Functional Recovery of Stroke Rats Induced by Granulocyte Colony-Stimulating Factor–Stimulated Stem Cells
Background— Stroke is a leading cause of death and disability worldwide; however, no effective treatment currently exists.
Methods and Results— Rats receiving subcutaneous granulocyte colony-stimulating factor (G-CSF) showed less cerebral infarction, as evaluated by MRI, and improved motor performance after right middle cerebral artery ligation than vehicle-treated control rats. Subcutaneous administration of G-CSF enhanced the availability of circulating hematopoietic stem cells to the brain and their capacity for neurogenesis and angiogenesis in rats with cerebral ischemia.
Conclusions— G-CSF induced increases in bone marrow cell mobilization and targeting to the brain, reducing the volume of cerebral infarction and improving neural plasticity and vascularization.
Received October 24, 2003; de novo received January 27, 2004; revision received April 29, 2004; accepted May 3, 2004.
Stroke is a leading cause of death and disability worldwide,1 with no effective treatment that enhances stroke recovery. A potential strategy for the treatment of stroke is transplantation of bone marrow stem cells.2,3 These cells appear to enter through the blood-brain barrier and selectively migrate to the ischemic hemisphere of the damaged brain to improve neurological recovery.2,3 However, because cell transplantation requires surgical intervention, it is clinically desirable to explore less invasive therapeutic procedures.
Administration of granulocyte colony-stimulating factor (G-CSF) is known to mobilize hematopoietic stem cells (HSCs) from bone marrow into peripheral blood.4 Peripheral blood–derived HSCs have been used in place of bone marrow cells in transplantation for the regeneration of nonhematopoietic tissues such as skeletal muscle and heart.5 G-CSF has been used extensively for >10 years in the treatment of neutropenia, as well as for bone marrow reconstitution and stem cell mobilization.6
In stroke treatment through administration of stem cells, 2 main determinants are critical for the colonization and transdifferentiation of stem cells into a variety of tissues: (1) ischemic tissue damage and (2) the number of circulating stem cells available.5 Under ischemic conditions, circulating stem cells appear to selectively migrate to ischemic regions to support plasticity and functional recovery of damaged tissue.5 The expression of stromal cell–derived factor-1 (SDF-1) and its receptor CXCR4 after focal cerebral ischemia7 led us to speculate that this chemokine may also signal adhesion and migration of HSCs to ischemic tissue. On this basis, we hypothesized that cerebral ischemia enhances HSC plasticity and provides an environment that enhances differentiation of HSCs into original lineage cell types of the damaged organ, such as endothelial cells and neurons. In this study we used a rat model to test the hypothesis that chemokines could mobilize HSCs in a manner similar to that in which they target inflammatory cells in nonneuronal damaged tissues. A sufficient number of HSCs, mobilized by G-CSF, could then home in on cerebral ischemic injuries to promote neuronal repair and recovery of function; this would provide a basis for the development of a noninvasive autologous therapy for cerebral ischemia.
In Vivo Brain Ischemia/Reperfusion
Under anesthesia with chloral hydrate (0.4 g/kg), ligations of the right middle cerebral artery (MCA) and bilateral common carotid arteries (CCAs) were performed by methods described previously8 to induce cerebral infarction. Briefly, the bilateral CCAs were clamped with nontraumatic arterial clips. With the use of a surgical microscope, the right MCA was ligated with a 10-0 nylon suture. Cortical blood flow was measured continuously with a laser-Doppler flowmeter (PF-5010, Periflux system, Perimed AB) in anesthetized animals. After 90 minutes of ischemia, the suture on the MCA and arterial clips on CCAs were removed to allow reperfusion. During recovery from the anesthesia, body temperature was maintained at 37°C with a heat lamp.
Experimental Animals and G-CSF Treatment
Adult male Sprague-Dawley rats (weight, 250 to 300 g; Experimental Animal Center, Tzu-Chi University, Hualien, Taiwan) were used in this study. One day after induction of cerebral ischemia, rats were injected subcutaneously with recombinant human G-CSF (50 μg/kg per day; Amgen Biologicals) once daily for 5 days.9 Control animals were subjected to cerebral ischemia and injected with saline.
Bromodeoxyuridine (BrdU), a thymidine analogue that is incorporated into the DNA of dividing cells during S-phase, was used for mitotic labeling (Sigma Chemical). The labeling protocol has been described previously.10 Pulse labeling was used to observe the time course of proliferative cells in the brain after cerebral ischemia. Experimental rats (including 15 G-CSF–treated rats and 15 control rats) were injected intraperitoneally with BrdU (50 mg/kg) every 4 hours for 12 hours before they were killed. Rats were killed at 7 days (n=10), 14 days (n=10), and 28 days (n=10) after cerebral ischemia. A cumulative labeling method was used to examine the population of proliferative cells during 14 days of cerebral ischemia. Rats (including 12 G-CSF–treated rats and 12 control rats) received daily injections of BrdU (50 mg/kg IP) for 14 consecutive days, starting the day after MCA ligation. These rats were euthanized 14 days after the last injection (n=24).
Neurological Behavioral Measurement
Behavioral assessments were performed 5 days before cerebral ischemia and 1, 7, 14, and 28 days subsequent to MCA ligation. The tests measured (1) body asymmetry and (2) locomotor activity. The baseline-tested scores were recorded to normalize those taken after cerebral ischemia. (1) The elevated body swing test was used to assess body asymmetry after MCA ligation and was evaluated quantitatively, as previously described.11 The frequency of initial head swing contralateral to the ischemic side of rat brain was counted in 20 continuous tests and was normalized as follows: percent recovery of neurological function=[1−(lateral swings in 20 tests −10)/10×100%. (2) For locomotor activity, rats were subjected to OPTO-VARIMAX (Columbus Instruments) activity monitoring for ≈2 hours for behavioral recording. Motor activity was counted as the number of beams broken by rat movement in the chamber. Two parameters of vertical movement were calculated: (1) vertical activity (the total number of beam interruptions that occurred in vertical sensors) and (2) vertical time (the time that rats spent in vertical movement).
MRI was performed in an imaging system (General Electric) at 3.0 T. Under anesthesia, the 6 to 8 coronal image slices were each 2 mm thick without any gaps. T2-weighted imaging (T2WI) pulse sequences were obtained with the use of a spin-echo technique (repetition time, 4000 ms; echo time, 105 ms) and were captured sequentially for each animal at 1, 7, and 28 days after cerebral ischemia. To measure the infarction area in the right cortex, we subtracted the noninfarcted area in the right cortex from the total cortical area of the left hemisphere. The area of infarct was drawn manually from slice to slice, and the volume was then calculated by internal volume analysis software (Voxtool, General Electric).
Immunohistochemistry of Brain Tissue
The brains of experimental rats were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde. For BrdU immunostaining, DNA was first denatured by incubating each section in 50% formamide of ×2 standard saline citrate at 65°C for 2 hours, then in 2N HCl at 37°C for 30 minutes, and finally rinsed in 0.1 mol/L boric acid with pH 8.5. Sections were then rinsed with Tris buffer and treated with 1% H2O2 to block endogenous peroxidase. The immunostaining procedure was performed with the use of the labeled streptavidin-biotin method (DAKO LASB-2 Kit, Peroxidase, DAKO). After deparaffinization, tissues slide were incubated with the appropriate diluted antibodies to BrdU (for nuclear identification; 1:400, Boehringer Mannheim) at room temperature for 1 hour. After they were washed with Tris-buffered saline containing 0.1% Tween-20, the specimens were sequentially incubated for 10 to 30 minutes with biotinylated anti-rabbit and anti-mouse (1:200; R&D Systems) immunoglobulins and peroxidase-labeled streptavidin. Staining was performed after a 10-minute incubation with a freshly prepared substrate-chromogen solution and then counterstained with hematoxylin. Quantification of BrdU-immunoreactive cells was performed on paraffin-embedded tissue sections and was counted digitally with the use of a ×60 objective lens (Carl Zeiss LSM510) via a computer imaging analysis system (Imaging Research). Cerebral cells with uniform nuclear BrdU immunostaining were counted as previously described.12
Laser-Scanning Confocal Microscopy for Double-Immunofluorescence Analysis
To identify the expression of cell type–specific markers in BrdU+ cells, double immunofluorescence was performed. The expressions of glial fibrillary acidic protein (GFAP), von Willebrand factor (vWF), microtubule-associated protein-2 (MAP-2), and neuronal nuclei (Neu-N) were tested. In BrdU-GFAP, BrdU-vWF, BrdU-MAP-2, and BrdU-Neu-N double immunofluorescence, mouse monoclonal anti-BrdU and fluorescein-conjugated sheep anti-mouse antibodies were used following the instructions of the Roche BrdU labeling and detection kit. Each coronal section was first treated with primary BrdU antibody conjugated with FITC (1:500; Jackson Immunoresearch) staining, followed by treatment with cell-specific antibodies: GFAP for astrocytes (1:400; Sigma), vWF for endothelial cells (1:400; Sigma), Neu-N for neuronal nuclei (1:200; Chemicon), and MAP-2 for neuronal dendrites (1:200; Boeringer-Mannheim) with Cy3 (1:500; Jackson Immunoresearch) staining. The tissue sections were analyzed with a Carl Zeiss LSM510 laser-scanning confocal microscope, and green (FITC) and red (Cy3) fluorochromes on the slides were excited by laser beam at 488 and 543 nm, respectively.
CXCR4 Immunoreactivity in Ischemic Brain
To verify the upregulation of CXCR4 in ischemic brain, rats received brain ischemia as mentioned previously. Then they were euthanized at 4 hours, 12 hours, 24 hours, and 3 days after cerebral ischemia. The ischemic area of each brain section was first confirmed by cresyl violet staining. Sections were incubated with anti-rat CXCR4 (1:100; Torrey Pines Biolab) for 2 hours at room temperature. After 3 washes with PBS, horseradish peroxidase–conjugated goat anti-rabbit IgG antibody (1:500; Santa Cruz Laboratories) was added to the sections and incubated for 1 hour at room temperature. Signals were visualized by applying substrate 3, 3′-diaminobenzidine (a brown color).
Neutralizing CXCR4 Antibody Treatment
To observe the blocking effect of CXCR4 antibody on G-CSF treatment in cerebral ischemia, rats received a daily subcutaneous injection of G-CSF (50 μg/kg IP) 1 day after cerebral ischemia for 5 consecutive days. They then received an intraperitoneal injection of a neutralizing rat CXCR4 antibody (Torrey Pines Biolab) on days 4 and 5 immediately after G-CSF treatment. Control rats received an injection of saline intraperitoneally. Each rat in this experimental set was also subjected to MRI and behavioral measurement at 1, 7, and 14 days after cerebral ischemia.
All measurements in this study were performed blindly. Student t tests were used to evaluate mean differences between the control group and the experimental group in terms of volume of lesion and cell numbers.
G-CSF Improves Neurological Behavior After Cerebral Ischemia
To evaluate neurological function in G-CSF–treated and saline-treated control animals (n=12 each) after cerebral ischemia, a body asymmetry trial was used to assess body swing before and after MCA ligation. Between 14 and 28 days after cerebral ischemia, rats subjected to G-CSF treatment exhibited significantly less body asymmetry than saline-treated controls (Figure 1A). Furthermore, examination of locomotor activity before and after cerebral ischemia indicated that in rats receiving G-CSF, both vertical activity and movement time significantly increased relative to controls between days 14 and 28 (Figure 1B, 1C).
G-CSF Decreases Infarction Volume
To sequentially observe and quantify the volume of cerebral infarction without euthanasia, MRI of 24 rats showing uniform cortical infarctions was performed 1, 7, and 28 days after the induction of cerebral ischemia. Cortical infarction in rats treated with G-CSF showed remarkable size reductions from day 7 to 28 (n=12) (Figure 2⇓A). By contrast, cortical infarction in control rats (n=12) showed only a small decrease in size during the same time period (Figure 2⇓B).
The 12 rats that received G-CSF treatment showed mild infarction 7 days after cerebral ischemia. Quantitative measurement of the infarction volume showed that infarction was significantly reduced from an average of 176±20 mm3 in saline-treated controls to 61±12 mm3 in G-CSF–treated animals (Figure 2⇑C). The area of the largest infarction significantly decreased from 21±3 mm2 in the controls to 8.4±1.3 mm2 in the treated rats (Figure 2⇑D), and infarcted areas were also significantly reduced from 6.3±0.4 slices per rat in the control animals to 3.1±0.2 slices per rat in the G-CSF–treated rats (Figure 2⇑E).
G-CSF Stimulates Stem Cell Mobilization and Homing to Brain After Cerebral Ischemia
To determine whether stem cells homed in on the injured brain tissue of G-CSF–treated rats, BrdU labeling was used to follow the engraftment of G-CSF–mobilized HSCs in the brain. BrdU-immunoreactive cells were detected mainly in the subventricular area of the lateral ventricle in both hemispheres of G-CSF–treated rats. Cumulative labeling of BrdU revealed a few BrdU-immunoreactive cells in the ipsilateral cortex near the infarcted boundary (Figure 3A, 3B, 3C, arrows) and subventricular region (Figure 3D, 3E, 3F, arrows). BrdU-immunoreactive cells were also found around the lumen of varying calibers of blood vessels in the perivascular portion (also in the endothelial cell lining of the vessel wall) (Figure 3G, 3H, 3I, arrows). BrdU pulse labeling was then used to quantify the BrdU-immunoreactive cells in the ischemic hemisphere of the G-CSF–treated rats at 7, 14, and 28 days after MCA ligation compared with those of saline-treated control rats (Figure 3J). In summary, G-CSF–treated ischemic rats exhibited significantly increased numbers of BrdU-immunoreactive cells in their ipsilateral hemispheres compared with saline-injected ischemic rats.
G-CSF Enhances Neurogenesis and Angiogenesis In Vivo
To determine whether mobilizing HSCs differentiated into neuronal, glial, or endothelial cells in the brains of G-CSF–treated rats, double-staining immunohistochemistry was performed. The result showed some BrdU+ cells (green for cell nucleus identification) colocalized with antibodies for Neu-N, MAP-2, GFAP, and vWF (red for cell type–specific markers) in the brains of G-CSF–treated rats (Figure 4A to 4L). Ischemic cortical areas of G-CSF–treated rats revealed an increase in BrdU+ cells coexpressing the neuronal phenotypes of Neu-N+ and MAP-2+ cells (Figure 4G to 4L) as well as the glial phenotype of GFAP+ cells (Figure 4A to 4C) compared with the saline-treated rats. Some BrdU+ cells showing vascular phenotypes (vWF+ cells) were also found around the perivascular and endothelial regions (Figure 4D to 4F) of the ischemic hemispheres of G-CSF–treated rats.
Increased Expression of CXCR4 in Ischemic Brain
Because of the potential importance of the chemokine SDF-1 in HSC-mediated repair of ischemic brain tissues, the pattern of CXCR4 expression in ischemic brains was analyzed by immunohistochemical staining (Figure 5). Significantly, a marked increase in expression of CXCR4 was detected in the ischemic region of G-CSF–treated rats at 4 as well as 12 hours after cerebral ischemia (Figure 5B, 5D) in comparison to the contralateral nonischemic side (n=6) or normal healthy controls (n=6) (Figure 5B, 5D, 5F). This elevated CXCR4 expression in the ischemic area returned to a normal level, however, 3 days after cerebral ischemia (data not shown). We also observed that CXCR4 was expressed in most cortical and vascular endothelial cells (Figure 5E).
Neutralizing CXCR4 Antibody Compromises the Effect Induced by G-CSF Treatment
To test whether the effect of G-CSF on stroke recovery is mediated through the binding of SDF-1 to its receptor CXCR4, we injected intraperitoneally a neutralizing rat CXCR4 antibody on days 4 and 5 of G-CSF treatment after induction of cerebral ischemia. MRI revealed that 14 days after cerebral ischemia, G-CSF–treated animals administered CXCR4 neutralizing antibody showed only a slight reduction in infarct volume compared with the large reduction observed in G-CSF–treated animals injected with saline (Figure 6A, 6B). Behavioral examinations, including body swing and locomotor activity tests, showed that addition of CXCR4 antibody significantly reduced the beneficial effects of G-CSF treatment (data not shown).
In this study, we demonstrated that subcutaneous injections of G-CSF, starting 1 day after cerebral ischemia and continuing for up to 5 days, enhance neural repair in rats suffering from cerebral ischemia. Infarction volume was markedly reduced, and there was also significant recovery of neurological dysfunction.
It is likely that the mechanisms providing therapeutic benefit in this study are multidimensional. First, we found that administration of G-CSF increased the mobilization of circulating HSCs to damaged areas of the brain, and this increase may in turn stimulate cell division in the penumbra of the ischemic brain. Second, it is possible that interaction of HSCs with ischemic tissue may lead HSCs and/or parenchymal cells to produce trophic factors13 that may contribute to the recovery of neural functions lost as a result of tissue injury.14 HSCs have been shown to constitutively express interleukins such as interleukin (IL)-1β, IL-8, and IL-16, fibroblast growth factor-2, vascular endothelial growth factor, insulin growth factor-1, granulocyte-monocyte colony-stimulating factor, and tumor necrosis factor-α.15 These cytokines may act as survival, growth, and/or differentiation factors for neuronal and vascular progenitor cells, which may in turn proliferate, migrate, and differentiate after brain injury and thus contribute to damage recovery processes. Neurotrophic factors have been shown to enhance neuronal sprouting,16 synaptogenesis,17 and neurotransmission18 and increase neurotransmitter release.19 In the case of the glial cell line–derived neurotrophic factor, its injection into the brain was found to greatly diminish infarction volume and improve neurological functions in rats suffering cerebral ischemia.20 Therefore, some G-CSF–mobilized HSCs could enter the cerebral ischemic region and interact with penumbral cells; this interaction may enhance the production of trophic factors such as glial cell line–derived neurotrophic factor and brain-derived neurotrophic factor, which may in turn promote repair3 of damaged parenchymal cells after stroke in rats.
In this study, more HSCs were found in the ischemic hemisphere than in the intact hemisphere of experimental rats. This suggests that disruption of the blood-brain barrier may facilitate selective entry of HSCs into the ischemic rather than the nonischemic contralateral hemisphere. It is interesting to note that in ischemic rat brain a number of neurotrophic factors are released, which have been shown to result in human bone marrow stromal cell growth factor production.3,21 Therefore, we speculate that ischemic damage to brain tissue may result in the release of trophic factors, which in turn may target HSCs to damaged tissues. Similarly, Chen and colleagues2 reported that intravenous administration of marrow stromal cells in rats results in their accumulation in the ischemic brain, and, in a model of hepatic injury, regenerated hepatic cells were shown to be of bone marrow origin.22 These findings suggest that the “injured” brain might specifically attract bone marrow–derived cells. It will be important to clarify which signaling molecules attract HSCs and direct their migration to damaged areas.
A recent report23 has indicated that SDF-1 is a strong chemoattractant for CD34+ cells that express CXCR4, the receptor for SDF-1, and plays an important role in HSC trafficking between peripheral circulation and bone marrow. Recently, Stumm et al7 demonstrated that focal cerebral ischemia causes an increase in SDF-1 expression in regions adjacent to the infarcted area. This lesion-induced upregulation of endothelial SDF-17 and the appearance of increased CXCR4 expression in the ischemic hemisphere in our study, 4 hours after ischemia, indicate that cerebro-endothelial SDF-1 could be a chemoattractant for peripheral blood cells. Lataillade and colleagues24 reported that a significant proportion of HSCs, mobilized by G-CSF, express CXCR4 receptors on their cell surface and that SDF-1 induces directional migration of HSCs. By attracting HSCs to the ischemic region, an SDF-1/CXCR4 interaction may be directly involved in vascular remodeling, angiogenesis, and neurogenesis, thereby alleviating stroke symptoms. In this study administration of anti-CXCR4 antibody compromised the effect induced by G-CSF treatment in rats with cerebral ischemia, resulting in poor neurological recovery after stroke. This chemotaxis may take place in a manner similar to the migration of leukocytes into damaged or inflamed tissues.25 In addition, HSCs migrating to the ischemic hemisphere could create local chemical gradients and/or localized chemokine accumulation, dictating a directional response in endothelial, neuronal, and glial progenitor cells.26 As a consequence of this autocrine regulatory pathway, endothelial and neuronal progenitor cells could mobilize and fuse with each other, a step required for subsequent formation of a structured network of branching vessels and neurons.27 In addition to mobilized HSCs, SDF-1 might also stimulate host endothelial progenitor cell differentiation from preexisting blood vessels and/or host endothelial progenitor cells derived from bone marrow.27 In addition to inducing HSC migration to ischemic regions, SDF-1 has also been shown to exert survival effects on cultured CD34+ cells26 and to regulate endothelial cell branching morphogenesis.28 Taken together, we therefore hypothesize that plasma levels of SDF-1, released from damaged tissues, may provide a host defense signal that in turn attracts mobilizing HSCs to repair the disordered tissue. This study also provides evidence that the ultimate degree of neurological improvement is dependent on the recruitment of sufficient HSCs to the damaged area of brain at an early stage after tissue injury.
In summary, we have shown that G-CSF administered to rats with cerebral ischemia can provoke new neuronal and vascular formation within infarcted regions of brain, attenuating tissue damage and effecting a reduction in infarction volume and improved neurological function. We propose that the G-CSF treatment may mobilize autologous HSCs into circulation, enhance their translocation into ischemic brain, and thus significantly improve lesion repair. We believe that the G-CSF therapeutic protocol reported here represents an attractive strategy for the development of a clinically significant noninvasive stroke therapy.
This work was supported in part by research grant AS92IMB3 from Academia Sinica, Taipei, Taiwan, and by a grant from the National Science Council (NSC92-2314-B-303-009), Taiwan. We thank Dr K. Deen for his critical reading of the manuscript.
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