Skeletal pain stands as a significant contributor to chronic suffering and long-term disability, frequently stemming from conditions like osteoarthritis, osteoporosis, age-related bone fractures, and bone cancer. The implications of skeletal pain extend beyond mere discomfort, often leading to a decline in functional status and an increase in both morbidity and mortality. Unfortunately, the incidence of skeletal pain frequently increases with age, making it a pressing concern for an aging global population.
The Challenge of Treating Skeletal Pain
Several factors contribute to the persistence of skeletal pain as a largely untreated worldwide health issue. One of the primary challenges lies in the limited availability of analgesic therapies that can effectively alleviate skeletal pain without causing significant unwanted side effects. Non-steroidal anti-inflammatory drugs and opiates, the most commonly used treatments for skeletal pain, can lead to renal, hepatic, respiratory, dependency, and/or functional status issues when used long term.
Furthermore, a limited understanding of the underlying mechanisms that drive skeletal pain hinders the development of targeted and effective treatments. This lack of understanding is, in large part, due to the paucity of preclinical skeletal pain models that closely mirror the severity and chronicity of human skeletal pain. The available surrogates for skeletal pain in preclinical models, such as limb use, weight bearing, nocifensive behaviors, or day/night activity, are often time and labor intensive compared to measuring skin hypersensitivity.
The Role of Skin Hypersensitivity in Skeletal Pain Assessment
Recent studies have indicated the presence of skin hypersensitivity in human patients and preclinical animal models of osteoarthritis, low back pain, and bone cancer pain. This has led to the exploration of whether measuring changes in skin hypersensitivity can serve as a reliable surrogate for measuring skeletal pain itself.
However, the relationship between skin hypersensitivity and skeletal pain remains unclear. While changes in the mechanical hypersensitivity of the skin can be detected in humans and animals with significant skeletal pain, it is uncertain whether these changes accurately reflect the intensity or nature of the underlying skeletal pain.
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Investigating the Link Between Skin Hypersensitivity and Skeletal Pain in a Murine Model
To delve deeper into this question, researchers have employed a murine model of cancer-induced bone pain (CIBP) to measure hypersensitivity of the skin and skeletal pain-related behaviors. This model involves injecting and confining GFP-transfected NCTC 2472 osteosarcoma cells unilaterally to the femur of C3H male mice, thereby generating skeletal pain.
In conjunction with this model, therapeutic administration of either anti-P2X3 or anti-NGF monoclonal antibodies was used to explore the effects on both skin hypersensitivity and skeletal pain-related behaviors.
Experimental Design and Methodology
The experiments were conducted on adult, male C3H/HeJ mice, approximately 8-9 weeks old and weighing 25-30 g at the time of tumor cell injection. These mice were chosen due to their histocompatibility with the NCTC clone 2472 sarcoma tumor line, which has been shown to form osteolytic lesions in bone following intramedullary injection.
The mice were housed in accordance with National Institutes of Health Guidelines and maintained in a vivarium at 22°C with a 12h alternating light-dark cycle, with ad libitum access to food and water.
Surgery was performed following induction of general anesthesia with ketamine/xylazine (100 mg/kg ketamine and 10 mg/kg xylazine; s.c.). An arthrotomy was performed, and a hole was drilled in the patellar groove of the femur using a pneumatic dental high-speed hand piece. A needle was inserted into the intramedullary canal to core a pathway for the sarcoma cells. Mice were then injected with Hanks buffered saline (HBSS) or 5 × 104 osteolytic murine sarcoma cells (NCTC 2472) transfected with green fluorescent protein (GFP) and suspended in HBSS. The injection site was sealed with a dental amalgam plug to confine the cells within the intramedullary canal, followed by irrigation with sterile saline. Muscles were secured back in position using a horizontal mattress suture to prevent patella displacement post-arthrotomy, and the incision was closed using wound clips.
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The anti-NGF antibody (mAb911) and the anti-P2X3 sequestering antibody (12D4) were administered at a dose of 10 mg/kg delivered i.p.
Animals were observed daily, and exclusion criteria included rapid weight loss (>20% in one week), patella displacement, lack of intra-femoral cancer cell growth by the end of the study, prolonged digestive abnormalities, severe ulcerative dermatitis or infected tumors, and paralysis.
Behavioral testing was performed at baseline (pre-sarcoma inoculation) and 21, 28, and 35 days post-sarcoma inoculation. Mice were assessed for cutaneous stimulus evoked-pain, spontaneous nocifensive behavioral indicators of pain, weight born by the ipsilateral hindlimb, and rearing activity.
Mechanical withdrawal thresholds were measured using calibrated von Frey monofilaments applied to the mid-plantar surface of the ipsilateral hindpaw. An algorithm was used to compute the 50% withdrawal threshold.
Time spent in nocifensive behavior was assessed by a blinded observer over a 300 second observation period. Nocifensive behaviors were defined as full guarding, reduced weight-bearing, tending to the affected limb, flinching the affected limb, and sporadic hopping.
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The percentage of weight borne by each limb was measured using a floor-instrumented dynamic weight bearing system.
The number of times an animal reared over a 300 second period was recorded as a measure of voluntary activity.
Immunofluorescence staining was performed on dorsal root ganglia (DRG) sections to assess the expression of P2X3 and TrkA.
Key Findings and Implications
The study revealed that anti-P2X3 significantly reduced hypersensitivity of skin, but had no significant effect on attenuating skeletal pain-related behaviors. In contrast, anti-NGF, which has been shown to reduce skeletal pain in both humans and animals models, attenuated both mechanical hypersensitivity of the skin and skeletal pain-related behaviors.
Animals injected with sarcoma cells and treated with vehicle developed significant mechanical hypersensitivity of the hind paw skin by day 21-post sarcoma cell injection, and paw withdrawal thresholds continued to decline for the duration of the study. This hypersensitivity did not extend to the tail, as tail flick latencies were not statistically different between any of the tested groups. Mechanical hypersensitivity was relieved when sarcoma injected animals were treated with either anti-P2X3 (on days 28 and 35) or anti-NGF (on days 21, 28, and 35).
These results suggest that while bone cancer can induce significant skeletal pain-related behaviors and hypersensitivity of the skin, relief of hypersensitivity of the skin is not always accompanied by attenuation of skeletal pain.