Quantifying Excitatory Synapses in Dorsal Horn After Facet Injury

Spinal cord sections were immunolabeled with mouse anti-synapsin (1:100; Synaptic Systems; Goettingen, Germany) and rabbit anti-homer (1:200; Synaptic Systems; Goettingen, Germany) to quantify synapse densities in the dorsal horn at day 7 after facet joint injury. Excitatory synapses were quantified by counting puncta with co-localized synapsin and homer labeling, using previously published methods.^{7 (link),16} Briefly, image stacks were acquired from the dorsal horn at 0.33μm increments up to 3μm of depth using a Zeiss LSM510 confocal microscope. The maximum intensity projection of each set of three sequential images was used to generate a single image (0.02mm² tissue area) corresponding to 1μm of tissue depth. The Puncta Analyzer plugin for ImageJ (National Institutes of Health; Bethesda, MD) was used to identify puncta exhibiting co-localization of synapsin and homer in each maximum intensity projection. The area of the tissue parenchyma, excluding any holes and/or gaps in the tissue sections, was quantified using a custom MATLAB code.^{7 (link)} The number of co-localized puncta in the dorsal horn was normalized to the corresponding tissue area for each image. The excitatory synapse number per area was then averaged across all rats in each group and compared between groups using Student’s t-test.