Laminectomy surgeries were performed as previously described21 : Mice were anesthetized by an intraperitoneal injection of ketamine‐xylazine (87mg/kg/13mg/kg, respectively) or meditomidin 0.5mg/kg, midazolam 5mg/kg, and fentanyl 0.05mg/kg. Anesthesia was reapplied as needed (every 60–120 minutes). After a double dorsal laminectomy over the fourth lumbar, L4, and L5 segments, mice were suspended using compact spinal cord clamps.30 A well around the opening was built using 2‐4% agarose to hold aCSF (as above, glucose omitted or alternatively, in mM: 148.2 NaCl, 3.0 KCl, 0.8 Na2HPO4, 0.2 NaH2PO4, 1.4 CaCl2, and 0.8 MgCl2). An imaging window free from dura was established in the imaging area using a bent hypodermic needle. In some experiments, astrocyte death was verified by addition of ethidium homodimer‐1 (1:500, 0.56mg/ml stock; Invitrogen, Carlsbad, CA).
In vivo imaging of the lumbar spinal cord was performed as previously described.21 , 31 Briefly, stacks were acquired using two‐photon microscopes (Olympus FV1000 MPE or FVMPE‐RS tuned to 910 nm for green fluorescent protein [GFP] alone and 980nm for GFP/OFP [orange fluorescent protein], equipped with a ×25/1.05 N.A. water‐dipping cone objective). All light was first filtered through a 690 nm short‐pass dichroic mirror. Fluorescence was collected using a G/R filter set (BA 495‐540, BA 570‐625) mounted in front of gallium arsenide phosphide photomultiplier tubes. Time‐lapse stacks were acquired at 3‐minute intervals for 3 hours (astrocyte loss) or at 15‐minute intervals for 6 hours (axon damage) with the following parameters: 35 images (zoom 1.8–2.0; pixel size: 0.28–0.38 µm) with 1‐µm z‐step. Diluted heat‐inactivated serum/plasma was applied every 30 minutes, supplemented with 20% of non‐heat‐inactivated HD serum as a complement source. Compared to experiments with acute brain slices, a higher concentration was needed, most likely because of restricted penetration and possibly the presence of complement blockers in vivo. Serum/plasma was present in the spinal opening for the initial 90 minutes of the experiment, after which it was replaced with aCSF (at 90 minutes and renewed every 60 minutes until the end of the experiment). Transient delivery of the NMO sample and complement source was designed to induce an acute, well‐defined injury and reduce consumption of sparse patient‐samples. In a set of control experiments using NMO1 samples and HD serum for up to 8 hours continuously, we found no qualitative difference between prolonged and transient applications, although, as expected, astrocyte depletion and the degree of axon swellings was more pronounced after longer exposure to patient IgG and complement. To confirm AQP4 specificity, we also performed experiments using a human IgG1 recombinant antibody rAb 7‐5‐53 reconstructed from a clonotypic plasma blast obtained from the CSF of an NMO patient (r‐AQP4‐IgG)9 . r‐AQP4‐IgG was applied at 1.5 μg/ml of IgG supplemented with 20% of non‐heat‐inactivated HD serum. The rAb ICOS‐5‐2, a divalent human IgG1 antibody of unknown specificity, developed from a chronic meningitis patient, served as an isotype control (r‐ctrl‐IgG).
We have previously demonstrated that phototoxicity does not appear to have a measurable impact on the health of spinal axons under such imaging conditions.21 , 22 , 31 , 32 Moreover, histological analysis confirmed NMO serum‐/plasma‐mediated astrocyte loss in the absence of oligodendrocyte loss in superficial spinal cord layers when no imaging was done (see below and cf. Fig 1 G,H). Additionally, axonal pathology could be confirmed in experiments in which only an initial and a final image (at 6 hours) were taken (cf. Fig 4 B). To rule out a spurious influence of the transgenic labeling of axons, we performed experiments without imaging using wild‐type (C57BL/6) and transgenic animals and antitubulin staining (see below). The analysis was performed by a scorer blind to the treatment conditions and genotypes and showed a comparable percentage of axonal swellings in spinal cords treated with NMO serum/plasma irrespective of genotype (data not shown).
In vivo imaging of the lumbar spinal cord was performed as previously described.
We have previously demonstrated that phototoxicity does not appear to have a measurable impact on the health of spinal axons under such imaging conditions.
