II. MATERIALS AND METHODS
15. In-vitro Angiogenesis assay
96 well plates were coated with 50 ul of BD matrigel. 50 ul of conditioned media from MSC, MSC/Ngn1, MSC+HGF, and MSC/Ngn1+HGF cell culture was added into the well. 2x104 mouse brain endothelial cells, bEND.3 cells suspended in 100ul of MSC culture media was added into the well.
The plate was incubated for 4 hrs at 37ºC and light microscopic images were acquired by Olympus microscope at 200X magnification. 10 random fields per well were selected for analysis. The experiment was done in duplet and repeated three times. The tube formation assay was analyzed by using the Angiogenesis analyzer program of ImageJ.
16. In-vitro NSC Proliferation assay
Secondary and tertiary neurospheres were dissociated into single cells and plated into 0.1 mg/ml PDL coated cover glasses. Conditioned media (in Serum-free media) from MSC, MSC/Ngn1, MSC+HGF, and MSC/Ngn1+HGF cell culture was obtained 48 hours of confluent culture. The culture was incubated for 16 hours in conditioned media and was fixed with 10% formalin for assessment of proliferation by Ki67 immunocytochemistry. Fresh MSC culture media was used as a control.
17. In-vitro Neuronal differentiation assay.
Secondary and tertiary neurospheres were dissociated into single cells and plated into 0.1 mg/ml PDL coated cover glasses. Conditioned media (in 10%
Serum containing media) from MSC, MSC/Ngn1, MSC+HGF, and MSC/Ngn1+HGF cell culture was obtained 48 hours of confluent culture. The culture was incubated for 3 days in conditioned media and was fixed with 10%
formalin for assessment of neuronal differentiation by Tuj1 immunocytochemistry. Fresh MSC culture media containing 10% serum was used as control.
18. Animal models and cell transplantation.
18.1. Rat model
Transient focal ischemia was induced by intraluminal filament occlusion of the middle cerebral artery (MCAo) according to a modified procedure originally described by Longa et al(Longa, Weinstein et al. 1989).
Male Sprague-Dawley rats (250 g) were anesthetized with intraperitoneal (i.p.) administration of ketamine (75 mg/kg) and xylazine hydrochloride (5 mg/kg).
The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed. A 4-0 monofilament nylon suture with a silicon-coated tip (Doccol Co., Sharon, MA, USA) was advanced from the ECA lumen into the ICA until it blocked the bifurcating origin of the MCA.
Two hours after MCAo, animals were re-anesthetized and reperfusion was performed by withdrawing the suture until the tip cleared the lumen of the ECA.
Rectal temperature was maintained at 37°C throughout the surgical procedure, using an electronic temperature controller linked to a heating pad (FHC, Bowdoinham, ME, http://www.fh-co.com). Animals showing similar scores (e.g., remaining on the Rotarod [Ugo Basile, Comerio, Italy, http://www.ugobasile.com] for more than 300 seconds during pre-training but
less than 10 seconds after surgery) and comparable infarct volumes in magnetic resonance imaging (MRI) were selected and randomly grouped. This way, we were able to scrupulously control the quality of animals and minimize variations among experimental groups. One month after MCAo, 1x106 cells in a total fluid volume of 10ul were intracranially transplanted into the striatum (anteroposterior [AP], 0.5; mediolateral [ML], 2.5; dorsoventral [DV], 5.0) and cortex (AP, 0.5; ML, 2.0; DV, 2.5) in the ipsilateral hemisphere (5 ul per site).
18.2. Mouse model
Adult (3-5 months old) Nestin-GFP and NG2-CreERTM/Nestin-GFP/Rosa-TdTomato double transgenic mice were used for assessing neurogenesis in local brain micro-environment. Transient middle cerebral artery occlusion (MCAo) was induced by an intraluminal suture method. Briefly, animals were anesthetized with isoflurane (3% for induction and 2% for maintenance) in a mixture of N2O: O2 (70:30) while being intubated and
mechanically ventilated. Rectal temperature was maintained at 37℃ throughout the surgical procedure. MCAo surgery and post-surgical care were performed as described in our previous report (Park, Marasini et al. 2014). After 60 minutes of occlusion, the nylon suture was gently removed from MCA to re-perfuse the MCA territory. One month after MCAo, 3x105 cells in a total fluid volume of 6ul PBS were intra-cranially transplanted into the striatum (anteroposterior [AP], 0.5; mediolateral [ML], 2.5; dorso-ventral [DV], 3.0.
19. Behavior assessment:
19.1 Behavirol Assement of the rat stroke model
Assessment of neurological severity test, Adhesive Removal Test and Rotarod Test were performed as described earlier (Kim, Yoo et al. 2008, Park, Marasini et al. 2014). For the Adhesive Removal Tests, an adhesive tape of 10 mm x10 mm was placed on the dorsal paw of each forelimb, and the time to remove each tape from the dorsal paw was measured. For the Rotarod Test, experimental animals were tested for their ability to run on a rotating cylinder that was accelerated from 4 to 40 rpm for 5 minutes. Two weeks before stroke induction, only animals capable of removing the adhesive tape within 10 seconds and remaining on the Rota-rod cylinder for more than 300 seconds were selected and included in the experiment. Data are presented as the mean latency to fall calculated from three trials. For the adhesive removal test, two square dots of adhesive-patch (100 mm2) were used as bilateral tactile stimuli
occupying the distal-radial region on the wrist of each forelimb. The time taken for each animal to remove the adhesive dot was recorded with a cut-off time of 300 sec. Data are presented as the average time to remove the patch from three trials.
19.2 Behavior assessment of the mouse stroke model, behavioral tests were carried out on 2nd, 4th, 7th, 14th, 21st and 28th day after MCA occlusion. Rotarod test evaluates the balance and coordination function (Terborg, Bramer et al.
2004). Prior to surgery, the mice were trained for balancing on the rotating drum (Acceler Rota-Rod 7650, UGO BASILE, Varese, Italy) for 5 days (3 trials per day) (Rogers, Campbell et al. 1997). The rotarod was accelerated from 4 to 40 r.p.m. for 250 sec as a preoperative baseline. Animals not achieving the baseline criteria were excluded from the subsequent study. The latency before falling off the accelerating drum was recorded with a maximum of 250 seconds. The longest latency from three consecutive trials for each testing day was extracted for data analysis using Sigma plot software (Systat Software Inc, San Jose, CA, USA).
Corner test is used to detect unilateral abnormalities of sensory and motor functions in the stroke model(Zhang, Schallert et al. 2002). A corner was made by placing two wooden cardboards (30 cm × 20 cm × 1 cm) at an angle of 30 degree. The mouse was made to enter the corner upon its placement at midway to the corner. As the mouse reached deep into the corner, both sides of
the vibrissae were stimulated together. Upon the stimulation of vibrissae, the mouse reared forward and upward and finally turned back towards the open end.
The direction towards which the mouse turned was recorded. A total of 10 trials were recorded per each animal pre-operatively and on indicated days. Sham animals did not show any preference in the direction while the ischemic mice showed marked preference in turning towards the non-impaired side (right turn).
The percentage of the right turn was analyzed as the indicator of the deficit.
Pole test is a simple behavior test used to assess motor dysfunction after stroke (Bouet, Freret et al. 2007). Mice were placed in the top of a 60 cm vertical pole with a diameter of 1 cm. The pole was placed in the home cage so that mice might prefer to descend to the floor of the cage. The recording was started when the animal began the turning movement. The time to turn completely downward (Tturn) and total time to descend to the floor (Ttotal) were recorded. When the animal paused while descending, the trial was repeated.
When the animal could not turn but instead descended with a lateral body position, then Ttotal was attributed to Tturn. When the animal fell off the pole immediately, the maximum scores for Tturn (10 sec) and Ttotal (15 sec) were assigned. The test was repeated for 3 trials per animal in each setting and the average Tturn and Ttotal were used for data analysis.
For the NSS test, a 0-5 grading scale was used, with a slight modification performed by Bederson et al. (Bederson, Pitts et al. 1986). Normal
mice were scored 0, while dead mice or mice unresponsive to stimulation were 3.0T X-Series Qasar Dual, Philips Healthcare, Amsterdam, Netherlands) equipped with a gradient system capable of 35 milliteslas/m. A fast-spin echo imaging sequence was used to acquire T2-weighted anatomical images of the rat brain in vivo using the following parameters: repetition time, 3,000 milliseconds (msec); effective echo time, 120 msec; field of view, 55 x 55 mm2; image matrix, 256 × 256; slice thickness, 1.5 mm; flip angle, 90°; pixel size, 0.21 x 0.21 mm2. A 300-mm diameter quadrature 16-ring birdcage coil arrangement was used for radiofrequency excitation, and a 40-mm diameter saddle coil was used for signal detection A total of 15 slices were scanned to cover the whole rat brain. For each
slice, the ischemic area from each T2-weighted image was marked manually and calculated using Osiris software (University of Geneva, Geneva, Switzerland).
Relative infarct volume (RIV) was normalized as described by Kim et al. (Kim, Yoo et al. 2008) and Neumann-Haefelin et al. (Neumann-Haefelin, Kastrup et al.
2000) using the equation RIV = (LT – (RT – RI)) x d, where LT and RT represented the areas of the left and right hemispheres in mm2, respectively, RI was the infarcted area in mm2, and d was the slice thickness (1.5 mm). RIV was expressed as a percentage of the right hemispheric volume. For mouse stroke models, MRI was performed using a 4.7-T animal MRI scanner (Biospec 47/40;
Bruker, Karlsruhe, Germany) located in the Korea Basic Science Institute (Ochang, Korea). The animal was placed on a non-magnetic holder equipped with a nose cone for the administration of anesthetic gas containing 2%
isoflurane in 70% N2O and 30% O2. T2-weighted images were obtained using the following parameters: repetition time (TR), 5,000 msec; echo time (TE), 90 msec; average, 4; acquisition matrix, 256 × 256; 8 slices with 1-mm thickness;
flip angle, 180°. T2*-weighted multi-slice images were acquired using the following parameters: TR, 561 msec; TE, 20 msec; average, 4; acquisition matrix, 256 × 256; 15 slices with 1-mm thickness; flip angle, 30°. The images were analyzed with Para-Vision Acquisition 5.1 (National Instruments, Austin, TX, USA).
21. Histological analysis 21.1 TTC staining
To confirm the proper MCA occlusion, animals were deeply anesthetized with a high dose of isoflurane gas 24 h after the MCAo surgery and were decapitated. The brains were extracted rapidly and coronally sliced into 2 mm sections on an ice-cold mouse mold (Leica Biosystems, Buffalo Grove, IL, USA). Each section was incubated in 2% of 2,3,5 triphenyl-tetrazolium chloride (TTC) in phosphate-buffered saline, pH 7.4 (PBS) for 10~12 min at 37oC in the dark. After the TTC solution was drained off, slices were fixed with 10%
formalin in PBS. The unstained area in the section was regarded as an infarct.
The images of slices were acquired with a stereoscopic microscope (SZX2-ILLB, Olympus Co. Tokyo, Japan)
21.2 Cresyl violet staining.
Animals were intra-cardially perfused with 10% formalin. Brains were carefully extracted and further fixed in 10% formalin overnight. After embedding in a paraffin block, five-micrometer coronal sections were mounted on slides and proceeded for cresyl violet staining as reported earlier(Tureyen, Vemuganti et al. 2004). Coronal sections representing the striatal region were used for analysis.
21.3 Immunohistochemistry
For immunohistochemical analysis, animals were anesthetized with ketamine (100 mg/kg) /xylazine (10 mg/kg) (Yuhan Co. Ltd., Seoul, Korea), perfused transcardially with ice-cold saline, and then fixed with 10% neutral buffered formalin (NBF). The brains were post-fixed in 10% NBF and embedded in paraffin. The paraffin blocks were serially sectioned to produce 5-mm thick sections, which were then deparaffinized and placed in boiled citrate buffer (pH 6.0) for 10 min. After blocking in 10% normal serum, the sections were incubated with antibodies against neuronal nuclei (NeuN; 1:500, Merck Millipore, Burlington, MA, USA), microtubule-associated protein 2 (MAP2;
1:500, Sigma-Aldrich, St. Louis, MO, USA), human mitochondrial antigen (1:100, Merck Millipore), IBA1(1:3000, WAKO), GFAP(1:500, Sigma), Tuj1 (1:500, Biolegend), APC-CC1(1:200, Abcam), CSPG (1:200, sigma), Pdgfrb (1:200, Abcam), Olig2 (1:200,EMD millipore), NG2 (1:200, EMD Millipore), Nestin (1:200, EMD Millipore) GFP (1:500, Abcam, Cambridge, UK), Tomato-lectin (1:500, Sigma), and ED1 (1:200, AbD Serotec, Kidlington, UK) at 4°C overnight. Antibody reactions were visualized using an ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Alternatively, brains were removed after perfusion and cryoprotected in 30% sucrose in 0.1 M phosphate buffer (pH 7.4) overnight. The cryopreserved brains were sectioned to produce 30 μm-thick sections which
were then incubated with Alexa Fluor 488- or 568-conjugated anti-IgG secondary antibodies (Life Technologies, Carlsbad, CA, USA). For quantification of Lectin-, GFAP-, IBA1-, ED1-, GFP-, DCx-positive cells in the ischemic penumbra, 5 mm coronal sections from the ischemic core (Antero-Posterior, + 1.2 mm to −0.8 mm from the bregma) were prepared from three animals. Both light microscopic and fluorescence images were acquired using an AxioScan.Z1 slide scanner (Zeiss, Jena, Germany), and the total number of immunoreactive cells in 1 mm2 region of interest in the peri-infarct area of the cortex and striatum were counted using ZEN software (Blue Edition, Zeiss). To trace TdTomato and GFP-positive cells in TdTomato and TdTomato-GFP reporter mice, 30 mm parasagittal sections were scanned with an AxioScan.Z1 equipped with an HBO lamp (HXP 120V, LEJ, Jena, Germany). The characteristics of TdTomato and GFP-positive cells in terms of their co-expression of various markers used were analyzed in the cortex, Striatum, sub-ventricular Zone (SVZ) and Sub-granular Zone of the dentate gyrus.
22. Statistical analysis
Statistical analyses were carried out using Sigmaplot (Systat Software Inc, San Jose, CA, USA). Data were analyzed by Student’s t test or one-way analysis of variance (ANOVA). Significant differences were further evaluated using Tukey’s honest significant difference post-hoc test. A p-value ˂ 0.05 was considered statistically significant. All data are expressed as mean ± S.E.
RESULTS
Part A. Chronic stroke animal models and therapeutic effects of MSC/Ngn1 cells in chronic stroke.
1. Characterization of chronic stroke animal models.
To generate a chronic stroke model of rat and mice, the intraluminal transient middle cerebral artery occlusion method was carried out in rats and mice (Fig 1A). Twenty four hours after reperfusion, TTC staining showed that both rat and mouse stroke models developed clear infarction (Non-stained area) in the ipsilateral striatum and dorsolateral cortex verifying proper occlusion of the Middle cerebral artery (Fig 1B) We also assessed the structural integrity of the ischemic brain over the same 28-day period using MRI. The hypointense areas in the ipsilateral hemisphere were evident at the acute phase of both rat and mouse models. In the chronic phase, rat MCAo showed an infarct cavity as shown by hypointense areas in the ipsilateral hemisphere (Fig 2A). However, In a mouse model of chronic stroke, MRI did not show any hypointense areas suggesting that the infarct cavity was not formed in a chronic stroke mouse model (Fig 2B). Sham-operated animals did not show any hypointense areas in the brain. Further, we evaluated the tissue integrity of chronic stroke animal models by Hematoxylin & Eosin and Cresyl-violet staining at 1 month after ischemia-reperfusion injury and compared with the histology of acute stroke i.e.,
3 days after reperfusion and sham-operated animals (Fig 2 C, D). Hematoxylin
& Eosin and Cresyl violet staining of coronal brain sections at the level of striatum grossly depicts the edematous ipsilateral hemisphere with the faintly stained ischemic area in acute stroke while atrophied ipsilateral hemisphere in chronic stroke. The tissue integrity of the chronic stroke rat brain was worse than a chronic stroke mouse brain. Furthermore, the presence of cells bearing pycnotic nuclei and vacuolated cytoplasm were readily present in the ischemic area of the acute stroke brain. In the chronic stroke brain, cells bearing non-neuronal morphology occupied the corresponding ischemic core region.
Fig 1. Induction of middle cerebral artery occlusion (MCAo) Rat and mouse model.
A. Transient cerebral ischemia was induced by inserting silicon-coated nylon suture (4-0 for rats and 6-0 for mice) via the right external carotid artery (ECA) through the internal carotid artery (ICA) to block middle cerebral artery (MCA).
B. Infarction resulted from the occlusion of MCA was evaluated by TTC staining 24 hours after reperfusion.
Fig 2. A general overview of a brain tissue structure of chronic stroke animal models. The changes in brain tissue integrity were monitored by MRI, H&E staining and cresyl violet staining from acute (day 3) to chronic phase of stroke (Day 30) in both rat and mouse model of stroke. MCA occlusion resulted in infarction of striatum and dorsolateral cortex of both rat and mouse brain in the acute phase of the stroke. In the chronic phase, rat MCAo resulted in the infarct cavity while chronic mouse MCAo resulted in atrophied ipsilateral brain hemisphere (A, B). Hematoxylin & Eosin and Cresyl violet staining showed significant loss of neural cells in acute stroke. Cells with Pycnotic nuclei and vacuolated cytoplasm are evident in ischemic core and penumbra regions. In the chronic phase, the infiltration of glial cells (non-neuronal morphology) is evident in ischemic core and penumbra regions of the ischemic brain (C, D).
Immunostaining against the neuronal marker, NeuN, and astroglial marker, GFAP showed large NeuN and GFAP non-reactive areas corresponding to ischemic region suggestive of on-going cell death mechanisms in acute stroke brain. In the chronic phase, there were sparsely distributed NeuN+ Neurons in the peri-infarct region while GFAP+ cells made a glial scar delineating ischemic and normal viable tissue. Iba1+ microglia/macrophages were readily observed in the ischemic region of acute stroke brain and persisted in the chronic phase of the stroke (Fig 3 A, B). In the chronic phase of ischemic stroke, there is the presence of growth inhibitory glial scar forming a fine border between ischemic core and normal viable brain tissue (4 A). This glial scar primarily consists of reactive astrocytes, activated macrophages/microglia, and extracellular matrix molecules, predominantly chondroitin sulfate proteoglycans (CSPGs). We found that, in the brain of chronic stroke brain, GFAP positive astrocytes became hypertrophied and elongated their processes from penumbra into the infarct core (4 B). These astrocytes strongly upregulated GFAP protein, a hallmark of astrogliosis responding to ischemic stroke. We were also able to observe that the pronounced change in astrocytic morphology and GFAP expression in reactive astrocytes was also accompanied by the upregulation of intermediate filament protein, Nestin. Double immune-histochemical studies showed that GFAP-positive reactive astrocytes in the glial scar expressed a high amount of extracellular matrix-like Neurocan and CSPGs, but did not overlap with ED1
positive activated microglia/macrophages. GFAP and ED1 positive cells along with extracellular matrix molecules neurocan and CSPGs formed a layer around the ischemic lesion, suggesting the presence of inhibitory glial scar in the chronic stroke rat brain. The glial scar is positioned in such a way that it forms a border around the injury site and acts as a neuroprotective barrier to evading inflammatory cells.
Fig. 3. Temporal changes in neural cells in acute (Day 3) and chronic stroke (Day 30). Immunostaining showed large NeuN and GFAP non-reactive areas corresponding to ischemic region suggestive of on-going cell death mechanisms in acute stroke brain. Iba1 immunostaining revealed activation of resident microglia and infiltration of macrophages into the ischemic brain in the acute phase of the stroke. In the chronic phase, there were sparsely distributed NeuN+
Neurons in the peri-infarct region while GFAP+ cells made a dense glial scar delineating ischemic and normal viable brain tissue. Iba1+
microglia/macrophages with activated morphology densely populated in the ischemic brain in the chronic phase of the stroke.
Fig. 4.Characterization of Glial Scar in chronic stroke. A. Low magnification image of a chronic stroke brain stained with GFAP & MAP2 and Region of interest for characterizing glial Scar. B. In the chronic phase of ischemic stroke, there is the presence of growth inhibitory glial scar forming a fine border between ischemic core and normal viable brain tissue. This glial scar primarily consists of reactive astrocytes (GFAP/Nestin+), activated macrophages/microglia (Iba1/ED1+) and extracellular matrix molecules, predominantly chondroitin sulfate proteoglycans (CSPGs). Note GFAP positive astrocytes became hypertrophied and elongated their processes from penumbra
into the infarct core.
Overall, the MCAo mice exhibited poor performance in behavioral tests during the first week after ischemia, but their performance improved spontaneously over the time. The MCAo animals consistently showed higher NSS score compared to the sham group through the testing period (Fig. 5A) In corner tests, normal or sham-operated mice displayed similar tendency in right and left turning behavior when the animals reached the corner. However, the
Overall, the MCAo mice exhibited poor performance in behavioral tests during the first week after ischemia, but their performance improved spontaneously over the time. The MCAo animals consistently showed higher NSS score compared to the sham group through the testing period (Fig. 5A) In corner tests, normal or sham-operated mice displayed similar tendency in right and left turning behavior when the animals reached the corner. However, the