Quantifying Proprioceptive Neuron Preservation

To confirm cellular preservation of proprioceptive sensory neurons, we counted parvalbumin (PV)-immunoreactive (IR) cellular profiles in dorsal root ganglia (DRGs) ipsilateral and contralateral to the nerve crush. We analyzed L4 DRGs from 10 animals with a crush injury of the right sciatic nerve 14 d before. The DRGs were embedded in Tissue-Tek OCT, serially cut (15 μm thickness) with a cryostat, and collected onto gelatin-coated glass slides. All sections were first blocked with 10% normal bovine serum for 1 h, followed by overnight incubation at 4°C with primary antibodies, as follows: rabbit anti-parvalbumin (1:1000; catalog #PV28, Swant; RRID:AB_2315235); and mouse biotinylated anti-NeuN (1:200; catalog #MAB377B, Millipore; RRID:AB_177621). After washes, donkey anti-rabbit IgG Alexa Fluor 488 (1:200; catalog #A-21206, Thermo Fisher Scientific; RRID:AB_2535792) and Alexa Fluor 488-streptavidin (1:200; catalog #S32356, Thermo Fisher Scientific) were used to reveal IR sites. After 2 h of incubation at room temperature, the sections were thoroughly washed, mounted on slides, and coverslipped with Fluoromount-G (SouthernBiotech). DRG sections were visualized with a fluorescence microscope (model BX51, Olympus); at least four sections per DRG were captured at 40× with a digital camera (model DP50, Olympus) and CellSens Digital Imaging software (version 1.9; Olympus), and were merged using ImageJ software. The number of positive parvalbumin and all NeuN neurons were manually counted. NeuN was used to ensure that all cellular profiles counted were from mid-cell cross sections that included the nucleus. We then estimated the proportion of NeuN⁺ neurons that were parvalbumin⁺ in DRGs ipsilateral and contralateral to the nerve crush. This procedure normalized differences in cell numbers depending on the level and orientation of L4 DRG section cut. We analyzed four to five sections per DRG in six control L4 DRGs with an average (±SD) of 1560.0 ± 398.9 neurons sampled per ganglia and eight injured L4 DRGs with an average of 1533.6 ± 282.4 neurons per ganglia. Ganglia in which sectioning and immunocytochemistry processing did not allow recovery of four to five sections with >200 NeuN neurons per section were not included in the quantitative analysis to avoid errors in percentage estimates because of uneven clustering of parvalbumin-IR neurons in DRG or other possible biases in sections with small numbers of neurons.