Limosilactobacillus reuteri administration alters the gut-brain-behavior axis in a sex-dependent manner in socially monogamous prairie voles.

Research on the role of gut microbiota in behavior has grown dramatically. The probiotic L. reuteri can alter social and stress-related behaviors - yet, the underlying mechanisms remain largely unknown. Although traditional laboratory rodents provide a foundation for examining the role of L. reuteri on the gut-brain axis, they do not naturally display a wide variety of social behaviors. Using the highly-social, monogamous prairie vole (Microtus ochrogaster), we examined the effects of L. reuteri administration on behaviors, neurochemical marker expression, and gut-microbiome composition. Females, but not males, treated with live L. reuteri displayed lower levels of social affiliation compared to those treated with heat-killed L. reuteri. Overall, females displayed a lower level of anxiety-like behaviors than males. Live L. reuteri-treated females had lower expression of corticotrophin releasing factor (CRF) and CRF type-2-receptor in the nucleus accumbens, and lower vasopressin 1a-receptor in the paraventricular nucleus of the hypothalamus (PVN), but increased CRF in the PVN. There were both baseline sex differences and sex-by-treatment differences in gut microbiome composition. Live L. reuteri increased the abundance of several taxa, including Enterobacteriaceae, Lachnospiraceae NK4A136, and Treponema. Interestingly, heat-killed L. reuteri increased abundance of the beneficial taxa Bifidobacteriaceae and Blautia. There were significant correlations between changes in microbiota, brain neurochemical markers, and behaviors. Our data indicate that L. reuteri impacts gut microbiota, gut-brain axis and behaviors in a sex-specific manner in socially-monogamous prairie voles. This demonstrates the utility of the prairie vole model for further examining causal impacts of microbiome on brain and behavior.

Social isolation alters behavior, the gut-immune-brain axis, and neurochemical circuits in male and female prairie voles.

The absence of social support, or social isolation, can be stressful, leading to a suite of physical and psychological health issues. Growing evidence suggests that disruption of the gut-immune-brain axis plays a crucial role in the negative outcomes seen from social isolation stress. However, the mechanisms remain largely unknown. The socially monogamous prairie vole (Microtus ochrogaster) has been validated as a useful model for studying negative effects of social isolation on the brain and behaviors, yet how the gut microbiome and central immune system are altered in isolated prairie voles are still unknown. Here, we utilized this social rodent to examine how social isolation stress alters the gut-immune-brain axis and relevant behaviors. Adult male and female prairie voles (n = 48 per sex) experienced social isolation or were cohoused with a same-sex cagemate (control) for six weeks. Thereafter, their social and anxiety-like behaviors, neuronal circuit activation, neurochemical expression, and microgliosis in key brain regions, as well as gut microbiome alterations from the isolation treatment were examined. Social isolation increased anxiety-like behaviors and impaired social affiliation. Isolation also resulted in sex- and brain region-specific alterations in neuronal activation, neurochemical expression, and microgliosis. Further, social isolation resulted in alterations to the gut microbiome that were correlated with key brain and behavioral measures. Our data suggest that social isolation alters the gut-immune-brain axis in a sex-dependent manner and that gut microbes, central glial cells, and neurochemical systems may play a critical, integrative role in mediating negative outcomes from social isolation.

Metagenome-Assembled Genome Sequences of Five Strains from the Microtus ochrogaster (Prairie Vole) Fecal Microbiome.

The prairie vole (Microtus ochrogaster) is an important model for the study of social monogamy and dual parental care of offspring. Characterization of specific host species-microbe strain interactions is critical for understanding the effects of the microbiota on mood and behavior. The five metagenome-assembled genome sequences reported here represent an important step in defining the prairie vole microbiome.

In-vitro evaluation of a ciprofloxacin- and ivacaftor-coated sinus stent against Pseudomonas aeruginosa biofilms.

BACKGROUND: We recently developed a novel ciprofloxacin-coated sinus stent capable of releasing antibiotics over a sustained period of time. Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that has synergistic bactericidal activity with ciprofloxacin and also enhances sinus mucociliary clearance. The objective of this study was to optimize and evaluate the efficacy of a ciprofloxacin- and ivacaftor-releasing biodegradable sinus stent (CISS) in vitro. METHODS: A CISS was created by coating ciprofloxacin/ivacaftor-embedded nanoparticles with an acrylate and ammonium methacrylate copolymer onto a biodegradable poly-L-lactic acid stent. In-vitro evaluation of the CISS included: (1) assessment of drug stability in nanoparticles by zeta potential, and drug-coating stability within the CISS using scanning electron microscopy (SEM); (2) determination of ciprofloxacin- and ivacaftor-release kinetics; and (3) assessment of anti-Pseudomonas aeruginosa biofilm formation by calculating relative optical density units (RODUs) compared with control stents at 590-nm optical density. RESULTS: The presence of drugs and a uniform coating on the stent were confirmed by zeta potential and SEM. Sustained drug release was observed through 21 days without an initial burst release. Anti-biofilm formation was observed after placing the CISS for 3 days onto a preformed 1-day P aeruginosa biofilm. The CISS significantly reduced biofilm mass compared with bare stents and controls (RODUs at 590-nm optical density; CISS, 0.31 ± 0.01; bare stent, 0.78 ± 0.12; control, 1.0 ± 0.00; p = 0.001; n = 3). CONCLUSION: The CISS maintains a uniform coating and sustained delivery of drugs providing a marked reduction in P aeruginosa biofilm formation. Further studies evaluating the efficacy of CISS in a preclinical model are planned.

Herbal dry extract BNO 1011 improves clinical and mucociliary parameters in a rabbit model of chronic rhinosinusitis.

BACKGROUND: Enhancing chloride (Cl- ) secretion in sinus epithelia represents a novel therapeutic approach to chronic rhinosinusitis (CRS). Herbal dry extract BNO 1011 enhances mucociliary clearance (MCC) via upregulation of Cl- secretion in sinonasal cultures in vitro and murine epithelium in vivo. The objective of this study is to evaluate whether the BNO 1011 improves MCC and clinical parameters in a rabbit model of CRS. METHODS: After the development of CRS in 30 New Zealand white rabbits, animals were randomly assigned to receive oral placebo (n = 10), BNO 1011 (low dose [LD], 25 mg/kg/daily) (n = 10), or BNO1011 (high dose [HD], 125 mg/kg/daily) (n = 10) for 4 weeks. Outcomes included sinus opacification (Kerschner's rabbit sinus CT grade), maxillary epithelial Cl- secretion (sinus potential difference [PD] assay), airway surface liquid (ASL) depth using micro-optical coherence tomography (µOCT), and submucosal gland density (SMD) on histopathology. Outcome parameters were analyzed by 2 blinded investigators. RESULTS: BNO 1011 significantly cleared sinus opacification (HD = 1.21 ± 0.63, LD = 1.26 ± 0.37,) compared to placebo (4.02 ± 0.92) (p = 0.009). BNO 1011 resulted in markedly greater mean sinus PD polarization (HD = -12.23 ± 1.4 mV, LD = -12.0 ± 3.0 mV) when compared to rabbits treated with placebo (-4.1 ± 1.1 mV) (p = 0.03). ASL depth was significantly improved when treated with HD (4.08 ± 0.06 µm) and LD (4.05 ± 0.06 µm) compared to placebo (3.5 ± 0.05 µm) (post hoc analysis, p < 0.0001). Histologically, epithelial thickness (HD = 10.0 ± 0.7 µm; LD = 13.7 ± 0.9 µm; placebo = 21.1 ± 2.3 µm; p < 0.005), subepithelial thickness (HD = 63.1 ± 6.6 µm; LD = 103.2 ± 6.7 µm; placebo = 113.3 ± 6.0 µm; p < 0.001), and SMD (HD = 22.2 ± 2.9%; LD = 31.8 ± 1.1%; placebo = 43.8 ± 1.7%; p < 0.0001) were noticeably better with the HD. CONCLUSION: Herbal dry extract BNO 1011 improves radiographic, histologic, and MCC parameters in a rabbit model of CRS.

Resveratrol and ivacaftor are additive G551D CFTR-channel potentiators: therapeutic implications for cystic fibrosis sinus disease.

BACKGROUND: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in defective Cl- transport and cause chronic bacterial infections in the upper and lower airways of cystic fibrosis (CF) patients. Ivacaftor is a CFTR potentiator that improves Cl- transport in CF patients with at least 1 copy of the G551D mutation. Resveratrol is also a potent CFTR potentiator that increases determinants of mucociliary transport. The objective of this study is to determine whether resveratrol and ivacaftor improve Cl- secretion in G551D CFTR over either agent alone. METHODS: Fisher rat thyroid cells (FRT) transfected with G551D CFTR and human sinonasal epithelial cells (HSNE) containing the CFTR G551D mutation were subjected to pharmacologic manipulation of transepithelial ion transport in Ussing chambers. Activity was further evaluated using whole-cell patch clamp methods in G551D FRT cells. RESULTS: In G551D FRT cells, resveratrol (100 µM) and ivacaftor (10 µM) significantly increased Cl- transport (change in short-circuit current, δISC = µA/cm2 ) compared with single-agent and dimethylsulfoxide vehicle controls (resveratrol + ivacaftor 4.97 ± 0.57 vs ivacaftor 0.74 ± 0.12 vs resveratrol 2.96 ± 0.52 vs control 0.74 ± 0.12; p < 0.001). Maximal Cl- secretion (20 µM forskolin) was also significantly enhanced (p < 0.0001). Activity was confirmed in G551D HSNE (resveratrol + ivacaftor 4.48 ± 0.39 vs ivacaftor 1.05 ± 0.11 vs. resveratrol 0.84 ± 0.3 vs control, 0.0 ± 0.02; p < 0.001), and whole-cell patch clamp analysis in G551D FRT cells (resveratrol + ivacaftor -2535 ± 179.3 pA vs ivacaftor -1408.9 ± 101.3 pA vs resveratrol; -766.2 ± 71.2 pA; p < 0.0001). CONCLUSION: Additive improvement in G551D CFTR-mediated Cl- secretion suggests that resveratrol could enhance ivacaftor therapy in these patients and improve CF-related rhinosinusitis.

Ivacaftor, a Cystic Fibrosis Transmembrane Conductance Regulator Potentiator, Enhances Ciprofloxacin Activity Against Pseudomonas aeruginosa.

BACKGROUND: Methods to improve the clinical efficacy of currently available antibiotics against multidrug resistant bacteria in cystic fibrosis (CF) chronic rhinosinusitis (CRS) are greatly needed. Ivacaftor, a cystic fibrosis transmembrane conductance regulator potentiator, was recently identified as having potentially beneficial off-target effects as a weak inhibitor of bacterial DNA gyrase and topoisomerase IV. The objective of the current study is to evaluate whether ivacaftor enhances the antimicrobial activity of ciprofloxacin against Pseudomonas aeruginosa. METHODS: The planktonic growth of the PAO-1 strain of P. aeruginosa was studied in the presence of ciprofloxacin and/or ivacaftor. Effects were measured according to optical density of cultured PAO-1 at 600 nm. For a static PAO-1 biofilm assay, the PAO-1 strain was inoculated and cultured for 72 h in the presence of the drugs. Formed PAO-1 biofilms were quantified by crystal violet staining and imaged with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). RESULTS: PAO-1 growth was significantly reduced in the presence of ivacaftor (8 or 16 µg/mL) and ciprofloxacin (0.02 or 0.05 µg/mL) compared to ciprofloxacin alone ( P < .001). Similarly, ivacaftor (8 or 16 µg/mL) showed a significant reduction of PAO-1 biofilms when treated with 0.05 µg/mL of ciprofloxacin. Significant synergism was noted between ciprofloxacin and 16 µg/mL of ivacaftor ( P < .0001) in reducing planktonic growth and biofilm formation. Quantitative measurements with crystal violet staining were correlated to CLSM and SEM images. CONCLUSION: Ivacaftor enhanced ciprofloxacin's antimicrobial activity against P. aeruginosa. Further studies evaluating the efficacy of ivacaftor/ciprofloxacin combination for P. aeruginosa for CF CRS are warranted.

l-Methionine anti-biofilm activity against Pseudomonas aeruginosa is enhanced by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor.

BACKGROUND: Biofilms may contribute to refractory chronic rhinosinusitis (CRS), as they lead to antibiotic resistance and failure of effective clinical treatment. l-Methionine is an amino acid with reported biofilm-inhibiting properties. Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator with mild antimicrobial activity via inhibition of bacterial DNA gyrase and topoisomerase IV. The objective of this study was to evaluate whether co-treatment with ivacaftor and l-methionine can reduce the formation of Pseudomonas aeruginosa biofilms. METHODS: P aeruginosa (PAO-1 strain) biofilms were studied in the presence of l-methionine and/or ivacaftor. For static biofilm assays, PAO-1 was cultured in a 48-well plate for 72 hours with stepwise combinations of these agents. Relative biofilm inhibitions were measured according to optical density of crystal violet stain at 590 nm. Live/dead assays (BacTiter-Glo™ assay, Promega) were imaged with laser scanning confocal microscopy. An agar diffusion test was used to confirm antibacterial effects of the drugs. RESULTS: l-Methionine (0.5 µM) significantly reduced PAO-1 biofilm mass (32.4 ± 18.0%; n = 4; p < 0.001) compared with controls. Low doses of ivacaftor alone (4, 8, and 12 µg/mL) had no effect on biofilm formation. When combined with ivacaftor (4 µg/mL), a synergistic anti-biofilm effect was noted at 0.05 µM and 0.5 µM of l-methionine (two-way analysis of variane, p = 0.0415) compared with corresponding concentrations of l-methionine alone. CONCLUSION: Ivacaftor enhanced the anti-biofilm activity of l-methionine against the PAO-1 strain of P aeruginosa. Further studies evaluating the efficacy of ivacaftor/l-methionine combinations for P aeruginosa sinusitis are planned.

Preclinical therapeutic efficacy of the ciprofloxacin-eluting sinus stent for Pseudomonas aeruginosa sinusitis.

BACKGROUND: The ciprofloxacin-coated sinus stent (CSS) has unique therapeutic potential to deliver antibiotics to the sinuses. The objective of this study is to evaluate the efficacy of the CSS stent in eliminating Pseudomonas aeruginosa infection in a rabbit model of sinusitis. METHODS: A ciprofloxacin-eluting sinus stent was created by coating ciprofloxacin/Eudragit RS100 on biodegradable poly-D/L-lactic acid (2 mg). After analyzing in-vitro inhibition of P aeruginosa (PAO-1 strain) biofilm formation, a total of 8 stents (4 shams, 4 CSSs) were placed unilaterally in rabbit maxillary sinuses via dorsal sinusotomy after inducing infection for 1 week with PAO-1. Animals were assessed 2 weeks after stent insertion with nasal endoscopy, sinus culture, computed tomography (CT) scan, histopathology, and scanning electron microscopy (SEM). RESULTS: PAO-1 biofilm formation was significantly reduced in vitro with exposure to the CSS (p < 0.0001). Insertion of the stent in PAO-1-infected rabbits for 2 weeks resulted in significant improvement in sinusitis according to endoscopy scoring (p < 0.0001) and CT scoring (p < 0.002). Histology and SEM revealed marked improvement in the structure of the mucosa and submucosa with no detection of biofilm structures in the CSS cohort. CONCLUSION: Although this study had a small sample size, we identified robust therapeutic efficacy of the CSS by reducing bacterial load and biofilm formation of P aeruginosa in a preclinical model of sinusitis after placement for 2 weeks.

Submucosal gland mucus strand velocity is decreased in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) may be initiated by innately impaired host defense mechanisms that predispose the upper airways to infection. Recent evidence suggests tethering of submucosal gland mucus strands represents an inciting event within cystic fibrosis (CF) airways, occurring prior to onset of chronic infection. Submucosal gland hypertrophy and defective mucociliary clearance (MCC) are present in actively inflamed sinuses, but mucus strand velocity may also be affected as a secondary event, further contributing to chronic disease. The objective of this study is to assess whether mucus strand velocity is decreased in patients with CRS. METHODS: Mucosal explants from patients with and without CRS were submerged in Ringer's solution mixed with fluorescent nanospheres. Methacholine was then added, and videos demonstrating strand growth and detachment were generated from a time-lapse of Z-stack images using a multiphoton confocal microscope. Dynamic mucus strands were identified and individual velocities quantified with the MTrackJ plug-in of ImageJ. RESULTS: Fifteen patients met criteria for ex vivo analysis of mucus strand velocities (CRS, n = 9 vs controls, n = 6). Mucus strands were recorded (pixels/second) streaming from the submucosal gland openings. Average mucus strand velocities were significantly decreased in patients with CRS (1.53 ± 0.67 vs controls, 4.86 ± 1.68 pixels/second; p < 0.001). CONCLUSION: This study is the first to report evidence of abnormal mucus strand velocity from submucosal glands in diseased sinonasal mucosa. Future pharmacologic studies targeting this critical component of MCC are warranted.

Protective and antifungal properties of Nanodisk-Amphotericin B over commercially available Amphotericin B.

OBJECTIVE: Amphotericin B (AMB), a potent antifungal agent, has been employed as topical and systemic therapy for sinonasal fungal infections. A novel formulation of nanodisc (ND) containing super aggregated AMB (ND-AMB) for the treatment of fungal infections has been recently developed to provide greater protection from AMB toxicity than current, clinically approved lipid-based formulations. The objective of the current study was to evaluate the safety and potency of ND-AMB for sinonasal delivery using an in vitro model. METHODS: Human sinonasal tissue was harvested during endoscopic sinus surgery and grown at air-liquid interface until well-differentiated. Cultures were exposed to ND-AMB vs AMB and changes in K+ permeability and resistance were measured and recorded via Ussing chamber assay. Ciliary beat frequency (CBF) was analyzed in parallel as well as cytotoxic assay. Potency was assessed using real-time PCR measurement of the Aspergillus fumigatus 18S rRNA. RESULTS: Ussing chamber studies revealed K+ currents that increased rapidly within 30 s of adding AMB (10 µg/mL) to the apical side, indicating apical membranes had become permeable to K+ ions. In contrast, negligible induction of K+ current was obtained following addition of ND-AMB [AMB = (107.7 ± 15.9) µA/cm2 AMB vs ND-AMB = (2.3 ± 0.7) µA/cm2 ND-AMB; P = 0.005]. ND-AMB also protected nasal epithelial cells from cytotoxicity of AMB (P < 0.05). There was no difference in ciliary beat frequency between the two groups (P = 0.96). The expression of A. fumigatus 18S rRNA with exposure of lower dose of ND-AMB was significantly lower compared to that with AMB (P < 0.05). CONCLUSIONS: Data from the present study suggests ND-AMB protects human nasal epithelia membranes from AMB toxicity by protecting against apical cell K+ permeability while maintaining uncompromised antifungal property compared to AMB. ND-AMB could provide a novel topical therapy for sinonasal fungal diseases.

Characterization of primary rat nasal epithelial cultures in CFTR knockout rats as a model for CF sinus disease.

OBJECTIVE: The objectives of the current experiments were to develop and characterize primary rat nasal epithelial cultures and evaluate their usefulness as a model of cystic fibrosis (CF) sinonasal transepithelial transport and CF transmembrane conductance regulator (CFTR) function. STUDY DESIGN: Laboratory in vitro and animal studies. METHODS: CFTR+/+ and CFTR-/- rat nasal septal epithelia (RNSE) were cultured on semipermeable supports at an air-liquid interface to confluence and full differentiation. Monolayers were mounted in Ussing chambers for pharmacologic manipulation of ion transport and compared to similar filters containing murine (MNSE) and human (HSNE) epithelia. Histology and scanning electron microscopy (SEM) were completed. Real-time polymerase chain reaction of CFTR+/+ RNSE, MNSE, and HSNE was performed to evaluate relative CFTR gene expression. RESULTS: Forskolin-stimulated anion transport (ΔIsc in µA/cm2 ) was significantly greater in epithelia derived from CFTR+/+ when compared to CFTR-/- animals (100.9 ± 3.7 vs. 10.5 ± 0.9; P < 0.0001). Amiloride-sensitive ISC was equivalent (-42.3 ± 2.8 vs. -46.1 ± 2.3; P = 0.524). No inhibition of CFTR-mediated chloride (Cl- ) secretion was exhibited in CFTR-/- epithelia with the addition of the specific CFTR inhibitor, CFTRInh -172. However, calcium-activated Cl- secretion (UTP) was significantly increased in CFTR-/- RNSE (CFTR-/- -106.8 ± 1.6 vs. CFTR+/+ -32.2 ± 3.1; P < 0.0001). All responses were larger in RNSE when compared to CFTR+/+ and CFTR-/- (or F508del/F508del) murine and human cells (P < 0.0001). Scanning electron microscopy demonstrated 80% to 90% ciliation in all RNSE cultures. There was no evidence of infection in CFTR-/- rats at 4 months. CFTR expression was similar among species. CONCLUSION: The successful development of the CFTR-/- rat enables improved evaluation of CF sinus disease based on characteristic abnormalities of ion transport. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E384-E391, 2017.

Assessment of acquired mucociliary clearance defects using micro-optical coherence tomography.

BACKGROUND: Dehydration of airway surface liquid (ASL) disrupts normal mucociliary clearance (MCC) in sinonasal epithelium, which may lead to chronic rhinosinusitis (CRS). Abnormal chloride (Cl- ) transport is one such mechanism that contributes to this disorder and can be acquired secondary to environmental perturbations, such as hypoxia at the tissue surface. The objective of this study was to assess the technological feasibility of the novel micro-optical coherence tomography (µOCT) imaging technique for investigating acquired MCC defects in cultured human sinonasal epithelial (HSNE) cells. METHODS: Primary HSNE cell cultures were subjected to a 1% oxygen environment for 12 hours to induce acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. Ion transport characteristics were assessed with pharmacologic manipulation in Ussing chambers. ASL, periciliary fluid (PCL), and ciliary beat frequency (CBF) were evaluated using µOCT. RESULTS: Amiloride-sensitive transport (ΔISC ) was greater in cultures exposed to hypoxia (hypoxia: -13.2 ± 0.6 µA/cm2 ; control: -6.5 ± 0.1 µA/cm2 ; p < 0.01), whereas CFTR-mediated anion transport was significantly diminished (hypoxia: 28.6 ± 0.3 µA/cm2 ; control: 36.2 ± 1.6 µA/cm2 ; p < 0.01), consistent with acquired CFTR dysfunction and sodium hyperabsorption. Hypoxia diminished all markers of airway surface function microanatomy as observed with µOCT, including ASL (hypoxia: 5.0 ± 0.4 µm; control: 9.0 ± 0.9 µm; p < 0.01) and PCL depth (hypoxia: 2.5 ± 0.1 µm; control: 4.8 ± 0.3 µm; p < 0.01), and CBF (hypoxia: 8.7 ± 0.3 Hz; control: 10.2 ± 0.3 Hz; p < 0.01). CONCLUSION: Hypoxia-induced defects in epithelial anion transport in HSNE led to predictable effects on markers of MCC measured with novel µOCT imaging. This imaging method represents a technological leap forward and is feasible for assessing acquired defects impacting the airway surface.

Sinus hypoplasia in the cystic fibrosis rat resolves in the absence of chronic infection.

BACKGROUND: Sinus hypoplasia is a hallmark characteristic in cystic fibrosis (CF). Chronic rhinosinusitis (CRS) is nearly universal from a young age, impaired sinus development could be secondary to loss of the cystic fibrosis transmembrane conductance regulator (CFTR) or consequences of chronic infection during maturation. The objective of this study was to assess sinus development relative to overall growth in a novel CF animal model. METHODS: Sinus development was evaluated in CFTR-/- and CFTR+/+ rats at 3 stages of development: newborn; 3 weeks; and 16 weeks. Microcomputed tomography (microCT) scanning, cultures, and histology were performed. Three-dimensional sinus and skull volumes were quantified. RESULTS: At birth, sinus volumes were decreased in CFTR-/- rats compared with wild-type rats (mean ± SEM: 11.3 ± 0.85 mm3 vs 14.5 ± 0.73 mm3 ; p < 0.05), despite similar weights (8.4 ± 0.46 gm vs 8.3 ± 0.51 gm; p = 0.86). CF rat weights declined by 16 weeks (378.4 ± 10.6 gm vs 447.4 ± 15.9 gm; p < 0.05), sinus volume increased similar to wild-type rats (201.1 ± 3.77 gm vs 203.4 ± 7.13 gm; p = 0.8). The ratio of sinus volume to body weight indicates hypoplasia present at birth (1.37 ± 0.12 vs 1.78 ± 0.11; p < 0.05) and showed an increase compared with CFTR+/+ animals by 16 weeks (0.53 ± 0.02 vs 0.46 ± 0.02; p < 0.05). Rats did not develop histologic evidence of chronic infection. CONCLUSION: CF rat sinuses are smaller at birth, but develop volumes similar to wild-type rats with maturation. This suggests that loss of CFTR may confer sinus hypoplasia at birth, but normal development ensues without chronic sinus infection.

Tolerance and pharmacokinetics of a ciprofloxacin-coated sinus stent in a preclinical model.

BACKGROUND: Chronic rhinosinusitis (CRS) is often associated with persistent bacterial infection despite the use of systemic antibiotics. Topically administered antibiotics are an alternative strategy, but require effective local concentrations, prolonged mucosal contact time, minor systemic absorption, and minimal depletion. The objectives of the current study were to analyze the in vitro release rate and in vivo drug delivery tolerance and pharmacokinetics of a ciprofloxacin-coated sinus stent (CSS). METHODS: The CSS (2 mg) was created from biodegradable poly-D/L-lactic acid. After analyzing in vitro release profile, CSSs were placed unilaterally in maxillary sinuses of 16 rabbits via dorsal sinusotomy. Animals were euthanized between 1 and 3 weeks postoperatively. Ciprofloxacin concentrations in the sinus tissue and plasmas were assessed using high-performance liquid chromatography. Radiological and histological evaluations were performed. RESULTS: In the in vitro release profile, an initial burst release was observed over the first 24 hours, followed by sustained release through the 14-day time point. In the rabbit model, ciprofloxacin was continuously released from the stent up to 3 weeks at doses >50 ng/mL. Histologic examination found no evidence of inflammation, epithelial ulceration, or bony reaction upon euthanization of the animals at 21 days. Computed tomography also demonstrated no signs of mucosal edema or opacification in the sinus. CONCLUSION: The CSS was safe in this preclinical model and sustained release was observed in both the in vitro and in vivo analyses. The innovative stent design coated with ciprofloxacin may provide a unique therapeutic strategy for chronic rhinosinusitis (CRS).

Sinus Microanatomy and Microbiota in a Rabbit Model of Rhinosinusitis.

Background: Rabbits are useful for preclinical studies of sinusitis because of similar physiologic features to humans. The objective of this study is to develop a rabbit model of sinusitis that permits assessment of microanatomy and sampling for evaluating shifts in the sinus microbiota during the development of sinusitis and to test how the mucociliary clearance (MCC) defect might lead to dysbiosis and chronic rhinosinusitis (CRS). Methods: Generation of CRS was accomplished with an insertion of a sterile sponge into the left middle meatus of New Zealand white rabbits (n = 9) for 2 weeks. After sponge removal, 4 rabbits were observed for another 10 weeks and evaluated for CRS using endoscopy, microCT, visualization of the functional micro-anatomy by micro-optical coherence tomography (µOCT), and histopathological analysis of the sinus mucosa. Samples were taken from the left middle meatus and submitted for microbiome analysis. Results: CT demonstrated opacification of all left sinuses at 2 weeks in all rabbits (n = 9), which persisted in animals followed for another 12 weeks (n = 4). Histology at week 2 showed mostly neutrophils. On week 14, significant infiltration of plasma cells and lymphocytes was noted with increased submucosal glands compared to controls (p = 0.02). Functional microanatomy at 2 weeks showed diminished periciliary layer (PCL) depth (p < 0.0001) and mucus transport (p = 0.0044) compared to controls despite a thick mucus layer. By 12 weeks, the thickened mucus layer was resolved but PCL depletion persisted in addition to decreased ciliary beat frequency (CBF; p < 0.0001). The mucin fermenting microbes (Lactobacillales, Bacteroidales) dominated on week 2 and there was a significant shift to potential pathogens (e.g., Pseudomonas, Burkholderia) by week 14 compared to both controls and the acute phase (p < 0.05). Conclusion: We anticipate this reproducible model will provide a means for identifying underlying mechanisms of airway-surface liquid (ASL) depletion and fundamental changes in sinus microbial communities that contribute to the development of CRS. The rabbit model of sinusitis exhibited diminished PCL depth with delayed mucus transport and significant alterations and shift in the sinus microbiome during the development of chronic inflammation.

High compliance vascular grafts based on semi-interpenetrating networks.

Current synthetic vascular grafts have poor patency rates in small diameter applications (<6 mm) due to intimal hyperplasia arising from a compliance mismatch between the graft and native vasculature. Enormous efforts have focused on improving biomechanical properties; however, polymeric grafts are often constrained by an inverse relationship between burst pressure and compliance. We have developed a new, semi-interpenetrating network (semi-IPN) approach to improve compliance without sacrificing burst pressure. The effects of heat treatment on graft morphology, fiber architecture, and resultant biomechanical properties are presented. In addition, biomechanical properties after equilibration at physiological temperature were investigated in relation to polyurethane microstructure to better predict in vivo performance. Compliance values as high as 9.2 ± 2.7 %/mmHg x 10-4 were observed for the semi-IPN graft while also maintaining high burst pressure, 1780 ± 230 mm Hg. The high compliance of these heat-treated poly(carbonate urethane) (PCU) and semi-IPN grafts is expected to improve long-term patency rates beyond even saphenous vein autografts by preventing intimal hyperplasia. The fundamental structure-property relationships gained from this work may also be utilized to advance biomedical device designs based on thermoplastic polyurethanes.