After surgery, we performed the mechanical allodynia test which was taken as a behavioral sign of BPA-induced neuropathic pain as described by Hou and Xu [41 (link)]. Briefly, rats were first placed in a transparent Plexiglas chamber on the mesh stand that was placed at about 30 cm over the floor. Before the start of each test, rats acclimatized themselves to the trial environment for approximately 15 min until they quieted down and stood on all four feet. The mechanical hypersensitivity was assessed by the electronic Von Frey aesthesiometer (IITC Life Science, Woodland Hills, CA, USA) with a polypropylene rigid probe. The rigid probe was employed vertically to the middle plantar surface of the right forepaw of each rat with increasing stress. The paw withdrawal threshold (PWT, g) was automatically recorded using the aesthesiometer when the paw was withdrawn. A withdrawal reaction was thought to be available only if the anterior limb was totally moved away from the wire mesh platform. Three repetitive measurements were carried out at an interval of 30 seconds. The average PWT of the three repetitive measurements was calculated for each rat on days 0 (pre-injury) and 1, 3, 5, 7 post-injury. The data of day 0 (pre-injury) served as baseline. All the mechanical allodynia tests were conducted with two investigators who were unwitting of the grouping and treatment.
Electronic von frey aesthesiometer
Manufactured by IITC Life Science
Sourced in United States
The Electronic von Frey aesthesiometer is a laboratory instrument used to measure sensory perception thresholds. It quantifies the mechanical sensitivity of the skin by applying a precise amount of force, allowing for the objective assessment of touch, pressure, and pain perception.
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17 protocols using electronic von frey aesthesiometer
1
Mechanical Allodynia Assessment in Rats
2
Detailed Preclinical Behavioral Assessments
3
Mechanical Withdrawal Threshold Assessment
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Mechanical withdrawal thresholds of spinal nocifensive reflexes were measured on the left paw using Electronic von Frey Aesthesiometer (IITC Life Science, Woodland Hills, CA, United States) with a plastic tip in an exclusive testing area (catalog number 76-0488, Harvard Apparatus, Holliston, MA, United States) 1 day before-SNL and 1, 2, 3, and 4 weeks after respective treatments, as described in our previous studies (Ji et al., 2018 (link); Shen et al., 2022b (link)). The average of six measurements per subject, taken at least 30 s apart, was calculated.
4
Evaluating Sensory, Locomotor, and Edema Responses
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An electronic von Frey aesthesiometer (IITC Life Science, Woodland Hills, CA USA) was used to measure mechanical threshold. To assess hindpaw weight-bearing, an incapacitance meter (Stoelting, Wood Dale, IL USA) was used. Horizontal locomotor activity was measured using a photobeam apparatus (Optovarimex, Columbus Instruments, Columbus, OH USA), in which 15 photobeams cross the width of a 20 × 40 × 23-cm clear Plexiglas rodent cage, with photobeams spaced 2.5 cm apart and 6 cm above the cage floor. To assess edema, maximum dorsal-ventral hindpaw thickness was measured with calipers.
5
Assessing Mechanical Sensitivity and Weight-Bearing in Rats
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All rats were acclimated to the test environment for 30 min. The paw withdrawal threshold was measured using an Electronic Von Frey Aesthesiometer (IITC Life Science). The rigid probe was perpendicularly applied to the plantar surface of the ipsilateral hind paw with increasing force until paw withdrawal or a flinching response. The average of 3 sec-time was recorded as the result of paw withdrawal mechanical threshold (PWMT) for each rat.24 The weight-bearing difference was tested using an Incapacitance Tester (MR-600; IITC Life Science). Rats were placed in a plexiglass chamber and each paw positioned separately on a load-bearing plate. The percentage of ipsilateral weight born was calculated according to the following: [(the weight distribution of the ipsilateral hind paw/the weight distribution of the ipsilateral plus contralateral hind paw) × 100%]. The mean value of 3 sec-time was determined as the result of the weight born for each rat.21 The operator was blinded to grouping.
6
Mechanical Paw Withdrawal Threshold Evaluation
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The same rats tested for SFL were used to test for mechanical paw withdrawal (PWT) 3, 2, and 1 day before surgery and at 3 h and 1, 2, 3, 4, 5, 7, 9, and 16 days after surgery. Rats were placed in individual Plexiglass chambers on a mesh floor and allowed to habituate for 30 min. Mechanical PWT was determined using an electronic von Frey aesthesiometer (IITC Life Science, Woodland Hills, CA). This consists of a handheld force transducer with a series of rigidity-graded, attachable 0.8 mm polypropylene tips. Starting with the least rigid tip, force was transversely applied for to the mid-plantar surface of the hind paw (adjacent to the site of surgical injury) until the occurrence of a rapid paw withdrawal response. Stimuli were applied for 2 to 3 s with an inter-stimulus interval of 5 min. The second and third trials were conducted using the tip that was two grades lower than the tip which elicited the first withdrawal response. Withdrawal thresholds (g) were expressed as the mean from three trials.
7
Mechanical Allodynia Evaluation in Mice
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Mechanical allodynia was evaluated using the Electronic von Frey Aesthesiometer (IITC, Woodland Hills, CA, USA), adapted from methods by Chaplan et al. (1994) (link). Mice were individually placed in the set-up of clear Plexiglass boxes placed on a wire-mesh platform. The automatic thin steel von Frey filament was positioned under the midplantar surface of the hindpaw. A gradual increase in force was applied until withdrawal of the paw was observed, measuring the maximum force of a mechanical stimulus to elicit a response. Withdrawal thresholds of force greater than 4.5 g was the cut-off point to avoid paw damage.
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Mechanosensitivity and Exploratory Behavior Protocols
9
Spinal Nociceptive Reflex Withdrawal
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The mechanical withdrawal thresholds of spinal nocifensive reflexes were evaluated on the left hind paw using an Electronic von Frey Aesthesiometer (IITC Life Science, Woodland Hills, CA, USA) with a plastic tip (catalog number 76-0488, Harvard Apparatus, Holliston, MA, USA) in a dedicated testing area at baseline (week 0) and at the end of the study (week 4) after the respective treatments. The average of six measurements per subject, taken at least 30s apart, was calculated.
10
Characterizing Mechanosensory Neuron Responses
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Once a single fiber had been adequately isolated the response to pressure with graded monofilaments was used to establish the threshold and extent of the receptive field. Monofilaments of increasing large caliber were tested up to 5× threshold. In early experiments calibrated Semmes Weinstein monofilaments (Bell-Krotosky 1987 ) were used to probe receptive fields, whereas in later recordings an IITC Electronic von Frey aesthesiometer® provided an analog voltage proportional to the applied force, which could be recorded by a computer. In both cases the monofilaments were applied manually with only enough pression to produce flexion. Monofilament pressure was applied for 5 s and a minimum of 10 repetitions were recorded with a 5 s pause between applications. This procedure enabled the unequivocal identification of RA and SA fibers. In experiments with the monofilament equipped with a strain gauge, a force approximately 4× threshold was applied to the receptive field and a minimum of 20 replications were recorded on a laboratory computer.
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