- Email: [email protected]
Intrastriatal glial cell line-derived neurotrophic factors for protecting dopaminergic neurons in the substantia nigra of mice with Parkinson disease**
NEURAL REGENERATION RESEARCH Volume 2, Issue 4, April 2007
Cite this article as: Neural Regen Res,2007,2(4),207-10
Basic Medicine
,QWUDVWULDWDO JOLDO FHOO OLQHGHULYHG QHXURWURSKLF IDFWRUV IRU SURWHFWLQJGRSDPLQHUJLFQHXURQVLQWKHVXEVWDQWLDQLJUDRI PLFHZLWK3DUNLQVRQGLVHDVH Chenghua Xiao1, Yanqiang Wang1, Hongmei Liu2, Hongjun Wang2, Junping Cao2, Dianshuai Gao2 1Department of Neurology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China 2Neurobiological Laboratory, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China
Abstract BACKGROUND: Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor (GDNF) injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE: To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease (PD), and analyze the action pathway. DESIGN: A controlled observation. SETTING: Neurobiological Laboratory of Xuzhou Medical College. MATERIALS: Twenty-four male Kunming mice of 7 8 weeks old were used. GDNF, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were purchased from Sigma Company (USA); LEICAQWin image processing and analytical system. METHODS: The experiments were carried out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group: GDNF 4-day group, phosphate buffer solution (PSB) 4-day group, GDNF 6-day group and L GDNF PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 solution (20 g/L, dispensed with 0.01 mol/L PBS) injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same On the 12th day after model establishment, the volume to the same injection at corresponding time points. midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase (TH) and calcium binding protein (CB) with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES: Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS: All the 24 mice were involved in the analysis of results. The numbers of TH+ and CB+ neurons in the GDNF 4-day group (54.33 6.92, 46.33 5.54) were obviously more than those in the PBS 4-day group (27.67 5.01, 21.50 5.96, P < 0.01). The numbers of TH+ and CB+ neurons in the GDNF 6-day group (75.67 5.39, 69.67 8.69) were obviously more than those in the PBS 6-day group (27.17 4.50, 21.33 5.72, P < 0.01) and those in the GDNF 4-day group (P < 0.01). CONCLUSION: Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression. Key Words: glial cell line-derived neurotrophic factor (GDNF); dopaminergic neurons; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
Chenghua Xiao , Master, professor, Associate Associate chief physician, Department of Neurology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China Supported by: the Natural Foundation of Science Department of Jiangsu No. Education, 02KJB310009* CH,Wang YQ,Liu Xiao HM,Wang HJ,Cao JP,Gao glial cell DS.Intrastriatal neurotrophic line-derived for protecting factors dopaminergic neurons in the substantia nigra of mice with disease.Neural Parkinson Regen Res 2007;2(4):207-10 www.sjzsyj.com/Journal/ 0704/07-04-207.html
Received:2007-02-15;Accepted:2007-03-25 (07-S-03-0286/YWY) Corresponding author: Chenghua Xiao, Department of Neurology, Affiliated Hospital of Xuzhou Medical College, 221002, Jiangsu Province, China E-mail: [email protected] Xuzhou
207
Xiao CH, et al. / Neural Regeneration Research,2007,2(4):207-10
,1752'8&7,21 Parkinson disease (PD) is a degenerative disease of central nervous system, and the main pathological change is the progressive degenerative death of dopaminergic neurons in substantia nigra, which can result in the decrease of dopamine contents in caudate nucleus and putamen[1-4]. Glial cell line-derived neurotrophic factor (GDNF) is one of the neurotrophic factors of dopaminergic neurons, since GDNF was separated from rat B49 glial cell line and purified by Lin et al in 1993, plenty of studies have confirmed that it has specific effects on promoting the survival and morphological differentiation of ventral mesencephalic dopaminergic neurons[5-10]. GDNF is the macromolecular substance of neuropeptide, and cannot get through blood brain barrier, thus it is a main problem how to enable it to pass through blood brain barrier and enter the central nervous system when GDNF is applied to treat PD. It has been proved in our laboratory that GDNF injection into substantia nigra of PD rats can obviously improve the PD symptoms and promote the survival of dopaminergic neurons[11,12]. Substantia nigra is deep in position and limited in range, the GDNF injection directly into substantia nigra has relatively greater damages with higher difficulty. Thus it is suspected whether GDNF injected into striatum, the target area of dopaminergic neuron, can protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport? In this study, PD models were constructed in mice using 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), and they were treated with intrastriatal injection of exogenous GDNF, then the changes of numbers of positive neurons of tyrosinehydroxylase (TH) and calcium binding protein (CB) in the compact part of substantia nigra were observed, and the protective effect of intrastriatal GDNF on dopaminergic neurons in substantia nigra were evaluated.
0$7(5,$/6$1'0(7+2'6 0DWHULDOV The experiments were carried out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. Twenty-four male Kunming mice of 7–8 weeks old, 20–25 g, were provided by the experimental animal center of Xuzhou Medical College [SYXK(Su)2002-0038]. Main reagents and apparatus: GDNF, MPTP, mice-anti-rat TH antibody (primary antibody), biotin-binding goat-anti-mice monoclonal antibody (second antibody), goat serum (stock solution) for blocking, and horseradish peroxidase (HRP) labeled biotin-avidin compound were all purchased from Sigma Company (USA); YL3A rotary microtome, pathological tissue rinsing and drying apparatus, Olympus light microscope, OlympusPM-CB20 208
photomicrographic system, LEICAQWin image processing and analytical system. 0HWKRGV (VWDEOLVKLQJ3'PRGHOV Mice models of PD were established by intraperitoneal injection of MPTP (30 mg/kg) at every 8:00 in the morning for 5 days continuously. *URXSLQJDQGDGPLQLVWUDWLRQ The mice were randomly divided into four groups: GDNF 4-day group (n=6), phosphate buffer solution (PSB) 4-day group (n=6), GDNF 6-day group (n=6) and PSB 6-day group (n=6). Mice in the GDNF 4 and 6-day groups were administrated with 1 L GDNF solution (20 g/L, dispensed with 0.01 mol/L PBS) injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. The mice were anesthetized by intraperitoneal injection of 100 g/L chloral hydrate, and the coordinates were selected according to the Paxinos and Watson atlas. The solution was drawn with 10 L Hamiton microsyringer, and injected at 0.5 L per minute, and the needle was kept for 5 minutes, then slowly withdrawn at 1 mm per minute. 6DPSOHREWDLQLQJDQGVHFWLRQSUHSDUDWLRQ The mice were anesthetized by intraperitoneal injection of 100 g/L chloral hydrate on the 12th day after model establishment, saline and formamint were infused via left ventricular intubation, then the midbrains were removed and fixed. The midbrains were dehydrated with alcohol in gradient, hyalinized with dimethylbenzene, immersed with wax and embedded. Serial coronal sections (6 m) of substantia nigra segments were prepared and collected in different groups. The cut cerebrosections were stuck to the cover glasses which were treated with poly lysine in advance, then baked at 37 for overnight. ,PPXQRKLVWRFKHPLFDOVWDLQLQJ The sections were dewaxed till dehydrate, endogenous peroxidase were removed by 3% hydrogen peroxide, repaired with microwave, and blocked with normal goat serum at 37 for 30 minutes, and then added in order by mice anti-TH antibody (1 3 000) or mice anti-CaBP antibody (1 1 000), biotin-binding goat-anti-mice Ig (1 50) and HRP labeled biotin-avidin compound. The sections were washed with 0.01 mol/L PBS for 3 times at the intervals of the procedures. Finally, CB and TH were stained with diaminobenzidine (DAB). 3RVLWLYHQHXURQVLQPLGEUDLQVXEVWDQWLDQLJUDFRXQWHGZLWK FRPSXWHUDLGHGLPDJHDQDO\WLFDOV\VWHP The TH+ and CB+ cells at corresponding time points were quantitatively analyzed with the computer-aided image analytical system ( 10, 20) respectively. The midbrain tissue section of each mouse was divided into 3 areas from rostral to caudal sides. The TH+ and CB+ neurons with
Xiao CH, et al. / Neural Regeneration Research,2007,2(4):207-10
obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. 6WDWLVWLFDODQDO\VLV The data were analyzed by the second author using SPSS 12.0 software with univariate analysis of variance, the means were compared, and P < 0.05 was considered as significant difference.
5(68/76 4XDQWLWDWLYHDQDO\VLVRIWKHH[SHULPHQWDODQLPDOV All the 24 mice were involved in the analysis of results. 1XPEHU RI SRVLWLYH QHXURQV LQ WKH PLGEUDLQ VXEVWDQWLD QLJUDRIPLFHLQHDFKJURXS (Table 1) Table 1 Comparison of TH+ and CB+ neurons in substantia nigra (x s, n =6, n) Group PBS 4-day PBS 6-day GDNF 4-day GDNF 6-day
TH+ neuron 27.67 27.17 54.33 75.67
5.01 4.50 6.92a 5.39bc
CB+ neuron 21.50 21.33 46.33 69.67
5.96 5.72 5.54a 8.69bc
PBS: phosphate buffer solution; GDNF: glial cell line-derived neurotrophic factor; TH; tyroxine hydroxylase; CB: calcium binding protein; aP < 0.01, vs. the PBS 4-day group; bP < 0.01, vs. the PBS 6-day group; cP < 0.01, vs. the GDNF 4-day group
The TH and CB neurons in the GDNF 4 and 6-day groups distributed in cluster, and the numbers and expressions of positive neurons were significantly increased as compared with those in the PBS 4 and 6-day groups (P < 0.01). The numbers of positive neurons had no significant differences between the PBS 4 and 6-day groups (P > 0.05), whereas those were significantly different between the GDNF 4 and 6-day groups (P < 0.01). The results indicated that at early MPTP damage, the intrastriatal injection of GDNF had positive effects on the survival, structural and functional recoveries of the involved dopaminergic neurons.
',6&866,21 PD is a chronic progressive disease, and suitable PD animal models have been established during the research on its pathogenesis and drug treatment, and the typical ones are 6-hydroxydopamine (6-OHDA) and MPTP. MPTP is a micromolecular substance without toxicity and side effects, it has high liposoluability, can get through the blood brain barrier easily, it can be activated by monoamine oxidase B (MAO-B) after absorbed by astrocyte, serotonergic neurons, etc., then converses to intermediary metabolite MPFP+, generates toxic MPP+ and releases to extracellular space, absorbed by the axon terminals of dopaminergic neurons
through presynaptic membrane dopamine transporter, retrograde transports to the cell body of dopaminergic neurons in substantia nigra, blocks the mitochondrial respiratory chain, and results in energy failure[13-17]. The following increased releases of reactive oxygen species (ROS), nitric oxide, and other free radicals, and the proliferation of reactive glial cells cause the death and apoptosis of dopaminergic neurons in substantia nigra[18-21]. In the substantia nigra striatum system, GDNF is one of the more than 20 cytokines, which have neurotrophic effects on dopaminergic neurons at present. Striatum targeted GDNF plays an important role in maintaining the maturity of dopaminergic neurons during early neurodevelopment and organic stress[22,23]. It has been demonstrated both in vivo and in vitro that dopaminergic neurons in substantia nigra are the potential neurotrophic molecules, they can block the progressive degeneration of dopaminergic neurons, and have the significance in treating PD[24-28]. Current studies have indicated that their neuroprotective effects may be associated with the proliferation of glial cells in vivo and GDNF related receptors activated signal pathway[29,30]. However, the shorter duration and limited increase amount of endogenous GDNF expression are difficult for the overall and lasting treatment of the damaged neurons. It has been found that the expression of GDNF mRNA and protein was first increased as early as 2 hours after ischemia-reperfusion in peri-infarct cerebral cortex and striatum, and showed a second increase at 72 hours[31]. In this study, intrastriatal GDNF was given to the MPTP-induced PD models on the 4th and 6th days, and the results showed that the positive TH and CB neurons were obviously different between the GDNF-treated group and control groups, there were also marked differences in the number, area and process length. It was indicated that intrastriatal GDNF could protect dopaminergic neurons and promote the survival, improve the anti-damage ability of the neurons, also accelerate the axonal growth. At early damage, the protective effect was more obvious as the reduced dosage of endogenous GDNF. In summary, GDNF can protect the dopaminergic neurons in midbrain substantia nigra of adult Kunming mice at early MPTP injury stage. As a neurotrophic factor for structural internal environment, GDNF has wide prospect in the PD treatment.
5()(5(1&(6 1. Granholm AC, Reyland M, Albeck D, et al. Glial cell line-derived neurotrophic factor is essential for postnatal survival of midbrain dopamine neurons. J Neurosci 2000;20(9):3182-90 2. Vernier P, Moret F, Callier S, et al. The degeneration of dopamine neurons in Parkinson's disease: insights from embryology and evolution of the mesostriatocortical system. Ann N Y Acad Sci 2004;1035:231-49 3. Lotharius J, Falsig J, van Beek J, et al. Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway. J Neurosci 2005;25(27):6329-42
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4. Boger HA, Middaugh LD, Huang P, et al. A partial GDNF depletion leads to earlier age-related deterioration of motor function and tyrosine hydroxylase expression in the substantia nigra. Exp Neurol 2006;202(2):336-47 5. Lin LF, Doherty DH, Lile JD, et al. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 1993;260(5111):1130-2 6. Cass WA, Peters LE, Harned ME, et al. Protection by GDNF and other trophic factors against the dopamine-depleting effects of neurotoxic doses of methamphetamine. Ann N Y Acad Sci 2006;1074:272-81 7. Burke RE. Postnatal developmental programmed cell death in dopamine neurons. Ann N Y Acad Sci 2003;991:69-79 8. Ugarte SD, Lin E, Klann E, et al. Effects of GDNF on 6-OHDA-induced death in a dopaminergic cell line: modulation by inhibitors of PI3 kinase and MEK. J Neurosci Res 2003;73(1):105-12 9. Oo TF, Kholodilov N, Burke RE. Regulation of natural cell death in dopaminergic neurons of the substantia nigra by striatal glial cell line-derived neurotrophic factor in vivo. J Neurosci 2003;23(12):5141-8 10. Buytaert-Hoefen KA, Alvarez E, Freed CR. Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF. Stem Cells 2004;22(5):669-74 11. Liu HM, Yu JK, Ding YX, et al. GDNF protects the DA neurons in the substantia nigra of PD model rats by activating astrocytes. Shenjing Jiepouxue Zazhi 2006;22(4):367-72 12. Ding YX, Liu HM, Wang HJ, et al. The protective mechanism of GDNF on dopaminergic neurons in substantia nigra of Parkinson disease rats. Xuzhou Yixueyuan Yuanbao 2006;26(1):8-13 13. Blandini F, Nappi G, Tassorelli C, et al. Functional changes of the basal ganglia circuitry in Parkinson's disease. Prog Neurobiol 2000;62(1):63-88 14. Bezard E, Gross CE, Foumier MC, et al. Absence of MPTP-induced neuronal death in mice lacking the dopamine transporter. Exp Neurol 1999;155(2):268-73 15. Gao HM, Liu B, Zhang W, et al. Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson's disease. FASEB J 2003;17(13):1954-6 16. Fornai F, Schluter OM, Lenzi P, et al. Parkinson-like syndrome induced by continuous MPTP infusion: convergent roles of the ubiquitin-proteasome system and alpha-synuclein. Proc Natl Acad Sci U S A 2005;102(9):3413-8 17. Wang T, Pei Z, Zhang W, et al. MPP+-induced COX-2 activation and subsequent dopaminergic neurodegeneration. FASEB J 2005;19(9):1134-6 18. Kurosaki R, Muramatsu Y, Michimata M, et al. Role of nitric
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(Edited by Zhang YB/Yin YL/Wang L)
Cite this article as: Neural Regen Res,2007,2(4),207-10
Basic Medicine
,QWUDVWULDWDO JOLDO FHOO OLQHGHULYHG QHXURWURSKLF IDFWRUV IRU SURWHFWLQJGRSDPLQHUJLFQHXURQVLQWKHVXEVWDQWLDQLJUDRI PLFHZLWK3DUNLQVRQGLVHDVH Chenghua Xiao1, Yanqiang Wang1, Hongmei Liu2, Hongjun Wang2, Junping Cao2, Dianshuai Gao2 1Department of Neurology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China 2Neurobiological Laboratory, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China
Abstract BACKGROUND: Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor (GDNF) injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE: To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease (PD), and analyze the action pathway. DESIGN: A controlled observation. SETTING: Neurobiological Laboratory of Xuzhou Medical College. MATERIALS: Twenty-four male Kunming mice of 7 8 weeks old were used. GDNF, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were purchased from Sigma Company (USA); LEICAQWin image processing and analytical system. METHODS: The experiments were carried out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group: GDNF 4-day group, phosphate buffer solution (PSB) 4-day group, GDNF 6-day group and L GDNF PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 solution (20 g/L, dispensed with 0.01 mol/L PBS) injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same On the 12th day after model establishment, the volume to the same injection at corresponding time points. midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase (TH) and calcium binding protein (CB) with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES: Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS: All the 24 mice were involved in the analysis of results. The numbers of TH+ and CB+ neurons in the GDNF 4-day group (54.33 6.92, 46.33 5.54) were obviously more than those in the PBS 4-day group (27.67 5.01, 21.50 5.96, P < 0.01). The numbers of TH+ and CB+ neurons in the GDNF 6-day group (75.67 5.39, 69.67 8.69) were obviously more than those in the PBS 6-day group (27.17 4.50, 21.33 5.72, P < 0.01) and those in the GDNF 4-day group (P < 0.01). CONCLUSION: Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression. Key Words: glial cell line-derived neurotrophic factor (GDNF); dopaminergic neurons; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
Chenghua Xiao , Master, professor, Associate Associate chief physician, Department of Neurology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China Supported by: the Natural Foundation of Science Department of Jiangsu No. Education, 02KJB310009* CH,Wang YQ,Liu Xiao HM,Wang HJ,Cao JP,Gao glial cell DS.Intrastriatal neurotrophic line-derived for protecting factors dopaminergic neurons in the substantia nigra of mice with disease.Neural Parkinson Regen Res 2007;2(4):207-10 www.sjzsyj.com/Journal/ 0704/07-04-207.html
Received:2007-02-15;Accepted:2007-03-25 (07-S-03-0286/YWY) Corresponding author: Chenghua Xiao, Department of Neurology, Affiliated Hospital of Xuzhou Medical College, 221002, Jiangsu Province, China E-mail: [email protected] Xuzhou
207
Xiao CH, et al. / Neural Regeneration Research,2007,2(4):207-10
,1752'8&7,21 Parkinson disease (PD) is a degenerative disease of central nervous system, and the main pathological change is the progressive degenerative death of dopaminergic neurons in substantia nigra, which can result in the decrease of dopamine contents in caudate nucleus and putamen[1-4]. Glial cell line-derived neurotrophic factor (GDNF) is one of the neurotrophic factors of dopaminergic neurons, since GDNF was separated from rat B49 glial cell line and purified by Lin et al in 1993, plenty of studies have confirmed that it has specific effects on promoting the survival and morphological differentiation of ventral mesencephalic dopaminergic neurons[5-10]. GDNF is the macromolecular substance of neuropeptide, and cannot get through blood brain barrier, thus it is a main problem how to enable it to pass through blood brain barrier and enter the central nervous system when GDNF is applied to treat PD. It has been proved in our laboratory that GDNF injection into substantia nigra of PD rats can obviously improve the PD symptoms and promote the survival of dopaminergic neurons[11,12]. Substantia nigra is deep in position and limited in range, the GDNF injection directly into substantia nigra has relatively greater damages with higher difficulty. Thus it is suspected whether GDNF injected into striatum, the target area of dopaminergic neuron, can protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport? In this study, PD models were constructed in mice using 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), and they were treated with intrastriatal injection of exogenous GDNF, then the changes of numbers of positive neurons of tyrosinehydroxylase (TH) and calcium binding protein (CB) in the compact part of substantia nigra were observed, and the protective effect of intrastriatal GDNF on dopaminergic neurons in substantia nigra were evaluated.
0$7(5,$/6$1'0(7+2'6 0DWHULDOV The experiments were carried out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. Twenty-four male Kunming mice of 7–8 weeks old, 20–25 g, were provided by the experimental animal center of Xuzhou Medical College [SYXK(Su)2002-0038]. Main reagents and apparatus: GDNF, MPTP, mice-anti-rat TH antibody (primary antibody), biotin-binding goat-anti-mice monoclonal antibody (second antibody), goat serum (stock solution) for blocking, and horseradish peroxidase (HRP) labeled biotin-avidin compound were all purchased from Sigma Company (USA); YL3A rotary microtome, pathological tissue rinsing and drying apparatus, Olympus light microscope, OlympusPM-CB20 208
photomicrographic system, LEICAQWin image processing and analytical system. 0HWKRGV (VWDEOLVKLQJ3'PRGHOV Mice models of PD were established by intraperitoneal injection of MPTP (30 mg/kg) at every 8:00 in the morning for 5 days continuously. *URXSLQJDQGDGPLQLVWUDWLRQ The mice were randomly divided into four groups: GDNF 4-day group (n=6), phosphate buffer solution (PSB) 4-day group (n=6), GDNF 6-day group (n=6) and PSB 6-day group (n=6). Mice in the GDNF 4 and 6-day groups were administrated with 1 L GDNF solution (20 g/L, dispensed with 0.01 mol/L PBS) injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. The mice were anesthetized by intraperitoneal injection of 100 g/L chloral hydrate, and the coordinates were selected according to the Paxinos and Watson atlas. The solution was drawn with 10 L Hamiton microsyringer, and injected at 0.5 L per minute, and the needle was kept for 5 minutes, then slowly withdrawn at 1 mm per minute. 6DPSOHREWDLQLQJDQGVHFWLRQSUHSDUDWLRQ The mice were anesthetized by intraperitoneal injection of 100 g/L chloral hydrate on the 12th day after model establishment, saline and formamint were infused via left ventricular intubation, then the midbrains were removed and fixed. The midbrains were dehydrated with alcohol in gradient, hyalinized with dimethylbenzene, immersed with wax and embedded. Serial coronal sections (6 m) of substantia nigra segments were prepared and collected in different groups. The cut cerebrosections were stuck to the cover glasses which were treated with poly lysine in advance, then baked at 37 for overnight. ,PPXQRKLVWRFKHPLFDOVWDLQLQJ The sections were dewaxed till dehydrate, endogenous peroxidase were removed by 3% hydrogen peroxide, repaired with microwave, and blocked with normal goat serum at 37 for 30 minutes, and then added in order by mice anti-TH antibody (1 3 000) or mice anti-CaBP antibody (1 1 000), biotin-binding goat-anti-mice Ig (1 50) and HRP labeled biotin-avidin compound. The sections were washed with 0.01 mol/L PBS for 3 times at the intervals of the procedures. Finally, CB and TH were stained with diaminobenzidine (DAB). 3RVLWLYHQHXURQVLQPLGEUDLQVXEVWDQWLDQLJUDFRXQWHGZLWK FRPSXWHUDLGHGLPDJHDQDO\WLFDOV\VWHP The TH+ and CB+ cells at corresponding time points were quantitatively analyzed with the computer-aided image analytical system ( 10, 20) respectively. The midbrain tissue section of each mouse was divided into 3 areas from rostral to caudal sides. The TH+ and CB+ neurons with
Xiao CH, et al. / Neural Regeneration Research,2007,2(4):207-10
obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. 6WDWLVWLFDODQDO\VLV The data were analyzed by the second author using SPSS 12.0 software with univariate analysis of variance, the means were compared, and P < 0.05 was considered as significant difference.
5(68/76 4XDQWLWDWLYHDQDO\VLVRIWKHH[SHULPHQWDODQLPDOV All the 24 mice were involved in the analysis of results. 1XPEHU RI SRVLWLYH QHXURQV LQ WKH PLGEUDLQ VXEVWDQWLD QLJUDRIPLFHLQHDFKJURXS (Table 1) Table 1 Comparison of TH+ and CB+ neurons in substantia nigra (x s, n =6, n) Group PBS 4-day PBS 6-day GDNF 4-day GDNF 6-day
TH+ neuron 27.67 27.17 54.33 75.67
5.01 4.50 6.92a 5.39bc
CB+ neuron 21.50 21.33 46.33 69.67
5.96 5.72 5.54a 8.69bc
PBS: phosphate buffer solution; GDNF: glial cell line-derived neurotrophic factor; TH; tyroxine hydroxylase; CB: calcium binding protein; aP < 0.01, vs. the PBS 4-day group; bP < 0.01, vs. the PBS 6-day group; cP < 0.01, vs. the GDNF 4-day group
The TH and CB neurons in the GDNF 4 and 6-day groups distributed in cluster, and the numbers and expressions of positive neurons were significantly increased as compared with those in the PBS 4 and 6-day groups (P < 0.01). The numbers of positive neurons had no significant differences between the PBS 4 and 6-day groups (P > 0.05), whereas those were significantly different between the GDNF 4 and 6-day groups (P < 0.01). The results indicated that at early MPTP damage, the intrastriatal injection of GDNF had positive effects on the survival, structural and functional recoveries of the involved dopaminergic neurons.
',6&866,21 PD is a chronic progressive disease, and suitable PD animal models have been established during the research on its pathogenesis and drug treatment, and the typical ones are 6-hydroxydopamine (6-OHDA) and MPTP. MPTP is a micromolecular substance without toxicity and side effects, it has high liposoluability, can get through the blood brain barrier easily, it can be activated by monoamine oxidase B (MAO-B) after absorbed by astrocyte, serotonergic neurons, etc., then converses to intermediary metabolite MPFP+, generates toxic MPP+ and releases to extracellular space, absorbed by the axon terminals of dopaminergic neurons
through presynaptic membrane dopamine transporter, retrograde transports to the cell body of dopaminergic neurons in substantia nigra, blocks the mitochondrial respiratory chain, and results in energy failure[13-17]. The following increased releases of reactive oxygen species (ROS), nitric oxide, and other free radicals, and the proliferation of reactive glial cells cause the death and apoptosis of dopaminergic neurons in substantia nigra[18-21]. In the substantia nigra striatum system, GDNF is one of the more than 20 cytokines, which have neurotrophic effects on dopaminergic neurons at present. Striatum targeted GDNF plays an important role in maintaining the maturity of dopaminergic neurons during early neurodevelopment and organic stress[22,23]. It has been demonstrated both in vivo and in vitro that dopaminergic neurons in substantia nigra are the potential neurotrophic molecules, they can block the progressive degeneration of dopaminergic neurons, and have the significance in treating PD[24-28]. Current studies have indicated that their neuroprotective effects may be associated with the proliferation of glial cells in vivo and GDNF related receptors activated signal pathway[29,30]. However, the shorter duration and limited increase amount of endogenous GDNF expression are difficult for the overall and lasting treatment of the damaged neurons. It has been found that the expression of GDNF mRNA and protein was first increased as early as 2 hours after ischemia-reperfusion in peri-infarct cerebral cortex and striatum, and showed a second increase at 72 hours[31]. In this study, intrastriatal GDNF was given to the MPTP-induced PD models on the 4th and 6th days, and the results showed that the positive TH and CB neurons were obviously different between the GDNF-treated group and control groups, there were also marked differences in the number, area and process length. It was indicated that intrastriatal GDNF could protect dopaminergic neurons and promote the survival, improve the anti-damage ability of the neurons, also accelerate the axonal growth. At early damage, the protective effect was more obvious as the reduced dosage of endogenous GDNF. In summary, GDNF can protect the dopaminergic neurons in midbrain substantia nigra of adult Kunming mice at early MPTP injury stage. As a neurotrophic factor for structural internal environment, GDNF has wide prospect in the PD treatment.
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(Edited by Zhang YB/Yin YL/Wang L)