Funding Source /Number Molecular Biology of Hemorrhagic Shock GM053789

1. Instrument and Surgical Field Preparation:
All experimental procedures are performed using aseptic techniques. Before beginning, the experimental area must be thoroughly cleaned and sterilized. The benchtop should be disinfected, allowed to air dry and then wiped down with 70% alcohol. Place a surgical blue pad and sterile field dressing in the experimental work area.
All materials and instruments are autoclave sterilized before use. Syringes and needles are received sterile. The researcher should be appropriately garbed in lab coat, surgical mask, and sterile surgical gloves.
Our surgical instruments are autoclave sterilized each evening. They are washed after surgery using antibacterial soap and water. They are allowed to dry on a clean surgical blue pad. They are then carefully placed into a sterilization pouch and sterilized for later use.
Turn on the hot bead sterilizer to ensure it reaches the appropriate temperature - 300-350&deg;F before starting the experiment. This sterilizer will be used to clean instruments between experimental procedures on respective mice. Get a stainless steel instrument tray and fill it 1/3 of the way with 70% ethanol. There should be enough 70% Ethanol to cover your surgical instruments. Turn on the circulating heating pad and place a surgical blue pad over it and then a sterile field dressing on top of that. This heater is used to ensure adequate warmth of the mouse during the experimental procedure and recovery period. Place all sterile instruments, gauze, syringes, needles and a 1.5mL microfuge tube on the sterile field dressing. Be careful when opening any sterile instruments and other items, do not to contaminate them by touching them. It is best to use sterile gloves when doing this setup procedure.
2. Pseudofracture Model - Experimental Procedures:
1. Induction of anaesthesia and positioning of the mouse.
Begin by administering an intraperitoneal injection of Pentobarbitol (70mg/kg). This is accomplished by first lifting the mouse from its cage using the proximal end of its tail. The mouse must then be weighed to calculate the correct dosage of anaesthesic. Next, place the animal on top of the cage while still holding its tail. Grab the scruff of the neck of the mouse with the thumb and middle finger on either side of the mouse just behind the forepaws. The index finger is used to pull the skin on the head/neck region back and partially immobilize the head. The mouse's tail is held between the little finger and the ring finger while the ring finger is pressed into the lumbar region of the mouse's spine. The anesthesia should take full effect within approximately 5 minutes, and should be confirmed with the reflex test.
After the animal is asleep, place the mouse in the supine position onto a plexiglass board and use a loose loop tape technique to immobilize them by taping their extremities. The loose loop technique simply entails cutting thin strips of tape and wrapping the tape loosely around the distal portion of each extremity. The tape is then stuck back to itself and the left over tape is attached to the board. This allows the experimental manipulation to be performed in a consistent way as well as ensuring the extremities of the mice assume a more natural position. The animal's abdominal, inguinal, and thigh regions are then shaved using our Oster A5 clippers (size 40 blade), to help maintain a sterile field. A 4x4 gauze is doused with betadine and the surgical area is then wiped to sterilize. A sterile field dressing is then folded and draped across the mouse to allow exposure of only the lower extremities and head.
After immobilization and sterilization, place a nose cone with isoflurane over the mouse's nose for a few seconds before beginning the experimental procedure. The nose cone consists of a 50cc conical tube filled with gauze. Half of the bottom end of the tube is cut out creating a space for the mouse's nose to fit into. A cap (bottom of a tissue storage container i.e. straight side wide mouth jar) is placed onto the cut end of the conical to ensure the isoflurane vapors remain in the tube when not in use. One mL of isoflurane should be added to the gauze within the cone. Meticulous attention should be paid to the mouse during use of anaesthesia and particularly use of isoflurane via the nose cone. Once the animal's respirations begin to slow, the experiment can begin, and the nose cone should be removed and closed.
If additional anesthesia is required throughout any part of the experiment, additional pentobarbital may be injected or isoflurane administered. Alternative methods of isofluorane administration are available and updated guidelines recommend the use of a precision vaporizer to allow better control of anesthesia, if feasible. However, close attention to the respiratory physiology of the mouse must be guaranteed with any of these methods.
2. Pseudofracture experiment.
Pseudo-fracture is a combination of soft tissue injury followed by bone solution injection to the injured muscle and is performed bilaterally to the hindlimbs. The bone solution must be prepared before any experimental manipulation of the recipient mice begins.
3. Bone solution preparation.
The bone solution prepared from one donor mouse will be enough for 3 recipient mice. A syngeneic donor mouse should be used that is age- and weight-matched to the recipient experimental mouse.
The donor mouse will be euthanized with inhaled isoflurane. The donor will then be taped to the plexiglass board with careful attention to tape the lower extremities only at the tip of the foot. The lower extremities must be carefully shaven and thoroughly covered in betadine, and then wiped with alcohol for sterility. Then the femur and tibia will be surgically removed in a sterile manner from both lower extremities. To remove the long bones from the lower extremity, make a surgical incision into the skin in the inguinal region, and continue to cut the skin along the length of the extremity, down to the ankle. Retract the skin, and dissect it away from the subcutaneous fascia and muscle both on the medial and lateral aspects for maximum exposure. Insert one blade of the scissors under the muscles that lie anterior and lateral to the tibia (tibialis anterior, extensor digitorum longus, extensor hallicus longus, peroneus longus, peroneus brevis) and slide the blade proximally and distally to lift the muscles cleanly away from the underlying bone. Repeat this technique on the posterior and medial underside of the tibia to separate the muscles underneath the tibia (gastrocnemius, soleus, plantaris, tibialis posterior, flexor digitorum longus, flexor hallicus longus). A minimal additional exertion should be used on the posterior side when sliding distally, in order to snap the fibula away from the tibia. At the proximal end of these leg muscles cut the tendons as close to their insertions as possible and retract the muscles distally, while pulling on the muscle group together in downward fashion a small tug will release the distal attachments of these muscles from the ankle joint. The ankle joint can be cut through directly with the scissors. Do not detach the tibia from the femur at this point! This connection gives additional leverage that will aid in the dissection of the femur from the hip joint. Follow a similar technique for release of the muscles from the femur and perform this posteriorly and both medially and laterally also if needed. Cut the distal attachment of these muscle groups to release them from the knee joint. Continue the dissection and follow underneath the muscles to dissect the end of the femur out of the hip joint. To aid in this dissection, the skin incision should be extended as far as required to achieve adequate exposure. Motion of the femur at the hip joint can help locate the proximal end of the femur during dissection.
Once the femur (with tibia attached) is removed from the donor mouse place them on a sterile 4x4gauze. The two bones can be separated at the knee joint by simply grasping each bone in a separate hand and gently twisting/rotating the two bones in opposite directions along their long axis. Gentle dissection and handling of these two bones is recommended as they can be easily fractured with any over-exertion of the manual manipulation. To remove the remaining tendon attachments from the long bones, use a dry sterile piece of 4x4 gauze wrapped around the length of the bone. Grasp this gauze tightly against the bone an pull it the full length of the bone several times, this will scrape the remaining tissues off the surface of the bone as they will adhere to the gauze and be quickly and easily pulled away from the bones. The collected bones should then be placed directly into a sterile 1.5mL microfuge tube and placed on ice for transport.
These four donor bones will be taken to a biological safety cabinet for preparation for the bone solution. The hood must be thoroughly disinfected before use, place a sterile field dressing over the work area within the hood. Place a sterile mortar and pestle, sterile 8mL tube and sterile Phosphate Buffered Saline (PBS) in the work area. A 1mL pipetteman, and respective tips should be available within the hood.
A sterile pair of forceps should be used to remove the harvested bones in a sterile fashion from the 1.5mL microfuge tube and placed into the mortar; the pestle will then be used to gently crush the bones. Phosphate Buffered Saline is the vehicle used to resuspend the bone fragments for injection. Pipette 1mL of PBS into the mortar and continue to crush the remaining fragments, with additional circular motions to ensure full resuspension. Then add another 1mL of PBS and continue crushing to create the 'bone solution'. The solution should have a pink hue, and there will be remnants left at the bottom of the mortar that cannot be fully broken up. Slowly pour the solution out of the mortar into the 8mL tube, to catch the maximum volume, while ensuring the larger remnants remain behind in the mortar. This bone solution should remain on ice until its experimental use and will be transferred to 1mL syringes for injection. A twenty gauge needle will be later attached to the 1mL syringe containing the bone solution in order to administer it to the mouse. This large needle size was chosen to ensure that all the bone fragments enter the region upon injection and don't block the needle.
Before injection to recipient mouse, a drop of the bone solution is placed on a MacConkey agar plate for culture (incubation for 24-48hours). This is to ensure sterility of the bone solution. The pH of the bone solution also must be checked to ensure a neutral pH.
4. Soft tissue injury.
This soft tissue injury is a bilateral hindlimb crush injury. The initial positioning of the mouse, with hind limbs slightly abducted and laterally rotated, helps in accessibility to the correct muscle group, the knee flexors (biceps femoris, semitendinosus and semimembranosus muscles). A large 18cm hemostat will be used to perform the crush injury. The force distribution of these applied hemostats was analyzed and found to be 270psi using Topaq Pressure Analysis System by Sensor Products Inc. The hemostat must be clamped around the posterior thigh musculature, midpoint along the femur, with its convex curve facing the femur. The hemostat should then be locked shut to the first click only, and remain for 30 seconds. Careful attention to the placement of this hemostat is important - it must not be clamped over the femur, to ensure it is not fractured. This is should be performed in a consistent manner each time to guarantee a reproducible injury between mice. This crush injury is performed on both lower extremities.
5. Bone solution injection.
The recipient experimental mouse will be anesthetized and prepared as previously described, and will have undergone soft tissue injury prior to this bone injection.
The bone solution will then be injected into the crushed thigh musculature of the recipient mouse bilaterally. Using a 20 gauge needle, 0.15mL of this solution will be injected into the posterior muscles of each thigh. Enter the needle through the skin approximately 2~3mm and pay attention to the distance of insertion of the needle - you will feel as the tip of the bevel just touches against the femur, this position is ideal. Inject the bone solution now. Withdraw the needle and quickly place a sterile gloved finger over the injection site to stop any back flow of bone solution out of the wound. Hold this finger there for several seconds.
The mouse will then be placed back into the cage and allowed full freedom of movement directly as anaesthesia subsides. Appropriate pain management must be administered as the mouse awakens.
6. Post-operative/recovery period.
The loose loop tape is removed and the animals are placed into a clean cage which is kept on a circulating heating pad for several hours post recovery. Appropriate warmth should be ensured with an additional heat lamp if required.
Food and water will be readily available. 
Analgesic must be administered as the animals begin to waken from the anesthesia in order to correctly manage pain. Buprenorphine (0.1mg/kg) is injected subcutaneously as the animals begin physical activity, but not before, so as not to compromise respiratory function.
Mice should be constantly monitored during anaesthesia, until recovery from anaesthesia or experimental endpoint. Mice should be also be very carefully monitored in the postoperative period and any additional pain medication required should be administered as needed. Monitor animal physical activity, respiratory status, food and water intake and any signs of distress (labored breathing, pain, changes in eating and drinking habits) should be addressed accordingly.
 Place your surgical instruments into 70% alcohol and wipe them with sterile gauze then put them into the microbead sterilizer for ~20 seconds for sterilization between animals. Remove the surgical instruments and spray them with 70% alcohol to help them cool off. Place them onto the sterile field dressing. Make sure there is no alcohol left on the instruments that will drip back into the next animals.
3. Comparative Bilateral Femur Fracture Model - Experimental Procedures:
The experimental mouse will be anesthetized and prepared as initially described. A large 18cm hemostat should be clamped around the hindlimb approximately 2-3mm above the knee joint with its convex surface facing the knee. Place your thumb on top of the clamped hemostat and your index finger between the hemostat and hip. Be sure to feel the femur before twisting so you know where you are breaking. Then twist hemostat counterclockwise while twisting your other hand clockwise. 
Repeat procedure with contralateral leg.
4. Multiple Trauma Model - Experimental Procedure for Combination of Pseudofracture with Hemorrhagic Shock:
Recipient mouse will be anesthetized and prepared as initially described. Mice that are to undergo hemorrhagic shock in combination with pseudo-fracture will have femoral artery cannulation before the pseudofracture procedure and then will be hemorrhaged from this catheter once the pseudo-fracture has been completed.



5. Secrets for Success:
General:
<ul>
<li>Make sure you take the extra effort to remain as sterile as possible.</li>
<li>Meticulous attention should be paid to the overall physiological condition of the mouse, including respiratory rate, during administration of anesthesia and particularly isoflurane via the nose cone.</li>
</ul>
Bone solution preparation:
<ul>
<li>Gentle manipulation of the bones - as can be easily fractured with over-exertion during simple manual manipulation!</li>
<li>Fibula removal from the tibia should be ensured upon dissection.</li>
<li>Begin by crushing bones gently at first - so as to keep all the fragments within the mortar!</li>
<li>Add only 1 mL PBS initially and continue crushing the remaining bone fragments, once a good suspension has formed, add the remaining 1mL.</li>
<li>Only the solution from the mortar and pestle should be collected - without the larger remaining tissues that could not be fully broken down - these be may block/get lodged in the needle upon injection into the recipient mouse.</li>
</ul>
Soft tissue injury:
<ul>
<li>Careful placement of this hemostat is important - not over the femur or it would be fractured. Clamp the hemostat over the correct musculature (posterior thigh - knee flexors) for reproducible injury.</li>
</ul>
Bone solution injection:
<ul>
<li>When injecting the solution, pay attention the distance of insertion of the needle - you will feel as the tip of the bevel just touches against the femur, this position is ideal.</li>
<li>Withdraw the needle and quickly place a finger over the injection site to stop any back flow of bone solution out of the wound. Hold finger there for several seconds.</li>
</ul>
6. Post Operative Concerns:
<ul>
<li>Check for infection in the leg.</li>
<li>Check bone solution culture.</li>
<li>Animal could have trouble using hind limbs as a result of experimental manipulation and associated inflammation. Manage pain appropriately.</li>
</ul>

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J., Vodovotz, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+exposure+of+bone+components+to+injured+soft+tissue+induces+Toll-like+receptor+4-dependent+systemic+inflammation+with+acute+lung+injury.">Local exposure of bone components to injured soft tissue induces Toll-like receptor 4-dependent systemic inflammation with acute lung injury.</a> Shock. 30, 686-691 (2008).</li><li>Bumann, M., Henke, T., Gerngross, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+haemorrhagic+shock+on+fracture+healing.">Influence of haemorrhagic shock on fracture healing.</a> Lang Arch Surg. 388, 331-338 (2003).</li><li>Santry, H. P., Alam, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+resuscitation:+past,+present,+and+the+future.">Fluid resuscitation: past, present, and the future.</a> Shock. 33, 229-241 (2010).</li><li>Hierholzer, C., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+the+early+phase+of+hemorrhagic+shock.">Molecular mechanisms in the early phase of hemorrhagic shock.</a> Lang Arch Surg. 386, 302-308 (2001).</li><li>Chaudry, I., Ayala, A., Ertel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhage+and+resuscitation:+immunological+aspects.">Hemorrhage and resuscitation: immunological aspects.</a> Am J Physiol. 259, 63-678 (1990).</li><li>Kawasaki, T., Hubbard, W. J., Choudhry, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trauma-hemorrhage+induces+depressed+splenic+dendritic+cell+functions+in+mice.">Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice.</a> J Immunol. 177, 4514-4520 (2006).</li><li>Kohut, L., Darwiche, S. S., Frank, A. M., Brumfield, J. M., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixed+volume+or+Fixed+Pressure:+A+Murine+Model+of+Hemorrhgaic+Shock.">Fixed volume or Fixed Pressure: A Murine Model of Hemorrhgaic Shock.</a> J Vis Exp. , (2010).</li><li>Menzel, C. L., Pfeifer, R., Darwiche, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+lower+extremity+damage+in+mice:+time+course+of+organ+damage+and+immune+response.">Models of lower extremity damage in mice: time course of organ damage and immune response.</a> J Surg Res. 166, e149-e156 (2011).</li><li>Pfeifer, R., Kobbe, P., Darwiche, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hemorrhage+in+the+induction+of+systemic+inflammation+and+remote+organ+damage:+Analysis+of+combined+pseudo-fracture+and+hemorrhagic+shock.">Role of hemorrhage in the induction of systemic inflammation and remote organ damage: Analysis of combined pseudo-fracture and hemorrhagic shock.</a> J Orthop Res. 29, 270-274 (2011).</li></ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee and the Research Conduct and Compliance Office of the University of Pittsburgh, an AALAS/AAALAC accredited institution. Animal sources include Jackson Laboratories and Charles Rivers Laboratories. All animals undergo extensive health assurance through each vendor as well as the University of Pittsburgh's internal animal health monitoring programs. This research is conducted in accordance with the US government principals for Use of vertebrate animals. The program is registered with the USDA, and has a letter of assurance with the Public Health Service Office of Laboratory Animal Welfare.

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of post-traumatic immune responses. The pseudofracture model immunologically mimics an extremity fracture environment through recreation of the features of a long bone fracture: injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.38,39 A biphasic immune response can be seen following pseudofracture trauma which consists of an early hyperinflammatory respon...

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pseudofracture: an acute peripheral tissue trauma model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Watch this Scientific Journal Video about Pseudofracture: An Acute Peripheral Tissue Trauma Model at JoVE.com

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

pseudofracture-an-acute-peripheral-tissue-trauma-model

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14.6K Views.  University of Pittsburgh. Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Video: Pseudofracture: An Acute Peripheral Tissue Trauma Model

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

A Saline/Bipolar Radiofrequency Energy Device As an Adjunct for Hemostasis in Solid Organ Injury/Trauma

Solid organ (liver, spleen, and kidney) hemorrhage is often life-threatening and can be difficult to stop in critically ill patients. Traditional techniques for arresting this ongoing bleeding include coagulation by high voltage electrocautery, topical hemostatic application, and the delivery of ignited argon gas. The goal of this study/video was to demonstrate the efficacy of a new energy device for arresting persistent solid organ hemorrhage.A novel instrument utilizing bipolar radiofrequency (RF) energy which acts to ignite/boil dripping saline from a simple handpiece is employed to arrest ongoing bleeding from solid organ injuries in a porcine model. This instrument is extrapolated from experience within elective hepatic resections. An escalating series of injuries to solid organs within a porcine model will be created. This will be followed by arresting hemorrhage with this novel energy device in sequence. A standard suction device will also be employed. This simple saline/RF energy instrument has the potential to arrest ongoing solid organ surface/capsular bleeding, as well as moderate hemorrhage associated with deep lacerations.

The goal of this publication is to demonstrate the potential application of a novel device using simulated solid organ injuries in a porcine model.

Solid organ (liver, spleen, and kidney) hemorrhage is often life-threatening and can be difficult to stop in critically ill patients. Traditional ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...