Osteopontin and its spatiotemporal relationship with glial cells in the striatum of rats treated with mitochondrial toxin 3-nitropropionic acid: possible involvement in phagocytosis

Background: Osteopontin (OPN, SPP1) is upregulated in response to acute brain injury, and based on its immunoreactivity, two distinct forms have been identified: intracellular OPN within brain macrophages and small granular OPN, identified as OPN-coated degenerated neurites. This study investigates the spatiotemporal relationship between punctate OPN deposition and astroglial and microglial reactions elicited by 3-nitropropionic acid (3-NP). Methods: Male Sprague-Dawley rats were intraperitoneally injected with mitochondrial toxin 3-NP and euthanized at 3, 7, 14, and 28 days. Quantitative and qualitative light and electron microscopic techniques were used to assess the relationship between OPN and glial cells. Statistical significance was determined by Student's t test or a one-way analysis of variance followed by Tukey's multiple comparisons test. Results: Punctate OPN-immunoreactive profiles were synthesized and secreted by amoeboid-like brain macrophages in the lesion core, but not by reactive astrocytes and activated microglia with a stellate shape in the peri-lesional area. Punctate OPN accumulation was detected only in the lesion core away from reactive astrocytes in the peri-lesional area at day 3, but had direct contact with, and even overlapped with astroglial processes at day 7. The distance between the OPN-positive area and the astrocytic scar significantly decreased from days 3 to 7. By days 14 and 28 post-lesion, when the glial scar was fully formed, punctate OPN distribution mostly overlapped with the astrocytic scar. Three-dimensional reconstructions and quantitative image analysis revealed numerous granular OPN puncta inside the cytoplasm of reactive astrocytes and brain macrophages. Reactive astrocytes showed prominent expression of the lysosomal marker lysosomalassociated membrane protein 1, and ultrastructural analysis confirmed OPN-coated degenerating neurites inside astrocytes, suggesting the phagocytosis of OPN puncta by reactive astrocytes after injury. Conclusions: Punctate OPN-immunoreactive profiles corresponded to OPN-coated degenerated neurites, which were closely associated with, or completely engulfed by, the reactive astrocytes forming the astroglial scar in 3-NP lesioned striatum, suggesting that OPN may cause astrocytes to migrate towards these degenerated neurites in the lesion core to establish physical contact with, and possibly, to phagocytose them. Our results provide novel insights essential to understanding the recovery and repair of the central nervous system tissue.