Early Postnatal Exposure To Airborne PM_2.5 Induces Autism-like Phenotypes In Rats And Its Related Mechanisms

Autism spectrum disorder（ASD）is a heterogeneous neurodevelopmental disorder,characterized by deficits in communication and social interaction,as well as restrictive and repetitive behaviors.The prevalence of ASD is increasing year by year,but the precise etiology remains unclear,and there is no cure for ASD by far.It has become a significant cause of mental illness in children,and ASD accounts for substantial financial burden to their families and the social.Although genetics plays an important role in these conditions,environmental exposures,particularly during the in utero or early postnatal stages,are increasingly being recognized as potential risk factors for ASD by a growing number of epidemiological evidence.However,the relevant research is more limited to epidemiological investigation,the direct laboratory evidence and the underlying mechanism is relatively scarce.Objective 1.Newborn SD rats were selected as the subjects,and fine particulate matter(PM_2.5)was used as the exposure substance to establish the animal model of early postnatal air pollution exposure.To observe whether such exposure can lead to changes in autism-like behavior of animals,and to study the pathophysiological characteristics of autism caused by air pollution.And providing evidence to the hypothesis of air pollution exposure is an important risk factor for the development of autism.2.To investigate the effect of PM_2.5 exposure on neuronal inflammation and ASD susceptibility gene in early postnatal rats,and to reveal the structure and function of PM_2.5 exposure induced neurodevelopment damage in rats,and to explore the possible mechanism of air pollution exposure leading to animal autism-like behavior.Methods 1.Before the establishment of the animal model of air pollution exposure,we collected the typical air pollutant PM_2.5 in the central urban area of Tianjin,and detached by ultrasonic and then lyophilized to prepare the exposure suspension.The concentration of PM_2.5 during the sampling period was monitored.We also detected the harmful components of PM_2.5,and 16 polycyclic aromatic hydrocarbons and 9 kinds of heavy metal elements were detected.To establish the model,litters were randomly divided into 4 groups: blank control（Control）;vehicle exposed（Saline）;2 mg/kg bw PM 2.5 exposures（Low）,and 20 mg/kg bw PM 2.5 exposure（High）.From postnatal day（PND）8,pups were administered PM 2.5 suspension or saline solution via intranasal instillation through both nostrils,once per day,for 2 weeks.2.To study the relationship between exposure of fine airborne particulate matter and autism,a battery of animal behavioral experiments in the laboratory setting were used to observe whether such exposure can lead to ASD-like phenotypes in rats.3.To observe the effect of PM_2.5 exposure on neuroinflammation and autism susceptibility genes in rats,we used ELISA to detect the expression of proinflammatory cytokines IL-1β,IL-6 and TNF in rat hippocampus and prefrontal cortex.And the expression of Gfap,Iba1,markers forastrocytes and microglia,the expression of Cntnap2,Shank3,Nrxn1,Mecp2 and Fmr1,autism susceptibility genes in of the hippocampus were examed by RT-q PCR,and western blot was used to validate the differentially expressed genes at the protein level.4.To observe the effect of neuronal synaptic development disorder in PM_2.5-induced autism-like behavior changes in rats,Nissl staining method was applied to verify the morphology of hippocampal neurons in PM_2.5 exposeed rats.And the changes of synaptic ultrastructure and synapses number in rats hippocampal were observed by transmission electron microscopy（TEM）,and the expression of synaptophysin was further detected by immunofluorescence.Results 1.The results show that the PM_2.5 mass concentration is 187.2 μg/m3 during the sampling period,reaching the level of heavy pollution.And the total concentration of polycyclic aromatic hydrocarbons was 1.61 μg/m3.The total concentration of heavy metal elements reached 3.882 μg/m3.2.The results of USVs analysis showed that the number of ultrasound vocalizations in rat pups exposed to high dose PM_2.5 decreased significantly,suggesting that high dose PM_2.5 exposure decreased the ability of communication in rats.Threechambered social experiment showed that the interaction time of the high-dose exposure group was significantly shorter than that of the other groups to social target.The results of olfactory habituation/dishabituation test showed a significant lower sniffing behavior to social order in the high dose exposure group.Together with the results of three-chambered test,they showed that the high dose PM_2.5 exposure induced a poor social interaction in male rats.The number of buried marble in the rats exposed to PM_2.5 was significantly less than that in the control group.And the results of the novel object recognition experiment showed that the time of interaction with the new objects in the PM_2.5 exposed group was significantly lower than that in the control group.These results suggest that PM_2.5 exposure lead to novel avoidance in early postnatal rats.In summary,PM_2.5 exposed neonatal male rats exhibit communication deficits,poor social interaction,and novelty avoidance,a series of autistic-like behaviors.3.The results of ELISA showed that the expression of IL-1β,IL-6 and TNF-α in hippocampus and cortex were up-regulated in PM_2.5 exposure rats,and the expression of Gfap and Iba1 in hippocampus of early postnatal rats was significantly increased by exposure to PM_2.5.The autism susceptibility gene Shank3 were significantly reduced in m RNA levels.In addition,western blotting was applied to validate the above results.And the results showed that GFAP and IBA1 were significantly increased at the protein level,while the expression of SHANK3 protein was also significantly decreased.These results suggest that PM_2.5 exposure exacerbates the inflammatory response of the nervous system in rats,and causes abnormal expression of autism susceptibility genes.4.The results of synaptic studies showed that the number of neurons in the hippocampal CA1 and CA3 regions is increased n the high dose PM_2.5 exposed group.The number of synapses in the high dose PM_2.5 exposed group was higher than that in the other two groups.And PM_2.5 exposure increased CA3 synaptophysin expression significantly.Conclusion PM_2.5 exposure led to ommunication deficits,poor social interaction,and novelty avoidance,a series of typical autism-like behavior changes in neonatal male rats.The inflammatory response,abnormalities of Shank3 expression and excessive formation of neural synapses in the brain may represent the underlying mechanisms.