SDF1 in the dorsal corticospinal tract promotes CXCR4+ cell migration after spinal cord injury

Abstract

Background: Stromal cell-derived factor-1 (SDF1) and its major signaling receptor, CXCR4, were initially described in the immune system; however, they are also expressed in the nervous system, including the spinal cord. After spinal cord injury, the blood brain barrier is compromised, opening the way for chemokine signaling between these two systems. These experiments clarified prior contradictory findings on normal expression of SDF1 and CXCR4 as well as examined the resulting spinal cord responses resulting from this signaling.

Methods: These experiments examined the expression and function of SDF1 and CXCR4 in the normal and injured adult mouse spinal cord primarily using CXCR4-EGFP and SDF1-EGFP transgenic reporter mice.

Results: In the uninjured spinal cord, SDF1 was expressed in the dorsal corticospinal tract (dCST) as well as the meninges, whereas CXCR4 was found only in ependymal cells surrounding the central canal. After spinal cord injury (SCI), the pattern of SDF1 expression did not change rostral to the lesion but it disappeared from the degenerating dCST caudally. By contrast, CXCR4 expression changed dramatically after SCI. In addition to the CXCR4+ cells in the ependymal layer, numerous CXCR4+ cells appeared in the peripheral white matter and in the dorsal white matter localized between the dorsal corticospinal tract and the gray matter rostral to the lesion site. The non-ependymal CXCR4+ cells were found to be NG2+ and CD11b+ macrophages that presumably infiltrated through the broken blood-brain barrier. One population of macrophages appeared to be migrating towards the dCST that contains SDF1 rostral to the injury but not towards the caudal dCST in which SDF1 is no longer present. A second population of the CXCR4+ macrophages was present near the SDF1-expressing meningeal cells.

Conclusions: These observations suggest that attraction of CXCR4+ macrophages is part of a programmed response to injury and that modulation of the SDF1 signaling system may be important for regulating the inflammatory response after SCI.

Figure 1

SDF1 and CXCR4 expression in the normal spinal cord. SDF1 is expressed throughout the dCST (a-g) a-b, Cross sections of SDF1-GFP mouse spinal cords show SDF1 in the meninges as well as in the dCST. Rhodamine labeled dextran injection into the sensorimotor cortex shows co-label of SDF1 and dextran dye confirming presence of SDF1-GFP in the dCST (c-g)(red:biotin, green:SDF1-EGFP, blue:Hoechst). e-g, Magnified images of the boxed area of c and d further demonstrating co-label of SDF1 and dCST label. h. The cognate receptor of SDF1, CXCR4, is found only in the ependymal layer (green:CXCR4-EGFP, blue:Hoechst). Scale bars a: 200 μm, b-d: 50 μm, h:20 μm

Figure 2

5 weeks post SCI, SDF1 is found in the dCST rostral to the injury and inside the lesion epicenter. Thoracic cross sections demonstrate SDF1-GFP in the dCST rostral (a, c), but not caudal (b, d) to the injury, however, the SDF1 is present in the meninges throughout (a-d)(green:SDF1-EGFP, blue:Hoechst). A longitudinal section that does not contain the dCST shows some migrating cells expressing SDF1, but most are trapped within the lesion epicenter by the GFAP+ scar (e, f). Longitudinal sections containing the dCST show the end of the tract at a cavitation noted by the high density of nuclei and the GFAP+ scar around its rim (g, h)(red:GFAP, green:SDF1-EGFP, blue:Hoechst) Scale bars: a-b, e: 200 μm, c-d, h: 50 μm, f: 25, g: 100 μm.

Figure 3

5 weeks post SCI, CXCR4 is found in 3 different areas of the spinal cord. Thoracic cross sections demonstrate CXCR4-GFP in the ependymal layer (a-d), peripherally toward the meninges (a-e) and in the dorsal funiculus rostral to the injury (a, c) but not caudal to it (b, d)(green:CXCR4-EGFP, blue:Hoechst). Longitudinal sections show CXCR4-GFP+ cells in the lesion epicenter as noted in f and in the cavitation in h, but also outside the lesion (f, h). Some CXCR4-GFP cells were found within the GFAP+ scar, but were distinct from the GFAP+ cells (g)(red:GFAP, green:CXCR4-EGFP, blue:Hoechst). Scale bars: a-b, f: 200 μm, c-d, e, h: 50 μm, g:25 μm

Figure 4

CXCR4-GFP cells appear in the periphery and in the dorsal funiculus in 2 weeks. At 24 hours post injury, CXCR4-GFP cells appear throughout the gray and white matter, but this expression is gone by 4 days post injury(a-f). The ependymal layer, which was full of CXCR4-GFP prior to injury, has fewer CXCR4-GFP cells post injury (a-b, d-e, g-h, j-k). A sprinkling of CXCR4-GFP cells are seen toward the meninges at 1 week(g-i), but the majority of the 2 apparently migrating populations arrive by 2 weeks(j, l-o). Caudal to the injury, no cells are again seen in the dorsal funiculus(k).(green:CXCR4-EGFP, blue:Hoechst). Scale bars: a, d, g, j-k: 200 μm, b-c, e-f, h-I, k-l: 50 μm, m-o: 25 μm.

Figure 5

CXCR4-GFP cells in the ependymal layer do not have neural progenitor markers. The CXCR4-GFP cells in the ependymal layer have long extended processes, but do not colocalize with GFAP+ processes at 24 hours post injury (a-b) or at 2 weeks (c-d)(red:GFAP, green:CXCR4-EGFP, blue:Hoechst). At 2 weeks, Nestin+ processes also extend from the ependymal layer, but do not colocalize with CXCR4-GFP(e-h). Occasionally, however, the Nestin+ processes are in close apposition to CXCR4-GFP processes (f) (red:Nestin, green:CXCR4-EGFP, blue:Hoechst). Scale bars: a-d: 20 μm, e-h: 50 μm.

Figure 6

CXCR4-GFP cells in the general periphery and in the dorsal funiculus express markers of hematopoetic lineage and not neural lineage. At 5 weeks (a, d) and at 2 weeks (b-c, e-f), the putative migrating cell populations are positive for both CD11b and NG2 identifying them as presumptive macrophages. No CXCR4-GFP cells colocalized with either GFAP or Nestin. Scale bars: d: 50 μm. [red:CD11b(a-c), NG2(d-f), GFAP(g-i), Nestin(j-l), green:CXCR4-EGFP, blue:Hoechst].