Supplemental Data

Files in this Data Supplement:

• Supplemental Figures and Tables - (pdf, 1.5 MB)
• Supplemental Video 1: 3D image of mouse microglia - CX3CR1^+/GFP mice were anesthetized and perfused with cold PBS. Brain tissues were dissected out and 300 µm brain slices were cut at 4°C in Hanks' solution immediately using a vibratome (Leica) and, were fixed in 4% paraformaldehyde for 24 h. Tissue slices were then washed with PBS and mounted on slides using Vectashield (Vector Laboratories, CA) and imaged using a confocal microscope (Leica). To reconstruct a 3D image of an entire microglia (cell body + processes), z-stacks of 40 optical sections (1.0 µm/section) were collected. (avi video, 90 MB)
• Supplemental Video 2: Wild type microglial response following tissue injury and hypoxia - CX3CR1^+/GFP mice were anesthetized and perfused with cold PBS. Brain tissues were dissected out and 300 µm brain slices were cut at 4°C in Hanks' solution immediately using a vibratome (Leica) and incubated in DMEM with 10% horse serum (Invitrogen, Carlsbad, CA). The maintenance of live cells in brain slices and acquisition of confocal microscopic images were performed using a Leica TCS SP scanning laser confocal microscope. Dynamic processes of microglial activation were acquired by collecting stacks of confocal optical sections at 15 min interval for 16 h. Each stack contained 30 sections spanning 60 µm in z dimension to provide adequate data for the reconstruction of the microglia. Wild type microglia have dense networks of fine, ramified cytoplasmic processes radiating from a relatively small cell body. Upon stimulation, the distal processes retract progressively toward the cell body. As processes shorten, proximal regions of processes become wider and appear brighter. In the late stage of transformation, microglia extend and then retract pseudopodia repeatedly. (avi video, 45 MB)
• Supplemental Video 3: Microglial response to tissue injury and hypoxia in brain tissue overexpressing MCP-1 - Brain tissue sections from huGFAP-MCP-1_hi tg⁺∙CX3CR1^+/GFP mice were prepared and incubated and, images collected in the same way as described in Video 2. Resting microglia in huGFAP-MCP-1_hi tg⁺ mice have morphologic features similar to that in wild type mice. Upon stimulation, the distal processes retract progressively toward the cell body. However, process retraction is rarely complete in tg⁺ mice. Instead, microspike-like protrusions are observed to form a feathery perimeter around the soma. Pseudopodia that extended from microglia are rarely observed. Distal processes become fragmented, while particulate debris accumulates. The cell bodies of microglia become smaller and spherical. Thus, over-expression of MCP-1/CCL2 in the CNS alters the microglial activation process, resulting in significantly impaired morphologic transformation of microglia. (avi video, 37 MB)