Establishment of a canine lens epithelial cell line.

Some immortalized lens epithelial cell lines have been established and are useful for molecular analysis. The establishment of additional cell lines must, however, enable a variety of in-vitro examinations. The objective of this study was to establish a new canine lens epithelial cell line by isolating CLC-1 cells from the lens tissue of a dog with cataracts. In CLC-1 cells, transforming growth factor beta (TGF-ß) treatment significantly decreased gene expression of an epithelial marker and elevated that of mesenchymal markers; these characteristics are similar to those of a human lens epithelial cell line. Interestingly, CLC-1 cells exhibited lower expression of an epithelial marker and higher expression of mesenchymal markers than an anterior lens capsule. These results suggest that CLC-1 cells were derived from a cell population that was committed to epithelial-mesenchymal transition in cataract lens tissue. In conclusion, CLC-1 cells could be useful for analyzing molecular pathogenesis in canine cataracts.

Certaines lignées de cellules épithéliales du cristallin immortalisées ont été établies et sont utiles pour analyse moléculaire. L'établissement de lignées cellulaires supplémentaires doit cependant permettre une variété d'examens in vitro. L'objectif de cette étude était d'établir une nouvelle lignée cellulaire épithéliale du cristallin canin en isolant les cellules CLC-1 du tissu du cristallin d'un chien atteint de cataracte. Dans les cellules CLC-1, le traitement par le facteur de croissance transformant bêta (TGF-ß) a significativement diminué l'expression génique d'un marqueur épithélial et élevé celle des marqueurs mésenchymateux; ces caractéristiques sont similaires à celles d'une lignée cellulaire épithéliale du cristallin humain. Fait intéressant, les cellules CLC-1 présentaient une expression inférieure d'un marqueur épithélial et une expression plus élevée de marqueurs mésenchymateux qu'une capsule antérieure du cristallin. Ces résultats suggèrent que les cellules CLC-1 étaient dérivées d'une population cellulaire qui était impliquée dans la transition épithéliale-mésenchymateuse dans le tissu du cristallin de la cataracte. En conclusion, les cellules CLC-1 pourraient être utiles pour analyser la pathogenèse moléculaire dans les cataractes canines.(Traduit par Docteur Serge Messier).

Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass.

Little is known about the pathological roles of sebaceous glands in canine skin diseases, as most examinations have been conducted with cultured human sebaceous epithelial cell lines. To our knowledge, there is no available canine sebaceous epithelial cell line. The purpose of this study was to establish a canine sebaceous epithelial cell line and characterize it. An eyelid mass in a dog was surgically resected for treatment, and it was histologically diagnosed as sebaceous epithelioma. Collected tissue was conducted for culture, and the growing epithelial-like cells were passaged. The cells showed continuous proliferation for over 6 months. After 40 passages, the cells were named CMG-1. Lipid droplets in the cytoplasm of CMG-1 cells were confirmed by Oil Red O staining. As reported in studies with human sebaceous epithelial cell lines, lipogenesis in CMG-1 cells was promoted by linoleic acid, whereas transforming growth factor-ß (TGF-ß) suppressed it. Additionally, real-time PCR revealed that the expression levels of chemokines and cytokines, including CC chemokine ligand (CCL)-2, CCL-20, CXCL-10, Tumor necrosis factor-α (TNF-α), Interleukin (IL)-1α, IL-1ß, and IL-8, were significantly increased in CMG-1 cells following treatment with lipopolysaccharide. In conclusion, we successfully established a new canine sebaceous epithelial cell line. Our data indicated that lipogenesis and inflammatory responses were quantitatively evaluable in this cell line. CMG-1 cells could be useful for the pathological analysis of sebaceous gland diseases in dogs.

Effects of Sedation by Intramuscular Administration of Medetomidine on Canine Abdominal Vascular System and Hepatic Parenchyma Imaging Using Enhancement Dynamic Computed Tomography.

This prospective crossover study compared the effects of intramuscular administration of medetomidine for sedation on parameters of the abdominal vascular system, measured by enhancement computed tomography (CT), to those of propofol-induced sevoflurane maintenance anesthesia, as a control, in five clinically healthy adult male beagle dogs (11.4-12.8 kg). Each animal underwent both protocols at a 1-week interval. The enhancement (HU) and time to peak enhancement on CT were measured for the aorta (AO), caudal vena cava (CVC), portal vein (PV), and hepatic parenchyma (HP). The contrast effects in the AO, PV, and HP were significantly delayed under medetomidine sedation compared to the control anesthesia protocol. Particularly, the contrast effect in the PV and HP was significantly delayed under sedation, appearing approximately 1 min after contrast medium injection. This delay likely reflects the peripheral vasoconstrictive effect of medetomidine. We noted a generally early high contrast enhancement of the CVC under medetomidine sedation, likely contributed by the induced bradycardia. Therefore, findings obtained on contrast enhancement CT under medetomidine sedation may be different from those obtained under propofol-induced sevoflurane maintenance anesthesia. These differences are important to consider when using the findings to inform diagnosis.

Combining non-contrast enhanced magnetic resonance thoracic ductography with vascular contrast-enhanced computed tomography to identify the canine thoracic duct.

Background: In humans, visualization of the thoracic duct by magnetic resonance imaging (MRI) has been attempted, and recent advances have enabled clinicians to visualize the thoracic duct configuration in a less invasive manner. Moreover, MRI does not require contrast media, and it enables visualization of morphological details of the thoracic structures. In veterinary practice, the thoracic duct has not been visualized three dimensionally in MRI. Aim: This study aimed to assess the performance of our magnetic resonance thoracic ductography (MRTD) technique to visualize the thoracic duct and the surrounding 3D anatomical structures by combining MRTD and vascular contrast-enhanced thoracic computed tomography (CT) images in dogs. Methods: Five adult male beagle dogs (11.4-12.8 kg) were included in this study. Sagittal and transverse T2-weighted images were scanned in MRI. Scanning in MRTD used a single-shot fast spin echo sequence with a respiratory gate. CT was performed after the intravenous injection of contrast medium. All MRTD and CT images were merged using a workstation. Results: The thoracic ducts were identified in MRTD images of all dogs, and the surrounding anatomical structures were located with the aid of contrast-enhanced thoracic CT. In all dogs, the thoracic ducts coursed along the right-dorsal side of the aorta, cranially from the L2 level. Thereafter, these bent to the left side at the aortic arch and curved at the left external jugular vein angle. A comparison of the number of thoracic ducts at each vertebra between transverse T2WI and MRTD did not reveal any significant differences for all vertebrae. Conclusion: The results from our study suggest that MRTD using the single-shot fast spin echo sequence could be a useful tool for visualization of the thoracic duct. Furthermore, the image merged from MRTD and vascular-enhanced images provided detailed anatomical annotation of the thorax. The MRTD protocol described in this study is safe and easily adaptable, without the need for contrast medium injection into the lymph system. In addition, the images fused from MRTD and vascular contrast-enhanced CT image of the thorax could provide detailed anatomical annotations for preoperative planning.

Effects of pupil size on canine visual evoked potential with pattern stimulation.

The purpose of this study was to investigate the effects of pupil diameter on canine visual evoked potentials with pattern stimulation (P-VEP). Atropine eye drop (1.0%) was applied to both eyes as a cycloplegic drug, and tafluprost eye drop (0.015%) was applied to one eye that was selected randomly for miosis (miosis group). The other eye did not receive tafluprost (mydriasis group). P-VEP was recorded at three pattern sizes. The P100 implicit time at a small pattern size in the mydriasis group was significantly prolonged compared to the miosis group. We hypothesized that the prolonged P100 implicit time under mydriatic conditions was due to increased spherical aberrations and concluded that mydriatic conditions affected P100 implicit time in canine P-VEP recordings.

Surgical removal of cataract in an Asiatic black bear (Ursus thibetanus) by phacoemulsification and aspiration.

A twenty-year-old male Asiatic black bear (Ursus thibetanus) presented at the Rakuno Gakuen University Animal Medical Center with a 10-year history of bilateral blindness and cataracts. Surgical treatment of bilateral cataracts by extracapsular lensextraction using phacoemulsification and aspiration (PEA) was performed under general anesthesia. An anterior capsulectomy was performed using micro iris scissors and micro anterior lens capsule forceps. The cataract was removed with PEA using the two-handed technique. After surgery, systemic corticosteroids, anti-inflammatory drugs and antibiotics were administered. After cataract removal, the bear had recovered vision, and good quality vision has been maintained to date (15 months). PEA can be a safe and effective treatment for cataracts that impair vision in bears.

Effect of medetomidine on tear flow measured by Schirmer tear test I in normal pigs.

Medetomidine, an α2-adrenoceptor agonist, was reported to decrease tear flow in some species. However, there are no reports about the effect of medetomidine on tear flow in pigs. The purpose of this study was to elucidate it. The study was performed in 10 clinically normal female Landrace pigs aged 3 months. Tear flow was measured by the Schirmer tear test (STT) I before (baseline) and 15 and 30 min after intramuscular administration of 80 µg/kg medetomidine. Compared to the STT I value at baseline, the value decreased significantly at 30 min after administration in both the left and right eyes. In pigs treated with medetomidine, an artificial tear solution or ophthalmic gel should be applied to protect the ocular surface.

Effect of intramuscular administration of medetomidine and xylazine on tear flow measured by the Schirmer tear test I in healthy cats.

OBJECTIVES: This study aimed to investigate the effects of intramuscular medetomidine and xylazine on tear flow in healthy cats. METHODS: Five cats each received medetomidine 10, 20, 40 and 80 µg/kg IM; xylazine 1.0, 2.0, 4.0 and 8.0 mg/kg IM; and physiological saline (2.0 ml IM) in a randomised order separated by intervals of at least 1 week. The Schirmer tear test (STT) I was performed in both eyes before and 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h after each dose. RESULTS: The STT I value decreased significantly at 0.5 and 1.0 h and at 0.75 and 1.0 h in both eyes after administration of medetomidine at 10 or 40 µg/kg. After administration of medetomidine 80 µg/kg, there was a significant decrease in the STT I reading at 0.75, 2 and 3 h in the left eye and 0.75, 1, 2 and 3 h in the right eye. The STT I value decreased significantly at: 0.5, 0.75, 1 and 2 h in the left eye and 0.75 h in the right eye after administration of xylazine 1.0 mg/kg; 0.5, 0.75, 1 and 2 h in the left eye and 0.5, 0.75, 1 and 3 h in the right eye after administration of xylazine 2.0 mg/kg; 0.5, 0.75, 1 and 2 h in both eyes after administration of xylazine 4.0 mg/kg; and 0.5, 0.75, 1, 2 and 3 h in the left eye and 0.75, 1, 2, 3 and 4 h in the right eye after administration of xylazine 8.0 mg/kg. CONCLUSIONS AND RELEVANCE: Both medetomidine and xylazine significantly decreased feline tear flow measured by STT I. Therefore, the ocular surface should be monitored carefully and protected appropriately in cats treated with these sedatives.

Measurement of visual acuity in Beagle dog by visual evoked potential with pattern stimulation.

We tried measurement of visual acuity in laboratory beagle using pattern stimulus visual evoked potential (P-VEP). We recorded P-VEP in 6 beagles which were corrected refractive power. The stimulus pattern size was 1.22 mm. The testing distance were 0.5, 1.0, and 2.0 m. The visual angles and spatial frequency were calculated from stimulus pattern size and distance. In all subjects, P-VEP was clearly recorded in all testing distance, and this result means that the eye could recognized grid pattern on the stimulus monitor. When stimulus monitor was set up 2.0 m, spatial frequency was 14.29 cpd. From our results, it was founded that the visual acuity in laboratory beagle which was corrected refractive power was 14.29 cpd and more.

Targeted ablation of p38α MAPK suppresses denervation-induced muscle atrophy.

The loss of skeletal muscle mass is a major cause of falls and fractures in the elderly, leading to compromised independence and a decrease in the quality of life. However, only a few therapeutic interventions leading to marginal clinical benefits in patients with this condition are currently available. Therefore, the demand to further understand the pathology of muscle atrophy and establish a treatment modality for patients with muscle atrophy is significant. p38α mitogen-activated protein kinase (p38α MAPK) is a ubiquitous signaling molecule that is implicated in various cellular functions, including cell proliferation, differentiation, and senescence. In the present study, we generated a mutant line in which p38α MAPK is specifically abrogated in muscle tissues. Compared with the control mice, these mutant mice are significantly resistant to denervation-induced muscle atrophy, suggesting that p38α MAPK positively regulates muscle atrophy. We also identified CAMK2B as a potential downstream target of p38α MAPK and found that the pharmacological inhibition of CAMK2B activity suppresses denervation-induced muscle atrophy. Altogether, our findings identify p38α MAPK as a novel regulator of muscle atrophy and suggest that the suppression of intracellular signaling mediated by p38α MAPK serves as a potential target for the treatment of muscle atrophy.

Morphological analyses of the retinal photoreceptor cells in the nocturnally adapted owl monkeys.

Owl monkeys are the only one species possessing the nocturnal lifestyles among the simian monkeys. Their eyes and retinas have been interested associating with the nocturnal adaptation. We examined the cellular specificity and electroretinogram (ERG) reactivity in the retina of the owl monkeys by comparison with the squirrel monkeys, taxonomically close-species and expressing diurnal behavior. Owl monkeys did not have clear structure of the foveal pit by the funduscope, whereas the retinal wholemount specimens indicated a small-condensed spot of the ganglion cells. There were abundant numbers of the rod photoreceptor cells in owl monkeys than those of the squirrel monkeys. However, the owl monkeys' retina did not possess superiority for rod cell-reactivity in the scotopic ERG responses. Scanning electron microscopic observation revealed that the rod cells in owl monkeys' retina had very small-sized inner and outer segments as compared with squirrel monkeys. Owl monkeys showed typical nocturnal traits such as rod-cell dominance. However, the individual photoreceptor cells seemed to be functionally weak for visual capacity, caused from the morphological immaturity at the inner and outer segments.

Aldehyde dehydrogenase activity helps identify a subpopulation of murine adipose-derived stem cells with enhanced adipogenic and osteogenic differentiation potential.

AIM: To identify and characterize functionally distinct subpopulation of adipose-derived stem cells (ADSCs). METHODS: ADSCs cultured from mouse subcutaneous adipose tissue were sorted fluorescence-activated cell sorter based on aldehyde dehydrogenase (ALDH) activity, a widely used stem cell marker. Differentiation potentials were analyzed by utilizing immunocytofluorescece and its quantitative analysis. RESULTS: Approximately 15% of bulk ADSCs showed high ALDH activity in flow cytometric analysis. Although significant difference was not seen in proliferation capacity, the adipogenic and osteogenic differentiation capacity was higher in ALDHHi subpopulations than in ALDHLo. Gene set enrichment analysis revealed that ribosome-related gene sets were enriched in the ALDHHi subpopulation. CONCLUSION: High ALDH activity is a useful marker for identifying functionally different subpopulations in murine ADSCs. Additionally, we suggested the importance of ribosome for differentiation of ADSCs by gene set enrichment analysis.

Single-Cell Phosphospecific Flow Cytometric Analysis of Canine and Murine Adipose-Derived Stem Cells.

This study aimed to demonstrate single-cell phosphospecific flow cytometric analysis of canine and murine adipose-derived stem/stromal cells (ADSCs). ADSCs were obtained from clinically healthy laboratory beagles and C57BL/6 mice. Cell differentiation into adipocytes, osteocytes, and chondrocytes was observed for the cultured canine ADSCs (cADSCs) and murine ADSCs (mADSCs) to determine their multipotency. We also performed single-cell phosphospecific flow cytometric analysis related to cell differentiation and stemness. Cultured cADSCs and mADSCs exhibited the potential to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, single-cell phosphospecific flow cytometric analysis revealed similar ß-catenin and Akt phosphorylation between mADSCs and cADSCs. On the other hand, it showed the phosphorylation of different Stat proteins. It was determined that cADSCs and mADSCs show the potential to differentiate into adipocytes, osteocytes, and chondrocytes. Furthermore, a difference in protein phosphorylation between undifferentiated cADSCs and mADSCs was identified.

Identification of rhodamine 123-positive stem cell subpopulations in canine hepatocellular carcinoma cells.

The majority of cases of chemotherapy for hepatocellular carcinoma (HCC) are not effective in human or veterinary medicine due to resistance against anticancer agents. In human medicine, hepatocellular carcinoma stem cells (HCSCs) were recently identified as cytokeratin 19 (CK19)-, cluster of differentiation (CD)-44-, and CD133-positive. However, there are few previous reports regarding canine HCSC (cHCSC). Additionally, to the best of our knowledge, the chemoresistance against anticancer agents of these cHCSCs has not been investigated. In the present study staining of cHCSCs was performed with rhodamine 123, a low-toxicity fluorescent dye for mitochondria, by flow cytometry. There were two subpopulations in the HCC cell line defined by their higher (RhoHi) and lower (RhoLo) fluorescence intensity of rhodamine 123. The RhoHi subpopulation demonstrated a higher Nanog gene expression, sphere-forming ability, and chemoresistance against gemcitabine. However, there was no significant difference between RhoHi and RhoLo regarding the proliferation rate and chemoresistance against mitoxantrone and doxorubicin. The present results indicate that the expression of rhodamine 123 identifies different stem cell subpopulations in a canine HCC cell line.

Comparison of postoperative pain and inflammation reaction in dogs undergoing preventive laparoscopic-assisted and incisional gastropexy.

This study compared the effects of postoperative pain and inflammation reaction after preventive laparoscopic-assisted gastropexy (LAG) and incisional gastropexy (IG) in 10 clinically normal Beagles. Surgical time, incision length, visual analog scale (VAS) score, University of Melbourne Pain Scale (UMPS) score, and plasma C-reactive protein (CRP), plasma cortisol (COR), and serum interleukin-6 (IL-6) levels were evaluated. The VAS and UMPS scores and COR and IL-6 levels were recorded at 0.5, 1, 2, 4, 8, 12, 18 and 24 hr after surgery. CRP level was recorded at 12, 24 and 48 hr after surgery. The VAS and UMPS scores showed no significant intergroup differences. Compared to IG, LAG had significantly lower surgical time (45 ± 9.91 min vs 64 ± 5.30 min; P<0.05), incision length (46 ± 8.21 mm vs 129 ± 19.49 mm; P<0.05), CRP level (12 hr after surgery; 4.58 ± 1.58 mg/dl vs 12.4 ± 1.34 mg/dl; P<0.01), and COR level (1 hr after surgery; 10.79 ± 3.07 µg/dl vs 15.9 ± 3.77 µg/dl; P<0.05). IL-6 levels showed no significant intergroup differences at any time point. However, LAG resulted in lower IL-6 levels than did IG at all postoperative time points. Neither procedure resulted in significant surgical complications. LAG produced lower surgical stress than did IG, suggesting that LAG is a safe, minimally invasive, and highly useful technique for preventing canine gastric dilatation-volvulus. Nevertheless, since this study used experimental models, its usefulness should be evaluated in future cases.

Aldehyde dehydrogenase activity identifies a subpopulation of canine adipose-derived stem cells with higher differentiation potential.

Adipose-derived stem cells (ADSCs) are abundant and readily obtained, and have been studied for their clinical applicability in regenerative medicine. Some surface antigens have been identified as markers of different ADSC subpopulations in mice and humans. However, it is unclear whether functionally distinct subpopulations exist in dogs. To address this issue, we evaluated aldehyde dehydrogenase (ALDH) activity-a widely used stem cell marker in mice and humans-by flow cytometry. Approximately 20% of bulk ADSCs showed high ALDH activity. Compared to cells with low activity (ALDHLo), the high-activity (ALDHHi) subpopulation exhibited a higher capacity for adipogenic and osteogenic differentiation. This is the first report of distinct ADSC subpopulations in dogs that differ in terms of adipogenic and osteogenic differentiation potential.

Craniocervical junction abnormalities with atlantoaxial subluxation caused by ventral subluxation of C2 in a dog.

Craniocervical junction abnormalities with atlantoaxial subluxation caused by ventral subluxation of C2 were diagnosed in a 6-month-old female Pomeranian with tetraplegia as a clinical sign. Lateral survey radiography of the neck with flexion revealed atlantoaxial subluxation with ventral subluxation of C2. Computed tomography revealed absence of dens and atlanto-occipital overlapping. Magnetic resonance imaging showed compression of the spinal cord and indentation of caudal cerebellum. The diagnosis was Chiari-like malformation, atlantoaxial subluxation with ventral displacement of C2, atlanto-occipital overlapping, and syringomyelia. The dog underwent foramen magnum decompression, dorsal laminectomy of C1, and ventral fixation of the atlantoaxial joint. Soon after the operation, voluntary movements of the legs were recovered. Finally, the dog could stand and walk without assistance. The dog had complicated malformations at the craniocervical junction but foramen magnum decompression and dorsal laminectomy for Chiari-like malformation, and ventral fixation for atlantoaxial subluxation resulted in an excellent clinical outcome.

Effect of refractive error on visual evoked potentials with pattern stimulation in dogs.

The purpose of this study was to investigate the effects of refractive error on canine visual evoked potentials with pattern stimulation (P-VEP). Six normal beagle dogs were used. The refractive power of the recorded eyes was measured by skiascopy. The refractive power was corrected to -4 diopters (D) to +2 D using contact lens. P-VEP was recorded at each refractive power. The stimulus pattern size and distance were 50.3 arc-min and 50 cm. The P100 appeared at almost 100 msec at -2 D (at which the stimulus monitor was in focus). There was significant prolongation of the P100 implicit time at -4, -3, 0 and +1 D compared with -2 D, respectively. We concluded that the refractive power of the eye affected the P100 implicit time in canine P-VEP recording.

Dark adaptation time in canine electroretinography using a contact lens electrode with a built-in light source.

The purpose of this study was to evaluate the dark adaptation time in canine electroretinography (ERG) using a contact lens electrode with a built-in LED. Twelve eyes of six normal laboratory beagle dogs were used and exposed to steady room light at 500 lux for 30 min for light adaption. ERG was recorded at different time points during dark adaptation in sedated and light-adapted beagles. The stimulus intensity was 0.0096 cd/m(2)/sec. The b-wave amplitude increased significantly until 25 min of dark adaptation, whereas no significant changes in amplitudes were observed after 30 min. Dark adaptation for more than 25 min would be necessary for accurate ERG in canine ERG using a contact lens electrode with a built-in LED.

Effect of sevoflurane concentration on visual evoked potentials with pattern stimulation in dogs.

The purpose of this study was to investigate the effects of sevoflurane concentration on canine visual evoked potentials with pattern stimulation (P-VEPs). Six clinically normal laboratory-beagle dogs were used. The minimum alveolar concentration (MAC) of sevoflurane was detected from all subjects by tail clamp method. The refractive power of the right eyes of all subjects was corrected to -2 diopters after skiascopy. For P-VEP recording, the recording and reference electrode were positioned at inion and nasion, respectively, and the earth electrode was positioned on the inner surface. To grasp the state of CNS suppression objectively, the bispectral index (BIS) value was used. The stimulus pattern size and distance for VEP recording were constant, 50.3 arc-min and 50 cm, respectively. P-VEPs and BIS values were recorded under sevoflurane in oxygen inhalational anesthesia at 0.5, 1.0, 1.5, 2.0, 2.5 and 2.75 sevoflurane MAC. For analysis of P-VEP, the P100 implicit time and N75-P100 amplitude were estimated. P-VEPs were detected at 0.5 to 1.5 MAC in all dogs, and disappeared at 2.0 MAC in four dogs and at 2.5 and 2.75 MAC in one dog each. The BIS value decreased with increasing sevoflurane MAC, and burst suppression began to appear from 1.5 MAC. There was no significant change in P100 implicit time and N75-P100 amplitude with any concentration of sevoflurane. At concentrations around 1.5 MAC, which are used routinely to immobilize dogs, sevoflurane showed no effect on P-VEP.