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.

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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. 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The authors declare no competing financial interests. KNW acknowledges unpaid consultation with NeuroChronix, Bessor Pharma, and USA Elixeria BioPharm, Inc.

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 ...

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

Research

JoVE Journal

Behavior

916 Views.  University of New Mexico Health Sciences Center. Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

Video: Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

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, ...

Mouse Model of Pressure Ulcers After Spinal Cord Injury

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.

Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients.

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony ...

Bioengineering

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Medicine

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Neuroscience

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

Effects of Manual Therapy on a Chronic Non-Specific Low Back Pain Murine Model

Manual Therapy for a Chronic Non-Specific Low Back Pain Rat Model

Chronic low back pain (CLBP) is a highly prevalent condition worldwide and a major cause of disability. The majority of patients with CLBP are diagnosed with chronic non-specific low back pain (CNLBP) due to an unknown pathological cause. Manual therapy (MT) is an integral aspect of traditional Chinese medicine and is recognized as Tuina in China. It involves techniques like bone-setting and muscle relaxation manipulation. Despite its clinical efficacy in treating CNLBP, the underlying mechanisms of MT remain unclear. In animal experiments aimed at investigating these mechanisms, one of the main challenges is achieving normative MT on CNLBP model rats. Improving the stability of finger strength is a key issue in MT. To address this technical limitation, a standardized procedure for MT on CNLBP model rats is presented in this study. This procedure significantly enhances the stability of MT with the hands and alleviates common problems associated with immobilizing rats during MT. The findings of this study are of reference value for future experimental investigations of MT.

This study proposes a standardized procedure for manual therapy on chronic low back pain model rats, which will be of reference value for future experimental research on manual therapy.

Chronic low back pain (CLBP) is a highly prevalent condition worldwide and a major cause of disability. The majority of patients with CLBP are diagnosed ...