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Cyclosporin A Attenuates the Decrease in Tyrosine Hydroxylase Immunoreactivity in Nigrostriatal Dopaminergic Neurons and in Striatal Dopamine Content in Rats with Intrastriatal Injection of 6-Hydroxydopamine
EXPERIMENTAL NEUROLOGY ARTICLE NO.
146, 526–535 (1997)
EN976575
Cyclosporin A Attenuates the Decrease in Tyrosine Hydroxylase Immunoreactivity in Nigrostriatal Dopaminergic Neurons and in Striatal Dopamine Content in Rats with Intrastriatal Injection of 6-Hydroxydopamine Kouji Matsuura,*,† Hirofumi Makino,† and Norio Ogawa* *Department of Neuroscience, Institute of Molecular and Cellular Medicine and †Third Department of Internal Medicine, Okayama University Medical School, 2-5-1 Shikatacho, Okayama 700, Japan
To explore new therapeutic strategies for Parkinson’s disease, we studied the possible protective effect of an immunosuppressant, cyclosporin A (CsA), treatment on changes in dopaminergic function in rats with intrastriatal injections of 6-hydroxydopamine (6OHDA). Four weeks after injection of 6-OHDA, dopamine (DA) and dihydroxyphenylacetic acid in the striatum were depleted by 70–80%, and repeated high-dose CsA (20 mg/kg) treatment for 1 week significantly protected against these depletions. Tyrosine hydroxylase immunoreactivity (TH-IR) of the cell bodies in the substantia nigra pars compacta (SNc) ipsilateral to the injection were lower than on the contralateral side at 4 weeks but not at 1 week after 6-OHDA injection. The number of TH-positive cell bodies in the SNc decreased to 64% but CsA treatment increased this to 87%. The staining of microglia in the SN with OX42 and Griffonia simplicifolia B4 isolectin was intense at 3 days and gradually decreased by 28 days after injection. At 3 and 7 days after injection, the microglial staining in the SN was prominent and equal both in the 6-OHDA group and in ascorbic acid (SA)-injected controls. By 28 days postinjection, the staining had decreased to control levels in the SA group but was still above the control in the 6-OHDA group. CsA treatment did not affect this staining in either group. These results suggest that CsA protects against 6-OHDA-induced injury of nigrostriatal DA neurons by a mechanism not involving microglia. r 1997 Academic Press
INTRODUCTION
Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder characterized by resting tremor, rigidity, bradykinesia/akinesia, and postural disturbance resulting from the idiopathic loss of dopaminergic neurons in the substantia nigra (SN). The observation of a decrease in striatal dopamine (DA) levels associated with the loss of dopaminergic neurons led to the introduction of levodopa treatment. 0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Levodopa is a highly effective treatment and is widely used, but chronic levodopa treatment causes severe clinical complications, including dyskinesia, wearing-off, and on–off phenomenon (29, 34). Moreover, the degeneration of dopaminergic neurons continues. Recently, several approaches have been proposed to slow, halt, or reverse the underlying progression of dopaminergic neuronal degeneration. These include the use of neurotrophic factors such as brain-derived neurotrophic factor (BDNF), GM1 ganglioside, free radical scavengers or the iron-chelator desferrioxamine (2–4, 7, 11, 18, 24, 25, 30, 38, 41–43, 45, 46). Several animal models of PD have been established which employ intranigral injections of 6-hydroxydopamine (6-OHDA) or systemic injection of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) to produce a rapid and selective lesion of nigrostriatal dopaminergic neurons. These differ in time course from PD where the slow progressive nature is due to a protracted, rather than acute, loss of nigrostriatal dopaminergic neurons. MPTP-treated mice have been used to evaluate the trophic effects of neuroactive substances on the nigrostriatal DA system because such mice show a moderate DA depletion in the striatum (ST) and loss of dopaminergic neurons in the SN (12, 17, 18, 33, 38–41). However, this model can show spontaneous improvement in indices of DA function (13, 35, 38). Intrastriatal injection of 6-OHDA provides a nerve terminal pruning and slowly progressive neurodegeneration model in which neurotrophins and other factors can be tested for their effects on residual DA neurons (3, 11, 37). It has been suggested that immunological reactions may play an important role in progressive neurodegenerative diseases such as PD, Alzheimer’s disease, and cerebrovascular disease (28, 32, 44, 53). Cyclosporin A (CsA) is an immunosuppressive drug widely used in organ transplantation and to treat autoimmune diseases. In previous studies (28) we have shown that repeated high-dose CsA (20 mg/kg) treatment significantly attenuated 6-OHDA-induced striatal dopamine depletion in mice,
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but the effect of CsA on the glial response was not studied. In the present work, we investigated the effects of CsA on the degeneration of dopaminergic neurons and on the response of perineuronal glial cells in rats after intrastriatal infusions of 6-OHDA. MATERIALS AND METHODS
Animal Experiments Adult male Sprague–Dawley rats weighing 280–320 g (Charles River Japan Inc., Kanagawa, Japan) at the beginning of the experiments were used (n 5 106). The animals were housed at constant room temperature (24 6 1°C) and humidity (55%) with a 12-h light–dark cycle (lights on 07:00–19:00) and were given free access to food and water. Intrastriatal injections of 10 µl of 6-OHDA solution [Sigma Chemical Co., St. Louis, MO; 2.0 mg/ml, in physiological saline containing 0.01% ascorbic acid (SA)] or SA as a control were performed under sodium pentobarbital anesthesia (35 mg/kg, ip). CsA (Sandoz, Basel, Switzerland), or the same volume of vehicle (V; 0.2% caster oil), was injected subcutaneously 24 h and 30 min before, 5 h after, and once daily on Days 1 to 6 after 6-OHDA (or SA) injection. Thus, experimental animals were divided into four groups: (1) SA 1 V, (2) SA 1 CsA (20 mg/kg), (3) 6-OHDA 1 V, and (4) 6-OHDA 1 CsA (20 mg/kg) (n 5 24 for each group). Animals were sacrificed 1 and 4 weeks postinjection and were divided into two groups. One group was used for histological analysis and another group was used for neurochemical analysis. Some animals were sacrificed 3 days after injection and were used for histological analysis. Surgical Protocol Following anesthesia, animals were placed in a stereotaxic frame with the tooth bar 5 mm above horizontal, and 6-OHDA (20 µg/10 µl) or vehicle was delivered from a microsyringe at a rate of 1 µl/min. Stereotaxic coordinates were 2.4 mm anterior to the bregma (AP), 2.5 and 3.5 mm lateral from the midline (ML), and 5.5 mm below the dura (DV), as well as AP 2.8 mm, ML 2.0 and 3.0 mm, DV 4.5 mm (four injections per rat; 2.5 µl at each point). The sites of insertion of the microsyringe were determined from the stereotaxic atlas of Pellegrino and Cushman (36). After injection, the microsyringe was left in place for 3 min and then withdrawn at a rate of 1 mm/min. Animals in the control group received 10 µl of SA in the manner described above. Neurochemical Analysis For biochemical studies, experimental rats (total n 5 48; n 5 6 for each group at 1 and 4 weeks postinjection) and 6 naive rats were sacrificed by microwave irradiation (5 kW, 1.2 s), the brain was removed, and
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the ST and SN were dissected. The concentrations of DA, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined by high-performance liquid chromatography (HPLC) with electrochemical detection according to published techniques (31). Briefly, tissues were homogenized in 0.2 M ice-cold perchloric acid. After centrifugation (3000g for 10 min at 4°C), supernatants were filtered (Millipore membrane type HC, 0.45-µm pores) and then injected directly into the HPLC system. Standard was measured often (about every 10 determinations) to allow correction for any loss or variation in the HPLC procedure. Histological Examination For morphological studies, experimental rats (total n 5 48; n 5 4 for each group at every time point) and 4 naive rats were anesthetized with an overdose of sodium pentobarbital and perfused through the left ventricle with saline followed by 300 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The brain was removed and immersed in the same fixative for 24 h at 4°C, after which the tissue was rinsed with 30% sucrose in 0.1 M phosphate buffer (pH 7.2) for 48 h and frozen on solid CO2. Frozen 20-µm coronal sections were cut on a cryostat and mounted one glass slides. The antibody against tyrosine hydroxylase (TH) (Eugene-Tech, Allendale, NJ) was used at a dilution of 1:1000. Antiglial fibrillary acidic protein (GFAP) (Chemicon, Temecula, CA) was used as a marker of astrocytes at a dilution of 1:1000. OX42 against a monomorphic determinant of CR3 antigens (Sera-lab, Sussex, UK) was used at a dilution of 1:500 as a marker of microglia. Immunocytochemistry was carried out at the level of the SN in order to evaluate the status of nigral neurons and perineuronal glial cells. Sections were immersed in methanol containing 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. The sections were rinsed in 10 mM phosphate-buffered saline (PBS) 3 times for 10 min and then preincubated for 15 min with 1% normal goat or horse serum in PBS containing 0.3% Triton X-100 and 1% bovine serum albumin (PBS-Tx). After rinsing, the sections were treated with anti-TH, anti-GFAP antibody or OX42 for 2 days at 4°C. After rinsing, they were incubated with biotinylated antibody (goat anti-rabbit IgG or horse anti-mouse IgG), at 1:200 dilution in PBS-Tx for 1 h at room temperature. They were rinsed and incubated for 1 h in the avidin–biotin peroxidase complex (Vectastain, ABC kit, Vector Laboratories, Burlingame, CA) at room temperature. After further rinsing, the peroxidase label was revealed by an intensified 3,38-diaminobenzidine (DAB) procedure (1). The sections were treated with 0.1 M phosphate buffer containing 0.05% DAB, 0.025% CoCl2, 0.02% nickel ammonium sulfate (NiSO4 (NH4 ) 2SO4 ), and 0.01% H2O2 for 5–7 min. Lectin histochemistry was carried out at the level of
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the SN. Sections were pretreated for 30 min in PBS-Tx with 0.1 mM MnCl2, CaCl2, and MgCl2 and incubated overnight at 4°C with peroxidase-labeled Griffonia simplicifolia B4 isolectin [GSA I-B4, L-5391; Sigma; a specific marker for microglia (26, 48, 50)], diluted to 20 µg/ml in PBS-Tx containing 0.1 mM MnCl2, CaCl2, and MgCl2. After three washes, lectin binding sites were localized using DAB/H2O2 with Co and Ni intensification. For quantitation, TH-immunopositive neurons with distinct nuclei were counted in six sections through the SN, matched for level as closely as possible from animal to animal. The degree of lesion (% survival) in the substantia nigra pars compacta (SNc) was determined by counting the number of tyrosine hydroxylase immunoreactivity (TH-IR) neurons on the lesioned side (right) relative to the number of TH-IR neurons on the unlesioned side (left). Statistical Analysis Data are presented as means 6 SEM. Significant differences between the group were evaluated using Mann–Whitney U tests. For all statistical tests the significance level was set at P , 0.05. RESULTS
Concentrations of DA, DOPAC, and HVA One and four weeks after post 6-OHDA injection, DA, DOPAC, and HVA in the ST and SN were measured and the ratios of the concentrations on the injection side (right side: R) to that on the control side (left side: L) for each rat were calculated (DA(R/L), DOPAC(R/L), HVA(R/L)). One week postinjection, there was no difference in DA(R/L), DOPAC(R/L), or HVA(R/L) between groups 3 and 4 in the ST or SN although these were significantly lower than those of naive rats in the ST (Table 1). But 4 weeks after injection, striatal DA(R/L) and DOPAC(R/L) in group 4 were both significantly larger than in group 3, which means that the CsA significantly attenuated the toxic effects of 6-OHDA on DA and DOPAC (Tables 1 and 2). There was no significant difference in striatal DA(R/L), DOPAC(R/L), or HVA(R/L) between 1 and 4 weeks after injection in group 3. Nigral DA(R/L) in group 3 was significantly lower than that in group 1 but there was no significant difference in DA(R/L) between groups 1 and 4 four weeks postinjection, indicating that the CsA also had some effect on nigral DA depletion (Table 3). Histological Changes The TH immunocytochemical staining of nigral neurons was not affected at 3 days and 1 week after injection in any group. TH-IR of the nigral cell bodies and dendrites in the SNc and of the dendrites in the
TABLE 1 The Effects of Intrastriatal 6-OHDA Injection on the Striatal Concentrations of DA, DOPAC, and HVA Group Normal (naive) 1 week 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA 4 weeks 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA
DA (R/L)
DOPAC (R/L)
HVA (R/L)
0.94 6 0.04
0.98 6 0.06
0.95 6 0.05
0.94 6 0.05 0.97 6 0.04 0.32 6 0.07 b,e 0.35 6 0.07 b,e
1.05 6 0.09 0.87 6 0.04 0.41 6 0.10 b,e 0.46 6 0.08 b,e
1.14 6 0.07 a 1.09 6 0.07 0.44 6 0.09 b,e 0.48 6 0.06 b,e
1.15 6 0.12 1.31 6 0.09 a 0.23 6 0.05 b,f 0.44 6 0.08 b,c,f
0.90 6 0.06 0.96 6 0.11 0.26 6 0.03 b,f 0.53 6 0.05 b,d,f
0.96 6 0.06 1.09 6 0.07 0.37 6 0.07 b,f 0.64 6 0.12 b
Note. Data are expressed as means (6SEM) ratio in injected side (right, R) to that in the contralateral side (left, L). The numbers of rats used/group is six at either 1 or 4 weeks postinjection. a P , 0.05, b P , 0.01 vs normal group; c P , 0.05, d P , 0.01 vs 6-OHDA 1 V (4 weeks) group; e P , 0.01 vs SA 1 V (1 week) group; f P , 0.01 vs SA 1 V (4 weeks) group.
substantia nigra pars reticulata (SNr) ipsilateral to the injection was lower than on the contralateral side 4 weeks after injection in group 3. Prior work has shown that, even if crossing nigrostriatal projections really exist, they are very sparse compared with ipsilateral projections (20), so that we could use the contralateral side as control. The count of TH-positive neurons in the SNc on the injected side was 63.5 6 2.4% of that on the contralateral side in group 3 and 86.9% 6 1.8% in group 4 (P 5 0.021) (Table 4). The loss of TH-IR was therefore significantly less in group 4, indicating that the CsA TABLE 2 The Effects of CsA on the Striatal DA Concentration after Intrastriatal 6-OHDA Injection Group Normal (naive) 1 week 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA 4 weeks 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA
Ipsilateral side
Contralateral side
6.64 6 0.12
7.10 6 0.22
6.87 6 0.50 6.05 6 0.42 1.95 6 0.45 b,e 2.36 6 0.43 b,e
7.33 6 0.49 6.27 6 0.35 6.39 6 0.34 7.05 6 0.44
7.96 6 0.48 8.03 6 0.31 a 1.36 6 0.32 b,f 2.88 6 0.49 b,c,f
7.04 6 0.38 6.27 6 0.47 6.30 6 0.48 6.83 6 0.72
Note. Data are expressed as means 6 SEM (ng/mg wet weight). The numbers of rats used/group is six at either 1 or 4 weeks postinjection. There was no significant difference in striatal DA concentration on contralateral side in each group. a P , 0.05, b P , 0.01 vs normal group; c P , 0.05, d P , 0.01 vs 6-OHDA 1 V (4 weeks) group; e P , 0.01 vs SA 1 V (1 week) group; f P , 0.01 vs SA 1 V (4 weeks) group.
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TABLE 3 The Effects of Intrastriatal 6-OHDA Injection on the Nigral Concentrations of DA and DOPAC
GFAP-positive astrocytes in any group at any time point studied (Fig. 4).
Group
DA (R/L)
DOPAC (R/L)
DISCUSSION
Normal (naive) 1 week 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA 4 weeks 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA
1.02 6 0.14
0.97 6 0.08
0.91 6 0.04 0.9 6 0.19 0.79 6 0.08 0.75 6 0.1
0.8 6 0.08 0.82 6 0.13 0.76 6 0.07 0.86 6 0.11
1.04 6 0.1 0.96 6 0.13 0.56 6 0.1 a 0.79 6 0.06
0.97 6 0.14 0.75 6 0.09 0.87 6 0.10 1.19 6 0.29
We have evaluated neurochemically and neuropathologically whether or not repeated high-dose CsA (20 mg/kg) treatment can protect the nigrostriatal dopaminergic system against neurodegeneration in 6-OHDA lesioned rats. Intrastriatal injection of 6-OHDA can cause a slowly progressive degeneration of the dopaminergic neurons (4, 5, 9, 11, 20, 21). High-dose CsA daily for 7 days significantly attenuated the decrease on the lesioned side in striatal DA(R/L), striatal DOPAC(R/L), nigral DA(R/L), and the number of TH-immunoreactive neurons in the SNc at 4 weeks after 6-OHDA lesions. This suggests that CsA treatment during the initial stages of neuronal damage either prevented degeneration or accelerated regeneration of the nigrostriatal DA neurons. There are some contradictory reports about the neurochemical changes in the ST of intrastriatal 6-OHDA lesioned rats. Altar et al. showed that the amounts of striatal [ 3H]mazindol binding and the levels of DA and its metabolites partially recover between 1 and 4 weeks after striatal injection of 6-OHDA (25 µg/1.5 µl in 0.1% SA) (5). Venero et al., however, found no recovery in the levels of striatal DA and its metabolites between 2 days and 4 weeks after striatal injection of 6-OHDA (25 µg/2.0 µl in 0.1% SA) (52). We found no spontaneous recovery of nigrostriatal amine content in this model. There was no significant difference in striatal DA(R/L), DOPAC(R/L), or HVA(R/L) between 1 and 4 weeks after injection in the untreated group injected with 6-OHDA (group 3). Although nigral DA(R/L) in this group was not significantly different from that in controls at 1 week after injection, it was significantly decreased at 4 weeks, indicating that progressive degeneration was occurring in this model. In our previous study, DA in the ST was depleted by 60% 7 days after cerebroventricular injection of 6-OHDA in mice, and repeated high-dose CsA (20 mg/kg) treatment significantly protected against this depletion (27). CsA also protected against striatal DA(R/L) depletion in the slowly progressive rat model of PD used here. In the SN, changes in TH-IR can be detected from 2 weeks after the injection (20) in accordance with our preliminary study (data not shown). TH-IR is a good and sufficient marker for evaluating the depletion of tissue DA levels in the nigrostriatal system (20). In this study, morphometric analysis quantitatively revealed the neuronal damage. At 4 weeks post 6-OHDA injection, the number of neurons was decreased by an average of 36% in group 3 and by 13% in group 4. Thus, CsA treatment for 7 days significantly attenuated the decrease in TH-IR in the SN at 4 weeks postinjection. These data
Note. Data are expressed as means (6SEM) ratio in injected side (right, R) to that in the contralateral side (left, L). HVA levels were undetectable in the substantia nigra. The numbers of rats used/group is six at either 1 or 4 weeks postinjection. a P , 0.05 vs SA 1 V (4 weeks) group.
attenuated damage to the dopaminergic neurons (Fig. 1). There was no decrease in the number of THimmunoreactive neurons in groups 1 and 2, indicating that neither SA nor CsA by itself had any effect on the number of TH-immunoreactive neurons in the SN. Microglia were stained with OX42 and GSA-IB4. OX42 is combined with CR3 antigens and GSA I-B4 determines a-D -galactose. We used both OX42 immunohistochemistry and another method, lectin histochemistry, as markers for microglia. All such staining was intense on Day 3, suggesting activation of the microglia. It was somewhat decreased but still elevated on Day 28 in the SN ipsilateral to the injection side compared to the staining in naive rats in group 3. The staining in group 1 (SA-injected controls) was as intense on Days 3 and 7 as that in group 3 (6-OHDA injected animals) but, in group 1, this staining had decreased to control levels by 28 days postinjection. CsA treatment did not affect this staining at any time point in either the SA or the 6-OHDA injection groups (Figs. 2 and 3). There was no significant change in TABLE 4 The Effects of CsA on the Number of Nigral THImmunoreactive Neurons 4 Weeks after Intrastriatal 6-OHDA Injection Group
Ipsilateral side
Contralateral side
6-OHDA 1 V 6-OHDA 1 CsA
245 6 22 334 6 43 a
390 6 31 385 6 44
Note. Data are expressed as means 6 SEM. The numbers of rats used/group is four. a P , 0.05 vs 6-OHDA 1 V group.
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FIG. 1. TH-IR in the SN 4 weeks (A–D) after 6-OHDA injections into the ST. (A,C) Contralateral to the injection side; (B,D) ipsilateral side to the injection. (A,B) Group 3 (6-OHDA 1 V); (C,D) group 4 (6-OHDA 1 CsA). The decrease in TH-IR ipsilateral to the injection side is less in group 4 (D) than in group 3 (B). A–D, original magnification, 325.
again indicate that there is progressive degeneration in this model, as in PD, and that CsA treatment significantly attenuated this degeneration. One might not expect CsA to cross the blood–brain barrier, but, following brain lesions, the barrier is impaired and CsA may cross (27, 32, 53). In contrast to the present data, CsA was found to enhance the toxicity of MPTP toward dopaminergic neurons in mice (19). And chronic administration of CsA to patients produces various central nervous system (CNS) side effects (8). These findings are in accord with the suggestion that CsA admininstered peripherally can affect the CNS. The intrastriatal injection model is advantagous for studies of the microglial and astrocytic response because there is no mechanical damage to the SN. In this study, a strong microglial reaction was observed in the SN after injection of either SA or 6-OHDA, although the 6-OHDA injection induced a more lasting reaction than did the SA. This reaction occurred during the initial neuronal degeneration stage but declined after about 4 weeks, even though neuronal degeneration was still progressing in the 6-OHDA injection group (20, 37). No
significant change in GFAP staining was observed in the SN. It might be expected that CsA protects dopaminergic neurons by suppressing microglial cytotoxicity which operates via mechanisms such as the production of reactive nitrogen intermediates, proteinases, cytokines and reactive oxygen intermediates (6). In this study, however, microglial activation in the SN was not affected by the CsA treatment, suggesting that the effects of CsA on the damaged DA system depend, to a large extent, on a direct action on the neurons, and not on the glial response. There are a number of reports on the neuroprotective effects of various substances on damaged dopamine neurons in vivo. These substances include: [1] neurotrophic factors such as BDNF (4), glial cell-lined derived neurotrophic factor (GDNF) (10, 51), fibroblast growth factor (FGF) (12, 33), and epidermal growth factor (EGF) (17); [2] monosialoganglioside GM1 and semi-synthetic sphingolipid (18, 38–43); [3] tocopherol (11); [4] the monoamine oxidase-B-inhibitor, deprenyl (15, 16, 49); [5] the iron chelator, desferrioxamine (7); and [6] the 1-amino-adamantanes, amantadine and
FIG. 2. Microglial reaction (OX42 staining) in the SN in naive rat or ipsilateral to the injection side following intrastriatal 6-OHDA or SA injections. Naive (A,J), 3 days (B,C,K,L) after SA injections and 3 days (D,E,M,N), 1 week (F,G), and 4 weeks (H,I) after 6-OHDA injections. (C,E,G,I,L,N) Sections from CsA-treated rats. At 3 days, the processes of microglia increased in number and were thick and poorly ramified compared to those in naive animals. CsA had no effect on OX42 staining at any time period studied. (J–N) Higher magnifications of A–E, respectively. A–I, original magnification, 325; J–N, original magnification, 350.
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FIG. 3. Microglial reaction (lectin labeling) in the SN in naive rat or ipsilateral to the injection side following intrastriatal 6-OHDA or SA injections. Naive (A,J) 3 days (B,C,K,L) after SA injections and 3 days (D,E,M,N), 1 week (F,G), and 4 weeks (H,I) after 6-OHDA injections. (C,E,G,I,L,N) Sections from CsA-treated rats. At 3 days, the processes of microglia increased in number and were thick and poorly ramified compared to those in naive animals. CsA had no effect on lectin staining at any time period studied. (J–N) Higher magnification of A–E, respectively. A–I, original magnification, 325; J–N, original magnification, 350.
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FIG. 4. GFAP immunohistochemistry in the SN in naive rat or ipsilateral to the injection side following intrastriatal 6-OHDA injections. Naive (A), 3 days (B,D), and 4 weeks (C,E) after 6-OHDA injections. (D and E) Sections from CsA-treated animals. No significant change compared with the naive rat was seen in any section. A–E, original magnification, 325.
memantine (23). The present results, however, provide the first evidence that CsA has a neuroprotective effect on dopaminergic neurons in rats following intrastriatal 6-OHDA injections. CsA binds specifically to cyclophilin and this complex inhibits the activity of calcineurin, the calcium-dependent, calmodulin-stimulated phosphoprotein phosphatase (14, 22). Since calcineurin has been reported to dephosphorylate DARPP-32 (a dopamine-regulated neuronal phosphoprotein) and to be richly distributed in the caudate putamen, SN, and striatonigral pathway, calcineurin may be important to the function of dopaminergic neurons (14, 47, 54, 55). Although further study is needed to clarify why CsA has a neuroprotective effect on dopaminergic neurons, it is possible that CsA may protect striatal DA and nigral TH-IR via inhibition of calcineurin activity. In conclusion, CsA may protect against dopaminergic neuronal damage in rats with intrastriatal 6-OHDA
injections, and this result adds further impetus to exploring the therapeutic potential of CsA in PD. ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture; and by Grants-in-Aid for the Research Committee on CNS degenerative diseases and Research Projects on Aging and Health from the Japanese Ministry of Health and Welfare. We thank Prof. Edith G. McGeer, Kinsmen Laboratory of Neurological Research, University of British Columbia, Canada, for criticism of the manuscript.
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EN976575
Cyclosporin A Attenuates the Decrease in Tyrosine Hydroxylase Immunoreactivity in Nigrostriatal Dopaminergic Neurons and in Striatal Dopamine Content in Rats with Intrastriatal Injection of 6-Hydroxydopamine Kouji Matsuura,*,† Hirofumi Makino,† and Norio Ogawa* *Department of Neuroscience, Institute of Molecular and Cellular Medicine and †Third Department of Internal Medicine, Okayama University Medical School, 2-5-1 Shikatacho, Okayama 700, Japan
To explore new therapeutic strategies for Parkinson’s disease, we studied the possible protective effect of an immunosuppressant, cyclosporin A (CsA), treatment on changes in dopaminergic function in rats with intrastriatal injections of 6-hydroxydopamine (6OHDA). Four weeks after injection of 6-OHDA, dopamine (DA) and dihydroxyphenylacetic acid in the striatum were depleted by 70–80%, and repeated high-dose CsA (20 mg/kg) treatment for 1 week significantly protected against these depletions. Tyrosine hydroxylase immunoreactivity (TH-IR) of the cell bodies in the substantia nigra pars compacta (SNc) ipsilateral to the injection were lower than on the contralateral side at 4 weeks but not at 1 week after 6-OHDA injection. The number of TH-positive cell bodies in the SNc decreased to 64% but CsA treatment increased this to 87%. The staining of microglia in the SN with OX42 and Griffonia simplicifolia B4 isolectin was intense at 3 days and gradually decreased by 28 days after injection. At 3 and 7 days after injection, the microglial staining in the SN was prominent and equal both in the 6-OHDA group and in ascorbic acid (SA)-injected controls. By 28 days postinjection, the staining had decreased to control levels in the SA group but was still above the control in the 6-OHDA group. CsA treatment did not affect this staining in either group. These results suggest that CsA protects against 6-OHDA-induced injury of nigrostriatal DA neurons by a mechanism not involving microglia. r 1997 Academic Press
INTRODUCTION
Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder characterized by resting tremor, rigidity, bradykinesia/akinesia, and postural disturbance resulting from the idiopathic loss of dopaminergic neurons in the substantia nigra (SN). The observation of a decrease in striatal dopamine (DA) levels associated with the loss of dopaminergic neurons led to the introduction of levodopa treatment. 0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Levodopa is a highly effective treatment and is widely used, but chronic levodopa treatment causes severe clinical complications, including dyskinesia, wearing-off, and on–off phenomenon (29, 34). Moreover, the degeneration of dopaminergic neurons continues. Recently, several approaches have been proposed to slow, halt, or reverse the underlying progression of dopaminergic neuronal degeneration. These include the use of neurotrophic factors such as brain-derived neurotrophic factor (BDNF), GM1 ganglioside, free radical scavengers or the iron-chelator desferrioxamine (2–4, 7, 11, 18, 24, 25, 30, 38, 41–43, 45, 46). Several animal models of PD have been established which employ intranigral injections of 6-hydroxydopamine (6-OHDA) or systemic injection of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) to produce a rapid and selective lesion of nigrostriatal dopaminergic neurons. These differ in time course from PD where the slow progressive nature is due to a protracted, rather than acute, loss of nigrostriatal dopaminergic neurons. MPTP-treated mice have been used to evaluate the trophic effects of neuroactive substances on the nigrostriatal DA system because such mice show a moderate DA depletion in the striatum (ST) and loss of dopaminergic neurons in the SN (12, 17, 18, 33, 38–41). However, this model can show spontaneous improvement in indices of DA function (13, 35, 38). Intrastriatal injection of 6-OHDA provides a nerve terminal pruning and slowly progressive neurodegeneration model in which neurotrophins and other factors can be tested for their effects on residual DA neurons (3, 11, 37). It has been suggested that immunological reactions may play an important role in progressive neurodegenerative diseases such as PD, Alzheimer’s disease, and cerebrovascular disease (28, 32, 44, 53). Cyclosporin A (CsA) is an immunosuppressive drug widely used in organ transplantation and to treat autoimmune diseases. In previous studies (28) we have shown that repeated high-dose CsA (20 mg/kg) treatment significantly attenuated 6-OHDA-induced striatal dopamine depletion in mice,
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but the effect of CsA on the glial response was not studied. In the present work, we investigated the effects of CsA on the degeneration of dopaminergic neurons and on the response of perineuronal glial cells in rats after intrastriatal infusions of 6-OHDA. MATERIALS AND METHODS
Animal Experiments Adult male Sprague–Dawley rats weighing 280–320 g (Charles River Japan Inc., Kanagawa, Japan) at the beginning of the experiments were used (n 5 106). The animals were housed at constant room temperature (24 6 1°C) and humidity (55%) with a 12-h light–dark cycle (lights on 07:00–19:00) and were given free access to food and water. Intrastriatal injections of 10 µl of 6-OHDA solution [Sigma Chemical Co., St. Louis, MO; 2.0 mg/ml, in physiological saline containing 0.01% ascorbic acid (SA)] or SA as a control were performed under sodium pentobarbital anesthesia (35 mg/kg, ip). CsA (Sandoz, Basel, Switzerland), or the same volume of vehicle (V; 0.2% caster oil), was injected subcutaneously 24 h and 30 min before, 5 h after, and once daily on Days 1 to 6 after 6-OHDA (or SA) injection. Thus, experimental animals were divided into four groups: (1) SA 1 V, (2) SA 1 CsA (20 mg/kg), (3) 6-OHDA 1 V, and (4) 6-OHDA 1 CsA (20 mg/kg) (n 5 24 for each group). Animals were sacrificed 1 and 4 weeks postinjection and were divided into two groups. One group was used for histological analysis and another group was used for neurochemical analysis. Some animals were sacrificed 3 days after injection and were used for histological analysis. Surgical Protocol Following anesthesia, animals were placed in a stereotaxic frame with the tooth bar 5 mm above horizontal, and 6-OHDA (20 µg/10 µl) or vehicle was delivered from a microsyringe at a rate of 1 µl/min. Stereotaxic coordinates were 2.4 mm anterior to the bregma (AP), 2.5 and 3.5 mm lateral from the midline (ML), and 5.5 mm below the dura (DV), as well as AP 2.8 mm, ML 2.0 and 3.0 mm, DV 4.5 mm (four injections per rat; 2.5 µl at each point). The sites of insertion of the microsyringe were determined from the stereotaxic atlas of Pellegrino and Cushman (36). After injection, the microsyringe was left in place for 3 min and then withdrawn at a rate of 1 mm/min. Animals in the control group received 10 µl of SA in the manner described above. Neurochemical Analysis For biochemical studies, experimental rats (total n 5 48; n 5 6 for each group at 1 and 4 weeks postinjection) and 6 naive rats were sacrificed by microwave irradiation (5 kW, 1.2 s), the brain was removed, and
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the ST and SN were dissected. The concentrations of DA, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined by high-performance liquid chromatography (HPLC) with electrochemical detection according to published techniques (31). Briefly, tissues were homogenized in 0.2 M ice-cold perchloric acid. After centrifugation (3000g for 10 min at 4°C), supernatants were filtered (Millipore membrane type HC, 0.45-µm pores) and then injected directly into the HPLC system. Standard was measured often (about every 10 determinations) to allow correction for any loss or variation in the HPLC procedure. Histological Examination For morphological studies, experimental rats (total n 5 48; n 5 4 for each group at every time point) and 4 naive rats were anesthetized with an overdose of sodium pentobarbital and perfused through the left ventricle with saline followed by 300 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The brain was removed and immersed in the same fixative for 24 h at 4°C, after which the tissue was rinsed with 30% sucrose in 0.1 M phosphate buffer (pH 7.2) for 48 h and frozen on solid CO2. Frozen 20-µm coronal sections were cut on a cryostat and mounted one glass slides. The antibody against tyrosine hydroxylase (TH) (Eugene-Tech, Allendale, NJ) was used at a dilution of 1:1000. Antiglial fibrillary acidic protein (GFAP) (Chemicon, Temecula, CA) was used as a marker of astrocytes at a dilution of 1:1000. OX42 against a monomorphic determinant of CR3 antigens (Sera-lab, Sussex, UK) was used at a dilution of 1:500 as a marker of microglia. Immunocytochemistry was carried out at the level of the SN in order to evaluate the status of nigral neurons and perineuronal glial cells. Sections were immersed in methanol containing 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. The sections were rinsed in 10 mM phosphate-buffered saline (PBS) 3 times for 10 min and then preincubated for 15 min with 1% normal goat or horse serum in PBS containing 0.3% Triton X-100 and 1% bovine serum albumin (PBS-Tx). After rinsing, the sections were treated with anti-TH, anti-GFAP antibody or OX42 for 2 days at 4°C. After rinsing, they were incubated with biotinylated antibody (goat anti-rabbit IgG or horse anti-mouse IgG), at 1:200 dilution in PBS-Tx for 1 h at room temperature. They were rinsed and incubated for 1 h in the avidin–biotin peroxidase complex (Vectastain, ABC kit, Vector Laboratories, Burlingame, CA) at room temperature. After further rinsing, the peroxidase label was revealed by an intensified 3,38-diaminobenzidine (DAB) procedure (1). The sections were treated with 0.1 M phosphate buffer containing 0.05% DAB, 0.025% CoCl2, 0.02% nickel ammonium sulfate (NiSO4 (NH4 ) 2SO4 ), and 0.01% H2O2 for 5–7 min. Lectin histochemistry was carried out at the level of
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the SN. Sections were pretreated for 30 min in PBS-Tx with 0.1 mM MnCl2, CaCl2, and MgCl2 and incubated overnight at 4°C with peroxidase-labeled Griffonia simplicifolia B4 isolectin [GSA I-B4, L-5391; Sigma; a specific marker for microglia (26, 48, 50)], diluted to 20 µg/ml in PBS-Tx containing 0.1 mM MnCl2, CaCl2, and MgCl2. After three washes, lectin binding sites were localized using DAB/H2O2 with Co and Ni intensification. For quantitation, TH-immunopositive neurons with distinct nuclei were counted in six sections through the SN, matched for level as closely as possible from animal to animal. The degree of lesion (% survival) in the substantia nigra pars compacta (SNc) was determined by counting the number of tyrosine hydroxylase immunoreactivity (TH-IR) neurons on the lesioned side (right) relative to the number of TH-IR neurons on the unlesioned side (left). Statistical Analysis Data are presented as means 6 SEM. Significant differences between the group were evaluated using Mann–Whitney U tests. For all statistical tests the significance level was set at P , 0.05. RESULTS
Concentrations of DA, DOPAC, and HVA One and four weeks after post 6-OHDA injection, DA, DOPAC, and HVA in the ST and SN were measured and the ratios of the concentrations on the injection side (right side: R) to that on the control side (left side: L) for each rat were calculated (DA(R/L), DOPAC(R/L), HVA(R/L)). One week postinjection, there was no difference in DA(R/L), DOPAC(R/L), or HVA(R/L) between groups 3 and 4 in the ST or SN although these were significantly lower than those of naive rats in the ST (Table 1). But 4 weeks after injection, striatal DA(R/L) and DOPAC(R/L) in group 4 were both significantly larger than in group 3, which means that the CsA significantly attenuated the toxic effects of 6-OHDA on DA and DOPAC (Tables 1 and 2). There was no significant difference in striatal DA(R/L), DOPAC(R/L), or HVA(R/L) between 1 and 4 weeks after injection in group 3. Nigral DA(R/L) in group 3 was significantly lower than that in group 1 but there was no significant difference in DA(R/L) between groups 1 and 4 four weeks postinjection, indicating that the CsA also had some effect on nigral DA depletion (Table 3). Histological Changes The TH immunocytochemical staining of nigral neurons was not affected at 3 days and 1 week after injection in any group. TH-IR of the nigral cell bodies and dendrites in the SNc and of the dendrites in the
TABLE 1 The Effects of Intrastriatal 6-OHDA Injection on the Striatal Concentrations of DA, DOPAC, and HVA Group Normal (naive) 1 week 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA 4 weeks 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA
DA (R/L)
DOPAC (R/L)
HVA (R/L)
0.94 6 0.04
0.98 6 0.06
0.95 6 0.05
0.94 6 0.05 0.97 6 0.04 0.32 6 0.07 b,e 0.35 6 0.07 b,e
1.05 6 0.09 0.87 6 0.04 0.41 6 0.10 b,e 0.46 6 0.08 b,e
1.14 6 0.07 a 1.09 6 0.07 0.44 6 0.09 b,e 0.48 6 0.06 b,e
1.15 6 0.12 1.31 6 0.09 a 0.23 6 0.05 b,f 0.44 6 0.08 b,c,f
0.90 6 0.06 0.96 6 0.11 0.26 6 0.03 b,f 0.53 6 0.05 b,d,f
0.96 6 0.06 1.09 6 0.07 0.37 6 0.07 b,f 0.64 6 0.12 b
Note. Data are expressed as means (6SEM) ratio in injected side (right, R) to that in the contralateral side (left, L). The numbers of rats used/group is six at either 1 or 4 weeks postinjection. a P , 0.05, b P , 0.01 vs normal group; c P , 0.05, d P , 0.01 vs 6-OHDA 1 V (4 weeks) group; e P , 0.01 vs SA 1 V (1 week) group; f P , 0.01 vs SA 1 V (4 weeks) group.
substantia nigra pars reticulata (SNr) ipsilateral to the injection was lower than on the contralateral side 4 weeks after injection in group 3. Prior work has shown that, even if crossing nigrostriatal projections really exist, they are very sparse compared with ipsilateral projections (20), so that we could use the contralateral side as control. The count of TH-positive neurons in the SNc on the injected side was 63.5 6 2.4% of that on the contralateral side in group 3 and 86.9% 6 1.8% in group 4 (P 5 0.021) (Table 4). The loss of TH-IR was therefore significantly less in group 4, indicating that the CsA TABLE 2 The Effects of CsA on the Striatal DA Concentration after Intrastriatal 6-OHDA Injection Group Normal (naive) 1 week 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA 4 weeks 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA
Ipsilateral side
Contralateral side
6.64 6 0.12
7.10 6 0.22
6.87 6 0.50 6.05 6 0.42 1.95 6 0.45 b,e 2.36 6 0.43 b,e
7.33 6 0.49 6.27 6 0.35 6.39 6 0.34 7.05 6 0.44
7.96 6 0.48 8.03 6 0.31 a 1.36 6 0.32 b,f 2.88 6 0.49 b,c,f
7.04 6 0.38 6.27 6 0.47 6.30 6 0.48 6.83 6 0.72
Note. Data are expressed as means 6 SEM (ng/mg wet weight). The numbers of rats used/group is six at either 1 or 4 weeks postinjection. There was no significant difference in striatal DA concentration on contralateral side in each group. a P , 0.05, b P , 0.01 vs normal group; c P , 0.05, d P , 0.01 vs 6-OHDA 1 V (4 weeks) group; e P , 0.01 vs SA 1 V (1 week) group; f P , 0.01 vs SA 1 V (4 weeks) group.
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TABLE 3 The Effects of Intrastriatal 6-OHDA Injection on the Nigral Concentrations of DA and DOPAC
GFAP-positive astrocytes in any group at any time point studied (Fig. 4).
Group
DA (R/L)
DOPAC (R/L)
DISCUSSION
Normal (naive) 1 week 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA 4 weeks 1. SA 1 V 2. SA 1 CsA 3. 6-OHDA 1 V 4. 6-OHDA 1 CsA
1.02 6 0.14
0.97 6 0.08
0.91 6 0.04 0.9 6 0.19 0.79 6 0.08 0.75 6 0.1
0.8 6 0.08 0.82 6 0.13 0.76 6 0.07 0.86 6 0.11
1.04 6 0.1 0.96 6 0.13 0.56 6 0.1 a 0.79 6 0.06
0.97 6 0.14 0.75 6 0.09 0.87 6 0.10 1.19 6 0.29
We have evaluated neurochemically and neuropathologically whether or not repeated high-dose CsA (20 mg/kg) treatment can protect the nigrostriatal dopaminergic system against neurodegeneration in 6-OHDA lesioned rats. Intrastriatal injection of 6-OHDA can cause a slowly progressive degeneration of the dopaminergic neurons (4, 5, 9, 11, 20, 21). High-dose CsA daily for 7 days significantly attenuated the decrease on the lesioned side in striatal DA(R/L), striatal DOPAC(R/L), nigral DA(R/L), and the number of TH-immunoreactive neurons in the SNc at 4 weeks after 6-OHDA lesions. This suggests that CsA treatment during the initial stages of neuronal damage either prevented degeneration or accelerated regeneration of the nigrostriatal DA neurons. There are some contradictory reports about the neurochemical changes in the ST of intrastriatal 6-OHDA lesioned rats. Altar et al. showed that the amounts of striatal [ 3H]mazindol binding and the levels of DA and its metabolites partially recover between 1 and 4 weeks after striatal injection of 6-OHDA (25 µg/1.5 µl in 0.1% SA) (5). Venero et al., however, found no recovery in the levels of striatal DA and its metabolites between 2 days and 4 weeks after striatal injection of 6-OHDA (25 µg/2.0 µl in 0.1% SA) (52). We found no spontaneous recovery of nigrostriatal amine content in this model. There was no significant difference in striatal DA(R/L), DOPAC(R/L), or HVA(R/L) between 1 and 4 weeks after injection in the untreated group injected with 6-OHDA (group 3). Although nigral DA(R/L) in this group was not significantly different from that in controls at 1 week after injection, it was significantly decreased at 4 weeks, indicating that progressive degeneration was occurring in this model. In our previous study, DA in the ST was depleted by 60% 7 days after cerebroventricular injection of 6-OHDA in mice, and repeated high-dose CsA (20 mg/kg) treatment significantly protected against this depletion (27). CsA also protected against striatal DA(R/L) depletion in the slowly progressive rat model of PD used here. In the SN, changes in TH-IR can be detected from 2 weeks after the injection (20) in accordance with our preliminary study (data not shown). TH-IR is a good and sufficient marker for evaluating the depletion of tissue DA levels in the nigrostriatal system (20). In this study, morphometric analysis quantitatively revealed the neuronal damage. At 4 weeks post 6-OHDA injection, the number of neurons was decreased by an average of 36% in group 3 and by 13% in group 4. Thus, CsA treatment for 7 days significantly attenuated the decrease in TH-IR in the SN at 4 weeks postinjection. These data
Note. Data are expressed as means (6SEM) ratio in injected side (right, R) to that in the contralateral side (left, L). HVA levels were undetectable in the substantia nigra. The numbers of rats used/group is six at either 1 or 4 weeks postinjection. a P , 0.05 vs SA 1 V (4 weeks) group.
attenuated damage to the dopaminergic neurons (Fig. 1). There was no decrease in the number of THimmunoreactive neurons in groups 1 and 2, indicating that neither SA nor CsA by itself had any effect on the number of TH-immunoreactive neurons in the SN. Microglia were stained with OX42 and GSA-IB4. OX42 is combined with CR3 antigens and GSA I-B4 determines a-D -galactose. We used both OX42 immunohistochemistry and another method, lectin histochemistry, as markers for microglia. All such staining was intense on Day 3, suggesting activation of the microglia. It was somewhat decreased but still elevated on Day 28 in the SN ipsilateral to the injection side compared to the staining in naive rats in group 3. The staining in group 1 (SA-injected controls) was as intense on Days 3 and 7 as that in group 3 (6-OHDA injected animals) but, in group 1, this staining had decreased to control levels by 28 days postinjection. CsA treatment did not affect this staining at any time point in either the SA or the 6-OHDA injection groups (Figs. 2 and 3). There was no significant change in TABLE 4 The Effects of CsA on the Number of Nigral THImmunoreactive Neurons 4 Weeks after Intrastriatal 6-OHDA Injection Group
Ipsilateral side
Contralateral side
6-OHDA 1 V 6-OHDA 1 CsA
245 6 22 334 6 43 a
390 6 31 385 6 44
Note. Data are expressed as means 6 SEM. The numbers of rats used/group is four. a P , 0.05 vs 6-OHDA 1 V group.
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MATSUURA, MAKINO, AND OGAWA
FIG. 1. TH-IR in the SN 4 weeks (A–D) after 6-OHDA injections into the ST. (A,C) Contralateral to the injection side; (B,D) ipsilateral side to the injection. (A,B) Group 3 (6-OHDA 1 V); (C,D) group 4 (6-OHDA 1 CsA). The decrease in TH-IR ipsilateral to the injection side is less in group 4 (D) than in group 3 (B). A–D, original magnification, 325.
again indicate that there is progressive degeneration in this model, as in PD, and that CsA treatment significantly attenuated this degeneration. One might not expect CsA to cross the blood–brain barrier, but, following brain lesions, the barrier is impaired and CsA may cross (27, 32, 53). In contrast to the present data, CsA was found to enhance the toxicity of MPTP toward dopaminergic neurons in mice (19). And chronic administration of CsA to patients produces various central nervous system (CNS) side effects (8). These findings are in accord with the suggestion that CsA admininstered peripherally can affect the CNS. The intrastriatal injection model is advantagous for studies of the microglial and astrocytic response because there is no mechanical damage to the SN. In this study, a strong microglial reaction was observed in the SN after injection of either SA or 6-OHDA, although the 6-OHDA injection induced a more lasting reaction than did the SA. This reaction occurred during the initial neuronal degeneration stage but declined after about 4 weeks, even though neuronal degeneration was still progressing in the 6-OHDA injection group (20, 37). No
significant change in GFAP staining was observed in the SN. It might be expected that CsA protects dopaminergic neurons by suppressing microglial cytotoxicity which operates via mechanisms such as the production of reactive nitrogen intermediates, proteinases, cytokines and reactive oxygen intermediates (6). In this study, however, microglial activation in the SN was not affected by the CsA treatment, suggesting that the effects of CsA on the damaged DA system depend, to a large extent, on a direct action on the neurons, and not on the glial response. There are a number of reports on the neuroprotective effects of various substances on damaged dopamine neurons in vivo. These substances include: [1] neurotrophic factors such as BDNF (4), glial cell-lined derived neurotrophic factor (GDNF) (10, 51), fibroblast growth factor (FGF) (12, 33), and epidermal growth factor (EGF) (17); [2] monosialoganglioside GM1 and semi-synthetic sphingolipid (18, 38–43); [3] tocopherol (11); [4] the monoamine oxidase-B-inhibitor, deprenyl (15, 16, 49); [5] the iron chelator, desferrioxamine (7); and [6] the 1-amino-adamantanes, amantadine and
FIG. 2. Microglial reaction (OX42 staining) in the SN in naive rat or ipsilateral to the injection side following intrastriatal 6-OHDA or SA injections. Naive (A,J), 3 days (B,C,K,L) after SA injections and 3 days (D,E,M,N), 1 week (F,G), and 4 weeks (H,I) after 6-OHDA injections. (C,E,G,I,L,N) Sections from CsA-treated rats. At 3 days, the processes of microglia increased in number and were thick and poorly ramified compared to those in naive animals. CsA had no effect on OX42 staining at any time period studied. (J–N) Higher magnifications of A–E, respectively. A–I, original magnification, 325; J–N, original magnification, 350.
531
FIG. 3. Microglial reaction (lectin labeling) in the SN in naive rat or ipsilateral to the injection side following intrastriatal 6-OHDA or SA injections. Naive (A,J) 3 days (B,C,K,L) after SA injections and 3 days (D,E,M,N), 1 week (F,G), and 4 weeks (H,I) after 6-OHDA injections. (C,E,G,I,L,N) Sections from CsA-treated rats. At 3 days, the processes of microglia increased in number and were thick and poorly ramified compared to those in naive animals. CsA had no effect on lectin staining at any time period studied. (J–N) Higher magnification of A–E, respectively. A–I, original magnification, 325; J–N, original magnification, 350.
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EFFECT OF CsA ON DOPAMINERGIC FUNCTION
FIG. 4. GFAP immunohistochemistry in the SN in naive rat or ipsilateral to the injection side following intrastriatal 6-OHDA injections. Naive (A), 3 days (B,D), and 4 weeks (C,E) after 6-OHDA injections. (D and E) Sections from CsA-treated animals. No significant change compared with the naive rat was seen in any section. A–E, original magnification, 325.
memantine (23). The present results, however, provide the first evidence that CsA has a neuroprotective effect on dopaminergic neurons in rats following intrastriatal 6-OHDA injections. CsA binds specifically to cyclophilin and this complex inhibits the activity of calcineurin, the calcium-dependent, calmodulin-stimulated phosphoprotein phosphatase (14, 22). Since calcineurin has been reported to dephosphorylate DARPP-32 (a dopamine-regulated neuronal phosphoprotein) and to be richly distributed in the caudate putamen, SN, and striatonigral pathway, calcineurin may be important to the function of dopaminergic neurons (14, 47, 54, 55). Although further study is needed to clarify why CsA has a neuroprotective effect on dopaminergic neurons, it is possible that CsA may protect striatal DA and nigral TH-IR via inhibition of calcineurin activity. In conclusion, CsA may protect against dopaminergic neuronal damage in rats with intrastriatal 6-OHDA
injections, and this result adds further impetus to exploring the therapeutic potential of CsA in PD. ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture; and by Grants-in-Aid for the Research Committee on CNS degenerative diseases and Research Projects on Aging and Health from the Japanese Ministry of Health and Welfare. We thank Prof. Edith G. McGeer, Kinsmen Laboratory of Neurological Research, University of British Columbia, Canada, for criticism of the manuscript.
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