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3.127 EFFECT OF UNILATERAL LESION WITH THE SELECTIVE PROTEASOME INHIBITOR LACTACYSTIN ON ROTATIONAL BEHAVIOR AND DA METABOLISM IN RATS
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Wednesday, 14 December 2011 / Parkinsonism and Related Disorders 18S2 (2012) S161–S234
3.127 EFFECT OF UNILATERAL LESION WITH THE SELECTIVE PROTEASOME INHIBITOR LACTACYSTIN ON ROTATIONAL BEHAVIOR AND DA METABOLISM IN RATS J. Konieczny, T. Lenda, M. Zapała, E. Lorenc-Koci. Department of Neuro-Psychopharmacology, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland A growing body of evidence suggests that proteasomal dysfunction may play an important role in the pathogenesis of Parkinson’s disease (PD). The aim of our study was to examine the time course of changes in rotational behavior and DA metabolism in rats lesioned unilaterally with lactacystin. Male Wistar rats were injected with lactacystin (2.5 mg/2 ml) into the left substantia nigra (SN) pars compacta. The animals were examined for spontaneous and the apomorphineinduced rotational behavior on days 1, 4, 7, 14 and 21 after surgery; then they were killed and the levels of DA and its metabolites were assayed in striatal and nigral homogenates using HPLC method. On the first few days after surgery, the lesioned rats displayed a spontaneous circling behavior contralateral to the lesioned side. Lactacystin evoked a progressive loss of DA and its metabolites, as well as a progressive acceleration of DA catabolism in the ipsilateral striatum and SN. After 21 days, the decline in DA level exceeded 90% both in the striatum and SN. However, after apomorfine treatment on 7th , 14th and 21st day after surgery no contralateral turning was observed in the lesioned rats. The present study shows that lactacystin produces biochemical changes characteristic of progressive degeneration of the DA system. However, the presence of spontaneous contralateral rotations on the first few days after lesion and lack of contralateral rotations after apomorphine suggests that lactacystin model differs from classical 6-OHDA model. The obtained results are discussed in the context of pathological changes observed in PD. 3.128 NEUROCHEMICAL RESPONSES AND DYNAMIC CHANGES OF GLIAL AND NEURONAL CELLS IN THE SUBSTANTIA NIGRA OF A LACTACYSTIN RAT MODEL L.W. Chen, L. Duan, X.Y. Jiao, Y.X. Ding, L. Zhang. The Fourth Military Medical University, Xi’an, China The full understanding of pathogenesis or neurochemical dynamics of Parkinson’s disease is critical for effective devising of novel neuroprotective and cell replacement therapies, and neurochemical responses and changes of glial and neuronal cells were studied in the substantia nigra of a lactacystin rat model. The animal model was prepared by a stereological microinjection of lactacystin (a proteasome inhibitor) 10 mg in 1 ml saline into the left medial forebrain bandle (MFB), and several neurochemical markers or quantitative changes were detected by immunohistochemistry, western blot and behavioral testing at 1W, 3W and 5W postlactacystin insult. It revealed that: 1. tyrosine hydroxylase (TH)-positive dopaminergic neurons went fast dying and nearly complete loss of TH staining in the substantia nigra along with some dysfunction motor behaviors; 2. abundant and strong iba1-postive microglial cells occurred and remained in the substantia nigra; 3. obvious increasing of GFAP-positive astrocyte and DJ-1 was also detected; and 4. responding dynamic changes of neural progenitor cell markers such as nestin and CD-133 and Wnt/beta-catenin signaling molecules was interestingly observed in the lesioned substantia nigra of this animal model. This study has provided new evidence on the neurochemical and dynamic properties of dopaminergic neurons and micro-environmental cell elements in the lactacystin-induced Parkinsonism rat model. Acknowledgement: This work was supported by grants from National Natural Science Foundation (81071609, 30970862 and 30772279) and Basic Research Program of China (2011CB504103).
3.129 THE ROLE OF NDFIP1 IN IRON-INDUCED CELL DAMAGE IN MES23.5 DOPAMINERGIC CELLS W. Jia, H. Xu, H. Jiang, J. Xie. Qingdao University, Qingdao, China Objective: Elevated iron accumulation has been reported in the substantia nigra (SN) in Parkinson’s disease (PD). Nedd4 family interacting protein 1 (Ndfip1) is an adaptor protein for Nedd4-mediated ubiquitination. It was reported Ndfip1 was a neuroprotective protein and Ndfip1-mediated protein ubiquitination might be a possible survival strategy in neuronal injury. However, the role of Ndfip1 in PD is not elucidated. The present study is to investigate the expression of Ndfip1 in 6-hydroxydopamine (6-OHDA) treated MES23.5 dopaminergic cells and the protective effect of Ndfip1 on iron-induced cell damage. Methods: Quantitative PCR was conducted to measure Ndfip1 mRNA levels. Mitochondrial transmembrane potential (DYm) was measured by flow cytometry using rhodamine123. Results: 1. The mRNA levels of Ndfip1 was decrease in 6-OHDA (10 mmol/L) treated MES23.5 cells. 2. DYm was decreased in MES23.5 cells after iron treatment. And over-expressed Ndfip1 in MES23.5 cells reversed this decrease of DYm induced by iron treatment. Conclusion: These results suggest the decreased expression of Ndfip1 might be associated with 6-OHDA-induced toxicity. And over-expression of Ndfip1 could protect MES23.5 cells from ironinduced cell damage, which is a key factor in PD. 3.130 NEUROCHEMICAL AND BEHAVIOURAL CHARACTERISATION OF SINGLE INTRANIGRAL INJECTION OF LPS INDUCED NEUROINFLAMMATORY RAT MODEL OF PD N. Sharma. Biophysics, Panjab University, Chandigarh, India Parkinson’s disease (PD) is characterised by slow & progressive degeneration of the dopaminergic neurons in SN and motor dysfunction. Recent studies have revealed an essential role for neuroinflammation that is initiated by microglial and infiltrated peripheral immune cells and their toxic products (cytokines, chemokines etc.) in pathogenesis of PD. LPS is a bacterial endotoxin and the most extensively utilized glial activator for the induction of inflammatory dnergic neurodegeneration. LPS model of PD represents a purely inflammation-driven animal model for the induction of nigrostriatal dopaminergic neurodegeneration. The aim of our study was to estimate the level of dopamine and its metabolites in mid brain at 2 different doses of single intranigral injection of LPS 2ug/2ul PBS and 5ug/5ul PBS using HPLC/ECD and then by selecting the more effective dose based on HPLC results. Behavioural characterisation of PD rats was done using several motor behavioral tests. Results: It was observed that decline in the level of dopamine in midbrain of rats injected with (5 ug/5 ul PBS) dose of LPS is greater than LPS (2 ug/2 ul PBS) suggesting that dopaminergic loss using LPS is dose dependent. LPS induced model of PD there is a significant alteration in the inactivation of dopamine where the turnover ratio of HVA to DA was significantly altered. Behavioral test (rotarod, actophotometer, cylinder test, body swing test, corridor test) also showed severe defect in motor coordination and balance indicating the establishment of neuroinflammatory model of PD. LPS administration resulted in significant microglial activation and sustained elevation of proinflammatory cytokines TNFa and IL-1b.
Wednesday, 14 December 2011 / Parkinsonism and Related Disorders 18S2 (2012) S161–S234
3.127 EFFECT OF UNILATERAL LESION WITH THE SELECTIVE PROTEASOME INHIBITOR LACTACYSTIN ON ROTATIONAL BEHAVIOR AND DA METABOLISM IN RATS J. Konieczny, T. Lenda, M. Zapała, E. Lorenc-Koci. Department of Neuro-Psychopharmacology, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland A growing body of evidence suggests that proteasomal dysfunction may play an important role in the pathogenesis of Parkinson’s disease (PD). The aim of our study was to examine the time course of changes in rotational behavior and DA metabolism in rats lesioned unilaterally with lactacystin. Male Wistar rats were injected with lactacystin (2.5 mg/2 ml) into the left substantia nigra (SN) pars compacta. The animals were examined for spontaneous and the apomorphineinduced rotational behavior on days 1, 4, 7, 14 and 21 after surgery; then they were killed and the levels of DA and its metabolites were assayed in striatal and nigral homogenates using HPLC method. On the first few days after surgery, the lesioned rats displayed a spontaneous circling behavior contralateral to the lesioned side. Lactacystin evoked a progressive loss of DA and its metabolites, as well as a progressive acceleration of DA catabolism in the ipsilateral striatum and SN. After 21 days, the decline in DA level exceeded 90% both in the striatum and SN. However, after apomorfine treatment on 7th , 14th and 21st day after surgery no contralateral turning was observed in the lesioned rats. The present study shows that lactacystin produces biochemical changes characteristic of progressive degeneration of the DA system. However, the presence of spontaneous contralateral rotations on the first few days after lesion and lack of contralateral rotations after apomorphine suggests that lactacystin model differs from classical 6-OHDA model. The obtained results are discussed in the context of pathological changes observed in PD. 3.128 NEUROCHEMICAL RESPONSES AND DYNAMIC CHANGES OF GLIAL AND NEURONAL CELLS IN THE SUBSTANTIA NIGRA OF A LACTACYSTIN RAT MODEL L.W. Chen, L. Duan, X.Y. Jiao, Y.X. Ding, L. Zhang. The Fourth Military Medical University, Xi’an, China The full understanding of pathogenesis or neurochemical dynamics of Parkinson’s disease is critical for effective devising of novel neuroprotective and cell replacement therapies, and neurochemical responses and changes of glial and neuronal cells were studied in the substantia nigra of a lactacystin rat model. The animal model was prepared by a stereological microinjection of lactacystin (a proteasome inhibitor) 10 mg in 1 ml saline into the left medial forebrain bandle (MFB), and several neurochemical markers or quantitative changes were detected by immunohistochemistry, western blot and behavioral testing at 1W, 3W and 5W postlactacystin insult. It revealed that: 1. tyrosine hydroxylase (TH)-positive dopaminergic neurons went fast dying and nearly complete loss of TH staining in the substantia nigra along with some dysfunction motor behaviors; 2. abundant and strong iba1-postive microglial cells occurred and remained in the substantia nigra; 3. obvious increasing of GFAP-positive astrocyte and DJ-1 was also detected; and 4. responding dynamic changes of neural progenitor cell markers such as nestin and CD-133 and Wnt/beta-catenin signaling molecules was interestingly observed in the lesioned substantia nigra of this animal model. This study has provided new evidence on the neurochemical and dynamic properties of dopaminergic neurons and micro-environmental cell elements in the lactacystin-induced Parkinsonism rat model. Acknowledgement: This work was supported by grants from National Natural Science Foundation (81071609, 30970862 and 30772279) and Basic Research Program of China (2011CB504103).
3.129 THE ROLE OF NDFIP1 IN IRON-INDUCED CELL DAMAGE IN MES23.5 DOPAMINERGIC CELLS W. Jia, H. Xu, H. Jiang, J. Xie. Qingdao University, Qingdao, China Objective: Elevated iron accumulation has been reported in the substantia nigra (SN) in Parkinson’s disease (PD). Nedd4 family interacting protein 1 (Ndfip1) is an adaptor protein for Nedd4-mediated ubiquitination. It was reported Ndfip1 was a neuroprotective protein and Ndfip1-mediated protein ubiquitination might be a possible survival strategy in neuronal injury. However, the role of Ndfip1 in PD is not elucidated. The present study is to investigate the expression of Ndfip1 in 6-hydroxydopamine (6-OHDA) treated MES23.5 dopaminergic cells and the protective effect of Ndfip1 on iron-induced cell damage. Methods: Quantitative PCR was conducted to measure Ndfip1 mRNA levels. Mitochondrial transmembrane potential (DYm) was measured by flow cytometry using rhodamine123. Results: 1. The mRNA levels of Ndfip1 was decrease in 6-OHDA (10 mmol/L) treated MES23.5 cells. 2. DYm was decreased in MES23.5 cells after iron treatment. And over-expressed Ndfip1 in MES23.5 cells reversed this decrease of DYm induced by iron treatment. Conclusion: These results suggest the decreased expression of Ndfip1 might be associated with 6-OHDA-induced toxicity. And over-expression of Ndfip1 could protect MES23.5 cells from ironinduced cell damage, which is a key factor in PD. 3.130 NEUROCHEMICAL AND BEHAVIOURAL CHARACTERISATION OF SINGLE INTRANIGRAL INJECTION OF LPS INDUCED NEUROINFLAMMATORY RAT MODEL OF PD N. Sharma. Biophysics, Panjab University, Chandigarh, India Parkinson’s disease (PD) is characterised by slow & progressive degeneration of the dopaminergic neurons in SN and motor dysfunction. Recent studies have revealed an essential role for neuroinflammation that is initiated by microglial and infiltrated peripheral immune cells and their toxic products (cytokines, chemokines etc.) in pathogenesis of PD. LPS is a bacterial endotoxin and the most extensively utilized glial activator for the induction of inflammatory dnergic neurodegeneration. LPS model of PD represents a purely inflammation-driven animal model for the induction of nigrostriatal dopaminergic neurodegeneration. The aim of our study was to estimate the level of dopamine and its metabolites in mid brain at 2 different doses of single intranigral injection of LPS 2ug/2ul PBS and 5ug/5ul PBS using HPLC/ECD and then by selecting the more effective dose based on HPLC results. Behavioural characterisation of PD rats was done using several motor behavioral tests. Results: It was observed that decline in the level of dopamine in midbrain of rats injected with (5 ug/5 ul PBS) dose of LPS is greater than LPS (2 ug/2 ul PBS) suggesting that dopaminergic loss using LPS is dose dependent. LPS induced model of PD there is a significant alteration in the inactivation of dopamine where the turnover ratio of HVA to DA was significantly altered. Behavioral test (rotarod, actophotometer, cylinder test, body swing test, corridor test) also showed severe defect in motor coordination and balance indicating the establishment of neuroinflammatory model of PD. LPS administration resulted in significant microglial activation and sustained elevation of proinflammatory cytokines TNFa and IL-1b.