Life Sciences 95 (2014) 22–28
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Effect of repeated allogeneic bone marrow mononuclear cell transplantation on brain injury following transient focal cerebral ischemia in rats Fumio Kamiya ⁎, Masayuki Ueda ⁎, Chikako Nito, Nobuo Kamiya, Toshiki Inaba, Satoshi Suda, Tomonari Saito, Kanako Muraga, Yasuo Katayama Department of Neurological Sciences, Graduate School of Medicine, Nippon Medical School, Japan
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Article history: Received 5 September 2013 Accepted 11 December 2013 Keywords: Bone marrow Cerebral ischemia Neuroprotection Allogeneic transplantation
a b s t r a c t Aims: Transplantation of bone marrow mononuclear cells (BMMCs) exerts neuroprotection against cerebral ischemia. We examined the therapeutic timepoint of allogeneic BMMC transplantation in a rat model of focal cerebral ischemia, and determined the effects of repeated transplantation outside the therapeutic window. Main methods: Male Sprague–Dawley rats were subjected to 90 minute focal cerebral ischemia, followed by intravenous administration of 1 × 107 allogeneic BMMCs or vehicle at 0, 3 or 6 h after reperfusion or 2 × 107 BMMCs 6 h after reperfusion. Other rats administered 1 × 107 BMMCs at 6 h after reperfusion received additional BMMC transplantation or vehicle 9 h after reperfusion. Infarct volumes, neurological deficit scores and immunohistochemistry were evaluated 24 or 72 h after reperfusion. Key findings: Infarct volumes at 24 h were significantly decreased in transplantation rats at 0 and 3 h, but not at 6 h, after reperfusion, compared to vehicle-treatment. Even high dose BMMC transplantation at 6 h after reperfusion was ineffective. Repeated BMMC transplantation at 6 and 9 h after reperfusion reduced infarct volumes and significantly improved neurological deficit scores at 24 and 72 h. Immunohistochemistry showed repeated BMMC transplantation reduced ionized calcium-binding adapter molecule 1, 4-hydroxy-2-nonenal and 8hydroxydeoxyguanosine expression at 24 and 72 h after reperfusion. Significance: Intravenous allogeneic BMMCs were neuroprotective following transient focal cerebral ischemia, and the therapeutic time window of BMMC transplantation was N3 h and b6 h after reperfusion in this model. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in ischemic brains, resulting in improved neuroprotection. © 2013 Elsevier Inc. All rights reserved.
Introduction Transplantation of bone marrow cells reportedly exerts neuroprotection against cerebral ischemia (Chen et al., 2001; Bliss et al., 2007). We previously showed that either transplantation of autologous bone marrow mononuclear cells (BMMCs) or bone marrow stromal cells (BMSCs) reduced infarct volume following transient focal cerebral ischemia in rats (Kamiya et al., 2008; Suda et al., 2011). However, it is difficult to prepare sufficient numbers of autologous BMMCs for transplantation at the acute phase of stroke, because collection of BMMCs requires surgical procedures. In addition, BMSCs also require enough time to culture cells, thus adding to the difficulty of preparing BMSCs at the acute phase of stroke. Moreover, we confirmed “allogeneic” and “autologous” BMMC transplantations were equally neuroprotective in a rat stroke
model (Kamiya et al., 2011). Therefore, allogeneic BMMCs may be a therapeutic option for immediate transplantation or repeated transplantation. The benefit of cell transfusion therapy for the acute phase of cerebral ischemia includes attenuation of inflammation and inhibition of antioxidant processes (Bliss et al., 2007; Yang et al., 2011). The present study was designed to determine the therapeutic time window of allogeneic BMMC transplantation in a rat model of transient focal cerebral ischemia, and to examine whether repeated BMMC transplantations outside the therapeutic time window achieved further neuroprotection. In addition, the mechanisms of neuroprotection were investigated in relation to inflammation and oxidative stress. Materials and methods Ischemia model
⁎ Corresponding authors at: Department of Neurological Sciences, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Tel.: +81 3 3822 2131; fax: +81 3 3822 4865. E-mail addresses: [email protected] (F. Kamiya), [email protected] (M. Ueda). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.12.016
All experimental procedures were approved by the institutional guidelines for animal use and care. A total of 134 male Sprague–Dawley rats, weighing 250 to 300 g, were used in this study. Anesthesia was
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performed with 5% halothane and maintained with 0.5 to 1.0% halothane in 70% N2O and 30% O2 mixture during all surgical procedures. The rats were subjected to transient focal cerebral ischemia for 90 min using an intraluminal suture technique (Ueda et al., 2013; Nito et al., 2011; Suda et al., 2011). Briefly, the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) on the right side were carefully exposed via a midline cervical incision. The ECA and the CCA were double-ligated using a silk suture. A silicon rubber-coated round-tip nylon surgical thread was inserted into the ICA (approximately 18 mm from the bifurcation) via a small puncture in the CCA to occlude the origin of the middle cerebral artery (MCA). A silk suture around the CCA was tightened to prevent bleeding from the puncture site. After 90 min of MCA occlusion, reperfusion was performed by withdrawing the suture. A PE-50 catheter was inserted into the tail artery for blood sampling and arterial blood pressure monitoring to confirm that physiological variables were within normal limits. Rectal temperature was maintained at 37.0 ± 0.5 °C during the procedure.
Magnetic resonance imaging Magnetic resonance imaging (MRI) analyses were performed using Unity-INOVA-300 system with a 7 T/18 cm horizontal magnet (Agilent Technologies., CA, USA), as described previously (Ueda et al., 2013; Nito et al., 2011). The continuous arterial spin labeling method (Williams et al., 1992), modified with a centrally encoded variable-tip-angle gradient echo technique (Ewing et al., 2003), was performed to obtain coronal images of cerebral blood flow (CBF) at the level of the bregma, to confirm successful MCA occlusion and CBF reduction.
BMMC extraction The femur bone was extracted from donor animals (n = 68) and bone marrow was obtained as described previously (Kamiya et al., 2008, 2013) and then heparinized. BMMCs were isolated by densitygradient centrifugation with Ficoll-Paque (Iihosi et al., 2004; Kamiya et al., 2008, 2013). The fraction containing BMMCs was stained using Turk's solution and trypan blue, and cells were microscopically counted using a Burker-Turk counting chamber. BMMCs (1 × 107) were then diluted in 1 ml phosphate-buffered saline (PBS) and stored on ice until transplantation. Because 1 × 107 of the BMMCs were sufficient for neuroprotection (Chen et al., 2001; Yang et al., 2011), this dose was prepared for all transplantation experiments in this study.
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Measurement of infarct volume To measure infarct volume, animals in experiment-1 were deeply anesthetized using 5% halothane, and were decapitated at 24 h after reperfusion (n = 8). The brains were carefully removed and cut into 2 mm-thick coronal sections. The slices were stained with 2,3,5triphenyltetra-zoliumchloride (TTC). The animals in experiment-2 were deeply anesthetized using 5% halothane and were perfusionfixed with 4% paraformaldehyde followed by a brief flush using heparinized saline at 24 or 72 h after reperfusion (n = 6). The brains were carefully removed and stored in 4% paraformaldehyde overnight, and 20-μm-thick coronal frozen sections were cut at 2 mm intervals on a cryostat. Each section was mounted on a poly-L-lysine coated slide glass, and stained with hematoxylin and eosin (HE). Infarct volumes were calculated by adding the total infarct areas on TTC or HE-stained sections using Image J software version 1.42q (NIH, Maryland, USA). Evaluation of motor function To evaluate motor function, neurological deficit scores were evaluated at 24 or 72 h after reperfusion using a five-point scale as previously described (Ren et al., 2011): 0: no neurological deficit, 1: failure to fully extend the right forepaw, 2: circling to the right, 3: falling to the right, and 4: unable to walk spontaneously. Immunohistochemistry Immunohistochemistry was performed using coronal frozen brain sections from experiment-2. Briefly, the sample sections were incubated with 10% goat serum in PBS to block nonspecific reactions, followed by incubation with rabbit polyclonal antibodies against ionized calcium-binding adapter molecule 1 (Iba-1) as a marker of activated microglia (1:500, Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse monoclonal antibody against 4-hydroxy-2-nonenal (4-HNE) as a marker of lipid peroxidation (1:50, Japan Institute for the Control of Aging, Shizuoka, Japan) or mouse monoclonal antibody against 8hydroxydeoxyguanosine (8-OHdG) as a marker of oxidative DNA damage (1:50, Japan Institute for the Control of Aging, Shizuoka, Japan) overnight at 4 °C. Then, the sections were processed with biotinylated goat anti-rabbit IgG or anti-mouse IgG (Vector Laboratories, CA, USA) at room temperature for 1 h, followed by avidin–biotin–peroxidase complex (Vector Laboratories) for 30 min. The labeled secondary antibodies were visualized using diaminobenzidine. Each process was followed by several brief washes with PBS. The Iba-1 positive cells in the cortical ischemic boundary zone were counted at three randomly chosen square fields (0.4 mm2).
BMMC transplantation
Statistical analysis
A PE-50 catheter was inserted into the left femoral vein of each recipient rat. In experiment-1, allogeneic BMMCs in PBS (1 × 107 cells in 1 ml of PBS) or vehicle (1 ml of PBS) were intravenously transplanted through the catheter at 0, 3 or 6 h after reperfusion (each n = 8). In addition, a high dose of allogeneic BMMCs (2 × 107 cells in 1 ml of PBS) were also transplanted at 6 h after reperfusion to confirm dose-dependent effects (n = 8). In experiment-2, another set of animals, administered 1 × 107 BMMCs or vehicle at 6 h after reperfusion, received additional BMMC transplantation of the same dose or vehicle at 9 h after reperfusion (each n = 6). Vehicle-treated animals in experiment-2 were administered 1 ml of PBS instead of BMMCs. A preliminary study showed the effects of transplantation on neuroprotection were equal between autologous and allogeneic BMMCs (Kamiya et al., 2011), thus allogeneic transplantation was used in the present study because the repeated transplantation in experiment-2 needs more BMMCs and too much autologous BMMC extraction is so invasive to the recipient rats.
Statistical analysis was performed using Statview version 5.0 software (SAS Institute, CA, USA). An analysis of variance (ANOVA) followed by Scheffe's post-hoc test was performed for comparisons of infarct volumes and immunohistochemical cell counts, and data were presented as mean ± standard deviations (SDs). A Mann–Whitney U-test was used to compare neurological deficit scores, and data were expressed as median and interquartile range. Statistical significance was set at p b 0.05. Results Therapeutic time window of allogeneic BMMC transplantation Infarct volumes assessed by TTC-stained sections at 24 h after reperfusion in the vehicle and single transplantation groups at 0, 3 and 6 h after reperfusion were 262 ± 81, 49.0 ± 7.7, 107 ± 39, and 195 ± 96 mm3, respectively. Infarct volumes were significantly decreased in the single
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transplantation groups at 0 (p b 0.001) and 3 h (p = 0.002), but not at 6 h (p = 0.13), compared with the vehicle group (Fig. 1A). Infarct volume in the high dose transplantation group at 6 h after reperfusion was 165 ± 51 mm3, without statistical significance compared with the vehicle group (p = 0.76) (Fig. 1A). Neurological deficit scores at 24 h after reperfusion in the vehicle group and single transplantation group at 0, 3 and 6 h after reperfusion were 2.5 (2–3), 1.0 (0.5–1.0), 1 (1–2) and 2 (2–3), respectively. Neurological deficit scores were significantly improved in the single transplantation group at 0 h (p b 0.05), but not at 3 (p = 0.052) and 6 h (p = 0.71) after reperfusion, compared with the vehicle group (Fig. 1B). The neurological deficit score in the high dose transplantation group at 6 h after reperfusion was 2.0 (1.5–2.5), without statistical significance compared with the vehicle group (p = 0.34) (Fig. 1B). This indicated that the effective therapeutic time window of allogeneic BMMC transplantation was at least 3 h and less than 6 h after reperfusion, and that even a high dose of allogeneic BMMC transplantation at 6 h after reperfusion was ineffective. Repeated transplantation and increased neuroprotection Fig. 2A shows representative HE-stained brain sections at 24 h after reperfusion. Infarct volumes assessed by HE-stained sections at 24 h after reperfusion in the vehicle and single transplantation groups at
6 h and repeated transplantation group at 6 and 9 h after reperfusion were 142 ± 32, 100 ± 27, and 56 ± 17 mm3, respectively (Fig. 2B). Infarct volumes at 24 h were not decreased in the single transplantation group (p = 0.07) and significantly decreased in the repeated transplantation group (p = 0.0008) compared with the vehicle group (Fig. 2B). Infarct volumes at 72 h after reperfusion in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 6 and 9 h after reperfusion were 110 ± 32, 75 ± 18, and 36 ± 8.0 mm3, respectively (Fig. 2B). Infarct volumes at 72 h were not decreased in the single transplantation group (p = 0.07) but were significantly decreased in the repeated transplantation group (p = 0.0006) compared with the vehicle group (Fig. 2B). Neurological deficit scores at 24 h in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 6 and 9 h were 2.5 (2–3), 2 (2–3) and 2 (1–2), respectively (Fig. 2C). Neurological deficit scores at 72 h in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 6 and 9 h were 3 (2–3), 2 (2) and 2 (1–2), respectively (Fig. 2C). Neurological deficit scores at 24 h after reperfusion were significantly improved in the repeated transplantation group (p = 0.036), but not in the single transplantation group (p = 0.71), compared with the vehicle group (Fig. 2C). Neurological deficit scores at 72 h after reperfusion were also significantly improved in the repeated transplantation group (p = 0.047), but not in the single transplantation group (p = 0.076), compared with the vehicle group (Fig. 2C). Immunohistochemistry
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BMMC Fig. 1. Therapeutic time window of allogeneic BMMC transplantation following transient focal cerebral ischemia. (A) Infarct volumes assessed by TTC-stained sections at 24 h after reperfusion were significantly decreased in the transplantation group at 0 and 3 h, but not at 6 h, after reperfusion, compared with vehicle-treatment (each n = 8). Infarct volume in the high dose transplantation group at 6 h after reperfusion was not statistically different from that in the vehicle group. *Significant difference from vehicle-treatment by ANOVA with Scheffe's post-hoc test (p b 0.05). (B) Neurological scores at 24 h were significantly decreased in the transplantation group at 0, but not at 3 and 6 h, after reperfusion, compared with vehicle-treatment (each n = 8). *Significant difference from vehicle-treatment by Mann–Whitney U-test (p b 0.05). Box plots indicate the median and interquartile range, and whiskers indicate the 10th and 90th percentiles.
Fig. 3A shows representative microphotographs of immunohistochemistry using specific antibodies against 4-HNE, 8-OHdG or Iba-1 at 24 and 72 h after reperfusion. Animals treated with vehicle or single BMMC transplantation at 6 h after reperfusion contained a large amount of Iba-1 labeled activated microglial cells, 4-HNE positive cells and 8-OHdG positive cells in the cortical ischemic boundary zone at 24 and 72 h after reperfusion, while repeated BMMC transplantation reduced the immunohistochemical expression of these cell types. Fig. 3B shows the number of Iba-1, 4-HNE and 8-OHdG positive cells at 24 h after reperfusion. The numbers of Iba-1 positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 95 ± 11, 67 ± 16, and 43 ± 16, respectively (Fig. 3B). The numbers of 4-HNE positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 133 ± 20, 110 ± 20, and 90 ± 14, respectively (Fig. 3B). The numbers of 8-OHdG positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 102 ± 23, 77 ± 19, and 70 ± 13, respectively (Fig. 3B). Fig. 3C shows the number of Iba-1, 4-HNE and 8-OHdG positive cells at 72 h after reperfusion. The numbers of Iba-1 positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 72 h after reperfusion were 97 ± 22, 69 ± 15, and 47 ± 11, respectively (Fig. 3C). The numbers of 4-HNE positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 122 ± 24, 91 ± 20, and 89 ± 8.0, respectively (Fig. 3C). The numbers of 8-OHdG positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 95 ± 13, 89 ± 10, and 75 ± 8.8, respectively (Fig. 3C). The number of Iba-1 positive cells at 24 h after reperfusion was significantly reduced in the repeated transplantation group (p = 0.012), but not in the single transplantation group (p = 0.063), compared with the vehicle group (Fig. 3B). The number of 4-HNE positive cells at 24 h after reperfusion was reduced in the repeated transplantation group (p = 0.010), but not in the single transplantation group (p = 0.12), compared with the vehicle group (Fig. 3B). The number of 8-OHdG positive cells at 24 h after reperfusion was reduced in the repeated transplantation group (p = 0.050), but not in the single transplantation group (p = 0.16), compared with the vehicle group
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Fig. 2. Repeated BMMC transplantations and increased neuroprotection. (A) Representative microphotographs of HE-stained brain sections in animals receiving vehicle, single BMMC transplantation (6 h) and repeated BMMC transplantation (6 and 9 h) at 24 h after reperfusion. Note small infarct areas in animals with repeated transplantations. (B) Infarct volumes assessed by HE-stained brain sections at 24 and 72 h after reperfusion were not significantly decreased in the transplantation group at 6 h after reperfusion, compared with vehicletreatment. Repeated transplantations at 6 and 9 h showed a significant reduction in infarct volumes (each n = 6). *Significant difference from vehicle-treatment by ANOVA with Scheffe's post-hoc test (p b 0.05). (C) Neurological scores at 24 and 72 h after reperfusion were not statistically different between animals receiving a single transplantation (6 h) or vehicle. Repeated transplantations at 6 and 9 h improved neurological deficit scores (each n = 6). *Significant difference from vehicle-treated rats by Mann–Whitney U-test (p b 0.05). Box plots indicate the median and interquartile range, and whiskers indicate the 10th and 90th percentiles.
(Fig. 3B). The number of Iba-1 positive cells at 72 h after reperfusion was reduced in the repeated transplantation group (p = 0.018), but not in the single transplantation group (p = 0.067), compared with the vehicle group (Fig. 3C). The number of 4-HNE positive cells at 72 h
after reperfusion was reduced in the repeated transplantation group (p = 0.035), but not in the single transplantation group (p = 0.71), compared with the vehicle group (Fig. 3C). The number of 8-OHdG positive cells at 72 h after reperfusion was reduced in the repeated
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Fig. 3. Immunohistochemical analysis. (A) Representative microphotographs of immunohistochemistry using antibodies against ionized calcium-binding adapter molecule 1 (Iba-1), 4hydroxy-2-nonenal (4-HNE) and 8-hydroxydeoxyguanoside (8-OHdG) at 24 and 72 h after reperfusion. Note decreased Iba-1, 4-HNE and 8-OHdG expressions in repeated transplantation animals. Scale bars indicate 25 μm. (B) Numbers of immunohistochemistry stained cells at 24 h after reperfusion. Labeled cells were not statistically different in number between single transplantation (6 h) and vehicle-treated groups (each n = 5). Repeated transplantations at 6 and 9 h had significantly reduced numbers of Iba-1, 4-HNE and 8-OHdG positive cells. *Significant difference from vehicle-treatment by ANOVA with Scheffe's post-hoc test (p b 0.05). (C) Numbers of immunohistochemistry stained cells at 72 h after reperfusion (each n = 5). Labeled cells were not statistically different in number between single transplantation (6 h) and vehicle-treated groups. Repeated transplantations at 6 and 9 h significantly reduced the numbers of Iba-1, 4-HNE and 8-OHdG positive cells. *Significant difference from vehicle-treatment by ANOVA with Scheffe's post-hoc test (p b 0.05).
transplantation group (p = 0.045), but not in the single transplantation group (p = 0.71), compared with the vehicle group (Fig. 3C). Discussion Stem cell therapy holds great promise in medicine and experimental data suggests it will have benefit in many clinical applications (Lees et al., 2012; Savitz et al., 2011). The effects of transplantation of different cell types, such as BMSCs, BMMCs, endothelial progenitor cells, umbilical cord stem cells, adipose stem cells and neural stem cells, have been investigated in rat models of stroke (Ohta et al., 2006; Savitz et al.,
2004; Bliss et al., 2007; Kawabori et al., 2013). Transplantation of either BMMCs or BMSCs was reported to have therapeutic effects in rat brain ischemia and spinal cord injury models (Shichinohe et al., 2010; Iihosi et al., 2004). Preparation of BMSCs requires a certain amount of time to culture the cells before transplantation, whereas BMMCs can be collected just prior to administration. Thus, the use of BMMCs might be absolutely advantageous in the treatment of acute phase transplantation. Several routes of administration to administer BMMCs in the ischemic stroke model have been reported, including intra-arterial, intravenous and intra-cerebral transplantations (Kawabori et al., 2011). Intravenous administration is the most non-invasive method and may be suitable for
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repeated transplantation. Therefore, we chose intravenous BMMC transplantation in this study. In the present study, infarct volumes assessed by TTC-stained sections were significantly smaller in the single transplantation groups (1 × 107 cells) at 0 and 3 h, but not at 6 h, after reperfusion, compared with the vehicle group. In addition, the mean infarct volume was smallest in the group that received 1 × 107 BMMC transplantation just after reperfusion, and delayed transplantation resulted in larger infarct volumes. The neurological deficit score was significantly improved only in the single transplantation group (1 × 107 cells) at 0 h after reperfusion, compared with the vehicle group. This indicated that earlier transplantation had greater neuroprotective potential in a rat model of transient focal cerebral ischemia, and that the effective therapeutic time window of early BMMC transplantation was at least 3 h but less than 6 h after reperfusion in this model. Earlier transplantation reportedly resulted in greater beneficial outcomes in the acute phase of cerebral ischemia (Iihosi et al., 2004; de Vasconcelos Dos Santos et al., 2010; Yang et al., 2011). However, BMMC administration immediately after reperfusion may not necessarily lead to the best period for transplantation. Early BMMC transplantation may be critical for the effects of antiinflammation and neuroprotection (Yoshihara et al., 2007; Bliss et al., 2007), while delayed BMMC transplantation may be involved in angiogenesis and neurogenesis after ischemia (Taguchi et al., 2004; NakanoDoi et al., 2010). In the present study, infarct volumes assessed by HE-stained sections were significantly smaller in the repeated transplantation group (6 and 9 h after reperfusion, each 1 × 107 BMMCs), but not in the single transplantation group (6 h after reperfusion, 1 × 107 BMMCs), compared with the vehicle group. In addition, neurological deficit scores were significantly improved in the repeated transplantation group, but not in the single transplantation group, compared with the vehicle group. To date, the effects of repeated BMMC transplantation on neuroprotection have not been investigated. Shehadah et al. has reported that multiple injections of human umbilical tissue-derived cells were not superior to a single injection in rat model of transient focal cerebral ischemia (Shehadah et al., 2013). However, the transplantations were initiated from day 1 after reperfusion in that study (Shehadah et al., 2013). In addition, Omori et al. showed that single high dose transplantation of human BMSCs was more beneficial than multiple transplantations in rat model of permanent focal cerebral ischemia (Omori et al., 2008). However, the transplantations were performed at 6, 24 and 48 h after ischemia (Omori et al., 2008). The animal models and transplanted cell sources in these studies were different from those in the present study. Therefore, previous negative data of multiple transplantations from these studies may not be directly comparable to the effects of repeated BMMC transplantations at a relatively acute stage in the present study. Because early BMMC transplantation is important for anti-inflammatory effects (Yoshihara et al., 2007; Bliss et al., 2007), repeated BMMC transplantations with relatively short intervals in the acute stage until 72 h after reperfusion might have therapeutic benefit. To confirm long lasting protective effects, the therapeutic effects of transplantation should be evaluated for a sufficiently long duration in future studies. Furthermore, repeated transplantation might reinforce the neuroprotective effect induced by endocrine secretion and prolong the duration of effect. Yang et al., reported that a single infusion of 1 × 107 cells and 3 × 107 cells at 24 h after stroke insult was equally beneficial (Yang et al., 2011), suggesting the presence of a plateau dose of BMMCs in a single infusion. Indeed, we confirmed low (1 × 107 cells) and high (2 × 107 cells) doses of BMMCs were equally ineffective at 6 h after reperfusion in the present study. Therefore, the protective effects of repeated transplantation at 6 and 9 h after reperfusion in the study were not dose-dependent. Van der Bogt et al. observed that intravenously transplanted BMMCs migrated to the injured area, also to the liver, spleen and bone marrow using in vivo bioluminescence imaging (van der Bogt et al., 2012). Furthermore, we previously confirmed that intravenously transplanted PKH26-labeled BMMCs were
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observed within the ischemic territory (Kamiya et al., 2008). Therefore, transplanted BMMCs might migrate to the infarct lesion in the present experiments. An intriguing potential repair mechanism is the ability of transplanted cells to attenuate stroke-induced inflammatory and/or immune responses (Bliss et al., 2007). The present study showed a reduction in the number of Iba-1, 4-HNE and 8-OHdG positive cells within the ischemic brain of the repeated transplantation group. Reduced Iba-1 expression indicates suppression of microglial activation, and suggests a reduction of local inflammation to the neural cells (Zhang et al., 2005). Since 4-HNE is used as an index of lipid peroxidation induced by free radicals after ischemic injury (Yoshino et al., 1997; Dalle-Donne et al., 2006), reduced 4-HNE expression is considered to result from the suppression of lipid peroxidation. In addition, reduced 8-OHdG accumulation might be caused by the suppression of free radical-induced oxidative DNA damage (Dalle-Donne et al., 2006). In a rat transient focal cerebral model, burst-like production of free radicals occurred after reperfusion (Peters et al., 1998), and contributed to ischemic neuronal cell death (Kitagawa et al., 1990). Therefore, repeated BMMC transplantation may reduce tissue oxidative stress following ischemia–reperfusion in this model. Conclusion The present study established the neuroprotective effects of intravenous administration of allogeneic BMMCs following transient focal cerebral ischemia, and demonstrated the therapeutic time window of early BMMC transplantation was at least 3 h and less than 6 h after reperfusion. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in the ischemic brain, resulting in greater neuroprotection. Conflict of interest statement No competing interest.
Acknowledgments This study was supported in part by a Grant-in-Aid for Scientific Research (c) 20591011 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Moeko Saito for technical assistance. References Bliss T, Guzman R, Daadi M, Steinberg GK. Cell transplantation therapy for stroke. Stroke 2007;38:817–26. Chen J, Li Y, Wang L, Zhang Z, Lu D, Lu M, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 2001;32: 1005–11. Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A. Biomarkers of oxidative damage in human disease. Clin Chem 2006;52:601–23. de Vasconcelos Dos Santos A, da Costa Reis J, Diaz Paredes B, Moraes L, Jasmin Giraldi-Guimarães A, Mendez-Otero R. Therapeutic window for treatment of cortical ischemia with bone marrow-derived cells in rats. Brain Res 2010;1306:149–58. Ewing JR, Wei L, Knight RA, Pawa S, Nagaraja TN, Brusca T, et al. Direct comparison of local cerebral blood flow rates measured by MRI arterial spin-tagging and quantitative autoradiography in a rat model of experimental cerebral ischemia. J Cereb Blood Flow Metab 2003;23:198–209. Iihosi S, Honmou O, Houkin K, Hashi K, Kocsis JD. A therapeutic window for intravenous administration of autologous bone marrow after cerebral ischemia in adult rats. Brain Res 2004;1007:1–9. Kamiya N, Ueda M, Igarashi H, Nishiyama Y, Suda S, Inaba T, et al. Intra-arterial transplantation of bone marrow mononuclear cells immediately after reperfusion decreases brain injury after focal ischemia in rats. Life Sci 2008;83:433–7. Kamiya F, Ueda M, Nito C, Kamiya N, Inaba T, Suda S, et al. Transplantation of allogeneic bone marrow mononuclear cells ameliorates brain injury following transient focal ischemia in rats. Program No. 193. Conference Abstracts of XXVth International Symposium on Cerebral Blood Flow, Metabolism and Function [CD-ROM]. Barcelona, Spain: International Society for Cerebral Blood Flow, Metabolism and Function; 2011. Kamiya N, Ueda M, Igarashi H, Nishiyama Y, Suda S, Okubo S, et al. In vivo monitoring of arterially transplanted bone marrow mononuclear cells in a rat transient focal brain ischemia model using magnetic resonance imaging. Neurol Res 2013;35:573–9.
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Kawabori M, Kuroda S, Sugiyama T, Ito M, Shichinohe H, Houkin K, et al. Intracerebral, but not intravenous, transplantation of bone marrow stromal cells enhances functional recovery in rat cerebral infarct: an optical imaging study. Neuropathology 2011;32: 217–26. Kawabori M, Kuroda S, Ito M, Shichinohe H, Houkin K, Kuge Y, et al. Timing and cell dose determine therapeutic effects of bone marrow stromal cell transplantation in rat model of cerebral infarct. Neuropathology 2013;33:140–8. Kitagawa K, Matsumoto M, Oda T, Niinobe M, Hata R, Handa N, et al. Free radical generation during brief period of cerebral ischemia may trigger delayed neuronal death. Neuroscience 1990;35:551–8. Lees JS, Sena ES, Egan KJ, Antonic A, Koblar SA, Howells DW, et al. Stem cell-based therapy for experimental stroke: a systematic review and meta-analysis. Int J Stroke 2012;7:582–8. Nakano-Doi A, Nakagomi T, Fujikawa M, Nakagomi N, Kubo S, Lu S, et al. Bone marrow mononuclear cells promote proliferation of endogenous neural stem cells through vascular niches after cerebral infarction. Stem Cells 2010;28:1292–302. Nito C, Ueda M, Inaba T, Katsura K, Katayama Y. FK506 ameliorates oxidative damage and protects rat brain following transient focal cerebral ischemia. Neurol Res 2011;33:881–9. Ohta T, Kikuta K, Imamura H, Takagi Y, Nishimura M, Arakawa Y, et al. Administration of ex vivo-expanded bone marrow-derived endothelial progenitor cells attenuates focal cerebral ischemia–reperfusion injury in rats. Neurosurgery 2006;59:679–86. Omori Y, Honmou O, Harada K, Suzuki J, Houkin K, Kocsis JD. Optimization of a therapeutic protocol for intravenous injection of human mesenchymal stem cells after cerebral ischemia in adult rats. Brain Res 2008;1236:30–8. Peters O, Back T, Lindauer U, Busch C, Megow D, Dreier J, et al. Increased formation of reactive oxygen species after permanent and reversible middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 1998;18:196–205. Ren X, Akiyoshi K, Dziennis S, Vandenbark AA, Herson PS, Hurn PD, et al. Regulatory B cells limit CNS inflammation and neurologic deficits in murine experimental stroke. J Neurosci 2011;31:8556–63. Savitz SI, Dinsmore JH, Wechsler LR, Rosenbaum DM, Caplan LR. Cell therapy for stroke. NeuroRx 2004;1:406–14. Savitz SI, Misra V, Kasam M, Juneja H, Cox Jr CS, Alderman S, et al. Intravenous autologous bone marrow mononuclear cells for ischemic stroke. Ann Neurol 2011;70:59–69.
Shehadah A, Chen J, Kramer B, Zacharek A, Cui Y, Roberts C, et al. Efficacy of single and multiple injections of human umbilical tissue-derived cells following experimental stroke in rats. PLoS One 2013;8:e54083. Shichinohe H, Kuroda S, Maruichi K, Osanai T, Sugiyama T, Chiba Y, et al. Bone marrow stromal cells and bone marrow-derived mononuclear cells: which are suitable as cell source of transplantation for mice infarct brain? Neuropathology 2010;30: 113–22. Suda S, Shimazaki K, Ueda M, Inaba T, Kamiya N, Katsura K, et al. Combination therapy with bone marrow stromal cells and FK506 enhanced amelioration of ischemic brain damage in rats. Life Sci 2011;89:50–6. Taguchi A, Soma T, Tanaka H, Kanda T, Nishimura H, Yoshikawa H, et al. Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesis in a mouse model. J Clin Invest 2004;114:330–8. Ueda M, Inaba T, Nito C, Kamiya N, Katayama Y. Therapeutic impact of eicosapentaenoic acid on ischemic brain damage following transient focal cerebral ischemia in rats. Brain Res 2013;1519:95–104. van der Bogt KE, Hellingman AA, Lijkwan MA, Bos EJ, de Vries MR, van Rappard JR, et al. Molecular imaging of bone marrow mononuclear cell survival and homing in murine peripheral artery disease. JACC Cardiovasc Imaging 2012;5:46–55. Williams DS, Detre JA, Leigh JS, Koretsky AP. Magnetic resonance imaging of perfusion using spin inversion of arterial water. Proc Natl Acad Sci U S A 1992;89:212–6. Yang B, Strong R, Sharma S, Brenneman M, Mallikarjunarao K, Xi X, et al. Therapeutic time window and dose response of autologous bone marrow mononuclear cells for ischemic stroke. J Neurosci Res 2011;89:833–9. Yoshihara T, Ohta M, Itokazu Y, Matsumoto N, Dezawa M, Suzuki Y, et al. Neuroprotective effect of bone marrow-derived mononuclear cells promoting functional recovery from spinal cord injury. J Neurotrauma 2007;24:1026–36. Yoshino H, Hattori N, Urabe T, Uchida K, Tanaka M, Mizuno Y. Postischemic accumulation of lipid peroxidation products in the rat brain: immunohistochemical detection of 4-hydroxy-2-nonenal modified proteins. Brain Res 1997;767:81–6. Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T. Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 2005;36:2220–5.
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Effect of repeated allogeneic bone marrow mononuclear cell transplantation on brain injury following transient focal cerebral ischemia in rats Fumio Kamiya ⁎, Masayuki Ueda ⁎, Chikako Nito, Nobuo Kamiya, Toshiki Inaba, Satoshi Suda, Tomonari Saito, Kanako Muraga, Yasuo Katayama Department of Neurological Sciences, Graduate School of Medicine, Nippon Medical School, Japan
a r t i c l e
i n f o
Article history: Received 5 September 2013 Accepted 11 December 2013 Keywords: Bone marrow Cerebral ischemia Neuroprotection Allogeneic transplantation
a b s t r a c t Aims: Transplantation of bone marrow mononuclear cells (BMMCs) exerts neuroprotection against cerebral ischemia. We examined the therapeutic timepoint of allogeneic BMMC transplantation in a rat model of focal cerebral ischemia, and determined the effects of repeated transplantation outside the therapeutic window. Main methods: Male Sprague–Dawley rats were subjected to 90 minute focal cerebral ischemia, followed by intravenous administration of 1 × 107 allogeneic BMMCs or vehicle at 0, 3 or 6 h after reperfusion or 2 × 107 BMMCs 6 h after reperfusion. Other rats administered 1 × 107 BMMCs at 6 h after reperfusion received additional BMMC transplantation or vehicle 9 h after reperfusion. Infarct volumes, neurological deficit scores and immunohistochemistry were evaluated 24 or 72 h after reperfusion. Key findings: Infarct volumes at 24 h were significantly decreased in transplantation rats at 0 and 3 h, but not at 6 h, after reperfusion, compared to vehicle-treatment. Even high dose BMMC transplantation at 6 h after reperfusion was ineffective. Repeated BMMC transplantation at 6 and 9 h after reperfusion reduced infarct volumes and significantly improved neurological deficit scores at 24 and 72 h. Immunohistochemistry showed repeated BMMC transplantation reduced ionized calcium-binding adapter molecule 1, 4-hydroxy-2-nonenal and 8hydroxydeoxyguanosine expression at 24 and 72 h after reperfusion. Significance: Intravenous allogeneic BMMCs were neuroprotective following transient focal cerebral ischemia, and the therapeutic time window of BMMC transplantation was N3 h and b6 h after reperfusion in this model. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in ischemic brains, resulting in improved neuroprotection. © 2013 Elsevier Inc. All rights reserved.
Introduction Transplantation of bone marrow cells reportedly exerts neuroprotection against cerebral ischemia (Chen et al., 2001; Bliss et al., 2007). We previously showed that either transplantation of autologous bone marrow mononuclear cells (BMMCs) or bone marrow stromal cells (BMSCs) reduced infarct volume following transient focal cerebral ischemia in rats (Kamiya et al., 2008; Suda et al., 2011). However, it is difficult to prepare sufficient numbers of autologous BMMCs for transplantation at the acute phase of stroke, because collection of BMMCs requires surgical procedures. In addition, BMSCs also require enough time to culture cells, thus adding to the difficulty of preparing BMSCs at the acute phase of stroke. Moreover, we confirmed “allogeneic” and “autologous” BMMC transplantations were equally neuroprotective in a rat stroke
model (Kamiya et al., 2011). Therefore, allogeneic BMMCs may be a therapeutic option for immediate transplantation or repeated transplantation. The benefit of cell transfusion therapy for the acute phase of cerebral ischemia includes attenuation of inflammation and inhibition of antioxidant processes (Bliss et al., 2007; Yang et al., 2011). The present study was designed to determine the therapeutic time window of allogeneic BMMC transplantation in a rat model of transient focal cerebral ischemia, and to examine whether repeated BMMC transplantations outside the therapeutic time window achieved further neuroprotection. In addition, the mechanisms of neuroprotection were investigated in relation to inflammation and oxidative stress. Materials and methods Ischemia model
⁎ Corresponding authors at: Department of Neurological Sciences, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Tel.: +81 3 3822 2131; fax: +81 3 3822 4865. E-mail addresses: [email protected] (F. Kamiya), [email protected] (M. Ueda). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.12.016
All experimental procedures were approved by the institutional guidelines for animal use and care. A total of 134 male Sprague–Dawley rats, weighing 250 to 300 g, were used in this study. Anesthesia was
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performed with 5% halothane and maintained with 0.5 to 1.0% halothane in 70% N2O and 30% O2 mixture during all surgical procedures. The rats were subjected to transient focal cerebral ischemia for 90 min using an intraluminal suture technique (Ueda et al., 2013; Nito et al., 2011; Suda et al., 2011). Briefly, the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) on the right side were carefully exposed via a midline cervical incision. The ECA and the CCA were double-ligated using a silk suture. A silicon rubber-coated round-tip nylon surgical thread was inserted into the ICA (approximately 18 mm from the bifurcation) via a small puncture in the CCA to occlude the origin of the middle cerebral artery (MCA). A silk suture around the CCA was tightened to prevent bleeding from the puncture site. After 90 min of MCA occlusion, reperfusion was performed by withdrawing the suture. A PE-50 catheter was inserted into the tail artery for blood sampling and arterial blood pressure monitoring to confirm that physiological variables were within normal limits. Rectal temperature was maintained at 37.0 ± 0.5 °C during the procedure.
Magnetic resonance imaging Magnetic resonance imaging (MRI) analyses were performed using Unity-INOVA-300 system with a 7 T/18 cm horizontal magnet (Agilent Technologies., CA, USA), as described previously (Ueda et al., 2013; Nito et al., 2011). The continuous arterial spin labeling method (Williams et al., 1992), modified with a centrally encoded variable-tip-angle gradient echo technique (Ewing et al., 2003), was performed to obtain coronal images of cerebral blood flow (CBF) at the level of the bregma, to confirm successful MCA occlusion and CBF reduction.
BMMC extraction The femur bone was extracted from donor animals (n = 68) and bone marrow was obtained as described previously (Kamiya et al., 2008, 2013) and then heparinized. BMMCs were isolated by densitygradient centrifugation with Ficoll-Paque (Iihosi et al., 2004; Kamiya et al., 2008, 2013). The fraction containing BMMCs was stained using Turk's solution and trypan blue, and cells were microscopically counted using a Burker-Turk counting chamber. BMMCs (1 × 107) were then diluted in 1 ml phosphate-buffered saline (PBS) and stored on ice until transplantation. Because 1 × 107 of the BMMCs were sufficient for neuroprotection (Chen et al., 2001; Yang et al., 2011), this dose was prepared for all transplantation experiments in this study.
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Measurement of infarct volume To measure infarct volume, animals in experiment-1 were deeply anesthetized using 5% halothane, and were decapitated at 24 h after reperfusion (n = 8). The brains were carefully removed and cut into 2 mm-thick coronal sections. The slices were stained with 2,3,5triphenyltetra-zoliumchloride (TTC). The animals in experiment-2 were deeply anesthetized using 5% halothane and were perfusionfixed with 4% paraformaldehyde followed by a brief flush using heparinized saline at 24 or 72 h after reperfusion (n = 6). The brains were carefully removed and stored in 4% paraformaldehyde overnight, and 20-μm-thick coronal frozen sections were cut at 2 mm intervals on a cryostat. Each section was mounted on a poly-L-lysine coated slide glass, and stained with hematoxylin and eosin (HE). Infarct volumes were calculated by adding the total infarct areas on TTC or HE-stained sections using Image J software version 1.42q (NIH, Maryland, USA). Evaluation of motor function To evaluate motor function, neurological deficit scores were evaluated at 24 or 72 h after reperfusion using a five-point scale as previously described (Ren et al., 2011): 0: no neurological deficit, 1: failure to fully extend the right forepaw, 2: circling to the right, 3: falling to the right, and 4: unable to walk spontaneously. Immunohistochemistry Immunohistochemistry was performed using coronal frozen brain sections from experiment-2. Briefly, the sample sections were incubated with 10% goat serum in PBS to block nonspecific reactions, followed by incubation with rabbit polyclonal antibodies against ionized calcium-binding adapter molecule 1 (Iba-1) as a marker of activated microglia (1:500, Wako Pure Chemical Industries, Ltd., Osaka, Japan), mouse monoclonal antibody against 4-hydroxy-2-nonenal (4-HNE) as a marker of lipid peroxidation (1:50, Japan Institute for the Control of Aging, Shizuoka, Japan) or mouse monoclonal antibody against 8hydroxydeoxyguanosine (8-OHdG) as a marker of oxidative DNA damage (1:50, Japan Institute for the Control of Aging, Shizuoka, Japan) overnight at 4 °C. Then, the sections were processed with biotinylated goat anti-rabbit IgG or anti-mouse IgG (Vector Laboratories, CA, USA) at room temperature for 1 h, followed by avidin–biotin–peroxidase complex (Vector Laboratories) for 30 min. The labeled secondary antibodies were visualized using diaminobenzidine. Each process was followed by several brief washes with PBS. The Iba-1 positive cells in the cortical ischemic boundary zone were counted at three randomly chosen square fields (0.4 mm2).
BMMC transplantation
Statistical analysis
A PE-50 catheter was inserted into the left femoral vein of each recipient rat. In experiment-1, allogeneic BMMCs in PBS (1 × 107 cells in 1 ml of PBS) or vehicle (1 ml of PBS) were intravenously transplanted through the catheter at 0, 3 or 6 h after reperfusion (each n = 8). In addition, a high dose of allogeneic BMMCs (2 × 107 cells in 1 ml of PBS) were also transplanted at 6 h after reperfusion to confirm dose-dependent effects (n = 8). In experiment-2, another set of animals, administered 1 × 107 BMMCs or vehicle at 6 h after reperfusion, received additional BMMC transplantation of the same dose or vehicle at 9 h after reperfusion (each n = 6). Vehicle-treated animals in experiment-2 were administered 1 ml of PBS instead of BMMCs. A preliminary study showed the effects of transplantation on neuroprotection were equal between autologous and allogeneic BMMCs (Kamiya et al., 2011), thus allogeneic transplantation was used in the present study because the repeated transplantation in experiment-2 needs more BMMCs and too much autologous BMMC extraction is so invasive to the recipient rats.
Statistical analysis was performed using Statview version 5.0 software (SAS Institute, CA, USA). An analysis of variance (ANOVA) followed by Scheffe's post-hoc test was performed for comparisons of infarct volumes and immunohistochemical cell counts, and data were presented as mean ± standard deviations (SDs). A Mann–Whitney U-test was used to compare neurological deficit scores, and data were expressed as median and interquartile range. Statistical significance was set at p b 0.05. Results Therapeutic time window of allogeneic BMMC transplantation Infarct volumes assessed by TTC-stained sections at 24 h after reperfusion in the vehicle and single transplantation groups at 0, 3 and 6 h after reperfusion were 262 ± 81, 49.0 ± 7.7, 107 ± 39, and 195 ± 96 mm3, respectively. Infarct volumes were significantly decreased in the single
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transplantation groups at 0 (p b 0.001) and 3 h (p = 0.002), but not at 6 h (p = 0.13), compared with the vehicle group (Fig. 1A). Infarct volume in the high dose transplantation group at 6 h after reperfusion was 165 ± 51 mm3, without statistical significance compared with the vehicle group (p = 0.76) (Fig. 1A). Neurological deficit scores at 24 h after reperfusion in the vehicle group and single transplantation group at 0, 3 and 6 h after reperfusion were 2.5 (2–3), 1.0 (0.5–1.0), 1 (1–2) and 2 (2–3), respectively. Neurological deficit scores were significantly improved in the single transplantation group at 0 h (p b 0.05), but not at 3 (p = 0.052) and 6 h (p = 0.71) after reperfusion, compared with the vehicle group (Fig. 1B). The neurological deficit score in the high dose transplantation group at 6 h after reperfusion was 2.0 (1.5–2.5), without statistical significance compared with the vehicle group (p = 0.34) (Fig. 1B). This indicated that the effective therapeutic time window of allogeneic BMMC transplantation was at least 3 h and less than 6 h after reperfusion, and that even a high dose of allogeneic BMMC transplantation at 6 h after reperfusion was ineffective. Repeated transplantation and increased neuroprotection Fig. 2A shows representative HE-stained brain sections at 24 h after reperfusion. Infarct volumes assessed by HE-stained sections at 24 h after reperfusion in the vehicle and single transplantation groups at
6 h and repeated transplantation group at 6 and 9 h after reperfusion were 142 ± 32, 100 ± 27, and 56 ± 17 mm3, respectively (Fig. 2B). Infarct volumes at 24 h were not decreased in the single transplantation group (p = 0.07) and significantly decreased in the repeated transplantation group (p = 0.0008) compared with the vehicle group (Fig. 2B). Infarct volumes at 72 h after reperfusion in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 6 and 9 h after reperfusion were 110 ± 32, 75 ± 18, and 36 ± 8.0 mm3, respectively (Fig. 2B). Infarct volumes at 72 h were not decreased in the single transplantation group (p = 0.07) but were significantly decreased in the repeated transplantation group (p = 0.0006) compared with the vehicle group (Fig. 2B). Neurological deficit scores at 24 h in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 6 and 9 h were 2.5 (2–3), 2 (2–3) and 2 (1–2), respectively (Fig. 2C). Neurological deficit scores at 72 h in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 6 and 9 h were 3 (2–3), 2 (2) and 2 (1–2), respectively (Fig. 2C). Neurological deficit scores at 24 h after reperfusion were significantly improved in the repeated transplantation group (p = 0.036), but not in the single transplantation group (p = 0.71), compared with the vehicle group (Fig. 2C). Neurological deficit scores at 72 h after reperfusion were also significantly improved in the repeated transplantation group (p = 0.047), but not in the single transplantation group (p = 0.076), compared with the vehicle group (Fig. 2C). Immunohistochemistry
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BMMC Fig. 1. Therapeutic time window of allogeneic BMMC transplantation following transient focal cerebral ischemia. (A) Infarct volumes assessed by TTC-stained sections at 24 h after reperfusion were significantly decreased in the transplantation group at 0 and 3 h, but not at 6 h, after reperfusion, compared with vehicle-treatment (each n = 8). Infarct volume in the high dose transplantation group at 6 h after reperfusion was not statistically different from that in the vehicle group. *Significant difference from vehicle-treatment by ANOVA with Scheffe's post-hoc test (p b 0.05). (B) Neurological scores at 24 h were significantly decreased in the transplantation group at 0, but not at 3 and 6 h, after reperfusion, compared with vehicle-treatment (each n = 8). *Significant difference from vehicle-treatment by Mann–Whitney U-test (p b 0.05). Box plots indicate the median and interquartile range, and whiskers indicate the 10th and 90th percentiles.
Fig. 3A shows representative microphotographs of immunohistochemistry using specific antibodies against 4-HNE, 8-OHdG or Iba-1 at 24 and 72 h after reperfusion. Animals treated with vehicle or single BMMC transplantation at 6 h after reperfusion contained a large amount of Iba-1 labeled activated microglial cells, 4-HNE positive cells and 8-OHdG positive cells in the cortical ischemic boundary zone at 24 and 72 h after reperfusion, while repeated BMMC transplantation reduced the immunohistochemical expression of these cell types. Fig. 3B shows the number of Iba-1, 4-HNE and 8-OHdG positive cells at 24 h after reperfusion. The numbers of Iba-1 positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 95 ± 11, 67 ± 16, and 43 ± 16, respectively (Fig. 3B). The numbers of 4-HNE positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 133 ± 20, 110 ± 20, and 90 ± 14, respectively (Fig. 3B). The numbers of 8-OHdG positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 102 ± 23, 77 ± 19, and 70 ± 13, respectively (Fig. 3B). Fig. 3C shows the number of Iba-1, 4-HNE and 8-OHdG positive cells at 72 h after reperfusion. The numbers of Iba-1 positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 72 h after reperfusion were 97 ± 22, 69 ± 15, and 47 ± 11, respectively (Fig. 3C). The numbers of 4-HNE positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 122 ± 24, 91 ± 20, and 89 ± 8.0, respectively (Fig. 3C). The numbers of 8-OHdG positive cells in the vehicle and single transplantation groups at 6 h and repeated transplantation group at 24 h after reperfusion were 95 ± 13, 89 ± 10, and 75 ± 8.8, respectively (Fig. 3C). The number of Iba-1 positive cells at 24 h after reperfusion was significantly reduced in the repeated transplantation group (p = 0.012), but not in the single transplantation group (p = 0.063), compared with the vehicle group (Fig. 3B). The number of 4-HNE positive cells at 24 h after reperfusion was reduced in the repeated transplantation group (p = 0.010), but not in the single transplantation group (p = 0.12), compared with the vehicle group (Fig. 3B). The number of 8-OHdG positive cells at 24 h after reperfusion was reduced in the repeated transplantation group (p = 0.050), but not in the single transplantation group (p = 0.16), compared with the vehicle group
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Fig. 2. Repeated BMMC transplantations and increased neuroprotection. (A) Representative microphotographs of HE-stained brain sections in animals receiving vehicle, single BMMC transplantation (6 h) and repeated BMMC transplantation (6 and 9 h) at 24 h after reperfusion. Note small infarct areas in animals with repeated transplantations. (B) Infarct volumes assessed by HE-stained brain sections at 24 and 72 h after reperfusion were not significantly decreased in the transplantation group at 6 h after reperfusion, compared with vehicletreatment. Repeated transplantations at 6 and 9 h showed a significant reduction in infarct volumes (each n = 6). *Significant difference from vehicle-treatment by ANOVA with Scheffe's post-hoc test (p b 0.05). (C) Neurological scores at 24 and 72 h after reperfusion were not statistically different between animals receiving a single transplantation (6 h) or vehicle. Repeated transplantations at 6 and 9 h improved neurological deficit scores (each n = 6). *Significant difference from vehicle-treated rats by Mann–Whitney U-test (p b 0.05). Box plots indicate the median and interquartile range, and whiskers indicate the 10th and 90th percentiles.
(Fig. 3B). The number of Iba-1 positive cells at 72 h after reperfusion was reduced in the repeated transplantation group (p = 0.018), but not in the single transplantation group (p = 0.067), compared with the vehicle group (Fig. 3C). The number of 4-HNE positive cells at 72 h
after reperfusion was reduced in the repeated transplantation group (p = 0.035), but not in the single transplantation group (p = 0.71), compared with the vehicle group (Fig. 3C). The number of 8-OHdG positive cells at 72 h after reperfusion was reduced in the repeated
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Fig. 3. Immunohistochemical analysis. (A) Representative microphotographs of immunohistochemistry using antibodies against ionized calcium-binding adapter molecule 1 (Iba-1), 4hydroxy-2-nonenal (4-HNE) and 8-hydroxydeoxyguanoside (8-OHdG) at 24 and 72 h after reperfusion. Note decreased Iba-1, 4-HNE and 8-OHdG expressions in repeated transplantation animals. Scale bars indicate 25 μm. (B) Numbers of immunohistochemistry stained cells at 24 h after reperfusion. Labeled cells were not statistically different in number between single transplantation (6 h) and vehicle-treated groups (each n = 5). Repeated transplantations at 6 and 9 h had significantly reduced numbers of Iba-1, 4-HNE and 8-OHdG positive cells. *Significant difference from vehicle-treatment by ANOVA with Scheffe's post-hoc test (p b 0.05). (C) Numbers of immunohistochemistry stained cells at 72 h after reperfusion (each n = 5). Labeled cells were not statistically different in number between single transplantation (6 h) and vehicle-treated groups. Repeated transplantations at 6 and 9 h significantly reduced the numbers of Iba-1, 4-HNE and 8-OHdG positive cells. *Significant difference from vehicle-treatment by ANOVA with Scheffe's post-hoc test (p b 0.05).
transplantation group (p = 0.045), but not in the single transplantation group (p = 0.71), compared with the vehicle group (Fig. 3C). Discussion Stem cell therapy holds great promise in medicine and experimental data suggests it will have benefit in many clinical applications (Lees et al., 2012; Savitz et al., 2011). The effects of transplantation of different cell types, such as BMSCs, BMMCs, endothelial progenitor cells, umbilical cord stem cells, adipose stem cells and neural stem cells, have been investigated in rat models of stroke (Ohta et al., 2006; Savitz et al.,
2004; Bliss et al., 2007; Kawabori et al., 2013). Transplantation of either BMMCs or BMSCs was reported to have therapeutic effects in rat brain ischemia and spinal cord injury models (Shichinohe et al., 2010; Iihosi et al., 2004). Preparation of BMSCs requires a certain amount of time to culture the cells before transplantation, whereas BMMCs can be collected just prior to administration. Thus, the use of BMMCs might be absolutely advantageous in the treatment of acute phase transplantation. Several routes of administration to administer BMMCs in the ischemic stroke model have been reported, including intra-arterial, intravenous and intra-cerebral transplantations (Kawabori et al., 2011). Intravenous administration is the most non-invasive method and may be suitable for
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repeated transplantation. Therefore, we chose intravenous BMMC transplantation in this study. In the present study, infarct volumes assessed by TTC-stained sections were significantly smaller in the single transplantation groups (1 × 107 cells) at 0 and 3 h, but not at 6 h, after reperfusion, compared with the vehicle group. In addition, the mean infarct volume was smallest in the group that received 1 × 107 BMMC transplantation just after reperfusion, and delayed transplantation resulted in larger infarct volumes. The neurological deficit score was significantly improved only in the single transplantation group (1 × 107 cells) at 0 h after reperfusion, compared with the vehicle group. This indicated that earlier transplantation had greater neuroprotective potential in a rat model of transient focal cerebral ischemia, and that the effective therapeutic time window of early BMMC transplantation was at least 3 h but less than 6 h after reperfusion in this model. Earlier transplantation reportedly resulted in greater beneficial outcomes in the acute phase of cerebral ischemia (Iihosi et al., 2004; de Vasconcelos Dos Santos et al., 2010; Yang et al., 2011). However, BMMC administration immediately after reperfusion may not necessarily lead to the best period for transplantation. Early BMMC transplantation may be critical for the effects of antiinflammation and neuroprotection (Yoshihara et al., 2007; Bliss et al., 2007), while delayed BMMC transplantation may be involved in angiogenesis and neurogenesis after ischemia (Taguchi et al., 2004; NakanoDoi et al., 2010). In the present study, infarct volumes assessed by HE-stained sections were significantly smaller in the repeated transplantation group (6 and 9 h after reperfusion, each 1 × 107 BMMCs), but not in the single transplantation group (6 h after reperfusion, 1 × 107 BMMCs), compared with the vehicle group. In addition, neurological deficit scores were significantly improved in the repeated transplantation group, but not in the single transplantation group, compared with the vehicle group. To date, the effects of repeated BMMC transplantation on neuroprotection have not been investigated. Shehadah et al. has reported that multiple injections of human umbilical tissue-derived cells were not superior to a single injection in rat model of transient focal cerebral ischemia (Shehadah et al., 2013). However, the transplantations were initiated from day 1 after reperfusion in that study (Shehadah et al., 2013). In addition, Omori et al. showed that single high dose transplantation of human BMSCs was more beneficial than multiple transplantations in rat model of permanent focal cerebral ischemia (Omori et al., 2008). However, the transplantations were performed at 6, 24 and 48 h after ischemia (Omori et al., 2008). The animal models and transplanted cell sources in these studies were different from those in the present study. Therefore, previous negative data of multiple transplantations from these studies may not be directly comparable to the effects of repeated BMMC transplantations at a relatively acute stage in the present study. Because early BMMC transplantation is important for anti-inflammatory effects (Yoshihara et al., 2007; Bliss et al., 2007), repeated BMMC transplantations with relatively short intervals in the acute stage until 72 h after reperfusion might have therapeutic benefit. To confirm long lasting protective effects, the therapeutic effects of transplantation should be evaluated for a sufficiently long duration in future studies. Furthermore, repeated transplantation might reinforce the neuroprotective effect induced by endocrine secretion and prolong the duration of effect. Yang et al., reported that a single infusion of 1 × 107 cells and 3 × 107 cells at 24 h after stroke insult was equally beneficial (Yang et al., 2011), suggesting the presence of a plateau dose of BMMCs in a single infusion. Indeed, we confirmed low (1 × 107 cells) and high (2 × 107 cells) doses of BMMCs were equally ineffective at 6 h after reperfusion in the present study. Therefore, the protective effects of repeated transplantation at 6 and 9 h after reperfusion in the study were not dose-dependent. Van der Bogt et al. observed that intravenously transplanted BMMCs migrated to the injured area, also to the liver, spleen and bone marrow using in vivo bioluminescence imaging (van der Bogt et al., 2012). Furthermore, we previously confirmed that intravenously transplanted PKH26-labeled BMMCs were
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observed within the ischemic territory (Kamiya et al., 2008). Therefore, transplanted BMMCs might migrate to the infarct lesion in the present experiments. An intriguing potential repair mechanism is the ability of transplanted cells to attenuate stroke-induced inflammatory and/or immune responses (Bliss et al., 2007). The present study showed a reduction in the number of Iba-1, 4-HNE and 8-OHdG positive cells within the ischemic brain of the repeated transplantation group. Reduced Iba-1 expression indicates suppression of microglial activation, and suggests a reduction of local inflammation to the neural cells (Zhang et al., 2005). Since 4-HNE is used as an index of lipid peroxidation induced by free radicals after ischemic injury (Yoshino et al., 1997; Dalle-Donne et al., 2006), reduced 4-HNE expression is considered to result from the suppression of lipid peroxidation. In addition, reduced 8-OHdG accumulation might be caused by the suppression of free radical-induced oxidative DNA damage (Dalle-Donne et al., 2006). In a rat transient focal cerebral model, burst-like production of free radicals occurred after reperfusion (Peters et al., 1998), and contributed to ischemic neuronal cell death (Kitagawa et al., 1990). Therefore, repeated BMMC transplantation may reduce tissue oxidative stress following ischemia–reperfusion in this model. Conclusion The present study established the neuroprotective effects of intravenous administration of allogeneic BMMCs following transient focal cerebral ischemia, and demonstrated the therapeutic time window of early BMMC transplantation was at least 3 h and less than 6 h after reperfusion. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in the ischemic brain, resulting in greater neuroprotection. Conflict of interest statement No competing interest.
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