Jinyoung Won

Melatonin treatment induces interplay of apoptosis, autophagy, and senescence in human colorectal cancer cells

In Asia, the incidence of colorectal cancer has been increasing gradually due to a more Westernized lifestyle. The aim of study is to determine the interaction between melatonin‐induced cell death and cellular senescence. We treated HCT116 human colorectal adenocarcinoma cells with 10 μm melatonin and determined the levels of cell death‐related proteins and evaluated cell cycle kinetics. The plasma membrane melatonin receptor, MT1, was significantly decreased by melatonin in a time‐dependent manner, whereas the nuclear receptor, RORα, was increased only after 12 hr treatment. HCT116 cells, which upregulated both pro‐apoptotic Bax and anti‐apoptotic Bcl‐xL in the early response to melatonin treatment, activated autophagic as well as apoptotic machinery within 18 hr. Melatonin decreased the S‐phase population of the cells to 57% of the control at 48 hr, which was concomitant with a reduction in BrdU‐positive cells in the melatonin‐treated cell population. We found not only marked attenuation of E‐ and A‐type cyclins, but also increased expression of p16 and p‐p21. Compared to the cardiotoxicity of Trichostatin A in vitro, single or cumulative melatonin treatment induced insignificant detrimental effects on neonatal cardiomyocytes. We found that 10 μm melatonin activated cell death programs early and induced G1‐phase arrest at the advanced phase. Therefore, we suggest that melatonin is a potential chemotherapeutic agent for treatment of colon cancer, the effects of which are mediated by regulation of both cell death and senescence in cancerous cells with minimized cardiotoxicity.

The effects of smartphone use on upper extremity muscle activity and pain threshold

[Purpose] The purpose of this study was to determine whether muscle activity and pressure-induced pain in the upper extremities are affected by smartphone use, and to compare the effects of phone handling with one hand and with both hands. [Subjects] The study subjects were asymptomatic women 20–22 years of age. [Methods] The subjects sat in a chair with their feet on the floor and the elbow flexed, holding a smartphone positioned on the thigh. Subsequently, the subjects typed the Korean anthem for 3 min, one-handed or with both hands. Each subject repeated the task three times, with a 5-min rest period between tasks to minimize fatigue. Electromyography (EMG) was used to record the muscle activity of the upper trapezius (UT), extensor pollicis longus (EPL), and abductor pollicis (AP) during phone operation. We also used a dolorimeter to measure the pressure-induced pain threshold in the UT. [Results] We observed higher muscle activity in the UT, AP, and EPL in one-handed smartphone use than in its two-handed use. The pressure-induced pain threshold of the UT was lower after use of the smartphone, especially after one-handed use. [Conclusion] Our results show that smartphone operation with one hand caused greater UT pain and induced increased upper extremity muscle activity.

Middle cerebral artery occlusion methods in rat versus mouse models of transient focal cerebral ischemic stroke.

Experimental stroke research commonly employs focal cerebral ischemic rat models (Bederson et al., 1986a; Longa et al., 1989). In human patients, ischemic stroke typically results from thrombotic or embolic occlusion of a major cerebral artery, usually the middle cerebral artery (MCA). Experimental focal cerebral ischemia models have been employed to mimic human stroke (Durukan and Tatlisumak, 2007). Rodent models of focal cerebral ischemia that do not require craniotomy have been developed using intraluminal suture occlusion of the MCA (MCA occlusion, MCAO) (Rosamond et al., 2008). Furthermore, mouse MCAO models have been widely used and extended to genetic studies of cell death or recovery mechanisms (Liu and McCullough, 2011). Genetically engineered mouse stroke models are particularly useful for evaluation of ischemic pathophysiology and the design of new prophylactic, neuroprotective, and therapeutic agents and interventions (Armstead et al., 2010). During the past two decades, MCAO surgical techniques have been developed that do not reveal surgical techniques for mouse MCAO model engineering. Therefore, we compared MCAO surgical methods in rats and mice. Forty-five male Sprague-Dawley rats, weighing 240–260 g, and thirty-four male C57BL/6 mice, weighing 20–25 g, were selected for this study. 4-0 and 6-0 monofilament nylon (AILEE Co., Busan, Korea) was used in MCAO surgery. The monofilament was cut into 5-cm (for rats) or 2-cm (for mice) pieces, and the tip of the monofilament was blunted by heating or poly-L-lysine coating (Sigma-Aldrich, St. Louis, MO, USA) (outer diameter of monofilament: 0.4–0.45 mm for rats and 0.15–0.18 mm for mice). All surgical instruments and materials were autoclaved, and the surgical procedure was performed under sterile conditions. This study was approved by the Ethics Committee for Animal Care and Use at Inje University (Approval No. 2012-29), which is certified by the Korean Association of Laboratory Animal Care. The animals were anesthetized with an intraperitoneal injection of Zoletil (tiletamine + zolazepam cocktail at 40 mg/kg or ketamine at 80 mg/kg) and xylazine (10 mg/kg) (Lee et al., 2012). After anesthetic induction, the animals were placed on a heating pad on a surgical table. During the surgical procedure, the body temperature was continuously monitored with a rectal probe and maintained at 36.5–37.0°C. The surgical region was disinfected with povidone-iodine or 70% alcohol. A midline neck incision was made, and the soft tissues over the trachea were gently retracted with a retractor. The common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated from the vagus nerve. Typically, the CCA bifurcates into the ECA and ICA, which flow toward the cranial and facial regions, respectively, and then the ICA bifurcates into the MCA and pterygopalatine artery (PPA). The occipital artery (OA) originates from the bifurcation point of the ECA which is placed on the side of the ICA (Figure 1A). Two closely spaced permanent knots were then placed at the distal part of the ECA (below the suprathyroid artery) to prevent the backflow of blood. A microvascular clamp was placed in the ICA and transiently proximal to the CCA junction. The tied section of the ECA was dissected using microscissors to insert the monofilament and reach the CCA junction, and a knot was placed below the arteriotomy in the ECA. The microvascular clamp placed in the ICA was removed to allow for filament insertion. The filament was carefully inserted, up to 18 to 20 mm for rats and 9 to 11 mm for mice, into the MCA from the CCA junction (Figure 1A; captured image). After confirmation of MCA blockage, the rat model allowed a blood supply from the CCA, whereas the mouse model allowed a blood supply after the occlusion period. After 60–90 minutes, the filament was carefully withdrawn until the tip was near the arteriotomy. Following removal of the filament, the knot was tightened in the ECA. When reperfusion was confirmed, the neck was sewn using surgical thread. To relieve pain and discomfort in the postoperative period, topical lidocaine gel was applied to the incision region, and the animal received 1.0 mL of normal saline subcutaneously as volume replenishment after the surgery. At 24 hours after the surgery, the animals were sacrificed and analyzed for brain infarction. All procedures had to be finished within 15 minutes, excluding the occlusion and reperfusion time (Figure 1). After 24 hours after reperfusion, infarct volume was calculated using 2% TTC staining method (Figure ​(Figure1C1C–E) (Bederson et al., 1986b; Park et al., 2012). Figure 1 Schematic representation of surgical procedure and quantification of infarct volume in subjected rodents. In summary, to develop standard and high-quality rodent models of stroke, several points should be taken in MCAO: (1) 0.40–0.45 mm outer diameter 4-0 monofilament nylon (for rats) and 0.15–0.18 mm outer diameter of 6-0 monofilament nylon (for mice) by heating or poly-L-lysine coating. (2) Thread insertion length: 18–20 mm (for rats) and 9–11 mm (for mice). (3) Operation period: maximum of 15 minutes. (4) Occlusion period: 60 minutes. (5) MCA occlusion allows CCA reperfusion for rats or bilateral CCA occlusion for mice.

Melatonin as a Novel Interventional Candidate for Fragile X Syndrome with Autism Spectrum Disorder in Humans

Fragile X syndrome (FXS) is the most common monogenic form of autism spectrum disorder (ASD). FXS with ASD results from the loss of fragile X mental retardation (fmr) gene products, including fragile X mental retardation protein (FMRP), which triggers a variety of physiological and behavioral abnormalities. This disorder is also correlated with clock components underlying behavioral circadian rhythms and, thus, a mutation of the fmr gene can result in disturbed sleep patterns and altered circadian rhythms. As a result, FXS with ASD individuals may experience dysregulation of melatonin synthesis and alterations in melatonin-dependent signaling pathways that can impair vigilance, learning, and memory abilities, and may be linked to autistic behaviors such as abnormal anxiety responses. Although a wide variety of possible causes, symptoms, and clinical features of ASD have been studied, the correlation between altered circadian rhythms and FXS with ASD has yet to be extensively investigated. Recent studies have highlighted the impact of melatonin on the nervous, immune, and metabolic systems and, even though the utilization of melatonin for sleep dysfunctions in ASD has been considered in clinical research, future studies should investigate its neuroprotective role during the developmental period in individuals with ASD. Thus, the present review focuses on the regulatory circuits involved in the dysregulation of melatonin and disruptions in the circadian system in individuals with FXS with ASD. Additionally, the neuroprotective effects of melatonin intervention therapies, including improvements in neuroplasticity and physical capabilities, are discussed and the molecular mechanisms underlying this disorder are reviewed. The authors suggest that melatonin may be a useful treatment for FXS with ASD in terms of alleviating the adverse effects of variations in the circadian rhythm.

The Incremental Induction of Neuroprotective Properties by Multiple Therapeutic Strategies for Primary and Secondary Neural Injury

Neural diseases including injury by endogenous factors, traumatic brain injury, and degenerative neural injury are eventually due to reactive oxygen species (ROS). Thus ROS generation in neural tissues is a hallmark feature of numerous forms of neural diseases. Neural degeneration and the neural damage process is complex, involving a vast array of tissue structure, transcriptional/translational, electrochemical, metabolic, and functional events within the intact neighbors surrounding injured neural tissues. During aging, multiple changes involving physical, chemical, and biochemical processes occur from the molecular to the morphological levels in neural tissues. Among many recommended therapeutic candidates, melatonin also plays a role in protecting the nervous system from anti-inflammation and efficiently safeguards neuronal cells via antioxidants and other endogenous/exogenous beneficial factors. Therefore, given the wide range of mechanisms responsible for neuronal damage, multi-action drugs or therapies for the treatment of neural injury that make use of two or more agents and target several pathways may have greater efficacy in promoting functional recovery than a single therapy alone.

Differential expression of caveolins and myosin heavy chains in response to forced exercise in rats

Exercise training can improve strength and lead to adaptations in the skeletal muscle and nervous systems. Skeletal muscles can develop into two types: fast and slow, depending on the expression pattern of myosin heavy chain (MHC) isoforms. Previous studies reported that exercise altered the distribution of muscle fiber types. It is not currently known what changes in the expression of caveolins and types of muscle fiber occur in response to the intensity of exercise. This study determined the changes in expression of caveolins and MHC type after forced exercise in muscular and non-muscular tissues in rats. A control (Con) group to which forced exercise was not applied and an exercise (Ex) group to which forced exercise was applied. Forced exercise, using a treadmill, was introduced at a speed of 25 m/min for 30 min, 3 times/day (07:00, 15:00, 23:00). Homogenized tissues were applied to extract of total RNA for further gene analysis. The expression of caveolin-3 and MHC2a in the gastrocnemius muscle of female rats significantly increased in the Ex group compared with the Con group (P<0.05). Furthermore, in the gastrocnemius muscle of male rats, the expression of MHC2x was significantly different between the two groups (P<0.05). There was an increased expression in caveolin-3 and a slightly decreased expression in TGFβ-1 in muscular tissues implicating caveolin-3 influences the expression of MHC isoforms and TGFβ-1 expression. Eventually, it implicates that caveolin-3 has positive regulatory function in muscle atrophy induced by neural dysfunction with spinal cord injury or stroke.

Pathophysiological and neurobehavioral characteristics of a propionic acid-mediated autism-like rat model

Autism spectrum disorder (ASD) is induced by complex hereditary and environmental factors. However, the mechanisms of ASD development are poorly understood. The purpose of this study was to identify standard indicators of this condition by comparing clinical, pathophysiological, and neurobehavioral features in an autism-like animal model. A total of 22 male Sprague-Dawley rats were randomly divided into control and 500 mg/kg propionic acid (PPA)-treated groups. Rats were subjected to behavioral tests, gene expression analyses, and histological analyses to detect pathophysiological and neurobehavioral alterations. Exploratory activity and non-aggressive behavior were significantly reduced in PPA-treated rats, whereas enhanced aggressive behavior during adjacent interactions was observed on day 14 after PPA administration. To evaluate gene expression after PPA administration, we analyzed hippocampal tissue using reverse transcription PCR. Glial fibrillary acidic protein was augmented in the PPA-treated group on day 14 after appearance of ASD-like behaviors by PPA administration, whereas octamer-binding transcription factor 4 expression was significantly decreased in the PPA-treated group. Histological evaluation revealed significantly reduced diameter and layer thickness of granule cells in PPA-treated rats compared with control rats. We conclude that PPA administration induced abnormal neural cell organization, which may have led to autism-like neurobehaviors, including increased aggressive behavior, reduced exploratory activity, and isolative and passive behaviors.

The Relationship between Autism Spectrum Disorder and Melatonin during Fetal Development

The aim of this review is to clarify the interrelationship between melatonin and autism spectrum disorder (ASD) during fetal development. ASD refers to a diverse range of neurodevelopmental disorders characterized by social deficits, impaired communication, and stereotyped or repetitive behaviors. Melatonin, which is secreted by the pineal gland, has well-established neuroprotective and circadian entraining effects. During pregnancy, the hormone crosses the placenta into the fetal circulation and transmits photoperiodic information to the fetus allowing the establishment of normal sleep patterns and circadian rhythms that are essential for normal neurodevelopment. Melatonin synthesis is frequently impaired in patients with ASD. The hormone reduces oxidative stress, which is harmful to the central nervous system. Therefore, the neuroprotective and circadian entraining roles of melatonin may reduce the risk of neurodevelopmental disorders such as ASD.