MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.
Apoptosis Regulatory Proteins ; Blotting, Western ; Ethanol ; Glioblastoma* ; Membranes ; NAD ; Tetrazolium Salts

Intracerebral hemorrhage (ICH) is one of the devastating types of stroke. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have potential benefits in recovery from brain damage following ICH. This study aimed to identify the beneficial effects of hUCB-MSCs and investigate whether they have anti-inflammatory effects on the ICH brain via neurotrophic factors or cytokines. hUCB-MSCs were transplanted into a collagenase-induced ICH rat model. At 2, 9, 16, and 30 days after ICH, rotarod and limb placement tests were performed to measure behavioral outcomes. ICH rats were sacrificed to evaluate the volume of lesion using H&E staining. Immunostaining was performed to investigate neurogenesis, angiogenesis, and anti-apoptosis at 4 weeks after transplantation. Inflammatory factors (TNF-alpha, COX-2, microglia, and neutrophils) were analyzed by immunofluorescence staining, RT-PCR, and Western blot at 3 days after transplantation. hUCB-MSCs were associated with neurological benefits and reduction in lesion volume. The hUCB-MSCs-treated group tended to reveal high levels of neurogenesis, angiogenesis, and anti-apoptosis (significant for angiogenesis). The expression levels of inflammatory factors tended to be reduced in the hUCB-MSCs-treated group compared with the controls. Our study suggests that hUCB-MSCs may improve neurological outcomes and modulate inflammation-associated immune cells and cytokines in ICH-induced inflammatory responses.
Animals ; Apoptosis ; Blotting, Western ; Brain ; Cerebral Hemorrhage* ; Cytokines ; Extremities ; Fluorescent Antibody Technique ; Humans ; Mesenchymal Stromal Cells* ; Microglia ; Models, Animal* ; Nerve Growth Factors ; Neurogenesis ; Rats ; Stroke ; Umbilical Cord*

BACKGROUND: Endotracheal intubation usually causes transient hypertension and tachycardia. The cardiovascular and arousal responses to endotracheal and endobronchial intubation were determined during rapid-sequence induction of anesthesia in normotensive and hypertensive elderly patients. METHODS: Patients requiring endotracheal intubation with (HT, n = 30) or without hypertension (NT, n = 30) and those requiring endobronchial intubation with (HB, n = 30) or without hypertension (NB, n = 30) were included in the study. Anesthesia was induced with intravenous thiopental 5 mg/kg followed by succinylcholine 1.5 mg/kg. After intubation, all subjects received 2% sevoflurane in 50% nitrous oxide and oxygen. Mean arterial pressure (MAP), heart rate (HR), plasma catecholamine concentration, and Bispectral Index (BIS) values, were measured before and after intubation. RESULTS: The intubation significantly increased MAP, HR, BIS values and plasma catecholamine concentrations in all groups, the peak value of increases was comparable between endotracheal and endobronchial intubation. However, pressor response persisted longer in the HB group than in the HT group (5.1 +/- 1.6 vs. 3.2 +/- 0.9 min, P < 0.05). The magnitude of increases in MAP and norepinephrine from pre-intubation values was greater in the hypertensive than in the normotensive group (P < 0.05), while there were no differences in those of HR and BIS between the hypertensive and normotensive groups. CONCLUSIONS: Cardiovascular response and arousal response, as measured by BIS, were similar in endobronchial and endotracheal intubation groups regardless of the presence or absence of hypertension except for prolonged pressor response in the HB group. However, the hypertensive patients showed enhanced cardiovascular responses than the normotensive patients.
Aged ; Anesthesia ; Arousal ; Arterial Pressure ; Heart Rate ; Humans ; Hypertension ; Intubation ; Intubation, Intratracheal ; Methyl Ethers ; Nitrous Oxide ; Norepinephrine ; Oxygen ; Plasma ; Succinylcholine ; Tachycardia ; Thiopental

Background: This study was performed to evaluate the effect of a wagon as a transport vehicle instead of the standard stretcher car to reduce children’s anxiety of separation from parents. The secondary goal was to evaluate whether this anxiolytic effect was related to age. Methods: We divided 80 children (age 2–7 years) into two groups. The stretcher group was transferred to the operating room on a conventional stretcher car, whereas the wagon group was transferred using a wagon. The level of anxiety was evaluated three times using the Modified Yale Preoperative Anxiety Scale (mYPAS): in the waiting area (T0), in the hallway to the operating room (T1), and before induction of anesthesia (T2). Results: The mYPAS score was significantly lower in the wagon group (36.7 [31.7, 51.7]) than in the stretcher group (51.7 [36.7, 83.3]) at T1 (P = 0.007). However, there was no difference in the mYPAS score between the two groups at T2 (46.7 [32.5, 54.2] vs. 51.7 [36.7, 75.0], respectively, P = 0.057). The baseline anxiety tended to be lower with increasing age (r = −0.248, P = 0.031). During transportation to the operating room, the increase in the mYPAS score (T1-T0) was greater as the age of children decreased in the stretcher group (r = −0.340, P = 0.034). However, no correlation was observed in the wagon group (r = −0.053, P = 0.756). Conclusion The wagon method decreased preoperative anxiety, suggesting that it may be a good alternative for reducing preoperative anxiety in children.

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of anti-cancer drugs. Neurotensin receptors (NTSRs) are widely distributed within the pain circuits in the central nervous system. The purpose of this study was to determine the role of NTSR1 by examining the effects of an NTSR1 agonist in rats with CIPN and investigate the contribution of spinal serotonin receptors to the antinociceptive effect. Methods: Sprague–Dawley rats (weight 150–180 g) were used in this study. CIPN was induced by injecting cisplatin (2 mg/kg) once a day for 4 days. Intrathecal catheters were placed into the subarachnoid space of the CIPN rats. The antiallodynic effects of intrathecally or intraperitoneally administered PD 149163, an NTSR1 agonist, were evaluated. Furthermore, the levels of serotonin in the spinal cord were measured by high-performance liquid chromatography. Results: Intrathecal or intraperitoneal PD 149163 increased the paw withdrawal threshold in CIPN rats. Intrathecal administration of the NTSR1 antagonist SR 48692 suppressed the antinociceptive effect of PD 149163 given via the intrathecal route, but not the antinociceptive effect of intraperitoneally administered PD 149163. Intrathecal administration of dihydroergocristine, a serotonin receptor antagonist, suppressed the antinociceptive effect of intrathecally administered, but not intraperitoneally administered, PD 149163. Injecting cisplatin diminished the serotonin level in the spinal cord, but intrathecal or intraperitoneal administration of PD 149163 did not affect this reduction. Conclusions NTSR1 played a critical role in modulating CIPN-related pain. Therefore, NTSR1 agonists may be useful therapeutic agents to treat CIPN. In addition, spinal serotonin receptors may be indirectly involved in the effect of NTSR1 agonist.

Despite increased evidence of bio-activity following far-infrared (FIR) radiation, susceptibility of cell signaling to FIR radiation-induced homeostasis is poorly understood. To observe the effects of FIR radiation, FIR-radiated materials-coated fabric was put on experimental rats or applied to L6 cells, and microarray analysis, quantitative real-time polymerase chain reaction, and wound healing assays were performed. Microarray analysis revealed that messenger RNA expressions of rat muscle were stimulated by FIR radiation in a dose-dependent manner in amount of 10% and 30% materials-coated. In 30% group, 1,473 differentially expressed genes were identified (fold change [FC] > 1.5), and 218 genes were significantly regulated (FC > 1.5 and p < 0.05). Microarray analysis showed that extracellular matrix (ECM)-receptor interaction, focal adhesion, and cell migration-related pathways were significantly stimulated in rat muscle. ECM and platelet-derived growth factor (PDGF)-mediated cell migration-related genes were increased. And, results showed that the relative gene expression of actin beta was increased. FIR radiation also stimulated actin subunit and actin-related genes. We observed that wound healing was certainly promoted by FIR radiation over 48 h in L6 cells. Therefore, we suggest that FIR radiation can penetrate the body and stimulate PDGF-mediated cell migration through ECM-integrin signaling in rats.
Actins ; Animals ; Cell Movement* ; Extracellular Matrix ; Focal Adhesions ; Gene Expression ; Homeostasis ; Infrared Rays ; Integrins ; Microarray Analysis ; Muscle, Skeletal* ; Platelet-Derived Growth Factor* ; Rats ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Wound Healing

Pruritus (itching) is classically defined as an unpleasant cutaneous sensation that leads to scratching behavior. Although the scientific criteria of classification for pruritic diseases are not clear, it can be divided as acute or chronic by duration of symptoms. In this study, we investigated whether skin injury caused by chemical (contact hypersensitivity, CHS) or physical (skin-scratching stimulation, SSS) stimuli causes initial pruritus and analyzed gene expression profiles systemically to determine how changes in skin gene expression in the affected area are related to itching. In both CHS and SSS, we ranked the Gene Ontology Biological Process terms that are generally associated with changes. The factors associated with upregulation were keratinization, inflammatory response and neutrophil chemotaxis. The Kyoto Encyclopedia of Genes and Genomes pathway shows the difference of immune system, cell growth and death, signaling molecules and interactions, and signal transduction pathways. Il1a , Il1b and Il22 were upregulated in the CHS, and Tnf, Tnfrsf1b, Il1b, Il1r1 and Il6 were upregulated in the SSS. Trpc1 channel genes were observed in representative itching-related candidate genes. By comparing and analyzing RNA-sequencing data obtained from the skin tissue of each animal model in these characteristic stages, it is possible to find useful diagnostic markers for the treatment of itching, to diagnose itching causes and to apply customized treatment.
Animals ; Biological Processes ; Chemotaxis ; Classification ; Cytokines ; Dermatitis, Contact* ; Gene Expression ; Gene Ontology ; Genome ; Hypersensitivity ; Immune System ; Interleukin-6 ; Mice* ; Models, Animal ; Neutrophils ; Pruritus* ; RNA* ; Sensation ; Sequence Analysis, RNA* ; Signal Transduction ; Skin* ; Transcriptome ; Transient Receptor Potential Channels ; Up-Regulation ; Wound Healing

Although atopic dermatitis (AD) is known to be a representative skin disorder, it also affects the systemic immune response. In a recent study, myoblasts were shown to be involved in the immune regulation, but the roles of muscle cells in AD are poorly understood. We aimed to identify the relationship between mitochondria and atopy by genome-wide analysis of skeletal muscles in mice. We induced AD-like symptoms using house dust mite (HDM) extract in NC/Nga mice. The transcriptional profiles of the untreated group and HDM-induced AD-like group were analyzed and compared using microarray, differentially expressed gene and functional pathway analyses, and protein interaction network construction. Our microarray analysis demonstrated that immune response-, calcium handling-, and mitochondrial metabolism-related genes were differentially expressed. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology pathway analyses, immune response pathways involved in cytokine interaction, nuclear factor-kappa B, and T-cell receptor signaling, calcium handling pathways, and mitochondria metabolism pathways involved in the citrate cycle were significantly upregulated. In protein interaction network analysis, chemokine family-, muscle contraction process-, and immune response-related genes were identified as hub genes with many interactions. In addition, mitochondrial pathways involved in calcium signaling, cardiac muscle contraction, tricarboxylic acid cycle, oxidation-reduction process, and calcium-mediated signaling were significantly stimulated in KEGG and Gene Ontology analyses. Our results provide a comprehensive understanding of the genome-wide transcriptional changes of HDM-induced AD-like symptoms and the indicated genes that could be used as AD clinical biomarkers.
Animals ; Biomarkers ; Calcium ; Calcium Signaling ; Citric Acid ; Citric Acid Cycle ; Cytokines ; Dermatitis, Atopic ; Gene Ontology ; Genome ; Metabolism ; Mice ; Microarray Analysis ; Mitochondria ; Muscle Cells ; Muscle Contraction ; Muscle, Skeletal ; Myoblasts ; Myocardium ; Oxidation-Reduction ; Protein Interaction Maps ; Pyroglyphidae ; Receptors, Antigen, T-Cell ; Skin