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the Department of Pharmacological Sciences, University Medical Center at Stony Brook, Stony Brook, NY.
Abstract
Background and Purpose— Microglial activation may contribute to the pathogenesis of the brain injury in intracerebral hemorrhage (ICH). We have reported that the tripeptide macrophage/microglial inhibitory factor (MIF), Thr-Lys-Pro, inhibits microglial activation and results in functional improvement when given before the onset of hemorrhage. In this study, we investigate the protection and efficacy of treatment when MIF is administered 2 hours after collagenase injection.
Methods— ICH was induced by injecting bacterial collagenase into the caudate nucleus; 100 μL MIF (500 μmol/L) was delivered via a micro-osmotic pump. Infusion of MIF or saline (control) was initiated 2 hours after collagenase injection and continued for 24 or 72 hours. Microglial activation and macrophage infiltration were assessed by 5-D-4 and F4/80 immunofluorescence, respectively. Production of reactive oxygen species was visualized by in situ detection of ethidium. Degenerating neurons were assessed by Fluoro-Jade B staining. Neurological deficits, brain injury volumes, and brain edema were assessed at 24 and 72 hours after MIF/saline treatment.
Results— MIF can inhibit microglial activation and macrophage infiltration, attenuate the numbers of ethidium-positive cells compared with the saline-treated control mice, reduce the injury volume, edema, and degenerating neurons, and improve the neurological functional outcome.
Conclusions— Activated microglia/macrophages are important contributors to brain injury after ICH. MIF could be a valuable neuroprotective agent for the treatment of ICH, if treatment is initiated soon after the onset of hemorrhage.
Key Words: free radicals intracerebral hemorrhage intracranial hemorrhage thrombolysis
Introduction
Stroke is the second most common cause of death in the world after heart disease and a leading cause of disability. Intracerebral hemorrhage (ICH) represents at least 10% of all stroke deaths.1 The prognosis of patients with ICH is poor, and the pathogenesis of damage after ICH remains poorly understood. Evidence of cell death caused by apoptosis2,3 and inflammation4–6 has recently been described. When ICH occurs, the blood-brain barrier becomes disrupted. As a result, macrophages and leukocytes infiltrate the brain parenchyma, and their presence has been proposed to constitute a primary mechanism of cell death.7 It is also possible that microglia contribute to the observed neuronal death, because the disruption of blood-brain barrier can activate microglia.
To investigate the role of microglia in the pathogenesis of brain injury in ICH, we use a mouse collagenase hemorrhage model.8–10 We have reported that extensive activation of microglia is evident around the site of ICH. The tripeptide macrophage/microglial inhibitory factor (MIF), Thr-Lys-Pro, can inhibit microglial activation11–13 and results in functional improvement and decreases in degenerating neurons when given before the onset of hemorrhage.9 In a more clinically relevant study, we investigate the protection when treatment with MIF is initiated 2 hours after collagenase injection.
Materials and Methods
Animal Procedures
C57BL/6 mice (Jackson Laboratories, Bar Harbor, Me) were cared for by the Department of Laboratory Animal Research with access to food and water ad libitum. The experiments were performed in accordance to the National Institutes of Health guide for care and use of laboratory animals and the guidelines established by the Institutional Animal Care and Use Committee at Stony Brook. All efforts were made to minimize the numbers of animals used and ensure minimal suffering of those animals.
ICH Model
The procedure for inducing ICH was previously described.8–10 A total of 54 adult mice 25 to 35 g were used. The mice were anesthetized by intraperitoneal injection of avertin (0.5 mg/g of body weight). To induce hemorrhage, mice were injected with collagenase (0.075U in 500 nL saline)8–10 unilaterally into the caudate putamen, using the stereotactic coordinates 0.5 mm posterior and 3.0 mm lateral of bregma, and 4.0 mm in depth. Collagenase was delivered over 5 minutes and the needle stayed in place for and additional 5 minutes to prevent any reflux.
Then, 100 μL MIF13 (500 μmol/L, Sigma) was delivered at a rate of 0.5 μL/h via a micro-osmotic pump (Durect) placed subcutaneously in the back of the animals. A brain infusion cannula connected to the pump was positioned at the coordinates mentioned. Infusion of MIF or saline (control) was initiated 2 hours after collagenase injection and continued for 24 or 72 hours. The mice were allowed to recover from surgery in a warm environment over a 3-hour period. Mice were carefully monitored for several hours after recovery from anesthesia. MIF-treated/saline-treated mice were euthanized 24 or 72 hours after MIF/saline treatment. The overall mortality rate in this study was <2%.
Experimental Groups
This study was performed in 4 parts. Each part consisted of a saline-treated control group and a MIF-treated group. All mice received collagenase injection and were euthanized after MIF/saline treatment on day 1 (n=34) or 3 (n=20) after neurological scoring.7 The experiments were divided into 4 parts. During part 1, microglial activation and macrophage infiltration were assessed by 5-D-4 and F4/80 immunofluorescence on days 1 and 3 (n=3/group). During part 2, hemorrhagic injury and degenerating neurons were analyzed on days 1 (n=6 to 8/group) and 3 (n=5/group). During part 3, production of reactive oxygen species (ROS) was visualized by in situ detection of ethidium on day 1 (n=5/group). During part 4, mice (n=5/group) were euthanized for brain water contents on days 1 and 3 after MIF/saline treatment.
Neurological Deficit
All MIF-treated/saline-treated mice were scored for neurological deficits using a modified 28-point neurological scoring system8–10 on days 1 (n=16 to 19/group) and 3 (n=10/group). The tests included body symmetry, gait, climbing, circling behavior, front limb symmetry, and compulsory circling. Each point was graded from 0 to 4, establishing a maximum deficit score of 24, by an observer blinded to the experimental treatment.
Hemorrhagic Injury Analysis
Mice were euthanized and their brains were removed, fixed, and dehydrated in 4% paraformaldehyde and 20% sucrose in phosphate-buffered saline (PBS). Injury volumes from MIF-treated/saline-treated mice (n=6 to 8/group for day 1, n=5/group for day 3) were digitally quantified by an observer blinded to the experimental treatment using the SPOT Advanced software v3.5.2 (Diagnostic Instruments Inc) on 50-μm coronal sections using a previously reported method of Luxol fast blue/cresyl violet staining.8–10 Hemorrhagic injury areas were summed from 6 to 8 coronal slices at different levels. Volumes in mm3 were calculated by multiplying the 0.5-mm slice thickness by the measured areas.8,9
Histology
Luxol fast blue/cresyl violet and Fluoro-Jade B staining were performed according to published protocols.14,15 Cells permeable to Fluoro-Jade B are marked for cell death. Degenerating neurons were counted in 3 fields immediately adjacent to the hematoma in at least 3 sections per animal using a magnification of x400 over a microscopic field of 0.01 mm2 and expressed as cells/mm2, because previously reported9 areas with large blood vessels were avoided. Five to 8 mice/group were analyzed by an observer blinded to the treatment.
In Situ Detection of O2– Production
Production of ROS after ICH was investigated by in situ detection of oxidized hydroethidine on day1 after ICH (n=5 each group).16,17 Hydroethidine, which functions as a redox-sensitive probe, is oxidized by superoxide to a fluorescent product, ethidium.18 Ethidium intercalates within the DNA of cells and nuclei fluoresce red. Hydroethidine (100 mg/mL in dimethyl sulfoxide; Molecular Probes) was diluted to 1 mg/mL in PBS just before use and sonicated. At selected time points after collagenase injection, mice were injected intraperitoneally with 300 μL of hydroethidine. Brains were harvested 1 hour later and frozen at –80°C. The brains were sectioned at 20 μm on a cryostat and mounted on glass slides. The brain sections were incubated with 2.5x10–3 mg/mL Hoechst 33258 (Molecular Probes) in PBS for 20 minutes in the dark and then rinsed with distilled water and coverslipped with Vectashield mounting medium (Vector Labs). Ethidium was visualized on a Nikon PCM 2000 confocal microscope (excitation, 510 nm; emission, 580 nm), and photographed using a digital camera system and double exposure to produce images of oxidized hydroethidine and Hoechst 33258. Fluorescence intensity and expression patterns of fluorescent ethidium in peri-ICH area were compared blindly among the groups. Cells with oxidized hydroethidine extending to the cytosol were counted under high magnification (x400) in 4 different sites randomly selected and averaged over the entire field. The percentage of these cells to the total cells stained with Hoechst 33258 was analyzed on coded samples.
Brain Edema Measurement
Mice (n=5/group) were euthanized by cervical dislocation 24 or 72 hours after MIF/saline treatment. The brains were removed and divided into 2 hemispheres along the midline. The cerebellum served as internal control. Brain samples were immediately weighed on an analytical balance (Denver Instrument Co) to obtain the wet weight. Brain samples were dried in a speed vacuum concentrator (Savant Instruments) for 24 hours to obtain the dry weight. Brain edema was expressed as (wet weight–dry weight)/wet weight of brain tissue.
Immunofluorescence
Free-floating sections were washed in PBS for 20 minutes, blocked in 5% normal serum, and incubated with 5-D-4 (recognizes activated microglia; 1:1000; Seikageiku) or F4/80 antibody (recognizes activated monocytes and macrophages/microglial cells; 1:100; Serotec), followed by Alexa488 (1:1000; Molecular Probes) or Cy3-conjugated (1:1000; Jackson ImmunoResearch) secondary antibody. Stained sections were examined using a Nikon PCM 2000 confocal microscope; the images were captured and analyzed using SPOT Advanced image software. Control sections were processed as noted, except that primary antibodies were omitted. They were devoid of specific staining. Activated microglia/macrophages were counted in 3 different fields immediately adjacent to the hematoma in at least 3 sections/animal using a magnification of x400 over a microscopic field of 0.01 mm2 and averaged and expressed as cells/0.01 mm2. Three mice/group were analyzed by an observer blinded to the experimental treatment.
Statistics
Statistical analysis was performed using 2-tailed Student t test. Statistical significance was set at P<0.05.
Results
Effect of MIF on Microglial Activation and Macrophage Infiltration After ICH
We reported that clearance of intracerebral hematomas elicited by collagenase injection takes place more slowly in tissue plasminogen activator (tPA)–/– mice.9 Because the hematomas manifested similarly and did not differ significantly in size until >24 hours after the collagenase injection,9 we proposed that hematoma persistence in the tPA–/– mice ensued from a lack of fibrinolysis (secondary to a failure of the missing tPA to activate plasmin, which cleaves fibrin) and/or from decreased phagocytic activity (because microglial activation is attenuated in tPA–/– mice19). To examine the latter, we evaluated the degree to which activated microglia and infiltrating macrophages become recruited to the site of injury. Collagenase-induced ICH caused activated microglia (5-D-4+, round cells; Figure 1) and infiltrating macrophages (F4/80+; Figure 2) to accumulate at the site of injury and up to 0.8 mm away at 24 and 72 hours after injection. Infusion of an inhibitor of microglial activation known as MIF11,13 2 hours subsequent to the collagenase injection blocked microglial activation and additionally inhibited macrophage accumulation, as evidenced by a dramatic reduction in activated microglia (day 1, from 6.1±1.7 to 0.9±0.2; day 3, from 7.1±1.2 to 1±0.3; mean±SD, numbers of cells/0.01 mm2; P<0.05, P<0.01, respectively; n=3 per group) and infiltrated macrophages (day 1, from 5.3±0.3 to 1.4±0.2; day 3, from 7.3±1.2 to 1.7±0.7; mean±SD, numbers of cells/0.01 mm2; both P<0.01; n=3 per group) in the border zone surrounding the site of injury (Figures 1 and 2).
Effect of MIF on ROS Production
ROS, a major factor in the pathogenesis of neuronal damage, is generated by microglia and macrophages. ROS production can be assessed by the deposition of oxidized hydroethidine (ethidium) as small red particles in the nucleus and cytosol. On day 1 after collagenase injection, the peri-ICH region exhibited significantly increased levels of oxidized hydroethidine (Figure 3A) compared with the contralateral (uninjected) side of the brain (Figure 3B). In MIF-treated mice, the number and intensity of ethidium-positive cells was decreased substantially (Figure 3C). Some cells exhibited significant levels of oxidized hydroethidine under normal physiological conditions and after ICH. These cells, however, appeared to be confined to the microvasculature and displayed morphology characteristic of endothelial cells (Figure 3D and 3E). Quantification revealed that MIF significantly attenuated the number of ethidium-positive cells in comparison to control ICH mice (saline-treated, 61.8±12.13; MIF-treated, 40.±12.03; mean±SD of percentage of oxidized hydroethidine-positive cells; P=0.023; n=5/group).
Effect of MIF on Stroke Volume, Edema, and Neuronal Cell Death
Because delivery of MIF after injury inhibited microglial activation and macrophage infiltration, we evaluated its effect on injury volume, edema, and the ensuing neuronal death. MIF reduced brain injury volume after ICH (day 1, from 11.7±4.2 mm3 to 6.7±2.5 mm3, P=0.034, n=6 to 8/group; day 3, from 12.7±2.6 mm3 to 7.8±1.8 mm3, P=0.009, n=5/group; Figure 4).
Brain edema is an important clinical complication of ICH. In our collagenase-induced ICH model, we measured brain water content. Compared with saline-treated controls, MIF after treatment reduced brain edema in the lesioned hemisphere after ICH (day 1, from 77.84±2.25% to 72.86±3.2%, P=0.02, n=5/group; day 3, from 79.84±2.08% to 74.88±2.21%, P=0.007, n=5/group; Figure 5). These results indicate that activated microglia/macrophages contribute to brain injury and edema after ICH.
To examine whether neuronal death was evident at the site of hemorrhage, we used Fluoro-Jade B histological staining. MIF reduced the number of degenerating neurons after ICH (day 1, from 295±32/mm2 to 242±37/mm2, n=5 to 8/group, P=0.024; day 3, from 415±41/mm2 to 318±16/mm2, n=5/group, P=0.004; Figure 6A).
Effect of MIF on ICH-Induced Neurobehavioral Deficits
ICH is usually accompanied by characteristic behavioral deficits. To determine whether the ICH pathological and molecular events that showed improvement after MIF treatment were paralleled by neurobehavioral recovery, repeated assessments of the animals were performed on days 1 and 3 after ICH. MIF significantly improved the neurobehavioral score of the animals compared with control animals; the score changed from 8.4±1.0 (n=16) to 6.8±1.3 (n=19) on day 1 (P=0.007) and from 7.6±1 (n=10) to 6.6±0.9 (n=10) on day 3 (P=0.03; Figure 6B). Active treatment with MIF improved all the tests evaluated. Preliminary results showed that MIF after treatment (100 μL at 500 μmol/L) delivered intravenously via the retro-orbital route also improved neurological function, further supporting the potential of MIF for clinical settings (data not shown).
Discussion
In this study, we used an ICH model pioneered by Clark et al.8 Collagenase, a proteolytic enzyme, is secreted at sites of inflammation by mononuclear cells and dissolves the extracellular matrix around capillaries and to open the blood-brain barrier. The injection of collagenase results in active intraparenchymal bleeding that models spontaneous ICH. The advantage of this model is that the hemorrhage is spontaneous in nature. The perifocal edema that occurs in the model may be either caused by ischemia resulting from vessel rupture or caused by the hematoma itself. Although bacterial collagenase induces significant inflammatory reactions, it has no direct effect on microglial activation.9
Using this model, we showed previously that ICH is characterized by accumulation of activated microglia and it persists in tPA–/– mice. We also reported that delivery of MIF before the induction of hemorrhage reduced the extent of neuronal damage.9 Whereas priming the system with MIF before the injury gave us a direction for investigation, we pursued a more relevant approach for treatment using MIF in this report to determine whether MIF administration after the induction of ICH also achieved tangible benefit.
Inflammation contributes to brain injury after ICH.4–7 Our results indicate that in addition to macrophage infiltration, microglial activation occurs in and around hematomas.9 The presence of activated microglia has been documented in another model of ICH in which rats are injected intracerebrally with autologous blood.5 Evidence, both in vitro20,21 and in vivo,22,23 indicates that activated microglia/macrophages constitute a source of ROS, which are a major factor in the pathogenesis of neuronal damage.24 ROS are mediators of ischemia/reperfusion injury.25 However, little is known about ROS in ICH. In the current study, increased ROS production was seen in the peri-ICH area, suggesting that ROS mediate acute ICH injury. ROS are detected in endothelial cells in normal and hemorrhagic conditions, suggesting that endothelial cells are also important in O2 production. This is consistent with a previous report that endothelial cells are a significant source of O2– after ischemia.17 Studies have shown that infusion of MIF delays microglial activation9,12 and is protective to neurons subjected to excitotoxic injury11 or facial nerve axotomy.12 This inhibition of timely activation results in a corresponding decrease in secretion of tumor necrosis factor-, tPA, and ROS by microglia/macrophages.11–13,22
Activated microglia exert cytotoxic effects in the brain through 2 very different, complementary processes.11 First, they promote neurotoxicity by releasing a variety of potentially noxious substances, and second, they function as phagocytes to eliminate injured neurons and debris,11 each of which is critical at a different time of disease progression. Consistent with the notion that MIF inhibits microglial activation and macrophage infiltration, when MIF is given 2 days before9 or 2 hours after collagenase injection, a reduced number of activated microglia and infiltrated macrophages is observed, indicating attenuation of microglial activation and macrophage migration. This attenuation results in decreased production of ROS, reduction of numbers of degenerating neurons, brain injury volume and edema, and improvements in neurological functional outcome on days 1 and 3 after ICH. However, longer-term therapeutic use of MIF may be unwarranted because it may eliminate neuroprotective benefits of microglia/macrophages as phagocytes13 and suppliers of neuroprotective molecules.26–29
In conclusion, our study provides support to the idea that activated microglia and macrophages are important contributors to brain injury after ICH. Our results also suggest that MIF could be a valuable neuroprotective agent for the treatment of ICH if treatment is initiated soon after the onset of hemorrhage. Further studies should be conducted to establish efficient routes of administration and define the maximum time after ICH at which treatment remains effective.
Acknowledgments
We thank members of the Tsirka laboratory and Dr Michael Frohman for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (RO1NS042168; S.E.T.) and a fellowship from the American Heart Association (0225701T; J.W.).
References
Qureshi AI, Tuhrim S, Broderick JP, Batjer HH, Hondo H, Hanley DF. Spontaneous intracerebral hemorrhage. N Engl J Med. 2001; 344: 1450–1460.
Matsushita K, Meng W, Wang X, Asahi M, Asahi K, Moskowitz MA, Lo EH. Evidence for apoptosis after intracerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab. 2000; 20: 396–404.
Gong C, Boulis N, Qian J, Turner DE, Hoff JT, Keep RF. Intracerebral hemorrhage-induced neuronal death. Neurosurgery. 2001; 48: 875–882.
Hickenbottom SL, Grotta JC, Strong R, Denner LA, Aronowski J. Nuclear factor-kappa B and cell death after experimental intracerebral hemorrhage in rats. Stroke. 1999; 30: 2472–2477.
Gong C, Hoff JT, Keep RF. Acute inflammatory reaction following experimental intracerebral hemorrhage in rat. Brain Res. 2000; 871: 57–65.
Castillo J, Davalos A, Alvarez-Sabin J, Pumar JM, Leira R, Silva Y, Montaner J, Kase CS. Molecular signatures of brain injury after intracerebral hemorrhage. Neurology. 2002; 58: 624–629.
Wang J and Tsirka SE. Contribution of extracellular proteolysis and microglia to intracerebral hemorrhage. Neurocrit Care 2005. In press.
Clark W, Gunion-Rinker L, Lessov N, Hazel K. Citicole treatment for experimental intracerebral hemorrhage in mice. Stroke. 1998; 29: 2136–2140.
Wang J, Rogove AD, Tsirka AE, Tsirka SE. Protective role of tuftsin fragment 1–3 in an animal model of intracerebral hemorrhage. Ann Neurol. 2003; 54: 655–664.
Thiex R, Mayfrank L, Rohde V, Gilsbach JM, Tsirka SA. The role of endogenous versus exogenous tPA on edema formation in murine ICH. Exp Neurol. 2004; 189: 25–32.
Rogove AD, Tsirka SE. Neurotoxic responses by microglia elicited by excitotoxic injury in the mouse hippocampus. Curr Biol. 1998; 8: 19–25.
Thanos S, Mey J, Wild M. Treatment of the adult retina with microglia-suppressing factors retards axotomy-induced neuronal degradation and enhances axonal regeneration in vivo and in vitro. J Neurosci. 1993; 13: 455–466.
Auriault C, Joseph M, Tartar A, Capron A. Characterization and synthesis of a macrophage inhibitory peptide from the second constant domain of human immunoglobulin G. FEBS Lett. 1983; 153: 11–15.
Geisler S, Heilmann H, Veh RW. An optimized method for simultaneous demonstration of neurons and myelinated fiber tracts for delineation of individual trunco- and palliothalamic nuclei in the mammalian brain. Histochem Cell Biol. 2002; 117: 69–79.
Schmued LC, and Hopkins KJ. Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res. 2000; 874: 123–130.
Kim GW, Noshita N, Sugawara T, Chan PH. Early decrease in DNA repair proteins, Ku70 and Ku86, and subsequent DNA fragmentation after transient focal cerebral ischemia in mice. Stroke. 2001; 32: 1401–1407.
Kim GW, Kondo T, Noshita N, Chan PH. Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice: implications for the production and role of superoxide radicals. Stroke. 2002; 33: 809–815.
Bindokas VP, Jordan J, Lee CC, Miller RJ. Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine. J Neurosci. 1996; 16: 1324–1336.
Rogove AD, Siao C, Keyt B, Strickland S, Tsirka SE. Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system. J Cell Sci. 1999; 112: 4007–4016.
Sankarapandi S, Zweier JL, Mukherjee G, Quinn MT, Huso DL. Measurement and characterization of superoxide generation in microglial cells: evidence for an NADPH oxidase-dependent pathway. Arch Biochem Biophys. 1998; 353: 312–321.
Thery C, Chamak B, Mallat M. Cytotoxic Effect of brain macrophages on developing. Eur J Neurosci. 1991; 3: 1155–1164.
Wu DC, Teismann P, Tieu K, Vila M, Jackson-Lewis V, Ischiropoulos H, Przedborski S. NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease. Proc Natl Acad Sci U S A. 2003; 100: 6145–6150.
Green SP, Cairns B, Rae J, Errett-Baroncini C, Hongo JA, Erickson RW, Curnutte JT. Induction of gp91-phox, a component of the phagocyte NADPH oxidase, in microglial cells during central nervous system inflammation. J Cereb Blood Flow Metab. 2001; 21: 374–384.
Facchinetti F, Dawson VL, Dawson TM. Free radicals as mediators of neuronal injury. Cell Mol Neurobiol. 1998; 18: 667–682.
Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab. 2001; 21: 2–14.
Elkabes S, DiCicco-Bloom EM, Black IB. Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function. J Neurosci. 1996; 16: 2508–2521.
Lee TH, Kato H, Chen ST, Kogure K, Itoyama Y. Expression disparity of brain-derived neurotrophic factor immunoreactivity and mRNA in ischemic hippocampal neurons. Neuroreport. 2002; 13: 2271–2275.
Miwa T, Furukawa S, Nakajima K, Furukawa Y, Kohsaka S. Lipopolysaccharide enhances synthesis of brain-derived neurotrophic factor in cultured rat microglia. J Neurosci Res. 1997; 50: 1023–1029.
Nakajima K, Honda S, Tohyama Y, Imai Y, Kohsaka S, Kurihara T. Neurotrophin secretion from cultured microglia. J Neurosci Res. 2001; 65: 322–331.