Anterior and posterior segment photography. An alternative approach using a dSLR camera adaptor

Purpose To describe a novel digital single lens reflex (dSLR) camera adaptor for anterior and posterior segment photography. Methods The adaptor was used to evaluate canine, feline, and equine patients presenting to Tufts Ophthalmology service. Anterior segment imaging was conducted with the adaptor mounted between a dSLR camera body (Canon 7D) and a macro lens (Canon EF‐S 60mm/f2.8). Posterior segment imaging was performed with the aid of an indirect ophthalmic lens mounted in front of the macro lens. Coaxial illumination during viewing was provided by a single white light‐emitting diode (LED) within the adaptor, while illumination during exposure was provided by the pop‐up flash or an accessory flash. Corneal and/or lens reflections were eliminated using a pair of linear polarizers, having their azimuths at right angles to one another. Results This dSLR camera adaptor provides quality high‐resolution, reflection‐free, images of both the anterior and posterior segments. It was easy to transport, assemble, and handle. The necessary adjustments, positioning, and focusing required for quality images were easily performed. Conclusion The described dSLR camera adaptor provides an alternative to existing imaging systems. High‐resolution image acquisition occurred at a fraction of the cost of established imaging system, particularly those devoted to the posterior segment.     

Establishing a reproducible method for the culture of primary equine corneal cells

Objective  To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation.
Procedures  Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining.
Results  All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogenous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery.
Discussion  This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.     

Determination of equine deep digital flexor muscle volume based on distances between anatomical landmarks

In equine medicine the use of Botox® is experimental. Dosages are determined from human treatment-protocols and limited numbers of equine studies. Determination of target-muscle volume can be helpful to extrapolate human dosages. The aim of the study was to calculate a formula enabling the estimation of the deep digital flexor muscle (DDFM) volume based on distances between anatomical landmarks.     

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.     

Insulin resistance in equine digital vessel rings: an in vitro model to study vascular dysfunction in equine laminitis

Reasons for performing study: One of the causes of equine laminitis is hyperinsulinaemia, which may be associated with endothelial dysfunction and insulin resistance of vessels. Hypothesis and objectives: Insulin resistance can be induced in palmar digital vessels by continued exposure to insulin in vitro. The objective was to evaluate this in vitro model for future studies. Methods: Palmar digital vessel segments were collected immediately after euthanasia from horses with normal insulin/glucose blood values. Four arterial and 4 venous rings (3 mm wide) were prepared and each ring mounted in a tissue bath, containing Tyrode's solution at 37°C, 2 g tension was applied and the rings allowed to equilibrate for 45 min. Of the 4 rings of each vessel type, one was used as a control. One each of the remaining 3 rings was used for incubation with insulin (to induce resistance), wortmannin (to block PI3‐kinase) and PD‐098059 (to block MAP‐kinase), respectively, for 30 min. After the incubation period, the rings were contracted with phenylephrine. When the response reached a plateau, a single dose of insulin was added to the baths and the response of each ring monitored for 30 min. Results: Insulin relaxed the control rings and those treated with PD 098059 but contracted those pretreated with insulin and wortmannin. Normal relaxation responses of the rings were converted to contractions by insulin resistance. Insulin resistance was confirmed by the qualitative response of insulin‐incubated and wortmannin‐incubated rings. Conclusions: This study demonstrated successful induction of insulin resistance in both arterial and venous rings. It also suggested that the MAP‐kinase pathway plays a minor role in controlling vasomotor tone under normal physiological conditions. Potential relevance: The study suggests that the induction of insulin resistance in equine palmar digital vessel rings is reliable and provides a good in vitro model for studying the vascular insulin resistance which may occur in equine laminitis.     

Anterior segment fluorescein angiography of the normal canine eye using a dSLR camera adaptor

Purpose To describe anterior segment fluorescein angiography (ASFA) of the normal canine eye using two different sedation/anesthetic protocols and a digital single lens‐reflex (dSLR) camera adaptor. Methods Dogs free of ocular and systemic disease were used for this study. Dogs received maropitant citrate (1.0 mg/kg SQ) and diphenhydramine (2.0 mg/kg SQ) 20 min prior to butorphanol [n = 6] (0.2 mg/kg IV) or propofol [n = 6] (4 mg/kg IV bolus, 0.2 mg/kg/min CRI). Standard color and red‐free images were obtained prior to administration of 10% sodium fluorescein (20 mg/kg IV). Image acquisition was performed using a dSLR camera (Canon 7D), dSLR camera adaptor, camera lens (Canon EF‐S 60 mm f/2.8 macro), and an accessory flash (Canon 580EXII). Imaging occurred at a rate of 1/s immediately following bolus for a total of 30 s, then at 1, 2, 3, 4, 5, and 10 min. Results Twelve dogs with a combined mean age of 5.1 years and various iris colors were imaged. Arterial, capillary, and venous phases were identified and time sequences recorded. Visibility of the vascular pattern was inversely related to iris pigmentation. Complete masking of blood flow was noted with heavily pigmented irises. Vessel leakage was noted in some eyes. Proper patient positioning and restricted ocular movements were critical in acquiring quality images. No adverse events were noted. Conclusion This study demonstrated that quality high resolution ASFA images were obtainable using a novel dSLR camera adaptor. ASFA of the normal canine eye is limited to irises, which are moderately to poorly pigmented. Use of general anesthesia produced higher quality images and is recommended for ASFA in the dog.     

Effects of recombinant equine growth hormone on in vitro biomechanical properties of the superficial digital flexor tendon of Standardbred yearlings in training

OBJECTIVE: To determine whether recombinant equine growth hormone (rEGH) would alter the in vitro biomechanical properties of the forelimb superficial digital flexor tendon (SDFT) in exercising young Standardbred horses. STUDY DESIGN: Randomized complete block design. ANIMALS: Twelve Standardbred yearlings. METHODS: Horses were trained for 12 weeks on a high-speed treadmill (10% positive incline). rEGH was administered intramuscularly (IM) daily (10 microg/kg during week 4; 20 microg/kg for weeks 5-9) to 6 horses (treated group), whereas 6 horses (control group) were administered an equivalent daily volume of sterile water IM. At 12 weeks, horses were euthanatized and left forelimb SDFTs were collected and stored (-70 degrees C). A section from the mid-region of the SDFT was held in cryoclamps with a 4 cm interspace distance and distracted at 10 mm/s until failure. The variables evaluated were maximal load at yield and failure, ultimate and yield tensile stress and strain, tendon stiffness, and mode of failure. Data were analyzed using unpaired, two-tailed, Student's t-test. Statistical significance was set at P < or =.05. RESULTS: Yield and ultimate tensile stress were significantly lower in the rEGH-treated horses compared with controls. There was a trend toward increased maximal displacement, increased ultimate tensile strain, and decreased tendon stiffness in rEGH-treated horses compared with controls. Tensile stress and cross-sectional area, and tensile stress and stiffness were significantly correlated at yield and failure points. CONCLUSIONS: rEGH, administered at the manufacturer's recommended dose rates to maturing Standardbred horses in training, does not significantly augment the in vitro biomechanical properties of the forelimb SDFT. CLINICAL RELEVANCE: Administration of rEGH to young horses in training is unlikely to enhance the physiologic adaptation of the SDFT to exercise stress.     

In vitro analysis of antiangiogenic activity of fungi isolated from clinical cases of equine keratomycosis

OBJECTIVE: The goal of this project was to explore the possibility that fungal organisms produce metabolites that inhibit angiogenesis. Procedures Fungal cultures were obtained from cases of keratomycosis, grown in Sabouraud's dextrose broth, and sterile filtered for use in experiments. The Matrigel assay was used to screen the filtrate samples for antiangiogenic activity. Matrigel is a basement membrane matrix that supports the differentiation of human umbilical vein endothelial (HUVE) cells into a capillary-like network of tubules. HUVE cells were cultured using standard techniques and passaged at confluence, with all cells being used at passage 3-6. HUVE cells (40 000 cells) were pipetted into each well of a 24-well tissue-culture plate coated with Matrigel. An aliquot of fungal media filtrate was added to each well and the plates allowed to incubate for 18 h, at which time they were evaluated for tubule formation. RESULTS: Two fungal isolates showed inhibition of tubule formation. The addition of 100, 200 and 400 &mgr;L of the fungal media filtrate from the first isolate (Fusarium sp. 99A34574) produced a consistent and dose-dependent inhibition of tubule formation. The second isolate (Aspergillus sp. 271599) did not show inhibition of tubule formation with 100 or 200 &mgr;L added to the wells, however, it did show inhibition at 400 &mgr;L/well. The remaining three isolates did not cause inhibition at any concentration. CONCLUSIONS: Our findings suggest that certain fungal organisms produce metabolites that inhibit tubule formation in vitro, and that these metabolites may play a significant role in altering the host vascular response to fungal infections of the cornea.     

Normal corneal thickness measurements in pigmented rabbits using spectral‐domain anterior segment optical coherence tomography

Objective  To document the thickness of the central cornea in pigmented rabbits using spectral‐domain anterior segment optical coherence tomography (AS‐OCT). Animals studied  Seventeen pigmented rabbits (6 male, 11 female, both eyes) were involved in this study. Procedures  Thirty‐four eyes from healthy pigmented rabbits underwent a complete ophthalmologic examination, including AS‐OCT. Eight radial scans, 6 mm in length and centered on the cornea, were obtained using the AS‐OCT. Corneal thickness was automatically calculated using pachymetry software. Measurements were displayed as the mean and standard deviation for each of the 17 regions defined by the software. The regions were the center (1 mm radius, area a), the inner ring (2.5 mm radius, area b), the outer ring (3 mm radius, area c), and the eight radial scan lines in eight directions (Superior (1), SN (2), Nasal (3), IN (4), Inferior (5), IT (6), Temporal (7), ST (8)) with an angle of 45° between each consecutive scan line (a, b 1–8, c 1–8). Results  There was no statistically significance difference in corneal thickness between gender, eye, and the eight directions (P = 0.804, P > 0.05, P > 0.05). There was a statistical difference between the thickness in areas a, b, and c (P < 0.05). The corneal thickness increased gradually from the center to the periphery of the 6 mm measured. The center corneal thickness was 387 ± 19.8 μm for OD and 384 ± 20.2 μm for OS. The corneal thicknesses of the thinnest point of the right eyes (OD) and the left eyes (OS) were 376 ± 20.2 and 370 ± 16.8 μm, respectively. There was positive correlation between the thinnest point and area a in both the right eyes (r = 0.892, P < 0.001) and the left eyes (r = 0.832, P < 0.001). Conclusions  This is the first documentation of the rabbit corneal thickness in vivo using the spectral‐domain AS‐OCT. Pigmented rabbit corneas were almost 150 μm thinner than human corneal values. Gender and eye were not associated with any statistical differences in central corneal thickness in this study.     

Isolation and cultivation of equine corneal keratocytes, fibroblasts and myofibroblasts

Objective  To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed.
Animal material  Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease.
Procedure  Equine corneal stroma was isolated using mechanical techniques and stromal sub-sections were then cultured. Customized media at different culture conditions was used to promote growth and differentiation of corneal stromal cells into keratocytes, fibroblasts and myofibroblasts.
Results  Cell culture techniques were successfully used to establish a method for the isolation and culture of equine corneal keratocytes, fibroblasts and myofibroblasts. Immunohistochemical staining for alpha-smooth muscle and F-actin was used to definitively differentiate the three cell types.
Conclusion  Equine corneal stromal keratocytes, fibroblasts and myofibroblasts can be predictably isolated and cultured in vitro using this protocol.     

Troponin assays in the assessment of the equine myocardium

In 2000, troponin assays were adopted as the test of choice for detection of myocardial injury in man. This decision was made after extensive testing and followed a 60 year search for a biomarker of myocardial damage with sufficient analytical sensitivity and specificity. This has led to proliferation of assays for use in human medicine, each requiring extensive testing and validation before it could be made available on the open market for human use. The search for ever‐more analytically sensitive assays and for a standard reference material continues. The adoption of troponin testing in veterinary medicine followed shortly after its development for use in man, providing a much‐needed means of detecting and monitoring myocardial damage in horses. However, application of these tests in veterinary medicine has exclusively involved use of assays designed for and clinically validated in human patients. There is no mandated requirement for test validation in veterinary medicine and, while many of these assays have been shown to be capable of detecting equine troponin, the wide diversity of available tests, lack of validation, absence of protocols for their use and lack of standardisation make their application problematic. The objective of this review article is to address this issue, offering guidance where data are available and encouraging caution where there are none. Ultimately, the overall goal of this review is to examine critically the use of troponin assays in the horse and to promote the accurate and appropriate interpretation of valid results.     

Anterior segment fluorescein angiography of the normal feline eye using a dSLR camera adaptor

Purpose To describe anterior segment fluorescein angiography (ASFA) of the normal feline eye using a digital single‐lens reflex (dSLR) camera adaptor. Animals Ten cats free of ocular and systemic disease were evaluated. Methods All cats received maropitant citrate (1.0 mg/kg SQ) and diphenhydramine (2.0 mg/kg SQ) 20 min prior to anesthesia using propofol (4 mg/kg IV bolus, 0.2 mg/kg/min CRI). Standard color and red‐free images were obtained prior to the administration of 10% sodium fluorescein (20 mg/kg IV). Imaging was performed using a dSLR camera (Canon 7D), dSLR camera adaptor, camera lens (Canon EF‐S 60 mm f/2.8 macro), and an accessory flash (Canon 580EXII). Imaging occurred at a rate of 1/second immediately following IV bolus of sodium fluorescein for a total of 30 s, then at 1, 2, 3, 4, 5, and 10 min. Results Ten cats with an average age of 3.7 ± 0.9 years and various iris colors were imaged. Arterial, capillary, and venous phases occurred 4.6, 7.8, and 8.9 s postinjection, respectively. Visibility of the vasculature was not impaired by the degree of iris pigmentation. Patency of a persistent pupillary membrane was noted in one cat. Vessel leakage was common, as well as, leakage into the aqueous humor. Proper patient positioning and restricted ocular movements were critical. No adverse events were noted. Conclusions This study demonstrates ASFA findings in normal feline eyes using a cost‐effective dSLR camera adaptor. Fluorescein leakage from vessels and into the aqueous humor was a common finding. Visibility of iris vasculature was not impaired by the degree of iris pigmentation.     

The effect of intralesional injection of bone marrow derived mesenchymal stem cells and bone marrow supernatant on collagen fibril size in a surgical model of equine superficial digital flexor tendonitis

Reasons for performing study: Collagen fibril size is decreased in repair tissue following tendon injury compared to normal tendon matrix in horses. Mesenchymal stem cells have been suggested to promote regeneration of tendon matrix rather than fibrotic repair following injury, although this concept remains unproven. Objectives: To explore the hypothesis that implantation of autologous mesenchymal stem cells derived from bone marrow into a surgically created central core defect in the superficial digital flexor tendon (SDFT) of horses would induce the formation of a matrix with greater ultrastructural similarities to tendon matrix than the fibrotic scar tissue formed in control defects. Methods: Tissue was collected 16 weeks after induction of injury and 12 weeks after treatment from normal and injured regions of control and treated limbs of 6 horses and examined using transmission electron microscopy. Collagen fibril diameters were measured manually with image analysis software and surface areas calculated. Three parameters assessed for normal and injured tissue were mass average diameter (MAD), collagen fibril index (CFI) and the area dependent diameter (ADD). Results: Normal regions from both treated and control limbs displayed higher MAD and CFI values, as well as a characteristic bimodal distribution in fibril size. Injured regions from both treated and control limbs displayed significantly lower MAD and CFI values, as well as a unimodal distribution in fibril size. There were no significant differences between treated and control limbs for any of the parameters assessed. Conclusions: Intralesional injection of autologous bone marrow derived mesenchymal stem cells had no measurable effect on the fibril diameter of collagen in healing tissue in the SDFT of this experimental model 16 weeks after injury. Potential relevance: Favouring matrix regeneration over fibrotic repair may not be the mechanism by which autologous mesenchymal stem cells assist healing of tendon injury.     

Effects of unilateral topical administration of 0.5% tropicamide on anterior segment morphology and intraocular pressure in normal cats and cats with primary congenital glaucoma

Objective To determine the effects of topical 0.5% tropicamide on anterior segment morphology (ASM) and intraocular pressure (IOP) in normal and glaucomatous cats. Animals used Normal cats and cats with inherited primary congenital glaucoma (PCG). Procedures Control IOP curves were performed in untreated normal and PCG cats. In the first experiment, tropicamide was applied OD in eight normal and nine PCG cats. IOP and pupillary diameter (PD) were measured at 0, 30, and 60 min, then hourly until 8 h post‐treatment. In a second experiment, six normal and seven PCG cats received tropicamide OD. High‐resolution ultrasound images were obtained at 0, 1, 5, and 10 h post‐treatment to measure ASM changes. IOP and PD were measured OD at 0, 1, 2, 3, 5, 7, and 9 h. Results In untreated normal cats IOP OU decreased throughout the day. In PCG cats IOP OU had wide fluctuations over time. In normal cats IOP response varied in the treated eye but did not change significantly in untreated eyes. IOP significantly increased from baseline in both eyes of all treated PCG cats. Increases in IOP were associated with some ASM changes. Cats with PCG had a significantly smaller angle recess areas, diminished ciliary clefts and decreased iris‐lens contact. ASM changes were not strongly correlated with IOP in all cats. Conclusions The ASM of PCG cats is markedly different from normal cats, and clinically significant increases in IOP OU occur in cats with PCG after tropicamide treatment. The mechanism for this increase remains unclear.     

马流感A/马/京防/74-1(H7N7)毒株HA基因的序列分析

流感病毒(Influenza Virus)根据血凝素(HA)和神经氨酸酶(NA)两种表面抗原蛋白分为不同的亚型,马流感I型(H7N7)和Ⅱ型(H3N8)是其中比较重要的两个亚型.本研究应用无特定病原体(SPF)鸡胚增殖马流感病毒A/马/京防/74-1(H7N7)毒株,TRIzol LS Reagent提取病毒RNA,RT-PCR扩增HA基因全片段,克隆到PMD18-T载体上,并进行了鉴定和序列测定.所获得的HA基因片段长1 727 bp,编码563个氨基酸残基.根据推导的氨基酸序列进行预测,有7个潜在的糖基化位点和16个半胱氨酸残基,通过序列分析推断,A/马/京防/74-1(H7N7)株病毒的HA来源于禽类,是一株通过基因重排出现的重组病毒.     

The effect of ocular blinkers on the horses' reactions to four different visual and audible stimuli: results of a crossover trial

Objective To determine the effect of ocular blinkers on driving horses’ reactions to visual and audible stimuli. Design Balanced crossover trial with horses randomly assigned to either wear blinkers or not wear blinkers first or second, then subjected to repeated sequences of four distinct stimuli (chain rattle, cap gun, umbrella opening, and shaking of an aluminum can containing coins). Two weeks later, this process was repeated with the ordering of wearing blinkers reversed. Animal studied Eight driving horses of various breeds. Procedures Responses were recorded quantitatively as inter‐beat times (the time between each heart beat in msec) and qualitatively via video recording. Statistical analysis of the first 10 s of inter‐beat times poststimulus assessed the effect of the presence of blinkers, order of blinker application and stimulus type using a linear regression model with a random effect for horse. Results Wearing blinkers is significantly associated with a decrease in the inter‐beat times (increase in heart rate) when they are worn by horses experiencing an unfamiliar sound. However, wearing blinkers is significantly associated with an increase in the inter‐beat times (decrease in heart rate) when worn by horses experiencing a primarily visual, familiar stimulus. Conclusions and Clinical Relevance Trained driving horses, when they wear blinkers, have a decrease in inter‐beat time (an increase in heart rate) when there is an unfamiliar sound. This may have relevance to horses who have limited vision caused by trauma or disease.