Name: urokinase-type plasminogen activator-induced mouse back pain model
Uploaded: 2023-09-01T12:00:00
Description: Watch this Scientific Journal Video about Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model at JoVE.com

Grant funding was provided by NIH HEAL UG3 NS123958. The housing facilities were inspected and accredited by AAALAC. Animals were housed in the Animal Resources Center (ARC) housing facility maintained by the laboratory staff and Division of Laboratory and Animal Resources (DLAR) staff. The procedures for behavioral testing are standard methods in the field as approved by the American Pain Society and the International Association for the Study of Pain. The method of euthanasia is consistent with recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

All animal procedures described are in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Studies were approved by the local Institutional Care and Use Committee (IACUC #23-201364-HSC) of the University of New Mexico Health Sciences Center. All studies comply with policies under the auspices of an OLAW Assurance of Compliance (A3002-01) on the use of animals in research, as described in Part III. II. Assurances and Certifications. Animals are housed in the Animal Resources Center (ARC) housing faci...

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This model of chronic back pain is simple to induce, and hypersensitivity established within 1 week can last for up to (and possibly beyond) 8 weeks. This allows for accurate study of the chronic pain state as opposed to other acute models that only last for a week or two. While we show the model in mice, the uPA-induced CBP model can also be established in rats2. An advantage of the model is that the prolonged time course provokes the development of anxiety- and depression-like behaviors, which a...

Low back pain is one of the most common causes of disability with 1 in 5 people suffering worldwide1. In spite of these efforts, few reliable animal models of back pain are popularly used in animal research in the pain field, especially in mice. Previous models have almost exclusively made use of rats for the induction of chronic back pain (CBP) such as those induced by injection of urinary plasminogen activator (uPA) into the lumbar facet joint2,3, injection of nerve growth factor (NGF) into trunk musculature4, or monosodium iodoacetate (MIA)5 or interleukin-1beta6 injection in the intravertebral disc. Of course, rats are preferred for these models mainly due to their larger size and ease of access for injection of inflammatory agents.
To be clear, mouse models of back pain do exist such as the SPARC-null mouse model of intervertebral-disc degeneration used for many years7, but these are more costly and time-consuming to establish than injection-based models. A recent mouse study established a model of lower back pain by combining NGF injection into low back muscles with vertical chronic restraint stress8. In the following protocol, we have adapted the uPA-induced CBP model from rats for mice2. Hypersensitivity is established within 1 week and persists up to 6-8 weeks. In addition, we establish that mice develop anxiety- and depression-like behaviors. Given the prevalence of back pain and the more common use of mice in molecular pain research, this durable model is readily established for use in the development of new treatment strategies for back pain relief.

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			<td>Scalpel Handle No. 3</td>
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			<td>Von Frey Filaments</td>
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urokinase-type plasminogen activator-induced mouse back pain model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Nociceptive-related behavioral testing and data analysis 
Evoked measures 
Hypersensitivity on the footpad develops within a day of urokinase injection. Within 1 week, the withdrawal threshold is significantly decreased and persists until euthanasia; this is shown through postsurgical week 4 (Figure 4A). Paw withdrawal latency is analyzed using the von Frey up-down method9 and the Hargreaves test. In the example plotted, mic...

Watch this Scientific Journal Video about Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model at JoVE.com

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

This protocol outlines a non-invasive method to induce chronic back pain in mice for up to 10 weeks, which can be performed quickly and reliably with minimal training. The most difficult aspect of this technique is to recognize how injecting ligament feels versus injecting muscle. Feeling resistance upon contact indicates ligament injection.No resistance means the needle could be in the muscle or abdominal cavity. To begin, take a sterile flat surface to place the mouse. Then, place a heating pad recovery station next to the surgery setup to immediately transfer mice after injection.Next, prepare the urokinase and a Hamilton syringe. Draw the solution beforehand to minimize the time the mouse spends under anesthesia. After anesthetizing the mouse, use two fingers to locate the lumbar spinal segments at L2 and L3 below the rib cage and verify the injection site.Place the tip of the Hamilton syringe next to the spine. Position the syringe at approximately 45-degree angle into the ligament immediately adjacent to the bone. Insert the needle tip gently, but firmly, into the ligament, and inject slowly until all five microliters have been injected.Remove the needle gently and slowly. Then, place the mouse in the heat recovery station until it wakes up and becomes mobile. Check the mice for one hour after surgery to ensure all normal motor function continues.After the surgery, check the weight and injection site of the mice daily for a week to prevent any infection or complications. Before testing, move each animal into a separate clear cubicle on the testing table with a screen top. Then, stimulate the foot pad with the 3.61 Von Frey filament that elicits four grams of force.A foot withdrawal of three to five stimuli is considered a positive response. Now, apply the next weaker filament in the series until the animal stops responding to the mechanical stimulation. Use the higher filament until a response is elicited, then switch back to the lower filament.Using a curve fitting algorithm, calculate the mechanical withdrawal threshold, which is the minimal amount of force needed to elicit a response 50%of the time. To perform the Hargreaves Test, place the mice in cubicles on a glass surface heated from below by an infrared emitter. Measure the time from applying infrared light stimulus on the mouse's hind paw to withdrawal as foot latency.For the Cold Plate Test, place the mice on the cold plate apparatus cooled to minus nine degrees Celsius. Record the time the mouse takes to lift its foot after being placed on the apparatus as the latency to withdraw the foot. To perform the Anxiety Test, position each animal into the place preference test box with a passageway between two chambers.One apparatus chamber should be brightly lit, while the opposite side should remain dark. Using a computer, monitor the animal's light and dark occupancy times and transitions during a 10-minute test. Next, place the model rodents or naive animals on the zero maze, or use the two-chamber light or dark place preference test.Assess the time spent in closed and open areas of the maze using a stopwatch. For the Sucrose Splash Depression Test, spray a 10 to 30%sucrose solution on the dorsal code and score the frequency, duration, and latency of grooming for 10 minutes. To perform novel object tests, acclimate mice individually to a clear plastic cage with an open top for one hour.Add two identical toy mini figures to opposite corners of the cage for five minutes. Acclimatize the animals on the testing day for one hour, and place the two identical mini figures in the same positions of the cage for five minutes before returning them to the home cage. Then, replace one of the original figures with a distinctly different novel object.After four hours, return the mice to the test cage and record the time spent exploring the objects. For motor function assessment, construct a tunnel from a paper towel tube cut lengthwise on one edge. Spread open the tunnel on a clean piece of printer paper.Hold the mice gently until they are calm. Apply non-toxic India ink on a mouse's paws by placing them into an ink well, stamp pad, or using a cotton swab. Release the mice at the tunnel entrance.Allow them to run through the tunnel and capture them at the end. Score paw prints based on stride length, stride width, and toe spread. Mechanical and thermal hypersensitivity assays demonstrated that the chronic back pain mice had significantly increased mechanical, heat, and cold sensitivity compared to the naive controls.Anxiety analysis showed that the total time spent in the light chamber of the light and dark box and the number of rearing events were significantly lower in mice with chronic back pain, indicating anxiety. During the Sucrose Splash Test, mice with chronic back pain exhibited lower grooming time and the number of grooming. However, the amount of time before grooming starts was significantly increased.Stride length between mice with chronic back pain and naive mice was significantly different. The most important thing to remember when attempting this procedure is that you are aiming for the ligaments around the spine, not the muscle. The chronic back pain model provides a reliable tool to assess a variety of novel research, pharmacologic, and other potential therapeutics.

urokinase-type-plasminogen-activator-induced-mouse-back-pain-model

Research

JoVE Journal

Behavior

Assessing Functional Performance in the Mdx Mouse Model

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.

Assessing Functional Performance in the Mdx Mouse Model

The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential ...

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

Treating Low Back Pain in Failed Back Surgery Patients with Multicolumn-lead Spinal Cord Stimulation

Failed back surgery syndrome (FBSS) refers to persistent, chronic pain following spinal surgery. Spinal cord stimulation with dorsal epidural leads can be used to treat back and leg pain in FBSS patients. This paper presents a detailed protocol for using spinal cord stimulation with surgical leads in FBSS patients. In our department, with the patient under general anesthesia, we place the lead in the epidural space by means of a small laminectomy at the 10th thoracic level. Placement of the lead is followed by a 1 month trial period with an externalized lead. If pain relief is greater than 50% at the end of this 1 month stimulation trial (required by Belgian reimbursement criteria), an internal pulse generator is then placed under the skin and connected to the lead in a second surgical procedure. We have demonstrated that using this technique in rigorously selected FBSS patients can significantly improve back pain, leg pain, patient activity, and quality of life for a sustained period of time.

This paper presents a method that uses spinal cord stimulation with a multicolumn lead to treat neuropathic low back pain in failed back surgery patients.

Failed back surgery syndrome (FBSS) refers to persistent, chronic pain following spinal surgery. Spinal cord stimulation with dorsal epidural leads can be ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical ...

Investigating Pain-Related Avoidance Behavior using a Robotic Arm-Reaching Paradigm

Avoidance behavior is a key contributor to the transition from acute pain to chronic pain disability. Yet, there has been a lack of ecologically valid paradigms to experimentally investigate pain-related avoidance. To fill this gap, we developed a paradigm (the robotic arm-reaching paradigm) to investigate the mechanisms underlying the development of pain-related avoidance behavior. Existing avoidance paradigms (mostly in the context of anxiety research) have often operationalized avoidance as an experimenter-instructed, low-cost response, superimposed on stimuli associated with threat during a Pavlovian fear conditioning procedure. In contrast, the current method offers increased ecological validity in terms of instrumental learning (acquisition) of avoidance, and by adding a cost to the avoidance response. In the paradigm, participants perform arm-reaching movements from a starting point to a target using a robotic arm, and freely choose between three different movement trajectories to do so. The movement trajectories differ in probability of being paired with a painful electrocutaneous stimulus, and in required effort in terms of deviation and resistance. Specifically, the painful stimulus can be (partly) avoided at the cost of performing movements requiring increased effort. Avoidance behavior is operationalized as the maximal deviation from the shortest trajectory on each trial. In addition to explaining how the new paradigm can help understand the acquisition of avoidance, we describe adaptations of the robotic arm-reaching paradigm for (1) examining the spread of avoidance to other stimuli (generalization), (2) modeling clinical treatment in the lab (extinction of avoidance using response prevention), as well as (3) modeling relapse, and return of avoidance following extinction (spontaneous recovery). Given the increased ecological validity, and numerous possibilities for extensions and/or adaptations, the robotic arm-reaching paradigm offers a promising tool to facilitate the investigation of avoidance behavior and to further our understanding of its underlying processes.

Avoidance is central to chronic pain disability, yet adequate paradigms for examining pain-related avoidance are lacking. Therefore, we developed a paradigm that allows investigating how pain-related avoidance behavior is learned (acquisition), spreads to other stimuli (generalization), can be mitigated (extinction), and how it may subsequently re-emerge (spontaneous recovery).

Avoidance behavior is a key contributor to the transition from acute pain to chronic pain disability. Yet, there has been a lack of ecologically valid ...

Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, animal models are needed to examine the mechanisms and effectiveness of novel therapies from a translational perspective. Several rodent models of back pain are used in current investigations. Surprisingly, however, no standardized behavioral test was validated to assess mechanical sensitivity in back pain models. This is critical to confirm that animals with presumed back pain present local hypersensitivity to nociceptive stimuli, and to monitor sensitivity during interventions designed to relieve back pain. The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. A test cage was fabricated specifically for this method; length x width x height: 50 x 20 x 7 cm, having a stainless-steel mesh on the top. This test cage allows the application of mechanical stimuli to the back. To perform the test, the back of the animal is shaved in the region of interest, and the test area is marked to repeat the test on different days, as needed. The mechanical threshold is determined with Von Frey filaments applied to the paraspinal muscles, utilizing the up-down method described previously. The positive responses include (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck (4) scratching or licking the back, and (5) escaping. This behavioral test (Back Mechanical Sensitivity (BMS) test) is useful for mechanistic research with rodent models of back pain for the development of therapeutic interventions for the prevention and management of back pain.

To develop novel therapeutic interventions for the prevention and management of back pain, animal models are required to examine the mechanisms and effectiveness of these therapies from a translational perspective. The present protocol describes the BMS test, a standardized method to assess back mechanical sensitivity in the rat.

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, ...

Elevated Plus Maze Test in an Angelman Syndrome Mouse Model

To begin, place the plus shaped maze on the testing platform just below the camera. Using the potentiometer on the wall, adjust the light intensity to 70 luxe at its center with the help of a luxometer with its sensor placed at the center of the maze during the adjustment. Open the software by double clicking on the viewer software icon and load the configuration for Elevated Plus Maze or EPM testing by clicking on the icon on the upper left side of the configuration tab.Enter the animal information into the corresponding fields of the experiment tab including animal id, genotype, gender, and experiment information. Verify that the zone's position, open arms, and closed arms are correctly configured. Using a visual control and computer mouse, ensure that the virtual outline zones match the corresponding EPM zones.Place the mouse cursor on the arrow icon on the upper left side of the acquisition tab. Remove the animal from the home cage and place it gently in the center of the EPM. Start the experiment by left clicking on the computer mouse.After five minutes, save the recorded data by clicking okay. Name the file and click save. Export the results to a csv file for offline analysis by clicking on the icon on the left vertical panel of the data analysis tab.In the EPM test, none of the parameters related to anxiety-like behavior were altered in maternal heterozygous Ube3A mutants.

Open Field Test in an Angelman Syndrome Mouse Model

Place the four open field, or OF, test boxes on the testing platform below the camera. Using the potentiometer on the wall, adjust the light intensity to 200 lux at the center of each OF test with the help of a luxometer with its sensor placed at the center of each box during the adjustment. Open the software by double clicking on the viewer software icon and load the configuration for EF testing by clicking on the icon on the upper left side of the configuration tab.Enter the animal information into the corresponding fields of the experiment tab. Verify that the zone's position matches the OF test boxes and adjust them if needed. Using a visual control and computer mouse, ensure that the virtual outline center and periphery zones match the corresponding OF test zones.Place the mouse cursor on the arrow icon on the upper left side of the acquisition tab. Remove four animals from the home cage and place them in the corner of each test box. Stop the experiment by left clicking on the computer mouse.In the OF test, the maternal heterozygous Ube3a mutant animals were significantly hypoactive as reflected by a shorter traversed distance, lower average speed, and longer resting time. However, none of the parameters related to anxiety-like behavior were altered in Ube3a mutants.

Tail Suspension Test in an Angelman Syndrome Mouse Model

Connect the tail suspension cage to the computer using a USB cable. Insert the USB dongle into the computer and start the software by double clicking on the BioTSD software icon. In the settings tab on the global, ensure the acquisition duration is set to 360 seconds.In the experiment tab, select the new list of subjects and create a new experiment file and a new list of tested subjects by following the instructions in the opened tab. Prepare the animals for the test by wrapping single-sided adhesive tape around three-fourths of the animal's tail starting from the base. Start the run by clicking start run then continue in the acquisition tab.Immediately after hanging the animal on the hook, click the start icon under the visualized position to acquire data. After testing all the animals, in the analysis tab, select the last four minutes of the acquisition for analysis. Select all valid runs in the analysis period and click analyze selected subjects.Choose the desired data format and click export selected data to export the collected data. In the tail suspension test, Ube3a mutant animals were immobile for significantly longer than their control littermates, indicating their depression-like behavior.