All our studies were approved by the Animal Experimentation Committee and the Gene Recombination Experimentation Committee at Sapporo Medical University.
Primary Dorsal Root Ganglion (DRG) culture
Rat DRG cultures were prepared and maintained as described previously, with minor modifications [Fex Svenningsen et al. 2003; Kikuchi et al. 2018; Kiryu-Seo et al. 2010]. Briefly, DRGs were dissected from the embryos of pregnant Sprague-Dawley rats on the 18–19th day of gestation. The DRGs were collected in L-15 medium and digested with 0.25% trypsin in Hank’s Balanced Salt Solution (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C for 30 minutes (min). Digestion was stopped using Dulbecco’s Modified Eagle’s Medium (Wako Co., Ltd., Tokyo, Japan) containing 10% fetal calf serum. The cells were then mechanically dissociated using a Pasteur pipette. The dissociated cells were resuspended in culture medium. (Neurobasal medium containing 2% B27, 0.3% GlutaMax, 10 ng/mL nerve growth factor, and penicillin/streptomycin; Thermo Fisher Scientific Inc.) One week after harvest, ascorbic acid was added to the medium. The cells were maintained for another 5–6 weeks with change of medium every 2–3 days. In this way, we maintained DRG cultures for 42 days in vitro (DIV) before observation.
To observe mitochondria in live cells, we used lentivirus prepared using ViraPower HiPerform Lentiviral Expression Systems (Invitrogen) following the manufacturer’s protocol and our previous report [Kikuchi et al. 2018]. Briefly, the vector containing the sequences targeting mitochondria with DsRed2 (Mito-DsRed2) was purchased from Clontech (Mountain View, CA, USA). The Mito-DsRed2 vector was amplified using polymerase chain reaction (PCR) using forward primer, 5’–CACCACCATGTCCGTCCTGACGCCGCTG–3’ and reverse primer 5’–ACTACAGGAACAGGTGGTGGCGGCCCTC–3’. The PCR products were subcloned into the pLenti6.3/V5 lentiviral vector using the TOPO cloning technique. The construct and packaging plasmids were transfected into 293 FT cells using Lipofectamine 3000 (Invitrogen). The viral supernatants were collected 48 hours after the transfection, filtered using 0.45-μm polyvinylidene membranes (Millipore, Billerica, MA, USA), and kept at -80°C until use.
Observation of mitochondria in DRG axons and oxygen-glucose deprivation
After three to four weeks of DRG culture, the lentivirus was added to the cells. Another two to three weeks after virus transfection, mitochondrial dynamics were measured using an inverted microscope (Axio Observer Z1, Carl Zeiss, Inc., Germany). This microscope has a micro-incubator (Tokai Hit Co., Ltd., Fujinomiya, Shizuoka, Japan) used to keep temperature at 37.0 ± 0.5°C and a 100% humid environment. Time-lapse images of mitochondrial dynamics were collected every 6 seconds for a total of 200 images. Before Oxygen-glucose deprivation (OGD) exposure, mitochondrial dynamics were recorded as pre-exposure (OGD-pre). After the observation of OGD-pre, the culture medium was then replaced with glucose-free medium and initial gas mixture containing 95% air and 5% CO2 was changed to 1% O2 and 5% CO2 regulated by N2 for OGD. After 1, 2, 3, 4, 5 and 6 hours (hr) OGD exposure (OGD-1hr—6hr), the behavior of mitochondria in the same axon were recorded and compared to that OGD-pre. For control, after the observation of same condition to OGD-pre (Cont-pre), the culture medium was then replaced normal DMEM, including glucose, and kept the initial gas mixture containing 95% air and 5% CO2. The timing to collect the mitochondrial dynamics was same to OGD-1hr—6hr (Cont-1hr—6hr). To count the numbers of motile mitochondria and measure the length of stationary mitochondria in the axon, a kymograph was generated using the sequential images obtained using Fiji.
Our experimental paradigm for in vitro OGD treatment of DRG cultures is shown in Fig. 1A.
Quantification of mitochondrial dynamics
Time-lapse images were processed using Fiji software, and kymographs were generated as described previously [Miller and Sheetz 2004]. We measured the number of motile mitochondria for 20 min and the lengths of stationary mitochondria in the kymograph. Stationary mitochondria were represented by vertical lines on the kymographs and motile mitochondria appeared as diagonal lines. The lengths of stationary mitochondria were measured as the vertical length labeled by DsRed2 at each stationary mitochondrion and converted to micrometers (Fig. 1B).
Transmission electron microscopy
We used cultured DRGs with decreasing length of axonal mitochondria by six-hour OGD exposure, along with sham-treated controls, to study the detailed structures of the axonal mitochondria. Both samples were fixed using 4% PFA containing 1% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4°C. After washing with distilled water, the enhanced samples were post-fixed in 1% OsO4 for 90 min at room temperature. The samples were then dehydrated in graded ethanol solutions, embedded in epoxy resin, and cut into ultrathin sections using an ultramicrotome (MT6000; Dupont, Wilmington, DE, USA). The ultrathin sections were then stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope (JEM-1400, JEOL Ltd., Tokyo, Japan).
To assess neural condition, nucleus in neural cell body was observed. Aspect ratio of axonal mitochondria is compared as [(major axis) / (minor axis)] with Fiji by manual tracing, to clear morphological change to sphere in mitochondria by OGD exposure [Picard et al. 2013].
Values are presented as means ± standard deviations (SD) in the text and error bars show means and the 95% confidence interval in figures. Statistical analysis was undertaken using one-way analyses of variance (one-wat ANOVAs) and Bonferroni corrections to compare three or more groups or T-test to compare two groups. The statistical analyses were performed using EZR ver.1.53 (Saitama Medical Center, Jichi Medical University, http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html, Saitama, Japan) [Kanda 2013].