We thank Kathy Gage, BS (Department of Anesthesiology, Duke University Medical Center, Durham, NC) for editorial assistance. NT acknowledges support from a DREAM Innovation Grant from Duke Anesthesiology and NIH/NIA R01 AG057525-01.

All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Institutional Animal Care &#38; Use Committee (IACUC) at Duke University.
1. Experimental Animals
<ol>
	<li>Keep mice in an air-conditioned environment with 12-h light/dark cycles and proper access to standard food and water. House no more than 5 littermates per cage, and avoid conditions that could encourage fighting.</li>
	<li>Use females C57BL6/J mice at 3 months of age (young) or 18 months of age (old).</li>
	<li>For parabiosis, acclimate 2 mice together for at least two weeks&#160;before surgery. Mice are either wild-type C57BL6/J or eGFP+.</li>
	<li>Check the body condition and overall appearance of mice daily.</li>
</ol>
2. Preoperative Setup for Parabiosis and Orthopedic Surgery
<ol>
	<li>Weigh the 2 mice.</li>
	<li>Administer general anesthesia via an anesthesia system at a constant O2 flow rate of 0.2 L/min. Use 5% isoflurane for induction in an induction chamber. 
	NOTE: The depth of anesthesia can be confirmed using a toe pinch.</li>
	<li>Place the mice on a heated pad in a supine position. Use a rectal probe to monitor body temperature throughout the surgical procedure.</li>
	<li>To maintain anesthesia, lower the inhaled concentration of isoflurane to 2.0% in 0.2 L/min through a facemask. Monitor to ensure adequate depth of anesthesia.</li>
	<li>Monitor physiologic parameters (respiratory rate, oxygen saturation, and heart beat) non-invasively using a pulse oximeter (optional).</li>
</ol>
3. Parabiosis Surgery (Protocol 1A)
<ol>
	<li>Administer analgesia (buprenorphine, SR/slow release,&#160;0.1 mg/kg subcutaneously) after induction and before surgical manipulation. Inject bupivacaine (0.25%) at the flanks just before opening. Apply eye lubricant. 
	NOTE: The parabiosis protocol can be performed independently from the tibial fracture.</li>
	<li>For all internal body suturing, use 4-0 polydioxanone suture material. For all external suturing, use 4-0 polypropylene suture material.</li>
	<li>Shave each of the 2 mice along a contiguous line from the elbow, the flank, and the knee on the side to be joined. Disinfect with iodine + 70% alcohol skin scrub over 3 alternating cycles in preparation for incision.&#160; 
	Used autoclaved instruments and maintain a sterile field. 
	NOTE: Place the wild-type mouse to the right of the eGFP mouse, and prepare the left flank of the wild-type mouse and the right flank of the eGFP mouse for surgery (Figure 1).</li>
	<li>On each mouse, use a scissor&#160;to make a skin incision along the flank, ranging proximal to the knee to just proximal to the elbow, and without disturbing the muscles underneath the skin.</li>
	<li>Join the triceps of the animals with 2 interrupted sutures.</li>
	<li>Join the body walls along the flanks with a running, continuous suture of 7-9 passes.</li>
	<li>Join the quadriceps of the animal with 2 interrupted sutures.</li>
	<li>Close the skin of the 2 parabionts with interrupted sutures.</li>
	<li>Allow the mice to awake in ambient air.</li>
	<li>To ensure success of this protocol, the health of the pairs must be maintained: monitor the pairs daily; perform body condition scoring twice weekly; and weigh the pairs twice weekly. 
	NOTE: Each pair is housed individually. 
	NOTE: If using regular buprenorphine (ie not SR/slow release) then administer 0.1 mg/kg subcutaneously in 1 mL of saline per mouse twice daily for 3 days.</li>
	<li>Allow 4 weeks of recovery time to ensure shared circulation between parabionts if performing tibial fracture surgery.</li>
</ol>
4. Tibial Fracture Surgery (Protocol 1B)
<ol>
	<li>Administer analgesia (buprenorphine, 0.1 mg/kg subcutaneously) after induction and before surgical manipulation. Inject bupivacaine (0.25%) at the surgical site proximal to the knee just before the opening. Apply eye lubricant.&#160;</li>
	<li>Shave the medial aspect of the right hind limb of the mouse to expose the surgical area, and disinfect with iodine + 70% alcohol skin scrub over 3 alternating cycles.&#160;Maintain a sterile surgical field throughout the procedure. To limit contamination; use autoclaved instruments and gloves; complete the surgery under a dissecting microscope (optional); and use a heating pad to maintain body temperature. 
	NOTE: If the protocol is performed on parabionts, only 1 mouse is fractured in the pair (the right tibia of the right mouse). See&#160;Figure 1B&#160;for a schematic diagram of the fracture on parabionts.</li>
	<li>Use a scissor to make a skin incision along the medial aspect of the right hind limb proximal to the knee down to the midshaft of the tibia on the right mouse.</li>
	<li>Expose the midshaft of the tibia and visually locate the diaphysis. Flex the knee and visualize the tibial plateau using the patella-femoral ligament as a landmark.</li>
	<li>Visualize the patellar tendon; manually drill by rolling thumb and index finger a 0.5-mm hole in the intramedullary canal using a 25-gauge needle. 
	NOTE: The drilled hole will run parallel along the tibia, in through the tibial plateau.</li>
	<li>Insert a 0.38-mm stainless steel pin through the hole about 15 mm into the medullary cavity until resistance is felt, and cut flush with the tibial plateau using a wire cutter (see Supplementary Video 1 for a 3D reconstruction).</li>
	<li>Using straight Bonn scissors, fracture the tibia midshaft (diaphysis). See Figure 1C for a schematic diagram of the fracture.</li>
	<li>Visually observe the fracture site and adjacent tissues to inspect for stabilization of the fracture sight.</li>
	<li>Close with dermal staples.</li>
	<li>Place the mice on heated pads to recover before returning them to a clean home cage. Inject 1 mL prewarmed (37 &#176;C) normal saline subcutaneously in each mouse for fluid replacement.</li>
	<li>Check mice daily for signs of lameness, infection, or bleeding. 
	NOTE: If using regular buprenorphine (ie not SR/slow release) then administer 0.1 mg/kg subcutaneously in 1 mL of saline daily for 3 days.</li>
</ol>

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The authors have nothing to disclose.

Fractures are a common clinical problem, and remain a leading cause of morbidity, particularly in the rapidly growing senior population. Here, we introduce a step-by-step protocol for a mouse model of tibial fracture to study mechanisms responsible for postoperative neuroinflammation and cognitive impairment. This model can be combined with parabiosis surgery to study neuro-immune interactions, tissue regeneration, and other signaling processes. Understanding these mechanisms will provide strategic targets to minimize the risks for postoperative complications and optimize outcomes.
Several orthopedic models have been developed to study bone repair in rodents25. We adopted and modified this tibial fracture procedure, originally described by Harry et al.8, to study effects of orthopedic surgery on brain function. We have also used this fracture model in combination with our parabiosis model to investigate factors that are responsible for bone healing and age-dependent tissue regeneration. When performed under volatile general anesthesia, this tibial fracture procedure requires only about 15 min per animal, results in zero to minimal mortality (depending on the age of the mouse and underlying genetic susceptibilities), and recapitulates common insults associated with long-bone fracture and orthopedic surgical trauma. Thus, this model is ideal for interrogating biologic pathways and performing longitudinal assessments. However, it is critical that the osteotomy and pinning are reproducible, and that damage to the soft tissue is consistent. Soft tissue damage can be modulated, for example by stripping the periosteum and pinching surrounding muscles to make the surgery more traumatic. Models of traumatic fracture induced by non-fixed blunt trauma or three-point bending would not provide such consistency or accuracy. These procedures often result in re-injury, which leads to prolonged inflammatory response. Conversely, models of fracture involving rigid fixation have a more moderate inflammation, which does not fully recapitulate the damage associated with orthopedic surgery26,27.
Other models using titanium-alloy pinning have been developed to closely mimic human arthroplasty and may be more relevant to interrogate prosthesis instability, osteolysis, and prosthesis-associated complications in mice28,29. Drill-hole models such as the one presented here, provide adequate stabilization, and mice can be tested in behavioral paradigms without significant deficits that could confound such tasks as fear conditioning or open field locomotion/anxiety testing6,7,11,15,19,20. However, rotational deformities can occur if the fixation is not properly locked. Some models use an external fixator, which provides superior stabilization but is challenging to implement in a mouse tibia, although it can be successfully implemented in a mouse femur27.
Cognitive impairments, including delirium and postoperative cognitive dysfunction, are common complications after orthopedic surgery for fracture repair, especially in elderly and frail patients30. This clinically relevant mouse model of tibial fracture surgery demonstrates that postoperative systemic cytokine release6,7,17, impaired blood-brain barrier function15,19, and altered microglial morphology16,22, contribute to the memory impairment, and may represent critical features of the postoperative neurologic complications seen in many patients after orthopedic surgery. It is important to note that other surgical procedures have been used to model postoperative cognitive dysfunction in mice. These include abdominal31,32,33 and vascular34 surgery, as well as superficial trauma35,36. The parabiosis technique is applicable to all of these models, which share similar endpoints, including inflammation, glial activation, and behavioral deficits, that may be mediated by common mechanisms.
Studies that include parabiosis have revealed novel roles for circulating factors that can impact cognitive function, neuroinflammation, and tissue rejuvenation in aged animals37,38,39,40,41,42. We have shown that parabiosis can be successfully combined with the tibial fracture model described here to interrogate regenerative pathways and study mechanisms involving blood-borne factors that influence healing and fracture repair12. Here, we demonstrated that the fracture-repair capacity of an aged animal can be rejuvenated when the aged animal is anastomosed to a young animal. This reversal of age was rooted in the engraftment of hematopoietic cells at the fracture site. Interestingly, such rejuvenation could also be achieved through transplantation of young bone marrow into aged mice. In this regard, bone marrow transplantation can be considered a more direct and simpler alternative approach to parabiosis. However, parabiosis is a more robust model for investigating the function of circulating cells and factors. We expect that a combination of parabiosis and orthopedic surgery models will play an important role in answering critical questions in perioperative care and aging biology.
In summary, we introduce a step-by-step protocol for a mouse model of tibial fracture to study mechanisms responsible for postoperative neuroinflammation and cognitive impairment after orthopedic surgical procedures. This model can be combined with a parabiosis procedure to study neuro-immune interactions, tissue regeneration, and other pathways. Defining these mechanisms will provide strategic targets to minimize the risks for postoperative complications and optimize outcomes.

Surgery has transformed the medical healthcare system and is continuously contributing to cutting edge technology, improved safety, and maintained quality of life. Unfortunately, surgery also induces pathophysiologic responses that can lead to postoperative complications including wound infections, neurologic impairments, and even mortality, particularly in elderly patients1,2. Orthopedic surgery is performed routinely, especially in older adults, to improve quality of life and repair common bone injuries. However, up to 50% of orthopedic surgery patients who are 65 years and older experience neurologic impairments such as postoperative delirium. This consistently correlates with poor prognosis, i.e., 5-fold increased risk for mortality at 6 months, persistent functional decline, increased nursing time per patient, increased length of hospital stay, and higher rates of nursing home placement3,4,5. Some risk factors, including advanced age, have been identified, but little is known about the mechanisms responsible for neurologic impairments after surgery.
Since fractures are very common in the elderly, we have established a mouse model of tibial fracture to determine the impact of peripheral trauma on postoperative recovery, including neuroinflammation and brain health (cognitive function)6,7. This model, originally described by Harry et al.8, consists of intramedullary pinning and tibial fracture under general anesthesia and analgesia, and thus mimics the skin injury, muscle trauma, and bone repair associated with common long-bone fractures and repair in humans. After this procedure, mice demonstrate changes in inflammatory markers similar to changes observed in humans9,10, as well as microglial activation in the hippocampus, which is associated with deficits in declarative memory and hippocampal neuroplasticity6,7,11. We have previously combined this fracture model with parabiosis. Parabiosis is a surgical model in which 2 mice are anastomosed, and thus share a circulatory system. This model has provided a breakthrough in the understanding of regulatory effects of circulating cells and humoral factors on organ function in the context of age and disease12,13,14. Using this approach we recently discovered systemic factors associated with age-dependent fracture healing12.
Here, we introduce a protocol that combines the tibial fracture model with parabiosis to study bone-to-brain age-dependent mechanisms, which are relevant to regenerative medicine and neuroimmunology. Protocol 1A describes the parabiosis procedure, and Protocol 1B details the tibial fracture procedure (Figure 1A). These can be performed independently or in combination, depending on the nature of the interrogation.

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			<td>Good for osteotomy, note to change regularly as becomes blunt</td>
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			<td height="21" style="height:21px;">Fine Scissors</td>
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			<td>Sharp scissors for cutting sutures</td>
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			<td height="21" style="height:21px;">22G x 3.5 In Quincke&#160; Spinal Needle</td>
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			<td style="width:175px;">405181</td>
			<td>Use inner rod for pinning</td>
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			<td height="21" style="height:21px;">Needle Holders</td>
			<td style="width:272px;">Fine science tools</td>
			<td>12001-13</td>
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		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Suture</td>
			<td style="width:272px;">Look</td>
			<td style="width:175px;">1079B</td>
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		<tr height="21">
			<td height="21" style="width:211px;height:21px;">C57BL6/J</td>
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			<td style="width:175px;">&#160;stock no. 000664</td>
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			<td height="63" style="width:211px;height:63px;">eGFP+ (expressing enhanced green fluorescent protein ubiquitously)</td>
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			<td style="width:175px;">&#160;stock no. 003291</td>
			<td></td>
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a mouse model of orthopedic surgery to study postoperative cognitive dysfunction and tissue regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

After transverse osteotomies were carefully performed under sterile conditions with steady insertion of the pins and proper suturing, mice showed no signs of lameness, infection, or bleeding in the left leg after surgery. Orthopedic healing was assessed using radiographic analysis and histomorphometry after Safranin-O staining (Figure 2). Radiographs of midshaft fractured tibiae indicated more tissue deposition in the fracture calluses of young mice than in the fracture calluses of aged mice. Fracture calluses were decalcified and embedded in paraffin in preparation for histological analysis. Sections were stained with Safranin-O and tissue deposition was quantified using histomorphometry analysis. Fracture calluses of young mice contained more bone and less fibrotic tissue than fracture calluses from aged mice.
Tibial fracture induces systemic and central inflammation6,7,11,15,16. Indeed, peripheral levels of pro-inflammatory cytokines and danger-associated molecular patterns (DAMPs) are rapidly elevated after orthopedic surgery, both in mice and humans7,17,18. This contributes to activation of microglial cells in the brain via several signaling mechanisms involving humoral, cellular, and neuronal pathways7,15,19,20,21. Following surgery, endothelial dysfunction, blood-brain barrier opening, and peripheral macrophage infiltration contribute to acute hippocampal neuroinflammation in wild-types and Ccr2RFP/+ Cx3cr1GFP/+ adult mice15,19, and have been associated with subsequent memory deficits that resemble human delirium and postoperative cognitive dysfunction15,19,22. This neuroinflammatory response is exacerbated in aged animals, with significant changes in microglial morphology, as detected by IBA-1 immunostaining (Figure 3).
Using the tibial fracture model described here, we also found transiently impaired hippocampal neurogenesis, as evidenced by a reduction in doublecortin (DCX) immunostaining in the dentate gyrus20. Electrophysiologic measurements of long-term potentiation (LTP), a surrogate for memory function, revealed a time-dependent disruption in neuroplasticity postoperatively11. Mice after surgery also display impairments in hippocampal-dependent memory function, for example using fear conditioning behavioral assessment (Figure 4). In fear conditioning, mice are placed in a chamber and exposed to an auditory cue followed by an aversive stimulus (i.e., footshock). Three days after tibial surgery, mice are tested in the conditioning chamber, this time without any auditory or aversive stimulation, and freezing behavior is recorded as an index of memory (for a detailed protocol see23).
Advanced age is a critical risk factor for memory decline. Yet, as we age, the capacity for tissue repair and regeneration also diminishes although these mechanisms remain poorly understood. Thus, we used the protocols herein described to perform heterochronic parabiosis (pairing young/old animals) and assessed fracture healing within the aged mouse (for a detailed parabiosis protocol see24). Blood-sharing between parabiotic pairs was confirmed, and found to be equal12. Exposure to a youthful circulation enhanced bone repair with earlier union, increased bone deposition, and decreased fibrosis (Figure 5)12. This rejuvenation of bone regeneration occurred independent of the endogenous osteocalcin-positive osteoblasts (Figure 5, brown cells), but rather relied on CD45-positive cells that migrated from the young parabiont (Figure 5, blue cells). These results indicate that the CD45-positive hematopoietic cells secrete a youthful and healthy niche, which is able to signal to the aged osteoblastic cells inducing them to become more active.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56701/56701fig1.jpg" /> 
Figure 1: Schematic representation of the parabiosis and tibial fracture surgery. (A) Timeline for performing parabiosis and tibial fracture (protocol 1A) or tibial fracture alone (protocol 1B). (B) Tibiae of 20-month-old mice in isochronic or heterochronic parabiotic pairs were fractured (right leg of the right mouse is fractured, as denoted with an X). Grey mice depict wildtype mice while green mice depict eGFP mice. (C) Schematic diagram of tibial fracture model with intramedullary pinning and mid-shaft fracturing. See also Supplementary Video 1 for a 3D reconstruction of the hind limb and pinning of the tibia. <a href="https://cloudflare.jove.com/files/ftp_upload/56701/56701fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56701/56701fig2v2.jpg" /> 
Figure 2: Tibial fracture healing within young and old mouse models. The tibiae of young or old mice were fractured and fracture calluses were investigated 21 days post injury. (A) Radiographic imaging and histologic staining (Safranin-O) were used to evaluate the fracture calluses 21 days post fracture. Staining shows collagenous tissues in blue and proteoglycans (contained within cartilage) in red. Dashed lines indicate approximate location of the fracture callus. (B) Histomorphometry was used to assess the amount of bone, cartilage, and fibrotic tissue deposited within the fracture callus 21 days post fracture. Data are expressed as mean &#177; 95% confidence intervals, *P&#60;0.05, statistically significant (one-way ANOVA, Dunnett&#8217;s test), scale bars represent 2 mm, and images were obtained using a microscope, 1.25x objective. n = 9 for each sample. <a href="https://cloudflare.jove.com/files/ftp_upload/56701/56701fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56701/56701fig3.jpg" /> 
Figure 3: Surgery induced age-dependent microglial activation in the hippocampus. Tibial fracture surgery induces greater hippocampal neuroinflammation in aged mice (20-month-old) compared to 4-month-old C57BL6/J mice. Brain-section staining with microglial marker IBA-1 shows more positive cells and morphologic changes in surgical groups 24 h after surgery. Images were obtained with an epifluorescent microscope with a magnification of 100x.; scale bar represents 10 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56701/56701fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56701/56701fig4.jpg" /> 
Figure 4: Impaired neurogenesis, long-term potentiation, and memory function after tibial fracture surgery. (A) DCX, a quantitative marker for neurogenesis, is significantly reduced in hippocampus dentate gyrus at 24 h after surgery. Images were obtained with a confocal laser-scanning microscope objective with a magnification of 10x; scale bar represents 10 &#181;m. (B) Electrophysiology in hippocampal slices from controls or mice 24 h after surgery. Long-term potentiation (LTP) was induced by high-frequency stimulation (HFS) and recorded over 1 h. Field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 stratum radiatum using an extracellular recording pipette filled with regular artificial cerebrospinal fluid. At 24 h after surgery, LTP induction is remarkably reduced compared to control mice. Data are expressed as mean &#177; s.e.m. n = 3, * p &#60; 0.05 1-way ANOVA. (C) Hippocampal-dependent memory function (defined as % of freezing using trace fear conditioning) is impaired in mice after surgery compared to controls and animals exposed to anesthesia only. Data are expressed as mean &#177; s.e.m. n = 9-10, * p &#60; 0.05 1-way ANOVA. <a href="https://cloudflare.jove.com/files/ftp_upload/56701/56701fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56701/56701fig5.jpg" /> 
Figure 5: Parabiosis surgery leads to rejuvenation of fracture healing, blood-sharing, and donor-cell engraftment. Isochronic and heterochronic parabiosis pairings were established and the aged mouse in each pair was fractured and assessed for bone healing. (A) Fracture calluses were investigated using radiographic imaging. Dashed lines indicate approximate location of the fracture callus. (B) Engraftment of eGFP+ cells was confirmed within the bone marrow. (C) Immunohistochemistry of the fracture callus was used to identify eGFP+ donor cells (blue) and osteocalcin+ osteoblastic cells (brown) from the parabiont. Scale bars represent 50 &#181;m and images were obtained using a 40x objective. <a href="https://cloudflare.jove.com/files/ftp_upload/56701/56701fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: 3D Reconstructionof the hind limb and pinning of the tibia. <a href="http://cloudflare.jove.com/files/ftp_upload/56701/parte_slow.wmv">Please click here to download this file.</a>

Watch this Scientific Journal Video about A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration at JoVE.com

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

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16.2K Views.  Duke University Medical Center. This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Video: A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

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

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; ...

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

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 ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

An Anatomical Study of Nerves at Risk During Minimally Invasive Hallux Valgus Surgery

The growing popularity of minimally invasive surgical (MIS) procedures makes it necessary that new anatomical references arise, to aid in tridimensional orientation and localization of structures that are not directly visible to the surgeon. This is especially critical for structures at risk like nerves or blood vessels. Optimization of the handling of cadaveric material and the combination of multiple techniques compensate for the limited availability of adequate specimens. The described protocol combines anatomical plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to help localize relevant structures, such as nerves, arteries, veins and to correctly position the portals during MIS procedures. Depiction of these structures in anatomy textbooks can differ from what is encountered in the surgical field; and for this reason, new anatomical studies with a surgical orientation are needed. However, this is a complex, time-consuming technique requiring specific training. The anatomical references described with the so-called 'clock method' provide the surgeon with an easy and reproducible system to locate the path of the nerves at risk in Hallux Valgus MIS procedures. This model can be extrapolated to many other minimally invasive surgical procedures.

Minimally invasive surgical (MIS) procedures rely on anatomical references to localize structures not directly visible to the surgeon. This manuscript describes a combined method of plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to locate the structures at risk during MIS procedures.

The growing popularity of minimally invasive surgical (MIS) procedures makes it necessary that new anatomical references arise, to aid in tridimensional ...

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 ...