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 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. 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 "Global Year about Back Pain" and 2022 the "Global Year for Translating Pain Knowledge to Practice." One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The features of this model are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP's naming 2022 as the Global Year for Translating Pain Knowledge to Practice.
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.
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 facility maintained by the laboratory staff and Division of Laboratory and Animal Resources (DLAR) staff. The method of euthanasia (100 µL of 59 mg/mL pentobarbital injection) is rapid and reliable and allows for the dissection and collection of various tissues for further research.
1. Animals
2. Model induction
Figure 1: Setup for urokinase CBP induction. (A) The Fine Science Tools baseplate recommended for mouse surgeries. The ribbed edges can have hooked string on them for holding the mouse in place. (B) Recovery station. An empty housing cage is recommended, half on the heating pad, half off. A clean cloth is placed on the bottom to give the mouse a comfortable resting area. (C) Anesthesia machine setup recommendation. Using a two-channel delivery system, set up one hose to the induction chamber and another to the surgery station. (D) A view of the mouse restraints. Two strings are knotted onto the ribbed edges of the base plate, then gently pulled across the mouse's neck and rear respectively. Make sure not to restrain the mouse too tightly so that it can still breathe normally. Please click here to view a larger version of this figure.
Figure 2: Urokinase injection induction of CBP. (A) A view of the injection site placement. As shown, feel with fingers to find the bottom of the mouse's ribcage for a reference point for L4-L5. (B) A view of the injection process, showing the angle for proper injection. (C) A 45° angle is preferable here, but adjust as needed to ensure the needle gets where it needs to. If needed, shave the injection site for better visualization. Please click here to view a larger version of this figure.
Figure 3: Diagram of the injection site. (A) A photograph of the location of the injection site. Ink is used here to indicate where the liquid will be entering the interspinous ligament between the L2 and L3 vertebrae. (B) A diagram showing the proper positioning of the needle and location of the injection site, shown from a side view. (C) A diagram showing a top-down view of the vertebrae, and injection sites for the interspinous ligaments. Injections will typically be on the interspinous ligaments next to the spine, but the needle can be inserted in the space between and intertransverse vertebrae as well. The use of blue dye in pilot trials is recommended as shown in (A). Please click here to view a larger version of this figure.
3. Behavioral assays
Figure 4: Mechanical and thermal hypersensitivity following CBP induction. Pain is measurable a week following model induction and persists for 8 weeks. (A) von Frey test. Mechanical threshold testing is performed with von Frey filaments applied to the footpad through a mesh top table with the up-down method as shown here over the course of 4 weeks. The naïve male threshold (green) is hidden beneath the blue line for the naïve female mice. The CBP mice (n = 4 males, 4 females) showed significantly increased mechanical sensitivity compared to the naïve controls (n = 2 males, 2 females). Two-way ANOVA (Dunnett's multiple comparisons test) was performed on these data: n = 4 per group. In post-hoc analyses, Bonferroni adjustment to all P-values for week-by-week comparisons of CBP versus Naïve yielded all 11 values < 0.0011. **** p < 0.0001. (B) Hargreaves test. The heat threshold was tested on the footpad with the Hargreaves test (50 °C). The CBP mice (n = 12 males, 12 females) showed significantly increased heat sensitivity compared to the naïve controls (n = 6 males, 6 females). Mann-Whitney two-tailed t-test was performed to test significance (p < 0.0001). (C) Cold sensitivity. The cold probe test was performed by placing mice on the cold plate apparatus cooled to -9 °C. Latency to withdraw was recorded as the time in seconds from placement of the mouse on the apparatus until the mouse begins foot lifting, licking, or shaking. In the data shown, a cold probe cooled to -9 °C was placed underneath the mouse's hind paw while the mouse is caged on top of a wire mesh. All mice were tested 1-3 weeks post injection. The CBP mice (n = 4 males, 6 females) showed significantly increased cold sensitivity compared to the naïve controls (n = 2 males, 4 females). Mann-Whitney two-tailed t-test was performed to test significance (p = 0.0002). Please click here to view a larger version of this figure.
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, mice with CBP (n = 4 males, 4 females) showed significantly increased mechanical sensitivity compared to controls (n = 2 males, 2 females). The model dissipates in weeks 6-7. This time course allows the evaluation of compounds to attenuate hypersensitivity at chronic time points, equivalent to years of pain experience in clinical patients. The reflexive von Frey test can be repeated several times in a single day when the efficacy of a short-lasting compound is being determined.
von Frey mechanical sensitivity test
The sensitivity of the footpad, and thus, the severity of the back pain model is quantified by the number of withdrawal events from graded von Frey filaments with defined tensile strength. Stimulation with the lowest fiber (0.008 g, 1.65) is usually not detectable except in severe cases of allodynia; the largest fiber (6.0 g, 4.74) is about the size of a blunt paper clip end, and though it can be felt by a mouse, a naïve mouse will usually not flinch upon application. Animals are free to voluntarily move their foot away from the stimulus. The mean occurrence of withdrawal events is expressed as the number of responses out of five, with zero indicating no withdrawal, and five indicating the maximum number of withdrawals. Responses to lower fibers compared to controls indicate increased sensitivity. After induction of this model, statistically significant mechanical hypersensitivity develops within a week and persists for multiple weeks post injection (Figure 4A).
Hargreaves thermal sensitivity threshold test
Withdrawal of the foot in seconds after exposure to the IR-induced heat stimulus is compared between (among) groups. A decrease in the withdrawal latency in seconds indicates heat hypersensitivity (Figure 4B). Animals are free to voluntarily move their foot away from the stimulus.
Cold probe thermal hypersensitivity
The back pain model induces cold hypersensitivity on the footpad. Animals are free to voluntarily move their foot away from the stimulus. A decrease in the withdrawal latency in seconds indicates cold hypersensitivity (Figure 4C). Withdrawal latency was taken with a stopwatch from when the cold stimulus was applied to the mouse exhibiting a withdrawal behavior, licking, flicking, or repeated lifting of the affected hind paw.
Non-evoked spontaneous pain measures
Figure 5: Altered non-evoked behavioral measures. (A) Anxiety in CBP model. Total time spent in the light chamber of the light/dark box and number of rearing events are significantly lower in mice with CBP, indicating anxiety. One-way ANOVA (Dunnett's multiple comparisons test) was performed on these data: (n = 4 CBP, n = 6 Naive). Tukey post-hoc test was performed to confirm differences between specific group means. * p < 0.05, ** p < 0.01. (B) Depression in CBP model. The total time spent grooming and the number of times groomed during the sucrose splash test are significantly lower in mice with CBP, whereas the amount of time before grooming starts was significantly increased. One-way ANOVA (Dunnett's multiple comparisons test) was performed on these data: (n = 4 CBP, n = 6 Naive). Tukey post-hoc test was performed to confirm differences between specific group means. * p < 0.05, *** p < 0.001. (C) Stride alterations in mice with CBP model. Stride length between mice with CBP and naïve mice was significantly different. One-way ANOVA (Dunnett's multiple comparisons test) was performed on these data: n = 4 per group. Tukey post-hoc test was performed to confirm differences between specific group means. ** p < 0.01. Please click here to view a larger version of this figure.
Light/dark test
A standard light/dark chamber can be used for anxiety analysis10. Mice with the back pain model are significantly less likely to spend time in the light chamber and have fewer rearing behaviors, which is indicative of the anxiety and the severity of the model. Alternatively, the elevated zero or plus mazes can be used to measure anxiety-like behaviors. Rodents with a pain model displaying anxiety-like behavior enter and spend less time in the open quadrants than naïve control animals (Figure 5A).
Depression-like behavior
The sucrose splash test can be utilized to assess depression-like behavior in the back pain model11. The test assesses the absence of grooming behavior as a sign of depression resulting from the induced pain model12,13. Mice with back pain groom significantly fewer times and for less time overall, as well as take longer to begin grooming (latency to groom) (Figure 5B).
Mobility motor behavior
Finally, the inkblot test can be used to differentiate gait differences between the model and control mice15. Mice with back pain show significantly shorter and wider strides compared to controls (Figure 5C). Measure the stride length for group comparisons, noting the differences in stride appearance.
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 are observed in patients with chronic lower back pain. Given the prevalence of back pain16, the ability to be performed in the more commonly used mouse models in molecular pain research is an advantage.
This induction of a pain model using uPA has been done similarly with an intraarticular injection to induce osteoarthritis3, but the method shown here (intraligament) can be done quicker and with far less risk of damage to the mouse. There is no suturing needed, and no risk of bleedout due to puncturing of the articular cavity. As it is a single injection with little preparatory work, it is also easy to blind technicians to groups for more accurate results.
The main limitation of this model involves the proper acclimation of the mice to the von Frey testing; in fact, it is recommended that mice be acclimated to their cubicle and the filaments at least a week prior to model induction to ensure that the flinch response is not due to the novelty of the environment they are in or due to seeing something moving beneath their feet.
It is critical that the injection be made into the ligaments to ensure maximal response to the urokinase and not into the muscle which produces about half the response. As the injection is close to the spine, it is still important to monitor all mice daily following injection for at least 1 week to ensure there are no complications from the procedure. If any mouse shows signs of lethargy, weight loss, paralysis, abnormal behavior, or excessive grooming of the injection site, consider removing the mouse from the study.
Future studies will examine the dorsal root ganglia (DRG) with in vitro patch clamp recordings. Flow analysis will determine the neuroimmune response and the presence of invading inflammatory cells in the DRG.
The authors have nothing to disclose.
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.
Animals and Consumables | |||
70% ethanol | Local Source | ||
BALB/c mice | Envigo | 20-25 g | |
Cotton balls | Fisher Scientific | 19-090-702 | |
Cotton-tipped applicators | Fisher Scientific | 19-062-616 | |
Isoflurane inhalant anesthetic | MedVet | RXISO-250 | |
Labeling tape | Fisher Scientific | NGFP7002 | |
Nitrile exam gloves | Fisher Scientific | ||
Oxygen tank | Local Source | ||
Surgical drape, Steri-Drape Utility Sheet, Absorbent Prevention | VWR | 76246-788 | cut into 15 x 15 cm pieces |
Tygon tubing with 3 mm inner diameter | Grainger | 22XH87 | |
Equipment | |||
#11 carbon steel scalpel blades | VWR | 21909-612 | |
Anesthesia induction chamber | Summit Medical Equipment Company | AS-01-0530-LG | |
Autoclave | Local Unit | ||
Biology Dumont #5 forceps | Fine Science Tools | 11252-30 | |
Glass bead sterilizer Germinator 500 | VWR | 102095-946 | |
IITC Life Sciences Series 8 Model PE34 Hot/Cold Plate Analgesia Meter | IITC | PE34 | |
Integra Miltex cotton & dressing pliers | Safco Dental Supply | 66-317 | |
OPTIKA CL31 double arm LED illuminator | New York Microscope Company | OPCL-31 | |
Plantar Test System with InfraRed Emitter, i. e. Hargreaves Apparatus | Ugo Basile | 37370-001 and 37370-002 | |
Scalpel Handle No. 3 | VWR | 25607-947 | |
Small animal heating pad | Valley Vet Supply | 47375 | |
Student Vannas spring scissors, straight blade | Fine Science Tools | 91500-09 | |
Table top animal research portable anesthesia workstation “PAM” | Patterson Scientific | AS-01-0007 | |
Von Frey Filaments | Ugo Basile | 37450-275 |