Angiogenic microspheres promote neural regeneration and motor function recovery after spinal cord injury in rats

Abstract

This study examined sustained co-delivery of vascular endothelial growth factor (VEGF), angiopoietin-1 and basic fibroblast growth factor (bFGF) encapsulated in angiogenic microspheres. These spheres were delivered to sites of spinal cord contusion injury in rats, and their ability to induce vessel formation, neural regeneration and improve hindlimb motor function was assessed. At 2-8 weeks after spinal cord injury, ELISA-determined levels of VEGF, angiopoietin-1, and bFGF were significantly higher in spinal cord tissues in rats that received angiogenic microspheres than in those that received empty microspheres. Sites of injury in animals that received angiogenic microspheres also contained greater numbers of isolectin B4-binding vessels and cells positive for nestin or β III-tubulin (P < 0.01), significantly more NF-positive and serotonergic fibers, and more MBP-positive mature oligodendrocytes. Animals receiving angiogenic microspheres also suffered significantly less loss of white matter volume. At 10 weeks after injury, open field tests showed that animals that received angiogenic microspheres scored significantly higher on the Basso-Beattie-Bresnahan scale than control animals (P < 0.01). Our results suggest that biodegradable, biocompatible PLGA microspheres can release angiogenic factors in a sustained fashion into sites of spinal cord injury and markedly stimulate angiogenesis and neurogenesis, accelerating recovery of neurologic function.

Figure 1. Characteristics of PLGA microspheres (PLGA-MS).

Microspheres were prepared as described in Methods. (a) Representative scanning electron micrographs of PLGA-MS, showing spherical microspheres with smooth surfaces devoid of irregularities. Scale bar = 20 μm. (b) Representative confocal micrographs of a microsphere containing FITC-BSA. Scale bar = 10 μm. (c) The fluorescence intensity profile was analyzed along a line running through the center of the FITC-BSA microsphere. Scale bar = 2 μm. (d) In vitro release profiles of microspheres loaded with VEGF, Ang-1, or bFGF at 37 °C in PBS (pH 7.4). Data are shown as the mean ± standard deviation (n = 3).

Figure 2

Release of different angiogenic factors from biodegradable PLGA microspheres in vivo. (a) Distribution of FITC-BSA microspheres at 3 days after injection into the injured spinal cord of rat. (b,c) Higher magnification of the area in the rectangle in (a). (b) Microspheres (green) and nuclei (blue) in spinal cord tissue. (c) Release and diffusion of FITC-BSA from PLGA microspheres into tissue. (d) Histogram showing in vivo release of angiogenic factors from microspheres into the spinal cord at 2, 4, and 8 weeks after spinal cord injury, based on ELISA of extracted tissues. Data are expressed as mean ± standard deviation (n = 4 for each group). Scale bar = 40 μm in (a) or 25 μm in (b,c).

Figure 3. Angiogenesis and recruitment of endogenous neuronal precursors in injured spinal cord at 4 weeks after injection of angiogenic microspheres.

(a–d) Representative photomicrographs of spinal cord sections after immunofluorescent labeling for nestin (red) and IB4-binding microvessels (green) in animals treated with (a) empty microspheres or (c) angiogenic microspheres. (b,d) Higher magnification of the area in rectangles in (a,c). Nestin-positive cells migrated into the injured spinal cord and localized within vessels in the lesion. (e–h) Representative photomicrographs of spinal cord sections after immunofluorescent labeling for βIII-tubulin (red) and IB4-binding microvessels (green) in animals treated with (e) empty microspheres or (g) angiogenic microspheres. (f,h) Higher magnification of the area in rectangles in (e–g). (i) Quantification of nestin-positive cells at the injury site; **P < 0.01 for the comparison of animals treated with empty or angiogenic microspheres. βIII-tubulin-positive cells migrated into the lesion and were closely associated with vessels. (j) Quantification of βIII-tubulin-positive cells at the injury site; **P < 0.01 for the comparison of animals treated with empty or angiogenic microspheres. (k) Quantification of IB4-binding vessels at the site of injury at 4 and 8 weeks after spinal cord injury; **P < 0.01 for the comparison of animals treated with empty or angiogenic microspheres. Data are expressed as mean ± SEM (n = 4 for each group). Scale bar = 400 μm in (a,c,e,g); or 100 μm in (b,d,f,h).

Figure 4. Angiogenesis with axonal growth and sprouting at the site of spinal cord injury at 8 weeks after injection of angiogenic microspheres.

(a–d) Representative photomicrographs of spinal cord sections after immunofluorescent labeling for NF (red) and IB4-binding microvessels (green) in animals treated with (a) empty microspheres or (c) angiogenic microspheres. (b,d) Higher magnification of the area enclosed by the rectangles in (a,c). NF-positive fibers closely surrounded and aligned with vessels in the lesion, even traversing its epicenter. (e–h) Representative photomicrographs of spinal cord sections after immunofluorescent labeling for 5-HT (red) and IB4-binding microvessels (green) in animals treated with (e) empty microspheres or (g) angiogenic microspheres. (f,h) Higher magnification of the area enclosed by the rectangles in (e,g). 5-HT-positive fibers were observed around vessels in the ventral horn, 3–5 mm caudal to the lesion epicenter. (i–l) Representative photomicrographs of spinal cord sections after immunofluorescent labeling for MBP (red) and IB4-binding microvessels (green) in animals treated with (i) empty microspheres or (k) angiogenic microspheres. (j–l) Higher magnification of the area in rectangles in (i–k). MBP-positive cells in white matter were closely associated with vessels. (m) Quantification of NF-positive fibers at the injury site; **P < 0.01 for the comparison of animals treated with empty or angiogenic microspheres. (n) Quantification of sprouted 5-HT-positive fibers caudal to the lesion; **P < 0.01 for the comparison of animals treated with empty or angiogenic microspheres. (o) Quantification of MBP-positive cells adjacent to the lesion; **P<0.01 for the comparison of animals treated with empty or angiogenic microspheres. Data are expressed as mean ± SEM (n = 4 for each group). Scale bar = 400 μm in (a,c,e,g,I,k); or 100 μm in (b,d,j,l); or 200 μm in (f,h).

Figure 5. Reduction in lesion volume and white matter degeneration at 8 weeks after injection of angiogenic microspheres.

(a) Representative photomicrographs of sections spanning from 3 mm rostral to 3 mm caudal to the lesion epicenter after staining with Luxol Fast Blue (LFB). (b) Quantification of LFB-positive spared white matter; *P < 0.05, **P < 0.01 for the comparison of animals treated with empty or angiogenic microspheres. (c,d) Sagittal sections stained with Nissl stain from animals treated with (c) empty microspheres or (d) angiogenic microspheres, showing cavity size in the contused spinal cord. (e) Higher magnification of the area in the rectangle in (d). (f) Quantification of cavity volume at the injury site; **P < 0.01 for the comparison of animals treated with empty or angiogenic microspheres. Data are expressed as mean ± SEM (n = 4 for each group). Scale bar = 1 mm in (a,c,d); or 200 μm in (e).

Figure 6. Transmission electron micrographs of the injury site at 12 weeks after injection of empty or angiogenic microspheres.

(a) Abundant myelinated axons with loose myelin sheaths (Ax) were observed in the lesion area in the control group. (b) In contrast to the control group, the group treated with angiogenic microspheres showed abundant myelinated axons with relatively compact myelin sheaths (Ax) in the lesion; many of these axons were associated with blood vessels (V). (c) Tissue from animals treated with angiogenic microspheres also showed a microvascular network in the lesion area. (d) Certain cytoplasmic extensions from astrocytes (asterisks) made contact with adjacent blood vessels and other cells, based on criteria described in Methods. Scale bar = 2 μm in (a,b,c); or 1 μm in (d).

Figure 7. Tractography images and fractional anisotropy (FA) map of the spinal cord of rats at 12 weeks after injury.

(a) Tractography image of the spinal cord in control animals treated with empty microspheres, showing the fracture of spinal fibers. (b,c) Coronal and transverse FA images of the injury epicenter in control animals. (d) Tractography image of the spinal cord in animals treated with angiogenic microspheres, showing more fibers in continuity. (e,f) Coronal and transverse FA images of the injury epicenter in animals treated with angiogenic microspheres. (g) Comparison of FA values along the spinal cord revealed significant differences between animals treated with empty or angiogenic microspheres at all locations. *P < 0.05 for the comparison of animals treated with empty or angiogenic microspheres. Data are expressed as mean ± SEM (n = 4 for each group). R, right; L, left; V, ventral; D, dorsal.

Figure 8. Microvasculature reconstruction promotes functional recovery.

Mean Basso-Beattie-Bresnahan scores over 12 weeks after spinal cord injury and microsphere injection are shown. *P < 0.05 for the comparison of animals treated with empty or angiogenic microspheres. Data are expressed as mean ± SEM (n ≥ 10 for each group).

Figure 9. Angiogenic microspheres up-regulate miR-210 expression and down-regulate ephrin-A3 expression in spinal cord tissues.

(a) Time course of expression of endogenous miR-210 after spinal cord injury and microsphere injection, based on real-time PCR. Results were normalized to those for SnRNAU6. (b) Expression of ephrin-A3 based on real-time PCR, showing down-regulation concomitant with up-regulation of miR-210 in animals treated with angiogenic microspheres at 4 and 8 weeks after injury [see panel (a)]. *P < 0.05, **P < 0.01 for the comparison of animals treated with empty or angiogenic microspheres. Data are expressed as mean ± SEM (n = 4 for each group).