Hyperalgesia

All prospective mechanosensitivity experiments with animals were conducted in accordance with the guidelines from the Canadian Council on Animal Care and approval of the Laval University Animal Care Committee. PWT was measured in adult male C57BL/6 mice using von Frey filaments 2 through 9. Mice were placed in acrylic chambers (5.5 × 10 cm) suspended above a wire mesh grid and allowed to acclimatize to the testing apparatus for 1 hour prior to experiments. When the mouse was not moving the von Frey filaments were pressed against the plantar surface of the paw until the filament buckled and held for a maximum of 3 seconds. A positive response was noted if the paw was sharply withdrawn on application of the filament. Flinching immediately upon removal of the filament was also considered a positive response as previously described [3 (link)]. Testing began with filament number 5 and progressed according to an up-down method. Mice were randomly assigned to be tested either with the method of Chaplan et al. [3 (link)] or SUDO. After the first measurement of PWT, a second measurement of PWT was conducted using the alternate test. In some prospective experiments, hyperalgesia was induced by intraplantar injection of capsaicin (0.5% w/v, 5 μl) or complete Freund’s adjuvant (CFA; 10 μl) in one hind paw under brief anesthesia with isoflurane (<3 minutes, 4% isoflurane). PWT was measured 3 hours after intraplantar injection of either compound and again 3 days after injection of CFA. The PWT estimates derived using SUDO or the method of Chaplan et al. [3 (link)] for each condition were compared using a paired t-test in Graphpad Prism 5.

All patients with T2DM were asked whether they had numbness, pain (prickling or stabbing, shooting, burning or aching pain), and paresthesia (abnormal cold or heat sensation, allodynia and hyperalgesia) in the toes, feet, legs or upper-limb. Then, an experienced physician performed the neurologic examination which included vibration, light touch, and achilles tendon reflexes on both sides in the knee standing position (as being either presence or weakening or loss). Vibration perception threshold (VPT) was assessed at the metatarsophalangeal joint dig I using a neurothesiometer (Bio- Thesiometer; Bio-Medical Instrument Co., Newbury, OH, USA). First, the patients were informed how to know the vibration sensation is felt by gradually turning the amplitude from zero to maximum, then the test began again from zero and they were asked to say the moment that they first felt it. Measurements were made on the planter aspect of the big toe bilaterally, three times consecutively for each big toe. The median of three readings is accepted as the VPT value of that measurement (35 (link)). Sensitivity to touch was also tested using a 5.07/10-g Semmes-Weinstein monofilament (SWM) at four points on each foot: three on the plantar and one on the dorsal side. The 10-g SWM was placed perpendicular to the skin and pressure was applied until the filament just buckled with a contact time of 2 s. Inability to perceive the sensation at any one site was considered abnormal (36 (link), 37 (link)). DPN was defined as VPT ≥25 V and/or inability to feel the monofilament (35 (link)), and then participants were divided into DPN group and no DPN group.
Ankle brachial index (ABI) was measured noninvasively by a continuous-wave Doppler ultrasound probe (Vista AVS, Summit Co., USA) with participants in the supine position after at least 5 min of rest. Leg-specific ABI was calculated by dividing the higher SBP in the posterior tibial or dorsalis pedis by the higher of the right or left brachial SBP (33 (link), 38 (link)). Patients were diagnosed as having PAD if an ABI value <0.9 on either limb (33 (link), 38 (link)).
DFU was defined as ulceration of the foot (distally from the ankle and including the ankle) associated with neuropathy and different grades of ischemia and infection (39 (link)).

The Von Frey filament test is used to measure allodynia and hyperalgesia and is a more direct way to measure visceral pain and hyperalgesia. Mice were individually housed and acclimatized to the modular animal enclosures (Stoelting, 57823) at least 3 days before the experiment. Tests were performed using Semmes–Weinstein Von Frey filaments (Stoelting, 58011) as previously described [70 (link), 71 (link)]. Filaments were applied to the abdomen or hind-paw ten times, the filament evoking a withdrawal response in at least 50% of applications was considered the threshold force (g).

All experimental procedures were approved by the Animal Care and Welfare Committee of Zhejiang University of Traditional Chinese Medicine, Zhejiang, China (approval number IACUC-20180319–12). Male Sprague-Dawley rats (weighing 200–230 g) used in this study were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences [SCXK(Hu)2018–0006] and housed at the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine [SYXK(Zhejiang)2018–0012]. All rats were fed standard rodent food, housed five per cage, and regularly cycled in light and dark. Previous studies have shown that there was a genderdependent difference in hyperalgesic priming, and PGE2 induced hyperalgesia was prolonged to 24 h in male rates treated 5 days prior with Car that did not induce hyperalgesia in female rats (Joseph et al., 2003 (link)). Therefore, only male rats were selected for the experiment in this study.