Rafael Resende Faleiros

Laminar inflammatory gene expression in the carbohydrate overload model of equine laminitis.

REASONS FOR PERFORMING STUDY There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis-related laminitis than the black walnut extract (BWE) model. OBJECTIVES To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. METHODS Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature>38.9°C (DEV group, n=8) or at onset of Obel grade 1 lameness (OG1 group, n=8). Control horses (CON group, n=8) were anaesthetised 24 h after NG administration of 6 l of deionised water. Laminar tissue was collected from horses while under anaesthesia, followed by humane euthanasia. Real time-quantitative PCR was used to assess laminar mRNA concentrations of genes involved in inflammatory signalling. RESULTS Increased mRNA concentrations (P<0.05) for IL-1β, IL-6, IL-12p35, COX-2, E-selectin and ICAM-1 were present in laminae from horses with OG1 lameness but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL-2, IL-4, IL-10, TNF-α, IFN-γ or COX-1 at either the DEV or OG1 time points. CONCLUSIONS There was a notable difference in the temporal pattern of inflammatory events between the BWE and CHO models, with the majority of laminar inflammatory events appearing to occur at or near the onset of lameness in the CHO model, whereas many of these events peak earlier in the developmental stages in the BWE model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leucocyte emigration and proinflammatory cytokine expression. POTENTIAL RELEVANCE The similar (although somewhat delayed) inflammatory response in the CHO model of laminitis indicates that inflammatory signalling is a consistent entity in the pathophysiology of laminitis.

Presence of mononuclear cells in normal and affected laminae from the black walnut extract model of laminitis.

REASONS FOR PERFORMING STUDY There is increasing evidence of involvement of inflammatory cells in acute laminitis. OBJECTIVE To immunolocalise monocytes/macrophages and B and T lymphocytes in the laminar tissue of normal horses and those with black walnut extract (BWE)-induced laminitis. METHODS Immunohistochemistry was used in archived laminar tissue samples from 20 horses divided equally into 4 groups: control animals (CON), and those administered BWE at 1.5 h (1.5H DTP group), at the onset of leucopenia (3H DTP group) and at the onset of lameness (LAM group). Antibodies against CD3, CD20 and CD163 were used to recognise lymphocytes (T and B) and monocytes/macrophages, respectively. RESULTS Mononuclear cells were present in laminar tissue of normal horses. The majority of CD3- and CD20-positive lymphocytes were localised around the deep dermal vessels but were also evident around vessels of the primary dermal laminae. CD163-positive macrophages were primarily perivascular in deep dermis or in dermal laminae. No changes in the number of laminar B or T lymphocytes occurred at any time point post BWE administration. However, increases (P=0.0016) in laminar CD163-positive cells occurred in the secondary dermal laminae (SDL) in the 1.5H DTP and 3H DTP groups, returning to basal values in LAM group. CONCLUSIONS Lymphocyte and macrophage populations are present in the laminar tissue of clinically normal horses and BWE administration induces an increase in CD163-positive macrophages in SDL. POTENTIAL RELEVANCE Both the host tissue population of mononuclear cells and the influx of monocytes may play an important role in the pathophysiological changes leading to laminar injury.

Experimental ischemia and reperfusion in equine small colon

The effects of ischemia and reperfusion were studied in the small colon of 12 horses. Under general anesthesia, arterial pressure and arterial hemoglobin oxygen saturation values were maintained constant and within the normal physiological range. After celiotomy, the small colon was exposed and three segments were demarcated. Total arterial venous ischemia was induced in two segments during 90 (group A) and 180 (group B) minutes. The third segment was the control group. Full-thickness biopsy specimens, for histopathology, were obtained at the end of the ischemia periods and at 90 (groups A and B) and 180 minutes (group A) of reperfusion. The mucosa and the submucosa were evaluated by semiquantitative morphological assessment such as epithelial detachment, edema and hemorrhage, and by quantitative morphological assessment such as percentual depth of mucosal loss (ML) and mucosal crypt:interstitium ratio (C:I). After ischemia, lesions such as ML, C:I, epithelial detachment and mucosal edema were more intense in group B when compared to group A. In both groups after reperfusion there were significant increases in ML, C:I, epithelial detachment and submucosal edema. The results showed aggravation of the mucosal lesions in the reperfusion period in equine small colon, and the model was considered adequate for experimental purposes.

Efeito da hidrocortisona sobre a lesão de reperfusão e reparação da mucosa após isquemia venosa experimental no jejuno de eqüinos

In order to evaluate the effect of hydrocortisone sodium succinate (HSS) for treatment of intestinal ischemia-reperfusion, 12 halothane-anesthetized horses were subjected to both venous and arteriovenous ischemia of the jejunum. After one hour of ischemia, HSS (4.0 mg/kg/IV) was administered to six animals (T group). The other six horses received saline (NT group). Biopsy specimens were obtained after one and two hours of ischemia, and one, two and 12 hours after reperfusion. These samples were evaluated to assess the degree of mucosal damage and infiltration of neutrophils, hemorrhage, and edema. The scores for neutrophil infiltration, edema and hemorrhage did not differ between T and NT groups in both models of ischemia. However, in the jejunum subjected to venous ischemia, the scores for mucosal lesion increased significantly (P<0.05) after two hours of reperfusion only in the NT group, indicating that HSS prevented reperfusion injury. The scores for mucosal damage were equivalent after 12 hours of reperfusion following arteriovenous ischemia in T and NT groups. In contrast, mucosal lesion due to venous ischemia were more severe in the NT group (P<0.01), indicating that intestinal repair was stimulated by HSS. These results indicate that HSS may be used as an adjuvant for treatment of intestinal isquemia and reperfusion lesions in the horse.

Sistemas osso-implante ex vivo utilizando haste intramedular polimérica para imobilização de fraturas femorais em bovinos jovens

The development of new devices using available and low cost materials may be an useful alternative for the treatment of long bone fractures in large animals. The aim of this study was to evaluate the mechanical strength of young bovine femur with diaphyseal fracture fixed with different polymeric intramedullary nails. Bending and compression tests using a universal machine were carried out in 4 distinct groups of 6 femurs from young calves. In one of the groups bones were intact while in the other three fractured bones were fixed using an intramedullary nail made of polypropylene, polyacetal or polyamide (one for each group). Considering bending and compression tests together, none of the used polymers offered strength to the fractured bones similar to the intact ones. The agreement of this findings with those from previous published in vivo results indicates that the employed ex vivo methods for mechanical assays may be useful in the search of stronger materials to be used in development of new devices.

Perfil eletroforético do proteinograma sérico de eqüinos com obstrução experimental do cólon menor

SUMMARY The changes in the serum proteinogram of equines submitted to ischemia and reperfusion of small colon by intraluminal distension were evaluated. Ten horses were submitted to standing laparotomy to induce a small colon obstruction for four hours. Five animals were instrumented without distention (control group – G1). In another five animals, mural ischemia was promoted placing a latex balloon distended with 40mmHg into the small colon (distended group – G2). Blood samples were collected before the surgical procedures (M1) and 4h after the balloon placement (M2) and 3h (M3) and 12h (M4) after reperfusion. After centrifugation and fractioning of the samples, the acute phase proteins were separated by SDS-PAGE and their concentrations were determined by computer densitometry. Nineteen proteins were separated by eletrophoresis and the molecular weights ranged from 185,000 to 14,000 Dalton (Da). The found molecular weights, related to the most known proteins, were: ceruloplasmin, 130,000 Da; C-reactive protein, 122,000 Da; transferrin, 85,000 Da;

Influência das pontas excessivas de esmalte dentário na digestibilidade e nutrientes de dietas de eqüinos

Para avaliar a influencia das pontas excessivas de esmalte dentario na digestibilidade dos nutrientes de dietas de equinos, utilizaram-se 13 animais alimentados com capim-elefante (Pennisetum purpureum) e racao comercial. Foram analisadas materia seca, proteina bruta, energia bruta, fibra em detergente neutro, fibra em detergente acido, hemicelulose e celulose nas fezes antes e duas semanas apos o desgaste corretivo das pontas excessivas de esmalte. A digestibilidade aumentou (P<0,001) em todas as variaveis estudadas apos o desgaste corretivo.

Falhas na utilização de poliacetal e poliamida em forma de haste intramedular bloqueada para imobilização de fratura femural induzida em bovinos jovens

In spite of the expressive development of veterinary orthopedics in the last years, long bone fractures in large animals remains a challenge for veterinary surgeons. This study is part of a proposal for development of a low-cost and easy-to-use polymeric interlocking nail designed to be used in newborns and young cattle. The objectives were to evaluate, in vivo, polyacetal and polyamide nails for immobilization of femoral fractures in calves. Five calves were submitted to general anesthesia and the left femur was fractured and then fixed using polyacetal or polyamide rods (nails) interlocked with four cortical screws (stainless steel) equally applied to the distal and proximal fracture line. In the postoperative period, calves were clinically assessed during 60 days by clinical and radiographic exams. Fractures occurred in four of the five polyacetal nails implanted in the first time and in two of the four polyamide nails implanted after the polyacetal nail failures. All failures occurred in the first 14 days after implantation. No rejection signs against the polymers were observed. In conclusion, polyacetal and polyamide did not have enough resistance to be used as intramedullary interlocking nails in this system designed to promote early femoral fractures immobilization in young calves.

Morphological abnormalities and apoptosis in lamellar tissue of equines after intestinal obstruction and treatment with hydrocortisone

Four experimental groups of equines were used in order to study morphological abnormalities and apoptosis in lamellar tissue. Group Cg (control) was composed of animals without any surgical procedure; group Ig (instrumented), animals that underwent enterotomy; group Tg (treated), animals that were subjected to intestinal obstruction and were treated with hydrocortisone; and group Ug (untreated), animals that were subjected to intestinal obstruction without treatment. The lamellar tissue was analyzed regarding the presence of tissue abnormalities and apoptosis. No morphological abnormalities were observed in animals of surgical groups, and no difference in apoptosis was observed between groups. It was concluded that intestinal obstruction allowed laminitis to develop, probably by systemic activation, and that the maneuvers performed in the enterotomy aggravated the process. Hydrocortisone did not aggravate the lesions of the lamellar tissue

Histopatologia das lâminas do casco de equinos com laminite aguda induzida e tratados com ketoprofeno, fenilbutazona e flunixin meglumine

ABSTRACT Experimental laminitis caused by carbohydrate overload was induced in 20 healthy horses. Seventy two hours after induction, samples of the laminar tissue were obtained by biopsy from the thoracic limbs digits for histopathology. The histological findings were compared to samples from horses without laminitis. Neutrophilic infiltrate was observed in 80% of the samples, congestion in 50%, hemorrhage in 35%, and hyperplasia of the arteriolar intima layer of the primary dermal lamina in 15%. Thrombi and intravascular blood clots were observed in 15% and 20% of the samples, respectively. Apparently, these findings were due to circulatory changes that resulted in edema, congestion, and hyperemia, followed by degeneration. In 70% of analyses performed on treated horses, the histological lesions were less severe than the clinical signs of lameness. It is concluded that: (i) the biopsy technique of laminar digital tissue from horses is viable; (ii) the artifacts generated by the biopsy technique do not compromise the histological analyses; and (iii) non-steroidal anti-inflammatory drugs do not avoid laminar lesions when administered after the beginning of clinical signs of laminitis. Keywords: equine, laminitis, digital histology, non-steroidal anti-inflammatory drugs