This research is an independent study and did not receive any funding.

The protocol adheres to the ethical guidelines set by the Institutional Animal Care and Use Committee (IACUC) at Beijing University of Chinese Medicine, Protocol number BUCM-4-2022062001-2109. The protocol uses Sprague Dawley (SD) rats (SCXK(Jing)2019-0008), aged 8-10 weeks and weighing 200 g-250 g.
1. Adaptation training
<ol>
	<li>Albino rats generally have a visual range of less than 60 cm15. During the modeling process, place the rats in opaque cages and keep them as far away as possible from non-modeled rats (at least, other rats should be outside the maximum visual range of the modeled rats) to prevent them from experiencing traumatic stress.</li>
	<li>Turn on the power and set the treadmill to 10 m/min with a 0&#176; incline. Place the rats on the treadmill and ensure the movements are as gentle as possible to avoid stress. Do not use additional stimulation; the rats will naturally crawl forward because they are unfamiliar with the environment.</li>
	<li>The training lasts for 15 min. After the training, clean up the feces and urine left by the rats and spray alcohol to eliminate the animal odor. Then, prepare the next rat for training. 
	NOTE: If a rat refuses to walk on the treadmill for 5 minutes, exclude it&#160;from the study.</li>
	<li>Continue adaptive training until the end of the 1st week.</li>
</ol>
2. Measuring the maximum weight-bearing capacity in rats
<ol>
	<li>Prepare two test tubes, a hook, and a loop fastener approximately 80 cm long. Place steel balls into the test tubes, ensuring that the initial combined weight of the test tubes, the hook, and the loop fastener is 150 g. Prepare test tubes with different total weights in 10 g increments, with the maximum weight being 350 g.</li>
	<li>A researcher uses both hands to fasten rats using gloves (Figure 1). Another researcher places the test tube on the back of the rat about 1 cm from the midline of the back. Since this step does not require movement of the rat, perform fixation by simply wrapping the hook and loop fastener around the rat without using additional measures to secure the tube.</li>
	<li>Change the tube until the rat&#39;s maximum weight is reached. When the rat reaches maximum weight-bearing, it cannot stand when prodded or the experimenter leaves. Reduce the weight by 10 g to allow the rat to stand. This weight represents the rat&#39;s maximum weight-bearing capacity.</li>
</ol>
3. Preparation of weight load
<ol>
	<li>Since secure fixation is required during training, secure the objects using 1-1.5 m elastic therapeutic tape based on rat size, which has strong adhesion. Use clay to adjust the weight of the test tubes to reduce the swinging of the load.</li>
	<li>After weighing the weight of the test tubes and the fixation objects, add an appropriate amount of clay into the test tubes to adjust them to the target weight. Use a glass stirring rod to evenly spread the clay inside the test tubes. Clay concentrated at one end of the test tube may cause it to detach easily during training.</li>
	<li>Adjust the total weight of the adjusted test tubes and clay at 50% of the rat&#39;s maximum weight-bearing capacity (Figure 1A). Exceeding this weight will make it difficult for the rat to complete the training. Do not adjust the weight of the load again until the end of the experiment.</li>
</ol>
4. Establishing steroid-induced osteonecrosis of the femoral head model
<ol>
	<li>Use the methylprednisolone combined with lipopolysaccharide (LPS+MPS) method for SONFH model establishment16,17. Perform intraperitoneal injection of LPS (20 &#181;g/kg) using a standard needle at the midline of the rat&#39;s abdomen once every 24 h, for a total of 2 injections.</li>
	<li>After the last LPS injection, inject MPS (40 mg/kg) into the unilateral gluteal muscle 24 h after regular feeding and continue injections every 24 h, alternating between the two gluteal areas, for a total of three injections.</li>
</ol>
5. Tension-free weight-bearing immobilization using elastic therapeutic tape and treadmill training
<ol>
	<li>Mark the rat&#39;s back midline at 1-2 cm on both sides; this is the reference point for the weight load. Ensure the marks are appropriately positioned to prevent the rat from tipping or hindering movement during treadmill training.</li>
	<li>With one investigator fixing the rat using gloves, place one hand under the rat&#39;s armpit to secure the forelimb and jaw while the other hand secures the hind limbs and tail. Avoid excessive force to prevent stress or suffocation (Figure 1B).</li>
	<li>Adhere the elastic therapeutic tape to the test tube without applying tension to secure the load on the rat&#39;s back. In the process of winding, do not stretch the elastic therapeutic tape to achieve tension-free fixation. 
	NOTE: The elastic therapeutic tape used in this model is designed for taping during intense physical activities, providing high adhesion and elasticity18. Due to its high elasticity, it greatly reduces the impact on the rat&#39;s movement while crawling.</li>
	<li>Ask the second investigator to fix the test tube to the rat&#39;s back parallel to the rat&#39;s spine, with the fixation position based on the markings made, i.e., 1-2 cm away from the midline on both sides of the rat&#39;s back.</li>
	<li>To minimize the suffering of the rat, reduce the impact on the rat&#39;s movement, and decrease mortality caused by fixation, perform fixation without applying tension. Stick the test tube with adjusted weight onto the elastic therapeutic tape, then wrap the test tube around the rat&#39;s body using a tension-free technique.</li>
	<li>Ensure tension-free immobilization and maintain the original length of the elastic therapeutic tape without stretching in order to prevent any adverse effects on rat breathing and movement (Figure 1C).</li>
	<li>After wrapping the test tube around the rat&#39;s body, place the rat in an open space or a single cage, monitoring its breathing and direction. If signs of rapid breathing or mouth opening appear, promptly release the rat to prevent suffocation.
	<ol>
		<li>Ideal immobilization allows the rat to move freely and perform activities like drinking, lifting forelimbs, and feeding (Figure 1D). If immobilization is unsatisfactory, release temporarily and re-immobilize after 20 min to minimize stress on the rat due to repeated immobilization stimuli.</li>
	</ol>
	</li>
	<li>Turn on the power and set the treadmill to 1 m/min with a 0&#176; incline. The treadmill training time is 30 min. Gently transfer the rat onto the treadmill and begin the timer.</li>
	<li>Do not provide additional stimulation to the rat. If the rat cannot complete the 30 min training, place it in a cage for a 10 min rest without removing the fixation. 
	NOTE: The elastic therapeutic tape used in this study has excellent stretchability and adhesiveness. When properly secured without tension, long-term fixation will not cause the test tube to detach or result in suffocation and death of the rat.</li>
	<li>After the training, clean up the feces and urine left by the rats and spray alcohol to eliminate the animal odor.</li>
	<li>Avoid the above steps being seen by other rats and handle the animals as gently as possible to prevent animals from vocalizing, thus causing traumatic stress to other rats.</li>
	<li>Immediately release the rat from fixation after completing training (Figure 1E). During release, use fingers to press down on the rat&#39;s fur to protect it, minimizing fur loss during release (Figure 1F).</li>
</ol>
6. Animal grouping
<ol>
	<li>To study the impact of weight-bearing training on SONFH, randomly divide 60 rats into four groups.</li>
	<li>In the Control group, do not establish the common steroid-induced osteonecrosis of the femoral head (SONFH) model or perform weight-bearing training. In the Control+Load group, carry out weight-bearing training only, without establishing the steroid-induced osteonecrosis of the femoral head model. In the Model group, establish the steroid-induced osteonecrosis of the femoral head model only. In the Model+Load group, perform weight-bearing training after establishing the steroid-induced osteonecrosis of the femoral head model.</li>
</ol>
7. Euthanasia and specimen collection
<ol>
	<li>Place the rats into a small animal euthanasia chamber and inject enough carbon dioxide to achieve a concentration higher than 5%. Once the rats lose consciousness, administer a dose of 0.3 mL/kg of body weight of a 20% potassium chloride solution, totaling 20 mL, via an intracardiac injection.</li>
	<li>Monitor the rats&#39; breathing and heartbeat to ensure they experience no pain or discomfort during euthanasia. Confirm death by checking for the absence of breathing and heartbeat. Spray alcohol to clean away any odor.</li>
	<li>Post-euthanasia, dissect the rat&#39;s hip region and extract the femoral head while keeping the femur intact. Perform a microCT scan following pretreatment, followed by Hematoxylin-Eosin (HE) staining.</li>
</ol>
8. Hematoxylin-Eosin staining
<ol>
	<li>After the modeling is completed, euthanize the rats. Remove the femurs for HE staining.</li>
	<li>Soak the rat femurs in 4% paraformaldehyde for 24 h to preserve biological tissues as specimens. Soak the rat femur specimens in 5% formic acid for decalcification for 5 days.</li>
	<li>Section the specimens into 5 &#181;m slices for HE staining. 
	NOTE: Empty lacunae refer to the situation in bone tissue19 where the lacunae, which should normally contain osteocytes, are devoid of these cells. It is an important indicator for assessing the health of bone tissue. In the pathological process of bone necrosis, insufficient blood supply may cause osteocyte death, leading to empty lacunae. In the diagnosis and evaluation of bone necrosis, the number and distribution of empty lacunae are important parameters for measuring the severity of the disease20,21.
	<ol>
		<li>Place rat femoral specimens into melted paraffin (56-60 &#176;C), first soaking in low-melting-point paraffin, if necessary, then gradually transfer to higher melting point paraffin, each time for about 1 h, to ensure thorough infiltration of the tissue with paraffin.</li>
		<li>Prepare embedding molds and pour melted paraffin into the molds to the appropriate depth. Retrieve the tissue samples from the paraffin using forceps after soaking, remove excess paraffin, and place them in molds. Cool to room temperature to solidify the paraffin, forming paraffin blocks.</li>
		<li>Using a paraffin microtome, cut the embedded paraffin blocks into 5 &#181;m thick sections.</li>
	</ol>
	</li>
	<li>Bake the rat femoral specimens with attached slices in a slide warmer for 1 h to enhance the adhesion between the slices and coverslip and reduce floating during subsequent staining processes.</li>
	<li>Immerse the baked slices sequentially in pure xylene to remove paraffin, followed by a series of dehydration through absolute ethanol, gradient ethanol (100%, 95%, 80%, 70%), and water, for 2 min each, completing the deparaffinization and rehydration process.</li>
	<li>Immerse the deparaffinized slices in a hematoxylin staining solution and stain for 10 min. Rinse with running water to remove unbound hematoxylin.</li>
	<li>Immerse the hematoxylin-stained sections in eosin staining solution and stain for 2 min to color the cytoplasm and connective tissue. Rinsed with a 1% acetic acid solution to fix the staining effect.</li>
	<li>Immerse the rat femoral bone specimen slices sequentially in 70%, 80%, 90%, 95%, and 100% ethanol, with each gradient for 2 min, gradually removing moisture.</li>
	<li>Immerse the rat femoral bone specimen slices in pure xylene for transparency, followed by rinsing with running water to remove the xylene. Apply neutral mounting resin, covering the slides, gently tapping to remove air bubbles, and allowing the mounting medium to solidify to complete the mounting process.</li>
	<li>Using the randomizr package in R, select 30 HE-stainedslides for each group and allocate 15 slides each to two researchers. Without informing them of the groupings, ask the researchers to calculate the empty lacunae rate of the slidesand collect the results for statistical analysis.</li>
</ol>
9. MicroCT analysis
<ol>
	<li>After the modeling is completed, euthanize the rats. Remove the femurs for Hematoxylin-Eosin staining.</li>
	<li>Soak the rat femurs in 4% paraformaldehyde for 24 h to preserve biological tissues as specimens. Place the rat femurs in the small animal-specific microCT device.</li>
	<li>Set the microCT scanning conditions as follows: X-ray voltage: 80 kV, Current: 100 &#181;A, Single exposure time: 50 ms, scanning resolution: 25 &#181;m. Perform scans at intervals of 0.5&#176;, focusing on the trabecular bone from beneath the cartilage to the upper part of the epiphysis as the region of interest.</li>
	<li>Define the area above the rat femoral neck as the region of interest.</li>
	<li>Perform coronal plane reconstruction of the scanning results using microCT device and collect bone morphometric measurements generated by the built-in software of the microCT device. Record the following observed indices: Total Volume (TV), Percent Bone Volume (BV/TV), Bone Surface/Volume Ratio (BS/BV), Structure Thickness (Tb.Th), Structure Separation (Tb.Sp), Number of Bone (Tb.N), Bone Density (BD).</li>
</ol>
10. Statistical analysis
<ol>
	<li>Present the data as the mean &#177; standard deviation (SD). Conduct statistical analyses for micro-CT and quantitative histologic evaluations using the independent sample t-test. Consider a p-value &#60; 0.05 as statistically significant.</li>
</ol>

Elastic Tape Rat Model of Steroid-Induced Femoral Head Osteonecrosis Enhancement

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+field+study+of+histologic+and+bacteriologic+characterization+of+femoral+head+separation+and+femoral+head+necrosis.">A field study of histologic and bacteriologic characterization of femoral head separation and femoral head necrosis.</a> Avian Dis. 64 (4), 571-581 (2020).</li><li>Fan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparisons+of+emu+necrotic+femoral+head+micro+structure+repaired+in+two+different+methods.">Comparisons of emu necrotic femoral head micro structure repaired in two different methods.</a> Acta Acad Med Sinicae. 38 (1), 16-21 (2016).</li><li>Naito, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+head+necrosis+and+osteopenia+in+stroke-prone+spontaneously+hypertensive+rats+(SHRSPs).">Femoral head necrosis and osteopenia in stroke-prone spontaneously hypertensive rats (SHRSPs).</a> Bone. 14 (5), 745-753 (1993).</li><li>Omokawa, S., Tamai, S., Ohneda, Y., Mizumoto, S., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+histopathological+study+on+the+femoral+head+necrosis+in+spontaneously+hypertensive+rats+(SHR).">A histopathological study on the femoral head necrosis in spontaneously hypertensive rats (SHR).</a> Nihon Seikeigeka Gakkai Zasshi. 66 (1), 69-82 (1992).</li></ol>

The author declares no conflicts of interest, affiliations, or collaborations that could potentially influence the objectivity or outcomes of this research.

Currently, various animals, such as rabbits25, rats26, mice27, pigs28, broilers29, ostriches8, and emus30 can be used to establish models of femoral head necrosis. Among them, rats, mice, and rabbits are the most commonly used species. The rat, as a model for femoral head necrosis, offers numerous advantages. Rats are easy to feed and breed, grow rapidly, ...

The administration of glucocorticoids is the most common risk factor for non-traumatic osteonecrosis of the femoral head (ONFH)1. Numerous pieces of evidence suggest that, in addition to glucocorticoids, the pressure load on the hip joint in patients is also associated with the occurrence of ONFH. Factors such as body weight and physical labor intensity are recognized as risk factors for ONFH2. Multiple clinical studies have demonstrated a close relationship between hip joint loading conditions and the timing and incidence of joint replacement3,4,5,6. Therefore, establishing a model that reflects the relationship between load bearing and steroid-induced osteonecrosis of the femoral head (SONFH) is important for a comprehensive investigation of this condition.
Large bipedal birds, such as ostriches and emus, serve as good models to simulate hip joint stress, resembling human leg loads7,8. However, maintaining large bird species is challenging, and the associated research costs are high. Spontaneously hypertensive rat models9,10 can exhibit higher rates of ONFH, but the compartment pressure load in the marrow generated by spontaneous hypertension differs significantly from mechanical pressure and is unsuitable for studying the impact of mechanical pressure on SONFH.
Small animal models are commonly used in research on SONFH. However, quadrupedal reptiles have lower hip joint stress, and their hip models cannot simulate the biomechanical environment of human hip joints during bipedal walking. Single limb immobilization models11 and partial unloading models12 are common, but both reduce limb loading. As humans are bipedal organisms with substantial lower limb loads while standing and walking, reducing the load in these models diminishes the connection between animal models and human diseases.
This study aims to establish a simple and cost-effective model for weight-bearing training to investigate the impact of weight-bearing training on steroid-induced osteonecrosis of the femoral head in rats. Currently, rat models have been used to study steroid-induced osteonecrosis of the femoral head13,14, but there is still no model that can provide long-term, safe fixation with minimal disruption to movement, which is also relatively simple and inexpensive. In this study, a high-adhesive fixation material is applied, adopting tension-free fixation, which maintains the rats&#39; mobility and reduces the suffering and even death caused by improper fixation.

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			<td>Servicebio,China</td>
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tension-free weight-bearing model of steroid-induced osteonecrosis of femoral head in rats

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure when walking and standing. In rat steroid-induced femoral head necrosis models, the biomechanical characteristics of the human hip under higher pressure often need to be simulated. Some scholars try to emulate the state of human hip pressure by making rats bear a certain weight, but fixing the weight-bearing object on the rat is tough. Rats can easily break free of immobilization, and sticking the weight on the rats with adhesive tape will cause the rats to suffocate or die from intestinal obstruction. Our research group used elastic therapeutic tape to perform tension-free immobilization of weight-bearing objects in rats so that the rats could breathe freely and not break away from the immobilization under weight-bearing conditions. Compared to the usual steroid-induced femoral head necrosis rat model, we found that this weight-bearing intervention can aggravate the progression of femoral head necrosis in rats.

Histopathology analysis 
Hematoxylin and eosin staining revealed that in the Control group and Control+Load group, bone trabeculae were intact and arranged regularly. Blood vessel endothelial cells were present in bone dimples, and the cell morphology appeared plump. In contrast, the Model group and Model+Load group exhibited fractured and disordered bone trabeculae, along with a significantly higher number of empty lacunae. The Model+Load group had more empty lacunae compared to the Model group. In...

Author Spotlight: Developing a Rat Model for Weight-Bearing Intervention to Investigate Osteonecrosis of the Femoral Head

The protocol shows building a simple and cost-effective rat weight-bearing training model for steroid-induced osteonecrosis of the femoral head using elastic therapeutic tape.

Tension-Free Weight-Bearing Model of Steroid-Induced Osteonecrosis of Femoral Head in Rats

Unlike humans, rats are animals that walk on all fours, while humans are bipedal animals that stand, and the hips are subjected to tremendous pressure ...

tension-free-weight-bearing-model-steroid-induced-osteonecrosis

Research

JoVE Journal

Medicine

414 Views.  Beijing University of Chinese Medicine. The protocol shows building a simple and cost-effective rat weight-bearing training model for steroid-induced osteonecrosis of the femoral head using elastic therapeutic tape.

Video: Author Spotlight: Developing a Rat Model for Weight-Bearing Intervention to Investigate Osteonecrosis of the Femoral Head

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

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 ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...