Objective To establish a stable PrP^Sc panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals＇ prion diseases. Methods Thirty brai...Objective To establish a stable PrP^Sc panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals＇ prion diseases. Methods Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP^Sc in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP^Sc were repeatedly detected by prp^SC-specific Western blots in half a year and 3 years later. Results PrPsc signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrPsc positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. TWenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of prpSc-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrPsc positive samples and glycoforms was almost unchanged. All normal hamsters＇ brain homogenates were PrP^Sc negative. Conclusion A PrP^Sc panel of prion disease can be established, which displays reliably stable prp^Sc-specific signals and glycoforms.展开更多
Objective To understand the infectious characteristics of a hamster-adapted scrapie strain 263K with five different routes of infection including intracerebral (i.e.), intraperitoneal (i.p.), intragastrical (i.g.), in...Objective To understand the infectious characteristics of a hamster-adapted scrapie strain 263K with five different routes of infection including intracerebral (i.e.), intraperitoneal (i.p.), intragastrical (i.g.), intracardiac and intramuscular (i.m.) approaches. Methods Hamsters were infected with crude- or fine-prepared brain extracts. The neuropathological changes, PrPSc deposits, and patterns of PK-resistant PrP were analyzed by HE stain, immunohistochemistry (IHC) assay and Western blot. Reactive gliosis and neuron loss were evaluated by glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) specific IHC. Results The animals inoculated in i.m. and Lp. ways with crude PrPSc extracts showed clinical signs at the average incubation of 69.212.8 and 65.5±3.9 days. Inoculation in i.c. and intracardiac ways with fine PrPSc extracts (0.00035 g) caused similar, but relative long incubation of around 90 days. Only one out of eight hamsters challenged in i.g way with low dosage (0.01 g) became ill after a much longer incubation (185 d), while all animals (4/4) with high dosage (0.04 g) developed clinical signs 105 days postinfection. The most remarkable spongiform degeneration and PrPSc deposits were found in brain stem among the five challenge groups generally. The number of GFAP-positive astrocytes increased distinctly in brain stems in all infection groups, while the number of NSE-positive cells decreased significantly in cerebrum, except i.c. group. The patterns of PK-resistant PrP in brains were basically identical among the five infection routes. Conclusion Typical TSE could be induced in hamsters by inoculating strain 263K in the five infection ways. The incubation periods in bioassays depend on infective dosage, administrating pathway and preparation of PrPSc. The neuropathological changes and PrPSc deposits seem to be related with regions and inoculating pathways.展开更多
基金supported by National Science and Technology Task Force Project (2006BAD06A13-2)National Basic Research Program of China (973 Program) (2007CB310505)+1 种基金Institution Technique R&D Grand (2008EG150300)National Natural ScienceFoundation of China (Grants 30571672, 30500018, 30771914, and 30800975)
文摘Objective To establish a stable PrP^Sc panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals＇ prion diseases. Methods Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP^Sc in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP^Sc were repeatedly detected by prp^SC-specific Western blots in half a year and 3 years later. Results PrPsc signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrPsc positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. TWenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of prpSc-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrPsc positive samples and glycoforms was almost unchanged. All normal hamsters＇ brain homogenates were PrP^Sc negative. Conclusion A PrP^Sc panel of prion disease can be established, which displays reliably stable prp^Sc-specific signals and glycoforms.
基金This work was supported by Chinese National Natural Science Foundation Grants 39928018,30070038 and 30130070, National High-tech Development Project (863 Project) 2001AA215391EU Project QLRT 2000 01441.
文摘Objective To understand the infectious characteristics of a hamster-adapted scrapie strain 263K with five different routes of infection including intracerebral (i.e.), intraperitoneal (i.p.), intragastrical (i.g.), intracardiac and intramuscular (i.m.) approaches. Methods Hamsters were infected with crude- or fine-prepared brain extracts. The neuropathological changes, PrPSc deposits, and patterns of PK-resistant PrP were analyzed by HE stain, immunohistochemistry (IHC) assay and Western blot. Reactive gliosis and neuron loss were evaluated by glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) specific IHC. Results The animals inoculated in i.m. and Lp. ways with crude PrPSc extracts showed clinical signs at the average incubation of 69.212.8 and 65.5±3.9 days. Inoculation in i.c. and intracardiac ways with fine PrPSc extracts (0.00035 g) caused similar, but relative long incubation of around 90 days. Only one out of eight hamsters challenged in i.g way with low dosage (0.01 g) became ill after a much longer incubation (185 d), while all animals (4/4) with high dosage (0.04 g) developed clinical signs 105 days postinfection. The most remarkable spongiform degeneration and PrPSc deposits were found in brain stem among the five challenge groups generally. The number of GFAP-positive astrocytes increased distinctly in brain stems in all infection groups, while the number of NSE-positive cells decreased significantly in cerebrum, except i.c. group. The patterns of PK-resistant PrP in brains were basically identical among the five infection routes. Conclusion Typical TSE could be induced in hamsters by inoculating strain 263K in the five infection ways. The incubation periods in bioassays depend on infective dosage, administrating pathway and preparation of PrPSc. The neuropathological changes and PrPSc deposits seem to be related with regions and inoculating pathways.
