2.6. Experimental Autoimmune Encephalomyelitis (EAE)

Age-matched (approximately 9-week-old) C57BL/6J WT females were exposed intranasally to SRM1649b PM or PBS control. Female mice are used because they are more susceptible to EAE than male mice (Constantinescu et al., 2011; Rahn et al., 2014). The mice were exposed to 40.0mg/mL PM or 800μg PM of SRM1649b PM in a total of 20μL per dose. Mice were anesthetized via inhalation with isoflurane for each intranasal treatment of SRM1649b PM and PBS control. Each mouse was anesthetized for a maximum of 15 minutes. EAE scoring was done prior to anesthesia via isoflurane.

Disease was induced on day 0 by injecting a myelin oligodendrocyte glycoprotein (MOG_35–55) emulsion and pertussis toxin. First, 500μL of pertussis toxin was injected intraperitoneally (IP) at 200ng per mouse. Next, 100μL of the MOG_35–55 emulsion was injected subcutaneously between the shoulder blades of each mouse. For the emulsion, 50mg MOG35–55 peptide (Tocris) was prepared and mixed in Complete Freund’s Adjuvant¹⁵ (CFA) (BD) augmented with 4mg/mL heat-killed M. tuberculosis (Difco) at a 1:1 ratio. The heat-killed M. tuberculosis was present in the emulsion at 200μg/mouse and MOG_35–55 peptide was present in the emulsion at 50μg/mouse. Mice were anesthetized with isoflurane and weighed prior to injection on day 0.

Additionally, on day 2, or 44–52 hours after the initial pertussis injection, the mice were injected with another 500μL of pertussis toxin IP as a booster. Mice were scored and weighed daily starting day 7 after induction and sacrificed on day 28. The mice were scored according to a standard procedure as follows: 0, no clinical symptoms; 0.5, partially limp/flaccid tail, 1, limp/flaccid tail; 2, hind limb weakness with incomplete paralysis, loss in coordinated movement, hind impaired righting reflex; 3, partial paralysis of hind limbs; 4, complete hind limb paralysis and 5, paraplegia or moribund. Mice were monitored and managed for pain and discomfort. Mice were analyzed solely for clinical endpoints.

Peak clinical score was calculated as the highest clinical score each mouse achieved during the experiment. Cumulative clinical score is the sum of all the clinical scores for each mouse throughout the experiment duration. For cumulative clinical score, mice that died due to disease were given a score of 5 and that score was carried until the end of the experiment. Day of onset represents the first day after EAE induction that a score greater than 0 is noted. The scores are calculated for each mouse on each day and in the case of peak score, the highest score is used, for cumulative score, the sum of the scores from day 7 to day 28 is used, and day of onset is the first day the mouse receives an EAE score. Each mouse has its own score that is included in analysis.

The mice were dosed with 40.0mg/mL PM of SRM1649b PM. The 40.0mg/mL PM or 800μg dose, equates to 26,666.7μg/m³, based on the amount of air a mouse would breathe in 24hrs. This is extremely high when considering the mass of PM per dose when compared to the 35.0μg/m³ 24-hour primary and secondary United States Environmental Protection Agency National Ambient Air Quality Standards for PM, however the dose chosen for the current study was based on the mass of PAHs present in PM per dose.

The nanogram PAH per milliliter of exposure based on a 20μL dose of 40.0mg/mL SRM1649b PM was calculated (Table 1). The calculations show the ng PAHs per exposure (0.02mL). The 8hr and 24hr air exposures were calculated based on the amount of air a mouse would breathe in 8hrs and 24hrs (Raşid et al., 2012) (Table 1). The calculated amounts were 0.01m³ for 8hrs and 0.03m³ for 24hrs. The permissible exposure level of PAHs set for humans by Occupational Safety and Health Administration (OSHA) is 0.2mg/m³ (200,000ng/m³) which is an 8hr time- weighted average permissible exposure limit (ATSDR, 2008). This demonstrates that the in vivo exposure the mice received is within the OSHA standard for humans and can be considered a relevant exposure.

The nanogram PAH per milliliter of exposure calculations are based on a 20μL dose of 40.0mg/mL SRM1649b PM). The calculations show the ng PAHs/mL of exposure (0.02mL). The 8hr and 24hr air exposures were calculated based on the amount of air a mouse would breath in 8hrs and 24hrs. The amounts were 0.01m³ for 8 hours and 0.03m³ for 24hrs (Raşid et al., 2012). The permissible exposure level of PAHs set for humans by OSHA is 0.2mg/m³ (200,000ng/m³) which is an 8-hr time-weighted average permissible exposure limit (ATSDR, 2008). This table demonstrates that the in vivo exposure the mice receive is within the OSHA standard for humans and can be considered a relevant exposure. Abbreviations: PAH, polycyclic aromatic hydrocarbon(s); SRM, standard reference material; OSHA, Occupational Safety and Health Administration.

The dosing regimens chosen in the current study aimed to account for long-term exposures, acute exposures around the time of disease induction, and every day exposures which would occur before and after disease induction. The mice received 40mg/mL PM (800μg in 20μL) of SRM1649b PM or PBS control intranasally 8 times starting at day −12 every 3 days until day 9 after induction for regular dosing. This was used to assess every day exposures and did not include additional doses at the time of disease induction. For chronic intranasal exposure, mice received 40.0mg/mL (800μg in 20μL) SRM1649b PM or PBS control intranasally 17 times starting at day −27 every 3 days until day 15 after induction. The mice were given intranasal doses on days 1 and 2 in addition to the pre-exposure. This regimen was used to assess long-term chronic exposures and two additional doses were added at the time of disease induction to assess whether chronic exposures to PM would alter severity of EAE. For acute exposure, mice received 4.0mg/mL (80μg in 20μL) or 40.0mg/mL (800μg in 20μL) SRM1649b PM or PBS control intranasally 10 times starting at day −7, every day until day 2 after induction. This regimen was used to assess a short exposure centered around the time of disease induction.

Mice were anesthetized via inhalation with isoflurane for each intranasal treatment of SRM1649b PM and PBS. Each mouse was anesthetized for less than 15 minutes. The mice were anesthetized one time per day and all injections or intranasal treatments were conducted at one time. EAE scoring was done prior to anesthesia via isoflurane. The effects of isoflurane anesthesia on mice has been well-studied. One study demonstrated that intermittent-repeated isoflurane exposure for 45 minutes at an interval of 3–4 days for a total of 6 times, had mild effects on the mice, based on the Mouse Grimace Scale in C57BL/6Rj mice (Hohlbaum et al., 2017). In the current study, the mice are anesthetized with isoflurane for a maximum of 15 minutes and the regular dosing regimen is similar. Moreover, another study showed that that 0.5% of isoflurane for 4 hours per day, 5 days a week, for 9 weeks, caused no change in body weight, organ weight, concentration of hepatic cytochrome P450 enzymes, or the rates of hepatic microsomal metabolism and concluded that since there was no evidence of organ toxicity or enhanced or inhibited microsomal metabolism, the regimen of isoflurane was relatively non-toxic under the conditions of the study (Rice et al., 1986). This dosing regimen is similar to the acute and chronic based on the daily use of isoflurane and the number of days the mice were anesthetized, but one clear difference is how long the mice are anesthetized for. These studies suggest that the isoflurane exposure to the mice is relatively non-toxic and has mild effects on the mice. On the other hand, it has been shown that 2hrs of 1.8% isoflurane in C57BL/6J mice led to attention deficit in the mice based on a battery of behavior tests (Yonezaki et al., 2015). In the current study, any potential effects of repeated isoflurane exposure in the EAE model are controlled for by using the PBS vehicle control in which all of the mice also get anesthetized. Tolerance to isoflurane was not observed over time and no treatment effects were observed. Therefore, the effects observed after SRM1649b PM treatment are likely not due to residual isoflurane effects but the PM exposure.

For AHR diet experiments, age matched female WT (C57BL/6J) mice were started on base diet (no AHR ligands) or I3C¹⁶ (enriched AHR ligands) at day −38. The base diet is a diet of essential amino acids that contains no AHR ligands and the diet enriched in AHR ligands is base diet supplemented with indole-3-carbinol, is a tryptophan derivative found in cruciferous vegetables and a precursor to high affinity AHR ligands (Hooper, 2011). Once exposed to stomach acid, I3C is converted into high affinity AHR ligands and can elicit anti-inflammatory and anti-cancer effects (Benson and Shepherd, 2011; Hooper, 2011). Mice received 4.0mg/mL in 200μL (800μg per dose or 40μg/g/day) PM or 1.0mg/mL in 200μL (200μg or 10μg/g/day) PM of SRM1649b PM or Saline control by oral gavage 8 times starting at day −12 every 3 days until day 9 after induction. The calculated dose of PM per mass of mouse was based on the average size of a mouse being 20 grams. Mice were anesthetized via inhalation with isoflurane for each gavage treatment