Hippo Signaling in the Endometrium.

The uterus is essential for embryo implantation and fetal development. During the estrous cycle, the uterine endometrium undergoes dramatic remodeling to prepare for pregnancy. Angiogenesis is an essential biological process in endometrial remodeling. Steroid hormones regulate the series of events that occur during such remodeling. Researchers have investigated the potential factors, including angiofactors, involved in endometrial remodeling. The Hippo signaling pathway discovered in the 21st century, plays important roles in various cellular functions, including cell proliferation and cell death. However, its role in the endometrium remains unclear. In this review, we describe the female reproductive system and its association with the Hippo signaling pathway, as well as novel Hippo pathway genes and potential target genes.

Estrogen Regulates the Expression and Localization of YAP in the Uterus of Mice.

The dynamics of uterine endometrium is important for successful establishment and maintenance of embryonic implantation and development, along with extensive cell differentiation and proliferation. The tissue event is precisely and complicatedly regulated as several signaling pathways are involved including two main hormones, estrogen and progesterone signaling. We previously showed a novel signaling molecule, Serine/threonine protein kinase 3/4 (STK3/4), which is responded to hormone in the mouse uterine epithelium. However, the role and regulation of its target, YES-associated protein (YAP) remains unknown. In this study, we investigated the expression and regulation of YAP in mouse endometrium. We found that YAP was periodically expressed in the endometrium during the estrous cycle. Furthermore, periodic expression of YAP was shown to be related to the pathway under hormone treatment. Interestingly, estrogen was shown to positively modulate YAP via endometrial epithelial receptors. In addition, the knockdown of YAP showed that YAP regulated various target genes in endometrial cells. The knockdown of YAP down-regulated numerous targets including ADAMTS1, AMOT, AMOTL1, ANKRD1, CTNNA1, MCL1. On the other hand, the expressions of AREG and AXL were increased by its knockdown. These findings imply that YAP responds via Hippo signaling under various intrauterine signals and is considered to play a role in the expression of factors important for uterine endometrium dynamic regulation.

Expression and Regulation of CD73 during the Estrous Cycle in Mouse Uterus.

Cluster of differentiation 73 (CD73, also known as ecto-5'-nucleotidase) is an enzyme that converts AMP into adenosine. CD73 is a surface enzyme bound to the outside of the plasma membrane expressed in several cells and regulates immunity and inflammation. In particular, it is known to inhibit T cell-mediated immune responses. However, the regulation of CD73 expression by hormones in the uterus is not yet clearly known. In this study, we investigated the expression of CD73 in ovariectomized mice treated with estrogen or progesterone and its regulation in the mouse uterus during the estrous cycle. The level of CD73 expression was dynamically regulated in the uterus during the estrous cycle. CD73 protein expression was high in proestrus, estrus, and diestrus, whereas it was relatively low in the metestrus stage. Immunofluorescence revealed that CD73 was predominantly expressed in the cytoplasm of the luminal and glandular epithelium and the stroma of the endometrium. The expression of CD73 in ovariectomized mice was gradually increased by progesterone treatment. However, estrogen injection did not affect its expression. Moreover, CD73 expression was increased when estrogen and progesterone were co-administered and was inhibited by the pretreatment of the progesterone receptor antagonist RU486. These findings suggest that the expression of CD73 is dynamically regulated by estrogen and progesterone in the uterine environment, and that there may be a synergistic effect of estrogen and progesterone.

Morphine Dependence is Attenuated by Treatment of 3,4,5-Trimethoxy Cinnamic Acid in Mice and Rats.

The effect of 3, 4, 5-trimethoxy cinnamic acid (TMCA) against morphine-induced dependence in mice and rats was investigated. Mice were pretreated with TMCA and then morphine was injected intraperitoneally; whereas rats were treated with TMCA (i.p.) and infused with morphine into the lateral ventricle of brain. Naloxone-induced morphine withdrawal syndrome and conditioned place preference test were performed. Moreover, western blotting and immunohistochemistry were used to measure protein expressions. Number of naloxone-precipitated jumps and conditioned place preference score in mice were attenuated by TMCA. Likewise, TMCA attenuated morphine dependent behavioral patterns such as diarrhea, grooming, penis licking, rearing, teeth chattering, and vocalization in rats. Moreover, the expression levels of pNR1and pERK in the frontal cortex of mice and cultured cortical neurons were diminished by TMCA. In the striatum, pERK expression was attenuated despite unaltered expression of pNR1 and NR1. Interestingly, morphine-induced elevations of FosB/ΔFosB+ cells were suppressed by TMCA (50, 100 mg/kg) in the nucleus accumbens sub-shell region of mice. In conclusion, TMCA could be considered as potential therapeutic agent against morphine-induced dependence.

Knockdown of PRKAR2B Results in the Failure of Oocyte Maturation.

BACKGROUND/AIMS: Cyclic adenosine monophosphate (cAMP)-dependent type 2 regulatory subunit beta (Prkar2b) is a regulatory isoform of cAMP-dependent protein kinase (PKA), which is the primary target for cAMP actions. In oocytes, PKA and the pentose phosphate pathway (PPP) have important roles during the germinal vesicle (GV) stage arrest of development. Although the roles of the PKA signal pathway have been studied in the development of oocyte, there has been no report on the function of PRKAR2B, a key regulator of PKA. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), immunohistochemistry, and immunofluorescence, we determined the relative expression of Prkar2b in various tissues, including ovarian follicles, during oocyte maturation. Prkar2b-interfering RNA (RNAi) microinjection was conducted to confirm the effect of Prkar2b knockdown, and immunofluorescence, qRT-PCR, and time-lapse video microscopy were used to analyze Prkar2b-deficient oocytes. RESULTS: Prkar2b is strongly expressed in the ovarian tissues, particularly in the growing follicle. During oocyte maturation, the highest expression of Prkar2b was during metaphase I (MI), with a significant decrease at metaphase II (MII). RNAi-mediated Prkar2b suppression resulted in MI-stage arrest during oocyte development, and these oocytes exhibited abnormal spindle formation and chromosome aggregation. Expression of other members of the PKA family (except for Prkaca) were decreased, and the majority of the PPP factors were also reduced in Prkar2b-deficient oocytes. CONCLUSION: These results suggest that Prkar2b is closely involved in the maturation of oocytes by controlling spindle formation and PPP-mediated metabolism.

Brain changes in overweight/obese and normal-weight adults with type 2 diabetes mellitus.

AIMS/HYPOTHESIS: Overweight and obesity may significantly worsen glycaemic and metabolic control in type 2 diabetes. However, little is known about the effects of overweight and obesity on the brains of people with type 2 diabetes. Here, we investigate whether the presence of overweight or obesity influences the brain and cognitive functions during early stage type 2 diabetes. METHODS: This study attempted to uncouple the effects of overweight/obesity from those of type 2 diabetes on brain structures and cognition. Overweight/obese participants with type 2 diabetes had more severe and progressive abnormalities in their brain structures and cognition during early stage type 2 diabetes compared with participants with normal weight. Relationships between each of these measures and disease duration were also examined. RESULTS: Global mean cortical thickness was lower in the overweight/obese type 2 diabetes group than in the normal-weight type 2 diabetes group (z = -2.96, p for group effect = 0.003). A negative correlation was observed between disease duration and global mean white matter integrity (z = 2.42, p for interaction = 0.02) in the overweight/obese type 2 diabetes group, but not in the normal-weight type 2 diabetes group. Overweight/obese individuals with type 2 diabetes showed a decrease in psychomotor speed performance related to disease duration (z = -2.12, p for interaction = 0.03), while normal-weight participants did not. CONCLUSIONS/INTERPRETATION: The current study attempted to uncouple the effects of overweight/obesity from those of type 2 diabetes on brain structures and cognition. Overweight/obese participants with type 2 diabetes had more severe and progressive abnormalities in brain structures and cognition during early stage type 2 diabetes compared with normal-weight participants.

Synergistic effect of melatonin and ghrelin in preventing cisplatin-induced ovarian damage via regulation of FOXO3a phosphorylation and binding to the p27Kip1 promoter in primordial follicles.

Premature ovarian failure during chemotherapy is a serious problem for young women with cancer. To preserve the fertility of these patients, approaches to prevent chemotherapy-induced ovarian failure are needed. In a previous study, we reported that melatonin treatment prevents the depletion of the dormant follicle pool via repression of the simultaneous activation of dormant primordial follicles by cisplatin. However, melatonin's protective effect was only partial and thus insufficient. In this study, we found that the hormone ghrelin enhances the protective effect of melatonin against cisplatin-induced ovarian failure in mouse model. Co-administration of melatonin and ghrelin more effectively prevented cisplatin-induced follicle disruption. Simultaneous treatment with melatonin and ghrelin almost restored the number of primordial follicles and the corpus luteum in cisplatin-treated ovaries, compared with single administration. We found melatonin and ghrelin receptors on the cell membrane of premature oocytes of primordial follicles. In addition, melatonin and ghrelin co-administration inhibited the cisplatin-induced phosphorylation of PTEN and FOXO3a that induces cytoplasmic translocation of FOXO3a. Inhibition of FOXO3a phosphorylation by melatonin and ghrelin increased the binding affinity of FOXO3a for the p27Kip1 promoter in primordial follicles. Co-administration of melatonin and ghrelin in cisplatin-treated ovaries restored the expression of p27Kip1 , which is critical for retention of the dormant status of primordial follicles. In conclusion, these findings suggest that melatonin and ghrelin co-administration is suitable for use as a fertoprotective adjuvant therapy during cisplatin chemotherapy in young female cancer patients.

Immune dysregulation in chronic stress: a quantitative and functional assessment of regulatory T cells.

OBJECTIVE: Chronic stress is closely related to immune dysfunction. Immune parameters have been analyzed in many ways in humans and animals under chronic stress. Recently, it has been proved that FoxP3+ regulatory T cells (Tregs) play a key role in immune regulation in vivo. However, it has not yet been elucidated how Tregs respond to chronic stress in vivo. Therefore, in the present study, we investigated the frequency of and functional changes in Tregs from mice under chronic stress. METHODS: Spleen cells were separated from C57/BL6 mice that had been exposed to immobilization stress for 3 weeks. The frequencies of FoxP3+ and CD4+ CD25+ cells were analyzed by flow cytometry. CD4+CD25- cells (effector T cells, Teffs), CD4+CD25+ cells (Tregs) and CD4- cells (antigen-presenting cells, APCs) were separated for the functional assessment of the proliferative activity of Teffs, the suppressive activity of Tregs and the feeder activity of APCs. RESULTS: The results showed that chronic immobilization stress significantly increased the frequencies of CD4+CD25+ and CD4+FoxP3+ cells. Chronic immobilization stress also enhanced the suppressive function of CD4+ CD25+ Tregs. On the other hand, the proliferative activity of Teffs and the feeder activity of APCs were decreased in the mice under chronic immobilization stress. CONCLUSION: Taken together, it is suggested that increased number and function of Tregs may actively contribute to the immune dysfunction in chronic immobilization stress, synergizing with the decreased function of Teffs and APCs.

Generation of a B2M homozygous knockout human somatic cell nuclear transfer-derived embryonic stem cell line using the CRISPR/Cas9 system.

Beta2-microglobulin (B2M) is a subunit of human leukocyte antigen class-I (HLA-I) heterodimer that mediates immune rejection through activation of cytotoxic T cells. B2M binding to HLA-I proteins is essential for functional HLA-I on the cell surface. Here, we generated a B2M homozygous knockout somatic cell nuclear transfer-induced embryonic stem cell (SCNT-ESC) line using CRISPR/Cas9-mediated gene targeting. B2M KO cell line, which does not express HLA-I molecules on cell surface, has pluripotency and differentiation ability to three germ layers. This cell line provides a useful cell source for investigating immunogenicity of allogeneic ESCs and their derivatives for tissue regeneration.

Modulation of sphingosine 1-phosphate and tyrosine hydroxylase in the stress-induced anxiety.

Stress causes endocrinological changes and leads to induce anxiety. It was determined the anxiety and stress-related endocrinological changes through the observation of the level of glucocorticoid and sphingolipid metabolites in serum after stress. Immobilized stress and electric shock was applied to rats for 7 days. This study investigated the induction of anxiety, changes of TH and pERK expression in cortex and amygdala after stress. Also it was determined the changes of glucocorticoid and anxiety when the rats were given stress after amygdala lesion. The stress-given rats spent a lesser percentage of time significantly in the open arm than the control rats. The elevated level of glucocorticoid after stress was suppressed in amygdala lesion group. The expression of TH in the amygdala was decreased, but the expression of TH was not changed in the cortex after stress. To investigate the changes in sphingolipid metabolites after stress, the levels of sphingosine and the phosphate form of sphingolipid (So-1-P) were analyzed in serum. The level of So-1-P was elevated after stress and anxiety was observed after the So-1-P infusion (100 pmol/10 µl/h, i.c.v., for 7 days). Continuous infusion of So-1-P for 7 days led to the significant decrease of TH expression in the amygdala. In conclusion, the results of this study indicate that the lesion of amygdala suppressed the stress-induced anxiety and elevation of glucocorticoid in serum. It was also observed that expression of TH in amygdala as well as increased levels of glucocorticoid in serum might be responsible biomarker, at least in part, of chronic stress. These results suggest that the elevation of So-1-P might be involved in induction of anxiety during stress by the modulation of dopaminergic system in amygdala.

Phytoceramide shows neuroprotection and ameliorates scopolamine-induced memory impairment.

The function and the role phytoceramide (PCER) and phytosphingosine (PSO) in the central nervous system has not been well studied. This study was aimed at investigating the possible roles of PCER and PSO in glutamate-induced neurotoxicity in cultured neuronal cells and memory function in mice. Phytoceramide showed neuro-protective activity in the glutamate-induced toxicity in cultured cortical neuronal cells. Neither phytosphingosine nor tetraacetylphytosphingosine (TAPS) showed neuroproective effects in neuronal cells. PCER (50 mg/kg, p.o.) recovered the scopolamine-induced reduction in step-through latency in the passive avoidance test; however, PSO did not modulate memory function on this task. The ameliorating effects of PCER on spatial memory were confirmed by the Morris water maze test. In conclusion, through behavioral and neurochemical experimental results, it was demonstrated that central administration of PCER produces amelioration of memory impairment. These results suggest that PCER plays an important role in neuroprotection and memory enhancement and PCER could be a potential new therapeutic agent for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Fumonisin B1-Induced Toxicity Was Not Exacerbated in Glutathione Peroxidase-1/Catalase Double Knock Out Mice.

Fumonisin B1 (FB1) structurally resembles sphingolipids and interferes with their metabolism leading to sphingolipid dysregulation. We questioned if FB1 could exacerbate liver or kidney toxicities in glutathione peroxidase 1 (Gpx1) and catalase (Cat) knockout mice. While higher serum levels of thiobarbituric acid reactive substances (TBARS) and sphinganine (Sa) were measured in Gpx1/Cat knockout mice (Gpx1/Cat KO) than wild type mice after 5 days of FB1 treatment, serum levels of alanine aminotransferase (ALT), sphingosine-1 phosphate (So-1-P), and sphinganine-1 phosphate (Sa-1-P) were found to be relatively low. Although Sa was highly elevated in Gpx1/Cat KO mice and wild mice, lower levels of So and Sa were found in both the kidney and liver tissues of Gpx/Cat KO mice than wild type mice after FB1 treatment. Paradoxically, FB1-induced cellular apoptosis and necrosis were hastened under oxidative stress in Gpx1/Cat KO mice.

Sirtuin 6 deficiency induces endothelial cell senescence via downregulation of forkhead box M1 expression.

Cellular senescence of endothelial cells causes vascular dysfunction, promotes atherosclerosis, and contributes to the development of age-related vascular diseases. Sirtuin 6 (SIRT6), a conserved NAD+-dependent protein deacetylase, has beneficial effects against aging, despite the fact that its functional mechanisms are largely uncharacterized. Here, we show that SIRT6 protects endothelial cells from senescence. SIRT6 expression is progressively decreased during both oxidative stress-induced senescence and replicative senescence. SIRT6 deficiency leads to endothelial dysfunction, growth arrest, and premature senescence. Using genetically engineered endothelial cell-specific SIRT6 knockout mice, we also show that down-regulation of SIRT6 expression in endothelial cells exacerbates vascular aging. Expression microarray analysis demonstrated that SIRT6 modulates the expression of multiple genes involved in cell cycle regulation. Specifically, SIRT6 appears to regulate the expression of forkhead box M1 (FOXM1), a critical transcription factor for cell cycle progression and senescence. Overexpression of FOXM1 ameliorates SIRT6 deficiency-induced endothelial cell senescence. In this work, we demonstrate the role of SIRT6 as an anti-aging factor in the vasculature. These data may provide the basis for future novel therapeutic approaches against age-related vascular disorders.

BIRC5 Expression is Regulated in Uterine Epithelium During the Estrous Cycle.

Baculoviral inhibitor of apoptosis repeat-containing 5 (Birc5), also known as survivin, is a member of the inhibitor of apoptosis (IAP) family of proteins and regulates the size of tissues through cell division control. The uterus is the most dynamically sized organ among tissues during the estrous cycle. Although Birc5 is expressed in some terminally differentiated cells, the regulation of its expression in the uterus remains unknown. We investigated the regulation of Birc5 expression in the mouse uterus. RT-PCR analysis showed that Birc5 was expressed in various tissues, including the uterus; the expression level of Birc5 was significantly higher at the diestrus stage. Immunohistochemistry and Western blotting analysis revealed that Birc5 was more active in luminal and glandular epithelium than in endometrial stroma. In ovariectomized mice, Birc5 expression in the uterus was gradually increased by estrogen treatment; however, progesterone injection decreased its expression. Estrogen-induced Birc5 expression was blocked by treatment with estrogen receptor antagonist, ICI 182, 780 and progesterone-reduced Birc5 expression was inhibited by the progesterone receptor antagonist RU486. These results suggest that Birc5 expression is dynamically regulated by a combination of estrogen and progesterone via their receptor-mediated signaling.

STK3/4 Expression Is Regulated in Uterine Endometrial Cells during the Estrous Cycle.

The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle.

Differential Regulation of TLE3 in Sertoli Cells of the Testes during Postnatal Development.

Spermatogenesis is a process by which haploid cells differentiate from germ cells in the seminiferous tubules of the testes. TLE3, a transcriptional co-regulator that interacts with DNA-binding factors, plays a role in the development of somatic cells. However, no studies have shown its role during germ cell development in the testes. Here, we examined TLE3 expression in the testes during spermatogenesis. TLE3 was highly expressed in mouse testes and was dynamically regulated in different cell types of the seminiferous tubules, spermatogonia, spermatids, and Sertoli cells, but not in the spermatocytes. Interestingly, TLE3 was not detected in Sertoli cells on postnatal day 7 (P7) but was expressed from P10 onward. The microarray analysis showed that the expression of numerous genes changed upon TLE3 knockdown in a Sertoli cell line TM4. These include 1597 up-regulated genes and 1452 down-regulated genes in TLE3-knockdown TM4 cells. Ingenuity Pathway Analysis (IPA) showed that three factors were up-regulated and two genes were down-regulated upon TLE3 knockdown in TM4 cells. The abnormal expression of the three factors is associated with cellular malfunctions such as abnormal differentiation and Sertoli cell formation. Thus, TLE3 is differentially expressed in Sertoli cells and plays a crucial role in regulating cell-specific genes involved in the differentiation and formation of Sertoli cells during testicular development.

Aminophylline and Ephedrine, but Not Flumazenil, Inhibit the Activity of the Excitatory Amino Acid Transporter 3 Expressed in Xenopus Oocytes and Reverse the Increased Activity by Propofol.

We investigated the effects of flumazenil, aminophylline, and ephedrine on the excitatory amino acid transporter type 3 (EAAT3) activity and the interaction with propofol. EAAT3 was expressed in the Xenopus oocytes. L-Glutamate-induced membrane currents were measured using the two-electrode voltage clamp at various drug concentrations. Oocytes were preincubated with protein kinase C- (PKC-) activator, or inhibitor, and phosphatidylinositol 3-kinase (PI3K) inhibitor. To study the interaction with propofol, oocytes were exposed to propofol, propofol + aminophylline, or ephedrine. Aminophylline and ephedrine significantly decreased EAAT3 activity. Aminophylline (95 µM) and ephedrine (1.19 µM) significantly decreased Vmax, but not Km of EAAT3, for glutamate. The phorbol 12-myristate-13-acetate-induced increase in EAAT3 activity was abolished by aminophylline or ephedrine. The decreased EAAT3 activities by PKC inhibitors (staurosporine, chelerythrine) and PI3K inhibitor (wortmannin) were not significantly different from those by aminophylline or ephedrine, as well as those by PKC inhibitors or PI3K inhibitor + aminophylline or ephedrine. The enhanced EAAT3 activities induced by propofol were significantly abolished by aminophylline or ephedrine. Aminophylline and ephedrine inhibit EAAT3 activity via PKC and PI3K pathways and abolish the increased EAAT3 activity by propofol. Our results indicate a novel site of action for aminophylline and ephedrine.

Fluoroscopic evaluation of diaphragmatic excursion during spontaneous breathing in healthy Beagles.

OBJECTIVE To evaluate dynamic movement of the diaphragm of clinically normal dogs by use of fluoroscopy and to obtain quantitative data of diaphragmatic excursion during spontaneous breathing. ANIMALS 8 healthy male Beagles with no history of respiratory tract disease. PROCEDURES Fluoroscopy was performed during stabilized respiratory conditions. The beam center was located at the level of the diaphragm, and diaphragmatic motion was recorded during 3 respiratory cycles in dogs positioned in left lateral, right lateral, and dorsal recumbency. Extent of excursion of the diaphragmatic cupula and both crura, difference in excursion between the left and right crura, and ratios of the excursions of the diaphragmatic cupula and left and right crura to the length of the eighth thoracic vertebra were determined. RESULTS Diaphragmatic crural excursion was symmetric for dogs in right lateral recumbency, and the crural excursion was approximately three-quarters of the vertebral length; however, crural excursion appeared to be asymmetric for dogs in left lateral recumbency. Mean ± SD difference in excursion between the right and left crura was 22.68 ± 8.68% for left lateral recumbency, 16.63 ± 9.22% for right lateral recumbency, and 18.11 ± 12.96% for dorsal recumbency. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the fluoroscopic view of a dog positioned in right lateral recumbency may allow better evaluation of the symmetry of diaphragmatic excursion, compared with results for other recumbency positions. This study provided quantitative data on the excursion of diaphragmatic movement observed by use of fluoroscopy in clinically normal Beagles.

Effect of position and time held in that position on ground-glass opacity in computed tomography images of dogs.

OBJECTIVE To evaluate effects of position, time in that position, and positive end-expiratory pressure on ground-glass opacity caused by physiologic atelectasis on lung CT images and to determine effects of recumbency position before CT. ANIMALS 6 healthy Beagles. PROCEDURES In a crossover study, dogs were placed in 4 positions (sternal, dorsal, right lateral, and left lateral recumbency) for 2 holding times (30 and 60 minutes). Dogs were then repositioned in sternal recumbency, and CT was performed at 2 positive end-expiratory pressures (0 and 15 mm Hg). Location, distribution, and degree of ground-glass opacities were evaluated on lung CT images. Volume and mean density of the lungs and ground-glass opacities as well as maximum density of ground-glass opacities were evaluated. RESULTS Ground-glass opacities were mainly observed in parts of the lungs that were dependent during the various positions before CT, except for sternal recumbency. Opacities were reversible and decreased or disappeared after lung inflation. Ground-glass opacities were observed most frequently and had greatest severity when dogs were positioned in left lateral recumbency before CT. Ground-glass opacities were negligible for dogs positioned in sternal recumbency before CT. CONCLUSIONS AND CLINICAL RELEVANCE Location and reversibility of ground-glass opacities may help clinicians distinguish whether they are attributable to atelectasis or a result of pathological changes. Dogs should be positioned in sternal recumbency to minimize the occurrence of ground-glass opacities, particularly when several procedures are performed before CT, which increases the time that a dog will remain in the same position.

Comparison of elastography, contrast-enhanced ultrasonography, and computed tomography for assessment of lesion margin after radiofrequency ablation in livers of healthy dogs.

OBJECTIVE To assess by use of various diagnostic imaging modalities acute changes in livers of healthy dogs after radiofrequency ablation (RFA) and determine the capability of each imaging modality to monitor ablation lesion changes. ANIMALS 6 healthy Beagles. PROCEDURES 12 ablation lesions were created in the liver of the dogs (2 lesions/dog). Ablation lesions were evaluated by use of conventional ultrasonography, strain elastography, and contrast-enhanced ultrasonography immediately after (time 0), 30 to 60 minutes after, and 3 days after RFA, and by use of CT 30 minutes and 3 days after RFA. Three dogs were euthanized shortly after RFA, and the other 3 dogs were euthanized on day 3. Lesion size measured by each imaging modality was compared with necropsy findings. RESULTS Immediately after RFA, clear margins were more visible with elastography and contrast-enhanced ultrasonography than with conventional ultrasonography, which had acoustic shadowing. On triphasic contrast CT, the ablation zone, which indicated necrosis and hemorrhage, was not enhanced and could be measured. Marked enhancement of the periablation rim was observed during the venous phase and was identified as granulation tissue. Size of the ablation area measured on enhanced CT images was strongly correlated with actual lesion size. CONCLUSIONS AND CLINICAL RELEVANCE For dogs of this study, CT was the most reliable method for lesion size determination. Although ultrasonographic imaging measurements underestimated lesion size, all modalities could be used to provide additional real-time guidance for RFA procedures of the liver as well as for other RFA procedures.