Formation of multinucleated giant cells and microglial degeneration in rats expressing a mutant Cu/Zn superoxide dismutase gene
© Fendrick et al; licensee BioMed Central Ltd. 2007
Received: 12 January 2007
Accepted: 28 February 2007
Published: 28 February 2007
Microglial neuroinflammation is thought to play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS). The purpose of this study was to provide a histopathological evaluation of the microglial neuroinflammatory response in a rodent model of ALS, the SOD1G93A transgenic rat.
Multiple levels of the CNS from spinal cord to cerebral cortex were studied in SOD1G93A transgenic rats during three stages of natural disease progression, including presymptomatic, early symptomatic (onset), and late symptomatic (end stage), using immuno- and lectin histochemical markers for microglia, such as OX-42, OX-6, and Griffonia simplicifolia isolectin B4.
Our studies revealed abnormal aggregates of microglia forming in the spinal cord as early as the presymptomatic stage. During the symptomatic stages there was prominent formation of multinucleated giant cells through fusion of microglial cells in the spinal cord, brainstem, and red nucleus of the midbrain. Other brain regions, including substantia nigra, cranial nerve nuclei, hippocampus and cortex showed normal appearing microglia. In animals during end stage disease at 4–5 months of age virtually all microglia in the spinal cord gray matter showed extensive fragmentation of their cytoplasm (cytorrhexis), indicative of widespread microglial degeneration. Few microglia exhibiting nuclear fragmentation (karyorrhexis) indicative of apoptosis were identified at any stage.
The current findings demonstrate the occurrence of severe abnormalities in microglia, such as cell fusions and cytorrhexis, which may be the result of expression of mutant SOD1 in these cells. The microglial changes observed are different from those that accompany normal microglial activation, and they demonstrate that aberrant activation and degeneration of microglia is part of the pathogenesis of motor neuron disease.
Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Loss of motor neurons results in muscle paralysis and ultimately death due to respiratory failure. 5–10% of ALS cases are familial inherited in an autosomal dominant pattern, and of familial ALS cases 20% have been linked to mutations located in the Cu/Zn superoxide dismutase 1 (SOD1) gene [1–4]. The discovery that SOD1 gene mutations are linked to motor neuron disease has facilitated development of transgenic rodent models to mimic human disease [1, 2, 5], and these have provided important leads towards understanding the molecular pathology of ALS. Since SOD1 is critically involved in eliminating superoxide, an undesirable byproduct of oxidative phosphorylation and a potential source of oxidative damage, the fact that transgenic animals with SOD1 mutations show unchanged or even elevated SOD1 activity has led to the conclusion that it is not a lack of enzymatic activity that contributes to disease development but rather some acquired toxic property of the enzyme [6, 7]. Thus the question arises, what are the cellular targets of this toxicity? Several studies have shown that expression of mutant SOD1 limited to motor neurons is insufficient to cause motor neuron degeneration [8, 9], and work by Cleveland and co-workers has generated findings, which show that toxicity to motor neurons requires damage from mutant SOD1 acting within nonneuronal cells  and, more specifically, that microglial cells are important for late stage disease development . These findings point towards a critical involvement of microglia in motor neuron disease development, yet the nature of microglial-neuronal interactions that lead to motor neuron degeneration remains unknown. One possibility, which has also been studied extensively in the context of other neurodegenerative diseases, notably Alzheimer's disease, is the notion of chronic and detrimental microglial neuroinflammation . According to this theory, activated microglia are seen as the main cellular source of inflammatory mediators in the CNS and as such are thought to be potentially neurotoxic [13, 14]. Chronic neuroinflammation is thought to be involved also in the pathogenesis of ALS based on a variety of in vivo and in vitro studies concerned with studying microglial activation using both human and animal tissues [15–20].
In order to learn more about the role of microglia in the pathogenesis of motor neuron disease, we set out to investigate microglial activation in the G93A SOD1 mutant rat during natural disease progression. The results reported here are unexpected in that they reveal a highly abnormal microglial reaction that does not meet the criteria of an anticipated, characteristic neuroinflammatory response.
Animal use protocols were approved by the University of Florida Institutional Use and Care of Animals Committee (IUCAC). All transgenic animals used in this study were male Sprague Dawley NTac:SD-TgN(SOD1G93A)L26H rats obtained from Taconic Farms where animals were screened extensively for infections prior to shipping. Upon arrival animals were housed under SPF conditions. Age-matched, wild type Sprague Dawley rats were purchased from Harlan. The time course of disease progression varied among individual animals, but in general once symptoms developed disease progression was quite rapid causing death of most animals by 5 months of age.
To examine microglial morphology, microglial markers were used at three stages of the disease: 1) presymptomatic stage, where animals had no apparent muscle weakness. Animals studied in this group were aged 74–84 days; 2) early symptomatic stage (onset), where animals first showed evidence of hind limb weakness. Animals studied in this group were aged 113–117 days; 3) late symptomatic (end stage), where animals were no longer able to right themselves after 30s. Animals studied in this group were aged 135–156 days. For each of the three disease stages, 4 transgenic and 4 age-matched wild type control animals were used.
Tissue processing and immunohistochemistry
Animals were deeply anesthetized with pentobarbital and perfused transcardially with phosphate buffer saline (PBS) followed by a fixative solution containing 4% paraformaldehyde in PBS. The spinal cord and brain were dissected out and fixed overnight in 4% paraformaldehyde at 4°C, transferred to 30% sucrose and then frozen. Lumbar spinal cord, cortical, and brainstem sections were cut in the coronal plane at 20 μm on a cryostat, mounted on slides and air dried. Sections were pretreated in PBS with 0.5% Triton X-100 for 15 min, blocked in 10% normal goat serum for 30 min and incubated overnight at room temperature in the primary antibody diluted in buffer. The primary antibodies included MRC OX-42 (Serotec, Cambridge, UK) and MRC OX-6 (Serotec, Cambridge, UK) at 1:500. The slides were rinsed in PBS and incubated in secondary antibody (1:500) for 1 h. Following incubation, slides were rinsed and Horseradish Peroxidase Avidin D was applied (1:500; Vector, Burlingame, CA) and incubated for 30 min. Slides were washed and immunoreactivity was visualized with 3,3'-diaminobenzidine (DAB)-H2O2 substrate. After a brief rinse, slides were dehydrated in increasing concentrations of ethanols, cleared in xylene, and coverslipped using Permount mounting medium (Fisher Scientific).
OX-42 immunoreactivity in the ventral spinal cord was quantified using Image Pro Plus software (version 4.5.1, Media Cybernetics, Carlsbad, CA). The area occupied by stained cells was highlighted and measured for each section of spinal cord (6 sections per animal) then expressed as a percentage of total area of ventral spinal cord. Using GraphPad Prism software (San Diego, CA) a t-test was performed to determine statistical significance between transgenic SOD 1 and control animals at each time point. A one-way ANOVA was performed to compare differences among the transgenic animals followed by a Tukey multiple comparison test.
Paraffin processing and lectin histochemistry
Animals were deeply anesthetized and transcardially perfused with phosphate buffer saline (PBS) followed by a fixative solution containing 4% paraformaldehyde. The spinal cord and brain were dissected out and fixed 2 h in 4% paraformaldehyde. The tissue was dehydrated through ascending alcohols, cleared in xylenes and embedded in paraffin. Serial 7 μm coronal sections were collected and mounted on slides. Sections were deparaffinized through xylenes, graded alcohols and rinsed in PBS. Next, the slides were trypsin treated (0.1% trypsin, 0.1% CaCl2) for 12 min at 37°C. Following a 10 min wash the slides were incubated overnight at 4°C in lectin GSA I-B4-HRP (Sigma Chemical Co.) diluted 1:10 in PBS containing cations (0.1 mM of CaCl2, MgCl2 and MnCl2) and 0.1% Triton X-100. After overnight incubation slides were briefly rinsed in PBS and visualized with 3,3'-diabimobenzidine (DAB)- H2O2 substrate. Sections were counterstained with cresyl violet, dehydrated through ascending alcohols, cleared in xylenes and coverslipped with Permount.
Development of microgliosis during natural disease progression in the spinal cord
Histopathology in the brain stem
Within vacuolated spaces rounded, shrunken microglia exhibiting nuclear fragmentation or shrinkage (pyknosis) were identified using lectin histochemical staining (Figs. 4H).
Microglia in midbrain and cerebral cortex
Prior work in ALS rodent models involving SOD1 mutations has generated clues about an involvement of glial cells. Damage to astrocytes has been described to occur concomitant with degeneration of motor neurons prompting the hypothesis that astrocytic damage promotes motor neuron degeneration . However, subsequent experiments showed that restricted expression of mutant SOD1 genes in astrocytes is not sufficient to cause motor neuron degeneration . Notwithstanding these findings, more recently it was determined using chimeric animals consisting of mixtures of normal cells and cell expressing human mutant SOD1 that nonneuronal cells containing mutant SOD1 are indeed required to cause damage to motor neurons, whereas wildtype nonneuronal cells promote motor neuron survival [10, 24]. In addition, recent work has shown that mutant SOD1 acting within microglial cells specifically is a primary determinant of late stage disease progression . These observations gain added significance when considered together with the current findings showing widespread microglial degeneration in the spinal cord gray matter of end stage animals, because it now seems clear that mutant SOD1 is particularly toxic to microglia and that SOD1-mediated microglial degeneration is linked to a terminal neurodegenerative disease state. Thus, loss of microglial cells could be very detrimental to neuronal survival . Future research may be directed towards elucidating the molecular mechanisms that underlie SOD1's selective microglial toxicity, and towards ways of inhibiting it as a strategy for new ALS treatments.
We use the term "cytorrhexis" to describe the kind of microglial degeneration we observed in SOD1G93A rats because it involves disintegration of the cell's cytoplasm rather than of its nucleus. Cytorrhexis has been used previously only to describe neuronal necrosis resulting from excitotoxicity , but extending its use to describe microglial cytoplasmic deterioration is appropriate since this form of cell death does not involve the nuclear disintegration (karyorrhexis) that is characteristic of apoptosis. Cytorrhexis therefore describes accidental, rather than programmed, microglial cell death. Our inability to detect large numbers of apoptotic microglia in the tissues studied indirectly supports the idea that cytorrhexis is the "preferred" mode of microglial cell death during the toxic disease state thought to be generated by mutant SOD1 expression. Finding widespread microglial degeneration in this particular animal model of neurodegenerative disease strongly supports the broader concept that microglial abnormalities characterize other neurodegenerative conditions as well [27–29].
Perhaps the earliest sign of an aberrant microglial response in SOD1 mutant rats is reflected in our observation of occasional microglial fusions in presymptomatic animals. We suspect that with disease onset these progress to produce the conspicuous multinucleated giant cells composed of many microglia fused into large syncytia. The occurrence of microglial giant cells throughout the lumbar spinal gray matter, as well as the brainstem, and especially their selective localization in the red nucleus, raises the intriguing possibility that their formation is related to the fact that these regions all give rise to fibers that project onto ventral motor neurons. It is conceivable therefore that a signal is transmitted retrogradely from ventral horn cells to these supraspinal regions to trigger formation of microglial fusions, consistent with the notion of disease spread from an initially affected region . However, at the same time the notable absence of microglial abnormalities and/or activation in the motor cortex reported here would argue against this idea. Additional studies providing more detailed mapping of the location of giant cells could be helpful in this regard.
Fusion of microglia into giant cells represents an anomalous type of cellular behavior, since microglia are normally "territorial" and exhibit strong contact inhibition. Microglial giant cells have never been described to occur in situ in rat brain, but they can form spontaneously in vitro using cultured microglia from a variety of species [30–33]. Multinucleated giant cells are a pathological hallmark in human brain during infectious diseases, most notably in HIV/AIDS encephalopathy [34, 35], and since microglia are the main cellular target of HIV-1 in the brain it is thought that presence of virus within microglia causes the cells to fuse with each other. However, the exact mechanisms that produce microglial fusions in the SOD1G93A rat are unknown and require additional studies. But regardless of the mechanism(s) involved, it seems clear from the current observations that fusion of microglia into giant cells is an abnormal cellular response that can progress further to produce frank microglial degeneration evident as cytorrhexis. It is unknown currently whether microglial cytorrhexis occurs in HIV encephalopathy, but the fact that neurodegenerative changes accompany advanced, untreated HIV encephalopathy (clinically evident as AIDS-dementia complex) raises the intriguing possibility that microglial degeneration may be part of the pathogenesis of cognitive dysfunction associated with HIV encephalitis. In addition, descriptions of an ALS-like syndrome in HIV-infected subjects [36–38] leads us to hypothesize that dysfunctional or degenerating microglia may be a common denominator in these two seemingly unrelated conditions.
The thought that microglial neuroinflammation promotes motor neuron degeneration can be traced back to the initial descriptions of activated microglia in human ALS tissues . Since then, the idea of detrimental neuroinflammation has received additional support from a variety of studies documenting microglial activation and increased production of proinflammatory substances in human ALS tissues, serum and CSF as well as in SOD1 transgenic mice [15, 20, 39–44]. With regard to the SOD1 transgenic rat, the only report thus far concerning neuroinflammation has described elevated gene expression of proinflammatory mediators in the spinal cord, as well as downregulation of the neuroprotective factor VEGF .
Our current findings provide a perspective on the pathogenesis of neurodegenerative disease that is different from the neuroinflammation theory which claims that chronic neuroinflammation leads to motor neuron degeneration . We propose that, rather than being overly activated, the brain's immune cells fail in performing normal, neuroprotective functions and that degeneration of microglia contributes to neurodegeneration. This may significantly change future approaches towards treatment by pharmacological and other means.
Supported by NIH grant NS49185.
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