Intra-carotid arterial transfusion of circulatory-derived autologous CD34+ cells in rodent after ischemic stroke — evaluate the impact of therapeutic time points on prognostic outcomes

This study tested the optimal time point for left intra-carotid arterial (LICA) administration of circulatory-derived autologous endothelial progenitor cells (EPCs) for improving the outcome in rat after acute ischemic stroke (IS).

artery transfusion of circulatory derived autologous EPCs into brain infarct area in old IS stroke patients [18]. The results of our study displayed that EPC therapy was safe.
However, the neurological and neuro-psychiatric functions as well as the brain perfusion status were found to have only minor improvement in those patients [18]. The results of these aforementioned unmet needs from our study [18] raise the consideration of a large randomized placebo-controlled trial to test the safety and efficacy of EPC therapy for patients after acute IS. In light of the above observation, an animal model of IS study, before applying for a clinical trial, focusing on the optimal time point for administration of EPCs that would offer the greatest benefit and the minimal side effect is extremely necessary. Accordingly, this study using a rat model of acute IS tried to find out the best time point of circulatory derived autologous EPC administration for reducing the brain infarct volume (BIV)/brain infarct area (BIA) and improving the neurological outcome in IS rat.

Procedure and protocol of acute IS induction
The protocol and procedure for the experimental model of acute IS have been described in our previous studies [17,19]. In detail, each animal was anesthetized by 2% inhalational isoflurane in a supine position on a warming pad (37 ºC). After exposure of the left common carotid artery (LCCA) through a transverse neck incision, a small arteriotomy was performed on the LCCA through which silicon rubber-coated monofilament (filament size 4-0, diameter 0.185 mm, length 30 mm; Co Ltd. AMBO) was carefully advanced into the distal left internal carotid artery for occlusion of the left middle cerebral artery, causing brain ischemia and infarction of the corresponding territory area.
The nylon monofilament was removed 50 mins after occlusion, followed by closure of the muscle and skin in layers. The animals were then cared for in a portable animal intensive care unit (ThermoCare®) with food and water for 24 hours.

Corner test for assessment of neurological function prior to and after IS induction
The sensorimotor functional test (corner test) was conducted for each rat at baseline and on days 0, 3, 7, 14, 28 and 60 after acute IS induction as we previously described [17,19].
In detail, the rat could walk through a tunnel and then turn into a 60-degree corner. To exit the corner, the rat could turn either left or right. The results were recorded by a technician blinded to the study design. This test was repeated 10 to 15 times with at least 30 seconds between each trial. We recorded the number of right and left turns from 10 successful trials for each animal and used the results for statistical analysis. All 10 animals in each group went through this neurological functional test.

Animal grouping and treatment protocol
Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-approved animal facility in our hospital, with controlled temperature and light cycles (24°C and 12/12 light/dark cycle). Pathogen-free, adult male Sprague-Dawley (SD) rats (n=70) weighing around 300-325g (Charles River Technology, BioLASCO Taiwan Co. Ltd., Taiwan) were utilized in the present study and were equally categorized into group 1 (sham-operated control), group 2 (acute IS), group 3 [acute IS + in the present study was based on our previous report [17]. For the molecular-cellular examination, six animals in each group were utilized.

Peripheral blood was collected and cultured for EPCs
The procedure and protocol for EPC isolation and culture were based on our previous reports [17] with some modifications. In brief, animals in groups 3 to 6 were anesthetized with inhalational 2.0% isoflurane by day 21 at group 3, by day 18 at group 4, by day 14 at group 5 and by day 14 at group 6 prior to IS induction and by day 7 after IS induction at group 7 to collect peripheral blood for culturing EPCs. Isolated mononuclear cells from peripheral blood were cultured in a 100-mm diameter dish with 10 mL DMEM culture medium containing 10% FBS for 21 days. Based on our previous method [17], flow cytometric analysis was performed for identification of cellular characteristics (i.e., EPC surface markers) after cell-labeling with appropriate antibodies on day 21 of cell cultivation prior to cell therapy (i.e., EPCs were transfused into LCCA on day 21 after cell culture).
Rats in groups 1 and 2 were anesthetized with inhalational 2.0% isoflurane and only received an identical procedure of peripheral blood sampling without further culture.

Procedure and protocol of brain magnetic resonance imaging (MRI) study
The procedure and protocol have been described in our previous report [17]. Magnetic resonance imaging (MRI) was performed at day 60 and after acute IS induction. Briefly, during MRI measurements, mice were anesthetized by 2% inhalational isoflurane with room air and placed in an MRI-compatible holder (Biospec 94/20, Bruker, Ettingen, Germany). Rectal temperature and respiration were monitored throughout the procedure to ensure normal physiological conditions were maintained. MRI data were collected using a Varian 9.4T animal scanner (Biospec 94/20, Bruker, Ettingen, Germany) with a rat surface array. The MRI protocol consisted of 40 T2-weighted images. Forty continuous slice locations were imaged with a field-of-view of 30 mm x 30 mm and an acquisition matrix dimension of 256 x 256 and slice thickness of 0.5 mm. The repetition time (TR) and echo time (TE) for each fast spin-echo volume were 4200 ms and 30 ms, respectively. A custom software, ImageJ (1.43i, NIH, USA), was used to process the region of interest (ROI). Planimetric measurements of images from MRI T2 were performed to calculate the stroke volumes of cortex. For brain MRI examination, four randomized animals in each group were utilized. The animals were euthanized on day 60 after brain MRI examination and the brain tissues were harvested for individual study.

Immunofluorescent (IF) staining of brain specimens
The procedure and protocol for IF staining have been reported in our previous studies [17][18][19]. For IF staining, rehydrated paraffin sections were first treated with 3% H 2 O 2 for 30 minutes and incubated with Immuno-Block reagent ( Fluor594-conjugated goat anti-mouse or rabbit IgG were used to localize signals. Sections were finally counterstained with DAPI and observed with a fluorescent microscope equipped with epifluorescence (Olympus IX-40). Three brain sections were analyzed for each rat. For quantification, three randomly selected high-power fields (HPFs; x400 for IF study) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.

Western blot analysis of brain specimens
Equal amounts (50 mg) of protein extracts were loaded and separated by SDS-PAGE using Biosciences) and exposed to Medical X-ray film (FUJI). For quantitation, ECL signals were digitized using Labwork software (UVP, Waltham, MA, USA). A standard control sample was loaded on each gel.

Vessel density in BIA
IHC staining of small blood vessels was performed with α-SMA (1:400) as primary antibody at room temperature for 1 h, followed by washing with PBS thrice. Ten minutes after the addition of anti-mouse-HRP conjugated secondary antibody, the tissue sections were washed with PBS thrice. Then 3,3' diaminobenzidine (DAB) (0.7 gm/tablet) (Sigma-Aldrich) was added, followed by washing with PBS thrice after one minute. Finally, hematoxylin was added as a counter-stain for nuclei, followed by washing twice with PBS after one minute. Three brain sections were analyzed in each rat. For quantification, three randomly selected HPFs (200x) were analyzed in each section. The mean number per HPF for each animal was then determined by summation of all numbers divided by 9.

Statistical analyses
Quantitative continuous data are expressed as mean ± SD. Statistical analysis was performed for continuous variables among groups by one-way ANOVA followed by Bonferroni multiple-comparison post hoc test for the between-group comparison. All analyses were conducted using SAS statistical software for Windows version 8.2 (SAS institute, Cary, NC, USA). A probability value <0.05 was considered statistically significant.

The brain infarct volume (BIV) at day 60 after IS and the time courses of corner test after acute IS (Figures 1 and 2)
At day 60 after acute IS, the brain MRI demonstrated that the BIV was lowest in group 1 (SC), highest in groups 2 (IS + culture medium 25λ) and significantly and progressively increased from groups 3 (IS + EPCs/at 3h after acute IS), 4 (IS + EPCs/at day 3 after acute IS), 5 (IS + EPCs/at day 7 after acute IS), 6 (IS + EPCs/at day 14 after acute IS) to 7 (IS + EPCs/at day 28 after acute IS) (Fig. 1).
The neurological function did not differ at baseline among seven groups. However, at day 3 after acute IS, the neurological function was significantly impaired in groups 2 to 7 than in group 1, but it showed no difference among the former six groups (Fig. 2).
On the other hand, at day 7 after IS, this parameter was significantly improved in groups 3 and 4 than in groups 2 and 5 to7, but it did not differ between groups 3 and 4 or among showed no difference between the groups 6 and 7 (Fig. 4).
Additionally, the cellular expressions of SDF-1α and CXCR4 were highest in group 3, lowest in group 1, significantly higher in group 4 than in groups 2, 5 to 7, significantly higher in group 5 to groups 2, 6 and 7 and significantly higher in groups 6 and 7 than in group 2, but they were similar between groups 6 and 7, suggesting an intrinsic response to ischemic stimulation that was significantly upregulated by EPCs treatment (Fig. 5).
To elucidate the angiogenesis/neovascularization in the brain infarct region, we performed IHC staining (i.e., α-SMA stain). As expected, the number of small vessel (i.e., diameter ≤ 25 µM) exhibited a similar pattern to that of the protein levels of CD31 and vWF among the groups (Fig. 6).

The protein expressions of inflammatory, oxidative stress, autophagic and apoptotic biomarkers in BIA at day 60 after acute IS (Figures 7 and 8)
The protein expressions of IL-1ß, TNF-α, NF-κB and MMP-9, four indicators of inflammation, were lowest in group 1 and highest in group 2, significant lower in group 4 and more significantly lower in group 3 than in groups 5 to 7, but they exhibited no difference among the groups 5 to 7 (Fig. 7).
Additionally, the protein expressions of NOX-1, NOX-2, oxidized protein and p22phox, four indicators of oxidative stress, displayed an identical pattern of inflammation among the groups (Fig. 7). Furthermore, the protein expressions of Beclin-1, Atg-5 and ratio of LC3B-II/LC3B-I, three indicators of autophagic biomarkers, exhibited an identical pattern of inflammation among the groups (Fig. 8).
Moreover, the protein expressions of mitochondrial-Bax, cleaved caspase 3 and cleaved PARP, three indices of apoptosis, showed an identical pattern to that of inflammation among the groups (Fig. 8).

Inflammatory cell expressions of GRPA+ and microglial cells in BIA at day 60
after acute IS (Figure 9) The results of IF microscopic findings demonstrated that the expressions of GFAP and microglial cells, two indicators of inflammatory cells specifically situated in brain tissue, was lowest in group 1, highest in group 2 and significantly and progressively increased from groups 3 to 7, but it displayed no difference between groups 5 and 6.

Discussion
This study which investigated the appropriate time point of intra-carotid administration of circulatory derived autologous EPCs for improving the neurological function and reducing the BIV yielded several preclinical striking implications. First, despite the difference in the degree of preservation of neurological function and brain architecture, the EPCs therapy at time intervals ≤ 14 days after IS still significantly preserved the neurological function and integrity of brain architecture in rat acute IS. Second, through different time intervals of administrating EPCs for rat after acute IS, this study identified that the optimal time point of EPC therapy for improving the neurological function and reducing the infarct zone was as early as possible (i.e., at 3 h after acute IS).
Intra-carotid administration of circulatory EPCs significantly improved neurological outcome and reduced the brain infarct size in rat after acute IS having been well recognized by our previous study [17]. Surprisingly, there is currently no data available to address the optimal timing of administration of EPCs after acute IS. In the current study, time courses of EPCs therapy for identifying the most appropriate time point was done in rat IS model. The most important finding in the present study was that 3 hours after acute IS was the best timing for EPC administration to improve the neurological function and preserve brain architecture. Our finding, in addition to supporting the finding of our previous study [5], highlights the common concept that cell therapy should be performed "as early as possible" for ischemia-related organ dysfunction.
Recently, we have performed a phase I clinical trial by utilizing the circulatory derived autologous CD34 + cells to treat those old IS patients [18]. Interestingly, although the safety of the cell therapy was confirmed [18], the therapeutic impact on improving the neurological and recognizing outcomes regrettably left much to be desired, attributing mainly to late treatment. Some previous clinical trials have also demonstrated that cell therapy for patients after acute myocardial infarction (AMI) failed to improve the heart function and clinical outcome [20,21]. In further, when we carefully analyzed the results from these clinical trials [20,21], a commonality of late administration of stem cells to patients after AMI was uncovered. An essential finding in the present study was that as we serially tested the time points of administration of EPCs at 3 h and days 3, 7, 14 and 28 in rat after acute IS, the later the EPC administration, the lesser effective degree of neurological improvement was found in the present study. Accordingly, our finding, in addition to explaining why those clinical studies [18,20,21] failed to prove the effectiveness of stem cell therapy for the improvement of the left ventricular (LV) function in patients after AMI, supports the common concept that only early administration of stem cells is the fundamentally mechanistic basis for improving ischemia-related organs, especially in setting of acute IS and AMI.
The underlying mechanisms for improving the ischemia-related LV dysfunction after the cell therapy have been well recognized mainly due to the angiogenesis and neovascularization, chemokine/paracrine effects, stem cell homing, anti-inflammatory response and immunomodulation but they are less likely due to the implanted stem cells to differentiate into the cardiomyocytes [22][23][24]. Of these factors, the angiogenesis is one of the most important factors for improving the ischemia-related organ dysfunction [16,17,[22][23][24]]. An essential finding in the present study was that the molecular-cellular levels of angiogenesis parameters were markedly upregulated after IS stroke (i.e., due to an intrinsic response) and further markedly upregulated by EPC treatment, especially in the time point of early administration of EPCs to the animals after acute IS. Of particular importance was that increases of angiogenesis biomarker were significantly associated with improving neurological function and reducing the brain infarct zone in those animals receiving early EPC treatment. Our findings were consistent with those of previous studies [16,17,[22][23][24].
A principal finding in the present study was that the inflammatory, oxidative-stress, apoptotic and autophagic biomarkers were substantially increased in acute IS animals than in those of SC animals. Additionally, the neurological impairment and brain infarct region were remarkably increased in these acute IS animals. Intriguingly, links between upregulations of inflammation, oxidative stress, apoptosis and autophagic biomarkers and impaired ischemia-related organ dysfunction have been fully investigated by the previous experimental studies [17,19,[22][23][24][25][26]. Along this line of thinking, our findings in addition to corroborating with the findings of previous studies [17,19,[22][23][24][25][26], could, at least in part, explain why the neurological status and BIV were notably deteriorated in those of acute IS animals without treatment. However, these molecular-cellular perturbations were significantly reversed in IS animals after receiving the early EPC treatment, once again suggesting that EPC treatment in an appropriate time point could effectively improve outcome in setting of acute IS. Accordingly, our preclinical findings may provide clinically relevant information and encourage us to utilize EPC therapy for those acute IS patients, especially when they have poor response or are refractory to conventional therapy.

Study Limitation And Conclusion
This study has limitations. First, the dosage of EPC to be utilized in the present study was based on our previous report [17] and no stepwise increase in the dosage of EPC therapy was performed in the current study. Accordingly, the optimal dosage of EPCs for rat after acute IS remains currently uncertain. Second, being void of comparison, this study did not provide information regarding whether the therapeutic effect of circulatory-derived EPCs is superior to bone marrow-derived EPCs for improving the neurological outcomes or vice versa in setting of acute IS. Third, despite extensive works were done in the present study, the exactly underlying mechanisms of improving the outcomes after EPC therapy for acute IS remains uncertain. Based on the results of the present study, Fig. 10 provides the schematically proposed mechanism for the impact of EPC therapy on mammalians after acute IS.
In conclusion, the results of the present study proved that early administration of EPCs for acute IS rodent would provide the best effect on improving the neurological outcome and protecting the brain architecture against acute IS damage.

Consent for publication
Not applicable.

Conflicts of interest
All authors have read the journal's policy on disclosure of potential conflicts of interest and the journal's authorship agreement. The authors declare that they have no conflicts of interest. The article has been reviewed by and approved by all named authors.  group 2 = ischemic stroke (IS); group 3 = EPC administration from LCCA at 3h after acute IS; group 4 = EPC administration from LCCA at day 3 after acute IS; group 5 = EPC administration from LCCA at day 7 after acute IS; group 6 = EPC administration from LCCA at day 14 after acute IS; group 7 = EPC administration from LCCA at day 28 after acute IS.    from LCCA at 3h after acute IS; group 4 = EPC administration from LCCA at day 3 after acute IS; group 5 = EPC administration from LCCA at day 7 after acute IS; group 6 = EPC administration from LCCA at day 14 after acute IS; group 7 = EPC administration from LCCA at day 28 after acute IS.

Figure 6
Expression of small vessel density in brain infarct zone at day 60 after acute IS A to G) Illustrating the microscopic finding (100x)   IS-EPCD3 = EPC administration from LCCA at day 3 after acute IS; IS-EPCD7 = EPC administration from LCCA at day 7 after acute IS; IS-EPCD14 = EPC administration from LCCA at day 14 after acute IS; IS-EPCD28 = EPC administration from LCCA at 36 day 28 after acute IS.

Figure 10
The underlying proposed mechanism of peripheral blood-derived endothelial progenitor cell (EPC) (i.e., CD34+ cells) therapy for improving the outcomes in patients or rodents after acute ischemic stroke. VEGF = vascular endothelial growth factors; NO = nitric oxide.