Person:

Hu, Kai

To study bilateral nerve changes in a newly developed novel mouse model for neurotrophic keratopathy by approaching the trigeminal nerve from the lateral fornix. Surgical axotomy of the ciliary nerve of the trigeminal nerve was performed in adult BALB/c mice at the posterior sclera. Axotomized, contralateral, and sham-treated corneas were excised on post-operative days 1, 3, 5, 7 and 14 and immunofluorescence histochemistry was performed with anti-β-tubulin antibody to evaluate corneal nerve density. Blink reflex was evaluated using a nylon thread. The survival rate was 100% with minimal bleeding during axotomy and a surgical time of 8±0.5 minutes. The blink reflex was diminished at day 1 after axotomy, but remained intact in the contralateral eyes in all mice. The central and peripheral subbasal nerves were not detectable in the axotomized cornea at day 1 (p<0.001), compared to normal eyes (101.3±14.8 and 69.7±12.0 mm/mm2 centrally and peripherally). Interestingly, the subbasal nerve density in the contralateral non-surgical eyes also decreased significantly to 62.4±2.8 mm/mm2 in the center from day 1 (p<0.001), but did not change in the periphery (77.3±11.7 mm/mm2, P = 0.819). Our novel trigeminal axotomy mouse model is highly effective, less invasive, rapid, and has a high survival rate, demonstrating immediate loss of subbasal nerves in axotomized eyes and decreased subbasal nerves in contralateral eyes after unilateral axotomy. This model will allow investigating the effects of corneal nerve damage and serves as a new model for neurotrophic keratopathy.

Osteoblast-derived vascular endothelial growth factor (VEGF) is important for bone development and postnatal bone homeostasis. Several studies have demonstrated that VEGF affects bone repair and regeneration; however, the cellular mechanisms by which it works are not fully understood. In this study, we investigated the functions of osteoblast-derived VEGF in healing of a cortical bone defect. In addition, how VEGF signaling modulates BMP2 functions during bone healing was also examined. To define the roles of osteoblast-derived VEGF in bone repair, a mouse tibial monocortical defect model was used. The effects of deleting Vegfa or Vefgr2 in osteoblast precursors and their descendants on the bone repair process were analyzed at various time points after surgery. To study how VEGF modulates the osteogenic activity of BMP2, BMP2, with or without the soluble VEGFR (sFlt1, VEGF decoy receptor), was delivered to the cortical defects in VE-cadherin-cre;tdTomato mice. The results indicate that osteoblast-derived VEGF is important at various stages during healing of the cortical defect. In the inflammation phase, osteoblast-derived VEGF controls neutrophil release into the circulation and macrophage-related angiogenic responses. VEGF is required, at optimal levels, for angiogenesis-osteogenesis coupling in areas where repair occurs by intramembranous ossification (IO). In this role, VEGF likely functions as a paracrine factor since deletion of Vegfr2 in osteoblast precursors and their progeny enhances osteoblastic maturation and mineralization. Furthermore, osteoblast- and hypertrophic chondrocyte-derived VEGF stimulates recruitment of blood vessels and osteoclasts, and promotes cartilage resorption at the repair site during the periosteal endochondral ossification stage. Finally, osteoblast-derived VEGF stimulates osteoclast formation in the final remodeling phase of the repair process. Our data also indicate that skeletal stem cells at different locations respond differently to BMP2, and that the osteogenic activity of BMP2 is modulated by extracellular VEGF. In the cortical defect, delivery of recombinant BMP2 inhibits intramembranous bone formation in the intramedullary space while it enhances endochondral bone formation in the injured periosteum. Inhibition of extracellular VEGF by sFlt1 reverses the inhibitory effects of BMP2 on intramembranous ossification-mediated bone repair. These findings add to the understanding of VEGF functions and provide a basis for clinical strategies to improve bone regeneration and treat cases of compromised bone healing.

The cornea is the shield to the foreign world and thus, a primary site for peripheral infections. However, transparency and vision are incompatible with inflammation and scarring that may result from infections. Thus, the cornea is required to perform a delicate balance between fighting infections and preserving vision. To date, little is known about the specific role of antigen-presenting cells in viral keratitis. In this study, utilizing an established murine model of primary acute herpes simplex virus (HSV)-1 keratitis, we demonstrate that primary HSV keratitis results in increased conventional dendritic cells (cDCs) and macrophages within 24 hours after infection. Local depletion of cDCs in CD11c-DTR mice by subconjuntival diphtheria toxin injections, led to increased viral proliferation, and influx of inflammatory cells, resulting in increased scarring and clinical keratitis. In addition, while HSV infection resulted in significant corneal nerve destruction, local depletion of cDCs resulted in a much more severe loss of corneal nerves. Further, local cDC depletion resulted in decreased corneal nerve infection, and subsequently decreased and delayed systemic viral transmission in the trigeminal ganglion and draining lymph node, resulting in decreased mortality of mice. In contrast, sham depletion or depletion of macrophages through local injection of clodronate liposomes had neither a significant impact on the cornea, nor an effect on systemic viral transmission. In conclusion, we demonstrate that corneal cDCs may play a primary role in local corneal defense during viral keratitis and preserve vision, at the cost of inducing systemic viral dissemination, leading to increased mortality.