Experimental spinal cord injury: quantitation of axonal damage by automated image analysis

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Abstract
The severity of acute experimental spinal cord injury in rats was assessed quantitatively with the aid of an automated image analyzer by measuring the amount of degenerating axons that had developed distal to the site of mechanical insult. Spinal cord injury was produced in adult male rats by epidural compression at T-11 with a Biemer vascular clip. On the 7th postoperative day, the animals were graded according to the degree of hindlimb motor deficit, as follows: Grade 0: normal (three rats); Grade 1: crawling with difficulty (10 rats); Grade 2: some voluntary movement (nine rats); and Grade 3: no voluntary movement (nine rats). The rats were then sacrificed. The L-6 segment was chosen for selective silver impregnation of degenerating axons by the Fink-Heimer method. Silver grains, representing degenerating axons and their terminals, were accumulated in the descending tracts and in Rexed's laminae VII and VIII. The extent of axonal damage was expressed by the percentage of the area occupied by silver grains in Rexed's lamina VIII. The area occupied by silver grains was 17.0% (mean) in Grade 0 rats, 22.3% +/- 2.63% (mean +/- standard deviation) in Grade 1 rats, 28.7% +/- 3.35% in Grade 2 rats, and 35.9% +/- 2.76% in Grade 3 rats. The severity in Grade 3 rats was close to that of rats with transected cords (37.6% +/- 0.89%). The differences among the groups were statistically significant (p less than 0.001). This method may serve as a useful tool for the objective assessment of therapeutic modalities in large series of small experimental animals.