Jehoon Yang

Fast isotopic exchange between mitochondria and cytosol in brain revealed by relayed 13C magnetization transfer spectroscopy.

In vivo 13C magnetic resonance spectroscopy has been applied to studying brain metabolic processes by measuring 13C label incorporation into cytosolic pools such as glutamate and aspartate. However, the rate of exchange between mitochondrial α-ketoglutarate/oxaloacetate and cytosolic glutamate/aspartate (Vx) extracted from metabolic modeling has been controversial. Because brain fumarase is exclusively located in the mitochondria, and mitochondrial fumarate is connected to cytosolic aspartate through a chain of fast exchange reactions, it is possible to directly measure Vx from the four-carbon side of the tricarboxylic acid cycle by magnetization transfer. In isoflurane-anesthetized adult rat brain, a relayed 13C magnetization transfer effect on cytosolic aspartate C2 at 53.2ppm was detected after extensive signal averaging with fumarate C2 at 136.1ppm irradiated using selective radiofrequency pulses. Quantitative analysis using Bloch–McConnell equations and a four-site exchange model found that VxE13–19 µmol per g per min (≫VTCA, the tricarboxylic acid cycle rate) when the longitudinal relaxation time of malate C2 was assumed to be within ±33% of that of aspartate C2. If VxEVTCA, the isotopic exchange between mitochondria and cytosol would be too slow on the time scale of 13C longitudinal relaxation to cause a detectable magnetization transfer effect.

Measuring N-acetylaspartate synthesis in vivo using proton magnetic resonance spectroscopy

N-Acetylaspartate (NAA) is an important marker of neuronal function and viability that can be measured using magnetic resonance spectroscopy (MRS). In this paper, we proposed a method to measure NAA synthesis using proton MRS with infusion of uniformly (13)C-labeled glucose, and demonstrated its feasibility in an in vivo study of the rat brain. The rate of (13)C-label incorporation into the acetyl group of NAA was measured using a localized, long echo-time proton MRS method. Signals from the (13)C satellites of the main NAA methyl protons at 2.02 ppm were continuously monitored for 10h. Quantification of the data based on a linear kinetic model showed that NAA synthesis rate in isoflurane-anesthetized rats was 0.19+/-0.02 micromol/g/h (mean+/-standard deviation, n=12).

Elevated endogenous GABA concentration attenuates glutamate–glutamine cycling between neurons and astroglia

In this study, the relationship between endogenous brain GABA concentration and glutamate–glutamine cycling flux (Vcyc) was investigated using in vivo 1H and 1H{13C} magnetic resonance spectroscopy techniques. Graded elevations of brain GABA levels were induced in rat brain after administration of the highly specific GABA-transaminase inhibitor vigabatrin (γ-vinyl-GABA). The glial-specific substrate [2-13C]acetate and 1H{13C} magnetic resonance spectroscopy were used to measure Vcyc at different GABA levels. Significantly reduced Vcyc was found in rats pretreated with vigabatrin. The reduction in group mean Vcyc over the range of GABA concentrations investigated in this study (1.0xa0±xa00.3–5.1xa0±xa00.5xa0μmol/g) was found to be nonlinear: ΔVcyc/Vcycxa0=xa0[GABA (μmol/g)]−0.35xa0−xa01.0 (r2xa0=xa00.98). The results demonstrate that Vcyc is modulated by endogenous GABA levels, and that glutamatergic and GABAergic interactions can be studied in vivo using noninvasive magnetic resonance spectroscopy techniques.

Detection of reduced GABA synthesis following inhibition of GABA transaminase using in vivo magnetic resonance signal of [13C]GABA C1.

Previous in vivo magnetic resonance spectroscopy (MRS) studies of gamma-aminobutyric acid (GABA) synthesis have relied on (13)C label incorporation into GABA C2 from [1-(13)C] or [1,6-(13)C(2)]glucose. In this study, the [(13)C]GABA C1 signal at 182.3 ppm in the carboxylic/amide spectral region of localized in vivo (13)C spectra was detected. GABA-transaminase of rat brain was inhibited by administration of gabaculine after pre-labeling of GABA C1 and its metabolic precursors with exogenous [2,5-(13)C(2)]glucose. A subsequent isotope chase experiment was performed by infusing unlabeled glucose, which revealed a markedly slow change in the labeling of GABA C1 accompanying the blockade of the GABA shunt. This slow labeling of GABA at elevated GABA concentration was attributed to the relatively small intercompartmental GABA-glutamine cycling flux that constitutes the main route of (13)C label loss during the isotope chase. Because this study showed that using low RF power broadband stochastic proton decoupling is feasible at very high field strength, it has important implications for the development of carboxylic/amide (13)C MRS methods to study brain metabolism and neurotransmission in human subjects at high magnetic fields.

Transplantation of Adipose Tissue-Derived Mesenchymal Stem Cells Prevents the Development of Lupus Dermatitis

MRL/lpr mice spontaneously develop high titers of anti-dsDNA antibodies and symptoms such as glomerular nephritis and organ weight gain. They also develop spontaneous skin inflammation similar to the cutaneous lesions common in human lupus erythematosus. This study aimed to compare the effects of long-term serial administration of human adipose tissue-derived mesenchymal stem cells (ASCs), CTLA4Ig-overexpressing ASCs, and cyclophosphamide treatment in MRL/lpr mice. MRL/lpr mice were divided into saline (C), cyclophosphamide (Y), ASC early (E), ASC late (L), and CTLA4Ig-overexpressing ASC (CT) treatment groups. Background-matched control MRL/MPJ mice treated with saline (N) were also compared. The treatment period was 5-23 weeks, except for the L group (15-23 weeks). Blood and tissue samples were collected when the mice were 24 weeks old. Organ weight, anti-dsDNA antibodies, urine protein, skin and kidney histologic abnormalities, and trabecular bone volume were evaluated. The Y group showed the greatest decrease in anti-dsDNA antibodies, organ weight, degree of kidney inflammation and glomerular infiltration of C3, and incidence rate of severe proteinuria; the E, L, and CT treatment groups showed better results than the C group. ASC transplantation reduced anti-dsDNA antibody levels significantly. Mice treated with ASCs or CTLA4Ig-ASCs starting from the early disease stage did not show dermatitis upon gross examination; they demonstrated significant improvement in hyperkeratosis, acanthosis, and inflammatory cell infiltration scores in histopathology. Micro-CT analysis revealed that cyclophosphamide treatment significantly decreased bone volume and increased bone spacing in the trabecular bone. Thus, we found that ASC and CTLA4-ASC treatments prevent lupus dermatitis development in MRL/lpr mice without adverse effects.

Therapeutic effects of CTLA4Ig gene-transduced adipose tissue-derived mesenchymal stem cell transplantation on established autoimmune thyroiditis.

This study aimed to identify the beneficial effects of adipose tissue-derived mesenchymal stem cells (ASCs) and ASCs that overexpress the CTLA4Ig gene (CTLA4Ig-ASCs) on established autoimmune thyroiditis and to examine changes in clinical chemistry parameters and the presence of humoral responses upon repeated long-term administration of autologous ASCs. This study also aimed to acquire desirable results in a preclinical study by using large-sized lab animals and applying ASCs that overexpress therapeutic genes. Experimental autoimmune thyroiditis was induced by immunization with thyroglobulin. Experimental dogs were divided into five groups: (i) ASC IT + IV, (ii) ASC IV, (iii) CTLA4Ig-ASC IT + IV, (iv) CTLA4Ig-ASC IV, and (v) control IT + IV (saline only), and they received intrathyroidal (IT; 10 million cells/250 μl saline per thyroid) administration one time or intravenous (IV; 20 million cells/5 ml) administration seven times within a 101-day period. Blood samples were collected every week, and thyroids were harvested on days 104-106. After serial ASC or CTLA4Ig transplantation, the levels of canine thyroglobulin autoantibodies (TgAA) in serum and the infiltration of T-lymphocytes between the follicles of the thyroid glands were decreased. The expression of FoxP3 in submandibular lymph nodes was significantly increased. Repeated long-term administration of autologous ASCs or CTLA4Ig-ASCs did not generate changes in clinical chemistry parameters or humoral responses. The TgAA test can detect autoimmune thyroiditis years before clinical signs of hypothyroidism occur. Thus, ASC and CTLA4Ig-ASC transplantation in that period can be attractive candidates to ameliorate autoimmune thyroiditis and prevent the development of hypothyroidism.

Intra-Arterially Delivered Mesenchymal Stem Cells Are Not Detected in the Brain Parenchyma in an Alzheimer's Disease Mouse Model.

Mesenchymal stem cells (MSCs) have a promising role as a therapeutic agent for neurodegenerative diseases such as Alzheimer’s disease (AD). Prior studies suggested that intra-arterially administered MSCs are engrafted into the brain in stroke or traumatic brain injury (TBI) animal models. However, a controversial standpoint exists in terms of the integrity of the blood brain barrier (BBB) in transgenic AD mice. The primary goal of this study was to explore the feasibility of delivering human umbilical cord-blood derived mesenchymal stem cells (hUCB-MSCs) into the brains of non-transgenic WT (C3H/C57) and transgenic AD (APP/PS1) mice through the intra-arterial (IA) route. Through two experiments, mice were infused with hUCB-MSCs via the right internal carotid artery and were sacrificed at two different time points: 6 hours (experiment 1) or 5 minutes (experiment 2) after infusion. In both experiments, no cells were detected in the brain parenchyma while MSCs were detected in the cerebrovasculature in experiment 2. The results from this study highlight that intra-arterial delivery of MSCs is not the most favorable route to be implemented as a potential therapeutic approach for AD.

Effects of Transplantation of CTLA4Ig-Overexpressing Adipose Tissue-Derived Mesenchymal Stem Cells in Mice With Sustained Severe Rheumatoid Arthritis.

CTLA4Ig has therapeutic potential for rheumatoid arthritis patients unresponsive to methotrexate (MTX) or TNF-α blockers. However, recombinant CTLA4Ig proteins are short acting and expensive. Adipose tissue-derived mesenchymal stem cells (ASCs) present an ideal stem cell source for practical regenerative medicine due to their abundant availability and their beneficial properties including immunomodulation, homing activity, paracrine effects, and differentiation ability. Therefore, we aimed to determine whether CTLA4Ig and human ASCs show synergistic effects on immunomodulation and clinical improvement of sustained severe rheumatoid arthritis in a mouse model. hASCs overexpressing CTLA4Ig (CTLA4Ig–hASC) were serially transplanted into mice with collagen-induced arthritis. Arthritic mice were subjected to four treatments based on their arthritis score on day 62 postimmunization: control (C group), hASC (H group), CTLA4Ig–hASC (CT group), and MTX (MTX group). A group of healthy mice was used as a normal control (N). Mice in the N and C groups were infused with 150 μl saline, and 2 × 106 hASCs or CTLA4Ig–hASCs in 150 μl of saline were intravenously administered to those in the H and CT groups, respectively, on days 63, 70, 77, and 84 after CII immunization. About 1 mg/kg of methotrexate was intraperitoneally administered to the MTX group three times a week for 4 weeks. Serial hASC and CTLA4Ig–hASC transplantation modulated various cytokines and chemokines related to the development of rheumatoid arthritis. Both treatments protected against destruction of cartilage, with CTLA4Ig–hASCs being most effective. Serum levels of CII autoantibodies and C-telopeptide of type II collagen were significantly low in the group transplanted with CTLA4Ig–hASCs. In vitro, ASC and CTLA4Ig–hASC treatment significantly decreased T-bet and GATA-3 expression in splenocytes from arthritic mice, and CTLA4Ig–hASC treatment significantly increased the ratio of Treg/Th17 (CD4+CD25+FoxP3+/CD4+CD25+RORγt) cells. Serial hASC and CTLA4Ig–hASC transplantation offers promising treatment for rheumatoid arthritis, and CTLA4Ig–hASCs showed stronger therapeutic effects than nontransduced hASCs.

Preventive effects of CTLA4Ig-overexpressing adipose tissue–derived mesenchymal stromal cells in rheumatoid arthritis

BACKGROUND AIMSnRheumatoid arthritis is a systemic autoimmune disorder. In this study, we first compared the therapeutic effects of syngeneic and xenogeneic adipose tissue-derived stem cells on a collagen-induced arthritis mouse model. Second, we investigated the synergistic preventive effects of CTLA4Ig and adipose tissue-derived mesenchymal stromal cells (ASCs) as a therapeutic substance.nnnMETHODSnArthritis was induced in all groups except for the normal, saline (N) group, using chicken type II collagen (CII). Animals were divided into C (control, saline), H (hASCs), M (mASCs) and N groups (experiment I) and C, H, CT (CTLA4Ig-overexpressing human ASC [CTLA4Ig-hASCs]) and N groups (experiment II), according to transplanted material. Approximately 2xa0× 10(6) ASCs or 150 μL of saline was intravenously administered on days 24, 27, 30 and 34, and all animals were killed on days 42 to 44 after CII immunization.nnnRESULTSnAnti-mouse CII autoantibodies were significantly lower in the H, M and CT groups than in the C group. Cartilage damage severity score and C-telopeptide of type II collagen were significantly lower in the CT group than in the C group. The serum levels of IL-6 were significantly lower in the H, M and CT groups than in the C group. The serum levels of keratinocyte chemoattractant were significantly lower in the CT group than the C group.nnnCONCLUSIONSnThere were similar effects of ASCs on the decrease of anti-mouse CII autoantibody levels between syngeneic and xenogeneic transplantations, and CTLA4Ig-hASCs showed synergistic preventive effects compared with non-transduced hASCs.

Correlation of quantitative dynamic contrast-enhanced MRI with microvascular density in necrotic, partial necrotic, and viable liver tumors in a rabbit model.

The purpose of this study was to examine the correlation of quantitative dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) with microvessel density (MVD) in necrotic, partial necrotic, and viable tumors using a rabbit VX2 liver tumor model. Nine rabbits were used for this study. The complete necrotic area (CNA), partial necrotic area (PNA), and viable tumor area (VTA) of liver tumors were experimentally induced by radiofrequency ablation (RFA). DCE-MRI data were processed based on the extended Kety model to estimate Ktrans,ve and vp parameters. The boundaries among CNA, PNA, and VTA were delineated based on H&E stain images, and MVD was assessed for each subregion of each VX2 tumor based. There were no correlations between ph-parameters (Ktrans,ve, and vp) and MVD for CNA. For PNA, the Ktrans values were positively correlated with the MVD (r=0.8124,p<0.0001). For VTA, we found a positive correlation between Ktrans values and the MVD (r=0.5743,p<0.05). Measuring from both the PNA and the VTA, mean Ktrans values were positively correlated with mean MVD (r=0.8470,p<0.0001). In a rabbit VX2 liver tumor model, Ktrans values correlated well with MVD counts of PNA and VTA in liver tumors. PACS number(s): 87.19.If MRI.The purpose of this study was to examine the correlation of quantitative dynamic contrast‐enhanced (DCE) magnetic resonance imaging (MRI) with microvessel density (MVD) in necrotic, partial necrotic, and viable tumors using a rabbit VX2 liver tumor model. Nine rabbits were used for this study. The complete necrotic area (CNA), partial necrotic area (PNA), and viable tumor area (VTA) of liver tumors were experimentally induced by radiofrequency ablation (RFA). DCE‐MRI data were processed based on the extended Kety model to estimate Ktrans,ve and vp parameters. The boundaries among CNA, PNA, and VTA were delineated based on H&E stain images, and MVD was assessed for each subregion of each VX2 tumor based. There were no correlations between ph‐parameters (Ktrans,ve, and vp) and MVD for CNA. For PNA, the Ktrans values were positively correlated with the MVD (r=0.8124,p<0.0001). For VTA, we found a positive correlation between Ktrans values and the MVD (r=0.5743,p<0.05). Measuring from both the PNA and the VTA, mean Ktrans values were positively correlated with mean MVD (r=0.8470,p<0.0001). In a rabbit VX2 liver tumor model, Ktrans values correlated well with MVD counts of PNA and VTA in liver tumors. PACS number(s): 87.19.If MRI