Expression of metalloproteinases and their inhibitors in degenerated and extruded intervertebral disks in chondrodystrophic dogs.

We analyzed the mRNA expression of matrix metalloproteinases (MMPs), metalloproteinases with thrombospondin motifs (ADAMTSs), and tissue inhibitors of metalloproteinases (TIMPs) in degenerated and herniated intervertebral disks (IVDs) in chondrodystrophic dogs. In degenerated IVDs, MMP3, 7, 13, and 14; ADAMTS4 and 5; and TIMP1-3 expression was significantly higher vs healthy controls (P<0.05). In herniated IVDs, MMP2, 3, 9, 13, and 14; ADAMTS4 and 5; and TIMP1 expression was significantly greater, and MMP7 expression was significantly lower vs degenerated IVDs (P<0.05). These results suggest that metalloproteinase may play a role in extracellular matrix degradation in IVD degeneration. Decreased MMP7 transcription may prevent proteoglycan degradation and reduces macrophage infiltration, which might affect the resorption process of herniated IVDs.

Hyperadrenocorticism Caused by a Pituitary Ganglioglioma in a Dog.

An 11-year-old castrated male Chihuahua dog was presented with complaints of polyuria, polydipsia, abdominal enlargement, and alopecia. Hyperadrenocorticism was diagnosed on the basis of clinical signs, blood tests, adrenocorticotropin-stimulation test results, and an elevated serum adrenocorticotropin concentration. Contrast-enhanced magnetic resonance imaging showed that the pituitary gland was enlarged, compatible with a pituitary macroadenoma. Pituitary-dependent hyperadrenocorticism was suspected, and transsphenoidal hypophysectomy was thus performed for complete resection of the tumor. After surgery, the serum adrenocorticotropin concentration normalized and the hyperadrenocorticism resolved. Histological and immunocytochemical analyses revealed a benign tumor composed of mature neuronal cells and glial cells, suggestive of a ganglioglioma with immunolabeling for adrenocorticotropin. Careful analysis of the resected tumor revealed no pituitary adenoma tissue. The clinical and histopathologic findings indicated that the ganglioglioma was directly responsible for the hyperadrenocorticism. This is the first case of hyperadrenocorticism caused by a ganglioglioma in a dog.

Transcatheter arterial embolization for treatment of hepatocellular carcinoma in a cat.

CASE DESCRIPTION: A 16-year-old 6.8-kg (15-lb) castrated male domestic shorthair cat was evaluated because of a 3 × 6-cm mass in the right medial lobe of the liver. CLINICAL FINDINGS: The cat had a history of frequent vomiting and anorexia along with 10% weight loss over the past year. TREATMENT AND OUTCOME: Transcatheter arterial embolization was selected because surgery (standard first-line treatment) was declined and only 1 vessel feeding the tumor was apparent on contrast-enhanced CT. A 4F sheath was placed in the left carotid artery, and a 3.3F guide catheter was advanced into the celiac artery. A 0.014-inch guidewire and 1.7F microcatheter were inserted into the hepatic artery through the guiding catheter and advanced into the feeding vessel. A mixture of polyvinyl alcohol particles and contrast agent was injected for embolization. A hypoechoic area in the tumor was identified on ultrasonography on posttreatment day 6, and necrotic and degenerated cells in the area were identified cytologically. By posttreatment day 71, vomiting had resolved and CT revealed decreased tumor size, but altered attenuation suggested a more solid mass on day 205. No feeding vessel for embolization was found on contrast-enhanced CT, so ultrasonic emulsification to remove the tumor was performed on day 231. No recurrence was seen on contrast-enhanced CT on day 420 or day 721. CLINICAL RELEVANCE: Findings suggested that transcatheter arterial embolization may be suitable for treating hepatic tumors in cats, but alternative approaches are needed in cats, compared with dogs, owing to anatomic differences.

Impact of normothermic preservation with extracellular type solution containing trehalose on rat kidney grafting from a cardiac death donor.

BACKGROUND: The aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx). METHODS: Post-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringer's (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4-6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2). RESULTS: Using the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion. CONCLUSIONS: Normothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.

Plasma concentration and cardiovascular effects of lidocaine during continuous epidural administration in dogs anesthetized with isoflurane.

The cardiovascular effects of continuous epidural administration (CEA) of lidocaine were investigated in anesthetized dogs. Loading epidural injections of 2, 4, or 6 mg/kg of lidocaine were followed by CEA with 1, 2, or 3 mg/kg/hr lidocaine, respectively, for 2 hr under 2.0% isoflurane anesthesia. Heart rate, direct blood pressure, cardiac index, and stroke volume decreased dose-dependently during CEA, whereas systemic vascular resistance did not significantly differ with dose, and no characteristic changes were observed in any groups. Plasma lidocaine concentration reached a steady state during CEA and increased in a dose-dependent manner. Circulatory suppression caused by lidocaine CEA was not attributable to peripheral vasodilation, but rather to the direct cardiac action of systemic lidocaine absorption from the peridural space.

Priming effects of lipopolysaccharide and inflammatory cytokines on canine granulocytes.

Granulocytes play a pivotal role in natural immunity. Under inflammatory conditions, granulocytes are universally primed by several agents, such as endotoxins and inflammatory cytokines. Primed granulocytes exert potent adhesiveness, chemotaxis, phagocytosis and reactive oxygen species (ROS) production, effectively eliminating invading agents. Reactivity against priming agents is known to vary with species; however, there have been few reports on the effects of priming agents on canine granulocytes. In the present study, we assayed the priming effects of lipopolysaccharide (LPS), recombinant canine tumor necrosis factor-alpha (rcTNF-alpha) and recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) on canine granulocyte function in vitro. Isolated recombinant canine were primed with various concentrations of LPS, rcTNF-alpha and rcGM-CSF, and CD11b expression was assayed. Furthermore, actin polymerization, phagocytosis and ROS production were then assayed at primer concentrations where enhancement of CD11b expression was observed. LPS did not enhance canine granulocyte function. Phagocytosis and actin polymerization were not enhanced by priming agents; however, rcTNF-alpha and rcGM-CSF enhanced CD11b expression and ROS production in canine granulocytes. These results suggest that priming effects are mainly reflected in CD11b expression and ROS production, with rcGM-CSF and rcTNF-alpha having a priming effect similar to that observed in humans.

Effects of lidocaine hydrochloride on canine granulocytes, granulocyte CD11b expression and reactive oxygen species production.

Lidocaine hydrochloride (Lido) is widely used for analgesia in veterinary medicine; however, in humans, it has been suggested that Lido attenuates granulocyte functions, such as adhesion and reactive oxygen species (ROS) production. Thus, Lido may affect canine granulocyte function; however, there have been no reports on the effects of Lido on canine granulocyte function. Thus, we studied the effects of Lido on canine granulocyte CD11b expression and ROS production. We further studied the effects of Lido on the priming of canine granulocyte CD11b expression and ROS production by recombinant canine granulocyte macrophage colony stimulating factor (rcGM-CSF). Isolated granulocytes were incubated with 3, 30 or 300 microg/ml Lido, or with Lido followed by priming with 5 ng/ml rcGM-CSF. CD11b was detected by the immune fluorescent antibody method, and the mean fluorescence intensity (MFI) was assayed by flow cytometry. ROS production was assessed by the peak time (PT) of ROS production and area under the luminol reaction curve (AUC), which represents total ROS production quantity against opsonized zymosan stimuli. Only 300 microg/ml Lido (tissue level observed by regional block) significantly attenuated both the MFI of CD11b and its enhancement by rcGM-CSF. Moreover, at this concentration, the AUC and its enhancement by rcGM-CSF were significantly attenuated by Lido; in contrast, Lido did not affect PT. In conclusion, Lido suppressed granulocyte adhesion to the endothelium and antiseptic capability by suppressing CD11b expression and/or ROS production. Particular care should thus be exercised when performing regional anesthesia block using Lido.

Relative nerve blocking properties of bupivacaine and ropivacaine in dogs undergoing brachial plexus block using a nerve stimulator.

In the present study, the efficacy of a nerve stimulator in performing brachial plexus block (BPB) in dogs was investigated. The nerve blocking effects of bupivacaine and ropivacaine for BPB were also compared. Twelve beagles were allocated to groups based on the following treatments: conventional BPB with 0.5% bupivacaine (0.5% BupiM group) or BPB with 0.5% bupivacaine, 0.5% ropivacaine or 0.75% ropivacaine and a nerve stimulator (the 0.5% BupiS, 0.5% RopiS and 0.75% RopiS groups, respectively). After BPB, nerve blocking effects were assessed based on sensory blockade in several cutaneous areas and knuckling. The ratio of full block (blockade in all cutaneous areas) for 0.5% BupiM was 25%, and that for 0.5% BupiS was significantly higher, 75% (p<0.05). For the 0.5% BupiS, 0.5% RopiS and 0.75% RopiS groups, the average duration of full block was 387, 184 and 275 min, respectively, and the average duration of knuckling was 703, 460 and 421 min, respectively. The duration of full block and knuckling for the two ropivacaine groups was shorter compared with that of the 0.5% BupiS group. In conclusion, when using bupivacaine and ropivacaine for BPB in dogs, it is worth noting that there are differences in onset time and duration and that effective perioperative analgesia can be achieved depending on the intended use.

Evaluation of immunological status in tumor-bearing dogs.

The purpose of this study was to evaluate immunological status in dogs with cancers at different stages, in comparison with normal dogs. The population of canine peripheral blood lymphocytes (cPBL), lymphocyte phenotypes, interleukin (IL)-6 activity and alpha 1-acid glycoprotein (alpha(1)-AG) level were analyzed. The tumor-bearing dogs had higher numbers of leukocytes than normal dogs, the count being higher in dogs with more advanced tumors. In the tumor-bearing dogs, differential leukocyte counts revealed higher percentages of inflammatory cells such as neutrophils, acidophils and monocytes, and lower numbers of CD4(+)T cells, than in normal dogs, the lymphocyte counts becoming much lower with tumor progression. In the tumor-bearing dogs, the CD8(+)T cell count at the early tumor stage was similar to that in normal dogs, but decreased with tumor progression, possibly reflecting the development of humoral immunity (Th2). Plasma IL-6 and TGF-beta activities were high in the tumor-bearing dogs. The plasma alpha(1)-AG concentration was also significantly high in the tumor-bearing dogs. Our findings suggest that assay of IL-6, TGF-beta and alpha(1)-AG may be very useful for prognostication in dogs with cancer, and that anti-tumor immunity is potently suppressed in such dogs.

Bulk cultures of canine peripheral blood lymphocytes with solid phase anti-CD3 antibody and recombinant interleukin-2 for use in immunotherapy.

Interleukin (IL)-2 can induce large numbers of lymphokine-activated killer cells in peripheral blood lymphocytes (PBL), but IL-2 alone cannot induce proliferation of a large number of canine (c) PBL. We used the solid phase anti-CD3 antibody and soluble recombinant (r) IL-2 in order to establish a large scale culture method for cPBL. The number of lymphocytes seeded (3 x 10 (7)) increased to 1 x 10(9) after incubation for 10 days. The phenotype of cultured cPBL cells (after 2 weeks) showed a CD4(+) or CD8(+) predominant cell population. The cultured cell solutions were administered with physiological saline intravenously to each dog. After transfusion of the cultured cells, the cPBL counts, especially the number of CD4(+), CD8(+) and CD4(-)CD8 (-)(DN) cells increased significantly in the recipient dogs. Natural killer (NK) cells, gammadeltaT cells and B cells were considered to be present in the DN cell population. The NK cells and gammadeltaT cells showed no adverse reaction to the transfusion of the activated cPBL. Therefore, it is necessary to recognize the B cells present in the DN cell population by detecting CD21(+) cells. In conclusion, the bulk culture system of cPBL with rIL-2 and solid phase anti-CD3 antibody may be useful for the development of novel immunotherapy in dogs.

Canine serum alkaline phosphatase isoenzymes detected by polyacrylamide gel disk electrophoresis.

Serum alkaline phosphatase (ALP) isoenzymes were studied in normal dogs using a commercially available polyacrylamide gel disk electrophoresis kit (PAG/disk kit). Serum samples taken from the dogs were incubated with neuraminidase, after which most showed ALP isoenzymes as two characteristic stained bands. To determine the origin of each band, ALP isoenzymes of serum and tissue extracts (liver, intestine and bone) were characterized by heating, wheat germ agglutinin (WGA) and levamisole treatments. The results suggested that the band detected on the anode was liver ALP (LALP) and that the band detected on the cathode represented bone ALP (BALP), and both were corticosteroid-induced ALP (CALP). The percentage of each ALP isoenzyme to total ALP activity was estimated by densitometry. The percentage of BALP was the highest in young dogs (age<1 year, 64.7% ), and this value decreased with age. In contrast, the percentage of LALP in young dogs (22.2%) was much lower than that in middle-aged dogs (ages 1 year to 7 years, 59.3%) and old dogs (ages>7 years, 50.4%). The present results suggested that a commercially available PAG/disk kit is capable of detecting three serum ALP isoenzymes in dogs, and further that it may have clinical applications in the evaluation of ALP isoenzymes in veterinary medicine.