Intra-luminal gene therapy in the porcine artery using a recombinant adeno-associated virus 9.

The ability to improve or restore blood flow and promote healing in ischemic tissue has many potential clinical applications. Augmentation by direct delivery of growth factors may further enhance results, but requires a method for sustained delivery. In this study, we have tested the ability of adeno-associated virus 9 (AAV9) delivered within the lumen of a porcine artery to transfect the vessel and produce a desired product. The marker chosen was green fluorescent protein (GFP) (Ke et al., 2011). In 4 farm pigs the cranial tibial artery was surgically exposed. The vessel was temporarily clamped proximally, and divided distally. A cannula was placed intraluminally, and the arterial segment was injected with 1×10E13 particles of AAV9.CB7.CI.GFP·WPRE.rBG. At 14days the transfected cranial tibial artery as well as the liver, spleen and kidneys were harvested. ELISA and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were used to analyze the artery for GFP production. Significant GFP expression was seen in all transfected cranial tibial vessels, as determined by both GFP protein production (ELISA) and mRNA (RT-qPCR). No GFP was identified in liver, spleen or kidney, nor in the no-GFP control animal artery. Adeno-associated virus 9 is an appropriate vector for gene therapy experiments in the porcine artery model. This vector, and the intraluminal deliver method described result in robust gene expression at 2weeks without evident systemic spill of the virus. The ability to limit delivery of the gene to an isolated segment of vessel is desirable for future research applications.

[Healing of free vascularized bone allotransplants: optimizing by short-term immunosuppression and host-derived neovascularization]. / Einheilung vaskularisierter Knochenallotransplantate : Optimierung durch Kurzzeit-Immunsuppression und der empfängerbasierten Neovaskularisation.

BACKGROUND: Living bone allotransplants (ATs) currently require long-term immunosuppression (IS), but this is impractical for extremity-preserving procedures. An alternative method to maintain viability of the transplant uses host-derived neoangiogeneic vessels combined with short-term IS. MATERIALS AND METHODS: Diaphyseal femoral defects in Dutch-Belted rabbits were reconstructed with a free microvascular AT from New Zealand White rabbits. Additionally, a host-derived intramedullary pedicled fascial flap was placed and short-term IS administered to two of four groups. Neovascularization and bone healing were quantified by microangiography and a custom radiographic score. RESULTS: Bone ATs with perfused fascial flaps achieved bone healing equivalent to autotransplant controls, even when they received IS only until host-derived neoangiogenesis replaced the original perfusion. Vascularized ATs without this combination achieved significantly inferior results. SUMMARY: This rabbit model demonstrated that increased bone turnover allows good healing but may temporarily weaken the allotransplant. However, by the more intense replacement of the graft with host-derived cells, this process may, in the long-term, ultimately result in a better transplant than an avascular graft.

MK-801 attenuates capillary bed compression and hypoperfusion following incomplete focal cerebral ischemia.

The effects of the N-methyl-D-aspartate (NMDA) antagonist MK-801 on capillary beds and CBF following 1 h of transient incomplete focal cerebral ischemia were studied by examining 133Xe CBF, capillary diameter, and area of perfused vasculature. Capillary diameter increased from a control of 5.24 +/- 0.37 to 8.62 +/- 0.57 microns (p less than 0.001) and area of perfused vasculature from 20,943 +/- 1,151 to 30,442 +/- 1,691 microns2/x 10 magnification field (p less than 0.001) with MK-801 1.0 mg/kg administered 30 min prior to ischemia. After flow restoration in control animals, there was a relative hypoperfusion with eventual normalization of CBF over 60 min. Alternatively, in MK-801 1.0 mg/kg animals, there was rapid normalization of CBF upon flow restoration without the postischemic hypoperfusion observed in controls. On histological analysis, there was consistently less neuronal edema in MK-801-treated animals. These results support the hypothesis that hypoperfusion following incomplete focal cerebral ischemia may be due in part to NMDA-mediated cellular edema with subsequent extravascular capillary bed compression.