StemCell Therapy

Mice

We used 510-week-old C57BL/6NCr male mice (SLC, Shizuoka, Japan) as the wild type (WT), and TRPV1-deficient (TRPV1/) and TRPA1-deficient (TRPA1/) male mice maintained on a C57BL/6NCr background46. Mice were housed in standard cages and maintained under a 12-h light/dark cycle at an ambient temperature of 242C with access to food and water ad libitum. All the animal care and experimental procedures were approved by our Institutional Animal Care and Use Committee and followed the National Institutes of Health and National Institute for Physiological Sciences guidelines (21A008), and carried out in compliance with the ARRIVE guidelines.

Diabetes was induced in mice by administering a single intraperitoneal dose of 150mg/kg STZ (Sigma-Aldrich) prepared freshly in 0.02M citrate buffer (pH 4.5) after a 24-h fasting period when they became 5weeks old. WT (non-DM), TRPV1/ (non-DM) and TRPA1/ (non-DM) mice received an equal volume of citrate-buffer vehicle. One week after administering STZ, the mice with consequent blood glucose concentrations of >400mg/dL were selected as WT (DM), TRPV1/ (DM) and TRPA1/ (DM) mice. Blood glucose levels were measured by Glutest Neo (Sanwa Kagaku Kenkyusho, Nagoya, Japan). Serum insulin concentration was measured by collecting blood from mice by cardiac puncture into a heparin-containing tube, collecting the supernatant immediately after centrifugation, freezing it at 80C and transporting it at low temperature to a testing contractor (Nikken Seil, Tokyo, Japan). The limit of detection for insulin levels was <0.1ng/mL.

While the mice were 510weeks old, hind paw withdrawal response to thermal stimuli of radiant heat was measured using a Plantar test (Catalog No. 57820; Ugo Basile, Comerio, Italy)47,48. The PWL at 5weeks of age was measured a few days before STZ and the buffer administration. We adjusted the two kinds of IR intensities to a PWL baseline of about 7s (IR=40) and 12s (IR=20). After 30min acclimation, paw withdrawal latencies (PWL) were measured 68 times per session, separated by a minimum interval of 5min. Paw withdrawals due to locomotion or weight shifting were not counted. Data are expressed as paw withdrawal latency in seconds.

Mice were killed after anesthesia with isoflurane, and dorsal root ganglia (DRG) were quickly harvested and placed on ice. The tissue was then immediately immersed in RNAlater Stabilization Solution (Invitrogen). After temporarily storing at 4C, RNAlater was removed, and an appropriate volume of Isogen II (Nippon Gene, Tokyo, Japan) was added to homogenize the ganglia with a Biomasher II apparatus (Nippi, Tokyo, Japan); they were completely homogenized and cells were lysed on ice. Then, total RNA was collected using Ethachinmate (Nippon Gene, Tokyo, Japan) and 75% isopropanol and RNA concentration was assayed using a NanoDrop One Microvolume UVVis Spectrophotometer (Thermo Fisher Scientific, United States). The RNA was reverse transcribed into cDNAs with ReverTra Ace qPCR Master Mix (Toyobo, Osaka, Japan) according to the manufacturers protocol. TRPV1, TRPA1, and 36B4 mRNA levels were assayed using a StepOnePlus Real-Time PCR System (Applied Biosystems) with SYBR Green Real Time PCR Master Mix Plus (Toyobo, Osaka, Japan) according to the manufacturers protocol. All data were analyzed using StepOne software (version 2.3; Life technologies).

The primer sequences used for qRT-PCR were as follows: TRPV1 (NM_001001445), 5-CCCGGAAGACAGATAGCCTGA -3 (forward) and 5-TTCAATGGCAATGTGTAATGCTG-3 (reverse); TRPA1 (NM_177781), 5-GTCCAGGGCGTTGTCTATCGG -3 (forward) and 5-CGTGATGCAGAGGACAGAGAT-3 (reverse); 36B4 (NM_007475.5), 5-AGATTCGGGATATGCTGTTGGC-3 (forward) and 5-TCGGGTCCTAGACCAGTGTTC-3 (reverse).

DRG were isolated and rinsed immediately in ice-cold phosphate-buffered saline (PBS; calcium- and magnesium-free) and put into an appropriate amount of protein lysis buffer (25mM TrisHCl [pH 7.6], 150mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40, and 1% protease inhibitor), and homogenized using a Biomasher II apparatus. The homogenates were then placed on ice for 30min to ensure complete lysis. Subsequently, the homogenates were centrifuged at 15,000 g for 30 min at 4 C and the supernatant was transferred to a new centrifuge tube. After measuring the protein concentration of each sample using a BCA Assay Kit (catalog No. 297-73101, Fujifilm Wako Chemicals), equal amounts of protein from the DRG were denatured at 95 C for 5 min and electrophoresed on 4%12% SDSpolyacrylamide gel. The proteins were transferred onto a poly(vinylidene fluoride) membrane. Nonspecific binding sites on the membranes were blocked using Tris-buffered saline (TBS) supplemented with 0.05% Tween 20 (Takara Bio, Shiga, Japan) (TBS-T) and bovine serum albumin (BSA) for 1 h at room temperature (RT), and incubated overnight at 4 C with rabbit anti-TRPV1 antibody (13000, Dr. Kido, Saga Medical School Faculty of Medicine, Saga University) and rabbit anti--actin antibody (13000, Cell Signaling Technology). Then anti-rabbit IgG HRP-linked secondary antibodies (11000, Cell Signaling Technology) were incubated with the membranes for 1 h at RT. Between respective steps, the immunoblots were rinsed with TBS-T 3 times for 10 min each time. All protein bands were labeled using an ECL kit (Amersham, United Kingdom) and then visualized using an ImageQuant LAS 4000 system (General Electric). The densities were normalized with respect to the -actin level.

According to a published method49 with some minor modifications described as follows, DRG were isolated from 5 to 10week old mice. In brief, resected DRG were collected in PBS (calcium- and magnesium-free) on ice, and then the tissues were incubated with 725g of collagenase type IX (catalog No. C9407, Sigma-Aldrich) in 250 L of Earles balanced salt solution (Sigma-Aldrich) containing 10% fetal bovine serum, MEM vitamin solution (1:100, Sigma-Aldrich), penicillinstreptomycin (1:200, Life Technologies), and GlutaMax (1:100, Life Technologies) at 37C for 30min. Next, the DRG neurons were dissociated by triturating the suspension through a fire-polished Pasteur pipette and filtering it through a 70m cell strainer (Flowmi). The isolated neurons were placed on 12mm diameter coverslips (Matsunami, Osaka, Japan) with 20 L of Earles balanced salt solution and used for experiments within 2h of isolation, maintaining them at 37C in a chamber under a humidified atmosphere of 95% O2 and 5% CO2.

Ca2+ transients were measured in isolated cultured DRG neurons incubated with 5mM Fluo-2-AM (Molecular Probes, Invitrogen) for 20min at 37C, and DRG were mounted in an open chamber and superfused with bath solution. The extracellular standard bath solution contained 140mM NaCl, 5mM KCl, 2mM MgCl2, 2mM CaCl2, 10mM HEPES, and 10mM glucose at pH 7.4, adjusted with NaOH. Cytosolic free Ca2+ concentrations were measured by dual-wavelength Fura-2 microfluorometry with excitation at 340/380nm and emission at 510nm. Fura-2 fluorescence was recorded with a CCD camera, CoolSnap ES (Roper Scientific/Photometrics). Data were acquired using imaging processing software IPlab (Solution Systems, Funabashi, Japan) and analyzed with ImageJ 1.53 (NIH). At the end of each experiment, ionomycin (5M) was applied in the presence of 20mM extracellular CaCl2 to obtain saturating levels of Ca2+ influx as Fmax. The population that did not respond to either molecular stimulus, responded to AITC alone, capsaicin alone, and the population responding to both stimuli were determined by the number of neurons responding to capsaicin and/or AITC divided by the number of neurons responding to ionomycin and expressed as a percentage.

Distal sciatic nerve tissue was prepared for imaging as previously described with slight modifications50. WT (non-DM), WT (DM) and TRPV1/ (non-DM) mice (5weeks after STZ administration or the same age) were perfused with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M phosphate buffer. We collected sciatic nerves from the same position for all mice, and these tissues were post-fixed for 4h, and maintained at 4C overnight. The samples were post-fixed in cold 2% OsO4 in PBS for 60min, dehydrated in a graded ethanol series and acetone and embedded in Quetol 812 epoxy resin (Nisshin EM Co.). The resin was incubated at 70C for 3 nights to ensure polymerization. Prior to TEM observation, semithin, 1m-thick sections were cut and stained with 1% toluidine blue for examination by light microscopy (AX80; Olympus). Ultrathin sections(70nm-thick) were prepared with an ultramicrotome (ULTRACUT S, Reichert-Nissei) and stained with uranyl acetate and lead citrate. The ultrathin sections were observed by TEM (HT7700; Hitachi High-Tech). Image analysis was performed with Image J software. The g-ratios were calculated by dividing the axon diameter by the diameter of the axon including the myelin. The diameter was calculated by the measured perimeter divided by .

The Thermal Gradient Ring (Catalog No. 35550; Ugo Basile) is an apparatus with 45cm inner diameter, 57cm outer diameter, and 24cm height. A camera is located on the upper side of the apparatus, which includes an infrared camera and an infrared transmissive inner wall. The cooling and heating devices were set so that the surface temperature range of the apparatus was from 10 to 55C. Floor surface temperature was monitored using a thermometer (HFT-51, Anritsu, Japan). Behavioral assays were performed between 9:00 and 17:00. All mice were acclimated for 30min in the thermal gradient apparatus with its floor at room temperature (2324C) before the day of the thermal gradient test. Mice were placed individually in the device in an innocuous mid-temperature zone. The behavioral data was videotaped for 60min and analyzed as spent time, travel distance or speed automatically using ANY-maze software. We defined preference temperature as the mean value using the zone temperature and spent time.

Data are presented as meansSEM. Statistical analysis was conducted using GraphPad Prism 9.2.0 (GraphPad Software, United State). Significant changes were identified using a two-tailed t test, at 95% confidence interval, or one-way ANOVA and two-way repeated measures ANOVA followed by a Bonferroni post hoc test with p<0.05 considered as significant (p: *<0.05, **<0.01, ***<0.01).

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Thermal gradient ring reveals thermosensory changes in diabetic peripheral neuropathy in mice | Scientific Reports - Nature.com

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