Objectives: This work aimed to study the effect(s) of environmental ozone (O3) exposure on the olfactory bulbs (OB) of Swiss Webster and C57BL/6J mouse strains, by using immunochemical (Western blot), and electrophysiological (patch clamp) techniques. In addition, to behavioural experiments (olfactory habituation/dishabituation test). Method: Swiss Webster and C57BL/6J mice were exposed to different O3 doses for different durations. Then, the effect of O3 exposure was evaluated by measuring the levels of oxidative stress biomarkers (4HNE and CYP1A1) in plasma and/or in the olfactory bulb (OB) samples, in control and O3 exposed mice. In addition, the excitability profiles of bulbar dopaminergic cells were measured in C57BL/6J mice. Moreover, the effect of O3 exposure on the olfactory response to social and non-social odours was also evaluated in the two strains. Finally, the body weight of the exposed mice was measured before and after O3 exposure for the two strains. Results: Concerning Swiss Webster mouse strain, O3 exposure to doses of 0.2 ppm 2 h/2 times/1 day, 0.5 ppm 30 min/once, 0.5 ppm 60 min/once and 0.2 ppm 2 h/2 times/2 days, did not cause any sufferance to the tested mice. The biochemical results showed a significant increase in the level of the oxidative stress biomarkers and OB total proteins levels in O3 exposed mice compared to the air exposed mice. In addition, the body weight of the O3 exposed mice was significantly reduced after the exposure. Furthermore, the biochemical results of C57BL/6J mice did show any significant differences in the plasma 4HNE and OB total proteins of mice exposed to 0.8 ppm 2h/2 times/5 days (GFP and WT), 0.5 ppm 30 min/once and 0.5 ppm 60 min/once O3 doses compared to air exposed mice. However, the OB 4HNE was statistically increased in the 0.8 ppm 2h/2 times/5 days O3 dose exposed WT mice. On the other hand, this marker was statistically insignificant in the other O3 doses exposed mice compared to control mice. Moreover, the CYP 1A1 was not detected in the GFP mice. Furthermore, the body weight of the all O3 exposed mice was statistically reduced after the exposure except for 0.5 ppm O3 doses exposed mice for 60 min once. However, the electrophysiological experiments showed a significant alteration in the spontaneous firing activity of bulbar DA neurones following O3 exposure, with overall reduction in the firing frequency, membrane resistance, rheobase and chronaxie with reorganization of spontaneous firing in bursts. Moreover, the variation in the number of the DA neurons between O3 and control mice was statistically insignificant. Regarding the behavioural experiment results, the effect of O3 exposure was different in the two strains. C57BL / 6J mice were less sensitive in comparison to Swiss Webster mice, while the latter showed high loss of olfactory sensitivity to environmental odours following O3 exposure. In addition, concerning Swiss Webster mice, the females were more vulnerable to O3 exposure than males. Conclusion: In the current study, O3 exposure caused an increase in the level of the proteins involved in the oxidative stress state in the OB of Swiss Webster mouse strain. While in C57BL/6J mouse strain this effect was not observed, but the electrophysiological studies showed a significant change in the firing properties of the OB DA cells. Also, the behavioural test showed deterioration in the olfactory function which was strain and sex dependant. Finally, O3 exposure caused a significant decrease in the body weight of the tested mice in both strains.
Scopo del lavoro: Lo scopo di questo lavoro è stato quello di studiare gli effetti dei danni provocati dalla applicazione di differenti dosi di ozono sul bulbo olfattorio (OB) di topi della linea Swiss Webster e C57BL/6J attraverso analisi immunochimica (Western Blot), elettrofisiologica (patch-clamp) e studi comportamentali (abituazione e disabituazione). Materiali e Metodi: topi dei ceppi Swiss Webster e C57BL / 6J sono stati esposti ad ozono per tempi e dosi differenti. Gli effetti dell’esposizione ad ozono sono stati valutati misurando i livelli di espressione di marker specifici dello stress ossidativo, quali 4HNE e CYP1A1 sia nell’OB che a livello plasmatico. Successivamente, questi valori sono stati paragonati a quelli ottenuti facendo lo stesso tipo di indagine sui topi di controllo, esposti all’aria. Quindi, sono state valutate le caratteristiche elettrofisiologiche di base dei neuroni dopaminergici del bulbo olfattorio dei topi C57BL/6J esposti ad ozono. Inoltre è stato valutato l’effetto dell’ozono sulla risposta olfattiva a odori sociali e non. Per tutti gli animali è stato inoltre valutato il peso prima e dopo i diversi trattamenti. Risultati: In topi del ceppo Swiss Webster, esposizioni all’ozono a dosi di, 0.2 ppm 2 h / 2 volte / 1 giorno, 0.5 ppm 30 min / una volta e 0.5 ppm 60 min / una volta, non hanno causato alcuna sofferenza ai topi. I risultati dell’analisi biochimica hanno mostrato un aumento significativo della proteina plasmatica 4HNE e della proteina bulbare 4HNE in topi esposti ad ozono rispetto ai topi di controllo. Il peso corporeo dei topi esposti era significativamente ridotto rispetto ai controlli. In topi del ceppo C57BL/6J, l’analisi biochimica non ha mostrato variazioni significative nei livelli di 4HNE nei topi esposti a ozono 0.8 ppm 2h / 2 volte / 5 giorni (GFP e WT), 0.5 ppm 30 min / una volta e 0.5 ppm 60 min / una volta rispetto ai topi di controllo esposti all'aria, la proteina CYP1A1 non è stata rilevata in questo ceppo di topi. Gli esperimenti elettrofisiologici hanno mostrato una significativa alterazione della scarica spontanea nei neuroni DA bulbari in seguito ad esposizione a ozono, con una riduzione complessiva della frequenza di scarica, e una riorganizzazione dell’attività spontanea in burst. Nessuna variazione significativa del numero di neuroni DA è stata osservata in seguito ad esposizione all'ozono. Gli studi comportamentali hanno mostrato effetti diversi nei due ceppi. Il C57BL/6J è risultato essere meno sensibile rispetto allo Swiss Webster. In quest’ultimo è stata messa in evidenza anche una diversità di genere nella risposta. Conclusioni: In questo lavoro, è stato dimostrato che l’esposizione ad ozono causa un aumento dei livelli di espressione delle proteine coinvolte nello stress ossidativo a livello del bulbo olfattorio dei topi Swiss Webster, ma non nei topi C57BL/6J. Anche le proprietà elettrofisiologiche dei neuroni dopaminergici dei topi C57BL/6J sono modificate in seguito alla esposizione a differenti dosi di ozono. Infine i test comportamentali hanno mostrato un deterioramento della funzione olfattiva in risposta alla esposizione all’ozono sesso- e ceppo-dipendente. I risultati indicano che i topi Swiss Webster sono più sensibili all'esposizione all'ozono rispetto C57BL/6J, mostrando attivazione dei marcatori di stress ossidativo a concentrazioni di ozono inferiori. Tuttavia, i C57BL/6J mostrano una significativa alterazione nella funzione dei neuroni DA bulbari che molto probabilmente compromette la funzione olfattiva.
Effects of environmental ozone exposure on mice olfactory bulb. Immunochemical, electrophysiological and behavioural studies
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2017
Abstract
Objectives: This work aimed to study the effect(s) of environmental ozone (O3) exposure on the olfactory bulbs (OB) of Swiss Webster and C57BL/6J mouse strains, by using immunochemical (Western blot), and electrophysiological (patch clamp) techniques. In addition, to behavioural experiments (olfactory habituation/dishabituation test). Method: Swiss Webster and C57BL/6J mice were exposed to different O3 doses for different durations. Then, the effect of O3 exposure was evaluated by measuring the levels of oxidative stress biomarkers (4HNE and CYP1A1) in plasma and/or in the olfactory bulb (OB) samples, in control and O3 exposed mice. In addition, the excitability profiles of bulbar dopaminergic cells were measured in C57BL/6J mice. Moreover, the effect of O3 exposure on the olfactory response to social and non-social odours was also evaluated in the two strains. Finally, the body weight of the exposed mice was measured before and after O3 exposure for the two strains. Results: Concerning Swiss Webster mouse strain, O3 exposure to doses of 0.2 ppm 2 h/2 times/1 day, 0.5 ppm 30 min/once, 0.5 ppm 60 min/once and 0.2 ppm 2 h/2 times/2 days, did not cause any sufferance to the tested mice. The biochemical results showed a significant increase in the level of the oxidative stress biomarkers and OB total proteins levels in O3 exposed mice compared to the air exposed mice. In addition, the body weight of the O3 exposed mice was significantly reduced after the exposure. Furthermore, the biochemical results of C57BL/6J mice did show any significant differences in the plasma 4HNE and OB total proteins of mice exposed to 0.8 ppm 2h/2 times/5 days (GFP and WT), 0.5 ppm 30 min/once and 0.5 ppm 60 min/once O3 doses compared to air exposed mice. However, the OB 4HNE was statistically increased in the 0.8 ppm 2h/2 times/5 days O3 dose exposed WT mice. On the other hand, this marker was statistically insignificant in the other O3 doses exposed mice compared to control mice. Moreover, the CYP 1A1 was not detected in the GFP mice. Furthermore, the body weight of the all O3 exposed mice was statistically reduced after the exposure except for 0.5 ppm O3 doses exposed mice for 60 min once. However, the electrophysiological experiments showed a significant alteration in the spontaneous firing activity of bulbar DA neurones following O3 exposure, with overall reduction in the firing frequency, membrane resistance, rheobase and chronaxie with reorganization of spontaneous firing in bursts. Moreover, the variation in the number of the DA neurons between O3 and control mice was statistically insignificant. Regarding the behavioural experiment results, the effect of O3 exposure was different in the two strains. C57BL / 6J mice were less sensitive in comparison to Swiss Webster mice, while the latter showed high loss of olfactory sensitivity to environmental odours following O3 exposure. In addition, concerning Swiss Webster mice, the females were more vulnerable to O3 exposure than males. Conclusion: In the current study, O3 exposure caused an increase in the level of the proteins involved in the oxidative stress state in the OB of Swiss Webster mouse strain. While in C57BL/6J mouse strain this effect was not observed, but the electrophysiological studies showed a significant change in the firing properties of the OB DA cells. Also, the behavioural test showed deterioration in the olfactory function which was strain and sex dependant. Finally, O3 exposure caused a significant decrease in the body weight of the tested mice in both strains.| File | Dimensione | Formato | |
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Phd thesis-07-11-2017 -Samah Batran.pdf
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Descrizione: Ph.D thesis Abdelrahim Batran Ali Samah
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