RAT L5/L6 SPINAL NERVE LIGATION MODEL

Animals

Studies are conducted in adult male and female Sprague Dawley rats (Envigo) in an AAALAC-accredited facility with approval from an Institutional Animal Care and Use Committee, and implemented in accordance with the Guide for the Care and Use of Laboratory Animals 8th Edition (National Research Council, 2011) with standards set by the National Institutes of Health.

L5/L6 surgery

The rat spinal nerve ligation (SNL) is an established model of neuropathic pain induced by a tight ligation of spinal nerves (Kim and Chung, 1992).

Under anesthesia, the lumbar (L) L5 and L6 spinal nerves are ligated while L4 is left untouched, inducing long-lasting mechanical and cold allodynia while minimizing motor impairments. Animals are carefully monitored throughout the experiment and rats showing motor deficits (e.g., foot drop) are removed from the study.

Study design

In the L5/L6 SNL model, mechanical allodynia using von Frey filaments and cold allodynia using acetone evaporation are assessed using appropriate group sizes determined by power analysis. Male and female rats (n=8-10) are evaluated in separate cohorts. Dose-responses are investigated, and vehicle and positive control groups are included to establish the validity of the experiment. The experimenter is blinded to the treatments, and dosing is performed by an independent experimenter.

The area of the hind paw tested with von Frey filaments or acetone is located in the center of the plantar surface, avoiding the foot pads (X on Figure 1A). Whenever possible, mechanical and cold allodynia are tested in the same cohort of animals (Figure 1B).

Experimental outcomes

Hind paw mechanical allodynia testing using von Frey filaments

Paw withdrawal threshold (PWT) is assessed using the up-down method (Chaplan et al., 1994) for both the ipsilateral and contralateral hind paws prior to surgery (Figure 1B). Beginning 24-hours after the L5/L6 SNL surgery, animals are primed by performing the standard up-down testing procedure on both hind paws. Post-surgery PWT baseline is then determined, and animals with consistent ipsilateral PWT ≤3.0g are included in the subsequent testing of the asset’s efficacy to treat mechanical allodynia in SNL rats. Animals are balanced across treatment groups based on body weight and post-surgery PWT.

Hind paw cold allodynia using acetone evaporation

The number of positive responses to acetone trials (Choi et al., 1994) is assessed for both the ipsilateral and contralateral hind paws prior to surgery (Figure 1B). Post-surgery, the number of positive responses to acetone trials is determined and animals with ipsilateral scores ≥2 are included in the subsequent testing of the asset’s efficacy to treat cold allodynia in SNL rats. Animals are balanced across treatment groups based on body weight and post-surgery number of positive responses to acetone trials.

This work was conducted by PsychoGenics Inc. (Paramus, NJ) in collaboration with PSPP, NINDS, NIH under contract # 75N95019D00026