Pain & Nociception — Animal Models & Behavioral Testing

The best model depends on your research question. For chronic inflammatory pain, CFA produces sustained hypersensitivity lasting 2–4 weeks. For neuropathic pain, SNI provides robust, long-lasting mechanical allodynia. The formalin test offers rapid screening with biphasic readouts distinguishing peripheral and central mechanisms. The hot plate test is optimal for acute supraspinal nociceptive threshold measurement and opioid screening.

Phase 1 (0–10 minutes) reflects direct chemical activation of peripheral C-fiber nociceptors and is sensitive to local anesthetics and peripherally acting analgesics. Phase 2 (15–60 minutes) involves central sensitization, spinal facilitation, and ongoing inflammatory mediator release — it is sensitive to centrally acting drugs including NSAIDs, opioids, and NMDA receptor antagonists. The interphase quiescent period (10–15 minutes) separates these mechanisms pharmacologically.

Mechanical allodynia in the SNI model develops within 24 hours of surgery and persists for at least 6 months, often longer. This prolonged time course makes SNI one of the most stable neuropathic pain models available. Cold hypersensitivity is also robust and long-lasting. The chronic nature allows longitudinal drug studies and investigation of pain chronification mechanisms.

Pain in mice is assessed through evoked and non-evoked measures. Evoked tests include von Frey filaments (mechanical threshold), Hargreaves test (thermal withdrawal latency), hot plate (supraspinal thermal nociception), and cold plate or acetone tests (cold allodynia). Non-evoked measures include spontaneous behaviors in the open field (locomotion, guarding), grimace scales, weight bearing, and conditioned place preference/aversion. Comorbidity assessments — elevated plus maze for anxiety and forced swim for depression — capture the affective dimension of chronic pain.

Central sensitization is an amplification of neural signaling in the spinal dorsal horn that produces pain hypersensitivity — touch becomes painful (allodynia) and painful stimuli feel more painful (hyperalgesia). CFA produces central sensitization by 24–48 hours post-injection. SNI induces rapid, persistent central sensitization. The formalin test Phase 2 involves transient central sensitization. The hot plate test does not produce central sensitization as it measures an acute supraspinal reflex.

CFA produces inflammatory pain in all standard laboratory mouse strains, but sensitivity varies. C57BL/6J is the most commonly used and best characterized strain. BALB/c mice show lower baseline mechanical thresholds and different inflammatory responses. CD-1 outbred mice offer genetic diversity but wider variability. Always run a pilot study when using CFA in a new strain and consider strain-specific differences in immune response and pain sensitivity.

The standard cutoff time is 30 seconds at 52–56 degrees C. This prevents thermal tissue damage to the paw while allowing sufficient dynamic range to detect both hyperalgesia (shorter latency) and analgesia (longer latency). Without a cutoff, analgesics could mask all pain responses and cause burns. The cutoff also serves as a data point — animals reaching cutoff are assigned the maximum value for statistical analysis.

Always include motor function controls alongside nociceptive assays. The rotarod test detects drug-induced motor impairment that could artificially increase hot plate latency or reduce formalin flinching. Open field locomotor activity identifies sedation. Grip strength testing rules out muscle relaxation. If a drug increases hot plate latency but impairs rotarod performance at the same dose, the apparent antinociception may be confounded by sedation rather than true pain relief.