Heloisa M.F. Mendes

Hydrocortisone decreases apoptosis in jejunum of horses subjected to experimental ischemia and reperfusion

In order to evaluate the effect of hydrocortisone on apoptosis in the jejunum of horses subjected to ischemia and reperfusion, ten horses were paired and grouped into two groups - treated (n=5) and non treated (n=5). Segments of the jejunum were used as controls (C), or as venous ischemia (VIsc), which were subjected to 2h of ischemia followed by 2 or 12h of reperfusion. C samples were collected at time zero (prior to ischemia) and VIsc samples were collected at 2h of ischemia and at 2 and 12h of reperfusion. TUNEL positive apoptotic cells were counted in 10 microscopical fields in deep mucosa from each horse throughout the time course. After 12h of reperfusion, the number of apoptotic cells in treated group were significantly lower than in untreated animals, indicating that hydrocortisone inhibits apoptosis. These results indicate that hydrocortisone has a beneficial effects favoring the maintenance of jejunal integrity in horses with ischemia and reperfusion injuries by preventing apoptotic cell death.

Avaliações macroscópica e histológica do reparo da cartilagem articular equina tratada com microperfurações do osso subcondral associadas ou não à injeção intra-articular de cartogenina

The aim of this study was to evaluate the joint cartilage repair by macroscopic (via arthroscopy) and histological (biopsy fragments) analyses in chondral defects induced into equine femoral trochlea treated by microperforation associated with or without intra-articular administration of kartogenin. Six horses weighing 342±1.58 kg (mean ± SD), aged approximately 7.2±1.30 years and with a body condition score of 7.1±0.75, were used. The horses underwent arthroscopy for induction of 1-cm2 chondral lesions in lateral femoral trochlea immediately treated by microperforation of the subchondral bone of both knees. Four weekly intra-articular injections of kartogenin (20μM) in one knee (treated group) and Ringer lactate solution in the contralateral joint (control group) were performed during the postoperative period. After 60 days, macroscopic evaluations were performed by video-arthroscopy, and biopsy samples of the repair tissue were taken for histopathological healing evaluation. No significant change was observed in macroscopic and histological scores for chondral healing between treated and untreated groups (P>0.05). The overall mean percentage of hyaline cartilage in both groups (17.5%) was consistent with other international studies using other types of chondral microperforation; however, no statistical differences were observed between groups (P>0.05). In conclusion, the therapy with kartogenin, according to the used protocol, did not produce any macroscopic and histological healing improvement in induced chondral lesions treated with microperforations in equine femoral trochlea.

Avaliação clínica e hematológica de equinos submetidos ao modelo de laminite por oligofructose, tratados ou não com um agente antagonista de receptores CXCR1/2

Leucocytes recruitment to tissues is an essential part of the innate immune response and an unregulated process can result in tissue damage. Thus, leucocytes infiltration has been implicated in the pathogenesis of acute laminitis. The objectives of this stud were to determine the effect of an antagonist for CXCR1/2, a chemokine receptor for neutrophils attraction on clinical signs and hematological parameters in horses given oligofructose to induce laminitis. Twelve horses were given oligofructose (10g/kg bw PO) in time 0 and divided into two groups: one treated (30mg/kg bw. ICXCR1/2 IV, times 6, 12, 18 e 24 h) and the other not treated. Cardiac and respiratory frequency, rectal temperature, mucous membrane colour, digital pulse, hoof sensitivity and Obels grade of lameness were recorded. Values for RBC, WBC and blood glucose, BUN, creatinin, ALT, AST, alkaline phosphatase, GGT, total bilirubin and serum protein concentrations were measured on times 0, 6, 12, 18, 24, 36, 48, 60 e 72 h. All the horses given oligofructose developed signs of endotoxemia like diarrhea, fever and leukocytosis and laminitis. Also, CXCR1/2 antagonist treatment did not cause any adverse effects. However, this substance when injected intravenously (30mg/kg) 6/6 hours in 4 applications, did not ameliorate clinical and hematological signs of endotoxemia.

Alterações morfométricas no plexo mioentérico do cólon menor equino distendido experimentalmente

The equine small colon is frequently affected by obstruction, and intestinal motility dysfunction is a common complication after its surgical treatment. This fact may be related to myoenteric plexus lesion caused by distention; however, little is known about the pathophysiology of this condition. The objective of this study was to evaluate the morphological alterations in the myoenteric inervation of segments of small colon of horses subjected to intraluminal distension with reduction of the microvascular perfusion (partial ischemia) of the intestinal wall. Nine horses were used to promote distension of on segment of small colon for 4 hours. Samples of intestinal wall were collected before and at the end of the distension, after 1.5 and 12 hours of reperfusion in the experimental segment and at the end of the procedure in a different distant segment. Samples were processed and histological sections were stained with cresyl violet for the morphometric studies. An image analyzer software was used to measure perimeter, diameter, and area of the neuronal body, nucleus and nucleolus of the neurons and the areas of the cytoplasm and nucleoplasm. Significant reductions (P<0.05) in the areas of the neuronal body and cytoplasm were detected at the end of intestinal distension, returning to the basal values during the reperfusion. In conclusion, intraluminal distension promoted changes in the morphology of the neurons of myoenteric plexus. These morphological modifications may be associated to the motility dysfunction frequently observed in clinical cases.

Apoptose no cólon menor eqüino submetido à isquemia e reperfusão experimentais

Intestinal ischemia and reperfusion are important factors for mortality in horses. The objective of this study was to detect and to quantify apoptosis in the mucosa of equine small colon in a model of ischemia and reperfusion. The small colon was surgically exposed in twelve horses, and two intestinal segments were demarcated and subjected to 90 (SI) or 180 (SII) minutes of complete arteriovenous ischemia. Intestinal samples were collected before ischemia (control), at its end and after 90 and 180 minutes of reperfusion. Samples were histological processed and stained by hematoxylin and eosin (SI and SII) and by the technique of TUNEL (SI). Digitized histological images were analyzed morphometrically to detect apoptotic cells and to determine the apoptotic index (AI). After 90 or 180 minutes of arteriovenous ischemia, an increase in apoptotic cells was verified when compared with the control group, although no difference could be detected between the different periods of ischemia (P<0.05). After the first 90 minutes of reperfusion, a decrease in AI occurred, similar in both segments, possibly due to lack of energy source promoted by ischemia. AI was maximized after 180 minutes of reperfusion (sample harvested only in SI) (P<0.05). In conclusion, apoptosis is an important cause of cellular mucosal death in equine small colon ischemic obstruction, occurring early in ischemia, and later (after 90 minutes) in the reperfusion period.

Utilização da glutamina no tratamento de lesões de isquemia e reperfusão no jejuno em eqüinos

ABSTRACT.- Pagliosa G.M., Alves G.E.S., Faleiros R.R., Matos J.R.T., Torres M.B., MendesH.M.F. & Costa R.S. 2009. [ Use of glutamine to treatment of ischemia and reperfusionlesions in equine jejune. ] Utilizacao da glutamina no tratamento de lesoes de isquemia ereperfusao no jejuno em equinos. Pesquisa Veterinaria Brasileira 29(3):246-252. Departamento deClinica e Cirurgia Veterinarias, Escola de Veterinaria, Universidade Federal de Minas Gerais, Ave-nida Antonio Carlos 6627, Caixa Postal 567, Belo Horizonte, MG 30123-970, Brazil. E-mail:geanepagliosa@gmail.comUnder general anesthesia and arterial pressure control, twelve horses were submittedsimultaneously to intraluminal distension and venous ischemia of jejune for 2 hours,followed by 12 hours of reperfusion. Six horses were treated with intravenous 2% glutamine(50mg/kg), and six others were treated with the same volume of saline solution 1h afterthe beginning of ischemia and 6 hours after reperfusion. Intestinal samples were collectedbefore and after 2 hours of ischemia and after 2 and 12 hours of reperfusion, and weresubmitted to hystopatological analyses, ultrastructural examination, measurement of areaand number of villi per mm

Histologic and inflammatory lamellar changes in horses with oligofructose-induced laminitis treated with a CXCR1/2 antagonist

With the hypothesis that blocking chemokine signaling can ameliorate acute laminitis, the aim was to evaluate the therapeutic effect of intravenous DF1681B, a selective antagonist for CXCR1 and CXCR2 (chemokine receptors), in an oligofructose equine laminitis model. To twelve mixed breed clinically healthy hoses with no previous history of hoof-related lameness was administered oligofructose (10g/kg given by nasogastric tube) and divided into two groups: treated (intravenous DF1681B at 30mg/kg 6, 12, 18, and 24h after oligofructose) and non-treated groups. Laminar biopsies were performed before and 12, 36, and 72h after administering oligofructose. Samples were stained with periodic acid-Schiff (PAS) and scored from 0 to 6 according to epidermal cell and basal membrane changes. The IL-1β, IL-6, and CXCL1 RNA expressions were determined by RT-PCR. Parametric and non-parametric tests were used to compare times within each group (P<0.05). The PAS grades and IL-1β and IL-6 RNA expression increased in the non-treated group, but remained constant in the treated horses. In conclusion, DF1681B therapy reduced laminar inflammation and epidermal deterioration in treated horses. CXCR1/2 blockage should be considered therapeutically for equine acute laminitis.

Efeito de anti-inflamatórios não esteroidais na apoptose de células epidermais lamelares de equinos com laminite

The aim of this study was to evaluate the influence of the administration of anti-inflammatory drugs (NSAIDs) ketoprofen, phenylbutazone and flunixin meglumine on the apoptotic index of the epithelial cells of lamellar tissue of horses with induced laminitis. 20 healthy horses were employed and underwent induction of laminitis by administration of starch, divided into four groups with induced laminitis (n = 5): saline, ketoprofen, phenylbutazone and flunixin meglumine. The lamellar tissue was collected by biopsy, fixed and stained with the TUNEL technique. All that were stained positive by this technique were added to the cell morphological identification of apoptosis. No significant difference was found in the apoptotic index between the groups treated with anti-inflammatory and controls (P> 0.05). It was concluded that the administration of NSAIDs after the prodromal phase of laminitis does not contribute to an intervention in the course of apoptosis in the lamellar tissue of horses with laminitis when compared to the untreated control group. Other studies with different evaluation periods are needed to clarify the effects of anti-inflammatory non-steroidal drugs in the pathophysiology of laminitis in horses, especially regarding the role of apoptosis.

Lipocalina associada à gelatinase de neutrófilos (NGAL) e calprotectina no tecido laminar de equinos após obstrução jejunal, tratados ou não com hidrocortisona

Laminitis is a severe hoof condition in horses that may cause intense suffering. In this study, leukocyte infiltration in hoof laminar tissue was investigated in horses subject to intestinal obstruction using immunohistochemistry to detect calprotectin, and zymography to detect neutrophil gelatinase associated lipocalin (NGAL). There were four groups: the Control Group (Gc), with seven horses, without surgical procedures; the Sham-operated Group (Gi), with five horses that were subjected to surgical procedure without intestinal obstruction; the No Treat group (Gnt), with four horses subjected to intestinal obstruction (jejunal distention using an intraluminal balloon) without treatment; and Treated group (Gt), with four horses subjected to intestinal obstruction and treated with hydrocortisone. Positive calprotectin imunostaining was detected in all experimental groups, with increase cell counts in horses of the distended group compared with the control group. NGAL expression was increased in Gd compared with Gc e Gi. The Gt did not differ from the others. In conclusion, small intestine distension can promote leukocyte infiltration in equine hoof laminar tissue, and NGAL zymography was considered a useful method for leukocyte tissue detection in horses. New studies will be conducted to verify the possible beneficial anti-inflammatory effects of hydrocortisone in hoof of horses with intestinal obstruction.

Standardized method to obtain dermo-epidermal junction samples from bovine hoof

As afeccoes podais em bovinos causam importante impacto economico negativo na bovinocultura. Pesquisas tem sido realizadas com o objetivo de avancar no entendimento dos processos ocorridos na juncao derme-epiderme do casco de bovinos com laminite e nos demais tecidos moles durante as lesoes infecciosas. Apesar disso, nao foram encontrados na literatura consultada estudos que descrevessem um metodo padronizado para a obtencao de amostras do tecido laminar do casco. Nesse contexto, foi necessario criar e estabelecer um metodo viavel para a colheita de amostras da juncao derme-epiderme, de modo a viabilizar o estudo de pos-graduacao que originou esta comunicacao. O objetivo e relatar um metodo padronizado, testado e bem-sucedido para obtencao de amostras da juncao derme-epiderme do casco de bovinos em suas regioes solear, axial e dorsal. Foram obtidos fragmentos transversais das unhas de vacas abatidas em frigorifico. A espessura desses fragmentos foi de 1,5cm, aproximadamente, e contemplava as regioes solear, axial e dorsal do casco. De forma sistematizada, amostras da juncao derme-epiderme de cada uma dessas regioes foram removidas, fixadas em formol, processadas e incluidas em parafina. A analise usando microscopia de luz demonstrou cortes histologicos integros e sem artefatos, que permitiram ampla avaliacao das estruturas tanto da derme quanto da epiderme. Concluiu-se que o metodo proposto viabiliza a obtencao de amostras de padrao e qualidade adequados ao estudo do tecido laminar do casco bovino.