Evaluation of a topical cyclosporine A prodrug on corneal graft rejection in rats.

PURPOSE: To assess the efficacy of a topical cyclosporine A (CsA), water-soluble prodrug, for promoting the survival of allogenic rat corneal grafts after penetrating keratoplasty (PKP). METHODS: Corneas of Brown-Norway rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided in three treatment groups: group I (PBS) and group II (0.26% Debio088) received drops five times per day. Group III received a daily intramuscular CsA injection (10 mg/kg/day). Blood CsA concentrations were measured on days 2 and 14. On day 4, 10, 13 after PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. An opacity score of greater than or equal to 3 was considered as a nonreversible graft rejection process. On day 14, the experimental eyes were processed for histology. RESULTS: On day 13, 12 of the 18 corneal transplants (67%) in group I showed irreversible graft rejection. Three of 18 transplants (19%) in group II and 5 of 16 transplants (28%) in group III showed irreversible graft rejection (p=0.013/p=0.019, OR=0.14/0.06 versus vehicle). Each mean clinical score for edema, opacity, and neovessels in group II were significantly lower than those of the grafts in group I (respectively p=0.010, p=0.013, p=0.024) and III except for neovessels (respectively p=0.002, p=0.001, p=0.057). Histology confirmed the clinical results. The mean CsA blood levels for groups II and III were, respectively 54+/-141 mug/l and 755+/-319 mug/l on day 2 and 14+/-34 mug/l and 1318+/-463 mug/l on day 14. CONCLUSIONS: Debio088 CsA prodrug drops given five times daily are as effective as intramuscular injection of 10 mg/kg/day for the prevention of acute corneal graft rejection in rats.

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury.

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3-treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine alpha-bungarotoxin and neurofilament antibody. Four weeks after surgery, most end-plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS-treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission.

Radiolabeled chimeric anti-CEA monoclonal antibody compared with the original mouse monoclonal antibody for surgically treated colorectal carcinoma.

Biodistribution and tumor uptake of a chimeric human-mouse monoclonal antibody (MAb) and the original mouse MAb have been comparatively studied. METHODS: Eighteen patients with suspected colorectal cancer scheduled for surgery underwent immunoscintigraphy with 123I-labeled chimeric anti-CEA MAb. Iodine-125 and 131I trace-labeled chimeric and original mouse MAb were simultaneously injected for biodistribution studies. RESULTS: Similar serum kinetics and a low immunogenicity were observed for both antibodies. Mean binding capacity to CEA measured in PBS after radiolabeling was identical for both MAbs and it was slightly decreased when measured in serum 1-4 hr after injection. Radiochromatograms of patients sera showed immune complex formation related to the amount of circulating CEA. Postoperative ex vivo radioactivity counting in tissue samples revealed similar antibody distributions with notably similar antibody uptakes in tumors. High tumor uptakes (between 0.02 to 0.06% injected dose per g) were observed in 3 of 13 patients operated for primary or metastatic colorectal cancer. CONCLUSION: In this dual-label technique, the radioiodinated anti-CEA IgG4 chimeric MAb and the original mouse IgG1 MAb were shown to have very similar behavior in colorectal cancer patients.

Thyroid hormone reduces the loss of axotomized sensory neurons in dorsal root ganglia after sciatic nerve transection in adult rat.

We have shown that a local administration of thyroid hormones (T3) at the level of transected rat sciatic nerve induced a significant increase in the number of regenerated axons. To address the question of whether local administration of T3 rescues the axotomized sensory neurons from death, in the present study we estimated the total number of surviving neurons per dorsal root ganglion (DRG) in three experimental group animals. Forty-five days following rat sciatic nerve transection, the lumbar (L4 and L5) DRG were removed from PBS-control, T3-treated as well as from unoperated rats, and serial sections (1 microm) were cut. The physical dissector method was used to estimate the total number of sensory neurons in the DRGs. Our results revealed that in PBS-control rats transection of sciatic nerve leads to a significant (P < 0.001) decrease in the mean number of sensory neurons (8743.8 +/- 748.6) compared with the number of neurons in nontransected ganglion (mean 13,293.7 +/- 1368.4). However, administration of T3 immediately after sciatic nerve transection rescues a great number of axotomized neurons so that their mean neuron number (12,045.8 +/- 929.8) is not significantly different from the mean number of neurons in the nontransected ganglion. In addition, the volume of ganglia showed a similar tendency. These results suggest that T3 rescues a high number of axotomized sensory neurons from death and allows these cells to grow new axons. We believe that the relative preservation of neurons is important in considering future therapeutic approaches of human peripheral nerve lesion and sensory neuropathy.

Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles.

PURPOSE: To study the kinetics of polylactide (PLA) nanoparticle (NP) localization within the intraocular tissues and to evaluate their potential to release encapsulated material. METHODS: A single intravitreous injection (5 micro L) of an NP suspension (2.2 mg/mL) encapsulating either Rh-6G (Rh) or Nile red (Nr) was performed. Animals were killed at various times, and the NPs localization within the intraocular tissues was studied by environmental scanning electron microscopy (ESEM), confocal microscopy, light microscopy histology, fluorescence microscopy, and immunohistochemistry. Eyes injected with blank NPs, free Rh, or PBS solution were used as the control. RESULTS: ESEM showed the flow of the NPs from the site of injection into the vitreous cavity and their rapid settling on the internal limiting membrane. Histology demonstrated the anatomic integrity of the injected eyes and showed no toxic effects. A mild inflammatory cell infiltrate was observed in the ciliary body 6 hours after the injection and in the posterior vitreous and retina at 18 to 24 hours. The intensity of inflammation decreased markedly by 48 hours. Confocal and fluorescence microscopy and immunohistochemistry showed that a transretinal movement of the NPs was gradually taking place with a later localization in the RPE cells. Rh encapsulated within the injected NPs diffused and stained the retina and RPE cells. PLA NPs were still present within the RPE cells 4 months after a single intravitreous injection. CONCLUSIONS: Intravitreous injection of PLA NPs appears to result in transretinal movement, with a preferential localization in the RPE cells. Encapsulated Rh diffuses from the NPs and stains the neuroretina and the RPE cells. The findings support the idea that specific targeting of these tissues is feasible. Furthermore, the presence of the NPs within the RPE cells 4 months after a single injection shows that a steady and continuous delivery of drugs can be achieved.

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.

Ocular antisense oligonucleotide delivery by cationic nanoemulsion for improved treatment of ocular neovascularization: an in-vivo study in rats and mice.

The efficacy of an antisense oligonucleotide (ODN17) cationic nanoemulsion directed at VEGF-R2 to reduce neovascularization was evaluated using rat corneal neovascularization and retinopathy of prematurity (ROP) mouse models. Application of saline solution or scrambled ODN17 solution on eyes of rats led to the highest extent of corneal neovascularization. The groups treated with blank nanoemulsion or scrambled ODN17 nanoemulsion showed moderate inhibition in corneal neovascularization with no significant difference with the saline and scrambled ODN17 control solution groups, while the groups treated with ODN17 solution or Avastin® (positive ODN17 control) clearly elicited marked significant inhibition in corneal neovascularization confirming the results reported in the literature. The highest significant corneal neovascularization inhibition efficiency was noted in the groups treated with ODN17 nanoemulsion (topical and subconjunctivally). However, in the ROP mouse model, the ODN17 in PBS induced a 34% inhibition of retinal neovascularization when compared to the aqueous-vehicle-injected eyes. A significantly higher inhibition of vitreal neovascularization (64%) was observed in the group of eyes treated with ODN17 nanoemulsion. No difference in extent of neovascularization was observed between blank nanoemulsion, scrambled ODN17 nanoemulsion, vehicle or non-treated eyes. The overall results indicate that cationic nanoemulsion can be considered a promising potential ocular delivery system and an effective therapeutic tool of high clinical significance in the prevention and forthcoming treatment of ocular neovascular diseases.

A test of Jessor's problem behavior theory in a Eurasian and a Western European developmental context.

PURPOSE: The current study tested the applicability of Jessor's problem behavior theory (PBT) in national probability samples from Georgia and Switzerland. Comparisons focused on (1) the applicability of the problem behavior syndrome (PBS) in both developmental contexts, and (2) on the applicability of employing a set of theory-driven risk and protective factors in the prediction of problem behaviors. METHODS: School-based questionnaire data were collected from n = 18,239 adolescents in Georgia (n = 9499) and Switzerland (n = 8740) following the same protocol. Participants rated five measures of problem behaviors (alcohol and drug use, problems because of alcohol and drug use, and deviance), three risk factors (future uncertainty, depression, and stress), and three protective factors (family, peer, and school attachment). Final study samples included n = 9043 Georgian youth (mean age = 15.57; 58.8% females) and n = 8348 Swiss youth (mean age = 17.95; 48.5% females). Data analyses were completed using structural equation modeling, path analyses, and post hoc z-tests for comparisons of regression coefficients. RESULTS: Findings indicated that the PBS replicated in both samples, and that theory-driven risk and protective factors accounted for 13% and 10% in Georgian and Swiss samples, respectively in the PBS, net the effects by demographic variables. Follow-up z-tests provided evidence of some differences in the magnitude, but not direction, in five of six individual paths by country. CONCLUSION: PBT and the PBS find empirical support in these Eurasian and Western European samples; thus, Jessor's theory holds value and promise in understanding the etiology of adolescent problem behaviors outside of the United States.

Antibiotic-induced modifications of the stiffness of bacterial membranes.

In the latest years the importance of high resolution analysis of the microbial cell surface has been increasingly recognized. Indeed, in order to better understand bacterial physiology and achieve rapid diagnostic and treatment techniques, a thorough investigation of the surface modifications induced on bacteria by different environmental conditions or drugs is essential. Several instruments are nowadays available to observe at high resolution specific properties of microscopic samples. Among these, AFM can routinely study single cells in physiological conditions, measuring the mechanical properties of their membrane at a nanometric scale (force volume). Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work we exploit such technique to characterize bacterial systems. We have performed an analysis of the mechanical properties of bacteria (Escherichia coli) exposed to different conditions. Such measurements were performed on living bacteria, by changing in real-time the liquid environment: standard phosphate buffered saline, antibiotic (ampicillin) in PBS and growth medium. In particular we have focused on the determination of the membrane stiffness modifications induced by these solutions, in particular between stationary and replicating phases and what is the effect of the antibiotic on the bacterial structure.

Physicochemical and biological characterization of single-walled and double-walled carbon nanotubes in biological media

To study the toxicity of nanoparticles under relevant conditions, it is important to reproducibly disperse nanoparticles in biological media in in vitro and in vivo studies. Here, single-walled nanotubes (SWNTs) and double-walled nanotubes (DWNTs) were physicochemically and biologically characterized when dispersed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA). BSA-SWNT/DWNT interaction resulted in a reduction of aggregation and an increase in particle stabilization. Based on the protein sequence coverage and protein binding results, DWNTs exhibited higher protein binding than SWNTs. SWNT and DWNT suspensions in the presence of BSA increased interleukin-6 (IL-6) levels and reduced tumor necrosis factor-alpha (TNF-α) levels in A549 cells as compared to corresponding samples in the absence of BSA. We next determined the effects of SWNTs and DWNTs on pulmonary protein modification using bronchoalveolar lavage fluid (BALF) as a surrogate collected form BALB/c mice. The BALF proteins bound to SWNTs (13 proteins) and DWNTs (11 proteins), suggesting that these proteins were associated with blood coagulation pathways. Lastly, we demonstrated the importance of physicochemical and biological alterations of SWNTs and DWNTs when dispersed in biological media, since protein binding may result in the misinterpretation of in vitro results and the activation of protein-regulated biological responses.

Enhanced oligonucleotide delivery to mouse retinal cells using iontophoresis.

PURPOSE: To study the combination of oligodeoxynucleotides (ODNs) intravitreous injection and saline transpalpebral iontophoresis on the delivery of ODNs to photoreceptors in the newborn rd1/rd1 mice. METHODS: Cathodal or anodal transpalpebral iontophoresis (1.43 mA/cm(2) for 5 min) was applied to eyes of postnatal day 7 (PN7) rd1/rd1 mice immediately before the intravitreous injection of ODNs. The effect of cathodal iontophoresis after ODNs injection was also evaluated. The influence of current intensity (0.5, 1.5, and 2.5 mA) was assayed with cathodal iontophoresis performed prior to ODNs injection. The duration of current-induced facilitation of ODNs delivery to photoreceptors was evaluated for 6 h following iontophoresis. One group of control eyes received cathodal iontophoresis prior to the intravitreous injection of phosphate buffered saline (PBS) or hexachlorofluorescein (Hex). The second control group received ODN or Hex intravitreous injection without iontophoresis. The penetration of fluorescent ODNs in the outer nuclear layer (ONL) was quantified by image analysis of the ONL fluorescence intensity on cryosection microphotographs. Integrity of ODN was assessed using acrylamide gel migration after its extraction from the retina of treated mice. The integrity of retinal structure, 1 and 24 h after iontophoresis, was analyzed using light and electron microscopy. RESULTS: Transpalpebral anodal or cathodal saline iontophoresis enhanced the penetration of ODNs in all retinal layers. Cathodal iontophoresis was more efficient than anodal iontophoresis in enhancing the tissue penetration of the injected ODN. Photoreceptor delivery of ODN was significantly higher when cathodal saline transpalpebral iontophoresis was applied prior than after the injection. The extent of enhanced tissue penetration decreased in parallel to the increased interval between iontophoresis application and the intravitreous injection. Current of 1.5 mA was safe and optimal for the delivery of ODNs to the ONL. One hour after iontophoresis followed by injection, ODN extracted from the retina of treated eyes remained intact. Histology and electron microscopy observations demonstrated that iontophoresis using the optimal parameters did not induce any permanent tissue alterations or structure damage. CONCLUSIONS: Saline transpalpebral iontophoresis facilitates the penetration of injected ODNs in photoreceptors for at least 3 h. This method may be considered for photoreceptor targeted gene therapy.

Surgical outcomes of Baerveldt aqueous shunt implantation versus combined phacoemulsification and Baerveldt aqueous shunt implantation

Purpose: To examine the efficacy and safety of Baerveldt shunt (BS) implantation compared to combined phacoemulsification and Baerveldt shunt implantation (PBS). This study was designed to detect a difference in IOP reduction of 20% (~4mmHg) between groups with 90% power. Methods: Sixty patients with medically uncontrolled glaucoma, prospectively underwent either or BS implantation with phacoemulsification (Group PBS; n=30) or BS implantation alone (group BS; n=30, pseudophakic eyes only). Groups were matched for age, glaucoma subtype and length of follow-up. Pre and post-operative measures recorded included patient demographics, visual acuity, IOP, number of glaucoma medications (GMs) and all complications. Success was defined as IOP≤21mmHg and 20% reduction in IOP from baseline with or without GMs. Results: Age of PBS and BS groups was 61 vs 62 years respectively (p=0.72*). There were no significant differences in preoperative baseline characteristics: PBS vs PB, mean IOP =25.5mmHg (standard deviation (SD); ±10.3mmHg) vs 26.1mmHg (SD ±10.6mmHg), p=0.81*; mean GMs=3.0 (SD ±1.1) vs 3.1 (SD ±1.0), p=0.83*; mean VA=0.3 vs 0.3, p=0.89*. At year one there were no significant differences observed between groups in post-operative IOP, GMs or VA, mean IOP =14.1mmHg (SD ±5.4mmHg) vs 11.5 mmHg (SD ±4.2mmHg), p=0.12*; mean GMs=1.6 (SD ±1.4) vs 1.1 (SD ±1.1), p=0.23*; mean VA=0.5 vs 0.4, p=0.46*. Complication rates were similar between the two groups (7% vs 14%). Success rate was lower in eyes with PBS (71%) than with BS (88%), however this did not reach statistical significance (p=0.95, log-rank test). * two-sample t-test Conclusions: There were no significant differences at year one in success or complication rates between PBS and BS groups suggesting that simultaneous phacoemulsification does not have a marked (difference of >4mmHg) effect on tube function. IOP reduction and success were less in the PBS group, a larger sample (n=120) would be required to investigate if there is a 10% difference in IOP reduction between groups, however it is unclear if this would be a clinically significant difference to justify separate surgeries.

Pathogenic role of basic calcium phosphate crystals in destructive arthropathies.

BACKGROUND: basic calcium phosphate (BCP) crystals are commonly found in osteoarthritis (OA) and are associated with cartilage destruction. BCP crystals induce in vitro catabolic responses with the production of metalloproteases and inflammatory cytokines such as interleukin-1 (IL-1). In vivo, IL-1 production induced by BCP crystals is both dependant and independent of NLRP3 inflammasome. We aimed to clarify 1/ the role of BCP crystals in cartilage destruction and 2/ the role of IL-1 and NLRP3 inflammasome in cartilage degradation related to BCP crystals. METHODOLOGY PRINCIPAL FINDINGS: synovial membranes isolated from OA knees were analysed by alizarin Red and FTIR. Pyrogen free BCP crystals were injected into right knees of WT, NLRP3 -/-, ASC -/-, IL-1α -/- and IL-1β-/- mice and PBS was injected into left knees. To assess the role of IL-1, WT mice were treated by intra-peritoneal injections of anakinra, the IL-1Ra recombinant protein, or PBS. Articular destruction was studied at d4, d17 and d30 assessing synovial inflammation, proteoglycan loss and chondrocyte apoptosis. BCP crystals were frequently found in OA synovial membranes including low grade OA. BCP crystals injected into murine knee joints provoked synovial inflammation characterized by synovial macrophage infiltration that persisted at day 30, cartilage degradation as evidenced by loss of proteoglycan staining by Safranin-O and concomitant expression of VDIPEN epitopes, and increased chondrocyte apoptosis. BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β. Similarly, treatment with anakinra did not prevent BCP crystal effects. In vitro, BCP crystals elicited enhanced transcription of matrix degrading and pro-inflammatory genes in macrophages. CONCLUSIONS SIGNIFICANCE: intra-articular BCP crystals can elicit synovial inflammation and cartilage degradation suggesting that BCP crystals have a direct pathogenic role in OA. The effects are independent of IL-1 and NLRP3 inflammasome.

Altering α-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism.

BACKGROUND: The envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo. METHODS: We pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals. RESULTS: In mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining. CONCLUSIONS: These results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer. ABBREVIATIONS: Armstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50).

Delivery of antisense oligonucleotide to the cornea by iontophoresis.

We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the design of specific antiangiogenic strategy in diseases of the cornea.