Assessment of Airway Sensitization Potential of Inhaled Trimellitic Anhydride by Monitoring the Elicitation Phase in a Mouse Model

Figures & data

TABLE 1 Protocols of sensitization, challenge and measurements for each group

Group	1st sensitization (Day 0)	2nd sensitization (Day 7)	Challenge (Day 14, for 15 min)	Measurements
Mouse IgE Test (Chemical: TMA)	25% TMA, 25 μl/Abdomen	12.5% TMA, 12.5 μl/Auricle	—	Total IgE on Day 14
EP/EP	25% TMA, 25 μl/Abdomen	12.5% TMA, 12.5 μl/Auricle	High concentration: 0.022 mg/l Low concentration: 0.0075 mg/l	AR on Days 15 & 16 besides the following examinations
ID/EP	3% TMA, 0.1 ml/Abdomen	12.5% TMA, 12.5 μl/Auricle	High concentration: 0.03 mg/l Low concentration: 0.0038 mg/l	AR on Day 14, Total IgE on Day 16, BALF analysis on Day 16, Lung pathology
Non-sensitized control	AOO, 25 μl/Abdomen	AOO, 12.5 μl/Abdomen	0.042 mg/l
ID/ID	3% TMA, 0.1 ml/Abdomen	3% TMA, 0.05 ml/Auricle (Day 5)	0.004 mg/l (Day 13)
IH/IH	0.02 mg/l for 45 min, 0.042 mg/l for 30 min with 3 hr interval	0.03 mg/l for 15 min	0.0063 mg/l (Day 18)	AR on Day 13 or 18 Total IgE on Day 13 or 18
OVA	8 ug OVA ip	8 μg OVA ip (Day 5)	0.5%OVA (Day 13, for 30 min)

Group: EP, Epicutaneous application of TMA on the abdomen or auricle; ID, Intradermal administration of TMA in the abdominal region or auricular region; IH, Inhalation of TMA dust; OVA, ovalbumin.

AR: Measurement of airway response during 5-min pre-challenge, 15-min challenge and 40-min post-challenge and for one hour on 24- and 48 hr post-challenge.

BALF: Bronchoalveolar lavage fluid.

FIG. 1 Penh values in pre-challenge, challenge, and post-challenge periods. Conscious mice were individually held in a restrained flow whole body plethysmograph and enhanced pause (Penh) was recorded every 1 min during 5 min pre-challenge, 15 min challenge, and 40 min post-challenge periods using an analytical recording system. Mice were challenged with fine TMA dust. Symbols represent airway responses of 6 mice/group.

TABLE 2 Enhanced pause (Penh) before, during and after challenge

	(Mean ± SD of 6 mice)
Group	Before challenge	During challenge	After challenge
EP/EP High concentration	0.452 ± 0.042	0.947 ± 0.374	1.013 ± 0.454
EP/EP Low concentration	0.562 ± 0.113	0.579 ± 0.154	0.581 ± 0.094
ID/EP High concentration	0.496 ± 0.051	0.731 ± 0.363	0.647 ± 0.180
ID/EP Low concentration	0.440 ± 0.038	0.471 ± 0.064	0.583 ± 0.180
Non-sensitized control	0.488 ± 0.038	0.523 ± 0.103	0.548 ± 0.077
ID/ID	0.458 ± 0.078	0.529 ± 0.054	0.903 ± 0.623
IH/IH	0.378 ± 0.065	0.611 ± 0.293	0.609 ± 0.098
OVA	0.412 ± 0.084	0.523 ± 0.139	0.485 ± 0.117

EP, Epicutaneous application of TMA on the abdomen or auricle; ID, Intradermal administration of TMA in the abdominal or auricular region; IH, Inhalation of TMA dust; OVA, ovalbumin.

Penh Before challenge (5 min) is the average of mean values of 6 mice and used as baseline data.

Penh During (15 min) and After (40 min) challenge are the means of peak values of 6 mice.

* p < 0.05 compared with Before challenge baseline data.

FIG. 2 Total serum IgE values in each group. Two days after inhalation challenge, 6 mice/group were anesthetized with pentobarbital and blood samples drawn from the posterior vena cava. Total serum IgE values were measured with a sandwich enzyme-linked immunosorbent assay (ELISA). ** p < 0.01 compared with non-sensitized control mice.

TABLE 3 BALF analyses in the EP/EP, ID/EP and Non-sensitized control groups

Group	Total cell counts (×10^4/ml)	Eosinophils (%)	Neutrophils (%)	Macrophages (%)
EP/EP High concentration	19.5 ± 9.0	16.7 ± 12.6**	1.2 ± 1.5	82.1 ± 13.7**
EP/EP Low concentration	15.3 ± 7.7	1.0 ± 1.5	2.3 ± 2.2	96.7 ± 2.3
ID/EP High concentration	11.0 ± 5.6	1.3 ± 1.9	0.8 ± 0.8	98.3 ± 1.0
ID/EP Low concentration	13.0 ± 5.7	0.6 ± 0.9	2.6 ± 1.8	96.8 ± 1.3
Non-sensitized control	11.3 ± 4.3	0.5 ± 0.8	1.0 ± 1.7	98.5 ± 2.0

EP, Epicutaneous application of TMA on the abdomen or auricle; ID, Intradermal administration of TMA in the abdominal or auricular region.

Total number of cells was counted using a hemocytometer. Two hundred cells in the smears of BALF were differentiated.

** p < 0.01 compared with Non-sensitized control group.

FIG. 3 Representative pulmonary region of lungs. Pulmonary regions from non-sensitized control group (A, B) and from EP/EP^hi group (C, D) 48 hr after challenge with TMA dust. A cell infiltration (including eosinophils) into the airspace and perivascular portions was observed only in TMA-sensitized and -challenged mice.