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Naloxone prevents microglia-induced degeneration of dopaminergic substantia nigra neurons in adult rats
Neuroscience Vol. 97, No. 2, pp. 285–291, 2000 285 Copyright 䉷 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
Naloxone prevents microglia-induced nigral degeneration
Pergamon PII: S0306-4522(00)00033-6 www.elsevier.com/locate/neuroscience
NALOXONE PREVENTS MICROGLIA-INDUCED DEGENERATION OF DOPAMINERGIC SUBSTANTIA NIGRA NEURONS IN ADULT RATS X. LU,* G. BING† and T. HAGG*‡ *Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 †Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, U.S.A.
Abstract—Resident microglia are involved in immune responses of the central nervous system and may contribute to neuronal degeneration and death. Here, we tested in adult rats whether injection of bacterial lipopolysaccharide (which causes inflammation and microglial activation) just above the substantia nigra, results in the death of dopaminergic substantia nigra pars compacta neurons. Two weeks after lipopolysaccharide injection, microglial activation was evident throughout the nigra and the number of retrogradely-labeled substantia nigra neurons was reduced to 66% of normal. This suggests that inflammation and/or microglial activation can lead to neuronal cell death in a well-defined adult animal model. The opioid receptor antagonist naloxone reportedly reduces release of cytotoxic substances from microglia and protects cortical neurons in vitro. Here, a continuous two-week infusion of naloxone at a micromolar concentration close to the substantia nigra, prevented most of the neuronal death caused by lipopolysaccharide, i.e. 85% of the neurons survived. In addition, with systemic (subcutaneous) infusion of 0.1 mg/d naloxone, 94% of the neurons survived. Naloxone infusions did not obviously affect the morphological signs of microglial activation, suggesting that naloxone reduces the release of microglial-derived cytotoxic substances. Alternatively, microglia might not cause the neuronal loss, or naloxone might act by blocking opioid receptors on (dopaminergic or GABAergic) neurons. Thus, local inflammation induces and the opioid antagonist naloxone prevents the death of dopaminergic substantia nigra neurons in adult rats. This may be relevant to the understanding of the pathology and treatment of Parkinson’s disease, where these neurons degenerate. 䉷 2000 IBRO. Published by Elsevier Science Ltd. Key words: cell death, inflammation, lipopolysaccharide, microglial activation, opioid, Parkinson’s disease.
Resident microglia are present throughout the CNS and constitute approximately 10% of the cells. 41 They are related to peripheral monocytes and macrophages and share a number of properties, including the expression of complement 3 receptor (CR3 59,67). Normal microglia in the CNS appear to be involved in immune surveillance or assistance in immune responses and reactions to injury. 23,58,69 After an injury to the CNS, microglia close to neuronal cell bodies proliferate, change morphology and become more like macrophages (“activated” microglia). 69,70 When neurons die, microglia become “reactive” and remove cell debris. 69,70 Reactive microglia may also actively contribute to cell death in the CNS, e.g., treatments that reduce microglial activation result in increased neuronal cell survival in a number of neuronal systems. 51,62,72,79 Injection of bacterial lipopolysaccharide (LPS) and the ensuing inflammation (and possibly the microglial activation) has been shown to induce neuronal cell death in the basal forebrain 8,76 and hippocampal formation 44,77 of adult rats. Microglia may also participate or contribute to degenerative events underlying neurological disorders, 25 including Parkinson’s disease. 46 This disease is characterized by the degeneration of dopaminergic neurons of the substantia nigra that project to the neostriatum and the resulting loss of dopamine, which causes many of the symptoms. 19 Microglial activation by LPS can cause the loss of developing dopaminergic neurons in vitro, 6 but see Ref. 80.
Activated and reactive microglia release cytotoxic substances, such as tumor necrosis factor a (TNFa), nitric oxide (NO) and tissue plasminogen activator, which may contribute to cell death in tissue cultures and in animals. 6,44,48,62,71 The opioid receptor agonist morphine primes and increases LPS-induced release of TNF by microglia in vitro, 11 most likely through mu type receptors. 63 It is thus of interest that the opioid receptor antagonist naloxone reportedly can reduce LPS-induced production of TNFa, NO and interleukin-1ß in vitro and protect cultured cortical neurons. 13,39 Naloxone also reportedly blocks morphineinduced morphological changes such as rounding which are associated with microglial activation. 16 Here, we tested in adult rats whether local injection of LPS could induce microglial activation in the substantia nigra pars compacta and whether this would lead to loss of the dopaminergic nigrostriatal neurons. Moreover, we tested whether local or systemic treatment with naloxone could reduce microglial activation and whether this would affect the neuronal death. EXPERIMENTAL PROCEDURES
Retrograde labeling of nigrostriatal neurons, supranigral lipopolysaccharide injections and naloxone treatments All animal procedures were approved by the Animal Care Committee of Dalhousie University and conformed to the Canadian Council on Animal Care. All efforts were made to minimize animal suffering and to reduce the number of animals used (adult Sprague–Dawley rats; 200 g, Charles River). All surgical procedures were performed under general anesthesia (0.5 ml i.m. injection of a mixture of ketamine at 62.5 mg/kg, xylazine at 3.25 mg/kg and acepromazine at 0.62 mg/kg) and aseptic conditions. The dopaminergic nigrostriatal neurons in the substantia nigra pars compacta were retrogradely labeled by stereotaxic injection of the fluorescent tracer 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindocarbocyanine
‡To whom correspondence should be addressed. Tel.: ⫹ 1-902-494-6622; fax: ⫹ 1-902-494-2670 or 494-1212. E-mail address: [email protected] (T. Hagg). Abbreviations: DiI, 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindocarbocyanine; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; PBS, phosphate-buffered saline; TH, tyrosine hydroxylase; TNFa, tumor necrosis factor a. 285
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Fig. 1. Naloxone prevents lipopolysaccharide-induced death of dopaminergic substantia nigra neurons. Coronal sections through the substantia nigra pars compacta (outlined by arrows) immunostained for the microglia marker complement receptor 3 (left column) reveal that compared to normal controls (A), microglia were activated after injection of lipopolysaccharide (LPS) above the substantia nigra (B) and that infusion of naloxone above the nigra did not obviously affect microglial activation (C). Adjacent sections immunostained for the dopaminergic marker TH (D–F) or viewed under epifluorescence for the retrograde tracer DiI (G–I) revealed that compared to controls (D, G), LPS caused some neuronal loss (E, H) and that naloxone rescued a number of neurons (F, I). Scale bar 200 mm.
(DiI; Molecular Probes, Junction City, OR, U.S.A.) into six sites throughout the striatum. 27 This procedure labels essentially all the nigrostriatal neurons of the pars compacta and all labeled neurons are dopaminergic, while only approximately 10% of the compacta neurons are not dopaminergic. 27 Two weeks later, microglial activation was induced by a unilateral injection of 5 mg of LPS (O111:B4; Sigma Chemical Company, St Louis, MO, U.S.A.) in 2 ml of phosphatebuffered saline (PBS) 0.5 mm above the rostral third of the right substantia nigra pars compacta (1.7 mm lateral, 4.8 mm caudal and 8.0 mm ventral from bregma, tooth bar set at ⫺3.3 mm). Immediately afterwards, the animals were implanted with the tip of a 0.36 mm diameter metal cannula above the substantia nigra (1.7 mm lateral, 4.1 mm caudal and 7 mm ventral from bregma). The cannula was connected to an Alzet 2002 minipump (12 ml per day; Alza, Palo Alto, CA, U.S.A.) filled with PBS (n 5) or PBS containing 4.4 ng/ day (10 ⫺6 M) naloxone (n 6; Sigma) for a 14-day infusion. 42 Other animals received a unilateral injection of LPS above the substantia nigra followed by a 14-day subcutaneous infusion of 0.1 mg/day (2.3 × 10 ⫺2 M) naloxone dissolved in 60% ethanol to enhance solubility (n 6) or as a control 60% ethanol (n 4), using an Alzet minipump (12 ml/day).
The CR3-stained sections were used to establish the extent of microglial activation in the substantia nigra pars compacta. The outlines of the compacta region were determined in the adjacent THstained sections by the distribution of the dopaminergic neurons and well-established landmarks. 27 The compacta region in each section was scored semi-quantitatively blind as to the animal treatment, on a scale of 0–5, with 1 being normal and 5 being most extensive microglial activation. To assess the severity of the local inflammation and the potential impact on the substantia nigra region, the mediolateral diameter of the LPS-induced cavitation around the injection site was measured as well as the distance between the ventral edge of the cavity and the substantia nigra. The TH-stained and DiI-labeled sections were used to determine the number of nigrostriatal neurons in the substantia nigra pars compacta. 27 The number of neurons on the non-injected side was used to calculate the percentage of surviving neurons on the LPSinjected side. Statistical analysis was performed with the Mann–Whitney U-test (ranked, non-parametric) using a probability level of P ⬍ 0.05 for determining statistical significance. RESULTS
Histology and quantification of dopaminergic neuron survival and microglial activation After 14 days, all animals were transcardially perfused with 4% paraformaldehyde, their brains postfixed and cryoprotected, and 30mm-thick coronal sections were cut throughout the nigral complex. 27 Every sixth section through the compacta region was processed for immunocytochemical detection of the monocyte/macrophage/microglial marker complement receptor 3 (OX42 or CD11b/c antibody, Cedar Lane, Hornby, Ontario, Canada) using a sensitive ABC–peroxidase method. 42 Adjacent sections were immunostained for detection of the dopaminergic neuronal marker tyrosine hydroxylase (TH; Mab 318, Chemicon, Temecula, CA, U.S.A. 42). All immunostained sections were mounted on glass slides, dehydrated and coverslipped in Permount. Other adjacent sections were mounted on glass slides and coverslipped in aqueous fluorescent mounting media for detection of DiI-labeled nigral neurons.
Supranigral lipopolysaccharide causes microglial activation and neuronal loss, and naloxone reduces neuronal loss in the substantia nigra: qualitative data After local injection of LPS and PBS infusion, activated microglia were readily identifiable throughout the substantia nigra pars compacta by their thicker processes and more rounded cell body (Fig. 1B). In addition, many ameboid microglia/macrophages could be seen in the region directly around the LPS injection site (not shown). The LPS injection often caused a 0.7–3.8 mm diameter lesion around the injection site but the edge of the lesion was 0.1–0.4 mm away from the substantia nigra pars compacta. In adjacent sections, the substantia nigra appeared to contain fewer TH-positive
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Fig. 2. Lipopolysaccharide causes neuronal death and naloxone rescues neurons throughout the substantia nigra pars compacta. (A) Semi-quantitative microglial activation scores (^S.E.M.) in sections throughout the rostrocaudal extent of the substantia nigra pars compacta immunostained for CR3 are illustrated. Normal is 1 (open circle, broken line) and 5 is most extensive activation. Microglial activation scores were the same in rats injected with lipopolysaccharide (LPS) above the substantia nigra (arrow in B) and infused for 14 days with PBS (open squares; n 5) or with naloxone (solid squares; n 6) above the nigra. (B) The number of retrogradely labeled DiI-positive neurons (as a percentage^S.E.M. of normal open circle, broken line) per individual section throughout the substantia nigra pars compacta (SNC) in the same rats as in (A), illustrate that naloxone rescues neurons throughout the substantia nigra (P ⬍ 0.0005). Microglial activation was also unaffected (C) and neuronal survival increased (D) in animals that had been injected with LPS above the nigra and infused subcutaneously (systemically) with naloxone dissolved in ethanol (solid triangles; n 6; P ⬍ 0.025) compared to ethanol alone (open triangles; n 4).
(Fig. 1E) or DiI-labeled (Fig. 1H) neurons than normal (Fig. 1D and G, respectively). After LPS injection plus infusion of naloxone dorsal to the substantia nigra the extent of microglial activation (Fig. 1C) did not appear different from that seen with LPS injection plus infusion of PBS (Fig. 1B). The extent of LPS-induced cavitation also did not appear different after naloxone treatment (not shown). However, in the presence of naloxone the number of dopaminergic nigrostriatal neurons appeared slightly greater (Fig. 1F, I) than with PBS treatment (Fig. 1E, H). Quantification of microglial activation and neuronal loss To better evaluate the potential differences between PBS and naloxone-treated animals the sections were used to obtain more quantitative measures. Compared to normal animals, microglial appearance of the contralateral substantia nigra of unilateral LPS-injected animals was unaltered. The microglial activation scores in the substantia nigra were greatest in the rostral half of the nigra of LPS-injected rats, i.e. closer to the injection site (Fig. 2A). The number of DiI-positive nigrostriatal neurons in animals injected with LPS and infused for 14 days close to the substantia nigra with PBS had decreased to 66 ^ 3% of that in the non-lesioned sides (P ⬍ 0.0025;
Mann–Whitney U-test). Neuronal cell loss was evident throughout the rostrocaudal extent of the substantia nigra (Fig. 2B). The numbers of neurons counted in TH-immunostained sections (not shown) was the same as the DiI-positive numbers. In LPS-injected animals that were infused with PBS containing 10 ⫺6 M naloxone directly above the substantia nigra for 14 days, microglial activation scores throughout the substantia nigra were not different from LPS-injected rats infused with PBS (Fig. 2A). In these LPS plus naloxonetreated animals, 85 ^ 3% of the DiI-positive neurons had survived (P ⬍ 0.0005 compared to LPS plus PBS treatment). The number of surviving neurons was greater throughout the nigral nucleus (Fig. 2B). When naloxone was administered systemically by a 14 day subcutaneous infusion, the LPSinduced microglial activation scores were not altered (Fig. 2C). In these animals, 94 ^ 7% of the neurons had survived, i.e. many more than the 74 ^ 3% counted after subcutaneous infusion of the 60% ethanol control infusion (P ⬍ 0.025). The rescue effect was detectable throughout the rostrocaudal extent of the nigra (Fig. 2D). The survival level with peripheral control infusions (74 ^ 3%) was not statistically different compared to that seen with PBS infusion above the substantia nigra (66 ^ 3%). The extent of LPS-induced cavitation
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around the injection site (1.7 ^ 0.4 mm in diameter) and the distance of the cavity edge from the nigra (0.18 ^ 0.04 mm) seen after PBS infusion was not statistically different from that seen after naloxone treatment (2.4 ^ 0.4 and 0.16 ^ 0.05, respectively). The diameter of the cavity and distance to the nigra seen after LPS and systemic control infusion (2.8 ^ 0.6 and 0.12 ^ 0.06) were not significantly different from those seen after systemic naloxone infusion (1.4 ^ 0.3 and 0.22 ^ 0.05). As the extent of microglial activation and LPS-induced cavitation showed high levels of variability, we conducted a correlation analysis with the extent of neuronal cell loss in the nigra. Neither the total activation (summed scores of all sections from an animal), nor the individual values of sections, nor the diameter of the cavity, nor the distance of the cavity from the nigra showed a significant correlation with the extent of neuronal cell loss in the substantia nigra pars compacta (not shown).
DISCUSSION
The results of this study provide evidence in adult rats that (i) LPS-induced inflammation and/or microglial activation is associated with the death of dopaminergic nigrostriatal neurons and that (ii) the opioid antagonist naloxone can improve the survival of these injured neurons.
Lipopolysaccharide-activated microglia may cause neuronal death by releasing cytotoxic substances The LPS-induced microglial activation appeared to be greatest in the rostral half of the substantia nigra pars compacta where the loss of dopaminergic neurons was most extensive. Conversely, microglial activation and cell loss were less extensive in the caudal regions, which were more distant from the LPS injection site. This suggests that LPS had diffused through the brain tissue to create a gradient of microglial activation and that the activated microglia around the dopaminergic cell bodies had caused the neuronal death. The death-inducing role of activated microglia has been suggested by findings in other CNS injury models, including the retina and hippocampal formation. 51,62,72,79 LPS injections have also been shown to induce neuronal cell death in the basal forebrain 8,76 and hippocampal formation 44,77 of adult rats. In those hippocampal studies, inducible NO synthase (iNOS) inhibitors reduced the cell loss, suggesting that excessive NO directly or indirectly caused the neuronal loss. In humans, microglia and inflammation may contribute to the neuronal degeneration seen in diseases such as Alzheimer’s and Parkinson’s. 25,34,45–47,57 The mechanisms by which activated microglia cause neuronal cell death in vivo are largely unknown. 69 In tissue cultures, microglia can release cytotoxic substances such as TNFa, NO and tissue plasminogen activator which are toxic to neurons. 2,6,44,48,62,71 In the brains of animals and humans, activated microglia are immunoreactive for TNFa and inducible NO synthase. 7,15,36 LPS can also increase the release of glutamate from cortical brain slices, 75 and from cultured monocytes 37 and microglia. 56 If this occurs in vivo, the higher levels of glutamate could have contribute to the neuronal cell loss in the substantia nigra pars compacta, similar to the cell loss seen with glutamate agonists. 49
Lipopolysaccharide may directly affect neurons or astrocytes in the substantia nigra The lack of a correlation between the microglial activation scores and neuronal death in individual sections through the nigra suggests that the degree of neurotoxicity by microglia may not be related to their morphological appearance. Alternatively, this result raises the possibility that microglial activation was not involved in the LPS-induced neuronal cell death. LPS can cause the disappearance of TH-positive substantia nigra neurons independent of NO, 9 perhaps also suggesting that activated microglia (which produce neurotoxic NO) are not involved. LPS might act directly on the nigral neurons in vivo. LPS plus TNF can induce iNOS and subsequent death of differentiated neuron-like PC12 cells 29 and LPS can induce iNOS in purified cultured cerebellar neurons. 64 LPS binds to CD14 which is expressed by macrophages and activated microglia 3,28,40,73 but not by neurons and other cells in the brain, although they potentially could acquire soluble CD14. 66 Neuroblastoma cells can respond to LPS despite very low or absent levels of CD14, 55 suggesting that other LPS-binding molecules exist. In fact, neurons reportedly have a membrane-associated histone H1-like protein that binds LPS. 4 On the other hand, LPS does not cause the loss of cultured mesencephalic dopaminergic neurons unless glial cells are present, 6 suggesting that LPS acts through activation of cells other than neurons. The responses of cultured astrocytes to LPS have been varied. LPS can cause astroglial dysfunction, 31 induce argininosuccinate synthetase, which is essential for NO synthesis, 65 induce iNOS, 12 and cause NO-dependent neuronal death in astroglial co-cultures. 26,68 However, in vivo, LPS has very little effect on these responses by astrocytes compared to microglia. 30,36 LPS can cause an increase in expression of mRNA of various neurotrophic factors by cultured astrocytes 1,22 and microglia. 50 If LPS induces such factors also in vivo, their beneficial effects on the dopaminergic neurons could mask some of the detrimental effects of LPS. Lastly, it is possible that LPS induces the synthesis and release of cytotoxic substances from yet other cells in the inflamed substantia nigra, including leukocytes other than macrophages. Naloxone may prevent neuronal loss by reducing release of cytotoxins from microglia Treatments with naloxone did not obviously affect the LPSinduced microglial activation but reduced the death of the dopaminergic nigrostriatal neurons. One possible explanation is that naloxone inhibited the release of cytotoxic substances from microglia without obviously altering their morphology. This possibility is consistent with recent findings in mixed glial cell cultures that pharmacological concentrations of naloxone (10 ⫺8 –10 ⫺6 M) reduce the LPS-stimulated release of the pro-inflammatory cytokine interleukin-1ß. 13 Naloxone (10 ⫺10 –10 ⫺6M) can also inhibit the release of TNF-a and the production of NO from activated microglia in vitro. 39 In cocultures of glia and embryonic mouse cortical neurons, naloxone can reduce LPS-induced microglial activation and neuronal death. 38 It is thus conceivable that naloxone also inhibits release of neurotoxic molecules from microglia in vivo, thereby leading to increased survival of the dopaminergic neurons in LPS-treated adult rats. The potential mechanisms could involve the blocking of endogenous opioids that act through mu opioid receptors, as macrophages of
Naloxone prevents microglia-induced nigral degeneration
transgenic mu receptor-deficient mice have a much reduced ability to phagocytose and produce TNF in response to morphine. 63 In vitro, naloxone also prevented the morphological changes of microglia that are associated with their activation by LPS or morphine. 16,38,39 The lack of an effect on microglial morphology here in adult rats might be due to a lower effective concentration of naloxone that was reached in vivo. Naloxone may prevent death of nigrostriatal neurons by blocking opioid receptor binding on dopaminergic and/or GABAergic neurons Alternatively, the protective effects of naloxone may not have been through receptors on microglia but by blocking endogenous opioids that act on neuronal receptors. In the neostriatum, mu, delta and kappa receptor binding sites are abundant. 43 Substantia nigra pars compacta neurons express kappa opioid receptor mRNA but only a few have mu receptors, 24 suggesting that the dopaminergic terminals in the striatum have kappa receptors. Kappa receptor agonists cause a decrease in dopamine release in the striatum 14,52 and, conversely, low doses of naloxone in the range used here can increase striatal dopamine release in adult rodents, as well as potentiate amphetamine- or carbachol-induced dopamine release. 20,21,35,78 Treatment with dopamine increases, and absence of dopamine or its receptors decreases, the synthesis of neurotrophic factors in the mouse striatum 5,53,60 and in cultured astrocytes. 32 Thus, it is possible that naloxone caused an increase in dopamine release and, thereby, an increase in the levels of protective neurotrophic factors. The neostriatum is the major source of GABAergic innervation of the substantia nigra and contains predominantly mu and kappa opioid receptor mRNA. 24 In the substantia nigra, predominantly mu type receptor binding is seen, 43 while few nigral neurons express mu receptor mRNA, suggesting that the binding is on the GABAergic afferents. Mu receptor activation by morphine increases the firing rate of the
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dopaminergic neurons in animals, 74 most likely by decreasing the activity of GABAergic interneurons in the nigra. 33 Naloxone would also act through the GABAergic afferents, which have the naloxone binding sites in the substantia nigra. 54 In apparent contrast, morphine has also been shown to increase GABA release in the substantia nigra of rats, although not in the striatum. 78 Thus, it is unresolved what effect naloxone might have had on GABAergic activity in the current study and how that would have affected survival of the dopaminergic neurons. Potential implications for Parkinson’s disease The present findings that LPS-induced inflammation causes the death of dopaminergic substantia nigra neurons, and that the opioid receptor antagonist naloxone can reduce this neuronal loss might be relevant to Parkinson’s disease where these neurons degenerate. The possible beneficial effects of anti-inflammatory treatments for degenerative neurological disorders are suggested by the finding that antiinflammatory drugs reduce the risk of developing Alzheimer’s disease. 45,47,61 Naloxone also might have function-improving effects since at low doses it can increase striatal dopamine in adult rodents. 20,21,35,78 This may seem paradoxical, as acute treatments with the opioid receptor agonist morphine increase the firing rate of dopaminergic neurons 74 and cause increases in striatal dopamine release. 14,17,18 However, chronic morphine treatments are not effective, as animals develop tolerance. 17 With these unresolved issues about dopamine levels in mind, the efficacy of naloxone for Parkinson’s disease could be considered, since it crosses the blood–brain barrier, has been approved, is currently used for a variety of other disorders, and has relatively few and mild side-effects. 10 Acknowledgements—We greatly appreciate the excellent technical support from Julie Bunker. This research was supported by the Parkinson Foundation of Canada and the Medical Research Council of Canada.
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Naloxone prevents microglia-induced nigral degeneration
Pergamon PII: S0306-4522(00)00033-6 www.elsevier.com/locate/neuroscience
NALOXONE PREVENTS MICROGLIA-INDUCED DEGENERATION OF DOPAMINERGIC SUBSTANTIA NIGRA NEURONS IN ADULT RATS X. LU,* G. BING† and T. HAGG*‡ *Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 †Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, U.S.A.
Abstract—Resident microglia are involved in immune responses of the central nervous system and may contribute to neuronal degeneration and death. Here, we tested in adult rats whether injection of bacterial lipopolysaccharide (which causes inflammation and microglial activation) just above the substantia nigra, results in the death of dopaminergic substantia nigra pars compacta neurons. Two weeks after lipopolysaccharide injection, microglial activation was evident throughout the nigra and the number of retrogradely-labeled substantia nigra neurons was reduced to 66% of normal. This suggests that inflammation and/or microglial activation can lead to neuronal cell death in a well-defined adult animal model. The opioid receptor antagonist naloxone reportedly reduces release of cytotoxic substances from microglia and protects cortical neurons in vitro. Here, a continuous two-week infusion of naloxone at a micromolar concentration close to the substantia nigra, prevented most of the neuronal death caused by lipopolysaccharide, i.e. 85% of the neurons survived. In addition, with systemic (subcutaneous) infusion of 0.1 mg/d naloxone, 94% of the neurons survived. Naloxone infusions did not obviously affect the morphological signs of microglial activation, suggesting that naloxone reduces the release of microglial-derived cytotoxic substances. Alternatively, microglia might not cause the neuronal loss, or naloxone might act by blocking opioid receptors on (dopaminergic or GABAergic) neurons. Thus, local inflammation induces and the opioid antagonist naloxone prevents the death of dopaminergic substantia nigra neurons in adult rats. This may be relevant to the understanding of the pathology and treatment of Parkinson’s disease, where these neurons degenerate. 䉷 2000 IBRO. Published by Elsevier Science Ltd. Key words: cell death, inflammation, lipopolysaccharide, microglial activation, opioid, Parkinson’s disease.
Resident microglia are present throughout the CNS and constitute approximately 10% of the cells. 41 They are related to peripheral monocytes and macrophages and share a number of properties, including the expression of complement 3 receptor (CR3 59,67). Normal microglia in the CNS appear to be involved in immune surveillance or assistance in immune responses and reactions to injury. 23,58,69 After an injury to the CNS, microglia close to neuronal cell bodies proliferate, change morphology and become more like macrophages (“activated” microglia). 69,70 When neurons die, microglia become “reactive” and remove cell debris. 69,70 Reactive microglia may also actively contribute to cell death in the CNS, e.g., treatments that reduce microglial activation result in increased neuronal cell survival in a number of neuronal systems. 51,62,72,79 Injection of bacterial lipopolysaccharide (LPS) and the ensuing inflammation (and possibly the microglial activation) has been shown to induce neuronal cell death in the basal forebrain 8,76 and hippocampal formation 44,77 of adult rats. Microglia may also participate or contribute to degenerative events underlying neurological disorders, 25 including Parkinson’s disease. 46 This disease is characterized by the degeneration of dopaminergic neurons of the substantia nigra that project to the neostriatum and the resulting loss of dopamine, which causes many of the symptoms. 19 Microglial activation by LPS can cause the loss of developing dopaminergic neurons in vitro, 6 but see Ref. 80.
Activated and reactive microglia release cytotoxic substances, such as tumor necrosis factor a (TNFa), nitric oxide (NO) and tissue plasminogen activator, which may contribute to cell death in tissue cultures and in animals. 6,44,48,62,71 The opioid receptor agonist morphine primes and increases LPS-induced release of TNF by microglia in vitro, 11 most likely through mu type receptors. 63 It is thus of interest that the opioid receptor antagonist naloxone reportedly can reduce LPS-induced production of TNFa, NO and interleukin-1ß in vitro and protect cultured cortical neurons. 13,39 Naloxone also reportedly blocks morphineinduced morphological changes such as rounding which are associated with microglial activation. 16 Here, we tested in adult rats whether local injection of LPS could induce microglial activation in the substantia nigra pars compacta and whether this would lead to loss of the dopaminergic nigrostriatal neurons. Moreover, we tested whether local or systemic treatment with naloxone could reduce microglial activation and whether this would affect the neuronal death. EXPERIMENTAL PROCEDURES
Retrograde labeling of nigrostriatal neurons, supranigral lipopolysaccharide injections and naloxone treatments All animal procedures were approved by the Animal Care Committee of Dalhousie University and conformed to the Canadian Council on Animal Care. All efforts were made to minimize animal suffering and to reduce the number of animals used (adult Sprague–Dawley rats; 200 g, Charles River). All surgical procedures were performed under general anesthesia (0.5 ml i.m. injection of a mixture of ketamine at 62.5 mg/kg, xylazine at 3.25 mg/kg and acepromazine at 0.62 mg/kg) and aseptic conditions. The dopaminergic nigrostriatal neurons in the substantia nigra pars compacta were retrogradely labeled by stereotaxic injection of the fluorescent tracer 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindocarbocyanine
‡To whom correspondence should be addressed. Tel.: ⫹ 1-902-494-6622; fax: ⫹ 1-902-494-2670 or 494-1212. E-mail address: [email protected] (T. Hagg). Abbreviations: DiI, 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindocarbocyanine; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; PBS, phosphate-buffered saline; TH, tyrosine hydroxylase; TNFa, tumor necrosis factor a. 285
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Fig. 1. Naloxone prevents lipopolysaccharide-induced death of dopaminergic substantia nigra neurons. Coronal sections through the substantia nigra pars compacta (outlined by arrows) immunostained for the microglia marker complement receptor 3 (left column) reveal that compared to normal controls (A), microglia were activated after injection of lipopolysaccharide (LPS) above the substantia nigra (B) and that infusion of naloxone above the nigra did not obviously affect microglial activation (C). Adjacent sections immunostained for the dopaminergic marker TH (D–F) or viewed under epifluorescence for the retrograde tracer DiI (G–I) revealed that compared to controls (D, G), LPS caused some neuronal loss (E, H) and that naloxone rescued a number of neurons (F, I). Scale bar 200 mm.
(DiI; Molecular Probes, Junction City, OR, U.S.A.) into six sites throughout the striatum. 27 This procedure labels essentially all the nigrostriatal neurons of the pars compacta and all labeled neurons are dopaminergic, while only approximately 10% of the compacta neurons are not dopaminergic. 27 Two weeks later, microglial activation was induced by a unilateral injection of 5 mg of LPS (O111:B4; Sigma Chemical Company, St Louis, MO, U.S.A.) in 2 ml of phosphatebuffered saline (PBS) 0.5 mm above the rostral third of the right substantia nigra pars compacta (1.7 mm lateral, 4.8 mm caudal and 8.0 mm ventral from bregma, tooth bar set at ⫺3.3 mm). Immediately afterwards, the animals were implanted with the tip of a 0.36 mm diameter metal cannula above the substantia nigra (1.7 mm lateral, 4.1 mm caudal and 7 mm ventral from bregma). The cannula was connected to an Alzet 2002 minipump (12 ml per day; Alza, Palo Alto, CA, U.S.A.) filled with PBS (n 5) or PBS containing 4.4 ng/ day (10 ⫺6 M) naloxone (n 6; Sigma) for a 14-day infusion. 42 Other animals received a unilateral injection of LPS above the substantia nigra followed by a 14-day subcutaneous infusion of 0.1 mg/day (2.3 × 10 ⫺2 M) naloxone dissolved in 60% ethanol to enhance solubility (n 6) or as a control 60% ethanol (n 4), using an Alzet minipump (12 ml/day).
The CR3-stained sections were used to establish the extent of microglial activation in the substantia nigra pars compacta. The outlines of the compacta region were determined in the adjacent THstained sections by the distribution of the dopaminergic neurons and well-established landmarks. 27 The compacta region in each section was scored semi-quantitatively blind as to the animal treatment, on a scale of 0–5, with 1 being normal and 5 being most extensive microglial activation. To assess the severity of the local inflammation and the potential impact on the substantia nigra region, the mediolateral diameter of the LPS-induced cavitation around the injection site was measured as well as the distance between the ventral edge of the cavity and the substantia nigra. The TH-stained and DiI-labeled sections were used to determine the number of nigrostriatal neurons in the substantia nigra pars compacta. 27 The number of neurons on the non-injected side was used to calculate the percentage of surviving neurons on the LPSinjected side. Statistical analysis was performed with the Mann–Whitney U-test (ranked, non-parametric) using a probability level of P ⬍ 0.05 for determining statistical significance. RESULTS
Histology and quantification of dopaminergic neuron survival and microglial activation After 14 days, all animals were transcardially perfused with 4% paraformaldehyde, their brains postfixed and cryoprotected, and 30mm-thick coronal sections were cut throughout the nigral complex. 27 Every sixth section through the compacta region was processed for immunocytochemical detection of the monocyte/macrophage/microglial marker complement receptor 3 (OX42 or CD11b/c antibody, Cedar Lane, Hornby, Ontario, Canada) using a sensitive ABC–peroxidase method. 42 Adjacent sections were immunostained for detection of the dopaminergic neuronal marker tyrosine hydroxylase (TH; Mab 318, Chemicon, Temecula, CA, U.S.A. 42). All immunostained sections were mounted on glass slides, dehydrated and coverslipped in Permount. Other adjacent sections were mounted on glass slides and coverslipped in aqueous fluorescent mounting media for detection of DiI-labeled nigral neurons.
Supranigral lipopolysaccharide causes microglial activation and neuronal loss, and naloxone reduces neuronal loss in the substantia nigra: qualitative data After local injection of LPS and PBS infusion, activated microglia were readily identifiable throughout the substantia nigra pars compacta by their thicker processes and more rounded cell body (Fig. 1B). In addition, many ameboid microglia/macrophages could be seen in the region directly around the LPS injection site (not shown). The LPS injection often caused a 0.7–3.8 mm diameter lesion around the injection site but the edge of the lesion was 0.1–0.4 mm away from the substantia nigra pars compacta. In adjacent sections, the substantia nigra appeared to contain fewer TH-positive
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Fig. 2. Lipopolysaccharide causes neuronal death and naloxone rescues neurons throughout the substantia nigra pars compacta. (A) Semi-quantitative microglial activation scores (^S.E.M.) in sections throughout the rostrocaudal extent of the substantia nigra pars compacta immunostained for CR3 are illustrated. Normal is 1 (open circle, broken line) and 5 is most extensive activation. Microglial activation scores were the same in rats injected with lipopolysaccharide (LPS) above the substantia nigra (arrow in B) and infused for 14 days with PBS (open squares; n 5) or with naloxone (solid squares; n 6) above the nigra. (B) The number of retrogradely labeled DiI-positive neurons (as a percentage^S.E.M. of normal open circle, broken line) per individual section throughout the substantia nigra pars compacta (SNC) in the same rats as in (A), illustrate that naloxone rescues neurons throughout the substantia nigra (P ⬍ 0.0005). Microglial activation was also unaffected (C) and neuronal survival increased (D) in animals that had been injected with LPS above the nigra and infused subcutaneously (systemically) with naloxone dissolved in ethanol (solid triangles; n 6; P ⬍ 0.025) compared to ethanol alone (open triangles; n 4).
(Fig. 1E) or DiI-labeled (Fig. 1H) neurons than normal (Fig. 1D and G, respectively). After LPS injection plus infusion of naloxone dorsal to the substantia nigra the extent of microglial activation (Fig. 1C) did not appear different from that seen with LPS injection plus infusion of PBS (Fig. 1B). The extent of LPS-induced cavitation also did not appear different after naloxone treatment (not shown). However, in the presence of naloxone the number of dopaminergic nigrostriatal neurons appeared slightly greater (Fig. 1F, I) than with PBS treatment (Fig. 1E, H). Quantification of microglial activation and neuronal loss To better evaluate the potential differences between PBS and naloxone-treated animals the sections were used to obtain more quantitative measures. Compared to normal animals, microglial appearance of the contralateral substantia nigra of unilateral LPS-injected animals was unaltered. The microglial activation scores in the substantia nigra were greatest in the rostral half of the nigra of LPS-injected rats, i.e. closer to the injection site (Fig. 2A). The number of DiI-positive nigrostriatal neurons in animals injected with LPS and infused for 14 days close to the substantia nigra with PBS had decreased to 66 ^ 3% of that in the non-lesioned sides (P ⬍ 0.0025;
Mann–Whitney U-test). Neuronal cell loss was evident throughout the rostrocaudal extent of the substantia nigra (Fig. 2B). The numbers of neurons counted in TH-immunostained sections (not shown) was the same as the DiI-positive numbers. In LPS-injected animals that were infused with PBS containing 10 ⫺6 M naloxone directly above the substantia nigra for 14 days, microglial activation scores throughout the substantia nigra were not different from LPS-injected rats infused with PBS (Fig. 2A). In these LPS plus naloxonetreated animals, 85 ^ 3% of the DiI-positive neurons had survived (P ⬍ 0.0005 compared to LPS plus PBS treatment). The number of surviving neurons was greater throughout the nigral nucleus (Fig. 2B). When naloxone was administered systemically by a 14 day subcutaneous infusion, the LPSinduced microglial activation scores were not altered (Fig. 2C). In these animals, 94 ^ 7% of the neurons had survived, i.e. many more than the 74 ^ 3% counted after subcutaneous infusion of the 60% ethanol control infusion (P ⬍ 0.025). The rescue effect was detectable throughout the rostrocaudal extent of the nigra (Fig. 2D). The survival level with peripheral control infusions (74 ^ 3%) was not statistically different compared to that seen with PBS infusion above the substantia nigra (66 ^ 3%). The extent of LPS-induced cavitation
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around the injection site (1.7 ^ 0.4 mm in diameter) and the distance of the cavity edge from the nigra (0.18 ^ 0.04 mm) seen after PBS infusion was not statistically different from that seen after naloxone treatment (2.4 ^ 0.4 and 0.16 ^ 0.05, respectively). The diameter of the cavity and distance to the nigra seen after LPS and systemic control infusion (2.8 ^ 0.6 and 0.12 ^ 0.06) were not significantly different from those seen after systemic naloxone infusion (1.4 ^ 0.3 and 0.22 ^ 0.05). As the extent of microglial activation and LPS-induced cavitation showed high levels of variability, we conducted a correlation analysis with the extent of neuronal cell loss in the nigra. Neither the total activation (summed scores of all sections from an animal), nor the individual values of sections, nor the diameter of the cavity, nor the distance of the cavity from the nigra showed a significant correlation with the extent of neuronal cell loss in the substantia nigra pars compacta (not shown).
DISCUSSION
The results of this study provide evidence in adult rats that (i) LPS-induced inflammation and/or microglial activation is associated with the death of dopaminergic nigrostriatal neurons and that (ii) the opioid antagonist naloxone can improve the survival of these injured neurons.
Lipopolysaccharide-activated microglia may cause neuronal death by releasing cytotoxic substances The LPS-induced microglial activation appeared to be greatest in the rostral half of the substantia nigra pars compacta where the loss of dopaminergic neurons was most extensive. Conversely, microglial activation and cell loss were less extensive in the caudal regions, which were more distant from the LPS injection site. This suggests that LPS had diffused through the brain tissue to create a gradient of microglial activation and that the activated microglia around the dopaminergic cell bodies had caused the neuronal death. The death-inducing role of activated microglia has been suggested by findings in other CNS injury models, including the retina and hippocampal formation. 51,62,72,79 LPS injections have also been shown to induce neuronal cell death in the basal forebrain 8,76 and hippocampal formation 44,77 of adult rats. In those hippocampal studies, inducible NO synthase (iNOS) inhibitors reduced the cell loss, suggesting that excessive NO directly or indirectly caused the neuronal loss. In humans, microglia and inflammation may contribute to the neuronal degeneration seen in diseases such as Alzheimer’s and Parkinson’s. 25,34,45–47,57 The mechanisms by which activated microglia cause neuronal cell death in vivo are largely unknown. 69 In tissue cultures, microglia can release cytotoxic substances such as TNFa, NO and tissue plasminogen activator which are toxic to neurons. 2,6,44,48,62,71 In the brains of animals and humans, activated microglia are immunoreactive for TNFa and inducible NO synthase. 7,15,36 LPS can also increase the release of glutamate from cortical brain slices, 75 and from cultured monocytes 37 and microglia. 56 If this occurs in vivo, the higher levels of glutamate could have contribute to the neuronal cell loss in the substantia nigra pars compacta, similar to the cell loss seen with glutamate agonists. 49
Lipopolysaccharide may directly affect neurons or astrocytes in the substantia nigra The lack of a correlation between the microglial activation scores and neuronal death in individual sections through the nigra suggests that the degree of neurotoxicity by microglia may not be related to their morphological appearance. Alternatively, this result raises the possibility that microglial activation was not involved in the LPS-induced neuronal cell death. LPS can cause the disappearance of TH-positive substantia nigra neurons independent of NO, 9 perhaps also suggesting that activated microglia (which produce neurotoxic NO) are not involved. LPS might act directly on the nigral neurons in vivo. LPS plus TNF can induce iNOS and subsequent death of differentiated neuron-like PC12 cells 29 and LPS can induce iNOS in purified cultured cerebellar neurons. 64 LPS binds to CD14 which is expressed by macrophages and activated microglia 3,28,40,73 but not by neurons and other cells in the brain, although they potentially could acquire soluble CD14. 66 Neuroblastoma cells can respond to LPS despite very low or absent levels of CD14, 55 suggesting that other LPS-binding molecules exist. In fact, neurons reportedly have a membrane-associated histone H1-like protein that binds LPS. 4 On the other hand, LPS does not cause the loss of cultured mesencephalic dopaminergic neurons unless glial cells are present, 6 suggesting that LPS acts through activation of cells other than neurons. The responses of cultured astrocytes to LPS have been varied. LPS can cause astroglial dysfunction, 31 induce argininosuccinate synthetase, which is essential for NO synthesis, 65 induce iNOS, 12 and cause NO-dependent neuronal death in astroglial co-cultures. 26,68 However, in vivo, LPS has very little effect on these responses by astrocytes compared to microglia. 30,36 LPS can cause an increase in expression of mRNA of various neurotrophic factors by cultured astrocytes 1,22 and microglia. 50 If LPS induces such factors also in vivo, their beneficial effects on the dopaminergic neurons could mask some of the detrimental effects of LPS. Lastly, it is possible that LPS induces the synthesis and release of cytotoxic substances from yet other cells in the inflamed substantia nigra, including leukocytes other than macrophages. Naloxone may prevent neuronal loss by reducing release of cytotoxins from microglia Treatments with naloxone did not obviously affect the LPSinduced microglial activation but reduced the death of the dopaminergic nigrostriatal neurons. One possible explanation is that naloxone inhibited the release of cytotoxic substances from microglia without obviously altering their morphology. This possibility is consistent with recent findings in mixed glial cell cultures that pharmacological concentrations of naloxone (10 ⫺8 –10 ⫺6 M) reduce the LPS-stimulated release of the pro-inflammatory cytokine interleukin-1ß. 13 Naloxone (10 ⫺10 –10 ⫺6M) can also inhibit the release of TNF-a and the production of NO from activated microglia in vitro. 39 In cocultures of glia and embryonic mouse cortical neurons, naloxone can reduce LPS-induced microglial activation and neuronal death. 38 It is thus conceivable that naloxone also inhibits release of neurotoxic molecules from microglia in vivo, thereby leading to increased survival of the dopaminergic neurons in LPS-treated adult rats. The potential mechanisms could involve the blocking of endogenous opioids that act through mu opioid receptors, as macrophages of
Naloxone prevents microglia-induced nigral degeneration
transgenic mu receptor-deficient mice have a much reduced ability to phagocytose and produce TNF in response to morphine. 63 In vitro, naloxone also prevented the morphological changes of microglia that are associated with their activation by LPS or morphine. 16,38,39 The lack of an effect on microglial morphology here in adult rats might be due to a lower effective concentration of naloxone that was reached in vivo. Naloxone may prevent death of nigrostriatal neurons by blocking opioid receptor binding on dopaminergic and/or GABAergic neurons Alternatively, the protective effects of naloxone may not have been through receptors on microglia but by blocking endogenous opioids that act on neuronal receptors. In the neostriatum, mu, delta and kappa receptor binding sites are abundant. 43 Substantia nigra pars compacta neurons express kappa opioid receptor mRNA but only a few have mu receptors, 24 suggesting that the dopaminergic terminals in the striatum have kappa receptors. Kappa receptor agonists cause a decrease in dopamine release in the striatum 14,52 and, conversely, low doses of naloxone in the range used here can increase striatal dopamine release in adult rodents, as well as potentiate amphetamine- or carbachol-induced dopamine release. 20,21,35,78 Treatment with dopamine increases, and absence of dopamine or its receptors decreases, the synthesis of neurotrophic factors in the mouse striatum 5,53,60 and in cultured astrocytes. 32 Thus, it is possible that naloxone caused an increase in dopamine release and, thereby, an increase in the levels of protective neurotrophic factors. The neostriatum is the major source of GABAergic innervation of the substantia nigra and contains predominantly mu and kappa opioid receptor mRNA. 24 In the substantia nigra, predominantly mu type receptor binding is seen, 43 while few nigral neurons express mu receptor mRNA, suggesting that the binding is on the GABAergic afferents. Mu receptor activation by morphine increases the firing rate of the
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dopaminergic neurons in animals, 74 most likely by decreasing the activity of GABAergic interneurons in the nigra. 33 Naloxone would also act through the GABAergic afferents, which have the naloxone binding sites in the substantia nigra. 54 In apparent contrast, morphine has also been shown to increase GABA release in the substantia nigra of rats, although not in the striatum. 78 Thus, it is unresolved what effect naloxone might have had on GABAergic activity in the current study and how that would have affected survival of the dopaminergic neurons. Potential implications for Parkinson’s disease The present findings that LPS-induced inflammation causes the death of dopaminergic substantia nigra neurons, and that the opioid receptor antagonist naloxone can reduce this neuronal loss might be relevant to Parkinson’s disease where these neurons degenerate. The possible beneficial effects of anti-inflammatory treatments for degenerative neurological disorders are suggested by the finding that antiinflammatory drugs reduce the risk of developing Alzheimer’s disease. 45,47,61 Naloxone also might have function-improving effects since at low doses it can increase striatal dopamine in adult rodents. 20,21,35,78 This may seem paradoxical, as acute treatments with the opioid receptor agonist morphine increase the firing rate of dopaminergic neurons 74 and cause increases in striatal dopamine release. 14,17,18 However, chronic morphine treatments are not effective, as animals develop tolerance. 17 With these unresolved issues about dopamine levels in mind, the efficacy of naloxone for Parkinson’s disease could be considered, since it crosses the blood–brain barrier, has been approved, is currently used for a variety of other disorders, and has relatively few and mild side-effects. 10 Acknowledgements—We greatly appreciate the excellent technical support from Julie Bunker. This research was supported by the Parkinson Foundation of Canada and the Medical Research Council of Canada.
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