Nerve Crush

Controlled optic nerve crush (CONC) was performed on adult mice as previously described [8 (link)]. The optic nerve was crushed approximately 3-6 mm behind the eye for 5 seconds using self closing forceps (Roboz RS-5027). Retinas were harvested at various time points after the procedure. RGC layer counts after CONC were performed on nissl stained retinas as described above. Unmanipulated contralateral eyes or contralateral eyes that had a sham surgery performed (no crush of the optic nerve) were used as control eyes.

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.

Loss of Ia/VGluT1 synapses requires a microglia reaction (Iba1⁺, CD11b⁺ cells) and activation of CCR2, which is also associated with infiltration of CD45⁺ CCR2⁺ CD11b⁺ peripheral macrophages and CD45⁺ CCR2⁺ CD3e⁺ T cells (see Introduction). The microglia reaction was analyzed by counting all Iba1⁺ cells in the ventral horn of the spinal cord at 3 d (n = 3), 5 d (n = 3), 7 d (n = 4), 14 d (n = 4), 21 d (n = 4), and 60 d (n = 2) after nerve crush injury at P10. To test for the presence of peripheral immune cells we tested for Iba1⁺, CD11b⁺, and/or CD45⁺ cells at 7, 14, 21, and 60 d after the injury. Transverse sections (50 μm thick) of L4-5 spinal cord were obtained in a sliding freezing microtome, collected free floating, blocked for 1 h with 10% normal donkey serum diluted in PBST, and incubated overnight at room temperature in a mixture containing a goat polyclonal antibody against Iba1 (1:500; Novus Biologicals; RRID:AB_521594) and a rat monoclonal antibody against CD11b (1:50; OX-43; catalog #M1/70.15.11.5.2-s, Developmental Hybridoma Bank) or a rabbit polyclonal antibody against CD45 (1:50; Abcam; RRID:AB_442810). Sections were washed in PBS and incubated for 2 h in donkey anti-goat IgG antibody conjugated to FITC and donkey anti-rat or anti-rabbit IgG conjugated to Cy3 (all secondary antibodies diluted 1:200 in PBST; Jackson ImmunoResearch). The sections were mounted on slides, coverslipped with Vectashield (Vector Laboratories), and imaged throughout with a confocal microscope (model FV1000, Olympus) using 1 μm z-steps and a 20× objective. Overlapping image tiles were captured to sample the whole spinal cord. At least three sections were imaged for each animal. We found no CD45⁺ cells, and therefore quantitative analyses focused on Iba1⁺ microglia. The ventral horn of each hemisection was manually selected by marking a region of interest (ROIs) limited dorsally by a straight horizontal line above the central canal and in all other directions by the border between gray and white matter. All Iba1⁺ cells inside these ROIs were counted using ImageJ software. Because the spinal cord changes in size with maturation, we calculated cell densities, as follows: the number of Iba1⁺ microglia divided by the ventral horn volume calculated from the ROI area multiplied by section thickness (50 μm). Densities ratios were obtained for each section between the side ipsilateral to the injury and the contralateral control side.