Activation of microglia, CNS resident immune cells, is a pathological hallmark 
of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting 
motor neurons. Despite evidence that microglia contribute to disease 
progression, the exact role of these cells in ALS pathology remains unknown. We 
immunomagnetically isolated microglia from different CNS regions of SOD1(G93A) 
rats at three different points in disease progression: presymptomatic, symptom 
onset and end-stage. We observed no differences in microglial number or 
phenotype in presymptomatic rats compared to wild-type controls. Although after 
disease onset there was no macrophage infiltration, there were significant 
increases in microglial numbers in the spinal cord, but not cortex. At disease 
end-stage, microglia were characterized by high expression of galectin-3, 
osteopontin and VEGF, and concomitant downregulated expression of TNFα, IL-6, 
BDNF and arginase-1. Flow cytometry revealed the presence of at least two 
phenotypically distinct microglial populations in the spinal cord. 
Immunohistochemistry showed that galectin-3/osteopontin positive microglia were 
restricted to the ventral horns of the spinal cord, regions with severe motor 
neuron degeneration. End-stage SOD1(G93A) microglia from the cortex, a less 
affected region, displayed similar gene expression profiles to microglia from 
wild-type rats, and displayed normal responses to systemic inflammation induced 
by LPS. On the other hand, end-stage SOD1(G93A) spinal microglia had blunted 
responses to systemic LPS suggesting that in addition to their phenotypic 
changes, they may also be functionally impaired. Thus, after disease onset, 
microglia acquired unique characteristics that do not conform to typical M1 
(inflammatory) or M2 (anti-inflammatory) phenotypes. This transformation was 
observed only in the most affected CNS regions, suggesting that overexpression 
of mutated hSOD1 is not sufficient to trigger these changes in microglia. These 
novel observations suggest that microglial regional and phenotypic heterogeneity 
may be an important consideration when designing new therapeutic strategies 
targeting microglia and neuroinflammation in ALS.