In Situ Cytokine Expression, Nitric Oxide and Reactive Oxygen Species Production During Primary Amoebic...

Primary amoebic meningoencephalitis (PAM), caused by Naegleria fowleri, is a rapidly progressive and fatal infection, and inflammation of the olfactory bulb plays a key role in disease pathogenesis. Although inflammatory and oxidative mediators have been studied in vitro, their production during the progression of PAM remains poorly understood. This study evaluated the temporal production of TNF-α, IL-1β, IL-6, nitric oxide (NO), and reactive oxygen species (ROS) in the olfactory bulbs of BALB/c mice from 1 to 7 days after intranasal infection with N. fowleri trophozoites. RT-PCR detected increased Tnf, Il1b, and Il6 transcript levels from day 3 post-infection. Western blot analysis complemented these findings by detecting the corresponding cytokine proteins from day 2, although their temporal patterns differed among the cytokines examined. Immunohistochemical analysis demonstrated that cytokine production was associated with the presence and accumulation of inflammatory cells in the olfactory bulb. In addition, ROS and NO production increased throughout disease progression, indicating the establishment of a pronounced oxidative microenvironment. These findings demonstrate that N. fowleri infection induces a sustained local inflammatory response characterized by cytokine overproduction and oxidative stress. Together with inflammatory cell infiltration, these mediators may contribute to extensive olfactory bulb damage and disease severity, ultimately leading to host death.