Quantification of surviving neurons after contusion, dislocation, and distraction spinal cord injuries using automated methods

Abstract

This study proposes and validates an automated method for counting neurons in spinal cord injury (SCI) and then uses it to examine and compare the surviving cells in common types of SCI mechanisms. Moderate contusion, dislocation, and distraction SCIs were surgically induced in Sprague Dawley male rats (n = 6 for each type of injury). Their spinal cords were harvested 8 weeks post injury with 5 normal weight-matched rats. The spinal cords were cut, stained with anti-NeuN antibody and fluorescent Nissl, and imaged in the dorsal and ventral horns at various distances to the epicenter. Neurons in the images were automatically counted using an algorithm that was designed to filter non-soma-like objects based on morphological characteristics (size, solidity, circular pattern) and check the remaining objects for the double-stained nucleus/cell body features (brightness variation, brightness distribution, color). To validate the automated method, some of the images were randomly selected for manual counting. The number of surviving cells that were automatically measured by the algorithm was found to be correlated with the values that were manually measured by 2 observers ( P < .001) with similar differences ( P > .05). Neurons in the dorsal and ventral horns were reduced after the SCIs ( P < .05). Dislocation and distraction, respectively, caused the most severe damage to the ventral horn neurons especially near the epicenter and the most extensive and uniform damage to the dorsal horn neurons ( P < .05). Our method was proved to be reliable, which is suitable for studying different types of SCI.