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Abstract

Stroke is a serious medical condition that causes long-term neurological disability in mainly elderly adults worldwide. Lack of therapy to improve functional recovery in the chronic phase of stroke is a major challenge for stroke research. Combining two hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF), our previous studies have demonstrated the neurovascular restorative efficacy of this treatment in the chronic phase of experimental stroke. Elevated plasma fibrinogen has been thought to serve as a predictor for ischemic stroke. Here we have determined the treatment frequency in reducing plasma fibrinogen and in restoring motor function in aged mice with chronic stroke. Our findings show that SCF + G-CSF treatment in chronic stroke decreases plasma fibrinogen and improves motor function in aged mice. No differences have been found between a 2-week treatment regimen and 7-day treatment in the plasma fibrinogen assay, while the 7-day treatment regimen displays a better recovery pattern with regard to motor function. This study provides new insight into understanding the potential contribution of SCF + G-CSF in both reducing the risk of recurrent ischemic stroke and enhancing stroke recovery.

Keywords

Stem cell factor (SCF)Granulocyte-colony stimulating factor (G-CSF)Hematopoietic growth factor Chronic stroke Stroke recovery Fibrinogen

Introduction

Stroke is the leading cause of neurological disability in adults worldwide. Stroke mainly affects the elderly (>65 years) and causes long-term neurological deficits, even lifelong disability. The chronic phase of stroke persists for a long period of time beginning at 3 months after the initial stroke, in most cases, and continuing up to the end of the individual's life. Enhancing stroke recovery in the chronic phase is a very important but underinvestigated field in stroke research.

Our previous work has demonstrated the efficacy of hematopoietic growth factors, stem cell factor (SCF), and granulocyte-colony stimulating factor (G-CSF) in brain repair in the chronic phase of experimental stroke. SCF in combination with G-CSF (SCF + G-CSF) has been shown to have a synergistic effect on hematopoietic stem cell mobilization (10). Our studies have revealed that SCF + G-CSF also synergistically regulates neurite outgrowth (25) and improves functional recovery in the chronic phase of experimental stroke (29). After determining the expression of receptors for SCF and G-CSF in the cortical neurons (31) and the possibility of SCF and G-CSF being able to cross the blood–brain barrier (30), we illustrated that SCF + G-CSF treatment in the chronic phase of stroke enhances synaptogenesis, axonal sprouting, and dendritic branching in the peri-infarctcavity cortex (8,9). SCF + G-CSF-improved functional recovery is dependent on the SCF + G-CSF-enhanced neural network remodeling in the peri-infarct-cavity cortex (8). Mushroom dendritic spines have been shown to be involved in forming functional neural circuits (19). Using live brain imaging, we found that the formation of mushroom-type dendritic spines in the ipsilesional cortex was increased by SCF + G-CSF in the chronic stroke brain of aged animals (9). However, it remains unclear whether a repeated treatment of SCF + G-CSF in the chronic phase of stroke would further enhance the recovery.

Fibrinogen is the key protein component of blood clots. High levels of plasma fibrinogen cause reduction of blood circulation because of increased plasma viscosity (23). The levels of plasma fibrinogen are increased in elderly humans (11), and increased fibrinogen in the blood has been shown to be involved in endothelial injury, cerebrovascular pathology, and neurovascular dysfunction (7,18). Elevated plasma fibrinogen has been considered to be a predictor of ischemic stroke or recurrent ischemic stroke (3,13,27). It remains to be determined, however, whether SCF + G-CSF treatment in chronic stroke alters the levels of plasma fibrinogen in the aged.

The purpose of this study was to determine the effects of SCF + G-CSF treatment in chronic stroke on reduction of plasma fibrinogen in aged animals and the efficacy of a repeated treatment of SCF + G-CSF for enhancing stroke recovery in the chronic phase.

Materials and Methods

Animals and Animal Model of Stroke

Forty-five 19- to 20-month-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME, USA) were subjected to cortical ischemia in the right hemisphere. Mice at this age are equivalent to a human age of 76–77 years. We chose this age for our study because the elderly population has a high incidence of stroke. The detailed information for making this mouse model of stroke is described elsewhere (8,9,21). Briefly, after being anesthetized with Avertin (0.4 g/kg, IP) (Sigma-Aldrich, St. Louis, MO, USA), the right common carotid artery (CCA) was permanently ligated through a midline incision in the neck. The right middle cerebral artery (MCA) was cauterized (World Precision Instruments, Sarasota, FL, USA) through a craniotomy made between the right eye and ear. The rectal temperature was monitored and maintained at 37°C throughout the surgery.

Groups and Treatment

The group size of our original design was 10~12 mice per group. However, a number of mice (a total of 16) died during either the anesthesia or the surgery, probably due to the advanced age of the animals. Surviving mice were randomly divided into three groups (n = 7–8): a vehicle control group, a group with 7-day treatment of SCF + G-CSF, and a group with 2-week treatment of SCF + G-CSF (5 days per week) at 3 months after induction of cortical ischemia. A group of age-matched mice without occlusion of CCA and MCA served as sham controls (n = 6).

Animals in the vehicle control group received subcutaneous injections of equal volumes of 0.9% saline and 5% dextrose (Baxter Healthcare Corporation, Deerfield, IL, USA). In the SCF + G-CSF-treated group, recombinant mouse SCF (200 μg/kg/day) (PeproTech, Rocky Hill, NJ, USA) and recombinant human G-CSF (50 μg/kg/day) (Amgen, Thousand Oaks, CA, USA) were subcutaneously injected.

Blood Collection and Enzyme-Linked Immunosorbent Assay

Blood samples were collected from the submandibular vein with a hematological tube (200–250 μl lavender tube with EDTA) (Becton Dickinson, Franklin Lakes, NJ, USA) at 3 days after the final injection of SCF + G-CSF. Blood samples harvested from young mice (n = 4, 2 months old) were used as young controls. Plasma was isolated, aliquoted, and stored at -80°C until use. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of fibrinogen in the plasma with a Mouse Fibrinogen ELISA Kit (Innovative Research, Novi, MI, USA) according to the manufacturer's instructions. Briefly, plasma samples and standards were added into the 96-well plate coated with fibrinogen capture antibody. After incubation with biotin-labeled anti-mouse fibrinogen primary antibody, avidin-conjugated horseradish peroxidase was added. 3,3’,5,5'-Tetramethylbenzidine (TMB) substrate for horseradish peroxidase was used for color development; the plate was placed into a microtiter plate spectrophotometer (Infinite M200; Tecan, Morrisville, NC, USA), and the optical density was determined at 450 nm. The total amount of fibrinogen for each well was measured based on the standard calibration curve.

Motor Function Testing

Motor function was evaluated with a Rota-Rod test 2 and 6 weeks posttreatment (8). Mice were placed on a rotating Rota-Rod beginning at 4 rpm and ending at 40 rpm. The maximum duration on the rotating Rota-Rod for each mouse was 5 min. Each mouse was tested three times a day for 5 consecutive days, and a 15-min break was given between the trials. The average latencies of each mouse to fall down from the rotating Rota-Rod were measured everyday and used for statistical analysis.

Statistical Analysis

Data analysis was based on a group size of six to seven because of a loss of additional mice during the experiment. The levels of plasma fibrinogen were statistically analyzed using one-way ANOVA followed by Tukey's post hoc test. For analyzing the Rota-Rod data, linear mixed effect models were fitted using SAS 9.3 (SAS Institute Inc., Cary, NC, USA) to primarily identify the effects of SCF + G-CSF treatment in increased latency to fall on Rota-Rod over time compared with the stroke vehicle controls. The covariance between observations from a single mouse was modeled by a heterogeneous Toeplitz structure, which generally demonstrated a better fit than the unstructured covariance matrix in terms of reducing the Akaike information criterion. Values of p < 0.05 were considered statistically significant. Data are presented as mean ± SEM.

Results and Discussion

Plasma fibrinogen assay revealed that SCF + G-CSF treatment in the chronic phase of stroke (cortical infarct) resulted in a significant reduction of plasma fibrinogen in aged mice. The levels of plasma fibrinogen were significantly decreased in both the 7-day treatment with SCF + G-CSF (p < 0.01) and 2-week treatment with SCF + G-CSF (p < 0.05) groups when compared to the stroke vehicle controls (Fig. 1). There was no significant difference in fibrinogen plasma concentration between SCF + G-CSF-treated stroke mice and nonstroke mice (age-matched sham controls and young intact controls) (Fig. 1).

Figure 1.

The levels of plasma fibrinogen 3 days after the final injection of SCF + G-CSF in the chronic phase of experimental stroke in aged mice. Note that both SCF + G-CSF-treated stroke animals show significant reductions in the concentration of plasma fibrinogen compared to stroke vehicle controls. MCAo, middle cerebral artery occlusion (experimental stroke); S + G 7d: SCF + G-CSF was injected continuously for 7 days; S + G 5 + 5d: SCF + G-CSF was injected continuously for 5 days followed by a 2-day break and additional 5-day injection. Data are mean ± SEM. **p < 0.01, *p < 0.05.

Circulating fibrinogen is the primary protein component of blood clots, and elevated levels of plasma fibrinogen have been found in elderly people (11) and in patients with ischemic stroke (3), atherosclerosis (13), cardiovascular disease (24), diabetes (4), and vascular dementia (26,28). In the present study, the concentration of plasma fibrinogen was not significantly different between young intact mice and aged sham mice (Fig. 1). This discrepancy compared to human studies might be due to differences in species. We also noted that stroke mice in the vehicle control group showed a trend toward increasing plasma fibrinogen in comparison with sham aged controls and intact young controls, but it did not reach significance due to a limited number of animals (Fig. 1).

It has been shown that high levels of plasma fibrinogen reduce blood flow due to increased plasma viscosity (23). Increased plasma fibrinogen causes an increase in permeability of endothelial cells (18), exacerbates blood–brain barrier (BBB) dysfunction (1), and leads to cerebrovascular dysfunction and cerebral hypoperfusion (7). Elevated plasma fibrinogen has been considered to be a predictor of ischemic stroke, recurrent ischemic stroke, advanced atherosclerosis, and vascular dementia (3,13,26–28). Reduction of plasma fibrinogen has been shown to decrease the risk for ischemic stroke, decrease infarct size in acute ischemic stroke (16), inhibit BBB permeability, and decrease neurovascular damage (7). The novel finding of the present study that SCF + G-CSF treatment in the chronic stroke phase reduces plasma fibrinogen in aged mice suggests a potential role of SCF + G-CSF in reducing the risk of recurrent ischemic stroke and neurovascular damage poststroke. It remains to be elucidated how SCF + G-CSF treatment in chronic stroke reduces plasma fibrinogen. Plasma fibrinogen is synthesized in the liver by hepatocytes. It is unlikely, however, that SCF + G-CSF inhibits hepatic production of fibrinogen because the receptors for SCF and G-CSF are not expressed in hepatocytes in normal adults (14,22). Whether SCF + G-CSF treatment could increase clearance of plasma fibrinogen is a question that could be addressed in future studies. In addition, a discrepancy has been noted between the present study and the findings reported by Morimoto and colleagues that a single intravenous or intracerebroventricular injection of G-CSF did not change the levels of plasma fibrinogen (17). These discrepancies may be related to methodological differences between the two studies. These different findings suggest that G-CSF in combination with SCF and/or repeated treatment with SCF + G-CSF may play a key role in reducing plasma fibrinogen.

Cerebral amyloid angiopathy (CAA) manifests as amyloid β (Aβ) deposition in the arteries of the cerebral cortex and leptomeninges. CAA occurs in the brain of the elderly. Recent evidence has shown that Ab specifically binds to fibrinogen and that fibrinogen is frequently localized to CAA (1,6,12,4). CAA is associated with vascular damage, which leads to ischemic or hemorrhagic stroke (2,5). A recent study showed an age-dependent neurovascular dysfunction and structural damage in a mouse model of CAA (2,5). Using a transgenic mouse model of inherited stroke and vascular dementia, we recently demonstrated that SCF + G-CSF treatment prevents development of CAA and restricts neurovascular degeneration in the brains of aged transgenic mice (15). The present study reveals a reduction of plasma fibrinogen by SCF + G-CSF treatment in aged mice with chronic stroke. Whether the decreased levels of plasma fibrinogen contribute to the SCF + G-CSF-inhibited CAA formation and neurovascular degeneration and whether SCF + G-CSF-reduced plasma fibrinogen is necessary for enhancing cerebrovascular functioning and neural network remodeling in aged brain of chronic stroke (9) remain to be clarified in future studies.

In the present study, motor function was evaluated by Rota-Rod testing at 2 and 6 weeks posttreatment. Our data showed that SCF + G-CSF-treated mice had significant improvement of motor functional recovery at both tested time points. At 2 weeks posttreatment, the increased latency to fall per day (the slope over time) in SCF + G-CSF-treatment groups (7-day treatment and 2-week treatment) was 3.39 s on average compared with the stroke vehicle controls (95% confidence interval: 0.20, 6.59; p = 0.038) (Fig. 2A, C). This finding indicates that SCF + G-CSF treatment in chronic stroke enhances motor learning and motor coordination. When analyzing each of the SCF + G-CSF treatment groups, both the 7-day treatment and 2-week treatment showed a similar trend toward increasing latency to fall, while they did not reach statistical significance.

Figure 2.

Motor function evaluation by a Rota-Rod test 2 and 6 weeks after SCF + G-CSF treatment in the chronic phase of experimental stroke in aged mice. (A) Rota-Rod test data 2 weeks after SCF + G-CSF treatment. (B) Rota-Rod test data 6 weeks after SCF + G-CSF treatment. (C) A table of comparisons between tested groups for determining the increased latency to fall per day. Note that SCF + G-CSF treatment significantly increases the latency to fall (or increases riding time on Rota-Rod) 2 and 6 weeks after treatment, suggesting motor functional improvement by the treatment. The 7-day treatment of SCF + G-CSF shows better motor functional recovery than those of vehicle treatment or 5 + 5-day treatment of SCF + G-CSF at 6 weeks posttreatment. Sham, sham operative group; Vehicle, stroke vehicle controls; S + G (7d): stroke mice with a 7-day treatment of SCF + G-CSF; S + G (5 + 5d): stroke mice with a 5-day treatment, 2-day break, and additional 5-day treatment of SCF + G-CSF. Data are mean ± SEM. **p < 0.01, *p < 0.05.

At 6 weeks posttreatment, SCF + G-CSF treatment (7-day treatment and 2-week treatment) also showed a significant increase in motor learning and motor coordination. Our findings revealed that the increased latency to fall per day (the slope over time) in the SCF + G-CSF treatment groups was 4.39 s on average compared to the stroke vehicle controls (95% confidence interval: 0.20, 8.57; p = 0.040) (Fig. 2B, C). When analyzing the two SCF + G-CSF treatment groups separately, we found that only the 7-day SCF + G-CSF treatment showed a significant increase in time on the Rota-Rod over time when compared with the stroke vehicle controls (95% confidence interval: 3.26, 11.96; p = 0.001), while the 2-week treatment group did not reach significance (Fig. 2B, C). Our findings did not support that repeated 2-week treatment of SCF + G-CSF has superior effects to a 7-day treatment paradigm on motor function improvement in aged mice with chronic stroke. At 6 weeks posttreatment, the 7-day treatment group showed a better pattern of motor learning and motor performance than those of the 2-week treatment group (95% confidence interval: 10.75, 2.39; p = 0.002).

The limitation of this study is the relatively small group size due to unexpected loss of aged mice during the experiment. The majority of them died during anesthesia and surgery for making the stroke model. This could be due to complications related to anesthesia and brain surgery in 19- to 20-month-old mice. The average life span for C57BL/6 mice is approximately 26 months. By the end of this study, the mice had almost achieved the end of life stage (~24 months). This could be an additional reason for losing the aged mice before the completion of this study. Through this study, we observed decreased plasma fibrinogen and improved motor function by SCF + G-CSF treatment in the chronic phase of stroke in aged mice. Repeated treatment for 2 weeks appears to not further enhance the effectiveness of SCF + G-CSF treatment in chronic stroke. Whether there is a causal relationship between the SCF + G-CSF-decreased plasma fibrinogen and improved motor function would be an open question for future studies.

Footnotes

Acknowledgment

This work was supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke (NINDS) (R01 NS060911), and George W. Perkins endowment. The authors declare no conflicts of interest.

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Fibrinogen Reduction and Motor Function Improvement by Hematopoietic Growth Factor Treatment in Chronic Stroke in Aged Mice: A Treatment Frequency Study

Abstract

Keywords

Introduction

Materials and Methods

Animals and Animal Model of Stroke

Groups and Treatment

Blood Collection and Enzyme-Linked Immunosorbent Assay

Motor Function Testing

Statistical Analysis

Results and Discussion

Footnotes

Acknowledgment

References