Yuko Kataoka-Sasaki

Intravenous infusion of mesenchymal stem cells promotes functional recovery in a model of chronic spinal cord injury.

Intravenous infusion of mesenchymal stem cells (MSCs) derived from adult bone marrow improves behavioral function in rat models of spinal cord injury (SCI). However, most studies have focused on the acute or subacute phase of SCI. In the present study, MSCs derived from bone marrow of rats were intravenously infused 10weeks after the induction of a severe contusive SCI. Open field locomotor function was assessed weekly until 20weeks post-SCI. Motor recovery was greater in the MSC-treated group with rapid improvement beginning in earlier post-infusion times than in the vehicle-treated group. Blood spinal cord barrier (BSCB) integrity was assessed by the intravenous infusion of Evans Blue (EvB) with spectrophotometric quantitation of its leakage into the parenchyma. In MSC-treated rats, BSCB leakage was reduced. Immunohistochemical staining for RECA-1 and PDGFR-β showed increased microvasculature/repair-neovascularization in MSC-treated rats. There was extensive remyelination around the lesion center and increased sprouting of the corticospinal tract and serotonergic fibers after MSC infusion. These results indicate that the systemic infusion of MSCs results in functional improvement that is associated with structural changes in the chronically injured spinal cord including stabilization of the BSCB, axonal sprouting/regeneration and remyelination.

Intravenous Preload of Mesenchymal Stem Cells Rescues Erectile Function in a Rat Model of Cavernous Nerve Injury

INTRODUCTION We evaluated the potential preventive effects and mechanisms of intravenously preloaded mesenchymal stem cells (MSCs) for erectile dysfunction (ED) in a cavernous nerve (CN) injury model. METHODS Male Sprague-Dawley (SD) rats were used for this study. Rats were randomized into two groups. One group was intravenously preloaded with MSCs (1.0 × 10(6) cells in 1 mL total fluid volume) and the other was infused with medium alone (1 mL Dulbeccos modified Eagles medium [DMEM]) for sham control, respectively. Crushed CN injury was induced immediately after infusion. The surgeon was blind to the experimental conditions (MSC or medium). MAIN OUTCOME MEASURES To assess erectile function, we measured the intracavernous pressure (ICP) and arterial pressure (AP) at 1 hour and 2 weeks after CN injury. After measuring the initial ICP/AP of pre-injury (normal) male SD rats, they were randomized into the two groups and infused with MSCs or medium. PKH26-labelled MSCs were used for tracking. To investigate the mRNA expression levels of neurotrophins in the major pelvic ganglia (MPG), we performed real-time quantitative real-time polymerase chain reaction. RESULTS The reduction of ICP/AP and area under the curve of ICP (ICP-AUC) in the MSC group was significantly lower than in the DMEM group (P < 0.05; P < 0.05) at 1 hour. The ICP/AP and ICP-AUC at 2 weeks post-injury in the MSC group was significantly higher than in the DMEM group (P < 0.01; P < 0.05). The preloaded PKH26-labelled MSCs were detected in the MPG and CN using confocal microscopy indicating homing of the cells to the injured nerve and ganglia. Glia cell-derived neurotrophic factor (GDNF) and neurturin, which are important neurotrophic factors for erection, had expression levels in MPG significantly higher in the MSC group than in the DMEM group (P < 0.01, 0.05). CONCLUSION Intravenous preload of MSCs before a CN injury may prevent or reduce experimental ED.

Intravenous infusion of mesenchymal stem cells inhibits intracranial hemorrhage after recombinant tissue plasminogen activator therapy for transient middle cerebral artery occlusion in rats

OBJECTIVE Reperfusion therapy with intravenous recombinant tissue plasminogen activator (rtPA) is the standard of care for acute ischemic stroke. However, hemorrhagic complications can result. Intravenous infusion of mesenchymal stem cells (MSCs) reduces stroke volume and improves behavioral function in experimental stroke models. One suggested therapeutic mechanism is inhibition of vascular endothelial dysfunction. The objective of this study was to determine whether MSCs suppress hemorrhagic events after rtPA therapy in the acute phase of transient middle cerebral artery occlusion (tMCAO) in rats. METHODS After induction of tMCAO, 4 groups were studied: 1) normal saline [NS]+vehicle, 2) rtPA+vehicle, 3) NS+MSCs, and 4) rtPA+MSCs. The incidence rate of intracerebral hemorrhage, both hemorrhagic and ischemic volume, and behavioral performance were examined. Matrix metalloproteinase-9 (MMP-9) levels in the brain were assessed with zymography. Quantitative analysis of regional cerebral blood flow (rCBF) was performed to assess hemodynamic change in the ischemic lesion. RESULTS The MSC-treated groups (Groups 3 and 4) experienced a greater reduction in the incidence rate of intracerebral hemorrhage and hemorrhagic volume 1 day after tMCAO even if rtPA was received. The application of rtPA enhanced activation of MMP-9, but MSCs inhibited MMP-9 activation. Behavioral testing indicated that both MSC-infused groups had greater improvement than non-MSC groups had, but rtPA+MSCs provided greater improvement than MSCs alone. The rCBF ratio of rtPA groups (Groups 2 and 4) was similar at 2 hours after reperfusion of tMCAO, but both were greater than that in non-rtPA groups. CONCLUSIONS Infused MSCs may inhibit endothelial dysfunction to suppress hemorrhagic events and facilitate functional outcome. Combined therapy of infused MSCs after rtPA therapy facilitated early behavioral recovery.

Synergic Effects of Rehabilitation and Intravenous Infusion of Mesenchymal Stem Cells After Stroke in Rats

Background Intravenous infusion of mesenchymal stem cells (MSCs) derived from adult bone marrow improves behavioral function in rat stroke models. Rehabilitation therapy through physical exercise also provides therapeutic efficacy for cerebral ischemia. Objective The purpose of this study was to investigate whether synergic effects of daily rehabilitation and intravenous infusion of MSCs has therapeutic effects after stroke in rats. Design This was an experimental study. Methods A permanent middle cerebral artery occlusion (MCAO) was induced by intraluminal vascular occlusion with a microfilament. Four experimental groups were studied: group 1 (vehicle only, n=10), group 2 (vehicle + exercise, n=10), group 3 (MSCs only, n=10), and group 4 (MSCs + exercise, n=10). Rat MSCs were intravenously infused at 6 hours after MCAO, and the rats received daily rehabilitation with treadmill running exercise for 20 minutes. Lesion size was assessed at 1, 14, and 35 days using magnetic resonance imaging. Functional outcome was assessed using the Limb Placement Test. Results Both combined therapy and MSC infusion reduced lesion volume, induced synaptogenesis, and elicited functional improvement compared with the groups without MSC infusion, but the effect was greater in the combined therapy group. Limitations A limitation of this study is that the results were limited to an animal model and cannot be generalized to humans. Conclusions The data indicate that the combined therapy of daily rehabilitation and intravenous infusion of MSCs improved functional outcome in a rat MCAO model.

Intravenous Infusion of Bone Marrow–Derived Mesenchymal Stem Cells Reduces Erectile Dysfunction Following Cavernous Nerve Injury in Rats

Introduction Intravenous preload (delivered before cavernous nerve [CN] injury) of bone marrow–derived mesenchymal stem cells (MSCs) can prevent or decrease postoperative erectile dysfunction (J Sex Med 2015;12:1713–1721). In the present study, the potential therapeutic effects of intravenously administered MSCs on postoperative erectile dysfunction were evaluated in a rat model of CN injury. Methods Male Sprague-Dawley rats were randomized into 2 groups after electric CN injury. Intravenous infusion of bone marrow–derived MSCs (1.0 × 106 cells in Dulbeccos modified Eagles medium 1 mL) or vehicle (Dulbeccos modified Eagles medium 1 mL) was performed 3 hours after electrocautery-induced CN injury. Main Outcome Measures To assess erectile function, we measured intracavernous pressure at 4 weeks after MSC or vehicle infusion. Histologic examinations were performed to investigate neuronal innervation and inhibition of smooth muscle atrophy. Green fluorescent protein–positive bone marrow–derived MSCs were used for cell tracking. To investigate mRNA expression levels of neurotrophins in the major pelvic ganglia (MPGs), quantitative real-time polymerase chain reaction was performed. Results The decrease of intracavernous pressure corrected for arterial pressure and area under the curve of intracavernous pressure in the bone marrow–derived MSC group was significantly lower than that in the vehicle group at 4 weeks after infusion (P < .05). Retrograde neuronal tracing indicated that the MSC group had a larger number of FluoroGold-positive neurons in the MPGs compared with the vehicle group. The ratio of smooth muscle to collagen in the MSC group was significantly higher than in the vehicle group. Green fluorescent protein–positive bone marrow–derived MSCs were detected in the MPGs and injured CNs using confocal microscopy, indicating homing of cells to the MPGs and injured CNs. Brain-derived neurotrophic factor and glial cell-derived neurotrophic factor expression levels in the MPGs were significantly higher in the MSC group than in the vehicle group (P < .01). Conclusion Intravenous infusion of bone marrow–derived MSCs after CN injury might have therapeutic efficacy in experimental erectile dysfunction. Matsuda Y, Sasaki M, Kataoka-Sasaki Y, et al. Intravenous Infusion of Bone Marrow–Derived Mesenchymal Stem Cells Reduces Erectile Dysfunction Following Cavernous Nerve Injury in Rats. Sex Med 2018;6:49–57.

ACTC1 as an invasion and prognosis marker in glioma

OBJECTIVE Glioma is a major class of brain tumors, and glioblastoma (GBM) is the most aggressive and malignant type. The nature of tumor invasion makes surgical removal difficult, which results in remote recurrence. The present study focused on glioma invasion and investigated the expression of actin, alpha cardiac muscle 1 (ACTC1), which is 1 of 6 actin families implicated in cell motility. METHODS mRNA expression of ACTC1 expression was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) in 47 formalin-fixed, paraffin-embedded glioma tissues that were graded according to WHO criteria: Grade I (n = 4); Grade II (n = 12); Grade III (n = 6); and Grade IV (n = 25). Survival was analyzed using the Kaplan-Meier method. The relationships between ACTC1 expression and clinical features such as radiological findings at the time of diagnosis and recurrence, patient age, Karnofsky Performance Scale status (KPS), and the MIB-1 index were evaluated. RESULTS The incidence of ACTC1 expression as a qualitative assessment gradually increased according to WHO grade. The hazard ratio for the median overall survival (mOS) of the patients with ACTC1-positive high-grade gliomas as compared with the ACTC1-negative group was 2.96 (95% CI, 1.03-8.56). The mOS was 6.28 years in the ACTC1-negative group and 1.26 years in the positive group (p = 0.037). In GBM patients, the hazard ratio for mOS in the ACTC1-positive GBMs as compared with the ACTC1-negative group was 2.86 (95% CI 0.97-8.45). mOS was 3.20 years for patients with ACTC1-negative GBMs and 1.08 years for patients with ACTC1-positive GBMs (p = 0.048). By the radiological findings, 42.9% of ACTC1-positive GBM patients demonstrated invasion toward the contralateral cerebral hemisphere at the time of diagnosis, although no invasion was observed in ACTC1-negative GBM patients (p = 0.013). The recurrence rate of GBM was 87.5% in the ACTC1-positive group; in contrast, none of the ACTC1-negative patients demonstrated distant recurrence (0.007). No remarkable relationship was demonstrated among ACTC1 expression and patient age, KPS, and the MIB-1 index. CONCLUSIONS ACTC1 may serve as a novel independent prognostic and invasion marker in GBM.

Intravenous infusion of mesenchymal stem cells reduces epileptogenesis in a rat model of status epilepticus

OBJECTIVE Status epilepticus (SE) causes neuronal cell death, aberrant mossy fiber sprouting (MFS), and cognitive deteriorations. The present study tested the hypothesis that systemically infused mesenchymal stem cells (MSCs) reduce epileptogenesis by inhibiting neuronal cell death and suppressing aberrant MFS, leading to cognitive function preservation in a rat model of epilepsy. METHODS SE was induced using the lithium-pilocarpine injection model. The seizure frequency was scored using a video-monitoring system and the Morris water maze test was carried out to evaluate cognitive function. Comparisons were made between MSCs- and vehicle-infused rats. Immunohistochemical staining was performed to detect Green fluorescent protein (GFP)+ MSCs and to quantify the number of GAD67+ and NeuN+ neurons in the hippocampus. Manganese-enhanced magnetic resonance imaging (MEMRI) and Timm staining were also performed to assess the MFS. RESULTS MSC infusion inhibited epileptogenesis and preserved cognitive function after SE. The infused GFP+ MSCs were accumulated in the hippocampus and were associated with the preservation of GAD67+ and NeuN+ hippocampal neurons. Furthermore, the MSC infusion suppressed the aberrant MFS in the hippocampus as evidenced by MEMRI and Timm staining. CONCLUSIONS This study demonstrated that the intravenous infusion of MSCs mitigated epileptogenesis, thus advancing MSCs as an effective approach for epilepsy in clinical practice.

Co-expression of tissue factor and IL-6 in immature endothelial cells of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are congenital abnormal clusters of capillaries that are prone to leaking and thought to result from a disorder of endothelial cells. The underlying pathology of CCM is not fully understood. We analyzed the expression of tissue factor (TF) and interleukin-6 (IL-6) in CCMs to determine the association of TF and IL-6 with clinical and pathological findings. Thirteen cases of operative specimens of sporadic CCMs were included in this study. The expression of messenger RNA of TF and IL-6 was assayed and the association with clinical factors was investigated. Then, the distribution of TF and IL-6 was examined with immunofluorescence. The mRNA expression of TF of CCMs was significantly higher than that of the control (p=0.017), and was correlated with the number of hemorrhage appearances (p=0.190, ρ=0.62). The mRNA expression level of IL-6 was significantly correlated with the mRNA expression level of TF (p=0.034, ρ=0.58). Examination of immunostained sections indicated that TF+ cells were also positive for IL-6, and distributed around normal endothelial cells. Moreover, the TF+/IL-6+ cells expressed CD31 and VEGFR2. The expressions of IL-6 and TF were correlated, and both were present in the same immature endothelial cells. TF is elevated in CCM and might mediate progressive events. These factors may play a prognostic role in CCM.

Actin, alpha, cardiac muscle 1 ( ACTC1 ) knockdown inhibits the migration of glioblastoma cells in vitro

BACKGROUND Recurrence is inevitable in glioblastomas (GBMs) and requires multifactorial processes. One of the factors that cause recurrence is the strong migratory capacity of GBM cells. We recently reported that actin, alpha, cardiac muscle 1 (ACTC1) could serve as a marker to detect GBM migration in clinical cases. OBJECTIVE This study aimed to clarify whether the knockdown of highly expressed ACTC1 can inhibit the migratory capacity of cells in the GBM cell line. METHODS ACTC1 expression was examined using immunocytochemistry and droplet digital polymerase chain reaction. The motility of GBM cells that were either treated with siRNA to knock down ACTC1 or untreated were investigated using a time-lapse study in vitro. RESULTS The relatively high ACTC1 expression was confirmed in a GBM cell line, i.e., U87MG. The ACTC1 expression in U87MG cells was significantly inhibited by ACTC1-siRNA (p < 0.05). A cell movement tracking assay using time-lapse imaging demonstrated the inhibition of U87MG cell migration by ACTC1 knockdown. The quantitative cell migration analysis demonstrated that the distance traversed during 72 h was 3607 ± 458 (median ± SD) μm by untreated U87MG cells and 3570 ± 748 μm by negative control siRNA-treated cells. However, the distance migrated by ACTC1-siRNA-treated cells during 72 h was significantly shorter (1265 ± 457 μm, p < 0.01) than the controls. CONCLUSION ACTC1 knockdown inhibits U87MG cell migration.

Functional recovery after the systemic administration of mesenchymal stem cells in a rat model of neonatal hypoxia-ischemia

The authors intravenously infused mesenchymal stem cells (MSCs) into a rat model of neonatal hypoxia-ischemia and found improvements in functional outcome, increased brain volume, and enhanced synaptogenesis. The results of this animal study suggest that the intravenous administration of MSCs should be further explored as a potential treatment for patients suffering from cerebral palsy after hypoxic-ischemic encephalopathy.