Shoichi Tachibana

Low Oxygen Tension Enhances Proliferation and Maintains Stemness of Adipose Tissue–Derived Stromal Cells

Abstract Recent evidence indicates that low oxygen tension or hypoxia alters the characteristics of stem cells. The actions of hypoxia are mediated through the hypoxia-inducible factor, a critical mediator of the cellular response to hypoxia. Adipose tissue–derived stromal cells (ASCs) are one of the most promising cell sources for tissue engineering applications. This study investigated the effect of hypoxia on ASCs in terms of the ability to proliferate and differentiate. ASCs were extracted from mice and maintained under hypoxic atmosphere (2% O2) for up to eight in vitro passages. The proliferation rate was examined as a growth curve, and the potency of differentiation was evaluated. To investigate the cell characteristics, we checked several stem-cell markers and growth factors. Compared with the normoxic state (20% O2), hypoxia enhances proliferation with an approximately six- to sevenfold higher ASC expansion over 6 weeks. The expression of Oct3/4 and Nanog (stem-cell marker) and the amount of secreted growth factors were increased under the hypoxic condition. These results suggest that low oxygen tension enhances proliferation and maintains stemness of ASCs. Thus, this study emphasizes the profitability of hypoxic culture for expansion of ASCs and maintenance of their undifferentiated state for further therapeutic use.

Effect of hydrogen gas on the survival rate of mice following global cerebral ischemia.

Global cerebral ischemia and reperfusion (I/R) often result in high mortality. Free radicals have been reported to play an important role in global cerebral I/R, and therefore, reduction of these might improve the outcome. Here, we investigated the effect of hydrogen gas (H2) (a strong free radical scavenger) on the survival rate of mice following global cerebral I/R. We further examined the histopathological outcome and also the brain water content (as a possible determinant of mortality). Male C57BL/6J mice were subjected to global cerebral I/R by means of 45-min bilateral common carotid artery occlusion (BCCAO). A total of 160 mice were divided into three groups: sham surgery (sham group), BCCAO without H2 (BCCAO group), and BCCAO treated with 1.3% H2 (BCCAO + H2 group). We observed that H2 treatment significantly (P = 0.0232) improved the 7-day survival rate of mice, from 8.3% (BCCAO group, n = 12) to 50% (BCCAO + H2 group, n = 10). Histopathological analysis revealed that H2 treatment significantly attenuated neuronal injury and autophagy in the hippocampal cornu ammonis 1 sector and also brain edema, after 24 h of reperfusion. The beneficial effects of H2 treatment on brain injury were associated with significantly lower levels of oxidative stress markers (8-hydroxy-2′-deoxyguanosine and malondialdehyde) in the brain tissue. Thus, we believe that H2 may be an effective treatment for global cerebral I/R. ABBREVIATIONS 8-OHdG—8-hydroxy-2′-deoxyguanosine BBB—blood-brain barrier BCCAO—bilateral common carotid artery occlusion CA1—cornu ammonis 1 CBF—cerebral blood flow I/R—ischemia and reperfusion LC3—microtubule-associated protein 1 light chain 3 MDA—malondialdehyde P-com A—posterior communicating artery ROS—reactive oxygen species

Therapeutic Effect of Exendin-4, a Long-Acting Analogue of Glucagon-Like Peptide-1 Receptor Agonist, on Nerve Regeneration after the Crush Nerve Injury

Glucagon-like peptide-1 (GLP-1) is glucose-dependent insulinotropic hormone secreted from enteroendocrine L cells. Its long-acting analogue, exendin-4, is equipotent to GLP-1 and is used to treat type 2 diabetes mellitus. In addition, exendin-4 has effects on the central and peripheral nervous system. In this study, we administered repeated intraperitoneal (i.p.) injections of exendin-4 to examine whether exendin-4 is able to facilitate the recovery after the crush nerve injury. Exendin-4 injection was started immediately after crush injury and was repeated every day for subsequent 14 days. Rats subjected to sciatic nerve crush exhibited marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). All these changes, except for the atrophy of TA, were improved significantly by the administration of exendin-4. Functional, electrophysiological, and morphological parameters indicated significant enhancement of nerve regeneration 4 weeks after nerve crush. These results suggest that exendin-4 is feasible for clinical application to treat peripheral nerve injury.

Protective effect of inhalation of hydrogen gas on radiation-induced dermatitis and skin injury in rats

The effect of inhalation of hydrogen-containing gas (1.3% hydrogen + 20.8% oxygen + 77.9% nitrogen) (HCG) on radiation-induced dermatitis and on the healing of healing-impaired skin wounds in rats was examined using a rat model of radiation-induced skin injury. An X-ray dose of 20 Gy was irradiated onto the lower part of the back through two holes in a lead shield. Irradiation was performed before or after inhalation of HCG for 2 h. Inhalation of HCG significantly reduced the severity of radiodermatitis and accelerated healing-impaired wound repair. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) showed that the proportion of apoptotic keratinocytes and the level of staining in the X-irradiated skin of rats that pre-inhaled HCG were significantly lower than that of rats which did not pre-inhale HCG. Cutaneous full-thickness wounds were then created in the X-irradiated area to examine the time-course of wound healing. X-irradiation significantly increased the time required for wound healing, but the inhalation of HCG prior to the irradiation significantly decreased the delay in wound healing compared with the control and post-inhalation of HCG groups. Therefore, radiation-induced skin injury can potentially be alleviated by the pre-inhalation of HCG.

Effects of platelet‐rich plasma‐containing fragmin/protamine microparticles in enhancing endothelial and smooth muscle cell growth and inducing collateral vessels in a rabbit model of hindlimb ischemia

The purpose of the study was to evaluate the effects of isogenous platelet-rich plasma (PRP)-containing fragmin/protamine microparticles (F/P MPs) as a delivery system for proteins in PRP on growth of endothelial and smooth muscle cells (SMCs) in vitro and as an alternative treatment for peripheral arterial disease (PAD) and critical limb ischemia. Frozen and thawed PRP contains high concentrations of growth factors that are adsorbed by F/P MPs. Human aorta endothelial cells (AECs) and SMCs were grown in a medium with PRP. Addition of F/P MPs significantly enhanced the proliferative effects of PRP on AECs and SMCs at 37 °C for >10 days. Intramuscular administration of phosphate-buffered saline (PBS; 2 mL, control), F/P MPs (12 mg in 2 mL PBS), PRP (2 mL), or PRP (2 mL) containing F/P MPs (12 mg) was then performed in a rabbit model of hindlimb ischemia prepared by resection of the left femoral artery. Blood flow and pressure were measured on days 0, 14, and 28, and angiography to assess arteriogenesis was performed on day 28. PRP-containing F/P MPs strongly induced functional collateral vessels in the rabbit model of hindlimb ischemia, indicating possible use of these microparticles in therapy for PAD.

Therapeutic effect of paroxetine on stress-induced gastric lesions in mice

Compared to the well-known anti-ulcerogenic properties of tricyclic antidepressants, the impact of selective serotonin reuptake inhibitors (SSRIs) on gastric mucosa is less clear. Human clinical trials have shown that SSRIs and non-steroidal anti-inflammatory drugs (NSAIDs) act synergistically and promote stomach ulcer formation and upper gastrointestinal tract bleeding. Acute SSRI treatment confers an additional risk for the formation of NSAID-induced gastric ulcers through increase in gastric acid secretion. Stress, which is often experienced by depressed patients, also deteriorates the gastric environment. Thus the potential for exacerbating stress-induced gastric lesions must be considered before prescribing SSRIs. Therefore, we evaluated the effects of paroxetine by using a water-immersion stress-induced stomach ulcer model of mice, by examining single vs. repeated paroxetine treatments for 8 and 22 days before stress induction. Repeated administration of paroxetine significantly decreased the area of stress-induced stomach lesions. Although stress significantly increased the serum corticosterone concentrations, the levels were not affected by the 8-day paroxetine treatment. We confirmed the anxiolytic and antidepressive effects of 8-day paroxetine treatment at 1 and 5 days after stress induction by using the elevated plus-maze and tail-suspension tests. We concluded that repeated paroxetine treatment significantly attenuates the stress-induced ulcerogenic process in the stomach.

A high concentration of triiodothyronine attenuates the stimulatory effect on hemin-induced erythroid differentiation of human erythroleukemia K562 cells.

Although thyroid hormone is a known stimulator of erythropoietic differentiation, severe anemia is sometimes observed in patients with hyperthyroidism and this mechanism is not fully understood. The aim of this study was to investigate the effect of triiodothyronine (T3) on hemin-induced erythropoiesis. Human erythroleukemia K562 cells were used as an erythroid differentiation model. Cell differentiation was induced by hemin and the effect of pre-incubation with T3 (0.1 to 100 nM) was analyzed by measuring the benzidine-positive rate, hemoglobin content, CD71 expression (transferrin receptor), and mRNA expression for transcription factors related to erythropoiesis and thyroid hormone receptors (TRs). Hemin, a promoter of erythroid differentiation, increased the levels of mRNAs for TRα, TRβ, and retinoid X receptor α (RXRα), as well as those for nuclear factor-erythroid 2 (NFE2), GATA-binding protein 1 (GATA1) and GATA-binding protein 2 (GATA2). Lower concentrations of T3 had a stimulatory effect on hemin-induced hemoglobin production (1 and 10 nM), CD71 expression (0.1 nM), and α-globin mRNA expression (1 nM), while a higher concentration of T3 (100 nM) abrogated the stimulatory effect on these parameters. T3 at 100 nM did not affect cell viability and proliferation, suggesting that the abrogation of erythropoiesis enhancement was not due to toxicity. T3 at 100 nM also significantly inhibited expression of GATA2 and RXRα mRNA, compared to 1 nM T3. We conclude that a high concentration of T3 attenuates the classical stimulatory effect on erythropoiesis exerted by a low concentration of T3 in hemin-induced K562 cells.

A rabbit model of fatal hypothyroidism mimicking “myxedema coma” established by microscopic total thyroidectomy

Myxedema coma (MC) is a life-threatening endocrine crisis caused by severe hypothyroidism. However, validated diagnostic criteria and treatment guidelines for MC have not been established owing to its rarity. Therefore, a valid animal model is required to investigate the pathologic and therapeutic aspects of MC. The aim of the present study was to establish an animal model of MC induced by total thyroidectomy. We utilized 14 male New Zealand White rabbits anesthetized via intramuscular ketamine and xylazine administration. A total of 7 rabbits were completely thyroidectomized under a surgical microscope (thyroidectomized group) and the remainder underwent sham operations (control group). The animals in both groups were monitored without thyroid hormone replacement for 15 weeks. Pulse rate, blood pressure, body temperature, and electrocardiograms (ECG) were recorded and blood samples were taken from the jugular vein immediately prior to the thyroidectomy and 2 and 4 weeks after surgery. The thyroidectomized rabbits showed a marked reduction of serum thyroxine levels at 4 weeks after the surgical procedure vs. controls (0.50±0.10 vs. 3.32±0.68 μg/dL, p<0.001). Additionally, thyroidectomized rabbits exhibited several signs of hypothyroidism such as hypothermia, systolic hypotension, bradycardia, and low voltage on ECGs, compared with controls. Of the 7 rabbits with severe hypothyroidism, 6 died from 4 to 14 weeks after the thyroidectomy possibly owing to heart failure, because histopathologic examinations revealed a myxedema heart. In summary, we have established a rabbit model of fatal hypothyroidism mimicking MC, which may facilitate pathophysiological and molecular investigations of MC and evaluations of new therapeutic interventions.