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.

<table><tbody><tr><td>Isoflurane</td><td>Piramal Healthcare</td><td>NDC 66794-017-25</td><td>Other volatile agents or injectable anesthesia can be also used</td></tr><tr><td>Buprenorphine</td><td>Reckitt-Benckiser Pharmaceuticals</td><td>NDC 12496- 6757-1</td><td>Optional and depending on individual Institutional Animal Care and Use Committee recommendations</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>04-355-451</td><td>70% solution for antiseptic treatment of skin and cleaning</td></tr><tr><td>10% povidone Iodine</td><td>Dynarex</td><td></td><td>For antiseptic treatment of skin</td></tr><tr><td>SomnoSuite</td><td>Kent Scientific</td><td>SS-01</td><td>Low Flow&amp;nbsp; Anesthesia system</td></tr><tr><td>MouseSTAT</td><td>Kent Scientific</td><td>PS1161</td><td>Pulse Oximeter &amp;amp; Heart Rate Monitor</td></tr><tr><td>Shaver</td><td>Wahl</td><td>9854L</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Scalpel Handle</td><td>Fine science tools</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Blades - #11</td><td>Fine science tools</td><td>10011-00</td><td></td></tr><tr><td>Adson Forceps</td><td>Fine science tools</td><td>11006-12</td><td>Needed for stripping the periosteum</td></tr><tr><td>Iris Forceps</td><td>Fine science tools</td><td>11066-07</td><td>Useful (1x2 teeth) to causing localized muscle/soft tissue trauma</td></tr><tr><td>Bonn Scissors (Straight)</td><td>Fine science tools</td><td>14084-08</td><td>Good for osteotomy, note to change regularly as becomes blunt</td></tr><tr><td>Fine Scissors</td><td>Fine science tools</td><td>14058-09</td><td>Sharp scissors for cutting sutures</td></tr><tr><td>22G x 3.5 In Quincke&amp;nbsp; Spinal Needle</td><td>BD</td><td>405181</td><td>Use inner rod for pinning</td></tr><tr><td>Needle Holders</td><td>Fine science tools</td><td>12001-13</td><td></td></tr><tr><td>Suture</td><td>Look</td><td>1079B</td><td></td></tr><tr><td>C57BL6/J</td><td>Jackson Laboratory</td><td>&amp;nbsp;stock no. 000664</td><td></td></tr><tr><td>eGFP+ (expressing enhanced green fluorescent protein ubiquitously)</td><td>Jackson Laboratory</td><td>&amp;nbsp;stock no. 003291</td><td></td></tr></tbody></table>

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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Medicine

16.3K Views.  Duke University Medical Center. Dit protocol beschrijft een muismodel van de orthopedische chirurgie die is gebruikt voor het bestuderen van de mechanismen van postoperatieve neuroinflammation en gedragsveranderingen, en wanneer deze wordt gecombineerd met parabiosis, om te studeren weefselregeneratie tijdens veroudering.

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

Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.

Mouse models have become increasingly popular in studies of behavioral neuroscience. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. This protocol describes the Pasta Matrix Reaching Task, which is a skilled motor task for use in mouse models of stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models ...

Behavior

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

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Developmental Biology

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

A Mouse Model of Direct Anastomosis via the Prespinal Route for Crossing Nerve Transfer Surgery

Crossing nerve transfer surgery has been a powerful approach for repairing injured upper extremities in patients with brachial plexus avulsion injuries. Recently, this surgery was creatively applied in the clinical treatment of brain injury and achieved substantial rehabilitation of the paralyzed arm. This functional recovery after the surgery suggests that peripheral sensorimotor intervention induces profound neuroplasticity to compensate for the loss of function after brain damage; however, the underlying neural mechanism is poorly understood. Therefore, an emergent clinical animal model is required. Here, we simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice. Neuroanatomical, electrophysiological, and behavioral experiments helped identify that the transferred nerves of these mice successfully reinnervated the impaired forelimb and contributed to accelerating motor recovery after brain injury. Therefore, the mouse model revealed the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

We simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice, contributing to the study of the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

Crossing nerve transfer surgery has been a powerful approach for repairing injured upper extremities in patients with brachial plexus avulsion injuries. ...

Neuroscience

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

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The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

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A Spinal Cord Injury Contusion Device for Accurate Mouse Models

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of contusion models of spinal cord injury by impacting the spinal cord results in similar pathologies to most spinal cord injuries in clinical practice. Accurate, reproducible, and convenient animal models of spinal cord injury are essential for studying spinal cord injury. We present a novel automated spinal cord injury contusion device for mice, the Guangzhou Jinan University smart spinal cord injury system, that can produce spinal cord injury contusion models with accuracy, reproducibility, and convenience. The system accurately produces models of varying degrees of spinal cord injury via laser distance sensors combined with an automated mobile platform and advanced software. We used this system to create three levels of spinal cord injury mice models, determined their Basso mouse scale (BMS) scores, and performed behavioral as well as staining assays to demonstrate its accuracy and reproducibility. We show each step of the development of the injury models using this device, forming a standardized procedure. This method produces reproducible spinal cord injury contusion mice models and reduces human manipulation factors via convenient handling procedures. The developed animal model is reliable for studying spinal cord injury mechanisms and associated treatment approaches.

Author Spotlight: Insight Into Innovations in Spinal Cord Injury Research

Presented here is a novel automated spinal cord injury contusion device for mice, which can accurately produce spinal cord injury contusion models with varying degrees.

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rmTBI Induction in Mice via a Closed-Head Injury Method

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...

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