TGF-alpha increases astrocyte invasion and promotes axonal growth into the lesion following spinal cord injury in mice - PubMed (original) (raw)

TGF-alpha increases astrocyte invasion and promotes axonal growth into the lesion following spinal cord injury in mice

Robin E White et al. Exp Neurol. 2008 Nov.

Abstract

Astrocytes respond to environmental cues and play a multifaceted role in the response to trauma in the central nervous system. As the most prevalent contributors to the glial scar, astrocytes are targeted as barriers to regeneration. However, there is also strong evidence that astrocytes are vital for neuroprotection and metabolic support after injury. In addition, consistent with their role during development, astrocytes may be capable of supporting the growth of injured axons. Therefore, we hypothesized that with appropriate stimulation, the reparative functions of endogenous astrocytes could be harnessed to promote axon growth and recovery after spinal cord injury. Transforming growth factor-alpha (TGF-alpha) is a mitogenic growth factor that is active on astrocytes and is poised to contribute to such a strategy. Recombinant TGF-alpha was administered intrathecally to adult C57BL/6 mice for two weeks following a moderate mid-thoracic spinal cord contusion. By three weeks post-injury, TGF-alpha infusion had not affected locomotor recovery, but promoted extensive axon growth and altered the composition of the lesion site. The center of the lesion in the treated mice contained greater numbers of new cells and increased astrocyte invasion. Despite the expression of inhibitory proteoglycans, there was a marked increase in axons expressing neurofilament and GAP-43 immunoreactivity, and the new axons were closely associated with increased laminin expression within and beyond the astrocyte matrix. The results demonstrate that astrocytes are dynamic players in the response to spinal cord injury, and the growth-supportive role of these cells can be enhanced by TGF-alpha infusion.

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Figures

Figure 1

Figure 1

TGF-α-infused mice have increased BrdU+ cells in the lesion center and dorsal horn compared to vehicle-infused mice, and some of these cells are astrocytes. Low (A–B) and high (C–D) power images of BrdU immunoreactivity at the lesion epicenter of TGF-α (A,C) and vehicle (B,D) infused animals. Boxes in (A) and (B) indicate area shown in (C) and (D), respectively. (E) Quantification of the distribution of BrdU+ cells at the lesion epicenter. Lesion = Lesion Core, DC = Dorsal Column, VSWM = Ventral Spared White Matter, LSWM = Lateral Spared White Matter, DH = Dorsal Horn. *p<0.05, **p<0.01. (F) High power image of BrdU and GFAP immunoreactivity caudal to the lesion epicenter, showing that some BrdU+ cells in the lesion center are astrocytes. Arrow = cell double-labeled with GFAP and BrdU. Scale = 100 µm (A–B), 20 µm (C–D), 10 µm (F).

Figure 2

Figure 2

TGF-α alters GFAP distribution and morphology. (A–C) Images of GFAP immunoreactivity throughout the length of the lesion in TGF-α- (A–C) and vehicle- (A’–C’) infused mice. (D) High power magnification images of GFAP immunoreactivity along the lesion border of TGF-α- (D) and vehicle- (D’) infused mice. Arrows in (D) show lacy, elongated astrocytic process in contrast to arrowheads in (D’) showing hypertrophied GFAP+ processes. (E–F) Quantification of percentage (E) and volume (F) of GFAP immunoreactivity throughout the length of the lesion. Scale = 100 µm (A–C), 10 µm (D). **p<0.01, ***p<0.001.

Figure 3

Figure 3

TGF-α infusion increases axons in the lesion core. NF immunoreactivity in the lesion core at the lesion epicenter of TGF-α- (A) and vehicle- (B) infused mice. (C–D) Confocal images of GFAP and NF immunoreactivity, showing NF+ axons extending past the glial border in TGF-α- (C) infused mice, but not vehicle- (D) infused mice. (E–F) Quantification of percentage (E) and volume (F) of GFAP immunoreactivity throughout the length of the lesion. Scale = 20 µm. *p<0.05, ***p<0.001.

Figure 4

Figure 4

The majority of axons in the lesion core and on the glial border are new axons or collaterals. (A) GAP-43 and NF immunofluorescence in the lesion core of a TGF-α- infused subject. (B) High power magnification image of GAP-43 and, NF immunoreactivity showing one axon double-labeled in the lesion core. Scale = 10 µm (A), 5 µm (B).

Figure 5

Figure 5

Axons within the lesion associate with either astrocytes or Schwann cells. (A–B) High power magnification images of NF+ axons associated with GFAP+ processes in the lesion core (A, Z-stack projection) and glial border (B). (C) High power magnification image of NF+ axons growing alongside p75+ processes in the dorsal root entry zone. Scale = 10 µm.

Figure 6

Figure 6

The EGFR is upregulated after injury and is located on astrocytes, but not Schwann cells. EGFR immunoreactivity in uninjured (A) and injured (B) mice two weeks following injury. The EGFR colocalizes with GFAP+ astrocytes in on the glial border (C), but is not located on Schwann cells infiltrating the lesion from the dorsal root entry zone (D). Scale = 100 µm (A–B), 10 µm (C–D).

Figure 7

Figure 7

TGF-α increases neurocan within the lesion, but does not inhibit axon growth. (A and B) Low power GFAP immunoreactivity caudal to the epicenter. Box = area shown in (A’-A’’’) and (B’-B’’’). High power magnification of GFAP (A’,B’), neurocan (A’’,B’’), and NF (A’’’,B’’’) immunoreactivity in the lesion core. White line dictates glial border in vehicle-infused mice (B’-B’’’). Scale = 100 µm (A–B), 20 µm (A’-A’’’, B’-B’’’).

Figure 8

Figure 8

TGF-α treatment increases laminin immunoreactivity in the lesion core. Low (A–B) and high (C–D) power images of laminin immunoreactivity at the lesion epicenter of TGF-α- (A,C) and vehicle- (B,D) infused mice. (E) Quantification of the proportional area of laminin immunoreactivity at the lesion epicenter. (F) High power confocal image of collagen IV and laminin in the lesion epicenter of TGF-α-treated mice. Scale = 100 µm (A–B), 20 μm (C–D,F). *p<0.05.

Figure 9

Figure 9

Axons comingle with laminin and are CGRP and 5-HT negative. (A) High power confocal image of NF and laminin in the lesion epicenter of a TGF-α-infused subject. (B,C) High power confocal image of NF, 5-HT, and CGRP in the dorsal root entry zone (B) and the lesion core (C). Arrows in (B) show axons double-labeled with CGRP, arrowheads in (B) show axons double-labeled with 5-HT, and asterisks in (B) show axons that label with all three markers. (D) Confocal z-stack projection of NF and the nuclear marker Draq5 in the lesion core of a TGF-α-treated mouse. Scale = 20 µm (A–C), 5 µm (D).

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