The authors are very thankful to the Federal Ministry of Education and Research (BMBF) of Germany for the funding of their work. Also, the authors thank Ines Lemser, Sylke Stahlberg, Ingolf R&#252;cknagel and all the other colleagues working in the team of the animal house (FLI, Germany) for their technical assistance with the bronchoscopies. They are very thankful to Maria-Christina Haase (FLI, Germany) for her help in providing literature. Furthermore, the authors wish to express their gratitude to Dr. Angela Berndt (FLI, Germany) and Nicolette Bestul (University of Wisconsin-River Falls) for critical reading of the manuscript.

Ethics Statement 
This study was carried out in strict accordance with European and National Law for the Care and Use of Animals. The protocol was approved by the Committee on the Ethics of Animal Experiments and the Protection of Animals in the State of Thuringia, Germany (Permit Number: 04-004/11). All experiments were performed under supervision of the authorized institutional Agent for Animal Protection. Bronchoscopy was strictly performed under general anesthesia. During the study, every effort was made to minimize discomfort or suffering.
General remarks 
The described techniques have been developed for calves of approximately 6-8 weeks of age, weighing about 60-80 kg. For the use in other large animal species or calves different in age and bodyweight, the techniques must be adapted to fit the size and weight and take into account the lung anatomy of the particular animal species. All equipment used must be sterile. Chlamydia psittaci is a&#160;zoonotic bacterium which can cause respiratory and general disease in humans. The non-avian C. psittaci strain DC15 used in the present protocol must be handled under biosafety level 2. All work with the pathogen and with infected animals must be performed wearing personal protective equipment, such as a respirator, a splash water proof jumpsuit, rubber boots and gloves. Wearing a suitable respirator is of great importance, since the natural route of infection for C. psittaci is aerogen. Packaging and labeling of samples must be disinfectant proof since all things must be treated with a disinfectant effective against chlamydiae according to the manufacturer&#8217;s instructions before leaving the animal housing unit.
1. Preparing the Animal for Bronchoscopy
<ol>
	<li>Determine the weight of the calf for dosage of anesthetics.</li>
	<li>Place an intravenous (IV) access in the left jugular vein.</li>
	<li>First, slowly inject xylazine (0.2 mg/kg bodyweight) over approximately 30 sec, then, after sedation occurs, inject ketamine (2.0 mg/kg bodyweight).&#160;</li>
	<li>Lift the animal onto the table and place it in right lateral recumbency. Once the animal is adequately positioned on the table, check if the IV access is still in place and readjust it if necessary. During anesthesia, regularly check the eyelid reflex to determine the depth of anesthesia.</li>
	<li>Once the animal is breathing steadily, have someone pull the tongue out and stretch the neck. Place a metal tube speculum in the animal&#8217;s mouth, using slight rotating movements. Push the speculum forward under sight control, using a flashlight, until the larynx is visible.</li>
	<li>Maintain anesthesia throughout the whole endoscopic procedure by injecting a bolus of 7 mg xylazine and 70 mg ketamine as needed.</li>
</ol>
2. Inoculation (Inoculation Sites: Figure 1)
<ol>
	<li>Prepare 3&#160;syringes with inoculum, containing 1, 2, and 5 ml of the inoculum.</li>
	<li>Insert a Teflon tube into the endoscope&#8217;s working channel. The tube should not protrude from the endoscope&#8217;s tip.</li>
	<li>Insert the endoscope through the metal speculum. Slight readjustments of the speculum might be necessary to enable the passing of the larynx. The Bronchus trachealis, which branches off to the right side, helps to align the picture on the monitor.</li>
	<li>Attach the syringe with 5 ml inoculum to the end of the Teflon tube. Navigate the tube into the branches where the inoculum shall be deposited and apply the desired amount (Right lung: Lobus medius: 0.5&#160;ml, Lobus accessorius: 0.5&#160;ml, Lobus caudalis: 0.5&#160;ml and 1.0&#160;ml; Left lung: Lobus cranialis, Pars cranialis: 0.5&#160;ml, Pars caudalis: 0.5&#160;ml, Lobus caudalis: 1.5&#160;ml). Attach the syringe with 1&#160;ml inoculum to the tube, then navigate to the Bronchus trachealis and deposit the inoculum (Lobus cranialis, Pars caudalis: 1.0&#160;ml). It is helpful to always approach the localizations in the same order.</li>
	<li>Remove the endoscope and the speculum.</li>
	<li>Spray 1 ml of the inoculum into each nostril with an actuator.</li>
	<li>Bring the animal back to the stable and place it in prone position for waking up. Do not leave the animal unattended or in the company of other animals until it has regained sufficient consciousness to maintain sternal recumbency. The recovery stable should be air-conditioned, since the animal&#8217;s ability for thermoregulation is decreased under general anesthesia. 
	NOTE: First clinical signs should occur about 24-36 hr after inoculation, depending on the pathogen used.</li>
</ol>
3. Sampling Procedures (Sampling Sites: Figure 2)
<ol>
	<li>Prepare the animal as described in steps 1.1-1.6.</li>
	<li>Bronchoalveolar lavage
	<ol>
		<li>Place 5 syringes each containing 20 ml of sterile isotonic saline, in a water-bath and allow them to warm up to approximately 38 &#176;C.</li>
		<li>Insert a lavage catheter into the endoscope&#8217;s working channel, then insert the endoscope into the metal speculum and navigate forward into the main bronchus of the left lung until the &#8220;wedge position&#8221; is reached where the endoscope cannot be pushed any further ahead.</li>
		<li>One after another, attach the syringes with the warm NaCl&#160;solution to the lavage catheter, instill the fluid and aspirate it directly. The bronchoalveolar lavage fluid must be stored in siliconized glass bottles and put on ice immediately after recovery to prevent the alveolar macrophages from attaching to the glass surface. Note both the amount of instilled saline and the amount of recovered fluid.</li>
		<li>Remove the lavage catheter from the working channel.</li>
	</ol>
	</li>
	<li>Bronchial brushing
	<ol>
		<li>Navigate the endoscope to the desired sampling location, in the described protocol this is the Bifurcatio tracheae.</li>
		<li>Cover the brush with the tube before inserting it into the endoscope&#8217;s working channel until the brush&#8217;s tip appears on the monitor.</li>
		<li>Push the brush with the plastic tube forward about 5&#160;cm and uncover it from the plastic tube by pushing the handle, then navigate it to the location that is to be brushed.</li>
		<li>Rub off epithelial calls by gently pushing and pulling the brush back and forth while navigating the endoscope to ensure contact between the brush and the wall of the bronchus. Stop rubbing when bleeding occurs. Cover the brush with the tube before pulling it out of the working channel.</li>
		<li>Prepare up to five smears on microscope slides by gently rolling the brush over the slide. Fixate the smears in cold methanol for 10 min, air dry and store at -20 &#176;C.</li>
		<li>The brush can be rinsed in various media, depending on the purpose of the sampled cells. If taking multiple brushings with the same brush, be sure to only rinse it in media which do not irritate the mucous membrane.</li>
	</ol>
	</li>
	<li>Transbronchial lung biopsy
	<ol>
		<li>Navigate the endoscope to the desired sampling location, in the described protocol this is the Pars caudalis of the Lobus cranialis. Before inserting the biopsy forceps into the working channel open and close it a couple of times to ensure that it is working smoothly.</li>
		<li>Push the biopsy forceps into the caudal branch of the Bronchus trachealis until a slight resistance occurs. Pull back 2-3 cm, open the forceps, push forward about 2 cm, close the forceps, pull back and remove the forceps from the working channel. This requires some practice.</li>
		<li>Carefully remove the tissue from the biopsy forceps, using a needle or small forceps. Depending on the further use of the tissue, store it in liquid nitrogen or a suitable fixation medium. This should happen right after removal to prevent autolytic processes.</li>
	</ol>
	</li>
	<li>Post-procedural treatment
	<ol>
		<li>Bring the animal back to the stable and place it in prone position for waking up. Do not leave the animal unattended or in the company of other animals until it has regained sufficient consciousness to maintain sternal recumbency. The recovery stable should be air-conditioned, since the animal&#8217;s ability for thermoregulation is decreased under general anesthesia.</li>
		<li>Monitor the animal closely for signs of pneumothorax for the next 24 hr. Provide feed and fresh water when the animal has regained full consciousness.</li>
	</ol>
	</li>
</ol>

The authors&#160;have nothing to disclose.

A bronchoscopic method of inoculation was developed and various bronchoscopic sampling methods were adapted to be used in large animals under experimental conditions. The described techniques are easy to learn, even for examiners with little experience in endoscopy. The process of bronchoscopy is minimally invasive and no adverse effects associated with the methods of the inoculation, as well as the described sampling methods (BAL, transbronchial lung biopsy, bronchial brushing), were ever seen in any of the animals. The complications associated with transbronchial lung biopsies in humans are bleeding and pneumothorax48, none of these were seen in the calves that underwent this procedure. The transbronchial lung biopsy is more time consuming and requires more equipment than the transcutaneous method, but it is less invasive and does not bear the risk of wound infection.
The visually controlled, endoscopic method of inoculation allows the deposition of a defined amount of pathogen at specific sites of the lung. Thus, it results in very consistent clinical and pathological findings in all inoculated animals32-34. However, it does not resemble all features of natural infection in calves. In a model of a respiratory C. psittaci infection, the described technique of inoculation led to lung lesions associated with the sites of pathogen placement34, whereas, in naturally acquired infections calves usually develop pneumonia of the apical lobes. This fact has to be taken into account when interpreting the relevance of experimental findings in the context of natural acquired lung infections in bovines.
Videoendoscopic BAL allows sampling a defined area of the lung. For experimental purposes, this is an advantage compared to the use of a nasal catheter under blind conditions. Due to the anatomy of the bovine lung, the blindly inserted catheter would be pushed to the right diaphragmatic lobe in most cases53,54 and the examiner has no influence on the area of the lung that is lavaged. Another advantage of the endoscopic BAL in anesthetized calves in lateral recumbency is the high average recovery rate of instilled fluid of more than 80%. A comparison with other studies reveals that, in standing, sedated calves, a recovery of 133.3 &#177; 1.6 ml 46 and 127.13 &#177; 3.53 ml&#160;45 after instillation of 240 ml fluid into the caudal lobe is reported. In sedated calves in sternal recumbency 51% of the instilled fluid could be recovered from the cranial lobe and 62% from the caudal lobe43. This means that approximately half of the instilled fluid could be recovered in upright position of the calf. Depending on the amount of BALF needed for further sample preparation, this might not leave enough material to carry out all desired experiments. BAL in cattle has been used by many research groups and many different parameters have been examined under various conditions. Most authors performed lavage of the basal lobes43,45,46, but the amount of fluid used for lavage differs between the research groups. This leads to inconsistency in dilution of the recovered cells, proteins and other substances, making it difficult to compare the findings from different publications. Therefore, for the use in cattle it is recommended to lavage with five fractions of 20 ml (i.e. 100 ml in total) body warm, isotonic saline, which are recovered immediately after instillation. When using a lavage catheter with a large diameter (i.e. &#62;2 mm), the volume of each fraction needs to be slightly increased, depending on the amount of fluid that will remain in the catheter.
The highly segmented anatomy of the bovine lung leads to a methodical limitation; results obtained from one part of the lung may not be true for the rest of the lung. Since there is no sight control of the whole lung area probed by transbronchial biopsy and lavage, the examiner cannot know whether the sampled areas were healthy or diseased. Therefore, it is very important to sample locations where the pathogen was inoculated before in order to have a higher recovery rate of the pathogen and to have a higher possibility of sampling diseased lung areas. Another limitation is the increased anesthetic risk in animals of poor clinical condition. The described methods should only be used in models of mild to moderate disease to keep the burden for the animals as low as possible. General anesthesia in ruminants should always be kept as short as possible, as the gas development in the rumen increases the anesthetic risk in these species. Animals must be placed in prone position immediately after bronchoscopy to allow the efflux of the developed gas and must be monitored closely until they are completely recovered from anesthesia. Also, the described techniques are not suitable for sampling intervals of less than 24 hr.
The described protocol can be adapted to other infectious agents. Endoscopic inoculation of various pathogens has been described, such as C. psittaci32-34, Pasteurella haemolytica38-40,42, Haemophilus somni55, and bovine viral diarrhea virus44. Also, the sites of pathogen deposit in the lung can be adapted to the desired model. When choosing the sampling sites, some important facts have to be taken into account: (i) Sampling sites should be chosen based on the locations of inoculation and on the expected pathological findings. (ii) When necropsy is to be performed care must be taken to leave enough unsampled lung areas for ex vivo sampling. (iii) Sampling site locations must be chosen so they can be reached with the equipment. Especially for transbronchial lung biopsy, there are limitations due to the length of biopsy forceps. (iv) The order of sampling is important, bronchial brushing and transbronchial lung biopsy might lead to minor bleeding, which would contaminate the BALF. Therefore, BALF must always be obtained first. When using the protocol in other species, the species-specific lung anatomy must be taken into account.

The Values of Large Animal Models in Biomedical Research
In modern interdisciplinary biomedical research, animal models are still indispensable to elucidate complex interactions &#8212; related to health or a disease status &#8212; within mammalian organisms. Despite 17 Nobel prizes being awarded to scientists that studied cattle, horses, sheep, or poultry as models for biomedical research1, nowadays the vast majority of animal experiments are undertaken with rodents, while less than 1% of the studies are working with domestic animals or livestock.
Small animals offer many practical advantages (i.e. low cost, genetic malleability, high throughput, availability of numerous genetic, and immunologic tools and kits), and genetically modified murine models are generally accepted to perform mechanistic studies discovering particular molecular pathways. In biomedical research of complex systems, the biological relevance and clinical usefulness of mice models is becoming more and more questionable. They could be misleading and bear the risk of oversimplification of biological complexity2-9.
Due to inter-species peculiarities, no single animal species will completely mirror the human situation, and the use of more than one model seems to be beneficial in an interdisciplinary biomedical research approach. In the context of translational medicine, large animals offer the opportunity to serve as comparative models providing results with high biological relevance of dual use for both human and animal health1. Remarkably, the human genome is more closely resembled by the bovine genome than by the genome of laboratory rodents. It also has been confirmed recently that, compared to other taxa, the genome of mice is much more rearranged10-12.
In a complex study design, use of livestock offers the unique opportunity of intra-individual, long-term studies by repeated collection of a variety of samples in vivo from one-and-the-same individuum without sacrificing the animal. Therefore, functional, inflammatory and morphological changes can be monitored in the same subject over a certain period of time13.
The Bovine Lung as a Suitable Respiratory Model
Due to the high number of significant differences in lung anatomy, respiratory physiology, and pulmonary immunology, mice do not reproduce many important pathophysiological aspects of human pulmonary disease. This must be taken into account when using them as animal models of respiratory disease2,9,14-16. Although peculiarities of anatomy and structure do exist for each mammalian lung, functional characteristics (i.e. lung volumes, airflows and respiratory mechanics) are better comparable between adult humans and calves due to similar body weights (50-100&#160;kg).
The species-specific characteristics of the bovine lung are summarized as follows: the left lung consists of two lobes (lobus cranialis, which is divided into two segments, and lobus caudalis), while the right lung consists of four lobes (lobus cranialis, lobus medius, lobus caudalis, and lobus accessorius). Unlike the lung anatomy of most other mammals, the bronchus of the right cranial lobe branches directly from the right lateral side of the trachea. With respect to subgross anatomy, the bovine lung presents a high degree of lobulation and a high percentage of interstitial tissue17,18 leading to a relatively low specific lung compliance and a higher pulmonic tissue resistance19. Therefore, the required breathing activity is rather high compared to other species20,21. The high degree of lobulation leads to strong independence of the segments. Thus, inflammatory processes are limited by connective tissue septa, and diseased and healthy segments often lie within the same lobe. Due to the lack of collateral airways, the bovine lung is particularly suited to mirror obstructive pulmonary dysfunctions13. Regarding the vasculature in the bovine lung, the small pulmonary arteries show very prominent smooth muscle layers. Therefore, the calf may also serve as a well-established animal model of pulmonary hypertension or vascular remodeling22-24.
With respect to respiratory infections, naturally occurring diseases exist in livestock that share many similarities with the comparable disease in man. Typical examples are bovine tuberculosis25, respiratory syncytial virus (RSV) infections in calves26-28, or naturally acquired Chlamydia infections29. Thus, large animal models do closely resemble the situation in the natural host. Therefore, they are most useful for studying host-pathogen interactions and the complex pathophysiology of the corresponding disease in human beings30,31.
As a biologically relevant model of respiratory Chlamydia psittaci infection, calves were chosen since bovines represent natural hosts for this pathogen32-35. Information obtained from this model, with respect to pathogenesis of the disease or possible transmission routes between animals and humans, will help to broaden our knowledge with impact for both cattle and man. The model can also help to verify generally accepted and alternative therapeutic options for the elimination of pulmonary C. psittaci infections, which is, again, of interest in both veterinary and human medicine.
Techniques Applied to and Specimens Obtainable from the Bovine Respiratory System
This paper describes and illustrates the techniques and diagnostic methods applicable to the bovine lung and used in our model to evaluate both the effects of the pathogen on the mammalian lung and the efficacy of therapeutic intervention.
Bronchoscopy has been performed in human medicine since the 1960s and is considered a safe procedure36. In calves, experimental bronchoscopy was described in 1968 for the first time37. The intrabronchial application of pathogens was suggested by Potgieter et al. as a reliable method to produce lower respiratory tract disease in calves38 and is now a widespread method in bovine research34,39,40. Intrabronchial inoculation of a defined amount of the pathogen under videoendoscopic control allows for selective placement of the infectious agent in the lung. This leads to consistent clinical and pathological findings in all animals34 and allows targeted sampling of lung regions that are expected to be altered due to pathogen exposure.
Bronchoalveolar lavage fluid (BALF) is a well-described indicator for the presence and severity of lung inflammation. The bronchoalveolar lavage (BAL) is a standard procedure in human medicine for the diagnosis of a variety of respiratory diseases41. In live cattle, BAL was introduced by Wilkie and Markham in the late seventies of the last century42. It was considered a safe and repeatable technique to study the lower respiratory tract of cattle. Due to the lack of sufficient data on BALF parameters in healthy animals, in 1988 Pringle et al. performed BAL on healthy calves with a flexible fiberoptic bronchoscope. The authors also pointed out the need to standardize BAL protocols under experimental conditions to acquire comparable results43. BAL is still used as an in vivo sampling method in calves44-46.
Bronchial brushing is commonly used in human medicine as a diagnostic tool to sample neoplastic lesions or for microbiological analysis36. For research purposes, primary cell cultures of epithelial cells harvested by cytological brushing can be obtained47. In cattle, the use of bronchial brushings for microbiological analysis has been described to characterize the microbial environment of the lung43.
Transbronchial lung biopsy provides lung tissue samples and is a valuable diagnostic tool for diffuse lung diseases in humans. Iatrogenic pneumothorax and procedure-related hemorrhage are complications associated with this technique. Their incidence is reported to be less than one percent in human patients48. Transbronchial lung biopsy is not a common method for the use in cattle, due to the high cost of the equipment required and the time needed to obtain biopsies. Instead, transcutaneous lung biopsies are more convenient under field conditions49-51.

<table border="0" cellpadding="0" cellspacing="0" width="1174">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Veterinary Video Endoscope</td>
			<td style="width:419px;">Karl Storz GmbH &#38; Co. KG, Tuttlingen, Germany</td>
			<td style="width:121px;">PV-SG 22&#8211;140</td>
			<td style="width:443px;">diameter: 9 mm, working channel: 2,2 mm, working length 140 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lavage catheter&#160;</td>
			<td>Karl Storz GmbH &#38; Co. KG, Tuttlingen, Germany</td>
			<td></td>
			<td>diameter: 2 mm; length: 180 cm, Luer-lock-adapter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acuator</td>
			<td>WEPA Apothekenbedarf GmbH &#38; Co KG, Hillscheid, Germany</td>
			<td>32660</td>
			<td>length: 60 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">biopsy forceps</td>
			<td>Karl Storz GmbH &#38; Co. KG, Tuttlingen, Germany</td>
			<td>REF 60180LT</td>
			<td>1.8 mm, serrated, oval</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnifix 20 ml, Luer-Lock</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>4617207V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cytology brush</td>
			<td>mtp GmbH, Neuhausen ob Eck, Germany</td>
			<td>110240-10&#160;</td>
			<td>working length 180 cm, brush length: 15 mm, diameter 1.8 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iv acess&#160;</td>
			<td>Henry Schein Vet GmbH, Hamburg, Germany</td>
			<td>370-211</td>
			<td>diameter 1.2 mm, length 43 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rompun 2% (xylazin)</td>
			<td>Bayer Vital GmbH, Leverkusen, Germany</td>
			<td></td>
			<td>0.2 mg/kg bodyweight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamin 10% (ketamine)</td>
			<td>bela-pharm GmbH &#38; Co. KG, Vechta, Germany</td>
			<td></td>
			<td>2.0 mg/kg bodyweight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isotonic saline solution</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>3200950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUB 6 waterbath</td>
			<td>CLF analytische Laborger&#228;te GmbH, Emersacker, Germany</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">metal tube speculum&#160;</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>diameter: 3.5 cm, length: 35 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">flashlight</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">siliconized glass bottles</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>siliconize with Sigmacote (Sigma-Aldrich Co. LLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnifix Luer 3 ml</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>4616025V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnifix Luer 5 ml</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>4616057V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sealing plugs</td>
			<td>Henry Schein Vet GmbH, Hamburg, Germany</td>
			<td>900-3057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inoculum</td>
			<td></td>
			<td>n/a</td>
			<td>dilute pathogen in 8mL buffer</td>
		</tr>
	</tbody>
</table>

the bovine lung in biomedical research: visually guided bronchoscopy, intrabronchial inoculation and in vivo sampling techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

Course of Disease
The effect of the pathogen on the animals&#8217; health can be assessed by clinical examination. In our respiratory infection models, animals were examined twice daily and clinical observations were recorded using a scoring system. Additional information was captured by performing other in vivo sampling methods, e.g. ,collection of blood and swabs or lung function measurement. Pathological examinations were carried out at different time points after inoculation to describe the progress of the infection32-34.
BALF Recovery Rate
The recovery rate of the instilled fluid was 83.05 &#177; 4.58% (mean &#177; SD).
Pathogen Detection
Recultivation of the pathogen can be performed from bronchial brushings. Also, PCR screening of various samples is possible to detect the pathogen, e.g. tissue biopsy, cytology brush sample, BALF-cells52 or pharyngeal swab. Visualization of the pathogen is possible by performing immunohistochemistry of frozen sections of the lung biopsies and sedimentation preparations of the BALF-cells (Figure 3). In previous experiments, PCR of blood samples and swabs (conjunctival, fecal, nasal) were performed to characterize the spreading and shedding of the pathogen32.
Markers of Local Inflammation of Lung Tissue
In the BALF, various parameters of lung inflammation can be studied. The total cell count and the proportion of neutrophils usually increase when lung inflammation is present. For cell differentiation, sedimentation preparations of BALF-cells can be stained according to Giemsa and differentiated using oil immersion (Figure 4). Cellular and liquid proportions of the BALF are separated by centrifugation (300 x&#160;g; 20 min). The BALF-supernatant contains various markers that change during inflammatory processes in the lung and can be studied under experimental conditions. Examples are total protein and eicosanoids29,34.
A schematic overview of the potential further use of the described samples is shown in Figure&#160;5.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51557/51557fig1highres.jpg" width="500" /> 
Figure 1.&#160;Scheme of the bovine lung with inoculation sites (yellow). The numbers indicate the order in which the inoculum is administered into the different bronchi. R: right; L: left. Right lung: 1 Lobus medius: 0.5&#160;ml, 2 Lobus accessorius: 0.5&#160;ml, 3 Lobus caudalis: 0.5&#160;ml and 4 1.0&#160;ml; Left lung: Lobus cranialis, 5 Pars cranialis: 0.5&#160;ml, 6 Pars caudalis: 0.5&#160;ml, 7 Lobus caudalis: 1.5&#160;ml, 8 Lobus cranialis, Pars caudalis: 1.0&#160;ml.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51557/51557fig2highres.jpg" width="500" /> 
Figure 2.&#160;Scheme of the bovine lung with inoculation sites (yellow) and sampling sites: bronchoalveolar lavage (blue), bronchial brushing (green), and lung biopsy (orange). Note that all samples are obtained from regions where the pathogen was deposited before. R: right, L: left.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51557/51557fig3highres.jpg" width="500" /> 
Figure 3.&#160;a) Lung biopsy from a calf inoculated with Chlamydia psittaci 4 days after inoculation (dpi), b) cellular sediment of BALF from a calf inoculated with C. psittaci 9 dpi. Immunohistochemical labeling for chlamydiae. Chlamydial inclusions (arrows) are present in the lung (a) and in alveolar macrophages (b). Hematoxylin counterstain.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51557/51557fig4highres.jpg" width="500" /> 
Figure 4.&#160;Cellular sediment of BALF from a calf inoculated with C. psittaci 9 dpi. Alveolar macrophages&#160;(#) are the predominant cell type in the BALF. The amount of neutrophil granulocytes&#160;(*) increases when inflammatory processes are present. Modified Pappenheim staining.
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/51557/51557fig5highres.jpg" width="600" /> 
Figure 5.&#160;Possible methods for sample preparation. <a href="https://www.jove.com/files/ftp_upload/51557/51557fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

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23.0K Views.  Friedrich-Loeffler-Institut. This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

Video: The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

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&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

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 ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...