| Parkinson'disease (PD) is a common chronic neurodegenerative disorder among elderly people. The main clinical manifestation is dyskinesia, accompanied with emotional disorders mostly, cognition dysfunctions, and even dementia. The neuropathologic hallmark of PD is progressive dopaminergic neurons loss in the substantia nigra. The neurochemistry characteristic is a marked reduction of dopamine (DA). The exact pathogenesis of this disease is still unknown. Currently, the research of PD depends on the PD animal models. Suitable animal models must be used for further research and better therapy of PD. Therefore, the development of animal models plays a key role in the research progression of PD.Since the biochemical and cellular changes in animals caused by MPTP are similar to those in human PD, MPTP has been applied widely in the PD model. MPTP mouse model is the most valuable animal model that displays the classic changes of neuropathology and neurobiochemistry of PD.SAMP8 (senescence-accelerated mouse prone 8) mouse has not only the early onset of senilty (4-6 months maturation period) and a short life span (10-17 months), but also characterized by learning and memory impairments, and affective disturbance in the aging process. It is thus a putative useful animal model to study aging or age-related disorders, such as AD. There is currently no animal model of PD that exhibits these conditions. In addition, the onset of PD closely related with aging, the PD patient has not only motor symptoms, but also other symptoms such as psychiatric and cognitive impairment. Application of the SAMP8 mouse to PD research may have several merits. But the sensitivity to MPTP/MPP+ neurotoxicity depends on the strain of mouse. Whether SAMP8 mouse is suitable for MPTP-PD animal model or not has not been reported by others. A great deal of study about the pathogenesis of PD has shown that the dopaminergic neurons in substantial nigra are very sensitive to the damage caused by oxidation and inflammation which have significant contribution to the etiology of this disorder. Recently, more and more studies for PD animal models and human PD suggest that the activation of the microglia plays an important role in dopaminergic neurodegeneration, including MPTP animal model. Some studies also show that medicine which inhibits microglial activation can relieve the degeneration of dopaminergic neurons in animal model of PD. It is also suggested that a large amount of NO induced by iNOS in activated microglia contributes to the death of dopaminergic neurons in MPTP toxicity and PD. In addition, NO induced by iNOS can regulate the expression of HO-1, which has been confirmed by studies in vivo and in vitro. The pathway of HO-1 has been considered as a major defense mechanism when neurons are exposed to the oxidative attack, and the expression of HO-1 has cytoprotective action through the anti-oxidative damage. But there is no research on the expression changes of iNOS, HO-1 and microglial activation under the same intervention conditions.Aging is the most prominent risk factor for PD. Some studies have shown that its neurotoxicity is age-dependent when MPTP was used. The roles of the microglia activation, iNOS and HO-1 contribute to the pathogenesis which aging correlated with the PD development is unknown.Therefore, we investigate whether or not MPTP can cause the damage to the nigrostriatal system of SAMP8 mouse for lays down the theoretical foundation by which the SAMP8 mouse may be utilized in the PD research. And we will furthermore explore the relationship between the microglia activation, expression of iNOS, HO-1 and the age-dependent dopaminergic system impairment. To investigate the role of aging in the PD occured and the possible mechanism, this may provide a new consideration for the pathogenesis and treatment of PD.1. Neurotoxicity effect of MPP+ on primary cultured mesencephalon neurons of SAMP8 mouse Objective: To investigate the midbrain neurons change of SAMP8 mouse followed by MPP+ treatment in vitro, which provide the theoretical foundation for the further study in vivo.Methods: SAMP8 mouse primary midbrain cell cultures were obtained from mice within 1 day after birth. On the sixth day after culturing, the cultures were treated with 100μmol/L of MPP+ for 6 hours, 9 hours, 12 hours and 24 hours, then the cells were fixed and followed by immunofluorescence or western-blot analysis for the expressed location and the level of TH respectively.Results: MPP+ led to the morphological changes of primary cultured midbrain neurons of SAMP8 mouse. The neurons had intact morphous with strong immunoreactivity, many and long dendrites in control group. Compared with the control group, the neurons had fewer and thinness dendrites with weak immunoreactivity in MPP+ group. MPP+ significantly decreased midbrain neurons numbers with marked decrease of TH protein levels after its treatment for 9h.Conclusion: There was neurotoxicity effect on primary cultured midbrain neurons of SAMP8 mouse followed by MPP+ treatment. It was showed that MPP+ led to marked decrease of neuronal numbers and TH protein levels, which is suggested that MPP+ can cause the DA neuronal degeneration in primary midbrain cell cultures.2. Acute damage of nigrostriatal system in MPTP-treated SAMP8 mouseObjective: Assess the changes of dopaminergic neuronal number in SN, DA levels in striatum and spontaneous motor activity in MPTP-treated SAMP8 mouse, and explore whether MPTP can cause damage to the nigrostriatal system of the SAMP8 mouse after acute intervention.Methods: 3-month-old male SAMP8 mice were divided into two groups: the MPTP group, which received MPTP (20 mg/kg) subcutaneous injection at 2-h intervals for 4 times, and the control group, which was treated with an equal volume of saline. Mice were sacrificed at 8 days after the first injection. Immunohistochemistry, high pressure liquid chromatography and behavior test were usded to investigate the changes of dopaminergic neuronal number in SN, DA levels in striatum and spontaneous motor activity of MPTP-treated SAMP8 mouse.Results: The spontaneous motor activity reached its lowest point at 6h and recovered at 48h after the first injection in the MPTP group. And the TH positive neuronal loss at 8 days was 42.39% (P<0.001), while striatal DA levels decreased at 8 days by 80.14% (P<0.001) in MPTP group.Conclusion: Damage occurred to the nigrostriatal in 3-month-old SAMP8 mice received four subcutaneous injections of MPTP (20 mg/kg) at 2h intervals. There were marked decreases in striatal DA levels and a loss of dopaminergic neurons in SN, with the behavior change shortly after injection in MPTP-SAMP8 mice. On the basis of the current findings, MPTP can lead to the damage of nigrostriatal system in SAMP8 mouse. This study lays down the theoretical foundation by which the SAMP8 mouse may be utilized in the research of PD.3. Age-related damage of nigrostriatal system in MPTP-treated SAMP8 mouseObjective: To investigated the different damage in the nigrostriatal system of 3-month-old and 6-month-old SAMP8 mice treated with MPTP and explore the role of aging in PD development.Methods: 3-month-old and 6-month-old male SAMP8 mice received MPTP (20 mg/kg) subcutaneous injection at 2-h intervals for 4 times, the control group was treated with an equal volume of saline. Mice were sacrificed at 6 h, 24 h, 3 days and 8 days after the first injection for the detection of DA neurons number and DA levels.Results: The spontaneous motor activity reached its lowest point at 6 h (P<0.05) and recovered at 48 h after the first MPTP injection in 3-month-old mice, but 6-month-old mice unrecovered through the study process (P<0.05).In 3-month-old mice, TH-positive neuronal loss was visible at 6 h (7.06%, P=0.235), 24 h (12.79%, P<0.05), 3 days (22.49%, P<0.01), and 8 days (42.39%, P<0.001), and the significant progression of dopaminergic neuronal loss occurred most prominently in the MPTP group from days 3 to 8 (3 d vs. 8 d, P<0.01). In 6-month-old mice, TH-positive neuronal loss was visible at 6 h (14.23%, P<0.05), 24 h (23.85%, P<0.01), 3 days (36.77%, P<0.001), and 8 days (45.90%, P<0.001), and the significant progression of dopaminergic neuronal loss occurred most prominently in the MPTP group from 24 h to 3 days (24h vs. 3 d, P<0.05). There was a significant difference in dopaminergic neuronal loss at 3 days after the first injection between the 6-month-old mice and 3-month-old mice (P<0.01). An earlier and severer dopaminergic neuronal loss was detected in the 6-month-old MPTP-SAMP8 mice.There was significant decrease of striatal TH-ir COD value in the 3-month-old MPTP group from 24 h to 3 days (24 h vs. 3 d, P<0.05), while this occured in the 6-month-old MPTP group from 6 h to 24h (P<0.05). There was a significant difference at 24h and 3 days after the first injection between the 6-month-old and 3-month-old mice (P<0.05). A more severe loss of TH-ir COD value was detected in 6-month-old MPTP-SAMP8 mice.Striatal DA levels decreased at 6 hours by 79.09%, at 24 hours by 80.33%, at 3 days by 83.86%, at 8 days by 80.14% (P<0.001) in 3-month-old MPTP group, and by 79.70%, 86.64%, 75.20%, 92.32% (P<0.001) respectively in 6-month-old MPTP group.Conclusion: The degeneration of DA neurons and its projected fibers in SAMP8 mouse caused by MPTP was time-dependent. There was age-related damage to the nigrostriatal system of SAMP8 mice treated with MPTP. Aging is a predisposing factor of PD.4. The possible mechanism about age-related damage of nigrostriatal system in MPTP-treated SAMP8 mouseObjective: To explore the relationship between the microglia activation, expression of iNOS, HO-1 and the age-dependent dopaminergic system impairment in MPTP-treated SAMP8 mice and reveal the possible mechanism which aging correlated with the PD onset.Methods: The choice and treatment of SAMP8 mice was similar to part 3. By immunohistochemistry and western-blot analysis, we detected the changes of TH, microglia, iNOS and HO-1.Results: MPTP caused the age-related loss of DA neurons number and decrease of TH protein levels in SAMP8 mice. There was significant progression of striatal cd11b protein in the both age MPTP group from 24 days to 3 days, but it abated at 8 days. A more marked increase of cd11b protein levels in the striatum at 24h and 3 days after the first injection in 6-month-old mice compared to those of 3-month-old mice. There was significant progression of striatal iNOS protein in the both age MPTP group from 24 days to 3 days, but it abated at 8 days. There was no significant difference in striatal iNOS protein levels at each time point between the 6-month-old mice and 3-month-old mice. Additionally, HO-1 positive cells were detected in striataum just only at 3 days, with the increase of HO-1 protein expression in both age groups. There was no significant difference in striatal HO-1 protein levels at each time point between the 6-month-old mice and 3-month-old mice. Western blot analysis showed no change of cd11b, iNOS and HO-1 protein levels in the midbrain after MPTP treatment compared to those of saline group in both age mice.Conclusion: There were pathological change of microglia and expressive change of iNOS chiefly in striatum of SAMP8 mouse after MPTP injection. The different microglia activition plays an important role in MPTP-induced age-related damage of SAMP8 mouse nigrostriatal system. Although iNOS participated in the damage of nigrostriatal system caused by MPTP, it was not the most important contributor to the age-related damage. The upregulation of HO-1 was ephemeral, and its neuroprotective effect was limited in this study.In a word, it is the first report to investigate the damage of the nigrostriatal system in the SAMP8 mouse induced by MPTP in vivo and in vitro. MPTP can cause parkinsonism characteristics such as marked decreases in striatal DA levels, the loss of DA neurons in the SN and the behavior change in SAMP8 mice. So it is suggested that the SAMP8 mouse can be utilized in the research of PD. MPTP can induce the age-related damage to the nigrostriatal system of SAMP8 mice, which suggested that aging is a predisposing factor of PD. And the different microglia activation may play an important role in PD occured which is aging correlated.
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