We thank Prof. Silvia Blemker for use of her Instron and Prof. George Christ for use of his surgical space as well as the 3D printed holder and grip. This work was supported by the UVA-Coulter Translational Research Partnership and the DoD (W81XWH-19-1-0157).

The authors have nothing to disclose.

The protocol is notable for several advantages. To our knowledge, it is the first published description of USLS in the rat model and will provide future investigators with reproducible steps for performing this procedure in the research setting. Second, we include a novel protocol for tensile testing of the native and surgical interface of the USL. The tensile testing protocol could be utilized in similar studies that investigate new tissue engineering approaches to augment native tissue repairs such as USLS. Moreover, the rat model itself is useful for the study of pelvic floor disorders due to ease of handling/boarding, short lifespan, and cost efficiency compared to larger animal models. Limitations of the protocol include an inability to assess one of the main complications of USLS, ureteral kinking. Despite this, we had no cases of presumed ureteral injury in this study. Another consideration is that the horizontal orientation of the pelvis, small fetal head-to-birth canal ratio, and lack of spontaneous prolapse in the rat model does limit some applicability of results to humans. However, the use of multiparous rats is a strength of this study since this accounts for the leading risk factor in the development of POP3.
The establishment of a successful protocol for hysterectomy and USLS in the Lewis rat will be a useful tool for future researchers investigating surgical components of POP, while minimizing variability in testing the mechanical behavior of the USL. Surgical animal models are beneficial in that they allow researchers to design clinically relevant experiments that control for parity, body mass, disease, and nutrition34 while mitigating the ethical risk of initial study in humans. Further, standardized models for POP allow researchers to bypass the limitations of human tissue collection. In particular, the tensile testing methods described in this protocol will enable consistency between studies. Previous rodent models tested the mechanical properties of the entire pelvic region, which includes the cervix, vagina, and the multiple pelvic support ligaments29,42. The methods described here allow for measurement of the USL in a way that maintains the native spinal and cervical attachments. It should be noted that the tensile testing methods do not assess the USL alone, but rather the USL in combination with its insertion at the sacrum and cervix. This is a strength of the study as it reflects the usual in situ forces to which the ligament is subjected. We acknowledge that the mechanical behavior of the isolated ligament would be different if it were tested ex vivo without its native attachments. This is especially true since the rat structures are small and limit the feasibility of collecting a sample suitable for ex vivo testing. The USLs do experience loading in multiple directions in situ, so the uniaxial nature of the test is a limitation, but using this method allows for meaningful comparisons between previous&#160;studies of rat USL mechanics29,42. While there is currently no widely accepted standard mechanical testing protocol, this model will be a useful tool for future tissue engineering studies in the field.
Several steps described in this protocol are critical to the health and well-being of the animals as well as the reproducibility of the USLS surgery and subsequent tensile testing. First, it is essential to obtain both the analgesic and the anti-inflammatory drugs described as the analgesic alone was found to be inadequate for pain management. The prophylactic antibiotic decreases the risk of surgical site infection and is the standard of care in human surgery. Regarding the USLS surgical procedure, avoiding damage to the ovaries and minimizing blood loss are essential for a successful surgery. Steps 1.3.3 and 1.3.4 describe separating the top of the uterine horn from the adjacent ovary; care should be taken to maintain this dissection on the side of the uterine horn to prevent disruption of delicate vessels around the ovary, which can result in excessive bleeding. Of note, other investigators have shown that ovarian function is preserved after removal of the uterine horns43. Moreover, if the ovaries are disrupted or removed, the overall collagen fibril architecture will be disturbed, altering the mechanical properties of its tissues44,45. Once the uterine horn is safely separated from the ovary, there is a clear plane of dissection allowing isolation of the uterine horn from the surrounding fat pads and vasculature. Despite the clear plane of dissection, the pedicles along the uterine horn should be secured with a clamp prior to transection with micro scissors. Contrary to surgical practice in humans, we have found that suture ligation of the hysterectomy pedicles is unnecessary, as clamping the pedicle prior to transection ensures adequate hemostasis. Step 1.3.6 of the protocol describes this careful process to minimize blood loss. As the hysterectomy is being performed, great care should be taken to identify the ureters as mentioned in steps 1.3.6 and 1.3.8. Understanding the anatomical proximity of the ureter is critical, as one of the most common complications associated with the USLs in humans is ureteral injury46.
In conclusion, we present a novel protocol for performing hysterectomy, uterosacral ligament suspension, and tensile testing of the USL in a rat model. We anticipate that our findings will assist future basic science investigators by providing a clear, reproducible description of these procedures and thereby allow for advancement of pelvic organ prolapse research.

Pelvic organ prolapse (POP) is a common pelvic floor disorder affecting millions of women worldwide with the potential to significantly impact many aspects of a woman&#39;s life, particularly with age1. Notably, approximately 13% of women in the United States will undergo surgery for prolapse or urinary incontinence2. A condition most common after pregnancy and childbirth, prolapse is characterized by the descent of pelvic organs, predominantly the various compartments of the vagina and/or uterus, beyond their normal position in the peritoneal cavity. This leads to bothersome symptoms of vaginal bulge or pressure, bowel, bladder, and sexual dysfunction, and overall reduced quality of life. Other risk factors for POP include obesity, tobacco use, chronic cough, and constipation3.
In healthy women, the pelvic floor organs are supported by the levator ani muscles, uterosacral ligaments (USLs), cardinal ligaments, connective tissue attachments to the pelvic sidewall and the distal structures of the perineal body4,5. The USLs are among the most important apical supportive structures for both the uterus and apical vagina, and thus, are often used in surgical correction of POP (Figure 1). Structural support from the USL stems from the dense collagenous connective tissue in the sacral region that transitions into closely packed smooth muscle. Due to this compositional gradient, the USL becomes interwoven with the uterine and vaginal musculature to provide sturdy support for the pelvic organs6,7. In the uterosacral ligament suspension (USLS), the USLs are secured to the vaginal vault following a hysterectomy, restoring the vagina and the surrounding structures to their anatomical position in the abdominal compartment. However, regardless of a transvaginal or laparoscopic route, the USLS procedure is plagued by a relatively high failure rate of up to 40% in some studies8,9. The recurrence rate of bothersome vaginal bulge symptoms at 5 years post-repair for apical compartment prolapse, such as USLs, was approximately 40% in a large multicenter randomized controlled trial9. In the same trial, retreatment for recurrent prolapse at 5 years was approximately 10%. The mechanism of this high failure rate has not been studied, but restoring the vagina and the surrounding structures to their anatomical position requires suture placement in the dense collagenous region of the USL10,11 rather than the smooth muscle region. Therefore, the high failure rate could be due to the mechanical and compositional mismatch of the surgically formed vagina-USL interface compared to the complete integration seen in the native cervical-USL attachment.
The economic impact of treating these disorders is also notable, with approximately $300 million spent annually in the US on ambulatory care12, and more than $1 billion spent annually in direct costs for surgical procedures13. Despite the vast economic resources dedicated to treating these conditions, the complications arising from many prolapse surgeries remain discouraging. For example, polypropylene mesh-based apical prolapse repairs, such as sacrocolpopexy, offer higher success rates compared to native tissue repairs14, but at the cost of potential complications such as mesh exposure or erosion. The FDA received nearly 3,000 complaints related to mesh complications between 2008 and 2010 alone. This culminated in an order by the FDA to halt the manufacture and sale of all transvaginally-placed mesh products for POP in April 201915. Therefore, there is a strong clinical need for materials other than polypropylene, and models with which to test them, that may augment native tissue prolapse repairs and increase success rates compared to traditional techniques with suture alone.
Since the FDA announcement in 2019, most pelvic surgeons have stopped using transvaginally-placed mesh for prolapse repairs, prompting investigators to seek new tissue engineering approaches to augment native tissue repairs16,17,18 such as with mesenchymal stromal cells (MSCs)9,20. With this shift in focus, there is an urgent need for the refinement of animal models that can assist with development of new materials; the challenge in this process is balancing clinical relevance with cost. To this end, basic science and clinical investigators studying pelvic organ prolapse have taken advantage of several animal models thus far, including rats, mice, rabbits, sheep, swine, and non-human primates19. The process of identifying an optimal animal model is challenging, as humans are bipedal, have no tail, and have a traumatic birth process compared to other mammalian species20. Swine21 have been utilized to simulate robotic sacrocolpopexy, while sheep have been used to simulate vaginal prolapse repairs22. These animal models, while clinically relevant, are limited in feasibility by cost and maintenance. Non-human primates have been used to study the pathogenesis of prolapse; squirrel monkeys in particular are one of the only species other than humans that can develop spontaneous prolapse, making them one of the most relevant animal models20. Non-human primates have also been used to study gynecologic surgical procedures such as sacrocolpopexy23 and uterine transplantation24. Similar to their sheep and swine counterparts, the primary limitation of non-human primates as an animal model of prolapse is the cost of maintenance, care, and boarding19.
Although the rodent pelvis is oriented horizontally with a much smaller head-to-birth canal size ratio compared to humans19, rats are suitable for small animal studies of USLS surgery since they have similar USL anatomy, cellularity, histological architecture, and matrix composition compared to the human USL25. Moreover, they are beneficial in terms of maintenance and boarding. Despite these beneficial attributes, there are no published reports of a rat model of USLS repair. Therefore, the aim is to describe a protocol for hysterectomy and USLS in the multiparous Lewis rat. This protocol will be beneficial for investigators who aim to study the pathophysiology and surgical components of POP using this accessible animal model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64311/64311fig01.jpg" /> 
Figure 1: Pelvic organ prolapse. (A) The normal orientation of organs in the peritoneal cavity and (B) the dramatic organ descension when prolapse occurs. Following hysterectomy, (C) uterosacral ligament suspension restores the vagina and surrounding structures to their proper anatomical position. <a href="https://www.jove.com/files/ftp_upload/64311/64311fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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development of a uterosacral ligament suspension rat model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Surgical feasibility and uterosacral suture placement 
There were no intraoperative complications related to hysterectomy or uterosacral ligament suspension in any of the animals. There was minimal bleeding during removal of the uterine horns, provided the adjacent vasculature was clamped prior to removal. Limited bleeding allowed for good visualization of the uterosacral ligaments for suture placement and prevented intra-operative bowel, rectum, ureteral, or bladder injury. Following placement of the sutures, the newly formed USL-vaginal vault junction prevented movement of the cervical/uterine stump as shown in Figure 5C. During the first three postoperative days, the animals were checked on daily, and then bi-weekly basis until the end of the experiment. With the extended-release opioid and NSAID analgesics administered at the time of surgery, additional analgesics were found to be unnecessary. Based on our experience with 16 animal surgeries (n = 8 for both control and USLS groups), a drop in weight should be expected in the first week following surgery with an average loss of 5.7 &#177; 1.4% from surgery day weight. As expected, the rats slowly gained weight over the subsequent 23 weeks, with an average weight gain of 15.1 &#177; 4.5% over the course of the experiment.
Mechanical testing of the USLS repair 
To demonstrate the functionality of the USLS repair, uniaxial tensile testing was performed. After euthanasia of the animal at the chosen post-operative timepoint, 24 weeks in this study, the surgical area should be carefully dissected to visualize the USL-vaginal vault junction as shown in Figure 6A. Compared to other methodologies for testing the rat USLs together with other supportive structures and pelvic organs29,42, the method described here is the first to test the rat USL in an isolated manner. The umbilical tape used in this study was strategically chosen for its flexibility as the tape compliance allowed for minimal disruption of the tissue during tensile testing preparation. Load displacement data, therefore, must be adjusted to account for the small amount of stretch contributed by the umbilical tape. Figure 9 provides an example of data obtained via tensile testing with Figure 9A providing an example of a typical stress-strain plot. Reporting of stress-strain data is recommended as this information is normalized and independent of the size of the specimens34 and can be better compared across studies. For the intact uterosacral ligament, we report structural properties such as ultimate load (2.9 &#177; 0.5 N) and stiffness (0.4 &#177; 0.1 N/mm) as well as normalized material properties such as ultimate stress (2.1 &#177; 0.4 MPa), ultimate strain (1.6 &#177; 0.5) and tangent modulus (4.0 &#177; 1.1 MPa). In the uniaxial tests performed on the rat reproductive organs and all their supportive tissue connections by Moalli et al., they reported an ultimate load at failure (13.2 &#177; 1.1 N) and stiffness (2.9 &#177; 0.9 N/mm) higher than the isolated USL29. The work done by Moalli et al. and other literature34,35 mention the high variability between tested specimens as shown in the data presented here. For the uterosacral ligament suspension repair, we found all structural material properties (stiffness, 0.33 &#177; 0.13 N/mm; ultimate load, 2.6 &#177; 1.3 N) and normalized material properties (ultimate stress, 1.8 &#177; 0.7 MPa; ultimate strain 1.3 &#177; 0.3; tangent modulus, 3.0 &#177; 0.9 MPa) to be lower than that of the native USL.

Watch this Scientific Journal Video about Development of a Uterosacral Ligament Suspension Rat Model at JoVE.com

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...

development-of-a-uterosacral-ligament-suspension-rat-model

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JoVE Journal

Medicine

3.6K Views.  University of Virginia. Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Video: Development of a Uterosacral Ligament Suspension Rat Model

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

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 ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...