Effects of nebulized ketamine on allergen-induced airway hyperresponsiveness and inflammation in actively sensitized Brown-Norway rats

Abstract

Since airway hyperresponsiveness (AHR) and allergic inflammatory changes are regarded as the primary manifestations of asthma, the main goals of asthma treatment are to decrease inflammation and maximize bronchodilation. These goals can be achieved with aerosol therapy. Intravenous administration of the anesthetic, ketamine, has been shown to trigger bronchial smooth muscle relaxation. Furthermore, increasing evidence suggests that the anti-inflammatory properties of ketamine may protect against lung injury. However, ketamine inhalation might yield the same or better results at higher airway and lower ketamine plasma concentrations for the treatment of asthma. Here, we studied the effect of ketamine inhalation on bronchial hyperresponsiveness and airway inflammation in a Brown-Norway rat model of ovalbumin (OVA)-induced allergic asthma. Animals were actively sensitized by subcutaneous injection of OVA and challenged by repeated intermittent (thrice weekly) exposure to aerosolized OVA for two weeks. Before challenge, the sensitized rats received inhalation of aerosol of phosphate-buffered saline (PBS) or aerosol of ketamine or injection of ketamine respectivity. Airway reactivity to acetylcholine (Ach) was measured in vivo, and various inflammatory markers, including Th2 cytokines in bronchoalveolar lavage fluid (BALF), as well as inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in lungs were examined. Our results revealed that delivery of aerosolized ketamine using an ultrasonic nebulizer markedly suppressed allergen-mediated airway hyperreactivity, airway inflammation and airway inflammatory cell infiltration into the BALF, and significantly decreased the levels of interleukin-4 (IL-4) in the BALF and expression of iNOS and the concentration of NO in the inflamed airways from OVA-treated rats. These findings collectively indicate that nebulized ketamine attenuated many of the central components of inflammatory changes and AHR in OVA-provoked experimental asthma, potentially providing a new therapeutic approach against asthma.

Figures

Figure 1

Effects of ketamine on ovalbumin-induced airway hyperresponsiveness in vivo. Airway reactivity to intravenous acetylcholine (Ach) was measured 16–18 hr after the last challenge, and is given as the increase in expiratory resistance (Re). (A) Responses were calculated as log percentages of the maximum Re increase above baseline following administration of different concentrations of Ach. (B, C, D) PC100, PC200 and PC400 represent the provocation concentration required to increase Re by 100, 200 and 400%, respectively. The log transformation linear regression U-test was used to examine pair-wise differences in concentration under the same % increase of Re above baseline, with the post-hoc Bonferroni method used to adjust the P-values. The data presented are given as the mean ± SEM (n = 6–8). # P < 0.05 vs. PBS control, * P < 0.05 vs. OVA control. PBS, negative control; OVA, positive control; 12.5 mg/ml, OVA-sensitized/12.5 mg/ml nebulized ketamine-exposed/OVA-challenged; 25 mg/ml, OVA-sensitized/25 mg/ml nebulized ketamine-exposed/OVA-challenged; 50 mg/ml, OVA-sensitized/50 mg/ml nebulized ketamine-exposed/OVA-challenged; 50 μg/kg, OVA-sensitized/50 μg/kg ketamine-injected i.p./OVA-challenged; 100 μg/kg, OVA-sensitized/100 μg/kg ketamine-injected i.p./OVA-challenged.

Figure 2

Effects of ketamine treatment on changes in total and differential cell numbers in BALF from ovalbumin-sensitized and -challenged rats. The total (A) and differential (B) cell numbers were counted in BALF from rats in the various treatment groups. Bars represent mean ± SEM (n = 6–8/group). # P < 0.05 vs. PBS control, * P < 0.05 vs. OVA control. The groups are as shown in Figure 1.

Figure 3

Effects of ketamine on histopathological changes seen in pulmonary sections from ovalbumin-sensitized and -challenged rats. Representative paraffin-embedded, Hematoxylin and Eosin-stained lung sections were prepared from the left lungs of experimental rats, showing bronchioles, lung alveoli and surrounding vessel structures. The groups are as shown in Figure 1. (A) PBS; (B1-B2) OVA; (C) 12.5 mg/ml; (D) 25 mg/ml; (E) 50 mg/ml; (F) 50 μg/kg; (G) 100 μg/kg. VL, vascular lumen; AL, airway lumen. Arrows demonstrate marked perivascular edema (B1, G), places where inflammation has destroyed a portion of the airway epithelium (B2, F), and eosinophils (B1, C, E). Arrowheads indicate peribronchial inflammation (B1, C, D, E, F, G), hemorrhage (B2, E, F, G) and congestion (B2). Sections were evaluated under light microscopy. Original magnification was ×40, and the scal bars represent 20 μM.

Figure 4

Effects of ketamine on inflammatory scores in lung tissues from ovalbumin-sensitized and -challenged rats. Lungs were sampled after measurement of airway reactivity and scored under the light microscope at a magnification of ×40. Bars represent mean ± SEM (n = 6–8/group). # P < 0.05 vs. PBS control, * P < 0.05 vs. OVA control. The groups are as shown in Figure 1.

Figure 5

Effects of ketamine on Interleukin-4 and Interleukin-13 protein levels in BALF from ovalbumin-sensitized and -challenged rats. IL-4 (A) and IL-13 (B) protein levels in BALF were measured by ELISA. Bars represent mean ± SEM (n = 6–8/group). # P < 0.05 vs. PBS control, * P < 0.05 vs. OVA control. The groups are as shown in Figure 1.

Figure 6

Effects of ketamine on NOS mRNA expression in pulmonary tissues from ovalbumin-sensitized and -challenged rats. (A) Expression of NOS mRNA in pulmonary tissues was detected by RT-PCR. Representative RT-PCR products for nNOS, iNOS, eNOS and β-actin (internal control) are shown. The groups are as shown in Figure 1. Lane 1, PBS; lane 2, OVA; lane 3, 12.5 mg/ml; lane 4, 25 mg/ml; lane 5, 50 mg/ml; lane 6, 50 μg/kg; lane 7, 100 μg/kg. (B) Semiquantitative densitometry was used to analyze iNOS and eNOS mRNA expression levels, normalized to that of β-actin. Bars represent mean ± SEM expressed as the relative density of NOS versus that of β-actin (n = 6–8/group). # P < 0.05 vs. PBS control; * P < 0.05 vs. OVA control.

Figure 7

Effects of ketamine on iNOS protein expression in pulmonary tissues from ovalbumin-sensitized and -challenged rats. Protein expression of iNOS in lung homogenates obtained from experimental rats was examined by Western blotting, with a representative example from each group shown. β-actin was detected as an internal control. The groups are as shown in Figure 1. (A) Lane 1, PBS; lane 2, OVA; lane 3, 12.5 mg/ml; lane 4, 25 mg/ml; lane 5, 50 mg/ml; lane 6, 50 μg/kg i.p,; lane 7, 100 μg/kg. (B) Densitometric analysis of iNOS protein levels in lungs obtained from rats in each group. The bars represent the mean ± SEM, with laser densitometry used to standardize iNOS protein levels with respect to that of β-actin (ratio of iNOS/β-actin, n = 6–8/group). # P < 0.05 vs. PBS control; * P < 0.05 vs. OVA control.

Figure 8

Effects of ketamine on nitric oxide production in pulmonary tissues from ovalbumin-sensitized and -challenged rats. Nitric oxide production was determined by measurement of nitrite, and the results are expressed as μmol/g protein. Bars represent mean ± SEM (n = 6–8/group). # P < 0.05 vs. PBS control; * P < 0.05 vs. OVA control. The groups are as shown in Figure 1.

Figure 9

HPLC determination of ketamine concentration in plasma from rats receiving nebulized ketamine. The retention times of ketamine and phenacetin (internal calibrator) in the utilized system were ~5.87 and 2.58 min, respectively. (A) Chromatograms of blank plasma supplemented with 500 μg/L of a ketamine standard. (B) Representative chromatogram of plasma sample collected from a normal rat at 0 min after inhalation of 25 mg/ml ketamine. (C) Plasma concentration-time profiles of ketamine over a period of 60 min after the rats received nebulized ketamine at concentration of 12.5, 25 or 50 mg/ml respectively. The values represent the mean ± SEM (n = 6/group).