2025 Volume 74 Issue 2 Pages 79-85
Previously, we reported that transplantation of regeneration-associated cells (RACs) via the ipsilateral external carotid artery reduced stroke volume in mice with permanent occlusion of the middle cerebral artery (MCA). However, intracarotid arterial transplantation is invasive and requires skill, and severe complications may occur, such as thromboembolism, infection, and decreased cerebral blood flow. This study aimed to investigate the efficacy of intravenous injection of RACs in reducing stroke volume and increasing anti-inflammatory and angiogenic factors in mice with focal cerebral ischemia. Mice with occluded MCAs received intravenous injections of phosphate-buffered saline (PBS) (control), low-dose RACs, or high-dose RACs. The proximal part of the left MCA was occluded to induce permanent focal ischemia. After 3 days, we administered PBS or low-dose (1 × 104 /50 µL) or high-dose RACs (1 × 105 /50 µL) through the tail vein and assessed the infarct volume on day 7. High-dose RACs significantly decreased infarct volume compared to PBS, whereas low-dose RACs showed no effect. The number of interleukin-10 (IL-10)-positive and vascular endothelial growth factor (VEGF)-positive cells in the peri-infarct area on day 7 was significantly higher in mice treated with low-dose and high-dose RACs than in the PBS control group. Intravenous injection of RACs can reduce ischemic stroke volume; however, a higher dose of RACs is required than the dose used in intraarterial transplantation. By assessing IL-10 and VEGF expression, the study sheds light on the underlying mechanisms of RAC therapy, revealing its potential anti-inflammatory and angiogenic properties in the treatment of cerebral ischemia.
Cerebral ischemia, characterized by vessel occlusion, results in death of neurons and glia because of lack of oxygen and glucose supply. These circumstances lead to a chain reaction of excitotoxicity, oxidative stress, disruption of the blood–brain barrier, and infiltration of inflammatory cells into the affected area, which damages the brain tissue.1,2,3,4 Despite extensive clinical trials on potential drugs targeting the pathological processes of ischemic stroke, the efficacy of most drugs in improving patient outcomes remains limited in patients with cerebral ischemia,5 underscoring the critical need for innovative therapies for cerebral ischemia.
Cell therapies are promising for stroke management, especially for patients who have not undergone thrombectomy or intravenous thrombolysis. Researchers have used stem cells, induced pluripotent stem cells, progenitor cells, and immature cells in animal cerebral ischemia models.6,7,8,9,10,11,12
Endothelial progenitor cells (EPCs) have the potential to be utilized in transplantation therapy. Asahara and colleagues were the first to isolate EPCs from the peripheral blood of adults and demonstrated their ability to promote angiogenesis and tissue regeneration.13,14 However, reports on EPCs are limited, partly because progenitor cells are difficult to collect for therapeutic use. Recently, we developed a new colony assay system to isolate mononuclear cells (MNCs) enriched with higher-grade EPCs.15 These are regeneration-associated cells (RACs), which contain not only EPCs, but also regulatory T cells and M2 macrophages.16 In our previous report, we revealed that the transplantation of RACs via the ipsilateral external carotid artery led to improvement in stroke volume following the permanent occlusion of the middle cerebral artery (MCA) in mice.16
However, intracarotid arterial transplantation of RACs is an invasive procedure that demands a high level of skill to be performed safely. In addition, there is a risk of severe complications such as thromboembolism, infection, and reduction in cerebral blood flow because of microstroke.17 However, in cell therapy, intravenous transplantation tends to lead to a greater accumulation of cells in peripheral organs than is likely with intraarterial transplantation.18,19
Therefore, the first aim of this study was to investigate the efficacy of intravenous injection of RACs in reducing infarct volume and increasing anti-inflammatory and angiogenic factors. Our second aim was to determine the quantity of cells required for treatment. We sought to determine whether a similar quantity of cells used for intraarterial transplantation16 was adequate for intravenous transplantation by evaluating both the same number of cells and ten times that number.
Ten-week-old C57BL/6 J male mice (mass 23–25 g) were obtained from CLEA Japan (Tokyo, Japan). The mice were inspected daily and given unrestricted access to food and water in a controlled environment in ventilated cages that contained soft wood chip bedding. Each cage housed five to seven mice. They were administered anesthesia using a combination of 4% isoflurane, 66% nitrous oxide, and 30% oxygen and were maintained with 1.5% isoflurane during all experiments.
Ethical approvalThe Animal Care and Use Committee of Tokai University School of Medicine granted approval for all experimental procedures and protocols (approval #12R-068).
Cell culture systemPeripheral blood (PB) was collected from the left ventricle of the heart of mice (800 µL per mouse) and subjected to density gradient centrifugation using Histopaque-1083 (1.083 g/mL; Sigma, St Louis, MO, USA) to isolate PB mononuclear cells (PB-MNCs). Cells were plated at a cell density of 5 × 105 cells /500 µL on a 24-well Primaria tissue culture plate [5 × 105 cells /500 µL per well (BD Falcon; BD Biosciences, San Jose, CA, USA)] in a defined serum-free medium (S0192-500ML StemLineII; Sigma) supplemented with five mouse recombinant proteins [SCF #300–07, TPO #300–18, Flt-3 ligand #300–19, vascular endothelial growth factor (VEGF) #100–20, and IL-6 #200–06 (PeproTech, Cranbury, NJ, USA)], as reported previously.15,20 Five days after culture initiation, non-adherent cells were eliminated by washing with phosphate-buffered saline (PBS). Adherent cells were collected using a solution of 2 mmol/L ethylenediaminetetraacetic acid in PBS. The harvested cells (RACs) were suspended in a solution of Iscove’s modified Dulbecco’s medium (Sigma Aldrich) at a concentration of 1 × 104 cells per 50 µL of solution.
Induction of focal cerebral ischemiaFocal cerebral ischemia was induced in mice under anesthesia. The left MCA was exposed by drilling a 2-mm hole at a site that was superior and lateral to the left foramen ovale. The proximal part of the left MCA was permanently blocked with a bipolar coagulator over a 1 mm segment distal to the origin of the lenticulostriate branch.21 To maintain the core body temperature of the mice at 37 °C, they were kept in a controlled environment with a humidity-regulated temperature of 32 °C during occlusion. The mice were placed in a controlled environment with regulated humidity and temperature for an additional 2 h after the incision was sutured, before being transferred back to their original cages for further experimentation. PBS (control), low-dose, or high-dose RACs were injected into the tail vein at 3 days post-occlusion (Fig. 1). The dosage of RACs for injection was set to 1.0 × 104 /mouse for the low dose and 1.0 × 105 /mouse for the high dose. Infarct volume was measured on day 7 after MCA occlusion (MCAO).
Experimental protocol.
Syngeneic transplantation was performed by transplanting low and high doses of regeneration-associated cells (RACs) on day 3 after middle cerebral artery occlusion (MCAO). On day 7 after MCAO, the mice brains were removed and assessed for brain infarction volume or immunohistochemistry.
For assessment of neurological deficits, neurological grading scores were evaluated from 0 to 3, based on the severity of symptoms, including forelimb flexion, resistance to lateral push, and circling behavior, before surgery and at 1 h and 7 days after MCAO as previously described.16 The body masses of mice were assessed on days 0, 3, and 7, and their body temperatures were measured both before and after MCAO.
Seven days after inducing MCAO in mice, the animals were briefly anesthetized again and killed. The brains were removed and cut into 1-mm-thick coronal sections. The sections were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin. Images were captured under a microscope, and measurement of the infarcted areas in the cerebral cortex and striatum was carried out using NIH ImageJ software. The stroke volume was determined by multiplying the measured areas by the gap between sections, which was assessed by a blinded examiner (E.N.). The brain sections were collected at 600-μm intervals, and the infarct volume was expressed as a percentage relative to the entire ipsilateral hemisphere volume.
Immunohistochemistry of factors relating to angiogenesis and anti-inflammation in tissuesTo assess the expression of factors related to angiogenesis and anti-inflammation in the tissues, immunohistochemistry was performed for VEGF and anti-inflammatory cytokine IL-10. Fixed sections were incubated in 5 mM hydrogen peroxide for 10 min before being exposed to 5% normal goat serum for an additional 10 min. Overnight incubation of the sections was carried out with polyclonal anti-goat VEGF (AF493NA; R&D Systems, Minneapolis, MN, USA) at a 50-fold dilution and monoclonal anti-rat IL-10 (ab33471; Abcam, Cambridge, UK) at a 200-fold dilution in a humidified chamber maintained at 4 °C. After thoroughly washing the sections with 0.01 M PBS (pH 7.2), they were incubated with Histofine Simple Stain Mouse Max PO (Nichirei, Tokyo, Japan) at a 100-fold dilution for 1 h. The bound antibodies were detected through the use of 3,3′-diaminobenzidine and hydrogen peroxide. Cells positive for VEGF and IL-10 in the peri-infarct regions of the frontal cortex and striatum, which were defined as regions within 0.6 mm from the edge of the ischemic region based on hematoxylin and eosin staining, were counted by one examiner (E.N.) blinded to the experimental protocol, in each of three predetermined areas (0.62 mm2) per high-power field (×400).
Statistical analysisStatistical analysis was conducted using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). The Kruskal–Wallis test was utilized to compare multiple groups. The significance level was set at P ≤ 0.05, with data presented as mean ± standard deviation.
The administration of high-dose RACs resulted in a considerable decrease in stroke volume (P < 0.05, Kruskal–Wallis test) when compared with that in the vehicle, whereas low-dose RACs showed no effect (PBS, n =15; low-dose RACs, n =8; high-dose RACs, n =10) (Fig. 2). The findings suggest that a higher dose of RACs is required to reduce the infarct volume in intravenous transplantation than in intraarterial transplantation.16 We also evaluated the physical findings of neurological deficits after treatment. Table 1 summarizes the physical parameters and neurological deficits. On day 7 after surgery, neurological deficit scores were not different among the PBS, low-dose RAC, and high-dose RAC groups. No significant differences were noted in body mass or body temperature among these three groups after MCAO at any point in time (Table 1).
Effect of high-dose RACs on infarct volume after MCAO.
The administration of a high dose of RACs (1 × 105 /50 µL) on day 3 after MCAO led to a significant reduction in infarct volume as compared to the phosphate-buffered saline (PBS) group (*P < 0.05, Kruskal–Wallis test). In contrast, the low dose of RACs (1 × 104 /50 µL) did not show any effect on the infarct volume when compared to the PBS group [PBS: n=15, RACs (1 × 104 /50 µL): n=8, and RACs (1 × 105 /50 µL): n=10].
| Time | Neurological deficit | Mass (g) | Temperature (°C) | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| PBS (n=15) |
High dose RACs (n=10) |
Low dose RACs (n=8) |
PBS (n=15) |
High dose RACs (n=10) |
Low dose RACs (n=8) |
PBS (n=15) |
High dose RACs (n=10) |
Low dose RACs (n=8) |
||
| Day 0 | Before surgery | 15 ± 0 | 15 ± 0 | 15 ± 0 | 24.34 ± 0.13 | 24.16 ± 0.20 | 23.76 ± 0.18 | 37.58 ± 0.17 | 37.83 ± 0.24 | 37.6 ± 0.21 |
| Day 0 | 1 h after MCAO | 11 ± 0.9 | 12.1 ± 0.9 | 12 ± 1.1 | 38.73 ± 0.17 | 38.5 ± 0.25 | 38.59 ± 0.15 | |||
| Day 7 | Killed | 14.8 ± 0.1 | 14.9 ± 0.1 | 14.9 ± 0.1 | 23.98 ± 0.18 | 24.02 ± 0.50 | 23.35 ± 0.21 | ND | ND | ND |
PBS: phosphate-buffered saline control group, RAC, regeneration-associated cell, MCAO: middle cerebral artery occlusion, ND: not determined.
We utilized immunohistochemistry to assess the expression levels of angiogenic and anti-inflammatory molecules in tissue samples. The impact of day 3 transplantation on tissue biology in the cerebral infarction area was assessed 7 days after MCAO. We analyzed the levels of the anti-inflammatory cytokine IL-10 and the angiogenic factor VEGF in the peri-infarct area at the early stage of recovery (day 7). Both high and low doses of RACs were found to significantly increase the number of VEGF-positive (Fig. 3) and IL-10-positive (Fig. 4) cells in the peri-infarct area compared to PBS (P < 0.05, Kruskal–Wallis test).
Effect of RACs on vascular endothelial growth factor (VEGF) expression in peri-infarct area.
Injection of both low (1 × 104 /50 µL) and high RACs (1 × 105 /50 µL) on day 3 after MCAO resulted in a significant increase in VEGF-positive cells in the peri-infarct area compared to PBS (*P < 0.05, Kruskal–Wallis test) [PBS: n=15, RACs (1 × 104 /50 µL): n=9, and RACs (1 × 105 /50 µL): n=10].
Effect of RACs on IL-10 expression in the peri-infarct region following MCAO.
The administration of both low (1 × 104 /50 µL) and high RACs (1 × 105 /50 µL) on day 3 after MCAO led to a significant increase in the number of IL-10-positive cells in the peri-infarct region when compared to the PBS group (*P < 0.05, Kruskal–Wallis test) [PBS: n=15, RACs (1 × 104 /50 µL): n=9, and RACs (1 × 105 /50 µL): n=10].
RACs are a novel cell population obtained from quality and quantity (QQ) controlled cultures of unfractionated MNCs.15,20 In this study, we showed that intravenous RAC delivery reduced the size of ischemic stroke.
The methodology for obtaining QQ controlled cultures is simple and safe. It can be applied not only to improve EPC expansion, but also to stimulate anti-inflammatory and angiogenic monocytes/macrophages and helper T lymphocytes, which facilitate the release of numerous protective and proangiogenic cytokines and growth factors.20 Fluorescence-activated cell sorting analysis has revealed that RACs contain expanded populations of progenitor cells (CD34+ or CD133+ cells) alongside M2 macrophages, which are known to release VEGF, and regulatory T-cells (CD4+/CD25+/Foxp3+), which are known to release IL-10.20 Expression of genes encoding vascular regeneration factors, including VEGF-B, angiopoietin-1 (Ang-1), leptin, IL-8, IL-10, and insulin-like growth factor 1, was much higher in QQ cultured MNCs than in the control populations.20
Although the ischemic volume was reduced by intravenous transplanted RACs when compared with PBS, only a high dose of RACs (1 × 105 /50 µL) was effective in reducing the ischemic stroke volume. Our findings suggest that the amount of RACs required to obtain neuroprotective effects by intravenous transplantation is larger than that required by intraarterial transplantation, as described in our previous report.16
Regarding the potential clinical application of RACs in patients with ischemic stroke, intravenous transplantation is an ideal route of delivery in terms of ease and clinical precedence. In our previous research, we injected labeled CD34-positive mononuclear blood cells through the tail vein of mice with unilateral hindlimb ischemia. Subsequent histological examination revealed numerous, proliferative labeled cells within the neovascularized ischemic hindlimb.13 Furthermore, we previously showed that intraarterial transplantation of RACs induced angiogenesis in the peripheral area of stroke.16 Therefore, it is suggested that EPCs administered from the tail vein spread to the peripheral organs and eventually reach the ischemic brain.
However, previous research has reported that most intravenously transplanted cells become trapped in filtering organs, such as the liver, spleen, and lungs.22,23,24 This could be one reason why a higher amount of RACs is required for stroke volume reduction following intravenous transplantation in cerebral ischemia. In addition, cell migration to the spleen may also produce additional therapeutic effects. The activation and recruitment of T cells and monocytes in the spleen can result in their migration to the brain, where they may contribute to tissue damage following a stroke.25 Intravenous administration of hematopoietic stem cells has been shown to decrease inflammatory reactions in the spleen, leading to a reduction in stroke volume in a mouse model of ischemic stroke.25
Compared with the use of intravenous transplantation, the use of intraarterial transplantation results in a significantly greater number of cells migrating into the infarct lesion.17 Although one of the merits of intraarterial transplantation is the capacity for higher cell engraftment in the brain, this might not be a disadvantage for RAC treatment because the mechanism of RAC therapy is reported to be a bystander effect rather than transdifferentiation. Trophic factors and cytokines derived from RACs ameliorate the damage following cerebral ischemia.16
RACs can activate anti-inflammatory and angiogenic monocytes, which subsequently release protective and pro-angiogenic cytokines and growth factors. IL-10 plays a significant role in anti-inflammatory polarization during ischemic stroke. Previous studies on ischemic stroke in mouse models have shown that IL-10 deficiency leads to increased infarct size and poor long-term outcomes.26 VEGF is upregulated and stimulates angiogenesis after ischemic stroke after cerebral ischemia in mice.27,28 Our research demonstrated that intravenous transplantation of RACs resulted in a higher number of IL-10- and VEGF-positive cells, suggesting that their neuroprotective effects against cerebral ischemia were attributed to their anti-inflammatory and angiogenic properties.
A limitation of this study is that we were unable to demonstrate any difference in the outcomes of functional tests between vehicle-treated mice and RAC-treated mice, despite finding a significant decrease in stroke size in the RAC-treated group. Neurological deficits were evaluated using neurological grading scores based on the severity of symptoms, including forelimb flexion, resistance to lateral push, and circling behavior, which were also used in our previous report.16 However, these tests may not be suitable for evaluating long-term outcomes because the recovery from neurological deficits in the MCAO model is so rapid that the neurological scores almost return to normal even in the vehicle group. Further studies using different functional tests to detect deficits in the chronic phase should be conducted to determine whether RACs can improve neurological deficits following cerebral ischemia. It has also been reported that the formation of glial scars by reactive astrocytes may obstruct axonal regeneration, thereby negatively affecting the functional outcomes during the late recovery phase after stroke.29 However, regulatory T-cells suppress the activation of astrocytes, and M2-type macrophages facilitate the scavenging of necrotic cells and tissue debris.30 In addition, they contribute to neural repair by producing trophic cytokines in the ischemic brain region. Therefore, it is improbable that the discrepancy between stroke volume and functional assessments is attributable to this factor. Finally, adverse effects of cell therapy for cerebral ischemia have been reported. For example, in vitro expansion of the cell population can introduce the risk of malignant transformation,31 whereas pulmonary embolism can occur following intravenous infusion because of trapped cells within the lungs.32 These potential adverse effects require further evaluation in future studies.
In conclusion, our study demonstrated the potential of intravenous transplantation of RACs as a therapeutic approach for reducing infarct volume and modulating anti-inflammatory and angiogenic responses in cerebral ischemia. By elucidating the efficacy and optimal dosage of RAC administration, our findings contribute to the advancement of cell-based therapies for stroke management. Further research is warranted to explore the clinical translation of intravenous RAC transplantation and its potential benefits for improving patient outcomes in ischemic stroke.
We thank S. Kohara, H. Yuzawa, N. Fujii, and Y. Takahari for their technical support. This work was partly supported by a Grant-in-Aid for Scientific Research (C) (grant no. 26461320) from the Japan Society for the Promotion of Science and the SENSHIN Medical Research Foundation in 2017. This work was partially supported by the Japan Regenerative Medicine Project of the Japan Agency for Medical Research and Development (grant no. 15bk0104012h003).
The authors have declared that no conflict of interest exists.
