Perivascular cells expressing platelet-derived growth factor receptor beta (PDGFR-β) have recently been implicated in fibrotic scar formation after acute brain injury, but their precise identity and detailed morphological characteristics remain elusive. This study sought to characterize and define the cellular phenotype of vascular-associated cells expressing PDGFR-β in the striatum of rats treated with the mitochondrial toxin 3-nitropropionic acid (3-NP). In the control striatum, PDGFR-β-positive cells were invariably localized on the abluminal side of smooth muscle cells of larger caliber vessels, and demonstrated morphological features typical of perivascular fibroblasts. PDGFR-β expression increased and expanded to almost all vessels, including microvessels in the lesion core, at 7 days after 3-NP injection. The cells expressing PDGFR-β had ultrastructural features of fibroblasts undergoing active collagen synthesis: large euchromatic nuclei with a prominent nucleolus, well-developed rough endoplasmic reticulum (rER) with dilated cisterns and extracellular collagen fibrils. By 14 days, PDGFR-β-positive cells had somata located at a distance from the vasculature, and their highly ramified, slender processes overlapped with those from other cells, thus forming a plexus of processes in the extravascular space of the lesion core. In addition, their ultrastructural morphology and spatial correlation with activated microglia/macrophages were elaborated by three-dimensional reconstruction. Using a correlative light- and electron-microscopy technique, we found that the intermediate filament proteins nestin and vimentin were induced in PDGFRβ-positive fibroblasts in the lesion core. Collectively, our data suggest that perivascular PDGFR-β-positive fibroblasts are distinct from other vascular cell types, including pericytes and contribute to fibrotic scar formation in the lesion core after acute brain injury. Nestin and vimentin play critical roles in the structural dynamics of these reactive fibroblasts.

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). 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. 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 lysosomal-associated membrane protein 1, and ultrastructural analysis confirmed OPN-coated degenerating neurites inside astrocytes, suggesting the phagocytosis of OPN puncta by reactive astrocytes after injury. 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.

Our aim was to examine the spatiotemporal profiles and phenotypic characteristics of neuron-glia antigen 2 (NG2) glia and their associations with neuroglial cells in striatal lesions due to the mitochondrial toxin 3-nitropropionic acid (3-NP). In control striatum, weak NG2 immunoreactivity was restricted to resting NG2 glia with thin processes, but prominent NG2 expression was noted on activated microglia/macrophages, and reactive NG2 glia in the lesion core after 3-NP injection. Activation of NG2 glia, including enhanced proliferation and morphological changes, had a close spatiotemporal relationship with infiltration of activated microglia into the lesion core. Thick and highly branched processes of reactive NG2 glia formed a cellular network in the astrocyte-free lesion core and primarily surrounded developing cavities 2–4 weeks post-lesion. NG2 glia became associated with astrocytes in the lesion core and the border of cavities over the chronic interval of 4–8 weeks. Immunoelectron microscopy indicated that reactive NG2 glia had large euchromatic nuclei with prominent nucleoli and thick and branched processes that ramified distally. Thus, our data provide detailed information regarding the morphologies of NG2 glia in the lesion core, and support the link between transformation of NG2 glia to the reactive form and microglial activation/recruitment in response to brain insults.