Validity of acute and chronic tactile sensory testing after spinal cord injury in rats

Abstract

Spinal cord injury (SCI) impairs sensory systems causing allodynia. Measuring the development of allodynia in rodent models of SCI is challenging due to spinal shock and marked motor impairments. Assessment of SCI-induced allodynia is not standardized across labs, making interpretation of results difficult. Therefore, we validated sensory threshold assessment after SCI and developed a novel assessment of allodynia prior to motor recovery in a rat SCI model. One hundred fifty-six Sprague-Dawley rats received T8 laminectomy or mild to moderate SCI using the OSU SCI device (0.3 mm to 1.3 mm cord displacement). To determine tactile thresholds, von Frey hairs (VFH) were applied in Up-Down or ascending order to the dorsal or plantar hindpaw. The most efficient and valid procedures that maintain high sensitivity and specificity were identified. Ten Up-Down VFH applications yielded stable thresholds; reducing the risk of threshold decay and unnecessary exposure to painful stimuli. Importantly, distraction of SCI-rats with food revealed differential decay of thresholds than when distraction is not provided. The new test uses dorsal VFH stimulation and is independent of trunk or hindlimb control. Acute dorsal VFH thresholds collected before recovery of hindlimb weight support accurately predicted plantar VFH thresholds measured at late timepoints (chi(2)=8.479; p<0.05). Thus, standardized testing early after SCI using the dorsal VFH test or later using 10 stimuli in the Up-Down test produces valid measures of tactile sensation across many SCI severities. Early detection of allodynia in experimental SCI will allow identification of mechanisms responsible for pain development and determine targets for therapeutic interventions.

Fig. 1

Presence (+) or absence (−) of paw withdrawal to 20 successive von Frey stimuli. In the Up–Down 20 and Dixon derived thresholds, the behavior of the hind paw to the stimulus influences the next force which is to be applied. When paw withdrawal is absent, stronger stimuli are applied until withdrawal returns. Then, lighter stimuli are delivered until the paw fails to withdraw. The perceptual threshold lies between the positive and negative responses. Once the threshold is crossed, this method should produce alternating positive and negative responses. The reported pain threshold for the Up–Down 20 method in this case would be 75.86 g (the lowest gram force that produced hind paw withdrawal at least 50% of its applications), while Dixon’s mathematical threshold estimate is bracketed by the positive and negative responses (53.17, solid line). Importantly, the precision of the derived threshold depends on the range of applied forces. Under normal conditions, there is a large range in applied forces due to the logarithmic design of the von Frey stimuli, producing less precise thresholds.

Fig. 2

Mean allodynic and non-allodynic tactile sensory thresholds grouped by percent white matter sparing (WMS) for von Frey Up–Down testing with 20 stimulus applications. The highest percentage of rats displaying allodynic thresholds (middle row) had <10% WMS. Note that the allodynic threshold remains constant regardless of the severity of SCI. Non-allodynic thresholds remained stable across all SCI severities despite a wide percentage of those responding (17–72%).

Fig. 3

Validation of Up–Down ‘20 von Frey hair testing protocol. Similar tactile sensory thresholds were derived when applying the Up–Down ‘20 VFH protocol or the gold standard Ascending method (n=12; R=0.90, p<0.001). Categorization of hind limb responses using Up–Down ‘20 and Ascending methods into allodynic or non-allodynic groups resulted in perfect agreement between the two testing paradigms (κ=1).

Fig. 4

Positive and negative responses to von Frey stimuli throughout a single testing session. (A) Representative example of non-allodynic response pattern during Up–Down 20 testing after spinal cord injury. The hind paw responds with alternating positive (+) and negative (−) responses successively, thereby bracketing the Dixon mathematical threshold estimate (solid line; 57.51). (B) Response patterns demonstrating 3 successive positive responses or threshold decay (denoted by the bracket) after SCI. Threshold decay drastically reduces the pain threshold and heightens sensitivity to tactile stimuli (Dixon estimate=20.99; solid line). It most often occurs in the latter half of the test session, during stimulation of the second hind paw. Normal oscillatory and decay patterns are seen irrespective of non-allodynic or allodynic classification.

Fig. 5

Maximizing distraction reduces the incidence of threshold decay. (C) Twenty-two percent of hind paws (n=292) tested with the Up–Down 20 method showed evidence of tactile threshold decay. In a subset of rats (n=11), tactile thresholds were measured with and without food distraction in the same SCI and naive rats. As expected, providing food distraction did not alter sensory thresholds. However, absence of food during sensory testing increased threshold decay by 28% (p<0.01). (A, B) Representative histograms depict a hind paw tested using Up–Down 20 methods 5 days apart with food in A and without food distraction in B. Note the robust threshold decay that occurs when food is not provided (string of positive (+) responses denoted by the bracket). The differential response that is elicited when distraction is or is not provided is indicative of some level of supraspinal processing.

Fig. 6

Early dorsal von Frey Hair (DVFH) sensory thresholds predict allodynic and non-allodynic tactile sensitivity across injury severities at later time points. (A) A side view of the technique to hold the rat while applying von Frey hairs to the dorsal surface of the hind paw. (B) Comparable sensory thresholds occurred between 7 dpo Dorsal VFH and 28 dpo Up–Down 20 measures after SCI. Remarkable similarity occurred for a wide range of SCI, with significant allodynia noted in more severe SCI (p<0.05 1.1 mm displacement vs. all other groups). (C) Strong agreement between the novel Dorsal VFH test and standard Up–Down methods is evident in a significant positive correlation (n=44; Pearson Coefficient=0.503, p<0.01, r²=0.67). Dashed lines show the cutoff threshold for allodynic sensation using either the new Dorsal VFH or Up–Down 20 techniques and indicate that allodynic thresholds determined at 7 dpo using the DVFH test predicts chronic below-level allodynia determined by Up–Down 20 methods.