One-pot synthesis of nanochain particles for targeting brain tumors.

To synthesize multi-component nanochains, we developed a simple 'one-pot' synthesis, which exhibited high yield and consistency. The nanochains particles consist of parent nanospheres chemically linked into a higher-order, chain-like assembly. The one-pot synthesis is based on the addition of two types of parent nanospheres in terms of their surface chemical functionality (e.g., decorated with PEG-NH2 or PEG-COOH). By reacting the two types of parent nanospheres at a specific ratio (∼2 : 1) for a short period of time (∼30 min) under rigorous stirring, nanochains were formed. For example, we show the synthesis of iron oxide nanochains with lengths of about 125 nm consisting of 3-5 constituting nanospheres. The chain-like shaped nanoparticle possessed a unique ability to target and rapidly deposit on the endothelium of glioma sites via vascular targeting. To target and image invasive brain tumors, we used iron oxide nanochains with the targeting ligand being the fibronectin-targeting peptide CREKA. Overexpression of fibronectin is strongly associated with the perivascular regions of glioblastoma multiforme and plays a critical role in migrating and invasive glioma cells. In mice with invasive glioma tumors, 3.7% of the injected CREKA-targeted nanochains was found in gliomas within 1 h. Notably, the intratumoral deposition of the nanochain was ∼2.6-fold higher than its spherical variant. Using MR imaging, the precise targeting of nanochains to gliomas provided images with the exact topology of the disease including their margin of infiltrating edges and distant invasive sites.

CNS innervation of posterior cricoarytenoid muscles: a transneuronal labeling study.

The CNS cell groups that project to neurons, which innervate the posterior cricoarytenoid muscles (PCA), were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the PCA of C8 spinal rats and after 5 days survival, brain tissue sections were processed for immunohistochemical detection of PRV. Retrogradely labeled motor neurons innervating the PCA were seen in the nucleus ambiguus and in the area ventral to it. Neurons innervating the PCA motoneurons were found throughout the ventral aspect of the medulla oblongata, in the nucleus tractus solitarius, and in the pons. Labeling was present in the midbrain periaquaductal gray, in the lateral and paraventricular hypothalamic nuclei, in the amygdaloid complex, in the hippocampus, and within the piriform cortex. In summary, the motor neurons that control PCA activity are innervated predominantly by a network of neurons that lie along the neuraxis, in the regions known to be involved in regulation of respiratory output and autonomic functions.

Medical student competence in eliciting a history for "chronic fatigue".

PURPOSE: We report an observational study of medical students' abilities in taking a complex history for which sleep disorders is one of several possible conditions. METHODS: Students are observed taking a focused history from a simulated patient whose chief complaint is "I am tired. I cannot get anything done." Nine groups of students (n = 360) completing the internal medicine core-clerkship were evaluated by one of three examiners. Students received full, partial, or no credit for each item on a uniform behavioral checklist, which included prompts for common medical and psychiatric disorders associated with chronic fatigue. RESULTS: Observed means were lowest for items pertaining to sleep behaviors and head trauma. Fewer than half of the students inquired about whether or not the person had difficulty falling asleep at night, family history of sleep apnea, and frequency and length of naps. In contrast, the majority of students inquired about heart disease, metabolic disorders, the use of illicit drugs, alcohol consumption, and the taking of medications. Examiners accounted for a significant source of variance in scores; yet the station discriminated among top and bottom students as measured by the Objective Structured Clinical Examination (OSCE) overall. No statistically significant differences were observed on the basis of clerkship site, primary care versus traditional-track students, time of year, or gender. CONCLUSION: A majority of students do not adequately cover issues relevant to sleep in contrast to other associated disorders when taking a focused history for chronic fatigue.

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 alpha3 subtype of the nicotinic acetylcholine receptor is expressed in airway-related neurons of the nucleus tractus solitarius, but is not essential for reflex bronchoconstriction in ferrets.

To assess the role of nicotinic acetylcholine receptors (nACh-R) in the transmission of afferent constricting inputs from bronchopulmonary receptors to the nucleus tractus solitarius (nTS) and in the mediation of reflex airway constriction, we performed a combined immunohistological and functional study. In ferrets, the expression of nAch-R on the nTS neurons activated by histamine stimulation of airway sensory receptors was studied using laser scanning confocal microscopy to co-immunolocalize c-fos encoded protein (cFos) and nACh-R alpha3 subunit. We observed that activation of airway sensory receptors by inhalation of aerosolized histamine, induced cFos expression in a subset of nTS neurons that also expressed the nAch-R alpha3 subtype. Furthermore, activation of nACh-R within the commissural subnucleus by nicotine, increased cholinergic outflow to the airways. These effects were diminished by prior administration of hexamethonium (nACh-R blocker) within the commissural subnucleus of the nTS. However, hexamethonium had no significant effects on airway reflex constrictions induced by lung deflation. These findings indicate that nACh-R are expressed by the nTS neurons receiving inputs from airway sensory receptors, activation of which by nicotine increases cholinergic outflow to the airways, but the nACh-R pathways are not required for reflex bronchoconstriction.

Alterations in respiratory behavior, brain neurochemistry and receptor density induced by pharmacologic suppression of sleep in the neonatal period.

UNLABELLED: The present study examined if drug suppression of active sleep (AS) in the neonate affected the development and expression of respiratory behavior. Secondly, we assessed brain neurochemistry and receptor density in specific supra-medullary brain regions to identify coincident biochemical alterations. Sprague-Dawley newborn rat pups were randomized and divided among six rat mothers (n=10/mother/group), each mother housed separately. Two untreated control (UC) groups received either no interventions or were fed milk vehicle twice daily and were handled similarly to the drug intervention animals. Pharmacological disruption of sleep was achieved by administration (2 groups of each) of either clonidine (CLO) 100 microm/kg, or scopolamine (SCO) 800 microm/kg, given orally twice daily for the first 7 days of life. On postnatal (P) days P10 and P19 of life, pups were assessed for metabolism, minute ventilation (VE), tidal volume (Vt) and frequency (f). On P21 (14 days after the end of drug exposure), pups from each condition were sacrificed and punch biopsies of the frontal cortex, hypothalamus, and hippocampus were examined for hydroxytryptophan (5-HT), and norepinepherine (NE) by HPLC. An equal number of pups were sacrificed and brains examined for muscarinic acetylcholine (mAch), alpha2-adrenergic and I1-imidazoline receptor density. RESULTS: Both CLO and SCO exposed animals had a lower V(t) and respiratory quotient than UC animals (p<0.01). CLO animals exhibited a higher f (p<0.01) and both CLO and SCO exhibited a lower V(t) (p<0.05) than the UC groups; VE was reduced in the SCO groups, compared with CLO and UC groups (p<0.01). Pattern of breathing in response to brief hypoxia exposure was altered for CLO and SCO. The normal decline in VE during sleep was not observed in CLO rats. Both drug exposures resulted in a comparable reduction in hypothalamic NE and 5-HT levels (p<0.05), while in the frontal cortex, and the hippocampus variable changes in NE and 5-HT, occurred. In CLO and SCO rats mAch receptors were increased in cortex, and reduced in hypothalamus; I1-imidazoline receptors were increased in hypothalamus and decreased in hippocampus (p<0.05 for each). In contrast, alpha2-adrenergic receptors were increased in cortex for both CLO and SCO, decreased in hypothalamus for CLO, and decreased in hippocampus for SCO (p<0.05 for each). CONCLUSIONS: these data show that drug-induced neonatal sleep suppression will alter ventilatory pattern, metabolism, and site-specific concentrations of adrenergic neurotransmitters and in receptor density, perhaps as a result of suppression of neonatal AS.

Effect of Pseudomonas infection on weight loss, lung mechanics, and cytokines in mice.

Poor growth, Pseudomonas aeruginosa endobronchitis, pulmonary inflammation, and decline of lung function are hallmarks of cystic fibrosis (CF), yet the relationship between these features is poorly understood. Because animal models of chronic bronchopulmonary infection with P. aeruginosa used to study pulmonary inflammation in CF have also been associated with weight loss, we sought to determine whether this weight loss was due to the inflammatory process and/or to changes in lung function. P. aeruginosa-laden agarose beads were instilled into the lungs of mice. Weight loss was greatest 3 d after Pseudomonas infection. Infected mice had a rapid though transient rise in absolute neutrophil counts, mTNF-alpha, mIL-1beta, mIL-6, mip-2, and KC in bronchoalveolar lavage fluid. There was no difference in lung resistance or lung compliance measured by body plethysmography between infected and control mice. Weight loss did correlate with the concentration of proinflammatory cytokine levels 3 d after inoculation of mice with Pseudomonas, and body composition analysis revealed loss of skeletal muscle mass. These results suggest that weight loss in P. aeruginosa-infected mice was associated with the inflammatory process and not with altered pulmonary responsiveness. These findings may provide insights into the cause of cachexia and weight loss seen in patients with CF.

Extended survival time following pseudorabies virus injection labels the suprapontine neural network controlling the bladder and urethra in the rat.

The central neurons that are involved in control of the urinary bladder and proximal urethra in adult male rats were identified by retrograde transport of the viral transneural tracer pseudorabies virus (PRV, Bartha strain). At 5 days post-injection, PRV-infected neurons were found in suprapontine central nervous system nuclei including ventrolateral periaqueductal gray, magnocellular division of the red nucleus, lateral hypothalamus and paraventricular nucleus, retrochiasmic region and suprachiasmatic nucleus. At days 6 and 7 PRV-infected neurons were observed in the amygdala, lateral septal nucleus, hippocampus, and frontal motor, piriform, and perirhinal cortices. These results identify the supraspinal neural networks that are involved in control of the lower urinary tract, and demonstrate the utility of long survival times to label higher-order neurons with PRV.

Moxonidine acting centrally inhibits airway reflex responses.

We examined the role of I1-imidazoline (I1-IR) receptors in control of airway function, by testing the effects of systemic administration of the I1-IR agonist moxonidine on reflex responses of tracheal smooth muscle (TSM) tone to either lung deflation or mechanical stimulation of intrapulmonary rapidly adapting receptors. Experiments were performed in either alpha-chloralose anesthetized or decorticate, paralyzed, and mechanically ventilated beagle dogs. Moxonidine (10-100 micrograms/kg) administered via three different routes (femoral vein, muscular branch of superior thyroid artery, and vertebral artery) attenuated TSM responses to stimulation of airway sensory nerve fibers by two different ways and caused a decrease in arterial pressure and heart rate. These effects were dose dependent and were significantly reversed by efaroxan (an I1-IR and alpha 2-adrenergic blocker) administered via the vertebral artery. Intravertebral efaroxan abolished the hemodynamic effects of moxonidine. Intravenous moxonidine (10-100 micrograms/kg) did not alter airway smooth muscle responses to electrical stimulation of the peripheral vagus nerve. In addition, in vitro moxonidine (1-100 micrograms/ml) had no effect on contractile responses to increasing doses of acetylcholine. These findings indicate that moxonidine may act at a central site to suppress reflex airway constriction, even when given into the systemic circulation. Given the presence of I1-IR sites and alpha 2-adrenergic receptors in brain regions participating in airway reflexes, these receptor classes may be involved in brainstem control of the cholinergic outflow to the airways.

I1-imidazoline receptors and cholinergic outflow to the airways.

We examined the role of I1-imidazoline receptors in the control of airway function, by testing the effects of systemic administration of the I1-imidazoline agonist moxonidine on reflex responses of tracheal smooth muscle (TSM) tone to either lung deflation or mechanical stimulation of intrapulmonary rapidly adapting receptors. Experiments were performed in either alpha-chloralose anaesthetized or decorticate, paralyzed and mechanically ventilated beagle dogs. Moxonidine (10-100 microg/kg) administered via three different routes (the femoral vein, muscular branch of superior thyroid artery, and vertebral artery) attenuated TSM responses to stimulation of airway sensory nerve fibers by two different ways, and caused a decrease in arterial pressure and heart rate. These effects were dose-dependent, and were significantly reversed by efaroxan (an I1-imidazoline and alpha2-adrenergic blocker) administered via the vertebral artery. Intravertebral efaroxan abolished the hemodynamic effects of moxonidine. Intravenous moxonidine (10-100 microg/kg) did not alter airway smooth muscle responses to electrical stimulation of the peripheral vagus nerve. In addition, in vitro moxonidine (1-100 microg/ml) had no effect on contractile responses to increasing doses of acetylcholine. These findings indicate that moxonidine may act at a central site to suppress reflex airway constriction, even when given into the systemic circulation. Given the presence of I1-imidazoline sites and alpha2-adrenergic receptors in brain regions participating in airway reflexes, these receptor classes may be involved in brainstem control of the cholinergic outflow to the airways.

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.

Selective hypoxic loading of the ventrolateral medulla inhibits cholinergic outflow to the airways.

In these studies we determined the effects of local hypoxia confined to the superficial structures of the ventral medulla on cholinergic outflow to the airways. Studies were performed in alpha-chloralose anesthetized, paralyzed and mechanically ventilated cats. Topical application or microinjection of sodium cyanide to the intermediate area of the ventrolateral medulla significantly decreased tracheal tone, measured by changes in pressure in a balloon placed in bypassed segment of the trachea (Ptseg). The changes were reversible. When parasympathetic activity was abolished by atropine methylnitrate and tracheal tone was restored with serotonin, cyanide acting centrally had no effect on tracheal pressure. These data indicate that local hypoxic loading confined to the ventrolateral medullary structures may significantly modulate parasympathetic outflow to the airways, and alter airway related protective reflex responses.

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.

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.

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.

Decreased energy metabolism in brain stem during central respiratory depression in response to hypoxia.

Metabolic changes in the brain stem were measured at the time when oxygen deprivation-induced respiratory depression occurred. Eucapnic ventilation with 8% oxygen in vagotomized urethan-anesthetized rats resulted in cessation of respiratory drive, monitored by recording diaphragm electromyographic activity, on average within 11 min (range 5-27 min), presumably via central depressant mechanisms. At that time, the brain stems were frozen in situ for metabolic analyses. By using 20-microns lyophilized sections from frozen-fixed brain stem, microregional analyses of ATP, phosphocreatine, lactate, and intracellular pH were made from 1) the ventral portion of the nucleus gigantocellularis and the parapyramidal nucleus; 2) the compact and ventral portions of the nucleus ambiguus; 3) midline neurons; 4) nucleus tractus solitarii; and 5) the spinal trigeminal nucleus. At the time of respiratory depression, lactate was elevated threefold in all regions. Both ATP and phosphocreatine were decreased to 50 and 25% of control, respectively. Intracellular pH was more acidic by 0.2-0.4 unit in these regions but was relatively preserved in the chemosensitive regions near the ventral and dorsal medullary surfaces. These results show that hypoxia-induced respiratory depression was accompanied by metabolic changes within brain stem regions involved in respiratory and cardiovascular control. Thus it appears that there was significant energy deficiency in the brain stem after hypoxia-induce respiratory depression had occurred.

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.

Nitric oxide and ventilatory response to hypoxia.

It is believed that hypoxia results in the release of neurotransmitters in the central nervous system, which can excite or inhibit breathing. Recent evidence indicates that nitric oxide (NO) is a physiological messenger molecule that may serve as a neurotransmitter in the CNS. In this study we examined (1) the localization of nitric oxide synthase (NOS) within the nucleus tractus solitarius, and (2) the role of the NO-cGMP pathway in the respiratory response to oxygen deprivation. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was used to determine the distribution of neurons that express NOS, an enzyme involved in NO formation. The NOS inhibitor N omega-nitro-L-arginine was used as tool to assess the NOS activity in the medulla, and to define the role of NO in the respiratory response to acute oxygen deprivation. In the rat and the cat brainstem, histochemical studies showed the presence of NADPH-diaphorase reactive neurons within subnuclei of the nucleus tractus solitarius which receive peripheral chemoreceptor inputs. Chronic pretreatment of rats with N omega-nitro-L-arginine (75 mg/kg, ip, twice daily for 7 days) caused a significant decrease in cGMP, and attenuated the ventilatory response to hypoxia. In anesthetized, paralyzed, vagotomized and artificially ventilated cats with intact carotid sinus nerves (n = 8), administration of N omega-nitro-L-arginine (30-100 mg/kg) attenuated the response to hypoxia, and caused the hypoxia induced roll-off of phrenic nerve activity to occur significantly earlier than when NOS activity was not inhibited. In sinoaortic denervated cats (n=9) blockage of NOS potentiated the decline of the phrenic nerve output. The data suggest that oxygen deprivation leads to activation of NO-cGMP pathway in the central nervous system, which contributes to the induction and maintenance of hypoxia-induced increase in respiratory output. In addition, these findings indicate that NO may inhibit inhibitory synaptic transmission that is triggered by CNS hypoxia, and this is not directly related to peripheral chemoreceptor inputs.