Name: evaluation of stem cell therapies in a bilateral patellar tendon injury model in rats
Uploaded: 2018-03-30T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats at JoVE.com

The authors would like to acknowledge Dr. Su, PhD, for her statistical analysis of the data. The authors also thank Dr. McClure, DVM, PhD DACLAM, for her advice on the anesthesia and pain management protocols used in the study. This project was supported by grants from Western University of Health Sciences Office of the Vice President for Research (12678v) and USDA Section 1433 funds (2090).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Western University of Health Sciences.
1. Isolation and Expansion of MSCs from Equine Umbilical Cord Matrix
<ol>
	<li>Obtain the placenta from an adult mare (pregnant) after observed foaling and aseptically isolate the umbilical cord from the placenta. Keep the umbilical cord in phosphate buffered saline (PBS) with 1% penicillin-streptomycin (P/S) at 4 &#176;C during transfer until pro...

Equine cells were selected for this project because we eventually intend to test candidate approaches in the management of natural tendinopathies in horses. Indeed, tendon injuries in horses are appealing as natural models of tendinopathy in man because of the biological similarity between the equine superficial digital flexor and Achilles tendon in humans41. The cell surface markers CD44, CD90, CD105, CD34, and MHC II were selected for immunophenotyping of cells, in accordance with the criteria r...

Tendon injury is one of the most common causes of significant pain and muscle atrophy across species1. In veterinary medicine, tendon and ligament injuries are of special interest in horses, as 82% of all injuries in race horses involve the musculoskeletal system, and 46% of those affect tendons and ligaments2,3. Scar tissue formation affects the biomechanical properties of healed tendons and explains the guarded prognosis for return to athletic use after flexor tendon injuries; re-injury occurs within 2 years in up to 67% of horses treated conservatively4. Regenerative medicine provides novel alternatives to a condition that challenges traditional treatments. Autologous stem cell therapy has produced some encouraging results5,6 but is limited by the morbidity associated with tissue collection, delayed administration due to processing/reprogramming of cells, and the influence of the patient&#39;s health status (such as age) on the properties of stem cells7,8. These limitations provide a rationale for investigating allogeneic stem cells as an off-the-shelf alternative. Fetal adnexa-derived cells are appealing candidates because they circumvent the ethical concerns and risk of teratoma formation associated with embryonic stem cells. Among fetal adnexa, umbilical cord matrix (UCM), also named Wharton&#39;s Jelly, is abundant and easy to collect.
Regardless of cell source, enhancing stemness is essential to establish a cell bank for allogenic regenerative medicine. From a functional standpoint, stemness can be defined as the potential for self-renewal and multi-lineage differentiation9. Evidence of stemness relies on proliferation and differentiation assays, along with expression of gene markers Oct4, Sox2, and Nanog9. One strategy to enhance stemness relies on the use of biomaterials to serve as void fillers and carriers enhancing proliferation and differentiation of UCM-MSCs. This approach eliminates concerns regarding manipulation of transcriptional factors to reprogram mature cells into induced pluripotent cells. Among biomaterials considered as potential carriers for stem cells, chitosan is appealing for its biocompatibility and degradability10. This natural aminopolysaccharide is formed by alkaline deacetylation of chitin, the second most abundant natural polysaccharide, primarily obtained as a subproduct of shellfish10. We have previously investigated interactions between MSCs and chitosan scaffolds, and observed the formation of spheroids11,12,13,14,15,16. We also reported on the superiority of chondrogenesis on chitosan matrices12,13,14,15,16,17,18. More recently, two independent studies described spheroids formation by adipose tissue and placenta tissue derived MSCs cultured on a chitosan film19,20. This formation of spheroids not only enhanced stemness, but also improved the retention of stem cells after in vivo implantation20.
The prevalence and morbidity of tendinopathy across species have prompted the development of experimental models to study the pathophysiology of tendinopathies and test new therapies such as stem cell injections. In horses, collagenase-induced tendonitis is a common model to demonstrate efficacy using MSCs in tendon repair21. The relevance of this approach is limited, as injections cause acute inflammatory changes, whereas clinical tendinopathies usually result from chronic overstrain22,23. In addition, chemical induction of tendon disease induces a healing response and does not replicate the impaired healing process present in clinical cases22,23. Excision of a segment of the superficial digital flexor tendon has been described as a surgical model of tendonitis in horses24. More recently, a minimally invasive approach was used to restrict the traumatic damage to the central core of the superficial digital flexor tendon25. Surgical models do not simulate the fatigue mechanism that may lead to natural tendon disease, and tend to lack reproducibility in the extent of damage created25. Regardless of the model, the morbidity and cost associated with equine models of tendon diseases are additional limitations, which justify an interest in rodent models as a first step for in vivo evaluation of novel therapies.
One of the main advantages of experimental models in rodents consists of the cost and ability to control inter-individual variability. Rodents can be standardized with respect to various physiological factors due to their rapid growth rates and relatively short life spans, limiting sources of variation and therefore reducing the number of animals required to detect differences. Strategies to induce tendon diseases in rodents have relied on chemical induction, but also on surgical creation of partial tendon defects21. Surgical models may simulate natural tendinopathies better than chemical models, but can lead to higher morbidity and catastrophic failure of the damaged tendon. In that respect, rats seem better candidates than mice for these models, as their size allows creation of larger defects, thereby facilitating evaluation of tissue healing. Sprague-Dawley rats have been used in experimental studies of tendinopathies in four major tendon groups: rotator cuff, flexor, Achilles, and patellar tendons26. Among these, models involving the patellar tendon are especially appealing because of the larger size of this tendon and the ease of accessing it27. The patellar tendon attaches the quadriceps muscle to the tibial tuberosity. Within this extensor mechanism, the patella is a sesamoid bone that directs the action of the quadriceps and delineates the proximal extent of the patellar tendon. The presence of bony anchors at the proximal and distal extents of the patellar tendon facilitates biomechanical tests. Models involving the patellar tendon typically rely on unilateral surgical defects, with a contralateral intact tendon serving as a control28,29. The most common patellar tendon defect model involves excising the central portion (1 mm in width) of the patellar tendon from the distal apex of the patella to the insertion of the tibial tuberosity, while the contralateral patellar tendon is left intact. Measures of outcomes have included histology, non-destructive biomechanical testing or biomechanical testing to failure, ultrasound imaging, ex vivo fluorescence imaging, gross observation, and functional tests28,30,31. Unilateral models do not allow comparison of a proposed treatment with conservative management of a similar injury within the same animal. Similarly, comparison between several treatments requires separate animals. A bilateral model would eliminate inter-individual variations and reduce the number of animals required for a study32. However, bilateral injuries may increase morbidity, and bilateral lameness could impede treatment evaluation. A few studies briefly report the use of bilateral patellar tendon defects in rats but focus on the effects of treatments rather than peri-operative management and morbidity of the model33,34.
This study&#39;s long-term goal is to develop a strategy to improve stemness and in vivo survival of UCM-MSCs destined to allogenic transplantation. To achieve this goal, we have recently reported improved stemness of UCM-MSCs by formation of spheroids on chitosan film and incubation under hypoxic environment35. These in vitro properties were associated with improved biomechanical properties of patellar tendon defects treated with conditioned UCM-MSCs. Based on these results, the rat bilateral patellar tendon defect model seems suitable to test candidate treatments for tendon injuries36. The purpose of the study reported here is to provide detailed protocols for isolation and characterization of UCM-MSCs, preparation of a biologic delivery system for stem cells, creation and treatment of bilateral patella tendon defects, and post-operative recovery and evaluation of tissue healing within the defects.

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evaluation of stem cell therapies in a bilateral patellar tendon injury model in rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

In the current study, results are presented as mean &#177; SD (standard deviation). Cells were isolated from the umbilical cords of 6 mares, and percentage of isolated cell lines expressing each cell surface marker under standard or chitosan conditioning were compared with a Friedman test, as a non-parametric analysis of variance with repeated measures. For tendon defect model creation, 8 rats were used for 7 days post-surgery assessment and 12 rats were used for 28 days assessment. Resul...

Watch this Scientific Journal Video about Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats at JoVE.com

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

The overall goals of this bilateral rat model are to optimize the use of animals and evaluate treatment candidates for tendon defects. This method can help answer any key questions in orthopedic field. Such as evaluation of cellular therapy on tendinopathy.The main advantage of this model is that it controls interindividual variation, thereby, reducing the number of animals required to detect differences between treatments. Visual demonstration of the model is critical because the creation of bilateral standardized patella tendon defects in rats is difficult to learn. The model requires a good knowledge of the local anatomy, strong basic surgical skills, and precision while working on small structures.To begin wash a harvested equine umbilical cord twice in a 50 milliliter tube using room temperature PBS containing antibiotics. Next, transfer the tissue to a 150 millimeter plate and section it into two inch long fragments. Then transfer the fragments to a 50 milliliter tube and wash them with PBS containing antibiotics to remove the blood.Do this two or three times at room temperature. Next, cut the fragmented parts of the umbilical cord longitudinally to expose the vessels that run its length. Using forceps and scissors, remove these vessels, which include two arteries and a vein.Now, from two or three fragments, collect the jelly-like matrix surrounding the vessels. Scrape it away with a scalpel. Then mince the jelly-like matrix, also known as Wharton's Jelley, into fine pieces.Next, transfer the jelly into a 50 milliliter tube with 15 milliliter of 0.1%collagenase type 1A in PBS and incubate the tissue at 37 degrees Celsius with gentle shaking for three to four hours until it is completely dissolved. Next, centrifuge the digested tissue and aspirate the supernatant. Now, we suspend the digested tissue in 15 milliliters of PBS and mix by pipetting.Then, repeat this wash with PBS three times using a slower centrifugation. After the last wash, resuspend the cells in 15 milliliters of PBS and then use a 100 micron cell strainer to filter the cells and centrifuge for five minutes at 150 times g to collect the cells. Resuspend the cells in 10 milliliters of freshly made culturing medium at 37 degrees Celsius.Transfer the suspension into a 25 square centimeter culture flask and incubate the cells, changing the medium every third day. When the cells reach 70 to 80%confluence, passage them, aspirate the medium, and wash the cells twice with room temperature PBS without antibiotics. Then use three milliliters of 0.25%trypsin edta to detach the cells.End the reaction with a six milliliter addition of warm medium. Collect the suspended cells and pellet them. Discard the supernatant and resuspend the pellet in one milliliter of medium.To complete the passage, reseed a tissue culture flask with 5, 000 cells per square centimeter and continue growing the culture. To begin, prepare fresh Chitosan solution. Then load each well of a 12 well plate with 500 microliters of solution.Swirl the plate to distribute the solution over all surfaces. Then, dry the plate under a laminar flow cabinet. After 16 to 24 hours, at thin film of chitosan will form in the plate wells.Neutralize this film by adding one milliliter of 0.5 normal sodium hydroxide to each well. Then, incubate the plate for two hours at room temperature. Later, aspirate the sodium hydroxide from each well and then wash each well with one milliliter of distilled water.Perform three water washes then wash each well with one milliliter of 70%ethanol once. After removing the ethanol wash, replace it with another milliliter of 70%ethanol and incubate the plate overnight in a laminar flow cabinet to sterilize the chitosan film. Then next day aspirate the remaining ethanol from each well and wash each well with PBS three times.Remove the PBS then further sterilize the chitosan film using an overnight direct exposure to ultraviolet light without the lid. Now, to form spheroids of umbilical cord derived mesenchymal stem cells, seed the wells with 20, 000 cells and incubate the plate under normal or hypoxic conditions. Place an anesthetized rat between two 0.5 liter bottles filled with warm water and cover the bottles with a cloth to keep the rat warm while not incurring skin injuries.To reduce risk of hypothermia, cover the body with bubble wrap. Next, to avoid skin trauma, apply hair remover cream over both stifles and use a tongue depressor to remove the cream and hair. Then, tape each limb to the table.Now, scrub the surgical site with chlorhexidine digluconate and rinse it with 70%ethanol. Do this three times and proceed with the surgery. With a sterile number 15 scalpel, incise the skin in a proximal to distal direction on the cranial medial aspect of the stifle.Start the incision about one centimeter proximal to the level of the patella and extend it approximately five millimeters distally to the tibial tubercle. Next, free the underlying subcutaneous tissue with the scalpel and reflect the skin to expose the patellar tendon. Now, align a 0.99 millimeter diameter kirschner wire against the patellar tendon between the distal aspect of the patella to the tibial tuberosity.Then, while keeping the wire stable, use the scalpel to make incisions on both sides of the wire, releasing a portion of tendon about one millimeter wide. Then resect the central section proximally and distally using fine iris scissors. In parallel, have an assistant prepare the clot.To do so, thaw the plasma sample and mix 20 microliters of it with half a million mesenchymal stem cells. Next, in a microcentrifuge tube, mix six microliters of 10%calcium chloride with 100 microliters of thawed plasma, this will activate that plasma. Then, mix the cell suspension into the activated plasma, this will cause the plasma to clot.Now, place the prepared clot within the patellar tendon defect. Limit the manipulation of the clot to minimize fluid release from the clot. Then, using five o polyglactin nine 10 sutures, carefully close the fascia with a cruciate pattern without interfering with the clot.Finish by closing the skin with an intradermal pattern. Cells were isolated from the umbilical cords of six mares and cultured under standard conditions or on chitosan films. The cells cultured on chitosan formed spheroids.The patellar tendon defect model was tested on many rats and a battery of behavioral tests were used to estimate the recovery. By 28 days post operative, the rats had returned to baseline strength after an initial deficit in strength measured at seven days post operatively. Upon histological examination with masson's trichrome stain, moderate arterial and capillary infiltration was noted along with moderate to extensive collagen formation whereas cartilage formation was not supported.Overall, the bilateral patellar tendon defect model limits interindividual variation without significant morbidity. Thus, reducing the number of animals needed for testing. After watching this video, you should have a good understanding of how to create a rat bilateral patellar tendon window defect model and how rats recover from this surgery.Once mastered, the procedure can be done under 30 minutes with three investigators. The first investigator prepares the cell, the second performs the surgery, while the third assists with the surgery and monitors anesthesia. Following this procedure, methods such biomechanical testing, histology and immunohistochemistry can be used in order to compare the effect of treatment on tendon healing.

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Research

JoVE Journal

Medicine

A Mouse Model of in Utero Transplantation

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

A Mouse Model of in Utero Transplantation

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

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

A 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 Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...