Animals and reagents

All animal procedures conformed to local ethical standards and ARRIVE guidelines. UK home office and Maltese national regulations were followed as appropriate. For RON experiments, nerves were dissected from juvenile (P8–12) or adult (P90–120) Wistar rats, adult (P80–110) PLP-GFP or C57 wild-type mice. Acute coronal vibratome-cut sections were cut in oxygenated, ice-cold cutting solution (in mM): NaCl, 92; KCl, 2.5; NaH 2 PO 4 , 1.2; MgSO 4 , 2; CaCl 2 , 2; NaHCO 3 , 30; glucose, 25; Hepes, 20; Na Pyruvate, 3; Thiourea, 2; Na Ascorbate, 5; pH, 7.4. Experiments were performed in artificial cerebrospinal fluid (aCSF), composition (in mM): NaCl, 126; KCl, 3; NaH 2 PO 4 , 2; MgSO 4 , 2; CaCl 2 , 2; NaHCO 3 , 26; glucose, 10; pH, 7.45, bubbled with 5% CO 2 /95% O 2 and kept at 37 °C. KCl (47 mM) was added to aCSF (NaCl replacement) for “High K+” experiments. For zero-Ca2+ aCSF, CaCl 2 was omitted, and 50 μM EGTA was added. For zero-Na+ aCSF, Na+ was replaced with NMDG. Oxygen–glucose deprivation was used as the model of ischemia for CAP and confocal imaging experiments: aCSF was replaced by a glucose-free aCSF (+10 mM sucrose to maintain osmolality) saturated with 95% N 2 /5% CO 2 . The chamber atmosphere was switched to 5% CO 2 /95% N 2 during OGD perfusion. Osmolarity of solutions was measured and adjusted as required. NBQX was purchased from Tocris (UK), PPDA and QNZ-46 from either Tocris or Abcam (UK), bafilomycin from Viva Bioscience (UK); all other reagents were from Sigma (UK) including carbenoxolone (CBX), 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB), sulfasalazine (SAS) and threo-beta-benzyloxyaspartate (TBOA).

In vivo treatment for in vitro recording

Adult C57 mice were injected i.p. with 200 µl of 50% DMSO solution containing 1 mM β-cyclodextrin +20 mg/kg QNZ-46, or a vehicle control without the QNZ-46. Injections were performed blind by animal house staff and mice were left for 240 min on a warming pad. Other than mild sedation in both groups attributed to the DMSO, the animals showed no signs of distress or aberrant behavior. RON or brain slices were collected as above, in solutions that did not contain QNZ-46.

Electrophysiology

RON CAPs were evoked and recorded with glass electrodes and the rectified area used to determine changes in conduction. CAPs were evoked via square-wave constant current pulses (Iso stim A320, WPI), amplified (Cyber Amp 320, Axon Instruments), subtracted from a parallel differential electrode, filtered (low pass: 800–10,000 Hz), digitized (1401 mini, Cambridge Electronic Design) and displayed on a PC running Signal software (Cambridge Electronic Design). Non-recoverable CAP loss from the RON indicates irreversible failure of axon function. Glutamate microelectrode biosensors (Sarissa Biomedical, Coventry, UK), amplified via a Duo-Stat ME-200+ potentiostat (Sycopel International, London, UK), were used to record glutamate concentration. Signals were differential to a null electrode and both active and null electrodes were gradually inserted into brain slice or RON, in the latter case through a small incision in the nerve sheath. An Ag/AgCl reference electrode was introduced into the bath. Sensors were calibrated in the chamber at the end of each experiment. Values from the null, sensor, and sensor-minus-null outputs were recorded at 0.5 Hz and subsequently converted into glutamate concentration (Supplementary Fig. 4). Recorded glutamate was high following electrode placement, presumably due to localized tissue damage (Supplementary Fig. 4), and declined to a low stable concentration over a 120–180 min (neonatal RON), 300–420 min (adult RON) or 180–240 min (slices) rest period before experiments were initiated. Nerves may have been pre-treated with 50 μM BAPTA-AM for zero-Ca2+ experiments (no difference was detected and data were pooled). Corpus callosum glutamate sensor experiments were conducted on 400 µm sections from adult Wistar rats, adult (P80–110) THY-1/YFP (Line H), GFAP-GFP or C57 wild-type mice. Slices were gradually warmed to 37 °C over ~60 min and rested for 120 min prior to use. Unless otherwise stated, preparations were maintained in an oxygenated (1.5 l/min) interface perfusion chamber (Harvard Apparatus Inc.) and continuously superfused (0.6–1 ml/min) with aCSF. Due to the oxygen-sensitivity of the glutamate biosensors, chemical anoxia (1 nM rotenone for neonates/25 µM antimycin-a for adults) + aglycemia was used as the model of ischemia for [glutamate] e recordings.

Two-photon confocal imaging

After deep isoflurane anesthesia and decapitation, the brain was rapidly removed into chilled aCSF supplemented with 75 mM sucrose and vibratome sectioned into coronal slices (400 μm thick) from the genu of the corpus callosum through the caudal extent of the hippocampus. Immediately after sectioning, slices were transferred to a Haas-type interface brain slice chamber (Harvard Apparatus, South Natick, MA) and allowed to recover at room temperature in aCSF for 60 min. Slices were transferred to a mini submerged chamber (0.5 ml) with a coverglass bottom (Warner Instrument Corporation, Hamden, CT) mounted on an upright BX50W1 Olympus Multiphoton microscope (Olympus, Tokyo, Japan) and perfused with room temperature aCSF at 3.5 ml/min. Final temperature control (37 ± 1 °C) was maintained using an in-line heater (Warner Instrument Corporation, Hamden, CT) equipped with a feedback thermistor placed in the chamber and the temperature raised gradually over 60 min. The multiphoton system housed Keplerian beam expanders with IR introduction light paths. A mode-locked MaiTai HP DeepSee laser system (Spectra-Physics) with a tuneable Ti: sapphire oscillator (690–1040 nm) was used as the excitation light source (pulse width < 100 fs; pulse repetition rate 80 Mhz) and controlled through an acousto-optical-modulator. The Group Velocity Dispersion was electronically compensated by a prism-coupled pre-chirper and the beam diameter adjusted by a Keplar telescope. Image acquisition was performed using the Olympus FluoView software.

Vesicular imaging was conducted on brain slices from transgenic animals using FM4-64. Corpus callosum slices were initially superfused with aCSF + 10 µM FM4-64 for 10 min. Slices were then subjected to a 50 mMK+ aCSF + 10 µM FM4-64 for 5 min, and subsequently returned to aCSF + 10 µM FM4-64 for a further 20 min. Next, slices were washed in aCSF (without FM4-64) for 15 min and a suitable region of the corpus callosum was identified. Finally, slices were exposed to 50 mMK+ aCSF for 10 min (or ischemia for 30 min) to promote vesicular fusion/FM4-64 unloading. Images were acquired every 10 s following laser excitation at 890 nm and collected using standard red and green filter settings. Mean pixel intensity within YFP(+) axons or GFP(+) astrocytes was determined using Olympus FluoView software, with the regions of interest determined by the green profile of axons/glia. Fluorescent emission scans during excitation at 405 nm were conducted using 10 nm bin width and the lambda-scan function, with 10 mM QNZ-46 in DMSO diluted 50:50 in immersion oil.

Single-photon imaging

Live imaging of NMDA-receptor-mediated myelin injury: PLP-GFAP RONs were exposed to FluoroMyelin Red (5%; 100 min, 5 °C in cutting solution) with or without drug, prior to dye wash-out and mounting in a temperature-controlled perfusion chamber (Warner Instruments, Hamden, CT, USA). RONs were superfused at 2 ml/min with aCSF with or without drug and imaged in a single plane on an inverted Nikon TE2000-U microscope. Imaging was achieved via spinning disk confocal imaging (Crest-Optics, X-light) and collected via MetaMorph software (Molecular Devices). FluoroMyelin Red and GFP was consecutively imaged following excitation of both at 488 nm using a 650 ± 50 nm BP filter (Thorlabs, Newton, New Jersey, USA) and a standard GFP filter set (Chroma Technology Corporation, Bellows Falls, VT, USA), respectively. RONs were imaged 20 min prior to switching to a 60-min period of OGD and then switched back for 60 min of recovery. Myelin decompaction was assessed using intensity line-plots drawn perpendicular to the longitudinal axis of individual myelin sheaths using ImageJ (NIH). Pre-OGD myelin sheath width was not significantly different in control and treated preparations and the data were pooled. Single-photon, laser scanning confocal images of QNZ-46 bath-loaded adult mouse ON and 200 μm coronal brain sections (P90–120) Wistar rats, (P80–110) PLP-GFP or C57 mice and brain sections from QNZ-46-injected mice, were collected using a Leica TCS SP8 microscope, using loading protocol and settings for GFP and FluoroMyelin Red comparable to those used for spinning disk above. QNZ-46 was imaged following excitation at 405 nm using filter settings standard for DAPI emission. Wide-field QNZ-46 imaging was performed using an Olympus epifluorescence microscope and a standard DAPI filter set; emission was quantified from the whole nerve section using ImageJ.

Measurement of myelinic vesicular-mediated Ca2+ increase: brain sections were prepared from adult (p100-p160) CD1 WT mice and dye loaded at room temperature (22 °C) for 2 h in the presence or absence of bafilomycin in continuously bubbled aCSF (5% CO 2 /95% O 2 ) containing 10 μM X-Rhod-1 AM and 1 μM DiOC68. Subsequently, sections were maintained in DiOC6-containing aCSF with or without bafilomycin until imaging. Dye-loaded sections were placed in a perfusion chamber and continuously superfused at 1 ml/min with bubbled aCSF or high-K aCSF (50 mMK+, see above). Single-photon laser scanning confocal images of corpus callosum myelinated axons were collected using a Leica TCS SP8 microscope at 37 °C. Excitation/emission wavelengths was 488/492–560 nm for DiOC6 and 561/585–655 nm for X-Rhod-1. Polygonal ROIs of DiOC6 and X-Rhod-1 dual-loaded axons were selected to exclude adjacent X-Rhod-1-loaded cell bodies. X-Rhod-1 intensity was normalized to the pre-high-K signal and data from individual mice were averaged for a single n (1–3 slices per mouse).

Transient middle cerebral artery occlusion

Eleven male Thy1-YFP and 15 C57 wild-type mice weighing 25−30 g were analgesized with buprenorphine (0.03 mg/kg b.w. i.p.) 2 h before surgery and subsequently anesthetized with isoflurane (3% initial, 1.0–1.5% maintenance) and 60% NO in O 2 . Animals were maintained normothermic (37 ± 0.5 °C) by means of a servo controlled heating blanket (Harvard Apparatus, Holliston, MA) with rectal temperature monitoring. Pulse oximetry (Spo2), heart rate, and respiratory rate were monitored continuously (STARR Life Sciences Corp., Allison Park, PA), along with systemic blood pressure via a Kent CODA® Standard tail-cuff blood pressure system (Kent Scientific Corporation, Torrington, USA). A fiber-optic probe (VP10M200ST, Moor Instruments Ltd, Axminster, UK) was affixed to the skull over the middle cerebral artery for measurement of regional cerebral blood flow using a moorVMS-LDF Laser Doppler System. Under microscope, the left common carotid, internal carotid, and external carotid arteries were exposed through a midline neck incision. The proximal portions of the left common carotid and the external carotid arteries were ligated and a 6–0 silicon-coated nylon suture (60SPRePK5-21910, Doccol Corporation, Massachusetts, USA) introduced into the internal carotid artery to occlude the middle cerebral artery at its origin. Mice were allowed to recover from anesthesia in a warm recovery cage throughout the duration of occlusion. After 60 min, reperfusion was obtained by withdrawal of the suture. Middle cerebral artery occlusion was confirmed by a sudden drop in relative cerebral blood flow to approximately 85% less than baseline measurement. Animals were subsequently administered buprenorphine for analgesia and placed in a recovery cage for 2 h before returning to their home cages with free access to food and water. Plasma glucose concentration was measured before tMCAO, 5 min and 24 h after reperfusion. Sham-treated animals received all surgical procedures but the filament was not inserted into the MCA. For animals pretreated with either QNZ-46 or vehicle (50:50;DMSO/β-cyclodextrin), the active drug or carrier was administered intraperitoneally 120 min before tMCAO.

Clinical evaluation

Body weight (g) was recorded prior to surgery and at 24 h for sham-, vehicle- and drug-treated groups. While a slight weight increase was observed in sham controls, experimental groups demonstrated weight loss after 24 h. Ischemia-induced weight loss was attenuated in drug-treated mice compared to vehicle group, although this was not significant (vehicle vs. drug-treated group: 5.30 ± 0.38 g vs. 4.58 ± 0.33 g).

Modified Bederson score

Neurological performance43 was assessed by two independent and blinded investigators 24 h post tMCAO according to the following scoring system: 0, no neurological deficit; 1, forelimb flexion; 2, decreased resistance to lateral push; 3, unidirectional circling; 4, longitudinal spinning; 5, no movement.

Corner test

The corner test detects abnormalities of sensory and motor function including vibrissae, forelimb and hindlimb use, and postural motor function44. The apparatus consisted of two cardboard boards each with a dimension of 30 × 20 × 1 cm3. The edges of the two boards were attached at a 30° angle with a small opening along the joint to encourage entry into the corner. The mouse was made to enter between the two boards facing the corner. As the animal progressed into the corner, both sides of the vibrissae were stimulated together and the mouse reared forward and upward, and then back to face the open end of the boards. The direction towards which the mouse turned was recorded for a total of ten trials per animal. Recordings were performed at baseline (prior to surgery) and again at 24 h reperfusion.

Wire hanging test

Motor function was evaluated using a wire hanging test for grip strength, balance, and endurance. This test is based on the latency of a mouse to fall off a metal wire upon exhaustion. The apparatus consisted of a 2-mm-thick metallic wire stretched between two poles held 50 cm above the ground, with a pillow in between to prevent injury upon falling. The mouse’s hindlimbs were covered with adhesive tape to prevent the animal from using all four paws. The mouse was trained to suspend its body by holding on to the wire. Pre-training was performed for 2 consecutive days before and on surgery day, prior to obtaining baseline values. The mouse was suspended for short intervals (10–20 s) for several trials and then returned to its cage. If the mouse fell before the end of the trial, it was immediately returned and allowed to grasp the wire. Mice were only returned to their home cage once training was completed. Experimental recordings were taken at baseline (prior to surgery) and again at 24 h reperfusion. Suspension time was measured for three trials per session with a maximum of 60 s per trial and a 30-s recovery period in between.

Infarct volume

Lesion volume was assessed 24 h post-MCAO following neurological assessment. Brains were removed, sectioned at 2 mm using a brain matrix and incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich) in saline for 20 min at 37 °C. Four slices per mouse were imaged and processed for infarct volume evaluation by blinded manual area tracing using digital imaging and image analysis software (ImageJ). For each section, the unstained area was defined as the ischemic lesion while red-stained areas delineated viable tissue43. Direct infarct volume was calculated by linear trapezoidal extrapolation (Cavalieri principle); for each brain sample, volume of infarction was calculated by integrating the area of damage at each stereotactic level and the slice thickness. An indirect/ corrected infarct volume was then calculated to compensate for the space-occupying effect of brain edema. For each brain, an edema index was calculated by dividing the sum of ipsilateral hemisphere volumes by the sum of contralateral hemisphere volumes. The actual infarct volume adjusted for edema was then determined by dividing the direct infarct volume by the edema index45.

Tissue processing and histological assessment

Thy-1-YFP mice subject to sham-, vehicle- and drug-treated tMCAO were killed (ketamine/xylazine), transcardially perfused (4% paraformaldehyde in PBS) and the brains were removed, post-fixed (24 h at 4 oC), cryopreserved (30% sucrose for 72 h at 4oC), embedded (low-melting point agarose, Sigma-Aldrich, A0576), and chilled. Free-floating 20 µm coronal sections (compresstome VF-300, Precisionary Instruments Inc., San Jose, California) were mounted onto gelatin-coated slides and YFP-imaged (EVOS Auto FL, ThermoFischer Scientific) using the appropriate green (496/518) filter set. The sections were immediately subsequently stained with 0.1% luxolfast blue (LFB, Solvent Blue 38, Sigma-Aldrich)/0.25% cresyl violet (Sigma-Aldrich) and myelin-imaged bright field. The mean optical density (OD) of the LFB stain was used as a measure of myelin integrity46. For each section, two ×20 and five ×60 non-overlapping images from homologous ipsilateral and contralateral external capsule were acquired and processed for OD using ImageJ software. OD calibration was carried out such that pixel values, usually in gray level units, were in OD. Each image was converted to 8-bit and the ROI was outlined manually. Any incidental border region of the cortex was excluded from analysis. OD was measured from ipsilateral and corresponding contralateral hemispheres and expressed as a ratio.

Electron microcopy

For brain sections: 24 h following the standard tMCAO protocol mice were anesthetized and transcardially perfused for 5 min with cold 4% PFA in 0.1 M Sørensen’s solution. The brain was then removed, sectioned at 2 mm using a brain matrix and incubated in 2% TTC for 20 min and assessed for lesion volume as above. Sections were then immersion-fixed in 2.5% gluteraldehyde/0.1 M Sørensen’s overnight and stored in Sørensen’s solution. The external capsule-motor cortex border region from both hemispheres was excised prior to post-fixation (1% osmium tetroxide), serially dehydration and epoxy infiltration. Ultrathin coronal sections (50–70 nm) were counterstained with uranyl acetate and lead citrate prior to blind examination using a Jeol 100CX electron microscope. Micrographs were analyzed for axon G-ratio by hand tracing (ImageJ, NIH) the external and internal myelin profile of all X-S axons within a micrograph and converting the area to idealized circles. Somata viability was assessed using a basic scoring system where one point was awarded for each of: (a) an intact cell membrane; (b) the presence of undamaged organelles such as mitochondria; (c) a normal nuclear morphology; and (d) the presence of clear cytoplasm. The external capsule-motor cortex border region of three vehicle-treated and three QNZ-46-treated mice were analyzed.

For RON: following either 30 min OGD (for long-sectioning: L-S) or 60 min OGD + 60 min recovery (for cross-sectioning: X-S), adult mouse nerves were immersion-fixed in 4% PFA in Sørensen’s for 5 min followed by 2.5% gluteraldehyde/0.1 M Sørensen’s overnight and storage in Sørensen’s. A minimum of four grid-sections in each of a minimum of three nerves were analyzed blind by hand including the tracing of axons and myelin profiles (X-S), axoplasmic vesicle counting (L-S) and focal myelin damage (L-S). G-ratio in X-S was calculated as above; vesicle counting was performed in L-S to allow vesicles to be distinguished from microtubules. Focal myelin injury was scored: 0 = normal compact myelin; 1 = one layer of myelin splitting; 2 = myelin bubbling involving multiple lamella; 3 = complete myelin breakdown.

Statistics

Data are mean ± SEM, significance determined by ttest or ANOVA with Holm−Šídák post hoc test as appropriate; Mann−Whitney U test was performed for nonparametric variables (Modified Bederson Score). CAP and biosensor recordings were stable over long periods of control recording and glutamate receptor antagonists had no effect under control conditions (Supplementary Fig. 6). For long periods of drug pre-treatment, control experiments were performed with the identical protocols to allow direct comparison. *P = <0.05 (rounded three decimal places), **P = <0.01 (rounded three decimal places), ***P = <0.001 (rounded four decimal places). Sample sizes for experimental groups are based on power calculations using established variability. Data from all completed experiments are included and no outliers were excluded. Two animals subjected to tMCAO died post-operatively and were not counted. Sham and test tMCAO trials were alternated and the experimenters were blinded to the contents of the injection; the micrographs generated from these experiments were analyzed blind (REF) and only unblinded once analysis was complete. All test experiments were intercalated with the relevant controls and where possible trials were conducted blind, including all in vivo treatments.

Data availability

All relevant data are available from the authors upon request.