Are l-glutamate and ATP cotransmitters of the peripheral chemoreflex in the rat nucleus tractus solitarius ?

Peripheral chemoreflex activation in awake rats or in the working heart-brainstem preparation (WHBP) produces sympathoexcitation, bradycardia and an increase in the frequency of phrenic nerve activity. Our focus is the neurotransmission of the sympathoexcitatory component of the chemoreflex within the nucleus of the tractus solitarius (NTS), and recently we verified that the simultaneous antagonism of ionotropic glutamate and purinergic P(2) receptors in the NTS blocked the pressor response and increased thoracic sympathetic activity in awake rats and WHBP, respectively, in response to peripheral chemoreflex activation. These previous data suggested the involvement of ATP and L-glutamate in the NTS in the processing of the sympathoexcitatory component of the chemoreflex by unknown mechanisms. For a better understanding of these mechanisms, here we used a patch-clamp approach in brainstem slices to evaluate the characteristics of the synaptic transmission of NTS neurons sending projections to the ventral medulla, which include the premotor neurons involved in the generation of the sympathetic outflow. The NTS neurons sending projections to the ventral medulla were identified by previous microinjection of the membrane tracer dye, 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate (DiI), in the ventral medulla and the spontaneous (sEPSCs) and tractus solitarius (TS)-evoked excitatory postsynaptic current (TS-eEPSCs) were recorded using patch clamp. With this approach, we made the following observations on NTS neurons projecting to the ventral medulla: (i) the sEPSCs and TS-eEPSCs of DiI-labelled NTS neurons were completely abolished by 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), an antagonist of ionotropic non-NMDA glutamatergic receptors, showing that they are mediated by L-glutamate; (ii) application of ATP increased the frequency of appearance of spontaneous glutamatergic currents, reflecting an increased exocytosis of glutamatergic vesicles; and (iii) ATP decreased the peak of TS-evoked glutamatergic currents. We conclude that L-glutamate is the main neurotransmitter of spontaneous and TS-evoked synaptic activities in the NTS neurons projecting to the ventral medulla and that ATP has a dual modulatory role on this excitatory transmission, facilitating the spontaneous glutamatergic transmission and inhibiting the TS-evoked glutamatergic transmission. These data also suggest that ATP is not acting as a cotransmitter with L-glutamate, at least at the level of this subpopulation of NTS neurons studied.

INTERMITTENT ACTIVATION OF PERIPHERAL CHEMORECEPTORS IN AWAKE RATS INDUCES Fos EXPRESSION IN ROSTRAL VENTROLATERAL MEDULLA-PROJECTING NEURONS IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS

Despite the well-established sympathoexcitation evoked by chemoreflex activation, the specific sub-regions of the CNS underlying such sympathetic responses remain to be fully characterized. In the present study we examined the effects of intermittent chemoreflex activation in awake rats on Fos-immunoreactivity (Fos-ir) in various subnuclei of the paraventricular nucleus of the hypothalamus (PVN), as well as in identified neurosecretory preautonomic PVN neurons. In response to intermittent chemoreflex activation, a significant increase in the number of Fos-ir cells was found in autonomic-related PVN subnuclei, including the posterior parvocellular, ventromedial parvocellular and dorsal-cap, but not in the neurosecretory magnocellular-containing lateral magnocellular subnucleus. No changes in Fos-ir following chemoreflex activation were observed in the anterior PVN subnucleus. Experiments combining Fos immunohistochemistry and neuronal tract tracing techniques showed a significant increase in Fos-ir in rostral ventrolateral medulla (RVLM)-projecting (PVN-RVLM), but not in nucleus of solitarii tract (NTS)-projecting PVN neurons. In summary, our results support the involvement of the PVN in the central neuronal circuitry activated in response to chemoreflex activation, and indicate that PVN-RVLM neurons constitute a neuronal substrate contributing to the sympathoexcitatory component of the chemoreflex. Published by Elsevier Ltd on behalf of IBRO.

Modulation of respiratory responses to chemoreflex activation by L-glutamate and ATP in the rostral ventrolateral medulla of awake rats

Moraes DJA, Bonagamba LGH, Zoccal DB, Machado BH. Modulation of respiratory responses to chemoreflex activation by L-glutamate and ATP in the rostral ventrolateral medulla of awake rats. Am J Physiol Regul Integr Comp Physiol 300: R1476-R1486, 2011. First published March 16, 2011; doi:10.1152/ajpregu.00825.2010.-Presympathetic neurons in the different anteroposterior aspects of rostral ventrolateral medulla (RVLM) are colocalized with expiratory [Botzinger complex (BotC)] and inspiratory [pre-Botzinger complex (pre-BotC)] neurons of ventral respiratory column (VRC), suggesting that this region integrates the cardiovascular and respiratory chemoreflex responses. In the present study, we evaluated in different anteroposterior aspects of RVLM of awake rats the role of ionotropic glutamate and purinergic receptors on cardiorespiratory responses to chemoreflex activation. The bilateral ionotropic glutamate receptors antagonism with kynurenic acid (KYN) (8 nmol/50 nl) in the rostral aspect of RVLM (RVLM/BotC) enhanced the tachypneic (120 +/- 9 vs. 180 +/- 9 cpm; P < 0.01) and attenuated the pressor response (55 +/- 2 vs. 15 +/- 1 mmHg; P < 0.001) to chemoreflex activation (n = 7). On the other hand, bilateral microinjection of KYN into the caudal aspect of RVLM (RVLM/pre-BotC) caused a respiratory arrest in four awake rats used in the present study. Bilateral P2X receptors antagonism with PPADS (0.25 nmol/50 nl) in the RVLM/BotC reduced chemoreflex tachypneic response (127 +/- 6 vs. 70 +/- 5 cpm; P < 0.001; n = 6), but did not change the chemoreflex pressor response. In addition, PPADS into the RVLM/BtC attenuated the enhancement of the tachypneic response to chemoreflex activation elicited by previous microinjections of KYN into the same subregion (188 +/- 2 vs. 157 +/- 3 cpm; P < 0.05; n = 5). Our findings indicate that: 1) L-glutamate, but not ATP, in the RVLM/BtC is required for pressor response to peripheral chemoreflex and 2) both transmitters in the RVLM/BtC are required for the processing of the ventilatory response to peripheral chemoreflex activation in awake rats.

Neonatal Clinical Evaluation, Blood Gas and Radiographic Assessment After Normal Birth, Vaginal Dystocia or Caesarean Section in Dogs

This study aimed to standardize signs and diagnostic criteria of respiratory function in newborn puppies delivered normally or after dystocia and caesarean operation. A total of 48 neonates were allocated into groups: eutocia (n = 20), dystocia (n = 8), caesarean (c)-section (n = 20). Neonatal health was assessed using the Apgar score and body temperature was determined at 0, 5 and 60 min after delivery. Venous blood gases (pO(2) and SO(2)) was measured immediately and 60 min after delivery, and a thoracic radiograph was made between 0 and 5 min of life. The c-section group had significantly lower Apgar scores at birth and 5 min. Hypothermia was present at 5 min in the eutocia and c-section groups, and at 60 min in all groups. The eutocia group had an irregular respiratory pattern in 78% of puppies at birth, 27.7% at 5 min and 21% at 60 min compared with 87.5%, 62.5% and 12.5% of the pups in the dystocia group where there was irregular respiratory rhythm, moderate to intense respiratory sounds with agonic episodes. The c-section group had respiratory alterations in 70%, 45% and 16% of puppies at 0, 5 and 60 min, respectively. Radiographic abnormalities were present in 17% of the pups in the eutocia group, 25% of the pups in the dystocia group and 30% of the pups in the c-section group, respectively. The c-section group had significantly lower SO(2) values at 60 min than at birth. All puppies had hypoxaemia, but a significant decrease was observed in the c-section group. Newborn puppies had tissue hypoxia and irregular respiratory pattern at birth. Caesarean-section puppies had lower vitality; however, all developed satisfactory Apgar scores at 5 min of life, regardless of the obstetric condition.

Pulp Vitality in Patients with Intraoral and Oropharyngeal Malignant Tumors Undergoing Radiation Therapy Assessed by Pulse Oximetry

Introduction: The aim of this study was to evaluate pulp oxygenation levels (%SpO(2)) in patients with malignant intraoral and oropharyngeal tumors treated by radiotherapy (RT). Methods: Pulp oxygenation levels were measured by pulse oximetry. Twenty patients were selected, and two teeth of each participant (n = 40) were analyzed, regardless of the quadrant and the area irradiated, at four different time points: TP1, before RI; TP2, at the beginning of RI with radiation doses between 30 and 35 Gy; TP3, at the end of RI with radiation dose! between 60 and 70 Gy; and TP4, 4 to 5 months after the beginning of cancer treatment. Results: Mean %SpO(2) at the different time points were 93% (TP1), 83% (TP2), 77% (TP3), and 85% (TP4). The Student`s t test showed statistically significant differences between TP1 and TP2 (P < .01), TP3 (P <.01), and TP4 (P <.01). TP3 was also statistically significantly different when compared with TP2 (P <.01) and TP4 (P <.01). No statistically significant difference could be observed between TP2 and TP4. Conclusion`s: Because the mean %SpO(2) before RI was greater than during and after therapy and values obtained 4 to 5 months after the beginning of RI were close to the initiation of RI, pulp tissue may be able to regain normal blood flow after RT. If the changes in the microcirculation of the dental pulp were indeed transitory, preventive endodontic treatment or extraction in patients who are currently undergoing or recently received RI and who show negative signs of pulp sensitivity may rot be necessary for pulpal reasons. (J Endod 2011;37:1197-1200)

Glucose transporter protein 1 expression in mucoepidermoid carcinoma of salivary gland: correlation with grade of malignancy

P>Mucoepidermoid carcinoma (MEC), the most common primary salivary malignancy, shows great variability in clinical behaviour, thus demanding investigation to identify of prognostic markers. Since Warburg`s studies, unrestricted cell growth during tumorigenesis has been linked to altered metabolism, implying hypoxic stimulation of glycolysis and diminished contribution of mitochondrial oxidative phosphorylation to cellular ATP supply. Hypothesizing that the study of MEC metabolic status could lead to the discovery of prognostic markers, we investigated by immunohistochemistry the expression of glucose transporter 1 (Glut-1), mitochondrial antigen and peroxiredoxin I (Prx I) in samples of MEC from different histological grades. Our results showed that mitochondrial antigen and Prx I were expressed in the majority of the MEC cases independent of the histological grade. In contrast Glut-1 expression increased significantly as the tumours became more aggressive. These results suggested that oxidative phosphorylation may contribute to ATP supply in all stages of MEC progression, and that the relative contribution of glycolysis over mitochondria for cellular ATP supply increases during MEC progression, favouring growth under low oxygen concentration. In addition, the observed high Prx I protein levels could provide protection to tumour cells against reactive oxygen species generated as a consequence of mitochondrial function and hypoxia-reoxygenation cycling. Altogether our findings suggest that upregulation of Glut-1 and Prx I constitute successful adaptive strategies of MEC cells conferring a growth advantage over normal salivary gland cells in the unstable oxygenation tumour environment.

Brain monoaminergic neurons and ventilatory control in vertebrates

Monoamines (noradrenaline (NA), adrenaline (AD), dopamine (DA) and serotonin (5-HT) are key neurotransmitters that are implicated in multiple physiological and pathological brain mechanisms, including control of respiration. The monoaminergic system is known to be widely distributed in the animal kingdom, which indicates a considerable degree of phylogenetic conservation of this system amongst vertebrates. Substantial progress has been made in uncovering the participation of the brain monoamines in the breathing regulation of mammals, since they are involved in the maturation of the respiratory network as well as in the modulation of its intrinsic and synaptic properties. On the other hand, for the non-mammalian vertebrates, most of the knowledge of central monoaminergic modulation in respiratory control, which is actually very little, has emerged from studies using anuran amphibians. This article reviews the available data on the role of brain monoaminergic systems in the control of ventilation in terrestrial vertebrates. Emphasis is given to the comparative aspects of the brain noradrenergic, adrenergic, dopaminergic and serotonergic neuronal groups in breathing regulation, after first briefly considering the distribution of monoaminergic neurons in the vertebrate brain. (C) 2008 Elsevier B.V. All rights reserved.

Effect of Mussel`s Gender and Size on a Stress Response Biomarker

In mussels, stress signals such as heat, osmotic shock and hypoxia lead to the activation of the phosphorylated p38 mitogen activated protein kinase (pp38-MAPK). This stress activated protein has been efficiently used as a biomarker to several natural and anthropogenic stresses. However, what has not been tested is whether differences in gender or size can affect the response of this biomarker. The present study tested whether there was variation in the expression of pp38-MAPK in mussels Perna perna of different gender and size classes when exposed to natural stress conditions, such as air exposure. The results show that gender does not affect the expression of pp38-MAPK. However, size does have an effect, where mussels smaller than 6.5 cm displayed significantly (p < 0.05) lower levels of pp38-MAPK when compared to those larger than 7 cm. Mussels are one of the most used bioindicator species and the use of biomarkers to determine the health status of an ecosystem has been greatly increasing over the years. The present study highlights the importance of using mussels of similar size classes when performing experiments using stress-related biomarkers.

MITOCHONDRIAL RESPIRATORY CHAIN AND CREATINE KINASE ACTIVITIES IN mdx MOUSE BRAIN

In this study we investigated energy metabolism in the mdx mouse brain. To this end, prefrontal cortex, cerebellum, hippocampus, striatum, and cortex were analyzed. There was a decrease in Complex I but not in Complex 11 activity in all structures. There was an increase in Complex III activity in striatum and a decrease in Complex IV activity in prefrontal cortex and striatum. Mitochondrial creatine kinase activity was increased in hippocampus, prefrontal cortex, cortex, and striatum. Our results indicate that there is energy metabolism dysfunction in the mdx mouse brain. Muscle Nerve 41: 257-260, 2010

A modified rat model of neonatal anoxia: Development and evaluation by pulseoximetry, arterial gasometry and Fos immunoreactivity

Neonatal anoxia is a worldwide clinical problem that has serious and lasting consequences. The diversity of models does not allow complete reproducibility, so a standardized model is needed. In this study, we developed a rat model of neonatal anoxia that utilizes a semi-hermetic system suitable for oxygen deprivation. The validity of this model was confirmed using pulse oximetry, arterial gasometry, observation of skin color and behavior and analysis of Fos immunoreactivity in brain regions that function in respiratory control. For these experiments, 87 male albino neonate rats (Rattus norvegicus, lineage Wistar) aged approximate 30 postnatal hours were divided into anoxia and control groups. The pups were kept in an euthanasia polycarbonate chamber at 36 +/- 1 degrees C, with continuous 100% nitrogen gas flow at 3 L/min and 101.7 kPa for 25 min. The peripheral arterial oxygen saturation of the anoxia group decreased 75% from its initial value. Decreased pH and partial pressure of oxygen and increased partial pressure of carbon dioxide were observed in this group, indicating metabolic acidosis, hypoxia and hypercapnia. respectively. Analysis of neuronal activation showed Fos immunoreactivity in the solitary tract nucleus, the lateral reticular nucleus and the area postrema, confirming that those conditions activated areas related to respiratory control in the nervous system. Therefore, the proposed model of neonatal anoxia allows standardization and precise control of the anoxic condition, which should be of great value in indentifying both the mechanisms underlying neonatal anoxia and novel therapeutic strategies to combat or prevent this widespread public health problem. (C) 2011 Elsevier B.V. All rights reserved.

INHIBITION OF THE CAUDAL PRESSOR AREA REDUCES CARDIORESPIRATORY CHEMOREFLEX RESPONSES

The caudal pressor area (CPA) is a brainstem area located close to the spinal cord. The activation of the CPA increases sympathetic activity and mean arterial pressure (MAP) by mechanisms dependent on the commissural nucleus of the solitary tract (commNTS) and rostroventrolateral medulla, however, the signals that activate the CPA to produce these responses are still unknown. Therefore, in the present study, we investigated the activity of glutamatergic and GABAergic mechanisms from the CPA and commNTS in rats exposed to hypoxia and the effects of the inhibition of CPA neurons on cardiorespiratory responses to peripheral chemoreceptor activation with i.v. sodium cyanide (NaCN). Male Sprague-Dawley rats (250-280 g, n=5-8/group) were used. In conscious rats, most of the commNTS neurons (66 +/- 11%) and part of the CPA neurons (36 +/- 7%) activated by hypoxia (8% O2) were glutamatergic (contained VGLUT2mRNA). Small part of the neurons activated during hypoxia was GABAergic (contained GAD-67mRNA) in the commNTS (9 +/- 4%) or the CPA (6 +/- 2%). In urethane anesthetized rats, the inhibition of CPA neurons with bilateral injections of muscimol (GABA-A agonist, 2 mM) reduced baseline MAP, splanchnic sympathetic nerve discharge (SND) and phrenic nerve discharge (PND). Muscimol into the CPA also reduced by around 50% the pressor and sympathoexcitatory responses and the increase in PND to peripheral chemoreceptor activation with NaCN (50 mu g/kg i.v.), without changing sympathetic baroreflex responses. These data suggest that CPA mechanisms facilitate cardiorespiratory responses to peripheral chemoreflex activation. Immunohistochemistry results also suggest that at least part of the CPA mechanisms activated by hypoxia is glutamatergic. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats

A rise in arterial PCO(2) stimulates breathing and sympathetic activity to the heart and blood vessels. In the present study, we investigated the involvement of the retrotrapezoid nucleus (RTN) and glutamatergic mechanisms in the Botzinger/C1 region (Botz/C1) in these responses. Splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane-anesthetized, sino-aortic-denervated, vagotomized, and artificially ventilated rats subjected to hypercapnia (end-expiratory CO(2) from 5% to 10%). Phrenic activity was absent at end-expiratory CO(2) of 4%, and strongly increased when end-expiratory CO(2) reached 10%. Hypercapnia also increased sSND by 103 +/- 7%. Bilateral injections of the GABA-A agonist muscimol (2 mM) into the RTN eliminated the PND and blunted the sSND activation (Delta = +56 +8%) elicited by hypercapnia. Injections of NMDA receptor antagonist AP-5 (100 mM), non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mM) or metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100 mM) bilaterally into the Botz/C1 reduced PND (Delta = +43 +/- 7%, +52 +/- 6% or +56 +/- 11%, respectively). MCPG also reduced sSND (Delta = +41 +/- 7%), whereas AP-5 and DNQX had no effect. In conclusion, the increase in sSND caused by hypercapnia depends on increased activity of the RTN and on metabotropic receptors in the Botz/C1, whereas PND depends on increased RTN activity and both ionotropic and metabotropic receptors in the Botz/C1.

REACTIVE OXYGEN SPECIES AND THE STRUCTURAL REMODELING OF THE VISUAL SYSTEM AFTER OCULAR ENUCLEATION

Redox processes associated with controlled generation of reactive oxygen species (ROS) by NADPH oxidase (Nox) add an essential level of regulation to signaling pathways underlying physiological processes. We evaluated the ROS generation in the main visual relays of the mammalian brain, namely the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG), after ocular enucleation in adult rats. Dihydroethidium (DHE) oxidation revealed increased ROS generation in SC and DLG between 1 and 30 days postlesion. ROS generation was decreased by the Nox inhibitors diphenyleneiodonium chloride (DPI) and apocynin. Real-time PCR results revealed that Nox 2 was upregulated in both retinorecipient structures after deafferentation, whereas Nox 1 and Nox 4 were upregulated only in the SC. To evaluate the role of ROS in structural remodeling after the lesions, apocynin was given to enucleated rats and immunohistochemistry was conducted for markers of neuronal remodeling into SC and DLG. Immunohistochemical data showed that ocular enucleation produces an increase of neurofilament and microtubule-associated protein-2 immunostaining in both SC and DLG, which was markedly attenuated by apocynin treatment. Taken together, the findings of the present study suggest a novel role for Nox-induced ROS signaling in mediating neuronal remodeling in visual areas after ocular enucleation. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Immobilized Kidney 28-kDa Endostatin- Related (KES28kDa) Fragment Promotes Endothelial Cell Survival

Background/Objective: Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys. Methods: Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. Results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 mu g (p < 0.05); 12.5 versus 3.15 mu g (p < 0.05). Immobilization of KES28kDa supports endothelial cell survival over the control p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers. Conclusion: These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival. Copyright (C) 2010 S. Karger AG, Basel

An orthotopic skull base model of malignant meningioma

Meningioma tumor growth involves the subarachnoid space that contains the cerebrospinal fluid. Modeling tumor growth in this microenvironment has been associated with widespread leptomeningeal dissemination, which is uncharacteristic of human meningiomas. Consequently, survival times and tumor properties are varied, limiting their utility in testing experimental therapies. We report the development and characterization of a reproducible orthotopic skull-base meningioma model in athymic mice using the IOMM-Lee cell line. Localized tumor growth was obtained by using optimal cell densities and matrigel as the implantation medium. Survival times were within a narrow range of 17-21 days. The xenografts grew locally compressing surrounding brain tissue. These tumors had histopathologic characteristics of anaplastic meningiomas including high cellularity, nuclear pleomorphism, cellular pattern loss, necrosis and conspicuous mitosis. Similar to human meningiomas, considerable invasion of the dura and skull and some invasion of adjacent brain along perivascular tracts were observed. The pattern of hypoxia was also similar to human malignant meningiomas. We use bioluminescent imaging to non-invasively monitor the growth of the xenografts and determine the survival benefit from temozolomide treatment. Thus, we describe a malignant meningioma model system that will be useful for investigating the biology of meningiomas and for preclinical assessment of therapeutic agents.