This work was supported by grants from National Natural Science Foundation of China (No. 81772368), Natural Science Foundation of Guangdong Province (No. 2017A030313496), and Guangdong Provincial Science and Technology Plan Project (No. 2016A020215225; No. 2017B090912007). The authors thank Dr. Fei Zhang, M.D. from the Department of Orthopaedic Surgery, The Eighth Affiliated Hospital of Sun Yat-sen University for his technical assistance during modification.

All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by The Third Affiliated Hospital of Sun Yat-sen University institutional animal care and use committee (permission number: 02-165-01). All the animal experiments were performed according to the ARRIVE guidelines.
1. Preoperative preparation
NOTE: Figure 1 shows the design of the surgical procedure.
<ol>
	<li>Rigidly immobilize the knee joint with a plastic plate and two metal screws at approximately 135&#176; flexion. 
	NOTE: Perform the surgery at the proximal femur and the distal tibia without violating the joint component.</li>
	<li>Prepare materials and instruments for internal fixation.
	<ol>
		<li>Construct a medical grade polypropylene plastic plates by cutting a 5 mL syringe (Figure 2a) using a surgical scissor to fit the following dimensions: length, 25 mm; width, 10 mm; thickness, 1 mm (Figure 2b). Smooth the perimeter of the plate with a scalpel vertically. Rinse the plate with sterile saline to wash off the debris by three times.
		<ol>
			<li>Sterilize with 75% ethanol for 4 h followed by irradiating with ultraviolet light for 3 h.</li>
		</ol>
		</li>
		<li>Pre-drilling holes in the plastic plate: Prepare a hand-held low-speed electric drill with a speed of about 0-4000 rpm (Figure 2c). Drill two holes at both ends of the plate, diameters are 1 mm and 0.9 mm, respectively (Figure 2d). Match both ends of the plate with M 1.4 mm x 8 mm and M 1.2 mm x 6 mm steel screws, respectively (Figure 2e).
		<ol>
			<li>Wipe with 75% ethanol and sterilize with UV light for 3 h before use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare surgical instruments: 1 straight Mosquito-Type&#8194;hemostatic clamp, 1 smooth curved forceps, 2 eyelid retractors, 1 needle-holder, 1 tissue&#8194;forceps, 1 suture&#8194;scissor, 1 micro tissue&#8194;scissor and 1 scalpel (Figure 2f). Sterilize the surgical instruments by autoclaving at 121.3 &#176;C for 20 min and drying.</li>
	<li>Experimental animals
	<ol>
		<li>Use Specific Pathogen Free (SPF) grade skeletally mature male Sprague-Dawley (or Wistar) rats, weighing between 250 - 350 g in the experiment. 
		NOTE: Choose either female or male rats for the experiment.</li>
		<li>Place the rats in cages and keep in a 12 h light/12 h dark cycle-controlled laboratory room. Provide adequate food and water.</li>
	</ol>
	</li>
</ol>
2. Surgery
<ol>
	<li>Adjust the temperature. Place a warming pad on a surgical platform in a thermostatic operating room.</li>
	<li>Anesthesia and skin preparation
	<ol>
		<li>Weigh the rat with an electronic scale and record.</li>
		<li>Restrain the rat and perform an intraperitoneal injection of sodium pentobarbital (30 mg/kg) to induced anesthesia. Assessing that the animal is sufficiently anesthetized using the toe pinch22. Administer the eyes with lubricant to protect the cornea from drying during surgery.</li>
		<li>Shave the lower body of the rat including the two hind limbs with an electric clipper and disinfect with a tincture of povidone iodine twice and 75% ethanol three times.</li>
		<li>Place the rat laterally, and cover with the surgical drape exposing one side hind leg and hip.</li>
		<li>Disinfect the surgical area again with povidone iodine.</li>
	</ol>
	</li>
	<li>Immobilize the knee joint with internal fixation using a mini-invasive technique. 
	NOTE: Keep the incision properly moist with sterile saline during the operation. The surgery usually requires two surgeons.
	<ol>
		<li>Mark the direction of skin incision. At the distal end of the femur greater trochanter, draw a line along the body surface projection of the muscle gap between the vastus lateralis and biceps femoris (Figure 3a). Incise the epidermis skin along the drawing line approximate 1.5 cm (Figure 3b).</li>
		<li>Bluntly dissect the muscle gap between vastus lateralis and biceps femoris with a tissue forceps until the femoral shaft is exposed approximately 1 cm in length (Figure 3c). Use the retractor to facilitate continuous separation of the muscle gap.</li>
		<li>Incise the epidermis skin approximate 1 cm along the body surface projection of the muscle gap between the tibialis anterior and fibularis longus on the distal lower extremity (Figure 3d). Bluntly dissect the muscle gap until the tibia is exposed approximately 1 cm in length (Figure 3e).</li>
		<li>Separate the soft tissues by the retractor and the smooth forceps, keep perpendicular and drill one 1.0 mm diameter hole into the femoral shaft at a speed of 1,500 rpm using an electric drill (Figure 3f). The proper drilling position is approximate 8 mm below the lower edge of the greater trochanter. Quickly press the wound to stop bleeding. 
		NOTE: Proper drilling diameter can avoid intraoperative fractures.</li>
		<li>Drill one 0.9 mm diameter hole into the tibia approximate 4 mm below the edge of the tibiofibular fusion (Figure 4a). Perform the drilling carefully to prevent the crushing of muscles or tendons.</li>
		<li>Use the straight Mosquito-Type&#8194;hemostatic clamp to form a submuscular course from the tibia hole to femur hole. The submuscular tunnel passes below the gastrocnemius in the tibia end and above the gluteus medius, below the biceps femoris in the femur end.</li>
		<li>Use one M 1.4 mm x 8 mm steel screw to secure one end of the plastic plate (with the 1.0 mm diameter hole) in the proximal femur (Figure 4b). Use one M 1.2 mm x 6 mm steel screw to secure another end of the plastic plate (with the 0.9 mm diameter hole) in the distal tibia (Figure 4c). Ensure the knee joint without varus deformity.</li>
	</ol>
	</li>
	<li>Close the wound: Suture the myofascia, deep fasciae, and subcutaneous tissue using 4-0 absorbable sutures (Figure 4d). Close the skin with Polyamide&#160;sutures (Figure 4f).</li>
</ol>
3. Postoperative management
<ol>
	<li>Apply postoperative analgesia through subcutaneous injection of Buprenorphine (0.03 mg/mL) at 0.05 mg/kg . Add 5 mg/mL Neomycin into drinking water for 5 days after the surgery.</li>
	<li>Inject the analgesia mixture (Buprenorphine and Carprofen) respectively at 0.05mg/kg and 5 mg/kg subcutaneously twice a day for at least 72 hours post-operation.</li>
	<li>Check whether the hind limb had over-edema in case of vascular injury. Made sure that the rats can walk normally in the case of nerve injury during surgery.</li>
</ol>
4. Postoperative examination
<ol>
	<li>Observe the healing of the surgical incision and physically examine the knee joint to evaluate early signs of infection every other day postoperatively. Check the degree of swelling of the ankle and metacarpophalangeal joint in case of continuous edema. 
	NOTE: Early postoperative infection can cause wound exudate, leg swelling, and delayed wound healing.</li>
	<li>Perform X-ray imaging of the hindlimb to ensure that correctly placed the screws on the first postoperative day. 
	NOTE: A Micro-CT scan analysis is another alternative option to display the proper location and the direction of the steel screws.</li>
	<li>Measure the passive range of motion (ROM) to evaluate the development of contracture. Take a knee joint ROM measurement at different time cohorts postoperatively as described previously20.
	<ol>
		<li>In brief, euthanize the rats and skin the hindlimbs. Remove the immobilizer and measure the knee joint angle using a mechanical arthrometer at two torques (667 or 1,060 g/cm)23.</li>
		<li>Calculate the ROM as a result of the total contracture, the myogenic contracture, and the arthrogenic contracture separately based on the investigation objectives24. 
		NOTE: Set different time cohorts (i.e., 1, 2, 4, 8, 16, and 32 weeks) according to the research objectives. The contralateral knee joint (non-operative or sham-operated) can serve as a control2.</li>
	</ol>
	</li>
	<li>Histological analysis of the posterior knee joint capsules.
	<ol>
		<li>Prepare the joint tissues. Dissect the knee joint tissue and fix it with 4% paraformaldehyde. Decalcify and embed it in paraffin as previously reported25. Cut the sections (5 &#956;m) at the medial midcondylar level in the sagittal plane. 
		NOTE: Choose to perform different evaluating staining including HE, aldehyde-fuchsin-Masson Goldner (AFMG), Elastica&#8211;Masson, or Immunohistochemistry staining for histological study in the joint capsule based on your study objectives15,26.</li>
		<li>Observe histomorphometric changes in the posterior knee joint capsules. Photograph the posterior region of the knee joint. Observe fibrous deposition and adhesion changes between the diaphysis-synovium junction and the meniscus6. 
		NOTE: Pathological changes of joint capsule are considered to be a pathogenic factor for knee joint contracture. Measure the length, the thickness, and the capsular areas of the posterior capsule as previously described according to the research content27.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This study aimed to elucidate a step-by-step knee joint immobilization method using a mini-invasive technique that permits rapid postoperative rehabilitation in animals after surgery. Conventionally, the muscle-gap separation approach is thought to be a minimally invasive technique in orthopedic surgery. As expected, we found that rats can return to a normal diet and activities just one day postoperatively, which was consistent with the previous study. Moreover, no artery or nerve injury occurred after the surgery, evide...

Joint contractures are defined as a restriction in the passive range of motion (ROM) of a diarthrodial joint1,2. The current therapies aiming to prevent and treat joint contracture have achieved some success3,4. However, the underlying molecular mechanism of acquired joint contracture remains largely unknown5. The etiology of joint contractures in different social communities is highly diverse and includes genetic factors, posttraumatic states, chronic diseases, and prolonged immobility6. It is widely accepted that immobility is a critical issue in the development of acquired joint contracture7. People who suffer from major joint contracture may ultimately result in physical disability8. Thus, a stable and reproducible animal model is necessary for investigating the potential pathophysiological mechanisms of acquired joint contracture.
The currently built immobilization-induced knee joint contracture models are mostly achieved by utilizing non-invasive plaster casts, external fixations, and internal fixations. Watanabe et al. reported the possibility of the use of plaster cast immobilization on rat knee joints9. By wearing a special jacket, one side of the lower limb joint of the rat is immobilized by a cast. The rat knee joint can remain fully flexed without any surgical trauma10,11. However, both the hip and ankle joint movements are also affected by this form of immobilization, which may increase the degree of muscle atrophy in quadriceps femoris or gastrocnemius12. In addition, edema and congestion of the hind limbs must be avoided by replacing the cast at set time points, which may affect the continuity of immobility. Another accepted method for the establishment of a knee joint contracture model is using external surgical fixation. Nagai et al. combined Kirschner wire and steel wire into an external fixator, which immobilized the knee joint to approximately 140&#176; of flexion13. In this method, a resin is used to cover the surface to prevent skin scratches. Although external fixation immobilization is robust and reliable14,15, percutaneous Kirschner wire pin tracks may increase the risk of infection16. In our own experience, using the external fixation technique may reduce the daily activity of rats due to an increase in the conditioned lick behavior.
Alternatively, Trudel et al. described a well-accepted model of joint contracture in the rat knee joint based on a surgical internal fixation17 (this method was modified from the one used by Evans and colleagues18). Notably, this method highlights the importance of utilizing a mini-incision technique to minimize the surgical wounds. The efficient development of joint contracture has been proved in this model19. However, the protocol on how to perform a minimal dissection to expose the bone surface is still unclear20. Also, the precise position where the screw is drilling is not fully understood. The implantation of the internal fixation through a subcutaneous or submuscular way is still controversial21. To solve these problems, we have modified this method by including an appropriate muscle-gap separation modus, which allows a mini-invasive exposure of the bone surface and the placement of the implantation through a submuscular channel. This protocol led to rapid postoperative rehabilitation in rats after surgery. Animals developed a limited joint range of motion after joint immobilization, which was consistent with morphological changes of capsular adhesion obtained from the histological analysis. We also describe an exact possible location of the drilled screws as confirmed by X-ray analysis or micro-CT analysis. Thus, this study aimed to describe in detail a minimal-invasive technique in a knee joint contracture model that was established by a muscle-gap separation modus combined with a mini-incision method. We believe that minimally invasive techniques can both reduce animal trauma and effectively mimic the pathological process of joint flexion contracture.

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			<td height="104" style="height:104px;width:223px;">Buprenorphine&#160;</td>
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			<td style="width:73px;">MCE</td>
			<td style="width:123px;">HY-B1227</td>
			<td style="width:172px;">analgesia&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cross screwdriver</td>
			<td style="width:73px;">STANLEY</td>
			<td style="width:123px;">PH0*125mm</td>
			<td style="width:172px;">tighten the screws</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:223px;">Electric drill</td>
			<td style="width:73px;">WEGO</td>
			<td style="width:123px;">185</td>
			<td style="width:172px;">drill hole(with stainless steel drill 0.9mm;1.0mm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Microsurgical instruments</td>
			<td style="width:73px;">RWD</td>
			<td style="width:123px;">/</td>
			<td style="width:172px;">Orthopaedic surgical instruments for animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Neomycin</td>
			<td style="width:73px;">Sigma</td>
			<td style="width:123px;">N6386</td>
			<td style="width:172px;">antibacterial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Sodium pentobarbital</td>
			<td style="width:73px;">Sigma</td>
			<td style="width:123px;">P3761</td>
			<td style="width:172px;">&#160;anaesthetize</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Stainless Steel screws</td>
			<td style="width:73px;">WEGO</td>
			<td style="width:123px;">m1.4*8; m1.2*6</td>
			<td style="width:172px;">screw(part of internal fixation)&#160;</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:223px;">Syringe&#160;</td>
			<td style="width:73px;">WEGO</td>
			<td style="width:123px;">3151474</td>
			<td style="width:172px;">use for plastic plate(part of internal fixation)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">&#956;-CT&#160;</td>
			<td style="width:73px;">ALOKA</td>
			<td style="width:123px;">Latheta LCT-200</td>
			<td style="width:172px;">in vivo CT scan</td>
		</tr>
	</tbody>
</table>

a mini-invasive internal fixation technique for studying immobilization-induced knee flexion contracture in rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

We observed that rats received minimally invasive surgery can return to the regular diet just one day postoperatively. In particular, the surgical incision has scarred without exudate (Figure 5a). The swelling of the ankle and metacarpophalangeal joints in the operative hindlimb has almost wholly disappeared two days postoperatively (Figure 5b) when compared with the contralateral side (Figure 5c). N...

Watch this Scientific Journal Video about A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats at JoVE.com

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

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Research

JoVE Journal

Developmental Biology

7.9K Views.  The Third Affiliated Hospital of Sun Yat-sen University. Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Video: A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...