(Stroke. 2001;32:544.)
© 2001 American Heart Association, Inc.
Original Contributions |
Reduction of Inflammatory Response in the Mouse Brain With Adenoviral-Mediated Transforming Growth Factor-ß1 Expression
From the Departments of Surgery (L.P., W.Y., X-M.C., A.L.B., G-Y.Y.) and Internal Medicine (B.J.R.), Medical School, University of Michigan, Ann Arbor; Institute of Neurology, Hua-Shan Hospital, Shanghai Medical University (People’s Republic of China) (L.P., X-M.C.); and Departments of Pediatrics, Neurology, and Anatomy, Medical School, University of Utah, Salt Lake City (A.L.B.).
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Abstract |
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Background and Purpose—Chemokines have been shown to play an important role in leukocyte and monocyte/macrophage infiltration into ischemic regions. The purpose of this study is to identify whether overexpression of the active human transforming growth factor-ß1 (ahTGF-ß1) can downregulate expression of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1
(MIP-1), and intercellular adhesion molecule-1 (ICAM-1)
and reduce ischemic brain injury.
Methods—Overexpression
of transforming growth factor-ß1 (TGF-ß1) was achieved through
adenoviral gene transfer. Five days after adenoviral transduction, the
mouse underwent 30 minutes of middle cerebral artery occlusion followed
by 1 to 7 days of reperfusion. TGF-ß1, MCP-1, MIP-1, and ICAM-1
were detected by enzyme-linked immunosorbent assay and
immunohistochemistry. Infarct areas and volumes were measured by cresyl
violet staining.
Results—MCP-1 and
MIP-1 expression is increased after middle cerebral artery
occlusion, and double-labeled immunostaining revealed
that MCP-1 is colocalized with neurons and astrocytes. Viral-mediated
TGF-ß1 overexpression was significantly greater at measured time
points, with a peak at 7 to 9 days. The expression of MCP-1 and
MIP-1, but not ICAM-1, was reduced in the mice overexpressing
ahTGF-ß1 (P<0.05).
Furthermore, infarct volume was significantly reduced in the mice
overexpressing ahTGF-ß1
(P<0.05).
Conclusions—This study
demonstrates that MCP-1 and MIP-1 expressed in the ischemic
region may play an important role in attracting inflammatory cells. The
reduction of MCP-1 and MIP-1, but not ICAM-1, in the mice
overexpressing ahTGF-ß1 suggests that the neuroprotective effect of
TGF-ß1 may result from the inhibition of chemokines during cerebral
ischemia and
reperfusion.
Key Words: cerebral ischemia, focal • cytokine • gene therapy • inflammation • mice
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inflammatory response is a common reaction of the brain parenchyma to ischemia and reperfusion1 and has been studied systematically by several research groups. Histologically, it is characterized by changes of leukocyte behavior in the microvessels. Many leukocytes roll and adhere to the postcapillary venule and capillary walls; these neutrophils then infiltrate and migrate outside the vascular walls and into the ischemic parenchyma.2 3 4 5 This inflammatory response is associated with expression of inflammatory mediators, including inflammatory cytokines,6 chemokines,7 and adhesion molecules.8 Our previous studies demonstrated that inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor-
(TNF-) are
increased after focal cerebral ischemia and that inhibiting
their actions could reduce ischemic brain
injury.9 10 We
have also shown that intercellular adhesion molecule-1 (ICAM-1) is
upregulated after ischemia and reperfusion, and this
upregulation is associated with the action of IL-1ß and
TNF-.8 11
However, IL-1ß and TNF- are poor attractants for
polymorphonuclear leukocytes and monocytes/macrophages. C-C
chemokines, such as monocyte chemoattractant protein-1 (MCP-1) and
macrophage inflammatory protein-1 (MIP-1), are
specifically involved in guiding monocytes/macrophages through
the parenchyma and toward the ischemic
area.7 12 However,
few chemokines have been studied in detail in experimental cerebral
ischemia. Transforming growth factor-ß1 (TGF-ß1) has been regarded as an important endogenous mediator that responds to ischemic injury in the central nervous system.13 Several studies demonstrated that TGF-ß1 mRNA expression was markedly increased during cerebral ischemia and reperfusion.14 15 16 17 Further studies demonstrated that both in vitro18 19 and in vivo16 20 21 22 administration of TGF-ß1 could attenuate ischemic brain injury. However, little is known of the mechanism through which TGF-ß1 acts during cerebral ischemia and postischemic reperfusion. Based on the actions of TGF-ß1 in the brain, the mechanisms through which TGF-ß1 acts may include the following: (1) modulation of the inflammatory cytokine cascade; (2) inhibition of T and B lymphocyte proliferation; (3) an antioxidative effect; and (4) an antiapoptotic effect. Although central or systemic administration of TGF-ß1 reduces ischemic brain injury in short-term experiments, several limitations are of concern. Activated TGF-ß1 is eliminated from the blood in a few minutes.23 Providing and maintaining an effective concentration of TGF-ß1 in the brain over a long time is difficult to achieve through systemic administration.
The specific aim of this study is to identify the mechanism
by which TGF-ß1 modulates ischemic brain injury. Using a
mouse temporary middle cerebral artery occlusion (MCAO) model, we
examined whether overexpression of the active human TGF-ß1
(ahTGF-ß1) gene can reduce the expression of MCP-1, MIP-1, and
ICAM-1 and then reduce ischemic brain injury. A better
understanding of the mechanisms through which TGF-ß1 acts may lead to
more effective therapies that limit brain injury during
ischemia and postischemic
reperfusion.
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Materials and Methods |
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Vector Construction and Intracerebral Injection
The construction of AdRSVlacZ, a human adenovirus serotype 5–derived adenoviral vector that transduces Escherichia coli ß-galactosidase, has been described elsewhere.24 25 A recombinant replication-defective (E1, E3 deleted) serotype 5 adenoviral vector was constructed for the overexpression of ahTGF-ß1. The human TGF-ß1 cDNA was generated as described,26 and both the 5' and 3' ends of the cDNA were modified with the use of Not 1 linkers. The resulting 2.0-kb ahTGF-ß1 cDNA fragment was cloned into the Not 1 site of the proviral plasmid pAdRSV4. The plasmid pAdRSVahTGF-ß1 was then used to generate recombinant adenoviral clones, as previously described.27 Individual clones of AdRSVahTGF-ß1 were isolated and subjected to 2 additional rounds of plaque purification before large-scale expansion and purification over CsCl gradients. High-titer stocks of AdRSVahTGF-ß1 were tested in vitro by infection of the TGF-ß1–deficient cell line 159153.2 followed by analysis of cell lysates and conditioned media by enzyme-linked immunosorbent assay (ELISA) (R&D Systems). The conditioned media was also analyzed for biological activity with the CCL4 mink lung epithelial cell DNA synthesis inhibition assay.28 Purification was done by CsCl banding and desalting over Sephadex G-50; high-titer stocks were stored at -20°C in PBS containing 5% glycerol and were diluted with complete media or additional PBS immediately before use. The AdRSVlacZ served as a control virus carrying a lacZ gene instead of TGF-ß1.
The institutional animal use and care committee approved procedures for the use of laboratory animals. Mature male CD-1 mice (Charles River, Wilmington, Mass) weighing 30 to 35 g were anesthetized with 4% chloral hydrate (400 mg/kg body wt IP). After induction of anesthesia, the mice were placed in a stereotaxic frame with a mouse holder (Kopf model 921, David Kopf Instruments), and a right burr hole was drilled in the pericranium 1 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture. A 28-gauge needle affixed to a Hamilton syringe was slowly inserted into the right lateral ventricle (3.0 mm deep from dura). One microliter of adenoviral suspension containing 1x1012 particles per milliliter was injected stereotaxically into the lateral ventricle at a rate of approximately 0.2 µL/min. The control animals were injected with the same amount of saline at the same injection rate. The needle was then withdrawn over 5 minutes. The hole was sealed with bone wax, and the wound was closed with a suture. The animals were allowed to recover in their cages.
Human TGF-ß1, MCP-1, and MIP-1
ELISA
Over the next 2 weeks, groups of 5 to 7 treated mice
were anesthetized with 4% chloral hydrate
intraperitoneally, as above, and killed, and their
brains were removed. These samples were quickly frozen in liquid
nitrogen until the recombinant human TGF-ß1 (rhTGF-ß1) ELISA was
performed. The brain was divided into contralateral and ipsilateral
hemispheres, and tissue samples were weighed, homogenized
in the lysis buffer, and centrifuged at
30 000g for 30 minutes. The
supernatants were collected for a commercially available rhTGF-ß1
ELISA (Quantikine, R&D Systems, Inc). All the supernatant samples were
assayed in duplicate. The optical density was determined by a
microplate reader set to 450 nm with the wavelength set to 540 nm.
Authentic rhTGF-ß1 was used to obtain a standard curve.
In addition, MCP-1 and MIP-1 concentrations were
determined with the use of MCP-1 and MIP-1 ELISA kits (Quantikine,
R&D Systems, Inc) according to the manufacturer’s
instructions.
Temporary Middle Cerebral Artery
Occlusion
Five days after adenoviral injection, the mice were
anesthetized with 1.5% isoflurane in 70%/30%
N2O/O2. A polyethylene
catheter (PE-10) was introduced into the left femoral artery for
continuous monitoring of arterial blood pressure, sampling
of blood gases, and pH analysis. Body temperature was
maintained at 37.0±0.5°C with a rectal temperature probe and a
regulated heating pad (YSI model 73ATD Indicating Controller, Yellow
Springs Instrument Co). Mean arterial blood pressure was
maintained at >90 mm Hg, and blood gases were analyzed
during each anesthesia period. The temporary MCAO method
has been described in our previous
studies.29 Briefly, the
internal carotid artery was isolated, and the pterygopalatine artery
was ligated. Then a 2-cm length of 5-0 rounded nylon (Dermalon)
suture with a slightly larger tip was gently advanced from the external
carotid artery to the beginning of the middle cerebral artery for a
distance of 10.0±0.5 mm. Reperfusion was performed by partially
withdrawing the suture from the internal carotid artery to the common
carotid artery. The occlusion lasted for 30 minutes, and reperfusion
was maintained for 1, 3, and 7 days. Ischemia
production was confirmed by surface cerebral blood flow (CBF)
measurement with the use of a laser-Doppler flowmeter (model
BPM2 System, Vasamedics Inc), as described
elsewhere.30 Baseline blood
flow recordings of these 3 regions were made 5 minutes before
the occlusion. Blood flow values were calculated and expressed as a
percentage of baseline values.
Infarct Volume
Three groups of mice (AdRSVahTGF-ß1 transduced,
AdRSVlacZ transduced, and saline treated; n=8 per group) were killed 1
day after temporary MCAO by decapitation. The brains were removed and
frozen immediately in 2-methylbutane at -42°C for a 5-minute
period. Cryostat sections (20 µm thick) distal from the frontal pole
were cut and mounted on slides. The sections were dried and then
stained with cresyl violet. With the use of NIH Image 1.62 software,
the ischemic lesion area was calculated as the difference in
area of the nonischemic hemisphere and the normal area in the
ischemic hemisphere. The infarct volume was calculated by
multiplying the infarct areas by the thickness of the
sections.
MCP-1 and ICAM-1 Immunohistochemistry
The experimental group design and the brain section
preparation were as described above. At specific time points, animals
were anesthetized and perfused transcardially with 20 U/mL
heparin in 0.1 mol/L PBS (pH 7.4) and 2%
paraformaldehyde solution. The brains were quickly
removed, post-fixed in 2% paraformaldehyde solution
overnight at 4°C, and immersed in 25% sucrose until they sank. The
brains were then embedded in Tissue-Tek O.C.T. (Sakura Finetek U.S.A.
Inc). Coronal sections (20 µm thick) were cut with the use of a
cryostat (model CM1800, Leica). Nonspecific binding sites were treated
with 15% normal rabbit serum for 30 minutes at room temperature.
Sections were incubated with 1:300 dilution of goat anti-mouse MCP-1
antibody (Santa Cruz Biotech) overnight at 4°C. Normal goat IgG was
used as a negative control. After treatment with 1%
H2O2 in 30%/70%
methanol/PBS solution, the sections were incubated with 1:500 dilution
of biotinylated rabbit anti-goat IgG antibody (Vector Laboratory) for
90 minutes at room temperature followed by an ABC process (ABC-Elite
Kit, Vector Laboratory). Finally, the sections were treated with stable
3,3'-diaminobenzidine tetrahydrochloride (Research Genetics) as
a peroxidase substrate. The sections were then rinsed in water, lightly
counterstained with hematoxylin, dehydrated through a graded series of
alcohols, cleared with xylene, and coverslipped with Permount mounting
media.
A double-labeled fluorescent immunohistochemical method was used in this study. After incubation with 5% normal donkey serum containing 2% bovine serum albumin and 0.1% saponin in PBS for 30 minutes at room temperature, the sections were incubated with 1:100 dilution of goat anti-mouse MCP-1 antibody (Santa Cruz Biotech) and 1:100 dilution of rabbit anti–glial fibrillary acidic protein (GFAP) antibody (for astrocytes; DAKO) or 1:100 dilution of rabbit anti–neuron-specific enolase (NSE) antibody (for neurons; Chemicon) overnight at 4°C. After they were washed, the sections were incubated with a 1:50 dilution of fluorescein isothiocyanate–conjugated donkey anti-goat IgG (Santa Cruz Biotech) and 1:50 dilution of rhodamine-conjugated donkey anti-rabbit IgG (Santa Cruz Biotech) for 1 hour at room temperature. Double-labeled immunostaining was evaluated with a fluorescence microscope (Nikon Microphoto-SA) with a filter cube (excitation filter, 450 to 490 nm; suppression filter, 515 to 560 nm) for fluorescein isothiocyanate labeling and another filter cube (excitation filter, 515 to 560 nm; suppression filter, 590 nm) for rhodamine. Photomicrographs were obtained by changing the filter cube without altering the position of the section and focus.
The procedure for ICAM-1 immunostaining was the same as for the MCP-1 immunostaining except for the primary antibodies. A rat monoclonal anti-mouse CD54 primary antibody (1:300 dilution; Caltag) was chosen. The total numbers of deep brown–stained ICAM-1–positive vessels were counted in 3 control sections located at +0.86, +0.38, and -0.10 mm from bregma. The counting was done manually to exclude the infiltrating cells occasionally expressing ICAM-1.
Statistical Analysis
All data are expressed as mean and SEM.
Parametric data among the AdRSVahTGF-ß1, AdRSVlacZ, and
saline control groups were evaluated with ANOVA followed by
Scheffé’s between-group comparison (Statview, Abacus Concepts Inc).
A value of P<0.05 was
considered statistically
significant.
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Results |
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During the temporary MCAO procedure, the animal’s body temperature was kept at 37±0.5°C. Physiological parameters, including blood pressure, blood PO2, PCO2, and blood pH, were all kept at normal levels, and there was no difference among the 3 groups (P>0.05; data not shown).
TGF-ß1 Expression
The level of rhTGF-ß1 in the mouse brain after
AdRSVahTGF-ß1 intraventricular injection was
measured by ELISA. In the normal mouse brain, rhTGF-ß1 concentration
was undetectable (<100 pg/g wet wt brain tissue). After cerebral
adenoviral transduction, the level of rhTGF-ß1 gradually increased
from day 3, peaked at days 7 to 9, and then decreased by day
14.31 The level of
rhTGF-ß1 was also measured in the temporary MCAO mice
(Figure 1
). These mice underwent a single
intraventricular injection of AdRSVahTGF-ß1 or
AdRSVlacZ or the same amount of saline 5 days before MCAO. There was no
statistical difference in the expression of rhTGF-ß1 between the
AdRSVlacZ-transduced and the saline control group mice
(P>0.05), but this expression
was slightly higher compared with the control (nonoperated) animals. In
the AdRSVahTGF-ß1–transduced mice, the levels of rhTGF-ß1 in the
right hemisphere (adenoviral-injected side) were 662±50, 1455±60, and
502±10 pg/g wet wt brain tissue 1, 3, and 7 days after temporary MCAO,
respectively. Meanwhile, the levels of rhTGF-ß1 in the left
hemisphere (ischemic side) were 839±107, 1139±119, and
299±116 pg/g wet wt tissue at the same time points after temporary
MCAO. The rhTGF-ß1 levels in both the ischemic hemisphere and
the contralateral hemisphere were similar
(P>0.05) 1 day after temporary
MCAO; however, the rhTGF-ß1 level in the contralateral hemisphere was
higher than that in the ischemic hemisphere 3 and 7 days after
temporary MCAO
(P<0.05).
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Surface CBF
Surface CBF was measured in the
AdRSVahTGF-ß1–transduced, AdRSVlacZ-transduced, and saline control
mice after temporary MCAO. CBF in the contralateral hemisphere during
occlusion was approximately 90% of baseline CBF. There were no
differences among the 3 groups of mice
(P>0.05). Surface CBF was
reduced in both the core (approximately 8% to 10% of baseline CBF)
and the perifocal area (approximately 25% of baseline CBF) in all 3
groups of animals at 5 minutes of occlusion. CBF recovered to >80% of
baseline flow after 5 to 10 minutes of reperfusion in both the
ischemic core and the perifocal area in these 3 groups of mice.
There was no difference between 30 minutes after reperfusion and 1 day
after reperfusion
(Figure 2;
P>0.05).
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Infarction Area and Volume
The infarct areas in the AdRSVahTGF-ß1–transduced,
AdRSVlacZ-transduced, and saline control groups 24 hours after
temporary MCAO are shown in
Figure 3A. The infarct areas in the
AdRSVahTGF-ß1–transduced mice on sections 2 to 12 were significantly
smaller compared with the AdRSVlacZ-transduced and saline control group
mice (P<0.05). Infarct volumes
in the AdRSVahTGF-ß1–transduced, AdRSVlacZ-transduced, and saline
control group 24 hours after temporary MCAO were 14±1, 21±2, and
22±3 mm3, respectively
(Figure 3B). There was no difference between the
AdRSVlacZ-transduced and saline control group
(P>0.05). However, infarct
volume in the AdRSVahTGF-ß1–transduced mice was smaller than in the
other 2 groups of mice
(P<0.05). The infarct volume
measurements 3 and 7 days after temporary MCAO in these 3 groups
paralleled the 1-day post–temporary MCAO group; there were no
further reductions observed after a longer reperfusion
time.
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Expression of MCP-1 and MIP-1 in Mouse
Brain
The levels of MCP-1 and MIP-1 in the brains were
determined by ELISA. In the normal brain, the levels of MCP-1 and
MIP-1 were <150 pg/g wet wt brain tissue. The levels of MCP-1 and
MIP-1 were slightly increased in the contralateral hemisphere in the
AdRSVahTGF-ß1–transduced, AdRSVlacZ-transduced, and saline-treated
groups of mice 1, 3, and 7 days after temporary MCAO. There were no
significant differences among the 3 groups
(Figure 4A and 4C;
P>0.05). The levels of MCP-1
and MIP-1 were greatly increased in the ipsilateral hemisphere 1 and
3 days after temporary MCAO in all 3 groups of mice and then gradually
decreased
(Figure 4B and 4D;
P<0.05). There was no
statistical significance between AdRSVlacZ-transduced and
saline-treated mice. However, MCP-1 and MIP-1 in the
AdRSVlacZ-transduced and saline control mice increased more than in the
AdRSVahTGF-ß1–transduced mice
(P<0.05).
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Immunohistochemistry showed that MCP-1 immunoreactivity
paralleled the ELISA results. Few cells staining positive for MCP-1
were detected in the contralateral hemisphere of the experimental
animals after temporary MCAO. The negative control sections showed no
immunopositive-staining cells. MCP-1 expression was observed 12 hours
after the onset of temporary MCAO in the ipsilateral hemisphere,
gradually increased at 24 to 48 hours, and then subsided
(Figure 5A and 5B). Double-labeled immunohistochemical
studies revealed that MCP-1 and NSE, and MCP-1 and GFAP were
colocalized in neurons and astrocytes
(Figure 5D and 5E). Colocalized positive-stained cells were
massively seen in the infarct hemisphere, which indicated that neurons
and astrocytes were the sources of MCP-1 during MCAO.
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Figure 6 showed that expression of MCP-1–positive cells was
increased in the ipsilateral hemisphere after 3 days of temporary MCAO.
However, the MCP-1 immunopositive-staining cells were attenuated in the
AdRSVahTGF-ß1–transduced mice compared with the AdRSVlacZ-transduced
and saline control groups. These MCP-1 immunopositive-staining cells
were mainly located around the ischemic bound zone and showed
neuron and astrocyte morphology.
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Immunostaining of
ICAM-1
There were a few ICAM-1–positive vessels in the
contralateral hemisphere among the AdRSVahTGF-ß1–transduced,
AdRSVlacZ-transduced, and saline control groups. The expression of
ICAM-1–positive vessels was increased in the ipsilateral hemisphere
after 24 hours of temporary MCAO. However, there were no statistically
significant differences among AdRSVahTGF-ß1–transduced,
AdRSVlacZ-transduced, and saline control groups (217±11 versus 259±53
and 241±39, respectively;
P>0.05). ICAM-1–positive
vessels were found in the ischemic core at the early time
points after temporary MCAO and in the perifocal region at later time
points, probably because of the death of a large number of brain cells
at the later time points
(Figure 7). The negative control sections showed no
ICAM-1–positive staining.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study demonstrates that (1) the rhTGF-ß1 gene can be locally overexpressed through intraventricular injection of AdRSVahTGF-ß1 and is overexpressed in the mouse brain for 2 weeks; (2) ischemic brain injury is reduced in the AdRSVahTGF-ß1–transduced mice; and (3) inflammatory mediators such as MCP-1 and MIP-1
are reduced in
the AdRSVahTGF-ß1–transduced mice. These findings suggest that the
neuroprotective effect of TGF-ß1 may be related to downregulating
inflammatory mediators during temporary cerebral ischemia.
Furthermore, AdRSVahTGF-ß1 gene transfer may provide a useful tool in
vivo for developing animal models of gene intracerebral
transduction. There are several advantages to introducing the TGF-ß1 gene into mouse brain with the use of an adenoviral vector. Recombinant adenoviruses can transduce multiple types of brain cells, including terminally differentiated cells such as neurons, glial cells, and ependymal cells.32 The adenoviral vector was injected on the right site of brain rather than the ischemic hemisphere because an inflammatory response to the adenovirus at the injection side might affect ischemic damage. Right intraventricular injection of the transgene resulted in a marked increase of target protein in the CSF or brain tissue.30 33 Our previous studies demonstrated the distribution of the lacZ gene (using X-Gal staining) in the AdRSVlacZ-transduced rodent brain.30 33 AdRSVahTGF-ß1 gene transfer produces a period of transient TGF-ß1 overexpression (approximately 2 weeks), which parallels the period of ischemic brain injury.31 As expected, our data show that AdRSVahTGF-ß1 could mediate transient overexpression of rhTGF-ß1 in murine brain. Expression increased at day 1, plateaued at days 7 to 9, and then gradually decreased. Henrich-Noack et al19 reported that the effect of TGF-ß1 during cerebral ischemia is dose dependent; a single dose of TGF-ß1 (4 ng ICV) shows a neuroprotective effect. In our study the ahTGF-ß1 level was >1 ng/g brain tissue at 5 days after adenoviral injection and persisted at detectable levels for another 5 days. A relatively low concentration of activated TGF-ß1 may effectively protect the brain from ischemic injury if the rhTGF-ß1 level is maintained for several days. TGF-ß1 was continuously overexpressed 7 days after temporary MCAO, although there was relatively low expression on the first day after temporary MCAO. This result suggests that adenoviral gene transfer can upregulate TGF-ß1 expression even during transient ischemia.
Our data demonstrate that the local overexpression of TGF-ß1 in the brain can reduce infarct volume measured 24 hours after temporary MCAO. Ischemic brain injury alters the pattern of gene expression, including the TGF-ß1 gene.34 35 36 Endogenous TGF-ß1 may play several roles during ischemic brain injury. TGF-ß1 reduces the activity and function of leukocytes during cerebral ischemia in the rabbit model of thromboembolic stroke.21 Moreover, TGF-ß1 may regulate inflammatory mediators such as IL-1ß in the ischemic brain37 and may also protect neurons from oxidative, apoptotic, and excitotoxic neuronal injury.19 38
No differences were observed in physiological parameters after temporary MCAO among the 3 groups, demonstrating that the adenoviral transduction does not cause visible toxic side effects. Surface CBF in the ischemic hemisphere of the AdRSVahTGF-ß1–transduced, AdRSVlacZ-transduced, and saline-treated mice all decreased to approximately 10% of baseline CBF after temporary MCAO, and no significant differences were detected among these groups, confirming our previous observation and suggesting that adenovirus transduction does not affect CBF and that the protective effect of TGF-ß1 during ischemia is not due to higher CBF from inadequate occlusion of the middle cerebral artery.
Focal cerebral ischemia elicits an inflammatory
response characterized by the infiltration and accumulation of
leukocytes and monocytes/macrophages and the secretion of
inflammatory
mediators.39 40 41
Chemokines were a new group of chemoattractants focusing on specific
leukocyte
populations42 43 :
the C-X-C or -chemokine (IP-10, MIP-2, and IL-8) and the C-C or
ß-chemokine (MIP-1, RANTES [regulated on activation, normal T
cells expressed and secreted], and MCP1/2/3). The
-chemokines showed a special chemoattraction and activation on
polymorphonuclear leukocytes, and the ß-chemokines chemoattract
monocytes, lymphocytes, eosinophils, and
basophils.43 MCP-1 is one of
the most potent ß-chemokines for human monocytes in vitro and can
induce the secretion of degradative
enzymes.44 Cultured human
fetal microglial cells and astrocytes can produce MCP-1 when stimulated
by lipopolysaccharide, IL-1ß, and TNF-. The microglial
cells also show increased migratory response to the
ß-chemokines.45 Gourmala
et al46 reported that MCP-1
mRNA was present in rat astrocytes surrounding the ischemic
tissue between 6 hours and 2 days after MCAO. After 4 days, MCP-1 mRNA
was found in macrophages and microglial cells in the infarct
tissue. Wang et al47
demonstrated that MCP-1 mRNA increased in the rat brain with temporary
MCAO or permanent MCAO at 6 hours and peaked between 12 and 48 hours;
increased MCP-1 protein expression was still detectable 5 days after
permanent MCAO. Kim et al48
found that both MCP-1 and MIP-1 mRNA were weakly expressed at 6
hours, peaked at 2 days, and were markedly attenuated at 4 days after
MCAO. Recently, Ivacko et
al49 demonstrated that MCP-1
mRNA and protein expression are increased in the neonatal rat brain
after hypoxia-ischemia injury. The temporal profile of
MCP-1 expression is in agreement with that of leukocyte accumulation in
ischemic brain parenchyma. In these models,
polymorphonuclear cells are detected 24 to 72 hours after
ischemia, followed at 7 to 16 days by monocyte and
macrophage
infiltration.4 However, the
expression of MCP-1 protein during ischemia and reperfusion is
still not completely elucidated. We identified that neurons and
astrocytes appear to be associated with MCP-1 expression during MCAO.
MCP-1 immunoreactivity was observed after 12 hours of MCAO, gradually
increased at 1 day, peaked at 2 and 3 days of MCAO, then subsided
(Figure 5
). This result suggests that MCP-1 expression might
correlate with necrosis since decreased synthesis of MCP-1 in the
ischemic core could be observed.
Increased expression of adhesion molecules has been demonstrated after cerebral ischemia and reperfusion. Several research groups reported that ICAM-1 and endothelial leukocyte adhesion molecule-1 (ELAM-1) are upregulated from 1 to 3 hours up to 1 week after temporary MCAO in rats.50 51 52 In the baboon MCAO model, Okada et al53 found that ICAM-1 and ELAM-1 upregulated and similarly localized to the endothelium of postcapillary microvasculature in the ischemic penumbra. Another supportive result is the inhibition of endothelial interactions with the leukocyte counterpart of ICAM-1 binding and the reduction in infarct size by 45% to 50% in the rat transient MCAO model.54 We did not find differences in ICAM-1 expression in the AdRSVahTGF-ß1–transduced, AdRSVlacZ-transduced, and saline-treated mice, suggesting that TGF-ß1 may not regulate ICAM-1 expression during ischemia and reperfusion.
In conclusion, this study describes a close correlation
between TGF-ß1 overexpression and neuroprotection against cerebral
ischemia and reperfusion. Inflammatory mediators such as MCP-1
and MIP-1, but not ICAM-1, are reduced in the rhTGF-ß1–transduced
mice, suggesting that TGF-ß1 may play a role in regulating chemokine
release and may attenuate ischemic brain injury. However,
overexpression of TGF-ß1 may directly reduce infarct volume and
thereby reduce chemokine expression as a secondary effect. Proving
which is cause and which is effect will require much additional
research in the future. On the basis of the potential role of TGF-ß1
as an anti-inflammatory cytokine, these results indicate
TGF-ß1 is a novel target molecule for the treatment of stroke and
further support a role for inflammatory response in the pathogenesis of
stroke.
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Acknowledgments |
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This study was supported by grants NS35089 (Dr Yang) and NS23870 (Dr Betz) from the National Institutes of Health. The authors thank Richard F. Keep for helpful discussion and Kathleen Donahoe for editorial assistance.
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Footnotes |
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Reprint requests to Guo-Yuan Yang, MD, PhD, University of Michigan, 5550 Kresge I/0532, 1500 E Medical Center Dr, Ann Arbor, MI 48109-0532.
Received August 1, 2000; revision received October 18, 2000; accepted October 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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