Name: lung fixation under constant pressure for evaluation of emphysema in mice
Uploaded: 2019-09-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice at JoVE.com

This work was supported in part by JSPS KAKENHI Grant Number 26461199 (T. Sato) and the Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Grant Number E2920 (T. Sato). The funder had no role in the design of the current methods and in writing the manuscript.

The following methods have been approved by the Animal Care and Use Committees of Juntendo University School of Medicine. The Guidelines for Proper Conduct of Animal Experiments, Science Council of Japan, June 1, 2006 were followed. There are three main steps in this method: 1) mouse dissection, 2) lung exsanguination, and 3) fixation of lung tissues assisted by specialized equipment. Typically, lung specimens are processed to embedment after 48 h of fixation12,15</sup...

<ol>
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The fixation procedure for rodent lungs presented here is not novel; however, this system has several advantages. Firstly, it can fix many lungs (maximum of 20) with the same condition at one time. The Society of Toxicologic Pathology states that the pressure for gravity instillation vary from 22&#8211;25 cmH2O22. Notably, several studies have performed lung fixation at a pressure of 25 cmH2O13,19,<sup clas...

COPD is one of the leading worldwide causes of death1. Cigarette smoke is the most important cause of COPD, but the mechanisms of pathogenesis remain incompletely defined. COPD demonstrates two main characteristics, including progressive limitation of airflow and an abnormal inflammatory response of the lung. Emphysematous disorder frequently occurs in the lungs of COPD patients2. The pathological findings of emphysema are characterized by alveolar wall destruction3. Several animal species have been used to generate COPD models in vivo (i.e., dogs, guinea pigs, monkeys, and rodents)4. However, the mouse has become the most commonly used in the construction of COPD models. This has many advantages, including its low cost, ability to be genetically modified, extensive genomic information availability, availability of antibodies, and ability to use a variety of mouse strains5. Presently, there is no mouse model that can mimic the full features of human COPD; thus, individual researchers must choose which model is most suitable for the specific COPD research6. The emphysematous mouse model is one of many COPD mouse models that are currently available. Additional models include the exacerbation mouse model, systemic co-morbidities model, and COPD susceptibility model7.
The emphysematous mouse model can be generated by several types of exogenous agents, including chemical agents and cigarette smoke exposure4. Chemical exposure (e.g., to elastase) produces a severe type of emphysema, while cigarette smoke results in mild emphysema8,9. Cigarette smoke is believed to be the main cause for the pathogenesis of COPD; therefore, the choice of cigarette smoke as a means to create a COPD mouse model is reasonable10. Many studies have used cigarette smoke to create emphysema in the mouse. For example, Nikula et al. successfully created an emphysematous mouse model from B6C3F1 female mice by exposing them to cigarette smoke for 7 or 13 months11. We have also established an emphysematous mouse model via senescence marker protein/SMP-30 KO mice12. It is crucial to perform a lung fixation method that can properly visualize this mild emphysema model by cigarette smoke exposure.
Various methods for lung fixation have been established13. However, there is no gold standard method of lung tissue fixation for evaluating emphysema14. Several studies from this lab have shown that the fixation system presented here is useful by creating a stable condition for evaluating emphysema12,15,16,17,18. The main advantage of the current system is that it can fix many lungs with the same condition at one time without lung collapse or deflation. The current lung fixation system uses some special equipment that allows lung specimens to be inflated at an appropriate constant pressure for a given period. This special equipment consists of three parts, including a lower container, upper container, and pump. Lung specimens are placed in the lower container that is connected to pressurized fixing agents, resulting in a 25 cmH2O pressure difference in the level of agents between the upper and lower containers19.

<table>
	<tbody>
		<tr>
			<td>10% formalin (formalin neutral buffer solution)</td>
			<td>Wako</td>
			<td>060-01667</td>
			<td></td>
		</tr>
		<tr>
			<td>Bent forceps</td>
			<td>Hammacher</td>
			<td>HSC187-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula, size 20G</td>
			<td>Terumo</td>
			<td>SR-FS2032</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula, size 22G</td>
			<td>Terumo</td>
			<td>SR-OT2225C</td>
			<td>Cannula to exsanguinate lung</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Hammacher</td>
			<td>HSC184-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtowel</td>
			<td>Nippon Paper Crecia (Kimberly Clark)</td>
			<td>61000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Nippon Paper Crecia (Kimberly Clark)</td>
			<td>62011</td>
			<td></td>
		</tr>
		<tr>
			<td>Lower container (acrylic glass material)</td>
			<td>Tokyo Science</td>
			<td>Custom-made</td>
			<td>Pressure equipment component</td>
		</tr>
		<tr>
			<td>Roller pump</td>
			<td>Nissin Scientific Corp</td>
			<td>NRP-75</td>
			<td>Pump machine to exsanguinate lung</td>
		</tr>
		<tr>
			<td>Roller pump RP-2000</td>
			<td>Eyela (Tokyo Rikakikai Co. Ltd)</td>
			<td>160200</td>
			<td>Pressure equipment pump</td>
		</tr>
		<tr>
			<td>Silicone tube &#216; 9 mm</td>
			<td>Sansyo</td>
			<td>94-0479</td>
			<td>Pressure equipment component</td>
		</tr>
		<tr>
			<td>Somnopentyl (64.8 mg/mL)</td>
			<td>Kyoritsu Seiyaku</td>
			<td>SOM02-YA1312</td>
			<td>Pentobarbital Sodium</td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Hammacher</td>
			<td>HSB014-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture thread, size 0</td>
			<td>Nescosuture</td>
			<td>GA01SW</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 mL</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 ml with needle</td>
			<td>Terumo</td>
			<td>SS-01T2613S</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>Terumo</td>
			<td>SS-10ESZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcock</td>
			<td>Terumo</td>
			<td>TS-TR1K01</td>
			<td></td>
		</tr>
		<tr>
			<td>Upper container (acrylic glass material)</td>
			<td>Tokyo Science</td>
			<td>Custom-made</td>
			<td>Pressure equipment component</td>
		</tr>
	</tbody>
</table>

lung fixation under constant pressure for evaluation of emphysema in mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

As described previously, the specialized equipment, which generates extended constant pressure, can be divided into three parts (Figure 3A). The lower part is the point at which to insert the lung sample (Figure 4A). The lung is connected via a cannula (20 G) to the tip of formalin flow using a three-way stop cock (Figure 4B). Pressure is generated from the different surface levels ...

Watch this Scientific Journal Video about Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice at JoVE.com

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

The constant pressure within the fixation device maintains the lungs in a reasonably inflated state generating a histological lung specimen suitable for evaluating mild cigarette smoke induced emphysema. The main advantage of this model is that it can fix many lungs at the same time using the same constant pressure without lung collapse or deflation. Demonstrating the procedure will be Naoko Arano a graduate student from my laboratory.After confirming a lack of response to reflex motion make an incision in the skin and muscle tissue along the medial line of the mouse, aiming for the cephalic region, and cut laterally to create a wider working space. After exposing the diaphragm layer puncture the tissue with forceps and open the thoracic space. Cut the sternal area allowing the lungs and heart to be visualized.And cut the left atrium and right ventricle. Insert a 24 gauge cannula connected to a perfusion pump into the right ventricle area and direct the cannula to the cephalic area until it reaches the pulmonary artery. Then, turn on the pump and perfuse the tissue with approximately 200 milliliters of PBS for one hour until all of the lung tissue has changed to a white color.At the end of the perfusion cut the connective tissue surrounding the trachea, lungs and heart, and use a suture to tie off the right main bronchus. Next, harvest all of the individual lobes of the right lung and place the heart and lobes of the left lung into individual 10 milliliter syringes containing an appropriate fixative. Retract the plunger creating a vacuum in the syringe to inflate the lungs and remove the lung from the syringe.Insert a 20 gauge cannula into the trachea and use a suture to secure the cannula in place. Connect a one milliliter syringe filled with fixative to the cannula and fill the lung with additional fixative to check for leaks. Be sure that the insertion site was secured to prevent the lung specimen from detaching from the pulmonary board during the fixation.Then transfer the tissue to the lung fixation pressure equipment, removing the sample and tying off the trachea with a knot at the end of the fixation period. The lower component of the lung fixation pressure equipment into which the lung sample is inserted is connected via 20 gauge cannula to the tip of the formalin flow with a three-way stopcock. Pressure is generated from the different surface levels of the fixative between the lower and upper compartments.The pressure difference is 25 centimeters of water but the height adjustment knob can be used to adjust the pressure to between 25 and 30 centimeters of water. A pump connects the lower and upper containers, preserving a 25 centimeter difference in the fixative surface height, and directs the flow of the agent. After 48 hours of fixation the lungs from SMP30 knockout mice exposed to ambient air do not exhibit a marked airspace enlargement.Significant airspace enlargement and alveolar wall destruction, however, are observed in SMP30 knockout mice exposed to chronic cigarette smoke. In addition, the mean linear intercept and destructive index of the lung specimens are significantly greater in the smoke exposed SMP30 knockout mice than in the air exposed animals. Our lab uses 10%formalin but researchers can use other fixation agents according to their experimental needs.As we have reported using the demonstrated fixation system lung samples can be harvested from various mouse emphysema models for their morphometric evaluation. As fixing agents can be hazardous always wear the appropriate personnel protective equipment and work in a well-ventilated room.

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Research

JoVE Journal

Medicine

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

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 ...

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...