A novel and simple method for endotracheal intubation of mice

Endotracheal intubation in mice is necessary for experiments involving intratracheal instillation of various substances, repeated pulmonary function assessments and mechanical ventilation. Previously described methods for endotracheal intubation in mice require the use of injection anaesthesia to immobilize the animal during the intubation procedure or the use of a volatile anaesthetic prior to intubation for immobilization. With these methods, the control of anaesthetic depth during the intubation procedure is absent. We describe a method for simple and rapid intratracheal intubation in mice for mechanical ventilation, using a self-built plastic support to facilitate the intubation procedure. General anaesthesia is maintained by means of inhalation through a non-rebreathing circuit connected to the plastic support. This set-up gives the operator control of anaesthetic depth and sufficient time to perform the intubation procedure. A purpose-made laryngoscopic blade is used to facilitate the intubation tube entering the trachea. The blade of the purpose-made laryngoscope is constructed as a retraction guide and is curved for easy handling. Under direct vision, the epiglottis is gently lifted by the laryngoscopic blade while the intubation tube is pushed into the trachea. Following this novel intubation technique, we were able to mechanically ventilate mice for at least 2 h without severely disturbing blood gases. Histological evaluation of the lungs and microscopic evaluation of the trachea and larynx showed no signs of trauma related to the intubation technique or mechanical ventilation.     

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.     

Video-assisted orotracheal intubation in mice

Orotracheal intubation in mice is a complicated technique because of the peculiar oropharyngeal anatomy and the difficulty in visualizing the laryngis aditus. Here we report a new and simple method for rapid endotracheal intubation by using a small bore, straight fibre-optic arthroscope. Under endoscope-assisted visualization of the laryngis aditus, a polyethylene cannula, inserted on a guide-wire in order to facilitate the introduction of the tip across the vocal cords, was advanced in the trachea. The success rate of intubation was 100%. We were also able to re-intubate the mice 4 and 8 weeks later without any major complications. We conclude that this method can be easily and safely used for studies where controlled pulmonary ventilation is necessary.     

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg–300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.     

Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells

Background

Chronic lung diseases are a major issue in public health. A serial pulmonary assessment using imaging techniques free of ionizing radiation and which provides early information on local function impairment would therefore be a considerably important development. Magnetic resonance imaging (MRI) is a powerful tool for the static and dynamic imaging of many organs. Its application in lung imaging however, has been limited due to the low water content of the lung and the artefacts evident at air-tissue interfaces. Many attempts have been made to visualize local ventilation using the inhalation of hyperpolarized gases or gadolinium aerosol responding to MRI. None of these methods are applicable for broad clinical use as they require specific equipment.

Methods

We have shown previously that low-field MRI can be used for static imaging of the lung. Here we show that mathematical processing of data derived from serial MRI scans during the respiratory cycle produces good quality images of local ventilation without any contrast agent. A phantom study and investigations in 85 patients were performed.

Results

The phantom study proved our theoretical considerations. In 99 patient investigations good correlation (r = 0.8; p ≤ 0.001) was seen for pulmonary function tests and MR ventilation measurements. Small ventilation defects were visualized.

Conclusion

With this method, ventilation defects can be diagnosed long before any imaging or pulmonary function test will indicate disease. This surprisingly simple approach could easily be incorporated in clinical routine and may be a breakthrough for lung imaging and functional assessment.     

Repetitive measurements of pulmonary mechanics to inhaled cholinergic challenge in spontaneously breathing mice.

Precise and repeatable measurements of pulmonary function in intact mice are becoming increasingly important for experimental investigations on various respiratory disorders including asthma. Here, we present validation of a novel in vivo method that, for the first time, combines direct and repetitive recordings of standard pulmonary mechanics with cholinergic aerosol challenges in anesthetized, orotracheally intubated, spontaneously breathing mice. We demonstrate that, in several groups of nonsensitized BALB/c mice, dose-related increases in pulmonary resistance and dynamic compliance to aerosolized methacholine are reproducible over short and extended intervals without causing detectable cytological alterations in the bronchoalveolar lavage or relevant histological changes in the proximal trachea and larynx regardless of the number of orotracheal intubations. Moreover, as further validation, we confirm that allergic mice, sensitized and challenged with Aspergillus fumigatus, were significantly more responsive to cholinergic challenge (P < 0.01) and exhibited marked eosinophilia and lymphocytosis in bronchoalveolar lavage fluids as well as significant pathological alterations in laryngotracheal histology compared with nonsensitized mice. We suggest that this approach will provide useful and necessary information on pulmonary mechanics in studies of various respiratory disorders in mice, including experimental models of asthma and chronic obstructive pulmonary disorder, investigations of pulmonary pharmacology, or more general investigations of the genetic determinants of lung function.     

What can computed tomography and magnetic resonance imaging tell us about ventilation?

This review provides a summary of pulmonary functional imaging approaches for determining pulmonary ventilation, with a specific focus on multi-detector x-ray computed tomography and magnetic resonance imaging (MRI). We provide the important functional definitions of pulmonary ventilation typically used in medicine and physiology and discuss the fact that some of the imaging literature describes gas distribution abnormalities in pulmonary disease that may or may not be related to the physiological definition or clinical interpretation of ventilation. We also review the current state-of-the-field in terms of the key physiological questions yet unanswered related to ventilation and gas distribution in lung disease. Current and emerging imaging research methods are described, including their strengths and the challenges that remain to translate these methods to more wide-spread research and clinical use. We also examine how computed tomography and MRI might be used in the future to gain more insight into gas distribution and ventilation abnormalities in pulmonary disease.     

High oxygen concentrations predispose mouse lungs to the deleterious effects of high stretch ventilation.

Mechanical ventilation is a necessary intervention for patients with acute lung injury. However, mechanical ventilation can propagate acute lung injury and increase systemic inflammation. The exposure to >21% oxygen is often associated with mechanical ventilation yet has not been examined within the context of lung stretch. We hypothesized that mice exposed to >90% oxygen will be more susceptible to the deleterious effects of high stretch mechanical ventilation. C57B1/6 mice were randomized into 48-h exposure of 21 or >90% oxygen; mice were then killed, and isolated lungs were randomized into a nonstretch or an ex vivo, high-stretch mechanical ventilation group. Lungs were assessed for compliance and lavaged for surfactant analysis, and cytokine measurements or lungs were homogenized for surfactant-associated protein analysis. Mice exposed to >90% oxygen + stretch had significantly lower compliance, altered pulmonary surfactant, and increased inflammatory cytokines compared with all other groups. Our conclusion is that 48 h of >90% oxygen and high-stretch mechanical ventilation deleteriously affect lung function to a greater degree than stretch alone.     

Imaging regional PAO2 and gas exchange

Several methods allow regional gas exchange to be inferred from imaging of regional ventilation and perfusion (V/Q) ratios. Each method measures slightly different aspects of gas exchange and has inherent advantages and drawbacks that are reviewed. Single photon emission computed tomography can provide regional measure of ventilation and perfusion from which regional V/Q ratios can be derived. PET methods using inhaled or intravenously administered nitrogen-13 provide imaging of both regional blood flow, shunt, and ventilation. Electric impedance tomography has recently been refined to allow simultaneous measurements of both regional ventilation and blood flow. MRI methods utilizing hyperpolarized helium-3 or xenon-129 are currently being refined and have been used to estimate local PaO(2) in both humans and animals. Microsphere methods are included in this review as they provide measurements of regional ventilation and perfusion in animals. One of their advantages is their greater spatial resolution than most imaging methods and the ability to use them as gold standards against which new imaging methods can be tested. In general, the reviewed methods differ in characteristics such as spatial resolution, possibility of repeated measurements, radiation exposure, availability, expensiveness, and their current stage of development.     

Repeated invasive lung function measurements in intubated mice: an approach for longitudinal lung research

Invasive lung function measurements are useful tools to describe respiratory disease models in mice but only result in one time-point measurements because of tracheostomy. We explored if intubation may overcome the need for tracheostomy thereby allowing invasive lung function monitoring of individual mice over time. Repeated invasive lung function measurements with Scireq(?) - FlexiVent or Buxco(?) - Forced Pulmonary Maneuvers(?) were performed three times in BALB/c mice with intervals of 10 days. Each lung function assessment following intubation was compared with a similar measurement in age-matched tracheostomized mice, the golden standard in lung function measurements. Tracheostomy and intubation gave similar results for resistance, elastance and compliance of the whole respiratory system as assessed by Flexivent. Likewise, Forced Pulmonary Maneuvers used to measure lung volumes such as total lung capacity, functional residual capacity, forced expiratory volume in 0.1 s and forced vital capacity, resulted in identical outcomes for both airway approaches. No interaction was found between the procedures for any of the pulmonary function variables. The observed changes over time were rather related to animal growth than to repetitive intubation. Eighty percent of the animals survived three consecutive intubations, which were hampered by transient breathing difficulties, weight loss and neutrophilic bronchoalveolar lavage immediately postextubation. Repetitive invasive lung function measurements by intubation are feasible and reproducible in healthy mice and results are comparable to the standard method. This may open new perspectives for longitudinal research in animal models of respiratory diseases.     

基于径向基函数神经网络的肺部加权频差电阻抗成像方法

目的 对肺通气过程进行床旁实时连续图像监控，是机械通气患者和临床医生的迫切需求。肺部电阻抗成像（EIT）可反映呼吸引起的胸腔电特性变化分布，在肺通气监测方面具有天然的优势。本文目的在于建立基于径向基函数神经网络（RBFNN）的肺部加权频差电阻抗成像（wfd-EIT）方法，实现对肺通气的高空间分辨率成像。方法 利用肺部wfd-EIT成像方法实时描绘胸腔电导率分布状况，再通过RBFNN将目标区域可视化并精准识别其边界信息。首先通过数值分析模拟，在各个激励频率利用COMSOL与MATLAB软件建立2 028个仿真样本，分为训练样本集和测试样本集，验证所提出成像方法的可行性和有效性。其次，为了验证仿真结果，建立肺部物理模型，选用具有低电导特性的生物组织模拟肺部通气区域，对其进行成像实验，并采用图像相关系数（ICC）和肺区域比（LRR）定量数据衡量成像方法的准确性。结果 wfd-EIT方法可以在任意时刻进行图像重建，并能够准确反映出目标区域的电特性分布；利用基于RBFNN的算法能够增强目标区域的成像精度，ICC可达0.94以上，更好地凸显其边界轮廓信息。结论 通过wfd-EIT成像方法，利用多频阻抗谱同步测量实现目标区域的快速可视化，并结合RBFNN网络逼近任意非线性函数的优点，实现对目标区域电特性变化的精准识别，为下一步进行临床肺通气的EIT图像监测奠定了理论和技术基础。     

Effect of ventilation rate on instilled surfactant distribution in the pulmonary airways of rats.

Liquid can be instilled into the pulmonary airways during medical procedures such as surfactant replacement therapy, partial liquid ventilation, and pulmonary drug delivery. For all cases, understanding the dynamics of liquid distribution in the lung will increase the efficacy of treatment. A recently developed imaging technique for the study of real-time liquid transport dynamics in the pulmonary airways was used to investigate the effect of respiratory rate on the distribution of an instilled liquid, surfactant, in a rat lung. Twelve excised rat lungs were suspended vertically, and a single bolus (0.05 ml) of exogenous surfactant (Survanta, Ross Laboratories, Columbus, OH) mixed with radiopaque tracer was instilled as a plug into the trachea. The lungs were ventilated with a 4-ml tidal volume for 20 breaths at one of two respiratory rates: 20 or 60 breaths/min. The motion of radiodense surfactant was imaged at 30 frames/s with a microfocal X-ray source and an image intensifier. Dynamics of surfactant distribution were quantified for each image by use of distribution statistics and a homogeneity index. We found that the liquid distribution depended on the time to liquid plug rupture, which depends on ventilation rate. At 20 breaths/min, liquid was localized in the gravity-dependent region of the lung. At 60 breaths/min, the liquid coated the airways, providing a more vertically uniform liquid distribution.     

Differential roles of p55 and p75 tumor necrosis factor receptors on stretch-induced pulmonary edema in mice

Ventilator-induced lung injury plays a crucial role in the outcome of patients with acute lung injury. Previous studies have shown a role for the cytokine tumor necrosis factor-alpha (TNF) in stretch-induced alveolar neutrophil recruitment, but the involvement of TNF in stretch-induced pulmonary edema is unclear. We investigated the effects of TNF through its individual p55 and p75 receptors on early pulmonary edema formation during high stretch ventilation, before neutrophil infiltration. Anesthetized wild-type or TNF receptor single/double knockout mice were ventilated with high tidal volume ( approximately 38 ml/kg) for 2 h or until they developed arterial hypotension. Pulmonary edema was assessed by physiological parameters including respiratory mechanics and blood gases, and by lavage fluid protein, lung wet:dry weight ratio, and lung permeability measurements using fluorescence-labeled albumin. High stretch ventilation in wild-type and TNF receptor double knockout animals induced similar pulmonary edema, and only 25-30% of mice completed the protocol. In contrast, the p55 receptor knockout mice were strongly protected from edema formation, with all animals completing the protocol. Myeloperoxidase assay indicated that this protective effect was not associated with decreased pulmonary neutrophil sequestration. The p75 receptor knockout mice, however, displayed increased susceptibility to edema formation, and no animals survived the full 2 h. These results demonstrate a novel role for TNF signaling (independent from its effects on neutrophil recruitment) specifically through the p55 receptor, in promoting high stretch-induced pulmonary edema, whereas p75 signaling may play an opposing role.     

Breathing and lung mechanics in hamsters: effect of pentobarbital anesthesia

An adaptation of the method reported by Skornik, Heimann, and Jaeger (Toxicol. Appl. Pharmacol. 59: 314-323, 1981) was used to evaluate pulmonary mechanics in intact awake hamsters. Lung volume changes were measured with a pressure plethysmograph, and pleural pressure was estimated by the use of a saline-filled esophageal catheter. We report data for normal awake hamsters studied at 18, 20, 22, 32, and 98 wk of age. Age-related differences were observed in tidal volume, dynamic compliance, and pulmonary resistance. To determine to what extent pulmonary mechanics are changed by anesthesia, hamsters were measured during spontaneous breathing while awake and while anesthetized. We found that anesthesia had a marked effect on the breathing pattern of normal hamsters. Twenty-five minutes after injection of pentobarbital sodium (70 mg/kg ip), tidal volume, dynamic compliance, pulmonary resistance, breathing frequency, and minute ventilation were 66, 40, 375, 60, and 41% of the corresponding awake values. Anesthesia always provoked a significant and dose-related decrease in tidal volume and an increase in respiratory period, together resulting in a profound decrease in minute ventilation. These significant differences from the awake values call into question the value of measurements in anesthetized animals. The methods described here yield reasonable and repeatable measurements and, because no anesthesia or surgery is required, they can be used in longitudinal studies when repeated measurements in the same animal over long periods of time can help define pathological changes or the effectiveness of various interventions.     

Role of aquaporin water channels in airway fluid transport, humidification, and surface liquid hydration

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport-related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54-56% efficient in wild-type mice, and reduced by only 3-4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3-5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 +/- 5 microm and [Na(+)] was 115 +/- 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by approximately 40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption.     

Assessing responses to cancer therapy using molecular imaging

Tumor responses to therapy in the clinic are still evaluated primarily from non-invasive imaging measurements of reductions in tumor size. This approach, however, lacks sensitivity and can only give a delayed indication of a positive response to treatment. Major advances in our understanding of the molecular mechanisms responsible for cancer, combined with new targeted clinical imaging technologies designed to detect the molecular correlates of disease progression and response to treatment, are set to revolutionize our approach to the detection and treatment of the disease. We describe here the imaging technologies available to image tumor cell proliferation and migration, metabolism, receptor and gene expression, apoptosis and tumor angiogenesis and vascular function, and show how measurements of these parameters can be used to give early indications of positive responses to treatment or to detect drug resistance and/or disease recurrence. Special emphasis has been placed on those applications that are already used in the clinic and those that are likely to translate into clinical application in the near future or whose use in preclinical studies is likely to facilitate translation of new treatments into the clinic.     

Murine nasal septa for respiratory epithelial air-liquid interface cultures

Air-liquid interface models using murine tracheal respiratory epithelium have revolutionized the in vitro study of pulmonary diseases. This model is often impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. We describe a simple technique to harvest the murine nasal septum and grow the epithelial cells in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their respective cultured epithelium at an air-liquid interface were compared by scanning electron microscopy (SEM). Immunocytochemistry for type IV beta-tubulin and zona occludens-1 (Zo-1) are performed to determine differentiation and confluence, respectively. To rule out contamination with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein (OMP) was performed. Transepithelial resistance and potential measurements were determined using a modified vertical Ussing chamber SEM reveals approximately 90% ciliated respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and potential confirming a confluent monolayer with electrically active airway epitheliumn containing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is a superior source when compared with the trachea. The nasal septum increases the yield of respiratory epithelial cells up to 8-fold.     

Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5'-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39(-/-) mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39(-/-) or cd73(-/-) mice with soluble apyrase or 5'-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5'-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.     

Thoracic traction on the trachea: mechanisms and magnitude

Both inspiratory increases and tonic thoracic traction (pull of the thorax) on the trachea [Ttx(tr)] have been shown to improve patency of the upper airway. To evaluate the origins and magnitude of Ttx(tr), we studied 15 anesthetized tracheotomized dogs. We divided the midcervical trachea and attached the thoracic stub to a strain gauge. Ttx(tr), esophageal pressure, and carinal displacement were observed during various conditions. These included unobstructed and obstructed spontaneous breathing, mechanical ventilation at various levels of positive end-expiratory pressure, and progressive hypercapnic stimulation. Observations during spontaneous breathing were performed before and after vagotomy. We found that inspiratory increases in Ttx(tr) were substantial, averaging 81 +/- 8 g force and increasing to 174 +/- 22 g force at an end-expiratory CO2 concentration of 10%. Ttx(tr) did not result simply from the pull of mediastinal and pulmonary structures transmitted through the carina. Changes in intrathoracic pressure acted independently to either draw the trachea into or push the trachea out of the thorax. Thus Ttx(tr) could be explained as the sum of mediastinal traction and force generated by changes in intrathoracic pressure.