J. Biomedical Science and Engineering, 2009, 2, 632-636
doi: 10.4236/jbise.2009.28092 Published Online December 2009 (http://www.SciRP.org/journal/jbise/
Published Online December 2009 in SciRes. http://www.scirp.org/journal/jbise
Normobaric hypoxia-induced brain damage in wistar rat
Ding-Yu Hu1,2, Qin Li1, Bo Li3, Rong-Ji Dai1, Li-Na Geng1, Yu-Lin Deng1*
1School of Life Science and Technology, Beijing Institute of Technology, Beijing, China;
2Department of Fire Engineering, The Chinese People’s Armed Police Force Academy, Langfang, Hebei, China;
3Beijing Vocational College of Electronic Science, Beijing, China.
Email: chem_hdy@yahoo.com.cn
Received 2 July 2009; revised 20 August 2009; accepted 28 August 2009.
The biochemical indicators of wistar rat under low oxy-
gen concentration, such as brain water content, necro-
sis, lactic acid and Na+-K+-ATPase, was detected to
evaluate normobaric hypoxia-induced brain damage
and to investigate the mechanism of wistar rat brain
injury. Histopathological changes in brain tissue in-
duced by hypoxia were investigated via hematoxylin
and eosin stain (HE). Hypoxia induced factor-1α
(HIF-1α) expression in brain was confirmed using im-
munohistochemistry. The results showed that the level
of lactic acid was positively correlated with the degree
of hypoxia, while concentration-dependent decrease in
total Na+-K+-ATPase activity was observed. Compared
with the control group, hypoxia group had a significant
difference on brain water content under severe hypoxic
conditions, the rate of brain necrosis increased obvi-
ously, followed by the increase of lactic acid level and
the decrease of Na+-K+-ATPase activity. Histopa-
thological analysis of brain confirmed that there was
neuronal cell death in hippocampal gyrus. HIF-1α ex-
pression enhanced the hypoxia adaptation capability of
the rat model through regulating the expressions of
multiple genes. Lactic acid, Na+-K+-ATPase and HIF-
1α played an important role in brain injury as a possi-
ble mechanism.
Keywords: Hypoxia; Brain Damage; HIF-1α; Rat
Hypoxia is an important pathobiological process in ma-
ny diseases and causes changing of body functions easily
[1,2]. Under airtight or demi-airtight environment, due to
the effects of organism metabolic and impairment of gas
exchange between the organization and environment,
quality of the air in the cabin gets worse gradually, con-
centration of oxygen drops rapidly and concentration of
carbon dioxide heightens rapidly. Hypoxia environment
emerges quickly after appreciably long time. Hypoxia
might lead to functional impairment, disturbance of
consciousness, reaction dullness, retardation at action,
damage of learning-memory function. Serious hypoxia
might cause pathological damage or even death. Study
on hypoxia mostly concentrated on hypoxic-ischemic
encephalopathy (HIE) [3,4], plateau hypoxia [5,6], learn-
ing-memory [7]; therapy of various diseases induced by
hypoxia and mechanisms [8,9], etc. Some studies had
upgraded to cell and molecular level.
Hypoxia-induced brain damage is a hot research area
of brain research. Brain damage may be induced by en-
ergy exhaustion in brain cell, overexpression of excita-
tory amino acids, oxygen free radical damageapoptosis
and inflammation, etc. The brain is susceptible to oxida-
tive stress. This is due to the high content of polyun-
saturated fatty acids, high rate of oxygen consumption,
regional high concentrations of iron, and relatively low
antioxidant capacity. These factors may predispose the
premature infant, apoplexy patients to brain damage.
Some of the mechanisms of hypoxia-induced brain da-
mage were tried to be elucidated but not clearly com-
pletely nowadays. More experimental data would be
needed. The investigation of the changes in energy me-
tabolites and brain damage during hypoxia and brain
hypoxic preconditioning might lead to the finding of an
effective way to protect the brain from hypoxia injury.
The goal of this study was to investigate the bio-
chemical effects of hypoxia on brain damage of rat
model in the airtight cabin and provide more data for
understanding the mechanism of brain damage. Brain
water content, necrosis area, the levels of lactic acid and
Na+-K+-ATPase activity were detected. HIF-1α (hypoxia
induced factor-1α) expression was confirmed using im-
munohistochemistry method. Histopathological changes
of brain in rat model induced by hypoxia were investi-
gated via hematoxylin and eosin stain (HE). All of rat
models were exposed to hypoxia for 2h at various con-
centrations of oxygen.
2.1 Animals
Male wistar rats weighing 180–200g (provided by Insti-
tute of Laboratory Animal Science, Chinese Academy of
Medical Science) were used in this study. Animals were
D. Y. Hu et al. / J. Biomedical Science and Engineering 2 (2009) 632-636 633
SciRes Copyright © 2009 JBiSE
allowed to acclimatize for at least 7 days prior to ex-
periment. Animals were housed at a room temperature of
22±2°C and a relative humidity of 50±10% with con-
trolled light (12-h light/12-h dark cycle, with the light
switched on at 7 a.m.). Food and water were available ad
libitum. All animals received humane care in compliance
with the Guide for the Care and Use of Laboratory Ani-
mals published by Beijing Administration Office of Lab-
oratory Animal.
2.2. Normobaric Hypoxia Equipment
Animals were placed in a custom-made 16-liter plastic
normobaric hypoxia chamber. Fresh soda lime was put
on the bottom of chamber. O2 and N2 cylinders were
linked with the chamber. The concentration of O2 was
controlled by infusing N2 at flow rate of 7.5L/min. The
concentrations of O2 and CO2 were monitored continu-
ously respectively [10]. 18%, 15%, 12%, 10%, 8%, 6%
O2 were designed and used in the experiment respec-
tively. Compared to hypoxia group, control group, which
exposed to normobaric normoxia (21% O2) without food
and water, was set up.
2.3. Water Content of Brain Tissue
After exposed to hypoxia for 2h, rats were anesthetized
with 1% pentobarbital (50 mg/kg of body weight, in-
traperitoneally) then killed by cervical dislocation. The
brain of each rat was isolated and weighted. Water con-
tent of brain tissue detected by lyopyilization was calcu-
lated as a measure of hypoxia-induced brain damage, i.e.
% water content = 100× ((wet brain weight-dry brain
weight) / wet brain weight) %.
2.4. Estimation of Brain Necrosis
After exposed to hypoxia for 2h, rats were anesthetized
with 1% pentobarbital (50 mg/kg of body weight, in-
traperitoneally), then killed by cervical dislocation. The
brain of each rat was isolated and coronally sectioned
into five slices (2 mm thick), and then those slices were
placed in 3% 2, 3, 5-triphenyltetrazolium chloride (TTC)
at 37 °C for 30 min. Those slices were dried on filter
paper and weighted respectively. Total damage sections
(grey section) were isolated and weighted. The relative
damage percentage was estimated by calculating the
brain damage area percentage by total slice (100×total
damage section / total slice).
2.5. Analysis of Lactic Acid and Na+-K+-ATPase
The levels of lactic acid and Na+-K+-ATPase activity in
rat model tissue were measured with kits (manufactured
by Nanjing Jiancheng Bio-engineering Institute) accord-
ing to the manufacturer’s instruction. After exposed to
hypoxia for 2h, rats were anesthetized with 1% pento-
barbital (50 mg/kg of body weight, intraperitoneally),
killed by cervical dislocation. The brain tissue was iso-
lated for biochemical examinations over an ice cube.
After weighting, the isolated tissue were collected in 0.1
M phosphate buffer (pH 7.4) and homogenized. The
homogenates were centrifuged at 2000 r min-1 or 1000 r
min-1 at 4 for 10 min. The supernatants were used for
analysis of lactic acid and Na+-K+-ATPase activity re-
spectively. The procedures of quantifying lactic acid and
Na+-K+-ATPase activity were carried out according to
the description of the kits. These indexes were evaluated
by means of measurement of optical density at 530nm,
636nm with a UV spectrophotometer respectively.
2.6. Histopathological Examination
In histopathological examination, rats were exposed to
6% O2 for 2h respectively and sacrificed by decapitation
whose brains were taken out and transferred to 4% para-
formaldehyde. Hippocampus sections were prepared (5
μm thick) and stained by hematoxylin and eosin. Stained
sections were evaluated qualitatively (light microscopy)
by an examiner blinded to experimental conditions.
2.7. HIF-1α Immunohistochemistry
After exposed to 6% O2 for 2h, the rats were anesthetized
with 1% pentobarbital (50 mg/kg of body weight, intrap-
eritoneally ) and perfused through the ascending aorta with
200 ml of 1% NaCl solution, followed with 200 ml of 4%
paraformaldehyde. The brain of each rat was isolated and
kept in the 4% paraformaldehyde solution until slicing.
The brains were dehydrated in 10% sucrose for 1 day and
then 30% sucrose solution for 2 days, till the brain sank to
the bottom of the bottle. Hippocampus section were cut at
35μm thickness on a freezing microtome and processed for
HIF-1α immunohistochemistry. The sections were rinsed
in PBS-T (add 1ml of tween 20 to 2L of phosphate buffer
saline), for three times. Then added with 3 ml of 1% H2O2
blocking solution at room temperature for 30 min. After
reaction, the slices were rinsed and then added with 2 ml of
5% BSA solution for 20 min. Added 1:200 dilution of rab-
bit anti-HIF-1α antibody, and weaved in the refrigerator for
24h. The reaction was followed by adding biotin labeled
monoclone mice anti-rabbit antibody. The slices were
rinsed, soaked in the SABC solution for 30 min. Then
DAB solution was used to stain for 10 min. The sections
were dehydrated in ascending alcohol concentrations,
cleared and covered in xylene. Rabbit anti-HIF-1α anti-
body, biotin labeled monoclone mice anti-rabbit antibody,
SABC and DAB solution were purchased from Boster
Biological Technology, LTD (Wuhan, Hubei, China).
2.8. Statistical Analysis
All results were expressed as mean±SEM. Statistical
analysis of data was performed by applying one-way
analysis of variance (ANOVA) followed by Tukey test.
The p values less than 0.05 were considered as statisti-
cally significant difference.
634 D. Y. Hu et al. / J. Biomedical Science and Engineering 2 (2009) 632-636
SciRes Copyright © 2009 JBiSE
3.1. Water Content of Brain Tissue
The increasing of water content of brain tissue was in-
duced by hypoxia. While exposed to 8%, 6% O2, water
contents in the rat brain were the highest. The volumes
of brain water content were 77.8% and 77.9% respec-
tively. When exposed in 10% O2 or more, the brain wa-
ter contents were located near 76.8%. Compared with
21% O2 group, there was significant difference in the
brain water content of 8%, 6% O2 group (Figure 1,
3.2. Brain Necrosis
Reduction reaction of TTC started under the effect of
chondriosome succinate dehydrogenase in competent
cell and then red stabile and indiffusible substance
would be formed, while reduction reaction of TTC did
not start in infarction section and the color of the section
would be grey. The method might be used to evaluate the
necrosis of brain. Results showed that brain infarction
ratio increased obviously under serious hypoxia condi-
tion (6% O2). For the brain infarction ratio, there was a
significant difference between group 21% O2 and group
6% O2 (Figure 2, p<0.05).
3.3. Lactic Acid
When the rat was exposed to 10% O2 or more, the level
of lactic acid in 10% brain homogenate tissue changed
from 1.23 mmol·L-1 to 1.26 mmol·L-1. while the rat
model group 6% O2 was exposed to 8% O2 or less, the
level of lactic acid increased significantly, it changed
from 1.26 mmol·L-1 to 4.2 mmol·L-1 (Figure 3). The
more serious the degree of hypoxia was, the higher the
Figure 1. Brain water content at various concentrations of
oxygen. (* p<0.05 compared with the group 21%).
lactic acid level was. It increased obviously at 10% O2 or
less. Lactic acid was accumulated in the brain sharply at
6% O2.
3.4. Na+-K+-ATPase
Hyperactivity of Na+-K+-ATPase was large enough to
maintain the ion homeostasis in the range of 21% ~12%
O2. The level of Na+-K+-ATPase activity decreased sig-
nificantly at serious hypoxia (8% O2) which induced
the function disorder of cell due to cell oedema and at-
rophia (Figure 4).
3.5. Histopathological Examination
After exposed to 6% O2 for 2h, the rat model was killed
and the brain was taken out. Hippocampus sections were
prepared for histopathological examination. Compared
with the control group, histopathological analysis of
brain confirmed that there was neuronal cell death in
Figure 2. Brain necrosis at various concentrations
of oxygen. (* p<0.05 compared with the group
21%15% 12% 10%8%6%Gro u p
Figure 3. Lactic acid levels in rat models at various
concentrations of oxygen.
D. Y. Hu et al. / J. Biomedical Science and Engineering 2 (2009) 632-636 635
SciRes Copyright © 2009
21% 15% 12% 10%8%6%
Oxygen concentration
-ATPase activity(mgprot/ml)
Figure 4. Na+-K+-ATPase activity in rat models at various
concentrations of oxygen.
hippocampal gyrus of hypoxia group (Figure 5A). Pre-
vious study indicated that there were neuronal cells
death in neuropile and cortex. When the concentration of
oxygen was 8%, 10% separately, histopathological a-
nalysis of brain showed that there was no cell death in
brain (Figure 5B and Figure 5C).
3.6. HIF-1α Immunohistochemistry
Compared to the control group, expression of HIF-1α in
rat hippocampus section was obvious in Figure 6. Ex-
periment confirmed that dilution ratio was an important
factor to complete the HIF-1α immunohistochemistry
successfully because of the instability and low abun-
dance of HIF-1α.
Figure 5. Representative photographs of histopathological
examnation (HE) in the rat hippocampus under hypoxia con-
ditions. (A: exposed to 6% O2 for 2h; B: exposed to 8% O2
for 2h; C: exposed to 10% O2 for 2h; D: exposed to normo-
baric normoxia for 2h without food and water).
Figure 6. HIF-1α immunohistochemistry photomicro-
graphs of rat hippocampus section (dilution 1:200, A: ex-
posed to 6% O2 for 2h; B: exposed to normobaric normoxia
for 2h without food and water).
Extreme hypoxia would cause acidosis easily and lead to
tissue oedema and cell death [11,12]. Results showed
that the levels of lactic acid increased significantly under
the severe hypoxia environment (6%~8% O2). It indi-
cated that hypoxia led to anaerobic metabolism and
metabolic acidosis. Pyruvic acid from glycolysis was
converted by anaerobic metabolism to lactic acid mostly.
Na+-K+-ATPase would transport Na+ ions and K+ ions
against their concentration gradient. The decrease of
Na+-K+-ATPase activity showed that the loss of ion ho-
meostasis occurred. It is generally believed that loss of
ion homeostasis played an important role in the patho-
genesis of brain cell damage. Extreme hypoxia-induced
perturbation of ion homeostasis led to the intracellular
accumulation of sodium and calcium ions, followed by
subsequent activation of proteases and phospholipases
and the formation of oxygen and nitrogen free radicals
[13]. Consequently, the events would cause changing of
functional and structural including cerebral edema,
eventually lead to cell death. Under hypoxia condition,
energy exhaustion would induce inhibition of Na+-
K+-ATPase activity and accumulation of lactic acid, fol-
lowed by acidosis and cell apoptosis.
HIF-1 is a transcriptional activator that regulates the
expression of multiple genes during continuous hypoxia
[14]. HIF-1 is composed of a constitutively expressed
HIF-1β and O2 regulated HIF-1α subunit. Previous stud-
ies confirmed that HIF-1 played a general role in coor-
dinating adaptive physiologic responses to hypoxia at
the level transcription. HIF-1α has also been implicated
in the coordinate transcriptional activation of genes en-
coding glycolytic enzymes in hypoxia cells, which pro-
vide an alternative means of energy product under con-
ditions of limited oxygen availability [15,16]. The re-
sults of immunohistochemistry confirmed that HIF-1α
was induced by hypoxia at 6% O2. Expression of HIF-1α
enhanced the hypoxia adaptation capability of the rat
model through regulating the expression of multiple
636 D. Y. Hu et al. / J. Biomedical Science and Engineering 2 (2009) 632-636
SciRes Copyright © 2009
The values of lactic acid are positively correlated with
the degree of hypoxia, while total Na+-K+-ATPase activ-
ity shows a concentration-dependent decrease. Com-
pared with the control group; hypoxia group has a sig-
nificant difference in brain water content under severe
hypoxia condition. The area of brain necrosis increases
sharply followed by the increase of lactic acid level and
the decrease of Na+-K+-ATPase activity, neuronal cell
death and HIF-1 expression appear in hippocampal gy-
rus obviously. Lactic acid, Na+-K+-ATPase and HIF-1α
played an important role as a possible mechanism in
brain injury.
This work was supported by Commission of Science Technology and
Industry for National Defense (Grant No. A2220060042) and the Na-
tional Natural Science Foundation of China (Grant No. 20705005).
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