Astaxanthin improves the developmental competence of in vitro-grown oocytes and modifies the steroidogenesis of granulosa cells derived from bovine early antral follicles.

In this study we investigated the effect of astaxanthin (Ax), which exhibits strong antioxidant activity, during invitro growth (IVG) on the developmental competence of oocytes and steroidogenesis of granulosa cells derived from early antral follicles. Bovine oocyte-cumulus-granulosa complexes collected from early antral follicles were cultured for 12 days in the presence or absence (control) of 500µM Ax. The viability of oocytes and antrum formation in the granulosa cell layer during IVG culture were greater in the presence than absence of Ax (P<0.05). Regardless of Ax treatment, 17ß-oestradiol production increased during IVG culture; however, progesterone production was significantly lower in the presence than absence of Ax (P<0.05). Reactive oxygen species levels were lower in Ax-treated oocytes than in controls after IVG (P<0.05). Although nuclear maturation and cleavage rates did not differ between the Ax-treated and control groups, Ax treatment led to weaker cathepsin B activity in oocytes and better blastocyst rates than in controls (P<0.05). Accordingly, Ax treatment during IVG increased the total number of cells in blastocysts (P<0.05). These results indicate that Ax supplementation of IVG medium improves the quality of bovine oocytes due to its antioxidative effects on growing oocytes and its suppression of the luteinisation of granulosa cells.

Investigation of exercise intensity during a freestyle wrestling match.

AIM: This study investigated changes in exercise intensity during a freestyle wrestling match. METHODS: Wrestling matches that consisted of three periods of 2 min were performed by nine elite collegiate male wrestlers. Exercise intensity was measured using heart rate (HR), blood lactate concentration ([BLa-]), and rating of perceived exertion (RPE). HR was continuously recorded during the match, and mean HR during each period was obtained. [BLa-] and RPE according to the 6-20-point Borg Perceived Exertion Scale were measured immediately after each period. RESULTS: HR during the wrestling match increased as a whole, but a continuous decrease in HR was observed during the first half of the second and third periods. This was likely caused by a cautious strategy involving reduced aggressive actions. HR significantly increased (P<0.05) from the first period (81 ± 6% of maximal HR [HRmax]) to the second period (88 ± 5% of HR(max)), and from the second to the third periods (92 ± 5% of HR(max)). [BLa-] significantly increased (P<0.05) from the first period (7.6 ± 2.0 mmol.L⁻¹) to the second period (10.4 ± 4.2 mmol.L⁻¹), but not from the second to the third periods (11.5 ± 3.1 mmol.L⁻¹). RPE significantly increased across the three periods (first, 13 ± 1; second, 15 ± 2; and third periods, 17 ± 2, P<0.05). CONCLUSION: Our results on changes in exercise intensity during each period and between periods would be helpful for making strategic decisions during wrestling matches and for planning daily training.

Importance of checking prehospital neurological findings to reveal incidence of spinal cord concussion.

OBJECTIVE: To clarify the incidence and characteristics of spinal cord concussion in an urban city in Japan. METHODS: The current retrospective study was conducted by reviewing the medical records of patients with spinal cord injuries admitted between January 2007 and December 2010. The subjects were divided into two groups: a spinal cord concussion group and a spinal cord injury group. RESULTS: There were 36 patients with spinal cord injuries admitted during this period. Among them, there were 8 patients with spinal cord concussions. No significant differences were apparent between the two groups with regard to sex, mechanism of injury, Glasgow Coma Scale, incidence of shock, inhospital Frankel classification, level of spinal cord injury, frequency of vertebral dislocation/fracture, surgery and the survival rate. However, the mean age in the spinal cord concussion group was lower than that in the spinal cord injury group. The systolic blood pressure, the heart rate and the diameter of minimum spinal canal in the concussion group were all higher than those in the spinal cord injury group. Half of the subjects in the spinal cord concussion group demonstrated immediate neurological improvement between prehospital and inhospital findings, however none of the subjects in the spinal cord injury group demonstrated such improvement. This difference was statistically significant. CONCLUSION: Spinal cord concussion is not rare in urban Japan. Checking neurological findings at the scene leads to the revelation of the true high incidence of spinal cord concussion among spinal cord injuries.

GAD67-GFP+ neurons in the Nucleus of Roller: a possible source of inhibitory input to hypoglossal motoneurons. I. Morphology and firing properties.

In this study we examined the electrophysiological and morphological properties of inhibitory neurons located just ventrolateral to the hypoglossal motor (XII) nucleus in the Nucleus of Roller (NR). In vitro experiments were performed on medullary slices derived from postnatal day 5 (P5) to P15 GAD67-GFP knock-in mouse pups. on cell recordings from GFP+ cells in NR in rhythmic slices revealed that these neurons are spontaneously active, although their spiking activity does not exhibit inspiratory phase modulation. Morphologically, GFP+ cells were bi- or multipolar cells with small- to medium-sized cell bodies and small dendritic trees that were often oriented parallel to the border of the XII nucleus. GFP+ cells were classified as either tonic or phasic based on their firing responses to depolarizing step current stimulation in whole cell current clamp. Tonic GFP+ cells fired a regular train of action potentials (APs) throughout the duration of the pulse and often showed rebound spikes after a hyperpolarizing step. In contrast, phasic GFP+ neurons did not fire throughout the depolarizing current step but instead fired fewer than four APs at the onset of the pulse or fired multiple APs, but only after a marked delay. Phasic cells had a significantly smaller input resistance and shorter membrane time constant than tonic GFP+ cells. In addition, phasic GFP+ cells differed from tonic cells in the shape and time course of their spike afterpotentials, the minimum firing frequency at threshold current amplitude, and the slope of their current-frequency relationship. These results suggest that GABAergic neurons in the NR are morphologically and electrophysiologically heterogeneous cells that could provide tonic inhibitory synaptic input to HMs.

Successful treatment for a case of near-fatal secondary adrenal insufficiency.

A thirty-six-year old female with shock was found to be unconsciousness a few days after developing a respiratory infection. Her past medical history included autoimmune hypothyroidism. Her state of shock was not controlled by massive fluid resuscitation with a vasopressor and antibiotics. However an infusion of 250 mg methylprednisolone dramatically improved her shock state. Further examination indicated secondary acute adrenal insufficiency. Adrenal insufficiency may complicate other endocrine disorders. Accordingly, a physician should consider hypoadrenocorticism, when patients are in a state of refractory shock in spite of massive infusion with a vasopressor especially in patients with other endocrine disorders.

Influence of thyroid hormones and transforming growth factor-ß1 on cystatin C concentrations.

Serum cystatin C concentrations are reported to increase in the hyperthyroid state. Serum concentrations of cystatin C and transforming growth factor-ß1 (TGF-ß1) were measured in patients with thyroid dysfunction, and the effects of 3,5,3'-tri-iodothyronine (T(3)) and TGF-ß1 on cystatin C production in human hepatoblastoma (Hep G2) cells were studied. Serum concentrations of cystatin C and TGF-ß1 were significantly higher in patients with Graves' disease compared with control subjects. Significantly positive correlations were observed between thyroid hormones and cystatin C, thyroid hormones and TGF-ß1, and TGF-ß1 and cystatin C in patients with thyroid dysfunction. Serum concentrations of cystatin C and TGF-ß1 decreased after treatment for hyperthyroidism. Cystatin C mRNA levels and cystatin C secretion were increased by T(3) and TGF-ß1 in cultured Hep G2 cells. These results suggest that serum cystatin C concentrations increase in patients with hyperthyroidism. The mechanisms for this may involve elevation of serum TGF-ß1 levels and the stimulatory effects of T(3) and TGF-ß1 on cystatin C production.

A case of pulmonary oedema induced by suffocation.

A 57-year old female civilian was suffocated by an intruder The victim (experienced severe dyspnoea) but violently resisted the assault. Two hours after this event, on admission to a Trauma Centre, she demonstrated left facial swelling with low percutaneous oxygen saturation. Chest X-ray and computed tomography demonstrated pulmonary oedema. This improved dramatically within a short time and she was discharged on the 5th hospital day. Pulmonary oedema induced by suffocation has been reported only rarely. The possible mechanisms by which pulmonary oedema might form after the relief of airway obstruction are discussed.

Isolation and characterization of the plant glsA promoter from Alstroemeria.

The differentiation of a vegetative cell and a generative cell is a critical event during pollen development. The Lilium GlsA is known to localize in pollen and is considered to be involved in development of the generative cell. Here, we cloned a glsA ortholog from Alstroemeria, a commercially important cut flower. The expression of AaglsA (Alstroemeria aurea glsA) transcripts increased gradually after pollen mitosis I (PMI) and reached a significant level when the generative cell started to elongate. Analysis of the promoter of AaglsA suggests that AaglsA expression is controlled by several cis-regulatory elements during pollen development. This is the first investigation of reproductive factors regulating male gametogenesis in Alstroemeria.

Noradrenergic excitation of a subpopulation of GABAergic cells in the basolateral amygdala via both activation of nonselective cationic conductance and suppression of resting K+ conductance: a study using glutamate decarboxylase 67-green fluorescent protein knock-in mice.

GABAergic interneurons play central roles in the regulation of neuronal activity in the basolateral nucleus of the amygdala (BLA). They are also suggested to be the principal targets of the brainstem noradrenergic afferents which are involved in the enhancement of the BLA-related memory. In addition, behavioral stress has been shown to impair noradrenergic facilitation of GABAergic transmission. However, the noradrenaline (NA) effects in the BLA have not been differentiated among medium- to large-sized GABAergic neurons and principal cells, and remain to be elucidated in terms of their underlying mechanisms. Glutamate decarboxylase 67 (GAD67) is a biosynthetic enzyme of GABA and is specifically expressed in GABAergic neurons. To facilitate the study of the NA effects on GABAergic neurons in live preparations, we generated GAD67-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the control of an endogenous GAD67 gene promoter. Here, we show that GFP was specifically expressed in GABAergic neurons in the BLA of this GAD67-GFP knock-in mouse. Under whole-cell patch-clamp recordings in vitro, we identified a certain subpopulation of GABAergic neurons in the BLA chiefly on the basis of the electrophysiological properties. When depolarized by a current injection, these neurons, which are referred to as type A, generated action potentials at relatively low frequency. We found that NA directly excited type-A cells via alpha1-adrenoceptors, whereas its effects on the other types of neurons were negligible. Two ionic mechanisms were involved in this excitability: the activation of nonselective cationic conductance and the suppression of the resting K+ conductance. NA also increased the frequency of spontaneous IPSCs in the principal cells of the BLA. It is suggested that the NA-dependent excitation of type-A cells attenuates the BLA output for a certain period.

Effect of high fat diet on NKT cell function and NKT cell-mediated regulation of Th1 responses.

Diet is one of the important factors that modulate immune responses. In the present study, we have examined the capacity of dietary lipids to modify immune responses in mice and we have investigated the contribution of glycolipid-reactive natural killer T (NKT) cells in this process. Mice fed, high fat diet (HFD; 21.2% fat, 0.20% cholesterol) for 3 weeks, as compared with mice fed standard fat diet (SFD; 4.3% fat, 0.03% cholesterol), showed significantly reduced interferon-gamma production in sera at 6 or 12 h after intraperitoneal injection of an NKT cell ligand, alpha-galactosylceramide. In contrast, production of interleukin-13 was significantly higher at 2 and 6 h in HFD fed mice compared with mice on SFD. No difference was detected in the serum interleukin-4 levels between these two groups of animals. The proportion of NKT cells in spleen and liver was reduced in mice fed HFD compared with those on SFD. In addition, activation of NKT cells assessed by up-regulation of CD69 was suppressed specifically in liver from mice fed HFD. Recall responses of conventional T cells and delayed-type hypersensitivity (Th1 type) against ovalbumin were significantly suppressed in mice fed HFD in comparison with those fed SFD. This suppression was not observed in CD1d-/- mice, suggesting that NKT cells in mice fed HFD played a role in suppressing Th1 responses. Taken together, our findings suggest a critical link between NKT cells, dietary lipid and adaptive immune responses.

Reduced carbohydrate intake in citrin-deficient subjects.

Citrin is the liver-type aspartate-glutamate carrier that resides within the inner mitochondrial membrane. Citrin deficiency (due to homozygous or compound heterozygous mutations in the gene SLC25A13) causes both adult-onset type II citrullinaemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). Clinically, CTLN2 is characterized by hyperammonaemia and citrullinaemia, whereas NICCD has a much more varied and transient presentation that can include multiple aminoacidaemias, hypoproteinaemia, galactosaemia, hypoglycaemia, and jaundice. Personal histories from CTLN2 patients have repeatedly described an aversion to carbohydrate-rich foods, and clinical observations of dietary and therapeutic outcomes have suggested that their unusual food preferences may be directly related to their pathophysiology. In the present study, we monitored the food intake of 18 Japanese citrin-deficient subjects whose ages ranged from 1 to 33 years, comparing them against published values for the general Japanese population. Our survey confirmed a marked decrease in carbohydrate intake, which accounts for a smaller proportion of carbohydrates contributing to the total energy intake (PFC ratio) as well as a shift towards a lower centile distribution for carbohydrate intake relative to age- and sex-matched controls. These results strongly support an avoidance of carbohydrate-rich foods by citrin-deficient patients that may lead to worsening of symptoms.

Abnormal response of the extremities to pain stimulation immediately after the return of spontaneous circulation from cardiopulmonary arrest.

OBJECTIVE: A study was undertaken to investigate retrospectively whether or not the appearance of an extensor reaction to pain stimulation immediately after the return of spontaneous circulation (ROSC) from cardiopulmonary arrest (CPA) is a sign of a favourable outcome. METHODS: Eighty-six patients, all of whom obtained ROSC and survived more than 1 week, were included in the study. They were divided into three groups: (1) patients with at least one abnormal response of the extremities to pain stimulation within 120 min after CPA (M = 2,3 group); (2) patients with no motor response to pain stimulation (M = 1 group); and (3) patients with only motor responses >3 on the Glasgow Coma Scale (M = 4-6 group). The following variables were investigated: prehospital setting, neurological findings on arrival, use of induced hypothermic therapy and outcome. The outcome was defined as the best cerebral performance achieved at any time within 1 month of the CPA and classified by analogy with cerebral performance category (CPC) into two groups: (1) good recovery, CPC 1-3; (2) poor recovery, CPC 4-5. RESULTS: The proportion of patients given induced hypothermic therapy in the M = 2,3 group was greater than in the M = 1 and M = 4-6 groups. Convulsions occurred more frequently in the M = 1 group than in the M = 2,3 and M = 4-6 groups. The proportion of patients making a good recovery in the M = 1 group (16%; 95% CI 6% to 26%) was smaller than in the M = 2,3 (81%; 95% CI 68% to 95%) and M = 4-6 groups (88%; 95% CI 72% to 100%). The proportion who survived in each group was similar (M = 1 group: 75% (95% CI 60% to 89%); M = 2,3 group: 90% (95% CI 81% to 100%); M = 4-6 group: 76% (95% CI 55% to 97%)). In the M = 2,3 group, four of the five subjects with convulsions did not regain consciousness while 93% (95% CI 83% to 100%) of the subjects in this group without convulsions did regain consciousness. CONCLUSION: An abnormal response of the extremities to pain stimulation immediately after ROSC tends to be a sign of a favourable outcome in patients without convulsions.

GABAergic phenotype of periglomerular cells in the rodent olfactory bulb.

Periglomerular (PG) cells in the rodent olfactory bulb are heterogeneous anatomically and neurochemically. Here we investigated whether major classes of PG cells use gamma-aminobutyric acid (GABA) as a neurotransmitter. In addition to three known subtypes of PG cells expressing tyrosine hydroxylase (TH), calbindin D-28k (CB), and calretinin (CR), we identified a novel PG cell population containing the GABAA receptor alpha5 subunit. Consistent with previous studies in the rat, we found that TH-positive cells were also labeled with antibodies against GABA, whereas PG cells expressing CB or the alpha5 subunit were GABA-negative. Using GAD67-GFP knockin mice, we found that all PG cell subtypes expressed GAD67-GFP. Calretinin labeled the major fraction (44%) of green fluorescent protein (GFP)-positive cells, followed by TH (16%), CB (14%), and the alpha5 subunit (13%). There was no overlap between these neuronal populations, which accounted for approximately 85% of GAD67-GFP-positive cells. We then demonstrated that PG cells labeled for TH, CB, or CR established dendrodendritic synapses expressing glutamic acid decarboxylase (GAD) or the vesicular inhibitory amino acid transporter, VGAT, irrespective of their immunoreactivity for GABA. In addition, CB-, CR-, and TH-positive dendrites were apposed to GABAA receptor clusters containing the alpha1 or alpha3 subunits, which are found in mitral and tufted cells, and the alpha2 subunit, which is expressed by PG cells. Together, these findings indicate that all major subtypes of PG cells are GABAergic. In addition, they show that PG cells provide GABAergic input to the dendrites of principal neurons and are interconnected with other GABAergic interneurons, which most likely are other PG cells.

Respiratory activity in brainstem of fetal mice lacking glutamate decarboxylase 65/67 and vesicular GABA transporter.

The respiratory neural network in the mammalian medulla oblongata shows rhythmic activity before birth. GABA and glycine are considered to be involved in control of respiratory rhythm. Recently we have demonstrated respiratory failure in glutamic acid decarboxylase (GAD) 67-deficient mice [Tsunekawa N, Arata A, Obata K (2005) Development of spontaneous mouth/tongue movement and related neural activity, and their repression in mouse fetus lacking glutamate decarboxylase 67. Eur J Neurosci 21:173-178]. To further evaluate the involvement of GABA and glycine in fetal respiratory function, we studied neural activities in brainstem-spinal cord blocks prepared from GAD65-/-:67-/- and vesicular GABA transporter (VGAT)-/-mice on embryonic day 14 (E14)-E15 and E18. In these knockout mice, the synthesis of GABA and the vesicular release of GABA and glycine are completely absent, respectively. Spontaneous respiratory discharges were observed in the ventral roots at the cervical cord (C) 4 level from wild-type mice but not from the knockout mice on E18. Administration of substance P induced C4 discharges in GAD65-/-:67-/- preparations but not in VGAT-/- preparations. C4 discharges were observed in the knockout mice on E14-E15, although the frequency was lower than that in the wild-type. Neuronal activities in the respiratory network of the E18 brainstem were recorded using a "blind" patch-clamp technique. Expiratory and inspiratory neurons with their characteristic firing patterns were observed in the wild-type fetuses. Strychnine reversed inspiratory-phase hyperpolarization to large depolarization in expiratory neurons. On the other hand, neurons in the same area of the knockout mice fired spontaneously without any rhythm. Substance P induced hyperpolarizing potentials in medullary neurons of GAD65-/-:67-/- mice. Further administration of strychnine induced large depolarizing potentials. Rhythmic activities were not observed in VGAT-/- mice even in the presence of substance P and strychnine. These results indicate that the lack of GABA and glycine impairs the function of the respiratory network in mouse fetuses and the impairment progresses with fetal age.

Calcium-dependent NMDA-induced dendritic injury and MAP2 loss in acute hippocampal slices.

Excessive glutamate receptor stimulation can produce rapid disruption of dendritic morphology, including dendritic beading. We recently showed that transient N-methyl-d-aspartic acid (NMDA) exposure resulted in irreversible loss of synaptic function and loss of microtubule associated protein 2 (MAP2) from apical dendrites. The present study examined the initiation and progression of dendritic injury in mouse hippocampal slices following this excitotoxic stimulus. NMDA exposure (30 microM, 10 min) produced irregularly shaped dendritic swellings, evident first in distal apical dendrite branches, and later (20-90 min) involving most proximal dendrites. Over the same time course, immunoreactivity for the microtubule-associated protein MAP2 was progressively lost from apical dendrites, and increased in CA1 somata. This damage and MAP2 loss was Ca2+-dependent, and was not reversible within the time course of these experiments (90 min post-NMDA washout). Formation of regularly-spaced, spherical dendritic varicosities (dendritic beading) was rarely observed, except when NMDA was applied in Ca2+-free ACSF. Under these conditions, beading appeared predominant in interneurons, as assessed from experiments with GAD67-GFP (Deltaneo) mice. Ca2+-removal was associated with significantly better preservation of dendritic structure (MAP2) following NMDA exposure, and other ionic fluxes (sensitive to Gd3+ and spermine) may contribute to residual damage occurring in Ca2+-free conditions. These results suggest that irregularly shaped dendritic swelling is a Ca2+-dependent degenerative event that may be quite different from Ca2+-independent dendritic beading, and can be a predominant type of injury in CA1 pyramidal neurons in slices.

Moyamoya disease associated with positive findings for rheumatoid factor and myeloperoxidase-anti-neutrophil cytoplasmic antibody.

A 39-year old female suddenly fell into a state of unconsciousness. She had no significantpast medical history. A computed tomography scan of the head demonstrated a massive left putaminal haemorrhage with a ventricular perforation, low density areas in the right frontal lobe, corona radiata and occipital lobe. A single emergency burr hole drainage of the haematoma was performed. Bilateral common carotid arteriograms showed stenosis of the right internal carotid artery and a complete obstruction of left internal carotid artery which were both accompanied by moyamoya vessels. The biochemical studies indicated serological positive findings for RF and MPO-ANCA. She was transferred to another hospital for nursing care in a vegetative state on the 163nd hospital day. This case indicates that immunological factors, inflammation or vasculitis might have possibly been associated with the development of either an obstruction or stenosis of the intracranial internal carotid arteries.

Dendritic morphogenesis of cerebellar Purkinje cells through extension and retraction revealed by long-term tracking of living cells in vitro.

Cerebellar Purkinje cells have the most elaborate dendritic trees among the neurons in the CNS. To investigate the dynamic aspects of dendritic morphogenesis of Purkinje cells, we performed a long-term analysis of living cells in cerebellar cell cultures derived from glutamate decarboxylase 67-green fluorescent protein mice. Most Purkinje cells had several primary dendrites during the 25-day culture period. Repeated observation of green fluorescent protein-expressing Purkinje cells over a period of 10-25 days in vitro demonstrated that not only extension, but also retraction of primary dendrites occurred during this culture period. Interestingly, both extension and retraction of primary dendrites were active between 10 and 15 days in vitro, and retraction of a primary dendrite occurred concomitantly with elongation of other primary dendrites in the same cell. Analysis of the morphological characteristics of the retracted primary dendrites demonstrated that shorter and less branched primary dendrites tended to retract. Furthermore, treatment with an inhibitor of calcium/calmodulin-dependent protein kinase II reduced the number of primary dendrites specifically during 5-15 days in vitro, the culture period when the extension and retraction of primary dendrites occurred actively. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-type glutamate receptors also reduced the number of primary dendrites during the same culture period, while inhibition of glutamate transporters increased the number. These findings suggest that the final morphology of Purkinje cells is achieved not only through extension, but also through retraction of their dendrites, and that calcium/calmodulin-dependent protein kinase II and neuronal activity are involved in this dendritic morphogenesis.