Animal Cells and Systems, 2013 Vol. 17, No. 3, 160169, http://dx.doi.org/10.1080/19768354.2013.805164 Combination effect of p-hydroxybenzyl alcohol and mesenchymal stem cells on the recovery of brain damage in a rat model of brain ischemia a a b b a b Phatcharida Kaengkan , Seung Eun Baek , Yong Won Choi , Kyung-Yoon Kam , Ji Yeong Kim ,Yan Ru Wu , c a Byung-Rok Do and Sung Goo Kang * a b Department of Biological Sciences, Institute of Basic Science, Inje University, Gimhae 621-749, Republic of Korea; Department of Occupational Therapy, Inje University, Gimhae 621-749, Republic of Korea; Bioengineering Institute, Hurim Biocell Inc., Seoul 153- 803, Republic of Korea (Received 13 March 2013; received in revised form 7 May 2013; accepted 9 May 2013) p-Hydroxybenzyl alcohol (HBA), an active component of Gastrodia elata blume, has been reported to provide neuroprotection by preventing brain damage. Transplantation of mesenchymal stem cell (MSC) has been shown to ameliorate ischemic brain injury in animals. To explore a way that might enhance neuroprotection after brain stroke, we investigated whether the transplantation of neural progenitor cells (NPCs) derived from MSCs from adipose tissue combined with HBA may enhance neuroprotective effects in an animal model of brain stroke. Intracarotid injection of combination therapy groups showed a significant reduction of infarct volume by 2,3,5-triphenylte- trazolium chloride staining and an improvement of neurological functions were observed at 3 days after middle cerebral artery occlusion (MCAO), compared to monotherapy groups. In our studies, immunohistochemistry showed that NPCs are more likely to enter a damaged brain than a contralateral nonischemic brain. Coadministration of HBA and NPCs enhanced the anti-apoptotic effect and reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells compared to the vehicle and NPCs at 7 days after MCAO. HBA and the combination of HBA NPCs induced the expression of genes encoding antioxidant proteins, including PDI, Nrf2, endogenous neurotrophic factor gene brain-derived neurotrophic factor, NGF, and VEGF, which enhances angiogenesis in an ischemic brain. These effects might be responsible for the survival of NPCs and improved functional behavior. Our finding indicates that combination therapy of HBA and NPCs enhances neuroprotection against ischemic brain injury. Keywords: p-hydroxybenzyl alcohol; mesenchymal stem cell; ischemia; combination therapy Introduction targeted to impede one aspect of the ischemic cascade, such as glutamate-induced excitotoxicity, free radical- Ischemic stroke is a leading cause of death and long- mediated injury, or inflammatory mechanisms. Thus, it lasting disability (De Keyser et al. 1999). It results from is logical to suggest that if one is able to interrupt the a transient or permanent reduction in cerebral blood propagation of these cascades, at least part of the brain flow in most cases, caused by the occlusion of a tissue can be protected (De Keyser et al. 1999; Cheng cerebral artery either by an embolus or by local et al. 2004; Fisher 2011). p-Hydroxybenzyl alcohol thrombosis (Dirnagl et al. 1999). The primary aim of (HBA) has been reported to be the main component of therapeutic intervention is to reduce the volume of Gastrodia elata blume (GEB) (Ha et al. 2000). It brain damage and thus to minimize neurological possesses many therapeutic properties including anti- impairment (Simon et al. 2001). Ischemic injury in oxidant, anti-excitotoxic, anti-inflammatory, and anti- the brain results in neuronal cell death, with character- apoptosis properties (Yu et al. 2005; Kam et al. 2011). istics of both necrosis and apoptosis (Lipton 1999; The improving effect of HBA after acute administra- Chen et al. 2001; Graham & Chen 2001). Ischemic tion on learning and memory processes was also neuronal cell death has classically been considered to reported (Hsieh et al. 1997). HBA pretreatment reduces be necrotic; however, the findings obtained based on infarct volume and increases the expression of gene the appearance of DNA fragmentation indicate that encoding antioxidant proteins which are protein dis- the apoptotic signaling pathway is activated and that ulfide isomerase (PDI) and 1-Cys peroxiredoxin (1-Cys apoptotic cell death is involved in ischemic brain Prx) genes (Yu et al. 2005). Also, the expression of the damage (Charriaut-Marlangue et al. 1996). transcription factor, nuclear factor erythroid 2-related Several neuroprotective drugs have been developed that have the potential to limit ischemic brain damage factor 2 (Nrf2), brain-derived neurotrophic factor and improve the outcome for patients. Traditionally, (BDNF), glial cell-derived neurotrophic factor the pathway for development and evaluation of a (GDNF), and myelin basic protein (MBP) genes neuroprotective drug was to develop a molecule increased with HBA treatment (Kam et al. 2011). *Corresponding author. Email: email@example.com # 2013 Korean Society for Integrative Biology TRANSLATIONAL MEDICINE Animal Cells and Systems 161 Studies in animal models of ischemic stroke have was seeded in noncoated 75-cm culture flasks with a 3 2 shown that stem cells transplanted into the brain can density of approximately 210 cells/cm . A fresh lead to functional improvement (Savitz et al. 2002; Bliss complete culture medium was added every three days. et al. 2007). Mesenchymal stem cells (MSCs) are AT-MSCs were induced to differentiate into NPCs by stromal cells found in a wide range of adult tissues the modified method of Woodbury et al. (2000). Cells including bone marrow. They can be readily isolated were cultivated in DMEM supplemented with 20% from a range of sources including bone marrow, FBS, 0.1% b-mercaptoethanol (BME, Sigma), adipose tissue, peripheral blood, and umbilical cord 1 nonessential amino acid (Gibco), and 2 mM blood (Maltman et al. 2011). They are considered as a L-glutamine (Gibco). The cells were cultured for 23 leading candidate for neurological regenerative therapy days. Twenty-four hours prior to neural induction, due to their immunological properties (Hoogduijn et al. media were substituted with preinduction medium 2010; Mauri et al. 2012). consisting of 1N2 supplement (Gibco), 20 ng/ml It was previously reported that combined treatment epithelial growth factor (EGF, Sigma), and 20 ng/ml with MSCs and neuroprotective agents enhanced basic fibroblast growth factor (bFGF, Sigma) in the N2 amelioration of ischemic brain damage in rats. Combi- medium (Gibco). For immunocytochemistry, cells were nation therapy with MSCs caused a significant reduc- washed with Phosphate buffered saline (PBS) and fixed tion of infarct volume, neurological score, apoptotic by treating with 4% paraformaldehyde at room tem- cells, enhanced MSC migration into the ischemic brain, perature for 30 min, followed by washing three times of and increased the number of engrafted MSCs com- PBS and PBS plus 1% Triton X-100. Cells were blocked pared with MSCs transplanted alone (Chen et al. 2002; with 5% bovine serum albumin (BSA) for 1 h and then Suda et al. 2011; Zhao et al. 2012). In the present study, were incubated with primary antibodies for 24 h at 48C. we explored the combined effect of HBA and neural The antibodies used in this study were b-tubulin III progenitor cells (NPCs) derived from mesenchymal (1:2000, Sigma) and Nestin (1:200, Millipore). The cells stem cells from adipose tissue (AT-MSCs) on neurolo- were washed three times with PBS and incubated for 30 gical function recovery and infarct volume by using a min at 48C with the secondary antibodies tetramethyl rat model of focal cerebral ischemia. rhodamine isothiocyanate (TRITC) (1:400, Sigma) and Fluorescein Isothiocyanate (FITC) (1:80, Sigma). Materials and methods Animals Experimental design Male Sprague-Dawley rats weighing 250300 g at the The following two experiments were performed in the time of surgery were used for the study. The animals present study. Experiment 1: the combination effect of were housed in pairs in cages in a temperature- neuroprotection of HBA and NPCs derived from controlled room (22938C) under a 12-h lightdark MSCs against damage from cerebral ischemia. HBA cycle. They had free access to food and tap water except (Sigma, USA) was dissolved in dimethyl sulfoxide the day before the operation. All experimental proce- (DMSO) and injected intraperitoneally. Four groups dures complied with current national and international were studied, including (1) vehicle group (5% DMSO, laws and recommendations. 1 ml/kg) (n3), (2) HBA group (HBA injection alone 25 mg/kg at 30 min after the onset of middle cerebral artery occlusion (MCAO) and once a day for 3 days Isolation of MSCs and induction of NPCs after MCAO (n3), (3) NPCs group (NPC 110 cell/ We previously reported the isolation of AT-MSCs and rat injection alone) (n3), and (4) HBA NPCs group the induction of NPCs (Hong et al. 2008). Human (combined injection of HBA and NPCs) NPCs (110 adipose tissue was collected from healthy donors by cells) suspended in PBS (n3). For combination liposuction. Adipose tissues were transported to the therapy, animals were injected with HBA at 30 min laboratory in saline solution within 2 h postsurgery. after the onset of MCAO and followed by NPCs MSCs were collected using a stem cell collector injection via the internal carotid artery (ICA) 1 h (Huricell, model no. HRD-1500, Hurim Biocell Co., after reperfusion. Animals were injected with HBA Korea). AT-MSCs were resuspended in the Dulbecco’s once a day for 3 days after MCAO. For intracarotid modified Eagle’s medium with low glucose (DMEM- (i.c.) injections, the ipsilateral carotid artery was LG, Gibco) supplemented with 100 U/ml penicillin, 100 exposed; NPCs in PBS were injected into the ICA mg/ml streptomycin, 3.7 mg/ml sodium bicarbonate, through a catheter located in the external carotid artery and 10% fetal bovine serum (FBS, Hyclone, South Logan, UT, USA). After cell counting, cell suspension (ECA). 162 P. Kaengkan et al. Ischemic surgery and infarct analysis Germany). NPCs were infected with GFP virus after transplantation. At preinduction, the media was sus- MCAO was achieved according to the methods de- pended in 5 ml PBS after adjustment to the respective scribed by others, with modifications (Longa et al. cell number (110 cells). Animals were injected with 1989). Briefly, the right common carotid artery was HBA at 30 min after the onset of MCAO and followed exposed and carefully dissected free of the vagus nerve. by NPCs injection via the ICA 1 h after reperfusion. The right external and internal carotid arteries were For i.c. injections, the ipsilateral carotid artery was also isolated. The ECA was then ligated at the distal exposed; NPCs in PBS were injected into the ICA end, which was cut-off. A 4-0 nylon thread precoated through a catheter located in the ECA. Rats were killed with silicon was aseptically introduced into the right at 3 days and 7 days after MCAO for TCC staining and carotid artery in an anterograde fashion toward the immunohistochemistry, respectively. The brain was carotid bifurcation. It was then directed distally up subjected to immunohistochemistry. The rats were through the right ICA to a distance of approximately sacrificed at 7 days after MCAO and then fixed by 20 mm from the carotid bifurcation to occlude the cardiac perfusion with 4% paraformaldehyde in 0.1 M origin of the middle cerebral artery. After 1 h, the phosphate buffer. Samples were embedded in embed- thread was withdrawn for reperfusion. All the animals ding medium for frozen tissue specimens (O.C.T. experienced ischemia for 1 h followed by reperfusion. compound, Sakura Finetek USA, Inc.) and frozen. The rectal temperature was maintained at 3790.58C Then the brains were sliced into coronal sections (10 throughout the surgical procedure by using a thermo- mm) by using a cryostat (HM 525, Thermo Scientific, statically controlled warming plate and overhead lamp. USA). After blocking with the normal serum for 2 h at Each group of animals was decapitated at 3 days room temperature, the sections were incubated with after MCAO, and the brains were removed carefully primary antibodies for 24 h at 48C. The antibodies used and placed in a metallic brain matrix for tissue slicing for immunohistochemistry in this study were against immediately after decapitation. The brain were dis- neuronal nuclei (NeuN) (1:700, Chemicon, USA) and sected into coronal sections of 2 mm in a metallic brain VEGF (1:200, Santa Cruz, USA), diluted in Tris- matrix, and then immersed sequentially in a 2% buffered saline (TBS) containing 1% BSA (w/v), and solution of 2,3,5-triphenyltetrazolium chloride (TTC) 0.3% Triton X-100. After rinsing in TBS, the sections in normal saline at 378C for 10 min, and then fixed in were incubated with secondary antibody-conjugated 10% formalin for 10 s. The infarct area in the brain FITC (1:300, Sigma, USA) and CY3 (1:500, Amer- section was measured by National Institutes of Health sham) for 2 h at room temperature. The sections were (NIH) Image software (Image J). The ischemic lesion then rinsed in TBS and mounted in an aqueous volume was calculated as the sum of ischemic lesions mounting medium. The resulting immunoreactivity from five brain slices from half of the hemisphere. was visualized by confocal laser scanning microscopy (Carl Zeiss, LSM 510 META). The numbers of GFP- positive cells in the ischemic area were counted in three Neurological deficit scores randomly selected microscopic fields under 200 The test was conducted by an investigator who was magnification in a blind fashion from the bregma blind to the experimental groups. All the rats were 0.00 mm to 2.00 mm (encompassing the majority evaluated using a modified neurological severity score of the lesion). (mNSS) (Chen et al. 2001). The mNSS was a composite of motor (muscle status and abnormal movement), sensory (visual, tactile, and proprioceptive), reflex, and Reverse transcription-polymerase chain reaction (RT- balance tests. Neurological function is graded on a scale PCR) analysis of 018 (normal score, 0; maximal deficit score, 18). The cortex and striatum of the lesioned side from each group were harvested for RT-PCR. Total RNA was extracted using the acid guanidinium isothiocyanate- NPCs labeling, transplantation, and phenol-chloroform method with Trizol (TRI) reagent, immunohistochemistry followed by extraction and precipitation with isopropyl Transplanted NPCs were infected by a recombinant alcohol. The cDNA was synthesized from equal adenovirus (Invitrogen, CA, USA) containing green amounts of total RNA with superscript III reverse fluorescent protein (GFP) gene with trace transplanted transcriptase, and polymerase chain reaction (PCR) cells in the lesion brain. GFP (810 pfu/ml) virus was was performed with high-fidelity Taq DNA polymerase. added to the media 3 days before transplantation. The expression level of the gene of interest was Resulting green fluorescence cells were visualized by corrected for that of the housekeeping gene, b-actin. The sequences of the primer pairs used were PDI: fluorescence microscopy (Carl Zeiss Axioskop2, Animal Cells and Systems 163 5?-TCT GGA GGA GGA GGA CAA C-3?,5?-TGG A fixed with 4% paraformaldehyde in phosphate buffer AA ACA CAT CGC TAT T-3?; Nrf2: 5?-AAG GAA G (pH 7.4) and treated with 0.3% H O in methanol for 2 2 CT GGA AAA CAT TG-3?,5?-AAA TGG TGC CCA 10 min, washed in PBS, and permeabilized in 0.1% AGA AAT TA-3?; BDNF: 5?-GGT CAC AGC GGC A Triton X-100 in 0.1% sodium citrate. Then the sections GA TAA AAA-3?,5?-TTG GGT AGT TCG GCA TTG were incubated with TdT enzyme in a humidified CGA-3?; NGF: 5?-ACA TCA AGG GCA AGG AGG T chamber for 60 min. Further incubation with a GA-3?,5?-TGA CAA AGG TGT GAG TCG TGG-3?; peroxidase-conjugated antibody was performed for 30 VEGF: 5?-GGACCCTGGCTTTACTGCT-3?,5?-TGG min at 378C. Finally, 3,3?-diaminobenzidine (DAB) was CTTTGGTGAGGTTTGAT-3? and b-actin: 5?-CCA T added and incubated for 10 min, followed by washing CA TGA AGT GTG ACG TT-3?,5?-CCA CCA ATC C with PBS for the coloration of apoptotic cells. The AC ACA GAG TA-3?. PCR products of different genes numbers of TUNEL-positive cells in the ischemic area were detected by electrophoresis on a 1.5% agarose gel were counted in three randomly selected microscopic containing ethidium bromide. Band intensities were fields under 200 magnification in a blind fashion. quantified by Image J and normalized with b-actin. Statistical analyses Terminal deoxynucleotidyl transferase-mediated dUTP Data are presented as the mean9SE. Student’s t-tests nick-end labeling (TUNEL) assay were used to compare the difference between the treatments and the vehicle group. The difference was To observe DNA strand breaks in nuclei, the apoptotic considered significant when pB 0.05. DNA fragmentation was analyzed using terminal deoxyribonucleotidyl transferase (TdT)-catalyzed in- corporation of fluorescein-labeled nucleotides to free Results 3?-OH ends of DNA in a template-independent manner Induction of neural stem cells from AT-MSCs using the In Situ Cell Death Detection Kit, POD (Roche Molecular Biochemicals, Inc., Mannheim, Immunocytochemical staining for neural markers was Germany), and was performed according to the performed to confirm the neural cells for b-tubulin III, manufacturer’s instructions. Briefly, the sections were a neuron-specific marker. Nestin expression is restricted Figure 1. Cell type-speciﬁc immunoﬂuorescence staining and the survival rate of NPCs differentiated from AT-MSCs. (A) Immunocytochemistry of NPCs to be transplanted was done with b-tubulin III, a neuronal marker and Nestin, a neural progenitor marker. (B) Immunohistochemistry of transplanted NPCs 7 days after MCAO at the cortex and striatum area. The ﬂuorescence microscope images (50) showed survival of GFP-positive cells after transplantation. (C) Quantitative analysis of the number of transplanted NPCs. Transplanted NPCs was signiﬁcantly increased in the combination of HBA NPCs treated group compared with the individual NPCs group. **pB0.01 vs. NPCs group. 164 P. Kaengkan et al. to neuroepithelial precursor cells in the neural tube and thus has been used as a cell type-specific marker for neural stem and progenitor cells (Figure 1A). The GFP- positive cells were evaluated in the ischemic zone of the treated animals. Transplanted NPCs were found in the peri-infarct region after the injection. A higher con- centration of i.c. transplanted NPCs was observed in the ipsilateral lesioned than in the contralateral (data not shown). Immunohistochemistry showed that GFP- positive cells in the combination group of HBA NPCs were higher than in the NPCs groups (Figure 1B, and C). Infarct volume The borders of the TTC stain enclosing the white infarct area were readily distinguishable in contrast to the red color of the normal area. Quantitative mea- surement of the infarct volume was performed in the section at 2.00 mm from the bregma point according to the rat brain atlas (Paxinos & Watson 2007). At 3 days after treatment, in the HBA alone group, NPCs alone group, and HBA NPCs group, the total infarct volumes were significantly decreased compared to the vehicle group. The infarct volumes in the vehicle- treated group and the groups treated with HBA, NPCs, and a combination of HBA NPCs were 53.8791.8, 46.0892.2, 43.2790.58, and 30.249 3.65% of the total area of the brain section, respec- tively. A significant decrease of the total infarct volume was also observed in the combination group of HBA NPCs compared to the vehicle, HBA alone, and NPCs alone groups (pB 0.05 and pB 0.01; Figure 2A and B). Figure 2. Combination effects of HBA and NPCs on the infarct volume at 3 days after MCAO. (A) TTC-stained Functional recovery 3 days after MCAO coronal section from vehicle, HBA (25 mg/kg), NPCs (1 10 ), and combination of HBA and NPCs. (B) The infarct The mNSS test showed that motor and sensory volume was calculated as the infarct areasthickness (2 mm) functions were impaired by ischemic insult. All treated and expressed as a percentage of the lesion half of the brain. groups showed no significant improvement at 1 day The combination of HBA and NPCs group shows a after ischemia; however, at 3 days after treatment, there signiﬁcantly lower infarct volume than the HBA and NPCs was significant functional recovery only in the combi- alone group 3 days after MCAO. Data are shown as means nation group of HBA NPCs compared to the vehicle SEM (n3) *pB0.05, **pB0.01 vs. individual vehicle group (pB 0.05; Figure 3). group, pB0.05 vs. individual HBA group, $pB0.05 vs. individual NPCs group. Anti-apoptotic effects combination of HBA NPCs (33911.93) groups dis- TUNEL-positive cells were frequently observed in the played a significant decrease in TUNEL-positive cells ischemic boundary area. Quantitative analysis of compared to the vehicle group (87912.53), but NPCs- TUNEL-positive cells showed that the HBA (87.679 alone-treated group (79.33914.57) failed to exert 14.50), NPCs (107.6794.16), and combination of antiapoptotic effects in this area. Moreover, the HBA HBA NPCs (8096.08) groups displayed a significant combined with the NPCs group had a significantly decrease in TUNEL-positive cells in the cortical area reduced number of TUNEL-positive cells compared to compared to the vehicle group (217915.95) at 7 days the NPCs-alone and vehicle groups (pB 0.05 and after focal cerebral ischemia. Striatal area TUNEL- positive cells showed that the HBA (53914) and pB 0.01; Figure 4A and B). Animal Cells and Systems 165 Figure 3. Combination effects of HBA and NPCs on functional behavioral recovery after MCAO. Neurological functional tests were performed at 1 and 3 days after MCAO. The combination of HBA and NPCs group shows signiﬁ- cantly lower mNSS score than the vehicle group 3 days after MCAO. Data are shown as means SEM (n3) *pB0.05 vs. individual vehicle group. Effect of HBA NPCs on gene expressions RT-PCR was performed to analyze the effects of HBA and NPCs on the expression of the PDI and Nrf2 genes that encode antioxidant proteins and endogenous neurotrophic factor BDNF, NGF genes. After a 1-h ischemic insult followed by 24 h of reperfusion, the ipsilateral whole brain of the lesioned area was subjected to RT-PCR analysis. The expression levels of PDI and Nrf2 genes were significantly elevated in the HBA NPCs group compared to the vehicle, HBA, and NPCs groups. The potential involvement of neurotrophic factors BDNF and NGF in the mechanism of neuroprotection by HBA and NPCs Figure 4. Combination effects of HBA and NPCs on and mRNA levels of neurotrophic factors were ana- TUNEL staining. (A) TUNEL staining in the cortex and lyzed by RT-PCR. The results showed increased striatum area of ischemic lesioned side of vehicle, HBA, expression of BDNF and NGF genes in the combina- NPCs, and combination of HBA NPCs treatment (200). tion of HBA NPC group (pB 0.05, pB 0.01, Figure (B) Quantitative analysis of the number of TUNEL-positive 5AD). cells. Data are the mean9SEM (n3) *pB0.05, **pB0.01 vs. individual vehicle group, $pB0.05 vs. individual NPCs group, $$pB0.01 vs. individual NPCs group. Immunohistochemistry of VEGF after NPCs transplantation Discussion Immunohistochemistry was performed at 7 days after It was previously reported that HBA from GEB extract the MCAO. The positive neuronal markers NeuN and protected the brain after neurotoxic insult. HBA has vascular endothelial growth factor marker VEGF received attention as a neuroprotective agent due to its (angiogenesis and the vasculogenesis marker) were anti-oxidative, anti-excitotoxic, and anti-apoptosis abil- evaluated in the cortical ischemic boundary zone of ities (Liu & Mori 1992; Dhiman et al. 2009; Yu et al. vehicle and treated animals (Figure 6A). Quantitative 2010; Yu et al. 2011). The effect of HBA on functional analysis showed that the mRNA levels of VEGF in the recovery may be related to the increased expression of cortex area of the combination of HBA NPCs neurotrophic factor genes (Yu et al. 2005; Kam et al. treated group were higher than in the vehicle and 2011). Neurotrophic factors are essential for neuronal NPCs groups (pB 0.05, pB 0.01, Figure 6B). survival and differentiation during development, as well 166 P. Kaengkan et al. Figure 5. Combination effects of HBA and NPCs on the gene expressions of PDI, Nrf2, and neurotrophic factors in the cortex and striatum of the lesioned area. (A and B) Speciﬁc gene expressions were determined in relation to the expression of b-actin using RT-PCR. (C and D) The levels of PDI, Nrf2, BDNF, and NGF gene expression were signiﬁcantly increased in the combination of HBA NPCs treated group compared with the individual vehicle group. Experiments were repeated three times and individual values are expressed as the mean9SEM. *pB0.05, **pB 0.01 vs. individual vehicle group, pB 0.05 vs. individual HBA group, pB0.01 vs. individual HBA group, $pB0.05 vs. individual NPCs group, $$pB0.01 vs. individual NPCs group. as for the maintenance of normal neuronal function in MCAO (Kam et al. 2011). Intracarotid transplanted adults (Snider 1994). Moreover, HBA could inhibit NPCs can survive and migrate in the brain environ- DNA degradation and be at least partially associated ment. NPCs are more likely to enter into a damaged with its anti-apoptotic actions (Yu et al. 2010). brain than into the contralateral nonischemic hemi- Stem cell transplantation has the potential to sphere. Chen et al. (2003) reported that the vast provide protection and regeneration after ischemic majority of intravenous administration of human injury (Kelly et al. 2004; Takahashi et al. 2008). mesenchymal stem cells (hMSCs) were localized to Many studies showed that the administration of the ischemic boundary zone, with some cells within NPCs and MSCs via different routes of delivery after vessels and with some cells detected in the contralateral experimental stroke reduced the infarct volume and hemisphere of brain. In our study, most intraarterial- improved functional recovery (Bliss et al. 2007). The injected cells migrated into the cortical zone of the experimental rat model has used two different hematic ischemic hemisphere which may help to improve administration routes, i.c. and intraventricular (i.v). functional recovery. Transplantation of NPCs and The results showed that both routes are equally injection with HBA after experimental stroke both effective in contributing to neurological recovery, reduced the infarct volume and improved functional decreasing cerebral damage, and improving protection recovery. The i.c. injection of NPCs alone into the mechanisms (Gutie ´rrez-Ferna ´ ndez et al. 2011). ischemic brain did not show significant functional Our results of posttreatment of HBA injection recovery after MCAO compared to the vehicle group. alone showed a significant difference in the reduction The significant improvement of functional recovery of the infarct volume compared to the vehicle group. was only found in rats treated with combination of The magnitude of the reduction was slightly similar to HBA NPCs 3 days after MCAO. The therapeutic our previous study of HBA pretreatment 3 days before potential of HBA on the middle cerebral artery is Animal Cells and Systems 167 Figure 6. Combination effects of HBA and NPCs on the expression of VEGF. (A) Immunohistochemistry showed NeuN positive neurons (green); a neuronal marker, VEGF positive (red); vascular endothelial growth factor marker and the double-labeling of NeuN (green), VEGF (red) in the cortical ischemic boundary zone at 7 days after MCAO (100). (B) The mRNA levels of VEGF in the cortex area were signiﬁcantly increased in the combination of HBA NPCs treated group compared with the individual vehicle, HBA, and NPCs group. **pB0.01 vs. individual vehicle group, pB0.05 vs. individual HBA group, $pB0.05 vs. individual NPCs group. probably related to its anti-apoptotic effect (Yu et al. the antioxidants PDI and Nrf2 and also increased the 2010). Posttreatment of HBA injection enhanced the expression of the endogenous neurotrophic factor genes anti-apoptotic effect and the combination of HBA BDNF and NGF after ischemia. This led to a more NPCs showed significantly reduced apoptotic cells in greatly decreased infarct volume and good functional both the cortical and striatal areas 7 days after MCAO recovery (Yu et al. 2005; Kam et al. 2011). Our compared to the NPCs alone and vehicle groups. observation that HBA combined with NPCs improved Previous and present studies showed that HBA the great behavior outcome may be due to the promo- tion of grafted NPCs that release trophic factors. injections increased the expression of genes encoding 168 P. Kaengkan et al. after reversible focal ischemia: an in situ DNA fragmen- Interaction of transplanted stem cells with the host in tation analysis. J Cereb Blood Flow Metab. 16:186195. the brain microenvironment may lead to production of Chen J, Li Y, Wang L, Lu M, Chopp M. 2002. Caspase trophic factors that promote plasticity and repair inhibition by Z-VAD increases the survival of grafted damaged brains (Chen et al. 2001; Chen et al. 2002). bone marrow cells and improves functional outcome after HBA treatment increases the effect on endogenous MCAO in rats. J Neurol Sci. 199:1724. Chen J, Sanberg PR, Li Y, Wang L, Lu M, Willing AE, neurotrophic activity in BDNF and NGF, which are Sanchez-Ramos J, Chopp M. 2001. Intravenous admin- neuroprotective in the rat model of ischemic stroke istration of human umbilical cord blood reduces beha- (Kokaia et al. 1995). This effect might be responsible for vioral deﬁcits after stroke in rats. Stroke. 32:26822688. the survival of NPCs which increased the number of Chen J, Zhang ZG, Li Y, Wang L, Xu YX, Gautam SC, Lu surviving engrafted NPCs in the ischemic brain and M, Zhu Z, Chopp M. 2003. Intravenous administration of human bone marrow stromal cells induces angiogen- improved functional behavior. Relative intensity of esis in the ischemic boundary zone after stroke in rats. increased antioxidants PDI and Nrf2 and endogenous Circ Res. 92:692699. neurotrophic factor genes BDNF and NGF corre- Cheng YD, Al-Khoury L, Zivin JA. 2004. Neuroprotection sponds with the localization of NPCs in the cortex of for ischemic stroke: two decades of success and failure. the brain. Therefore, these data suggest that NPCs may Neurotherapeutics. 1:3645. increase endogenous levels of PDI, Nrf2, BDNF, and De Keyser J, Sulter G, Luiten PG. 1999. Clinical trials with neuroprotective drugs in acute ischaemic stroke: are we NGF in the combination treatment. doing the right thing? Trends Neurosci. 22:535540. Immunostaining for endogenous rat VEGF showed Dhiman SB, Kamat JP, Naik DB. 2009. Antioxidant activity an increase in VEGF immunoreactivity within the and free radical scavenging reactions of hydroxybenzyl cortical ischemic boundary in rats treated with the alcohols. Biochemical and pulse radiolysis studies. Chem combination of HBA NPCs compared to the im- Biol Interact. 182:119127. munoreactivity in the control rats. Our data demon- Dirnagl U, Iadecola C, Moskowitz MA. 1999. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. strate that treatment of stroke with a combination of 22:391397. HBA NPCs might enhance angiogenesis in an is- Fisher M. 2011. New approaches to neuroprotective drug chemic brain by increasing endogenous rat VEGF development. Stroke. 42:S24S27. expression. Angiogenesis is induced by a variety of Graham SH, Chen J. 2001. Programmed cell death in cerebral growth factors including VEGF which can be secreted ischemia. J Cereb Blood Flow Metab. 21:99109. Gutie ´rrez-Ferna ´ ndez M, Rodr´ ıguez-Frutos B, Alvarez-Grech by MSCs. Angiogenesis is associated with improved J, Vallejo-Cremades MT, Expo ´ sito-Alcaide M, Merino J, neurological recovery from stroke. Newly formed Roda JM, D´ ıez-Tejedor E. 2011. Functional recovery vessels improve tissue perfusion around the ischemic after hematic administration of allogenic mesenchymal boundary zone and the enhancement of angiogenesis stem cells in acute ischemic stroke in rats. Neuroscience. promotes functional recovery in rats after stroke 175:394405. Ha JH, Lee DU, Lee JT, Kim JS, Yong CS, Kim JA, Ha JS, (Villars et al. 2000; Chen et al. 2003). Injection of Huh K. 2000. 4-hydroxybenzaldehyde from Gastrodia NPCs not only replace tissue, but also augment elata B1. is active in the antioxidation and GABAergic function, evoke the expression of growth and trophic neuromodulation of the rat brain. J Ethnopharmacol. factors from endogenous cells, and enhance plasticity 73:329333. of the remaining tissue, particularly tissue within the Hong IK, Jeong NH, Kim JR, Do B-R, Kim HK, Kang SG. boundary zone of an ischemic lesion. We also speculate 2008. Differentiation of dopaminergic and cholinergic neurons from mesenchymal-like stem cells derived from that treatment of an HBA injection may improve the the adipose tissue. Dev Reprod. 12:3139. viability of transplanted NPCs and thereby enhance Hoogduijn MJ, Popp F, Verbeek R, Masoodi M, Nicolaou A, neuroprotection against ischemic injury. Thus, these Baan C, Dahlke M-H. 2010. The immunomodulatory results suggest that combining HBA NPCs is a properties of mesenchymal stem cells and their use for valuable strategy to enhance NPCs graft survival and immunotherapy. Int Immunopharmacol. 10:14961500. Hsieh M-T, Wu C-R, Chen C-F. 1997. Gastrodin and p- to promote functional recovery. hydroxybenzyl alcohol facilitate memory consolidation and retrieval, but not acquisition, on the passive avoid- ance task in rats. J Ethnopharmacol. 56:4554. Acknowledgments Kam KY, Yu S, Jeong N, Hong J, Jalin A, Lee S, Choi Y, Lee This work was supported by the 2012 Inje University research C, Kang S. 2011. p-Hydroxybenzyl alcohol prevents brain grant. injury and behavioral impairment by activating Nrf2, PDI, and neurotrophic factor genes in a rat model of brain ischemia. Mol Cells. 31:209215. References Kelly S, Bliss TM, Shah AK, Sun GH, Ma M, Foo WC, Masel J, Yenari MA, Weissman IL, Uchida N, et al. 2004. Bliss T, Guzman R, Daadi M, Steinberg GK. 2007. Cell Transplanted human fetal neural stem cells survive, transplantation therapy for stroke. Stroke. 38:817826. migrate, and differentiate in ischemic rat cerebral cortex. Charriaut-Marlangue C, Margaill I, Represa A, Popovici T, Proc Natl Acad Sci USA. 101:1183911844. Plotkine M, Ben-Ari Y. 1996. Apoptosis and necrosis Animal Cells and Systems 169 Kokaia Z, Zhao Q, Kokaia M, Elme ´r E, Metsis M, Smith Suda S, Shimazaki K, Ueda M, Inaba T, Kamiya N, Katsura M-L, Siesjo BK, Lindvall O. 1995. Regulation of brain- K-I, Katayama Y. 2011. Combination therapy with bone marrow stromal cells and FK506 enhanced amelioration derived neurotrophic factor gene expression after tran- of ischemic brain damage in rats. Life Sci. 89:5056. sient middle cerebral artery occlusion with and without Takahashi K, Yasuhara T, Shingo T, Muraoka K, Kameda brain damage. Exp Neurol. 136:7388. M, Takeuchi A, Yano A, Kurozumi K, Agari T, Miyoshi Lipton P. 1999. Ischemic cell death in brain neurons. Physiol Y, et al. 2008. Embryonic neural stem cells transplanted Rev. 79:14311568. in middle cerebral artery occlusion model of rats Liu J, Mori A. 1992. Antioxidant and free radical scavenging demonstrated potent therapeutic effects, compared to activities of Gastrodia elata Bl. and Uncaria rhynchophylla adult neural stem cells. Brain Res. 1234:172182. (Miq.) jacks. Neuropharmacology. 31:12871298. Villars F, Bordenave L, Bareille R, Ame ´de ´e J. 2000. Effect of Longa EZ, Weinstein PR, Carlson S, Cummins R. 1989. human endothelial cells on human bone marrow stromal Reversible middle cerebral artery occlusion without cell phenotype: role of VEGF? J Cell Biochem. 79: craniectomy in rats. Stroke. 20:8491. 672685. Maltman DJ, Hardy SA, Przyborski SA. 2011. Role of Woodbury D, Schwarz EJ, Prockop DJ, Black IB. 2000. Adult mesenchymal stem cells in neurogenesis and nervous rat and human bone marrow stromal cells differentiate system repair. Neurochem Int. 59:347356. into neurons. J Neurosci Res. 61:364370. Mauri M, Lentini D, Gravati M, Foudah D, Biella G, Costa Yu SJ, Kim JR, Lee CK, Han JE, Lee JH, Kim HS, Hong JH, B, Toselli M, Parenti M, Coco S. 2012. Mesenchymal Kang SG. 2005. Gastrodia elata blume and an active stem cells enhance GABAergic transmission in co- component, p-hydroxybenzyl alcohol reduce focal is- cultured hippocampal neurons. Mol Cell Neurosci. chemic brain injury through antioxidant related gene 49:395405. expressions. Biol Pharm Bull. 28:10161020. Paxinos G, Watson C. 2007. The rat brain in stereotaxic Yu SS, Zhao J, Lei SP, Lin XM, Wang LL, Zhao Y. 2011. coordinates. San Diego: Academic Press. 4-hydroxybenzyl alcohol ameliorates cerebral injury in Savitz SI, Rosenbaum DM, Dinsmore JH, Wechsler LR, rats by antioxidant action. Neurochem Res. 36:339346. Caplan LR. 2002. Cell transplantation for stroke. Ann Yu SS, Zhao J, Zheng WP, Zhao Y. 2010. Neuroprotective Neurol. 52:266275. effect of 4-hydroxybenzyl alcohol against transient focal Simon L, Szila ´gyi G, Bori Z, Orbay P, Nagy Z. 2001. (-)-D- cerebral ischemia via anti-apoptosis in rats. Brain Res. deprenyl attenuates apoptosis in experimental brain 1308:167175. ischaemia. Eur J Pharmacol. 430:235241. Zhao Y, Guan Y, Xu Y, Li Y, Wu W. 2012. Sodium Ferulate Snider WD. 1994. Functions of the neurotrophins during combined with bone marrow stromal cell treatment nervous system development: what the knockouts are ameliorating rat brain ischemic injury after stroke. Brain teaching us. Cell. 77:627638. Res. 1450:157165.
Animal Cells and Systems
– Taylor & Francis
Published: Jun 1, 2013
Keywords: p -hydroxybenzyl alcohol; mesenchymal stem cell; ischemia; combination therapy