4-hydroxy-2-nonenal as a marker of the oxidative stress in brains of dogs with canine distemper / 4-hidroxi-2-nonenal como marcador de estresse oxidativo no cérebro de cães com cinomose

Canine Distemper is a disease caused by Canine morbillivirus (CM), a pantropic virus that can affect the central nervous system (CNS), causing demyelination. However, the pathogenesis of this lesion remains to be clarified. Brain samples of 14 naturally infected dogs by CM were analyzed to evaluate the presence of oxidative stress and demyelination. RT-PCR assay was performed to confirm a diagnosis of canine distemper in the brain, immunohistochemistry anti-CM was used to localize the viral proteins in the tissue, and anti-4-hydroxy-2-nonenal (4-HNE) was a marker of a product of lipid peroxidation. The results showed the presence of viral proteins in the demyelinated area with the presence of 4-HNE. Our results suggest that the CM virus infection causes oxidative stress leading to lipid peroxidation, which causes tissue damage and demyelination. In conclusion, oxidative stress plays a significant role in canine distemper pathogenesis in the CNS.(AU)

A cinomose canina é uma doença causada pelo Morbilivírus canino (CM), um vírus pantrópico que pode afetar o sistema nervoso central (SNC), causando desmielinização. No entanto, a patogênese dessa lesão não está totalmente esclarecida. RT-PCR e imuno-histoquímica foram realizadas para confirmação do diagnóstico de cinomose em amostras de encéfalo de 14 cães naturalmente infectados. Após confirmação, foi realizada uma avaliação do estresse oxidativo por imuno-histoquímica com uso de anti-4-hidroxi-nonenal (4HNE) como marcador de produtos resultantes da peroxidação lipídica. Os resultados sugerem que a infecção pelo CM causa estresse oxidativo no tecido, levando a peroxidação lipídica, a qual causa danos ao tecido, culminando com desmielinização. Conclui-se que o estresse oxidativo tem papel importante na patogênese da cinomose canina no sistema nervoso central.(AU)

Puma (Puma concolor) predating sheep in Uberlândia, Brazil: physical injuries, stress and myopathy / Predação de ovelhas por onça-parda (Puma concolor) em Uberlândia, Brasil: lesões macroscópicas, estresse e miopatia

Puma (Puma concolor) is in Brazil among the major species involved in human-wildlife conflicts, primarily due to the predation of sheep and goats in farms. A puma might kill from one to several animals in a single predation episode, and free-ranging flocks of goats and sheep are usually affected. These attacks are rarely witnessed and direct observation of predation is rare. We herein report three predation episodes and the cause of death of sheep kept inside paddocks in a farm located besides a major town of Minas Gerais State, Brazil. Death of 31 animals was attributed to a puma as determined by circumstantial evidence, necropsy of 16 animals, laboratory findings and direct observation of the predator at the occurrence site. However, death of only eight animals was caused by traumatic injury mainly at the cervical region. The remaining animals lacked lethal traumatic injuries and post mortem examination indicated circulatory collapse (congestion of internal organs). Two surviving animals had a great increase in creatinine phosphokinase blood levels and thus death of remaining animals was attributed to stress and myopathy caused by fear and chasing of sheep trapped inside paddocks. Measures taken after the third predation episode (illumination of paddocks, pasture height control, vegetation clearance around the paddocks and closure of animals in stalls at night) prevented further attacks.

A onça-parda (Puma concolor) é uma das principais espécies relacionadas a conflitos entre humanos e animais selvagens no Brasil, principalmente devido predação de ovinos e caprinos em fazendas. Este felino selvagem pode matar de um a vários animais em um único episódio de predação, sendo geralmente, cabras e ovelhas criados extensivamente os mais afetados. Esses ataques raramente são testemunhados e por isso a observação direta da predação também é rara. Neste trabalho são relatados três episódios de predação e causa mortis de ovinos mantidos em piquetes em uma fazenda vizinha à Uberlândia, Minas Gerais, Brasil. A morte de 31 animais foi atribuída a uma onça-parda, conforme provas circunstanciais, exame necroscópico de 16 animais, achados laboratoriais e a observação direta do predador no local da ocorrência. Entretanto, a morte de apenas oito animais foi causada por lesão traumática, principalmente na região cervical. Os demais não apresentaram lesões traumáticas letais e nestes o exame post-mortem indicou colapso circulatório (congestão dos órgãos internos). Dois animais sobreviventes apresentaram acentuado aumento dos níveis sanguíneos de creatinina fosfoquinase, permitindo assim atribuir a morte dos demais animais ao estresse e miopatia provocadas pelo medo e perseguição das ovelhas dentro dos piquetes. Após o terceiro episódio de predação medidas preventivas foram adotadas e a iluminação dos piquetes, o controle da altura do capim nos piquetes, a redução da vegetação ao redor dos piquetes e a manutenção dos animais em baias fechadas durante a noite impediram novos episódios, pelo menos até o momento.

Correlation between systolic function and presence of myofibroblasts in doxorubicin-induced cardiomyopathy / Correlação entre função sistólica e presença de miofibroblastos na cardiomiopatia induzida pela doxorrubicina

ABSTRACT: Cardiotoxicity induced by doroxubicin generates systolic disfunction and myocardial remodeling with presence of myofibroblasts. These cells are thought to be attracted to the injured heart to avoid the development of congestive heart failure. The objective of this study was to evaluate the systolic dysfunction generated by doxorubicin through Doppler echocardiography, and its correlation with the presence of myofibroblasts in the myocardium. Twenty-five New Zealand White rabbits were divided into two groups (control, and treated with doxorubicin). The drug was administered for six weeks; Doppler echocardiography was performed before the first, and after the last administration of doxorubicin. Immuno detection of myofibroblasts was performed by immunohistochemistry. The treated group exhibited significant reduction in systolic function as assessed by Doppler echocardiography, and increased frequency of myofibroblasts, which were present in similar amounts in the left ventricle, interventricular septum, and right ventricle. There was a significant negative correlation between number of myofibroblasts in the interventricular septum and in the left ventricle with systolic function indices, which reveals that the higher the number of fibroblasts, the worst systolic function is in rabbits treated with doxorubicin. Increase in myofibroblast numbers was not sufficient to preserve systolic function.

RESUMO: A cardiotoxicidade induzida pela doxorrubicina gera disfunção sistólica e remodelamento miocárdico, com presença de miofibroblastos. Acredita-se que essas células sejam atraídas para a não evolução do quadro de insuficiência cardíaca congestiva. O objetivo do presente trabalho foi avaliar a função sistólica gerada pela doxorrubicina por meio da ecodopplercadiografia e correlacioná-la com a presença de miofibroblastos no miocárdio. Foram utilizados 25 coelhos da raça Nova Zelândia, alocados em dois grupos (controle e tratados com doxorrubicina). O fármaco foi administrado por seis semanas e a ecodopplercardiografia foi realizada no momento zero e após a última administração da doxorrubicina. A imunodetecção dos miofibroblastos foi realizada por imuno-histoquímica. Houve redução significativa na função sistólica, observada na ecodopplercardiografia e aumento na imundetecção dos miofibroblastos nos animais tratados, na mesma intensidade no ventrículo esquerdo, septo interventricular e ventrículo direito. Houve correlação negativa significativa entre o número de miofibroblastos no septo interventricular e no ventrículo esquerdo com os índices de função sistólica, revelando que quanto mais miofibroblastos presentes, pior é a função sistólica de coelhos tratados com doxorrubicina. O aumento do número de miofibroblastos não foi suficiente para manutenção da função sistólica.

Ketorolac eye drops reduce inflammation and delay re-epithelization in response to corneal alkali burn in rabbits, without affecting iNOS or MMP-9.

PURPOSES: To assess the effects of 0.5% ketorolac tromethamine without preservatives on the expression of iNOS and MMP-9 in alkali burn ulcers. METHODS: Twelve eyes of 120-day-old male rabbits were treated (TG) every 6 h with 0.5% ketorolac tromethamine and 12 other eyes were treated with saline solution (CG), immediately after the occurrence of ulcers by 1 M sodium hydroxide (NaOH). Re-epithelialization was monitored using fluorescein every 6 h. After 24 h, six corneas (n=6) of each group were collected (M1). The others (n=6) were collected after reepithelialization (M2). At both moments, the inflammatory infiltrate and the conditions of the newly formed epithelium were histologically analyzed. iNOS and MMP-9 were evaluated by immunohistochemistry. RESULTS: Mean epithelialization time in TG was 55 ± 0.84 h. In CG, it was 44 ± 1.06 h (p=0.001). At M1, corneas of TG had lower inflammatory exudation compared with (p <0.001). At M2, TG revealed discrete inflammatory exudation (p>0.05) and lower numbers of epithelial layers compared with CG. The mean iNOS in stromal cells did not differ in TG over both moments compared with CG (p>0.05) At M2, the central corneal region expressed more iNOS in both groups compared with the peripheral region. No significant differences were observed in iNOS scores of epithelial immunostaining between the groups and across M1 and M2 (p=0.69). Epithelial immunostaining scores for MMP-9 did not differ in TG compared with CG (p=0.69). The average immunostaining score of MMP-9 in stromal cells showed no differences between groups or moments. There was no correlation between immunostaining of iNOS and MMP-9 or between the amount of inflammatory cells and immunostaining of iNOS. CONCLUSIONS: Use of 0.5% keratolac tromethamine reduced inflammation and delayed reepithelialization in a cornea alkali burn model without impacting the expression of iNOS or MMP-9.

Ketorolac eye drops reduce inflammation and delay re-epithelization in response to corneal alkali burn in rabbits, without affecting iNOS or MMP-9 / Colírio de cetorolaco reduz inflamação e atrasa a epitelização em resposta a queimadura por álcali em coelhos, sem afetar iNOS ou MMP-9

Purposes: To assess the effects of 0.5% ketorolac tromethamine without preservatives on the expression of iNOS and MMP-9 in alkali burn ulcers. Methods: Twelve eyes of 120-day-old male rabbits were treated (TG) every 6 h with 0.5% ketorolac tromethamine and 12 other eyes were treated with saline solution (CG), immediately after the occurrence of ulcers by 1 M sodium hydroxide (NaOH). Re-epithelialization was monitored using fluorescein every 6 h. After 24 h, six corneas (n=6) of each group were collected (M1). The others (n=6) were collected after reepithelialization (M2). At both moments, the inflammatory infiltrate and the conditions of the newly formed epithelium were histologically analyzed. iNOS and MMP-9 were evaluated by immunohistochemistry. Results: Mean epithelialization time in TG was 55 ± 0.84 h. In CG, it was 44 ± 1.06 h (p=0.001). At M1, corneas of TG had lower inflammatory exudation compared with (p <0.001). At M2, TG revealed discrete inflammatory exudation (p>0.05) and lower numbers of epithelial layers compared with CG. The mean iNOS in stromal cells did not differ in TG over both moments compared with CG (p>0.05) At M2, the central corneal region expressed more iNOS in both groups compared with the peripheral region. No significant differences were observed in iNOS scores of epithelial immunostaining between the groups and across M1 and M2 (p=0.69). Epithelial immunostaining scores for MMP-9 did not differ in TG compared with CG (p=0.69). The average immunostaining score of MMP-9 in stromal cells showed no differences between groups or moments. There was no correlation between immunostaining of iNOS and MMP-9 or between the amount of inflammatory cells and immunostaining of iNOS. Conclusions: Use of 0.5% keratolac tromethamine reduced inflammation and delayed reepithelialization in a cornea alkali burn model without impacting the expression of iNOS or MMP-9. .

Objetivos: Avaliarem-se os efeitos do cetorolaco de trometamina 0,5%, sem conservante, sobre a expressão da iNOS e da MMP-9, em córneas com úlceras químicas. Métodos: Doze olhos de coelhos machos, 120 dias de idade, foram tratados (GT ), a cada 6 horas, com o cetorolaco de trometamina 0,5% e outros 12 com solução salina (GC), imediatamente à ocorrência de úlceras por hidróxido de sódio (NaOH) 1 mol/L. A reepitelização foi monitorada por fluresceína a cada seis horas. Decorridas 24 horas, seis córneas (n=6) de cada grupo foram colhidas (primeiro momento). As demais (n=6) o foram após a sua reepitelização (segundo momento). Em ambos os momentos, avaliaram-se o infiltrado inflamatório e as condições do epitélio neoformado (HE). Por imuno-histoquímica, avaliou-se a imunomarcação de iNOS e de MMP-9. Resultados: A média do tempo de epitelização no GT foi de 55 ± 0,84 horas. No GC, ela foi de 44 ± 1,06 horas (p=0,001). Às 24 horas, as córneas do GT apresentaram menor exsudação inflamatória (p<0,01). No segundo momento, o GT mostrou discreta exsudação inflamatória (p>0,05) e menor número de camadas epiteliais comparativamente ao GC. A média de imunomarcação de iNOS em células do estroma não diferiu do GT, em ambos os momentos (p>0,05). No segundo momento, a região central da córnea expressou mais iNOS, comparativamente à periférica, em ambos os grupos. Não se observaram diferenças significativas nos escores de imunomarcação epitelial de iNOS entre os grupos e os momentos (p=0,69). Os escores de imunomarcação epitelial para MMP-9 não diferiram entre os grupos (p=0,69). A média de imunomarcação da MMP-9 em células do estroma não exibiram diferenças entre os grupos e momentos da avaliação (p=0,32). Não houve correlação entre a imunomarcação de iNOS e de MMP-9, assim como quanto ao quantitativo de células inflamatórias e à imunomarcação de iNOS. Conclusões: Cetorolaco 0,5% reduziu a inflamação e atrasou a epitelização na queimadura corneal ...

Immunodetection of cells with a CD44+/CD24- phenotype in canine mammary neoplasms.

BACKGROUND: Cancer stem cells (CSCs) are able to self-renew and to form metastases. Using flow cytometry, CSCs were detected in canine mammary tumors as cells CD44(+) and CD24(-). The aim of this study was to detect these CSCs by immunohistochemistry and correlate their frequency with canine mammary neoplasm grade and histopathological type.130 mammary neoplasm samples were selected from tissue blocks at the Department of Pathology at UNESP and classified according to (BJVP 4:153-180, 2011). These samples were composed by adenomas, lymph node metastases, solid carcinomas grades II and III, tubular, papillary and carcinomas in mixed tumor grades I, II and III. Immunohistochemistry was performed with antibodies against CD44 and CD24. Linear regression was performed using Pearson's correlation test. RESULTS: The value at CD44 was positive and CD24 becomes zero was 46.75%. Cells with a CD44(+)/CD24(-) phenotype were detected in 40 out of 130 samples with an advantage of high grade tumors (II and III) and metastases among tubular, papillary and carcinomas in mixed tumors. In these samples, percentages of cells stained by CD44 and CD24 antibodies were 62.2% and 0%, respectively. Published reports usually correlate grade III tumors with the expression of CD44 but not with CD24 expression. Studies using flow cytometry have found CSC frequencies similar to those found in our study. CONCLUSIONS: Immunohistochemistry was found to be a reliable technique for the detection of CSCs in canine mammary neoplasms, and the frequency of these cells positively correlates with grades II and III tumors (poor prognosis).

Doxorubicin induced dilated cardiomyopathy in a rabbit model: an update.

Dilated cardiomyopathy (DCM) is characterized by chamber dilation and cardiac dysfunction. Because of the poor prognosis, models are needed for the investigation of and development of new therapeutic approaches, as well as stem cell therapy. Doxorubicin (DOX), used as chemotherapeutic agent, is reported to be cumulative cardiotoxic causing DCM. The aim of the study was to investigate the onset of systolic dysfunction using echocardiography in rabbits receiving two different doses of DOX (1mg/kg twice a week and 2 mg/kg once a week). Twenty rabbits were treated with doxorubicin in two different doses for 6 weeks and compared with a control group treated with NaCl 0.9%. The effect of doxorubicin on the myocardium was investigated with histological analysis and scanning electron microscopy of left ventricle (LV), as well as in the interventricular septum (IVS) and right ventricle (RV). The results showed a high mortality rate for rabbits receiving 2 mg/kg once a week. A significant reduction in systolic function was present in animals treated with DOX after 6 weeks, with decreased ejection fraction and shortening fraction. Histology and electron microscopy revealed vacuolization, intracytoplasmic granulation, necrosis and interstitial fibrosis in LV, as well as in the IVS and RV. Doxorubicin induced changes are present in the LV, RV and IVS, and the administration at the dose of 1 mg/kg twice a week for only 6 weeks is safe and sufficient to induce DCM in rabbits.

Influence of apoptosis on the cutaneous and peripheral lymph node inflammatory response in dogs with visceral leishmaniasis.

In canine visceral leishmaniasis (CVL), the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir for the protozoon Leishmania (L.) chagasi and the skin is the main site of contamination by the vector insect. Some protozoa use apoptosis as an immunological escape mechanism. The aim of this study was to correlate the presence of apoptosis with the parasite load and with the inflammatory response in the skin and lymph nodes of dogs naturally infected with Leishmania (L.) chagasi. Thirty-three dogs from the municipality of Araçatuba (São Paulo, Brazil) were used, an endemic area for CVL. Muzzle, ear and abdominal skin and the popliteal, subscapular, iliac and mesenteric lymph nodes of symptomatic (S), oligosymptomatic (O) and asymptomatic (A) dogs were analyzed histologically. The parasite load and percentage apoptosis were evaluated using an immunohistochemical technique. Microscopically, the lymph nodes presented chronic lymphadenitis and the skin presented plasmacytic infiltrate and granulomatous foci in the superficial dermis, especially in the ear and muzzle regions. The inflammation was most severe in group S. The parasite load and apoptotic cell density were also greatest in this group. The cause of the lymphoid atrophy in these dogs was correlated with T lymphocyte apoptosis, thus leaving the dogs more susceptible to CVL. The peripheral lymph nodes presented the greatest inflammatory response. Independent of the clinical picture, the predominant inflammatory response was granulomatous and plasmacytic, both in the skin and in the peripheral lymph nodes. The ear skin presented the greatest intensity of inflammation and parasite load, followed by the muzzle skin, in group S. The ear skin area presented a non-significant difference in cell profile, with predominance of macrophages, and a significant difference from group A to groups O and S. It was seen that in these areas, there were high densities of parasites and cells undergoing apoptosis, in group S. The association between apoptosis and parasite load was not significant in the lymph nodes, but in the muzzle regions and at the ear tips, a positive correlation was seen between the parasite load and the density of cells undergoing apoptosis. The dogs in group S had the highest parasite load and the greatest number of apoptotic cells, thus suggesting that the parasite had an immune evasion mechanism, which could be proven statistically in the skin.

Expression of matrix metalloproteinases, type IV collagen, and interleukin-10 in rabbits treated with morphine after lamellar keratectomy.

PURPOSE: To study the effects of topical administration of 1% morphine on corneal analgesia in rabbits submitted to lamellar keratectomy and to assess the expression of matrix metalloproteinase-1, metalloproteinase-2, metalloproteinase-9 (MMPs), type IV collagen, and interleukin-10 (IL-10) during the treatment. METHODS: Morphine group (MG) received 50 µL of topical 1% morphine four times daily, while the control group received saline instead. Corneal touch threshold (CTT) and the wound area were assessed until corneal healing. Corneal samples were processed for routine histology, immunohistochemistry, zymography, and ELISA. RESULTS: Following keratectomy, CTT increased significantly from 6 to 96 h time points. Mean corneal re-epithelization rate and scores of leukocyte infiltration did not differ significantly between treatment groups. Immunolabeling pattern for MMP-1, MMP-9, and type IV collagen was similar in both treatment groups. In the MG, zymography indicated significantly higher levels of active MMP-2 on days 6 and 12; and in the latent MMP-9, on days 3 and 6, and in the active MMP-9, on day 6. Latent MMP-2 and MMP-9, and active MMP-9 decreased to values close to those of healthy corneas on day 12, but levels of active MMP-2 remained significantly elevated in the MG. IL-10 levels measured on days 1-6 were reduced as compared to those of healthy corneal tissue and returned to levels close to those of healthy corneas on day 12. CONCLUSION: Topical morphine promoted corneal analgesia for up to 4 days and did not delay corneal re-epithelization. The re-establishment of MMPs and IL-10 to levels close to baseline values at the end of the study and the expression of type IV collagen in both groups reinforce that, with caution, 1% morphine can be used after lamellar keratectomy in rabbits.

Immune response pattern of the popliteal lymph nodes of dogs with visceral leishmaniasis.

The present study aimed to estimate the cell response and parasite load in the popliteal lymph nodes of dogs with visceral leishmaniasis (VL), comparing these findings with the clinical staging of the disease. From the necropsy, 33 dogs were classified as symptomatic (S), asymptomatic (A), or oligosymptomatic (O). Cytology and histopathology were used to determine any presence of microscopic lesions and immunohistochemistry, for parasite load. Dog hyperimmune serum was used as the primary antibody. The inflammatory infiltrate in lymph nodes consisted of macrophages and plasmocytes. The granulomas invaded the trabecular and sinusoid regions and sometimes compressed the lymphocytes of the cortical region (atrophy) and medullary cord cells. Parasite load intensity was unrelated to the density of the macrophages infiltrating the lymph node. Significant differences in parasite load (P < 0.05) were observed between the three groups of infected dogs. Follicular hyperplasia of the cortical region occurred among A and O, while follicular atrophy predominated among S. The parasite load was the greatest among S, followed by O. It can be concluded that, regardless of clinical condition, the most evident cell response consisted of macrophages and plasmocytes. Lymphoid atrophy was observed among animals with intense granulomatous reaction and high parasite load, such as among the symptomatic dogs (P < 0.05). Likewise, the oligosymptomatic dogs also presented high density of parasites in the lymph nodes. Thus, we can confirm that dogs with clinical manifestations of VL have an immune system that is less effective for controlling infection by Leishmania chagasi, thereby favoring parasite multiplication.