(Stroke. 2000;31:1735.)
© 2000 American Heart Association, Inc.
Original Contributions |
Contribution of Microglia/Macrophages to Expansion of Infarction and Response of Oligodendrocytes After Focal Cerebral Ischemia in Rats
From the Department of Internal Medicine and Therapeutics, Division of Strokology (T.M., K. Kitagawa, T.O., K. Kuwabara, Y.Y., M.H., M.M.), and Department of Neurology (T.Y., M.M.), Osaka University Graduate School of Medicine (Japan).
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Abstract |
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Background and Purpose—The purpose of this study was (1) to examine the contribution of microglia and macrophages with their interleukin-1ß production and (2) to assess the vulnerability and response of oligodendrocytes in cerebral infarction.
Methods—Male Wistar rats were subjected to permanent occlusion of the left middle cerebral artery. Expansion of ischemic infarction and response of oligodendrocytes were investigated together with accumulation of inflammatory cells, production of interleukin-1ß, and disruption of the blood-brain barrier. Apoptotic cell death was inferred from fragmented DNA and the expression of proapoptotic Bax protein.
Results—During expansion of infarction, amoeboid microglia and extravasation of serum albumin were observed not only in the infarcted area but also in the adjacent surviving area, whereas macrophages accumulated along the boundary and granulocytes migrated into the center of the infarction. Both amoeboid microglia and macrophages produced interleukin-1ß, an inflammatory cytokine, during an early ischemic period. Furthermore, macrophages within the infarcted tissue expressed Bax protein and subsequently showed fragmented nuclear DNA. Oligodendrocytes were detected in the infarcted area even after 24 hours following middle cerebral artery occlusion, but they subsequently developed fragmented DNA. A week after onset of ischemia, oligodendrocytes were found to be accumulated in the intact area bordered with the infarct together with reactive astrocytes.
Conclusions—-Our results suggest the importance of amoeboid microglia, macrophages, and their interleukin-1ß production in gradual expansion of cerebral infarction. Resident oligodendrocytes may be resistant to ischemic insults, and oligodendrocytes accumulated at the border of the infarction may participate in tissue repair after cerebral infarction.
Key Words: apoptosis • cerebral infarction • interleukins • macrophages • microglia • oligodendrocytes • rats
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Introduction |
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It is known that cerebral infarction expands slowly over 24 hours from the core to the peripheral area after occlusion of a middle cerebral artery (MCA) in the rodent,1 where the peripheral area is a potential target for therapeutic intervention.2 3 However, the molecular mechanism of the expansion of infarction to the peripheral area remains unclear, although several studies have focused on the role of inflammatory cells, including granulocytes and microglia.4 5 6 To clarify the type of inflammatory cells directly involved in the expansion of infarction, we first examined the relationship between expansion of the ischemic lesion and the appearance of inflammatory cells together with inflammatory cytokines in the rat brain after occlusion of the left MCA. In the mouse brain, oligodendrocytes have been shown to be more resistant to ischemic insults than neurons,7 but it is still unknown whether oligodendrocytes are resistant to ischemia in other rodent brains because they are considered susceptible to hypoxia and ischemia, at least in the developing brain.8 9 It has been difficult to detect oligodendrocytes with conventional histological technique. However, we have used a sensitive in situ hybridization method and succeeded in finding oligodendrocytes in paraffin-embedded sections of adult rodent brains.7 We therefore investigated the vulnerability of oligodendrocytes in ischemic mouse brain by using the in situ hybridization technique.
Another purpose of this study was to investigate the dynamics of tissue repair after cerebral infarction. After the process of cerebral infarction is completed in 2 to 3 days after vessel occlusion, the process of tissue repair or remodeling is activated, and the infarction is eventually encircled by glial scar a few weeks later.6 For resorption of infarcted tissue, macrophages and granulocytes invading the infarcted tissue have to succumb following their efforts in the injured tissue. We used in situ end labeling of fragmented DNA and immunohistochemistry for Bax, an inducer of apoptosis, to investigate whether those macrophages and granulocytes were removed through apoptosis after they invaded the infarction. Another important aspect of tissue repair is the formation of glial scar that reestablishes the interface in the glial-pial boundary.10 While participation of astrocytes is well known in formation of glial scar, the role of oligodendrocytes has not drawn attention. We therefore investigated whether oligodendrocytes were involved in this process by using the in situ hybridization technique.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Animals
Forty-seven adult male Wistar rats weighing 250 to 300 g (Charles River Laboratory, Kanagawa, Japan) were used. They were fed standard laboratory chow and had free access to water before and after the surgical procedure. Animals were cared for according to the guidelines of the Animal Center of the Osaka University Graduate School of Medicine.
Surgical Procedure
Each animal was anesthetized with halothane, and
occlusion of the left MCA was accomplished according to Koizumi et
al11 and Longa et al.12 Briefly, the left
common carotid artery was exposed through a midline incision, and the
internal carotid artery was isolated and carefully separated. A 4-0
nylon monofilament, whose tip was rounded by heating, was introduced
from the bifurcation of the internal carotid artery and advanced until
resistance was felt. Rats were observed postoperatively, and those with
spastic paralysis of the right forelimb and circling to the right
during the ischemic period were taken for
histological examination. Rectal temperature was
monitored routinely to maintain it at 37.0±0.5°C during the surgical
procedure.
Immunohistochemistry
For morphological examinations with paraffin sections, 3
nonoperated control rats and 33 ischemic rats at 3 hours (n=3),
6 hours (n=5), 16 hours (n=5), 24 hours (n=5), 48 hours (n=5), 72 hours
(n=4), 96 hours (n=3), and 7 days (n=3) after MCA occlusion were
perfusion-fixed with saline and then Zamboni’s solution or 4%
paraformaldehyde solution under deep pentobarbital
anesthesia. Brains were removed and postfixed in the same
fixative for 5 hours at 4°C. Then each tissue block was dehydrated,
embedded in paraffin, and cut into 4-µm-thick coronal sections.
Another 11 rats were used for examinations with frozen sections. In
nonoperated control rats (n=3), 24-hour ischemia rats (n=4),
and 48-hour ischemia rats (n=4), the brain was cryoprotected
after perfusion-fixation and cut into 10-µm-thick coronal sections
with a cryostat. The peroxidase-antiperoxidase method for light
microscopy was used for the immunohistochemical procedure for
albumin, microtubule-associated protein 2 (MAP2), glial
fibrillary acidic protein (GFAP), and myeloperoxidase
(MPO).13 Briefly, each deparaffinized and rehydrated
section was incubated with 10% nonimmunized goat serum, with anti-MAP2
(Sigma-Aldrich Co; 1:200), anti-albumin (Organon
Teknik Co; 1:400), anti-GFAP (Dako; 1:400), or anti-MPO (Dako; 1:100)
antibody at 4°C overnight, with an appropriate antiserum (1:20) for 1
hour, and with the peroxidase-antiperoxidase complex (Miles-Yeda Ltd;
1:100) for 1 hour. Proliferating cells were detected by using a
monoclonal mouse anti–proliferating cell nuclear antigen (PCNA)
coupled with horseradish peroxidase (HRP) (Dako). Immunoreactions for
interleukin-1ß (IL-1ß) and Bax, a Bcl-2 family protein, were also
performed with an anti–IL-1ß antibody14 (NIBSC; 1:200)
to detect the production of IL-1ß and with an anti-Bax
antibody (1:200) to detect apoptotic cell death of
microglia/macrophages, respectively, by using an avidin-biotin
complex method. Monoclonal anti-rat CD11b/c antibody (OX-42, Serotec;
1:500) was used to detect microglia as well as macrophages in
the frozen section. The paraffin sections were finally reacted with
0.05% 3'3-diamonobenzidine in the presence of 0.01%
H2O2. The frozen sections
used for double staining with fragmented DNA were finally reacted with
3-amino-9-ethylcarbazole for visualization of immunoreaction for OX-42.
The control sections were incubated with nonimmune serum or
ascites.
Lectin Histochemistry
We used griffonia simplicifolia B4 isolectin (GSA I-B4) for
detection of resident and reactive
microglia/macrophages.15 The rehydrated sections
were incubated in PBS containing cations (0.1 mmol/L of
CaCl2, MgCl2, and
MnCl2 ) for at least 10 minutes before
application of GSA I-B4 HRP conjugates (Sigma-Aldrich Co). Aliquots
of the conjugates were diluted 1:10 in PBS containing the cations and
0.1% Triton X-100. After incubation overnight at 4°C, the slides
were washed with PBS, and the peroxidase reaction was accomplished by
incubation in the
3'3-diaminobenzidine-H2O2
substrate medium.
In Situ Hybridization Histochemistry
Rat cDNA encoding proteolipid protein (PLP) mRNA in pGEM3
plasmid was used, and RNA probes were transcribed by RNA polymerases
from appropriate linearized plasmids containing T3 or SP6
promoters using digoxigenin-UTP (DIG RNA Labeling Mixture,
Boehringer Mannheim).7 RNA probes were generated
in both antisense and sense directions, and sense probes were used as
controls. The deparaffinized and rehydrated sections were treated with
10 µg/mL of proteinase K in Tris-EDTA buffer for 10 minutes at
37°C, followed by the second fixation with 4%
paraformaldehyde. To quench the endogenous
alkaline phosphatase activity, the sections were immersed in 0.2N HCl
and then were acetylated in 0.1 mol/L triethanolamine-HCl with
acetic anhydride. The sections were finally dehydrated with ethanol and
chloroform. The probes in the hybridization solution, containing 50%
deionized formamide, 10% dextran sulfate, 1x Denhardt’s solution,
200 µg/mL tRNA, 10 mmol/L Tris-HCl (pH 8.0), 0.6 mol/L NaCl, and
0.25% SDS, were denatured by heating up to 85°C for 3 minutes. Each
section was covered with the hybridization solution and then with a
cover glass, placed on a hotplate at 85°C for 3 minutes, and
incubated at 55°C overnight. After hybridization, the sections were
immersed in 5x SSC at 55°C to dislodge cover glasses, then washed in
50% formamide and 2x SSC at 65°C for 30 minutes. The sections were
then treated with RNase A (Boehringer Mannheim) for 30 minutes
at 37°C and washed in 50% formamide with 2x SSC at 65°C for 30
minutes. After that, the sections were incubated with the blocking
buffer for 1 hour and then reacted with alkaline
phosphatase–conjugated Fab fragment derived from an antiserum to
digoxigenin (Boehringer Mannheim, 1:500) overnight. To
visualize the reaction product, the sections were incubated with
4-nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indoyl-phosphate for 24 to 48 hours at 4°C in a
dark, moist chamber.
Terminal
Deoxynucleotidyltransferase–Mediated
dUTP-Biotin Nick End-Labeling Staining
The terminal
deoxynucleotidyltransferase
(TdT)–mediated dUTP-biotin nick end-labeling (TUNEL) procedure was
performed according to the method of Gavrieli et al.16 In
brief, deparaffinized and rehydrated sections were treated with
proteinase K (10 µg/mL in Tris-EDTA buffer), immersed in 2%
H2O2 for 5 minutes to
inactivate endogenous peroxidase, and then,
after a rinse with water, immersed in TdT with biotin-11-dUTP
(Sigma-Aldrich Co) and incubated at 37°C for 1 hour. The sections
were incubated with 2% bovine serum albumin for 10 minutes for
blocking, which was followed by reaction with the avidin-biotin complex
(Vector Laboratories, Inc) for 45 minutes. Finally, the sections were
incubated with diaminobenzidine and 0.01%
H2O2. In the frozen
sections used for double staining with immunohistochemistry, cells
incorporating dUTP were visualized with metal-enhanced
diaminobenzidine, which provided a blue-black reaction product.
Statistical Analysis
All values are presented as mean±SD. The number of
cells was compared between different time points with repeated-measures
ANOVA and Fisher’s protected least significant difference post hoc
test. P<0.05 was considered statistically significant.
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Results |
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Inflammatory Cells in Gradual Expansion of the Ischemic Lesion to the Periphery of the MCA Territory
In the center of the MCA territory including the striatum and the frontal cortex, the immunoreactivity for MAP2 was already lost 3 hours after MCA occlusion. In the periphery of the MCA territory, however, ischemic neuronal damage detected as loss of the MAP2 immunoreactivity gradually expanded from 6 to 48 hours to the border between the MCA and anterior cerebral artery territory (Figure 1
). This gradual expansion of the
ischemic lesion was typically observed in the parietal cortex.
Therefore, the sections including parietal cortex were used for
investigation of the contribution of microglia/macrophages to
expansion of cerebral infarction. In the sham-operated rats,
extravasation of albumin was absent and microglial cells were
not observed in the cerebral cortex. In the core of the infarction in
the cortex, extravasation of albumin was first observed 3 hours
after MCA occlusion and expanded into the whole ischemic
hemisphere 16 hours after MCA occlusion. Microglial cells were first
detected 6 hours after MCA occlusion; they were of the resting type
with ramified shape (ramified microglia) and persisted in the
ischemic hemisphere for 7 days. However, at 16 and 24 hours
after MCA occlusion, activated microglial cells of the amoeboid
type (amoeboid microglia), as identified by their enlarged size, stout
processes, and intense staining, were observed not only in the
infarcted tissue but also in the area adjacent to infarction with
preserved immunoreactivity for MAP2 (Figure 2). The lectin binding to microglia and
macrophages was confirmed by the immunohistochemical reaction
with OX-42 (Figure 2E1 and 2E2). The amoeboid microglia showed
positive immunoreaction for PCNA in the adjacent sections (Figure 3). It seems that PCNA-positive cells
were mostly microglia/macrophages, but a few PCNA-positive
cells existed in the microvessels, as reported by Abumiya et
al.17 Those areas, adjacent to the infarction at 16 and 24
hours, subsequently lost the immunoreaction for MAP2 and became
infarcted at 48 hours after MCA occlusion (Figure 1).
Macrophages that were morphologically discriminated from
microglia by their large nuclei and granulocytes that were detected
with the immunoreaction for MPO were always absent in the area with
preserved immunoreaction for MAP2 (Figure 2). Between 16 and 24
hours after MCA occlusion, however, both macrophages and
granulocytes started to emerge in the infarcted tissue.
Macrophages were mainly accumulated in the boundary within the
infarction, whereas granulocytes were distributed widely from the
boundary zone to the infarcted core. The immunoreaction for IL-1ß was
hardly observed in the brain sections of sham-operated rats and of rats
subjected to MCA occlusion for 6 hours. Between 16 and 24 hours after
MCA occlusion, the immunoreaction for IL-1ß was visible near the
border of infarction. Amoeboid microglia and macrophages were
found to express IL-1ß in the adjacent sections (Figure 3),
but ramified microglia possessing IL-1ß were absent. At 48 hours
after MCA occlusion, the immunoreaction for IL-1ß began to fade, and
at 7 days there was no immunoreaction for IL-1ß in the
ischemic hemisphere. The distribution of amoeboid microglia,
macrophages, and granulocytes is schematically
presented in relation to gradual expansion of infarction and
extravasation of serum albumin in Figure 4.
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,macrophage; and 
, granulocyte. C1/D1 and C2/D2 are
photographs of the areas identified with squares 1 and 2 in A at a
higher magnification, respectively. E1 and E2 are photographs of the
area in the frozen sections corresponding to squares 1 and 2 along the
infarct border, respectively. Square 2 had preserved immunoreaction for
MAP2, but square 1 already lost the immunoreactivity for MAP2. While
macrophages and granulocytes (arrows in C1 or E1 and arrowheads
in D1, respectively) accumulated in the ischemic lesion in
square 1, the area with preserved immunoreaction for MAP2 in square 2
was devoid of macrophages and granulocytes (C2 and D2).
However, activated microglia with amoeboid shape were abundant
in square 2 (arrowheads in C2 or E2) even with immunoreaction for MAP2.
Bar=250 µm in A and B. Bar=50 µm in C1, C2, D1, D2, E1,
and E2.
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)
were used as markers to identify equivalent areas in 2 adjacent
sections, respectively. A, C, and E, Lectin histochemistry. B,
Immunoreaction for PCNA. D and F, Immunoreaction for IL-1ß.
Bar=100 µm.
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In contrast to early destruction of the neuron-specific marker, the
signals for oligodendrocytes were preserved in the infarcted tissue
even 24 hours after MCA occlusion (Figure 5). Consistent with our
previous observation,7 the mRNA signals for PLP were
present in the oval-shaped cells, oligodendrocytes, mainly in the
white matter. However, the mRNA signals for PLP started to decrease in
the center of the MCA territory, including the lateral
caudoputamen, at 16 hours after MCA occlusion, but the
signals were still present and colocalized with macrophages
and granulocytes in the infarcted tissue for up to 48 hours after MCA
occlusion (Figure 5). Up to 24 hours after MCA occlusion, cells
with fragmented DNA detected by the TUNEL method largely appeared to be
damaged neurons, but scattered oligodendrocytes were also clearly shown
to have DNA fragmentation within the infarction (Figure 5). The
mRNA signals for PLP completely disappeared in the infarcted
tissue 96 hours after MCA occlusion; however, they were still
present in the corpus callosum even at 7 days after MCA occlusion
(Figure 6).
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Glial Responses in the Tissue Repair Process After Cerebral
Infarction
Expansion of cerebral infarction was complete by 48 hours after
MCA occlusion. At this point, the infarcted tissue consisted of damaged
cells and invading cells, including microglia/macrophages and
granulocytes. The immunohistochemical reaction for Bax protein and the
TUNEL method showed abundant positive nuclei in the infarct.
Macrophages in the center of the infarct started to express Bax
protein at 24 hours and began to exhibit DNA fragmentation at 48 hours
after MCA occlusion (Figure 7). Double
staining with TUNEL and immunoreaction for OX-42 revealed that the
ratio of macrophages to TUNEL-positive cells was 27.0±5.4% in
the center of the infarction at 48 hours after MCA occlusion. Reactive
astrocytes started to increase in the peripheral area of
infarction at 24 hours after MCA occlusion, but they were rarely
detected in the center of the infarcted tissue. At the rim of the
infarct, ramified microglia and oligodendrocytes were also accumulated
and colocalized with reactive astrocytes at 7 days after MCA occlusion
(Figure 8).
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P<0.05
vs other groups. Bar=50 µm. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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The penumbral area has been considered a therapeutic target in focal cerebral ischemia.2 3 The concept of penumbra was based on functional and biochemical changes,18 but progressive vascular compromise may also be involved in expansion of the lesion.19 20 It takes hours to days to detect ischemic damage in the penumbral area with the conventional histological method.21 In the present study we used the immunoreaction for MAP2 to detect expansion of the ischemic lesion after MCA occlusion because our previous studies demonstrated that MAP2 was a specific and sensitive marker for ischemic neuronal damage.7 13 On the basis of the present findings in the periphery of the MCA territory of the cerebral cortex, we concluded that macrophages and granulocytes did not play major roles in the progression of ischemic neuronal damage for up to 16 hours. During this early ischemic period after MCA occlusion, neuronal insults, including glutamate toxicity22 and spreading depression,23 may be more important than inflammatory cells for progression of the ischemic lesion. However, expansion of the ischemic lesion was still observed from 16 to 48 hours after MCA occlusion. During this period, amoeboid microglia became visible in the area where the immunoreaction for MAP2 was still preserved, but this area subsequently became infarcted. Immunoreaction for PCNA suggested the activation and proliferative response of amoeboid microglia. Furthermore, the accumulation of macrophages was observed mainly along the border within the infarction. Both amoeboid microglia and macrophages produce IL-1ß, an inflammatory cytokine, and it is likely that those inflammatory cells contributed to expansion of infarction, especially in the late ischemic period after MCA occlusion. The presence of IL-1ß in the ischemic brain and aggravation of ischemic damage have been reported.24 25 Several recent studies also suggested that inflammatory cytokines such as IL-1ß and tumor necrosis factor
(TNF-) were expressed
mainly in microglia/macrophages,14 26 but there is
a possibility that neurons and astrocytes generate those
cytokines after ischemia.27 The
contribution of inflammatory cells in expansion of infarction at 16 and
24 hours after MCA occlusion is also consistent with the report
emphasizing the pathogenic role of inducible nitric oxide synthase
expressed in inflammatory cells after focal cerebral
ischemia.28 However, it should be taken into
account that the contribution of inflammatory cells may be different in
nonhuman primates, in which the delimiting lesion is fixed relatively
early.29 Furthermore, the effect of suppression of
activated microglia/macrophages in cerebral infarction
remains unclear, although depletion of granulocytes has been shown to
reduce infarct size.30 Consistent with our previous findings in
mice,7 the present study with in situ hybridization
histochemistry suggested that oligodendrocytes were resistant
against ischemic insult, although some reports have shown
opposite findings.9 Because mRNA for PLP is exclusively
localized in the perinuclear region of oligodendrocytes in the
brain,31 the preservation of mRNA signals for PLP at least
indicates preservation of the integrity of the cytoplasmic membrane.
This technique has been applied for identification of oligodendrocytes
in human brain sections32 and in murine brain sections
subjected to demyelination and remyelination.33
Oligodendrocytes were often detected in the infarction 24 and 48 hours
after MCA occlusion. Colocalization of oligodendrocytes with
macrophages and granulocytes in the infarcted tissue suggested
the possibility that oligodendrocytes were destroyed by invading
inflammatory cells in the infarcted tissue through an apoptotic
mechanism, as suggested by the presence of fragmented DNA in
oligodendrocytes. It has been reported that cytokines such as
TNF- and IL-1ß were involved in microglia-mediated
apoptosis in cultured oligodendrocytes.34 35
Apoptotic death of oligodendrocytes has been also observed
after spinal cord injury.36 After completion of the expansion of infarction, both resorption of the infarcted tissue and glial scar formation occur for tissue repair or remodeling. Two major cellular components that migrated into the infarcted area to eliminate tissue debris were macrophages and granulocytes. However, these cells also had to be destroyed or recruited for tissue resorption. After a peripheral nerve injury, activated proliferating microglia have been reported to exhibit nuclear DNA fragmentation by 7 days after injury.37 In the present study we suspect that macrophages accumulated in the infarction underwent apoptosis after expressing Bax protein and DNA fragmentation. Surprisingly, approximately a quarter of TUNEL-positive cells within the infarction showed microglia/macrophages phenotype with double staining. Although the TUNEL approach does not unequivocally define apoptosis, accumulation of Bax protein in macrophages has been reported to mediate apoptotic cell death induced by nitric oxide and morphine.38 39 Whether those macrophages in infarction express caspase activity, a better marker for the presence of apoptosis, remains to be determined. It remains unclear how granulocytes were removed from the infarcted tissue before resorption. It has long been believed that glial cells participate in the glial scar formation at the rim of the infarct.40 41 Reactive astrocytes and activated microglia have been shown to participate in the formation of glial scars.10 Consistent with our previous findings in mice,7 the present study clearly showed that oligodendrocytes also accumulated, together with astrocytes and microglia, along the rim of the infarct in the rat after MCA occlusion. Although controversy exists regarding whether or not all astroglial reactions are beneficial for regeneration,42 our findings suggested that oligodendrocytes colocalized with reactive astrocytes could contribute to the tissue repair process, glial scar formation, and nerve regeneration. It is also possible that accumulation of oligodendrocytes is an attempt to provide myelin when axonal regeneration occurs.
In conclusion, we demonstrated the possibilities that (1) microglia/macrophages contributed to the gradual expansion of cerebral infarction through production of IL-1ß; (2) oligodendrocytes were resistant to ischemic insult; (3) once macrophages invaded the infarction, they eventually underwent apoptosis; and (4) oligodendrocytes were a component of glial scar formation together with astrocytes and microglia. The possibility of therapeutic control of activated microglia/macrophages to limit cerebral infarction will be pursued in the future.
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Acknowledgments |
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This study was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture and by a Sasagawa research-promoting grant. The authors thank Y. Imaeda and Y. Inoue for secretarial assistance.
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Footnotes |
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Reprint requests to Takuma Mabuchi, MD, Department of Internal Medicine and Therapeutics (A-8), Division of Strokology, Osaka University Graduate School of Medicine, 2–2 Yamadaoka, Suita-city, Osaka 565-0871.
Received November 10, 1999; revision received March 30, 2000; accepted April 4, 2000.
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Editorial Comment
Guest Editors Department of Molecular and Experimental Medicine The Scripps Research Institute La Jolla, California
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Cellular inflammation, triggered by ischemia, is a significant contributor to the final pathology of ischemic stroke. Garcia and colleaguesR1 R2 identified the early involvement of polymorphonuclear (PMN) leukocytes and the subsequent invasion of monocytes into ischemic brain after middle cerebral artery occlusion (MCAO). But, the potential contributions of resident cells of the ischemic brain to the "secondary injury" triggered by ischemia has received little attention until recently. Mabuchi and colleagues, in their very fine survey, examined the responses of resident microglia and oligodendrocytes in the ischemic territory to MCAO in the Wistar rat. By 16 to 24 hours after MCAO, ameboid microglia and macrophages within the ischemic tissue expressed the cytokine IL-1ß, while oligodendrocytes were found in the central injured region and were apparently increased in number in the peripheral regions by 7 days. Activation of microglia, and the accumulation of the relatively ischemia-resistant oligodendrocytes to the ischemic territory, suggest their participation in the injury process.
Microglia, primary immune-competent cells of the central nervous system, are derived from cells of the monocyte/macrophage lineage in the bone marrow.R3 R4 With breakdown of the blood brain barrier which accompanies cerebral ischemia, brain inflammation, trauma, and toxic injury, perivascular microglia and infiltrating monocytes/macrophage from the circulation are activated. These cells share in common many phenotypic markers and responses.R5 R6 Furthermore, following MCAO, and after transmigration of PMN leukocytes from the circulation has begun, mononuclear cells accumulate within the injured territory by 24 hours, in response to specific chemokines.R2 R7
Microglial cell activation and proliferation is stimulated by
macrophage colony-stimulating factor 1 (CSF-1),R8
an astrocyte-derived growth factor,R9 and by transforming
growth factor ß2 (TGF-ß2), a neuron-derived factor, in the presence
of CSF-1.R10 Certain chemokines can recruit and
activate specific subsets of inflammatory cells, including
mononuclear cells, to sites of tissue damage, which may enhance
progression of endothelial cell, astrocyte, and
neuronal injury.R11 R12 Tumor necrosis factor and
interleukin-1ß, derived from neurons, astrocytes, and
activated inflammatory cells, contribute to the
ischemic injury in experimental preparations.R13
Chemokines and their receptors have been detected in microglia,
neurons, and astrocytes under physiologic conditions, and following
hypoxia microglial generation of chemokines is stimulated by
excitotoxic stress, CSF-1, or TGF-ß2.R11 The present
data suggest that microglial activation in this setting may occur
during the evolution of the injury and the expansion of the
infarct.
Similarly, the topographical distribution of oligodendrocytes implies potential participation in reactive processes. Microglia/macrophages may also participate in the repair and remodeling of the tissue after or during development of the infarct.R14 R15
The panorama of microglial and oligodendrocytic response to focal ischemia following central nervous system ischemia suggests that detectable activation is not immediate but requires preparatory steps that include cytokine and chemokine secretion, and undoubtedly more. The contributions that activated microglia may make to the development of the injury have not been explored. It will be of interest to determine whether microglia with genetically altered phenotypes may also alter the evolution of the injury after MCAO. This is particularly interesting, as microglia may serve immune and other functions, belonging to a family of cells with potentially diverse functional roles. The precise derivation of microglia in the ischemic regions, whether they arrive from the circulation and when, are important questions. This information and the reactivity of oligodendrocytes to the ischemic insult, could provide leads for limiting expansion of the injury. To what degree these cells contribute to later attempts at repair of the tissue will provide ample basis for future studies.
Received November 10, 1999; revision received March 30, 2000; accepted April 4, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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