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A morphological analysis of the motor neuron degeneration and microglial reaction in acute and chronic in vivo aluminum chloride neurotoxicity
Journal of Chemical Neuroanatomy 17 (2000) 207 – 215 www.elsevier.com/locate/jchemneu
A morphological analysis of the motor neuron degeneration and microglial reaction in acute and chronic in vivo aluminum chloride neurotoxicity Bei Ping He a, Michael J. Strong a,b,* a
Neurodegeneration Research Group, The John P. Robarts Research Institute, The Uni6ersity of Western Ontario, London, Ont., Canada N6A 5A85 b The Department of Clinical Neurological Sciences, The Uni6ersity of Western Ontario, Rm 7 0F 10, UC-LHSC, 339 Windermere Rd., London, Ont., Canada N6A 5A5 Received 18 April 1999; received in revised form 17 June 1999; accepted 26 September 1999
Abstract The monthly intracisternal inoculation of aluminum chloride (AlCl3) to young adult New Zealand white rabbits induces motor neuron degeneration marked by intraneuronal neurofilamentous aggregates similar to that observed in amyotrophic lateral sclerosis (ALS). However, in contrast to ALS, this process occurs in the experimental paradigm in the absence of a glial response. In addition, whereas ALS is a fatal disorder, the cessation of aluminum exposure leads to both clinical and neuropathological recovery. Because microglia can influence neuronal regeneration, we have examined the effect of both acute and chronic aluminum exposure on microglial activation in vivo. We have studied microglial morphology in young adult New Zealand white rabbits receiving either single (1000 mg) or repeated sublethal (100 mg monthly) intracisternal inoculums of AlCl3. In addition, rabbits receiving 1000 mg AlCl3 inoculums were studied following an unilateral sciatic axotomy 48 h prior to the AlCl3 exposure. Our studies demonstrate that microglial activation in vivo is inhibited by AlCl3 exposure, and that a correlation exists between the extent of microglia suppression and the potential for recovery. This suggests that microglial activation is an important determinant of neuronal injury. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Aluminum; Amyotrophic lateral sclerosis; Microglia; Motor neuron; Rabbit
1. Introduction In amyotrophic lateral sclerosis (ALS), the degeneration of upper and lower motoneurons results in a clinical syndrome of progressive weakness, culminating in respiratory failure and death. There are three variants of ALS, including a classical sporadic variant, a familial variant (accounting for 5 – 10% of ALS), and a western Pacific variant. The cause of ALS is unknown, although mutations in copper/zine superoxide dismutase (SOD-1) have been observed in approximately 15% of familial ALS cases (Rosen et al., 1993; Siddique and Deng, 1996). The unifying link amongst these three variants is the selective degeneration of motor neurons, * Corresponding author. Tel.: +1-519-6633874; fax: + 1-5196633609. E-mail address: [email protected] (M.J. Strong)
often accompanied by the development of both neurofilamentous aggregates and ubiquitinated inclusions. In addition to this, microglial activation has been recognized in regions of neuronal degeneration, including the motor cortex, nuclei of motoneurons and ventral horn of the spinal cord (McGeer et al., 1993). The observation of significant microglial activation in transgenic mice harboring SOD-1 mutations lends support to the hypothesis that microglia may play a key role in this degenerative process (Hall et al., 1998). In this study, we have examined the role of microglia in an experimental model of motor neuron degeneration possessing many of the morphological and ultrastructural features of ALS (Wakayama et al., 1996). Microglia, central nervous system resident macrophages, undergo profound functional changes in response to pathogenic stimuli, including activation from resting ramified microglia to amoebic, proliferat-
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ing macrophages capable of participating in neuronal injury. It has been thought that microglial activation could be involved in mediating local immune responses in the CNS and in a number of neurodegenerative processes (Davis et al., 1994). However, these responses may be either beneficial or harmful (Kreutzberg, 1996). Morphologically, reactive microglia demonstrate enlarged cytoplasm with shortened and thickened processes (McGeer et al., 1993). At the same time, reactive microglia in ALS upregulate the immunoglobulin receptor Fc gamma R1, the complement receptor type 3 and 4, the class II major histocompatibility complex molecules HLA–DR, HLA – DP and HLA – DQ and common determinants of the class I HLA – A,B,C complex (Kawamata et al., 1992). In addition to this upregulation of cell surface markers, activated microglia release several secretary products, including proteinases, cytokines, reactive oxygen intermediates, and reactive nitrogen intermediates (Banati et al., 1993). We have previously shown that the acute neurotoxicity of AlCl3 to spinal motoneurons in young adult New Zealand white rabbits is markedly enhanced by a sciatic axotomy 48 h preceding aluminum exposure (Strong and Gaytan-Garcia, 1996). Although the mechanism of the induction of this pathology is unclear, a prominent microglial proliferative response and activation occurs in response to axotomy (Streit et al., 1988). In a model of chronic AlCl3 neurotoxicity, induced by repeated monthly intracisternal inoculations of AlCl3, many of the clinical, histological and ultrastructural characteristics of ALS are reproduced (Strong and Garruto, 1991a; Wakayama et al., 1996). However, in contrast to ALS, these clinical and neuropathological features are reversible upon cessation of AlCl3 exposure, and there is a failure to induce a glial response (Strong et al., 1995). Because microglia can participate directly in neuronal injury and recovery (Thanos et al., 1993), we have now examined the relationship between the extent of neurofilamentous degeneration induced by acute AlCl3 exposure, the potential for recovery following chronic aluminum exposure, and the microglial response to the induction of neuronal pathology. Microglia were identified on the basis of lectin labeling (Sasaki et al., 1991) and morphological characteristics.
2. Materials and methods
2.1. Animals and inoculation All procedures were performed in accordance with the Canadian Council on Animal Care Guidelines. In total, 52 New Zealand white rabbits (Don Riemans Fur Ranch, Ste. Agathe, Ontario), at the age of 5 – 6 weeks, were used in the experiments.
2.1.1. Acute axotomy studies As previously described (Gaytan-Garcia et al., 1996), for acute AlCl3 toxicity studies a unilateral sciatic neurectomy was performed under ketamine (40 mg/kg) and acepromazine (1 mg/kg) anesthesia on 12 rabbits. Forty eight hours following the neurectomy, six rabbits were inoculated intracisternally (cisterna magna) with 1000 mg chromatographically pure AlCl3 (ICN Laboratories, Lisle, IL) in 100 ml 0.9% NaCl. Six control rabbits were inoculated with 100 ml 0.9% NaCl. Forty eight h post inoculum, the rabbits were killed by sodium pentobarbital injection. 2.1.2. Chronic studies For the chronic AlCl3 inoculation experiment, a total of 40 rabbits were intracisternally inoculated (cisterna magna) with either 100 mg AlCl3 in 100 ml 0.9% NaCl (n= 32) or 100 ml 0.9% NaCl (n= 8) at intervals of 28 days for a total of 267 days as previous described (Strong et al., 1995). Amongst them, following the 2nd inoculum, three rabbits were allowed to survive until 267 days without further AlCl3 exposure and another five killed by sodium pentobarbital injection. After the 4th inoculum, three animals were allowed to survive until the termination of the study and another six were killed. Following the 6th inoculum, three animals were kept alive until 267 days and six animals were allowed to survive for 156 days. The remaining rabbits continued to receive AlCl3 inoculations every 28 days until day 267. Control rabbits (n= 2) were killed at each time interval. This paradigm yielded four subsets of rabbits: a control, NaCl-inoculated population autopsied at each 56-day time interval; a AlCl3-inoculated subgroup receiving inoculations every 28 days for the duration of the study; a AlCl3 subgroup receiving AlCl3 every 28 days for defined intervals with subsequent survival free of AlCl3 exposure to the completion of the study (recovery subgroup); and a subgroup of AlCl3-inoculated rabbits autopsied at defined 56-day intervals. 2.2. Staining The lumbar enlargement, cervical and thoracic regions of the spinal cord and the brain were immersion fixed in 10% neutral buffered formalin. The remaining tissue was flash frozen in isopentane in liquid nitrogen for future studies. Paraffin-imbedded 6-mm sections were cut for the lectin (biotinylated Ricinus Communis Agglutinin I; RCA, Vector Laboratories, USA) and mouse monoclonal anti 200 kDa neurofilament (Boehringer Mannheim Biochemica, Germany) single or double staining. Lectin has been observed to be a reliable marker for microglia, although endothelial cells can be labeled (Sasaki et al., 1991). We observed no alteration in the RCA-1 staining patterns of microglia
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treated AlCl3 in vitro (data not shown). As previously described, anti-neurofilament 200 kD (NFH) antibodies reliably demonstrated diseased motoneurons in AlCl3inoculated rabbits (Strong et al., 1995; Wakayama et al., 1996). In the present study, we have failed to label rabbit microglia with several mouse monoclonal antibodies, including CD18, CD23, CD68, CD49d, MAC387, GM–CSFR, HLA – DR and anti-rabbit monoclonal antibody: CD11b. Following deparaffinization using routine methodologies, sections were digested with 1% trypsin in 0.01 M phosphate buffered saline (PBS, pH 7.4) for 15 min at 37°C. After incubation with 0.3% hydrogen peroxide (H2O2) and then 5% BSA, each for 20 min at room temperature, the sections were incubated over night with the lectin at the dilution of 1:5000 at 4°C. Control sections were incubated with PBS without the RCA-1. After incubation, RCA was detected by the Elite avidin-biotin complex (Vector Laboratories, USA) with 0.05% 3-3%-diaminobenzidine tetrachloride (DAB) (Sigma Chemical company, USA) as a peroxidase substrate (5 mg DAB in 5 ml of PBS+ 1.7 ml of 30% H2O2 + 5 mg nickel chloride). The slides processed only for RCA were then counterstained with hematoxylin, dehydrated, and coverslipped using permount. The slides processed for double labeling RCA and antiNFH were washing with PBS. The sections were then incubated with 5% BSA in PBS for 20 min at room temperature and followed by an overnight incubation with anti-NFH antibody at dilution of 1:1000 at 4°C. Control sections were incubated with PBS without first or secondary antibody. After incubation, anti-NFH was detected by the avidin – biotin complex – AP (Vector Laboratories, USA) with alkaline phosphatase (Alkaline phosphatase substrate kit, Vector laboratories, USA) as an enzyme marker. When preparing the substrate solution, 1 mM levamisole was added into 0.1 M Tris –HCl (pH 8.2) buffer in order to block endogenous alkaline phosphatase activity. The sections were dehydrated and coverslipped, using Permount without any counterstain.
2.3. Cell counting Cell counting was performed by an investigator blinded to their treatment (BH). Microglia counting was based on the RCA-1 positive structures with the morphology of the cell body observed by light microscopy using four sections from each case. In the acute axotomy series, sections were taken from the L5 spinal level, corresponding to the level at which chromatolytic neurons were observed. The same level was studied in the chronic toxicity series. Microglia counting was performed for the nucleus motoris medialis and lateralis in each ventral horn. As a control for the presence of neurofilamentous pathology, we also per-
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formed cell counts on the inferior vestibular nucleus. This sensory nucleus was chosen as a control region given previous studies that have demonstrated a resistance of these neurons to aluminum toxicity (Wisniewski et al., 1967; Bugiani and Ghetti, 1982; Strong et al., 1995). The number of microglia in each animal was presented as mean from four counting areas of 0.13 mm2 in the sections of the lumbar cord or brain. Statistical analysis was performed using the Student’s t-test. The NFH positive structures were counted from an average of five cross sections of the spinal cord of each animal. The data are presented as the absolute numbers in total. In order to determine the spatial relationship between the microglia and diseased motoneurons, NFH positive neuronal structures were subgrouped as possessing 0, one, two, or more than two attached microglial perikaryon or processes. Microglial attachment to a motoneuron was defined as microglia clearly adjacent to the motoneuron perikaryon.
3. Results
3.1. Acute-axotomy model Fig. 1 shows the number of microglia in the ventrolateral region of the lumbar cord section. The number of microglia increased significantly in the ventrolateral area ipsilateral to the axotomy at day 4 post-axotomy in both AlCl3- and NaCl-inoculated groups, compared with that of the contralateral side. However, the number of microglia present ipsilateral to the axotomy was significantly less following AlCl3 inoculation than following NaCl-inoculation (PB 0.05). There was no statistical difference (P\0.05) in microglia number in the ventrolateral region contralateral to axotomy, compared with the microglia number in the same region in the control animal. Microglia stained intensely with RCA-1 (Fig. 2). Four days after sciatic neurectomy, in the ventrolateral area of the lumbar cord segment from NaCl-inoculated animals, the morphology of the microglia ipsilateral to the neurectomy was not obviously different from those observed in the contralateral region. After AlCl3 inoculation, microglia showed thinner, less complex branching patterns (Fig. 2B), compared to control animals (Fig. 2A). As previously described, AlCl3-inoculated rabbits developed intraneuronal neurofilamentous aggregates and neuroaxonal spheroids within spinal motor neurons. However, the number of neurons developing neurofilamentous aggregates was increased ipsilateral to the axotomy (Strong and Gaytan-Garcia, 1996). Neuroaxonal spheroids tended to be localized to predominantly the ventrolateral region of the anterior horns. Occa-
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Fig. 1. The number of microglia in the ventrolateral area of the lumber cord section at 4 days after sciatic nerve cut and 2 days after 1000 mg AlCl3 in 100 ml 0.9% NaCl intracisternal exposure. The microglial number in the side ipsilateral to the axotomy of both NaCl (n =6) and AlCl3-inoculated (n =6) groups is significantly greater than that in the contralateral side (P B 0.05). However, the increased microglial number in the lesioned side of saline control group is significantly more than that of AlCl3 exposure group (* P B0.05).
sional diffuse anti-NFH immunoreactivity was observed in control spinal motor neurons, in the absence of perikaryal aggregates. Microglial process attachment to anti-NFH immunoreactive neurons ipsilateral to the sciatic axotomy was consistently greater in NaCl-inoculated than in AlCl3-inoculated rabbits (Fig. 3). Although neurofilamentous aggregate bearing neurons and neuroaxonal spheroids were observed following AlCl3 inoculation, the majority of these cells exhibited less than two microglial processes attaching.
3.2. Chronic model In the ventrolateral regions of the lumbar cord, basophilic inclusions were observed in the cytoplasm of motoneurons. Some motoneurons also exhibited features of chromatolysis. However, we did not observe cytoplasm inclusions and features of chromatolysis in neurons of the nucleus vestibularis in either AlCl3-inoculated or control rabbits. The number of microglia in the ventrolateral region of the lumbar cord sections was significantly decreased
after AlCl3 inoculation compared to that of NaCl-inoculated animals (Fig. 4). The number of microglia in the recovery groups (those animals killed at day 267 without further AlCl3 inoculation after the 2nd, 4th, or 6th inoculation) was greater than that in AlCl3 inoculation groups (PB 0.01). The number of microglia in the region of the inferior vestibular nucleus was also significantly decreased after AlCl3 inoculation compared to that of NaCl-inoculated animals (Fig. 5). The number of microglia in the recovery groups was greater than that in AlCl3-inoculated groups (PB 0.01). In the saline control and recovery rabbits, microglia appeared very well ramified (Fig. 2C, G) and were evenly distributed in the ventral horn of the lumbar segment as well as in the area of the inferior vestibular nucleus. However, after AlCl3 inoculation, microglia showed thinner, less complex branching patterns (Fig. 2D,E,H). The blood vessels have been clearly stained with RCA-1. There was no apparent difference in the intensity of RCA-1 staining of blood vessels between AlCl3and NaCl-inoculated groups.
Fig. 2. (A – B) presents axotomized motoneurons. The motoneurons, one of them is NFH positive (red staining) after AlCl3 inoculation (B), show eccentric nuclei and are slightly swollen, features typical of the axotomized cell. Several microglia (black staining) have been found to closely contact to these motoneurons. In NaCl-inoculated rabbit (A), activated microglia (arrows) possess thicker processes. However, following acute AlCl3 inoculation (B), microglia show thinner branches (arrows). Figures (C – H) are taken from the chronic AlCl3 experiment. At lower magnification, microglia in NaCl-inoculated animals (C) are more obvious than those in AlCl3-inoculated ones (D). Figure E show a single microglia (arrow) closely related to a neuroaxonal spheroid (illustration from day 56 post-inoculation). (F) Following the 6th inoculation in the chronic AlCl3 experiment, neuroaxonal spheroids demonstrated less microglial attachment, and in many instances, there were no associated microglia. (G) A microglia (arrow) from the lumbar section of an NaCl-inoculated rabbit. Several processes branch out from its cell body. After the 4th AlCl3-inoculation (H), microglia (arrow) show thinner and less complex process, compared with those in control and recovery groups. Arrowheads show some of RCA positive vascular vessels. No obvious difference was observed in the RCA staining intensity in vessels in the lumbar cord and nucleus vestibularis between AlCl3- and NaCl-inoculated groups. Scale Bar: 25 mm (A, B), 160 mm (C, D), 25 mm (E, F), 15 mm (G, H).
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Fig. 2.
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NFH positive structures were also mainly located in the ventrolateral region of the spinal cord. Some positive structures could be enlarged axons where the neurofilament aggregated. Microglia could be noted to be close to or attached to, NFH positive structures (Fig. 2E) in the spinal cord of AlCl3-inoculated or NFH negative motoneurons in the spinal cord of both AlCl3inoculated and control animals. NFH positive structure did not, however, always possess a close spatial relationship with microglia (Fig. 2F). In fact, microglial processes seldom surrounded the NFH positive structures totally. The relationship between the induction of neurofilamentous aggregates and the proximity microglial processes is presented in Fig. 6.
4. Discussion Motoneurons are uniquely sensitive to the toxicity of AlCl3 both in vivo and in vitro (Strong and Garruto, 1991b; Strong et al., 1995). In vivo, following the inoculation of either organic or inorganic aluminum compounds, affected neurons develop neurofilamentous aggregates in the neuronal soma, axons and dendrites. Acute high dose AlCl3 exposure induces widespread neurofibrillary tangle-like perikaryal inclusions throughout the CNS (Bugiani and Ghetti, 1982), and culminates in a fulminant disease course. In contrast, chronic low-dose AlCl3 intoxication brings about progressive spasticity with spinal motoneuron degeneration
and pathologically confirmed neurogenic muscular atrophy (Strong et al., 1995), resembling that observed in human ALS (Wakayama et al., 1996). However, in contrast to the almost uniformly fatal ALS, the pathological changes of motoneurons following chronic AlCl3 exposure are, to some extent, reversible following cessation of AlCl3 exposure (Strong et al., 1995). While the most obvious reason is the removal of the pathological stimulus in this chronic model, it is also possible that the response of AlCl3-exposed spinal motoneurons is modified by either intrinsic or extrinsic factor(s) as yet unknown, such as microglia. As discussed, activated microglia are capable of expressing many immuno-antigens found on peripheral macrophages (Kawamata et al., 1992) and of participating in cytotoxicity (Banati et al., 1993). Following peripheral nerve injury, resting microglia become active (Graeber et al., 1988) and proliferate and migrate towards the perikarya of axotomized motoneurons (Streit et al., 1988; Kreutzberg et al., 1989). Inhibition of this response is associated with an enhanced axonal regeneration following axotomy (Thanos et al., 1993). Although microglia can respond to very minor pathological stimuli, and both acute and chronic AlCl3 exposure induces significant neuronal injury, we have observed a failure of a microglial response in AlCl3 neurotoxicity. Firstly, a significant reduction in microglial number was noted in the lumbar segment of the spinal cord and in the area of the inferior vestibular nucleus following chronic AlCl3 inoculation. Although
Fig. 3. Post-axotomy analysis of microglial attachment to NFH positive structures. The figure shows the total number of NFH positive cells and spheroids in cord sections at 4 days after sciatic axotomy and 2 days after either high dose AlCl3 intracisternal exposure or NaCl inoculation. A fewer NFH positive structures were observed in the NaCl-inoculated animals compared to AlCl3-inoculated animals. The number of diseased motoneurons to which two or more microglia attach is less than observed in control.
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Fig. 4. Chronic AlCl3-inoculation microglial analysis. Animals were intracisternally inoculated with 100 mg AlCl3 in 100 ml 0.9% NaCl at intervals of 28 days for a total of 267 days (n= 6), or autopsied at day 51 (n =5), day 107 (n = 6) or day 156 (n = 6). At each time interval, three rabbits were allowed to survive without further AlCl3 exposure and another two were inoculated with 100 ml 0.9% NaCl. At each autopsy interval, the lumbar ventral horns of AlCl3-inoculated animals showed significantly less microglial cells than saline controls (P B 0.001). In the absence of further AlCl3 exposure (recovery), microglial number becomes similar to that of saline control animals.
Fig. 5. Chronic AlCl3-inoculation microglial analysis. The number of microglia in vestibularis nucleus after AlCl3 inoculation showed significantly less than that after NaCl inoculation (PB 0.001). In the recovery group, the microglial number becomes similar to that of NaCl inoculated animals.
we do not know the cause of this, applying AlCl3 after a sciatic axotomy resulted in a reduction of the microglial numbers in the ipsilateral ventral horn of the lumbar cord segment, suggesting that AlCl3 does inhibit microglial proliferation. Secondly, while there were many diseased motoneurons in the spinal cord of rab-
bits after either chronic or acute AlCl3 exposure, microglia still showed thinner, less complex branching patterns. This further supports the suppressive role of AlCl3 to microglial transformation. Thirdly, in the chronic AlCl3 experiments, the majority of NFH positive cells were not closely surrounded by microglia,
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Fig. 6. The number of NFH positive cells and spheroids in cord sections after lower dose AlCl3 intracisternal exposure. There are more NFH positive structures at day 107 and day 156 after AlCl3 inoculation. However, most of them are not closely related with microglia. Rare NFH immunoreactive structures were observed in both saline control and recovery rabbits.
suggesting microglial migration was also hindered. In the acute-axotomy group, we also failed to see microglia enveloping NFH positive cells or spheroids after aluminum exposure. Although the mechanisms of AlCl3 suppression of microglial activation are not understood, the suppression is not directly related or due to the presence of degenerating neurons. In the present study, we found a suppression of microglia in the area of the inferior vestibular nucleus in which no obvious neurodegeneration was observed. Although there is very little evidence to indicate directly that microglia may be toxic to neurons in vivo, in vitro microglia have the capacity to develop into cytotoxic macrophages (Frei et al., 1978). Co-culture of neurons with activated microglia increases neuronal death (Thery et al., 1991; Chao et al., 1992), and microglia can phagocytose neuronal debris (Torvik, 1972). Upon cellular activation in vitro, microglia release proteases that could potentially result in degradation of the extracellular matrix and thereby compromise neuronal function (Colton et al., 1993). Abundant nitrite, a product of the free radical nitric oxide, was detected in activated microglia and microglia/neuron co-cultures but not in astrocyte or neuron cultures alone (Chao et al., 1992). The latter implied that microglia were the principal source of the nitric oxide and that activated microglia might kill neurons via a nitric oxide mediated mechanism. Although it has been shown that microglia upregulate inducible nitric oxide synthase in vitro following Zeolite (aluminosilicate par-
ticles) exposure (Garrel et al., 1994), it is not known if AlCl3 can have the same effect. Microglia are also the major cellular source of the immunoregulatory cytokines such as interleukin-1 (IL-1) (Giulian et al., 1985), IL-5 (Sawada et al., 1993), and tumor necrosis factor-a (TNF-a) (Sawada et al., 1989). It is of interest, therefore, that motor neuron death in neurodegeneration may be TNF-a-mediated (Choe et al., 1998; Ghezzi et al., 1998). Because we previously observed both clinical and neuropathological recovery following chronic AlCl3 exposure, and now report the inhibition of microglial proliferation and activation in the same model, we are proposing that the failure to induce a microglial response is permissive to recovery in the chronic AlCl3 model. Further in vitro experiments are in process to determine the effect of AlCl3 on microglial function.
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A morphological analysis of the motor neuron degeneration and microglial reaction in acute and chronic in vivo aluminum chloride neurotoxicity Bei Ping He a, Michael J. Strong a,b,* a
Neurodegeneration Research Group, The John P. Robarts Research Institute, The Uni6ersity of Western Ontario, London, Ont., Canada N6A 5A85 b The Department of Clinical Neurological Sciences, The Uni6ersity of Western Ontario, Rm 7 0F 10, UC-LHSC, 339 Windermere Rd., London, Ont., Canada N6A 5A5 Received 18 April 1999; received in revised form 17 June 1999; accepted 26 September 1999
Abstract The monthly intracisternal inoculation of aluminum chloride (AlCl3) to young adult New Zealand white rabbits induces motor neuron degeneration marked by intraneuronal neurofilamentous aggregates similar to that observed in amyotrophic lateral sclerosis (ALS). However, in contrast to ALS, this process occurs in the experimental paradigm in the absence of a glial response. In addition, whereas ALS is a fatal disorder, the cessation of aluminum exposure leads to both clinical and neuropathological recovery. Because microglia can influence neuronal regeneration, we have examined the effect of both acute and chronic aluminum exposure on microglial activation in vivo. We have studied microglial morphology in young adult New Zealand white rabbits receiving either single (1000 mg) or repeated sublethal (100 mg monthly) intracisternal inoculums of AlCl3. In addition, rabbits receiving 1000 mg AlCl3 inoculums were studied following an unilateral sciatic axotomy 48 h prior to the AlCl3 exposure. Our studies demonstrate that microglial activation in vivo is inhibited by AlCl3 exposure, and that a correlation exists between the extent of microglia suppression and the potential for recovery. This suggests that microglial activation is an important determinant of neuronal injury. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Aluminum; Amyotrophic lateral sclerosis; Microglia; Motor neuron; Rabbit
1. Introduction In amyotrophic lateral sclerosis (ALS), the degeneration of upper and lower motoneurons results in a clinical syndrome of progressive weakness, culminating in respiratory failure and death. There are three variants of ALS, including a classical sporadic variant, a familial variant (accounting for 5 – 10% of ALS), and a western Pacific variant. The cause of ALS is unknown, although mutations in copper/zine superoxide dismutase (SOD-1) have been observed in approximately 15% of familial ALS cases (Rosen et al., 1993; Siddique and Deng, 1996). The unifying link amongst these three variants is the selective degeneration of motor neurons, * Corresponding author. Tel.: +1-519-6633874; fax: + 1-5196633609. E-mail address: [email protected] (M.J. Strong)
often accompanied by the development of both neurofilamentous aggregates and ubiquitinated inclusions. In addition to this, microglial activation has been recognized in regions of neuronal degeneration, including the motor cortex, nuclei of motoneurons and ventral horn of the spinal cord (McGeer et al., 1993). The observation of significant microglial activation in transgenic mice harboring SOD-1 mutations lends support to the hypothesis that microglia may play a key role in this degenerative process (Hall et al., 1998). In this study, we have examined the role of microglia in an experimental model of motor neuron degeneration possessing many of the morphological and ultrastructural features of ALS (Wakayama et al., 1996). Microglia, central nervous system resident macrophages, undergo profound functional changes in response to pathogenic stimuli, including activation from resting ramified microglia to amoebic, proliferat-
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ing macrophages capable of participating in neuronal injury. It has been thought that microglial activation could be involved in mediating local immune responses in the CNS and in a number of neurodegenerative processes (Davis et al., 1994). However, these responses may be either beneficial or harmful (Kreutzberg, 1996). Morphologically, reactive microglia demonstrate enlarged cytoplasm with shortened and thickened processes (McGeer et al., 1993). At the same time, reactive microglia in ALS upregulate the immunoglobulin receptor Fc gamma R1, the complement receptor type 3 and 4, the class II major histocompatibility complex molecules HLA–DR, HLA – DP and HLA – DQ and common determinants of the class I HLA – A,B,C complex (Kawamata et al., 1992). In addition to this upregulation of cell surface markers, activated microglia release several secretary products, including proteinases, cytokines, reactive oxygen intermediates, and reactive nitrogen intermediates (Banati et al., 1993). We have previously shown that the acute neurotoxicity of AlCl3 to spinal motoneurons in young adult New Zealand white rabbits is markedly enhanced by a sciatic axotomy 48 h preceding aluminum exposure (Strong and Gaytan-Garcia, 1996). Although the mechanism of the induction of this pathology is unclear, a prominent microglial proliferative response and activation occurs in response to axotomy (Streit et al., 1988). In a model of chronic AlCl3 neurotoxicity, induced by repeated monthly intracisternal inoculations of AlCl3, many of the clinical, histological and ultrastructural characteristics of ALS are reproduced (Strong and Garruto, 1991a; Wakayama et al., 1996). However, in contrast to ALS, these clinical and neuropathological features are reversible upon cessation of AlCl3 exposure, and there is a failure to induce a glial response (Strong et al., 1995). Because microglia can participate directly in neuronal injury and recovery (Thanos et al., 1993), we have now examined the relationship between the extent of neurofilamentous degeneration induced by acute AlCl3 exposure, the potential for recovery following chronic aluminum exposure, and the microglial response to the induction of neuronal pathology. Microglia were identified on the basis of lectin labeling (Sasaki et al., 1991) and morphological characteristics.
2. Materials and methods
2.1. Animals and inoculation All procedures were performed in accordance with the Canadian Council on Animal Care Guidelines. In total, 52 New Zealand white rabbits (Don Riemans Fur Ranch, Ste. Agathe, Ontario), at the age of 5 – 6 weeks, were used in the experiments.
2.1.1. Acute axotomy studies As previously described (Gaytan-Garcia et al., 1996), for acute AlCl3 toxicity studies a unilateral sciatic neurectomy was performed under ketamine (40 mg/kg) and acepromazine (1 mg/kg) anesthesia on 12 rabbits. Forty eight hours following the neurectomy, six rabbits were inoculated intracisternally (cisterna magna) with 1000 mg chromatographically pure AlCl3 (ICN Laboratories, Lisle, IL) in 100 ml 0.9% NaCl. Six control rabbits were inoculated with 100 ml 0.9% NaCl. Forty eight h post inoculum, the rabbits were killed by sodium pentobarbital injection. 2.1.2. Chronic studies For the chronic AlCl3 inoculation experiment, a total of 40 rabbits were intracisternally inoculated (cisterna magna) with either 100 mg AlCl3 in 100 ml 0.9% NaCl (n= 32) or 100 ml 0.9% NaCl (n= 8) at intervals of 28 days for a total of 267 days as previous described (Strong et al., 1995). Amongst them, following the 2nd inoculum, three rabbits were allowed to survive until 267 days without further AlCl3 exposure and another five killed by sodium pentobarbital injection. After the 4th inoculum, three animals were allowed to survive until the termination of the study and another six were killed. Following the 6th inoculum, three animals were kept alive until 267 days and six animals were allowed to survive for 156 days. The remaining rabbits continued to receive AlCl3 inoculations every 28 days until day 267. Control rabbits (n= 2) were killed at each time interval. This paradigm yielded four subsets of rabbits: a control, NaCl-inoculated population autopsied at each 56-day time interval; a AlCl3-inoculated subgroup receiving inoculations every 28 days for the duration of the study; a AlCl3 subgroup receiving AlCl3 every 28 days for defined intervals with subsequent survival free of AlCl3 exposure to the completion of the study (recovery subgroup); and a subgroup of AlCl3-inoculated rabbits autopsied at defined 56-day intervals. 2.2. Staining The lumbar enlargement, cervical and thoracic regions of the spinal cord and the brain were immersion fixed in 10% neutral buffered formalin. The remaining tissue was flash frozen in isopentane in liquid nitrogen for future studies. Paraffin-imbedded 6-mm sections were cut for the lectin (biotinylated Ricinus Communis Agglutinin I; RCA, Vector Laboratories, USA) and mouse monoclonal anti 200 kDa neurofilament (Boehringer Mannheim Biochemica, Germany) single or double staining. Lectin has been observed to be a reliable marker for microglia, although endothelial cells can be labeled (Sasaki et al., 1991). We observed no alteration in the RCA-1 staining patterns of microglia
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treated AlCl3 in vitro (data not shown). As previously described, anti-neurofilament 200 kD (NFH) antibodies reliably demonstrated diseased motoneurons in AlCl3inoculated rabbits (Strong et al., 1995; Wakayama et al., 1996). In the present study, we have failed to label rabbit microglia with several mouse monoclonal antibodies, including CD18, CD23, CD68, CD49d, MAC387, GM–CSFR, HLA – DR and anti-rabbit monoclonal antibody: CD11b. Following deparaffinization using routine methodologies, sections were digested with 1% trypsin in 0.01 M phosphate buffered saline (PBS, pH 7.4) for 15 min at 37°C. After incubation with 0.3% hydrogen peroxide (H2O2) and then 5% BSA, each for 20 min at room temperature, the sections were incubated over night with the lectin at the dilution of 1:5000 at 4°C. Control sections were incubated with PBS without the RCA-1. After incubation, RCA was detected by the Elite avidin-biotin complex (Vector Laboratories, USA) with 0.05% 3-3%-diaminobenzidine tetrachloride (DAB) (Sigma Chemical company, USA) as a peroxidase substrate (5 mg DAB in 5 ml of PBS+ 1.7 ml of 30% H2O2 + 5 mg nickel chloride). The slides processed only for RCA were then counterstained with hematoxylin, dehydrated, and coverslipped using permount. The slides processed for double labeling RCA and antiNFH were washing with PBS. The sections were then incubated with 5% BSA in PBS for 20 min at room temperature and followed by an overnight incubation with anti-NFH antibody at dilution of 1:1000 at 4°C. Control sections were incubated with PBS without first or secondary antibody. After incubation, anti-NFH was detected by the avidin – biotin complex – AP (Vector Laboratories, USA) with alkaline phosphatase (Alkaline phosphatase substrate kit, Vector laboratories, USA) as an enzyme marker. When preparing the substrate solution, 1 mM levamisole was added into 0.1 M Tris –HCl (pH 8.2) buffer in order to block endogenous alkaline phosphatase activity. The sections were dehydrated and coverslipped, using Permount without any counterstain.
2.3. Cell counting Cell counting was performed by an investigator blinded to their treatment (BH). Microglia counting was based on the RCA-1 positive structures with the morphology of the cell body observed by light microscopy using four sections from each case. In the acute axotomy series, sections were taken from the L5 spinal level, corresponding to the level at which chromatolytic neurons were observed. The same level was studied in the chronic toxicity series. Microglia counting was performed for the nucleus motoris medialis and lateralis in each ventral horn. As a control for the presence of neurofilamentous pathology, we also per-
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formed cell counts on the inferior vestibular nucleus. This sensory nucleus was chosen as a control region given previous studies that have demonstrated a resistance of these neurons to aluminum toxicity (Wisniewski et al., 1967; Bugiani and Ghetti, 1982; Strong et al., 1995). The number of microglia in each animal was presented as mean from four counting areas of 0.13 mm2 in the sections of the lumbar cord or brain. Statistical analysis was performed using the Student’s t-test. The NFH positive structures were counted from an average of five cross sections of the spinal cord of each animal. The data are presented as the absolute numbers in total. In order to determine the spatial relationship between the microglia and diseased motoneurons, NFH positive neuronal structures were subgrouped as possessing 0, one, two, or more than two attached microglial perikaryon or processes. Microglial attachment to a motoneuron was defined as microglia clearly adjacent to the motoneuron perikaryon.
3. Results
3.1. Acute-axotomy model Fig. 1 shows the number of microglia in the ventrolateral region of the lumbar cord section. The number of microglia increased significantly in the ventrolateral area ipsilateral to the axotomy at day 4 post-axotomy in both AlCl3- and NaCl-inoculated groups, compared with that of the contralateral side. However, the number of microglia present ipsilateral to the axotomy was significantly less following AlCl3 inoculation than following NaCl-inoculation (PB 0.05). There was no statistical difference (P\0.05) in microglia number in the ventrolateral region contralateral to axotomy, compared with the microglia number in the same region in the control animal. Microglia stained intensely with RCA-1 (Fig. 2). Four days after sciatic neurectomy, in the ventrolateral area of the lumbar cord segment from NaCl-inoculated animals, the morphology of the microglia ipsilateral to the neurectomy was not obviously different from those observed in the contralateral region. After AlCl3 inoculation, microglia showed thinner, less complex branching patterns (Fig. 2B), compared to control animals (Fig. 2A). As previously described, AlCl3-inoculated rabbits developed intraneuronal neurofilamentous aggregates and neuroaxonal spheroids within spinal motor neurons. However, the number of neurons developing neurofilamentous aggregates was increased ipsilateral to the axotomy (Strong and Gaytan-Garcia, 1996). Neuroaxonal spheroids tended to be localized to predominantly the ventrolateral region of the anterior horns. Occa-
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Fig. 1. The number of microglia in the ventrolateral area of the lumber cord section at 4 days after sciatic nerve cut and 2 days after 1000 mg AlCl3 in 100 ml 0.9% NaCl intracisternal exposure. The microglial number in the side ipsilateral to the axotomy of both NaCl (n =6) and AlCl3-inoculated (n =6) groups is significantly greater than that in the contralateral side (P B 0.05). However, the increased microglial number in the lesioned side of saline control group is significantly more than that of AlCl3 exposure group (* P B0.05).
sional diffuse anti-NFH immunoreactivity was observed in control spinal motor neurons, in the absence of perikaryal aggregates. Microglial process attachment to anti-NFH immunoreactive neurons ipsilateral to the sciatic axotomy was consistently greater in NaCl-inoculated than in AlCl3-inoculated rabbits (Fig. 3). Although neurofilamentous aggregate bearing neurons and neuroaxonal spheroids were observed following AlCl3 inoculation, the majority of these cells exhibited less than two microglial processes attaching.
3.2. Chronic model In the ventrolateral regions of the lumbar cord, basophilic inclusions were observed in the cytoplasm of motoneurons. Some motoneurons also exhibited features of chromatolysis. However, we did not observe cytoplasm inclusions and features of chromatolysis in neurons of the nucleus vestibularis in either AlCl3-inoculated or control rabbits. The number of microglia in the ventrolateral region of the lumbar cord sections was significantly decreased
after AlCl3 inoculation compared to that of NaCl-inoculated animals (Fig. 4). The number of microglia in the recovery groups (those animals killed at day 267 without further AlCl3 inoculation after the 2nd, 4th, or 6th inoculation) was greater than that in AlCl3 inoculation groups (PB 0.01). The number of microglia in the region of the inferior vestibular nucleus was also significantly decreased after AlCl3 inoculation compared to that of NaCl-inoculated animals (Fig. 5). The number of microglia in the recovery groups was greater than that in AlCl3-inoculated groups (PB 0.01). In the saline control and recovery rabbits, microglia appeared very well ramified (Fig. 2C, G) and were evenly distributed in the ventral horn of the lumbar segment as well as in the area of the inferior vestibular nucleus. However, after AlCl3 inoculation, microglia showed thinner, less complex branching patterns (Fig. 2D,E,H). The blood vessels have been clearly stained with RCA-1. There was no apparent difference in the intensity of RCA-1 staining of blood vessels between AlCl3and NaCl-inoculated groups.
Fig. 2. (A – B) presents axotomized motoneurons. The motoneurons, one of them is NFH positive (red staining) after AlCl3 inoculation (B), show eccentric nuclei and are slightly swollen, features typical of the axotomized cell. Several microglia (black staining) have been found to closely contact to these motoneurons. In NaCl-inoculated rabbit (A), activated microglia (arrows) possess thicker processes. However, following acute AlCl3 inoculation (B), microglia show thinner branches (arrows). Figures (C – H) are taken from the chronic AlCl3 experiment. At lower magnification, microglia in NaCl-inoculated animals (C) are more obvious than those in AlCl3-inoculated ones (D). Figure E show a single microglia (arrow) closely related to a neuroaxonal spheroid (illustration from day 56 post-inoculation). (F) Following the 6th inoculation in the chronic AlCl3 experiment, neuroaxonal spheroids demonstrated less microglial attachment, and in many instances, there were no associated microglia. (G) A microglia (arrow) from the lumbar section of an NaCl-inoculated rabbit. Several processes branch out from its cell body. After the 4th AlCl3-inoculation (H), microglia (arrow) show thinner and less complex process, compared with those in control and recovery groups. Arrowheads show some of RCA positive vascular vessels. No obvious difference was observed in the RCA staining intensity in vessels in the lumbar cord and nucleus vestibularis between AlCl3- and NaCl-inoculated groups. Scale Bar: 25 mm (A, B), 160 mm (C, D), 25 mm (E, F), 15 mm (G, H).
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Fig. 2.
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NFH positive structures were also mainly located in the ventrolateral region of the spinal cord. Some positive structures could be enlarged axons where the neurofilament aggregated. Microglia could be noted to be close to or attached to, NFH positive structures (Fig. 2E) in the spinal cord of AlCl3-inoculated or NFH negative motoneurons in the spinal cord of both AlCl3inoculated and control animals. NFH positive structure did not, however, always possess a close spatial relationship with microglia (Fig. 2F). In fact, microglial processes seldom surrounded the NFH positive structures totally. The relationship between the induction of neurofilamentous aggregates and the proximity microglial processes is presented in Fig. 6.
4. Discussion Motoneurons are uniquely sensitive to the toxicity of AlCl3 both in vivo and in vitro (Strong and Garruto, 1991b; Strong et al., 1995). In vivo, following the inoculation of either organic or inorganic aluminum compounds, affected neurons develop neurofilamentous aggregates in the neuronal soma, axons and dendrites. Acute high dose AlCl3 exposure induces widespread neurofibrillary tangle-like perikaryal inclusions throughout the CNS (Bugiani and Ghetti, 1982), and culminates in a fulminant disease course. In contrast, chronic low-dose AlCl3 intoxication brings about progressive spasticity with spinal motoneuron degeneration
and pathologically confirmed neurogenic muscular atrophy (Strong et al., 1995), resembling that observed in human ALS (Wakayama et al., 1996). However, in contrast to the almost uniformly fatal ALS, the pathological changes of motoneurons following chronic AlCl3 exposure are, to some extent, reversible following cessation of AlCl3 exposure (Strong et al., 1995). While the most obvious reason is the removal of the pathological stimulus in this chronic model, it is also possible that the response of AlCl3-exposed spinal motoneurons is modified by either intrinsic or extrinsic factor(s) as yet unknown, such as microglia. As discussed, activated microglia are capable of expressing many immuno-antigens found on peripheral macrophages (Kawamata et al., 1992) and of participating in cytotoxicity (Banati et al., 1993). Following peripheral nerve injury, resting microglia become active (Graeber et al., 1988) and proliferate and migrate towards the perikarya of axotomized motoneurons (Streit et al., 1988; Kreutzberg et al., 1989). Inhibition of this response is associated with an enhanced axonal regeneration following axotomy (Thanos et al., 1993). Although microglia can respond to very minor pathological stimuli, and both acute and chronic AlCl3 exposure induces significant neuronal injury, we have observed a failure of a microglial response in AlCl3 neurotoxicity. Firstly, a significant reduction in microglial number was noted in the lumbar segment of the spinal cord and in the area of the inferior vestibular nucleus following chronic AlCl3 inoculation. Although
Fig. 3. Post-axotomy analysis of microglial attachment to NFH positive structures. The figure shows the total number of NFH positive cells and spheroids in cord sections at 4 days after sciatic axotomy and 2 days after either high dose AlCl3 intracisternal exposure or NaCl inoculation. A fewer NFH positive structures were observed in the NaCl-inoculated animals compared to AlCl3-inoculated animals. The number of diseased motoneurons to which two or more microglia attach is less than observed in control.
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Fig. 4. Chronic AlCl3-inoculation microglial analysis. Animals were intracisternally inoculated with 100 mg AlCl3 in 100 ml 0.9% NaCl at intervals of 28 days for a total of 267 days (n= 6), or autopsied at day 51 (n =5), day 107 (n = 6) or day 156 (n = 6). At each time interval, three rabbits were allowed to survive without further AlCl3 exposure and another two were inoculated with 100 ml 0.9% NaCl. At each autopsy interval, the lumbar ventral horns of AlCl3-inoculated animals showed significantly less microglial cells than saline controls (P B 0.001). In the absence of further AlCl3 exposure (recovery), microglial number becomes similar to that of saline control animals.
Fig. 5. Chronic AlCl3-inoculation microglial analysis. The number of microglia in vestibularis nucleus after AlCl3 inoculation showed significantly less than that after NaCl inoculation (PB 0.001). In the recovery group, the microglial number becomes similar to that of NaCl inoculated animals.
we do not know the cause of this, applying AlCl3 after a sciatic axotomy resulted in a reduction of the microglial numbers in the ipsilateral ventral horn of the lumbar cord segment, suggesting that AlCl3 does inhibit microglial proliferation. Secondly, while there were many diseased motoneurons in the spinal cord of rab-
bits after either chronic or acute AlCl3 exposure, microglia still showed thinner, less complex branching patterns. This further supports the suppressive role of AlCl3 to microglial transformation. Thirdly, in the chronic AlCl3 experiments, the majority of NFH positive cells were not closely surrounded by microglia,
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Fig. 6. The number of NFH positive cells and spheroids in cord sections after lower dose AlCl3 intracisternal exposure. There are more NFH positive structures at day 107 and day 156 after AlCl3 inoculation. However, most of them are not closely related with microglia. Rare NFH immunoreactive structures were observed in both saline control and recovery rabbits.
suggesting microglial migration was also hindered. In the acute-axotomy group, we also failed to see microglia enveloping NFH positive cells or spheroids after aluminum exposure. Although the mechanisms of AlCl3 suppression of microglial activation are not understood, the suppression is not directly related or due to the presence of degenerating neurons. In the present study, we found a suppression of microglia in the area of the inferior vestibular nucleus in which no obvious neurodegeneration was observed. Although there is very little evidence to indicate directly that microglia may be toxic to neurons in vivo, in vitro microglia have the capacity to develop into cytotoxic macrophages (Frei et al., 1978). Co-culture of neurons with activated microglia increases neuronal death (Thery et al., 1991; Chao et al., 1992), and microglia can phagocytose neuronal debris (Torvik, 1972). Upon cellular activation in vitro, microglia release proteases that could potentially result in degradation of the extracellular matrix and thereby compromise neuronal function (Colton et al., 1993). Abundant nitrite, a product of the free radical nitric oxide, was detected in activated microglia and microglia/neuron co-cultures but not in astrocyte or neuron cultures alone (Chao et al., 1992). The latter implied that microglia were the principal source of the nitric oxide and that activated microglia might kill neurons via a nitric oxide mediated mechanism. Although it has been shown that microglia upregulate inducible nitric oxide synthase in vitro following Zeolite (aluminosilicate par-
ticles) exposure (Garrel et al., 1994), it is not known if AlCl3 can have the same effect. Microglia are also the major cellular source of the immunoregulatory cytokines such as interleukin-1 (IL-1) (Giulian et al., 1985), IL-5 (Sawada et al., 1993), and tumor necrosis factor-a (TNF-a) (Sawada et al., 1989). It is of interest, therefore, that motor neuron death in neurodegeneration may be TNF-a-mediated (Choe et al., 1998; Ghezzi et al., 1998). Because we previously observed both clinical and neuropathological recovery following chronic AlCl3 exposure, and now report the inhibition of microglial proliferation and activation in the same model, we are proposing that the failure to induce a microglial response is permissive to recovery in the chronic AlCl3 model. Further in vitro experiments are in process to determine the effect of AlCl3 on microglial function.
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