Article Figures & Data
Figures
- Figure 1.
SPARC is expressed by mature ramified microglia in the brain. A, At P6 migratory, amoeboid developing microglia expressing Iaba-1 (red, closed arrowhead) enter the brain parenchyma and are detected as clusters around the lateral ventricle (LV) that rarely coexpress SPARC (green), which is enriched in radial glial processes (green). B, C, From P9 to P12, microglia migrate away from the LV into the brain, differentiate into a ramified morphology, and begin to express SPARC (open arrowhead; inset). D, In the P6 cerebral cortex, SPARC is primarily expressed in radial glia processes (green, arrow) and is absent from Iba-1-expressing immature microglia (arrowhead, red). E, F, From P9 to P12, microglia invade the cortex and become ramified, and by P12, SPARC is coexpressed with Iba-1 (yellow-orange, open arrowheads) in all cortical microglia. In the adult brain, SPARC is expressed by Iba-1-positive microglia in all regions, including the cerebral cortex (G; Cx); the corpus callosum (H; CC); the CA1 region of the hippocampus (CA1; I); the cerebellum (J; Cb), where SPARC is also detected in Bergmann glia (Iba-1-negative); and the thalamus (K; Th), one of the few adult regions where Iba-1-negative astrocytes express SPARC (green). L, SPARC is not detected in Iba-1-positive macrophages in the adult choroid plexus (CP). Scale bars: 20 μm.
- Figure 2.
SPARC is downregulated in microglia and upregulated in astrocytes following photothrombotic stroke. A–D, Using VIP immunohistochemistry, SPARC is initially absent from the initial infarct (A; demarcated by dotted line) but is readily detected at all levels of the lesion core and perilesion zone at 1 week after photothrombotic stroke (B; rostral edge of lesion shown, arrowhead), and is elevated at the center of the lesion and cells migrating to the lesion (C, D; arrows) until at least 4 weeks post injury. At 1 week post lesion (E), SPARC appears diffusely throughout the lesion core, and although SPARC (green) colocalizes (yellow) in Iba-1-expressing (red) ramified microglia (F; arrowhead) in the uninjured cortex, SPARC is not in activated hypertrophic microglia (G; red, open arrowhead) in the perilesion zone (region, asterisk in E, H, K). I, SPARC is normally undetectable in GFAP-expressing (red) cortical astrocytes (arrowhead), but is upregulated (H, J) in hypertrophic astrocytes at the outer margin of the perilesion zone (open arrowhead). K–M, SPARC (green/white) is also upregulated on cells closely associated with laminin-expressing (red) blood vessels in the lesion core and perilesion zone. Scale bars: A–D, E, J, K, H, 100 μm; F, G, I, J, L–M, 10 μm.
- Figure 3.
SPARC is lost in reactive microglia following NMDA excitotoxic lesion of the OB. A–C, In the contralateral unlesioned OB (B) SPARC (green) is coexpressed by ramified Iba-1-expressing (red) microglia in all neuronal layers of the OB, with the glomerular layer (GL) and external plexiform (EPL) layer shown. D–F, High magnification of boxed area in A, showing SPARC coexpressed by Iba-1-expressing ramified microglia (arrowheads). G–I, Twenty-four hours after infusion of NMDA into the ipsilateral OB, at the peak of excitotoxic neuronal death, Iba-1-positive microglia become hypertrophic and reactive (I), at which point SPARC immunoreactivity is no longer detectable in microglia (H). J–L, High magnification of boxed area in G, showing SPARC-negative hypertrophic microglia (open arrowheads). Scale bars: (in C) A–C, 100 μm; (in I) G–I, 100 μm; (in F) D–F, 20 μm; (in L) J–L, 20 μm.
- Figure 4.
The absence of SPARC differentially regulates microglial expansion, process length, and branching in cortical gray and white matter in the uninjured SPARC-null cerebral cortex. A, B, GFP-expressing ramified microglia are evenly distributed, but Iba-1-positive cells (C) are more dense (n = 3) in SPARC nulls than control (+/+) (p < 0.01) and significantly less dense in the white matter of the corpus callosum (p < 0.05) compared with control mice. D, Microglial morphology was analyzed by visualizing 3D deconvolved images of GFP+ microglia in SPARC null/CX3CR1-GFP double transgenic mice and skeletonizing to analyze for process outgrowth (microns per cell) and number of primary branch points (E, arrowhead). F–I, SPARC-null microglia in the gray matter (G; GM) and white matter (I; WM) have increased process outgrowth (J; p < 0.05, Student's t test) and, in white matter, have more primary branch points (K; p < 0.01) than control microglia.
- Figure 5.
SPARC-null mice do not develop smaller lesions following photothrombotic stroke. Seven days after inducing photothrombotic stroke (7 DPI). A, Using immunofluorescence for GFP or Iba-1 (green) and Gal-3 (red) in staggered sections through the lesion site reveals a similar location and extent of lesion in SPARC nulls (−/−) and controls (+/+). (Asterisk indicates sections that were used in calculations, but removed for figure construction). Black vertical bars indicate distance from bregma. The lesion site could be clearly delineated with a combination of DAPI+ nuclei (B, D) GFAP (C, E) in the astrogliotic lesion penumbra. F, At 7DPI, lesion volumes in both SPARC-null (−/−) and control (+/+) mice were variable but not significantly different, with lesion volume shrinking in both genotypes by 32 DPI, but still not significantly different from each other. G, Indicates the cortical area within which all lesions were located. Scale bars: A, 100 mm; C, E, 60 mm.
- Figure 6.
In SPARC nulls, microgliosis is increased and functional recovery is enhanced, following photothrombotic stroke. A, B, Seven days after photothrombotic stroke lesion, Gal-3 (red, used to indicate a high level of microglial activation) is not expressed by GFP-expressing (green) ramified microglia in the contralateral cortex (A; detail in insets), but is (B) expressed by a small proportion of hypertrophic activated microglia in the perilesion zone (peri), and is expressed by all amoeboid GFP-CX3CR1-positive cells in the lesion core (C). D–F, In CX3CR1-GFP/SPARC control mice 1 week after surgery, both Gal-3 immunoreactivity and GFP-CX3CR1 signal are increased in the lesion core (images are representative maximal lesion core cross section). G–I, In SPARC nulls, Gal-3-expressing cells and GFP-expressing cells in the lesion core and prelesion area appear to be significantly enhanced. J, Gal-3 immunoreactivity (IR) and GFP-CX3CR1 normalized detection are significantly increased in the SPARC-null CNS relative to the wild-type (assayed area 1.5 × 1 mm centered on lesion as shown in D and G; n = 6–7, p < 0.05 in both cases). K, In the forelimb preference cylinder test, both SPARC-null and control mice showed a decrease in contralateral forelimb function following photothrombotic lesion to the forelimb sensorimotor cortex. SPARC-null mice demonstrated increased functional recovery by 32 d (p < 0.01, Student's t test with Welch's correction), SEM. Scale bars: A–C, 40 μm; D–I, 500 μm.
- Figure 7.
SPARC inhibits microglial proliferation in vivo and in vitro. Two days following photothrombotic stroke surgery, cortical microglia adjacent to the lesion in SPARC controls (A) become activated (more amoeboid) and proliferate (coexpress proliferating cell nuclear antigen, PCNA; red, arrowheads), a response which appears enhanced at this early stage lesion in the (B) SPARC null. C, Proliferation of microglia, assessed by coexpression of PCNA with GFP-CX3CR1, is significantly increased in the SPARC-null cerebral cortex (images are representative; assayed area 1.5 × 1 mm centered on lesion; p < 0.05) relative to the wild-type controls. D, SPARC is not expressed in vitro by partially activated NFκB-expressing microglia derived from postnatal mixed glial cultures, but is expressed in vitro by the majority of (E) adult-derived microglia. F–H, Microglial cultures derived from SPARC-null adult mice are significantly higher in density after DIV 8, when compared with wild-type microglia. H, SPARC-null adult-derived microglia proliferate at a significantly greater rate than wild-type microglia, as assessed by thymidine analog (EdU) uptake over 48 h. This effect is diminished by addition of exogenous SPARC to the culture media. Scale bars: 20 μm. All error bars are SEM.
