Establishing and functional testing of a canine corneal construct

Purpose  To provide a model to be used for in vitro studies on drug effects in dogs, this study was conducted to establish a protocol for the construction of a three-dimensional corneal construct. Primary canine corneal cells and a rabbit corneal epithelial (RCE) cell line were used in comparison.
Methods  The corneal construct was assembled step by step in membrane inserts of a six-well plate over a total of 5 weeks, including culture at the air–liquid interface to allow a differentiation of the epithelial cells. The constructs were studied histologically.
Single cell cultures of canine corneal cells as well as RCE cells were stimulated with lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS). Treatment with different concentrations of dexamethasone was used to test its effects on the cellular prostaglandin E₂ (PGE₂) production. The same experiments were repeated with the corneal constructs and the reactions compared. Expression of the glucocorticoid receptor (GR) in the constructs was studied using immunohistochemistry.
Results  A protocol for the construction of a vital corneal construct was established and the morphological similarity to the canine cornea in vivo shown. The GR protein was detected in all three cell types of the constructs. Stimulation with LPS and SDS led only in the corneal constructs to a significantly increased PGE₂ production, which could be reduced by dexamethasone.
Conclusions  The corneal construct is an interesting system to test drug effects on corneal cells. It allows studies on a cornea-like system including all three major cell types.     

Effects of anti-inflammatory drugs and preservatives on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture

Objective To determine the effects of commonly used ophthalmic corticosteroids, suprofen, polysulfated glycosaminoglycan and preservatives on morphologic characteristics and migration of canine corneal epithelium grown in cell culture. Animals studied Corneal epithelial cells harvested from the corneas of euthanized dogs were propagated in cell culture. Procedures Canine corneal epithelium was grown in tissue culture. The cells were treated with different corticosteroids, polysulfated glycosaminoglycan, suprofen or preservatives at different concentrations after a defect was created in the monolayer. Cellular morphologic characteristics and closure of the defect were compared between test drugs and controls. Results Morphologically the cells treated with dexamethasone were essentially the same as controls. Prednisolone and hydrocortisone caused rounding and shrinkage of the cells. Both suprofen and polysulfated glycosaminoglycan caused no apparent changes in morphologic characteristics at the lowest concentrations tested, but at higher concentrations there was a concentration‐dependent degree of rounding and shrinkage. Benzylkonium chloride and thimerosal caused rounding and shrinkage of all the cells at all concentrations tested. Dexamethasone, hydrocortisone, and suprofen did not inhibit epithelial migration over the defects at the lowest concentrations tested. All other drugs and concentrations inhibited cellular migration. Conclusion Dexamethasone affected the morphologic characteristics and migration of corneal epithelial cells less than hydrocortisone and prednisolone; therefore, dexamethasone may be the drug of choice when a corticosteroid is indicated and an epithelial defect is present. Suprofen and polysulfated glycosaminoglycan caused a concentration‐dependent effect on morphologic characteristics and migration. The preservatives caused severe changes and inhibited migration of the canine corneal epithelial cells at all concentrations and may therefore contribute to poor epithelialization of ulcers treated with preservative‐containing drugs.     

Isolation,proliferation and characterization of endometrial canine stem cells

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.     

Optimization of adipogenic differentiation conditions for canine adipose-derived stem cells

BackgroundCanine adipose-derived stem cells (cADSCs) exhibit various differentiation properties and are isolated from the canine subcutaneous fat. Although cADSCs are valuable as tools for research on adipogenic differentiation, studies focusing on adipogenic differentiation methods and the underlying mechanisms are still lacking.ObjectivesIn this study, we aimed to establish an optimal method for adipogenic differentiation conditions of cADSCs and evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) and estrogen receptor (ER) signaling in the adipogenic differentiation.MethodsTo induce adipogenic differentiation of cADSCs, 3 different adipogenic medium conditions, MDI, DRI, and MDRI, using 3-isobutyl-1-methylxanthine (M), dexamethasone (D), insulin (I), and rosiglitazone (R) were tested.ResultsMDRI, addition of PPARγ agonist rosiglitazone to MDI, was the most significantly facilitated cADSC into adipocyte. GW9662, an antagonist of PPARγ, significantly reduced adipogenic differentiation induced by rosiglitazone. Adipogenic differentiation was also stimulated when 17β-estradiol was added to MDI and DRI, and this stimulation was inhibited by the ER antagonist ICI182,780.ConclusionsTaken together, our results suggest that PPARγ and ER signaling are related to the adipogenic differentiation of cADSCs. This study could provide basic information for future research on obesity or anti-obesity mechanisms in dogs.     

Tissue distribution of dexamethasone in canine ocular compartments following topical application of dexamethasone-21-isonicotinate and oxytetracycline HCl

Objective  To study the dexamethasone (DXM) concentration at different time points in various compartments of the canine eye following topical application of DXM-21-isonicotinate and oxytetracycline hydrochloride
Animals studied  Thirty dogs to be euthanized for reasons not related to this study were selected and their ocular health status evaluated. Selected animals were treated with DXM-oxytetracycline ointment and euthanized after 6, 11 or 16 h.
Procedure  The concentration of DXM was determined in the following compartments of the eye: third eyelid, cornea, aqueous humor, iris, lens, vitreous body and choroid/retina. The DXM concentration in the eye was measured by radioimmunoassay. The applied amount of DXM was 0.04 mg in 0.2 mL ointment. Dogs were treated once with Corti Biciron^® eye ointment (DXM-21-isonicotinate and oxytetracycline hydrochloride, S & K Pharma, Perl, Germany) and were euthanized 6, 11 and 16 h after treatment.
Results  At 6 h following topical application the mean DXM concentration was highest in the anterior structures of the eye (third eyelid: 18 ng/g, cornea: 36 ng/g). The concentration in the posterior structures was below detection level. A decreased DXM concentration in the anterior structures was measured 11 and 16 h after treatment.
Conclusion  It could be demonstrated that therapeutically relevant concentrations of DXM after a single topical administration are only achieved in anterior structures of the eye. A dosing interval of 6–11 h is recommended to achieve therapeutic drug concentration in those structures. The posterior structures of the eye are not reached by topical administration.     

犬TfR胞外区密码子的优化、真核表达及与犬细小病毒VP2蛋白结合

为制备大量具有天然活性的犬胞外区可溶性转铁蛋白受体(sTfR),本试验通过密码子优化提高sTfR在真核细胞中的表达水平.利用RT-PCR方法从犬肝脏中扩增sTfR编码基因,依据该基因编码的氨基酸序列,参照人偏爱的密码子,对该基因进行密码子优化并由公司合成.利用peDNA3.1-CD5质粒分别构建野生型和密码子优化的sTfR基因真核表达载体,经磷酸钙介导使其在HEK293T细胞中进行表达,利用Western-blotting鉴定表达产物,通过ELISA检测重组犬sTfR蛋白与犬细小病毒VP2蛋白的结合活性.结果显示本试验扩增的犬sTfR基因与GenBank该基因序列的同源性为100%;通过在HEK 293T细胞中进行瞬时表达,结果显示密码子优化可以明显提高sTfR基因在HEK 293T细胞中的表达水平,提高了75%.同时表达的sTfR蛋白能够与犬细小病毒VP2蛋白进行特异结合,表明表达的重组sTfR蛋白具有天然活性.     

清开灵对LPS诱导仔猪发热相关调节因子动态变化的影响

采用LPS人工复制仔猪发热模型,攻毒5h后按要求给于清开灵。监测各时间段的体温以及在0,5,30,72h各时段采血用ELISA法检测PGE2、cAMP、α-MSH和AVP的含量。结果显示:在注射LPS时,各组PGE2、cAMP、AVP、α-MSH的含量无显著性差异(P>0.05);在注射LPS 5h后,与空白组相比,模型组PGE2、cAMP、AVP和α-MSH的含量升高;在30h时,与模型组相比,清开灵各组PGE2、cAMP含量降低,AVP含量升高,且差异显著(P<0.05);在72h时,清开灵各组PGE2、cAMP、AVP和α-MSH的含量与空白组比较差异不显著(P>0.05)。体温与上述相关因子存在显著相关性。结果表明:清开灵有明显的解热作用,其机制可能是通过诸多调节因子共同调节的作用。     

In vitro anti‐tubulin effects of mebendazole and fenbendazole on canine glioma cells

Benzimidazole anthelmintics have reported anti‐neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC₅₀) (±SD) obtained from performing the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay after treating J3T, G06‐A, and SDT‐3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550 ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC₅₀ showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.     

An in vitro evaluation of storage media for the preservation of canine packed red blood cells

The effect of four different red blood cell storage media on in vitro parameters of stored canine red blood cells was studied. The storage media included citrate-phosphate-dextrose-adenine (CPDA-1), two additive solutions, and an additive solution modified by the addition of plasma. Biochemical and hematologic parameters, including red cell adenosine triphosphate (ATP); 2,3-diphosphoglycerate (2,3-DPG); pH; percent hemolysis; and supernatant sodium, potassium, and glucose were assessed immediately following preparation of the red cell concentrate and after 35 and 42 days of storage at 4 degrees C. All parameters changed significantly (p < 0.05) during storage. Significant differences due to effect of the storage media were also seen at each time period. After 35 days and 42 days of storage, CPDA-1 maintained the highest pH, potassium, and sodium values, and had the lowest 2,3-DPG, ATP (p=0.052), and glucose values. No differences were seen in hemolysis after 35 days of storage. No additional benefit was noted from the addition of plasma to the additive solution. The additive solutions compared favorably with CPDA-1.     

Radiosensitivity of canine osteosarcoma cells transfected with wild‐type p53 in vitro*

The p53 gene is one of the important tumour suppressor genes that are involved with the cell survival signal pathway. One of the major functions of the p53 protein is to organize cell cycle regulation and induction of apoptosis for cellular genetic stability. It has been documented that more than 50% of all human cancers include a p53 mutation. We evaluated the difference in radiosensitivity between upregulating the expression of canine wild‐type p53 (cp53) in cultured osteosarcoma (D17) cells and naive D17 cells in vitro. We found that upregulating transfected cp53 D17 cells increased their radiation sensitivity in vitro, and there was a significant decrease (P < 0.009) in survival between cp53‐transfected D17 cells and naive D17 cells. In this experiment, a p53 enhancement ratio (p53ER) reached approximately 3.0 at high doses. The transfected cp53 D17 cells were significantly more radiosensitive at all doses evaluated than naive D17 cells, except at 1 Gy where too few data points were available. The p53ER increased rapidly at doses less than 4 Gy, achieving a maximum of about 3.0 for doses of 4 Gy and above. This study shows the enhanced radiosensitivity of the transfected p53 at clinically relevant doses.     

光动力学作用对犬乳腺肿瘤细胞IL-6和IL-2影响的试验

用HMME-PDT作用于犬乳腺肿瘤细胞株CHMm后,台盼蓝染色绘制各组细胞的生长曲线,放射免疫法检测PDT后不同时间点检测细胞IL-6、IL-2含量的变化。台盼蓝染色PDT组与对照组比较细胞死亡率明显上升,PDT后随时间延长IL-2含量增加,IL-6含量降低。说明PDT对犬乳腺肿瘤细胞株CHMm的杀伤效应可能是由于细胞IL-6、IL-2含量的变化而引起的。     

Comparison of Optisol-GS and neomycin-polymyxin B-gramicidin ophthalmic solution for corneal storage in the dog

The objective of the research was to compare the efficacy of Optisol-GS (OGS, Bausch & Lomb Surgical, Irvine, CA, USA) with triple antibiotic ophthalmic solution (neomycin-polymyxin B-gramicidin, NPG; Bausch & Lomb, Tampa, FL, USA) in preserving the viability of corneal endothelial cells. The study subjects were thirty young to middle-aged dogs with no gross corneal pathology that had been euthanized by pentobarbital overdose for reasons unrelated to this project. Corneal tissues were harvested, analyzed, and randomly assigned to treatment groups: one of two media (OGS or NPG), and one of five storage times (1, 7, 14, 21, or 35 days). Six corneas were stored in each medium for each time period. Corneal endothelial cell viability was evaluated pre- and poststorage by vital staining (trypan blue and alizarin red S), and endothelial cell morphology was evaluated with scanning electron microscopy. Storage in NPG caused significant loss (100%) of endothelial cells after all storage times. OGS storage maintained a high level of endothelial cell viability up to 21 days (98.9% ± 1.3% viability). A significant decrease in percentage viability was also found for OGS-stored corneas between 21 and 35 days, when endothelial cell viability decreased to 61.4% ± 45.9%. The conclusions are that NPG storage at −20 °C is a very poor choice of media for corneal tissue banking if graft clarity is the goal. Storage in Optisol-GS at 4 °C for up to 21 days resulted in significantly higher percentages of viable endothelial cells. Optisol-GS storage should facilitate corneal preservation for canine keratoplasty patients.     

Effects of ibrutinib on proliferation and histamine release in canine neoplastic mast cells

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE‐dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI‐1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC₅₀ values ranging between 0.1 and 1 μM in primary MCT cells and between 1 and 3 μM in C2 and NI‐1 cells. In C2 cells, the combination “ibrutinib + midostaurin” produced synergistic growth‐inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE‐dependent histamine release in primary MCT cells, with IC₅₀ values ranging from 0.05 to 0.1 μM in NI‐1 cells, and from 0.05 to 1 μM in primary MCT cells. In summary, ibrutinib exerts anti‐proliferative effects in canine neoplastic MCs and counteracts IgE‐dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.     

The effects of moxifloxacin ophthalmic solution 0.5% or gatifloxacin ophthalmic solution 0.3% treatment on corneal wound healing in pigmented rabbits following anterior keratectomy

Purpose These studies examined corneal healing rates, Type‐IV collagen and zonula occludens membrane‐associated protein (ZO‐1) expression, as well as aqueous PGE₂ and IL‐1β concentrations in pigmented rabbits treated with either moxifloxacin 0.5%, gatifloxacin 0.3% or BSS^® following anterior keratectomy. Methods Anterior keratectomy surgery was followed by topical administration with commercial ophthalmic formulations of either moxifloxacin or gatifloxacin or BSS^® (TID for 96 h). Images of the fluorescein‐stained healing corneas were analyzed for wound area. At 48 or 96 h following surgery, aqueous humor samples were collected and analyzed for the inflammatory mediators PGE₂ and IL‐1β using an ELISA. The corneas were subsequently evaluated using both scanning and transmission electron microscopy. In a second parallel study, corneas were evaluated at both 48 and 96 h for Type‐IV collagen and ZO‐1 expression using immunohistochemistry. Results Fluorescein‐stained corneal images at 96 h postsurgery demonstrated that 90% ± 8% re‐epithelialization for moxifloxacin, 81% ± 14% for gatifloxacin, and 88 ± 6% for BSS^® (P > 0.05). PGE₂ levels in the aqueous humor of fluoroquinolone treated eyes were reduced at 48 h compared to BSS^® treated eyes. IL‐1β was undetectable in all samples. No differences in Type‐IV collagen or ZO‐1 expression were observed between any treatment groups. There were no differences between groups in histological appearance or in ultrastructural healing processes. Conclusions These studies demonstrated that the commercial ophthalmic formulations of moxifloxacin and gatifloxacin were similar to each other in their effects on the levels of aqueous humor PGE₂ and rates of corneal wound re‐epithelialization.