Title page
Contents
Part 1 Expression of Heat shock protein 25 in the mouse cerebellum is not induced by heat shock stimuli 6
Introduction 7
Materials and Methods 10
1. Animals 10
2. Heat shock induction 10
3. Immunohistochemistry 11
4. Western Blotting analysis 12
5. Image capture and photography 12
Results 14
1. Hsp70 expression in brain stem and cerebellum according to the post-heatshock acquisition times 14
2. Hsp25 expression in brain stem and cerebellum according to the post-heatshock acuisition times 15
3. Quantitative analysis of Hsp25 and Hsp70 in both regions by western blotting 15
Discussion 19
References 22
Abstract 28
Part 2 Developmental changes of Heat shock protein 25 in the Mongolian gerbil cerebellum 30
Introduction 31
Materials and Methods 34
2.1. Animals 34
2.2. Tissue preparation 34
2.3. Immunohistochemistry 35
2.4. Western blot analysis 35
2.5. Double immunofluorescent study 36
2.6. Image capture and photography 37
Results 39
1. Immunological localization of Hsp25 during postnatal development of the gerbil cerebellum 39
2. Intracellular localization of Hsp25 in the developing gerbil cerebellum 39
3. Absence of the parasagittal band formations in the Hsp25 immunoreactive Purkinje cells 40
4. Quantitative analysis of Hsp25 expression of the developing gerbil cerebellum by Western blotting 41
Discussion 47
Reference 49
Abstract 54
국문초록 56
Fig. 1. High-power photomicroscope of Hsp25(Fig. 1A~1D) and Hsp70 (Fig.1E~1H) immunoreactivity in motor neurons of facial nuclei in Balb/C mouse brain stem followed by 0 hr(Acute), 1hr, 4hr post-heatshock acquisition time. A, E, control;B, F, acute;C, G, 1h; D,H,4h;Scalebar=50㎛ 16
Fig. 2. High-power view of Hsp25(Fig.2A~2D) and Hsp70 (Fig.2E~2H) immunoreactivity in the Purkinje cell layers of Lobule IX, X of Balb/C mouse cerebellum followed by 0 hr (Acute), 1hr, 4hr post-heatshock acquisition time. A, E, control;B, F, acute;C, G, 1h ; D,H,4h;Scalebar=50㎛ 17
Fig. 3 Western blot analysis of Hsp25 and Hsp70 in Balb/C mouse brain stem and cerebellum followed by 0 hr(Acute), 1hr, 4hr post-heatshock acquisition time compared by control group. Hsp70 expression level of Balb/C mouse cerebellum significantly increased by heatshock stimuli. But, It shows no significant change in Hsp25 expression levels after 18
Part 2 Developmental changes of Heat shock protein 25 in the Mongolian gerbil cerebellum 6
Fig. 1 Sagittal views at midline of the developmental gerbil cerebellum reveals varient spatio-temporal pattern of Hsp25 expression. In the gerbil cerebellum at postnatal day (PD) 0, Hsp25 immunoreactivity is not obvious in entire region(Fig.1A). Beginning at PD 5, Hsp25 immunohistochemstry deposits reaction product in Purkinje cells. At PD 5, Hsp25 42
Fig. 2 Highly magnified sagittal view of Hsp25 immunohistochemistry in the part of anterior zone (LobulesI~V) of the gerbil cerebellum. In the gerbil cerebellum at PD 0, there are no obvious Hsp25 immunoreactive cells (Fig. 2A). From PD 5, Hsp25 showed a prominent cytoplasmic localization in Purkinje cells in the cerebellum (Fig. 2B~2I). Both the neuronal 43
Fig. 3 Double immunofluoroscent study for Hsp25 (Fig. 3A, 3D, 3G, 3J), calbindin immunoactivity (Fig. 3B, 3E, 3H, 3K) and their merged images (Fig. 3C, 3F, 3I, 3L) respectively. As increasing postnatal days, Purkinje cells of the gerbil cerebellum shows more and more correspondance between immunoreactivity of Hsp25 and calbindin, as a 44
Fig. 4 Coronal views of the cerebellar lobules I, II reveals no specific band formations of Hsp25 immunoreactive dendrites in the molecular layer of the gerbil cerebellum. the Molecular layer of the gerbil cerebellum showed a non-specific diffuse pattern of immunoreactivity because of dendritic Hsp25 expression of Purkinje cells. Scale bar=250㎛ 45
Fig. 5 Western blotting analysis of cerebellar homogenates according to diverse postnatal stages of the gerbil revealed that, consistent with the result of immunohistochemistry, little Hsp25 was present before the PD 8 cerebellum of the gerbil, with abruptly increased levels just after PD 10. The second surge of Hsp25 level was in PD 20. and there is no 46