Pediatr Neonatol 2009;50(5):208−216
O RI G I N A L A RT I CL E
Human Umbilical Cord Blood Cells Protect Against
Hypothalamic Apoptosis and Systemic Inflammation
Response During Heatstroke in Rats
Won-Shiung Liu1, Chun-Ta Chen1, Ning-Hui Foo1, Hsuan-Rong Huang1,
Jhi-Joung Wang2, Sheng-Hsien Chen2,3*, Te-Jen Chen1*
1
Department of Pediatrics, Chi Mei Medical Center, Tainan, Taiwan
Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan
3
Department of Obstetrics and Gynecology, Chi Mei Medical Center, Tainan, Taiwan
2
Received: Nov 25, 2008
Revised: Jan 7, 2009
Accepted: Jan 19, 2009
KEY WORDS:
cytokines;
heatstroke;
human umbilical
cord blood cells;
inflammation;
rat
Background: Intravenous administration of human umbilical cord blood cells
(HUCBC) has been shown to improve heatstroke by reducing arterial hypotension as
well as cerebral ischemia and damage in a rat model. To extend these findings, we
assessed both hypothalamic neuronal apoptosis and systemic inflammatory
responses in the presence of HUCBCs or vehicle medium immediately after initiation of heatstroke.
Methods: Anesthetized rats, immediately after the initiation of heat stress, were
divided into two groups and given either serum-free lymphocyte medium (0.3 mL
per rat, intravenously) or HUCBCs (5 × 106 in 0.3 mL serum-free lymphocyte medium,
intravenously). Another group of rats were exposed to room temperature (26ºC)
and used as normothermic controls. Heatstroke was induced by exposing the anesthetized rats to a high ambient temperature of 43ºC for 68 minutes.
Results: After the onset of heatstroke, animals treated with serum-free lymphocyte
medium displayed hyperthermia, hypotension, bradycardia, hypothalamic neuronal
apoptosis and degeneration, and up-regulation of systemic inflammatory response
molecules including serum tumor necrosis factor-alpha, soluble intercellular adhesion molecule-1 and E-selectin. Heatstroke-induced hypotension, bradycardia,
hypothalamic neuronal apoptosis and degeneration, and increased systemic inflammatory response molecules were significantly inhibited by HUCBC treatment.
Although heatstroke-induced hyperthermia was not affected by HUCBC treatment,
the serum levels of the anti-inflammatory cytokine interleukin-10 were significantly increased by HUCBC therapy during hyperthermia.
Conclusions: These findings suggest that HUCBC transplantation may prevent the
occurrence of heatstroke by reducing hypothalamic neuronal damage and the
systemic inflammatory responses.
*Corresponding authors. Department of Obstetrics and Gynecology; Department of Pediatrics, Chi Mei Medical Center,
901 Chung Hwa Road, Yung Kang City, Tainan, Taiwan.
E-mail: [email protected]
©2009 Taiwan Pediatric Association
HUCBCs protect heatstroke rats
209
1. Introduction
2. Materials and Methods
Based on the understanding of the pathophysiology
of heatstroke, it has been proposed that heatstroke
is a form of excessive hyperthermia (core temperature rising above 40ºC) associated with a systemic
inflammatory response that leads to multi-organ dysfunction in which central nervous system disorders
predominate.1
It is generally believed that the anterior hypothalamus preoptic area is an essential thermoregulatory center in the brain. A recent report
has demonstrated that heatstroke rats display increased levels of markers for cellular ischemia (e.g.
glutamate, lactate-to-pyruvate ratio and nitrite)
and damage (e.g. glycerol), and enhanced expression of inducible nitric oxide synthase in the hypothalamus.2 In addition, various serum molecules
including tumor necrosis factor-alpha (TNF-α),3 soluble intercellular adhesion molecule-1 (sICAM-1),4,5
and E-selectin6−8 have been demonstrated to be
involved in the pathophysiology of systemic inflammatory response syndrome.9−11 Hyperthermia,
which occurs during heatstroke, may facilitate the
leakage of endotoxin from the intestine to the systemic circulation and result in excessive activation
of leukocytes and endothelial cells.1 Thus, heatstroke is characterized by the release of TNF-α and
other cytokines, up-regulation of cell-surface adhesion molecules, and release of soluble cellsurface adhesion molecules (e.g. E-selectin and
1CAM-1).
Human umbilical cord blood cells (HUCBCs)
have emerged as an alternative to bone marrow
because of their greater availability, lower risk of
mediating viral transmission, and weaker immunogenicity.12 Evidence has accumulated to show that
HUCBC transplantation is a promising new therapeutic method against neurodegenerative diseases
such as stroke, traumatic brain injury and spinal
cord injury, as well as blood diseases.13−16 We have
demonstrated that HUCBC therapy may resuscitate
rats with heatstroke by reducing circulatory shock
and cerebral ischemia.17 However, it is unknown
whether the hypothalamic neuronal damage and
the systemic inflammatory responses that occur
during heatstroke can be reduced by HUCBC
administration.
This study possessed two objectives. The first
was to assess whether neuronal degeneration and
apoptosis in the hypothalamus, both of which occur
during heatstroke, could be reduced by HUCBC
administration. The second was to assess whether
heatstroke-induced up-regulation of TNF-α, E-selectin
and ICAM-1 in the peripheral blood stream could
be ameliorated by HUCBC administration.
2.1. Animals
Adult Sprague-Dawley rats (weighing 294 ± 15 g)
were obtained from the Animal Resource Center of
the National Science Council of the Republic of
China (Taipei, Taiwan). The animals were housed
four per group at an ambient temperature (Ta) of
22 ± 1ºC, with a 12-hour light/dark cycle. Pellet rat
chow and tap water were available ad libitum. All
protocols were approved by the Animal Ethics
Committee of the Chi Mei Medical Center (Tainan,
Taiwan) in accordance with the Guide for the Care
and Use of Laboratory Animals of the National
Institutes of Health, and the guidelines of the
Animal Welfare Act. Adequate anesthesia was
maintained to abolish the corneal reflex and pain
reflexes induced by tail pinching throughout all
experiments (approximately 8 hours in duration) by
a single intraperitoneal dose of urethane (1.4 g/kg
body weight). At the end of the experiments, control rats and any rats that had survived heatstroke
were killed with an overdose of urethane.
2.2. Surgery and physiological parameter
monitoring
The right femoral artery and vein of rats were
cannulated with polyethylene tubing (PE50), under
urethane anesthesia, for blood pressure monitoring
and drug administration. The core temperature (Tco)
was monitored continuously by a thermocouple,
while mean arterial pressure (MAP) and heart rate
(HR) were monitored continuously with a pressure
transducer.
2.3. Induction of heatstroke
The Tco of the anesthetized animals were maintained at about 36ºC with an infrared light lamp,
except during the heat stress experiments.
Heatstroke was induced by placing the animals in
a folded heating pad maintained at 43ºC by circulating hot water. The time at which the MAP
decreased irreversibly from the peak was taken
as the onset of heatstroke.18 After the onset of
heatstroke, the heating pad was removed, and
the animals were allowed to recover at room temperature (26ºC). Our pilot results showed that the
latency for onset of heatstroke in vehicle-treated
rats was 68 ± 2 minutes (n = 8). Therefore, in the
following groups of heatstroke rats, all animals
were exposed to 43ºC for exactly 68 minutes and
were then allowed to recover at room temperature
(26ºC).
210
2.4. Experimental groups
Animals were assigned randomly to one of three
groups. One group of rats, treated with an intravenous dose of vehicle solution (0.3 mL serum-free
lymphocyte medium per rat) was exposed to a Ta
of 26ºC, and the physiological parameters were
continuously recorded for up to 480 minutes (or to
the end of the experiments). This group of rats was
used as normothermic controls. The second group
was treated with the same dose of vehicle solution
after the initiation of heat exposure (Ta 43ºC for
68 minutes) and was used as vehicle-treated heatstroke animals. The third group of rats was treated
with an intravenous dose of HUCBC (5 × 106 in
0.3 mL serum-free lymphocyte medium) immediately after the initiation of heat exposure (Ta 43ºC
for 68 minutes). The last two groups of rats were
exposed to heat exposure (43ºC) for exactly 68
minutes to induce heatstroke and were then allowed to recover at room temperature (26ºC).
Physiological parameters and survival time (interval between the initiation of heat exposure and
animal death) were observed for up to 480 minutes
(or to the end of the experiments).
2.5. Preparation of HUCBCs
Human HUCBCs were obtained from the freshly
collected buffy coat fraction from healthy donors
at the Chi Mei Medical Center (Tainan, Taiwan).
This project was approved by the Institutional
Review Board of Chi Mei Medical Center. HUCBCs
were isolated by centrifugation over a Ficoll-Paque
(Pharmacia, Uppsala, Sweden) density gradient at
400× g for 30 minutes at room temperature in a
Sowall RT600 B (Du Pont, CO., Wlimington, DE, USA).
The cells collected at the interface were washed
three times in serum-free Roswell Park Memorial
Institute-1640 (BRL, Grand Island, NY, USA) and subsequently resuspended in serum-free lymphocyte
medium (Gibco, BRL). For intravenous administration, a 26-gauge needle was inserted into the rat’s
tail vein, and cells (0.3 mL) were delivered over a
1-minute period.
W.S. Liu et al
scale of 0−3, modified from the grading system of
Pulsinelli et al,19 in which 0 is normal, 1 indicates
approximately 30% of the neurons are damaged, 2
indicates that approximately 60% of the neurons
are damaged, and 3 indicates that 100% of the
neurons are damaged. Each hemisphere was evaluated independently by an examiner blinded to the
experimental conditions.
The TUNEL assay was performed using the same
hypothalamic tissue used in histological verification.
Color was developed using 3,3⬘-diaminobenzidine
tetrachloride (Sigma Chemical Co., St. Louis, MO,
USA). Sections were treated with xylene and ethanol to remove paraffin and for dehydration. They
were then washed with phosphate buffered saline
(PBS) and incubated in 3% hydrogen peroxide solution for 20 minutes. The sections were treated with
5 μg/mL proteinase k for 2 minutes at room temperature, and rewashed in PBS (0.1 M, pH 7.4). The
sections were then treated with a TUNEL reaction
mixture (terminal deoxynucleotidyl transferase,
nucleotide mixture, Roche, Mannheim, Germany)
at 37ºC for 1 hour, and the sections were washed
with distilled water. They were then incubated in
anti-fluorescein, check antibody-conjugated with
horseradish peroxidase at room temperature for
30 minutes, washed and visualized using the avadinbiotin complex (ABC) technique and 0.05% 3,3⬘diaminobenzidine tetrachloride as a chromogen. The
numbers of TUNEL-positive cells were counted by
a pathologist at 200× magnification, 30 fields per
section. Blinding was performed for the pathologist’s
grading of results.
2.7. Measurement of serum TNF-a,
ICAM-1 and IL-10 levels
Blood samples were collected, immediately separated, and stored at −80ºC until they could be
assayed. We used commercially available ELISA
kits for the determination of serum TNF-α, ICAM-1
and IL-10 levels (Quantikine, R&D Systems Inc.
Minneapolis, MN, USA) according to the manufacturer’s instructions.
2.8. Measurement of E-selectin
2.6. Neuronal damage score and apoptosis
At the end of the experiments, animals were killed
by an overdose of urethane, and the brains were
fixed in situ and left in the skull in 10% neutralbuffered formalin for at least 24 hours prior to removal from the skull. The brain was removed and
embedded in paraffin blocks. Serial sections (10 μm
thick) through the hypothalamus were stained with
hematoxylin and eosin for microscopic evaluation.
The extent of neuronal damage was scored on a
Rat peripheral polymorphonuclear (PMN) cells
were isolated from the whole blood of rats and
treated with heparin (100 units/mL). Erythrocytes
were allowed to sediment for 30 minutes after the
addition of 3 mL of 6% dextran (weight/volume in
PBS) to 10 mL blood. After sedimentation, the
plasma containing leukocytes was centrifuged
twice at 300g for 5 minutes each. The precipitates
were mixed with 70% osmolality-adjusted Percoll
and centrifuged at 30,000g for 30 minutes at 26ºC.
HUCBCs protect heatstroke rats
211
The PMN-rich layer was fractionated. Each fraction was washed twice with Hanks’ balanced salt
solution, and the cell number was counted. The
purity of the PMNs was determined to exceed 95%
by Giemsa staining. Cells (1 × 106 cells/tube) were
incubated with a rabbit polyclonal antibody to
CD62E (ab18981; Abcam PIC332 Cambridge, UK) or
control. After washing, the cells were stained with
a secondary antibody (goat polyclonal to rabbit
IgG-H&L [FITC]•[ab6717]; Abcam PIC). Cells were
incubated for 1 hour at 4ºC and washed. The cells
were mixed with oligosaccharides and incubated
for 20 minutes, and then co-incubated with KM93
for 60 minutes. The fluorescence intensity of cells
was analyzed with a FACStar (Becton Dickinson).
2.9. Statistical analysis
All data are expressed as means ± standard deviation.
One-way analysis of variance with Tukey’s multiple
comparisons test was used for serum markers. The
Wilcoxon test was used for histological assessment.
Significant differences were established at p < 0.05.
For all statistical analyses, SPSS software version
10.0 (SPSS Inc., Chicago, IL, USA) was used.
3. Results
3.1. HUCBC treatment attenuates
hypotension and improves
survival during heatstroke
Table 1 summarizes the survival time for vehicletreated and HUCBC-treated rats during heatstroke.
Table 1 Effects of heat exposure (43ºC for 68 minutes)
on survival time in rats treated with vehicle
medium or human umbilical cord blood cells
(5 × 106/0.3 mL, intravenously) immediately
after the initiation of heat exposure
Treatment group
Normothermic controls
Vehicle-treated heatstroke rats*
HUCBC-treated heatstroke rats*
Survival time (min)
> 480 (n = 8)
22 ± 2 (n = 8)†
214 ± 23 (n = 8)‡
Except for the normothermic controls, data are means ± standard deviation followed by the number of animals used in
parentheses. Group 1 was terminated about 480 minutes
after the initiation of experiment (or at the end of experiment) by an over-dose of an anesthetic, so they should survive more than 480 minutes. *For both groups exposed to
43ºC, the heat stress was withdrawn at 68 minutes and the
rats were allowed to recover at room temperature (26ºC).
†
p < 0.05 compared with normothermic controls; ‡p < 0.05
compared with vehicle-treated heatstroke rats; HUCBC =
human umbilical cord blood cells.
The survival time values were 20−24 minutes (n = 8)
and 209−237 minutes (n = 8) for vehicle-treated
and HUCBC-treated rats, respectively. The HUCBCtreated heatstroke rats had significantly longer
survival time compared with vehicle-treated heatstroke rats (p < 0.05).
Figure 1 shows the effect of heat exposure
(43ºC for 68 minutes) on Tco, MAP and HR in rats
treated with vehicle medium and in rats treated
with HUCBCs. As shown in this figure, 22 minutes
after the termination of heat exposure in the vehicle-treated group, the values of both MAP and HR
were significantly lower than those of normothermic
controls (p < 0.05). On the other hand, the values
for Tco in the vehicle-treated heatstroke rats were
significantly higher than those of the normothermic controls. Heatstroke-induced hypotension and
bradycardia, but not hyperthermia, were significantly
reduced by HUCBC treatment.
3.2. HUCBC treatment attenuates
hypothalamic apoptosis and neuronal
degeneration during heatstroke
Figure 2 summarizes the effects of heat exposure
on the number of TUNEL-positive cells in the hypothalamus of normothermic controls, vehicletreated heatstroke rats, and HUCBC-treated rats.
At 22 minutes after the onset of heatstroke, the
number of TUNEL-positive cells of the hypothalamus was greater in vehicle-treated heatstroke rats
than in the normothermic controls. However, increase of TUNEL-positive cells in the hypothalamus
of heatstroke rats was greatly attenuated by
HUCBC. A typical example of TUNEL staining of the
hypothalamus is shown in the top panel of Figure 2.
As shown in Table 2, after the onset of heatstroke,
the hypothalamic neuronal damage scores were
higher in animals treated with vehicle compared
with the normothermic controls. Histopathological
verification revealed that heatstroke caused cell
body shrinkage, pyknosis of the nucleus, loss of Nissl
substance, and disappearance of the nucleus in the
hypothalamus of vehicle-treated rats (Figure 3).
However, HUCBC treatment had neuroprotective
effects (Figure 3).
3.3. HUCBC treatment up-regulates serum
IL-10 levels but down-regulates serum
E-selectin, ICAM-1 and TNF-a levels
during heatstroke
Figure 4 shows the serum levels of E-selectin,
ICAM-1, TNF-α and IL-10 among the three experimental groups. Compared with the normothermic
controls, vehicle-treated heatstroke rats had
higher levels of E-selectin, ICAM-1 and TNF-α at 22
212
W.S. Liu et al
Onset
Injection
Injection
45
MAP (mmHg)
Ta (°C)
40
35
30
25
0
20
40
60
45
40
*
*
*
*
† †
*
* *
−60 −40 −20
100
0
20
40
60
80
*
* *
*
35
600
*
* * † †
500
400
300
200
100
0
30
−60 −40 −20
0
20
40
60
100
700
*
*
Tco (°C)
80
HR (beats·min−1)
20
−60 −40 −20
160
140
120
100
80
60
40
20
0
−20
Injection
80
100
Time (min)
* *
−60 −40 −20
0
20
40
60
80
100
Time (min)
Figure 1 Effects of heat stress (ambient temperature [Ta] 43ºC for 68 minutes) on core temperature, mean arterial
pressure and heart rate. Open circles, values at Ta of 43ºC in eight rats treated with vehicle immediately after the
onset of heat exposure. Solid circles, values at Ta of 43ºC in eight rats treated with human umbilical cord blood cells
(5 ˜ 106/0.3 mL, intravenously) immediately after the initiation of heat exposure. Another eight rats were used as
normothermic controls (solid triangles). Values are means ± standard deviation. *p < 0.05 compared with the normothermic controls; †p < 0.05 compared with the vehicle-treated group (at 43ºC). HR = heart rate; MAP = mean arterial
pressure; Ta = ambient temperature; Tco = core temperature.
minutes after the onset of heatstroke. The increase in the serum levels of these three markers
caused by heatstroke were significantly reduced
by HUCBC therapy. However, compared with the
vehicle-treated rats, HUCBC-treated rats had
higher serum levels of IL-10 at 22 minutes after
the onset of heatstroke.
4. Discussion
Heatstroke is defined as a condition in which the
core temperature is elevated to a critical level
that induces multi-organ damage and dysfunction.20−22 The severity of illness depends on
the degree of hyperthermia and its duration.23 The
current approach for treatment of heatstroke is
whole-body cooling.1 However, heatstroke is often
fatal following adequate body cooling.24,25 Tissue
damage continues to develop despite cooling of the
whole body to the normal body temperature in 25%
of heatstroke patients.26 It has also been shown
that normal volunteers can passively endure a core
temperature of about 42ºC with none or minimal
tissue injury.27,28 Indeed, as demonstrated in the
present study, in the absence of whole-body cooling,
HUCBC treatment significantly prevented the occurrence of heatstroke syndromes without affecting the induced hyperthermia. The same contention
has been previously proposed.29 Thus, it appears
that tissue ischemia and hypoxia, rather than
hyperthermia, are the main causes of heatstroke.
Evidence has accumulated to suggest that thermoregulatory deficits may occur during heatstroke.
For example, unanesthetized, unrestrained heatstroke mice displayed hypothermia when exposed
to room temperature.2,30−32 The hypothermia that
occurred after onset of heatstroke may have resulted from neuronal apoptosis and cell degeneration in the hypothalamus (as demonstrated in the
present study). In the current study, we also demonstrated the protective effects of HUCBCs in reducing heatstroke-induced hypothalamic neuronal
apoptosis and degeneration.
HUCBCs protect heatstroke rats
213
A
B
Figure 2 Histological examination of neuronal damage.
The photomicrographs of the hypothalamus in a normothermic control rat (A), a heatstroke rat treated with
vehicle (B), or a heatstroke rat treated with human
umbilical cord blood cells (5 ˜ 106/0.3 mL, intravenously)
(C) immediately after the initiation of heat stress.
Twenty-two minutes after 68-minute heat exposure, the
hypothalamus of the rat treated with vehicle showed cell
body shrinkage, pyknosis of the nucleus, loss of Nissl
substance, and disappearance of the nucleolus (B). By
contrast, administration of human umbilical cord blood
cells reduced neuronal damage, as shown in panel C.
Magnification ˜ 400.
C
Table 2 Effects of heat exposure (43ºC for 68 minutes) on neuronal damage score values of the
hypothalamus in rats treated with vehicle
medium or human umbilical cord blood cells
(5 ˜ 106/0.3 mL, intravenously) immediately
after the initiation of heat exposure
Treatment group
Normothermic controls
Vehicle-treated heatstroke rats
HUCBC-treated heatstroke rats
Neuronal damage
score (0−3)
(0, 0.75)
2 (2, 2)*
1 (0.25, 0.75)†
Values are medians with the first and third quartile in parentheses for eight rats per group. To determine neuronal damage score, the animals were killed after 68 minutes of heat
exposure.
HUCBCs have emerged as an alternative to bone
marrow because of greater availability, weaker immunogenicity and lower risk of mediating viral transmission.12 Moreover, HUCBCs are only localized by
immunohistochemistry and polymerase chain reaction analysis in injured tissue and spleen.33 When
HUCBCs are transplanted into the neonatal subventricular zone at least some of them differentiate
into neuronal and glial phenotypes within this neurogenic region.34 Upon administration of HUCBCs after
stroke,13 traumatic brain injury14 and heatstroke,17
there were significant improvements in behavioral
indicators or survival. After treatment with HUCBCs,
the neuronal damage that occurred during heatstroke was greatly reduced.17 Future studies are
required to ascertain whether heatstroke-induced
hypothalamic neuronal damage can be repaired by
HUCBC therapy.
The plasma levels of inflammatory cytokines such
as TNF-α and interleukin-1β are elevated in persons
with heatstroke.35−38 Studies in rats and rabbits
have also shown that heatstroke induces systemic
and brain production of TNF-α and interleukin1β.39,40 The increase in the plasma levels of these
proinflammatory cytokines is associated with the
severity of heatstroke. Evidence has also suggested
that IL-10 may have a therapeutic potential in acute
and chronic inflammatory disease. For example, IL10-knockout mice have an increased likelihood of
inflammatory illness41 and higher mortality rates
214
W.S. Liu et al
HS + Vehicle
HS + HUCBC
Hypothalamus
NC
800
TUNEL-positive cells
*
600
Figure 3 TUNEL-positive cells. *The number of TUNELpositive cells in the hypothalamic sections 22 minutes
after the termination of heat stress was significantly
(p < 0.05; n = 8) increased in vehicle-treated rats compared
with normothermic controls. †The number of TUNELpositive cells in the hypothalamic sections 22 minutes
after the termination of heat stress was significantly
(p < 0.05; n = 8) decreased in heatstroke rats treated with
human umbilical cord blood cells (5 ˜ 106/0.3 mL, intravenously) compared with vehicle-treated rats. HS = heatstroke; HUCBC = human umbilical cord blood cells;
NC = normothermic controls.
400
†
200
0
NC
HS + HUCBC
(5 × 106/
0.3 mL, i.v.)
*
100
250
200
150
100
†
50
0
E-selectin expression
(Mean fluorescence intensity)
Serum TNF-α (pg/mL)
300
HS +
vehicle
500
*
80
60
†
40
20
0
1000
*
400
Serum ICAM-1 (pg/mL)
Serum IL-10 (pg/mL)
†
300
200
*
100
0
800
600
400
†
200
0
NC
HS + PBMC
(5 × 106/
0.3 mL, i.v.)
HS + HUCBC
(5 × 106/
0.3 mL, i.v.)
NC
HS + PBMC
(5 × 106/
0.3 mL, i.v.)
HS + HUCBC
(5 × 106/
0.3 mL, i.v.)
Figure 4 Serum levels of tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecule (ICAM), E-selectin and
interleukin (IL)-10 among the three experimental groups. *Serum levels of TNF-α, IL-10, E-selectin and ICAM-1 determined 22 minutes after the termination of heat stress were significantly (p < 0.05; n = 8) increased in vehicle-treated
rats compared with normothermic controls. †Serum levels of TNF-α, E-selectin and ICAM-1 determined 22 minutes
after the termination of heat stress were significantly (p < 0.05; n = 8) decreased and the serum levels of IL-10 were
significantly (p < 0.05; n = 8) increased in heatstroke rats treated with human umbilical cord blood cells (5 ˜ 106/0.3 mL,
intravenously) compared with vehicle controls. HS = heatstroke; HUCBC = human umbilical cord blood cells; ICAM = intercellular adhesion molecule; IL = interleukin; NC = normothermic controls; TNF-α = tumor necrosis factor-alpha.
HUCBCs protect heatstroke rats
after experimental sepsis.42 Exogenous administration of recombinant IL-10 protects mice from lethal
endotoxemia by reducing TNF- release.43 In endotoxemic mice, neutralization of endogenously produced
IL-10 results in an increased production of proinflammatory cytokines and enhanced mortality.44
In the present study, we showed that administration
of HUCBC increased the serum levels of IL-10 and
decreased the levels of TNF-α, and prolonged the survival time during heatstroke. In addition, glucocorticoids,45 interleukin-1 receptor antagonists2 and
TNF-α may restore tissue blood flow and homeostatic
function and limit multi-organ dysfunction and death
in heatstroke. In terms of survival time, our previous
studies2,45 have shown that cortisone and an IL-1 receptor antagonist have similar potency to HUCBCs in
treating heatstroke. Therefore, we need to evaluate the therapeutic effects of the combination of
HUCBCs and anti-inflammatory or anti-cytokine drugs
during heatstroke in future studies.
TNF-α plays critical roles as a mediator of inflammatory responses.3 The expression of adhesion molecules is also induced by TNF-α, resulting in increased
leukocyte-endothelial adherence and activation of
leukocytes.46 Activation of leukocyte adhesion to the
endothelium and migration of leukocytes into the
tissue where cytotoxic chemicals are released causes
tissue injury.47 The adhesion molecule ICAM-1 mediates firm adhesion between leukocytes and endothelial cells and contributes to the migration of
leukocytes from post-capillary venues into the reperfused tissue.47,48 During inflammation, endothelial
cells express ICAM-1, which initiates adhesion and
transendothelial migration of circulating leukocytes.47 The serum TNF-α and ICAM-1 levels can be
considered as markers for the systemic inflammatory
response because they indirectly reflect the wholebody production of TNF-α and ICAM-1 in various
organs.46,49−51 CD62E (E-selectin) is an endothelial
cell-specific selectin that is expressed on cytokineinduced endothelial cells only after activation by
proinflammatory cytokines.52,53 E-selectin has been
associated with the blood vessel endothelium in diverse inflammatory situations.54 Yoshida et al reported that E-selectin expressed in endothelial cells
becomes associated with the actin cytoskeleton during leukocyte adhesion, suggesting that E-selectin
engages in transmembrane signaling upon ligand
binding.55 The serum levels of TNF-α, E-selectin and
ICAM-1 were upregulated in patients with heatstroke.1
The present results further demonstrated that the
increased serum levels of these molecules during
heatstroke in a rat model could be significantly reduced by HUCBC treatment. Considering these
observations together, it is apparent that the heatstroke-induced systemic inflammatory response
can be attenuated by HUCBC therapy.
215
5. Conclusions
In summary, our results demonstrate that, in the
absence of whole-body cooling, HUCBC treatment
significantly prevents the occurrence of heatstroke
syndrome without affecting the induced hyperthermia. Heatstroke-induced hypothalamic neuronal
apoptosis and degeneration, and the systemic inflammation response (as demonstrated by increased
serum levels of TNF-α, E-selectin and ICAM-1) can be
significantly prevented by HUCBC treatment. These
findings indicate that HUCBC transplantation may
improve heat tolerance by reducing the occurrence
of hypothalamic neuronal apoptosis and degeneration, and the systemic inflammatory response.
Acknowledgments
The study was supported in part by research grants
from the National Council of the Republic of China
(NSC95-2314-B-384-018, NSC96-2314-B-384-006-MY3
and NSC96-2314-B-384-003-MY3) and technically in
part by HealthBanks Biotech Co. Ltd.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Bouchama A, Knochel JP. Heat stroke. N Engl J Med 2002;
346:1978−88.
Shen KH, Lin CH, Chang HK, Chen WC, Chen SH. Premarin
can act via estrogen receptors to rescue mice from heatstroke-induced lethality. Shock 2008;30:668−74.
Morecroft JA, Spitz L. The role of inflammatory mediators in
necrotizing enterocolitis. Semin Neonatol 1997;2:273−80.
Menger MD, Vollmar B. Adhesion molecules as determinants
of disease: from molecular biology to surgical research.
Br J Surg 1996;83:588−601.
Boldt J, Wollbruck M, Kuhn D, Linke LC, Hempelmann G. Do
plasma levels of circulating soluble adhesion molecules
differ between surviving and nonsurviving critically ill
patients? Chest 1995;107:787−92.
Lasky LA. Selectin-carbohydrate interactions and the initiation
of the inflammatory response. Annu Rev Biochem 1995;64:
113−39.
Kansas GS. Selectins and their ligands: current concepts
and controversies. Blood 1999;88:3259−87.
Varki A. Selectin ligands. Proc Natl Acad Sci USA 1994;91:
7390−97.
Etzioni A. Adhesion molecules: their role in health and
disease. Pediatr Res 1996;39:191−8.
Endo S, Inada K, Kasai T, et al. Levels of soluble adhesion molecules and cytokines in patients with septic multiple organ
failure. J Inflamm 1995;46:212−9.
Goda K, Tanaka T, Monden M, Miyasaka M. Characterization of
an apparently conserved epitope in E- and P-selectin identified by dual-specific monoclonal antibodies. Eur J Immunol
1999;29:1551−60.
Lewis ID. Clinical and experimental uses of umbilical cord
blood. Intern Med J 2002;32:601−9.
Chen J, Sanberg PR, Li Y, et al. Intravenous administration of
human umbilical cord blood reduces behavioral deficits
after stroke in rats. Stroke 2001;32:2682−8.
216
14. Lu D, Sanberg PR, Mahmood A, et al. Intravenous administration of human umbilical cord blood reduces neurological
deficit in the rat after traumatic brain injury. Cell Transplant
2002;11:275−81.
15. Saporta S, Kim JJ, Willing AE, Fu ES, Davis CD, Sanberg PR.
Human umbilical cord blood stem cells infusion in spinal
cord injury: engraftment and beneficial influence on
behavior. J Hematother Stem Cell Res 2003;12:271−8.
16. Willing AE, Lixian J, Milliken M, et al. Intravenous versus
intrastriatal cord blood administration in a rodent model
of stroke. J Neurosci Res 2003;73:296−307.
17. Chen SH, Chang FM, Tsai YC, Huang KF, Lin MT. Resuscitation
from experimental heatstroke by transplantation of human
umbilical cord blood cells. Crit Care Med 2005;33:1377−83.
18. Chen SH, Chang FM, Tsai YC, Huang KF, Lin CL, Lin MT. Infusion
of human umbilical cord blood cells protect against cerebral ischemia and damage during heatstroke in the rat. Exp
Neurol 2006;199:67−76.
19. Pulsinelli WA, Levy DE, Duffy TE. Regional cerebral blood
flow and glucose metabolism following transient forebrain
ischemia. Ann Neurol 1982;11:499−509.
20. Epstein Y, Moran DS, Shapiro Y, Sohar E, Shemer J. Exertional
heat stroke: a case series. Med Sci Sports Exerc 1999;31:
224−8.
21. Epstein Y, Sohar E, Shapiro Y. Exertional heatstroke: a preventable condition. Isr J Med Sci 1995;31:454−62.
22. Shibolet S, Coll R, Gilat T, Sohar E. Heatstroke: its clinical picture and mechanism in 36 cases. Q J Med 1967;36:525−48.
23. Shapiro Y, Rosenthal T, Sohar E. Experimental heatstroke:
model in dogs. Arch Intern Med 1973;131:688−92.
24. Dematte JE, O’Mara K, Buescher J, et al. Near-fatal heat
stroke during the 1995 heat wave in Chicago. Ann Intern Med
1998;129:173−81.
25. Knochel JP, Reed G. Disorder of heat regulation. In: Clinical
Disorder of Fluid and Electrolyte Metabolism, 5th ed; Maxwell
MH, Kleeman CR, Martin RG, eds. New York: McGraw-Hill,
1994:1549−90.
26. Bouchama A, Cafege A, Devol EB, Labdi O, el Assil K, Seraj M.
Ineffectiveness of dantrolene sodium in the treatment of
heatstroke. Crit Care Med 1991;19:176−80.
27. Bynum GD, Pandolf KB, Schuette WH, et al. Induced hyperthermia in sedated humans and the concept of critical
thermal maximum. Am J Physiol 1978;235:R228−36.
28. Pettigrew RT, Galt JM, Ludgate CM, Horn DB, Smith AN. Circulatory and biochemical effects of whole-body hyperthermia.
Br J Surg 1974;61:727−30.
29. Chang CK, Chang CP, Chiu WT, Lin MT. Prevention and repair
of circulatory shock and cerebral ischemia/injury by various agents in experimental heatstroke. Curr Med Chem
2006;13:3145−54.
30. Leon LR, Blaha MD, DuBose DA. Time course of cytokine,
corticosterone, and tissue injury responses in mice during
heat strain recovery. J Appl Physiol 2006;100:1400−9.
31. Chatterjee S, Premachandran S, Bagewadikar RS, Bhattacharya
S, Chattopadhyay S, Poduval TB. Arginine metabolic pathways determine its therapeutic benefit in experimental
heatstroke: role of Th1/Th2 cytokine balance. Nitric Oxide
2006;15:408−16.
32. Chatterjee S, Premachandran S, Sharma D, Bagewadikar RS,
Poduval TB. Therapeutic treatment with L-arginine rescues
mice from heat stroke-induced death: physiological and
molecular mechanisms. Shock 2005;24:341−7.
33. Vendrame M, Cassady J, Newcomb J, et al. Infusion of human
umbilical cord blood cells in a rat model of stroke dosedependently rescues behavioral deficits and reduces infarct volume. Stroke 2004;35:2390−5.
34. Zigova T, Song S, Willing AE, et al. Human umbilical cord blood
cells express neural antigens after transplantation into the
developing rat brain. Cell Transplant 2002;11:265−74.
W.S. Liu et al
35. Bouchama A, Parhar RS, el Yazigi A, Sheth K, al Sedairy S.
Endotoxemia and release of tumor necrosis factor and
interleukin 1 alpha in acute heatstroke. J Appl Physiol
1991;70:2640−4.
36. Chang DM. The role of cytokines in heat stroke. Immunol
Invest 1993;22:553−61.
37. Bouchama A, al Sedairy S, Siddiqui S, Shail E, Rezeig M.
Elevated pyrogenic cytokines in heatstroke. Chest 1993;
104:1498−502.
38. Hammami MM, Bouchama A, Shail E, al Sedairy S. Elevated
serum level of soluble interleukin-2 receptor in heatstroke.
Intensive Care Med 1998;24:988.
39. Lin MT, Liu HH, Yang YL. Involvement of interleukin-1 receptor mechanisms in development of arterial hypotension in
rat heatstroke. Am J Physiol 1997;273:H2072−7.
40. Lin MT, Kao TY, Su CF, Hsu SS. Interleukin-1 beta production
during the onset of heat stroke in rabbits. Neurosci Lett
1994;174:17−20.
41. Rennick DM, Fort MM, Davidson NJ. Studies with IL-10-/- mice:
an overview. J Leukoc Biol 1997;61:389−96.
42. Berg DJ, Kuhn R, Rajewsky K, et al. Interleukin-10 is a central regulator of the response to LPS in murine models of
endotoxic shock and the Shwartzman reaction but not
endotoxin tolerance. J Clin Invest 1995;96:2339−47.
43. Gerard C, Bruyns C, Marchant A, et al. Interleukin 10 reduces
the release of tumor necrosis factor and prevents lethality in
experimental endotoxemia. J Exp Med 1993;177:547−50.
44. Standiford TJ, Strieter RM, Lukacs NW, Kunkel SL. Neutralization of IL-10 increases lethality in endotoxemia: cooperative effects of macrophage inflammatory protein-2 and
tumor necrosis factor. J Immunol 1995;155:2222−9.
45. Liu CC, Chien CH, Lin MT. Glucocorticoids reduce interleukin-1
concentration and result in neuroprotective effects in rat
heatstroke. J Physiol 2000;527:333−43.
46. Wyble CW, Desai TR, Clark ET, Hynes KL, Gewertz BL. Physiologic concentrations of TNFalpha and IL-1beta released
from reperfused human intestine upregulate E-selectin and
ICAM-1. J Surg Res 1996;63:333−8.
47. Menger MD, Vollmar B. Adhesion molecules as determinants
of disease: from molecular biology to surgical research.
Br J Surg 1996;83:588−601.
48. Hogg N, Bates PA, Harvey J. Structure and function of
intercellular adhesion molecule-1. Chem Immunol 1991;50:
98−115.
49. Chen LW, Egan L, Li ZW, Greten FR, Kagnoff MF, Karin M. The
two faces of IKK and NF-kappaB inhibition: prevention of
systemic inflammation but increased local injury following
intestinal ischemia-reperfusion. Nat Med 2003;9:575−81.
50. Kuzu MA, Koksoy C, Kuzu I, Gurhan I, Ergun H, Demirpence E.
Role of integrins and intracellular adhesion molecule-1 in
lung injury after intestinal ischemia-reperfusion. Am J Surg
2002;183:70−4.
51. Olanders K, Sun Z, Borjesson A, et al. The effect of intestinal ischemia and reperfusion injury on ICAM-1 expression,
endothelial barrier function, neutrophil tissue influx, and
protease inhibitor levels in rats. Shock 2002;18:86−92.
52. Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Gimbrone
MA Jr. Identification of an inducible endothelial-leukocyte
adhesion molecule. Proc Natl Acad Sci USA 1987;84:
9238−42.
53. Phillips ML, Nudelman E, Gaeta FC, et al. ELAM-1 mediates
cell adhesion by recognition of a carbohydrate ligand, sialylLex. Science 1990;250:1130−32.
54. Bevilacqua MP, Nelson RM, Mannori G, Cecconi O.
Endothelial-leukocyte adhesion molecules in human disease. Annu Rev Med 1994;45:361−78.
55. Yoshida M, Westlin WF, Wang N, et al. Leukocyte adhesion
to vascular endothelium induces E-selectin linkage to the
actin cytoskeleton. J Cell Biol 1996;133:445−55.