Figure 4

TUNEL assay of brain sections from rabbits treated with 2.5 μg/kg of Stx2. (A) Condensed dark brown cells were detected in the dentate gyrus of the hippocampus at the onset of paralysis. TUNEL-positive neurons showing shrunken cytoplasm, pycnotic nuclei, and/or apoptotic bodies increased at a later stage in the following other regions: CA1 of the hippocampus (B) and cerebral cortex (C). Apoptotic glial cells in pons (D) and small vessels (E) with condensed cytoplasm and apoptotic bodies were also observed at a later stage. (F) TUNEL assay without TdT showed no positive staining. (G) Quantitative evaluation of TUNEL-positive cells per 10 fields in the hippocampus, cerebellum, and other cerebral regions, including the basal ganglia, thalamus, and cerebral cortex, and (H) TUNEL-positive vessels per 100 fields in the cerebral parenchyma at the onset of neurological symptoms and at a later stage when compared with control rabbits treated with PBS(-) (n = 2 in each group). The number of apoptotic cells significantly increased in the hippocampus at both onset and a later stage, and in granular layers of the cerebellum and other cerebral regions at a late stage. TUNEL-positive vessels were significantly elevated at a later stage. *: P < 0.01 and **: P < 0.05. (Original magnification: (A) and (F) 200×, (B) 800×, (C) and (E) 600×, (D) 400×)