Kuniaki Harada

I.V. infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat.

I.V. delivery of mesenchymal stem cells prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischemia models. Administration of the brain-derived neurotrophic factor to the infarction site has also been demonstrated to be neuroprotective. To test the hypothesis that brain-derived neurotrophic factor contributes to the therapeutic benefits of mesenchymal stem cell delivery, we compared the efficacy of systemic delivery of human mesenchymal stem cells and human mesenchymal stem cells transfected with a fiber-mutant F/RGD adenovirus vector with a brain-derived neurotrophic factor gene (brain-derived neurotrophic factor-human mesenchymal stem cells). A permanent middle cerebral artery occlusion was induced by intraluminal vascular occlusion with a microfilament. Human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells were i.v. injected into the rats 6 h after middle cerebral artery occlusion. Lesion size was assessed at 6 h, 1, 3 and 7 days using MR imaging, and histological methods. Functional outcome was assessed using the treadmill stress test. Both human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells reduced lesion volume and elicited functional improvement compared with the control sham group, but the effect was greater in the brain-derived neurotrophic factor-human mesenchymal stem cell group. ELISA analysis of the infarcted hemisphere revealed an increase in brain-derived neurotrophic factor in the human mesenchymal stem cell groups, but a greater increase in the brain-derived neurotrophic factor-human mesenchymal stem cell group. These data support the hypothesis that brain-derived neurotrophic factor contributes to neuroprotection in cerebral ischemia and cellular delivery of brain-derived neurotrophic factor can be achieved by i.v. delivery of human mesenchymal stem cells.

Intravenous infusion of immortalized human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat

Intravenous infusion of bone marrow cells has demonstrated therapeutic efficacy in animal models of cerebral ischemia and spinal cord injury. We intravenously delivered human mesenchymal stem cells (SH2+, SH3+, CD34-, and CD45-) immortalized with a human-telomerase gene (hTERT-MSCs) and transfected with eGFP or LacZ into rats 12 h after induction of transient middle cerebral artery occlusion (MCAO), to study their potential therapeutic benefit. hTERT-MSCs were delivered at 12 h after lesion induction. Lesion size was assessed using MR imaging and spectroscopy, and histological methods. Functional outcome was assessed using the Morris water maze and a treadmill test. Intravenous delivery of hTERT-MSCs reduced lesion volume and the magnitude of the reduction and functional improvement was positively correlated with the number of cells injected. The reduction of lesion size could be assessed in vivo with MRI and MRS and was correlated with subsequent histological examination of the brain. This work demonstrates that highly purified hTERT-MSCs reduce cerebral infarction volume and improve functional outcome.

Therapeutic benefits by human mesenchymal stem cells (hMSCs) and Ang-1 gene-modified hMSCs after cerebral ischemia

Transplantation of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has been reported to ameliorate functional deficits after cerebral artery occlusion in rats. Although several hypotheses to account for these therapeutic effects have been suggested, current thinking is that both neuroprotection and angiogenesis are primarily responsible. In this study, we compared the effects of hMSCs and angiopoietin-1 gene-modified hMSCs (Ang-hMSCs) intravenously infused into rats 6 h after permanent middle cerebral artery occlusion. Magnetic resonance imaging and histologic analyses revealed that rats receiving hMSCs or Ang-hMSCs exhibited comparable reduction in gross lesion volume as compared with the control group. Although both cell types indeed improved angiogenesis near the border of the ischemic lesions, neovascularization and regional cerebral blood flow were greater in some border areas in Ang-hMSC group. Both hMSC- and Ang-hMSC-treated rats showed greater improved functional recovery in the treadmill stress test than did control rats, but the Ang-hMSC group was greater. These results indicate the intravenous administration of genetically modified hMSCs to express angiopoietin has a similar effect on reducing lesion volume as hMSCs, but the Ang-hMSC group showed enhanced regions of increased angiogenesis at the lesion border, and modest additional improvement in functional outcome.

Intravenous administration of glial cell line-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in the adult rat

Intravenous administration of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has been reported to ameliorate functional deficits after cerebral artery occlusion in rats. Several hypotheses to account for these therapeutic effects have been suggested, and current thinking is that neuroprotection rather than neurogenesis is responsible. To enhance the therapeutic benefits of hMSCs potentially, we transfected hMSCs with the glial cell line‐derived neurotrophic factor (GDNF) gene using a fiber‐mutant F/RGD adenovirus vector and investigated whether GDNF gene‐modified hMSCs (GDNF‐hMSCs) could contribute to functional recovery in a rat permanent middle cerebral artery occlusion (MCAO) model. We induced MCAO by using intraluminal vascular occlusion, and GDNF‐hMSCs were intravenously infused into the rats 3 hr later. MRI and behavioral analyses revealed that rats receiving GDNF‐hMSCs or hMSCs exhibited increased recovery from ischemia compared with the control group, but the effect was greater in the GDNF‐hMSC group. Thus, these results suggest that intravenous administration of hMSCs transfected with the GDNF gene using a fiber‐mutant adenovirus vector may be useful in the cerebral ischemia and may represent a new strategy for the treatment of stroke.

Therapeutic benefits of angiogenetic gene-modified human mesenchymal stem cells after cerebral ischemia

Intravenous transplantation of human mesenchymal stem cells (hMSCs) expanded from adult bone marrow ameliorates functional deficits in rat cerebral infarction models. Several hypotheses to account for the therapeutic mechanisms have been suggested, but angiogenesis is thought to be of critical importance. Recently, we have reported the therapeutic benefits of hMSCs which have been transfected with the angiopoietin-1 gene in a rat permanent middle cerebral artery occlusion (MCAO) model. To potentially enhance the therapeutic effects of angiopoietin-1 gene-modified hMSC (Ang-hMSC), we transfected hMSCs with the angiopoietin-1 gene and the VEGF gene, and investigated whether the combination of Ang-1 and VEGF gene-modified hMSCs (Ang-VEGF-hMSC) contribute to functional recovery in a rat MCAO model. We induced MCAO using intraluminal vascular occlusion, and hMSCs, Ang-hMSCs, VEGF-hMSCs or Ang-VEGF-hMSCs were intravenously infused 6 h later. MRI and behavioral analyses revealed that rats receiving Ang-VEGF-hMSCs showed the greatest structural-functional recovery as compared to the other groups. These results suggest that intravenous administration of hMSCs transfected with the angiopoietin-1 and VEGF gene using a fiber-mutant adenovirus vector may represent a new strategy for the treatment of ischemia.

In vivo quantification of the metabolites in normal brain and brain tumors by proton MR spectroscopy using water as an internal standard

Metabolite concentrations in normal adult brains and in gliomas were quantitatively analyzed by in vivo proton magnetic resonance spectroscopy (MRS) using the fully relaxed water signal as an internal standard. Between January 1998 and October 2001, 28 healthy volunteers and 18 patients with gliomas were examined by in vivo proton MRS. Single voxel spectra were acquired using the point-resolved spectroscopic pulse sequence with a 1.5-T scanner (TR/TE/Ave = 3000 ms/30 ms/64). The calculated concentrations of N-acetyl-aspartate (NAA), creatine (Cre), choline (Cho), and water (H2O) in the normal hemispheric white matter were 23.59 +/- 2.62 mM (mean +/- SD), 13.06 +/- 1.8 mM, 4.28 +/- 0.8 mM, and 47280.96 +/- 5414.85 mM, respectively. The metabolite concentrations were not necessarily uniform in different parts of the brain. The concentrations of NAA and Cre decreased in all gliomas (p < 0.001). The NAA/Cho and NAA/H2O ratios can distinguish the normal brain from gliomas, and low-grade astrocytoma from high-grade group (p < 0.001). The concentration of taurine (Tau) in medulloblastomas was 29.64 +/- 5.76 mM. This is the first quantitative analysis of Tau in medulloblastoma in vivo and confirms earlier in vitro findings.

Optimization of a therapeutic protocol for intravenous injection of human mesenchymal stem cells after cerebral ischemia in adult rats.

The systemic injection of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has therapeutic benefits after cerebral artery occlusion in rats, and may have multiple therapeutic effects at various sites and times within the lesion as the cells respond to a particular pathological microenvironment. However, the comparative therapeutic benefits of multiple injections of hMSCs at different time points after cerebral artery occlusion in rats remain unclear. In this study, we induced middle cerebral artery occlusion (MCAO) in rats using intra-luminal vascular occlusion, and infused hMSCs intravenously at a single 6 h time point (low and high cell doses) and various multiple time points after MCAO. From MRI analyses lesion volume was reduced in all hMSC cell injection groups as compared to serum alone injections. However, the greatest therapeutic benefit was achieved following a single high cell dose injection at 6 h post-MCAO, rather than multiple lower cell infusions over multiple time points. Three-dimensional analysis of capillary vessels in the lesion indicated that the capillary volume was equally increased in all of the cell-injected groups. Thus, differences in functional outcome in the hMSC transplantation subgroups are not likely the result of differences in angiogenesis, but rather from differences in neuroprotective effects.

Therapeutic benefits of human mesenchymal stem cells derived from bone marrow after global cerebral ischemia.

Although intravenous delivery of mesenchymal stem cells (MSCs) prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat middle cerebral artery occlusion models, there are few reports of MSC treatment in global cerebral ischemia. We utilized a global cerebral ischemia model induced by arresting the heart with a combination of hypovolemia and intracardiac injections of a cold potassium chloride solution in order to study the potential therapeutic benefits of human mesenchymal stem cells (hMSCs) on global cerebral ischemia. hMSCs were intravenously injected into the rats 3 h after resuscitation from cardiac arrest. The effects on structural and functional outcome of hMSC were assessed at 5 h and 1, 3, and 7 days using magnetic resonance spectroscopy (MRS), histology, and cognitive functional analysis. Intravenous delivery of hMSCs reduced the Lac/Cr ratios, nuclear DNA fragmentation, neuronal loss, and elicited functional improvement compared with the control sham group. Enzyme-linked immunosorbent assay (ELISA) of the hippocampus revealed an increase in BDNF in hMSC-treated group. These data suggest that intravenous delivery of hMSC may have a therapeutic effect in global cerebral ischemia.

Age-Related Changes in Intramyocellular Lipid in Humans by in vivo 1H-MR Spectroscopy

Background: It is considered that the increasing intramyocellular lipid (IMCL) affects health risks and muscle attenuation. Though body fat increases significantly with age in lean humans, it is not known whether IMCL increases or not. In this study, we investigated the changes with age in IMCL concentrations in skeletal muscles using 1H-MR spectroscopy and studied them in relation to body fat percentage, waist-hip ratio, and blood components. Methods: Twenty-four lean young (age 21.2 ± 1.9, BMI 21.5 ± 1.8) and 23 lean old (age 70.9 ± 2.4, BMI 21.7 ± 1.3) subjects took part in the study. Subjects were grouped by gender into age- and BMI-matched young and old groups. The 1H-MRS was obtained from the tibialis anterior (TA), medial gastrocnemius (MG) and soleus (SOL) muscles. Results: The IMCL content in SOL and MG in the old was found to be higher (p < 0.01) than that in the young. No age difference in IMCL content in TA was found. IMCL concentrations in SOL were higher than those in MG and TA in the order of SOL > MG > TA (p < 0.01). IMCL content correlated significantly with waist-hip ratio in all skeletal muscles. A significant relationship was observed between percent body fat and IMCL in TA and MG (p < 0.05). However, no correlation was found between IMCL content in each muscle and BMI. The IMCL content in all skeletal muscles significantly correlated with HbA1c, triglyceride, total cholesterol and LDL cholesterol concentrations. Conclusion: These results suggest that increased IMCL in both lean older men and women might be related to body composition, blood lipids and lipoprotein profiles, and that this might affect muscle attenuation.

Systemic Oxidative Stress Is Associated With Lower Aerobic Capacity and Impaired Skeletal Muscle Energy Metabolism in Patients With Metabolic Syndrome

OBJECTIVE Systemic oxidative stress is associated with insulin resistance and obesity. We tested the hypothesis that systemic oxidative stress is linked to lower aerobic capacity and skeletal muscle dysfunction in metabolic syndrome (MetS). RESEARCH DESIGN AND METHODS The incremental exercise testing with cycle ergometer was performed in 14 male patients with MetS and 13 age-, sex-, and activity-matched healthy subjects. Systemic lipid peroxidation was assessed by serum thiobarbituric acid reactive substances (TBARS), and systemic antioxidant defense capacity was assessed by serum total thiols and enzymatic activity of superoxide dismutase (SOD). To assess skeletal muscle energy metabolism, we measured high-energy phosphates in the calf muscle during plantar flexion exercise and intramyocellular lipid (IMCL) in the resting leg muscle, using 31P- and 1proton-magnetic resonance spectroscopy, respectively. RESULTS Serum TBARS were elevated (12.4 ± 7.1 vs. 3.7 ± 1.1 μmol/L; P < 0.01), and serum total thiols and SOD activity were decreased (290.8 ± 51.2 vs. 398.7 ± 105.2 μmol/L, P < 0.01; and 22.2 ± 8.4 vs. 31.5 ± 8.5 units/L, P < 0.05, respectively) in patients with MetS compared with healthy subjects. Peak VO2 and anaerobic threshold normalized to body weight were significantly lower in MetS patients by 25 and 31%, respectively, and inversely correlated with serum TBARS (r = −0.49 and r = −0.50, respectively). Moreover, muscle phosphocreatine loss during exercise was 1.4-fold greater in patients with MetS (P < 0.05), and IMCL content was 2.9-fold higher in patients with MetS (P < 0.01), indicating impaired skeletal muscle energy metabolism, and these indices positively correlated with serum TBARS (r = 0.45 and r = 0.63, respectively). CONCLUSIONS Systemic oxidative stress was associated with lower aerobic capacity and impaired skeletal muscle energy metabolism in patients with MetS.