Marcin Nowak

The carbon dioxide laser: an alternative surgery technique for the treatment of common cutaneous tumors in dogs

BackgroundTumors of the skin and subcutaneous tissue are the largest group of canine neoplasms. Total excision is still the most effective method for treatment of these skin tumors. For its universal properties the carbon dioxide (CO2) laser appears to be an excellent surgical instrument in veterinary surgery. Laser techniques are alternatives to traditional methods for the surgical management of tumors. The aim of this study was to compare various types of laser techniques in skin oncologic surgery: excision, ablation and mixed technique and to suggest which technique of CO2 laser procedure is the most useful in particular case of tumors in dogs.FindingsThe study was performed on 38 privately-owned dogs with total number of 40 skin tumors of different type removed by various CO2 laser operation techniques from 2010–2013. The treatment effect was based on the surgical wound evaluation, the relative time of healing and possible local recurrence of the tumor after 3 months post surgery. Local recurrence was observed in two cases. The study showed that in 30 cases time needed for complete resection of lesions was less than 10 minutes. Time of healing was longer than 12 days in 6 cases (42.8%) with tumor excision and in 14 cases (87.5%) where excision with ablation technique was performed.ConclusionsThe advantages of the CO2 laser surgery were better hemostasis, precision of working, non-contact dissection, less instruments at the site of operation and minimum traumatization of the surrounding tissues.

Ozonotherapy in an Induced Septic Shock. I. Effect of Ozonotherapy on Rat Organs in Evaluation of Free Radical Reactions and Selected Enzymatic Systems

The confirmed advantageous effects of oxygen/ozone therapy in several clinical conditions stimulated experimental studies on effects of the therapy in rats with an induced septic shock. The studies were conducted on adult male rats of Wistar strain. Four groups of the animals, each of 15 rats, included: I—control group, (C); II—animals intraperitoneally administered with O2/O3 (CO), III—rats given of Escherichia coli endotoxin (lipopolysaccharide—LPS) (CL), IV—rats administered with the lipopolysaccharide plus administered with the oxygen/ozone mixture (OL). Activities of catalase and superoxide dismutase and of free radical reactions were estimated. The exposure to LPS augmented activities of SOD and of catalase in liver, lungs and heart. In all the examined organs LPS induced significant changes in levels of free radicals. Except of the lungs, parallel administration of the rats with LPS and ozone/oxygen revoked development of the alterations. The obtained results point to a strong, stabilizing and regenerative effect of ozonotherapy.

alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA) appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay), apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy) were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

Early morphological and functional alterations in canine hepatocytes due to α-amanitin, a major toxin of Amanita phalloides.

The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and death angel (A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of α-amanitin (α-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of α-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure.

T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo

Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo.

Comparison of the biopsy and cytobrush techniques for diagnosis of subclinical endometritis in mares

BackgroundEndometritis is a major cause of infertility in the mare. Therefore, the diagnosis of this disease is very important in veterinary practice. The objective of this study was to compare bacteriological and cytological results obtained from the mare uterus using biopsy (EB) and cytobrush (CB) techniques and relating these findings to the presence of polymorphonuclear cells (PMNs) in endometrial tissue as the gold standard for detection of endometritis. In particular, we tested the hypothesis that endometrial cytology and microbiology data obtained from material collected using the EB and CB techniques are similar, so that the CB technique could preferentially be used to detect subclinical endometritis in clinical practice.MethodsA total of 69 mares suspected of subclinical endometritis because of previous reproductive history and 15 maiden mares were enrolled in this study. Material collected from both EB and CB was smeared on sterile glass slides for cytological examinations and on culture media for microbiological examinations. Bacteriological cultures and cytological samples were classified as negative (no growth or mixed cultures of more than three microorganisms; <2% PMNs) or positive (pure growth of microorganisms; >2% PMNs) for endometritis.ResultsPositive growth was observed in 43% of CB samples and in 54% of EB samples (difference not significant). The growth of β-hemolytic streptococci was always connected with positive cytology. This relationship was not observed for growth of E. coli or for non-pathogenic flora. The sensitivity of bacterial growth and cytology from EB was 0.63 and 0.73 respectively. The sensitivities of bacterial growth and cytology from CB were 0.50 and 0.71 respectively.ConclusionMicrobiological and cytological results obtained from CB are similar to those obtained from EB and based on these findings the CB technique may be recommended for collection of materials from the mare’s uterus in clinical practice.

Cases with manifestation of chemodectoma diagnosed in dogs in Department of Internal Diseases with Horses, Dogs and Cats Clinic, Veterinary Medicine Faculty, University of Environmental and Life Sciences, Wroclaw, Poland

In the period of 3 years, 9 tumours of chemodectoma were supravitally diagnosed and histopathologically verified in dogs. In this period 15 351 dogs were admitted to the Clinic of Dogs and Cats and 2 145 dogs were examined in the cardiological outpatient clinic for dogs. This tumour is located in a typical place - at the base of the heart. Most frequently the tumour manifested in older boxers. Only in one case such a tumour was diagnosed in another breed of dogs. The tumours ranged in size between 3 and 16 cm in diameter. The principal sign accompanying tumours of cardiac base involved dyspnoea but in 3 cases the tumours yielded no clinical signs. All the diagnoses were additionally verified using immunohistochemical examination. We used antibodies to chromogranin A (clone DAK-A3 1:100), synaptophysin (clone SY38 1:20) and neuron-specific enolase (clone BBS/NC/VI-H14 1:150). An immunohistochemical examination is vital for the diagnosis since it allows to differentiate histologically distinct types of neoplasia which may locate in the same site and may manifest a similar histological pattern.

Immunohistochemical localization of somatostatin and pancreatic polypeptide in smokers with chronic pancreatitis

Recent studies have demonstrated a significant role of tobacco smoking in the development of chronic pancreatitis. Although there are published papers on the effects of cigarette smoking on insulin secretion in patients, no data are available on the effects of smoking on pancreatic endocrine cells secreting somatostatin and pancreatic polypeptide. The aim of the study was to evaluate the effects of cigarette smoking on endocrine pancreatic function by immunolocalization of somatostatin and pancreatic polypeptide in the pancreas from smokers and non-smoking patients with chronic pancreatitis in comparison with healthy controls. The LSAB2-HRP technique with polyclonal antibodies was used for the immunolocalization of somatostatin and pancreatic polypeptide in histological preparations of the pancreas. The intensity of immunohistochemical reaction was calculated with digital image analysis. The study demonstrated increased numbers of somatostatin (D) secreting cells and pancreatic polypeptide (PP) cells and their altered location in pancreatic islets and parenchyma of smoking patients with chronic pancreatitis, as compared to non-smoking patients and healthy controls. Smoking patients showed significantly higher immunostaining of the hormones in the pancreas compared to non-smoking patients and healthy persons. This study indicates that smoking may play a significant role in the development of endocrine disturbances in the development of chronic pancreatitis.

Preliminary reference values for electrocardiography, echocardiography and myocardial morphometry in the European brown hare (Lepus europaeus)

The study aimed at defining reference values for electrocardiographic (ECG) and echocardiographic parameters as well as macroscopic dimensions of the heart and microscopic dimensions of cardiomyocytes in the European brown hare. The studies were conducted on 30 adult, clinically healthy hares of either sex caught in Poland. ECG and echocardiography were performed supravitally on anaesthetized hares. After euthanasia, gross and microscopic myocardial and cardiomyocyte dimensions were determined. Heart rate amounted to 140 ± 37.5 beats/min, the leading rhythm involved the sinus rhythm. P wave time was 26 ± 5 ms, PQ time was 80 ms, QRS time was 29 ± 3.5 ms, and ST was 97.5 ± 7 ms. Echocardiography determined a left ventricular wall end-diastolic diameter of 8.6 ± 2.0 mm and an intraventricular septum end-diastolic diameter of 5.75 ± 1.0 mm. The thickness of the interventricular septum corresponded to that of the free wall of the left ventricle, a finding consistent with physiological hypertrophy. Preliminary reference values were established for echocardiography. The findings were similar to those obtained at necropsy. The ECG and echocardiographic studies represent the first supravital examination of cardiac function in the hare. The obtained results illustrate adaptation of hares myocardium to its mode of life. The cardiac findings resemble the athletes heart syndrome described in humans. The findings may prove useful in further studies on the physiology of the cardio-vascular system in the hare.

Type of Inflammation Differentially Affects Expression of Interleukin 1β and 6, Tumor Necrosis Factor-α and Toll-Like Receptors in Subclinical Endometritis in Mares.

Mares that fail to conceive or lose their embryos, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). In this study, the question was addressed: does SE fully activate selected mechanisms of innate immunity in mares? For this aim, expression of mRNAs for Toll-like Receptor 2 and 4 (TLR 2/4), interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF) was examined in control mares versus either mares suffering from chronic endometritis (ChE) or subacute suppurative endometritis (SSE). The concentrations of IL-1β, IL-6 and TNF-α in supernatants from endometrial tissue cultures after 4 h incubation were measured using the enzyme immunoassay (EIA) method. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. Based on histopathological assessment, mares were classified as suffering from ChE, SSE or as being healthy. In addition, immuno-localization of both TLR2 and TLR4 as well as TNF-α was investigated in the equine endometria. The mRNA expression of TLR2 (P < 0.01), IL-1β (P < 0.0001), IL-6 (P < 0.0001) and TLR4 and TNF (P < 0.05) was up-regulated in endometria of mares suffering from SSE compared with unaffected mares. Concentrations of IL-6 and TNF-α were increased only in mares exhibiting SSE, compared with unaffected (P < 0.01 for both) and ChE mares (P < 0.05 for both). Immuno-localization of TNF-α and TLRs was confirmed, both in unaffected and SE-affected endometria, and was present in the luminal and glandular epithelia and stromal cells. The severity of inflammation impacts the immune response and fosters activation of innate immunity mechanisms, as observed in the endometria of mares. The intracellular localization of TLRs and TNF-α in the endometria indicates a key role of endometrial epithelial and stromal cells in the immune response and inflammation.