Chung-Tien Lin

Subconjunctival Injection of Bevacizumab (Avastin) on Corneal Neovascularization in Different Rabbit Models of Corneal Angiogenesis

PURPOSE Bevacizumab is a potent recombinant humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF). The purpose of this study was to evaluate the therapeutic effect of subconjunctival injection of bevacizumab on corneal neovascularization (NV) in different rabbit models. METHODS Several rabbit models of corneal NV were used, including (1) a corneal micropocket assay with VEGF pellet, (2) a corneal micropocket assay with basic fibroblast growth factor (b-FGF) pellets, (3) mechanical limbal injury-induced corneal NV, and (4) an alkali-induced model of corneal NV. Subconjunctival injections of bevacizumab (0.25-2.5 mg) were applied twice per week for 2 to 8 weeks. Digital photographs of the cornea were analyzed to determine the length of corneal NV and the area of cornea covered by NV as a percentage of the total corneal area. Immunohistochemical staining with anti-human IgG antibody labeled with Cy3 was used to determine the detection of intracorneal distribution of bevacizumab after injection. RESULTS Subconjunctival injection of bevacizumab caused significant inhibition of corneal NV formation as measured by length or surface area in all animal models (P<0.05). No significant ocular complications were found. Staining of bevacizumab was found in the corneal stroma for 3 to at least 14 days in the different rabbit models. CONCLUSIONS Subconjunctival injection of bevacizumab is effective in inhibiting corneal NV in several rabbit models. Bevacizumab may diffuse into the corneal stroma and persist for a few days after injection. It may be useful in preventing corneal NV in the acute phase of various kinds of corneal inflammation.

Secreted frizzled-related protein 2 (SFRP2) is highly expressed in canine mammary gland tumors but not in normal mammary glands

Canine mammary gland tumor (MGT) is the commonest tumor in female dogs and a good animal model of human breast cancer. A group of newly identified genes encoding secreted frizzled-related proteins (SFRP) have been implicated in apoptosis regulation and tumorigenesis. Canine mammary tissues from 50 spontaneous MGTs and 10 normal mammary glands (MGs) were obtained from surgically excised specimens and analyzed for expression of SFRP2, β-catenin, and cyclin D1. By RT-PCR and in situ hybridization, SFRP2 gene was found abundantly expressed in neoplastic mammary tissues but not in normal mammary tissues, suggesting that SFRP2 may contribute as a tumor marker in canine MGTs. By immunohistochemical staining, the immunoreactivity of the SFRP2 protein was detected in more diverse areas than SFRP2 mRNA expression, including nuclei or/and cytoplasm and extracellular matrix of the tumor. In tumor masses, β-catenin lost its tight association with the membrane and diffused into the nucleus. The expression of β-catenin (79.4% positive) and cyclin D1 (71.4% positive) was also increased in MGTs. In the course of tumor progression, SFRP2 mRNA (p < 0.05) and β-catenin protein (p < 0.01) steadily increased but not in cyclin D1. The level of SFRP2 was linearly correlated with its downstream target β-catenin (p < 0.05), but not correlated with cyclin D1 (p < 0.5). As revealed in this study, the exclusive overexpression of SFRP2 in canine MGTs suggests that SFRP2 is a potential candidate gene for further investigation of mammary tumorigenesis and complex etiology of the canine model of mammary neoplasms.

Comparison of the Bacteriostatic Effects, Corneal Cytotoxicity, and the Ability to Seal Corneal Incisions among Three Different Tissue Adhesives

Purpose: To compare the bacteriostatic effects, corneal cytotoxicity, and ability to seal corneal incisions among fibrin glue and 2 commercially available cyanoacrylate derivatives: N-butyl cyanoacrylate and methoxypropyl cyanoacrylate. Methods: The bacteriostatic activities of these tissue glues were verified by measuring the zones of bacterial growth inhibition surrounding the adhesive droplets on agar plates inoculated with Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, or Mycobacterium chelonae. Corneal cytotoxicity was tested by a direct contact method by using cultured bovine corneal epithelial cells, keratocytes, and corneal endothelial cells challenged with droplets of adhesives. Each of the cells was treated with droplets of adhesives. The ability to seal corneal incisions was verified by calculating the maximum intraocular pressure resistant to leakage of rabbit corneal stab wounds sealed with tissue adhesives. Results: Methoxypropyl cyanoacrylate and N-butyl cyanoacrylate showed bacteriostatic effects against S. aureus, S. pneumoniae, and M. chelonae but not P. aeruginosa and E. coli. In contrast, fibrin glue had no such effects against either Gram-positive or -negative bacteria (P < 0.01). Methoxypropyl cyanoacrylate showed the highest levels of corneal cytotoxicity, followed by N-butyl cyanoacrylate. Fibrin glue, however, showed minimal cytotoxicity (P < 0.01). Methoxypropyl cyanoacrylate and N-butyl cyanoacrylate also displayed a greater ability to seal corneal incisions than that of fibrin glue (P < 0.01). Conclusions: The bacteriostatic effects, corneal cytotoxicity, and ability to seal corneal incisions differed among the 3 compounds tested. These different properties should be considered when choosing tissue adhesives during corneal surgery.

Secreted Frizzled Related Protein 2 (sFRP2) Decreases Susceptibility to UV-Induced Apoptosis in Primary Culture of Canine Mammary Gland Tumors by NF-κB Activation or JNK Suppression

Tumor formation can result from a decrease in cell death, as well as an increase in cell proliferation. In spite of the high incidence of mammary gland tumors (MGTs) in female dogs, the understanding of its etiology is still poor. Consistent with several proto-oncogenes (such as Wnt) for the mammary gland, sFRP2 is expressed in canine MGTs which is normally silent in the mammary gland. To elucidate the roles of SFRP2 in the tumorigenesis of MGTs, apoptosis regulation mediated by sFRP2 was investigated by overexpression of sFRP2 in MGT cells. DNA fragmentation and TUNEL assays showed a decreased susceptibility of the cells to UV-induced apoptosis in the context of sFRP2 overexpression. To analyze the pathways through which sFRP2 transduces anti-apoptosis signals, multiple-color immunofluorescence staining, immunoprecipitation, and immunoblotting were carried out. sFRP2 was found co-localized in the extracellular matrix of MGTs and the tyrosine phosphorylation of FAK was enhanced. Moreover, JNK was suppressed and NF-kB was activated in the cells expressing sFRP2 after UV-induced apoptosis analyzed by immunoblotting and electrophoretic mobility shift assay (EMSA). Taken together, these results suggest that sFRP2 exerts its anti-apoptotic function in mammary cancer cells through NF-κB activation or JNK suppression.

In-Vitro Effects of Dexamethasone on Cellular Proliferation, Apoptosis, and Na+-K+-ATPase Activity of Bovine Corneal Endothelial Cells

Purpose: To assess the in-vitro effects of dexamethasone (DEX) on the proliferation, apoptosis, and Na+-K+-ATPase activity of bovine corneal endothelial cells. Methods: Bovine corneal endothelial cells were cultured with DEX ranging from 10−10 to 10−3 M. The effect of DEX on the proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Apoptosis and necrosis were detected by staining with fluorescein-conjugated annexin V and propidium iodide, followed by flow cytometry. The effect of DEX on Na+-K+-ATPase activity was evaluated using non-isotopic methods. Results: DEX did not affect cellular proliferation or induce apoptosis/necrosis from 10−10 to 10−5 M. At 10−4 and 10−3 M, DEX significantly decreased proliferation and increased apoptosis and/or necrosis. DEX significantly increased the Na+-K+-ATPase activity from 10−8 to 10−6 M, with the maximal effect at 10−6 M (p < 0.01); this effect was inhibited by RU38486, an antiglucocorticoid molecule. Conclusions: Bovine corneal endothelial cells express glucocorticoid receptor (GR) mRNA and protein. DEX decreases cell proliferation and induces cellular apoptosis and/or necrosis at high concentrations. DEX also increases the Na+-K+-ATPase activity at certain concentrations.

In Vivo Confocal Microscopic Findings of Corneal Wound Healing after Corneal Epithelial Debridement in Diabetic Vitrectomy

PURPOSE To study healing of corneal wounds using in vivo confocal microscopy in patients who received corneal epithelial debridement during pars plana vitrectomy for proliferative diabetic retinopathy and to investigate risk factors for delayed healing. DESIGN Prospective, observational case series. PARTICIPANTS Forty-four eyes of 40 patients were enrolled. METHODS In vivo confocal microscopy was used to evaluate selected images of the corneal basal and apical surface epithelial cells and subbasal nerves before surgery, weekly for the first month, and at 3 and 6 months after surgery. Slit-lamp biomicroscopy was carried out at the same time. Multiple linear regression analysis of selected potential risk factors was performed to investigate the main determinants of delayed corneal healing. MAIN OUTCOME MEASURES Healing rate of corneal epithelial cells and subbasal nerves and factors influencing the healing. RESULTS By slit-lamp biomicroscopy, corneal epithelial defects were found in 22.8% of eyes at 2 weeks and in 5.4% at 1 month after surgery. In vivo confocal microscopy demonstrated incomplete healing of basal epithelial cells in 72.1%, 15.2%, and 0% of eyes and incomplete healing of surface apical epithelial cells in 81.1%, 9.1%, and 0% of eyes at 1, 3, and 6 months after surgery. The percentage of subbasal nerves regaining preoperative appearance was 0%, 6.8%, and 89.3% at 1, 3, and 6 months after surgery. Regression analysis revealed infusion of silicone oil (P = 0.020) and C(3)F(8) (P = 0.017) resulted in delayed healing by slit-lamp biomicroscopy; age (P = 0.028), diabetic treatment regimen (P = 0.014), and scleral buckling (P = 0.001) correlated with delayed recovery of basal cells by in vivo confocal microscopy. The latter 2 factors also were related to delayed reconformation of apical cells (P = 0.011 and 0.004, respectively). Neither healing of apical and basal cells showed a significant correlation to findings by slit-lamp biomicroscopy (r = 0.19 and 0.09). CONCLUSIONS Healing of corneal epithelial wounds in diabetic eyes is slow. Both the basal and apical epithelial layers were involved in the slow healing process. Age, diabetic treatment regimen, and several intraoperative factors may alter healing rates. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Regulation of tristetraprolin during differentiation of 3T3-L1 preadipocytes

Tristetraprolin is a zinc‐finger‐containing RNA‐binding protein. Tristetraprolin binds to AU‐rich elements of target mRNAs such as proto‐oncogenes, cytokines and growth factors, and then induces mRNA rapid degradation. It was observed as an immediate‐early gene that was induced in response to several kinds of stimulus, such as insulin and other growth factors and stimulators of innate immunity such as lipopolysaccharides. We observed that tristetraprolin was briefly expressed during a 1–8 h period after induction of differentiation in 3T3‐L1 preadipocytes. Detailed analysis showed that tristetraprolin mRNA expression was stimulated by fetal bovine serum and differentiation inducers, and was followed by rapid degradation. The 3′UTR of tristetraprolin mRNAs contain adenine‐ and uridine‐rich elements. Biochemical analyses using RNA pull‐down, RNA immunoprecipitation and gel shift experiments demonstrated that adenine‐ and uridine‐rich element‐binding proteins, HuR and tristetraprolin itself, were associated with tristetraprolin adenine‐ and uridine‐rich elements. Functional characterization confirmed that tristetraprolin negatively regulated its own expression. Thus, our results indicated that the tight autoregulation of tristetraprolin expression correlated with its critical functional role in 3T3‐L1 differentiation.

The different effects of early and late bevacizumab (Avastin) injection on inhibiting corneal neovascularization and conjunctivalization in rabbit limbal insufficiency.

PURPOSE To compare the effects of early, mid, and late subconjunctival injection of bevacizumab on corneal neovascularization (NV) and conjunctivalization in a rabbit limbal insufficiency model. METHODS Limbal insufficiency was created surgically, and subconjunctival injections of 2.5 mg bevacizumab twice weekly for 1 month were started immediately (early group), 1 week (mid group), and 1 month after injury (late group). The corneal epithelial alterations, stromal opacity, centricity, extent, and PICS (percentage of involved corneal surface) of the corneal NV were evaluated. The expression of cytokeratins K3, K4, K12, K13, and MUC5 by the corneal surface cells was examined by immunohistochemistry. RESULTS Bevacizumab significantly inhibited corneal NV in the early and mid, but not the late, treatment groups at 4 weeks after treatment (PICS: P < 0.01 in the early group, P < 0.01 in the mid group, and P > 0.05 in the late group). Early and mid treatment produced significant inhibition of corneal alteration (P < 0.01 in the early group and P = 0.03 in the mid group) and stromal opacity (P < 0.01 in the early group and P = 0.02 in the mid group) at 4 months after treatment but not in the late group. The immunohistochemistry of cytokeratin on the corneal surface cells at 1 month after treatment was K3(+), K4(-), K12(+), K13(-), and MUC5(-) in the early group; K3(+), K4(+), K12(+), K13(+), and MUC5(-) in the mid group; and K3(+), K4(+), K12(-), K13(+), and MUC5(+) in the late treatment group. CONCLUSIONS Early and mid bevacizumab injection inhibited corneal NV, epithelial alteration, and stromal opacity in limbal insufficiency, but late treatment did not. Early treatment with bevacizumab seems to be clinically beneficial in the management of limbal injury such as chemical burn.

Synergistic antiviral effect of Galanthus nivalis agglutinin and nelfinavir against feline coronavirus.

Abstract Feline infectious peritonitis (FIP) is a fatal disease in domestic and nondomestic felids caused by feline coronavirus (FCoV). Currently, no effective vaccine is available for the prevention of this disease. In searching for agents that may prove clinically effective against FCoV infection, 16 compounds were screened for their antiviral activity against a local FCoV strain in Felis catus whole fetus-4 cells. The results showed that Galanthus nivalis agglutinin (GNA) and nelfinavir effectively inhibited FCoV replication. When the amount of virus preinoculated into the test cells was increased to mimic the high viral load present in the target cells of FIP cats, GNA and nelfinavir by themselves lost their inhibitory effect. However, when the two agents were added together to FCoV-infected cells, a synergistic antiviral effect defined by complete blockage of viral replication was observed. These results suggest that the combined use of GNA and nelfinavir has therapeutic potential in the prophylaxis and treatment of cats with early-diagnosed FIP.

Occurrence of epilepsy at different zeitgeber times alters sleep homeostasis differently in rats.

STUDY OBJECTIVES Controversial sleep disruptions (e.g., poor nighttime sleep and daytime somnolence) are common in epilepsy patients. Sleep is known to be regulated by homeostatic factors, which mediate sleep propensity, and the circadian oscillator, a clocklike mechanism. However, it is unknown how epileptic episodes that occur at different zeitgeber times (ZTs) alter sleep regulation. This study was designed to elucidate the sleep disruptions associated with epilepsy and their underlying mechanisms by delivering kindled epilepsy at different ZTs: ZT0, ZT6, and ZT13. DESIGN Kindled epilepsy was induced at 3 different ZTs, and sleep-wake activities were analyzed before and after full-blown seizure. Ribonuclease protection assay, radioimmunoassay, and immunohistochemistry were respectively employed to determine the levels of interleukin-1 mRNA, corticosterone, and PER1 protein. SETTING The experiments were performed at Neurophysiology Laboratory at National Taiwan University. PARTICIPANT AND INTERVENTIONS: Male Sprague-Dawley rats were implanted with electroencephalograph (EEG) electrodes, a bipolar stimulating electrode, and a guide cannula. Kindling stimuli were delivered via a bipolar electrode placed in the right central nucleus of the amygdala. MEASUREMENT AND RESULTS Kindled epilepsy occurring at ZT0 and ZT13 predominantly affected homeostatic factors, whereas ZT6-kindling stimuli altered the circadian oscillator. ZT0-kindling decreased rapid eye movement (REM) and non-REM (NREM) sleep, which was mediated by corticotrophin-releasing hormone, but did not alter the rhythm of sleep-wake fluctuation. On the other hand, ZT13-kindling enhanced interleukin-1 and consequently increased NREM sleep without altering the sleep-wake fluctuation. Nevertheless, the expression of PER1 protein in suprachiasmatic nucleus of the hypothalamus and the circadian rhythm of sleep fluctuation were respectively advanced 6 h and 2 h when kindling stimulation was delivered at ZT6. Shifts of sleep circadian rhythm and PER1 oscillation induced by ZT6-kindling were blocked by administration of hypocretin receptor antagonist SB334867 into the SCN, indicating the involvement of hypocretin. CONCLUSION These observations suggest that the occurrence of epilepsy at different ZTs alters sleep processes differently. CITATION Yi PL; Chen YJ; Lin CT; Chang FC. Occurrence of epilepsy at different zeitgeber times alters sleep homeostasis differently in rats. SLEEP 2012;35(12):1651-1665.