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doi: 10.1227/NEU.0b013e31827fcd83

Cerebellar Purkinje Cells Exhibit Increased Expression of HMGB-1 and Apoptosis in Congenital Hydrocephalic H-Tx Rats

Watanabe, Mitsuya MD; Miyajima, Masakazu MD, PhD; Ogino, Ikuko PhD; Nakajima, Madoka MD, PhD; Arai, Hajime MD, PhD

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BACKGROUND: Highly integrated anatomic and functional interactions between the cerebrum and the cerebellum during development have been reported. In our previous study, we conducted a proteome analysis to identify the proteins present in the congenital noncommunicating hydrocephalus in the cerebellum. We found higher expression of high-mobility group box-1 protein (HMGB-1) in hydrocephalic H-Tx rats.

OBJECTIVE: We studied the expression pattern of HMGB-1 in the cerebellum.

METHODS: We studied congenital hydrocephalic H-Tx rats aged 1 day and 7 days along with age-matched nonhydrocephalic H-Tx and Sprague-Dawley rats as controls. Gene and protein expressions of HMGB-1 in the cerebellum were assayed by real-time polymerase chain reaction and Western blotting, respectively; furthermore, immunohistochemical analyses were performed by using HMGB-1 (indicator of apoptosis), single-stranded DNA; adhesion factor related to cell migration, HNK-1; and the Purkinje cell-specific antibody, calbindin.

RESULTS: Cytoplasmic HMGB-1 expression observed in Purkinje cells in the 1-day-old hydrocephalic group was stronger than that in the nonhydrocephalic and Sprague-Dawley groups. Double fluorescent staining with single-stranded DNA confirmed that Purkinje cells were undergoing apoptosis. HNK-1 expression was lower in the Purkinje cell layer in the 7-day-old rats in the hydrocephalic group, and Purkinje cells were disrupted in comparison with the control groups. Morphological changes in the cerebellum were observed in the 7-day-old rats in the hydrocephalic group in comparison with the control groups.

CONCLUSION: Our results suggest that cerebellar neuronal cell damage in the early postnatal period may be related to the higher expression of HMGB-1 in the Purkinje cells.

ABBREVIATIONS: ANOVA, analysis of variance

GAPDH, glyceraldehyde 3-phosphate dehydrogenase

HMGB-1, high mobility group box-1

PCR, polymerase chain reaction

RAGE, receptor for advanced glycation end product

SD, Sprague Dawley

ssDNA, single-stranded DNA

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