Evidence of Biofilm and Persister Cell Formation in Revision Rhinoplasty.

Background: Postoperative rhinoplasty infection can lead to serious cosmetic deformity, loss of structural integrity to the nose, and functional deficiencies. Understanding the factors contributing to postoperative infection is important. Microbial biofilms and persister cells play an important role in health care-associated infections. The objective of this study is to identify microbial biofilm and persister cells in the nasal soft tissue of patients undergoing revision rhinoplasty. Methods: Fourteen patients undergoing rhinoplasty were recruited for this study. Nasal soft tissue was removed during rhinoplasty and preserved in 2% paraformaldehyde/2.5% glutaraldehyde. High-resolution images were then obtained from these nasal soft tissue samples. Results: Three samples were positive for the presence of microbial persister cells or biofilms. All samples came from patients undergoing revision rhinoplasty. These patients had between one to six previous rhinoplasty procedures and one patient had previous injectable nasal filler. Conclusions: Biofilms and persister cells are able to form in nasal soft tissue of revision rhinoplasty patients in the absence of an implant and may contribute to increased postoperative infection risk.

Laryngeal adductor function following potassium titanyl phosphate laser welding of the recurrent laryngeal nerve.

OBJECTIVES/HYPOTHESIS: Recurrent laryngeal nerve (RLN) transection injuries may occur during thyroidectomy and other surgical procedures. Laser nerve welding has been shown to cause less technique-related axonal damage than the traditional suture method. We compared functional adductor results using these two methods of RLN repair. STUDY DESIGN: Animal model. METHODS: Canine hemilarynges underwent pretreatment testing of laryngeal adductor function, followed by RLN transection and repair using potassium titanyl phosphate (KTP) laser welding (n = 8) or microneural suture (n = 16) techniques. Six months later, adductor function was measured again and expressed as a proportion of the pretreatment value. RESULTS: The mean laryngeal adductor pressure ratios were 82.4% (95% confidence interval [CI]: 72.8%-92.0%) for the laser repair group and 55.5% (95% CI: 49.4%-61.6%) for the suture control group, with a difference of 26.9% (95% CI: 15.3%-38.5%). Both spontaneous and stimulated glottic closure was observed in the laser welding and microsuture repair groups. CONCLUSIONS: Laser nerve welding resulted in greater strength of adduction than suture repair of an acutely transected RLN. Suture anastomosis may traumatize more axons than the laser. Stronger vocal fold adduction is associated clinically with better protection from aspiration and improved voice outcomes. KTP laser welding should be considered for anastomosis of the RLN and other nerves. LEVEL OF EVIDENCE: NA Laryngoscope, 130:1764-1769, 2020.

Correction: A model of chronic, transmissible Otitis Media in mice.

[This corrects the article DOI: 10.1371/journal.ppat.1007696.].

A model of chronic, transmissible Otitis Media in mice.

Infection and inflammation of the middle ears that characterizes acute and chronic otitis media (OM), is a major reason for doctor visits and antibiotic prescription, particularly among children. Nasopharyngeal pathogens that are commonly associated with OM in humans do not naturally colonize the middle ears of rodents, and experimental models in most cases involve directly injecting large numbers of human pathogens into the middle ear bullae of rodents, where they induce a short-lived acute inflammation but fail to persist. Here we report that Bordetella pseudohinzii, a respiratory pathogen of mice, naturally, efficiently and rapidly ascends the eustachian tubes to colonize the middle ears, causing acute and chronic histopathological changes with progressive decrease in hearing acuity that closely mimics otitis media in humans. Laboratory mice experimentally inoculated intranasally with very low numbers of bacteria consistently have their middle ears colonized and subsequently transmit the bacterium to cage mates. Taking advantage of the specifically engineered and well characterized immune deficiencies available in mice we conducted experiments to uncover different roles of T and B cells in controlling bacterial numbers in the middle ear during chronic OM. The iconic mouse model provides significant advantages for elucidating aspects of host-pathogen interactions in otitis media that are currently not possible using other animal models. This natural model of otitis media permits the study of transmission between hosts, efficient early colonization of the respiratory tract, ascension of the eustachian tube, as well as colonization, pathogenesis and persistence in the middle ear. It also allows the combination of the powerful tools of mouse molecular immunology and bacterial genetics to determine the mechanistic basis for these important processes.

AMPA/kainate receptors mediate axonal morphological disruption in hypoxic white matter.

We used acute brain slices to investigate the hypothesis that oxygen-glucose deprivation (OGD) induced loss of axon function and neurofilament labeling are correlated to axonal morphological disruption in the corpus callosum of adult brain. Coronal brain slices including corpus callosum were prepared from adult mice. White matter immunohistochemical properties and conduction along axons remained stable over 12 h after preparation. White matter injury was assessed by recording compound action potentials (CAPs) across corpus callosum, combined with immunofluorescence for axonal neurofilaments and by bright field microscopy of myelin profiles in semi-thin sections. OGD for 30 min resulted in irreversible loss of the CAPs, formation of axon heads and bulbs, and swelling of myelin profiles in slices examined 1h after OGD. In slices followed for 9 h after OGD, there was complete loss of neurofilament labeling and myelin profiles. Because overactivation of AMPA/kainate receptors mediates axon structural and functional disruption in hypoxic corpus callosum slices, we tested whether blockade of AMPA/kainate receptors reduced OGD-induced axonal morphological disruption. NBQX (30 microM), an AMPA/kainate receptor antagonist, prevented OGD-induced formation of axon heads and bulbs, swelling of myelin profiles, loss of neurofilament staining and preserved axonal morphology. These results expand our previous findings that the AMPA/kainate receptor activation contributes to axonal morphological disruption, as well as loss of electrical function.

A possible role for nitric oxide in osteoclastogenesis associated with cholesteatoma.

HYPOTHESIS: This study was designed to investigate the potential role of nitric oxide in cholesteatoma-induced bone resorption, in vitro and in vivo. BACKGROUND: Cholesteatoma is a disease of inflammatory bone resorption in the middle ear leading to hearing loss and vestibular dysfunction. Inappropriate activation of osteoclasts causes the morbidity associated with this disease. Previous studies suggest nitric oxide may be an important mediator of osteoclast function. METHODS: A murine model of cholesteatoma induced bone resorption was used to demonstrate nitric oxide synthase (NOS) gene expression and the effect of a NOS inhibitor. An in vitro osteoclast culture method was used to demonstrate the effect of nitric oxide on isolated osteoclasts. Osteoclast development was assayed by counting the number of mature osteoclasts; activity was assayed by measuring the amount of resorbed bone. RESULTS: Quantitative reverse transcriptase-polymerase chain reaction results demonstrated the temporal expression of all three NOS isoforms in vivo. NOS I demonstrated very low levels of expressions throughout the duration of the study with no change in expression in response to keratin implant. Similarly, NOS III also demonstrated low levels of expression and no change in response to keratin. NOS II was highly upregulated in response to keratin throughout the duration of the study. In vitro, pharmacological nitric oxide donors--sodium nitroprusside and S-nitroso-N-acetyl-D,L-penicillamine--dose-dependently stimulated osteoclast resorption. Alone, interferon gamma (IFNgamma)--but not IL-1beta or TNFalpha--generated nitrite in vitro. A cytokine cocktail of IL-1beta, TNFalpha, and IFNgamma synergistically enhanced nitrite production. Nitrite production was blocked by the addition of aminoguanidine (AG), suggesting that AG-inhibited cytokine mediated nitrite production. However, in an in vivo model of cholesteatoma-induced bone resorption, the osteoclast response of AG-treated mice was not statistically different from untreated controls. CONCLUSIONS: All three NOS isoforms were expressed in an in vivo model of cholesteatoma-induced bone resorption with significant upregulation of NOS II throughout the study. Exogenously administered nitric oxide dose-dependently enhanced osteoclast activation in vitro. The pro-inflammatory cytokines, IL-1beta, TNFalpha, and IFNgamma, synergistically induce nitrite production, which was abrogated by treatment with the nitric oxide synthase inhibitor, AG. Although AG suppresses nitrite production in vitro, treatment had no effect on osteoclast response in vivo, suggesting that the effects of inflammatory cytokines on osteoclast response were mediated through other pathways.

Zoledronic acid inhibits osteoclastogenesis in vitro and in a mouse model of inflammatory osteolysis.

This study assessed effects of the bisphosphonate zoledronic acid (ZLNA) on osteoclastogenesis. To assess the effect of ZLNA on osteoclast formation in vitro, we cultured mouse bone marrow cells under conditions that promote osteoclastogenesis. Administered at concentrations from 10(-6) to 10(-9) mol/L, ZLNA led to a dose-dependent inhibition of osteoclastogenesis. Combined TUNEL staining and histochemical staining for tartrate-resistant acid phosphatase showed that ZLNA induced apoptosis in osteoclasts and monocytic precursor cells. To study the effects of ZLNA in vivo, we placed keratin particles onto the surface of the parietal bone of mice to induce localized inflammatory bone resorption. Three experimental groups received daily subcutaneous injections of ZLNA (1, 3, or 10 microg/kg body weight) from 4 days before surgery until 5 days after keratin implantation. The ZLNA significantly reduced osteoclast recruitment in a dose-dependent manner, but did not affect the degree of inflammation or the mineral apposition rate.

Anatomical evidence of microbial biofilms in tonsillar tissues: a possible mechanism to explain chronicity.

CONTEXT: Bacteria within biofilms are resistant to host defenses and antibiotics. The presence of bacterial biofilms within the tissue and crypts of inflamed tonsils may explain the chronicity and recurrent characteristics of some forms of tonsillitis. OBJECTIVE: To determine if microbial biofilms occur within clinically abnormal tonsils. DESIGN: In this study, we evaluated the histomorphological appearance of 19 human tonsils for evidence of biofilm formation using light and transmission electron microscopy. SUBJECTS: Human tissues were collected during surgical tonsillectomy. Fifteen specimens were removed because of a history of repeated infections, and 4 were removed because of hypertrophy and obstruction. INTERVENTIONS: No interventions were used in this study. MAIN OUTCOME MEASURE: Histological and ultrastructural evidence of bacterial biofilms within the crypts of tonsils. RESULTS: Gram-positive and gram-negative bacteria were seen within otherwise acellular deposits among crypts of 11 of 15 infected tonsils. Regions of accumulated bacteria possessed the ultrastructural appearance of typical amorphous polysaccharide biofilm matrix. Small clusters of bacterial colonies were seen in 3 of 4 tonsils removed because of hypertrophy. CONCLUSIONS: There is strong anatomical evidence for the presence of bacterial biofilms in chronically diseased tonsils. Because sessile bacteria within biofilms are resistant to host defenses and antibiotics, bacterial biofilms within tonsils may explain the chronicity and recurrent nature of some forms of tonsillitis.

Nitric oxide synthase I mediates osteoclast activity in vitro and in vivo.

Bone resorption is responsible for the morbidity associated with a number of inflammatory diseases such as rheumatoid arthritis, orthopedic implant osteolysis, periodontitis and aural cholesteatoma. Previous studies have established nitric oxide (NO) as a potentially important mediator of bone resorption. NO is a unique intercellular and intracellular signaling molecule involved in many physiologic and pathologic pathways. NO is generated from L-arginine by the enzyme nitric oxide synthase (NOS). There are three known isoforms of NOS with distinct cellular distributions. In this study, we have used mice with targeted deletions in each of these isoforms to establish a role for these enzymes in the regulation of bone resorption in vivo and in vitro. In a murine model of particle induced osteolysis, NOS I-/- mice demonstrated a significantly reduced osteoclast response. In vitro, osteoclasts derived from NOS I-/- mice were larger than wild type controls but demonstrated decreased resorption. Although NOS I has been demonstrated in osteoblasts and osteocytes as a mediator of adaptive bone remodeling, it has not previously been identified in osteoclasts. These results demonstrate a critical role for NOS I in inflammatory bone resorption and osteoclast function in vitro.

Evidence for microbial biofilms in cholesteatomas.

BACKGROUND: Sessile bacteria within biofilms are highly resistant to eradication by antimicrobial agents. Previously, we have shown that the most common organisms cultured from experimentally induced cholesteatomas are biofilm formers. Additionally, the keratin "matrix" of a cholesteatoma is an ideal environment for the support of biofilm formation. OBJECTIVE: To determine if microbial biofilms occur within the keratin matrix of infected cholesteatomas. DESIGN: We evaluated the histomorphologic characteristics of 24 human and 22 experimental cholesteatomas for evidence of biofilm formation using light and transmission electron microscopy. SUBJECTS: Human tissues were collected during surgical eradication of existing cholesteatomas. Twenty-two gerbil cholesteatomas were either spontaneously occurring or induced by external auditory canal ligation and harvested several months later. RESULTS: Gram-positive and gram-negative bacteria were seen within acellular deposits among the keratin accumulations in 21 of 22 gerbil and 16 of 24 human cholesteatomas. Regions of accumulated bacteria possessed the ultrastructural appearance of typical amorphous polysaccharide biofilm matrix. CONCLUSIONS: There is strong anatomic evidence for the presence of bacterial biofilms in experimental and human cholesteatomas. The existence of bacterial biofilms within cholesteatomas may explain the clinical characteristics of infected cholesteatomas, that is, persistence and recurrence of infection, with surgical eradication being the only effective treatment.

MeroGel hyaluronic acid sinonasal implants: osteogenic implications.

OBJECTIVES/HYPOTHESIS: The study sought to evaluate the effects of MeroGel (Medtronic Xomed Surgical Products, Jacksonville, FL) hyaluronic acid sinonasal implants by using a mouse model. We expected that this material would dissolve, elicit minimal inflammation, and leave intact mucosa. However, pilot data showed that MeroGel may be osteogenic. The present work focuses on the description and quantification of these bony changes. STUDY DESIGN: Animal experiment. METHODS: The effects of MeroGel were evaluated using sinonasal and calvarial implants in C57BL/6 mice. The calvarial experiments employed both light microscopy (bone histomorphometry) and fluorochrome labeling (mineral apposition). Each implant study was subdivided into normal control animals, sham-operated control animals, a foreign body control, and MeroGel implants. MeroGel was also placed within subcutaneous pockets remote from bone. All mice were killed 1 month after surgery. Both percentage of bone area within the sinonasal cavity and total calvarial bone area were quantified and compared. Fluorochrome images and the subcutaneous implants were examined qualitatively. RESULTS: MeroGel placement engendered new bone formation within the nasal lumen of mice. Compared with normal control animals, sham-operated control animals, and a foreign body control, this increase was statistically significant. The calvaria from MeroGel-implanted mice were thickened as compared with normal animals, sham-operated control animals, and a foreign body control. Tetracycline labeling demonstrated mineral apposition along the dorsal and ventral surfaces of the calvaria and within enlarged marrow spaces. MeroGel placed subcutaneously did not engender bone formation. CONCLUSIONS: MeroGel, a unique esterified form of hyaluronic acid, induced bone formation within the sinonasal cavity and on the calvaria of mice implanted with it. Our experiments suggest that, when placed adjacent to traumatized, remodeling bone, MeroGel may have osteogenic potential.