Efficacy of antivascular photodynamic therapy using benzoporphyrin derivative monoacid ring A (BPD-MA) in 14 dogs with oral and nasal tumors.

Antivascular photodynamic therapy (PDT) suppresses tumor growth and prolonged the survival in solid tumor-bearing mice. The purpose of this study was to assess the efficacy of antivascular PDT using BPD-MA for treatment of oral and nasal tumors in 14 dogs. At 15 min after initiating intravenous infusion of 0.5 mg/kg benzoporphyrin derivative monoacid ring A, tumors were irradiated with laser light at 690 nm emitted by a diode laser. The 1-year survival rate of 7 dogs with oral tumors was 71%. The 1-year survival rate of 7 dogs with nasal tumors was 57%. Imaging of each tumor was performed by using angiographic computed tomography before and after each antivascular PDT. Contrast-enhanced tumors were observed before antivascular PDT, but these tumors were not enhanced with contrast medium following antivascular PDT. Antivascular PDT is suggested to be a promising method for dogs with oral and nasal tumors that cannot be effectively treated with current antitumor therapies.

Phenotypic analysis and effects of sequential administration of activated canine lymphocytes on healthy beagles.

We attempted to accumulate the basic data for evaluation of activated lymphocyte therapy for small animal medicine. The peripheral blood mononuclear cells (PBMCs) collected from healthy dogs were activated using anti-CD3 antibody and human recombinant (hr) interleukin (IL)-2 and reactivated using hr interferon (IFN)-alpha and hr IL-2. The property of obtained cells was compared with PBMCs. The number of cells was shown to have increased approximately>50 -fold by cultivation. The proportion of CD8+ cells was significantly increased, the cytotoxicity of the cultured cells was revealed to have been reinforced. Additionally, CD56 mRNA levels tended to have increased. The cells obtained by this method were confirmed to be activated lymphocytes. Furthermore, we investigated the effects of sequential administration of the obtained cells to healthy dogs. By sequential administration of the activated lymphocytes, the cell proliferative activity, proportion of CD4+ cells and CD8+ cells, and serum IFN-gamma concentration were shown to have increased, and no severe adverse effects were observed. Consequently, activated lymphocytes could be induced using anti-CD3 antibody and IL-2 in healthy dogs, and sequential administration of activated lymphocytes reinforced the recipient's immunity.

Relative quantification of canine CD56 mRNA expression by real-time polymerase chain reaction method in normal tissues and activated lymphocytes.

Real-time PCR was optimized for the quantification of canine CD56 mRNA expression. This study was conducted to easily quantify canine CD56 expression and to identify its expression in normal tissues, peripheral blood mononuclear cells and activated lymphocytes in dogs. This assay revealed the highest level of CD56 mRNA expression in the normal canine brain, followed by the lung, kidney and liver. CD56 mRNA expression level in peripheral blood mononuclear cells was considerably lower; among activated lymphocytes in vitro, CD56 mRNA expression was increased.

Antitumor effects and blood flow dynamics after photodynamic therapy using benzoporphyrin derivative monoacid ring A in KLN205 and LM8 mouse tumor models.

Photodynamic therapy (PDT) using benzoporphyrin derivative monoacid ring A (BPD-MA) induces direct tumor cell damage and microvascular injury. We administered BPD-MA at 3h or 15min before laser irradiation to KLN205 and LM8 tumors in murine models. Tumor growth delay was induced more effectively by 15-min-interval PDT than by 3-h-interval PDT. Vascularity and blood perfusion was significantly decreased by 15-min-interval PDT. We observed death of all tumor cells, except peripheral cells, in the 3-h-interval PDT group, and death of cells around the damaged tumor vasculature in the 15-min-interval PDT group. Thus, 15-min-interval PDT enhanced the antitumor effect by damaging tumor vasculature.

Cardiovascular effects of continuous propofol infusion in horses.

We examined the influence of propofol infusion on cardiovascular system at the rate of 0.14, 0.20 and 0.30 mg/kg/min in six adult Thoroughbred horses. The cardiovascular parameters were heart rate (HR), mean arterial pressure (MAP), mean right atrial pressure (MRAP), stroke volume (SV), cardiac output (CO), systemic vascular resistance (SVR), pre-ejection period (PEP) and ejection time (ET). In order to keep the ventilation conditions constantly, intermittent positive pressure ventilation was performed, and the partial arterial CO(2) pressure was maintained at 45 to 55 mmHg during maintenance anesthesia. SV showed a significant dose-dependent decrease however, CO did not show significant change. SVR decreased significantly at higher dose. PEP was prolonged and PEP/ET increased significantly at the highest dose. From these results, it became clear that SV decreases dose-dependently due to decrease of cardiac contractility during anesthesia with continuous propofol infusion in horses. On the other hand, since MAP and CO did not show significant changes, total intravenous anesthesia with propofol was suggested to be suitable for long-term anesthesia in horses.

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis.

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.

Intracellular localization and concentration as well as photodynamic effects of benzoporphyrin derivative monoacid ring A in four types of rodent tumor cells.

The relative sensitivities of different tumor cells to photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA) were compared in the four tumor cells. A good correlation was observed between the cell survival at 0.1 microg/ml of BPD-MA and sensitizer uptake/10(6) cells (r = -0.99) or the plating efficiency of cells (r = 0.99). At 3 h after the irradiation, a significant difference was observed in the proportion of apoptotic cells among the four tumor cells (p = 0.024). In conclusion, cell responses to PDT depend on the several factors such as the cell line, photosensitizer dose, and fluence.

Matrix metalloproteinase inhibitor RECK expression in canine tumors.

Matrix metalloproteinases (MMPs) selectively degrade the extracellular matrix, and they have been reported to play an important role in tumor invasion, metastasis and angiogenesis. These enzymes are closely related to tumor malignancy and patient survival time. Recently, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene was identified as an endogenous membrane-anchored MMP inhibitor. The down-regulation of RECK has been implicated in tumor progression. In this study, the expression levels of the RECK messenger ribonucleic acid (mRNA) in various spontaneously developed canine tumors were investigated by using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and the correlation between RECK and clinicopathological factors, as well as MMP-9 expression were analyzed. The median age of 36 dogs investigated in this study was 9 years old (range, 1-15 years old). Quantitative RT-PCR could detect low levels of expression of RECK mRNA in the tumor samples. The expression levels of RECK mRNA in some tumor tissue samples were significantly lower than those in normal tissue samples. No significant associations of RECK with clinicopathological factors were observed. Using the Mann-Whitney U test, the expression level of the MMP-9 mRNA was observed to be significantly correlated to RECK expression (p<0.05).

The minimum infusion rate (MIR) of propofol for total intravenous anesthesia after premedication with xylazine in horses.

To investigate an adequate infusion rate of propofol for total intravenous anesthesia (TIVA) in horses, the minimum infusion rate (MIR) comparable to the minimum alveolar anesthetic concentration (MAC) of inhalation anesthetic was determined under constant ventilation condition by intermittent positive pressure ventilation (IPPV). In addition, arterial propofol concentration was measured to determine the concentration corresponding to the MIR (concentration preventing reaction to stimulus in 50% of population, Cp(50)). Further, 95% effective dose (ED(95)) was estimated as infusion rate for acquiring adequate anesthetic depth. Anesthetic depth was judged by the gross purposeful movement response to painful stimulus. MIR and Cp(50) were 0.10 +/- 0.02 mg/kg/min and 5.3 +/- 1.4 microg/ml, respectively. ED(95) was estimated as 0.14 mg/kg/min (1.4MIR).

Ventilatory failure and successful management for a dog with severe cervical meningioma.

A 12-year-old intact male mongrel dog with a weight of 22 kg was referred with a complaint of progressive tetraparesis. Cervical myelography revealed an intradural-extramedullary mass at the second cervical vertebra. After computed tomography (CT) under general anesthesia, the patient showed dyspnea and cyanosis caused by insufficient movement of the chest wall. Positive pressure ventilation was therefore initiated. Hemilaminectomy and partial mass removal were performed 12 hr after the CT. The mass was histopathologically diagnosed as meningioma. Gradual weaning from the mechanical ventilation lasted for 80 hr after the operation. The patient eventually recovered from the ventilatory failure and the tetraparesis at approximately 6 and 14 days after the operation, respectively.

Molecular cloning of canine membrane-anchored inhibitor of matrix metalloproteinase, RECK.

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5% and 91.9% homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.

Hindbrain decompression in a dog with scoliosis associated with syringomyelia.

A 6-month-old female Border Collie was examined because of a 1-month history of progressive curvature of the cervical portion of the vertebral column. Radiography revealed severe cervical and thoracic scoliosis. Cervical syringomyelia and hydrocephalus were observed by means of magnetic resonance imaging. Suboccipital craniotomy and laminectomy of the first cervical vertebra were performed, and substantial improvement in the scoliosis and syringomyelia was observed 3 months after surgery. No recurrences were seen during the first year after surgery.

Mechanism of macrophage activation by chitin derivatives.

In order to analyze the detailed mechanisms responsible for macrophage activation by chitin derivatives, resident peritoneal macrophages were prepared and stimulated with chitin, chitosan and low-molecular weight chitosan. Our findings were as follows: (i) chitosan induced apoptosis of peritoneal macrophages, but this did not occur when chitin or water soluble low-molecular weight chitosan were used; (ii) chitosan treatment induced activation markers, such as the major histocompatibility complex (MHC) class I, class II, Fc receptors, transferrin receptor, mannose receptor, Fas, and macrophage inflammatory protein (MIP)-2, whereas chitin and low molecular weight soluble chitosan induced only the expression of MHC class I and II molecules; (iii) apoptosis induced by chitosan was mediated by the Fas signaling pathway, in response to phagocytosis via the mannose receptor. We conclude that since chitosan activates macrophages, this may be the mechanism by which it accelerates wound healing.

Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells.

The bone marrow harbors a population of mesenchymal stem cells (MSCs) that possess the potential to differentiate into bone, cartilage, and fat, and along other tissue pathways. To date, MSCs from various species have been studied. Despite the bovine experimental model being widely used in experiments in vivo and in vitro, only a limited amount of information regarding bovine MSCs is available. The aim of this study was to isolate and induce the multilineage mesenchymal differentiation of bovine MSCs, thereby initiating further research on these cells. Bovine MSCs were isolated from eight calves, and osteogenic, chondrogenic, and adipogenic differentiation was induced by using a combination of previously reported protocols for other species. The level of differentiation was evaluated by histological examination and by analyzing the expression of tissue-specific genes by a quantitative "real time" reverse transcription/polymerase chain reaction technique. Following osteoinduction, the isolated fibroblast-like cells transformed into cuboidal cells and formed alkaline-phosphatase-positive colonies; during differentiation, these colonies transformed into mineralized nodules. In addition, osteogenesis was followed by osteocalcin and collagen type I mRNA expression. Chondrogenesis was confirmed by the demonstration of collagen type II, aggrecan, and sox9 mRNA expression in the cells stimulated by transforming growth factor beta1 in monolayer culture. After being cultured in an adipogenesis-inducing medium, the MSCs responded by the accumulation of lipid vacuoles and the expression of adipocyte-specific genes. We have therefore demonstrated that cells harvested from bovine bone marrow are capable of in vitro extensive multiplication and multilineage differentiation, making them a relevant and invaluable model in the field of stem cell research.

Odontogenic cysts in three dogs: one odontogenic keratocyst and two dentigerous cysts.

Odontogenic cysts, which showed cystic radiolucency in the jaw bone by radiographic examination and computed tomography, were enucleated by operation in 3 dogs. One dog had a odontogenic keratocyst in the incisive bone of the right maxilla and another 2 cases revealed dentigerous cysts in the mandible. These cyst walls were enucleated or transpired by semiconductor laser. Afterwards, osteogenesis was confirmed at the defective part of jaw bone by extirpation of the cyst in all cases, and no recurrence has been noted in any cases. Odontogenic cyst is a disease which should be treated by surgical extirpation or transpiration.

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system.

OBJECTIVE: Pluripotent mesenchymal stem cells (MSC) have been isolated and well characterized from several tissue sources, including bone marrow stroma. MSC from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated MSC from bovine bone marrow and induced chondrogenesis in order to establish a new experimental model of stem cell research. METHODS: Bone marrow was harvested from 8 calves. For inducing chondrogenesis, MSC were cultured in pellet culture system in a chemically defined medium supplemented with 0 and 10 ng/mL of transforming growth factor beta1 (TGF-beta1). Chondrogenic differentiation was evaluated by histological, immunohistochemical, and in situ hybridization techniques. The degrees of genes expression were measured by quantitative RT-PCR. RESULTS: Metachromatic alcian blue staining and immunoreactivity for type II collagen were detected in both pellet groups (0 and 10 ng/mL TGF-beta1) after 7 days of culturing. In situ hybridization demonstrated strong expression of type II collagen and aggrecan mRNAs in the round cells located at the center region of pellets and at densely organized areas. On the other hand, type I collagen mRNA was strongly expressed in the superficial layer of the pellets. After 20 days of pellet culture, expression of type II collagen mRNA in the cells which were not treated by TGF-beta1 was 1.7-fold higher compared with that treated by TGF-beta1. CONCLUSION: Independent, spontaneous chondrogenesis of bovine MSC in pellet culture occurred without addition of any external bioactive stimulators, namely factors from TGF-beta family, which were previously considered necessary.

Regeneration of cartilage tissue by autologous chondrocytes transplantation for cartilage defects in a experimental bovine model.

To evaluate the effects of chondrocytes transplantation on the regeneration of cartilage by intraarticular injection or injection into blood clots at cartilage defects, eight full-thickness cartilage defects were created surgically on the articular surface of each femoral trochlea of two calves. Autologous chondrocytes were isolated individually from the cartilage pieces collected at the creation of defects. And isolated cells were cultured in monolayers for proliferation. Cells were injected into synovial fluid (Group 2, n=11) or into the blood clots at the cartilage defects (Group 3, n=5) of the left femoropatellar joint on weeks 2 and 3, respectively after the operation. The defects (Group 1, n=16) of right femoropatellar joint were left untreated in the control group. After 14 weeks, repaired tissues were evaluated based on gross and histological examinations. In Group 3, more repaired tissues and a better interface between the repaired tissue and host cartilage were observed compared with the results for Groups 1 and 2. Moreover, cartilaginous tissue were observed more in defects of Group 3 than in defects of other groups. In conclusion, the present study suggests that the injection of cells into the blood clot at a cartilage defect might be applicable for the regeneration of damaged cartilage.

Effects of ascorbic acid on proliferation and biological properties of bovine chondrocytes in alginate beads.

Bovine chondrocytes were cultured in monolayers and alginate beads with or without ascorbic acid (Asc) for 16 days. Cell proliferation was examined every 4 days by staining with Hoechst 33258 dye. The gene expression of aggrecan, and collagen type I and II was analyzed at 16 days by reverse transcription and polymerase chain reaction. Cell morphology and the production of extracellular matrix (ECM) were evaluated by cytochemical, immunocytochemical and electron microscopical methods. Cells were continuously cultured in alginate beads with Asc for 2 months, and the cell morphology and ECM were examined. The proliferation of chondrocytes was significantly stimulated with Asc in both monolayers and alginate beads at 16 days. Expression of the collagen type I gene in both cultures was increased, and that of the collagen type II gene in alginate beads was decreased, by Asc. There were no significant cytochemical and immunocytochemical differences between the cultures in alginate beads with or without Asc at 16 days. In alginate beads cultured with Asc for 2 months, proliferating cells were observed mainly at the periphery of the beads, and glycosaminoglycan and collagen type II were found around the cells. These results suggest that Asc stimulated the proliferation of chondrocytes and maintained the chondrogenic properties of the cells in an alginate beads culture.

Efficacy of enamel matrix protein applied to spontaneous periodontal disease in two dogs.

Enamel matrix protein (EMP) was applied for regeneration of periodontal tissue in 2 dogs with spontaneous periodontal disease. Case 1 had bony resorption around the root and root apex of the maxillary fourth premolars. Case 2 had vertical resorption of bone between the mandibular first and second molars. A flap was formed in the buccal gingiva, and EMP was applied onto the surface of the exposed root. One or 4 months postoperatively, increased bone level and clinical attachment were recognized. EMP was therefore suggested to be effective to induce regeneration of periodontal tissues in the cases with periodontal disease.

Clinical observations during induction and recovery of xylazine-midazolam- propofol anesthesia in horses.

To evaluate clinical usefulness of xylazine (1.0 mg/kg)-midazolam (20 microg/kg)-propofol (3.0 mg/kg) anesthesia in horses, 6 adult Thoroughbred horses were examined. The quality of induction varied from poor to excellent and 5 out of 6 horses presented myotonus in the front half of the body. However, paddling immediately after induction observed in other reports of equine propofol anesthesia was not observed. Recovery time was 35.3 +/- 9.3 min and the quality of recovery was calm and smooth in all horses. Respiration rate decreased after induction and hypoxemia was observed during lateral recumbency. Heart rate also decreased after induction, however mean arterial blood pressure was maintained above approximately 100 mmHg.