Bernadette O. Erokwu

CO2-induced c-fos expression in the CNS catecholaminergic neurons

In these studies we examined c-fos expression in catecholaminergic neurons following exposure of unanesthetized rats to hypercapnic stress. Breathing a gas mixture with elevated CO2 (15% CO2, 21% O2 and 64% N2, or 15% CO2 balance O2) for 60 min, induced activation of the c-fos gene in widespread regions of the CNS, as indicated by the expression of Fos-like immunoreactive protein (Fos). Similar results were obtained in carotid body denervated animals. Colocalization studies of tyrosine hydroxylase (TH) and Fos protein revealed that in the brainstem, 73 to 85% of noradrenaline-containing cells expressed Fos immunoreactivity. Double-labeled neurons were found in the ventrolateral medullary reticular formation (A1 noradrenaline cells), in the dorsal aspect of medulla oblongata (A2 noradrenaline cells), in the ventrolateral pons (A5 noradrenaline cells), and in the locus coeruleus (A6 noradrenaline cells). However, over 90% of TH-immunoreactive neurons in the mesencephalon and diencephalon (dopaminergic cells) did not express Fos-like immunoreactivity in response to CO2. These results indicate that the brainstem noradrenaline-containing neurons are part of the neuronal networks that react to hypercapnic exposure.

Magnetic Resonance Imaging of Multifunctional Pluronic Stabilized Iron-Oxide Nanoparticles in Tumor-Bearing Mice

We are investigating the magnetic resonance imaging characteristics of magnetic nanoparticles (MNPs) that consist of an iron-oxide magnetic core coated with oleic acid (OA), then stabilized with a pluronic or tetronic block copolymer. Since pluronics and tetronics vary structurally, and also in the ratio of hydrophobic (poly[propylene oxide]) and hydrophilic (poly[ethylene oxide]) segments in the polymer chain and in molecular weight, it was hypothesized that their anchoring to the OA coating around the magnetic core could significantly influence the physical properties of MNPs, their interactions with biological environment following intravenous administration, and ability to localize to tumors. The amount of block copolymer associated with MNPs was seen to depend upon their molecular structures and influence the characteristics of MNPs. Pluronic F127-modified MNPs demonstrated sustained and enhanced contrast in the whole tumor, whereas that of Feridex IV was transient and confined to the tumor periphery. In conclusion, our pluronic F127-coated MNPs, which can also be loaded with anticancer agents for drug delivery, can be developed as an effective cancer theranostic agent, i.e. an agent with combined drug delivery and imaging properties.

Increased expression of fibronectin and the α5β1 integrin in angiogenic cerebral blood vessels of mice subject to hypobaric hypoxia

The extracellular matrix (ECM) is an important regulator of angiogenesis and vascular remodeling. We showed previously that angiogenic capillaries in the developing CNS express high levels of fibronectin and its receptor alpha5beta1 integrin, and that this expression is developmentally downregulated. As cerebral hypoxia leads to an angiogenic response, we sought to determine whether angiogenic vessels in the adult CNS re-express fibronectin and the alpha5beta1 integrin. Ten-week old mice were subject to hypobaric hypoxia for 0, 4, 7 and 14 days, and fibronectin/integrin expression examined. Fibronectin and the alpha5 integrin subunit were strongly upregulated on capillaries in the hypoxic CNS, with the effect maximal at the earliest time point examined (4 days). Immunofluorescent studies demonstrated that the alpha5 integrin was expressed by angiogenic endothelial cells. In light of the defined angiogenic role for fibronectin in other systems, this work suggests that induction of fibronectin-alpha5beta1 integrin expression may be an important molecular switch driving angiogenesis in the hypoxic CNS.

The paraventricular nucleus of the hypothalamus influences respiratory timing and activity in the rat

In this study we sought to determine the role of the paraventricular nucleus of the hypothalamus (PVN) in modulating respiratory output. Experiments were performed in urethane anesthetized, vagotomized and mechanically ventilated Wistar rats. Electromyographic activity of the diaphragm (D[EMG]) was recorded and used to define the respiratory effects of PVN stimulation. The ventilation rate and volume were pre-adjusted so that baseline activity was 30% of the activity observed upon addition of 7% CO2 in O2. Microinjection of L-glutamate (4 nmol, 100 nl) into the PVN produced an increase in peak D(EMG), and an increase in frequency of D(EMG) discharge. Changes in respiratory timing were mainly due to shortening of expiratory time (0.66 +/- 0.06 s vs. 0.90 +/- 0.10 s; mean +/- SEM; P < 0.05), while inspiratory time was less affected (0.48 +/- 0.04 vs. 0.51 +/- 0.04 s; P > 005). The rate of rise of D(EMG) increased by 101 +/- 28% from the baseline (P < 0.05). In addition, neuroanatomical tracing studies suggest the presence of direct connection between PVN and phrenic motoneurons. The results indicate that PVN neurons participate in regulation of breathing activity and in coordination of cardiovascular and respiratory functions.

KETONES SUPPRESS BRAIN GLUCOSE CONSUMPTION

The brain is dependent on glucose as a primary energy substrate, but is capable of utilizing ketones such as beta-hydroxybutyrate (beta HB) and acetoacetate (AcAc), as occurs with fasting, prolonged starvation or chronic feeding of a high fat/low carbohydrate diet (ketogenic diet). In this study, the local cerebral metabolic rate of glucose consumption (CMRglu; microM/min/100g) was calculated in the cortex and cerebellum of control and ketotic rats using Patlak analysis. Rats were imaged on a rodent PET scanner and MRI was performed on a 7-Tesla Bruker scanner for registration with the PET images. Plasma glucose and beta HB concentrations were measured and 90-minute dynamic PET scans were started simultaneously with bolus injection of 2-Deoxy-2[18F]Fluoro-D-Glucose (FDG). The blood radioactivity concentration was automatically sampled from the tail vein for 3 min following injection and manual periodic blood samples were taken. The calculated local CMRGlu decreased with increasing plasma BHB concentration in the cerebellum (CMRGlu = -4.07*[BHB] + 61.4, r2 = 0.3) and in the frontal cortex (CMRGlu = -3.93*[BHB] + 42.7, r2 = 0.5). These data indicate that, under conditions of ketosis, glucose consumption is decreased in the cortex and cerebellum by about 10% per each mM of plasma ketone bodies.

The excitatory amino acid glutamate mediates reflexly increased tracheal blood flow and airway submucosal gland secretion.

In six decerebrated and in eight alpha-chloralose anesthetized, paralyzed and mechanically ventilated beagle dogs, we have studied involvement of glutamate and glutamate receptors in transmission of excitatory inputs from the airway sensory receptors to the nucleus tractus solitarius and from this site to airway-related vagal preganglionic cells that regulate the tracheal circulation and the submucosal gland secretion. Stimulation of airway sensory fibers by lung deflation-induced reflex increase in tracheal blood flow and submucosal gland secretion. These responses were diminished by prior administration of AMPA/kainate receptor antagonist CNQX into the fourth ventricle (n=6). Furthermore, topical application or microinjection of AMPA/kainate receptor blockers, into the region of the ventrolateral medulla, where airway-related vagal preganglionic neurons are located, abolished the reflex changes in tracheal submucosal gland secretion (n=8); in these dogs mucosal blood flow was not measured). These findings indicate that reflex increase in tracheal blood flow and submucosal gland secretions are mediated mainly via release of glutamate and activation of the AMPA/kainate subtype of glutamate receptors.

The role of the medullary raphe nuclei in regulation of cholinergic outflow to the airways

In these studies we determined the role of the medullary midline nuclei on a cholinergic outflow to the airways by examining the response of tracheal tone and lung resistance to pharmacological stimulation. Studies were performed on alpha-chloralose-anesthesized, paralyzed and mechanically ventilated cats, and ferrets. L-glutamate microinjection into the medullary midline neurons significantly decreased tracheal tension, and reduced lung resistance. These effects were abolished by prior topical application of methysergide, a broad spectrum serotonin receptor antagonist. Stimulation of the medullary raphe nuclei was also associated with a significant increase in the phrenic nerve output, and a decrease in arterial blood pressure. The results indicate that the medullary midline neurons are involved in regulation of cholinergic outflow to the airways, and raise the possibility that alterations in the serotonergic pathways may cause airway dysfunction.

Increased adiposity in the retinol saturase-knockout mouse

The enzyme retinol saturase (RetSat) catalyzes the saturation of all‐irans‐retinol to produce (R)‐all‐trans‐13,14‐dihydroretinol. As a peroxisome pro‐liferator‐activated receptor (PPAR) γ target, RetSat was shown to be required for adipocyte differentiation in the 3T3‐L1 cell culture model. To understand the mechanism involved in this putative proadipogenic effect of RetSat, we studied the consequences of ablating RetSat expression on retinoid metabolism and adipose tissue differentiation in RetSat‐null mice. Here, we report that RetSat‐null mice have normal levels of retinol and retinyl palmitate in liver, serum, and adipose tissue, but, in contrast to wild‐type mice, are deficient in the production of all‐trans‐13,14‐dihydroretinol from dietary vitamin A. Despite accumulating more fat, RetSat‐null mice maintained on either low‐fat or high‐fat diets gain weight and have similar rates of food intake as age‐ and gender‐matched wildtype control littermates. This increased adiposity of RetSat‐null mice is associated with up‐regulation of PPARγ, a key transcriptional regulator of adipogenesis, and also its downstream target, fatty acid‐binding protein 4 (FABP4/aP2). On the basis of these results, we propose that dihydroretinoids produced byRetSat control physiological processes that influence PPARγ activity and regulate lipid accumulation in mice.—Moise, A. R., Lobo, G. P., Erokwu, B., Wilson, D. L., Peck, D., Alvarez, S., Domínguez, M., Alvarez, R., Flask, C. A., de Lera, A. R., von Lintig, J., Palczewski, K. Increased adiposity in the retinol saturase‐knockout mouse. FASEB J. 24, 1261–1270 (2010). www.fasebj.org

The brainstem network involved in coordination of inspiratory activity and cholinergic outflow to the airways

The respiratory rhythm modulates cholinergic outflow to the tracheal smooth muscle through the parasympathetic nerves. To determine the basis of this modulation, we combined the retrograde tracer technique to identify bulbospinal cells projecting to phrenic motoneurons, and the transneuronal labeling method to visualize medullary neurons that innervate airway-related vagal preganglionic cells. Following injections of fluorogold into the ventral horns of the cervical spinal cord and injections of pseudorabies virus (PRV) into the wall of the extrathoracic trachea of superior cervical ganglioctomized Sprague-Dawley rats. A large number of the double-labeled cells were identified along the ventral aspect of the medulla oblongata. Most frequently, double-labeled neurons were seen in the medial tegmental field, particularly in the parapyramidal region, within the gigantocellular nuclei, and the caudal raphe nuclei. Less frequently, double-labeled neurons were found in the ventrolateral medulla. No double-labeled cell was observed in the dorsal aspect of the medulla oblongata. This study indicates that a subset of medullary neurons that project to phrenic motoneurons also innervate the airway-related vagal preganglionic cells, allowing the coupling of inspiratory activity and parasympathetic outflow to the airways.

The role of excitatory amino acids in airway reflex responses in anesthetized dogs

In these studies we examined the role of excitatory amino acids (EAAs) neurotransmission in communicating sensory inputs to the airway-related vagal preganglionic neurons, by examining the effects of either NMDA or AMPA/kainate receptor blockade on reflex and chemical responses of tracheal smooth muscle. Experiments were performed in chloralose anesthetized, paralyzed and mechanically ventilated beagle dogs (n = 18), under hyperoxic, normocapnic, and normohydric conditions. Topical application or microinjection of NMDA receptor blockers, into the region of the ventrolateral medulla where airway-related vagal preganglionic neurons are located, insignificantly decreased the reflex changes in tracheal tone. However, topical application or microinjection of AMPA/kainate subtype of glutamate receptor selective antagonists markedly reduced reflex increase in tracheal tone induced by (1) lung deflation, (2) stimulation of laryngeal cold receptors, and (3) activation of peripheral or central chemoreceptors. These effects were potentiated by prior NMDA receptor blockade. Findings indicate that an increase in central cholinergic outflow to the airways by a variety of excitatory afferent inputs is mediated via activation of EAA receptors, mainly AMPA/kainate subtype of glutamate receptors.