Neonatal inflammatory pain and systemic inflammatory responses as possible environmental factors in the development of autism spectrum disorder of juvenile rats

Jin Hwan Lee, Alyssa R. Espinera, Dongdong Chen, Ko-Eun Choi, Asha Yoshiko Caslin, Soonmi Won, Valentina Pecoraro, Guang-Yin Xu, Ling Wei, Shan Ping Yu

Autism spectrum disorder (ASD) affects many children and juveniles. The pathogenesis of ASD is not well understood. Environmental factors may play important roles in the development of ASD. We examined a possible relationship of inflammatory pain in neonates and the development of ASD in juveniles. Acute inflammation pain was induced by 5 % formalin (5 μl/day) subcutaneous injection into two hindpaws of postnatal day 3 to 5 (P3-P5) rat pups. Western blot, immunohistochemical, and behavioral examinations were performed at different time points after the insult. Formalin injection caused acute and chronic inflammatory responses including transient local edema, increased levels of inflammatory cytokines, TNF-α, and IL-1β in the blood as well as in the brain, and increased microglia in the brain. One day after the pain insult, there was significant cell death in the cortex and hippocampus. Two weeks later, although the hindpaw local reaction subsided, impaired axonal growth and demyelization were seen in the brain of P21 juvenile rats. The number of bromodeoxyuridine (BrdU) and doublecortin (DCX) double-positive cells in the hippocampal dentate gyrus of P21 rats was significantly lower than that in controls, indicating reduced neurogenesis. In the P21 rat's brain of the formalin group, the expression of autism-related gene neurexin 1 (NRXN1), fragile X mental retardation 1 (FMR1), and oxytocin was significantly downregulated, consistent with the gene alteration in ASD. Juvenile rats in the formalin group showed hyperalgesia, repetitive behaviors, abnormal locomotion, sleep disorder, and distinct deficits in social memory and social activities. These alterations in neuroinflammatory reactions, gene expression, and behaviors were more evident in male than in female rats. Importantly, an anti-inflammation treatment using indomethacin (10 mg/kg, i.p.) at the time of formalin injections suppressed inflammatory responses and neuronal cell death and prevented alterations in ASD-related genes and the development of abnormal behaviors. These novel observations indicate that severe inflammatory pain in neonates and persistent inflammatory reactions may predispose premature infants to development delays and psychiatric disorders including ASD. The prevention of pain stimuli and prompt treatments of inflammation during development appear vitally important in disrupting possible evolution of ASD syndromes.