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Regular Articles |
From the Departments of Pathology*
and Anatomy
and Histology,
Institute for Biomedical
Research, University of Sydney, Sydney, New South Wales, Australia.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-D-galactose expression and reactive
morphological changes in microglia during FMCM. These results suggest
that disruption of the CNS milieu by entry of plasma
constituents, or circulating malaria parasites in the absence
of an immune response, by themselves are insufficient to induce
the reactive microglial changes that are characteristic of FMCM. In
addition, dexamethasone-sensitive event(s), presumably
associated with immune system activation, occurring within the
first few days of malaria infection are essential for the development
of reactive microglia and subsequent fatal neurological
complications.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of the events so far observed within the CNS parenchyma, changes in
microglia are the earliest, occurring within 2 to 3 days
postinoculation (p.i.), that is, at least 3 days before the onset of
cerebral symptoms and 4 days before death.6
Morphological
changes included retraction of ramified processes, soma enlargement, an
increasingly amoeboid appearance, and vacuolation. There was also
redistribution of activated microglia toward retinal vessels, in
particular toward the venous side of the vascular endothelium. Another
striking feature was the up-regulation of -D-galactose residues on
microglia, made evident by an increase in Griffonia
simplicifolia (GS) lectin staining. Consistent with previous
findings,9,10
only a small population of microglia in the
uninfected adult mouse retina were GS lectin-positive.6
However, there was a striking increase in the focal density of
GS-positive microglia, indicative of their activation,11
during the progression of the disease. In a "resolving" model of
murine cerebral malaria,4
the microglial changes were
transient and much less pronounced.12
Our previous studies using the retinal whole mount technique have shown
a strong relationship between microglial activation, by morphological
criteria, and events occurring at the blood-brain barrier
(BBB) during FMCM.6
Microglia redistribute toward the
retinal vessels with compromised barrier properties, changes in
microglial morphology are initiated in those microglia juxtaposed with
the retinal vasculature, and tumor necrosis factor- (TNF-)
production by microglia is most commonly found in areas close to
the cerebral vessels.8
Comparable microglial changes,
including evidence of activation, in human cerebral malaria have been
reported.13,14
A possible initiating event for microglial changes is the early increase in BBB permeability seen in FMCM.4,15,16 To determine whether the breakdown in barrier properties alone is sufficient to initiate microglial morphological and distributional changes in the absence of parasite inoculation, we now have experimentally induced an increase in BBB permeability through intracarotid injection of arabinose.17 This procedure produces vasodilatation and endothelial cell dehydration, stretching endothelial cell membranes, and increasing the permeability of intercellular tight junctions.18
Considering protein concentration in the CNS extracellular space is about 200-fold less than that of the plasma,19 it has been hypothesized that exposure to plasma constituents as a result of increased BBB permeability may play a role in the modulation of microglia function and phenotype.20 For example, the expression by microglia of CD4 and sialic acid binding receptor are regulated by exposure to an inducing agent in serum.20 Microglia may alter their morphology in response to increased BBB permeability because they phagocytose foreign protein and thereby act as sinks to preserve the CNS microenvironment.21 Furthermore, circulating factors in the blood, including kinins and amino acids such as glutamine and glycine, may cause stress or injury to brain cells and subsequently modulate microglial activity.22,23
Microglia are also responsive to products of the immune system.
Inflammatory mediators and/or high concentrations of potassium from
damaged cells provide signals necessary to promote microglial
activation and release of reactive oxygen species.24
In
addition, microglia can be stimulated by substances such as
lipopolysaccharide (LPS) and interferon-
(IFN-) to secrete
cytokines such as interleukin (IL)-1,25
IL-6,26
and TNF-.27,28
Therefore, in a
second series of experiments we have compared the microglial changes
seen in the FMCM model with those in the same model after treatment
with dexamethasone, an anti-inflammatory and immunosuppressive
agent.29
Previously, we have shown that dexamethasone can
protect FMCM mice from death due to cerebral complications without
affecting the growth of the parasite.30
However, to be
effective the dexamethasone must be administered 2 to 3 days before the
expression of cerebral symptoms.
Using a combination of dexamethasone intervention, experimental permeabilization of the BBB, and the retinal whole mount technique we have carried out a detailed study of potential events leading to the early activation of microglia during FMCM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBA/T6 mice (6–8 weeks old) weighing 20–25 g were obtained from the Blackburn Animal House, University of Sydney. A minimum of 6 mice from each experimental group was sacrificed at the times indicated and their retinae were dissected as previously described,31 then prepared for GS lectin histochemistry and nucleoside diphosphatase (NDPase) histochemistry as detailed below. For all parameters and treatments studied, two independent observers examined the retinal preparations.
FMCM Inoculation Procedure
Mice were given i.p. injections of 106 erythrocytes parasitized with Plasmodium berghei ANKA, suspended in 200 µl of phosphate buffered saline (PBS). Before sacrifice, blood smears were taken and the parasitemias were determined.4,32 To ensure that the mouse/parasite combination followed the usual course of the disease, an inoculated group of mice from each series was allowed to progress through the course of infection until death, which invariably occurred between days 6 and 8 p.i.
Visualization of Microglia with GS Lectin and NDPase Histochemistry
As described previously, microglia and the retinal vasculature
were visualized using the GS isolectin B4 (GS lectin), which binds to
-D-galactose.6,10,33
Microglia also were visualized
using NDPase histochemistry.6,8
Dexamethasone Treatment
FMCM mice were given 300-µl injections of Dexadreson V (75 mg/kg; Intervet International, Boxmeer, The Netherlands) subcutaneously on day 0 and day 1 p.i., a procedure which totally protects them against cerebral complications, or on day 3 and day 4 p.i., which greatly ameliorates, but does not completely abolish, those symptoms.30 Mice were sacrificed at day 7 p.i. (the terminal stage of the disease in untreated mice, which die with cerebral complications), day 13 p.i. (a time point between the terminal stage of the disease in untreated mice and the time of death from hemolysis in dexamethasone-treated mice), or the time of death in treated mice (day 15–22 p.i.). Retinal whole mounts were prepared from these mice. Before sacrifice, parasitemias were determined and behavioral observations were made and these were compared with the untreated FMCM group.
Intracarotid Injection of Arabinose
CBA/T6 mice were anesthetized with an i.p. injection of Avertin (12 µl/g body weight) and their necks were shaved with electric clippers. An incision was made centrally over the trachea and the carotid artery was exposed by separating the sternohyoid, sternomastoid, and omohyoid muscles. The tissue fascia in the area was removed, displaying the carotid sheath which invests the common carotid artery, internal jugular vein, vagus nerve, and ansa cervicalis. Arabinose (200 µl of 1.6 mol/L solution in isotonic saline; Sigma, St. Louis, MO) with 2% (w/v) Evans blue solution was injected slowly into the carotid, the needle was removed and a small piece of Gelfoam (Upjohn, Rydalmere, Australia) was applied to allow clotting. Polymyxin B (1 µg/ml; Calbiochem, La Jolla, CA) was included in the injection mixture to counter any possible effects of endotoxin contamination. Once the bleeding had stopped the wound was sutured.
At 12, 24, or 36 hours after co-injection of arabinose and Evans blue, 6 mice were sacrificed by CO2 asphyxiation and the contralateral and ipsilateral brain segments and retinae were examined macroscopically for leakage of Evans blue-labeled albumin. Successful intracarotid injections showed Evans blue-albumin staining of the cerebral hemisphere and retina ipsilateral to the injection site. Several controls were performed to be certain that the observations were due to osmotic barrier opening only and not to other effects, such as damage to the carotid artery or excessive blood loss. First, coronal brain sections were stained for hematoxylin and eosin. Mice showing ischemic damage (most often seen as the appearance of shrunken neurons) were eliminated from the study. Second, mice showing neurological symptoms (loss of balance or paralysis) after they regained consciousness were also excluded from the study.
Evaluation of Microglial Density
Outlines of retinae were drawn using a graticule in the 10x eyepeice (Olympus, Tokyo, Japan). The density of GS lectin-positive or NDPase-positive microglia within each 1-mm2 region was counted, at a magnification of 40x. These values were then multiplied by a magnification correction factor (x16) to give cells/mm2 and converted into a grading scale. A representative retina was mapped for each time point. This sampling technique provides a general estimate of changes in microglial density within the retina during the progression of the disease and illustrates whether this microglial response occurs over the whole retina or in focal regions.
Analysis of Microglial Distribution
Photomicrographs of retinae were taken at 800x total magnification using an Olympus Vanox microscope equipped with Normarski optics. Photographic montages were assembled and drawings of the retinal microglia and vasculature were made on transparencies. This technique illustrates whether there is a change in the association of microglia with each other or with vessels at a particular level of the vascular tree within the retina during the progression of the disease.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with earlier observations, FMCM mice died 7 days p.i.
with P. berghei ANKA, exhibiting cerebral symptoms
including hemiplegia, convulsions, and coma.4,34
We
previously have shown that morphological changes in microglia begin
very early in FMCM, at days 2–3 p.i., which is 3 to 4 days before the
onset of cerebral symptoms.6
These changes include
retraction of ramified processes, soma enlargement, and the adoption of
an amoeboid morphology with vacuoles (arrows in Figure 1, A and B
). Such changes were at maximum
frequency and severity at the terminal stage (day 7 p.i.) of FMCM
(Figure 1, A
-C).
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) and NDPase
histochemistry (arrow, Figure 1E
). However, the changes were mild
compared with those found at day 7 p.i. in FMCM mice not treated
with dexamethasone (compare Figure 1A
with 1D, and Figure 1B
with 1E).
Mice treated with dexamethasone on days 0 and 1 p.i. displayed no
cerebral symptoms at any stage, including on day 7 p.i., but died
between days 15 and 22 p.i. At day 7 p.i., microglia
displayed the ramified morphology typical of resting
microglia.6
This amelioration of microglial morphological
changes could be found using both GS lectin histochemistry (arrow,
Figure 1G
) and NDPase histochemistry (arrow, Figure 1H
). These
observations are summarized in Table 1
.
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-D-Galactose Expression
Focal increases in the density of GS lectin-labeled microglia
occur during the progression of FMCM, probably indicating a change in
the activation status of these cells.6,11,35
In
dexamethasone-treated mice there was a substantial decrease in the
number of GS lectin-labeled microglia at day 7 p.i. compared with
untreated FMCM mice (86 cells/mm2; compare Figure 1C
with 1F and 1I). The earlier the mice were treated with
dexamethasone, the fewer microglia were labeled with the lectin. In
mice treated with dexamethasone on days 3 and 4 p.i. with P.
berghei ANKA and sacrificed at day 7 p.i., there was an
increase in density of microglia in some focal regions (48
cells/mm2
compared with uninfected mice, 17
cells/mm2). However, the density in the
dexamethasone-treated mice was much less than that found in FMCM mice
not treated with this agent (compare Figure 1C
with 1F). A low density
of GS lectin-labeled microglia was seen in mice treated with
dexamethasone on days 0 and 1 p.i. (21
cells/mm2). This extent of labeling was similar
to that of uninfected mice (17
cells/mm2).6
It was evident in mice
treated with dexamethasone that there was an amelioration of
morphological changes concomitant with the reduction in up-regulation
of -D-galactose residues on microglia (Figure 1)
. This finding is in
agreement with the suggestion that changes in microglial morphology are
correlated with changes in phenotype.36
Morphological Changes in Microglia in Response to an Experimentally Induced Increase in BRB Permeability
In agreement with previous observations,5
opening of
the blood-retinal barrier (BRB) in retinae ipsilateral to the
injection site was evident by the gross leakage of Evans blue-albumin
complex from the retinal vessels, resulting in poor vessel delineation
and high background fluorescence in the surrounding parenchyma. In
contrast, minimal staining was noted in the contralateral retina.
Infusion of vehicle (isotonic saline plus polymyxin B) did not result
in an increase in BRB permeability in either ipsilateral retinae or
contralateral retinae.5
Mice showing any neurological
symptoms after they regained consciousness or with cerebral tissue
showing histological damage were excluded from the study (see Materials
and Methods for details). Microglia from mice that had suffered
ischemic damage as a result of damage to the carotid artery were
characterized by their lack of processes and a cell diameter >15 µm
(Figure 2A)
.
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later. In response
to an intracarotid injection of vehicle, retinal microglia on the
ipsilateral side displayed a ramified morphology, typical of resting
microglia, at 12 (Figure 2C)
, 24 (Figure 2E)
, and 36 hours after i.c.
injection (Figure 2G)
, suggesting that the injection procedure itself
did not cause major changes in microglial morphology.
In contrast, at 12 hours post-i.c. injection of arabinose, microglia
from the ipsilateral retina displayed tortuous processes with prominent
distensions and a hypertrophied cell body (Figure 2D)
. From 24 hours
post-i.c. injection of arabinose, numerous microglia displayed shorter
processes and further enlargement of the cell body (Figure 2F)
in
addition to those morphological changes found at 12 hours. These
morphological changes persisted until the last observed time point (36
hours post-i.c. injection of arabinose, Figure 2H
). These observations
are summarized in Table 2
.
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Because NDPase labels the total population of retinal microglia,
NDPase histochemistry was used to investigate whether an
experimentally-induced increase in BRB permeability causes an increase
in total numbers of microglia. There was no consistent increase in the
numbers of NDPase-labeled microglia at each time point. The average
density of microglia in different areas of the retina in
arabinose-treated mice, 12 to 36 hours after injection, varied between
90 and 94 cells/mm2
(Figure 3, A
-C). Furthermore, there was no
obvious proliferative response in microglia that were redistributed
toward the retinal vessels at 12 (Figure 3D)
, 24 (Figure 3E)
, or 36
hours (Figure 3F)
after injection of arabinose. The average density of
NDPase-labeled microglia during FMCM ranged between 99 and 118
cells/mm2.6
Microglia could not be
visualized with the GS lectin in mice given an intracarotid injection
of arabinose, because arabinose interferes with the binding of the
lectin to the galactose residues.
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Figure 4
shows diagrammatically the
microglial distributional changes in the ipsilateral retina after an
intracarotid injection of arabinose. Microglia found in the retina
ipsilateral to the i.c. injection of vehicle and in control animals
were seen in a regular array over the entire tissue (Figure 4A)
.
However, from 12 hours post-i.c. administration of arabinose, it was
evident that there was a closer association of microglia with each
other and with vessels at all levels of the vascular tree. Microglia
were similarly distributed at 24 hours post-i.c. arabinose. In
contrast, at 36 hours post-i.c. arabinose, there appeared to be many
more microglia in close association with the arterial side of the
vascular tree (Figure 4B)
. This finding contrasts with the
redistribution of microglia during FMCM, where microglia were found
most closely associated with the venous side of the vascular
tree.8
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microglia Are Not Activated by Circulating Malaria Parasites in the Absence of an Immune Response
Observations from the present study lead us to suggest an
association between the immune response to the malaria parasite, the
activation status of microglia, and the fatal neurological
complications of FMCM. From Figure 1
it is evident that the earlier the
infected mice were treated with dexamethasone, the less activated the
microglia became, and this coincided with less severe neurological
symptoms. Microglial activation and neurological symptoms were totally
abrogated in mice treated with dexamethasone at the time of parasite
inoculation. Neither treatment of FMCM mice with dexamethasone at days
0 and 1 p.i., nor treatment at days 3 and 4 p.i., affected
the growth of the parasite. These results suggest that parasite
antigens and soluble factors released into the circulation after
parasite growth, in the absence of an immune response, are insufficient
to active microglia.
Site of Dexamethasone Action
Because the dexamethasone-sensitive event(s) occur early in the
pathogenic process, it has been suggested that the drug acts at the
stage of T cell activation.30
Given the key role played by
microglia in the pathogenesis of FMCM,6
(and these
results), an alternative mechanism is that dexamethasone ameliorates
cerebral complications by preventing the early involvement of
microglia. Dexamethasone has been shown to inhibit TNF- production
by LPS-stimulated murine and human microglia in vitro by
reducing translation of this cytokine,38,39
TNF- plays
an essential role in the pathogenesis of FMCM,40
and
microglia produce this cytokine during the course of the
disease.8
Castano and colleagues,41
however,
found that proliferation and activation of microglia during Wallerian
degeneration were not affected by dexamethasone treatment. From these
observations they hypothesized that microglia do not respond to
glucocorticoids in vivo. Thus, a direct effect of
dexamethasone on microglia in vivo in FMCM remains to be
unequivocally established.
Microglia Display Shorter and Thicker Processes and Redistribute toward the Vasculature in Response to Plasma Constituents
As suggested by Perry and colleagues,42,43 activation of microglia may be a consequence of increased permeability of the CNS barrier to macromolecules. In regions of the CNS where the BBB is absent, or after a breakdown in the BBB, microglia express molecules that are normally either absent or present at a low level.20 For example, the expression of CD4 and sialoadhesin, a macrophage receptor for sialic acid, on rat microglia is in part regulated by exposure to constituents of the plasma.20
Previous studies15,16
have shown a general increase in
permeability of the BRB in FMCM as early as day 2 to 3 p.i. This
coincides with the observed microglial responses, so it is possible
that movement of plasma molecules across the CNS barrier initiates the
morphological changes and accumulation of microglia toward the retinal
vessels. This hypothesis is supported by the coincidence between the
decrease in BRB permeability and the decrease in GS lectin labeling and
microglial morphological changes in malaria-infected,
dexamethasone-treated animals compared with their counterparts not
treated with the drug (Figure 1)
. Microglia from mice treated with
dexamethasone after the initial increase in BBB/BRB permeability (days
2–3 p.i.) displayed both an up-regulation of -D-galactose residues
on their surface and changes in morphology compared with their
counterparts in uninfected mice. However, these changes were mild
compared with FMCM mice not treated with dexamethasone and, likewise,
the neurological symptoms were less severe. Furthermore, dexamethasone
treatment (days 0 and 1 p.i.) before the permeability changes
completely ameliorated the increase in BBB permeability, up-regulation
of -D-galactose residues and microglial morphological changes, and
the development of any cerebral symptoms.
The role of plasma proteins in the initiation of the microglial
response is further supported by the changes induced by an intracarotid
injection of arabinose (Figures 2–4)
, because an experimentally
induced increase in BBB/BRB permeability resulted in changes in
morphology and distribution of microglia. It should be noted, however,
that there was a major difference between microglial changes that
resulted from intracarotid injection of arabinose and those observed
during FMCM. In the former case, there was a preferential
redistribution of microglia toward the arterial side of the vascular
tree (Figure 4B)
, whereas microglia from FMCM mice showed preferential
redistribution toward the venous side.6
This contrast is
most likely due to the differences in the agent that modifies
permeability in these different situations. Because the arabinose
solution is injected into the carotid artery, the arabinose passes
through the arterial side of the vascular tree before the venous side.
Therefore, the arterial vasculature will be subjected to a higher
concentration of arabinose. As a result, microglia may redistribute
toward the area with the highest extravascular concentration of
arabinose or plasma constituents. In contrast, inflammatory agents that
cause an increase in vascular permeability are more likely to affect
the venous side of the vascular tree. Indeed, observations in retinal
whole mounts have shown a more pronounced leakage of plasma proteins
into the parenchyma on the venous side of the vascular tree in
FMCM15
and experimental allergic
encephalomyelitis.44
Furthermore, there is a loss of
astrocyte ensheathment of vessel segments, predominantly on the venous
side of the circulation, at the terminal stage of FMCM.5
This loss of astrocytes is not observed following an experimentally
induced increase in BBB permeability.5
During FMCM,
microglia may be recruited to the site of astrocyte damage to
phagocytose astrocytic debris and foreign protein that has entered
through the damaged BBB. A phagocytic role for microglia is consistent
with the adoption of the amoeboid/vacuolated morphology that is
routinely observed during FMCM.
In summary, we have investigated the response of microglia to circulating malaria parasites in the absence of an immune response and to disruption of the CNS microenvironment with plasma constituents. These are the stimuli most likely to modulate microglial activity during FMCM. Changes in microglial morphology, such as thickening and distension of microglial processes and redistribution of microglia toward the vascular endothelium, can be initiated by an increase in permeability of the CNS barrier to macromolecules. However, an increase in BBB permeability is not sufficient to induce reactive microglia with an amoeboid and/or vacuolated microglia like those seen during FMCM. In contrast, dexamethasone-sensitive event(s), probably related to immune activation, occurring within the first few days of infection are necessary for the development of reactive microglial changes and subsequent fatal neurological complications.
From the current study we conclude that a likely course of events in FMCM is as follows: Malaria parasites produce a vasoactive factor that leads to increased BBB permeability. This in turn results in the movement of plasma constituents into the CNS, which initiates the redistribution of microglia and astrocytes5 toward blood vessels. Modest morphological changes of microglia, denoting a departure from their resting state, would result from exposure to, and phagocytosis of, extravasated plasma constituents and/or cell debris. These mild microglial changes are necessary, but not sufficient, for the development of cerebral pathology. Rather, concurrent dexamethasone-sensitive immunopathological events are the most crucial, because once they are initiated the disease becomes irreversible and invariably fatal. Further reactive microglial changes, unique to animals that go on to develop irreversible cerebral complications, might be caused by malarial exoantigens entering the CNS through the compromised BBB. These reactive changes also could be induced, or reinforced, by cytokines derived from circulating leukocytes, or monocytes adhering to the cerebral microvascular endothelium. The secretion of various neuroactive and neurotoxic factors by peripheral or local CNS immunocompetent cells, such as monocytes and microglia, respectively, may interfere with CNS functions, contributing to coma and death. Tumor necrosis factor8,40 and metabolites of the kynurenine pathway of tryptophan metabolism45 may be of particular importance in causing derangement of CNS functions.
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Acknowledgements |
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Footnotes |
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Supported by grants to N. H. and T. C.-L. from the National Health and Medical Research Council and Sydney University Research Grants Scheme. I. M. was supported by an Australian Postgraduate Award.
Accepted for publication October 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression in the brain during fatal murine cerebral malaria: evidence for production by microglia and astrocytes. Am J Pathol 1997, 150:1473-1486[Abstract]
by microglia and astrocytes in culture. Brain Res 1989, 491:394-397[Medline]
-D-galactosyl epitopes markedly affects the quantity of four major proteins secreted by macrophages. J Leukocyte Biol 1992, 52:80-83[Abstract]
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