Styrofoam

A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin (ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Subsequently, skin (10 × 10 cm²) was placed (not fixed, not kept in medium) on a styrofoam lid that was covered with aluminium foil to provide (at least partial) backpressure. Suction chambers with 5 openings (Ø = 5 mm) on the orifice plate were attached to skin, topped with a styrofoam lid and pressed with 1 kg weight in order to avoid movement of the plate. A pressure of 200–250 millimeter (mm) mercury (Hg) (ex vivo) or 150–200 mm Hg (in vivo) caused the skin to be drawn through the openings creating typical suction blisters of different size within 6–8 h (ex vivo) and 1–2 h (in vivo). Suction blister fluid (~110 µl/5 blisters) was collected using a syringe with a needle. Cells within the fluid were counted and placed on adhesion slides for staining and analysis. Blister roof epidermis was cut with a scissor, fixed with ice-cold acetone (10 minutes) and used for staining. For comparison and control, epidermal sheets were prepared from unwounded skin biopsy punches (Ø = 6 mm; 3.8% ammonium thiocyanate (Carl Roth GmbH + Co. KG, Germany) in PBS (Gibco, Thermo Fisher, Waltham, MA, USA), 1 h, 37 °C). Removal of the blister roof created a wound area. Biopsies (Ø = 6 mm) from wounded and unwounded areas were cultivated for 12 days in either duplicates or triplicates in 12 well culture plates and Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco) and were cultured at the air-liquid interphase. Medium was changed every second day.

Litters chosen for testing contained more than seven pups for BTBR (7.8±1.05), B6 (7.1±0.58) and FVB/NJ (7.5±0.34), and more than five pups for 129X1 (5.5±0.40; a strain known for small litters). One female and one male from each litter of BTBR, B6, FVB/NJ and 129X1 mice (n = 10 litters each strain) were used for baseline measurements of the ultrasonic vocalizations from pnd 2 to 12. Body weights and body temperatures of pups were measured after the ultrasonic vocalization test on pnd 2, 4, 6, 8 and 12. On each day of testing, each pup was placed into an empty plastic container (diameter, 5 cm; height 10 cm), located inside a sound-attenuating styrofoam box, and assessed for USVs during a five minute test. At the end of the five minute recording session, each pup was weighed and its axillary temperature measured by gentle insertion of the thermal probe in the skin pocket between upper foreleg and chest of the animal for about 30 seconds (Microprobe digital thermometer with mouse probe, Stoelting Co., Illinois, USA). No differences in patterns of calling were detected in a comparison of male and female pups, therefore data were collapsed across sex.
An Ultrasound Microphone (Avisoft UltraSoundGate condenser microphone capsule CM16, Avisoft Bioacoustics, Berlin, Germany) sensitive to frequencies of 10–180 kHz, recorded the pup vocalizations in the sound-attenuating chamber. The microphone was placed through a hole in the middle of the cover of the styrofoam sound-attenuating box, about 20 cm above the pup in its plastic container. The temperature of the room was maintained at 22±1°C. Vocalizations were recorded using Avisoft Recorder software (Version 3.2). Settings included sampling rate at 250 kHz; format 16 bit. For acoustical analysis, recordings were transferred to Avisoft SASLab Pro (Version 4.40) and a fast Fourier transformation (FFT) was conducted. Spectrograms were generated with an FFT-length of 1024 points and a time window overlap of 75% (100% Frame, Hamming window). The spectrogram was produced at a frequency resolution of 488 Hz and a time resolution of 1 ms. A lower cut-off frequency of 15 kHz was used to reduce background noise outside the relevant frequency band to 0 dB. Call detection was provided by an automatic threshold-based algorithm and a hold-time mechanism (hold time: 0.01 s). An experienced user checked the accuracy of call detection, and obtained a 100% concordance between automated and observational detection. Parameters analyzed for each test day included number of calls, duration of calls, qualitative and quantitative analyses of sound frequencies measured in terms of frequency and amplitude at the maximum of the spectrum.
Waveform patterns of calls were examined in depth in twenty sonograms collected from every strain, one from each of the pups tested. The sonograms were one minute in length and selected from recordings at postnatal day 8. We classified 3633 BTBR calls, 2333 B6 calls, 1806 129X1 calls and 2575 FVB/NJ calls. Each call was identified as one of 10 distinct categories, based on internal pitch changes, lengths and shapes, using previously published categorizations [21] (link), [22] (link), [24] (link). Classification of USVs included ten waveform patterns described below, and illustrated visually in Figure 2 and S1 and as audiofiles (Sounds S1, S2, S3, S4, S5, S6, S7, S8, S9, S10) in Supporting Information.
Inter-rater reliability in scoring the call categories was 98%. Call category data were subjected to two different analyses: a) strain-dependent effects on the frequency and duration of the vocalizations emitted by each subject at pnd 8 b) strain-dependent effects on the probability of producing calls from each of the ten categories of USV, as described below under Statistical analysis.

The waste Styrofoam used in this study was electronic containers (WPS-1) and food containers (WPS-2). Four types of WPS-based adhesives were prepared from WPS-1 and WPS-2 and then modified with MDI and MA (Figure 1). The WPS was cleaned with AquaDes and dried at room temperature. After that, the WPS was crushed into particles. The dissolution of WPS particles was adopted from a published work with some modifications [16 (link)]. Approximately 200 g (20% w/v) of WPS particles was dissolved in 1000 mL of co-solvents consisting of 100 mL of ethyl acetate, 100 mL pf methylene chloride, 150 mL of butyl acetate, 60 mL of propanol, 90 mL of butanol, and 500 mL of toluene. The dissolution process was performed at 60 °C under continuous stirring at 100 rpm for 10 min. After all the WPS particles were dissolved, around 40 g (20% w/v) of MDI based on the WPS solids was poured into the mixture. The cross-linking reaction was performed at 60 °C under continuous stirring at 100 rpm for 30 min. The modification with MA was performed following the above procedure.