Lidocaine effects on corneal endothelial cell ultrastructure

The purpose of this study was to determine whether intracameral commercial lidocaine 2% induces alterations on the rabbit corneal endothelium. Forty white rabbits received different substances inside the anterior chamber: group (G)1, no substance; G2 and G3 received lidocaine 2% with preservative in aqueous solution; G4 and G5, lidocaine 2% with preservative in gel solution; G6 and G7, the anesthetic preservative (metilparahydroxybenzoate 0.1%); and G8 and G9, lidocaine 2% without preservative in aqueous solution. The animals from G2, 4, 6 and 8 were sacrificed after 1 h, and from G3, 5, 7 and 9 after 24 h after injection of the substance inside the anterior chamber. The corneas were clinically evaluated and assessed by transmission and scanning electron microscopy. G1, 2, 6, 7, 8 and 9 animals had very similar characteristics in clinical, ultrastructural and morphometric evaluations; the G3 and G4 animals showed discrete edema and one animal in G5 had intense corneal edema. We conclude that lidocaine 2% with preservative induces few ultrastructural alterations in the corneal endothelial cells.     

Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells

Background: Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells. Objectives: The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood. Methods: Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti‐CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146‐positive cells were stained with fluorescein‐conjugated Ulex europaeus agglutinin 1 (UEA‐1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA‐anticoagulated whole blood samples from 10 healthy client‐owned dogs. Results: The anti‐CD146–coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA‐1–positive cells were obtained from whole blood, while >85–90% of cultured canine aortic endothelial cells were UEA‐1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10–40 μm in size. Using immunomagnetic isolation, 43.4 ± 15.6 CECs/mL (range 24–70/mL) were isolated from canine whole blood samples. Conclusions: Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.     

Density of corneal endothelial cells and corneal thickness in eyes of euthanatized horses

OBJECTIVE: To determine density of corneal endothelial cells and corneal thickness in eyes of euthanatized horses. SAMPLE POPULATION: 52 normal eyes from 26 horses. PROCEDURE: Eyes were enucleated after horses were euthanatized. Eyes were examined to determine that they did not have visible ocular defects. Noncontact specular microscopy was used to determine density of corneal endothelial cells. Corneal thickness was measured, using ultrasonic pachymetry or specular microscopy. RESULTS: Mean density of corneal endothelial cells was 3,155 cells/mm2. Cell density decreased with age, but sex did not affect cell density. Values did not differ significantly between right and left eyes from the same horse. Cell density of the ventral quadrant was significantly less than cell density of the medial and temporal quadrants. Mean corneal thickness was 893 microm. Sex or age did not affect corneal thickness. Dorsal and ventral quadrants were significantly thicker than the medial and temporal quadrants and central portion of the cornea. We did not detect a correlation between corneal thickness and density of endothelial cells in normal eyes of horses. CONCLUSIONS AND CLINICAL RELEVANCE: Density of corneal endothelial cells decreases with age, but corneal thickness is not affected by age or sex in normal eyes of horses. The technique described here may be useful for determining density of endothelial cells in the cornea of enucleated eyes. This is clinically relevant for analyzing corneal donor tissue prior to harvest and use for corneal transplantation.     

Density of corneal endothelial cells, corneal thickness, and corneal diameters in normal eyes of llamas and alpacas

OBJECTIVE: To determine density of corneal endothelial cells, corneal thickness, and corneal diameters in normal eyes of llamas and alpacas. ANIMALS: 36 llamas and 20 alpacas. PROCEDURE: Both eyes were examined in each camelid. Noncontact specular microscopy was used to determine density of corneal endothelial cells. Corneal thickness was measured, using ultrasonographic pachymetry. Vertical and horizontal corneal diameters were measured, using Jameson calipers. RESULTS: Values did not differ significantly between the right and left eyes from the same camelid. There was no significant effect of sex on density of corneal endothelial cells or corneal thickness in either species. Mean density of endothelial cells was 2,669 cells/mm2 in llamas and 2,275 cells/mm2 in alpacas. Density of endothelial cells decreased with age in llamas. Polymegathism was observed frequently in both species. Mean corneal thickness was 608 microm for llamas and 595 microm for alpacas. Corneal thickness and density of endothelial cells were negatively correlated in llamas. Older (> 36 months old) llamas had significantly larger horizontal and vertical corneal diameters than younger llamas, and older alpacas had a significantly larger vertical corneal diameter than younger alpacas. CONCLUSIONS AND CLINICAL RELEVANCE: Density of corneal endothelial cells is only slightly lower in camelids than other domestic species. Density of endothelial cells decreases with age in llamas. Age or sex does not significantly affect corneal thickness in normal eyes of llamas and alpacas. Specular microscopy is useful for determining density of corneal endothelial cells in normal eyes of camelids.     

猪脐静脉血管内皮细胞的分离及体外培养研究

为建立一种操作简单并且能获得大量猪血管内皮细胞的分离培养方法,本研究采集新生健康长白猪脐带,取其脐静脉血管,灌注0.1%的Ⅰ型胶原酶,于37℃水浴消化10 min后收集内皮细胞,培养于含20%胎牛血清的M199培养基中,长满单层后用胰酶消化传代培养。并对培养的细胞形态学、相关抗原和其他特征以及对病毒的易感性进行鉴定。结果表明,Ⅰ型胶原酶消化后可获得大量内皮细胞,细胞培养后的形态学观察显示细胞贴壁生长良好,呈典型的铺路石状排列;第Ⅷ因子相关抗原和细胞摄取乙酰化低密度脂蛋白均呈阳性,内皮细胞比率可高达100%。传3代后的生长曲线显示细胞传代后延迟期小于1 d,对数生长期约3 d,第5 d进入平台期,第7 d开始脱落死亡。病毒感染试验表明所获得的内皮细胞对猪瘟病毒高度易感,病毒生长与常用的PK-15没有区别。上述研究结果表明本研究建立的血管内皮细胞分离培养方法简便有效,为大量制备原代血管内皮细胞提供了可靠的方法。     

Isolation,characterization, and functional analysis of ferret lymphatic endothelial cells

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases – the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-I ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles LECs might play in health and disease in animal models.     

Effect of epidermal growth factor on healing of corneal endothelial cells in cats

Epidermal growth factor was injected intracamerally into the anterior chamber of the right eye of 9 cats. The central portion of the cornea in 8 of the 9 cats that had been cryoinjured. Effect of epidermal growth factor on the repair of endothelial cells in cats was evaluated by endothelial specular microscopy. Endothelial cell density and corneal thickness were studied quantitatively, as a measure of endothelial cell function. The repair process also was evaluated qualitatively by studying morphologic changes, developing as a result of reendothelialization and return to normal function. Seemingly, differences between rate of healing of cryoinjured eyes injected with epidermal growth factor and that in nontreated eyes were not significant (P = 0.86). The endothelial repair process was characterized by enlargement and migration of adjacent noninjured cells.     

Intravascular papillary endothelial hyperplasia of the conjunctiva in a horse

Objective Vascular tumors of the conjunctiva in the horse are rare. We present a unique case of an intravascular papillary endothelial hyperplasia of the conjunctiva. Animal studied Horse. Procedures Case report. A 6‐year‐old‐mare presented with a red mass in the conjunctiva of the left eye. After complete ophthalmologic examination the lesion was excised. The tissue was processed for light microscopy and studied histopathologically. Results Pathologic examination revealed a nonencapsulated vascular lesion composed of confluent vascular spaces filled by multiple papillary structures composed of a central collagenous core lined by hyperplastic endothelial cells. There was neither atypical endothelial cell nor mitotic activity. Conclusions Intravascular papillary endothelial hyperplasia is a benign proliferative lesion that should be differentiated from malignant vascular tumors.     

Use of specular microscopy to determine corneal endothelial cell morphology and morphometry in enucleated cat eyes

The purpose of this study was to investigate the effect of age on endothelial morphology and morphometry in cats. The corneal endothelium was studied using a contact specular microscope. A total of 18 cats (Felis catus Linnaeus, 1758) were evaluated in this study. The subjects were divided into three groups of six cats each in function of age: G1 (1 to 3 months old), G2 (5 to 12 months old), and G3 (24 to 40 months old). The examination presented data as endothelial cell density (ECD), average cell area, corneal thickness, polymegathism, and pleomorphism. Results revealed ECD decrease in corneas of normal cats with age, as well as a corresponding increase in endothelial cell area and pleomorphism. The present work suggests that the endothelial parameters evaluated change with advancing age.     

Serum vascular endothelial growth factor in dogs with haemangiosarcoma and haematoma

Splenic haemangiosarcomas are frequently seen in dogs. Because of their bad prognosis differentiation from other benign splenic lesions are of prognostic importance.     

Expression of vascular endothelial growth factor in canine oral malignant melanoma

Serum, plasma and tissue expression of vascular endothelial growth factor (VEGF) was measured in 20 dogs previously diagnosed histologically with oral melanoma. The concentrations of VEGF in serum and plasma were significantly higher in dogs with melanoma than in a control population (P ≤ 0.002). Concentrations of VEGF in the serum and plasma of dogs with melanoma were highly correlated (r = 0.867). Ninety‐five per cent of melanoma tissues expressed VEGF. Two staining patterns were detected: diffuse and granular cytoplasmic staining. High blood concentrations of VEGF were correlated to a shorter survival time in dogs receiving definitive therapy (P = 0.002). Survival times were significantly longer in dogs receiving definitive therapy versus palliative therapy (median 496 versus 97 days, P = 0.007). Blood concentrations of VEGF were associated with stage (P < 0.05). Dogs with oral melanoma have increased serum, plasma and tissue concentrations of VEGF. Increased expression of VEGF may be a reasonable target for future therapy of canine oral melanoma.     

人端粒酶催化亚基和SV40大T抗原永生化猪脐静脉血管内皮细胞

为建立稳定的猪血管内皮细胞系,用于猪瘟病毒(CSFV)的致病机理研究提供理想的细胞模型,本研究利用逆转录病毒分别转导人端粒酶催化亚基(hTERT)基因或SV40大T抗原(SV40 LT)基因进入猪脐静脉血管内皮细胞(SUVEC),通过G418或嘌呤霉素筛选阳性克隆,并进行细胞传代研究和细胞形态学及表型鉴定。分别获得了两种方法建立的永生化猪血管内皮细胞系SUVEC-hT和SUVEC-ST。两种细胞系均呈铺路石样单层排列生长,具有高度的增殖活性,在传代70代后不表现任何衰老细胞的特征,并保持接触生长抑制。SUVEC-hT细胞系持续表达hTERT、CD31、CD34和von Willebrand factor,并能摄取Dil标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)。SUVEC-ST细胞系持续表达SV40 LT、CD31和CD34,并能摄取Dil-Ac-LDL。两个细胞系均保持正常的核型并对CSFV敏感。结果表明通过表达外源的hTERT或SV40 LT,SUVEC已被永生化,并且保留了血管内皮细胞的主要生物学特征。     

趾叶炎小鼠血管内皮生长因子表达的研究

采用组织化学和免疫组化的染色方法,检测趾叶炎趾部组织中血管内皮生长因子表达水平、炎性细胞、肥大细胞及其脱颗粒的动态变化,探讨血管内皮生长因子(VEGF)在趾叶炎发病中的作用。结果表明,二磷酸组织胺注射后炎症组小鼠趾部血管内皮生长因子在真皮网状层、血管及腺体周围和部分肥大细胞内,在14 h表达达到一个峰值,而后表达减弱,72 h~15 d表达迅速增强;炎症组小鼠趾部肥大细胞及其脱颗粒和炎性细胞数量,随二磷酸组织胺注射时间的延长,急剧增多,均极显著高于正常组(P(0.01),且其变化趋势均与血管内皮生长因子表达趋势相吻合;同时在真皮出现了许多增生的小动脉,管壁增厚,管腔内有血栓形成。结果说明血管内皮生长因子参与小鼠趾叶炎的发病过程。