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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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatosensory+behavioral+alterations+in+a+NGF-induced+persistent+low+back+pain+model.">Somatosensory behavioral alterations in a NGF-induced persistent low back pain model.</a> Behavioural Brain Research. 418, 113617 (2022).</li><li>Suh, H. R., Cho, H. -. Y., Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+model+of+intervertebral+disc+degeneration+by+the+intradiscal+application+of+monosodium+iodoacetate+(MIA)+in+rat.">Development of a novel model of intervertebral disc degeneration by the intradiscal application of monosodium iodoacetate (MIA) in rat.</a> The Spine Journal. 22 (1), 183-192 (2022).</li><li>Kim, H., Hong, J. Y., Lee, J., Jeon, W. -. J., Ha, I. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1&#946;+promotes+disc+degeneration+and+inflammation+through+direct+injection+of+intervertebral+disc+in+a+rat+lumbar+disc+herniation+model.">IL-1&#946; promotes disc degeneration and inflammation through direct injection of intervertebral disc in a rat lumbar disc herniation model.</a> The Spine Journal. 21 (6), 1031-1041 (2021).</li><li>Millecamps, M., Tajerian, M., Sage, E. H., Stone, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+signs+of+chronic+back+pain+in+the+SPARC-null+mouse.">Behavioral signs of chronic back pain in the SPARC-null mouse.</a> Spine. 36 (2), 95-102 (2011).</li><li>La Porta, C., Tappe-Theodor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+impact+of+psychological+and+psychophysical+stress+on+low+back+pain+in+mice.">Differential impact of psychological and psychophysical stress on low back pain in mice.</a> Pain. 161 (7), 1442-1458 (2020).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. 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S., Vetter, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+used+to+evaluate+pain+behaviors+in+rodents.">Methods used to evaluate pain behaviors in rodents.</a> Frontiers in Molecular Neuroscience. 10, 284 (2017).</li><li>David, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis-dependent+and+-independent+effects+of+fluoxetine+in+an+animal+model+of+anxiety/depression.">Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression.</a> Neuron. 62 (4), 479-493 (2009).</li><li>Yalcin, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-dependent+history+of+mood+disorders+in+a+murine+model+of+neuropathic+pain.">A time-dependent history of mood disorders in a murine model of neuropathic pain.</a> Biological Psychiatry. 70 (10), 946-953 (2011).</li><li>Madathil, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-specific+overexpression+of+insulin-like+growth+factor-1+protects+hippocampal+neurons+and+reduces+behavioral+deficits+following+traumatic+brain+injury+in+mice.">Astrocyte-specific overexpression of insulin-like growth factor-1 protects hippocampal neurons and reduces behavioral deficits following traumatic brain injury in mice.</a> PloS One. 8 (6), e67204 (2013).</li><li>Sugimoto, H., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+protocol+of+footprint+analysis+and+hanging+box+test+for+mice+applied+the+chronic+restraint+stress.">Low-cost protocol of footprint analysis and hanging box test for mice applied the chronic restraint stress.</a> Journal of Visualized Experiments: JoVE. (143), e59027 (2019).</li><li>Hassan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+chronic+low+back+pain+phenotypic+domains+and+characteristics+accounting+for+individual+variation:+a+systematic+review.">Identifying chronic low back pain phenotypic domains and characteristics accounting for individual variation: a systematic review.</a> Pain. , (2023).</li></ol>

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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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

730 ビュー.  University of New Mexico Health Sciences Center. このプロトコルは、マウスに最大10週間慢性腰痛を誘発する非侵襲的な方法を概説しており、最小限のトレーニングで迅速かつ確実に実行できます。このテクニックの最も難しい側面は、靭帯を注入することと筋肉を注入することがどのように感じるかを認識することです。接触時の抵抗感は靭帯注入の兆候です。抵抗がないということは、針が筋肉や腹腔内にあ?...