Alexandre Lima de Andrade

Healing and expression of growth factors (TGF-β and PDGF) in canine radial ostectomy gap containing platelet-rich plasma

OBJECTIVES To evaluate bone healing in an experimental radial ostectomy in dogs treated with autologous platelet-rich plasma (PRP), through histological, densitometric, radiographic studies, as well as expression of growth factors in the ostectomy gap. METHODS Twenty-one dogs were randomly divided into either a control or a PRP group. All underwent unilateral ostectomy of the radius to generate a gap of 2.0 mm, that was stabilized with external skeletal fixation. The ostectomy gap was either filled with PRP or left empty as a control. The radiographic and densitometric studies were performed after surgery, then at intervals until 60 days during the post-surgery period. Histological and immunohistochemical evaluations were performed at seven or 60 days post-surgery. Analyses were performed using a statistical analysis system, and the level of significance was set at p <0.05. RESULTS The median radiographic healing score in the PRP group increased significantly between day 0 and day 60. Furthermore, at 60 days, the median healing score and the proportion of healed ostectomies in the control group (score 1; 1/6 healed) and the PRP group (score 5; 4/5 healed) were significantly different. There were differences between groups in radiographic and densitometric values at days 45 and 60. The histological evaluation showed advanced bone healing at 60 days in the PRP group and signs of delayed union in the control group. CLINICAL RELEVANCE Platelet-rich plasma can be used as an adjuvant therapy because it may promote better bone healing of a radial ostectomy treated with external skeletal fixation in dogs.

Immunohistochemical approach to the pathogenesis of clinical cases of bovine Herpesvirus type 5 infections.

Meningoencephalitis by Herpesvirus type 5 (BoHV-5) in cattle has some features that are similar to those of herpetic encephalitis in humans and other animal species. Human Herpesvirus 3 (commonly known as Varicella-zoster virus 1), herpes simplex viruses (HSV), and equid Herpesvirus 1 (EHV-1) induce an intense inflammatory, vascular and cellular response. In spite of the many reports describing the histological lesions associated with natural and experimental infections, the immunopathological mechanisms for the development of neurological disorder have not been established. A total of twenty calf brains were selected from the Veterinary School, University of São Paulo State, Araçatuba, Brazil, after confirmation of BoHV-5 infection by virus isolation as well as by a molecular approach. The first part of the study characterized the microscopic lesions associated with the brain areas in the central nervous system (CNS) that tested positive in a viral US9 gene hybridization assay. The frontal cortex (Fc), parietal cortex (Pc), thalamus (T) and mesencephalon (M) were studied. Secondly, distinct pathogenesis mechanisms that take place in acute cases were investigated by an immunohistochemistry assay. This study found the frontal cortex to be the main region where intense oxidative stress phenomena (AOP-1) and synaptic protein expression (SNAP-25) were closely related to inflammatory cuffs, satellitosis and gliosis, which represent the most frequently observed neurological lesions. Moreover, MMP-9 expression was shown to be localized in the leptomeninges, in the parenchyma and around mononuclear infiltrates (p < 0.0001). These data open a new perspective in understanding the role of the AOP-1, MMP-9 and SNAP-25 proteins in mediating BoHV-5 pathogenesis and the strategies of host-virus interaction in order to invade de CNS.

Microbiota conjuntival de cães sadios da cidade de Araçatuba (SP)

Purpose: To identify the microorganisms of the ocular conjunctiva in healthy dogs in Aracatuba (SP) city, in summer and winter. Methods: Forty dogs, male and female, age ranging from 2 to 5 years were used. After ocular wash, samples were collected from the inferior conjunctival sac in order to isolate and identify anaerobic, aerobic bacteria and fungi. Results: Staphylococcus aureus and Sthaphylococcus b-haemolyticus and Penicilium sp. were the most frequently observed microorganisms. Conclusion: There was ocular conjunctiva microbiota variation between the studied seasons. Staphylococcus b-haemolyticus was observed only during the winter.

Bilateral lamellar keratoplasty in descemetocele treatment in dog with botulism by use of equine renal capsule and conjunctival pedicle graft

A 3-year-old, male mixed breed dog with botulism and bilateral descemetocele was submitted to lamellar keratoplasty with equine renal capsule preserved in glycerin in the right eye and conjunctival pedicle graft in the left eye. The evolution was satisfactory in both eyes, but better in the eye receiving the equine renal capsule, because the corneal transparence was more evident in that eye. On the other hand, the surgical period was more quickly in the eye receiving the equine renal capsule because the preparation of the conjunctival pedicle before the keratoplasty was not necessary.

Fractal dimension and Shannon's entropy analyses of the architectural complexity caused by the inflammatory reactions induced by highly crystalline poly(vinyl alcohol) microspheres implanted in subcutaneous tissues of the Wistar rats

The results of the histopathological analyses after the implantation of highly crystalline PVA microspheres in subcutaneous tissues of Wistar rats are here in reported. Three different groups of PVA microparticles were systematically studied: highly crystalline, amorphous, and commercial ones. In addition to these experiments, complementary analyses of architectural complexity were performed using fractal dimension (FD), and Shannons entropy (SE) concepts. The highly crystalline microspheres induced inflammatory reactions similar to the ones observed for the commercial ones, while the inflammatory reactions caused by the amorphous ones were less intense. Statistical analyses of the subcutaneous tissues of Wistar rats implanted with the highly crystalline microspheres resulted in FD and SE values significantly higher than the statistical parameters observed for the amorphous ones. The FD and SE parameters obtained for the subcutaneous tissues of Wistar rats implanted with crystalline and commercial microparticles were statistically similar. Briefly, the results indicated that the new highly crystalline microspheres had biocompatible behavior comparable to the commercial ones. In addition, statistical tools such as FD and SE analyses when combined with histopathological analyses can be useful tools to investigate the architectural complexity tissues caused by complex inflammatory reactions.

Uso da flunixina meglumina tópica no tratamento de uveítes em cães

The purpose of this study was to compare the effects of flunixin meglumine and dexamethasone topically for control of uveitis in dogs. Clinical evaluation and assessment of aqueous protein concentration were performed. Seventeen dogs with anterior uveitis secondary to variable ethiologies were studied. The animals were allotted in two groups and treated during 15 days. The flunixin meglumine was more efficient for induction of decrease in aqueous protein concentration, and control of uveitis when compared to dexamethasone. Thus, a parenteral solution of flunixin meglumine can be employed for the treatment of inflamatory uveal diseases.

Effects of antiproteolytic agents on corneal epithelial viability and matrix metalloproteinase-2 and metalloproteinase-9 activity in alkali-burned corneas of rats

OBJECTIVE To evaluate the effects of agents on corneal re-epithelization and metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) activities in corneas of rats submitted to ulceration. ANIMALS STUDIED Ninety eight healthy rats. PROCEDURES Corneal ulcers were created using 1N NaOH in their left eye. Eyes were treated every 6 h with 1% ethylenediaminetetraacetic acid (EDTA), 3% chondroitin sulfate (CS), 10% N-acetylcysteine NAc and saline (S) at 6-h intervals. Corneas were stained with fluorescein and photographed at the same time points. Following 20 h and 40-42 h of corneal injury, corneas were processed for scanning electron microscopy (SEM) to quantify microvilli density, and MMPs activities were analyzed using zymography. RESULTS The percentage of wound area and the time in hours for corneal re-epithelization did not differ significantly among treatment groups (P > 0.05). In first and the second moments, latent MMP-2 was significantly elevated in the eyes treated with NAC and CS (P < 0.001). Active MMP-2 did not change significantly among treatment groups in the first moment (P > 0.05); significantly higher activity was observed in the second moment in the eyes treated with CS (P <0.001). In the second moment, latent MMP-9 decreased significantly in eyes treated with EDTA and S (P < 0.01). Microvilli corneal density did not change significantly between healthy subjects and treatment groups (P > 0.05). CONCLUSION Any of the studied substances did not accelerate corneal re-epithelization and did not add protection to the corneal microvilli. Significant higher levels of active form of MMP-2 in 3% chondroitin sulfate-treated group may indicate that the agent acts as substrate for such enzyme. At the end of the experiment, 1% EDTA was the most efficient agent to inhibit significantly the latent form of MMP-9. However, any of the substances add benefit over saline on reducing the proteolytic activity in the cornea of rats after alkali injury.

Anestesia peribulbar com ropivacaína como alternativa ao bloqueio neuromuscular para facectomia em cães

A peribulbar block technique was developed and its clinical efficacy was compared with neuromuscular blockade in dogs undergoing cataract surgery. Twelve dogs of different breeds were randomly and equally allocated in two groups. After given acepromazine (0.05mg/kg, IV), anesthesia was induced with propofol (5mg/kg, IV) and maintained with isoflurane in oxygen during spontaneous breathing. A peribulbar block with 0.75% ropivacaine was performed in G1 dogs while partial neuromuscular blockade with pancuronium (0.01mg/kg IV) was provided in G2 dogs. Globe position scores, oculocardiac reflex, and intra-ocular pressure (mmHg) were evaluated at pre-defined intervals during surgery. Peribulbar blocks were successful performed according to posterior peribulbar block described in humans. In both groups, the globe was centralized and globe position scores did not differ between groups. The intra-ocular pressure was significantly lower in G1 after the block (10.7±0.6 vs 14.7±0.6). There were no electrocardiographycal changes attributed to the oculocardiac reflex. In conclusion, satisfactory surgical conditions were provided by the peribulbar block. This technique can be used as an alternative to the use of neuromuscular blocking agents in dogs undergoing cataract surgery, with the advantage of providing analgesia of the eye.

Repair of lamellar scleral lesions in dogs with preserved equine renal capsule: short report

Purpose: To evaluate the use of equine renal capsule preserved in glycerin to repair lamellar scleral lesions in dogs. Methods: Twelve healthy mongrel dogs, male and female, weighing 12 kg were used. The study was both clinical and morphological, and performed on the first, third, seventh, 15 th , 30 th and 60 th day after surgery. Temporal canthotomy was performed after standard preoperative and general anesthesia. Conjunctival and scleral square incisions of 0.5x0.5 cm were carried out in a one o’clock position, near the limbus. A fragment of hydrated biological prosthesis, of the same shape, was sutured with 7-0 Vicryl ® in an interrupted suture. Results: The clinical evaluation showed blepharospasm/photophobia until the 7 th day after surgery. Conjunctival edema appeared up to the 5 th day after surgery. Mucoid ocular discharge was sustained until the 10 th day after surgery. Hyperemia was observed until the end of the evaluation period. There were no signs of graft extrusion in all animals. The anterior and posterior segments did not show clinical signs of inflammation. The optical microscopy morphological evaluation showed an inflammatory exudation with acute aspects in the early and intermediate periods, and inflammatory exudation with chronic aspects in the late periods. There was incorporation of the implant by the recipient’s sclera. Conclusion: These results suggest that the equine renal preserved capsule could be a useful alternative tissue to repair lamellar corneal lesions in dogs and humans.

Osteogenic potential of mesenchymal cells derived from canine umbilical cord matrix co-cultured with platelet-rich plasma and demineralized bone matrix

Canine mesenchymal cells (MSCs) derived from Whartons jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.