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Differentiation between canine chronic enteropathy (CCE) and intestinal lymphoma is a diagnostic challenge as histopathology might fail to yield unequivocal results. Detection of clonal rearrangements of the T-cell-receptor gamma (TCRG) chain and IG heavy chain (IGH) V-J genes offer a useful solution. In this retrospective study, histopathology samples of 35 CCE patients and 7 healthy Beagle dogs underwent clonality testing. Patients suffered either from inflammatory bowel disease (IBD), food responsive diarrhea (FRD) or protein loosing enteropathy secondary to IBD (PLE/IBD). Healthy Beagles served as controls (CO). Canine IBD activity index (CIBDAI) and histopathological WSAVA-grading differed significantly (p<0.001) between groups. CIBDAI improved significantly after appropriate therapy (p < 0.0001). Intestinal biopsies of all CO showed polyclonal patterns for B- and T-cell primers. All samples from CCE patients showed polyclonal patterns for the B-cell primers. Targeting TCRG, 4 patients showed a monoclonal or oligoclonal pattern of the lymphocytic infiltrates in the duodenum and/or colon. Clinical improvement was observed in all dogs. Although a small cell lymphoma cannot be excluded in view of the short follow up duration, a false positive result, in the sense of a canonical rearrangement or unspecific amplification due to a antigenic stimulation in a non-neoplastic inflammatory process is possible., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background/aim: Our aim was to investigate the crosstalk between tumor and immune cells (M2 macrophages) and its effects on cyclo-oxygenase-2 (COX2) regulation in canine mammary tumors (CMT)., Materials and Methods: Sh1b CMT cells and human BT474 mammary or HT29 colon cancer cells were co-cultured with canine peripheral blood mononuclear cells (PBMCs) or with macrophage-like differentiated THP1 monocytes (dTHP1). Intracellular COX2 expression by PBMCs, dTHP1 and cancer cells was evaluated by flow cytometry., Results: Co-culturing of Sh1b and canine PBMCs induced COX2 overexpression in CMT cells. In turn, COX2 expression by PBMCs, mostly CD68 + macrophages, was attenuated by co-culture with Sh1b (p=0.0001). In accordance, co-culture with dTHP1 prompted intracellular production of COX2 in both Sh1b CMT cells and HT29 human colon cancer cells and reduced production of COX2 in BT474 human mammary cancer cells. The intracellular COX2 expression from dTHP1 decreased when treated with conditioned medium from cultured Sh1b and HT29 cancer cells., Conclusion: Bidirectional COX2 regulation between cancer and monocytes/macrophages might shape a tolerogenic tumor microenvironment in CMT., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Inflammatory bowel disease (IBD) is common in dogs. Despite the known importance of intestinal lymphocytes in its pathogenesis, little is known about the role of peripheral blood lymphocytes (PBLs) in IBD., Objectives: The aims of this study were (1) comparison of PBLs analyzed by flow cytometry (FCM) in IBD dogs and healthy controls and (2) comparison of PBLs in IBD dogs at the time of diagnosis and in dogs in clinical remission., Animals: Whole blood samples of 19 IBD dogs at the time of diagnosis and blood samples of 6 dogs in clinical remission were collected. Ten healthy dogs served as controls., Methods: In this prospective observational study, PBLs were analyzed with multicolor FCM by staining with a panel of anticanine and cross-reactive monoclonal antibodies against T- and B-cell differentiation antigens, including CD45, CD3, CD4, CD8α, CD8β, TCRαβ, TCRγδ, CD79αcy, and CD21., Results: The IBD patients' PBLs had significantly decreased percentages of TCRγδ + T lymphocytes (median: healthy dogs, 3.32; IBD dogs, 0.97; P = 0.03) and CD21 + B cells (median: healthy dogs, 27.61; IBD dogs, 17.26; P = 0.04). There were no significant differences in PBLs between pretreatment and follow-up samples., Conclusions and Clinical Importance: The differences between PBLs in healthy and IBD dogs analyzed by FCM indicate an imbalance of lymphocytes with different immunologic functions and emphasize the potential value of this technique in a larger cohort of dogs. The PBLs did not differ between IBD dogs before treatment and clinically well-controlled dogs after treatment., (Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Accumulating evidence suggests that epileptic seizures originating from the temporal lobe (TL) occur in cats. Typically, affected animals have clinically focal seizures with orofacial automatisms including salivation, facial twitching, lip smacking, chewing, licking, and swallowing. Motor arrest and autonomic and behavioral signs also may occur. Many affected cats have magnetic resonance imaging (MRI) changes within the hippocampus or histopathologically confirmed hippocampal sclerosis or necrosis. From the 1950s to the 1980s, cats frequently were used as animal models for neurophysiological experiments and electrophysiological studies, from which important basic knowledge about epilepsy originated, but which has been rarely cited in clinical veterinary studies. These studies were reviewed. Experimental research on cats showed the widespread anatomical connections among TL structures. The ictal clinical signs originating from the hippocampus, amygdala, or lateral temporal cortex are similar, because of their dense interconnections. The ictal signs can be divided into autonomic, somatic, and behavioral. For research purposes, a 6-stage system was established, reflecting the usual sequential progression from focal to generalized seizure: attention response (1), arrest (2), salivation, licking (3), facial twitching (4), head turning or nodding (5), and generalized clonic convulsions (6). Knowledge of this data may help in recognizing low-stage (stage 1 or stage 2) epileptic seizures in clinical practice. Early experimental research data are in accordance with recent clinical observations regarding ictal clinical signs of TL epileptic seizures in cats. Furthermore, the research data supports the idea that TL epilepsy represents a unique clinical entity with a specific seizure type and origin in cats., (Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Both humans and their most important domestic animals harbor IgE and a similar IgE receptor repertoire and expression pattern. The same cell types are also involved in the triggering or regulation of allergies, such as mast cells, eosinophils or T-regulatory cells. Translational clinical studies in domestic animals could therefore help cure animal allergies and at the same time gather knowledge relevant to human patients. Dogs, cats and horses may spontaneously and to different extents develop immediate type symptoms to pollen allergens. The skin, nasal and bronchial reactions, as well as chronic skin lesions due to pollen are in principle comparable to human patients. Pollen of various species most often causes allergic rhinitis in human patients, whereas in dogs it elicits predominantly eczematous lesions (canine atopic dermatitis), in horses recurrent airway obstruction or hives as well as pruritic dermatitis, and in cats bronchial asthma and so-called cutaneous reactive patterns (eosinophilic granuloma complex, head and neck pruritus, symmetric self-induced alopecia). In human allergy-specific IgE detection, skin tests or other allergen provocation tests should be completed. In contrast, in animals IgE and dermal tests are regarded as equally important and may even replace each other. However, for practical and economic reasons intradermal tests are most commonly performed in a specialized practice. As in humans, in dogs, cats and horses allergen immunotherapy leads to significant improvement of the clinical symptoms. The collected evidence suggests that canines, felines and equines, with their spontaneous allergies, are attractive model patients for translational studies.

Background: Many dogs suffering from inflammatory bowel disease (IBD) are presented to veterinary clinics. These patients are diagnosed based on a history of chronic gastrointestinal signs and biopsy-confirmed histopathologic intestinal inflammation. Intestinal intraepithelial lymphocytes (IEL) are part of the first line of defense in the gastrointestinal immune system. Alterations in IEL subsets may play a role in the pathogenesis of IBD., Hypothesis: The aim of this study was to characterize the phenotypes of IEL in dogs with IBD compared with healthy control dogs., Animals: Intestinal intraepithelial lymphocytes subpopulations of control dogs (n = 5) obtained from endoscopic biopsies (EB) were compared to those obtained from full thickness biopsies (FTB) on the same day. In addition, the phenotypes of IEL from FTB of control dogs (n = 10) were compared with EB of IBD dogs (n = 10). Each participant was scored clinically using the canine inflammatory bowel disease activity index (CIBDAI), and all samples were graded histopathologically. Three-color flow cytometry of isolated IEL was performed using monoclonal antibodies against T- and B-lymphocyte subpopulations., Results: No significant differences in the composition of IEL subpopulations were found in control dogs based on method of biopsy. The IBD dogs had significantly higher CIBDAI and histopathologic scores compared with control dogs and their IEL contained a significantly higher frequency TCRγδ T-cells., Conclusions and Clinical Importance: Endoscopic biopsies provide suitable samples for 3-color flow cytometry when studying canine intestinal IEL and IBD patients show significant changes of major T-cell subsets compared to healthy control dogs., (Copyright © 2014 by the American College of Veterinary Internal Medicine.)

We report an evaluation of the treatment and outcome of cats with suspected primary epilepsy. Phenobarbital therapy was used alone or in combination with other anti-epileptic drugs. Outcome after treatment was evaluated mainly on the basis of number of seizures per year and categorised into four groups: seizure-free, good control (1-5 seizures per year), moderate control (6-10 seizures per year) and poor control (more than 10 seizures per year). About 40-50% of cases became seizure-free, 20-30% were considered good-to-moderately controlled and about 30% were poorly controlled depending on the year of treatment considered. The duration of seizure events after treatment decreased in 26/36 cats and was unchanged in eight cats. The subjective severity of seizure also decreased in 25 cats and was unchanged in nine cats. Twenty-six cats had a good quality of life, nine cats an impaired quality of life and one cat a bad quality of life. Despite being free of seizures for years, cessation of treatment may lead to recurrence of seizures in most cats.

Background: Treatment-resistant complex partial seizures (CPS) with orofacial involvement recently were reported in cats in association with hippocampal pathology. The features had some similarity to those described in humans with limbic encephalitis and voltage-gated potassium channel (VGKC) complex antibody., Hypothesis/objectives: The purpose of this pilot study was to evaluate cats with CPS and orofacial involvement for the presence of VGKC-complex antibody., Animals: Client-owned cats with acute orofacial CPS and control cats were investigated., Methods: Prospective study. Serum was collected from 14 cats in the acute stage of the disease and compared with 19 controls. VGKC-complex antibodies were determined by routine immunoprecipitation and by binding to leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2), the 2 main targets of VGKC-complex antibodies in humans., Results: Five of the 14 affected cats, but none of the 19 controls, had VGKC-complex antibody concentrations above the cut-off concentration (>100 pmol/L) based on control samples and similar to those found in humans. Antibodies in 4 cats were directed against LGI1, and none were directed against CASPR2. Follow-up sera were available for 5 cats in remission and all antibody concentrations were within the reference range., Conclusion and Clinical Importance: Our study suggests that an autoimmune limbic encephalitis exists in cats and that VGKC-complex/LGI1 antibodies may play a role in this disorder, as they are thought to in humans., (Copyright © 2012 by the American College of Veterinary Internal Medicine.)

In contrast to infections with enterohaemorrhagic E. coli (EHEC), which are thought to be classical zoonosis, the zoonotic potential of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is still widely unknown. The aim of our study was to determine the frequency of EHEC and ESBL-producing Enterobacteriaceae in domestic animals (dogs and cats) in the Tyrol. Among 228 fecal samples of dogs (n = 92) and cats (n = 136) three samples (1.3%) were positive in the EHEC-ELISA. In two of the three cases isolation of the organism was not possible, the third sample of a two-year-old crossbreed bitch yielded EHEC O103:H2. In twelve of 228 (5.3%) fecal samples 13 ESBL-producing Enterobacteriaceae (in ten cats and two dogs) were found.These animals mainly derived from homes for animals (ten animals, 83%). 75% of the isolates belonged to the CTX-M-1-group, 8% to the CTX-M-2-group and 17% to the CTX-M-9-group. One isolate was positive for CTX-M-1 and CTX-M-9. Typing of the 13 ESBL-producing isolates by multilocus sequence typing (MLST) showed ten different sequence types, which points out the importance of the horizontal transfer of mainly plasmid-coded ESBL genes. Transmission of EHEC and ESBL-producing Enterobacteriaceae from domestic animals to humans is possible, corroborated by the fact that the EHEC serotype found in one dog and the sequence types detected by MLST in several dogs and cats were previously reported to occur in severe human infection.

The main aim of this study was to identify interictal epileptiform discharges in a group of dogs with seizures of known aetiology (symptomatic epilepsy, SE) and in dogs with idiopathic epilepsy (IE). Propofol was used for chemical restraint in all dogs. We found electroencephalographic (EEG) changes that could be considered epileptiform discharges (EDs) in 5 out of 40 dogs (12.5%). The EEG changes identified were spikes in four cases and periodic epileptiform discharges in one case. All EDs were seen in the SE group. We conclude that the interictal electroencephalographic examinations of propofolanaesthetised dogs suffering from IE and SE rarely show epileptic discharges and that the diagnostic value of such EEGs in the work-up for epilepsy seems to be low as epileptic discharges were unlikely to be detected. However, positive findings are more likely to be connected with SE. We found frequent, transient EEG phenomena (spindles, K-complexes, vertex waves, positive occipital sharp transients of sleep, cyclic alternating patterns), which are non-epileptic but their differentiation from epileptic phenomena is challenging.

Angiogenesis, which is essential for malignancies to progress, depends on various signalling proteins including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2). Microvessel density (MVD) is frequently used to evaluate angiogenesis. This study assessed the relationship between expression of VEGF, VEGFR-1 and VEGFR-2, MVD and the survival time in dogs with lymphoma. VEGF, VEGFR-1 and VEGFR-2 expression was evaluated immunohistochemically and microvessel profiles were counted in 34 lymphoma samples. Seventy-nine percent of the samples showed high VEGF expression and 62% were highly positive for VEGFR-1; VEGFR-2 immunoreactivity was mostly negative. Dogs treated with chemotherapy had a median survival time of 266days, but no significant relationships were found between overall survival time, MVD and expression of VEGF, VEGFR-1 or VEGFR-2. In this study, VEGF its receptors and the MVD were no prognostic factors in dogs with lymphoma., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chronic kidney disease (CKD) may affect excretion and metabolism of vitamins but data for dogs are limited. In this study, blood vitamin levels were investigated in 19 dogs with chronic renal failure. High performance liquid chromatography was used to quantify retinol, retinyl esters, tocopherol, thiamine, riboflavin, pyridoxal-5'-phosphate, ascorbic acid and 25-hydroxycholecalciferol concentrations, whereas cobalamin, folate, biotin and pantothenic acid were measured by microbiological methods. Levels of retinol, retinyl palmitate, ascorbic acid, and vitamins B1, B2 and B6 were increased compared to healthy dogs. Dogs with CKD showed decreased concentrations of 25-hydroxycholecalciferol and folate. Alpha-tocopherol, biotin, pantothenate and cobalamin levels were not significantly different between controls and dogs with CKD. Whether lower vitamin D and folate concentrations in dogs with CKD justify supplementation has to be evaluated in future studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To facilitate comparative oncology trials we compared the biological and molecular homologies of canine (dog; Canis lupus familiaris) and human tumor-associated antigens ErbB-1 and -2. Further, we investigated whether they could serve as targets for anti-ErbB-1 (cetuximab) and anti-ErbB-2 antibodies (trastuzumab), which are highly relevant in human clinical oncology. Immunohistochemistry of canine mammary cancer showed ErbB-1 overexpression in 3/10 patients and ErbB-2 in 4/10. We report 91% amino acid homology for ErbB-1 and 92% for ErbB-2 between canine and human molecules. Modeling of canine on human ErbB-1 revealed that the cetuximab epitope only differs by 4 amino acids: Lys443 is replaced by Arg, Ser468 by Asn, Gly471 by Asp, and Asn473 by Lys in canines. The trastuzumab binding site is identical in human and canine ErbB-2 apart from a single amino acid change (Pro557 to Ser). Binding of cetuximab and trastuzumab to canine mammary carcinoma cells CF33, CF41, Sh1b and P114 was confirmed by flow cytometry. Both antibodies significantly inhibited canine tumor cell proliferation partly due to growth arrest in G(0)/G(1) phase. We explain the lower efficiency on the tested canine than on human SKBR3 and A431 cells, by a 2-log lower expression level of the canine ErbB-1 and -2 molecules. Our results indicate significant homology of human and canine Erb-1 and -2 tumor associated antigens. The fact that the canine homologues express the cetuximab and trastuzumab epitopes may facilitate antibody-based immunotherapy in dogs. Importantly, the striking similarities of ErbB-1 and -2 molecules open up avenues towards comparative strategies for targeted drug development., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Seventeen cats were presented with acute onset of complex partial seizures with orofacial involvement (salivation, facial twitching, lip smacking, chewing, licking or swallowing), motor arrest (motionless starring) and behavioural changes. In 11 cats hippocampal necrosis (HN) was confirmed by histopathology. In a further six cats hippocampal changes were suggested by magnetic resonance imaging. The mean monitoring time of eight cats which were not euthanased in the acute phase of the disease, was 408 days (60-908): four cats are still alive. In all surviving cases, the owners reported a good quality of life. We conclude that an acute cluster of complex partial seizures with orofacial involvement are often associated with HN and that HN is not necessarily a fatal condition. Supportive and antiepileptic therapy can result in remission. The long-term outcome can be good to excellent; therefore, euthanasia should be avoided in the acute phase of the signs., (Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.)

A subcutaneous continuous glucose monitoring system (GlucoDay; Menarini Diagnostics) based on microdialysis was investigated for its clinical applicability in veterinary medicine. Ten diabetic dogs, referred as clinically stable, were equipped with this system and sent home for a maximum observation period of 48 hours. Time of insulin administration, feeding and other events were written in a diary and plotted afterwards in the glucose graph. Implantation of the microdialysis fibre, acceptance of the device and evaluation of individual canine glucose profiles were without complication. Based on the monitoring data, recommended treatment adjustments were given to the referring veterinarians in all 10 dogs; hypoglycaemic or prolonged hyperglycaemic episodes were detected in six dogs.

Comparative oncology aims at speeding up developments for both, human and companion animal cancer patients. Following this line, carcinoembryonic antigen (CEA, CEACAM5) could be a therapeutic target not only for human but also for canine (Canis lupus familiaris; dog) patients. CEACAM5 interacts with CEA-receptor (CEAR) in the cytoplasm of human cancer cells. Our aim was, therefore, to phylogenetically verify the antigenic relationship of CEACAM molecules and CEAR in human and canine cancer.Anti-human CEACAM5 antibody Col-1, previously being applied for cancer diagnosis in dogs, immunohistochemically reacted to 23 out of 30 canine mammary cancer samples. In immunoblot analyses Col-1 specifically detected human CEACAM5 at 180 kDa in human colon cancer cells HT29, and the canine antigen at 60, 120, or 180 kDa in CF33 and CF41 mammary carcinoma cells as well as in spontaneous mammary tumors. While according to phylogenicity canine CEACAM1 molecules should be most closely related to human CEACAM5, Col-1 did not react with canine CEACAM1, -23, -24, -25, -28 or -30 transfected to canine TLM-1 cells. By flow cytometry the Col-1 target molecule was localized intracellularly in canine CF33 and CF41 cells, in contrast to membranous and cytoplasmic expression of human CEACAM5 in HT29. Col-1 incubation had neither effect on canine nor human cancer cell proliferation. Yet, Col-1 treatment decreased AKT-phosphorylation in canine CF33 cells possibly suggestive of anti-apoptotic function, whereas Col-1 increased AKT-phosphorylation in human HT29 cells. We report further a 99% amino acid similarity of human and canine CEA receptor (CEAR) within the phylogenetic tree. CEAR could be detected in four canine cancer cell lines by immunoblot and intracellularly in 10 out of 10 mammary cancer specimens from dog by immunohistochemistry. Whether the specific canine Col-1 target molecule may as functional analogue to human CEACAM5 act as ligand to canine CEAR, remains to be defined. This study demonstrates the limitations of comparative oncology due to the complex functional evolution of the different CEACAM molecules in humans versus dogs. In contrast, CEAR may be a comprehensive interspecies target for novel cancer therapeutics.

In the present study 125 cats with recurrent seizures were analysed. The main goal was to investigate the aetiology and compare primary epilepsy (PE) with secondary epilepsy (SE) regarding signalment, history, ictal pattern, clinical and neurological findings. Seizure aetiology was classified as PE in 47 (38%) and SE in 78 (62%) cats. SE was caused mainly by intracranial neoplasia (16), hippocampal necrosis (14), toxicosis (eight), and encephalitis (seven). A significant difference between PE and SE was found in: age, body weight, duration of seizure, occurrence of status epilepticus and neurological deficits. Status epilepticus, altered interictal neurological status and seizure onset over the age of 7 years indicated SE more frequently than PE. If the seizures occurred during resting conditions and rapid running occurred the aetiology was more likely to be PE than SE., (Copyright © 2010 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.)

Lyme arthritis in dogs can be induced under experimental and natural conditions. However, the veterinary relevance of canine borreliosis is still under extensive investigation. The prevalence of symptoms is clearly low, although the risk of tick exposure is high. Current research focuses on case definitions, methods for diagnosing clinical disease in dogs, and discrimination between an immune response to a natural infection and an immune response to vaccination. In this experimental study, 23 dogs raised under tick-free conditions were allocated to two groups. The 11 dogs in the first group were vaccinated with a commercial borrelia vaccine and subsequently developed detectable antibody titers. The 12 dogs in the second group were walked on two consecutive days in an area where ticks were endemic. On day 5 after exposure, engorged ticks were removed from the 12 dogs and were analyzed for Borrelia DNA by a real-time PCR assay. Blood samples were taken before exposure/vaccination and at defined time points thereafter. Antibody responses were evaluated using an immunofluorescence antibody test (IFAT) and Western blotting. Seven dogs from which Borrelia-positive ticks were removed seroconverted and developed individual immune responses. Blood and urine samples taken from the tick-exposed group at weeks 1 and 3 for real-time PCR analysis and culture were always negative for bacterial DNA. In conclusion, despite serological evidence of infection/immunization, no clinical signs of disease were observed. The antibody patterns in a single Western blot did not permit differentiation between the different antigen sources (vaccine versus natural infection). However, repeated Western blot analyses may be useful for the confirmation of infection or vaccination status, since the time courses of the levels of specific antibodies seem to be different.

Serial blood glucose measurements are currently regarded as the 'gold standard' for evaluating glycaemic control of canine diabetic patients. The aim of this study was to investigate a subcutaneous continuous glucose monitoring system based on microdialysis. Analyses were performed by taking interstitial glucose samples from two different anatomical regions (interscapular region [IR], thoracic region [TR]) in six healthy Beagle dogs during induced hypoglycaemia and hyperglycaemia, the feeding period and a period of stable glucose values. For comparison, plasma glucose concentration was measured simultaneously by a hexokinase-based automated chemistry analyser. The mean absolute differences ranged from 11.7 mg/dL (SD 23.5 mg/dL, TR) to 5.3 mg/dL (SD 19.2 mg/dL, IR) with a correlation of r=0.43-0.92 (P<0.01). Sensitivity for the detection of hypoglycaemia was 85.0% TR and 93.3% IR, and the specificity was 99.5% TR and 99.7% IR, respectively. In a modified Clarke error grid analysis, 99.3% TR and 99.7% IR of the values fell into zone A and B. Collapse of the microdialysis fibre was a recurrent problem although this was easily detectable by fluctuations in system pressure. The microdialysis system reflects physiological as well as induced variations in blood glucose in a valid manner., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Canine osteosarcoma, an aggressive cancer with early distant metastasis, shows still despite good chemotherapy protocols poor long term survival. The aim of our study was to determine whether sorafenib, a novel multikinase inhibitor, has any effect on D-17 canine osteosarcoma cells. A cell proliferation kit was used for detecting surviving cells after treatment for 72 h with sorafenib or carboplatin or their combination. A significant decrease of neoplastic cells was observed after incubation with 0.5-16 microM sorafenib or with 80-640 microM carboplatin. Using immunocytochemistry for activated caspase 3 to evaluate apoptosis, we found significantly more positive cells in the sorafenib treated groups. Paradoxically, expression of the nuclear proliferation marker Ki-67 was also significantly higher in sorafenib treated cells. The drug sorafenib showed potent antitumour activity against D-17 canine osteosarcoma cells in vitro, suggesting a potential as a therapeutic tool in the treatment of bone cancer in dogs., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Acute myeloid leukemia (AML) in dogs is a rare disease with poor prognosis. In most subjects, palliative treatment or euthanasia is performed. A 3.5-year-old male castrated labrador with AML-M7, which was treated with induction polychemotherapy (8 cycles) using vincristine (0.5 mg/m(2)/cycle), daunorubicin (20 mg/m(2)/cycle), cytosine arabinoside (ARA-C, 100 mg/m(2)/cycle) and prednisolone (1 mg/kg/day) is reported. Treatment was well tolerated and complete remission was achieved. Postinduction chemotherapy consisted of ARA-C, daunorubicin and prednisolone. After 3, 5 and 18 months, the subject relapsed. Each relapse was treated with ARA-C (up to 1,000 mg/m(2)) and etoposide or daunorubicin. Again, no severe side-effects occurred and the disease was controlled, with 37 chemotherapy-cycles (ARA-C, 3 x 1,000 mg/m(2)/cycle), for 24 months. Based on a literature-search, this is the first report documenting a long-term response of canine AML, probably resulting from the high-dose ARA-C. Clinical trials using high-dose ARA-C are now required to confirm antileukemic efficacy in canine leukemias.

Cytolinker proteins stabilize cells mechanically, regulate cytoskeleton dynamics, and provide scaffolds for signaling molecules. For plectin, the prototype of these proteins, an unusual diversity of isoforms has been reported, which show distinct expression patterns, subcellular localizations, and functions. Plectin has been shown to have important functions in skin and muscle, but little is known about its role in neural cells. To address this issue, we generated two knock-out mouse lines, one which was selectively lacking plectin 1c (P1c), the major isoform expressed in neural cells, and another in which plectin was conditionally deleted in neuronal precursor cells. Using isoform-specific antibodies, we found P1c to be expressed late in development and to associate with postsynaptic dendrites of central nervous system neurons, motorneurons of spinal cord, sciatic nerve axons, and Schwann cells. Motor nerve conduction velocity was found significantly reduced in sciatic nerve from P1c-deficient as well as from conditional knock-out mice. This defect was traceable to an increased number of motor nerve fibers with small cross-sectional areas; the thicknesses of axons and of myelin sheaths were unaffected. This is the first report demonstrating an important role of plectin in a major nerve function.

Objective: To establish the radiosensitivity and effect of irradiation on vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) expression in the canine mastocytoma cell line C2., Sample Population: Canine mastocytoma cell line C2., Procedures: C2 cells were irradiated with single doses of 2, 4, 6, and 8 Gy. The 3-(4, 5-di-methyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and proliferation assays with (methyl-hydrogen 3) thymidine were used for radiosensitivity experiments. Expression of VEGFR was determined via flow cytometry and apoptotic rate via annexin assay. Human and canine VEGF ELISA kits were evaluated in crossover assay experiments, and the canine kit was used thereafter., Results: C2 cells secreted VEGF constitutively. Radiation did not induce a significant increase in VEGF secretion, regardless of radiation dose. Consistently, radiation did not up-regulate VEGFR. Cell survival rates decreased in a dose-dependent manner. The apoptotic cell fraction had a dose-dependent increase that reached its maximum 24 to 48 hours after radiation., Conclusions and Clinical Relevance: The C2 cell line was radiosensitive, and a fraction (up to 40%) of cells died via apoptosis in a dose-dependent manner. In response to radiation, C2 cells did not upregulate VEGF production or VEGFR. Further studies are needed to determine whether tumor control could be improved by combining radiotherapy with VEGFR inhibitors or apoptosis-modulating agents.

Objective: To describe the activity pattern of the vastus lateralis muscle in dogs at walk measured by surface electromyography (EMG) in relation to kinematics and kinetics of the pelvic limb., Study Design: Experimental., Animals: Malinois dogs (n=11)., Methods: Dogs (mean +/- SD age, 5.5 +/- 2.9 years; weight, 27.3 +/- 3.8 kg; shoulder height, 62.7 +/- 3.3 cm) walked on a treadmill system with integrated force plates, which allowed simultaneous analysis of kinematics, kinetics, and EMG data from all limbs. The maxima, minima, and their time of occurrence in the motion cycle of the EMG and the pelvic limb kinematics and kinetics were calculated; correlations between joint movement patterns, ground reaction forces (GRF), and activity pattern of the muscle group were investigated., Results: The vastus lateralis muscle had an activity pattern with 2 peaks and a close positive correlation with GRF. The 1st peak occurred in early stance, followed by a decrease in activity during mid-stance. The 2nd peak occurred directly before the quick activity decrease in late stance phase, reaching its minimum early in swing phase., Conclusions: These results suggest that the vastus lateralis muscle supports the vertical position and elevation of the pelvis during stance and push-off. During early stance, the muscle acts as a coantagonist to the hamstring muscle group and the gastrocnemius muscle, and restrains flexion during the late stance., Clinical Relevance: Results of this study could enhance diagnosis of locomotor system disorders and facilitate monitoring effects of treatments (e.g., therapeutic exercises) on gait ability and muscle function.

The Pax5 gene encodes the B-cell specific activator protein (BSAP), a member of the highly conserved paired box (PAX)-domain family of transcription factors and a key regulator in the development and differentiation of B-cells. Pax5 serves as a valuable B-cell marker in the classification of human lymphoma patients as it is restricted to lymphomas of B-cell lineage. In dogs, detection of Pax5 protein in lymphoma tissue has not been reported. Therefore, we have investigated the expression and detection of BSAP using a monoclonal anti-Pax5 antibody (anti-BSAP, clone 24) in canine lymphoma tissue samples to evaluate its diagnostic relevance as a B-cell marker. A series of 25 lymph nodes from 23 canine non-Hodgkin lymphoma patients, a reactive canine lymph node, and a normal non-reactive canine lymph node, were evaluated. All B-cell non-Hodgkin lymphomas (15) were found to express Pax5 protein. In addition, there was a strong correlation between Pax5 and CD79a expression. Three CD3 positive and five CD3 and CD79a positive lymphomas were immunophenotypically negative for anti-Pax5, indicating a T-cell lineage. In conclusion, anti-Pax5 antibody may offer an excellent B-cell marker in canine lymphomas.

In the present study, 240 cases of dogs with seizures were analysed retrospectively. The aim was to examine the underlying aetiology and to compare primary or idiopathic epilepsy (IE) with symptomatic epilepsy (SE) concerning signalment, history, ictal pattern, clinical and neurological findings. The diagnosis of symptomatic epilepsy was based on confirmed pathological changes in haematology, serum biochemistry, cerebrospinal fluid (CSF) analysis and morphological changes of the brain by CT/MRI or histopathological examination. Seizure aetiologies were classified as idiopathic epilepsy (IE, n = 115) and symptomatic epilepsy (SE, n = 125). Symptomatic epilepsy was mainly caused by intracranial neoplasia (39) and encephalitis (23). The following variables showed significant difference between the IE and SE group: age, body weight, presence of partial seizures, cluster seizures, status epilepticus, ictal vocalisation and neurological deficits. In 48% of the cases, seizures were found to be due to IE, while 16% were due to intracranial neoplasia and 10% to encephalitis. Status epilepticus, cluster seizures, partial seizures, vocalisation during seizure and impaired neurological status were more readily seen with symptomatic epilepsy. If the first seizure occurred between one and five years of age or the seizures occurred during resting condition, the diagnosis was more likely IE than SE.

Microvessel density is a frequently used parameter of angiogenesis, which is a complex multistep process necessary for tumor progression. The aim of this study was to compare the microvessel density of normal lymph node biopsies with those diagnosed with lymphoma in dogs. Furthermore, we sought to determine if there was any correlation between microvessel density and vascular endothelial growth factor expression in canine lymphoma, representing a potential target for anti-angiogenic therapy. Combined immunohistochemistry (von Willebrand factor) and lectin histochemistry was used to highlight microvessels in 40 untreated canine lymphomas and 14 normal lymph nodes. To evaluate microvessel density, the number of profiles of blood vessels per unit area was calculated. Fifty image fields (a total area of 5.68 mm(2)) were sampled for each specimen in a systematic random, way. We found a significant difference between the microvessel densities (MVD) of normal and neoplastic lymph nodes (177+/-35 versus 241+/-72 microvessel profiles/mm(2)). Classifying lymphoma samples according to the working formulation and the Kiel classification system revealed no significant differences in MVD between different grade malignancies. Immunohistochemical demonstration of the proangiogenic protein vascular endothelial growth factor showed expression in 60% of canine lymphomas, although there was no correlation between microvessel density and vascular endothelial growth factor expression. As an increase in tumor angiogenesis was observed in lymphoma samples compared to normal canine lymph node tissue, additional anti-angiogenic therapy, besides conventional chemotherapy as a lymphoma treatment may be effective. The optimal target among many pro-angiogenic factors has yet to be elucidated.

Desmopressin is a synthetic analogue of the hypothalamic peptide vasopressin and binds to specific pituitary vasopressin (V3) receptors. The V3-receptor is overexpressed in pituitary corticotrope tumors and the injection of desmopressin induces a marked ACTH and cortisol release in human patients with pituitary- (PDH), but not adrenal tumor (AT) dependent hyperadrenocorticism. In this prospective study, we investigated the effects of desmopressin on serum cortisol levels in 80 dogs suspected of Cushing's syndrome. The aim was to find a sensitive and specific test to exclude AT. According to standard tests the dogs were divided into 3 groups (group 1=other disease, n=27; group 2=PDH, n=46; group 3=AT, n=7). Desmopressin was injected as an i.v. bolus of 4microg and serial blood samples were collected before and after 30, 60 and 90min. Desmopressin significantly stimulated cortisol release in dogs with PDH (median 51%, range -24 to 563%; p<0.0001), whereas no increase was seen in dogs with AT (median -12%, range -44 to 5%; p=0.063) and in controls (median +7%, range -36 to 196%; p=0.131). Using a cut off value of 10% increase over baseline, it was possible to exclude AT in 75% of patients. The results of this study suggest that the desmopressin test could be a useful tool in differentiating pituitary from adrenal dependent Cushing's syndromes. Additional dogs with adrenocortical tumor must be tested in order to recommend its use in clinical practice.

Infections with Encephalitozoon cuniculi in rabbits are observed at increasing frequency and are known as opportunistic infections in immunocompromised humans. 191 pet rabbits with suspected encephalitozoonosis, presented at the Animal Hospital of the Veterinary University of Vienna (Austria), were included in this study. Rabbits were serologically examined for antibodies against E. cuniculi (144 positive out of 184 rabbits with suspected encephalitozoonosis compared to 14 positive out of 40 clinically healthy rabbits tested as part of a standard health check) and Toxoplasma gondii (8 positive out of 157). Of the 144 seropositive rabbits with clinical signs, 75% showed neurological symptoms, 14.6% demonstrated phacoclastic uveitis and 3.5% suffered from renal failure. 6.9% of the animals had combined symptoms. Vestibular disease dominated within the rabbits that showed neurological symptoms. Polymerase chain reaction (PCR) could not detect parasite DNA in urine or cerebrospinal fluid (CSF), but did so in 4 out of 5 samples of liquefied lens material in cases with phacoclastic uveitis due to lens capsule rupture. Additionally further diagnostic procedures, such as inspection of the external ear canal (N=69), radiography of the tympanic bullae (N=65) were performed to rule out differential diagnosis. 54.2% of the patients exhibiting neurological symptoms recovered within a few days, while 87.5% of the rabbits suffering from renal failure died or had to be euthanized.

Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation and has a pivotal role in tumour angiogenesis. The expression of VEGF and its receptors VEGFR-1 and VEGFR-2 was examined immunohistochemically in 43 specimens of canine lymphoma and in six normal lymph nodes. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect VEGF protein and mRNA, respectively. VEGF protein was expressed by 60% of the tumours with diffuse cytoplasmic labelling of the neoplastic cells. Endothelial cells, macrophages and plasma cells were also immunolabelled. VEGFR-1 was expressed by variable numbers of neoplastic cells in 54% of lymphoma specimens. VEGFR-1 was also expressed by macrophages, plasma cells, reticulum cells, and vascular endothelial cells. Macrophages and lymphocytes in germinal centres of normal lymph nodes were also immunoreactive with anti-VEGF and VEGFR-1. Most tumours did not express VEGFR-2 but in 7% of sections there was focal labelling of neoplastic and endothelial cells, with a cytoplasmic and perinuclear pattern. The observed variability in expression of VEGF and its receptors probably relates to the fact that lymphoma is a heterogeneous lymphoproliferative tumour. Individual differences in VEGF and VEGFR expression must be taken into account when VEGF and VEGFR-targeted approaches for anti-angiogenic therapy are considered in dogs.

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and a potential autocrine growth factor for neoplastic cells in various malignancies. In the present study, we have investigated expression of VEGF and VEGF receptors in canine mastocytomas and the canine mastocytoma cell line C2. As assessed by immunostaining of tissue sections and cytospin slides, primary neoplastic mast cells (MC) and C2 cells were found to express the VEGF protein. In Northern blot and RT-PCR experiments, C2 cells expressed VEGF mRNA in a constitutive manner. VEGF mRNA expression in C2 cells was counteracted by LY294002 and rapamycin, suggesting involvement of the PI3-kinase/mTOR pathway. Moreover, C2 cells were found to express VEGF receptor-1 (Flt-1) and VEGF receptor-2 (KDR). However, recombinant VEGF failed to promote (3)H-thymidine uptake in C2 cells, and a neutralizing anti-VEGF antibody (bevacizumab) failed to downregulate spontaneous proliferation in these cells. In addition, rapamycin decreased the expression of VEGF in C2 cells at the mRNA and protein level without suppressing their proliferation. Together, canine mastocytoma cells express VEGF as well as VEGF receptors. However, despite co-expression of VEGF and its receptors, VEGF is not utilized as an autocrine growth regulator by canine mastocytoma cells.

Avian influenza A virus subtype H5N1 was transmitted to domestic cats by close contact with infected birds. Virus-specific nucleic acids were detected in pharyngeal swabs from 3 of 40 randomly sampled cats from a group of 194 animals (day 8 after contact with an infected swan). All cats were transferred to a quarantine station and monitored for clinical signs, virus shedding, and antibody production until day 50. Despite unfamiliar handling, social distress, and the presence of other viral and nonviral pathogens that caused illness and poor health and compromised the immune systems, clinical signs of influenza did not develop in any of the cats. There was no evidence of horizontal transmission to other cats because antibodies against H5N1 virus developed in only 2 cats.

A decisive role in cancer development has been attributed to cyclooxygenase-2 (COX-2) activity, but the significance of COX-2 inhibitors in cancer treatment still needs to be thoroughly investigated. We studied the influence of meloxicam, a non-steroidal antiinflammatory drug with preferential inhibitory effects on COX-2 compared to COX-1, on canine osteosarcoma (D-17) cells. We demonstrated that D-17 cells expressed mRNA and COX-2 protein. Treatment with meloxicam induced a time- and dose-dependent inhibition of cellular growth. To determine if apoptosis plays a role in meloxicam-induced cell death, we performed agarose gel electrophoresis and found a DNA-ladder pattern, typically seen in apoptosis, as well as early apoptotic changes by Annexin V tests. Furthermore, electron microscopy revealed ultrastructural alterations typical of apoptosis. Quantification of apoptotic cells by immunohistochemical staining of caspase 3 confirmed the results. However, further studies with meloxicam are necessary to assess its potential use for treatment of osteosarcomas in dogs.

Cyclooxygenase (COX) inhibitors, already widely used to reduce fever, inflammation and pain, are under increasing consideration as potential agents for the prevention and treatment of neoplasia. As COX-2 was detected in human and canine osteosarcomas, we have evaluated the effect of the preferential COX-2 inhibitor meloxicam on an established D-17 canine osteosarcoma cell line, which expressed, as well as COX-1 and COX-2 also COX-3 (as demonstrated by Western blot). An XTT proliferation kit was used to assess surviving cells after drug treatment. At low concentrations (1, 2, 4 and 10 microm) meloxicam caused an increase in cell numbers while a marked anti-proliferative effect was observed at higher concentrations (100, 200 microm) after 3 days and also 3 weeks of incubation. The chemotherapeutic drug doxorubicin showed a cytotoxic effect at all concentrations (60-1920 nm). Exposure of tumour cells to combinations of meloxicam and doxorubicin revealed synergistic effects (with 240 nm doxorubicin), as well as sub-additive and antagonistic results, especially if combined with concentrations of meloxicam typically found in serum. Care should be taken in concluding, on the basis of one in vitro study, that meloxicam does not have a role in the treatment of canine osteosarcomas given that the results from in vivo studies may differ.

Tick-borne encephalitis (TBE) is caused by a Flavivirus and transmitted by ticks. It is known in dogs for nearly 30 years and the number of TBE cases is increasing. In addition to fever, cerebrocortical, thalamic, and brainstem symptoms occur simultaneously. Not all TBE infections in dogs lead to clinical signs but peracute/lethal as well as subacute and chronic courses have been reported. TBE is a seasonal disease, depending on climate related tick activity. Infected ticks are spreading the virus over central Europe with a tendency to expand to new endemic areas in western Europe.

Background: Adding sodium bicarbonate to lidocaine to enhance its efficacy during peripheral nerve block is controversial. The authors studied the effect of adding sodium bicarbonate to lidocaine with and without epinephrine versus equivalent alkalinization by sodium hydroxide (NaOH) on onset, degree, and duration of peripheral nerve block., Methods: Part I examined alkalinization by sodium bicarbonate versus NaOH to pH 7.8 on 0.5% lidocaine, with and without epinephrine (1:100,000), prepared from crystalline salt. Part II examined 0.5% and 1.0% commercial lidocaine solutions, with and without epinephrine, either unalkalinized or alkalinized with sodium bicarbonate or NaOH. With NaOH, pH was adjusted to 7.8, but with sodium bicarbonate, no pH adjustments were made to simulate clinical conditions., Results: In part I, addition of either NaOH or sodium bicarbonate to 0.5% lidocaine without epinephrine produced a faster onset than did unalkalinized lidocaine, without effecting degree or duration of block. In solutions with epinephrine there were no differences in onset, degree, or duration between lidocaine alkalinized with sodium bicarbonate versus NaOH. In part II, addition of sodium bicarbonate or NaOH to 1.0% commercial lidocaine without epinephrine did not accelerate onset compared with the unalkalinized solution. However, adding sodium bicarbonate decreased the degree and duration of block by 25% and more than 50%, respectively, compared with lidocaine unalkalinized and alkalinized with NaOH. With epinephrine, sodium bicarbonate hastened onset without effecting degree and duration compared with the unalkalinized solution., Conclusions: With 1% commercial lidocaine without epinephrine, sodium bicarbonate decreases the degree and duration of block. However, in solutions with epinephrine, sodium bicarbonate hastens onset, without effecting degree or duration.

Background: N-butyl tetracaine has local anesthetic and neurolytic properties. An injection of this drug at the rat sciatic notch produces rapid onset and nerve impairment lasting > 1 week. This study aimed to elucidate the structure-activity relation of various tetracaine derivatives to design better neurolytic agents., Methods: N-alkyl tetracaine salts (n = 2-6) were synthesized, and their ability to elicit sciatic nerve impairment of sensory and motor functions in vivo was tested in rats. A single dose (0.1 ml at 37 mM) was administered close to the sciatic nerve at the sciatic notch. Regeneration was assessed morphologically in transverse sections of treated nerves. Finally, the drug potency in blocking Na+ currents was studied under voltage-clamp conditions., Results: N-ethyl and N-propyl tetracaine derivatives were non-neurolytic and elicited complete sciatic nerve block lasting 3-7 h. In contrast, N-butyl, N-pentyl, and N-hexyl tetracaine derivatives were strong neurolytic agents and elicited functional impairment of sciatic nerve for > 1 week. All derivatives were strong Na+ channel blockers, more potent than tetracaine if applied intracellularly. External drug application showed marked differences in their wash-in rate: tetracaine > N-hexyl > N-butyl > N-ethyl tetracaine. All derivatives were trapped within the cytoplasm and showed little washout within 7 min., Conclusions: When n-alkylation is 4-6, n-alkyl tetracaine appeared as a strong neurolytic agent. Neurolytic derivatives retained their local anesthetic activity and elicited rapid onset of nerve block after injection. Such derivatives are potential local anesthetic-neurolytic dual agents for chemical lesions of the sciatic nerve.

Mechanosensitive A beta-fibers (n = 29) and nociceptive A delta- (n = 6) and C-fibers (n = 10) of the rat sciatic nerve were superfused with lidocaine (LID, 0.1-1.4 mM) in vivo. The [LID] to abolish single electrically stimulated impulses (tonic blockade) in axons was 0.2 to 0.8 mM for A beta-, 0.1 to 0.6 mM for A delta- and 0.1 to 1.4 mM for C-fibers. Within each of the fiber groups there was no dependence of blocking [LID] on conduction velocity; slower fibers were no more susceptible than faster ones. Mean blocking concentrations differed between groups, with C-fibers having an IC50 = 0.80 +/- 0.32 mM (+/- S.E.), significantly higher (P < .05, ANOVA) than A beta-fibers (IC50 = 0.41 +/- 0.15 mM) and A delta-fibers (IC50 = 0.32 +/- 0.18 mM). The [LID] causing 50% impulse failure in A beta-fibers during a 200-Hz, 10-stimulus train (phasic blockade) ranged from 0.2 mM to 0.7 mM; the mean IC50 equaled 0.28 mM (n = 17). Stimulation of nociceptive A delta-fibers (n = 4) and C-fibers (n = 5) at 5 or 10 Hz for 10 pulses produced no phasic block at [LID]s (0.1-0.5 mM) below those required for tonic blockade. Uptake of 14C-lidocaine by the nerve, measured in vivo under conditions identical with those for electrophysiology, showed that: a) little drug was in the segments of nerve beyond the superfusion chamber, b) lidocaine was uniformly distributed in the nerve within the chamber, c) the intraneural lidocaine content was identical with that in nerves equilibrated in vitro. The results show a lack of monotonic dependence of sensitivity to local anesthetic on fiber diameter, but do suggest that mean susceptibility to nerve block by lidocaine differs for fibers grouped by, and perhaps according to, function.

Background and Objectives: Pregnant patients need less local anesthetic in order to obtain the same quality of functional block as nonpregnant patients. Our goal was to demonstrate a similarly increased functional susceptibility to local anesthetics in the awake pregnant rat during peripheral nerve block and to investigate the pharmacokinetic and/or pharmacodynamic mechanisms responsible for this phenomenon., Methods: Radiolabeled lidocaine uptake was determined in vivo during sciatic nerve block with 0.1 ml of 1% lidocaine in the nerves of nine pregnant and five nonpregnant female rats and six male rats at the return of deep pain sensation, assessed by withdrawal of the hindlimb from a brief squeeze of a digit with serrated forceps. During recovery from complete functional block, the time at which deep pain returned and the amount of lidocaine in the nerve at that time were compared among the three groups of rats. Lidocaine content was also determined in vitro after exposure of ensheathed sciatic nerves from pregnant and nonpregnant rats to a 0.2% lidocaine bath for specified times., Results: Full block of function developed in all groups within 6 minutes of the lidocaine injection and lasted significantly longer in pregnant rats than in nonpregnant and male rats (49.0 +/- 3.3 vs 34.0 +/- 3.1 and 32.0 +/- 1.3 minutes mean +/- SEMI, respectively. At the time of deep pain return, the intraneural lidocaine content of pregnant rats was significantly lower than that of nonpregnant and male rats (2.2 +/- 0.25 vs 3.9 +/- 0.7 and 3.7 +/- 0.6 nmoles/mg of wet nerve, respectively). No difference in lidocaine uptake kinetics between P and NP nerves was observed in vitro., Conclusions: Block of peripheral neural function is prolonged in pregnant rats, and lidocaine content in the nerve is lower at a specific stage of neural block. These results are consistent with a pharmacodynamic mechanism for increased susceptibility to lidocaine neural block during pregnancy.

Background: The quest for a drug that would provide analgesia with minimal motor deficiency, through the selective inhibition of impulses in small-diameter fibers, was brightened by a previous report of veratridine's C-fiber-selective actions on the isolated rabbit vagus nerve. The goal of the present research was to demonstrate the same actions on rat sciatic nerve in vitro and to observe the functionally differential blockade in the rat in vivo., Methods: Sciatic nerves were removed from rats, mounted in a recording chamber, wherein a 1-cm length of the ensheathed nerve was superfused with the plant alkaloid veratridine (2 microM) in bicarbonate-buffered Liley's solution, and the compound action potential (CAP) was stimulated supramaximally to give A- and C-fiber elevations. Onset, steady-state, and recovery from veratridine effects were assayed for a range of stimulus frequencies. Open-field behavior and quantitative neurological assessments of proprioception, motor function, and nociception were tested in 15 trained rats after injection near the sciatic nerve of 0.1 ml veratridine at 0.5, 0.7, and 1.0 mM each plus epinephrine (1:200,000)., Results: Veratridine inhibited the C-fiber component of the CAP in a frequency-dependent manner. At 0.1 Hz the CAP was 65% of the control amplitude, 50% at 0.5 Hz, and 40% at 5 Hz. A-fiber elevations were unattenuated at stimulus frequencies as high as 50 Hz. Steady-state inhibition was reached 5 min after drug administration, and recovery from the effects was 30% complete by 15 min of drug washout. Proprioception, measured as a "hopping" or "placing" reaction, was inhibited dose dependently by maximum degree and for durations of, respectively, 0.5 mM, 61%, 180 min; 0.7 mM, 100%, 360 min; and 1 mM, 100%, 420 min. Extensor postural thrust, as a measure of motor function, was inhibited by and for 0.5 mM, 77%, 240 min; 0.7 mM, 99%, 390 min; and 1 mM, 100%, 420 min. Analgesia, as a prolonged withdrawal latency to a noxious thermal stimulus, had the following profile: 0.5 mM, 10%, 30 min; 0.7 mM, 52%, 150 min; and 1 mM, 66%, 150 min., Conclusions: Despite the fact that veratridine gave a C-fiber preferential blockade in the isolated sciatic nerve, heightened analgesia over motor block was not achieved in vivo. Indeed, just the opposite occurred. If preferential C-fiber blockade also occurs in vivo, then its traditionally expected correlation with analgesia must be re-examined.

Background: Neurolytic agents such as phenol (5% to 10%) and absolute alcohol have long been used clinically to destroy the pathogenic nerve regions that manifest pain. Both phenol and alcohol are highly destructive to nerve fibers. However, these agents exert only weak local anesthetic effects and therefore are difficult to administer to alert patients without pain. This report describes a tetracaine derivative that displays both local anesthetic and neurolytic properties. Studies with such a compound may lead to the design of neurolytic agents that are more effective and more easily administered than phenol and alcohol., Methods: A tetracaine derivative, N-butyl tetracaine quaternary ammonium bromide, was synthesized, and its ability to elicit sciatic nerve block of sensory and motor functions in vivo was tested in rats. A single dose of 0.1 ml N-butyl tetracaine at 37 mM was injected into the sciatic notch. Transverse sections of treated sciatic nerves were subsequently examined to determine the neurolytic effect of this drug. Finally, the local anesthetic properties of N-butyl tetracaine were studied in vitro; both tonic inhibition and use-dependent inhibition of Na+ currents in neuronal GH3 cells were characterized under whole-cell voltage-clamp conditions., Results: N-butyl tetracaine at 37 mM (equivalent to 1.11% tetracaine-hydrochloric acid concentration) elicited prolonged sciatic nerve block of the withdrawal response to noxious pinch in rats for more than 2 weeks. The withdrawal response was fully restored after 9 weeks. Parallel to sensory block, motor functions of the hind legs were similarly blocked by this drug. Morphologic examinations 3 and 5 weeks after a single injection of drug revealed degeneration of many sciatic nerve fibers, consistent with the results of functional tests. Finally, N-butyl tetracaine was found to be a potent Na+ channel blocker in vitro. It produced strong tonic and use-dependent inhibition of Na+ currents with a potency comparable to that of tetracaine., Conclusions: A single injection of N-butyl tetracaine produces ultralong sciatic nerve block in rats. This compound possesses both local anesthetic and neurolytic properties and may prove useful as a neurolytic agent in pain management.

Impulse activity in axons generates aftereffects on membrane excitability that can alter the conduction velocity of subsequently conducted impulses. We used a computerized stimulus pattern (a 1 Hz stimulus period followed by a period of repeated short bursts at 200 Hz) to assess in vivo activity-dependent changes in conduction latency of functionally identified rat cutaneous afferents conducting in the A beta range. Several different parameters of activity dependence were measured: burst supernormality, the average increase in conduction latency following conditioning with a single preceding impulse during high frequency burst stimulation; burst subnormality, the average latency increase during each burst; depression, a long-term increase in latency caused by the high frequency stimulation. The data show that different mechanosensitive A beta afferents with overlapping resting conduction velocities exhibit activity-dependent changes in conduction latency that are characteristic of their particular functions.

Background: Use of long-acting local anesthetics that elicit complete neural blockade for more than 3 h often is desirable in pain management. Unfortunately, clinically available local anesthetics are in general not suitable for prolonged analgesia. This report describes the organic synthesis and functional testing of a lidocaine derivative that appears to fulfill the criteria of long-acting local anesthetics., Methods: A lidocaine derivative, N-beta-phenylethyl lidocaine quaternary ammonium bromide, was synthesized, and its ability to inhibit Na+ currents in cultured rat neuronal GH3 cells was tested in vitro under whole-cell voltage clamp conditions. Neurologic evaluation of sciatic nerve block of sensory and motor functions in vivo was subsequently performed in rats., Results: N-beta-phenylethyl lidocaine was found to be a potent Na+ channel blocker in vitro. It produced both tonic and use-dependent blocks of Na+ currents that exceeded lidocaine's effects by a factor of > 2 (P < 0.05). In vivo, N-beta-phenylethyl lidocaine elicited a prolonged and complete sciatic nerve block of the motor function and the withdrawal response to noxious pinch that was 3.6- and 9.3-fold longer than that of lidocaine (P < 0.001), respectively., Conclusions: In an attempt to elicit prolonged local anesthesia, a quaternary ammonium derivative of lidocaine containing a permanent charge and an additional hydrophobic component was synthesized. Complete sciatic neural blockade of more than 3 h was achieved with this derivative. Of note, sensory blockade was prolonged to a greater extent than motor blockade. The approach used in this study may prove useful for developing new drugs applicable in pain management.

Background: During peripheral nerve block, local anesthetic (LA) penetrates within and along the nerve to produce the observed functional deficits. Although much is known about the kinetics and steady-state relation for LA inhibition of impulse activity in vitro in isolated nerve, little is known about the relation between functional loss and intraneural LA content in vivo. This study was undertaken to investigate the relation of functional change to intraneural LA., Methods: A sciatic nerve block was performed in rats with 0.1 ml 1% lidocaine radiolabeled with 14C. The total intraneural uptake of LA was determined at different times after injection, and the distribution of lidocaine along the nerve was assayed at different stages of functional block. Drug content was also compared with equilibrium lidocaine uptake in the isolated rat sciatic nerve., Results: Total intraneural lidocaine in vivo increased to near steady-state in about 3 min, stabilizing at approximately 14.3 nmol/mg wet tissue for about 12 min before decreasing to zero at 70 min after injection. Although intraneural lidocaine was 1.6% of the injected dose during full block, only 0.3% was left when deep pain sensation returned and 0.065% was still detected when functions fully recovered. Despite these large differences in total lidocaine content, the longitudinal distribution remained constant. Intraneural lidocaine concentrations obtained at full block and partial recovery could be achieved in vitro by equilibration in 0.7-0.9 and 0.2-0.3 mM lidocaine, respectively., Conclusions: During peripheral nerve block only a small amount of injected LA penetrates into the nerve. The intraneural content of LA correlates with the depth of functional block.

The non-obese diabetic (NOD) mouse, a model of Type 1 diabetes in humans, has proven useful for the study of genetic, immunologic and epidemiologic aspects of inherited diabetes. Behavioral evidence of hyperalgesia may also be present in the NOD mouse but has not been described. This study examined NOD mice with (NOD+) and without (NOD-) insulin-dependent diabetes, and control strain (ILI) mice for evidence of hyperalgesia to a noxious thermal stimulus. Interestingly, both NOD+ and NOD- mice showed reduced mean hindpaw withdrawal latencies when compared with non-diabetic ILI mice. NOD+ and NOD- mice were also abnormal in their general appearance, activity level, posture, gait and muscle bulk when compared with ILI mice. These findings raise the possibility that hyperalgesia in insulin-dependent NOD mice, or insulin-dependent humans with Type 1 diabetes, may be independent of diabetes and due to a primary disturbance within sensory pathways.

Background: Quantitative behavioral testing is necessary to establish a reproducible measure of differential functional blockade during regional anesthesia. Methods for assessment of the neurologic status (mental status, posture, gait, proprioception, motor function, autonomic function, and nociception) in veterinary neurology were adapted for the rat and used to monitor functional changes separately during a sciatic nerve block., Methods: Sprague-Dawley rats were acclimated to laboratory routine before the study so that lidocaine (0.1 ml, 1%) could be injected near the sciatic notch without any chemical restraint. The onset, duration, and magnitude of functional losses were monitored. Proprioceptive integrity was evaluated by assessing the response to tactile placing and the hopping response. Extensor postural thrust, a test for postural reactions in small animals, was assessed on a digital balance and found adequate for quantifying motor function. Analgesia was assessed by measuring withdrawal response latencies to noxious thermal stimulation (51 degrees C) and to superficial and deep noxious pinches. Autonomic function was monitored by measuring skin temperature. Contralateral limb function was used as an internal control, and injection of saline was used as an external control in separate, control animals., Results: Onset of postural and gait abnormalities were observed as early as 40 s after injection. On each occasion proprioceptive impairment was detected first, followed by impairment of motor function and nociception. Complete absence of proprioception occurred from 10 to 30 min (n = 9) and of motor function at 30 min after injection (n = 10); both functions were fully recovered by 120 min. A unilateral increase in skin temperature on the foot was detected by 1 min; had reached its maximum change, 5.3 +/- 0.7 degrees C, at 10 min; and had returned to control levels at 60 min after injection (n = 12). Withdrawal response to cutaneous or superficial pain was absent in all ten animals from 5 to 30 min whereas the response to deep pain was absent in all ten animals at 20 min only. The response to noxious stimulation recovered at 90 min. Attention was paid to the temporal relation of the impairment of various functions., Conclusions: Quantitative observations of the onset, offset, and intensity of differential functional impairment or block over time will make it possible to establish the doses and conditions for local anesthetics that result in differential nerve block and will permit comparison of these changes among different drugs and "clinical" protocols.

Cutaneous afferents exhibit changes in excitability after impulse activity that are correlated with functional modality but are independent of axonal diameter, as studied in 39 cold fibers and 51 nociceptors of the rat. Latency of conducted impulses was used to indicate changes in axonal excitability caused by electrical stimulation. Stimuli were applied both at fixed frequencies and at the time intervals of impulses previously recorded during response to natural stimulation. Latency increased following both these forms of electrical stimulation, as well as after natural stimulation of the receptive fields. The latency increase was correlated with the number of impulses and the frequency of the preceding discharge in all of 4 nociceptors and 13 cold fibers studied for this feature. Increase of latency by electrical or natural stimulation led to reduced responsiveness to natural stimulation. The magnitude and time course of latency changes were correlated with fiber modality. In 32 nociceptors the latency increased continuously with time during a stimulus train, whereas in 21 cold fibers there was only an initial increase in latency over the first few seconds, after which the latency remained at a plateau even as the firing response continued. Paralleling this slowing, impulse failure occurred more frequently during repetitive stimulation in both A delta and C nociceptors than in velocity-matched cold fibers of either class. Based on the magnitude of latency increases during stimulus trains at different frequencies, two distinct patterns were discerned in A nociceptors: "Type II" fibers slowed significantly more than "Type I" or cold fibers. The results support the hypotheses (1) that the pattern of latency changes during activity are signatures for the modality in a given fiber; and (2) that endogenous, activity-dependent processes of the axon contribute to adaptation and encoding in cutaneous sensory afferents.

Ethanol is well known to enhance the potencies of a range of anesthetics. However, its effects on steroid anesthesia have not been reported. Because ethanol often is added to the delivery vehicle to increase steroid solubility, it is important to evaluate its contribution to steroid anesthesia. Intravenous injection of ethanol and anesthetic steroid(s) produced marked analgesia and a marked increase in the number of mice exhibiting the loss of righting response (LRR). Ethanol alone at these low doses was merely sedative, and steroid alone was hypnotic without any analgesic benefit. The nocifensive response to tail clamp was used as a measure of analgesia. Progesterone or the anesthetic steroid, 3 alpha-hydroxy-5 alpha- pregnan-20-one (3 alpha) alone reduced, but did not abolish the nocifensive response. Progesterone (80 mg/kg) or 3 alpha (3 mg/kg) administered together with ethanol (1.1 g/kg) abolished the nocifensive response (analgesia) and resulted in LRR within 10 s. The onset of both effects was rapid, and the duration was greater than 8 min. With 3 alpha alone, the reduced nocifensive behavior fully returned before the mouse regained the righting response. Thus, the evaluation of the nocifensive response is not precluded by LRR. The rapid onset of LRR and analgesia suggest that 3 alpha and progesterone, not their metabolites, are responsible for the observed activities.