Dendrimer delivery of an anti-VEGF oligonucleotide into the eye: a long-term study into inhibition of laser-induced CNV, distribution, uptake and toxicity.

We have performed a long-term study into the use of a lipophilic amino-acid dendrimer to deliver an anti-vascular endothelial growth factor (VEGF) oligonucleotide (ODN-1) into the eyes of rats and inhibit laser-induced choroidal neovascularization (CNV). In addition, the uptake, distribution and retinal tolerance of the dendrimer plus oligonucleotide conjugates were examined. Analysis of fluorescein angiograms of laser photocoagulated eyes revealed that dendrimer plus ODN-1 significantly inhibited (P<0.05) the development of CNV for 4-6 months by up to 95% in the initial stages. Eyes similarly injected with ODN-1 alone showed no significant difference (P>0.05) in mean severity score at 2 months (2.86+/-0.09), 4 months (2.15+/-0.17) or 6 months (2.7+/-0.12) compared to the vehicle-injected controls. Furthermore, we showed that intravitreally injected ODN-1 tagged with 6-fam was absorbed by a wide area of the retina and penetrated all of the retinal cell layers to the retinal pigment epithelium. Ophthalmological examinations indicated that the dendrimers plus ODN-1 conjugates were well tolerated in vivo, which was later confirmed using immunohistochemistry, which showed no observable increase in antigens associated with inflammation. We conclude that the use of such dendrimers may provide a viable mechanism for the delivery of therapeutic oligonucleotides for the treatment of angiogenic eye diseases.

Generation of transgenic mice with mild and severe retinal neovascularisation.

AIM: To generate a mouse model for slow progressive retinal neovascularisation through vascular endothelial growth factor (VEGF) upregulation. METHODS: Transgenic mice were generated via microinjection of a DNA construct containing the human VEGF165 (hVEGF) gene driven by a truncated mouse rhodopsin promoter. Mouse eyes were characterised clinically and histologically and ocular hVEGF levels assayed by ELISA. RESULTS: One transgenic line expressing low hVEGF levels showed mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation, which remained stable up to 3 months postnatal. Histologically, there were some disturbance and thinning of inner and outer nuclear layers, with occasional focal areas of neovascularisation. By contrast, three other lines expressing high hVEGF levels presented with concomitantly severe phenotypes. In addition to the above, clinical features included extensive neovascularisation, haemorrhage, and retinal detachment; histologically, focal to extensive areas of neovascularisation associated with retinal folds, cell loss in the inner and outer nuclear layers, and partial retinal detachment were common. CONCLUSIONS: The authors generated four hVEGF overexpressing transgenic mouse lines with phenotypes ranging from mild to severe neovascularisation. These models are a valuable research tool to study excess VEGF related molecular and cellular changes and provide additional opportunities to test anti-angiogenic therapies.

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy.

Neovascularisation (NV) within the eye often results in visual loss. Vascular endothelial growth factor (VEGF) has been implicated in the development of ocular NV. Previous studies have shown that VEGF antagonists successfully suppressed retinal and choroidal NV in animal models. However, the systemic approach and transient nature of the delivery systems used in these studies hinder therapeutic application. To achieve stable and localised ocular anti-angiogenic therapy, we explored the use of recombinant adeno-associated virus (rAAV)-mediated secretion gene therapy (SGT). In this study, we generated a rAAV vector encoding soluble VEGF receptor 1, sFlt-1 (AAV-CMV.sflt) and determined its ability to inhibit cautery-induced corneal NV and laser-induced choroidal NV. Delivery of AAV-CMV.sflt into the anterior chamber resulted in transgene expression in the iris pigment epithelium and corneal endothelium, which reduced the development of corneal NV in the stroma of cauterised rats by 36% compared with cauterised control groups (P = 0.009). Subretinal delivery of AAV-CMV.sflt near the equator of the eye also suppressed choroidal NV at the laser lesions around the optic nerve by 19% (P = 0.002), indicating that there was diffusion of the secreted anti-angiogenic protein across the retina. Both results suggest that the long-term suppression of ocular NV is possible through the use of stable rAAV-mediated SGT.

Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization.

Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.

Transplantation of fresh-frozen menisci: an experimental study in dogs.

Transplantation of 25 fresh-frozen medial menisci was studied in 15 adult dogs. Before implantation, the allografts were deep-frozen and stored at -70 degrees C for 6 weeks to 18 months. The animals were killed 2 to 8 months postoperatively, and their knees and transplants were examined macroscopically and histologically. Complete healing of the allografts was found in 19 knees, incomplete in 3, and healing by massive fibrovascular tissue in 3 knees. Some shrinkage of the transplants taken 2 to 4 months after the surgery was observed; however, the 6- and 8-month specimens appeared grossly normal. Histologically, all transplants displayed a characteristic decrease in the number of cells, but this was significantly less pronounced in the 6- and 8-month specimens. Some degenerative alterations were found in all transplanted knees, but was obviously less pronounced in areas covered by the allografts and in the 6- and 8-month specimens. It is concluded that the transplantation of the fresh-frozen menisci could be successful; although the transplants are subjected to a remodeling process, they appear to function normally and protect the articular cartilage. The technique of conservation by freezing at -70 degrees C offers the advantage of an effective meniscal banking.

Isolated dislocation of the carpal scaphoid: two case reports.

Two unusual cases of isolated dislocation of the carpal scaphoid are presented. They were treated in a conservative way, and both of them achieved good functional results.

[Fracture of a transfixed intramedullary nail in a patient with pseudoarthrosis of the femoral diaphysis]. / Prelom transfiksiranog intramedularnog klina u bolesnika s pseudoartrozom dijafize femura.

This is a case report on a patient with a multiple femur's fracture where a transfixed intramedullar nailing of Grosse-Kempf type has been done. "The nail dynamization" was not carried out before the beginning of vertical loading. Seven months after the full vertical loading had begun, at a sudden torsian movement of the extremity, the nail had broken. Analyzing the cause of the fixative material fracture, a conclusion has been made that, apart from the presence of adequate trauma, the nail fracture happened also due to the development of pseudoarthrosis of femur and the unfulfilled "nail dynamization".

Allograft meniscus transplantation in the dog.

We studied transplantation of a fresh meniscus in 25 knees in 15 adult dogs. On 2 tables and with 2 surgical teams the medial menisci were explanted, exchanged and implanted into the opposite dog's knee. The animals were killed 4-12 months postoperatively, and the transplants were studied histologically. Complete healing in the host's capsular tissues was found in 18 knees, incomplete healing in 3, and healing by massive fibrovascular scar tissue in 4 knees. In the 4- and 5-month specimens the transplants were found to be narrower, thinner, and of changed color and consistency, while in the 8- and 12-month specimens most of the allografts appeared grossly normal. Histologically, the allografts had normal general microarchitecture, but a marked decrease in the number of cells. In the 8- and 12-month specimens the cells had increased but the number of cells in general was still less than in the controls. Although some degenerative changes of the medial tibial articular cartilage were noted in most knees, there were less changes beneath the allografted meniscus.

[The size and suitability of blood vessels for transplantation into the knee of the dog]. / Velicina i podobnost krvnih sudova kolena u psa za transplantacione zahvate.

An investigation of the size and suitability for the transplantation procedures of the knee blood vessels was made in 20 adult dogs with 40 extremities using the technique of injection of the arterial network through the femoral artery with the mixture of the methacrylic resin (SIGMAL R "Galenika", Belgrade) and the red ink. When the polymerisation of injected methacrylic resin was completed the specimens were exposed to either chemical or biological maceration of the tissues. Thirty-five specimens were chemically macerated using either 30% solution of hydrochloride acid or 40% potassium hydroxide. With this method bones were also destroyed and only the methacrylic vascular cast remained unaffected. Five specimens were exposed to the biological maceration using the insects (Dermestes vulpinus). This method proved to be better as the bones elements were not destroyed which allowed more precise study of vessel relations and topography. Calibre of the arteries was measured using the micromethod MEBA (Zagreb) with the accuracy of 0.01 mm. The main arterial vessels of the knee joint on the medial side are aa. saphena, genus descendes (branches of the femoral artery, then a. genus descendens (branches of the femoral artery, then a. genus medialis (branch of the popliteal artery), and on the lateral side are a. caudalis femoris distalis, (branch of the femoral artery), a genus lateralis (branch of the popliteal artery) and recurrent branches of the a. tibialis cranialis. Calibre of the femoral and popliteal arteries was over 2 mm which means that they are suitable for macrosurgical sutures, while the calibre of all other arteries was under 2 mm, and it is concluded that they are suitable only for microsurgical sutures.

[Transplantation of the meniscus in dogs--preliminary report]. / Transplantacija meniskusa u psa--preliminarno saopstenje.

A preliminary investigation of the allogenic transplantation of the meniscus without the vascular bundle was carried out in 4 adult dogs with 8 knees. In one operation session on 2 dogs at 2 operative tables and with two operative teams an identical surgical procedure in general anaesthesia was done. A medial parapatellar incision of the knee was used, the medial capsule and the collateral ligament near its proximal insertion were severed. Explanted medial menisci were exchanged and the meniscus from the other dog was put in position of the explanted one and fixed with 3 tangentially placed sutures (posterior, medial and anterior) which were taken out through the capsule and teid extra-articularly; there was no immobilisation, and after 3 weeks the identical operation was done on the other knee. The animals were sacrificed 4-5 months after the operation, the knees were dissected and observed. In all 8 knees transplanted menisci were completely healed around their periphery, however their appearance was rather changed. They were narrower, thinner, and of different colour and consistency. The transplanted menisci were removed and conserved in 10% (formalin) for the further investigation. It is concluded that the allogenic meniscal transplantation without the vascular bundle is possible and that the further investigation of this problem is justified.

[Specifics in the reconstruction of individual elements in replantation of the forelimb in dogs]. / Specificnosti rekonstrukcije pojedinih elemenata pri replantaciji podlakta u psa.

The experiment was carried out on the four extremities of six, sexually mature dogs of a different race. Gradual reconstruction of the anatomic structures was performed as a single act after the section of the elements. To reach stability of the forearm we employed different osteosynthetic techniques and succeeded to reconstruct blood vessels, nerves, ligaments and the skin, afterwards. Numerous techniques of suturing material were used. The animals were sacrificed six months after being operated and were exposed to microscopic observation and histological analysis. The results obtained suggested the necessity of a firm osteosynthesis of the forearm bones, use of less traumatic suturing material for suturing both blood vessels and fasciculi when preparating nerves.

[Revascularization of the forelimb in dogs]. / Revaskularizacija podlakta u psa.

Revascularization of the forearm was performed on six mature dogs of a different race. Arteria mediana was cut 2 cm above the fork of the arteria radialis and later sutured with a nonresorptive monofilament thread/Ethilonr/7-0 and 8-0 in diameter. The same process was repeated on the cefalica vein. Arteriographies were performed on the operated extremities, pre-operatively, immediately after operation and while an animal being sacrificed. The subjects were sacrificed six months after the operation. Then microscopic and histological analyses were carried out to assess the quality of the blood vessels at the suturing place. At the place of the blood vessels reconstruction, lumen did not get narrower which indicated that all the vessels were in a perfect functional condition. In a number of dogs a higher collagenic proliferation was noticed, in the vicinity of the blood vessels sutured. In the regenerative process of the arterial wall tunica mediana almost took a predominant position, like in the intact arteria. All the three strata were present with no relevant deviations in structure. In the cefalica vein the regeneration of the connective tissue support of the smooth muscles cells was insufficient. Based on the analysis of the results obtained it might be concluded that after revascularization of the forearm of the dog the regeneration of the blood vessels made the segment functionally capable with the structure not different from the normal one which existed in the blood vessels of the same part of the body.