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Granulocyte colony-stimulating factor induces sensorimotor recovery in intracerebral hemorrhage
Brain Research 1041 (2005) 125 – 131 www.elsevier.com/locate/brainres
Research report
Granulocyte colony-stimulating factor induces sensorimotor recovery in intracerebral hemorrhage Hee-Kwon Parka,1, Kon Chua,b,c,1, Soon-Tae Leea,b,1, Keun-Hwa Junga,b, Eun-Hee Kima, Kyung-Bok Leea, Young-Mok Songd, Sang-Wuk Jeonge, Manho Kima,b, Jae-Kyu Roha,b,T a
Department of Neurology, Stroke and Neural Stem Cell Laboratory, Clinical Research Institute, Seoul National University Hospital, 28, Yongon-Dong, Chongro-Gu, Seoul, 110-744, South Korea b Program in Neuroscience, Neuroscience Research Institute of SNUMRC, Seoul National University, Seoul, South Korea c Center for Alcohol and Drug Addiction Research, Seoul National Hospital, Seoul, South Korea d Department of Neurology, Dankook University Hospital, Chonan City, South Korea e Department of Neurology, Ilsan Paik Hospital, Inje University, Goyang City, South Korea Accepted 16 November 2004 Available online 23 March 2005
Abstract Granulocyte colony-stimulating factor (G-CSF) has been used in the treatment of neutropenia in hematologic disorders. The neuroprotective and anti-inflammatory effects of G-CSF were reported in various neurological disease models. In this study, we examined whether G-CSF induces functional recovery after intracerebral hemorrhage (ICH). ICH was induced using collagenase injection in adult rats. Either G-CSF (50 Ag/kg, i.p.) or saline was given from 2 h after ICH and every 24 h for 3 days. 72 h after ICH induction, the rats were sacrificed for histological analysis and measurement of brain edema. Behavioral tests were performed before and 1, 7, 14, 21, 28, and 35 days after ICH. We also measured the blood–brain barrier (BBB) permeability using Evans blue dye injection method. G-CSF-treated rats recovered better on rotarod and limb placing tests, starting from 14 days throughout 5 weeks after ICH. The brain water content and BBB permeability of G-CSF-treated group decreased in the lesioned hemispheres compared with those of ICH-only group. In G-CSF-treated group, the number of TUNEL+, myeloperoxidase+, and OX42+ cells was smaller than that of ICH-only group in the periphery of hematoma. These findings suggest that G-CSF induces long-term sensorimotor recovery after ICH with reduction of brain edema, inflammation, and perihematomal cell death. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of nervous system Topic: Neurotrophic factors: biological effects Keywords: G-CSF; Intracerebral hemorrhage; Brain edema; Inflammation; Atrophy
1. Introduction Intracerebral hemorrhage (ICH) represents at least 10– 15% of all strokes in the Western population [20]. Medical
T Corresponding author. Fax: +822 3672 4949. E-mail address: [email protected] (J.-K. Roh). 1 The first three authors contributed equally to this work. 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.11.067
therapy for patients with ICH is limited to only supportive care or invasive evacuation of hematoma in selective patients. Previous studies on ICH indicated that brain injury can be caused by inflammatory mediators released from the blood and leukocytes [2,8,13,18,19], characterized by the induction of parenchymal inflammation within hours of ICH occurrence. It is initiated by adherence of leukocytes to damaged brain endothelia and subsequent brain entry [19]. TNF-a, matrix metalloproteinases (MMPs; especially
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MMP-12), adhesion molecules, and glutamate levels have been reported to be up-regulated after ICH [8,19]. Granulocyte colony-stimulating factor (G-CSF), a 20kDa protein, is a member of the cytokine family of growth factors. It stimulates the proliferation, survival, and maturation of the cells committed to the neutrophilic granulocyte lineage [10]. G-CSF expands the monocyte/macrophage subset and promotes an anti-inflammatory pattern conferring protection in murine endotoxemia [9]. Moreover, T cell allogenic and mitogenic reactivities are inhibited in G-CSFtreated individuals [15], corresponding to a reduced interferon-g production capacity [11]. It is known that G-CSF exerts anti-inflammatory and pro-Th2 effects via JAK-STAT signaling to trigger an imbalance in the cytokines produced by lymphocytes, resulting in the reduction of interferon-g and TNF-a release and increase in the levels of antagonists for inflammatory cytokines [2,6,9,27]. This could reduce cytokine toxicity, neutrophil activation, and infiltration [12]. The anti-inflammatory effect of G-CSF provided durable protection from experimental autoimmune encephalomyelitis (EAE) [27]. G-CSF administration modulates and enhances endogenous bone marrow stem cells to acquire neuronal characteristics [5] and have anti-apoptotic and neuroprotective effects on neuronal cells [22]. In the present study, we examined the two hypotheses. First, G-CSF treatment may have anti-inflammatory action in the setting of ICH. Second, G-CSF treatment may also reduce the brain edema and perihematomal cell death following ICH and improve functional outcome.
2. Materials and methods 2.1. Induction of intracerebral hemorrhage and G-CSF treatment All the procedures were performed following an institutionally approved in accordance with NIH Guide for the Care and Use of Laboratory Animals. 163 male Sprague– Dawley rats (Daehan Bio, Seoul, R.O.K.) weighing 200 to 220 g were used in these experiments. Rats were grouped as ICH-only group (n = 72), G-CSF-treated ICH group (n = 78), sham-operated group (n = 9), and G-CSF-treated normal group (n = 4). Experimental ICH was induced by the stereotaxic intrastriatal administration of bacterial collagenase type IV (Sigma), as described elsewhere [4,12,13,21]. In brief, after intraperitoneal injections of 1% ketamine (30 mg/kg; Sigma) and xylene hydrochloride (4 mg/kg; Sigma), rats were placed in a stereotaxic frame (David Kopf instruments, Tujunga, CA), and a burr hole was made. 30-gauge Hamilton syringe needle was inserted into the striatum. ICH was induced by the administration of 1 Al containing 0.23 CDU of collagenase type IV (Sigma) over 5 min. After completion of collagenase infusion, craniotomies were sealed with bone wax, and rats were allowed to recover.
The recombinant human G-CSF (50 Ag/kg, Kirin, Japan), dissolved in 2 ml of 0.9% saline, was administered intraperitoneally, 2 h after ICH induction and daily afterward for 3 days. Saline was also given to the ICH-only group during the same period. Physiologic parameters, including mean arterial blood pressure, blood gases, and glucose concentration, were measured during the experiment. 2.2. Behavioral testing Behavioral testing (n = 15, respectively) was performed weekly using the rotarod and modified limb placing tests (MLPT), which were monitored by two individuals blinded to rat treatment status. In the rotarod test [4,12], the rats were placed on the accelerating rotarod cylinder, and the time the animals remained on the rotarod was measured. The animals were trained for 3 days before stereotaxic operation. The maximum duration (in seconds) on the device was recorded with 3 rotarod measurements 1 day before ICH induction. Motor test data are presented as percentages of the maximal duration compared with the baseline control (before ICH). The MLPT is a modified version of a test previously described [4,12,13]. The test consists of two limb-placing tasks that assess the sensorimotor integration of the forelimb and the hindlimb by checking responses to tactile and proprioceptive stimulation. First, the rat is suspended 10 cm over a table and the stretch of the forelimbs towards the table is observed and evaluated: normal stretch, 0 points; abnormal flexion, 1 point. Next, the rat is positioned along the edge of the table, with its forelimbs suspended over the edge and allowed to move freely. Each forelimb (forelimb—second task, hindlimb— third task) is gently pulled down and retrieval and placement are checked. Finally, the rat is placed towards the table edge to check for lateral placement of the forelimb. The three tasks are scored in the following manner: normal performance, 0 points; performance with a delay (2 s) and/or incomplete, 1 point; no performance, 2 points. 7 points means maximal neurological deficit and 0 points means normal performance. 2.3. Measuring brain water contents and BBB permeability Three days after operation, rats were anesthetized and sacrificed by decapitation (n = 12 respectively). The brains were removed immediately and divided into two hemispheres along the midline then the cerebellum was removed. The brain samples were immediately weighed on an electronic analytical balance to obtain the wet weight and then dried in a gravity oven at 100 8C for 24 h to obtain the dry weight. Water contents were expressed as a percentage of wet weight [4,13]. To evaluate vascular permeability, a quantitative fluorescent detection of extra-vasated Evans blue dye was used as described elsewhere [2]. Briefly, Evans blue (3 ml/kg of 2% in 0.9% normal saline) was injected into
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the rats via a tail vein over 3 min on the 3rd day after ICH induction in both groups (n = 6 respectively). Rats were sacrificed on the next day after Evans blue injection. Brains were rapidly removed after perfusion with normal saline and sectioned coronally into a 4-mm rostral block from the collagenase injection site. After separating right and left hemispheres, each segment was weighed and placed in 10-fold volume of 50% trichloroacetic acid solution. Following homogenization and centrifugation, the supernatant was diluted with ethanol (1:25) and was measured with a luminescence spectrometer. The tissue content of Evans blue was quantified from a linear standard curve derived from known amounts of the dye and was normalized to sample weight. 2.4. Histological analysis Histological analysis was done both in G-CSF-treated group and ICH-only group, at earlier timing (3 days after ICH, n = 9 respectively; evaluation of cell death and inflammation) and later time period (6 weeks after ICH, n = 9 respectively; evaluation of hemispheric atrophy). Rats were perfused by cardiac puncture with 100 ml cold saline and 100 ml of 4% paraformaldehyde in 0.1 mol/l phosphatebuffered saline. Brains were removed and cryopreserved for cryostat sectioning at 30-Am thicknesses. Immunostains were processed as described previously [4,12,13]. We performed immunostaining with antibodies to myeloperoxidase (MPO; 1:200, DAKO, A0398) and OX42 (1:100, Chemicon, MAB1405), followed by fluorescent microscopy (Nikon, MD, USA) for evaluation of inflammatory infiltrates. The axial sections through the center (3 sections per animal; 1 mm width) of the hemorrhagic lesion were analyzed. We counted marker-specific cells in the whole section (3 sections per each antibody staining). The MPO+ or OX42+ cells were identified and counted. Total counts in these sections were converted into cell densities with respect to the entire hemisphere and compared between the groups.
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injection (n = 9 respectively). The TUNEL procedure for in situ detection of DNA fragmentation was performed as described elsewhere [4,7,13,14], using a TUNEL kit (Oncogene, QIA33). Cell density counts were performed on sections counterstained with toluidine blue. 2.7. Spectrophotometric assay of intracerebral hemorrhage Cerebral hemorrhage was quantified with a previously described spectrophotometric assay [1,3,25]. Hemispheric brain tissue was obtained from normal rats subjected to complete transcardial perfusion to remove intravascular blood. Incremental volumes of homologous blood (0 to 200 Al) were added to each hemispheric sample with PBS to reach a total volume of 3 ml, followed by homogenization for 30 s, sonication on ice for 1 min, and centrifugation at 13,000 rpm for 30 min. Drabkin’s reagent (1.6 ml; Sigma) was added to 0.4-ml aliquots and allowed to stand for 15 min at room temperature. Optical density was measured and recorded at 540 nm with a spectrophotometer (Molecular Devices, CA, USA). Measurements from perfused brains subjected to ICH were compared with this standard curve to obtain data in terms of hemorrhage volume (Al). Rats were assigned to 5 different groups with different sacrifice timing and G-CSF treatment starting point (n = 6, respectively); early group (measured at 6 h after ICH; ICH-only vs. 2 h GCSF) and late group (measured at 3 days after ICH; ICH only vs. 2 h G-CSF vs. 6 h G-CSF). 2.8. Statistical analysis All data in this study are presented as mean F standard deviations. Data were analyzed by repeated measures of analysis of variance, and unpaired Student’s t test, if they were normally distributed (Kolmogorov–Smirov test, P N 0.05). Otherwise, we used Mann–Whitney U test and specified the test used. Two-tailed probability value of b0.05 was considered significant.
3. Results 2.5. Morphometric measurement of hemispheric atrophy 3.1. Physiological parameters For determining hemispheric atrophy, three sections through the needle entry site were Nissl stained at 6 weeks after ICH (n = 9 respectively). The total hemispheric areas of each section were traced and measured using image analyzer. The morphometric analyses involved computer-assisted hand delineation of the area of the striatum, cerebral cortex, and ventricle as well as the whole hemisphere [12]. 2.6. TUNEL assay Rats used for TUNEL staining were sacrificed with an overdose of sodium pentobarbital at 72 h after collagenase
Moderate leukocytosis was observed in G-CSF-treated group (4080 F 172 cells/Al vs. 2330 F 386 cells/Al; P b 0.01, Mann–Whitney U test) at 1 day after ICH. Other physiological parameters were not different before and 30 min after ICH (Table 1). 3.2. G-CSF improved sensorimotor deficits The G-CSF-treated group showed better performance on the rotarod test, persisting for up to 5 weeks than the control group (Fig. 1). From the 2nd week, the G-CSF-treated group showed better results on MLPT (Fig. 1). The sensorimotor
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Table 1 Physiologic parameters before and 30 min after ICH induction ICH-only group Baseline pH PaO2, mm Hg PaCO2, mm Hg Rectal temperature, 8C MABP, mm Hg Serum glucose, mg/dl
7.38 109.1 39.3 36.8 88 94
F F F F F F
0.03 10.5 1.3 0.1 3 4
G-CSF-treated group After ICH
Baseline
7.37 102.3 40.1 36.8 85 86
7.39 108.1 39.8 36.6 86 89
F F F F F F
0.02 7.2 1.2 0.4 3 7
F F F F F F
After ICH 0.02 6.5 1.1 0.2 3 8
7.37 101.1 39.4 36.6 88 89
F F F F F F
0.02 5.7 1.4 0.4 1.8 4
Values are means F SD. One-way analysis of variance revealed no significant inter-group difference for any variable. ICH indicates intracerebral hemorrhage, and MABP, mean arterial blood pressure.
improvement was maintained throughout the entire experimental period. Sham-operated rats (n = 9) showed only slight sensorimotor abnormality at 1 day after surgery, and those deficits disappeared from 7 days after surgery. When G-CSF was given to normal, unoperated rats (n = 4), its effect on the sensorimotor testing was not found. 3.3. G-CSF decreased brain edema, BBB permeability, and hemispheric atrophy Brain water content of the lesioned (left) hemisphere was 80.47 F 0.35% in ICH-only group and 77.93 F 0.49% in G-CSF-treated group ( P b 0.01, t test) and those of nonlesioned (right) hemisphere was 78.94 F 0.62% in ICHonly group and 77.25 F 0.41% in G-CSF-treated group ( P b 0.05, t test; Fig. 2A). Quantitative analysis of Evans blue dye penetration demonstrated that leakage of the G-CSFtreated group was significantly lower than that of the control group in the lesioned hemisphere (5.7 F 1.5 Ag/g vs. 22.87 F 1.87 Ag/g, P b 0.01, Fig. 2B). Hemispheric area analysis at 6 weeks after ICH showed a significant atrophy of the lesioned hemisphere in both groups. The G-CSFtreated group showed lesser hemispheric (13.1 F 3.2% [GCSF] vs. 24.2 F 4.0% [ICH-only]), striatal (14.9 F 5.2%
vs. 28.3 F 3.6%), and cortical (4.0 F 2.9% vs. 7.4 F 3.1%) atrophy compared with the ICH-only group significantly ( P b 0.01; Fig. 2C). The volumes of hemorrhage at 3 days after ICH were 22.3 F 3.5 Al (ICH-only), 24.9 F 5.1 Al (2 h G-CSF), and 22.7 F 4.9 Al (6 h G-CSF), which were not statistically different (Fig. 3B; P N 0.05). To analyze the possible early effect of G-CSF on the collagenase activity (associated with hematoma formation in this experiment), we performed an additional spectrophotometric assay of hematoma volume. The hematoma volumes at 6 h after ICH were 45.9 F 4.9 Al (ICH-only) and 42.6 F 7.5 Al (2 h G-CSF), which also showed no difference ( P = 0.68; Fig. 3B). 3.4. G-CSF treatment decreased inflammation and perihematomal cell death The TUNEL staining revealed a high density of positively stained cells within the hemorrhage lesion itself as well as in the surrounding periphery (Fig. 4). TUNEL+ cells were mostly found in the perihematomal areas and rarely found in the contralateral side. Quantitative analysis showed regional TUNEL+ cell differences between the groups. The ICH-only group (337.50 F 24.95 cells/mm2)
Fig. 1. Behavioral tests. In the rotarod test (A) and modified limb placing test (B), the G-CSF-treated ICH group showed a better performance from 14 days onwards, and these benefits continued up to 5 weeks ( P b 0.01). n = 15 per group. *P b 0.05, **P b 0.01 (ANOVA) compared with ICH-only group.
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Fig. 2. Hemispheric atrophy and brain water content. G-CSF treatment reduces brain water content (A) with blood–brain barrier permeability (B) in lesioned and contralateral hemispheres at 3 days after ICH and also reduces the degree of progressive hemispheric atrophy at 6 weeks after ICH (C). Values are mean F SD. *P b 0.05, **P b 0.01 compared with ICH-only group.
showed significantly more TUNEL+ cells than the G-CSFtreated group (141.88 F 36.03 cells/mm2; P b 0.01, t test; Fig. 4). In G-CSF-treated group, MPO+ neutrophils
(136.84 F 12.25 cells/mm2 [G-CSF] vs. 199.98 F 16.32 cells/mm2 [ICH-only]; P b 0.01) or OX42+ microglial cells (25.79 F 5.59 cells/mm2 [G-CSF] vs. 89.31 F 10.58 cells/
Fig. 3. Spectrophotometric assay of intracerebral hemorrhage. (A) The linear relationship between blood volume and hemoglobin absorbance in the spectrophotometric assay allowed this to be used as a standard curve for calculating hemorrhage volumes in G-CSF-treated brains. (B) Collagenase-induced hemorrhage volumes (mean F SD) were not different with the timing of G-CSF treatment. Hemorrhage volume was smaller when measured at 3 days than 1 day after ICH.
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Fig. 4. Evaluations of inflammatory cells and perihematomal cell death. (A, C, E) G-CSF-treated group, (B, D, F) ICH-only group. Detection of TUNEL reaction product (A, B), myeloperoxidase (MPO)-positive cells (C, D), and OX-42-positive cells (E, F) in the perihematomal area 3 days after ICH. (G) Quantification of inflammatory cells and TUNEL+ cells. Values are mean F SD. n = 9 per group. **P b 0.01 compared with ICH-only group. Scale bar = 100 Am.
mm2 [ICH-only]; P b 0.01) were less found around the perihematomal striatum, compared with ICH-only group at 3 days after ICH (Fig. 4).
4. Discussion These experiments were undertaken to test whether the administration of G-CSF may have a therapeutic effect on ICH. In this study, G-CSF reduces brain edema, perihematomal cell death, and inflammation after ICH with sensorimotor improvement. Furthermore, G-CSF treatment reduces the progressive hemispheric atrophy. The mechanism how the G-CSF reduced the brain edema is unknown. Previous studies on ICH indicated that the inflammatory mediators released from the infiltrating leukocytes and activated microglia caused brain edema and the functional deficit after ICH [4,8,13,18,19]. Rodent and human studies have shown that G-CSF-induced antiinflammatory effect is associated with a shift toward Th2 from Th1, resulting in a reduction of Th1-oriented cytokine production such as TNF-a and IFN-g [17,23,27]. In our study, systemic administration of G-CSF reduced the ICHassociated pathologic findings, suggesting that the beneficial effect of G-CSF on brain edema might be mediated by its anti-inflammatory effect. However, whether this results from a local effect of G-CSF crossing the BBB cannot be excluded. In addition, the collagenase-induced ICH model is
not physiologic and raise a concern that collagenase itself may induce excess inflammation. However, our previous results showed that direct collagenase toxicity was not the major cause of inflammation or cell death [4]. Despite of the leukocytosis in the peripheral blood, the number of infiltrating microglia and neutrophils was less in G-CSF-treated group than in ICH-only group. The leukocytosis but impaired leukocyte accumulation in the damaged tissue has been previously described on MPO+ cells [22,24,26]. It is suggested that the G-CSF-stimulated neutrophils may not necessarily migrate into the injured tissue or aggravate inflammation. The reduction of BBB leakage and the reduction of cytokines (e.g. TNF-a) also could decrease the infiltrating leukocytes [24]. Effect of the inflammatory cytokines in ICH is required to further determine the role of G-CSF. G-CSF has been known to have anti-apoptotic effects in animal models of myocardial infarction and cerebral ischemia [16,22]. In the present study, G-CSF treatment reduced perihematomal cell death with long-term hemispheric atrophy after ICH. Perihematomal cell loss after ICH can be caused by apoptosis, mediated by thrombin of the hematoma [14]. In ischemia model, G-CSF achieved a neuroprotective effect mediated by G-CSFR and JAKSTAT3 pathway [22], which might also be involved in the perihematomal cell death in the ICH model. However, the hypothesis that G-CSF can reduce the apoptotic cascade after ICH via JAK-STAT3 cascade remains to be defined.
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Research report
Granulocyte colony-stimulating factor induces sensorimotor recovery in intracerebral hemorrhage Hee-Kwon Parka,1, Kon Chua,b,c,1, Soon-Tae Leea,b,1, Keun-Hwa Junga,b, Eun-Hee Kima, Kyung-Bok Leea, Young-Mok Songd, Sang-Wuk Jeonge, Manho Kima,b, Jae-Kyu Roha,b,T a
Department of Neurology, Stroke and Neural Stem Cell Laboratory, Clinical Research Institute, Seoul National University Hospital, 28, Yongon-Dong, Chongro-Gu, Seoul, 110-744, South Korea b Program in Neuroscience, Neuroscience Research Institute of SNUMRC, Seoul National University, Seoul, South Korea c Center for Alcohol and Drug Addiction Research, Seoul National Hospital, Seoul, South Korea d Department of Neurology, Dankook University Hospital, Chonan City, South Korea e Department of Neurology, Ilsan Paik Hospital, Inje University, Goyang City, South Korea Accepted 16 November 2004 Available online 23 March 2005
Abstract Granulocyte colony-stimulating factor (G-CSF) has been used in the treatment of neutropenia in hematologic disorders. The neuroprotective and anti-inflammatory effects of G-CSF were reported in various neurological disease models. In this study, we examined whether G-CSF induces functional recovery after intracerebral hemorrhage (ICH). ICH was induced using collagenase injection in adult rats. Either G-CSF (50 Ag/kg, i.p.) or saline was given from 2 h after ICH and every 24 h for 3 days. 72 h after ICH induction, the rats were sacrificed for histological analysis and measurement of brain edema. Behavioral tests were performed before and 1, 7, 14, 21, 28, and 35 days after ICH. We also measured the blood–brain barrier (BBB) permeability using Evans blue dye injection method. G-CSF-treated rats recovered better on rotarod and limb placing tests, starting from 14 days throughout 5 weeks after ICH. The brain water content and BBB permeability of G-CSF-treated group decreased in the lesioned hemispheres compared with those of ICH-only group. In G-CSF-treated group, the number of TUNEL+, myeloperoxidase+, and OX42+ cells was smaller than that of ICH-only group in the periphery of hematoma. These findings suggest that G-CSF induces long-term sensorimotor recovery after ICH with reduction of brain edema, inflammation, and perihematomal cell death. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of nervous system Topic: Neurotrophic factors: biological effects Keywords: G-CSF; Intracerebral hemorrhage; Brain edema; Inflammation; Atrophy
1. Introduction Intracerebral hemorrhage (ICH) represents at least 10– 15% of all strokes in the Western population [20]. Medical
T Corresponding author. Fax: +822 3672 4949. E-mail address: [email protected] (J.-K. Roh). 1 The first three authors contributed equally to this work. 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.11.067
therapy for patients with ICH is limited to only supportive care or invasive evacuation of hematoma in selective patients. Previous studies on ICH indicated that brain injury can be caused by inflammatory mediators released from the blood and leukocytes [2,8,13,18,19], characterized by the induction of parenchymal inflammation within hours of ICH occurrence. It is initiated by adherence of leukocytes to damaged brain endothelia and subsequent brain entry [19]. TNF-a, matrix metalloproteinases (MMPs; especially
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MMP-12), adhesion molecules, and glutamate levels have been reported to be up-regulated after ICH [8,19]. Granulocyte colony-stimulating factor (G-CSF), a 20kDa protein, is a member of the cytokine family of growth factors. It stimulates the proliferation, survival, and maturation of the cells committed to the neutrophilic granulocyte lineage [10]. G-CSF expands the monocyte/macrophage subset and promotes an anti-inflammatory pattern conferring protection in murine endotoxemia [9]. Moreover, T cell allogenic and mitogenic reactivities are inhibited in G-CSFtreated individuals [15], corresponding to a reduced interferon-g production capacity [11]. It is known that G-CSF exerts anti-inflammatory and pro-Th2 effects via JAK-STAT signaling to trigger an imbalance in the cytokines produced by lymphocytes, resulting in the reduction of interferon-g and TNF-a release and increase in the levels of antagonists for inflammatory cytokines [2,6,9,27]. This could reduce cytokine toxicity, neutrophil activation, and infiltration [12]. The anti-inflammatory effect of G-CSF provided durable protection from experimental autoimmune encephalomyelitis (EAE) [27]. G-CSF administration modulates and enhances endogenous bone marrow stem cells to acquire neuronal characteristics [5] and have anti-apoptotic and neuroprotective effects on neuronal cells [22]. In the present study, we examined the two hypotheses. First, G-CSF treatment may have anti-inflammatory action in the setting of ICH. Second, G-CSF treatment may also reduce the brain edema and perihematomal cell death following ICH and improve functional outcome.
2. Materials and methods 2.1. Induction of intracerebral hemorrhage and G-CSF treatment All the procedures were performed following an institutionally approved in accordance with NIH Guide for the Care and Use of Laboratory Animals. 163 male Sprague– Dawley rats (Daehan Bio, Seoul, R.O.K.) weighing 200 to 220 g were used in these experiments. Rats were grouped as ICH-only group (n = 72), G-CSF-treated ICH group (n = 78), sham-operated group (n = 9), and G-CSF-treated normal group (n = 4). Experimental ICH was induced by the stereotaxic intrastriatal administration of bacterial collagenase type IV (Sigma), as described elsewhere [4,12,13,21]. In brief, after intraperitoneal injections of 1% ketamine (30 mg/kg; Sigma) and xylene hydrochloride (4 mg/kg; Sigma), rats were placed in a stereotaxic frame (David Kopf instruments, Tujunga, CA), and a burr hole was made. 30-gauge Hamilton syringe needle was inserted into the striatum. ICH was induced by the administration of 1 Al containing 0.23 CDU of collagenase type IV (Sigma) over 5 min. After completion of collagenase infusion, craniotomies were sealed with bone wax, and rats were allowed to recover.
The recombinant human G-CSF (50 Ag/kg, Kirin, Japan), dissolved in 2 ml of 0.9% saline, was administered intraperitoneally, 2 h after ICH induction and daily afterward for 3 days. Saline was also given to the ICH-only group during the same period. Physiologic parameters, including mean arterial blood pressure, blood gases, and glucose concentration, were measured during the experiment. 2.2. Behavioral testing Behavioral testing (n = 15, respectively) was performed weekly using the rotarod and modified limb placing tests (MLPT), which were monitored by two individuals blinded to rat treatment status. In the rotarod test [4,12], the rats were placed on the accelerating rotarod cylinder, and the time the animals remained on the rotarod was measured. The animals were trained for 3 days before stereotaxic operation. The maximum duration (in seconds) on the device was recorded with 3 rotarod measurements 1 day before ICH induction. Motor test data are presented as percentages of the maximal duration compared with the baseline control (before ICH). The MLPT is a modified version of a test previously described [4,12,13]. The test consists of two limb-placing tasks that assess the sensorimotor integration of the forelimb and the hindlimb by checking responses to tactile and proprioceptive stimulation. First, the rat is suspended 10 cm over a table and the stretch of the forelimbs towards the table is observed and evaluated: normal stretch, 0 points; abnormal flexion, 1 point. Next, the rat is positioned along the edge of the table, with its forelimbs suspended over the edge and allowed to move freely. Each forelimb (forelimb—second task, hindlimb— third task) is gently pulled down and retrieval and placement are checked. Finally, the rat is placed towards the table edge to check for lateral placement of the forelimb. The three tasks are scored in the following manner: normal performance, 0 points; performance with a delay (2 s) and/or incomplete, 1 point; no performance, 2 points. 7 points means maximal neurological deficit and 0 points means normal performance. 2.3. Measuring brain water contents and BBB permeability Three days after operation, rats were anesthetized and sacrificed by decapitation (n = 12 respectively). The brains were removed immediately and divided into two hemispheres along the midline then the cerebellum was removed. The brain samples were immediately weighed on an electronic analytical balance to obtain the wet weight and then dried in a gravity oven at 100 8C for 24 h to obtain the dry weight. Water contents were expressed as a percentage of wet weight [4,13]. To evaluate vascular permeability, a quantitative fluorescent detection of extra-vasated Evans blue dye was used as described elsewhere [2]. Briefly, Evans blue (3 ml/kg of 2% in 0.9% normal saline) was injected into
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the rats via a tail vein over 3 min on the 3rd day after ICH induction in both groups (n = 6 respectively). Rats were sacrificed on the next day after Evans blue injection. Brains were rapidly removed after perfusion with normal saline and sectioned coronally into a 4-mm rostral block from the collagenase injection site. After separating right and left hemispheres, each segment was weighed and placed in 10-fold volume of 50% trichloroacetic acid solution. Following homogenization and centrifugation, the supernatant was diluted with ethanol (1:25) and was measured with a luminescence spectrometer. The tissue content of Evans blue was quantified from a linear standard curve derived from known amounts of the dye and was normalized to sample weight. 2.4. Histological analysis Histological analysis was done both in G-CSF-treated group and ICH-only group, at earlier timing (3 days after ICH, n = 9 respectively; evaluation of cell death and inflammation) and later time period (6 weeks after ICH, n = 9 respectively; evaluation of hemispheric atrophy). Rats were perfused by cardiac puncture with 100 ml cold saline and 100 ml of 4% paraformaldehyde in 0.1 mol/l phosphatebuffered saline. Brains were removed and cryopreserved for cryostat sectioning at 30-Am thicknesses. Immunostains were processed as described previously [4,12,13]. We performed immunostaining with antibodies to myeloperoxidase (MPO; 1:200, DAKO, A0398) and OX42 (1:100, Chemicon, MAB1405), followed by fluorescent microscopy (Nikon, MD, USA) for evaluation of inflammatory infiltrates. The axial sections through the center (3 sections per animal; 1 mm width) of the hemorrhagic lesion were analyzed. We counted marker-specific cells in the whole section (3 sections per each antibody staining). The MPO+ or OX42+ cells were identified and counted. Total counts in these sections were converted into cell densities with respect to the entire hemisphere and compared between the groups.
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injection (n = 9 respectively). The TUNEL procedure for in situ detection of DNA fragmentation was performed as described elsewhere [4,7,13,14], using a TUNEL kit (Oncogene, QIA33). Cell density counts were performed on sections counterstained with toluidine blue. 2.7. Spectrophotometric assay of intracerebral hemorrhage Cerebral hemorrhage was quantified with a previously described spectrophotometric assay [1,3,25]. Hemispheric brain tissue was obtained from normal rats subjected to complete transcardial perfusion to remove intravascular blood. Incremental volumes of homologous blood (0 to 200 Al) were added to each hemispheric sample with PBS to reach a total volume of 3 ml, followed by homogenization for 30 s, sonication on ice for 1 min, and centrifugation at 13,000 rpm for 30 min. Drabkin’s reagent (1.6 ml; Sigma) was added to 0.4-ml aliquots and allowed to stand for 15 min at room temperature. Optical density was measured and recorded at 540 nm with a spectrophotometer (Molecular Devices, CA, USA). Measurements from perfused brains subjected to ICH were compared with this standard curve to obtain data in terms of hemorrhage volume (Al). Rats were assigned to 5 different groups with different sacrifice timing and G-CSF treatment starting point (n = 6, respectively); early group (measured at 6 h after ICH; ICH-only vs. 2 h GCSF) and late group (measured at 3 days after ICH; ICH only vs. 2 h G-CSF vs. 6 h G-CSF). 2.8. Statistical analysis All data in this study are presented as mean F standard deviations. Data were analyzed by repeated measures of analysis of variance, and unpaired Student’s t test, if they were normally distributed (Kolmogorov–Smirov test, P N 0.05). Otherwise, we used Mann–Whitney U test and specified the test used. Two-tailed probability value of b0.05 was considered significant.
3. Results 2.5. Morphometric measurement of hemispheric atrophy 3.1. Physiological parameters For determining hemispheric atrophy, three sections through the needle entry site were Nissl stained at 6 weeks after ICH (n = 9 respectively). The total hemispheric areas of each section were traced and measured using image analyzer. The morphometric analyses involved computer-assisted hand delineation of the area of the striatum, cerebral cortex, and ventricle as well as the whole hemisphere [12]. 2.6. TUNEL assay Rats used for TUNEL staining were sacrificed with an overdose of sodium pentobarbital at 72 h after collagenase
Moderate leukocytosis was observed in G-CSF-treated group (4080 F 172 cells/Al vs. 2330 F 386 cells/Al; P b 0.01, Mann–Whitney U test) at 1 day after ICH. Other physiological parameters were not different before and 30 min after ICH (Table 1). 3.2. G-CSF improved sensorimotor deficits The G-CSF-treated group showed better performance on the rotarod test, persisting for up to 5 weeks than the control group (Fig. 1). From the 2nd week, the G-CSF-treated group showed better results on MLPT (Fig. 1). The sensorimotor
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Table 1 Physiologic parameters before and 30 min after ICH induction ICH-only group Baseline pH PaO2, mm Hg PaCO2, mm Hg Rectal temperature, 8C MABP, mm Hg Serum glucose, mg/dl
7.38 109.1 39.3 36.8 88 94
F F F F F F
0.03 10.5 1.3 0.1 3 4
G-CSF-treated group After ICH
Baseline
7.37 102.3 40.1 36.8 85 86
7.39 108.1 39.8 36.6 86 89
F F F F F F
0.02 7.2 1.2 0.4 3 7
F F F F F F
After ICH 0.02 6.5 1.1 0.2 3 8
7.37 101.1 39.4 36.6 88 89
F F F F F F
0.02 5.7 1.4 0.4 1.8 4
Values are means F SD. One-way analysis of variance revealed no significant inter-group difference for any variable. ICH indicates intracerebral hemorrhage, and MABP, mean arterial blood pressure.
improvement was maintained throughout the entire experimental period. Sham-operated rats (n = 9) showed only slight sensorimotor abnormality at 1 day after surgery, and those deficits disappeared from 7 days after surgery. When G-CSF was given to normal, unoperated rats (n = 4), its effect on the sensorimotor testing was not found. 3.3. G-CSF decreased brain edema, BBB permeability, and hemispheric atrophy Brain water content of the lesioned (left) hemisphere was 80.47 F 0.35% in ICH-only group and 77.93 F 0.49% in G-CSF-treated group ( P b 0.01, t test) and those of nonlesioned (right) hemisphere was 78.94 F 0.62% in ICHonly group and 77.25 F 0.41% in G-CSF-treated group ( P b 0.05, t test; Fig. 2A). Quantitative analysis of Evans blue dye penetration demonstrated that leakage of the G-CSFtreated group was significantly lower than that of the control group in the lesioned hemisphere (5.7 F 1.5 Ag/g vs. 22.87 F 1.87 Ag/g, P b 0.01, Fig. 2B). Hemispheric area analysis at 6 weeks after ICH showed a significant atrophy of the lesioned hemisphere in both groups. The G-CSFtreated group showed lesser hemispheric (13.1 F 3.2% [GCSF] vs. 24.2 F 4.0% [ICH-only]), striatal (14.9 F 5.2%
vs. 28.3 F 3.6%), and cortical (4.0 F 2.9% vs. 7.4 F 3.1%) atrophy compared with the ICH-only group significantly ( P b 0.01; Fig. 2C). The volumes of hemorrhage at 3 days after ICH were 22.3 F 3.5 Al (ICH-only), 24.9 F 5.1 Al (2 h G-CSF), and 22.7 F 4.9 Al (6 h G-CSF), which were not statistically different (Fig. 3B; P N 0.05). To analyze the possible early effect of G-CSF on the collagenase activity (associated with hematoma formation in this experiment), we performed an additional spectrophotometric assay of hematoma volume. The hematoma volumes at 6 h after ICH were 45.9 F 4.9 Al (ICH-only) and 42.6 F 7.5 Al (2 h G-CSF), which also showed no difference ( P = 0.68; Fig. 3B). 3.4. G-CSF treatment decreased inflammation and perihematomal cell death The TUNEL staining revealed a high density of positively stained cells within the hemorrhage lesion itself as well as in the surrounding periphery (Fig. 4). TUNEL+ cells were mostly found in the perihematomal areas and rarely found in the contralateral side. Quantitative analysis showed regional TUNEL+ cell differences between the groups. The ICH-only group (337.50 F 24.95 cells/mm2)
Fig. 1. Behavioral tests. In the rotarod test (A) and modified limb placing test (B), the G-CSF-treated ICH group showed a better performance from 14 days onwards, and these benefits continued up to 5 weeks ( P b 0.01). n = 15 per group. *P b 0.05, **P b 0.01 (ANOVA) compared with ICH-only group.
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Fig. 2. Hemispheric atrophy and brain water content. G-CSF treatment reduces brain water content (A) with blood–brain barrier permeability (B) in lesioned and contralateral hemispheres at 3 days after ICH and also reduces the degree of progressive hemispheric atrophy at 6 weeks after ICH (C). Values are mean F SD. *P b 0.05, **P b 0.01 compared with ICH-only group.
showed significantly more TUNEL+ cells than the G-CSFtreated group (141.88 F 36.03 cells/mm2; P b 0.01, t test; Fig. 4). In G-CSF-treated group, MPO+ neutrophils
(136.84 F 12.25 cells/mm2 [G-CSF] vs. 199.98 F 16.32 cells/mm2 [ICH-only]; P b 0.01) or OX42+ microglial cells (25.79 F 5.59 cells/mm2 [G-CSF] vs. 89.31 F 10.58 cells/
Fig. 3. Spectrophotometric assay of intracerebral hemorrhage. (A) The linear relationship between blood volume and hemoglobin absorbance in the spectrophotometric assay allowed this to be used as a standard curve for calculating hemorrhage volumes in G-CSF-treated brains. (B) Collagenase-induced hemorrhage volumes (mean F SD) were not different with the timing of G-CSF treatment. Hemorrhage volume was smaller when measured at 3 days than 1 day after ICH.
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Fig. 4. Evaluations of inflammatory cells and perihematomal cell death. (A, C, E) G-CSF-treated group, (B, D, F) ICH-only group. Detection of TUNEL reaction product (A, B), myeloperoxidase (MPO)-positive cells (C, D), and OX-42-positive cells (E, F) in the perihematomal area 3 days after ICH. (G) Quantification of inflammatory cells and TUNEL+ cells. Values are mean F SD. n = 9 per group. **P b 0.01 compared with ICH-only group. Scale bar = 100 Am.
mm2 [ICH-only]; P b 0.01) were less found around the perihematomal striatum, compared with ICH-only group at 3 days after ICH (Fig. 4).
4. Discussion These experiments were undertaken to test whether the administration of G-CSF may have a therapeutic effect on ICH. In this study, G-CSF reduces brain edema, perihematomal cell death, and inflammation after ICH with sensorimotor improvement. Furthermore, G-CSF treatment reduces the progressive hemispheric atrophy. The mechanism how the G-CSF reduced the brain edema is unknown. Previous studies on ICH indicated that the inflammatory mediators released from the infiltrating leukocytes and activated microglia caused brain edema and the functional deficit after ICH [4,8,13,18,19]. Rodent and human studies have shown that G-CSF-induced antiinflammatory effect is associated with a shift toward Th2 from Th1, resulting in a reduction of Th1-oriented cytokine production such as TNF-a and IFN-g [17,23,27]. In our study, systemic administration of G-CSF reduced the ICHassociated pathologic findings, suggesting that the beneficial effect of G-CSF on brain edema might be mediated by its anti-inflammatory effect. However, whether this results from a local effect of G-CSF crossing the BBB cannot be excluded. In addition, the collagenase-induced ICH model is
not physiologic and raise a concern that collagenase itself may induce excess inflammation. However, our previous results showed that direct collagenase toxicity was not the major cause of inflammation or cell death [4]. Despite of the leukocytosis in the peripheral blood, the number of infiltrating microglia and neutrophils was less in G-CSF-treated group than in ICH-only group. The leukocytosis but impaired leukocyte accumulation in the damaged tissue has been previously described on MPO+ cells [22,24,26]. It is suggested that the G-CSF-stimulated neutrophils may not necessarily migrate into the injured tissue or aggravate inflammation. The reduction of BBB leakage and the reduction of cytokines (e.g. TNF-a) also could decrease the infiltrating leukocytes [24]. Effect of the inflammatory cytokines in ICH is required to further determine the role of G-CSF. G-CSF has been known to have anti-apoptotic effects in animal models of myocardial infarction and cerebral ischemia [16,22]. In the present study, G-CSF treatment reduced perihematomal cell death with long-term hemispheric atrophy after ICH. Perihematomal cell loss after ICH can be caused by apoptosis, mediated by thrombin of the hematoma [14]. In ischemia model, G-CSF achieved a neuroprotective effect mediated by G-CSFR and JAKSTAT3 pathway [22], which might also be involved in the perihematomal cell death in the ICH model. However, the hypothesis that G-CSF can reduce the apoptotic cascade after ICH via JAK-STAT3 cascade remains to be defined.
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