(Stroke. 1997;28:2252-2258.)
© 1997 American Heart Association, Inc.
Articles |
Ischemic Stroke Injury Is Reduced in Mice Lacking a Functional NADPH Oxidase
From the Departments of Cardiovascular Research (C.E.W., J.M., G.R.T) and Immunology (S.P.G., W.C.D., J.R., J.T.C.), Genentech Inc, South San Francisco, Calif, and the Department of Pediatrics (M.C.D.), Indiana University, School of Medicine, Indianapolis, Ind.
Correspondence to Dr Claire E. Walder, Eisai London Research Laboratories Limited, Bernard Katz Building, University College London, Gower Street, London WC1E 6BT UK. E-mail cwalder{at}elrl.co.uk
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Abstract |
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Background and Purpose Free radicals account for a significant proportion of the brain damage that occurs during ischemic stroke. Using mutant mice (X-CGD) with a dysfunctional phagocytic NADPH oxidase, we investigated the role of this superoxide-generating enzyme as a mediator of the reperfusion injury in a mouse model of middle cerebral artery occlusion.
Methods Transient (2 hour) middle cerebral artery occlusion was performed in X-CGD or wild-type litter mates (8- to 10-week-old). After 22 hours of reperfusion, brains were harvested and infarct volume delineated using 2,3,5-triphenyltetrazolium chloride. To elucidate the origin of the damaging NADPH oxidase, transient ischemia was also performed in X-CGD or wild-type mice transplanted with wild-type C57 Bl/6J or X-CGD bone marrow, respectively.
Results The infarct volume induced by transient ischemia was significantly less in X-CGD mice (29.1±5.6 mm3; n=13) than wild-type littermates (54.0±10.6 mm3; n=10; P<.05). The elimination of a functional NADPH oxidase from either the circulation or the central nervous system, by performing the appropriate bone marrow transplant experiments, did not reduce the infarct size induced by transient ischemia. This suggests that in order to confer protection against transient ischemia and reperfusion, a putative neuronal and circulating NADPH oxidase need to be inactivated.
Conclusions Brain injury was reduced in mice lacking a functional NADPH oxidase in both the central nervous system and peripheral leukocytes, suggesting a pivotal role for the NADPH oxidase in the pathogenesis of ischemia-reperfusion injury in the brain.
Key Words: mice • cerebral ischemia • neutrophils • reperfusion • free radicals
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Introduction |
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Despite the obvious need to reperfuse ischemic brain tissue, which is now possible using tissue-type plasminogen activator1 after embolic stroke, there is evidence to suggest that reperfusion injury is likely to occur if reflow is delayed.2 Under normal conditions, brain mitochondria constantly produce low levels of O2.– and hydrogen peroxide, which are effectively scavenged by endogenous SOD, glutathione peroxidase, and catalase. Reflow to a previously ischemic region results in a massive increase in oxygen levels that together with a perturbed antioxidant defense mechanism results in the overproduction of oxygen free radicals. Since oxygen radicals have a very short half-life and their concentration in vivo is low, detection and quantitation are very difficult. Despite this, a role for oxygen radicals in mediating reperfusion injury in the brain was implicated some years ago.3 4 Attenuation of ischemic damage and the associated edema in a rat stroke model was observed after treatment with SOD or catalase, conjugated to polyethylene glycol to increase their half-life5 6 or SOD entrapped in liposomes to reduce clearance and increase blood-brain barrier permeability.7 Since SOD and catalase are able to scavenge oxygen free radicals, this further implicated oxygen radicals in this destructive role. The use of transgenic mice that overexpress the Cu-ZnSOD confirmed these earlier findings when smaller infarct volumes were demonstrated after transient, but not permanent, MCA occlusion.8 9 Although it is clear that oxygen free radicals do play a role in mediating reperfusion injury in the brain, their source remains to be elucidated. Not only can a number of different free radicals be generated (eg, O2.–, HO., and NO) from a number of different enzyme systems (eg, xanthine oxidase, NO synthase, and NADPH oxidase) but they can also originate from a number of different cell types. One in particular, the neutrophil, is able to release a variety of cytotoxic products including oxygen radicals and has been implicated in reperfusion injury in a number of different animal models of stroke.
Neutrophils have been observed within the vessels and the parenchyma of ischemic brain after reperfusion.10 11 Depletion of neutrophils from the circulation using anti-neutrophil antibodies reduces the brain injury associated with ischemia and reperfusion,12 suggesting a causative role for neutrophils. Furthermore, antibodies directed against endothelial ICAM-113 or the neutrophil ß2-integrins CD11a/CD18 and CD11b/CD1814 15 (the ligands for ICAM-1) all result in reductions in brain injury in animal models of stroke.16 Confirmation of these effects comes from the observation that ICAM-1 knockout mice have a smaller infarct volume when compared with their wild-type control littermates in an intraluminal suture model of MCA occlusion.17 Although this evidence supports a role for neutrophils in mediating tissue damage, it does not distinguish between the potential of these leukocytes for physical obstruction,10 11 release of proteolytic enzymes,18 or generation of free radicals.19
The purpose of this study was to investigate whether O2.– generated by NADPH oxidase, a major source of oxygen radicals from the neutrophil, are involved in mediating stroke-induced brain injury. NADPH oxidase is a complex multi-component electron transport enzyme that transfers electrons from NADPH to molecular oxygen to form O2.–. The terminal electron donor is cytochrome b558, which consists of two subunits, p22phox and gp91phox. Mutations in either of these subunits or two other essential components of NADPH oxidase, p47phox and p67phox, result in a dysfunctional enzyme that causes CGD, a disorder characterized by life-threatening microbial infections and inflammatory granuloma.20 A mouse model of the X-linked CGD (X-CGD) has been generated with a nonfunctional allele for the gp91phox subunit of the phagocytic NADPH oxidase using targeted homologous recombination in murine ES cells.21 These X-CGD mice are viable when housed in a protective environment with normal growth and development and have the same number of circulating neutrophils that are able to migrate to the peritoneal cavity after injection of thioglycollate.21 However, the neutrophils of these mice are unable to release O2.– after stimulation with PMA, as detected by cytochrome c reduction and the NBT assay.21 These mice were used to elucidate the role of the O2.–-generating NADPH oxidase in focal cerebral ischemia and reperfusion.
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Materials and Methods |
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MCA occlusion was carried out in 8- to 10-week-old male wild-type C57 Bl/6J (C57 Bl/6J, Jackson Laboratory, Bar Harbor, Me), X-CGD mutant mice (C57 Bl/6JxE129, Genentech Inc, South San Francisco, Calif), or wild-type littermates (WT: C57 Bl/6JxE129, Genentech Inc, South San Francisco, Calif). All animal procedures were approved by the Genentech Institutional Animal Care and Use Committee. Anesthesia was induced with 4% halothane and maintained with 1.0% to 1.5% halothane in 60% nitrous oxide and 40% oxygen. Body temperature was maintained at 37°C throughout surgical procedure and recovery by use of a homeothermic blanket (Harvard Apparatus, Inc) and heat lamp. The femoral artery was cannulated for blood pressure recording (Grass model 7D polygraph, Grass Instruments), and a 60 µL blood sample was taken immediately before occlusion for blood gas analysis (pH, Pco2, and Po2; model 238 blood gas analyzer, Ciba Corning). Transient MCA occlusion was performed using the method of Zea Longa et al,22 modified for use in mice. Briefly, the right CCA, ECA, and ICA were exposed. An 11 mm length of 5-0 nylon monofilament suture, with the tip flamed to round the end, was advanced from the ECA into the lumen of the ICA until it blocked the origin of the MCA. In addition, the ipsilateral CCA was occluded. After 2 hours of recovery, animals were briefly reanesthetized with halothane to allow withdrawal of the suture and release of the ipsilateral CCA occlusion, thus permitting reperfusion of the MCA territory. Twenty-two hours after reperfusion, the mice were euthanized with pentobarbital sodium, and the brains were harvested rapidly and placed in iced saline for 1 to 2 minutes. The forebrains were sliced into four 2-mm sections and placed in a 1% solution of 2,3,5-triphenyltetrazolium chloride for 15 minutes at 37°C to delineate the infarct. The brain slices were placed into 10% buffered formalin and photographed the following day. The volume of infarct was determined using computerized planimetry (NIH Image).
At reperfusion, after being randomly assigned, C57 Bl/6J mice were treated with either anti-mouse ICAM-1 MAb (n=7; n=15) (clone 3E2, Pharmingen) or hamster IgG isotype–matched control (n=8; n=14) at a dose of 2 or 5 mg/kg IP. Similarly, X-CGD (n=19) and WT (n=14) mice underwent the same procedure of 2 hours of transient MCA plus ipsilateral CCA occlusion followed by 22 hours of reperfusion. However, these animals were not treated with any drugs at reperfusion or any other time. These mice had no observable phenotypic differences and were of identical weight (23.6 g±0.5 and 22.4±1 g for WT and X-CGD mice, respectively). All animals were euthanized at 24 hours, and their brain infarct volumes were determined as above.
In order to establish a lethal dose of radiation for this strain of
mice (C57 Bl/6JxE129) irradiated at such a young age (4- to
5-week-old), female X-CGD heterozygotes were irradiated with either 7.5
(n=9) or 10.5 (n=6) Gy of 
radiation from a 137Cs source
without bone marrow transplant. Blood cell counts were monitored weekly
by taking 40 µL of blood from the orbital sinus for counting on a
Serrono Baker hematology analyzer (Serrono
Diagnostics Inc).
Bone marrow preparations were made from a pool of cells taken from one
femur of three different donors. The femurs were flushed with
Dulbecco's modified Eagle's medium and the cells filtered through a
layer of 40-micron nylon mesh (Nitex, Western Industrial Sales). The
cells were resuspended in Dulbecco's modified Eagle's medium and a
viability count was done. C57 Bl/6J mice (4- to 5-week-old;
n=50) were irradiated with a lethal radiation dose of 10.5 Gy of
radiation from a 137Cs source and rescued by a tail-vein
injection of bone marrow (106 cells in 0.2 mL) prepared
from male X-CGD or WT donors (Table 1
). A
small sample of mice from each group (n=3) was used to measure blood
cell counts weekly to monitor the success of the graft. Forty
microliters of blood was taken from the orbital sinus for counting on a
Serrono Baker hematology analyzer. Male X-CGD (n=26) or WT
(n=26) mice (4- to 5-week-old) were similarly irradiated with a lethal
radiation dose. Bone marrow (106 cells in 0.2 mL) prepared
from C57 Bl/6J mice was given to these mice by a tail-vein
injection (Table 1). Blood cell counts were only measured at 4 and 5
weeks in these mice (n=6).
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At 8 to 10 weeks of age, C57 Bl/6J mice reconstituted with donor bone marrow from X-CGD or WT mice (n=17 and n=16, respectively) were subject to the same protocol of transient (2 hours) MCA plus CCA occlusion to determine the role of the NADPH oxidase from the circulating leukocytes at inducing damage. Similarly, the converse experiment was performed to elucidate the role of NADPH oxidase from potential sources in the brain (neurons, glial cells or endothelial cells), ie, X-CGD or WT mice given bone marrow from C57 Bl/6J mice (n=22 and n=10, respectively). Brain infarct volume was analyzed by the same method of triphenyltetrazolium chloride staining as described previously. Successful transplantation of animals that had a bone marrow transplant before MCA occlusion was confirmed by measuring NADPH oxidase activity of circulating neutrophils. For these determinations, a 200 to 500 µL blood sample was collected via cardiac puncture under sodium pentobarbital anesthesia, immediately before harvesting the brain. Erythrocytes were lysed by incubation with 170 mmol/L NH4Cl, 9 mmol/L KHCO3, 97.3 mmol/L Na4EDTA, pH 7.3, for 3 minutes at 22°C, and leukocytes were harvested by centrifugation. Intracellular production of oxidants was measured using a flow cytometric assay of DHR fluorescence. Cells were incubated in phosphate-buffered saline containing 5 mmol/L glucose 0.1% gelatin, and 1 µmol/L DHR for 10 minutes at 37°C. Cells were then treated with 275 U/mL catalase before the addition of 100 ng/mL PMA or DMSO as a control. After a 20-minute incubation at 37°C, samples were read on a FACScan and analyzed using Cell-Quest software.
All values are expressed as mean±SEM. Statistical significance was determined by Student's unpaired t test, or two-way ANOVA for blood cell counts after bone marrow transplant. In both cases, a value P<.05 was considered to be significant.
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Results |
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In a murine model of acute ischemic stroke whereby the MCA was occluded for 2 hours followed by 22 hours of reperfusion, neutrophil involvement was demonstrated by the 44% reduction of infarct volume after treatment with a MAb directed against ICAM-1 (5 mg/kg). This appears to be a dose-dependent effect since the lower dose of 2 mg/kg was not effective (Fig 1
). When X-CGD mice were subjected to
identical periods of brain ischemia and reperfusion, the
resulting infarct volumes were significantly reduced by 46% when
compared with their WT littermates (Fig 2). This reduction in brain injury could
not be accounted for by dissimilarities in blood pressure, heart rate,
blood gases, or pH between the groups, since there were no significant
differences in these parameters immediately before
occlusion (Table 2).
Histological analysis showed that the number of
neutrophils migrating into the infarct in the X-CGD mice at 24 hours
post-ischemia (6.9±1.1/36 random 1000x fields of 1 coronal
section, n=13) was no different from that of the infarct of the
wild-type littermates (7.4±1.5/36 random 1000x fields of 1 section,
n=10), demonstrating that it is not simply the presence of neutrophils
that is detrimental but the ability of neutrophils to release oxygen
free radicals.
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Only 1 of 9 female heterozygotes (age 4- to 5-weeks) irradiated with 7.5 Gy died. This occurred after 3 weeks. The remaining 8 animals survived for at least 7 weeks after irradiation. After nadir values of 0.3±0.1x103/mm3, 7.6±0.5x106/mm3, and 200±25x103/mm3, white blood cell, red blood cell, and platelet counts returned to 6.9±1.1x103/mm3, 9.7±0.2x106/mm3, and 1059±41x 103/mm3, respectively, after 7 weeks. In contrast, all 6 female heterozygote mice irradiated with 10.5 Gy died within 1 week of irradiation, demonstrating that 10.5 Gy is a lethal radiation dose resulting in complete or nearly complete myloablation.
Twenty five percent C57 Bl/6J mice that were irradiated
with 10.5 Gy and rescued with a bone marrow transplant from donor X-CGD
or WT died between 7 and 14 days after irradiation. Blood cell counts
from a sample of surviving mice (n=3 per group) showed that white blood
cells, red blood cells, and platelets returned to normal levels by
4 to 5 weeks after treatment (Fig 3).
When the surviving C57 Bl/6J mice that had been transplanted
with bone marrow from X-CGD mice were subjected to 2 hours of transient
MCA occlusion 4 to 5 weeks after this procedure, the infarct volume
induced after 24 hours was 51.1±5.6 mm3. This was no
difference in the damage induced in the control C57 Bl/6J
animals that had been transplanted with WT bone marrow (53.3±5.1
mm3) (Fig 4). The lack of
difference in the ischemia- reperfusion-induced damage was
unlikely to be due to differences in weight (19.5±0.3 and 19.2±0.6 g
for WT and X-CGD, respectively) or blood gas values between the two
groups, since these were the same at the time of occlusion (Table 2
).
Blood taken from a cardiac puncture at the end of the experiment and
subjected to a DHR assay verified that there was very little or no
NADPH oxidase activity in unstimulated cells (DMSO) from either C57
Bl/6J mice given X-CGD or WT marrow or in stimulated cells (PMA)
from the same recipients and X-CGD donors. However, cells from the C57
Bl/6J recipients and WT donors showed NADPH oxidase activity
upon stimulation with PMA, thus confirming successful bone marrow
transplant in all cases (Fig 5A).
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When the converse experiments were performed, ie, X-CGD or WT
were irradiated with 10.5 Gy and rescued with normal bone marrow from
C57 Bl/6J donor mice, 38% died between 7 and 14 days (12 WT and
8 X-CGD died). This loss is not surprising, since the mice are still
immature (4- to 5-week-old) and therefore likely to be more sensitive
to this procedure. The animals that did survive went on to recover and
leukocyte, erythrocyte, and platelet counts mirrored those of the
previous experiment at 5 weeks after irradiation and bone marrow
transplant (6.0±0.5x103/mm3 leukocyte,
9.5±0.5x106/mm3 erythrocyte, and
1024±53x103/mm3 platelets at 5 weeks;
n=6). Four to five weeks after the irradiation and subsequent
transplantation of normal bone marrow, the animals (WT and X-CGD) were
subjected to 2 hours of transient MCA occlusion followed by 22 hours of
reperfusion. In this experiment, the infarct volumes were equivalent in
the two groups of animals (50.1±5.1 and 47.9±6.0 mm3
for WT and X-CGD, respectively) (Fig 4). Similarly, weights (20.7±0.5
and 20.5±0.5 g for WT and X-CGD respectively) and blood gas values
(Table 2) for each group at the time of occlusion were the same and are
therefore unlikely to have influenced the volume of damage. Blood
removed at the end of the experiment and subjected to the DHR assay
once again showed little or no NADPH oxidase activity in animals from
either group that was unstimulated (DMSO). However, both groups of
animals showed NADPH oxidase activity after stimulation with PMA, again
confirming successful bone marrow transplant (Fig 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrates that transient focal ischemia in X-CGD mice results in a smaller lesion volume than that induced in WT littermates, suggesting that O2.– generated from the NADPH oxidase enzyme plays a major role in mediating ischemia-reperfusion injury in the brain. The contribution of free radicals to the ischemic damage found in this study supports the findings of others who have demonstrated a role for oxygen radicals by showing attenuation of damage in the presence of either exogenous or endogenous free radical scavengers.5 6 7 8 Although effective in reducing brain injury, these studies have not addressed the origin of the reactive oxygen species, since the exogenous and endogenous free radical scavengers neutralize reactive species from all sources. In this paper we have shown that O2.– ions generated by NADPH oxidase contribute to the pathogenesis of ischemia-reperfusion injury.
There is considerable evidence suggesting that neutrophils contribute to the damage associated with ischemia and reperfusion in the brain. Attenuation of brain injury after 2 hours of transient ischemia by an anti–ICAM-1 MAb in the present study confirms these findings and demonstrates that the damage induced in this model is neutrophil dependent. It has been suggested that neutrophils play an important role in the development of ischemia-reperfusion injury by releasing a variety of cytotoxic products, including oxygen radicals. A recent report by Matsuo et al23 provides evidence supporting a role for neutrophils in radical production during ischemia and reperfusion of the rat brain. Therefore, since activated neutrophils are known to release vast quantities of O2.– generated by NADPH oxidase, it is likely that in the present study O2.– from that particular source are implicated.
We set out to try to elucidate the source of the damaging NADPH oxidase–derived O2.– by giving lethally irradiated wild-type C57 Bl/6J mice bone marrow from X-CGD mice before transient ischemia and reperfusion. If neutrophil NADPH oxidase–derived O2.– were the source of the damaging effect, then we would expect the lesion volume in these animals to be attenuated. This was not the case, but the reason for the lack of protection in this scenario is uncertain. It is quite possible that release of O2.– from neutrophils is only one of many contributing factors. The fact that neutrophils are still present in these experiments means that they are able to bind and physically obstruct the vessels contributing to the no-reflow phenomenon,24 where, despite patent major vessels, the microvascular perfusion has been disrupted. In addition, reactive oxygen species have been reported to rapidly increase the ability of endothelial ICAM-1 to bind neutrophils.25 These animals have potential sources of functional NADPH oxidase in the brain. It is therefore conceivable that cell types such as neurons, glial cells, and endothelial cells are all still able to generate O2.–. Therefore, O2.– from these sources could potentially increase the binding ability of neutrophils, thus increasing damage. In addition, microglia, the resident macrophages of the brain parenchyma,26 may be another source of free radicals since macrophages are known to express NADPH oxidase. Consequently, it may be that the NADPH oxidase must be rendered nonfunctional from all sources in order for a protective effect on the ischemic insult to be observed.
Although, it was expected that the neutrophils would be the primary source of the damaging ROS, released either upon contact with the postcapillary endothelium27 or after extravasation into the parenchyma, we could not rule out the possibility that oxygen radicals may be produced by the NADPH oxidase from a number of other sources.
NADPH oxidase is believed to be restricted to cells of hematopoietic
origin, including neutrophils, eosinophils, monocytes, and
macrophages. Eosinophils have not been observed in cerebral
infarcts and monocytes/macrophages do not infiltrate into the
tissue until several days after the insult in the rat,28
suggesting that during the acute phase these leukocytes are not the
primary source of damaging ROS. Reverse transcription–polymerase chain
reaction, Northern blotting, and immunohistochemical analysis
have revealed the presence of some components of the NADPH oxidase
complex in a number of other cell types, including cultured
fibroblasts,29 vascular smooth muscle
cells,30 type I cells of the carotid body,31
neuroepithelial bodies of the lung,32
glomerular mesangial cells,33 and
human umbilical vascular endothelial
cells.34 Although these cells express some components of
NADPH oxidase, it is uncertain as to whether they undergo a respiratory
burst and generate ROS. It is possible that the
gp91phox component of the NADPH oxidase complex
may have additional functions including oxygen sensing and
H+ conductance.32 35 Neurons and glial cells
are known to produce ROS, but the presence of NADPH oxidase in these
cell types is also currently unknown. This would potentially be another
source of O2.–, and experiments to determine
this are currently ongoing. When we performed the bone marrow
transplant experiment in such a way as to produce mice with a
dysfunctional NADPH oxidase in the brain (ie, X-CGD mice were
given bone marrow from wild-type C57 Bl/6J mice) before
transient ischemia and reperfusion, protection was still not
afforded, suggesting that inactivating nonleukocyte NADPH oxidase alone
is not sufficient to prevent such brain injury. It is possible that the
irradiation and bone marrow transplant had other effects that affected
the susceptibility to neutrophils in stroke. However, if the control
animals±irradiation and bone marrow transplant in Figs 1 and 4 are
compared, we see that irradiation and bone marrow transplant had no
significant effect on outcome after cerebral ischemia.
It is quite likely that O2.–are originating from a number of different sources. In addition to NADPH oxidase, highly reactive and potentially damaging ROS may also be generated by xanthine oxidase from endothelial cells36 and/or neuronal NO synthase37 both of which result in exacerbation of ischemic brain injury.38 39 NO is able to react with O2.–to generate peroxynitrite and other oxygen radicals,40 which may subsequently be responsible for the brain damage. In conclusion, regardless of their source, we have shown that O2.–generated by the NADPH oxidase account for a significant amount of the brain injury that occurs after the interruption and re-establishment of cerebral blood flow. The enzyme may be present in cells other than neutrophils, and multiple sources may be present within the brain. These studies open up the possibility that inhibitors of this enzyme may prove to be an important therapeutic tool for the treatment of stroke.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr Dinauer is a recipient of research funding from Genentech Inc, South San Francisco, Calif. The authors wish to thank M.T. Butt (Pathology Associates International) for histological analysis of brain sections.
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Footnotes |
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Reviews of this article were directed by Dr Hermes Kontos.
At the time this study was conducted, Dr Walder, Dr Green, Walter Darbonne, Joanne Mathias, Julie Rae, Dr Curnutte, and Dr Thomas and were full-time employees and stockholders of Genentech Inc.
Received May 9, 1997; revision received July 29, 1997; accepted July 29, 1997.
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