All animal experiments were performed under the following conditions; Room temperature 23 ± 2°C, relative humidity 60% ± 10%, and an alternating 12-hours light–dark cycle (8 AM to 8 PM). Animals were quarantined and acclimatized before experiments at least for a 1-week period standard diet (Oriental Yeast Industries, Tokyo, Japan) and water were available ad libitum. The care and handling of the animals were in accordance with the National Institute of Health guidelines. All procedures involving animals were carried out in accordance with the Institutional Guidelines.
For fasting treatment, mice and rats had access to only water. For the ketogenic diet, Diet D10070801 (Research Diets, New Brunswick, NJ, USA), consisting of 67% fat, 17% protein, and 0% carbohydrate was used.
Rat model of toluene 2, 4-diisocyanate (TDI) induced Nasal Allergy
Nasal Allergy models were performed by the method described previously with slight modifications . Six-week-old male Brown Norway rats (n = 6–8 in each experiment) were purchased from Japan SLC, Inc. Five μl of a 10% solution of TDI (Sigma-Aldrich, St. Louis, MO, USA) in ethyl acetate (Sigma-Aldrich, St. Louis, MO, USA) was intranasally applied to the bilateral nasal cavities once a day for five consecutive days (sensitization). This sensitization procedure was then repeated after a 1-day interval. Seven days after the latest sensitization, and prior to being evaluated, 5 μl of 10% TDI solution was applied again to the nasal cavaties to provoke nasal allergy-like behaviors. Sensitized rats without provocation by TDI solution served as the control group.
Nasal allergy-like behaviors were evaluated by counting the amount of nasal rubbing and sneezing during a 20 minute period immediately after TDI application. Changes in nasal secretions were evaluated by measuring the weight of cotton thread 60 minutes after TDI application.
The total plasma IgE levels were determined after TDI application. Blood was collected from the abdominal aorta. Plasma samples were obtained by centrifugation and stored at -30°C until use. The total plasma IgE level was determined by using Rat IgE ELISA Kit (Shibayagi Co., Ltd, Gumma, Japan) according to the manufacturers instruction.
After nasal secretion was measured, rats were applied to histological examination of the nasal cavity and mucosa of maxilloturbinate. Rats were euthanized by cervical dislocation under anesthetized with sodium pentobarbital. The head was separated from the neck and the whole anterior face sections were prepared using a multipurpose cryosection preparation kit (Section Lab Co., Ltd, Hiroshima, Japan) according to the method of Kawamoto . In brief, anterior face of tissue was frozen in isopentane (-160°C) cooled with dry ice and then freeze-embedded with SCEM compound (Section Lab Co., Ltd, Hiroshima, Japan). Twenty-μm thick sections were cut with cryomicrotome (CM3050S, Leica Co., Ltd, Heidelberg, Germany) and collected with cryofilm (Section Lab Co., Ltd, Hiroshima, Japan). For hematoxylin and eosin (H&E) and Toluidine blue (pH4.1) staining, tissue sections were fixed by 4% paraformaldehyde after drying section and mounted with SCMM (Section Lab Co., Ltd, Japan). For immunohistochemistry, the sections were incubated with a polyclonal antibody to mast cell tryptase (Santa Cruz, CA, USA) at 4°C overnight, followed by labeling with Alexa 488-conjugated goat anti-rabbit IgG (Life Technologies, Inc. Grand Island, NY, USA) and Hoechst 33342 (Dojindo Co., Ltd, Kumamoto, Japan) at RT for 30 min. These sections were observed with microscope (BZ-9000, Keyence Co., Ltd., Tokyo, Japan) or a confocal microscope (LSM 710, Carl Zeiss, Inc. Heidelberg, Germany).
Passive cutaneous anaphylaxis (PCA)
Six-week old male Wistar rats (Japan Clea, Inc. Tokyo, Japan) were used (n = 7–27 in each time point). The homologous antiserum used was prepared according to the method described. Briefly, rats were sensitized by intramuscular injection of 0.2 ml of 10 mg/mL egg albumin (Sigma-Aldrich, St. Louis, MO, USA) to both femurs and intraperitoneal administration of Bordetellapertussis inactive microorganism suspension (B. pertussis, Kitasato Institute Research Center for Biologicals, Saitama, Japan). On day 14, animals were bled and antiserum was collected. Separated antiserum was stored at -80°C.
Rat homologous antiserum (100 μL) was injected into the shaved back skin of rats. After 24 h, sensitized rats were injected with 0.5 mL of mixed solution of 0.5% Evan blue (Fluka, Buchs, Switzerland) and 1% egg albumin (1:1) through the tail vein. Thirty minutes later, rats were sacrificed and the dye content of the shaved dorsal skin was determined quantitatively. Pieces of the dorsal skin containing extravasated dye were removed and soaked overnight in stoppard glass tubes containing 1 ml of mixed solution of 0.6 N H3PO4/acetone (13:5). The absorbance of supernatant was measured at 620 nm using a spectrophotometer and the concentration of the dye in the shaved dorsal skin (μg/site) was determined. Fasting duration was determined as the time between food removal and antigen challenge.
Systemic anaphylactic shock
Ten-week old male BALB/c mice (Japan Clea, Inc. Tokyo, Japan) were used (n = 6–12 in each experiment). For the evaluation for IgE dependent anaphylaxis, mice were sensitized with an intraperitoneal injection of 125 μg egg albumin dissolved in aluminum gel. Seven days later, mice were challenged by intraperitoneal injection of 125 μg egg albumin dissolved in aluminum gel . For IgE independent anaphylaxis, anaphylactic shock was evoked by intraperitoneal injection of 6.5 mg/kg compound 48/80 (Sigma-Aldrich, St. Louis, MO, USA) in saline solution . Immediately after the antigen or compound 48/80 challenge, rectal temperature was measured with a digital thermometer (Tateyama kagaku industry Co.,Ltd, Toyama, Japan).
Preparation of rat peritoneal mast cells
Eleven-week old male Sprague Dawley rats (Japan Clea, Inc. Tokyo, Japan) were used (n = 4 in each treatment). Rat peritoneal mast cells (RPMC) were collected by rinsing the peritoneal cavities of normal rats with 20 ml of mast cell medium (MCM, containing 150 mM NaC1, 3.7 mM KC1, 3.0 mM Na2HPO4, 3.5 mM KH2PO4, 5.6 mM dextrose,0.1% gelatin, 0.1% (w/v) bovine serum albumin) containing 10 U/ml heparin under anesthesia with pentobarbital. Peritoneal cells were sedimented twice at 50 x G for 7 minutes at 4°C and re-suspended in MCM. RPMC were then washed twice with siraganian buffer [119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 25 mM piperazine-N,N0-bis(2-ethanesulfonic acid)(PIPES), pH 7.2] and resuspended in siraganian buffer. RPMC (5 × 104).
The degree of degranulation was determined by measuring the release of β-hexosaminidase . The enzymatic activity of β-hexosaminidase in supernatants and sonicated disrupted cell pellets was measured with p-nitrophenyl N-acetyl-β-d-glucosaminide (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M sodium citrate (pH 4.5) for 60 minutes at 37°C. The reaction was stopped by the addition of 1 M Na2CO3 glycine. The release of the product, 4-p-nitrophenol, was detected by absorbance at 405 nm. The extent of degranulation was calculated by dividing 4-p-nitrophenol absorbance in the supernatant by the sum of absorbance in the supernatant and disrupted cell pellet.
To induce RPMC degranulation, cells were incubated with 1 μg/mL compound 48/80 at 37°C for 10 minutes. For the evaluation of the effect of D-BHB on mast cell degranulation, cells were preincubated with various concentrations of D-BHB for 10 minutes, and then incubated with compound 48/80.
Measurement of intracellular calcium
The [Ca2+]i in RPMC was measured with the fluorescent Ca2+ indicator, Fura-2/AM (Invitrogen, Carlsbad, CA, USA). Cells were plated onto glass bottom culture dishes. The cell was loaded with the indicator by incubation with 2.5 μM of Fura-2/AM in in Standard Solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM dextrose) for 4 h at room temperature (22–24°C). After loading, cells were washed three times with Standard solution and incubated for further 30 minutes to ensure de-esterification. Fluorescence recordings were performed using two set-ups, employing either video-imaging or a single detector system. For video-imaging, a coverslip with dye-loaded cells was mounted in a chamber fixed on the stage of an inverted fluorescence microscope (IX71, Olympus Co., Tokyo, Japan) equipped with UV objective lens (UApo 20X3/340, Olympus Co., Tokyo, Japan). The emission signal passed through a band pass filter (500 ± 10 nm) and was detected by a 12 bit CCD camera (C-8214, Hamamatsu Photonics K.K., Hamamatsu, Japan). Fluorescence intensities at 510 nm with excitation at 340 and 380 nm were recorded at 5 s intervals. The [Ca2+]i in an individual cell was calculated from the ratio of fluorescence images acquired at excitation at 340 nm to those at 380 nm. All experiments were performed at room temperature (22–24°C).
Increases in [Ca2+]i were evoked by 1 μg/mL compound 48/80 for 240 sec. For the evaluation of the effect of D-BHB on [Ca2+]i, cells were stimulated with compound 48/80 under 100 mM D-BHB.
Serum and intraperitoneal D-BHB
Eleven-week old male Sprague Dawley rats (Japan Clea, Inc. Tokyo, Japan) were used (n = 4 in each treatment). The concentration of D-BHB in collected serum and intraperitoneal fluid was measured using Ketone test A (Sanwa kagaku kenkyusho Co., Ltd., Nagoya, Japan) according to the manufacturers instruction.
We used the Student’s t-test for comparison of the two groups and the Dunnett test for multiple comparisons. The Pearson product–moment correlation coefficient was used to evaluate association between anaphylactic response and changes in serum D-BHB concentration. Differences between measured variables were considered significant if the resultant P value was 0.05 or less. Analysis was performed using the JMP version 8 (SAS Institute Inc., Cary, North Carolina, USA).