NO₂Inhalation Contributes To The Development And Progression Of Ischemic Stroke And Vascular Denmentia In Rat

As important as TSP, PM10and SO2, NO2, the major component of nitrogen oxides (NOx), also represents an important urban pollutant in most developed cities of the world. People are widely exposed to NO2discharged from multiple sources including automobile exhaust, fossil fuel burning industries, gas cooking stoves, tobacco smoking and some others in occupational environment. Peak levels of up to0.2parts per million (ppm) are encountered in the outdoors, particularly along kerbsides in downtown areas with heavy motor vehicular traffic. Indoors NO2concentrations are often greater than those found outdoors, with peak levels exceeding2ppm in homes with unvented sources of combustion. In garages, ferries, skating ice rinks and kitchens with gas cookers, it can even reach up to4ppm. Herein, NO2-related health risks have become the global focal point. Due to the inhalation route of exposure, respiratory damage effect of NO2was always considered to be a key concern either in epidemiological or laboratory studies, while other effects were always ignored.Recently, some epidemiological literatures linked NO2pollution with increasing risk of cardiovascular diseases and neurological disorders, which implied that lung is not the only target of NO2. To explore the toxicological effects of NO2on the heart and brain, a role for oxidative stress, inflammatory responses and cell apoptosis in the both tissues of rats treated with different concentrations of NO2(0,5,10and20mg/m3) was investigated. The pathological change was observed by Hematoxylin and eosin (HE) staining, and the number of apoptotic cells was quantified by TUNEL method; The activity or contents of Cu/Zn-SOD, Mn-SOD, GPx, MDA and NO were measured with test kits, while PCO were measured by DNPH assay; the levels of TNF-a and Ⅱ-1β were examined by ELISA; the mRNA expression of oncogenes and apoptosis-related genes were examined through real-time RT-PCR analysis. For the heart, mild pathology occurred after7-d exposure (6h/d); marked oxidative stresses were induced as reduction/induction of antioxidants (Cu/Zn-SOD, Mn-SOD and GPx) activity and increasing formation of MDA and PCO; mRNA and protein biomarkers of inflammation (TNF-α and IL-1β) were up-regulated, and p53mRNA expression, bax/bcl-2ratio and the mean number of TUNEL-positive myocytes were increased as well. For the brain, observable adverse effects were induced encompassing mild brain pathology, increased neuronal apoptosis, altered antioxidants (Cu/Zn-SOD, Mn-SOD, GPx and NO) activity and increasing formation of PCO after7-day exposure (6h/day); NO2inhalation also induced augment of oncogenes (c-fos, c-jun) levels, and deregulation of apoptosis-related genes (p53, bax and bcl-2) expression. With all above data, the present report provided essential information for the characterization of the cardiotoxic and neurotoxic hazard of NO2, which is required in response to the general concern about the vulnerability of the cardiovascular and neurological system to it.Ischemic stroke is the second leading cause of death and the most frequent disease leading to disability in the world, with a high incidence affecting up to0.2%of the population every year. In China, the rate of ischemic stroke increased by almost9%every year. In USA, stroke rates in five to44-year-olds rose by about a third within10years. Worryingly, the risk factors for ischemic stroke have not been fully clarified, and the effective means to control its morbidity and mortality have not been found. In the last decade, an increasing body of epidemiologic literatures has provided compelling evidence to link outdoor air pollution to stroke mortality, with positive associations being observed for NO2, sulfur dioxide (SO2), ozone (O3), carbon monoxide (CO) and particulate matter (PM). Moreover, it was reported that NO2could also influence the outcome of stroke patients. These effects, however, are not conclusive given the limited number of studies, their small size and their methodological constraints, which also leads to some contrary results. Moreover, relative molecular mechanisms cannot be elucidated by epidemiologic studies. Here the correlativity between NO2and ischemic stroke was determined. The hemorheological parameters were measured using YDA-IV blood viscosity apparatus; the rat model of ischemic stroke was set up using MCAO method; the behavioral deficits were measured through symptom scoring; the infarct volume were measured by TTC method; the pathological change and quantification of apoptosis cell number were measured by HE and TUNEL methods respectively. First, we found that blood viscosity, red blood cell (RBC) aggregation-, electrophoresis-and rigidity-index in healthy rats were increased after exposure to5mg/m3NO2for one-and three-month, which were very similar with the clinical phenomenon observed from ischemic stroke patients. Then, we set up stroke rat model and exposed them to NO2at the same concentration for one week, and found that NO2exposure time-dependently delayed neurological structure and function recovery of MCAO (middle cerebral artery occlusion) rat, and worsened pathological injuries and apoptosis induced by MCAO operation. Trough these studies, we confirmed the relevance of NO2exposure and ischemic stroke.Endothelial and inflammatory responses are two common cellular pathomechanisms involved in ischemic brain damage. To elucidate the detailed mechanisms of ischemic stroke induced by NO2inhalation, we treated Wistar rats with NO2at various concentrations and determined the messenger RNA (mRNA) and protein expression of endothelin-1(ET-1), nitric oxide synthases (NOSs), cyclooxygenase-2(COX-2), and intercellular adhesion molecule1(ICAM-1) in the cortex, all of which are the endothelial and inflammatory biomarkers in stroke. The results showed that NO2elevated the levels of ET-1, iNOS, COX-2and ICAM-1mRNA and protein but inhibited the expression of eNOS and nNOS in a concentration-dependent manner. Then, we set up stroke rat model and exposed them to the lowest dose (5mg/m3) of NO2for one week, and found that endothelial injuries and inflammation were induced in cortex by MCAO treatment and exacerbated by followed NO2inhalation. The related mRNA and protein expression were examined via real-time RT-PCR technique as well as Western blotting method. In the present study, the upregulation of ET-1expression in rat cortex suggests the reduction of cerebral blood and a tendency to ischemic injuries via endothelial dysfunction following NO2inhalation. NO2inhalation-induced excessive expression of iNOS, COX-2and ICAM-1, as well as release of Ⅱ-1β and TNF-a could promote blood-borne inflammatory cell adherence and infiltration. Consequently, leukocytes exacerbated brain injury by physically obstructing capillaries and reducing blood flow during reperfusion and/or by migrating into the brain parenchyma and releasing cytotoxic products. Our data implicates the occurrence and development of ischemic injuries in rat brains via endothelial and inflammatory mechanisms after NO2exposure. Expression of eNOS, COX-2and ICAM-1protein after sub-chronic NO2exposure showed the same response as that observed after one-week exposure at higher concentrations, providing evidence that endothelial nitric oxide synthase (eNOS), cyclooxygenase-2(COX-2) and intercellular adhesion molecule1(ICAM-1) can be potential indicators for NO2-induced ischemic stroke.As above reported, NO2could delay the recovery of nerve function after stroke, which implied a possible risk of vascular dementia (VaD) with NO2inhalation, which is often a common cognitive complication resulting from stroke. However, the effect and detailed mechanisms have not been fully elucidated. In the present study, synaptic mechanisms, the foundation of neuronal function and viability, were investigated in both model rats of ischemic stroke and healthy rats after NO2exposure. Transmission electron microscope (TEM) observation showed that5mg/m3NO2exposure not only exacerbated the ultrastructural impairment of synapses in stroke model rats, but also induced neuronal damage in healthy rats. Meantime, we found that the expression of synaptophysin (SYP) and postsynaptic density protein95(PSD-95), two structural markers of synapses in ischemic stroke model were inhibited by NO2inhalation; and so it was with the key proteins mediating long-term potentiation (LTP), the major form of synaptic plasticity. On the contrary, NO2inhalation induced the expression of nearly all these proteins in healthy rats in a concentration-dependent manner. All the proteins expression were examined by Western blotting method. Our results indicated that NO2exposure could increase the risk of VaD through inducing excitotoxicity in healthy rats but weakening synaptic plasticity directly in stroke model rats.In conclusion, this work revealed the damage effect of NO2inhalation on the heart and brain, confirmed an association between NO2inhalation and increased risk for ischemic stroke as well as VaD, elucidated the related molecular mechanisms and explored the molecular markers for indicating these effects, which will help us open up therapeutic approaches to prevent, ameliorate, or treate heart and brain disorders resulting from NO2exposure in its polluting areas.