CCL2-ir levels in the cortex and CC of normal, saline and LPS injected mice
CCL2-ir density in the cortex and CC of normal and saline injected mice The CCL2-ir labeling was distinctively visualized in the immunostained sections (Fig. 1), while no visible positive structure was observed in the control stain (Fig. 1A, B). Most of CCL2-ir positive structures were observed surrounding the vasculature like profiles in both the cortex (Fig. 1C, D) and the CC (E, F arrows). In both the cortex and the CC, some neuron-like cells were encountered, as characterized by the clear DAPI marked nuclei that are centered in CCL2-ir positive ovoid or fusiform profiles (C~F insets). It appears that more neuron-like cells are scattered in I –III layers of the cortex (Fig. 1C, D) or in the cingulate cortex (Fig. 6). In the CC, CCL2-ir labeling is predominantly situated in bilateral parts of the CC, the areas between cingulate cortex and lateral ventricles (Fig. 1E, F aligned arrows). The staining was resulted from the combination of two CCL2 antibodies (e.g. PA5-34505 and AB25124, see Table 1), as illustrated in supplementary figure 1.
Statistical comparison of CCL2-ir intensities in normal and saline GM/WM Firstly, the comparison of CCL2-ir intensity found in the cortex GM and the CC WM showed a significant difference (Fig. 2A, p < 0.05). Secondly, the intensity of CCL2-ir staining in the CC including an ependymal layer (CC+Epend) was significantly higher than that in the CC without an ependymal layer (Fig. 2B, p < 0.05). The CCL2-ir intensity in the CC+Epend was significantly higher than the CCL2-ir intensity in the cortex (Fig. 2C, p < 0.01). Meanwhile, CCL2-ir intensity in the cortex or CC of the normal vs saline injected mice showed no obvious difference (Fig. 2D), which indicates the latter can represent the normal cases.
CCL2-ir intensities in cortex and CC of LPS injected mice It seemed that more CCL2-ir labeling overlapped with neuron-like structures (Fig. 1D insets) in the cortex of LPS injected mice. In all rostral, middle and caudal parts of the CC in the LPS injected mice, more CCL2-ir labeling appeared to be confined in the blood vessels (Fig. 1G-J, opened arrows). This kind of labeling was seen less often in the cortex compared to the CC.
Statistical comparison of CCL2-ir intensities in the saline vs LPS injected mice The intensity of CCL2-ir labeling in the cortex (Fig. 2E) of the LPS injected mice was significantly higher than that in the saline injected cases (Fig. 2E, p < 0.05). While in the CC, the difference of CCL2-ir intensity between saline and LPS treated mice was evident, and nearly reached to a statistically significant level (Fig. 2E, p = 0.054). Similarly, CCL2-ir intensity in the CC+Epend of the LPS group was obviously higher than that in the saline group (Fig. 2E, p = 0.074).
Cellular CCL2-ir distribution in brain of saline and LPS injected mice
Co-localization of CCL2-ir and GFAP-like labeling in the cortex and CC The CCL2-ir and GFAP-like double labeled structures were stained in the cortex and the CC of the normal or saline injected mice (Fig. 3), and the double labeling was usually in areas surrounding vasculature-like (arrows) structures (Fig. 3A, C, arrow-arrowheads, inset). However, the CCL2-ir and GFAP labeling appeared to not always overlap (Fig. 3A GFAP Merged). Co-localization of CCL2 and GFAP immunoreactivity was also observed in the ependymal layers attached to the CC (Fig. 5A, arrow-arrowhead).
Following systemic LPS, co-localization of CCL2 (Fig. 3 arrows) and GFAP-like immunoreactivity was also mainly observed in areas surrounding the vasculature-like structures in both the cortex (Fig. 3B) and the CC (D, insets, arrow-arrowheads). And most of the double labeling in the CC was scattered in regions between the cingulate cortex and the lateral ventricle (Fig. 3C, D). Similarly, the CCL2-ir and GFAP double labeling was also seen abutting or inside the ependymal layer attached to the CC (Fig. 5B, arrow-arrowheads).
Co-localization of CCL2-ir and Iba-1-like labeling in the cortex and CC The co-localization of CCL2-ir (arrows) and Iba-1 (arrowheads) labeling was seldomly visualized in the cortex and CC of the normal or saline injected animals (Fig. 4), likewise, the double labeling was occasionally seen in areas surrounding the vasculature-like (arrows) structures (Fig. 4C inset, arrow-arrowheads). Sporadically, CCL2-ir and Iba-1 double labeled cell was seen in choroid plexus tissue that seems be invaginated from the lateral ventricle (Fig. 5C, arrow-arrowhead).
Following systemic LPS, Iba-1 positive cells with hypertrophic soma and/or hyper-ramified pseudopodia were seen in the cortex and the CC (Fig. 4B, D, opened arrowheads), and more amoeba like Iba-1 positive cells were observed in the CC (Fig. D, framed areas and insets, opened arrowheads). Some hyper-ramified pseudopodia (Fig. 4D, arrow-arrowhead) or amoeba like Iba-1 labeled cells were also CCL2-ir positive (Fig. 4D, framed areas and inset, arrow-arrowheads, Fig. 5E, D opened arrowheads). Meanwhile, co-localization of CCL2-ir (Fig. 5, arrows) and Iba-1 (arrowheads) were identified in or adjacent to the ependymal layers (arrow-arrowheads).
Co-localization of CCL2-ir and NeuN-like labeling in rat cortex and CC In the normal rat brain, most of the CCL2-ir positive (Fig. 6A, arrows) and NeuN labeled cells (arrowheads) were located at I ~ III layer of the cortex (Fig. 6A, insets and arrow-arrowheads). Following systemic LPS, more CCL2-ir (Fig. 6B, arrows) and NeuN (arrowheads) co-localized cells were observed in layer II and III of the cortex (arrow-arrowheads). The CCL2-ir positive neuron like cells (Fig. 6C, D arrows) were also seen in the cingulate cortex above the CC (framed areas and insets, arrow-arrowheads). In addition, the CCL2-ir positive neuron like cells (Fig. 6C, D framed areas and insets, arrow-arrowheads) were encountered in the CC of both normal (C) and LPS treated (D) rats. The result of NeuN immunostaining was from rats, since the data collected from the rats were more consistent.
Co-localization of CCL2-ir and Ly6g or MPO immunostaining in the cortex and CC Clusters of MPO like immunoreactivity were only visualized in the meninges area, or occasionally seen in the choroid plexus in-betweens the hippocampus of saline or LPS injected mice. Even following systemic LPS, neither MPO nor Ly6g positive cells were observed in the brain parenchyma or the CC+Epend.
Statistical comparison of double labeling between saline and LPS injected mice The double labeled structures (arrow-arrowheads) in the cortex, the CC and the CC+Epend were counted and recorded (Fig. 3-5). The number of double labeled cells from normal, saline and LPS injected mice (Fig. 7A - E) or rats (F), were summarized, statistically analyzed and shown in Fig. 7.