature , followed
by a biotinlated horse anti - mouse secondary antibody for 1 h (1∶
200 , Vector , Burlingame , CA) . Endogenous peroxidase activity was
blocked with 3 % H2O2 for 5 min. Visualization was performed using
Vectastain ABC Elite with 0. 5 g/ L 3 , 3′- diaminobenzidine en2
hanced with 15 g/ L ammonium nickel sulfate , 2 g/ L beta - D glu2
cose , 0. 4 g/ L ammonium chloride and 0. 01 g/ L beta - glucose oxi2
dase (Sigma) .
Infarct volume measurement The area displaying loss of
MAP2 staining were measured using Micro Image(Olympus , Tokyo ,
Japan) and the volumes calculated according to the Cavalieri Principle
using the following formula : V = ΣA ×P ×T,where V = total vol2
ume , ΣA is the sum of the areas measured , P is the inverse of the
sections sampling fraction , and T is the section thickness. The inves2
tigator measuring the MAP2 negative areas and calculating the vol2
umes was blinded to the treatment. The MAP2 negative areas reflect2
ed infarcts , whereas the MAP2 positive volume of the contralateral
hemisphere minus the MAP2 positive volume of the ipsilateral hemi2
sphere reflected tissue loss[11 ] .
Neuropathology score Brain injury in different regions was e2
valuated using a semi - quantitative neuropathologic scoring sys2
tem[12 ] . The cortical injury was graded from 0 - 4 , 0 being no observ2
able injury and 4 confluent infarction encompassing most of the hemi2
sphere. The damage in hippocampus , striatum and thalamus were as2
sessed both with respected to hypotrophy ( shrin2kage) (0 - 3) and
observable cell injury/ infarction (0 - 3) resulting in a neuropathology
score for each brain region (0 - 6) . The total score (0 - 22) was the
sum for all four regions.
Fluorometric assay of Caspase - 3 like activity Animals
from both NAC and vehicle (1 h after LPS , 2 h before HI and imme2
diately after HI) treated groups were sacrificed by decapitation 24 h
after HI ( n = 6 per group) . The brains were rapidly dissected out on
a bed of ice. The parietal cortex was dissected out from each hemi2
sphere and 9 volumes of ice - cold homogenization buffer was added
[15 mmol/ L Tris - HCl , pH 7. 6 , 320 mmol/ L sucrose , 1 mmol/ L
DTT,1 mmol/ L MgCl2 and 3 mmol/ L EDTA - K, 0. 5 % protease
inhibitor cocktail (Sigma) ] . Homogenization was performed by soni2
cation. Samples of homogenate (50 μL) were mixed with 50μL of
extraction buffer as described earlier[13 ] . Cleavage of Ac - DEVD -
AMC (Enzyme Systems Pro2ducts , Livermore , CA) was measured at
37 ℃using a spectramax Gemini micro plate fluorometer (Molecular
Devices , Sunnyvale , CA) with an excitation wave length of 380 nm
and an emission wave length of 460 nm , and expressed as pmol AMC
released per mg protein and minute.
Immunoblotting of Caspase - 3 Samples whole homogenate
were mixed with NuPAGE LDS 4 × sample buffer and l reducing a2
gent and heated (70 ℃) for 10 min. Individual samples were run on
4 %- 12 % NuPAGE Bis - Tris gels (Novex , San Diego , CA) and
transferred to reinforced nitrocellulose (Schleicher & Schuell , Das2
sel , Germany) membranes. After blocking with 30 mmol/ L Tris -
HCl (pH 7. 5) , 100 mmol/ L NaCl and 0. 1 % Tween 20 (TBS - T)
containing 5 % fat - free milk powder for 1 h at room temperature ,
the membranes were incubated with rabbit anti - Caspase - 3 poly2
clonal antibody(1∶1000 , Santa Cruz , CA ) for 1 h followed by incu2
bated with the peroxidase - labeled goat anti - rabbit secondary anti2
bodies ( 1 ∶2000 ) for 30 min at room temperature ( Vector ,
Burlingame , CA) . Immuno2reative species were visualized using the
Super Signal Western Dura subatrate ( Perce , Rockford , IL) and a
LAS 1000 cooled CCD camera (Fujifilm , Tokyo , Japan) . Immunore2
ative bands were quantified using the Image Gauge software ( Fuji2
film , Tokyo , Japan) .
Statistics ANOVA followed by Fisher′s PLSD post hoc test was
used when comparing the brain volume. The Mann - Whitney U -
test was used when comparing the pathological score in different brain
regions. Unpaired t test was used when compared Caspase - 3 like
activity. The significant level was assigned at P < 0. 05.
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实用儿科临床杂志第21 卷第6 期2006 年3 月 J Appl Clin Pediatr , Vol . 21 No. 6 March 2006
© 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
Results
Brain damage was evaluated by two independent methods , in2
farct volume measurement and neuropathology score. The brain injury
after LPS - sensitized HI insult included cortex , hippocampus , thala2
mus and striatum in the ipsilateral hemisphere and no injury in the
contralateral hemisphere (Figure 1) . The total infarct volume (mean
±SEM) was (24. 2 ±4. 1) mm3 in vehic - letreated group , and
(22. 8 ±3. 9) mm3 in low dose NAC - treated group ( P = 0. 776) ,
and(5. 3 ±1. 6) mm3 in high dose NAC - treated group(77 % reduc2
tion , P < 0. 001) (Table 1) . The tissue loss was reduced from (88.
3 ±10. 2) mm3 in vehic - letreated group to (29. 4 ±6. 1) mm3 in
high dose NAC - trea2ted group (67 % reduction , P < 0. 001) , but
there was no significant reduction in the low dose NAC treated group
compared with that of vehicle treated group ( P = 0. 725) . The injury
in different brain regions was evaluated by pathological score showed
that the injury in all the regions were significantly reduced after high
dose NAC treatment ( Table 2) . Caspase - 3 like activity was in2
creased significantly in the ipsilateral hemisphere at 24 h after HI in2
sult compared with the contralateral hemisphere (Table 3) ,in accor2
dance with our earlier finding[13 ] . The activity decreased significantly
after high dose NAC treatment (53 % reduction , P < 0. 001) . The
immunobloting showed that 32 kD proformof Caspase - 3 was cleaved
and produced 29 kD and 17 kDfragments at 24 h after HI in the ipsi2
lateral hemisphere. The 17 kD band ,active form of Caspase - 3 , was
much less in the high dose NAC treated group than that of vehicle
treated group (Figure 2) .
Table 1 Brain Infarct and Tissue Lossat at 7 Days after HI( …x ±s)
Vehicle 25 mg/ kg NAC 200 mg/ kg NAC
Infarct volume 24. 2 ±4. 1 22. 8 ±3. 9 5. 3 ±1. 6
3
Tissue loss volume 86. 3 ±10. 2 83. 8 ±10. 0 29. 4 ±6. 1
3
Compared with vehicle group 3
P < 0. 001
Table 2 Pathological Score in Different Brain Regions at 7 Days after HI
( …x ±s)
Cortex Hippocampus Striatum Thalamus
Vehicle 2. 9 ±0. 4 2. 8 ±0. 3 2. 0 ±0. 3 1. 6 ±0. 2
200 mg/ kg NAC 1. 0 ±0. 3 △ 1. 3 ±0. 3 △ 1. 3 ±0. 2
3
0. 6 ±0. 2
3
Compared with vehicle group 3
P < 0. 01 , △P < 0. 001
Table 3 Caspase - 3 Activity at 24 hours after HI( …x ±s)
Vehicle 200 mg/ kg NAC
Ipsilateral 16. 1 ±2. 4 7. 5 ±2. 6 △
Contralateral 2. 6 ±0. 3 2. 4 ±0. 3
Compared with vehicle group △P < 0. 001
Discussions
Even though HIBD exists as an important etiological factor of
neurodevelopmental deficit , however , the etiology of neurological
handicaps is considered to be multifactorial. Infections / inflammation
and depression are likely to be important alone or in combination.
The extent of neuronal cell injury in the rats brain suffered from post2
natal HI was significantly increased by antenatal exposure to LPS[14 ] .
Clinical study also showed that antenatal infection and depression at
birth confer a synergistic effect with a substantially (78 fold) high risk
of cerebral palsy[15 ] ,suggesting that infectious products may sensitive
the fetus to additional insults. Animal studies showed that LPS given
4 h prior to HI sensitized the brain to injury in 7 - day - old
rats[16 - 18 ] . In this study , LPS was given 3 days before HI and the
brain injury was much more pronounced than that of the HI alone
(authors′unpublished data) . It indicates that bacterial endotoxin can
sensitize the immature brain to a secondary insult from 4 h to 3 days
before HI.
NAC has been used as an antioxidant because NAC is a precur2
sor of glutathione. Glutathione is well known to protect brain from is2
chemic injury[19 ] . Treatment with NAC resulted in significant increase
glutathione content in both cortex and striatum regions. Excessive ni2
tric oxide (NO) production after HI may induce cellular injury in var2
ious ways , including reaction with superoxide to form highly reactive
peroxynitrite[10 ] . NAC has been shown to be beneficial in HIBD as it
inhibits NO production and further reduce the apoptotic cell loss[4 ,8 ] .
In additional , pretreatment with NAC attenuates LPS - induced in2
flammation and cytokine production[20 ] . It has been shown that NAC
has neuroprotective effect against ischemic brain injury in adult ani2
mal mod2xels[4 - 8 ] . In this study , NAC did not protect brain from in2
jury at low dose (20 mg/ kg) administered before and after reperfu2
sion. Other study showed both low dose (50 mg/ kg) and high dose
(500 mg/ kg) did not have protective effect in the adult stroke model.
The protective dose was between 150 and 250 mg/ kg[4 ] . Because
NAC has a short half life and crosses the blood brain barrier , we
chose to use repeated[5 ] .
MAP2 , one kind of cytoskeletal proteins , has been used as a
sensitive indicator of neuronal injury. The decrease in MAP2 im2
munoreactivity is considered to be one sensitive marker of brain in2
jury[9 ] . In this study , the infarct size was measured by staining with
MAP2 antibody. We found that the loss of MAP2 immunoreactivity
was only in the ipsilateral hemisphere and high does of NAC could
prevent the loss of MAP2 immunoreactivity but not low dose. It indi2
cates that the neuroprotection of NAC was dose dependent.
Apoptotic and necrotic mechanisms account for neuronal death
after cerebral HI. A large number of neurons die via the apoptotic
mitochondrial pathway. Caspase - 3 has been identified as a key pro2
tease in the execution of apoptosis and appears to be an important
downstream event after HI in the immature brain. Caspase - 3 activa2
tion correlated well with DNA damage and tissue injury after HI[21 ] .
In our model , Caspase - 3 like activity was increased significantly in
the ipsilateral hemisphere at 24 h after HI insult compared with the
contralateral hemisphere , and decreased significantly after high dose
NAC treatment. It indicates that neuroprotective effect of NAC in2
volved Caspase - 3 inhibition. To our knowledge , this is first report
to show NAC has effect on Caspase - 3 activation.
Since the safety and efficacy record of NAC in other diseases as
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实用儿科临床杂志第21 卷第6 期2006 年3 月 J Appl Clin Pediatr , Vol . 21 No. 6 March 2006
© 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
well as crosses the blood - brain barrier[5 ] and its ability to protect
brain injury when given before and after ischemic insult , it would be
a very attractive candidate for treatment of neonatal brain injury in
clinical.
Figure 1 Representative MAP2 Staining at Dorsal Hippocampus Level .
Pups were Treated with Vehicle or Different Dose of NACas Indica2ted in
LPS Sensitized HI Model . Pronounced Protection Was Achieved in High
Dose NAC Treatment Pups
Figure 2 Representative Caspase - 3 Immunoblotting. The 32 kD Pro2
form Caspse - 3 Was Cleaved and Produced 29 kD and 17 kD Products in
Ipsilateral Hemisphere (L) . Cleavage Product Was Decreased after High
Dose NAC Treatment
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·381 ·
实用儿科临床杂志第21 卷第6 期2006 年3 月 J Appl Clin Pediatr , Vol . 21 No. 6 March 2006
© 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net, 百拇医药
[ Received Date] 2006 - 02 - 13
[ Foundation Item] Supported by the National Natural Science Foundation of
China (30571972)
[ Biography] NIE Chun - xia ,female ,chief doctor ,postgraduate student ,major
in neonatal brain damage.
Therapy Effect of N- Acetylcysteine on Lipopolysaccharide - Sensitized Neonatal
Rat with Hypoxic - Ischemic Brain Damage
NIE Chun - xia , WANG Xiao - yang , ZHU Chang - lian
(Department of Pediatrics ,the Third Hospital Affiliated to Zhengzhou University ,Zhenghou 450052 ,China)
Abstract : Objective To evaluate the effect of N - acetylcysteine (NAC) on lipopolysaccharide (LPS) - sensitized neonatal rats with hypoxic
- ischemic brain damage (HIBD) and possible mechanism except the antioxidant . Methods With the total number of 98 Wistar pups at postnatal
day 8 of either sex was used in this study. There were 86 pups which were divided into three groups to evaluate the brain injury :vehicle group ( n =
29) ,low dose (25 mg/ kg) ( n = 31) and high dose NAC (200 mg/ kg) ( n = 26) treatment group. The pups were injected with LPS(0. 1 mg/ kg)
intraperitoneally 3 days before hypoxic - ischemic (HI) insult . Multiple dose of NAC (25 mg/ kg or 200 mg/ kg) or vehicle was injected intraperi2
toneally before and after HI. Brain injury was evaluated 7 days after HI. For the Caspase - 3 activity and immunoblotting analysis , the samples were
collected at 24 h after HI treated either with vehicle or high dose NAC ( n = 6 per group) . Results The brain injury volume was significantly re2
duced by high dose NAC (200 mg/ kg) treatment compared with that of vehicle (77 % reduction , P < 0. 001) . The tissue loss was reduced 67 % ( P
< 0. 001) in high dose NAC treated group compared with that of vehicle. However ,there was no significant reduction of brain injury in the low dose
NAC treatment group compared with vehicle group. Caspa2se - 3 like activity measurement showed that the activity decreased 53 % after high dose
NAC treatment ( P < 0. 001) compared with that of vehicle treatment . The immunoblots showed that the active formof Caspase - 3 , 17 kDa band ,
was abolished by the high dose NAC treatment . Conclusions NAC treatment attenuate LPS - sensitized neonatal HI brain injury is dose dependent .
The neuroprotective effect involves Caspase - 3 inhibition.
J Appl Clin Pediatr ,2006 ,21( 6) :378 - 381
Key words : hypoxic - ischemic ;neuroprotection ;N - acetylcysteine ;lipopolysaccharides
N2乙酰半胱氨酸对内毒素增敏的新生大鼠缺氧缺血性脑损伤的治疗作用
聂春霞,王小阳,朱长连
(郑州大学第三附属医院儿科,郑州450052)
[摘要] 目的 探讨N2乙酰半胱氨酸(NAC) 对内毒素(LPS) 增敏的新生大鼠缺氧缺血性脑损伤(HIBD) 的防治效果和作用机
制。方法 8 日龄Wistar 大鼠98 只,性别不拘, 其中86 只随机分为3 组,安慰剂组(29 只) 、小剂量(25 mg/ kg) (31 只) 和大剂量(200
mg/ kg) NAC 治疗组(26 只) 。新生大鼠予腹腔注射LPS(0. 1 mg/ kg) ,3 d 后结扎左侧颈总动脉并吸入7. 7 %的氧气40 min 制备成
LPS 增敏的HIBD 动物模型。在应用LPS 后1 h、缺氧缺血(HI) 前2 h、HI 后0、24 h 腹腔注射NAC(25 mg/ kg 或200 mg/ kg) 或同体积
生理盐水,HI 后7 d 评价脑损伤的程度。余下12 只分别给予大剂量NAC (6 只) 和安慰剂(6 只) 并在HI 后24 h 测定脑组织Caspase2
3 的活性和蛋白印迹。结果 大剂量NAC 治疗组脑梗死体积较安慰剂组减少77 % ( P < 0. 001) , 而组织丢失体积均减少67 % ( P
< 0. 001) 。小剂量NAC 治疗组脑梗死及脑组织丢失体积与对照组相比无明显差别。HI 后24 h 大剂量NAC 治疗组Caspase23 活性
较安慰剂组显著降低(53 % , P < 0. 001) 。结论 NAC 对LPS 增敏的新生大鼠HIBD 的防治作用呈剂量依赖性,其神经保护作用与
抑制Caspase - 3 相关。
实用儿科临床杂志,2006 ,21 (6) :378 - 381
[关键词] 缺氧缺血;脑神经保护;N2乙酰半胱氨酸;脂多糖类
[中图分类号] R722. 1 [文献标识码] A [文章编号] 1003 - 515X(2006) 06 - 0378 - 04
Introduction
Hypoxic - ischemic brain damage (HIBD) is an important cause
of neurodevelopmental impairment and disability despite marked im2
provements in perinatal practice aimed at improving neonatal out2
comes. The causes of the perinatal HIBD are complicated , but as2
phyxia and inflammation seem to be important risk factors. The princi2
pal mechanisms are initiated by energy depletion , followed by activa2
tion of glutamate receptors. The cascade of subsequent events to neu2
ronal death involved accumulation of cytosolic calcium and activation
of a variety of calcium - medita2ted deleterious events , including es2
pecially generation of free radicals[1 ] . A growing body of evidence
supports the beneficial effects of antioxidants[2 ,3 ] . N - acetylcysteine
(NAC ,Sigma ,Germany ,01039) , a precursor of glutathione and a po2
tent antioxidant , has been identified to reduce the oxidative stress and
inflammation , and protect against brain damage in adult animal mod2
els[4 - 8 ] . Previous study has shown that brain injury is related with
brain maturity[9 ] . There is no report to show the effect of NACon im2
mature brain injury. The purpose of this study is to investigate the ef2
fect of NAC on LPS - sensitized hypoxic2ischemic (HI) neonatal brain
injury and possible mechanisms except antioxidant.
Materials and methods
Materials Eight - day - old Wistar rat (from Charles Ri2ver ,
Sulzfeld ,Germany ,weight 15 - 20 g) pups of either sex were injected
with LPS (0. 1 mg/ kg) ( E. coli 055 :B5 ,Sigma Germany) intraperi2
toneally. Unilateral hypoxic - ischemia was induced on postnatal day
11 according to the Rice - Vannucci model and modified[10 ] . Pups
were anesthetized with halothane (5 %for induction , 1. 5 % - 3. 5 %
for maintenance) in a mixture of nitrous oxide and oxygen (1∶1) and
the duration of anesthesia was < 5 min. The left common carotid
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实用儿科临床杂志第21 卷第6 期2006 年3 月 J Appl Clin Pediatr , Vol . 21 No. 6 March 2006
© 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
artery was cut between double ligatures of prolene sutures (6 - 0) .
After the surgical procedure the wounds were infiltrated with a local
anesthesia , and the pups were allowed to recover for 1 h. The pups
were placed in a chamber perfused with a humidified gas mixture (7.
7 % oxygen in nitrogen) for 40 min. The temperature in the incubator
and the temperature of the water used to humidify the gas mixture
were kept at 36 ℃. After hypoxic exposure the pups were returned to
their dams until were sacrificed.
Experimental groups The rat pups were divided into three
groups when evaluated the brain injury :1. vehicle group ( n = 29) ;
2. low dose NAC (25 mg/ kg) treatment group ( n = 31) ;3. high dose
NAC (200 mg/ kg) treatment group ( n = 26) . Multiple dose of
saline or NAC (25 mg/ kg or 200 mg/ kg) were given intraperitoneally
1 h after LPS injection , 2 h before carotid artery ligation , 0 h and 24
h after HI respectively. For the mechanism study , the rat pups were
divided into vehicle and high dose NAC (200 mg/ kg) treatment
groups ( n = 6 per group) . Multiple injections of saline or NAC were
given intraperitoneally as above but not 24 h. Tissue preparation and
MAP2 immunostaining. The pups were deeply anesthetized and perfu2
sion - fixed with 5 %formaldehyde in 0. 1 mol / L PBS at 7 days after
HI. The brains were rapidly removed and immersion - fixed in 5 %
formaldehyde at 4 °C for 24 h. After dehydration with graded ethanol
and xylene , the brains were paraffin - embedded and cut into 6μm
frontal sections. Every 100th section was stained with an antibody a2
gainst microtubule - associated protein 2 (MAP2) . Antigen recovery
was performed by heating the sections in 0. 01 mol/ L boiling sodium
citrate buffer (pH 6. 0) for 10 min. Nonspecific binding was blocked
for 30 min with 4 % horse serum in PBS. Anti - MAP2 (clone HM-
2 , 1∶2000 , Sigma) incuba2ted for 1 h at room temperature , followed
by a biotinlated horse anti - mouse secondary antibody for 1 h (1∶
200 , Vector , Burlingame , CA) . Endogenous peroxidase activity was
blocked with 3 % H2O2 for 5 min. Visualization was performed using
Vectastain ABC Elite with 0. 5 g/ L 3 , 3′- diaminobenzidine en2
hanced with 15 g/ L ammonium nickel sulfate , 2 g/ L beta - D glu2
cose , 0. 4 g/ L ammonium chloride and 0. 01 g/ L beta - glucose oxi2
dase (Sigma) .
Infarct volume measurement The area displaying loss of
MAP2 staining were measured using Micro Image(Olympus , Tokyo ,
Japan) and the volumes calculated according to the Cavalieri Principle
using the following formula : V = ΣA ×P ×T,where V = total vol2
ume , ΣA is the sum of the areas measured , P is the inverse of the
sections sampling fraction , and T is the section thickness. The inves2
tigator measuring the MAP2 negative areas and calculating the vol2
umes was blinded to the treatment. The MAP2 negative areas reflect2
ed infarcts , whereas the MAP2 positive volume of the contralateral
hemisphere minus the MAP2 positive volume of the ipsilateral hemi2
sphere reflected tissue loss[11 ] .
Neuropathology score Brain injury in different regions was e2
valuated using a semi - quantitative neuropathologic scoring sys2
tem[12 ] . The cortical injury was graded from 0 - 4 , 0 being no observ2
able injury and 4 confluent infarction encompassing most of the hemi2
sphere. The damage in hippocampus , striatum and thalamus were as2
sessed both with respected to hypotrophy ( shrin2kage) (0 - 3) and
observable cell injury/ infarction (0 - 3) resulting in a neuropathology
score for each brain region (0 - 6) . The total score (0 - 22) was the
sum for all four regions.
Fluorometric assay of Caspase - 3 like activity Animals
from both NAC and vehicle (1 h after LPS , 2 h before HI and imme2
diately after HI) treated groups were sacrificed by decapitation 24 h
after HI ( n = 6 per group) . The brains were rapidly dissected out on
a bed of ice. The parietal cortex was dissected out from each hemi2
sphere and 9 volumes of ice - cold homogenization buffer was added
[15 mmol/ L Tris - HCl , pH 7. 6 , 320 mmol/ L sucrose , 1 mmol/ L
DTT,1 mmol/ L MgCl2 and 3 mmol/ L EDTA - K, 0. 5 % protease
inhibitor cocktail (Sigma) ] . Homogenization was performed by soni2
cation. Samples of homogenate (50 μL) were mixed with 50μL of
extraction buffer as described earlier[13 ] . Cleavage of Ac - DEVD -
AMC (Enzyme Systems Pro2ducts , Livermore , CA) was measured at
37 ℃using a spectramax Gemini micro plate fluorometer (Molecular
Devices , Sunnyvale , CA) with an excitation wave length of 380 nm
and an emission wave length of 460 nm , and expressed as pmol AMC
released per mg protein and minute.
Immunoblotting of Caspase - 3 Samples whole homogenate
were mixed with NuPAGE LDS 4 × sample buffer and l reducing a2
gent and heated (70 ℃) for 10 min. Individual samples were run on
4 %- 12 % NuPAGE Bis - Tris gels (Novex , San Diego , CA) and
transferred to reinforced nitrocellulose (Schleicher & Schuell , Das2
sel , Germany) membranes. After blocking with 30 mmol/ L Tris -
HCl (pH 7. 5) , 100 mmol/ L NaCl and 0. 1 % Tween 20 (TBS - T)
containing 5 % fat - free milk powder for 1 h at room temperature ,
the membranes were incubated with rabbit anti - Caspase - 3 poly2
clonal antibody(1∶1000 , Santa Cruz , CA ) for 1 h followed by incu2
bated with the peroxidase - labeled goat anti - rabbit secondary anti2
bodies ( 1 ∶2000 ) for 30 min at room temperature ( Vector ,
Burlingame , CA) . Immuno2reative species were visualized using the
Super Signal Western Dura subatrate ( Perce , Rockford , IL) and a
LAS 1000 cooled CCD camera (Fujifilm , Tokyo , Japan) . Immunore2
ative bands were quantified using the Image Gauge software ( Fuji2
film , Tokyo , Japan) .
Statistics ANOVA followed by Fisher′s PLSD post hoc test was
used when comparing the brain volume. The Mann - Whitney U -
test was used when comparing the pathological score in different brain
regions. Unpaired t test was used when compared Caspase - 3 like
activity. The significant level was assigned at P < 0. 05.
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实用儿科临床杂志第21 卷第6 期2006 年3 月 J Appl Clin Pediatr , Vol . 21 No. 6 March 2006
© 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
Results
Brain damage was evaluated by two independent methods , in2
farct volume measurement and neuropathology score. The brain injury
after LPS - sensitized HI insult included cortex , hippocampus , thala2
mus and striatum in the ipsilateral hemisphere and no injury in the
contralateral hemisphere (Figure 1) . The total infarct volume (mean
±SEM) was (24. 2 ±4. 1) mm3 in vehic - letreated group , and
(22. 8 ±3. 9) mm3 in low dose NAC - treated group ( P = 0. 776) ,
and(5. 3 ±1. 6) mm3 in high dose NAC - treated group(77 % reduc2
tion , P < 0. 001) (Table 1) . The tissue loss was reduced from (88.
3 ±10. 2) mm3 in vehic - letreated group to (29. 4 ±6. 1) mm3 in
high dose NAC - trea2ted group (67 % reduction , P < 0. 001) , but
there was no significant reduction in the low dose NAC treated group
compared with that of vehicle treated group ( P = 0. 725) . The injury
in different brain regions was evaluated by pathological score showed
that the injury in all the regions were significantly reduced after high
dose NAC treatment ( Table 2) . Caspase - 3 like activity was in2
creased significantly in the ipsilateral hemisphere at 24 h after HI in2
sult compared with the contralateral hemisphere (Table 3) ,in accor2
dance with our earlier finding[13 ] . The activity decreased significantly
after high dose NAC treatment (53 % reduction , P < 0. 001) . The
immunobloting showed that 32 kD proformof Caspase - 3 was cleaved
and produced 29 kD and 17 kDfragments at 24 h after HI in the ipsi2
lateral hemisphere. The 17 kD band ,active form of Caspase - 3 , was
much less in the high dose NAC treated group than that of vehicle
treated group (Figure 2) .
Table 1 Brain Infarct and Tissue Lossat at 7 Days after HI( …x ±s)
Vehicle 25 mg/ kg NAC 200 mg/ kg NAC
Infarct volume 24. 2 ±4. 1 22. 8 ±3. 9 5. 3 ±1. 6
3
Tissue loss volume 86. 3 ±10. 2 83. 8 ±10. 0 29. 4 ±6. 1
3
Compared with vehicle group 3
P < 0. 001
Table 2 Pathological Score in Different Brain Regions at 7 Days after HI
( …x ±s)
Cortex Hippocampus Striatum Thalamus
Vehicle 2. 9 ±0. 4 2. 8 ±0. 3 2. 0 ±0. 3 1. 6 ±0. 2
200 mg/ kg NAC 1. 0 ±0. 3 △ 1. 3 ±0. 3 △ 1. 3 ±0. 2
3
0. 6 ±0. 2
3
Compared with vehicle group 3
P < 0. 01 , △P < 0. 001
Table 3 Caspase - 3 Activity at 24 hours after HI( …x ±s)
Vehicle 200 mg/ kg NAC
Ipsilateral 16. 1 ±2. 4 7. 5 ±2. 6 △
Contralateral 2. 6 ±0. 3 2. 4 ±0. 3
Compared with vehicle group △P < 0. 001
Discussions
Even though HIBD exists as an important etiological factor of
neurodevelopmental deficit , however , the etiology of neurological
handicaps is considered to be multifactorial. Infections / inflammation
and depression are likely to be important alone or in combination.
The extent of neuronal cell injury in the rats brain suffered from post2
natal HI was significantly increased by antenatal exposure to LPS[14 ] .
Clinical study also showed that antenatal infection and depression at
birth confer a synergistic effect with a substantially (78 fold) high risk
of cerebral palsy[15 ] ,suggesting that infectious products may sensitive
the fetus to additional insults. Animal studies showed that LPS given
4 h prior to HI sensitized the brain to injury in 7 - day - old
rats[16 - 18 ] . In this study , LPS was given 3 days before HI and the
brain injury was much more pronounced than that of the HI alone
(authors′unpublished data) . It indicates that bacterial endotoxin can
sensitize the immature brain to a secondary insult from 4 h to 3 days
before HI.
NAC has been used as an antioxidant because NAC is a precur2
sor of glutathione. Glutathione is well known to protect brain from is2
chemic injury[19 ] . Treatment with NAC resulted in significant increase
glutathione content in both cortex and striatum regions. Excessive ni2
tric oxide (NO) production after HI may induce cellular injury in var2
ious ways , including reaction with superoxide to form highly reactive
peroxynitrite[10 ] . NAC has been shown to be beneficial in HIBD as it
inhibits NO production and further reduce the apoptotic cell loss[4 ,8 ] .
In additional , pretreatment with NAC attenuates LPS - induced in2
flammation and cytokine production[20 ] . It has been shown that NAC
has neuroprotective effect against ischemic brain injury in adult ani2
mal mod2xels[4 - 8 ] . In this study , NAC did not protect brain from in2
jury at low dose (20 mg/ kg) administered before and after reperfu2
sion. Other study showed both low dose (50 mg/ kg) and high dose
(500 mg/ kg) did not have protective effect in the adult stroke model.
The protective dose was between 150 and 250 mg/ kg[4 ] . Because
NAC has a short half life and crosses the blood brain barrier , we
chose to use repeated[5 ] .
MAP2 , one kind of cytoskeletal proteins , has been used as a
sensitive indicator of neuronal injury. The decrease in MAP2 im2
munoreactivity is considered to be one sensitive marker of brain in2
jury[9 ] . In this study , the infarct size was measured by staining with
MAP2 antibody. We found that the loss of MAP2 immunoreactivity
was only in the ipsilateral hemisphere and high does of NAC could
prevent the loss of MAP2 immunoreactivity but not low dose. It indi2
cates that the neuroprotection of NAC was dose dependent.
Apoptotic and necrotic mechanisms account for neuronal death
after cerebral HI. A large number of neurons die via the apoptotic
mitochondrial pathway. Caspase - 3 has been identified as a key pro2
tease in the execution of apoptosis and appears to be an important
downstream event after HI in the immature brain. Caspase - 3 activa2
tion correlated well with DNA damage and tissue injury after HI[21 ] .
In our model , Caspase - 3 like activity was increased significantly in
the ipsilateral hemisphere at 24 h after HI insult compared with the
contralateral hemisphere , and decreased significantly after high dose
NAC treatment. It indicates that neuroprotective effect of NAC in2
volved Caspase - 3 inhibition. To our knowledge , this is first report
to show NAC has effect on Caspase - 3 activation.
Since the safety and efficacy record of NAC in other diseases as
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实用儿科临床杂志第21 卷第6 期2006 年3 月 J Appl Clin Pediatr , Vol . 21 No. 6 March 2006
© 1994-2006 China Academic Journal Electronic Publishing House. All rights reserved. http://www.cnki.net
well as crosses the blood - brain barrier[5 ] and its ability to protect
brain injury when given before and after ischemic insult , it would be
a very attractive candidate for treatment of neonatal brain injury in
clinical.
Figure 1 Representative MAP2 Staining at Dorsal Hippocampus Level .
Pups were Treated with Vehicle or Different Dose of NACas Indica2ted in
LPS Sensitized HI Model . Pronounced Protection Was Achieved in High
Dose NAC Treatment Pups
Figure 2 Representative Caspase - 3 Immunoblotting. The 32 kD Pro2
form Caspse - 3 Was Cleaved and Produced 29 kD and 17 kD Products in
Ipsilateral Hemisphere (L) . Cleavage Product Was Decreased after High
Dose NAC Treatment
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