The contribution of heart rate to the oxygen consumption of the chicken embryo during cold- or hypoxia-hypometabolism.

In embryos, cooling and hypoxia cause a decrease in oxygen consumption ( [Formula: see text] ); we asked what was the relative contribution of heart rate (HR) and of the 'not-HR' factor (the product of stroke volume and arterial-venous O2 difference) to the drop in [Formula: see text] . Data of HR (with subcutaneous electrodes) and [Formula: see text] (by an open-flow methodology) were collected simultaneously on chicken embryos close to end-incubation. Over the last four days of incubation (E16-E20) differences in HR contributed about 30% of the differences in resting [Formula: see text] among embryos. At E20, progressive cooling from 38 to 8°C decreased [Formula: see text] entirely because of the decrease in HR, with minimal compensation of the 'not-HR' component. The same pattern during cooling occurred in younger embryos (age E16), in E20 embryos simultaneously exposed to hypoxia (15% O2) and in E20 normoxic embryos which were incubated in hypoxia (15% O2). Differently, in E20 embryos in normothermia, progressive hypoxia (15%, 10% or 5% O2) lowered [Formula: see text] largely because of the reduction in the 'not-HR' component. We conclude that at end incubation during hypometabolism the changes in HR contribute very differently to the decrease in [Formula: see text] , from about the totality of it during cold to only about 10-20% during hypoxia, depending on its severity. It follows that during cold-hypometabolism, but not during hypoxic hypometabolism, the changes in HR are a good index of the changes in [Formula: see text] . The close relationship between [Formula: see text] and HR during cold-hypometabolism may permit estimates of the changes in [Formula: see text] from the changes in HR in infants undergoing therapeutic hypothermia.

Aerobic scope in chicken embryos.

We investigated the aerobic scope of chicken embryos, that is, the margin of increase of oxygen consumption ( [Formula: see text] ) above its normal value. [Formula: see text] was measured by an open-flow methodology at embryonic ages E3, E7, E11, E15, E19 and at E20 at the internal (IP) and external pipping (EP) phases, at the normal incubation temperature (Ta=38°C), in hypothermia (Ta=30°C) and in hyperthermia (Ta=41 and 44°C). In the cold, Q10 averaged ~2 at all ages, except in IP and EP when lower values (~1.5) indicated some degree of thermogenesis. In hyperthermia (38-44°C) Q10 was between 1 and 1.4. Hyperthermia had no significant effects on [Formula: see text] whether the results combined all ages or considered individual age groups, except in IP (in which [Formula: see text] increased 8% with 44°C) and EP embryos (+13%). After opening the air cell, which exposed the embryo to a higher O2 pressure, hyperthermic [Formula: see text] was significantly higher than in normothermia in E19 (+13%), IP (+22%) and EP embryos (+22%). We conclude that in chicken embryos throughout most of incubation neither heat nor oxygen availability limits the normal (normoxic-normothermic) values of [Formula: see text] . Only close to hatching O2-diffusion represents a limiting factor to the embryo's [Formula: see text] . Hence, embryos differ from postnatal animals for a nearly absent aerobic scope, presumably because their major sources of energy expenditure (growth and tissue maintenance) are constantly maximized.

Human Dental Pulp Cells Differentiate toward Neuronal Cells and Promote Neuroregeneration in Adult Organotypic Hippocampal Slices In Vitro.

The adult mammalian central nerve system has fundamental difficulties regarding effective neuroregeneration. The aim of this study is to investigate whether human dental pulp cells (DPCs) can promote neuroregeneration by (i) being differentiated toward neuronal cells and/or (ii) stimulating local neurogenesis in the adult hippocampus. Using immunostaining, we demonstrated that adult human dental pulp contains multipotent DPCs, including STRO-1, CD146 and P75-positive stem cells. DPC-formed spheroids were able to differentiate into neuronal, vascular, osteogenic and cartilaginous lineages under osteogenic induction. However, under neuronal inductive conditions, cells in the DPC-formed spheroids differentiated toward neuronal rather than other lineages. Electrophysiological study showed that these cells consistently exhibit the capacity to produce action potentials, suggesting that they have a functional feature in neuronal cells. We further co-cultivated DPCs with adult mouse hippocampal slices on matrigel in vitro. Immunostaining and presto blue assay showed that DPCs were able to stimulate the growth of neuronal cells (especially neurons) in both the CA1 zone and the edges of the hippocampal slices. Brain-derived neurotrophic factor (BDNF), was expressed in co-cultivated DPCs. In conclusion, our data demonstrated that DPCs are well-suited to differentiate into the neuronal lineage. They are able to stimulate neurogenesis in the adult mouse hippocampus through neurotrophic support in vitro.

Effects of dexmedetomidine on cardiorespiratory regulation in spontaneously breathing newborn rats.

BACKGROUND: Dexmedetomidine, a selective α2-adrenoceptor agonist, is a new sedative agent. OBJECTIVE: To examine the dexmedetomidine-associated changes in cardiorespiratory indices in spontaneously breathing newborn rats. METHODS: An abdominal catheter to administer drugs and subcutaneous electrodes to record electrocardiographic data were inserted into 2- to 4-day-old rats under isoflurane anesthesia; the rats were then placed in individual chambers. After recovery from the anesthesia, the rats received intraperitoneal administrations of normal saline (NS, vehicle), dexmedetomidine (50 µg·kg(-1)), or dexmedetomidine (50 µg·kg(-1)) followed 5 min later with NS or the selective α2-adrenoceptor antagonist atipamezole (1 mg·kg(-1)) (n = 10 in each group). Cardiorespiratory indices were recorded for each animal throughout the experiment. RESULTS: Dexmedetomidine administration significantly decreased heart rate (HR) and minute ventilation (V'E) (P < 0.05) compared with control, whereas NS administration did not. The decrease in HR and V'E after dexmedetomidine administration was significantly less in rats that received atipamezole (P < 0.05) than in those that received NS after dexmedetomidine administration. The dexmedetomidine-associated V'E depression was attributed to a significant decrease in respiratory frequency (fR) but not tidal volume (VT ). The change in fR was reversed by atipamezole administration, which itself induced no significant changes in HR and fR. CONCLUSION: In spontaneously breathing immature rats, dexmedetomidine administration significantly reduced HR and V'E. Because atipamezole fully reversed decreases in fR and therefore V'E, dexmedetomidine-related respiratory suppression occurs predominantly through α2-adrenoceptor-related suppression of fR.

Respiratory sinus arrhythmia in spontaneously breathing, unanesthetized newborn and adult Wistar rats.

We examined respiratory sinus arrhythmia (RSA) and possible interaction with respiratory frequency (fR) and heart rate (HR) in spontaneously breathing, unanesthetized newborn Wistar rats (2- to 5-day-old; n = 54) and the adult rats (8-week-old; n = 34). Instantaneous heart rate (inst-HR) was calculated as the reciprocal of the inter-beat-interval. For each breath, RSA was determined as the difference between the maximum and minimum inst-HR value. The absolute RSA or RSA% (RSA per HR) were calculated as the average RSA of 10 consecutive breaths. RSA (or RSA%) in the newborn rats was significantly lower than that in the adult rats. Correlation coefficient between RSA (or RSA%) and 1/fR or HR/fR, but not HR, was significant in newborn rats, whereas only that between RSA (or RSA%) and HR was significant in adult rats. The power spectrum density of heartbeat fluctuation was detectable in both age groups. The present findings suggest that RSA exists and could be influenced by fR, rather than HR, in newborn rats.

Effect of dexmedetomidine on cardiorespiratory regulation in spontaneously breathing adult rats.

PURPOSE: We examined the cardiorespiratory effect of dexmedetomidine, an α2- adrenoceptor/imidazoline 1 (I1) receptor agonist, in spontaneously breathing adult rats. METHODS: Male rats (226-301 g, n = 49) under isoflurane anesthesia had their tail vein cannulated for drug administration and their tail artery cannulated for analysis of mean arterial pressure (MAP), pulse rate (PR), and arterial blood gases (PaO2, PaCO2, pH). After recovery, one set of rats received normal saline for control recording and was then divided into three experimental groups, two receiving dexmedetomidine (5 or 50 µg·kg-1) and one receiving normal saline (n = 7 per group). Another set of rats was divided into four groups receiving dexmedetomidine (50 µg·kg-1) followed 5 min later by 0.5 or 1 mg∙kg-1 atipamezole (selective α2-adrenoceptor antagonist) or efaroxan (α2-adrenoceptor/I1 receptor antagonist) (n = 6 or 8 per group). Recordings were performed 15 min after normal saline or dexmedetomidine administration. RESULTS: Compared with normal saline, dexmedetomidine (5 and 50 µg·kg-1) decreased respiratory frequency (fR, p = 0.04 and < 0.01, respectively), PR (both p < 0.01), and PaO2 (p = 0.04 and < 0.01), and increased tidal volume (both p = 0.049). Dexmedetomidine at 5 µg·kg-1 did not significantly change minute ventilation (V'E) (p = 0.87) or MAP (p = 0.24), whereas dexmedetomidine at 50 µg·kg-1 significantly decreased V'E (p = 0.03) and increased MAP (p < 0.01). Only dexmedetomidine at 50 µg·kg-1 increased PaCO2 (p < 0.01). Dexmedetomidine (5 and 50 µg·kg-1) significantly increased blood glucose (p < 0.01), and dexmedetomidine at 50 µg·kg-1 increased hemoglobin (p = 0.04). Supplemental atipamezole or efaroxan administration similarly prevented the 50 µg·kg-1 dexmedetomidine-related cardiorespiratory changes. PRINCIPAL CONCLUSION: These results suggest that dexmedetomidine-related hypoventilation and hypertension are observed simultaneously and occur predominantly through activation of α2-adrenoceptors, but not I1 receptors, in spontaneously breathing adult rats.

Expression of nischarin, an imidazoline 1 receptor candidate protein, in the ventrolateral medulla of newborn rats.

The activation of imidazoline 1 (I1) receptors is suggested to stimulate the respiratory drive in newborn rats. Here, we immunohistochemically examined whether nischarin, an I1 receptor candidate protein, is expressed in the ventrolateral medulla, where cardiorespiratory centers are located. Newborn rats (age, 3-5 days) were deeply anesthetized with isoflurane; the brainstem was dissected, sectioned sagittally, and labeled with nischarin. Nischarin-associated signals were observed broadly throughout the newborn rat brainstem, including at motor nuclei (motor trigeminal nucleus and facial nucleus), sensory nuclei (lateral superior olive, medial and spinal vestibular nuclei, cuneate nucleus, spinal trigeminal nucleus, and solitary nucleus), and the rostral and caudal ventrolateral medullar regions. In particular, the rostral ventrolateral medulla included a layer of aggregated nischarin expression along the ventral surface, and the layer was in close contact with GFAP-positive processes. In addition, some Phox2b-positive neurons were positive for nischarin in the region. Our results reveal nischarin expression in the newborn rat brainstem and suggest that I1 receptor activation at the level of the ventrolateral medulla contributes to central chemoreception and respiratory control in newborn rats.

Enzyme-digested Colla Corii Asini (E'jiao) prevents hydrogen peroxide-induced cell death and accelerates amyloid beta clearance in neuronal-like PC12 cells.

As an aging-associated degenerative disease, Alzheimer's disease is characterized by the deposition of amyloid beta (Aß), oxidative stress, inflammation, dysfunction and loss of cholinergic neurons. Colla Corii Asini (CCA) is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging. CCA might delay aging-induced degenerative changes in neurons. In the present study, we evaluated antioxidant activity, cytoprotective effects, and Aß removability of enzyme-digested Colla Corii Asini (CCAD). Oxygen radical absorbance capacity (ORAC) activity assay showed that, as compared to gelatins from the skin of porcine, bovine and cold water fish, CCA exhibited the highest ORAC activity. The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage. Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008, 2013, 2017 and gelatin from cold water fish skin, CCA manufactured in 2008 presented the smoothest surface structure. We further tested the protective effects of CCAD (manufactured in 2008) and enzyme-digested gelatin from cold water fish skin (FGD) on hydrogen peroxide (H2O2)-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells. Presto blue assay showed that both FGD and CCAD at 0.5 mg/mL increased cell viability in H2O2-treated neuronal-like PC12 cells. The protection of CCAD was significantly superior to that of FGD. Acetylcholinesterase (AchE) assay showed that both FGD and CCAD inhibited AchE activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1% and 74.5% of that in non-treated cells, respectively. The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine. Although CCAD inhibited AchE activity in neuronal-like PC12 cells, CCAD prevented H2O2-induced abnormal deterioration of AchE. ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aß1-42-treated neuronal-like PC12 cells, suggesting that CCAD can enhance Aß clearance. Our results suggest that CCA might be useful for preventing and treating Alzheimer's disease.

The cessation of breathing in the chicken embryo during cold-hypometabolism.

The avian embryo toward end-incubation combines gas exchange through the chorioallantoic membrane (CAM) and pulmonary ventilation (V˙E). The main experiments examined breathing activity during cold-hypometabolism. Chicken embryos close to hatching were prepared for simultaneous measurements of oxygen consumption ( [Formula: see text] ) and carbon dioxide production ( [Formula: see text] ; open-flow methodology) and breathing frequency (f; barometric technique). As ambient (Ta) and egg temperature (Tegg) dropped, breathing eventually ceased at ∼18°C, when [Formula: see text] and [Formula: see text] were 22-28% of the normothermic values. With the eggshell experimentally covered to reduce CAM gas exchange breathing ceased at slightly lower [Formula: see text] and [Formula: see text] (17-18% of normothermia). Once breathing had stopped, egg exposure to hypoxia (10% or 5% O2) or hypercapnia (3% or 8% CO2) did not resume breathing, which recovered with re-warming. In normothermia, 10% O2 caused hypometabolism and tachypnea; differently, in 5% O2 [Formula: see text] dropped as much as with hypothermia and breathing stopped, to recover upon return in air. Correlation analysis among Ta, Tegg, [Formula: see text] , [Formula: see text] and f during cooling and re-warming indicated that f followed more closely the changes in [Formula: see text] and, especially, in [Formula: see text] than the changes in Ta or Tegg. Some considerations suggest that in this experimental model the cessation of breathing in hypothermia or severe hypoxia may be due to hypometabolism, while the lack of chemo-responses may have a different mechanistic basis.

The hypometabolic response to repeated or prolonged hypoxic episodes in the chicken embryo.

Hypoxia (hx) in embryos causes a drop in oxygen consumption ( [Formula: see text] ) that rapidly recovers upon return to normoxia. We asked whether or not this pattern varies with the embryo's hypoxic history. The [Formula: see text] of chicken embryos in the middle (E12) or at end-incubation (E19) was measured by an open-flow methodology during 15-min epochs of moderate (15% O2) or severe hx (10% O2). Each hx-epoch was repeated or alternated with air by various modalities (air-hx-air-hx-air-hx-air, air-2·hx-air-2·hx-air, air-5·hx-air), in randomized sequences. The hx drop in [Formula: see text] was larger with severe than with moderate hx; however, in either case, its magnitude was essentially independent of the preceding hx history. E19 embryos had hx drops in [Formula: see text] of the same magnitude whether their incubation was in air or in moderate hx from E4 to E19. A different protocol (air-12·hx-air) gave variable results; with moderate hx, the [Formula: see text] response was similar to that of the other hx regimes. Differently, with severe hx most embryos progressively decreased [Formula: see text] and eventually died. We interpret these data on the basis of what is known on the 'compensatory partitioning' between costs of growth and maintenance. With moderate hx presumably each episode caused an energy shortfall absorbed entirely by the blunted growth. Hypoxic events of this type, therefore, should have no long-term functional effects other than those related to the small birth weight. Differently, the aerobic energy shortfall with severe hypoxia probably impinged on some maintenance functions and became incompatible with survival.

Stem cell therapy for central nerve system injuries: glial cells hold the key.

Mammalian adult central nerve system (CNS) injuries are devastating because of the intrinsic difficulties for effective neuronal regeneration. The greatest problem to be overcome for CNS recovery is the poor regeneration of neurons and myelin-forming cells, oligodendrocytes. Endogenous neural progenitors and transplanted exogenous neuronal stem cells can be the source for neuronal regeneration. However, because of the harsh local microenvironment, they usually have very low efficacy for functional neural regeneration which cannot compensate for the loss of neurons and oligodendrocytes. Glial cells (including astrocytes, microglia, oligodendrocytes and NG2 glia) are the majority of cells in CNS that provide support and protection for neurons. Inside the local microenvironment, glial cells largely influence local and transplanted neural stem cells survival and fates. This review critically analyzes current finding of the roles of glial cells in CNS regeneration, and highlights strategies for regulating glial cells' behavior to create a permissive microenvironment for neuronal stem cells.

Role of rostral medulla in serotonin-induced changes of respiratory rhythm in newborn rat brainstem-spinal cord preparations.

Abnormalities of the serotonin (5-hydroxytryptamine, 5-HT) system may induce respiratory disorders. We examined which regions in the rostral medulla are important for the effect of 5-HT on the frequency of respiratory-like nerve (fR-like) activity by transecting the preparations at different levels near the facial nucleus (nVII) in newborn rat brainstem-spinal cord preparations. The fR-like activity at the fourth cervical ventral root (C4) of the Pons-medulla-spinal cord preparations in 2-3-day-old rats (n=25) was monitored at 26°C, and the change in fR-like activity in response to application of 10µM 5-HT before and after transection was compared among three groups, in which nVII was retained (group A, n=10), partially retained (group B, n=7), or eliminated (group C, n=8) by the transection. Before transection, the resting fR-like activity (set to 100%) and stimulant effect of 5-HT (+101-143%) were similar among the groups. After transection, resting fR-like activity increased in all groups, but the facilitatory effects of 5-HT on the fR-like activity were abolished in groups A and C (fR-like activity of -4% and +7%, respectively). In group B, 5-HT became inhibitory (fR-like activity of -28%). In conclusion, a distinct part of the rostral medulla in the absence of pontine influences may mediate the inhibitory effects of 5-HT on the respiratory rhythm.

TRPV1 receptor expression in cardiac vagal afferent neurons of infant rats.

We examined in infant rats whether cardiac afferent neurons in the nodose ganglia (NG) express transient receptor potential vanilloid 1 (TRPV1) receptors. Anesthetized rats (9-11 days old) were injected with 2 µl of a fluorescent retrograde tracer (Fluoro-Gold, FG) into the ventricular wall through the anterior epicardial surface. After 2 days, the NG were excised under anesthesia for identification of FG-labeled neurons and immunohistochemical analysis. Of 3858 NG neurons, 5% (202 neurons) were labeled with FG. Among the FG-labeled neurons, 180 (89%) were TRPV1 positive, of which 123 co-expressed 200-kDa neurofilaments (NF200), which is specific for myelinated nerve fibers. Among the FG-labeled neurons that expressed both TRPV1 and NF200, 37 had relatively large cell diameters (>26 µm) (range: 12-38 µm). In conclusion, in infant rats, most cardiac afferent neurons (both myelinated and unmyelinated) in the NG may express TRPV1 receptors. Although functional properties such as those related to the arterial baroreflex may vary among the neurons, our results suggest that, in immature animals, TRPV1 receptors help convey cardiac sensations and control autonomic reflexes.

Direct inhibition of the transient voltage-gated K(+) currents mediates the excitability of tetrodotoxin-resistant neonatal rat nodose ganglion neurons after ouabain application.

The purpose of the present study was to determine the relationship between the responses of transient and sustained K(+) currents, and action potentials to ouabain, and to compare the immunoreactive expression of alpha Na(+)-K(+)-ATPase isoforms (α(1), α(2) and α(3)) in neonatal rat small-diameter nodose ganglion neurons. We used perforated patch-clamp techniques. We first confirmed that the neurons (n=20) were insensitive to 0.5 µM tetrodotoxin (TTX). Application of 1 µM ouabain 1) decreased the transient K(+) currents in 60% of neurons and the sustained K(+) currents in 20%, 2) increased voltage-gated transient and sustained K(+) currents in 20% of neurons, and 3) had no effect on transient K(+) currents in 20% of neurons and on sustained K(+) currents in 60%. Thirteen of the neurons were of a rapidly adapting type, and the remaining 7 were of a slowly adapting type. In 6 rapidly adapting type neurons (46%), their activity was not significantly altered by ouabain application, but in 4 rapidly adapting type neurons, the activity increased. In the remaining 3 rapidly adapting type neurons, ouabain application hyperpolarized the resting membrane potential. The slowly adapting type 7 neurons each showed increased activity after 1 µM ouabain application. The α(1) isoform of Na(+)-K(+)-ATPase was identified as the predominant immunoreactive isoforms in small-diameter nodose ganglion neurons. These results suggest that the increased activity of small-diameter nodose ganglion neurons seen after application of 1 µM ouabain is mediated by direct inhibition of the transient K(+) current.

Flecainide inhibits the stimulatory effect of veratridine on the response of airway mechanoreceptors to maintained inflations in rats.

AIMS: the purpose of the present study was to investigate (a) whether maintained inflations result in the inhibition of slowly adapting pulmonary stretch receptor (SAR) discharge to elicit an abrupt change in receptor activity and (b) whether pretreatment with veratridine, a Na(+) channel opener, and/or flecainide, a Na(+) channel blocker, alters the responses of SAR properties to maintained inflations. MAIN METHODS: we measured the properties of SAR activity during maintained inflations at different pressures in 31 anesthetized, artificially ventilated rats with unilateral vagotomy. KEY FINDINGS: During maintained inflations (approximately 5, 10 and 15 cmH(2)O) for about 5s, the procedures did not produce the induction of inhibition of either 16 low-threshold SARs (firing during both inflation and deflation) or 15 high-threshold SARs (firing during inflation only). In these preparations, the excitatory responses of SARs to maintained inflations at three different pressures were markedly enhanced after administration of veratridine (50 µg/kg), but under such conditions, the inhibition of SAR discharges was not observed. In the same SAR preparations, after flecainide treatment (9 mg/kg) sufficient for the blockade of veratridine (50 µg/kg)-induced SAR stimulation, maintained inflations at higher pressures (10 and 15 cmH(2)O) greatly inhibited SAR discharges. Under these conditions, the inhibition of SAR discharges was not observed during maintained inflations at 5 cmH(2)O. SIGNIFICANCE: These results suggest that neither low-threshold SARs nor high-threshold SARs in the rat lung are deactivated during maintained inflations at higher pressures.