BALB/c mice were housed under standard pathogen-free conditions in a temperature- and humidity-controlled room with food and water provided. Euthanasia procedures were conducted in accordance with University of Michigan’s Unit for Laboratory Animal Management policies. Carbon dioxide at 30%–70% flow rate of chamber volume per minute was used as a primary method, and terminal bleeding was used as a secondary method.

Sex as a biological variable: Both male and female mice between the ages of 4 and 6 weeks were employed in this study. We did not observe differences between male and female mice.

Study approval: All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan under PRO00011478.

TEWL was taken at baseline and at 15-min intervals throughout each experiment. TEWL was measured with a Tewameter™ Nano (Courage + Khazaka gmbh, Germany) placed on the surface of the skin. After an equilibration time of 25–30 s, five sets of 5-s measurements were captured. The paired software (MPA plus, Courage + Khazaka) was used to analyze each set of measurements and compute one mean value. TEWL was taken at the ear, paw, and abdomen of mice to find the most consistent place to measure ( Figure 1A ). Once the ear was selected as the location for all future measurements, captured by pinning between the finger and the tewameter ( Figure 1A ), all subsequent data represent an ear measurement unless otherwise specified.

The mice were heated under a heat lamp (Model HL-1B 120v; Braintree Scientific) for 10 min before tail intravenous (IV) injection. The experimental mice received an IV injection in the tail with a volume of 50 μL per mouse of histamine (Fisher Scientific, AAL0919814) in phosphate-buffered saline (PBS; Cytiva HyClone, SH30256.01) at a concentration of 200 mg/mL ( Figure 1B ). Control mice received an injection of 50 μL of PBS. Physiological responses of TEWL, temperature, and reaction score were taken at baseline and at 15-min intervals for 60 min. After the trial, the mice were anesthetized with isoflurane (ULAM) and blood samples were collected for MCPT-1 enzyme-linked immunosorbent assay (ELISA) prior to euthanasia.

All mice were sensitized with IV injections in the tail vein with 200 μL/100 μg per mouse of anti-dinitrophenyl (DNP)-IgE (Millipore Sigma, D8406) and PBS at a concentration of 50 μL/mL ( Figure 1C ). Prior to each challenge, all mice were starved for 5 h before experimental mice were challenged via oral gavage with a 50 mg/kg of DNP-albumin (Sigma-Aldrich, A6661) dissolved in 250 μL of PBS. The control mice were gavaged with 250 μL of PBS per mouse. Physiological responses of TEWL, temperature, and reaction score were taken at baseline and at 15-min intervals for 60 min. After the trial, the mice were anesthetized with isoflurane (ULAM) and blood samples were collected for MCPT-1 ELISA prior to euthanasia.

All mice were sensitized to ovalbumin (OVA) via intraperitoneal injection with a solution of 50 μg of OVA (Sigma-Aldrich, 9006-59-1) and 100 μL/kg of alum (InvivoGen, 21645-51-2) into 250 μL of PBS per mouse ( Figure 1D ). Two weeks following sensitization, the mice were challenged every 2–3 days for a total of seven challenges (23, 24). Prior to each challenge, all mice were starved for 5 h before experimental mice were orally gavaged 50 mg/kg of OVA dissolved in 250 μL of PBS, and the control mice were gavaged with 250 μL of PBS. Physiological responses of TEWL, temperature, and reaction score were taken at baseline and every 15 min for 60 min. After the trial, the mice were anesthetized with isoflurane (ULAM) and blood samples were collected for MCPT-1 ELISA prior to euthanasia.

Core temperature was measured via a lubricated rectal probe (Model RET-3; Physitemp Instruments Inc.) following 5-s equilibration after insertion (Model Bat-12; Physitemp Instruments Inc). Reaction score was judged at each 15-min interval. The mice were assigned a score of 1 through 5 according to standard scoring, where 1 = excessive itching, 2 = hunching, 3 = labored breathing, 4 = moribund, and 5 = death ( Figure 1E ) (23). Diarrhea score is recorded binarily with 0 meaning no diarrhea occurred and 1 meaning it has.

Baseline TEWL was taken under various experimental control conditions. Mice were heated under a heat lamp (Model HL-1B 120v; Braintree Scientific) for either 5, 10, or 15 min and their TEWL and temperature were recorded every 15 min for 60 min. The mice were also placed under isoflurane until they were unconscious. The mice had their TEWL and temperature taken every 15 min for 60 min. Tail vein IV injections via a 26G ½ inch needle (Exel International, 14-841-32) and intragastric gavage via a reusable feeding needle (GloMed Inc., NC1299558) were also evaluated for their effect on baseline TEWL.

Following the last anaphylaxis event of each trial, blood was obtained from each mouse via cardiac puncture. The blood was placed into an ice bucket for 30 min before centrifugation at 9,000 rpm for 10 min to collect serum. The serum was stored at −80 °C until analysis. MCPT-1 levels were quantified using the Mouse MCPT-1 (mMCP-1) Uncoated ELISA kit (Invitrogen, 88-7503) according to the manufacturer’s instructions. At the end of the procedure, once the stop solution was added, the plate was read at 450 nm using the GloMax® Explorer plate reader (Promega, GM3500) and concentrations were calculated using a standard curve.

All statistical analyses were performed using GraphPad Prism (GraphPad Software, 10.4.1). Data were assessed for normality using the Shapiro–Wilk test. Comparisons between two groups were conducted using unpaired two-tailed t-tests for normally distributed data or the Mann–Whitney U test for non-normally distributed data. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference test was used. For the area under the curve (AUC) analysis, we calculated the area of the TEWL results above the baseline set for the food challenge by measurement at time 0 for each mouse. Where relevant, simple linear regressions were fit to XY data with an R ² and p-value reported. Data are presented as mean ±standard error of the mean (SEM) or median with interquartile range (IQR) as appropriate. Statistical significance was defined as p<0.05.

To determine which part of the murine body would have TEWL results most reflective of human physiology (17), three body parts were tested: ear (mean, 17.7 g/m²/h), abdomen (mean, 21.8 g/m²/h), and paw (mean, 54.1 g/m²/h) ( Figure 2A ). The ear was most similar in TEWL to the human volar forearm data taken from OFCs as previously reported by our group (17) and was convenient to take measurements from. The abdomen posed the risk of the mouse urinating on the instrument and was not viable for repeated measures. Paw TEWL values were significantly greater than the ear and abdomen TEWL values ( Figure 2A ). All TEWL measurements aside from those explicitly labeled with a body site ( Figure 2A ) were conducted using the ear only.

To establish whether sex impacted TEWL measurement, the TEWL of both male (mean, 18.7 g/m²/h) and female (mean, 17.9 g/m²)/h) mice was compared with no significant differences observed ( Figure 2B ). TEWL from control mice over the course of seven non-reactive food challenges over a 17-day period [akin to the repeated reactions used in the active systemic anaphylaxis (ASA) studies later] was examined to determine that TEWL values were stable over time with no significant differences ( Figure 2C ). To confirm that both tewameters report similar measurements, a device comparison showed that minimal variation between tewameters was conducted (means, 9.8 and 9.9 g/m²/h) ( Figures 2D, E ).

To identify the impact on TEWL of the heat lamp used during tail vein IV injections, mice were warmed under the heat lamp for 5, 10, and 15 min before core temperature and TEWL were taken in parallel at 5-min intervals for 15 min after warming ( Figures 3A–F ). While core temperature increased after heating, TEWL did not vary significantly.

To analyze the effect of isoflurane anesthesia, TEWL and temperature measurements were taken before (mean = 38.7 °C; 12.9 g/m²/h), during (mean = 36.9 °C; 2.5 g/m²/h), and after (mean = 38.7 °C; 13.6 g/m²/h) anesthesia ( Figures 3G, H ). Time from anesthesia onset to TEWL measurement was 2 min. To measure the effect of oral gavage on TEWL, TEWL was taken before (mean = 13.7 g/m²/h) and after (mean = 13.4 g/m²/h) oral gavage of PBS ( Figure 3I ). To identify the effect of tail IV injection on TEWL, TEWL was taken before (mean = 15.8 g/m²/h) and after (mean = 15.5 g/m²/h) tail IV injection of PBS ( Figure 3J ). Time from gavage or injection to TEWL measurement was 1 min. Overall, anesthesia clearly impacted TEWL measurements, so anesthesia was only used after challenge for terminal bleeding to avoid affecting TEWL measurements during anaphylaxis. Heating, gavage, and IV injections appeared to have no significant effect on TEWL and so were used in subsequent anaphylaxis models.

To identify the impact of a non-IgE-mediated anaphylaxis-like event on the TEWL of mice, the TEWL and temperature of the control and histamine groups were measured at baseline and in 15-min intervals after challenge with histamine ( Figures 4A, B ). The greatest change in TEWL occurred at the 15-min time point (mean control = 0.12 g/m /h; histamine = 5.7 g/m²/h), while temperature decreased more slowly and was minimally different at 15 min (mean control = −0.01°C; histamine = −0.47°C) and continued to decline at a significant rate for the duration of the trial ( Figures 4C, D ). Maximum anaphylaxis reaction score (mean control = 0, histamine = 3.4) and diarrhea status (mean control = 0, histamine = 0) were also recorded throughout the trial for the control and histamine mice as a secondary confirmation of anaphylaxis ( Figures 4E, F ). TEWL change at 15 min did not correlate with temperature change at 15 min ( Figure 4G ) but did correlate with anaphylaxis reaction score ( Figure 4H ). Serum was obtained via terminal bleed from mice following oral challenge and tested for MCPT-1 of the control (mean = 1,708.86 pg/mL) and histamine (mean = 1,468.61 pg/mL) mice ( Figure 4I ) and did not correlate with TEWL change at 15 min ( Figure 4J ), as expected.

To identify the impact of passive systemic anaphylaxis (PSA) on the TEWL of mice, TEWL and temperature were measured at baseline and in 15-min intervals post-oral challenge with DNP-challenged mice ( Figures 5A, B ). The TEWL and temperature change of DNP mice (mean TEWL change at 15 min = 3.48 g/m²/h; mean temperature change at 15 min = −0.55°C) changed the most at the 15-min time point, compared to that of the control mice (mean 0.91 g/m²/h; 0.03°C) ( Figures 5C, D ). Anaphylaxis reaction score (mean control = 0; DNP = 1.7) and diarrhea status were also recorded throughout the trial to corroborate the reaction status ( Figures 5E, F ). TEWL change (baseline TEWL subtracted from TEWL of mice at 15 min) correlated with temperature change ( Figure 5G ) as well as with anaphylaxis reaction score ( Figure 5H ). Serum MCPT-1 was obtained from control (mean = 1,210.83 pg/mL) and DNP (mean = 5,995.14 pg/mL) mice via terminal bleed following oral challenge ( Figure 5I ) and did not correlate with the TEWL change ( Figure 5J ).

To identify the impact of ASA on the TEWL of mice, TEWL and temperature of OVA-sensitized mice were measured at baseline and in 15-min intervals post-oral challenge with OVA (only data from challenge number 7 used) ( Figures 6A, B ). The TEWL and temperature change of OVA-challenged mice (mean TEWL change at 15 min = 3.61 g/m²/h; mean temperature change at 15 min = −1.4°C) changed the most at the 15-min time point, compared to that of the control mice (mean TEWL change at 15 min = 0.11 g/m²/h; mean temperature change at 15 min = −0.97°C) ( Figures 6C, D ). Anaphylaxis reaction score and diarrhea status were also recorded for control (maximum anaphylaxis reaction score = 0; diarrhea status = 0) and OVA (mean maximum anaphylaxis reaction score = 2.84; diarrhea status = 0.72) mice throughout the trial to corroborate the reaction’s severity ( Figures 6E, F ). TEWL change at 15 min correlated with temperature change ( Figure 6G ) as well as with anaphylaxis reaction score ( Figure 6H ). Serum MCPT-1 was obtained via terminal bleed following oral challenge in control (mean = 3,738.46 pg/mL) and OVA mice (mean = 12,648.7 pg/mL) ( Figure 6I ), which trended toward a correlation with TEWL change ( Figure 6J ).

To identify the impacts of varying types of anaphylaxes on TEWL measurements, histamine models (non-IgE-mediated) and DNP and OVA (IgE-mediated) models were compared by TEWL results ( Figure 7A ), TEWL change at 15 min ( Figure 7B ), temperature ( Figure 7C ), and temperature change at 15 min ( Figure 7D ). TEWL increased significantly for all models, whereas other markers such as anaphylaxis reaction severity and temperature change varied greatly depending on which model was utilized. In addition, in order to evaluate the total change in TEWL during the entire challenge time course, we calculated the AUC for TEWL in each challenge. We then correlated the values with temperature change at 15 min, maximum anaphylaxis severity score, and MCPT-1 results ( Supplementary Figure S1 ). To determine the effect of anaphylaxis on TEWL during earlier challenges, prior to the seventh trial, TEWL change at 15 min, temperature change at 15 min, maximum anaphylaxis severity score, and diarrhea status were recorded and plotted from Trial 4 through Trial 6 ( Supplementary Figure S2 ).