Biophotonic Therapy with Fluorescent Light Energy Decreases Facial Erythema, Improves Signs and Symptoms of Rosacea, and Increases Patient Satisfaction: A Postmarket Study.

BACKGROUND: Rosacea is a difficult-to-manage chronic inflammatory skin condition reported to have a negative psychosocial impact on patients. Novel approaches are sought to target the many signs and symptoms of the condition while also improving the quality of life of patients. OBJECTIVE: We assessed the efficacy of the Kleresca® biophotonic platform (KLOX Technologies Inc., Laval, Canada), which creates fluorescent light energy (FLE), to induce a novel form of photobiomodulation for treating rosacea. We also assessed patient satisfaction with their facial appearance and concerns about perceptions of others before and after treatment. METHODS: Nine patients were treated once a week for four weeks with FLE. Patients and the treating clinician completed questionnaires throughout and after the treatment to grade the rosacea signs and symptoms and capture patients' perceptions of the treatment and their condition. RESULTS: FLE significantly reduced the inflammatory erythematous reaction of the face, improved flushing and erythema associated with rosacea, and had a positive impact on patients' self-perception and emotional wellbeing. CONCLUSION: Our results support FLE as an effective, noninvasive treatment modality for rosacea.

Fluorescent light energy modulates healing in skin grafted mouse model.

Skin grafting is often the only treatment for skin trauma when large areas of tissue are affected. This surgical intervention damages the deeper dermal layers of the skin with implications for wound healing and a risk of scar development. Photobiomodulation (PBM) therapy modulates biological processes in different tissues, with a positive effect on many cell types and pathways essential for wound healing. This study investigated the effect of fluorescent light energy (FLE) therapy, a novel type of PBM, on healing after skin grafting in a dermal fibrotic mouse model. Split-thickness human skin grafts were transplanted onto full-thickness excisional wounds on nude mice. Treated wounds were monitored, and excised xenografts were examined to assess healing and pathophysiological processes essential for developing chronic wounds or scarring. Results demonstrated that FLE treatment initially accelerated re-epithelialization and rete ridge formation, while later reduced neovascularization, collagen deposition, myofibroblast and mast cell accumulation, and connective tissue growth factor expression. While there was no visible difference in gross morphology, we found that FLE treatment promoted a balanced collagen remodeling. Collectively, these findings suggest that FLE has a conceivable effect at balancing healing after skin grafting, which reduces the risk of infections, chronic wound development, and fibrotic scarring.

Effective removal of solar lentigines by combination of pre- And post-fluorescent light energy treatment with picosecond laser treatment.

We present a novel treatment for removal of SL that is efficient and enables removal or lesions not immediately visible. Kleresca® FLE technology combined with picosecond laser treatment removes SL lesions and improves skin quality and appearance.

N-acetylcysteine Decreases Fibrosis and Increases Force-Generating Capacity of mdx Diaphragm.

Respiratory muscle weakness occurs due to dystrophin deficiency in Duchenne muscular dystrophy (DMD). The mdx mouse model of DMD shows evidence of impaired respiratory muscle performance with attendant inflammation and oxidative stress. We examined the effects of N-acetylcysteine (NAC) supplementation on respiratory system performance in mdx mice. Eight-week-old male wild type (n = 10) and mdx (n = 20) mice were studied; a subset of mdx (n = 10) received 1% NAC in the drinking water for 14 days. We assessed breathing, diaphragm, and external intercostal electromyogram (EMG) activities and inspiratory pressure during ventilatory and non-ventilatory behaviours. Diaphragm muscle structure and function, cytokine concentrations, glutathione status, and mRNA expression were determined. Diaphragm force-generating capacity was impaired in mdx compared with wild type. Diaphragm muscle remodelling was observed in mdx, characterized by increased muscle fibrosis, immune cell infiltration, and central myonucleation. NAC supplementation rescued mdx diaphragm function. Collagen content and immune cell infiltration were decreased in mdx + NAC compared with mdx diaphragms. The cytokines IL-1ß, IL-6 and KC/GRO were increased in mdx plasma and diaphragm compared with wild type; NAC decreased systemic IL-1ß and KC/GRO concentrations in mdx mice. We reveal that NAC treatment improved mdx diaphragm force-generating capacity associated with beneficial anti-inflammatory and anti-fibrotic effects. These data support the potential use of NAC as an adjunctive therapy in human dystrophinopathies.

Fluorescent light energy: A new therapeutic approach to effectively treating acne conglobata and hidradenitis suppurativa.

The Kleresca® biophotonic platform utilizing fluorescent light energy (FLE) effectively treated both acne conglobata and hidradenitis suppurativa. FLE decreased the presence of inflammatory nodules, cysts, and associated erythema. It also supported a healing response to improve skin texture. FLE offers a new treatment approach to recalcitrant inflammatory skin conditions.

FLUORESCENT LIGHT ENERGY: The Future for Treating Inflammatory Skin Conditions?

Background: We have previously reported clinical efficacy with a novel form of photobiomodulation-a biophotonic platform inducing fluorescent light energy (FLE)-in both disease-affected and healthy skin; however, the cellular mechanisms remain largely unknown. Objective: This study investigated the cellular mechanism of action of FLE on key skin and immune cells. Methods: We examined the effects of FLE on the clinical presentation of inflammation in a representative patient with acne vulgaris. The effect of FLE and an FLE-mimicking control lamp on collagen production from primary human dermal fibroblast (HDF) cells was assessed in the presence and absence of the proinflammatory cytokine, interferon gamma (IFN-γ). Cytokine production was assessed from HDF and human epidermal keratinocytes (HEK) exposed to M1 macrophage-conditioned media following illumination with either a blue light-emitting diode (LED) or FLE. Finally, the effects of FLE on angiogenesis were assessed in human aortic endothelial (HAE) cells. Results: FLE reduced inflammatory lesions and associated redness in the representative acne patient. Following the resolution of inflammation there was an overall enhancement of the skin's texture. FLE enhanced collagen production from nonstressed HDF cells, decreased the inflammatory profile of HDF and HEK cells, and enhanced angiogenesis in HAE cells. Conclusion: Our results suggest FLE is capable of enhancing collagen production, modulating cutaneous inflammation, and encouraging angiogenesis. While further research is required, our findings have important implications for approaches to treating inflammatory skin conditions and achieving better aesthetic outcomes.

Recovery of respiratory function in mdx mice co-treated with neutralizing interleukin-6 receptor antibodies and urocortin-2.

KEY POINTS: Impaired ventilatory capacity and diaphragm muscle weakness are prominent features of Duchenne muscular dystrophy, with strong evidence of attendant systemic and muscle inflammation. We performed a 2-week intervention in young wild-type and mdx mice, consisting of either injection of saline or co-administration of a neutralizing interleukin-6 receptor antibody (xIL-6R) and urocortin-2 (Ucn2), a corticotrophin releasing factor receptor 2 agonist. We examined breathing and diaphragm muscle form and function. Breathing and diaphragm muscle functional deficits are improved following xIL-6R and Ucn2 co-treatment in mdx mice. The functional improvements were associated with a preservation of mdx diaphragm muscle myosin heavy chain IIx fibre complement. The concentration of the pro-inflammatory cytokine interleukin-1ß was reduced and the concentration of the anti-inflammatory cytokine interleukin-10 was increased in mdx diaphragm following drug co-treatment. Our novel findings may have implications for the development of pharmacotherapies for the dystrophinopathies with relevance for respiratory muscle performance and breathing. ABSTRACT: The mdx mouse model of Duchenne muscular dystrophy shows evidence of hypoventilation and pronounced diaphragm dysfunction. Six-week-old male mdx (n = 32) and wild-type (WT; n = 32) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (xIL-6R; 0.2 mg kg-1 ) and corticotrophin-releasing factor receptor 2 agonist (urocortin-2; 30 µg kg-1 ) subcutaneously over 2 weeks. Breathing and diaphragm muscle contractile function (ex vivo) were examined. Diaphragm structure was assessed using histology and immunofluorescence. Muscle cytokine concentration was determined using a multiplex assay. Minute ventilation and diaphragm muscle peak force at 100 Hz were significantly depressed in mdx compared with WT. Drug treatment completely restored ventilation in mdx mice during normoxia and significantly increased mdx diaphragm force- and power-generating capacity. The number of centrally nucleated muscle fibres and the areal density of infiltrates and collagen content were significantly increased in mdx diaphragm; all indices were unaffected by drug co-treatment. The abundance of myosin heavy chain (MyHC) type IIx fibres was significantly decreased in mdx diaphragm; drug co-treatment preserved MyHC type IIx complement in mdx muscle. Drug co-treatment increased the cross-sectional area of MyHC type I and IIx fibres in mdx diaphragm. The cytokines IL-1ß, IL-6, KC/GRO and TNF-α were significantly increased in mdx diaphragm compared with WT. Drug co-treatment significantly decreased IL-1ß and increased IL-10 in mdx diaphragm. Drug co-treatment had no significant effect on WT diaphragm muscle structure, cytokine concentrations or function. Recovery of breathing and diaphragm force in mdx mice was impressive in our studies, with implication for human dystrophinopathies.

Diaphragm Muscle Weakness Following Acute Sustained Hypoxic Stress in the Mouse Is Prevented by Pretreatment with N-Acetyl Cysteine.

Oxygen deficit (hypoxia) is a major feature of cardiorespiratory diseases characterized by diaphragm dysfunction, yet the putative role of hypoxic stress as a driver of diaphragm dysfunction is understudied. We explored the cellular and functional consequences of sustained hypoxic stress in a mouse model. Adult male mice were exposed to 8 hours of normoxia, or hypoxia (FiO2 = 0.10) with or without antioxidant pretreatment (N-acetyl cysteine, 200 mg/kg i.p.). Ventilation and metabolism were measured. Diaphragm muscle contractile function, myofibre size and distribution, gene expression, protein signalling cascades, and oxidative stress (TBARS) were determined. Hypoxia caused pronounced diaphragm muscle weakness, unrelated to increased respiratory muscle work. Hypoxia increased diaphragm HIF-1α protein content and activated MAPK, mTOR, Akt, and FoxO3a signalling pathways, largely favouring protein synthesis. Hypoxia increased diaphragm lipid peroxidation, indicative of oxidative stress. FoxO3 and MuRF-1 gene expression were increased. Diaphragm 20S proteasome activity and muscle fibre size and distribution were unaffected by acute hypoxia. Pretreatment with N-acetyl cysteine substantially enhanced cell survival signalling, prevented hypoxia-induced diaphragm oxidative stress, and prevented hypoxia-induced diaphragm dysfunction. Hypoxia is a potent driver of diaphragm weakness, causing myofibre dysfunction without attendant atrophy. N-acetyl cysteine protects the hypoxic diaphragm and may have application as a potential adjunctive therapy.

Sensorimotor control of breathing in the mdx mouse model of Duchenne muscular dystrophy.

KEY POINTS: Respiratory failure is a leading cause of mortality in Duchenne muscular dystrophy (DMD), but little is known about the control of breathing in DMD and animal models. We show that young (8 weeks of age) mdx mice hypoventilate during basal breathing due to reduced tidal volume. Basal CO2 production is equivalent in wild-type and mdx mice. We show that carotid bodies from mdx mice have blunted responses to hyperoxia, revealing hypoactivity in normoxia. However, carotid body, ventilatory and metabolic responses to hypoxia are equivalent in wild-type and mdx mice. Our study revealed profound muscle weakness and muscle fibre remodelling in young mdx diaphragm, suggesting severe mechanical disadvantage in mdx mice at an early age. Our novel finding of potentiated neural motor drive to breathe in mdx mice during maximal chemoactivation suggests compensatory neuroplasticity enhancing respiratory motor output to the diaphragm and probably other accessory muscles. ABSTRACT: Patients with Duchenne muscular dystrophy (DMD) hypoventilate with consequential arterial blood gas derangement relevant to disease progression. Whereas deficits in DMD diaphragm are recognized, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to perform an analysis of respiratory control in a model of DMD, the mdx mouse. In 8-week-old male wild-type and mdx mice, ventilation and metabolism, carotid body afferent activity, diaphragm muscle force-generating capacity, and muscle fibre size, distribution and centronucleation were determined. Diaphragm EMG activity and responsiveness to chemostimulation was determined. During normoxia, mdx mice hypoventilated, owing to a reduction in tidal volume. Basal CO2 production was not different between wild-type and mdx mice. Carotid sinus nerve responses to hyperoxia were blunted in mdx, suggesting hypoactivity. However, carotid body, ventilatory and metabolic responses to hypoxia were equivalent in wild-type and mdx mice. Diaphragm force was severely depressed in mdx mice, with evidence of fibre remodelling and damage. Diaphragm EMG responses to chemoactivation were enhanced in mdx mice. We conclude that there is evidence of chronic hypoventilation in young mdx mice. Diaphragm dysfunction confers mechanical deficiency in mdx resulting in impaired capacity to generate normal tidal volume at rest and decreased absolute ventilation during chemoactivation. Enhanced mdx diaphragm EMG responsiveness suggests compensatory neuroplasticity facilitating respiratory motor output, which may extend to accessory muscles of breathing. Our results may have relevance to emerging treatments for human DMD aiming to preserve ventilatory capacity.

Restoration of pharyngeal dilator muscle force in dystrophin-deficient (mdx) mice following co-treatment with neutralizing interleukin-6 receptor antibodies and urocortin 2.

NEW FINDINGS: What is the central question of this study? We previously reported impaired upper airway dilator muscle function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Our aim was to assess the effect of blocking interleukin-6 receptor signalling and stimulating corticotrophin-releasing factor receptor 2 signalling on mdx sternohyoid muscle structure and function. What is the main finding and its importance? The interventional treatment had a positive inotropic effect on sternohyoid muscle force, restoring mechanical work and power to wild-type values, reduced myofibre central nucleation and preserved the myosin heavy chain type IIb fibre complement of mdx sternohyoid muscle. These data might have implications for development of pharmacotherapies for DMD with relevance to respiratory muscle performance. The mdx mouse model of Duchenne muscular dystrophy shows evidence of impaired pharyngeal dilator muscle function. We hypothesized that inflammatory and stress-related factors are implicated in airway dilator muscle dysfunction. Six-week-old mdx (n = 26) and wild-type (WT; n = 26) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (0.2 mg kg-1 ) and corticotrophin-releasing factor receptor 2 agonist (urocortin 2; 30 µg kg-1 ) over 2 weeks. Sternohyoid muscle isometric and isotonic contractile function was examined ex vivo. Muscle fibre centronucleation and muscle cellular infiltration, collagen content, fibre-type distribution and fibre cross-sectional area were determined by histology and immunofluorescence. Muscle chemokine content was examined by use of a multiplex assay. Sternohyoid peak specific force at 100 Hz was significantly reduced in mdx compared with WT. Drug treatment completely restored force in mdx sternohyoid to WT levels. The percentage of centrally nucleated muscle fibres was significantly increased in mdx, and this was partly ameliorated after drug treatment. The areal density of infiltrates and collagen content were significantly increased in mdx sternohyoid; both indices were unaffected by drug treatment. The abundance of myosin heavy chain type IIb fibres was significantly decreased in mdx sternohyoid; drug treatment preserved myosin heavy chain type IIb complement in mdx muscle. The chemokines macrophage inflammatory protein 2, interferon-γ-induced protein 10 and macrophage inflammatory protein 3α were significantly increased in mdx sternohyoid compared with WT. Drug treatment significantly increased chemokine expression in mdx but not WT sternohyoid. Recovery of contractile function was impressive in our study, with implications for Duchenne muscular dystrophy. The precise molecular mechanisms by which the drug treatment exerts an inotropic effect on mdx sternohyoid muscle remain to be elucidated.

Assessment of renal function in the anaesthetised rat following injection of superparamagnetic iron oxide nanoparticles.

A recent study showed that a significant fall in mean arterial pressure (MAP) occurred following intravenous injection of two novel superparamagnetic iron oxide nanoparticles (SPIONs), MF66 and OD15. To assess if this was caused by excessive glomerular clearance, the effect of both particles on renal function was studied. Experiments were performed on sodium pentobarbital anaesthetised male Wistar rats (250-350 g). Twenty-minute urine clearances were taken followed by an i.v. bolus of MF66, OD15 (2 mg·kg-1), or dH2O (0.4 mL·kg-1). MF6 or OD15 injection resulted in a significant transient drop in MAP and renal blood flow by approximately 33% and 50% (P < 0.05). The absolute excretion of sodium was significantly increased (P < 0.05) by almost 80% and 70% following OD15 and MF66, respectively. Similarly, fractional excretion of sodium was increased by almost 80% and 60% following OD15 and MF66, respectively. The glomerular filtration rate was not significantly affected, but urine flow increased nonsignificantly by approximately 50% and 66% following i.v. injection of OD15 and MF66, respectively. SPIONs produce a decrease in blood pressure and a natriuresis; however, the rate of fluid filtration in the kidney was not significantly affected.

Pharmacokinetics and bio-distribution of novel super paramagnetic iron oxide nanoparticles (SPIONs) in the anaesthetized pig.

Manufactured nanomaterials have a variety of medical applications, including diagnosis and targeted treatment of cancer. A series of experiments were conducted to determine the pharmacokinetic, biodistribution and biocompatibility of two novel magnetic nanoparticles (MNPs) in the anaesthetized pig. Dimercaptosuccinic acid (DMSA) coated superparamagnetic iron oxide nanoparticles (MF66-labelled 12 nm, core nominal diameter and OD15 15 nm); at 0.5, or 2.0 mg/kg) were injected intravenously. Particles induced a dose-dependent decrease in blood pressure following administration which recovered to control levels several minutes after injection. Blood samples were collected for a 5-h period and stored for determination of particle concentration using particle electron paramagnetic resonance (pEPR). Organs were harvested post-mortem for magnetic resonance imaging (MRI at 1.5 T field strength) and histology. OD15 (2.0 mg/kg) MNP had a plasma half-life of approximately 15 min. Both doses of the MF66 (0.5 and 2.0 mg/kg) MNP were below detection limits. MNP accumulation was observed primarily in the liver and spleen with MRI scans which was confirmed by histology. MRI also showed that both MNPs were present in the lungs. The results show that further modifications may be required to improve the biocompatibility of these particles for use as diagnostic and therapeutic agents.

Respiratory Control in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic disease caused by defects in the dystrophin gene resulting in loss of the structural protein dystrophin. Patients have reduced diaphragm functional capacity due to progressive muscle weakness. Respiratory morbidity in DMD is further characterised by hypoxaemic periods due to hypoventilation. DMD patients die prematurely due to respiratory and cardiac failure. In this study, we examined respiratory function in young adult male mdx (dystrophin deficient) mice (C57BL/10ScSn-Dmd(mdx)/J; n = 10) and in wild-type controls (WT; C57BL/10ScSnJ; n = 11). Breathing was assessed in unrestrained, unanaesthetised animals by whole-body plethysmography. Ventilatory parameters were recorded during air breathing and during exposure to acute hypoxia (F(i)O(2) = 0.1, 20 min). Data for the two groups of animals were compared using Student's t tests. During normoxic breathing, mdx mice had reduced breathing frequency (p = 0.011), tidal volume (p = 0.093) and minute ventilation (p = 0.033) compared to WT. Hypoxia increased minute ventilation in WT and mdx animals. Mdx mice had a significantly increased ventilatory response to hypoxia which manifest as an elevated % change from baseline for minute ventilation (p = 0.0015) compared to WT. We conclude that mdx mice have impaired normoxic ventilation suggestive of hypoventilation. Furthermore, mdx mice have an enhanced hypoxic ventilatory response compared to WT animals which we speculate may be secondary to chronic hypoxaemia. Our results indicate that a significant respiratory phenotype is evident as early as 8 weeks in the mdx mouse model of DMD.

Chronic Intermittent Hypoxia Blunts the Expression of Ventilatory Long Term Facilitation in Sleeping Rats.

We have previously reported that chronic intermittent hypoxia (CIH), a central feature of human sleep-disordered breathing, causes respiratory instability in sleeping rats (Edge D, Bradford A, O'halloran KD. Adv Exp Med Biol 758:359-363, 2012). Long term facilitation (LTF) of respiratory motor outputs following exposure to episodic, but not sustained, hypoxia has been described. We hypothesized that CIH would enhance ventilatory LTF during sleep. We examined the effects of 3 and 7 days of CIH exposure on the expression of ventilatory LTF in sleeping rats. Adult male Wistar rats were exposed to 20 cycles of normoxia and hypoxia (5 % O(2) at nadir; SaO(2) ~ 80 %) per hour, 8 h per day for 3 or 7 consecutive days (CIH, N = 7 per group). Corresponding sham groups (N = 7 per group) were subjected to alternating cycles of air under identical experimental conditions in parallel. Following gas exposures, breathing during sleep was assessed in unrestrained, unanaesthetized animals using the technique of whole-body plethysmography. Rats were exposed to room air (baseline) and then to an acute IH (AIH) protocol consisting of alternating periods of normoxia (7 min) and hypoxia (FiO(2) 0.1, 5 min) for 10 cycles. Breathing was monitored during the AIH exposure and for 1 h in normoxia following AIH exposure. Baseline ventilation was elevated after 3 but not 7 days of CIH exposure. The hypoxic ventilatory response was equivalent in sham and CIH animals after 3 days but ventilatory responses to repeated hypoxic challenges were significantly blunted following 7 days of CIH. Minute ventilation was significantly elevated following AIH exposure compared to baseline in sham but not in CIH exposed animals. LTF, determined as the % increase in minute ventilation from baseline following AIH exposure, was significantly blunted in CIH exposed rats. In summary, CIH leads to impaired ventilatory responsiveness to AIH. Moreover, CIH blunts ventilatory LTF. The physiological significance of ventilatory LTF is context-dependent but it is reasonable to consider that it can potentially destabilize respiratory control, in view of the potential for LTF to give rise to hypocapnia. CIH-induced blunting of LTF may represent a compensatory mechanism subserving respiratory homeostasis. Our results suggest that CIH-induced increase in apnoea index (Edge D, Bradford A, O'halloran KD. Adv Exp Med Biol 758:359-363, 2012) is not related to enhanced ventilatory LTF. We conclude that the mature adult respiratory system exhibits plasticity and metaplasticity with potential consequences for the control of respiratory homeostasis. Our results may have implications for human sleep apnoea.

Prenatal stress-induced alterations in major physiological systems correlate with gut microbiota composition in adulthood.

Early-life adverse experiences, including prenatal stress (PNS), are associated with a higher prevalence of neurodevelopmental, cardiovascular and metabolic disorders in affected offspring. Here, in a rat model of chronic PNS, we investigate the impact of late gestational stress on physiological outcomes in adulthood. Sprague-Dawley pregnant dams were subjected to repeated restraint stress from embryonic day 14 to day 20, and their male offspring were assessed at 4 months of age. PNS induced an exaggeration of the hypothalamic-pituitary-adrenal (HPA) axis response to stress, as well as an elevation of blood pressure and impairment of cognitive function. Altered respiratory control was also observed, as demonstrated by increased variability in basal respiratory frequency and abnormal frequency responses to both hypoxic and hypercapnic challenges. PNS also affected gastrointestinal neurodevelopment and function, as measured by a decrease in the innervation density of distal colon and an increase in the colonic secretory response to catecholaminergic stimulation. Finally, PNS induced long lasting alterations in the intestinal microbiota composition. 16S rRNA gene 454 pyrosequencing revealed a strong trend towards decreased numbers of bacteria in the Lactobacillus genus, accompanied by elevated abundance of the Oscillibacter, Anaerotruncus and Peptococcus genera in PNS animals. Strikingly, relative abundance of distinct bacteria genera significantly correlated with certain respiratory parameters and the responsiveness of the HPA axis to stress. Together, these findings provide novel evidence that PNS induces long-term maladaptive alterations in the gastrointestinal and respiratory systems, accompanied by hyper-responsiveness to stress and alterations in the gut microbiota.

Biodistribution and pharmacokinetic studies of SPION using particle electron paramagnetic resonance, MRI and ICP-MS.

AIM: Superparamagnetic iron oxide nanoparticles (SPIONs) may play an important role in nanomedicine by serving as drug carriers and imaging agents. In this study, we present the biodistribution and pharmacokinetic properties of SPIONs using a new detection method, particle electron paramagnetic resonance (pEPR). MATERIALS & METHODS: The pEPR technique is based on a low-field and low-frequency electron paramagnetic resonance. pEPR was compared with inductively coupled plasma mass spectrometry and MRI, in in vitro and in vivo. RESULTS: The pEPR, inductively coupled plasma mass spectrometry and MRI results showed a good correlation between the techniques. CONCLUSION: The results indicate that pEPR can be used to detect SPIONs in both preclinical and clinical studies.

Chronic nitric oxide synthase inhibition does not impair upper airway muscle adaptation to chronic intermittent hypoxia in the rat.

Nitric oxide (NO) is an important modulator of striated muscle function. Nitric oxide synthase (NOS) expression and activity is altered by hypoxia and NO is implicated in respiratory muscle remodeling following chronic sustained hypoxia. We sought to determine if NO is implicated in upper airway dilator muscle adaptation to chronic intermittent hypoxia (CIH). Thirty-two adult male Wistar rats (284±13, mean±SD) were exposed to alternating bouts of hypoxia (90 s; 5% O2 at the nadir) and normoxia (210 s; 21% O2) for 12 cycles per hour, 8h/day for 3 weeks. Sham animals were exposed to normoxia in parallel. Half of the animals in both groups received the nNOS inhibitor-L-NNA (2mM) in the drinking water throughout the study (N=8 for all groups). Sternohyoid (pharyngeal dilator) muscle contractile and endurance properties were determined ex vivo. Sternohyoid muscle myosin heavy chain (MHC) isoform composition and cross-sectional area was determined by fluorescence microscopy. Chronic nNOS blockade did not alter sternohyoid muscle peak force or force-frequency relationship in sham or CIH-treated animals. In contrast, chronic nNOS blockade significantly decreased sternohyoid muscle endurance with equivalent effects in sham and CIH-treated rats. Our results suggest that NO is an important modulator of sternohyoid muscle endurance. However, our data provide no evidence to suggest that NO is implicated in upper airway muscle adaptation to CIH.

Effect of chronic intermittent hypoxia on the reflex recruitment of the genioglossus during airway obstruction in the anesthetized rat.

We sought to test the hypothesis that chronic intermittent hypoxia (CIH)-a feature of sleep-disordered breathing in humans-impairs reflex recruitment of the genioglossus (GG, pharyngeal dilator) during obstructive airway events. Adult male Wistar rats were exposed to 20 cycles of normoxia and hypoxia (5% O2 at nadir) per hour, 8h a day for 7 days (CIH, N=7). The sham group (N=7) were exposed to normoxia in parallel. Following gas treatments, rats were anesthetized with an i.p. injection of urethane (1.5g/kg; 20%, w/v). Fine concentric needle electrodes were inserted into the GG and the costal diaphragm. Discriminated GG motor unit potentials and whole electromyograph (EMG), together with arterial blood pressure and arterial O2 saturation, were recorded during quiet basal breathing and during nasal airway occlusion. Airway occlusion significantly increased GG EMG activity in all animals; but there was no difference in the reflex response to airway occlusion between sham and CIH-treated animals (+105±22% vs. +105±17%, mean±SEM for area under the curve of integrated GG EMG, % increase from baseline, p=0.99). Occluded breaths were characterized by a significant increase in the firing frequency of phasically active units and the recruitment of large motor units that were quiescent under basal conditions. Though there are reports of impaired control of the upper airway following CIH in the rat, we conclude that reflexly evoked motor discharge to the GG is not affected by 7 days of CIH, a paradigm that we have shown increases apnea index in sleeping rats.

Reactive oxygen species mediated diaphragm fatigue in a rat model of chronic intermittent hypoxia.

Respiratory muscle dysfunction documented in sleep apnoea patients is perhaps due to oxidative stress secondary to chronic intermittent hypoxia (CIH). We sought to explore the effects of different CIH protocols on respiratory muscle form and function in a rodent model. Adult male Wistar rats were exposed to CIH (n = 32) consisting of 90 s normoxia-90 s hypoxia (either 10 or 5% oxygen at the nadir; arterial O2 saturation ∼ 90 or 80%, respectively] for 8 h per day or to sham treatment (air-air, n = 32) for 1 or 2 weeks. Three additional groups of CIH-treated rats (5% O2 for 2 weeks) had free access to water containing N-acetyl cysteine (1% NAC, n = 8), tempol (1 mM, n = 8) or apocynin (2 mM, n = 8). Functional properties of the diaphragm muscle were examined ex vivo at 35 °C. The myosin heavy chain and sarco(endo)plasmic reticulum Ca(2+)-ATPase isoform distribution, succinate dehydrogenase and glyercol phosphate dehydrogenase enzyme activities, Na(+)-K(+)-ATPase pump content, concentration of thiobarbituric acid reactive substances, DNA oxidation and antioxidant capacity were determined. Chronic intermittent hypoxia (5% oxygen at the nadir; 2 weeks) decreased diaphragm muscle force and endurance. All three drugs reversed the deleterious effects of CIH on diaphragm endurance, but only NAC prevented CIH-induced diaphragm weakness. Chronic intermittent hypoxia increased diaphragm muscle myosin heavy chain 2B areal density and oxidized glutathione/reduced glutathione (GSSG/GSH) ratio. We conclude that CIH-induced diaphragm dysfunction is reactive oxygen species dependent. N-Acetyl cysteine was most effective in reversing CIH-induced effects on diaphragm. Our results suggest that respiratory muscle dysfunction in sleep apnoea may be the result of oxidative stress and, as such, antioxidant treatment could prove a useful adjunctive therapy for the disorder.