Supplementary MaterialsSupplementary material Suppl_for_CT_1763_R2. in the expression of trophic factors; vascular endothelial growth factor, glial cellCderived neurotrophic factor, and fibroblast growth factor were densely expressed in samples cultured with SS ( 0.01). In addition, SS-MSCs revealed different cell cycleC or aging-associated messenger RNA expression in a later passage, and -galactosidase staining showed the senescence of MSCs observed during culture expansion was lower in MSCs cultured with SS than those cultured with NS or FBS ( 0.01). Several proteins related to the activity of receptors, growth factors, and cytokines were more prevalent in the serum of stroke patients than in that of normal subjects. Neurogenesis and angiogenesis were markedly increased in rats that had received SS-MSCs ( 0.05), and these rats showed significant behavioral improvements RB ( 0.01). Our results indicate that stroke induces a process of recovery via the activation of MSCs. Culture methods for MSCs using SS obtained during the acute phase of a stroke could constitute a novel MSC activation method that is feasible and efficient for the neurorestoration of stroke. = 9, 30.4 18.1 d) and from healthy normal subjects (= 8). Aliquots of serum were stored at ?70 C until ready for use. Patient basal characteristics are provided in Table NVP-BGJ398 inhibition 1. Table 1. Patients Baseline Characteristics. Value(% of female)4 (44.4%)4 (50%)0.819NIHSS (SD)16.71 (4.89)Infarct volume, mL (SD)13.57 (8.73)Risk factors, + ln(and are the initial and final cell numbers, respectively7. The population doubling time (PDT) was calculated using the equation test and fold change was cut off at 1.5. Molecular function analysis was performed using FunRich9. Middle Cerebral Artery Occlusion Model We induced transient middle cerebral artery occlusion (tMCAO) using a previously described intraluminal vascular occlusion method that was modified in our laboratory10. NVP-BGJ398 inhibition Briefly, anesthesia was induced in male Sprague-Dawley rats (7 to 8 wk, 250 to 300 g, Orient Bio Inc., Seongnam, South Korea) with 4% isoflurane and maintained with 1.5% isoflurane in 70% N2O and 30% O2. The temperature was maintained at 37.0 C to 37.5 C (measured rectally) with heating pads throughout the surgery and occlusion period. A 4-0 surgical monofilament nylon suture with a rounded tip was advanced from the left common carotid artery into the lumen of the internal carotid artery until it blocked the origin of the middle cerebral artery. Reperfusion was allowed 90 min after tMCAO by withdrawing the suture NVP-BGJ398 inhibition until the tip cleared the lumen of the common carotid artery. Rats with hemorrhagic transformation or subarachnoid hemorrhage caused by rupture NVP-BGJ398 inhibition of the intracranial artery and rats without observable neurological deficits following MCAO were excluded from further analyses. The regional cerebral blood flow (rCBF) in the MCA territory was measured transcranially by laser Doppler flowmetry (Moor Instruments, Wilmington, DE, USA) via probes placed on ipsilateral hemisphere. CBF was recorded continuously during 10 min of baseline recording, 10 min of ischemia, and 15 min of reperfusion. The reduction in CBF was calculated as percentage of baseline. Rats that failed to show at least 70% rCBF reduction were also excluded from further analyses. In a separate experiment, physiological parameters (blood pressure, pH, pCO2, pO2, Na, K, Ca, glucose, hematocrit, and hemoglobin) were measured at 4 different time points (before MCAO, at 10 min after MCAO, at 10 min after reperfusion, and after treatments, = 5 per group). Femoral artery cannulation was performed for arterial pressure monitoring and arterial blood sampling with heparin tube. The arterial blood pressure was continuously monitored during operation, and arterial blood samples were obtained 5 min prior to ischemia (baseline), 10 min following reperfusion, and 10 min following treatment for blood gas analysis (i-STAT, Abbott Diagnostics, Lake Forest, IL, USA). hMSC Transplantation hMSCs were harvested after being cultured in DMEM with 10% FBS, 10% NS, or 10% SS by passage 3 to 5 5. hMSCs (2 106 cells) were administrated 1 d after tMCAO. The control group received PBS after tMCAO. The suspended hMSCs were slowly injected with a 1-mL syringe into the tail vein of the rats. In this study, a total of 40 rats were equally randomized into 4 groups: the PBS, FBS-hMSCs, NS-hMSCs, and SS-hMSCs. Four animals (2 animals in the PBS group, 1 animal in the NS-hMSCs and SS-hMSCs groups, respectively) died within 24 h after tMCAO, and these animals were excluded. Two animals without observable neurological deficits were excluded in the FBS-hMSCs group. One animal with subarachnoid hemorrhage was excluded each in the NS-hMSCs and SS-hMSCs groups. A total of 32 animals.