Combined VEGF/PDGF improves olfactory regeneration after unilateral bulbectomy in mice

Abstract

The olfactory receptor neurons lining the nasal cavity have a remarkable capacity to regenerate throughout life. They are replenished continuously and their axons make new connections within the olfactory bulb. However, some factors such as head trauma and skull base surgery damage the olfactory nerve which lead to olfactory dysfunction. Losing the sense of smell has considerable effects on quality of life and life-expectancy. Therefore, there is a clear need to find a treatment for olfactory dysfunction. One such potential treatment is growth factor therapy which showed promising results in the spinal cord and brain injuries. The aim of the present study was to investigate whether combined delivery of two growth factors, vascular endothelial growth factor and platelet-derived growth factor treatment can improve the olfactory neurons regeneration in mice. The degeneration of the olfactory neurons was induced by unilateral bulbectomy. The treatment group received 1.5 µg of the combined growth factors intranasally, while the control injured group received saline. Growth factor treatment significantly increased the number of immature neurons at 5 and 7 days post injury and also the number of mature olfactory neurons at 10 and 14 days post bulbectomy. Regenerating axons extended over a larger volume in the operated cavity in the treatment group compared to control group at 14 days post bulbectomy. The growth factor treatment also significantly reduced astrocytic glia scar in the operated cavity. The results indicate that the combined delivery of the growth factors has the potential to improve olfactory dysfunction.

Keywords: astrocytes; axon; glial scar; growth factors; neuron; olfactory bulb.

Figure 1

Widespread death of primary olfactory neurons in response to unilateral bulbectomy. Panels are coronal sections through the nasal cavity of postnatal OMP-ZsGreen (OMP-ZsG) mice, dorsal is to the top. Primary olfactory neurons are green and nuclei are blue (DAPI). (A) Three days after unilateral bulbectomy, the olfactory receptor neurons expressing ZsGreen on the bulbectomy side (left nasal cavity, NC-l) underwent degeneration and the number of neurons was reduced compared to the epithelium lining the nasal cavity on right control unoperated side (NC-r). Higher magnification views of the primary olfactory neurons (indicating by arrows) in unoperated side (B) and in the operated (C) indicated degenerating neurons at 3 days after bulbectomy. The olfactory epithelium of control animal with no injury depicted in D. E is higher magnification of D. Scale bar: 500 μm for A, 50 μm for B–D and 30 μm for E. OE: Olfactory epithelium; OB: olfactory bulb; LP: lamina propria.

Figure 2

Combined vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) treatment enhanced olfactory neurons regeneration after unilateral bulbectomy. Panels are coronal sections through the nasal cavity of postnatal olfactory marker protein (OMP)-ZsGreen mice, dorsal is to the top. Primary olfactory neurons are green and nuclei are blue (DAPI or 4′,6-diamidino-2-phenylindole). (A, C, E) factory epithelium of the operated side of the control injured group and (B, D, F) the growth factor treatment group post bulbectomy. Dotted line shows the thickness of the epithelium. (G) The thickness of olfactory epithelium at 3 days and 5 days post injury (n = 6, each time points) was significantly reduced in growth factor animals (GF) compared to control injured animals, however there was a significant increase (*P < 0.05) in the thickness of epithelium at 10 days and 14 days post bulbectomy in the growth factor treatment vs. control injured group. (H) Growth factor treatment significantly increased the number of mature olfactory neurons (300 µm, length of epithelium) at 10 and 14 days post injury (n = 6 at each time points; *P < 0.05, vs. control injured group). There was a significant difference in number of olfactory receptor neurons (H) and the olfactory epithelium thickness (G) between both injured animal groups and animals with no injury across all time points (P < 0.01), indicating the successful unilateral bulbectomy which led to the degeneration of the olfactory epithelium (two asterisks were not shown in the graphs to prevent over-crowding). Scale bar is 50 μm for A–F. OE: Olfactory epithelium; ORN: olfactory receptor neuron.

Figure 3

Combined VEGF and PDGF treatment enhanced immature olfactory neurons generation. Panels are coronal sections through the nasal cavity, illustrating operated side of olfactory epithelium of OMP-ZsGreen mice; dorsal is to the top. Immunolabeling of the olfactory epithelium with anti-growth associated protein-43 (GAP-43) (magenta) showed the treatment group had more GAP-43 positive neurons at 5 days (C, D) and 7 days (G, H) compared to the control injured group (5 days: A, B; 7 days: E, F). (I) There were significantly more GAP-43 positive neurons (300 µm, length of epithelium) at 5 and 7 days post bulbectomy (*P < 0.05) (n = 6 at each time point) in the treatment group vs. control injured, and the treatment vs control no injury groups (only at 7 days). Nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bar is 50 µm for A–H.

Figure 4

The growth factor (GF) treatment reduced astrocytic glial scars and improved axonal fasciculation. Panels are coronal sections through the olfactory bulb of operated animals. Astrocytes were immunolabelled with anti-glial fibrillary acidic protein (GFAP) (red); axons are green. At 14 days after bulbectomy, the regenerating axons entered the bulbar cavity with larger axon bundles in treated animals (A, B, arrows). The operated cavity contained less astrocytes in the growth factor treatment animals (A, C) compared to the control injured animals (B, D, arrows), the vertical dotted line indicated the thickness of the astrocytic scar. (E–J) Higher magnification of the area highlighted with arrows in A, B. At 14 days post bulbectomy, the regenerating axons formed a large axon bundle in the treatment group (E, dashed line) compared to the control injured animals with a few smaller axon bundles (F, dashed line). The astrocytes were tightly wrapped around the axon bundles in the control injured animals (H, J, arrowheads), while they were distributed more in the peripheral part of the plexus with more gaps between cells allowing axonal extension (G, I, arrowheads and arrows). (K) There were significantly less astrocytes in the operated cavity of the treated animals compared to control injured animals (n = 6, **P < 0.01). Scale bars: 500 μm for A–D and 50 μm for E–J.

Figure 5

Combined PDGF and VEGF enhanced olfactory axon extension. Panels are coronal sections through the bulbar cavity of operated animals. Primary olfactory neurons are green and nuclei are blue (DAPI or 4′,6-diamidino-2-phenylindole). (A) 14 days post bulbectomy, the regenerating plexus (arrowhead) in GF treated animals extended to the caudal region of cavity to the level of forebrain cortex (Ct) on the unoperated side. (B) In control injured animals, the plexus was not observed (arrowhead) at the level of the forebrain cortex on the unoperated side. (C) After 14 days post bulbectomy, the regenerating olfactory axons in the growth factors treatment projected significantly deeper into the cavity left by the bulbectomy in comparison to the control injured animals (*P < 0.05). Nuclei are stained with DAPI (blue). Scale bar is 500 μm for A, B. OB: Olfactory bulb; GF: growth factor.