Hyperoxic and hyperbaric-induced cardioprotection: role of nitric oxide synthase 3.

OBJECTIVE: The relative contributions of the fraction of inspired oxygen (FIO2) and atmospheric pressure (ATM) to cardioprotection are unknown. We determined whether the product of FIO2 x ATM (oxygen partial pressure) controls the extent of hyperoxic+hyperbaric-induced cardioprotection and involves activation of nitric oxide synthase (NOS). METHODS: Adult Sprague Dawley rats (n = 10/gp) were treated for 1 h with (1) normoxia+normobaria (21% O2 at 1 ATM), (2) hyperoxia+normobaria (100% O2 at 1 ATM), (3) normoxia+hyperbaria (21% O2 at 2 ATM) and (4) hyperoxia+hyperbaria (100% O2 at 2 ATM). RESULTS: Infarct size following 25 min ischemia and 180 min reperfusion was decreased following hyperoxia+normobaria and normoxia+hyperbaria compared with normoxia+normobaria and further decreased following hyperoxia+hyperbaria treatment. l-NAME (200 microM) reversed the cardioprotective effects of hyperoxia+hyperbaria. Nitrite plus nitrate content was increased 2.2-fold in rats treated with normoxia+hyperbaria and hyperoxia+hyperbaria. NOS3 protein increased 1.2-fold and association of hsp90 with NOS3 four-fold in hyperoxic+hyperbaric rats. CONCLUSIONS: Cardioprotection conferred by hyperoxia+hyperbaria is directly dependent on oxygen availability and mediated by NOS.

Real-time reverse transcriptase polymerase chain reaction: an improvement in detecting mRNA levels in mouse cranial tissue.

BACKGROUND: Quantitation of messenger RNA levels has traditionally been carried out by Northern blot analysis. While this is regarded as the standard method, it is time-consuming and requires large quantities of RNA. Reverse-transcriptase polymerase chain reaction is a semiquantitative method that has been used as a more rapid and sensitive alternative to Northern blotting. Real-time reverse-transcriptase polymerase chain reaction is a quantitative technique that is gaining widespread acceptance as a rapid and reliable way of quantifying mRNA. Since both techniques are currently being used to evaluate gene expression in the murine cranial suture model, the present study was performed to compare the sensitivity and variability of real-time to conventional reverse-transcriptase polymerase chain reaction in this model. METHODS: Mouse brain RNA was isolated and amplified using real-time and conventional methods. For the real-time method, a serial 10-fold dilution of RNA, ranging from 1 fg to 100 ng, was performed. For the conventional method, the minimum amount of RNA needed for consistent polymerase chain reaction amplification was determined. Transforming growth factor beta-1 and beta-actin RNA transcripts were measured using both techniques. RESULTS: One femtogram of RNA could be detected by the real-time method, although 10 fg were required to reliably detect differences; 500 ng of RNA was required for consistent polymerase chain reaction amplification using the conventional method. The variability of real-time reverse-transcriptase polymerase chain reaction when expressed as a coefficient of variation (SD as a percentage of the mean) ranged from 0.23 to 2.6 percent for all genes tested, as compared with 9 to 70 percent for conventional reverse-transcriptase polymerase chain reaction. CONCLUSIONS: Real-time reverse-transcriptase polymerase chain reaction was used successfully to detect mRNA from different mouse genes. The real-time method is much more sensitive in detecting small amounts of mRNA than both Northern blot analysis and conventional polymerase chain reaction. The variability of the real-time method is more than 10-fold lower compared with the conventional method performed in the authors' laboratory for all genes tested.

Outcomes of tongue-lip adhesion for neonatal respiratory distress caused by Pierre Robin sequence.

Tongue-lip adhesion can be effective in relieving tongue-based airway obstruction in the neonatal period. However, longitudinal evaluation of these patients demonstrates they require additional procedures for ultimate control of their airway, feeding, and orthognathic relationship. Charts of patients with Pierre Robin sequence who underwent unsuccessful nonoperative treatment and tongue-lip adhesion procedures by the senior author between 1989 to 1999 were reviewed. In particular, secondary interventions were quantified and qualified to determine if tongue-lip adhesion was a definitive treatment of the pathology caused by this sequence. Eleven patients with ages ranging from 2 to 6 weeks underwent initial tongue-lip adhesion. Seven patients were syndromic (3 Stickler's, 1 Goldenhar's, 1 Fragile X, 1 Miller's, 1 chromosome 15 rearrangement), and four had isolated Pierre Robin sequence. Two patients had dehiscence of their initial repair within 2 weeks, for a primary surgical success rate of 82%, consistent with other published reports. A detailed follow-up of these patients revealed that 10 of 11 (91%) required additional intervention for airway or feeding issues. Of those requiring secondary intervention, an additional 1.9 secondary procedures were performed per patient to achieve control of the airway, feeding, and orthognathic problems. More than half of the patients needed gastrostomy tubes to overcome feeding difficulties. Five patients necessitated secondary surgery for recurrent airway obstruction within 4 months of the initial surgical treatment (four distractions and one repeat tongue-lip adhesion). Four others required distraction at an older age for orthognathic indications. Two patients were successfully treated with tongue-lip adhesion only. Tongue-lip adhesion has a high initial success rate for correction of neonatal airway obstruction. However, long-term follow-up indicates a high incidence of secondary intervention requirements. In retrospect, perhaps tongue-lip adhesion should be considered a temporizing procedure for most patients with Pierre Robin sequence.

To distract or not to distract: an algorithm for airway management in isolated Pierre Robin sequence.

Approaches advocated for treatment of airway obstruction among neonates with Pierre Robin sequence include positioning, tongue-lip adhesion, mandibular distraction, and tracheostomy, with no established guidelines regarding which modality is appropriate for a specific patient. This report proposes an algorithm for the management of neonatal upper airway obstruction among patients with isolated Pierre Robin sequence. Data for 21 patients with isolated Pierre Robin sequence who were treated by one surgeon during a 9-year period were reviewed. Eighteen patients presented during the first 1 week of life and three patients presented late, between 12 and 33 months of age. Follow-up periods ranged from 9 to 70 months (median, 33 months). Successful airway management was achieved with positioning alone for 10 patients, with tongue-lip adhesion for seven of nine patients, with tracheostomy for two patients, and with mandibular distraction for three patients. Changes in the maxillary-mandibular discrepancy were significant with natural mandibular growth during the first 1 year of life (p < 0.0001). Oromotor studies performed 3 months or more after tongue-lip adhesion reversal (n = 9) demonstrated no appreciable deficits in tongue function, relative to other children with cleft lips/palates. A multidisciplinary team should evaluate all patients with isolated Pierre Robin sequence, to fully assess the maxillary-mandibular relationship, anatomically define the site of airway obstruction, and identify feeding difficulties. Patients should be evaluated for episodes of desaturation occurring spontaneously, during feeding, or during sleeping. Patients with desaturation should be further evaluated with double endoscopy (nasoendoscopy and bronchoscopy). If the airway obstruction is localized to the tongue base alone and cannot be controlled with positioning, then tongue-lip adhesion is the initial treatment of choice, because such patients demonstrate significant mandibular growth during the first 1 year of life. Mandibular distraction among neonates is reserved for failures of tongue-lip adhesion in which isolated tongue-base airway obstruction is documented. Neither of the patients who experienced failure of tongue-lip adhesion in this series would have been a candidate for distraction with the algorithm presented. Avoiding routine neonatal distraction serves to avoid facial scarring, nerve and tooth bud injury, and potential disturbances of intrinsic mandibular growth. Patients with persistent respiratory difficulties beyond age 9 months require reevaluation for multiple sites of airway obstruction. Mandibular distraction may be one of several modalities required to avoid tracheostomy for such patients.

Microfocal CT: a method for evaluating murine cranial sutures in situ.

INTRODUCTION: The murine model is a well-established surrogate for studying human cranial suture biology. In mice, all sutures with the exception of the posterior frontal (PF) suture remain patent throughout life. Histology is regarded as the gold standard for analyzing sutures. On this basis, PF suture fusion begins on day of life 25 and is complete by day 45. Cranial suture histology, however, requires sacrifice of the animal to obtain tissue for analysis. As a result, knowledge of the kinetics of cranial suture fusion is based on a patchwork analysis of many sutures from many different animals. The behavior of a single suture through time is unknown. Our goal is to develop a noninvasive means to repeatedly image mouse cranial sutures in vivo. As a first step, the present study was performed to evaluate microfocal computer tomography (micro-CT) technology for the use of capturing images of a mouse cranium in situ. METHODS: The micro-CT system consists of a microfocal X-ray source and a large format CCD camera optically coupled to a high-resolution X-ray image intensifier, digitally linked to a computer. The PF and sagittal sutures lie in continuity along the midline of the skull. Holes were drilled in the calvaria on both sides of the PF and sagittal sutures of a 45-day-old euthanized mouse. A micro-CT scan of this animal was performed and hundreds of cross-sectional images were generated for the cranium. These images were used to reconstruct three-dimensional volumetric images of the entire cranium. Comparisons were made between (1). the gross specimen and the three dimensional reconstructions; (2). two-dimensional coronal images obtained by micro-CT and those obtained by histology. RESULTS: Analysis of PF and sagittal sutures demonstrated the following: (1). The drilled holes were accurately rendered by micro-CT, when compared to both the gross specimen and the histology. (2). The sagittal suture was found to be patent by both micro-CT and histology. (3). The PF suture is fused by histology, but unexpectedly, the PF suture appears incompletely fused by micro-CT. By micro-CT, however, the anterior and endocranial regions appear more extensively fused than the remainder of the PF suture, a finding consistent with published histologic analysis. CONCLUSIONS: We successfully imaged 45-day-old mouse cranial sutures in situ using micro-CT technology. Precise correlation between histologic sections and radiologic images is difficult, but convincing similarities exist between the gross specimen and images from micro-CT and histology. PF suture fusion in a 45-day-old animal appears different by micro-CT than by histology. One possible explanation for this apparent discrepancy is that suture fusion in histology is determined based on the appearance of bone morphology and not tissue density, as the specimens are necessarily decalcified to section the bone. Micro-CT, on the other hand, distinguishes tissues on the basis of density. Newly forming bone may require bone matrix formation prior to complete calcification; PF suture in 45-day-old mice may be morphologically complete but incompletely ossified. Studies correlating histologic and micro-CT assessment of suture development are underway. Micro-CT appears to be a promising method for noninvasive imaging of mouse cranial suture.

Airway management in patients with isolated Pierre Robin sequence during the first year of life.

Managing the airway of patients with Pierre Robin sequence is diagnostically and therapeutically challenging. Like many other pathologies, Pierre Robin sequence is best managed with a multidisciplinary team. Providing a comprehensive evaluation is important to detect patients who may have silent events during activities of early life. Children with airway obstruction should have a complete assessment to anatomically define the site of airway obstruction. Therefore, management options can be targeted to the specific deficiency. The present article outlines a treatment protocol for patients who present with isolated Pierre Robin sequence. The authors propose that initial diagnostic and therapeutic interventions remain relatively noninvasive. Using this protocol, more than 80% of patients with isolated Pierre Robin sequence who the authors have examined for airway obstruction in the neonatal period have been effectively treated with positioning or tongue-lip adhesion. While more aggressive and successful techniques have been reported, the authors have not found them necessary for the majority of patients with isolated Pierre Robin sequence. The following review details their approach to patients with Pierre Robin sequence.