Intervertebral Disc Regeneration Using Stem Cell/Growth Factor-Loaded Porous Particles with a Leaf-Stacked Structure.

Although biological therapies based on growth factors and transplanted cells have demonstrated some positive outcomes for intervertebral disc (IVD) regeneration, repeated injection of growth factors and cell leakage from the injection site remain considerable challenges for human therapeutic use. Herein, we prepare human bone marrow-derived mesenchymal stem cells (hBMSCs) and transforming growth factor-ß3 (TGF-ß3)-loaded porous particles with a unique leaf-stack structural morphology (LSS particles) as a combination bioactive delivery matrix for degenerated IVD. The LSS particles are fabricated with clinically acceptable biomaterials (polycaprolactone and tetraglycol) and procedures (simple heating and cooling). The LSS particles allow sustained release of TGF-ß3 for 18 days and stable cell adhesiveness without additional modifications of the particles. On the basis of in vitro and in vivo studies, it was observed that the hBMSCs/TGF-ß3-loaded LSS particles can provide a suitable milieu for chondrogenic differentiation of hBMSCs and effectively induce IVD regeneration in a beagle dog model. Thus, therapeutically loaded LSS particles offer the promise of an effective bioactive delivery system for regeneration of various tissues including IVD.

Evaluation of treatment with a combination of mycophenolate mofetil and prednisolone in dogs with meningoencephalomyelitis of unknown etiology: a retrospective study of 86 cases (2009-2017).

BACKGROUND: Combination therapy with glucocorticoids and adjunctive immunomodulating drugs has been generally accepted as a standard treatment regimen for meningoencephalomyelitis of unknown etiology (MUE). We hypothesized that treatment with MMF as an adjunctive agent along with glucocorticoids would be effective and well-tolerated protocol in dogs with MUE. Eighty-six dogs with MUE between May 2009 and June 2017 were included (59 females and 27 males; mean age of 5.93 years; mean body weight of 3.83 kg). The medical records of dogs with MUE treated with prednisolone and MMF were retrospectively evaluated to determine the therapeutic response, survival time, and treatment-related adverse effects. RESULTS: A partial or complete response (CR) was recorded for 75 dogs. The overall median survival time from the initiation of treatment was 558 days. Dogs that showed CR with no relapse over the treatment period (from diagnosis to death) had significantly longer median survival times. A significantly higher mortality hazard ratio of 4.546 was recorded in dogs that failed to achieve CR. The interval between the onset of clinical signs and the clinical presentation was not significantly associated with CR, relapse rate, and survival time. Adverse effects included gastrointestinal upsets in 26 dogs (30.23%), sporadic infections in 17 dogs (19.77%), and pancreatitis in seven dogs (8.14%). CONCLUSIONS: The results suggest that adjunctive MMF treatment for MUE is safe and comparable to other immunosuppressive protocols. The treatment should focus on the achievement of CR and preventing relapse for successful management.

Oxygen-Releasing Microparticles for Cell Survival and Differentiation Ability under Hypoxia for Effective Bone Regeneration.

Sufficient oxygen delivery into tissue-engineered three-dimensional (3D) scaffolds to produce clinically applicable tissues/organs remains a challenge for researchers and clinicians. One potential strategy to overcome this limitation is the use of an oxygen releasing scaffold. In the present study, we prepared hollow microparticles (HPs) loaded with an emulsion of the oxygen carrier perfluorooctane (PFO; PFO-HPs) for the timely supply of oxygen to surrounding cells. These PFO-HPs prolonged the survival and preserved the osteogenic differentiation potency of human periosteal-derived cells ( hPDCs) under hypoxia. hPDCs seeded onto PFO-HPs formed new bone at a faster rate and with a higher bone density than hPDCs seeded onto phosphate buffered saline-loaded control HPs. These findings suggest that PFO-HPs provide a suitable environment for the survival and maintenance of differentiation ability of hPDCs at bony defects without vascular networks until new blood vessel ingrowth occurs, thus enhancing bone regeneration. PFO-HPs are a promising system for effective delivery of various functional cells, including stem cells and progenitor cells, to regenerate damaged tissues/organs.

Development of Porous Beads to Provide Regulated BMP-2 Stimulation for Varying Durations: In Vitro and In Vivo Studies for Bone Regeneration.

It is commonly accepted that the sustained release of bone morphogenetic protein-2 (BMP-2) can enhance bone regeneration and minimize its safety issues. However, little is known regarding the appropriate duration of BMP-2 stimulation for sufficient osteogenic differentiation and new bone formation because of the short half-life of BMP-2 in the physiological environment and the lack of a well-defined delivery matrix that can regulate the release period of BMP-2. In this study, we prepared porous poly(lactic-co-glycolic acid) (PLGA) beads with different surface pore sizes that can regulate the release period of BMP-2 (i.e., 7, 17, and 30 days) while providing the BMP-2 concentration required for bone regeneration. Our findings in both in vitro cell culture and in vivo animal studies using these BMP-2-loaded beads demonstrate that release of BMP-2 within 7 days affects only the initial differentiation of human periosteum-derived cells (hPDCs) and does not significantly enhance their subsequent differentiation into mature functional cells. However, extending the duration of BMP-2 stimulation over 17 days can provide a suitable environment for osteogenic differentiation of hPDCs and new bone formation.

Immunomodulatory properties and in vivo osteogenesis of human dental stem cells from fresh and cryopreserved dental follicles.

In our previous study, dental follicle tissues from extracted wisdom teeth were successfully cryopreserved for use as a source of stem cells. The goals of the present study were to investigate the immunomodulatory properties of stem cells from fresh and cryopreserved dental follicles (fDFCs and cDFCs, respectively) and to analyze in vivo osteogenesis after transplantation of these DFCs into experimental animals. Third passage fDFCs and cDFCs showed similar expression levels of interferon-γ receptor (CD119) and major histocompatibility complex class I and II (MHC I and MHC II, respectively), with high levels of CD119 and MHC I and nearly no expression of MHC II. Both fresh and cryopreserved human DFCs (hDFCs) were in vivo transplanted along with a demineralized bone matrix scaffold into mandibular defects in miniature pigs and subcutaneous tissues of mice. Radiological and histological evaluations of in vivo osteogenesis in hDFC-transplanted sites revealed significantly enhanced new bone formation activities compared with those in scaffold-only implanted control sites. Interestingly, at 8 weeks post-hDFC transplantation, the newly generated bones were overgrown compared to the original size of the mandibular defects, and strong expression of osteocalcin and vascular endothelial growth factor were detected in the hDFCs-transplanted tissues of both animals. Immunohistochemical analysis of CD3, CD4, and CD8 in the ectopic bone formation sites of mice showed significantly decreased CD4 expression in DFCs-implanted tissues compared with those in control sites. These findings indicate that hDFCs possess immunomodulatory properties that involved inhibition of the adaptive immune response mediated by CD4 and MHC II, which highlights the usefulness of hDFCs in tissue engineering. In particular, long-term preserved dental follicles could serve as an excellent autologous or allogenic stem cell source for bone tissue regeneration as well as a valuable therapeutic agent for immune diseases.

Operative Techniques and Preliminary Outcomes Following Percutaneous Endoscopic Thoracolumbar Pediculectomy in Dogs.

OBJECTIVE: To examine the technical feasibility of percutaneous endoscopic pediculectomy using a lateral approach and to evaluate its use for decompression and examination of the thoracic and lumbar spinal canals in small dogs. STUDY DESIGN: Experimental study. ANIMALS: Clinically normal adult dogs (n=10). METHODS: After optimizing the technique in cadavers, percutaneous endoscopic pediculectomy was performed using a lateral approach to the thoracic (T12) or lumbar (L2) vertebrae in 5 dogs each. Using fluoroscopic guidance, a K-wire, dilator, and (cannula) working sleeve were placed. A window for visualizing the spinal cord and floor of the spinal canal was created using a specialized drill, rongeurs, trephine, and elevator. Gait and neurologic status were monitored postoperatively, and computed tomography (CT) and magnetic resonance imaging (MRI) performed. RESULTS: All procedures were completed successfully (T12, 45 ± 13 minutes; L2, 59 ± 14 minutes) with clear observation of the spinal cord and floor of the spinal canal. Normal ambulation was maintained in 9 dogs. One dog in the L2 group had ipsilateral hind limb weakness that resolved within 4 days. One dog in the L2 group suffered a fracture of the accessory process. One dog each in the T12 and L2 groups had hyperintense lesions on T2-weighted MRI images, suspicious of focal edema, which reduced at 4 weeks after initial examination, possibly reflecting gliosis. CONCLUSION: Pediculectomy using a percutaneous endoscopic thoracolumbar lateral approach is feasible, provides a good view of the spinal canal, and may be an alternative for treatment of disk disease in dogs.

BMP-2-immobilized PCL 3D printing scaffold with a leaf-stacked structure as a physically and biologically activated bone graft.

Although three-dimensional (3D) printing techniques are used to mimic macro- and micro-structures as well as multi-structural human tissues in tissue engineering, efficient target tissue regeneration requires bioactive 3D printing scaffolds. In this study, we developed a bone morphogenetic protein-2 (BMP-2)-immobilized polycaprolactone (PCL) 3D printing scaffold with leaf-stacked structure (LSS) (3D-PLSS-BMP) as a bioactive patient-tailored bone graft. The unique LSS was introduced on the strand surface of the scaffold via heating/cooling in tetraglycol without significant deterioration in physical properties. The BMP-2 adsorbed on3D-PLSS-BMPwas continuously released from LSS over a period of 32 d. The LSS can be a microtopographical cue for improved focal cell adhesion, proliferation, and osteogenic differentiation.In vitrocell culture andin vivoanimal studies demonstrated the biological (bioactive BMP-2) and physical (microrough structure) mechanisms of3D-PLSS-BMPfor accelerated bone regeneration. Thus, bioactive molecule-immobilized 3D printing scaffold with LSS represents a promising physically and biologically activated bone graft as well as an advanced tool for widespread application in clinical and research fields.

Surgical Correction of a Sinus Venosus Atrial Septal Defect with Partial Anomalous Pulmonary Venous Connections Using Cardiac Computed Tomography Imaging and a 3D-Printed Model.

Sinus venosus atrial septal defects (SVASDs), concurrent with partial anomalous pulmonary venous connections (PAPVCs), are a rare congenital heart disease in dogs. Surgical correction is essential when clinical signs or significant hemodynamic changes are present. We aimed to report on the successful surgical correction of an SVASD with PAPVCs, using a computed tomography (CT)-based customized 3D cardiac model. A 10-month-old male poodle was referred for corrective surgery for an ASD. Echocardiography confirmed a hemodynamically significant left-to-right shunting flow through an interatrial septal defect and severe right-sided heart volume overload. For a comprehensive diagnosis, a CT scan was performed, which confirmed an SVASD with PAPVCs. A customized 3D cardiac model was used for preoperative decision-making and surgical rehearsal. The defect was repaired using an autologous pericardial patch under a cardiopulmonary bypass (CPB). Temporary pacing was applied for sinus bradycardia and third-degree atrioventricular block. The patient recovered from the anesthesia without further complications. The pacemaker was removed during hospitalization and the patient was discharged without complications 2 weeks post-surgery. At the three-month follow-up, there was no shunting flow in the interatrial septum and the right-sided volume overload had been resolved. The cardiac medications were discontinued, and there were no complications. This report indicates the validity of surgical correction under CPB for an SVASD with PAPVCs, and the advantages of utilizing a CT-based 3D cardiac model for preoperative planning to increase the surgical success rate.

Comparison of bone subtraction CT angiography with standard CT angiography for evaluating circle of Willis in normal dogs.

BACKGROUND: Bone subtraction computed tomography angiography (BSCTA) is a useful alternative technique for improving visualization of vessels surrounded by skull bone. However, no studies have compared computed tomography angiography (CTA) and BSCTA for improving the visibility of canine cerebral blood vessels. OBJECTIVES: To evaluate the potential benefit of BSCTA for better delineation of brain arteries of the circle of Willis (CoW) in dogs by comparing BSCTA with non-subtraction computed tomography angiography (NSCTA). METHODS: Brain CTA was performed for nine healthy beagle dogs using a bolus tracking method with saline flushing. A total dose of 600 mgI/kg of contrast agent with an iodine content of 370 mgI/mL was injected at a rate of 4 ml/s. Bone removal was achieved automatically by subtracting non-enhanced computed tomography (CT) data from contrast CT data. Five main intracranial arteries of the CoW were analyzed and graded on a scale of five for qualitative evaluation. RESULTS: Scores of basilar artery, middle cerebral artery, and rostral cerebral artery in the BSCTA group were significantly higher than those in the NSCTA group (p = 0.001, p = 0.020, and p < 0.0001, respectively). Scores of rostral cerebellar artery (RcA) and caudal cerebral artery (CCA) did not differ significantly between the two groups. However, scores of RcA and CCA in the BSCTA group were higher than those in the NSCTA group. CONCLUSIONS: BSCTA improved visualization of intracranial arteries of the CoW with close contact to bone. Thus, it should be recommended as a routine scan method in dogs suspected of having brain vessel disease.

Quantitative Analysis of Brain CT Perfusion in Healthy Beagle Dogs: A Pilot Study.

Brain computed tomography (CT) perfusion is a technique that allows for the fast evaluation of cerebral hemodynamics. However, quantitative studies of brain CT perfusion in veterinary medicine are lacking. The purpose of this study was to investigate the normal range of perfusion determined via CT in brains of healthy dogs and to compare values between white matter and gray matter, differences in aging, and each hemisphere. Nine intact male beagle dogs were prospectively examined using dynamic CT scanning and post-processing for brain perfusion. Regional cerebral blood volume (rCBV), regional cerebral blood flow (rCBF), mean transit time, and time to peak were calculated. Tissue ROIs were drawn in the gray matter and white matter of the frontal, temporal, parietal, and occipital lobes; caudate nucleus; thalamus; piriform lobe; hippocampus; and cerebellum. Significant differences were observed between the white matter regions and gray matter regions for rCBV and rCBF (p < 0.05). However, no significant differences were identified between hemispheres and between young and old groups in brain regions. The findings obtained in this study involving healthy beagle dogs might serve as a reference for regional CT perfusion values in specific brain regions. These results may aid in the characterization of various brain diseases in dogs.

Optimizing contrast protocol for bone-subtraction CT angiography of intracranial arteries in normal dogs using 160-slice CT.

BACKGROUND: Bone-subtraction computed tomography angiography (CTA) (BSCTA) is a new technique designed to overcome the limitation of three-dimensional CTA, where the vessels surrounded by bone and calcification can be obscured. An optimal contrast CT protocol for intracranial artery visualization with BSCTA has yet to be established in dogs. OBJECTIVES: The purpose of this study was to determine the optimal contrast protocol of CTA for visualizing intracranial artery using an automatic bone-subtraction technique in dogs. METHODS: Brain CTA was performed four times for each of nine healthy beagle dogs to cover all the contrast protocols: two different contrast iodine concentrations (300 and 370 mgI/mL) and two different contrast media injection rates (2 and 4 mL/s). Bone removal post-processing was performed automatically by subtracting the non-enhanced CT data from the contrast CT data using a dedicated workstation. The bone-subtracted intracranial vessels were analysed for quantitative and qualitative evaluation. RESULTS: Quantitative evaluation showed significantly higher CT attenuation values for the group with a 370 mgI/mL iodine content at a rate of 4 mL/s than the two groups with a 300 mgI/mL iodine content at the rates of 2 and 4 mL/s (p < 0.001). Qualitative assessment revealed significantly higher mean scores for the 370 mgI/mL groups than the 300 mgI/mL groups and significantly higher mean scores for the 4 mL/s groups than the 2 mL/s groups (p < 0.05). CONCLUSIONS: The optimal contrast protocol for BSCTA suggests that high iodine material concentration and high injection rate should be used for strong arterial attenuation and great visualization of the intracranial arterial structure in dogs.

Successful treatment of canine infective endocarditis caused by Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens is a gram-positive bacterial species that is utilised as a probiotic in humans and animals. There are no reports of infective endocarditis (IE) in dogs. An 8-year-old, spayed, female Maltese presented with a 1-month history of fever, depression, weight loss, and hindlimb lameness. Laboratory test results indicated non-regenerative anaemia, neutrophilia, hyperglobulinemia, and proteinuria. Echocardiography revealed vegetation on the septal leaflet of the mitral valve and thromboemboli in the left atrium. Consecutive blood culture results revealed that the blood samples were consistently positive for Bacillus amyloliquefaciens, which is generally considered a probiotic bacterial species for animals. Broad-spectrum antibiotics (amoxicillin-clavulanic acid and cefotaxime) and anticoagulants (clopidogrel and rivaroxaban) were administered for 4 months. The clinical signs were responsive to antibiotic treatment. After 4 months, the dog was no longer febrile and the size of the thromboemboli in the left atrium had decreased. Bacteria were no longer isolated in blood cultures after antibiotic therapy. To the best of our knowledge, this is the first case report of canine IE caused by bactaeremic infection with Bacillus amyloliquefaciens.

Bioactive Porous Particles as Biological and Physical Stimuli for Bone Regeneration.

Even though bony defects can be recovered to their original condition with full functionality, critical-sized bone injuries continue to be a challenge in clinical fields due to deficiencies in the scaffolding matrix and growth factors at the injury region. In this study, we prepared bone morphogenetic protein-2 (BMP-2)-loaded porous particles as a bioactive bone graft for accelerated bone regeneration. The porous particles with unique leaf-stacked morphology (LSS particles) were fabricated by a simple cooling procedure of hot polycaprolactone (PCL) solution. The unique leaf-stacked structure in the LSS particles provided a large surface area and complex release path for the sufficient immobilization of BMP-2 and sustained release of BMP-2 for 26 days. The LSS was also recognized as a topographical cue for cell adhesion and differentiation. In in vitro cell culture and in vivo animal study using a canine mandible defect model, BMP-2-immobilized LSS particles provided a favorable environment for osteogenic differentiation of stem cells and bone regeneration. In vitro study suggests a dual stimulus of bone mineral-like (leaf-stacked) structure (a physical cue) and continuously supplied BMP-2 (a biological cue) to be the cause of this improved healing outcome. Thus, LSS particles containing BMP-2 can be a promising bioactive grafting material for effective new bone formation.

Comparative analysis of vocalizations of thoroughbred mares (Equus caballus) between estrus and diestrus.

We investigated the differences between vocalizations of mares in estrus and diestrus and determined the spectrographic parameters to discriminate estrus from diestrus. Thoroughbred brood mares (n=89) were exposed to a teasing procedure for 3 min, and we recorded all vocalizations emitted from them. Among the mares, 56.5% of estrus and 78.6% of diestrus mares emitted calls toward an approaching stallion, indicating that there was higher tendency in the occurrence rate of vocal responses in diestrus than estrus mares. We analyzed the spectrographic data of the mares (25 estrus and 22 diestrus mares) emitting calls in the form of a squeal toward an approaching stallion. Based on broad bandwidth spectrographic analysis, the duration and third formant of the call have a significant effect on discriminating estrus from diestrus.

Behavioral reactivity of Jindo dogs socialized at an early age compared with non-socialized dogs.

The aim of this study was to determine whether socialized Jindo puppies would show different behavioral reactivity from non-socialized puppies. Puppies (n=12), 7 weeks of age, were divided into socialized and non-socialized groups. The socialized group from the 7th until 13th week after birth was provided a socialization program, and the non-socialized group was reared in a semi-isolated environment without being exposed to the program. At 13 weeks after birth, both groups were adopted by new families and raised as a family pet until adulthood. Both groups were tested in 5 behavioral tests at 7, 9, 11, 13 and 60 weeks of age, and their behavioral responses to the tests were recorded using video cameras. The contact, fearful and playful behaviors toward each behavioral test were scored on a scale of 1 to 5 points. Using all of the score data, a principal component analysis (PCA) extracted three primary factors: 'social reactivity towards humans and a dog', 'playful reactivity towards novel stimuli and a dog' and 'fearful reactivity towards social stimuli'. The three extracted factors were compared between the socialized and non-socialized groups in each test session (weeks). Based on the results, the socialized Jindo puppies in the test session at the 9th week after birth, in contrast to the non-socialized puppies, exhibited a higher intensity of playful reactivity towards novel stimuli and a dog. However, there were no effects of the socialization program on the Jindo puppies in terms of social reactivity towards humans and a dog as well as fearful reactivity towards social stimuli.

Thymic Hematoma Found in a Wild Eurasian Otter (Lutra lutra).

Thymic hematomas are uncommon in animals, and the few reported cases are all in dogs. In May 2019, we necropsied a wild Eurasian otter (Lutra lutra) in Junju, Republic of Korea, and found a thymic hematoma without any signs of trauma or hemorrhage in other organs, except for a hemothorax. This study describes the macroscopic and microscopic examinations of a thymic hematoma in an Eurasian otter.

Development of bone regeneration strategies using human periosteum-derived osteoblasts and oxygen-releasing microparticles in mandibular osteomyelitis model of miniature pig.

Hypoxia and limited vascularization inhibit bone growth and recovery after surgical debridement to treat osteomyelitis. Similarly, despite significant efforts to create functional tissue-engineered organs, clinical success is often hindered by insufficient oxygen diffusion and poor vascularization. To overcome these shortcomings, we previously used the oxygen carrier perfluorooctane (PFO) to develop PFO emulsion-loaded hollow microparticles (PFO-HPs). PFO-HPs act as a local oxygen source that increase cell viability and maintains the osteogenic differentiation potency of human periosteum-derived cells (hPDCs) under hypoxic conditions. In the present study, we used a miniature pig model of mandibular osteomyelitis to investigate bone regeneration using hPDCs seeded on PFO-HPs (hPDCs/PFO-HP) or hPDCs seeded on phosphate-buffered saline (PBS)-HPs (hPDCs/PBS-HP). Osteomyelitis is characterized by a series of microbial invasion, vascular disruption, bony necrosis, and sequestrum formation due to impaired host defense response. Sequential plain radiography, computed tomography (CT), and 3D reconstructed CT images revealed new bone formation was more advanced in defects that had been implanted with the hPDCs/PFO-HPs than in defects implanted with the hPDCs/PBS-HP. Thus, PFO-HPs are a promising tissue engineering approach to repair challenging bone defects and regenerate structurally organized bone tissue with 3D architecture.

Ultrasonographic evaluation of skin thickness in small breed dogs with hyperadrenocorticism.

The purpose of this study was to propose a standard for differentiation between normal dogs and patients with hyperadrenocorticism (HAC) by measuring skin thickness via ultrasonography in small breed dogs. Significant changes in skin thickness of patients treated with prednisolone (PDS) or patients with HAC treated with trilostane were evaluated. Skin thickness was retrospectively measured on three abdominal digital images obtained from small breed dogs weighing ＜ 15 kg that underwent abdominal ultrasonography. Mean skin thickness of normal dogs was 1.03 ± 0.25 mm (mean ± SD). Both the HAC and PDS groups showed significantly thinner skin than that in the normal group. Seven of the 10 HAC patients treated with trilostane had increased skin thickness. The area under the curve value of 0.807 was based on the receiver operating characteristics (ROC) curve for differentiating normal dogs from HAC patients. Sensitivity was 76% and specificity was 73% when skin thickness was less than the 0.83 mm cutoff value. In conclusion, measurement of skin thickness in small breed dogs by using ultrasonography is likely to provide clinical information useful in differentiating HAC patients from normal dogs. However, exposure to PDS, trilostane, and other conditions may have a significant effect on skin thickness.

Novel vertebral computed tomography indices in normal and spinal disorder dogs.

This study was carried out to derive and evaluate reference computed tomography (CT)-based indices for normal canine spine. CT and magnetic resonance images were acquired from 12 clinically normal Beagle dogs (normal group) and 50 dogs with 56 spinal disorders (patient group). Image acquisition regions were cervical spine (C2-T1), thoracic spine (T1-T13), and lumbar spine (L1-L7). Measured indices were: the ratios of width to height of the spinal cord including the dura matter (CR) and of the vertebral foramen (FR), and the ratio of the cross-sectional area of the spinal cord to that of the vertebral foramen (CFAR). Reliability analysis was performed to evaluate intermodality agreement. Student's t-tests and receiver operating characteristic curves were used to discriminate the normal and patient groups on CT. Intermodality agreements of the normal and patient groups were acceptable to excellent. The highest discriminating levels of CR at the vertebral body level and the intervertebral disc space level were 1.25 or more and 1.44 or more, respectively. FR and CFAR had the highest discriminating level at the cervical region. This report presents quantitative information on canine spinal morphometry; the obtained indices may be helpful for CT screening of dogs with spinal disorders.

Usefulness of transthoracic lung ultrasound for the diagnosis of mild pneumothorax.

The aim of the present study was to investigate the diagnostic accuracy of ultrasonography in the detection of mild pneumothorax using computed tomography (CT) in dogs. Nine adult healthy beagles were included in the study. A thoracic tube was inserted into pleural space at the left thoracic wall, and each dog underwent the examinations in the order of CT, lung ultrasonography, and radiography before the infusion of room air into the pleural space. Two, 3, and 5 mL/kg infusions of room air were sequentially introduced into the pleural space and CT, lung ultrasound, and radiography examinations were performed. Sonographic signs included A-lines, stratosphere, lung sliding, lung point, lung pulse, and reverse sliding signs. Radiographs were evaluated for the absence or presence of a pneumothorax. Lung ultrasound results were more accurate than radiography results for the detection of mild pneumothorax. The overall sensitivity of the sonographic reverse sliding sign was higher than that of other sonographic signs, and its specificity was 100% for detection of mild pneumothorax. Thus, the reverse sliding sign is useful when using lung ultrasonography for diagnosis of mild pneumothorax.