Change Of Microglia In The Lumbar Spinal Cord And Motor Cortex Of SOD1-G93A Transgenic Mice

Objective: Amyotrophic lateral sclerosis (ALS) is a fatal neurodeg- enerative disease, characterized by progressive muscular atrophy, weakness and pyramidal signs. Patients usually die in 3-5 years after the onset due to bulbar paralysis, respiratory muscle paralysis or lung infection. It is characterized mainly by the loss of upper motor neurons (MN) or lower MNs or both, and activation of astrocytes and microglia. 5-10% of ALS cases are familial (fALS), the majority of cases (90-95%) are sporadic (sALS). The clinical manifestation of sporadic and familial ALS are very similar. Approximately 20% of fALS and 4% of sALS are linked to mutations in the gene encoding for the ubiquitous copper/zinc superoxide dismutase (SOD1). Among this the G93A (glycine to alanine substitution at the 93rd codon) site is the most common mutant. SOD1-G93A transgenic mice display progressive motor neuron degeneration and similar to the cases of human ALS. So the mutant SOD1-G93A mice are the most common animal model to investigate ALS in the world.In vivo and in vitro studies using human SOD1 (hSOD1) transgenic mice have demonstrated that human mutant SOD1 (hmSOD1) induces vulnerability of motor neurons through aberrant protein aggregation, mitochondrial dysfunction, oxidative stress, cytoskeletal abnormalities, defective axonal transport, glutamate excitotoxicity, inadequate growth factor signaling, and neuroinflammation.Neuroinflammation is a term coined to describe cellular and molecular processes which encompasses activation of microglia and astrocytes and infiltration of peripheral immune cells. It occurs in the central nervous system (CNS) in a variety of pathological conditions including neurodegenerative diseases, such as ALS. Neuroinflammation is a pathological feature present in ALS patients and in the mutant hSOD1 mouse model. Gliosis is a consistent feature of neuroinflammation in neurodegenrative diseases. In ALS, the areas of neuroinflammation are composed of primarily astrocytes and microglia, and to a much lesser extent, T-lymphocytes. The cumulative evidence suggests that inflammation plays a central role in ALS, however the nature of microglial -neuronal interactions that lead to motor neuron degeneration remains unknown. One possibility that has been extensively studied is the notion that chronic and detrimental microglial neuroinflammation contributes to neuronal degeneration.This study was developed using the widely studied SOD1-G93A transgenic mouse model of ALS. Immunohistochemistry and laser confocal microscope were used to investigate the microglial change in shape and number in the lumbar spinal cord and motor cortex of SOD1-G93A transgenic mice at different stages. Western blotting was used to study the expression of microglia-specific marker with the disease progressed. The aim was to study the change of microglia in the disease progression, discuss its possible role in motor neuron degeneration, and provide potential targets for ALS therapy.Methods:1 Breeding of transgenic miceAll animals were kept at constant temperature and humidity and sterile conditions (specific pathogen free, SPF), fed with sterilized SPF rodents feed particles. Experiments were carried out according to the rugulations of laboratory animal management of HeBei province. Animals were maintained with ad libitum access to water and diet, three or five mice per cage. Transgenic SOD1-G93A mice and their non-transgenic littermates were generated by breeding hemizygous male B6SJL-Tg (SOD1-G93A) 1 Gur/J mice to female B6SJLF1 hybrids, both of which were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and originally produced by Gurney et al. The genetic offspring mice were identified at three weeks of age by genotyping for human SOD1 with tissue from tail.2 Motor function score and experimental groups 2.1 Motor function ScoreThe mice were evaluated for signs of motor deficit daily, beginning at 50 days of age, with the following 4 point scoring system. (1). 4 point, Normal, no sign of motor dysfunction. (2). 3 point, Hind limb tremors are evident when suspended by the tail. (3). 2 point, Gait abnormalities are present. (4). 1 point, Dragging of at least one hind limb. (5). 0 point, Inability to right itself within 30s when placed on either side or its back.2.2 Experimental groupsMice were divided into three groups including pre-clinical (60 days), clinical onset (about 100-120 days), and end stage (about 130-150 days), based on the pathogenous law of SOD1-G93A mice. Each time point included model groups and littermate control groups. SOD1-G93A transgenic mice were model groups, non-transgenic littermate mice were control groups. Each group had 6 female mice at each time point. Pre-clinical group was 60-day-old mice, without clinical symptoms and no weight loss. Clinical onset was defined when the mice began to lose weight, and show gait abnormalities. When the mice lost > 20% of their body weight, and could not upright itself in 30 seconds after being placed on either side or its back, it was scored as end stage.3 Methods3.1 SamplingTo monitor disease progression, the animals were inspected, scored and weighed every day. After anesthesia with 10% chloral hydrate (350mg/kg), SOD1-G93A transgenic mice were sacrificed at pre-clinical (60 days), clinical onset (about 100-120 days) or end stage (about 130-150 days), and the non-transgenic littermates were also sacrificed respectively in the corresponding times. Spinal cord and motor cortex were preserved in the fixative after transcardially perfusion with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in PBS, or freshly dissected, put into 1.5ml centrifuge tubes, froze in liquid nitrogen, and then stored in -80℃refrigerator.3.2 Western blottingTen milligram of whole tissue extracts were prepared using a total protein extraction kit following the manufacturer's instruction and quantified using the BCA method. Thirty micrograms of protein from each sample was run on 10% SDS-PAGE gels, and blotted onto PVDF membranes. After blocking in 5% skim milk for 1 hour, primary antibodies including rabbit anti-CD11b (1:200), mice anti-GAPDH (1:200) or goat anti-SOD1 (1:200) was added respectively and the memebranes were incubated overnight at 4℃. Next day, the membranes were rinsed with TPBS for 5 times, 5 minutes every time. Then anti-rabbit, anti-mice or anti-goat fluorescence-labeled secondary antibody (1:3000) was added. After incubation for 1 h at room temperature, the membranes were rinsed for 4 times with TPBS, and 1 time with PBS, 5 minutes every time. The bands of interest were detected using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). Band intensity was quantified, normalized by the GAPDH band, and statistically analyzed.3.3 ImmunohistochemistrySpinal cord and motor cortex tissue fixed by 4% paraformaldehyde, were cut into 20μm free-floating sections using a vibratome (Leica VT 1000S). The sections were rinsed for 3 times in 0.01 M PBS, 5min every time and then treated with 3% hydrogen peroxidase in methanol for 15 min. After rinsing in 0.01M PBS for 3 times, 5min every time, the sections were perforated with 0.3% Triton X-100 for 15 min. After blocking with 10% horse serum for 1h at room temperature, the sections were incubated overnight at 4°C with antibody against Iba1 (1:200). After rinsing, the sections were subsequently incubated with a biotin-conjugated secondary antibody (goat anti-rabbit IgG, 1:200) for 2h at room temperature, followed by horseradish peroxidase conjugated streptavidin for 1h at room temperature and treatment with 0.03% diamino- benzidine as a chromogen for 10min. The sections were spread onto the slides, dehydrated in ethanol, cleared in xylene, and mounted.3.4 Confocal microscopy The lumbar spinal cord fixed by 4% paraformaldehyde was cryoprotected in a 30% sucrose/4% paraformaldehyde solution for 12h. Buried in OCT embedding medium, the spinal cord tissue was frozen and sliced into 30μm sections using a Leica CM1850 freezing microtome. Similar to immunohistochemistry, the sections were rinsed, perforated, blocked and incubated with Iba1 antybody, then incubated with FITC-labelled goat anti-rabbit secondary antibodies for 1h at room temperature. After rinsing with PBS, Slides were mounted with antifade solution and analyzed by fluorescent confocal microscopy (Olympus FV1000).3.5 Microglial countingNikon 50i research microscope was used. Five fields from three mice in each group were analysed at 400×magnification. Iba1 positive cells with entire cell body were counted in the lumbar anterior horn and motro cortex of SOD1-G93A transgenic mice and their littermates at pre-clinical, onset and end stages.3.6 Statistical analysisResults were expressed as means±S.D. Statistical analyses were performed using one-way ANOVA with SPSS 13.0.4 statistical software. Differences were considered significantat at P < 0.05.Results:1 Identification of transgenic miceThe PCR amplification products of geneome DNA from offspring mice demonstrates: a 324 bp band, which is the PCR product of IL-2, and a 236 bp band, which is the PCR product of hSOD1 gene.2 Clinical manifestationSOD1-G93A transgenic mice did not show any clinical changes at 60 days of age and scored 4. At about 80-90 days of age, the transgenic mice showed tremor of their hind limbs when suspended by tail and scored 3. At about 100-120 days of age, one or two hind limbs of SOD1-G93A mice began to weak and showed muscle atrophy, and gait abnormality was observed. At the beginning of gait abnormality, the mice were scored 2. Then, muscle weakness progressed and hind limbs couldn't support the body. Subsequently, at least one hind limb paralyzed and was dragged when walking. At this time, the mice were scored 1. At about 130-150 days of age, the SOD1-G93A mice can't upright itself within 30sec and lost weight >20%. The mice were scored 0 and considered as end stage. The above manifestations were not observed with non-transgenic littermates that were scored 4 all the time.3 Changes of microglia3.1 Westen blotting results of CD11b (specific maker of microglia) showed low expression of CD11b in the lumbar spinal cord of non-transgenic mice and no difference was observed from 60 to 150 days of age (P>0.05). In the lumbar spinal cord of SOD1-G93A transgenic mice, CD11b expression increased slightly at pre-clinical stage and dramatically at clinical onset stage, and reached maximum at end stage (P<0.05). However, in the motor cortex of SOD1-G93A transgenic mice, no significant difference of CD11 expression was observed compared with non-transgenic littermates (P>0.05).3.2 Immunohistochemistry for Iba1 (another specific maker of microglia) showed that few Iba1-positive microglia with a small cell body and few prominences were observed in the lumbar ventral horn of littermates, and there was no significant difference from 60 to 150 days of age. At the pre-clinical stage of SOD1-G93A mice, Iba1-positive microglia increased slightly, and became obvious at onset and end stages, showing large cell body and thick prominences. Similarly, Iba1-positive microglia increased slightly at the onset and end stages in the posterior horn, which is much less than the anterior horn. However, the number of Iba1-positive microglia in the motor cortex of SOD1-G93A mice did not change compared with non-transgenic littermates.Conclusions:There are microglia proliferations in the disease progression of SOD1-G93A mice, which occur prior to the clinical manifestation, and become obvious at onset stage and reach a peak at the end stage. Microglia proliferation is in accordance with the vulnerable region, and the proliferative extent is proportional to the severity of disease. Microglial activation in the lumbar spinal cord of SOD1-G93A mice may contribute to the injury of motor neurons.