Effect of cold irritation on peripheral white blood cell and
ICAM-1, IL-1β expression
of brain tissue in rat
Tian Jin-Zhou,1,2 Yin Jun-Xiang,1, Shi Jing,1 Zhang Liu-Tong,2
Chen Yu-Jing,1 Zhang Lei-Ming1
Neurology Centre, Dongzhimen Hospital, Beijing University of
Chinese Medicine, Beijing P,R,China.
2 Department of Preclinical Medicine, Hubei College of
Traditional Chinese Medicine, Wuhan, P,R,China
3 Department of Neurology, Affiliated Lianyungang Hospital of
Nanjing University of Chinese Medicine, P,R,China
Background: Some studies have showed that hypothermia is
a neuroprotective factor for cerebral ischemic injury.
Inflammation reaction plays a very important role in
pathomechanism of neuron degeneration disease induced by
cerebral ischemia. However whether there is relationship
between cold irritation and inflammation reaction is not
well known. Objective: To explore effect of cold
irritation on peripheral white blood cell and ICAM-1, IL-1β
expression of brain tissue in rat with cerebral ischemia.
Methods: Model rats were put into low temperature water
(0º) for cold irritation for 5 minutes one time every day
for 20 days. MCAO rats were subjected to middle cerebral
artery occlusion (MCAO) using an intraluminal suture method
with permanent ligation of the ipsilateral common carotid
artery. We assessed count of the peripheral white blood
cells.The brains of all rats were cut at 1, 3, and 5 days
after cerebral ischemia and frozen brain tissues were
continuously sliced and stained immunohistochemically with
Intercellular Adhesion Molecule-1 (ICAM-1) or Interleukin-1(IL-1)
antibody. Results: Cold irritation model rats were
associated with increased leukocyte at 1 and 3 days post-ischemia,
increased ICAM-1-positive vessels at 1, 3, and 5 days, and
increased interleukin-1(IL-1) at 3 and 5 days. Vascular
pathology of the hippocampus at electron microscope levels
showed that the blood vessel has inflammation infiltration
at 1, 3 and 5 days. Conclusion:
These data demonstrate that
cold irritation significantly increased endothelial adhesion
molecule expression, leukocyte infiltration, and vascular
pathology of the hippocampus in rat. The mechanism of neuron
injury may be related to the inflammation reaction induced
by cold irritation.
Keywords: cold irritation, inflammation reaction,
interleukin-1, intercellular adhesion molecule
large number of studies have been conducted in recent years that
have consistently shown hypothermia to be an effective means of
reducing cerebral ischemic injury in both global and focal
models of stroke. Interleukin -1L (IL-1) begins to increase at
15 min and peak at 1-2 h later after transient middle cerebral
artery occlusion (tMCAO).1
Polymorphonuclear leukocytes(PMNLs, or neutrophils), another
central component, play an important role in the progress of
inflammatory response. Neutrophils appear within hours of focal
cerebral ischemia, peaking in 1-2 days later. And then they are
replaced by monocytes/macrophages at 3-7 days.2
At the same time, adhesion molecules which can be locked
on the surfaces of leukocytes and endothelial cells were one key
step of leukocyte rolling, margination and transendothelial
migration. 2 Intercellular
Adhesion Molecule-1 (ICAM-1) was expressed in microvessels at
1-3 h, and peaked at 24- 48 h and lasting for about 7 days2
in brain of rats with focal cerebral
hours hypothermia during ischemia can significantly reduce the
brain infarct volume of rats at 1, 3 and 7 days following 2 h
occlusion of the middle cerebral artery3
(MCA). Inflammation reaction plays a very important role
in pathomechanism of neuron degeneration disease induced by
cerebral ischemia. While whether cold irritation (0º) shows
neuroprotective function or neuron damage in brain of rats is
still not known. Whether there is relationship between cold
irritation and inflammation reaction is also not known. So the
purpose of this study is to explore the effect of cold
irritation on brain tissue and peripheral white blood cell and
ICAM-1, IL-1β expression in brain of a rat with cold irritation.
Materials and methods
Experimental Procedures. Animal protocols were approved by the
Stanford University Administrative Panel on Laboratory Animal
Care. Institutional guidelines were followed in all protocols.
All animal experiments were conducted in accordance with the NIH
guide for the care and use of laboratory animals (NIH
publication 80- 23). All efforts were made to minimize animal
suffering, and only the smallest number of animals were used to
generate reliable scientific data.
Animals and Experimental groups. Adult male Sprague-Dawley rats,
weighing 200-250 g, were obtained from the Experimental Animal
Center of Beijing University, China. The environment condition:
air temperature was 22±2º and air humidity was 60%. They were
maintained under controlled lighting (lights on 07:00–19:00 h)
and given free access to water and the commercial laboratory
rodent diet. Twelve hours prior to experiment, the rats were
fasted, but allowed free access to water. They were randomly
divided into three groups (72 rats per group): 1, 3 and 5 days
survival. Ice water group, MCAO group and normal group.
Preparing of cold irritation model rats. Model rats were put
into low temperature water (0º) for cold irritation for 5
minutes one time every day for 20 days. During the progress of
cold irritation rats was ensured keeping their head or nose
above water, and at the same time the body of rats were
submersed into the cold water. Adding other ice into the cold
water so that keeping water temperature. Rats were put mouse
cages and permitted access to water and the commercial
laboratory rodent diet after cold irritation.
Preparing of Middle Cerebral Artery Occlusion rats. Male Sprague-Dawley
rats weighing between 290 and 320 g (Charles River, Wilmington,
Del) were anesthetized with 3% halothane by facemask and were
subsequently maintained with 1% halothane in 200 ml/ min oxygen
and 800 ml/min air. Depth of anesthesia was assessed every 15
min by hind-limb pinch. A thermistor probe was inserted 50 mm
into the rectum and rectal temperature was maintained between
36.5º and 37.5º during ischemia. ECG leads were placed to
monitor heart rate and respirations. Physiological parameters
were monitored every 15 min and maintained in the normal range
throughout surgery. The MCA was occluded using an intraluminal
suturepre viously used by our lab.4,5 In brief, a midline
incision was made in the neck to expose the common carotid
(CCA), external carotid (ECA), internal carotid (ICA), and
pterygopalatine (PPA) arteries.
The CCA, ECA, and PPA were ligated with a 6-0 silk suture.
Ischemia was induced by inserting an uncoated, 30-mm long
segment of 3-0 nylon monofilament suture (tip rounded by flame)
19-20 mm from the bifurcation of the CCA to induce ischemia in
the arterial territory supplied by the MCA. After 2 h of
ischemia, the suture was removed and the animal was allowed to
recover. The rats must be fed for 20 days.
Plasma measurements and Counts of WBC. Measurement of the plasma
concentration of various metabolites and hormones was carried
out, either in trunk blood collected at time of death during ad
libitum feeding (leptin) or in tail-tip samples obtained after
overnight food deprivation, six at 09:00 h or 30 min after rats
were given 2 g/kg glucose in water by gavage.6 Six rats per
group were respectively anesthetized at 1, 3 and 5 days post-ischemia,
counts of the peripheral white blood cells was assessed.
Tissue Fixation. At the completion of the experiment, animals
were killed at the specified time points with 10% Chloral
Hydratea halothane overdose and prepared for histological
analysis. Animals were perfused intracardially with normal
saline followed by 4% paraformaldehyde 10% formalin. Brains were
quickly removed and sliced into 30-mm-thick coronal sections.
Brain slices were then fixed in 10% buffered formalin (pH 7.4)
for 1 week. For ICAM- , IL-1 immunohistochemistry, animals were
perfused with normal saline only. Brains were quickly removed
and cryopreserved in 20% sucrose/ phosphate-buffered saline (PBS)
solution for 24 h. Brains were then sliced into 30- m-thick
coronal sections,flashfrozen on dry ice in OCT (Miles) and
stored at -70º until use. 25-μm-thick sections were cut on
cryostat from the frozen brain slices and placed on Superfrost
Plus slides (Fischer Scientific). Sections were air dried for 24
h and then fixed for 10 min in 75% acetone/25% ethanol prior to
Freeze Sectioning. Tissue blocks from five rats were
cryoprotected by soaking in a 30% sucrose solution in PB until
they sank. Parallel series of 50 μm thick coronal sections were
then obtained on a freezing microtome. For cytoarchitectonic
reference, one series of sections was mounted onto gelatin-coated
glass slides, air-dried, stained with cresyl violet, dehydrated
and coverslipped. Other series of sections from which the
present material was taken were soaked in a buffered 20%
ethylenglycol solution and stored at –20°C.
Immunohistochemistry for Light Microscopy.
Prior to beginning the immunohistochemical protocol, a series of
three to eight coronal sections that as a group covered a
variety of rostrocaudal levels of the brain and brainstem were
selected from each brain. Sections included samples from a wide
variety of coronal levels across the cerebral hemispheres,
thalamus, hypothalamus, mesencephalon, pons, rostral medulla
oblon gata and cerebellum; the olfactory bulbs were not included.
Sections were thoroughly rinsed in PB at 4°C for 48 h. Sections
were then pretreated with a 1% hydrogen peroxide solution in
phosphatebuffered saline (PBS) solution for 20 min, rinsed, and
subsequently blocked with 10% horse serum + 3% bovine serum
albumin + 0.5% Triton X-100 in 0.1 M PBS. Sections were
incubated for 48 h at room temperature, either in mouse
monoclonal IgG 142 (1:400, Namur, Belgium), or mouse monoclonal
IgG CR-50 (1:400, RIKEN, Japan). A biotinylated horse anti-mouse
IgG (Pierce, Rockford, IL, 1:200) was used as secondary antibody.
ICAM-1 staining was performed on fresh frozen tissue using a
murine monoclonal Ab (1A29, Serotec, 1:50). Immunoreagents were
diluted in 0.1 M PBS containing 3% normal horse serum and 0.1%
Triton X-100. Sections were subsequently incubated in avidin–biotinylated
horseradish peroxidase complex (ABC, Vector Laboratories,
Burlingame, CA) in 0.1 M PBS for 1 h, and developed with 0.01%
H2O2 + 0.04% 3,3'-diaminobenzidine tetrahydrochloride (DAB) in
acetate buffer pH 6. In some experiments, we enhanced the
opacity of the reaction product by including 2.5% nickel sulfate
in the developer medium (Ni-DAB). Multiple rinses in PBS were
performed between each of the above steps. The specificity of
the monoclonal antibodies used is well characterized.8 In
addition, each experiment included a control section processed
without the primary antibodies and this always resulted in the
absence of immunostaining. The concentration of primary
antibodies was tested and optimized in preliminary experiments.
Sections were mounted on gelatin-coated glass slides and air-dried.
Some sections were lightly counterstained with cresyl violet.
All sections were finally dehydrated in graded alcohols, cleared
in xylene, and coverslipped with DePeX. Ultrathin Re-sectioning
and Electron Microscopy. Cells with identifiable cellular
morphology and evident Reelin immunostaining were selected (n =
6) from the semithin sections. These cells were photographed and
their location recorded on detailed camera lucida drawings.
Under a stereomicroscope, the tissue region (-2 mm2) containing
each cell was then dissected, flatmounted in Araldite, and re-sectioned
in ultrathin (60– 80 nm) sections.9 Some ultrathin sections were
intensified with lead citrate (0.4%), while other sections were
left without intensification. Sections were visualized at
1000–100 000x using a JEOL JEM 1010 transmission electron
microscope. Sections were imaged for analysis with a Bioscan
digital imaging system (Gatan, Pleasanton, CA, USA). For the
purpose of illustration, the regions of interest were directly
photographed on film.
Data analysis. From ICAM-1 IL-1-stained tissue, immunopositive
vessels were counted in a blinded fashion from four adjacent
fields (100×) in each of the four regions described above.
Immunopositive vessels were expressed as mean number of vessels
per field. All data was collected from single sections taken
through the central region of the infarct.
Statistical analysis. Statistical analyses for continuous data
were performed using a one-way analysis of variance followed by
a multiple comparison procedure (Bonferroni post-hoc test). All
data are expressed as mean±S.E.M. P<0.05 was considered
Changes of counts of WBC. Counts of the peripheral white
blood cells in cold irritation group and MCAO group
increased at 1 and 3 days after ischemia. There were
significantly different compared with normal group (*P<0.05,
**P<0.01). But there were not significantly different
compared between cold irritation group and MCAO group(P>0.05)(Table
Expression of ICAM-1 in CA1 region of hippocampus.
Expression of ICAM-1 in CA1 region of hippocampus increased
at 1, 3, and 5 days after ischemia in cold irritation group
and MCAO group. The optical density mean (OD Mean) were
significantly different compared with normal group (*P<0.05,
**P<0.01). But there were not significantly different
compared between cold irritation group and MCAO group(P>0.05)
(Table 2, Figure 1).
Expression of IL-1 in CA1 region of hippocampus. Expression
of IL-1 in CA1 region of hippocampus increased at 3 and 5
days after ischemia, but not at 1 day in cold irritation
group and MCAO group.The optical density means (OD Mean)
were significantly different compared with normal group
(*P<0.05, **P<0.01). But there were not significantly
different compared between cold irritation group and MCAO
group(P>0.05) (Table 3, Figure 2).
Vascular pathology of the hippocampus at electron microscope.
Vascular pathology of the hippocampus at electron microscope
levels showed that there was inflammation infiltration
in brain microvessel at 1, 3 and 5 days, and there was a
great deal of inflammation infiltration at 3 days. There
were angiostegnosis, swelling of endothelial cell
mitochondria and micrangium periphery astrocyte cellular
Hypothermia is an neuroprotective factor against cerebral
ischemic injury. This study showed that cold irritation can
increase positive expression of ICAM-1 in brain vessels at 1, 3,
and 5 days, and increase positive expression of interleukin-1(IL-1)
at 3 and 5 days. Vascular pathology of the hippocampus at
electron microscope levels showed that there were a great deal
of inflammation infiltration in brain microvessels at 1, 3 and 5
Leukocyte adhesion to endothelium is necessary for initiation of
the peripheral immune response. Once activated, leukocytes bind
to endothelial ICAM-1 through their CD11/CD18 leukointegrin.
ICAM-1 constitutively expressed at low level on vascular
endothelium, but during the progress of ischemic insult and
subsequent reperfusion, ICAM-1 has been shown to be dramatically
upregulated in brain microvasculature.10-14 ICAM-1 mRNA has been
detected at 1 h post-ischemia, reaching peak levels at 10 h
after reperfusion.15,16 Several studies have now shown that
antagonists to adhesion molecules effectively reduce reperfusion
injury after MCAO.17,18 Additionally, ICAM-1-deficient mice have
been shown to be resistant to cerebral ischemic injury.19,20
Recently, Mabuchi and colleagues conducted a study which showed
that microglia and macrophages, with their IL-1L production,
contributed significantly to the expansion of infarct following
focal cerebral ischemia in rats.21
We explored the effect of cold irritation on the number of
peripheral white blood cell and the protein expression of
ICAM-1, IL-1ƒÀ in brain tissue of rats. Results indicated that
cold irrittaion can lead to neuron damage, increase endothelial
adhesion molecule expression and enhance leukocyte infiltration
in brain of rats. The mechanism of neuron damage may be related
to the activation of inflammation reaction induced by cold
We are thankful to Mr. Congshun Song and Mr. Pengwen Wan for
their help in experiment and manuscript preparation. This work
was supported by the National Key Basic Research Program of
China(973 Grant No:2003CB517104)
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