Brain cytokine and chemokine mRNA expression in mice induced by intranasal instillation with ultrafine carbon black

Ambient air ultrafine particles (UFPs) have gained enormous attention to many researchers with recent evidence showing them to have more hazardous effects on human health than larger ambient particles. Studies focusing the possibility of effects on brain are quite limited. To examine the effect of ultrafine carbon black (ufCB) on mice brain, we instilled 125 microg of 14 nm or 95 nm CB into the nostrils of 8-week-old male BALB/c mice, once a week for 4 weeks. Four hours after the last instillation, we collected olfactory bulb and hippocampus and detected the expression of cytokine and chemokine mRNA by quantitative real-time PCR method. In this study, we found the induction of proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha and chemokines (monocyte chemoattractant protein-1/CCL2, macrophage inflammatory protein-1 alpha/CCL3), and monokine induced interferon-gamma/CXC chemokine ligand (CXCL9) mRNA in brain olfactory bulb, not in the hippocampus of mice instilled with 14 nm ufCB intranasally. We suggest that the intranasal instillation of ufCB may influence the brain immune function depending on their size. To our knowledge, this is the first study to demonstrate region-specific brain cytokine and chemokine mRNA-induction in mice triggered by intranasal instillation of specific-sized ufCB, in a physiologically relevant condition.     

Sexual dimorphism in the GABAergic control of gonadotropin release in intact rats

GABA is a potent regulator of gonadotropin release both in male and female rats. We reported 24 h profiles of GABA release in the medial preoptic area (MPO) where gonadotropin-releasing hormone (GnRH) surge generator resides in female rats. In this article, we review the sex difference in 24 h profiles of GABA release. GABA release is high and episodic in male rats without any time dependency, but female rats showed a surge-like secretion of GABA in the early morning of the proestrous day. GABA release rapidly decreased until the afternoon of the day of proestrus followed by the preovulatory luteinizing hormone (LH) surge. The peak time of GABA episodes changes with estrous cycle in female rats. Fitting with the double cosinor method demonstrated that the acrophase of the GABA release in proestrous female rats occurs in the early morning, whereas the acrophases in diestrous females, estrous females and males occur at various time of day. Proestrous female rats showed significant difference in the peak time and acrophase of the GABA release compared with other estrous stages of female and male rats. These results demonstrated further sexual dimorphism of GABA release in the MPO, suggesting that coupling between the GABA release and the circadian clock may be a determining factor in the sex difference of the hypothalamo-pituitary-gonadal (HPG) axis in rats.     

Sexual dimorphism of GABA release in the medial preoptic area and luteinizing hormone release in gonadectomized estrogen-primed rats

We showed marked sex differences in the GABA outflow in the medial preoptic area of intact rats. To further determine the sexually dimorphic effects of estrogen on the GABA outflow, an in vivo microdialysis study was performed in gonadectomized rats 3-5 days after the estrogen- or cholesterol-priming. Dialysates and sequential blood samples (150 microl each) were simultaneously collected under freely moving conditions. Serum estradiol concentrations at 72 and 84 h after the estrogen capsule implantation were approximately 75 pg/ml in both sexes. Ovariectomized estrogen-primed (OVX+E(2)) rats showed high GABA outflow from the late night through the morning, which was significantly declined until the onset of surge like secretion of luteinizing hormone (LH) in the afternoon (N=7). Ovariectomized cholesterol-primed (OVX+C) rats consistently showed low GABA outflow and high serum LH concentration (N=8). Conversely, orchidectomized estrogen-primed (ORX+E(2)) rats showed high and episodic GABA outflow without any daily changes (N=7), which was significantly greater than orchidectomized cholesterol-primed (ORX+C; N=8) and OVX+C rats. Only OVX+E(2) rats showed significant daily changes in the GABA outflow and serum LH concentration. Fitting with the double cosinor method demonstrated that the acrophase of the GABA outflow in OVX+E(2) rats occurs in the early morning, whereas the acrophases in OVX+C, ORX+C, and ORX+E(2) rats occur at various times of day. The present findings suggest that sex-specific effects of estrogen on the daily GABA release in the medial preoptic area may be involved in the sex difference of LH release in rats.     

Modulation of neurological related allergic reaction in mice exposed to low-level toluene

The contributing role of indoor air pollution to the development of allergic disease has become increasingly evident in public health problems. It has been reported that extensive communication exists between neurons and immune cells, and neurotrophins are molecules potentially responsible for regulating and controlling this neuroimmune crosstalk. The adverse effects of volatile organic compounds which are main indoor pollutants on induction or augmentation of neuroimmune interaction have not been fully characterized yet. To investigate the effects of low-level toluene inhalation on the airway inflammatory responses, male C3H mice were exposed to filtered air (control), 9 ppm, and 90 ppm toluene for 30 min by nose-only inhalation on Days 0, 1, 2, 7, 14, 21, and 28. Some groups of mice were injected with ovalbumin intraperitoneally before starting exposure schedule and these mice were then challenged with aerosolized ovalbumin as booster dose. For analysis of airway inflammation, bronchoalveolar lavage (BAL) fluid were collected to determine inflammatory cell influx and lung tissue and blood samples were collected to determine cytokine and neurotrophin mRNA and protein expressions and plasma antibody titers using real-time RT-PCR and ELISA methods respectively. Exposure of the ovalbumin-immunized mice to low-level toluene resulted in (1) increased inflammatory cells infiltration in BAL fluid; (2) increased IL-5 mRNA, decreased nerve growth factor receptor tropomyosin-related kinase A and brain-derived neurotrophic factor mRNAs in lung; and (3) increased IgE and IgG(1) antibodies and nerve growth factor content in the plasma. These findings suggest that low-level toluene exposure aggravates the airway inflammatory responses in ovalbumin-immunized mice by modulating neuroimmune crosstalk.     

Effect of long-term exposure to low-level toluene on airway inflammatory response in mice

Volatile organic compounds are the main substances causing multiple chemical sensitivity reactions in human. Our laboratory has previously showed that the exposure of low-level formaldehyde causes immunogenic and neurogenic inflammatory responses in mice. The aim of the present study was to investigate the effect of long-term, low-level toluene exposure on airway inflammatory responses in mice lung. We exposed female C3H mice to filtered air (0ppm) or 50ppm of toluene for 6h/day on 5days/week for 6 or 12 weeks in the whole body exposure chamber. One day following the last toluene exposure, we collected bronchoalveolar lavage fluid from each mouse and examined cellular infiltration and production of cytokines, chemokines, neurotrophins and substance P by using ELISA method. We found that the number of total cells and macrophages increased significantly in both 6 and 12-week-exposed mice. In addition, the production of interferon-gamma and substance P were decreased significantly and nerve growth factor was not affected in both 6 and 12-week-exposed mice. In contrast, neurotrophin-3 production in bronchoalveolar lavage fluid was significantly increased only in 12-week-exposed mice. Our findings suggest that long-term (12-week) exposure of mice to low-level toluene modulates airway inflammatory response via neurological signaling.     

Determination of toluene in brain of freely moving mice using solid-phase microextraction technique

For the purpose of measuring the pharmacokinetics of inhaled toluene in the brain of mice, we developed a method for the direct detection of toluene by gas chromatography/mass spectrometry (GC/MS). The method uses a solid-phase microextraction (SPME) fiber inserted into the mouse' hippocampus (CA1) through a cannula fixed onto the animal. BALB/c mice were exposed to 0, 0.9, 9, 50 and 90 ppm toluene for 30 min. The toluene level detected near the hippocampus by this method after exposure to 0.9 ppm toluene in air showed no significant difference from the level found in the non-exposure control group; however, the toluene level increased significantly after exposure to concentrations of 9, 50 and 90 ppm. These increases were concentration-dependent. In addition, the pharmacokinetics of toluene in the brain of mice exposed to 50 ppm toluene showed that the toluene level decreased rapidly after the exposure, and returned to control levels after 60 min. This study describes the method which has successfully detected toluene levels in the brain of conscious, free-moving mice for the first time.     

Profiles of in vivo gamma-aminobutyric acid release in the medial preoptic area of intact and castrated male rats

We have suggested that gamma-aminobutyric acid (GABA) in the hypothalamus plays a tonic inhibitory role in the control of the luteinizing hormone (LH) release in intact male rats. To assess whether feedback from the testis alters the inhibitory GABAergic tone in the medial preoptic area (MPO) of male rats, an in vivo microdialysis study was performed in gonadally intact (n = 10), castrated (n = 12) and castrated testosterone-primed (n = 10) male rats. The microdialysis samples were collected and sequential blood samples were also obtained at 1-hour intervals. GABA in the dialysate was determined by high-performance liquid chromatography system and serum LH concentration was determined by radioimmunoassay. Episodic GABA release in the MPO was observed in all three groups of male rats, although castrated male rats showed lower GABA release (2.3 +/- 0.3 ng/h) than intact and castrated testosterone-primed male rats (4.0 +/- 0.5 and 4.6 +/- 1.0 ng/h, respectively). Conversely, castrated male rats showed higher serum LH concentration (7.31 +/- 0.46 ng/ml) than intact and castrated testosterone-primed male rats (0.71 +/- 0.04 and 0.53 +/- 0.07 ng/ml, respectively). In addition, intravenous infusion of bicuculline significantly increased serum LH in intact male rats, whereas bicuculline did not alter serum LH concentrations in castrated male rats. These results are consistent with the hypothesis that the feedback of testosterone stimulates GABA release in the region of the GnRH cell bodies and dendrites in male rats.     

Effect of ultrafine carbon black particles on lipoteichoic acid-induced early pulmonary inflammation in BALB/c mice

We studied the interaction effects of a single intratracheal instillation of ultrafine carbon black (CB) particles and staphylococcal lipoteichoic acid (LTA) on early pulmonary inflammation in male BALB/c mice. We examined the cellular profile, cytokine and chemokine levels in the bronchoalveolar lavage (BAL) fluid, and expression of chemokine and toll-like receptor (TLR) mRNAs in lungs. LTA produced a dose-related increase in early pulmonary inflammation, which was characterized by (1) influx of polymorphonuclear neutrophils (PMNs) and (2) induction of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha/CCL3, but no effect on monocyte chemoattractant protein (MCP)-1/CCL2 at 24 h after instillation. Levels of some proinflammatory indicators and TLR2-mRNA expression were significantly increased by 14 nm or 95 nm CB (125 microg) and low-dose LTA (10 microg) treatment compared to CB or LTA alone at 4 h after instillation. Notably, PMN levels and production of IL-6 and CCL2 in the 14 nm CB + LTA were significantly higher than that of 95 nm CB + LTA at 4 h after instillation. However, at 24 h after instillation, only PMN levels were significantly higher in the 14 nm CB + LTA than 95 nm CB + LTA but not the cytokines and chemokines. These data show additive as well as synergistic interaction effects of 14 nm or 95 nm ultrafine CB particles and LTA. We suggest that early pulmonary inflammatory responses in male BALB/c mice may be induced in a size-specific manner of the CB particles used in our study.     

Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure

Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1 beta, TNF-alpha, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 microg) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 mug of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1 beta mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-alpha mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 beta mRNA expressions synergistically with LTA in the mice olfactory bulb.