Developing a mouse model of acute encephalopathy using low-dose lipopolysaccharide injection and hyperthermia treatment

Abstract

Acute encephalopathy (AE) is mainly reported in East Asia and, in most cases, results from pediatric viral infections, leading to fever, seizure, and loss of consciousness. Cerebral edema is the most important pathological symptom of AE. At present, AE is classified into four categories based on clinical and pathophysiological features, and cytokine storm-induced AE is the severest among them. The pathogenesis of AE is currently unclear; this can be attributed to the lack of a simple and convenient animal model for research. Here, we hypothesized that the induction of systemic inflammation using lipopolysaccharide (LPS) injection followed by hyperthermia (HT) treatment can be used to develop an animal model of cytokine storm-induced AE. Postnatal eight-day-old mouse pups were intraperitoneally injected with low-dose LPS (50 or 100 mg/kg) followed by HT treatment (41.5 C, 30 min). Histological analysis of their brains was subsequently performed.Fluorescein isothiocyanate assay combined with immunohistochemistry was used to elucidate blood-brain barrier (BBB) disruption. LPS (100 mg/kg) injection followed by HT treatment increased BBB permeability in the cerebral cortex and induced microglial activation. Astrocytic clasmatodendrosis was also evident. The brains of some pups exhibited small ischemic lesions, particularly in the cerebral cortex. Our results indicate that a low-dose LPS injection followed by HT treatment can produce symptoms of cytokine storm-induced AE, which is observed in diseases, such as acute necrotizing encephalopathy and hemorrhagic shock and encephalopathy syndrome. Thus, this mouse model can help to elucidate the pathogenetic mechanisms underlying AE.

Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons

“…First, we tested whether degenerating astrocytes with clasmatodendrosis, an irreversible morphological change of damaged astrocytes, [20][21][22][23] in the AE model mice were FJC(+). 16 There were no FJC(+) cells in the control brain (Fig. 5A).…”

“…In the AE model mice, GFAP(+) astrocytes are injured and exhibit the symptom of clasmatodendrosis, an irreversible morphological change of dying astrocytes. 16,[20][21][22][23] The GFAP signal of astrocytes located in the cortical gray matter was much weaker than in the cortical white matter, both in the control and in the AE mice. When protocol 1 was used, the GFAP signal in the cortical gray matter was greatly reduced by FJC treatment, and it was difficult to detect GFAP(+)/FJC(+) degenerating astrocytes.…”

“…To analyze the astrocytes with clasmatodendrosis, acute encephalopathy (AE) model mice were prepared as previously described. 16 Briefly, lipopolysaccharide (LPS; Escherichia coli serotype O127:B8; Sigma-Aldrich) dissolved in phosphate buffered saline (PBS) was injected IP at a dose of 100 µg/kg into postnatal day 8 (P8) mice. Two hours after LPS injection, hyperthermia treatment was performed for 30 min to maintain the rectal temperature at >39C using an electric heater.…”

“…In the AE model mice, not only astrocytes but also neurons are also dying at 6 hr after hyperthermia treatment. 16 Thus, FJC(+) cells with and without processes might be degenerating neurons and degenerating other cell types including astrocytes, respectively. Moreover, FJC(+) cells increased with time after 6-AN injection, and some of them had processes.…”