Chlorine dioxide

To prepare a mechanically strong membrane, it was reinforced by filter paper and the gelatin was cross-linked with glutaraldehyde. As the cellulose in the filter paper does not react with ClO₂ from the point of our experiments, it is an inert material.
10 ml of 10 % aqueous gelatin solution was mixed rapidly with 0.5 ml of 25 % glutaraldehyde solution at room temperature, and a filter paper disk (diameter: 10 cm) was soaked with the mixture. Then the disk was placed between two glass plates covered with polyethylene foils. Spacers were applied to produce a 0.5 mm thick membrane. After a 2 hour setting time the filter paper reinforced gelatin membrane was removed from the form and it was placed into distilled water overnight before the measurements.

A video fear-conditioning system and software (Med Associates, Burlington, VT, USA) were used to measure freezing in the ICR mice while in a conditioning chamber (dimensions: 30.5 × 24.1 × 21.0 cm), which was cleaned in between testing with Vimoba, a chlorine dioxide solution. Freezing was defined as no movement other than respiration.^{28 (link)} Mice were moved to a holding room adjacent to the test room and acclimated for 30 min (min) under 175–177 lux room lighting before testing.
For fear-conditioning procedures, mice were moved to the test room under the same conditions (175–177 lux) and then placed in the conditioning chamber for 30 s. After an initial one-half min of habituation (baseline), ICR mice were presented with six conditioned stimulus–unconditioned stimulus (CS–US) pairings (for example, tone–footshock pairings) separated by a 30-s interval. Each tone (80 dB, 3000 Hz) lasted 30 s. Mice were presented with the electric footshock at 0.7 mA during the last 2 s of each 30-s tone. The number of CS–US pairings and footshock intensity was used because ICR mice only present an appreciable increase in freezing behavior by the fourth CS–US presentation, demonstrating acquisition of conditioned fear learning (see Figures for illustrated examples). At the end of the fear-conditioning protocol, mice were placed in their home cages in a separate holding room before being returned to their housing facility.
The following day, mice were placed in the conditioning chamber with a white smooth floor contextual insert that was positioned over the grid floor and a white curved wall contextual insert (context B). Vanilla extract (McCormick, Sparks, MD, USA) was used as a distinctive olfactory cue in the conditioning chamber for the short-term extinction training. After 30 s of habituation in the chamber, mice were presented with 20 tones (80 dB, 3000 Hz) with durations of 30 s, each separated by a 30-s interval. After 20 min and 30 s, mice were returned to a holding room and their home cages.
The extinction protocol was repeated the following day using the same procedure and drug administration. For contextual fear conditioning, an identical paradigm was used except that all tones were omitted on all days of the experiment, and the extinction context was identical to that of the conditioning context. In overtraining experiments, mice were conditioned as described above for 5 consecutive days with the exact same procedure. The short-term extinction training procedure thereafter was identical to that described for the single conditioning experiments.

The gaseous ClO₂ was obtained using a novel gaseous chlorine dioxide treatment system developed by our previous study, and the schematic of the ClO₂(g) treatment system is shown in Figure 1 [25 (link)]. Briefly, 0.4 mL of 20% NaClO₂ solution was injected in equal amounts into four absorbent pads (1 cm × 1 cm × 1 cm; CoSoFth Co., Jinjiang, China), which were affixed to the inside wall of a self-made treatment chamber (15 L). Then, CO₂ gas was generated using 5 ± 0.2 g of dry ice (BaiQuan Co., Fuzhou, China). The CO₂ gas was absorbed by the absorbent pads and dissolved in the solution to form H₂CO₃, which then reacted with NaClO₂ to produce ClO₂ gas. The equations for ClO₂ gas generation are as follows. The intensity of chlorine dioxide gas is calculated by the cumulative exposure of chlorine dioxide (mg·L⁻¹).
$${\mathrm{C}\mathrm{O}}_2\left(\mathrm{g}\right)+{\mathrm{H}}_2\mathrm{O}\leftrightarrow {\mathrm{H}}_2{\mathrm{C}\mathrm{O}}_3$$
$$2{\mathrm{H}}_2{\mathrm{C}\mathrm{O}}_3+5{\mathrm{N}\mathrm{a}\mathrm{C}\mathrm{l}\mathrm{O}}_2\to 2{\mathrm{H}}_2\mathrm{O}+2{\mathrm{N}\mathrm{a}}_2{\mathrm{C}\mathrm{O}}_3+4{\mathrm{C}\mathrm{l}\mathrm{O}}_2\left(\mathrm{g}\right)+\mathrm{N}\mathrm{a}\mathrm{C}\mathrm{l}$$