Dissection microscope

Mouse Spleen T cells were centrifuged with Phosphate Buffer Saline (PBS) at 300 × g for 10 min. Pellet was resuspended in TexMACs (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) cell culture media (%3 human AB serum and 1% Pen/Strep). 500,000 cells in 100 µL were added into a microplate already coated with a monoclonal antibody specific for mouse IFN-γ. 1000 nM SARS-CoV-2 virus Peptivator pool (SARS-CoV-2 S, N, and M protein peptide pool) (Miltenyi Biotech, GmbH, Bergisch Gladbach, Germany) were added into each well including mouse spleen T cells. The microplate was incubated in a humidified 37 °C CO₂ incubator. After 48 h incubation, IFN-γ secreting cells were determined with Mouse IFNγ ELISpot Kit (RnDSystems, USA) according to the manufacturer’s instructions. The spots were counted under the dissection microscope (Zeiss, Germany).

At the end of the experiments, mice were sacrificed in deep isoflurane anesthesia and the ipsilateral nerve stump (common peroneal and tibial nerve) of the sciatic nerve (proximal to the nerve injury), the sural nerve, and the L4 and L5 DRG were dissected for quantitative real-time-PCR (qRT-PCR) using a dissection microscope (Zeiss, Oberkochen, Germany) at the following time points: baseline, 5, 7, 15 days after SNI (n = 6 mice/age-group). Tissue from naïve mice served as controls. Tissue was shock frozen in liquid nitrogen and was stored at −80°C before further processing. For immunohistochemistry the ipsilateral nerve stump (common peroneal and tibial nerve) of the sciatic nerve and the sural nerve (n = 5 per group) were dissected 7 days after SNI. Tissue was embedded in Tissue Tek, optimal cutting temperature (OCT) medium (Sakura, Staufen, Germany), frozen in 2-methylbutane cooled in liquid nitrogen and stored at −80°C until further processing.

Damage to the DG region of the hippocampus often prevents the growth of new born cells during the critical period of memory formation, so ultrastructural changes in the DG were observed using transmission electron microscopy. The DG tissue was extracted under a dissection microscope (Zeiss, Germany) and 1-mm³ tissue blocks were generated that were fixed and subsequently treated as described previously [32 (link), 33 (link)]. Morphological changes in nerve cells and synapses were observed using an AMTXR60 digital camera attached to a JEM 1400 transmission electron microscope. We identified mitochondria by the presence of distinctive cristae and a double membrane and mature synapses by the presence of the following features on at least one section: a postsynaptic density, at least three synaptic vesicles within 100 nm of the presynaptic membrane and a clearly defined synaptic cleft.

All procedures involving animals were approved by the Ohio State Institutional Animal Care and Use Committee in accordance with institutional and national guidelines. Nestin-GFP mice were provided by Grigori Enikolopov at Cold Spring Harbor Laboratory [31 (link)]. All mice were housed in a 12 h light–dark cycle with food and water ad libitum. For isolation of the dentate gyrus (DG), adult mice (6–9 week old) were anesthetized with an intraperitoneal injection of ketamine (87.5 mg/kg) and xylazine (12.5 mg/kg) before perfusion with PBS. Following perfusion, the brain was removed and placed in cold Neurobasal A medium (Gibco 10-888-022) on ice. After bisecting the brain along the midsagittal line, the cerebellum and diencephalic structures were removed to expose the hippocampus. Under a dissection microscope (Zeiss), the DG was excised using a beveled syringe needle and placed in ice cold PBS without calcium or magnesium (Gibco 10-010-049). DGs from mice were first mechanically dissociated with sterile scalpel blades and then enzymatically dissociated with a pre-warmed papain (Roche 10108014001)/dispase (Stem Cell Technologies 07913)/DNase (Stem Cell Technologies NC9007308) (PDD) cocktail at 37 °C for 20 min. Afterwards, the tissue was again mechanically disrupted by trituration for 1 min. Dissociated cells were collected by centrifugation at 500 g for 5 min.