Neuropathological Examination of Mice Chronically Exposed to Secondhand Smoke

Abstract

Introduction Around 21.6–35% of military personnel are smokers, while 12.26% of them have been regularly exposed to second-hand smoke (SHS). Second-hand smoke is considered an important risk factor for neurological diseases because it can induce oxidative stress, DNA damage, and disrupt DNA repair pathways. Material and Methods The brain of air (sham) or SHS exposed mice was cryoperserved, sectioned, and placed on a glass slide before immunoprobing them with antibodies to observe for oxidative DNA damage (8-oxoG), oxidative DNA repair (8-oxoguanine DNA glycosylase 1, Ogg1; apurinic/apyrimidinic endonuclease, Ape1), and inflammatory (glial fibrillary acidic protein) proteins. Results Nissl staining of the prefrontal cortex (PFCTX) revealed the presence of dark, shrunken cells, hippocampal thinning, and the presence of activated astrocytes in SHS exposed mice. 8-oxoG staining was also more prominent in the PFCTX and hippocampus (HIPP) of SHS exposed mice. Ogg1 staining was reduced in the PFCTX and CA3 hippocampal neurons of SHS exposed mice, whereas it was more prominent in CA1 and CA4 hippocampal neurons. In contrast, Ape1 staining was more prominent in the PFCTX and the HIPP of SHS exposed mice. Conclusions These studies demonstrate that oxidative DNA damage (8-oxoG) was elevated and oxidative DNA repair (Ape1 and Ogg1) was altered in the brain of SHS exposed mice. In addition, activated astrocytes (i.e., glial fibrillary acidic protein) were also observed in the brain of SHS exposed mice. Therefore, SHS induces both oxidative DNA damage and repair as well as inflammation as possible underlying mechanism(s) of the cognitive decline and metabolic changes that were observed in chronically exposed mice. A better understanding of how chronic exposure to SHS induces cognitive dysfunction among military personnel could help improve the combat readiness of U.S. soldiers as well as reduce the financial burden on the DOD and veterans’ families.

Behavioral and Cognitive Performance Following Exposure to Second-Hand Smoke (SHS) from Tobacco Products Associated with Oxidative-Stress-Induced DNA Damage and Repair and Disruption of the Gut Microbiome

“…Antibodies. The immunohistochemistry was carried out using a mouse monoclonal antibody (15A3) raised against 8-hydroxyguanosine (8-OHdG) from Santa Cruz Biotechnology (Dallas, TX, USA), anti-APE1 polyclonal antibodies from ThermoFisher and an antibody to the senescence marker p21 from Abcam (ab107099) as described in [ 25 , 30 ]. The quantification of the results of the immunohistochemical analyses for oxidative-stress-induced markers in the CA1 region of the hippocampus and prefrontal cortex are described in Table 1 [ 30 ].…”

“…The stained sections were imaged on a Leica DM100 microscope (Deerfield, IL, USA) at various magnifications. Three representative images from the prefrontal cortex of air- and SHS-exposed mice were manually counted, the counts averaged and the data analyzed using R as previously described [ 30 ].…”

“…Higher magnifications of the prefrontal cortex ( boxes: 34.7x). Stained sections from air- and SHS-exposed mice ( n = 3/tx) were manually counted for β-Gal staining, as previously described [ 30 ]. There was no effect of SHS on β-Gal staining in either brain region.…”

“…In a follow-up study, we reported increased levels of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), a marker of oxidative DNA damage and a biomarker of DNA damage following exposure to cancer-causing agents [ 29 ], generated following oxidative-stress-induced damage to 2′-deoxyguanosine in the prefrontal cortex of SHS as compared to air controls and a trend towards increased levels in the CA1 area of the hippocampus [ 30 ]. In the prefrontal cortex, levels of the oxidative DNA repair marker AP endonuclease 1 (APE1), which is involved in the repair of oxidative DNA damage, were also higher in SHS than air-exposed mice [ 30 ]. SHS might also increase various markers of cell senescence in the brain following the oxidative DNA damage, such as β-galactosidase [ 31 , 32 , 33 ].…”