Kukoamine A activates Akt/GSK-3ß signaling pathway to inhibit oxidative stress and relieve myocardial ischemia-reperfusion injury

Purpose: Myocardial ischemia/reperfusion (MI/R) injury refers to a pathological condition of treatment of myocardial infarction. Oxidative stress and inflammation are believed to be important mechanisms mediating MI/R injury. Kukoamine A (KuA), a sperm, is the main bioactive component extracted from the bark of goji berries. In this study, we wanted to investigate the possible effects of KuA on MI/R injury. Methods: In this experiment, all rats were divided into sham operation group, MI/R group, KuA 10 mg + MI/R group, KuA 20 mg + MI/R group. After 120 min of ischemia/reperfusion treatment, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximal rates of rising and fall of left ventricular pressure (±dp/dtmax), and ischemic area were detected. Serum samples of rats in each group were collected. The enzyme activities of catalase (CAT), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), levels of malondialdehyde (MDA), CK muscle/brain (CK-MB), tumor necrosis factor (TNF), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were detected using enzyme-linked immunosorbent assay (ELISA). The apoptosis of myocardium in each group was detected according to the instructions of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expressions of mammalian target of glycogen synthase kinase-3ß (GSH-3ß) and protein kinase B (Akt) mRNA level in myocardial tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR). Results: MI/R rats showed a significant increase in oxidative stress and inflammation. In addition, we showed that KuA significantly improved the myocardial function such as LVSP, left ventricular ejection fraction, +dp/dt, and -dp/dt. Here, it attenuated dose-dependent histological damage in ischemia-reperfused myocardium, which is associated with the enzyme activities of SOD, GSH-PX, and levels of MDA, IL-6, TNF-α, L-1ß. Conclusions: KuA inhibited gene expression of Akt/GSK-3ß, inflammation, oxidative stress and improved MR/I injury. Taken together, our results allowed us to better understand the pharmacological activity of KuA against MR/I injury.

Polymyositis associated with brainstem signs in a dog

Background: Polymyositis is a generalized inflammatory myopathy which can lead to rhabdomyolysis. This affection may have several origins, including degenerative, metabolic, autoimmune, infectious, inflammatory, ischemic, traumatic, by drug use, induced by toxins and also of idiopathic origin. Diagnosis is made with seric dosage, electrodiagnostic tests and muscle biopsy. Lesions in the rostral oblong medulla may affect the central vestibular system, and there may be signs such as opisthotonos, nystagmus, and strabismus. The aim of this report is to describe a case of a mixed breed dog with manifestation of polymyositis associated with brainstem signs of probable idiopathic origin. Case: A 5-year-old mixed breed male dog was attended with opisthotonos episodes for 2 days, and pelvic limbs extension and thoracic limbs flexion that lasted 10 to 20 min at intervals of approximately 1 h. The animal was anorexic and had also presented one episode of emesis. Upon neurological examination, ventromedial strabismus and Horner's syndrome was observed on the right side, besides vertical nystagmus, flaccid tetraparesis and absence of proprioception in the four limbs. Biochemical analyses revealed creatine kinase (CK) increased (2,433.9 UI/L - reference: 1.5-28.4 UI/L), and urinalysis showed dark color and presence of occult blood without, however, erythrocyturia. Electrocardiogram (ECG) showed QS wave and deviation of the electrical axis. Treatment with prednisolone (1 mg/kg, BID), phenobarbital (2 mg/kg, BID), maropitant citrate (1 mg/kg in 2 doses), and crystalloid fluid therapy (50 mL/kg/day) were prescribed. On the 4th day, the dog was more active and feeding without a tube, so it recommended keep the treatment at home. On the 10th day, the animal had proprioception present on the 4 limbs and normorexia. Biochemical analyses and urinalysis showed no alterations, but normochromic normochromic anemia with thrombocytopenia and leukocytosis by neutrophilia showed in blood count exam. PCR to Ehrlichia canis, Hepatozoon sp., and Babesia canis resulted negative. On the 15th day, blood count, biochemical analyses and urinalysis showed no alterations. Neurological examination revealed only positional vertical nystagmus. which remained as a sequel. Discussion: Polymyositis may be accompanied by rhabdomyolysis, characterized by acute muscle necrosis, increased CK and myoglobinuria. The animal had polymyositis of acute onset, with myoglobinuria and elevated CK values, whose presentation included myalgia and muscle weakness. In humans, polymyositis is accompanied by changes in electrocardiographic tracing without clinical alterations. In dogs, the first report that showed cardiac involvement was compatible with myocarditis. The changes in ECG in the present case was attributed to failure in myocardial electrical conduction. The patient also showed signs of brainstem and central vestibular system injuries. Stress myopathy, intoxication, snakebite, infectious, and metabolic diseases were discarded leading to a clinical suspicion as idiopathic origin. Similar to a published case, the patient of this report received symptomatic and supportive treatment, being discharged from the hospital 20 days after the onset of clinical signs. Thus, polymyositis may be accompanied by signs indicative of brainstem injury. Patients with rhabdomyolysis require intense monitoring due to the high risk of developing acute renal failure. Since no causative agent was identified, symptomatic treatment combined with the prevention of possible complications were fundamental for the maintenance of the animal's life.

Fratura de úmero em uma curicaca (Theristicus caudatus) - manejo anestésico e cirúrgico / Humerus fracture in a buff-necked ibis (Theristicus caudatus) - anesthesia and surgical procedures

Background: The medical science of birds, with the exception of aviculture, has a very short history compared to other subdisciplines of veterinary medicine. With this in mind, the current work aims to report the case of a buff-necked ibis with an open fracture of the left humerus, presenting the surgical treatment and anesthetic protocol used, in order to contribute to the avian medical literature. Case: An adult buff-necked ibis (Theristicus caudatus) was referred to the University Veterinary Hospital with an open fracture of the left humeral shaft, 7 days after rescue. During the physical examination, a skin lesion was identified in the fracture area, with signs of low vascularization, devitalization, necrotic tissues, and purulent secretions being noted. On radiographic examination, the fracture was classified as comminuted, with exposure of the left humeral shaft. After evaluating the limb, it was decided to amputate the wing, and 24 h later, the patient was referred to the operating room after fasting for 4 h. As pre-anesthetic medication, ketamine (20 mg/kg) and midazolam (1 mg/kg) were administered, both intramuscularly. Orotracheal intubation was performed, after which the tracheal tube was connected to a Baraka-type gas-free system and the supply of isoflurane was started through a universal vaporizer, diluted in 100% oxygen. For transoperative analgesia, brachial plexus block was performed using 2% lidocaine (2 mg/kg). During the surgical procedure, an incision was made in the skin and subcutaneous tissue in the middle third of the left humerus, and detachment of the greater deltoid muscle was performed with a periosteal elevator, followed by excision of the tensor propatagialis. In the ventrodorsal region, circular ligation of the brachial vein, ulnar vein and artery, and median-ulnar nerve was carried out, and disarticulation of the scapulacoracoid-humeral region. Subsequently, abolition of dead space and a myorrhaphy were performed, followed by demorrhaphy. In the immediate post-operative period, morphine (5 mg/kg), meloxicam (0.1 mg/kg), and enrofloxacin (10 mg/kg) were administered intramuscularly. The patient was discharged from the hospital 6 h after the end of the surgical procedure. Discussion: Interest in the conservation of wild birds is one of the causes of the increased demand for anesthetic and surgical procedures in these species. However, it is a challenge for professionals in the field. The use of analgesics is recommended for reasons of well-being, but also because of the possibility of reducing the concentration of inhalational anesthetics in surgical procedures. Ketamine associated with midazolam promotes sufficient sedation and muscle relaxation in the patient, enabling safe preoperative management, in addition to reducing the amount of inhaled anesthetics used during the transoperative period. Isofluorane promotes safe general anesthesia for birds and has an advantage over injectable drugs, as it provides better dynamic control of anesthetic depth in these species. The brachial plexus block performed is a simple procedure that promotes quality anesthesia and analgesia in the perioperative period. The choice for amputation was due to the absence of musculature for closure, severe skin, muscle, and bone devitalization, and the infectious process in the region, factors that would prevent osteosynthesis. Although amputation through the bone is preferable, the disarticulation technique was used due to the absence of a healthy proximal humeral fragment. The patient's death can be explained by the poor nutritional status the bird was in, as it presented an open fracture with severe contamination, a concomitant injury that occurred during the possible trauma, and the excessive time between the day of the trauma and the day of medical attendance. However, the surgical and anesthetic procedures were adequate and satisfactory for the patient. The importance of identifying and treating diseases secondary to contaminated fractures in these species is emphasized.

Penile fractures in young bulls raised in feedlots in Southern Brazil

Background: Penile fracture is a pathology of young cattle that perform precocious and disordered breeding. The incompatibility of height between males and females and sodomy between males cause a great pressure on the sigmoid flexure and retractor muscle of the penis, which are the main causes and sites of organ injury. This study aimed to describe the epidemiological and pathological aspects of penile fractures observed in young bulls raised in pre-export feedlots (PEFs) in southern Brazil. Cases: In 2 PEFs located in the municipalities of Pelotas (property 1) and Capão do Leão (property 2), 3 male cattle [1 from property 1 and 2 from property 2] presented subcutaneous edema in the foreskin and perineum, associated with dysuria. The evolution of the clinical picture was approximately 20 days in all cases, with evolution to death. The bovine necropsied on property 1 had an increased volume and inguinal edema, involving the penis and scrotal sac. Necrosis of the subcutaneous tissue and local musculature was also observed. The testicles were surrounded by the necrotic tissue, and the right testis was swollen, with flaccid parenchyma adhering to the tunica albuginea. In the necropsy of 1 bull from property 2, an increase in the inguinal volume was observed, with an extensive area of necrosis and edema extending from the prepuce to the caudal musculature of the scrotal sac. There were also marked varicosis in the sigmoid flexure and necrosis of the adjacent region, without the involvement of the corpus cavernosum. During the necropsy of the 2 young bulls, fragments of organs from the abdominal, thoracic, and brain cavities were collected and fixed in 10% buffered formalin. From the bull of the property 2, an anatomical piece consisting of the penis, prepuce, and testicles was also collected and fixed in 10% buffered formalin. After 48 h, the tissue samples were cleaved, embedded in paraffin, cut into 3-µm-thick sections, and stained using hematoxylin and eosin (HE). A histological evaluation of the penile lesions in both cattle revealed intense hemorrhage, congestion, and necrosis of the muscles and tissues adjacent to the corpus cavernosum. In addition to areas of dystrophic calcification, neutrophil and macrophage infiltration was also observed. In the bull from the property 1, an intense edema and proliferation of fibrous tissue surrounding the urethra were noted. There were also marked tubular degeneration and intense infiltration of neutrophils, lymphocytes, and macrophages in the inner portion of the tunica albuginea. Discussion: In the present cases, the diagnosis was based on epidemiological data associated with clinical signs and pathology. The macroscopic lesions observed were probably due to the involvement of blood vessels adjacent to the penis, which suffered trauma during sodomy mating among cattle. These lesions have been described in other reports of this pathology and in diseases, such as acropostitis-phimosis, fibropapilloma of the glans, preputial abscess, and urolithiasis, and the differential diagnosis of these diseases must be carried out, as they have different etiologies. In the bulls of the present study, no lesions were observed in the corpus cavernosum, and this condition was attributed to the presence of varicosis and accumulation of urine in the prepuce, due to the difficulty in exposing the penis. Histologically, there were intense hemorrhage, congestion, and necrosis of the muscles and tissues adjacent to the corpus cavernosum, with the infiltration of neutrophils and macrophages, and areas of dystrophic calcification. The presence of necrotic lesions in tissues adjacent to the penis may be related to hypoxia, vascular lesions, or the action of chemical elements present in the urine. In both cases, vascular lesions were present, which were attributed to the main triggering factor for the disease.

Remote ischemic conditioning improves rat brain antioxidant defense in a time-dependent mechanism

ABSTRACT Purpose To clarify the best protocol for performing remote ischemic conditioning and to minimize the consequences of ischemia and reperfusion syndrome in brain, the present study aimed to evaluate different time protocols and the relation of the organs and the antioxidant effects of this technique. Methods The rat's left femoral artery was clamped with a microvascular clamp in times that ranged from 1 to 5 minutes, according to the corresponding group. After the cycles of remote ischemic conditioning and a reperfusion of 20 minutes, the brain and the left gastrocnemius were collected. The samples were used to measure glutathione peroxidase, glutathione reductase and catalase levels. Results In the gastrocnemius, the 4-minute protocol increased the catalase concentration compared to the 1-minute protocol, but the latter increased both glutathione peroxidase and glutathione reductase compared to the former. On the other hand, the brain demonstrated higher catalase and glutathione peroxidase in 5-minute group, and the 3-minute group reached higher values of glutathione reductase. Conclusions Remote ischemic conditioning increases brain antioxidant capacity in a time-dependent way, while muscle presents higher protection on 1-minute cycles and tends to decrease its defence with longer cycles of intermittent occlusions of the femoral artery.

A new experimental model of intrinsic denervation in ileum from wistar rats through intramural microinjections of benzalkonium chloride / Um novo modelo experimental de desnervação intrínseca em íleo de ratos wistar através de micro injeções intramurais de cloreto de benzalcônio

Extensive literature is available about the intrinsic denervation of segments of the digestive tube through the application of CB in the serosa of the viscera. However, this technique has some disadvantages like causing peritonitis, flanges and high mortality, limiting its use in humans. The aim of the present study was to evaluate the feasibility of benzalkonium chloride (CB) to induce intrinsic chemical denervation, through applications of CB in the intramural ileum of wistar rats, as well as deepen the knowledge about the evolution of neuronal injury caused in the process. We used 40 rats, divided into two groups (control-GC and benzalkonium-GB) of 20 animals each, divided into four sub-groups according to the time of postoperative assessment of 24, 48 hours, 30 and 90 days. The animals were submitted to intramural microinjections of sterile saline solution 0.9% (GC) or benzalkonium chloride (GB) in ileal portion, and subsequent histopathological analysis and immunohistochemistry for evaluation of neuronal injury. A significant decrease (p<0.05) was found of the neuronal myenteric count over time in groups, GB3, GB4 and GB2. The specific positive immunolabeling for H2AX and Caspase-3 confirmed the results obtained in the histopathological evaluation, denoting the ignition of irreversible cell injury in 24 hours, evolving into neuronal apoptosis in 48 hours after application of the CB 0.3%. Under the conditions in which this work was conducted, it can be concluded that the application of CB 0.3% by means of microinjections intramural in the ileal wall is able to induce intrinsic chemical denervation of the diverticulum of wistar rats and that the main mechanism of neuronal death is induction of apoptosis.(AU)

Existe vasta literatura sobre a desnervação intrínseca de segmentos do tubo digestório através da aplicação de CB na serosa da víscera. Entretanto, essa técnica tem a desvantagem de causar peritonite, formação de bridas e alta mortalidade, não sendo factível para eventuais utilizações em humanos. O objetivo do presente estudo foi avaliar a viabilidade do Cloreto de benzalcônio (CB) induzir desnervação química intrínseca, por meio de aplicações intramurais em íleo de ratos wistar, além de aprofundar o conhecimento sobre a evolução da lesão neuronal causada neste processo. Foram utilizados 40 ratos, distribuídos em dois grupos (controle- GC e benzalcônio- GB) de 20 animais cada, subdivididos em quatro subgrupos de acordo com o tempo de avaliação pós-operatória de 24, 48 horas, 30 e 90 dias. Os animais foram submetidos à microinjeções intramurais de solução salina estéril 0,9% (GC) ou de cloreto de benzalcônio (GB) em porção ileal, e posterior análise histopatológica e imuno-histoquímica, para avaliação da lesão neuronal. Houve diminuição significativa (p<0,05) na contagem neuronal mientérica ao longo do tempo nos grupos GB2, GB3 e GB4. A imunomarcação específica positiva para H2AX e Caspase-3 confirmou os resultados obtidos na avaliação histopatológica, denotando início da lesão celular irreversível em 24 horas, evoluindo para apoptose neuronal em 48 horas após a aplicação do CB 0,3%. Nas condições em que este trabalho foi conduzido, é possível concluir que a aplicação de CB 0,3% por meio de microinjeções intramurais na parede ileal é capaz de induzir desnervação química intrínseca da porção ileal de ratos wistar e que o principal mecanismo de morte neuronal é a indução de apoptose.(AU)

A new experimental model of intrinsic denervation in ileum from wistar rats through intramural microinjections of benzalkonium chloride / Um novo modelo experimental de desnervação intrínseca em íleo de ratos wistar através de micro injeções intramurais de cloreto de benzalcônio

Extensive literature is available about the intrinsic denervation of segments of the digestive tube through the application of CB in the serosa of the viscera. However, this technique has some disadvantages like causing peritonitis, flanges and high mortality, limiting its use in humans. The aim of the present study was to evaluate the feasibility of benzalkonium chloride (CB) to induce intrinsic chemical denervation, through applications of CB in the intramural ileum of wistar rats, as well as deepen the knowledge about the evolution of neuronal injury caused in the process. We used 40 rats, divided into two groups (control-GC and benzalkonium-GB) of 20 animals each, divided into four sub-groups according to the time of postoperative assessment of 24, 48 hours, 30 and 90 days. The animals were submitted to intramural microinjections of sterile saline solution 0.9% (GC) or benzalkonium chloride (GB) in ileal portion, and subsequent histopathological analysis and immunohistochemistry for evaluation of neuronal injury. A significant decrease (p<0.05) was found of the neuronal myenteric count over time in groups, GB3, GB4 and GB2. The specific positive immunolabeling for H2AX and Caspase-3 confirmed the results obtained in the histopathological evaluation, denoting the ignition of irreversible cell injury in 24 hours, evolving into neuronal apoptosis in 48 hours after application of the CB 0.3%. Under the conditions in which this work was conducted, it can be concluded that the application of CB 0.3% by means of microinjections intramural in the ileal wall is able to induce intrinsic chemical denervation of the diverticulum of wistar rats and that the main mechanism of neuronal death is induction of apoptosis.(AU)

Existe vasta literatura sobre a desnervação intrínseca de segmentos do tubo digestório através da aplicação de CB na serosa da víscera. Entretanto, essa técnica tem a desvantagem de causar peritonite, formação de bridas e alta mortalidade, não sendo factível para eventuais utilizações em humanos. O objetivo do presente estudo foi avaliar a viabilidade do Cloreto de benzalcônio (CB) induzir desnervação química intrínseca, por meio de aplicações intramurais em íleo de ratos wistar, além de aprofundar o conhecimento sobre a evolução da lesão neuronal causada neste processo. Foram utilizados 40 ratos, distribuídos em dois grupos (controle- GC e benzalcônio- GB) de 20 animais cada, subdivididos em quatro subgrupos de acordo com o tempo de avaliação pós-operatória de 24, 48 horas, 30 e 90 dias. Os animais foram submetidos à microinjeções intramurais de solução salina estéril 0,9% (GC) ou de cloreto de benzalcônio (GB) em porção ileal, e posterior análise histopatológica e imuno-histoquímica, para avaliação da lesão neuronal. Houve diminuição significativa (p<0,05) na contagem neuronal mientérica ao longo do tempo nos grupos GB2, GB3 e GB4. A imunomarcação específica positiva para H2AX e Caspase-3 confirmou os resultados obtidos na avaliação histopatológica, denotando início da lesão celular irreversível em 24 horas, evoluindo para apoptose neuronal em 48 horas após a aplicação do CB 0,3%. Nas condições em que este trabalho foi conduzido, é possível concluir que a aplicação de CB 0,3% por meio de microinjeções intramurais na parede ileal é capaz de induzir desnervação química intrínseca da porção ileal de ratos wistar e que o principal mecanismo de morte neuronal é a indução de apoptose.(AU)

Microbiological, Physicochemical, and Histological Analyses of Broiler Carcasses with Cachexia

Broiler carcasses presenting cachexia, as determined by the federal inspection agency, were submitted to microbiological, physicochemical, and histological analyses. Over a 2-month period, 278 carcasses from straight-run flocks of 43- to 48-d-old Cobb broilers from two different farms were condemned due to cachexia and subjected to gross examination in the final inspection sector. Out of the 278 carcasses, 131 did not present any gross signs of infection, and were submitted to microbiological analyses (n=83), physicochemical analyses (n=28), and viscera and muscle samples of 20 carcasses were evaluated by histology. The microbiological results of cachectic carcasses complied with the current legislation on the consumption of fresh poultry meat. Lower lipid and higher protein, moisture, and volatile matter contents, and higher pH (p 0.05) were determined in the cachectic than in the control carcasses from a same flock, but no ash content differences. All carcasses were negative for hydrogen sulfide. The histological analysis showed that65% of the cachectic carcasses did not have any muscle injury, 20% showed mild hyalinization and 15% moderate proliferation of fibrous connective tissue. The findings of this study indicate the importance of further technical and scientific studies on the utilization of cachectic broiler carcasses for the manufacture of edible products, rather than whole carcasses rendering, thereby preventing the resulting economic losses.(AU)

Microbiological, Physicochemical, and Histological Analyses of Broiler Carcasses with Cachexia

Broiler carcasses presenting cachexia, as determined by the federal inspection agency, were submitted to microbiological, physicochemical, and histological analyses. Over a 2-month period, 278 carcasses from straight-run flocks of 43- to 48-d-old Cobb broilers from two different farms were condemned due to cachexia and subjected to gross examination in the final inspection sector. Out of the 278 carcasses, 131 did not present any gross signs of infection, and were submitted to microbiological analyses (n=83), physicochemical analyses (n=28), and viscera and muscle samples of 20 carcasses were evaluated by histology. The microbiological results of cachectic carcasses complied with the current legislation on the consumption of fresh poultry meat. Lower lipid and higher protein, moisture, and volatile matter contents, and higher pH (p 0.05) were determined in the cachectic than in the control carcasses from a same flock, but no ash content differences. All carcasses were negative for hydrogen sulfide. The histological analysis showed that65% of the cachectic carcasses did not have any muscle injury, 20% showed mild hyalinization and 15% moderate proliferation of fibrous connective tissue. The findings of this study indicate the importance of further technical and scientific studies on the utilization of cachectic broiler carcasses for the manufacture of edible products, rather than whole carcasses rendering, thereby preventing the resulting economic losses.

UTILIZAÇÃO DE CÉLULA-TRONCO MESENQUIMAL ASSOCIADA AO EXERCÍCIO COMO ABORDAGEM TERAPÊUTICA PARA OTIMIZAR A REGENERAÇÃO DE NERVO ISQUIÁTICO EM CAMUNDONGOS

Lesões nervosas periféricas (PNI) podem causar dor neuropática, perda de massa muscular e um longo tempo para reabilitação. A capacidade regenerativa e a recuperação funcional da dependem do grau da lesão, da idade e da distância entre o dano do nervo e o músculo-alvo. Principalmente, essa regeneração é pobre e limitada. O exercício é usado em ensaios clínicos para reduzir a atrofia muscular e estimular a plasticidade. A terapia com células-tronco mesenquimais (MSC) promove a produção de neurotrofinas, substratos e um microambiente ideal para o reparo de tecidos. Nós analisamos os efeitos regenerativos da terapia com MSC e exercícios de natação (SW) no modelo de compressão do nervo isquiático de camundongo. As MSC foram injetadas (300ul, 1x106, ip.) 7 dias após a lesão e o SW foi iniciado 14 dias após a lesão, realizado 3 vezes por semana durante 20 minutos. Foram estudados 4 grupos: DMEM (inoculação de meio de cultura), DMEM + SW (meio de cultura e exercício), MSC (apenas inoculação de células-tronco mesenquimais), MSC + SW (associação de exercício e terapia celular). A análise funcional (LSS e SFI) foi feita antes da cirurgia, um dia após e semanalmente durante 8 semanas, quando os animais foram sacrificados para avaliação morfológica (microscopia de luz e eletrônica). Nossos resultados mostraram que a terapia combinada com MSC e SW apresentou um maior número de fibras mielinizadas e não mielinizadas e um grande número de fibras dentro da razão G ideal. Os resultados mostraram maior sobrevida dos neurônios motores e sensoriais e melhor recuperação das funções motoras. Concluímos que o uso de células-tronco mesenquimais e a natação aceleram a regeneração axonal promovendo a recuperação precoce das funções motoras e sensoriais.

Peripheral nervous (PN) injuries can cause neuropathic pain, loss of muscle mass, and a long time to rehabilitation. The PN regenerative capacity and functional recovery are dependent on the degree of PN injuries, age, and the distance from the nerve damage to the target muscle. Mostly, this regeneration is poor and limited. Exercise is used in clinical trials to reduce muscle atrophy and stimulate plasticity. The mesenchymal stem cells (MSC) therapy combines regenerative and tissue replacement to promote neurotrophins production and substrates for regeneration. We analyzed the regenerative effects of MSC therapy and swimming exercise (SE) in the mouse sciatic nerve compression model. MSC was injected (300ul, 1x106,ip.) 7 days after injury. SE was started 14 days after injury, performed 3 times a week for 20 minutes. We studied 4 groups: DMEM (cell culture medium injected), DMEM+SW (DMEM and SE), MSC (mesenchymal stem cells injected), MSC+SW (combined SE and cell therapy). Functional analysis (LSS and SFI) was made before surgery, one day after, and weekly for 8 weeks when animals were euthanized for morphological assessment (light microscopy and electronica). Our results showed that combined MSC therapy and SE presented a higher number of myelinated and unmyelinated fibers and a large number of fibers within the ideal G-ratio. The results showed motor and sensory neurons survival and better recovery of motor and sensory functions. We conclude that the use of mesenchymal stem cells and swimming accelerates axonal regeneration promoting early recovery of motor and sensory functions.

Avaliações clínico-patológica e imunohistoquímica de lesões cardíacas em gatos (Felis catus) com doença renal crônica

A troponina I (cTnI) é uma proteína expressa unicamente pelas células do miocárdio, e portanto um marcador sensível e específico de lesão cardíaca em humanos, cães, gatos e equinos. A troponina I cardíaca não se expressa no músculo esquelético humano durante o desenvolvimento fetal, após trauma do músculo esquelético ou durante a regeneração desse tipo de músculo. Ao contrário da CK-MB, a troponina I cardíaca é altamente específica para o tecido miocárdico, não é detectável no sangue de pessoas sadias, mostra um aumento proporcionalmente bem maior acima dos valores limite, nos casos de infarto do miocárdio pode permanecer elevada por 7 a 10 dias após o episódio agudo. Para a espécie felina, os valores de referência normais para cTnI sérica variam de 0,03 a 0,16 ng / ml . Sabe-se que o coração e os rins mantêm a homeostase hemodinâmica por meio de uma estreita relação que regula o débito cardíaco, o volume e o tônus vascular. Com base nestas informações, o objetivo deste estudo foi avaliar a ocorrência de danos aos cardiomiócitos de felinos com doença renal crônica (DRC) através da dosagem sérica de cTnI e detectar lesões em cardiomiócitos que, por vezes não apresentam sinais morfológicos de agressão, além de demonstrar que as áreas agredidas podem ser maiores que o evidenciado na coloração hematoxilina & eosina (H.E.). Foram utilizados 20 felinos com DRC (12 fêmeas e 8 machos) a partir do estágio II, de acordo com o estadiamento proposto pela IRIS, independente de raça, sexo ou idade. Anterior à utilização destes animais, foi solicitada autorização prévia do tutor para realização de exame clínico seguido de colheita do sangue. Como critério de inclusão, os pacientes deveriam apresentar azotemia (Ref.: 1,6mg/dL). Foram excluídos os animais que apresentavam doença cardíaca primária ou outras condições que pudessem levar ao aumento da cTnI sérica, tais como cardiomiopatia hipertrófica, hipertireoidismo, neoplasia cardíaca primária ou metastática. A avaliação dos níveis séricos de cTnI foi realizada nos 20 gatos; destes, 10 apresentaram valores elevados de troponina sérica acima de 0,16ng/mL. Dos 10 animais que apresentaram os valores de cTnI sérica elevados (Ref.:0,03 0,16ng/mL), dois encontravam-se no estágio II e oito animais no estágio IV da IRIS. Para o exame histológico e imuno-histoquímico, foram coletadas regiões do ápice, septo interventricular, ventrículo esquerdo, ventrículo direito, músculo papilar esquerdo, músculo papilar direito, átrio direito e átrio esquerdo do coração. O exame imuno-histoquímico revelou, em dois animais, marcada perda de troponina em grandes grupos de cardiomiócitos e, em 9 animais houve variação de sutil a leve no padrão de marcação pelo anticorpo antitroponina C com miócitos isolados apresentando perda marcada da imunorreatividade. Em alguns casos havia diversos pequenos grupos de miócitos que tinham marcada diminuição de imunorreatividade principalmente nas regiões do ápice e músculo papilar; essas áreas correspondiam a células com alterações imperceptíveis ou muito discretas no H.E. Os resultados da avaliação imuno-histoquímica confirmaram a presença de alterações regressivas, ou seja, a técnica foi capaz de detectar lesões muito precoces (regiões do miocárdio sem sinais morfológicos de agressão), e demonstrou que as áreas agredidas/lesadas eram maiores que o evidenciado no H.E. O uso de troponinas como importante indicador imunohistoquímico de lesão cardíaca já foi demonstrado caninos, bovinos e ovinos, entretanto não havia estudos em felinos e, no presente estudo, todos os cardiomiócitos com características histológicas de morte celular, não apresentaram marcação anti-troponina, o que demonstra alta sensibilidade do marcador para a espécie. A partir desses dados, observa-se que 80% dos animais que apresentaram elevação nos níveis séricos de cTnI encontravam-se no estágio IV, ou seja, quanto maior o estágio da DRC, maior a probabilidade de apresentar lesão miocárdica. Outro dado observado nos 10 animais que apresentaram valores de cTnI elevados, foi que destes, 4 eram machos e 6 eram fêmeas e, dos dois animais dentro do estágio II, havia um macho e uma fêmea. Já no estágio IV, havia três machos e cinco fêmeas, o que sugere não haver correlação entre o sexo e o grau de lesão miocárdica em felinos com DRC. Este estudo demonstra que a utilização de biomarcadores de lesão miocárdica pode auxiliar os veterinários a aprimorarem o julgamento clínico nas decisões sobre condutas terapêuticas nos pacientes com DRC.

roponin I (cTnI) is a protein expressed solely by myocardial cells, and therefore a sensitive and specific marker of cardiac injury in humans, dogs, cats and horses. Cardiac troponin I is not expressed in human skeletal muscle during fetal development, after trauma of the skeletal muscle, or during regeneration of such muscle. Unlike CK-MB, cardiac troponin I is highly specific for myocardial tissue, is not detectable in the blood of healthy people, shows a proportionally larger increase above threshold values, in cases of myocardial infarction may remain elevated for 7 10 days after the acute episode. For feline species, normal reference values for serum cTnI range from 0.03 to 0.16 ng / ml. The heart and kidneys are known to maintain hemodynamic homeostasis through a close relationship that regulates cardiac output, volume and vascular tone. Based on this information, the aim of this study was to evaluate the occurrence of damage to feline cardiomyocytes from chronic kidney disease (CKD) by measuring serum cTnI and to detect lesions in cardiomyocytes that sometimes show no morphological signs of aggression, besides demonstrate that the areas affected may be larger than that evidenced by hematoxylin & eosin (HE) staining. Twenty CKD felines (12 females and 8 males) from stage II were used, according to the proposed IRIS staging, regardless of breed, sex or age. Prior to the use of these animals, prior authorization from the guardian was requested for clinical examination followed by blood collection. As inclusion criteria, patients should have azotemia (Ref .: 1.6mg / dL). Animals with primary heart disease or other conditions that could lead to increased serum cTnI, such as hypertrophic cardiomyopathy, hyperthyroidism, primary or metastatic cardiac neoplasia, were excluded. Serum cTnI levels were evaluated in 20 cats; Of these, 10 had elevated serum troponin values - above 0.16 ng / mL. Of the 10 animals that had elevated serum cTnI values (Ref.:0.03 - 0.16ng / mL), two were in stage II and eight were in IRIS stage IV. For histological and immunohistochemical examination, regions of the apex, interventricular septum, left ventricle, right ventricle, left papillary muscle, right papillary muscle, right atrium and left atrium of the heart were collected. Immunohistochemical examination revealed marked loss of troponin in large groups of cardiomyocytes in two animals, and in 9 animals there was subtle to slight variation in the pattern of antitroponin C antibody labeling with isolated myocytes showing marked loss of immunoreactivity. In some cases there were several small groups of myocytes that had markedly decreased immunoreactivity mainly in the apex and papillary muscle regions; these areas corresponded to cells with imperceptible or very slight changes in HE. The results of the immunohistochemical evaluation confirmed the presence of regressive changes, ie, the technique was able to detect very early lesions (myocardial regions without morphological signs of aggression), and demonstrated that the areas injured / injured were larger than that evidenced in HE. The use of troponins as an important immunohistochemical indicator of cardiac injury has already been demonstrated in canines, cattle and sheep, however there were no studies in felines and, in the present study, all cardiomyocytes. with histological characteristics of cell death, did not show anti-troponin labeling, which demonstrates high marker sensitivity for the species. From these data, it is observed that 80% of the animals that presented increase in serum cTnI levels were in stage IV, that is, the higher the stage of CKD, the greater the probability of presenting myocardial injury. Another finding observed in the 10 animals with high cTnI values was that of these 4 were male and 6 were female and of the two animals within stage II there was a male and a female. In stage IV, there were three males and five females, suggesting that there is no correlation between sex and the degree of myocardial injury in cats with CKD. This study demonstrates that the use of myocardial injury biomarkers may help veterinarians to improve clinical judgment in decisions about therapeutic management in patients with CKD.

Indicators of stress in tilapia subjected to different stunning methods / Indicadores de estresse em tilápias submetidas a diferentes métodos de atordoamento

Nile tilapia is one of the main fish species cultivated in the World. However, there are few studies evaluating the effects of stunning methods on the stress of this fish. The aim of this study was to evaluate the effect of electrical stunning (ES), CO2 narcosis (CN), and iced water (IW) on indicators of stress in tilapia, e.g., muscle adenosine triphosphate (ATP), enzymatic antioxidants (catalase, lactate dehydrogenase and glutathione reductase) and plasma cortisol. The muscle ATP was highest (P 0.05) in fish stunned by CN. However, the cortisol level was higher (P 0.05) in tilapia stunned by IW and CN. The levels of catalase in the white muscles were higher (P 0.05) of catalase were observed in tilapia stunned by ES and IW. Lactate dehydrogenase levels in the white muscles were higher (P 0.05) in fish stunned by ES and lower (P 0.05) in fish stunned by CN. In the red muscles, lactate dehydrogenase levels were higher (P 0.05) in fish stunned by IW in comparison to ES and CN. Therefore, tilapia stunned using CN causes less stress compared to ES or IW and is in accordance with good practices of animal welfare.(AU)

A tilápia do Nilo é uma das principais espécies cultivadas no mundo. No entanto, existem poucos estudos avaliando os efeitos de métodos de atordoamento no estresse deste peixe. O objetivo deste estudo foi avaliar o efeito do atordoamento elétrico (AE), narcose por CO2 (NC) ou água e gelo (AG) nos indicadores de estresse de tilápias, tais como: adenosina trifosfato (ATP), enzimas antioxidantes (catalase, lactato desidrogenase e glutationa redutase) e cortisol plasmático. O ATP muscular foi mais alto (P 0.05) nos peixes atordoados por NC. No entanto, o nível de cortisol foi mais alto (P 0.05) nos peixes atordoados por AG e NC. Os níveis de catalase no músculo branco foi mais alto (P 0.05) nos peixes atordoados por AE em relação aos peixes atordoados por NC e AG. Para o músculo vermelho, os níveis mais altos (P 0.05) de catalase foram observados nas tilápias atordoadas por AE e AG. Os níveis de lactato desidrogenase no músculo branco foram mais altos (P 0.05) nos peixes atordoados por AE e mais baixos (P 0.05) nos peixes atordoados por NC. No músculo vermelho, os níveis de lactato desidrogenase foram mais altos (P 0.05) nos peixes atordoados por AG em comparação ao AE e NC. Portanto, tilápias atordoadas usando NC causam menos estresse em comparação ao AE e AG e está de acordo com as boas práticas de bem estar animal.(AU)

Indicators of stress in tilapia subjected to different stunning methods / Indicadores de estresse em tilápias submetidas a diferentes métodos de atordoamento

Nile tilapia is one of the main fish species cultivated in the World. However, there are few studies evaluating the effects of stunning methods on the stress of this fish. The aim of this study was to evaluate the effect of electrical stunning (ES), CO2 narcosis (CN), and iced water (IW) on indicators of stress in tilapia, e.g., muscle adenosine triphosphate (ATP), enzymatic antioxidants (catalase, lactate dehydrogenase and glutathione reductase) and plasma cortisol. The muscle ATP was highest (P 0.05) in fish stunned by CN. However, the cortisol level was higher (P 0.05) in tilapia stunned by IW and CN. The levels of catalase in the white muscles were higher (P 0.05) of catalase were observed in tilapia stunned by ES and IW. Lactate dehydrogenase levels in the white muscles were higher (P 0.05) in fish stunned by ES and lower (P 0.05) in fish stunned by CN. In the red muscles, lactate dehydrogenase levels were higher (P 0.05) in fish stunned by IW in comparison to ES and CN. Therefore, tilapia stunned using CN causes less stress compared to ES or IW and is in accordance with good practices of animal welfare.

A tilápia do Nilo é uma das principais espécies cultivadas no mundo. No entanto, existem poucos estudos avaliando os efeitos de métodos de atordoamento no estresse deste peixe. O objetivo deste estudo foi avaliar o efeito do atordoamento elétrico (AE), narcose por CO2 (NC) ou água e gelo (AG) nos indicadores de estresse de tilápias, tais como: adenosina trifosfato (ATP), enzimas antioxidantes (catalase, lactato desidrogenase e glutationa redutase) e cortisol plasmático. O ATP muscular foi mais alto (P 0.05) nos peixes atordoados por NC. No entanto, o nível de cortisol foi mais alto (P 0.05) nos peixes atordoados por AG e NC. Os níveis de catalase no músculo branco foi mais alto (P 0.05) nos peixes atordoados por AE em relação aos peixes atordoados por NC e AG. Para o músculo vermelho, os níveis mais altos (P 0.05) de catalase foram observados nas tilápias atordoadas por AE e AG. Os níveis de lactato desidrogenase no músculo branco foram mais altos (P 0.05) nos peixes atordoados por AE e mais baixos (P 0.05) nos peixes atordoados por NC. No músculo vermelho, os níveis de lactato desidrogenase foram mais altos (P 0.05) nos peixes atordoados por AG em comparação ao AE e NC. Portanto, tilápias atordoadas usando NC causam menos estresse em comparação ao AE e AG e está de acordo com as boas práticas de bem estar animal.

EFEITOS DO FENILPROPRIONATO DE NANDROLONA EM LESÕES ÓSSEAS POR ESCAREAÇÃO INDUZIDAS EXPERIMENTALMENTE NA TÍBIA DE RATOS WISTAR (Rattus norvegicus)

O Fenilproprionato de nandrolona (FPP) é um esteroide andrógeno anabolizante (EAA), de baixa atividade androgênica, e aprimorada ação anabólica, com atuação no sistema musculoesquelético como ganho de massa muscular, óssea e resistência a fraturas. Acredita-se que eles estimulam a diferenciação e proliferação dos osteoblastos além da inibição de osteoclastos, agindo por meio de fatores de crescimento promovendo formação óssea periosteal precoce e densa. Objetivou-se explorar os possíveis efeitos positivos do tratamento com Fenilproprionato de Nandrolona no reparo de fraturas e qualidade óssea, comparando o processo de consolidação da lesão criada na tíbia de ratos Wistar, dos animais submetidos ao tratamento com Fenilproprionato de Nandrolona com aqueles que receberam placebo e o controle, através de análise histológica descritiva e ensaio biomecânico, buscando, principalmente, uma alternativa menos cruenta no tratamento de uniões retardadas e não-uniões. Foram utilizados 50 ratos Wistar submetidos à lesão cirúrgica no terço proximal de ambas as tíbias, com o auxílio de uma broca de aço cirúrgico de 1,5mm de diâmetro. Após o procedimento cirúrgico, os animais foram divididos em cinco grupos que receberam injeções por via intramuscular: grupo G10 - FPP na dose de 10mg/kg; grupo G20 - FPP na dose de 20mg/kg; grupo GPI recebera placebo de óleo mineral no volume de 0,03 mL, volume equivalente a dose de 10 mg/kg de FPP, e o grupo GPII que recebera placebo de óleo mineral no volume de 0,06 mL, volume equivalente a dose de 20 mg/kg de FPP; e grupo GC que foi o controle não recebendo qualquer tratamento, apenas submetido a lesão. Os cinco grupos foram subdivididos em dois subgrupos, de 05 animais cada, com eutanásia programada aos oito e 16 dias de pós-operatório, respectivamente. Os resultados da análise histológica descritiva apresentaram diferença estatística entre os grupos tratados com nandrolona em relação aos demais nos parâmetros avaliados neoformação óssea e atividade osteoblastica, e não apresentou diferença estatística quanto a atividade osteoclastica. Já os resultados do ensaio biomecânico foram favoráveis nos grupos tratados com nandrolona G10 e G20 com 16 dias em relação aos demais quanto a força máxima e rigidez. Desta forma, conclui-se que a nandrolona foi capaz de promover maior neoformação óssea e propiciou maior rigidez óssea e resistência a força máxima na dose de 10mg/kg em ratos Wistar estudados.

Fenilproprionation of nandrolone (FPP) is an anabolic androgenic steroid (EAA), with low androgenic activity and enhanced anabolic action, in musculoskeletal system such as muscle mass gain, mechanics and resistance to fractures. It is believed that they stimulate the differentiation and proliferation of osteoblasts in addition to inhibiting osteoclasts, acting through growth factors promoting early and dense periosteal bone formation. Objective to explore the possible positive effects of treatment with nandrolone phenylproprionate, on fracture repair and bone quality, comparing the application process of the lesion created on the tibia of Wistar rats, of animals used for treatment with nandrolone phenylproprionate. with those who received placebo and control, it mainly seeks a less cruel alternative in the treatment of retardad union and non-unions. Fifty Wistar rats were used that used surgical lesions in the proximal third of areas such as the tibia, with the aid of a 1.5 mm diameter surgical steel drill. After the surgical procedure, the animals were divided into five groups that received intramuscular injections: group G10 - FPP at a dose of 10mg / kg; group G20 - FPP at a dose of 20mg / kg; GPI group receives a placebo of mineral oil without volume of 0.03 mL, volume equivalent to a dose of 10 mg / kg of FPP, and the GPII group receives a placebo of mineral oil without volume of 0.06 mL, volume equivalent to a dose 20 mg / kg of FPP; The CG group that was the control did not receive any treatment, only submitted to injury. The five groups were subdivided into two subgroups, of 05 animals each, commuting scheduled for eight and 16 postoperative days, respectively. The results of the descriptive histological analysis showed a statistical difference between the groups treated the groups treated with nandrolone in relation to the others in the parameters evaluated for boné formation and osteoblast activity, and there was no statistical difference in terms os osteoclastic activity. The results of the biomechanical test were favoreble in the groups treated with nandrolone G10 and G20 with 16 days compared to the others regarding maximum strength and stiffness. Thus, it is concluded thet nandrolone was able to promote greater bone neofomation and provided greater bone rigidity and resistence to maximum strenght at a dose of 10mg/kg in Wistar rats studied.

Serum dosage of CPK-MB in dogs with ST deviation by chemiluminescence / A dosagem sérica de CPK-MB em cães com desvio ST por quimiluminescência

Although frequently in humans, hypoxic and ischemic heart diseases are poorly documented in dogs, with only few reports of acute myocardial infarction (AMI) in this species. Some electrocardiographic findings might suggest myocardium hypoxia/ischemia, like ST segment elevation or depression, but there are no studies showing whether deviations in ST segment are associated to myocardial injury and serum increase of creatine phosphokinase (CPK-MB). In order to investigate possible myocardial cells injury in poor perfusion conditions, 38 dogs were studied, 20 with normal electrocardiogram and 18 with ST segment elevation or depression, recorded in lead II, at a paper speed of 50 mm/sec and N sensibility (1mV=1cm). Serum measurement of creatine phosphokinase isoenzyme MB (CPK-MB) in normal dogs (group 1) determined control values (in ng/mL), which were compared to those obtained from dogs with deviation (group 2), which allowed confirmation or not of myocardial injury. CPK-MB mean values obtained from dogs in groups 1 and 2 were 0.540ng/ml (SD±0.890)ng/mL and 0.440ng/mL (SD±1.106), respectively. At a significance level of 5%, the relation of CPK-MB with age, mass and total creatine phosphokinase (CPK-T) was not significant in groups 1 and 2. CPK-MB showed no difference, at 5% level, between groups 1 and 2. In conclusion, it is possible to use the human chemiluminescent immunometric assay kit in canine species and that hypoxia/ischemia revealed by ST segment deviation does not mean significant myocardium injury.(AU)

Embora frequente em humanos, as doenças hipóxicas e isquêmicas do coração são pouco relatadas em cães, com poucos relatos de infarto agudo do miocárdio (IAM) nesta espécie. Alguns achados no eletrocardiograma podem sugerir hipóxia/isquemia miocárdica, como a elevação ou depressão do segmento ST, mas não há estudos que mostram se os desvios do segmento ST estão associados a lesões miocárdicas e aumento sérico da creatinafosfoquinase (CPK-MB). A fim de investigar possíveis lesões nas células miocárdicas em condições de má perfusão, 38 cães foram estudados, 20 com eletrocardiograma normal e 18 com elevação ou depressão do segmento ST, registrados em papel, na derivação II, velocidade de 50 mm/s e sensibilidade N (1mV = 1cm). A mensuração da creatinafosfoquinase isoenzima MB (CPK-MB) em cães normais (grupo 1) determinou os valores controle (em ng/ml), que foram comparados com os obtidos a partir de cães com desvio (grupo 2), permitindo a confirmação ou não da lesão miocárdica. Os valores médios de CPK-MB obtidos de cães nos grupos 1 e 2 foram 0,540ng/ml (DP±0,890) e 0,440ng / ml (DP ± 1.106), respectivamente. A um nível de significância de 5%, a relação de CPK-MB com a idade, massa e creatinofosfoquinase total (CPK-T) não foi significativa nos grupos 1 e 2. Não houve diferenças na CPK-MB, ao nível de 5%, entre os grupos 1 e 2. Conclui-se que é possível utilizar o kit de ensaio imunométrico por quimioluminescência humano na espécie canina e que a hipoxia/isquemia revelada pelos desvios do segmento ST, não significa lesão miocárdica.(AU)

AVALIAÇÃO DE MATRIZES DESCELULARIZADAS PARA REGENERAÇÃO MUSCULAR

RESUMO MIRANDA, C.M.F.C. Titulo em português: Avaliação de Matrizes Descelularizadas para Regeneração Muscular. [Título em inglês: Evaluation of Decellularized Matrices for Muscle Regeneration]. 2018. 132 f. Tese (Doutorado em Ciências) Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, 2018. Matrizes extracelulares descelularizadas (dMEC) têm sido utilizadas com sucesso como biomateriais biológicos para regeneração tecidual, já que apresentam vantagens como semelhança estrutural com os tecidos naturais, biocompatibilidade , biodegradabilidade, capacidade de sinalização e homeostase tecidual. O primeiro objetivo deste trabalho foi avaliar a eficácia de quatro protocolos para descelularização de músculo esquelético murino, objetivando a produção de matrizes extracelulares (MEC) acelulares para potenciais aplicações na engenharia de tecidos. Músculos tibiais foram coletados e congelados a -20°C ou armazenados a temperatura ambiente, seguido por descelularização em soluções contendo reagentes químicos EDTA + Tris, SDS e Triton X-100, os quais foram aplicados em diferentes sequências e analisados de acordo com modificações macroscópicas causadas nos tecidos, remoção de conteúdo celular e genético das matrizes e na capacidade de preservar a composição protéica e estrutura tridimensional da MEC. Observou-se a otimização da remoção de conteúdo celular, além da preservação da ultraestrutura e composição da MEC nos protocolos com congelação prévia a -20°C e descelularização inicial com a solução SDS, quando comparado àqueles que iniciaram com EDTA + Tris. O segundo objetivo deste trabalho foi comparar a biocompatibilidade da dMEC de músculo murino e placenta canina em modelo murino de injúria muscular. As dMEC foram produzidas a partir da placenta canina (1% SDS, 5 mM EDTA + 0,05% Tripsina, 1% Triton X-100) e do músculo esquelético tibial de ratos Wistar (Congelação a -20°C, 1% SDS, 5 mM EDTA + 50mM Tris, 1% Triton X-100). A lesão muscular foi realizada a partir de uma incisão de 1cm e divulsão na região média do músculo tibial; então, as dMEC foram implantadas. Um grupo cirúrgico, sem o implante, foi usado como um controle. Os animais foram eutanasiados depois de 3, 15 e 45 dias. Análise histológica e imunohistoquímica foram realizadas para avaliar a morfologia tecidual e a presença de infiltrado inflamatório e células em proliferação (CD163 e PCNA). No 3° dia após a implantação, uma intensa reação inflamatória (CD163+) e proliferação celular (PCNA+) foram observadas, adjacentes ao biomaterial, em ambas as matrizes. No 15° dia, o biomaterial de MEC placentária ainda preservou o infiltrado celular (CD163+ e PCNA+) e iniciou o processo de absorção do biomaterial, enquanto que a dMEC do músculo já estava completamente absorvida. Finalmente, depois dos 45 dias, ambos os biomateriais foram absorvidos e o músculo estava completamente cicatrizado, sem fibrose. Conclui-se que congelar as amostras a -20°C anteriormente ao processamento e submeter à incubação inicial em solução de 1% SDS (Protocolo 2B) favoreceu a descelularização de músculo esquelético murino. Além disso, dMEC xenogênicas e alogênicas foram reabsorvidas e apresentaram processos de integração distintos em modelo não-crítico de lesão muscular esquelética, sugerindo que eventuais alterações sistêmicas, observadas a longo prazo, sejam avaliadas, bem como, a validação da utilização destas matrizes em modelos de perda muscular volumétrica. Palavras-chave: Descelularização, biomaterial, músculo esquelético, placenta, biocompatibilidade.

ABSTRACT MIRANDA, C.M.F.C. Título em inglês: Evaluation of Decellularized Matrices for Muscle Regeneration. [título em português: Avaliação de Matrizes Descelularizadas para Regeneração Muscular]. 2018. 132 f. Tese (Doutorado em Ciências) Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, 2018. Decelularized extracellular matrices (dECM) have been successfully used as biological biomaterials for tissue engineering, since they present advantages such as structural similarity with natural tissues, biocompatibility, biodegradability, signaling capacity and tissue homeostasis. The first aim of this study was to evaluate the efficacy of four protocols for the decellularization of murine skeletal muscle for the production of acellular extracellular matrices (ECM) for potential applications in tissue engineering. Tibial muscle was frozen at -20°C or stored at room temperature, followed by decellularization in solutions containing EDTA + Tris, SDS and Triton X-100 chemical reagents, which were applied in different sequences and analyzed according to macroscopic modifications , removal of cellular and genetic content and the ability to preserve the ECM composition and three-dimensional structure. Thus, the optimization of cellular removal was observed, as well as the preservation of the ECM ultrastructure and composition in the protocols with freezing at -20°C and initial decellularization with the SDS solution, when compared to those starting with EDTA + Tris. The second aim of this study was to compare the biocompatibility of the dECM of murine muscle and canine placenta in murine model of muscle injury. Decellularized ECM were produced from the canine placenta (1% SDS, 5 mM EDTA + 0.05% Trypsin, 1% Triton X-100) and the tibial muscle of Wistar rats (freezing at -20°C, 1% SDS , 5 mM EDTA + 50 mM Tris, 1% Triton X-100). The muscle injury was performed followed a 1 cm incision and divulsion in the middle region of the tibial muscle, where the dECM were implanted. A surgical group, without the implant, was used as a control. The animals were euthanized after 3, 15 and 45 days. Histological and immunohistochemistry analyses were performed to evaluate the tissue morphology and the presence of inflammatory infiltrate and cells in proliferation (CD163 and PCNA). On the 3rd day after implantation, an intense inflammatory reaction (CD163+) and cell proliferation (PCNA+) were observed, adjacent to the biomaterial, in both matrices. On 15th day, the placenta biomaterial still preserved the cellular infiltrate (CD163+ and PCNA+) and started the biomaterial absorption process, while the muscle biomaterial was already fully absorbed. Finally, after 45th day, both biomaterials were absorbed and the muscle was completely healed, without fibrosis. It was concluded that freezing samples at -20°C prior to processing and subjecting to the initial incubation in 1% SDS solution (Protocol 2B) favored the decellularization of murine skeletal muscle. In addition, xenogeneic and allogeneic dECM were resorbed and presented a distinct integration process in a non-critical model of skeletal muscle, suggesting that potential systemic changes, observed in the long term evaluation, as well as the validation of the use of these matrices in volumetric muscle loss models must be tested. Keywords: Decellularization, biomaterial, skeletal muscle, placenta, biocompatibility

CARACTERIZAÇÃO ANATOMOPATOLÓGICA DAS LESÕES DE FILÉS DE TILÁPIA-DO-NILO (Oreochromis niloticus) INDUZIDAS POR Streptococcus agalactiae E Francisella noatunensis orientalis

O território brasileiro dispõe de características territoriais, hídricas e climáticas favoráveis à expansão de criações intensivas de espécies de peixes água doce com destaque para a tilápia-do-Nilo. O filé de tilápia-do-Nilo aparece como preferência nacional no consumo desse pescado e também têm grande participação no mercado internacional. Poucos estudos de peixes são relacionados às enfermidades que resultem em lesões nos cortes ou subprodutos no meio produtivo. No período de maio a setembro de 2017 foram coletados 70 filés de tilápia-do-Nilo (Oreochromis niloticus) que foram descartados na linha de inspeção. As amostras foram analisadas macroscopicamente e processadas rotineiramente para histologia. Foi realizado exame de reação em cadeia da polimerase (PCR) para Streptococcus agalactiae e PCR em tempo real (qPCR) para Francisella noatunensis subsp. orientalis (Fno). As principais lesões macroscópicas foram pontos pretos multifocais, cavitações e áreas brancas não delimitadas. Microscopicamente verificou-se miosite piogranulomatosa e necrótica em diversos graus e distribuição. Das amostras analisadas, 67% foram positivas para Streptococcus agalactiae e Francisella noatunensis orientalis. As taxas de descarte variaram de 0,2 até 2,0 % da filetagem considerando os lotes acometidos pelos dois agentes encontrados.

Fish represents an important and healthy source of protein for human nutrition. The fillet of Nile tilapia (Oreochromis niloticus) has an important part in the international fish meat trade. However, there are few studies on the causes of injury and condemnation of the Nile tilapia fillets in the inspection line of Federal Meat Inspection Services at slaughterhouses. From May to September 2017, we examined macroscopically and sampled 70 fillets of Nile tilapias, discarded as improper for human consumption at inspection line. We processed the samples for histopathology and additionally ran a polymerase chain reaction (PCR) for Streptococcus agalactiae and Real Time PCR (qPCR) for Francisella noatunensis subsp. orientalis (Fno). The main gross lesions were black multifocal foci, cavities and more dense non-descript white areas. The microscopic findings were pyogranulomatous and necrotizing myositis of varying degrees and distribution. Out of all analised samples, 67% were positive for Streptococcus agalactiae, Fno or both. Discard rates varied from 0.2-2.0% of the total filleting, considering the batches affected by the two found agents.

ESTUDO COMPARATIVO DOS EFEITOS DO VENENO BRUTO DE SERPENTES Crotalus durissus terrificus (LAURENTI, 1768) E Crotalus durissus collilineatus (AMARAL, 1926) EM LESÕES TECIDUAIS OCASIONADAS PELA INTOXICAÇÃO EXPERIMENTAL EM RATOS WISTAR

Estudo comparativo dos efeitos do veneno bruto de serpentes Crotalus durissus terrificus (Laurenti, 1768) e Crotalus durissus collilineatus (Amaral, 1926) em lesões teciduais ocasionadas pela intoxicação experimental em ratos wistar Crotalus durissus é a única espécie do gênero que habita o Brasil e é responsável pelo maior número de mortes por acidentes ofídicos. O veneno crotálico é uma mistura complexa de proteínas ativas, enzimas, toxinas e polipeptídeos. Esses componentes possuem ações neurotóxicas, miotóxicas, nefrotóxicas, hepatotóxicas e coagulantes, no entanto, estudos mostram variações no efeito do veneno. O objetivo deste estudo foi quantificar, qualificar e diferenciar as lesões teciduais sistêmicas produzidas pelo veneno das serpentes Crotalus durissus terrificus e Crotalus durissus collilineatus em ratos Wistar. Utilizou-se ratos machos Wistar (n = 60). Os animais foram alocados em três grupos (n = 20 cada): Grupo Controle (GC); Grupo C. durissus terrificos (CdtG); e, grupo de serpentes C. durissus collilineatus (CdcG). Após a eutanásia, cérebro, pulmão, coração, fígado, jejuno, cólon, baço, rins, músculo esquelético e testículos foram coletados para análises histopatológicas e um escore de lesão por animal foi estabelecido. No sistema nervoso central, escores significativos das lesões (p <0,05) foram observados no CdcG e no CdtG. O maior escore lesional foi observado no CdtG. No tecido muscular, o CdcG apresentou maior escore de lesão no coração, enquanto no músculo esquelético o maior escore foi encontrado no CdtG. No rim, lesões significativas foram observadas no CdcG e no CdtC com o escore lesional médio mais alto no CdcG. O maior escore de lesão hepática foi no CdtG. No pulmão, maior lesional elevado foi observado apenas no CdcG. No baço, baixos escores lesionais foram evidenciados. Nos intestinos, o cólon apresentou baixos escores de lesão, porém diferenças significativas (p <0,05) foram observadas. O único órgão que apresentou diferença significativa (p <0,05) entre os três grupos foi o jejuno. Nos testículos escores lesionais significativos foram encontrados em ambos os grupos. Nossos resultados mostram que o veneno crotálico tem um efeito deletério em todos os sistemas orgânicos. Ao abordar um paciente com suspeita de picada de cascavel, o clínico deve considerar variações nos sinais clínicos em virtude da composição, bem como diferenças no desempenho de cada subespécie em diferentes tecidos. Os dados obtidos em nosso estudo fornecem subsídios para novas pesquisas sobre a toxicidade do veneno crotálico das diferentes subespécies do gênero Crotalus durissus. Palavras-chave: Escores Lesionais, Cascavéis e Histopatologia

Comparative study of the effects of the raw venom of snakes Crotalus durissus terrificus (Laurenti, 1768) and Crotalus durissus collilineatus (Amaral, 1926) on tissue injury caused by experimental intoxication in wistar rats Crotalus durissus is the only species of the genus that inhabits Brazil and is responsible for the highest number of deaths from ophidian accidents. Crotalic venom is a complex mixture of active proteins, enzymes, toxins, and polypeptides. These components have neurotoxic, myotoxic, nephrotoxic, hepatotoxic and coagulant actions, however, studies show variations in the effects of the poison. The objective of this study was to quantify, characterize and differentiate the systemic tissue lesions produced by venom of Crotalus durissus terrificus and Crotalus durissus collilineatus snakes in Wistar rats. Male Wistar rats (n = 60) were used. The animals were allocated into three groups (n = 20 each): Control Group (CG); C. durissus terrificus group (CdtG); and C. durissus collilineatus group (CdcG). After euthanasia, brain, lung, heart, liver, jejunum, colon, spleen, kidneys, skeletal muscle and testes were collected for histopathological analysis and a lesion score per animal was established. In the central nervous system, significant lesion scores (p <0.05) were observed in the CdcG and CdtG. The highest lesion score was observed in the CdtG. In muscle tissue, the CdcG presented a higher lesion score in the heart, whereas in the skeletal muscle the highest score was found in the CdtG. In the kidney, significant lesions were observed in the CdcG and CdtC with the highest mean lesion score in the CdcG. The highest hepatic injury score was in the CdtG. In the lungs, a higher lesion score was observed only in the CdcG. In the spleen, low lesion scores were evidenced. In the intestines, the colon presented low lesion scores, but significant differences (p <0.05) were observed. The only organ that presented significant differences (p <0.05) between the three groups was the jejunum. In the testis, significant lesion scores were found in both groups (p<0,05). Our results show that crotalic venom has a deleterious effect on all organic systems. When addressing a patient whit a suspected rattlesnake bit, the clinician should consider variations in clinical signs by virtue of the composition as well as differences in the performance of each subspecies in different tissues. The data obtained in our study provide subsidies for new research on the toxicity of crotalic venom of the different subspecies of the genus Crotalus durissus.

AVALIAÇÃO DE MARCADORES SÉRICOS DE LESÃO MUSCULAR E PROTEÍNAS DE FASE AGUDA EM CAVALOS DA RAÇA CAMPOLINA SUBMETIDOS À PROVA DE MARCHA

A realização de atividade física tanto em humanos quanto em animais proporciona uma boa saúde física para os praticantes. As atividades e cultura esportivas estão cada vez em evidência e, é sabido animais de alto rendimento exigem, a cada dia, animais mais condicionados, pois maior será sua valorização. Atualmente, existe pouca informação relacionada as respostas fisiológicas do organismo em cavalos atletas submetidos ao concurso de marcha. A resposta de fase aguda (RFA) é um mecanismo fisiopatológico de proteção muito rápido caracterizado pela alteração na concentração sérica de certas proteínas quando as células são submetidas a algum estresse capaz de quebrar sua homeostase e, antecede o surgimento dos sinais clínicos. É importante dados a fim de compreender como o cavalo se comporta perante um estresse fisiológico induzido pelo exercício. O objetivo deste trabalho foi avaliar o efeito da marcha sobres os marcadores séricos de lesão muscular e proteínas de fase aguda. Foram utilizados 20 equinos, de ambos os sexos, da raça Campolina, com idade entre 3,5 e 12,5 anos, pesando em torno de 400 Kg. Participaram do estudo animais atletas clinicamente saudáveis, sob inspeção do exame físico e laboratorial. Amostras de sangue (soro) foram coletadas 24 horas antes do concurso de marcha repouso (T0), imediatamente após o término do exercício, em torno de 5 minutos (T5m), trinta minutos e 12 horas após o concurso (T30m e T12h, respectivamente). Após a realização de todos os procedimentos foram observados diferenças significativas entre alguma situação (momento, interação momento sexo ou sexo) para a ureia, creatinina, aspartato aminotransferase, creatina quinase e nas proteínas de fase aguda (proteína plasmática total, albumina, haptoglobina, glicoproteína ácida e ceruloplasmina. Sendo assim, podemos concluir que os cavalos avaliados estavam aptos a competir em provas de marcha uma vez que após a realização do exercício todas as variáveis analisadas apresentavam um decréscimo podendo chegar a valores bem semelhantes ao observado no repouso, não evidenciando lesões.

Performing physical activity on both humans and animals provides good physical health for practitioners. Sports activities and culture are constantly in evidence and, it is well known that high-yielding animals require more and more conditioned animals every day, since their value will be greater. Currently, there is little information related to the physiological responses of the organism in horses submitted to the running contest. The acute phase response (RFA) is a pathophysiological mechanism of very rapid protection characterized by the alteration in the serum concentration of certain proteins when the cells are subjected to some stress capable of breaking their homeostasis and precedes the appearance of the clinical signs. Importance is given to understanding how the horse behaves in the face of an exercise-induced physiological stress. The aim of this study was to evaluate the effect of gait on serum markers of muscle injury and acute phase proteins. Twenty horses of both sexes of the Campolina breed, aged between 3.5 and 12.5 years, weighing around 400 kg were used. The animals were clinically healthy, under physical and laboratorial examination. Samples of blood were collected 24 hours before the end - of - exercise contest, at approximately 5 minutes (T5m), 30 minutes and 12 hours after the contest (T30m and T12h, respectively). After performing all procedures, significant differences were observed between any situation (moment, sex or sex moment interaction) for urea, creatinine, aspartate aminotransferase, creatine kinase and acute phase proteins (total plasma protein, albumin, haptoglobin, glycoprotein acid and ceruloplasmin, so we can conclude that the evaluated horses were able to compete in gait tests since after the exercise all the analyzed variables presented a decrease, reaching values very similar to that observed at rest, not showing any lesions

Evaluation of oxidative stress in mice subjected to aerobic exercise / Avaliação do estresse oxidativo em camundongos submetidos ao exercício físico aeróbico

PURPOSE: To evaluate the influence of aerobic exercise on oxidative stress in mice. METHODS: The study included twenty female mice Mus musculus-Swiss divided into two groups: sedentary control (GA) and exercise (GB), each containing ten animals. All animals underwent an adaptation period of seven days isolated in individual boxes. After this period, the animals in the exercise group (GB) were trained in angled running wheel with circumference of 25 cm assembled on an articulated axle during five minutes for three consecutive days. On the fourth day, they underwent an exercise program of one session lasting 45 minutes. The evaluation of oxidative stress was performed by determining the levels of malondialhyde derived of lipid peroxidation by the TBA method. The samples were read in a spectrophotometer at 535 nm. RESULTS: No significant difference was observed in the intergroup comparison of MDA levels in the tissues evaluated. A significant difference was observed in the intragroup comparison of MDA levels in the control group (p = 0.0201).The Tukeys' post hoc test indicated significantly lower values of MDA in the smooth muscle in relation to plasma. In the analysis of variance in the exercise group, a significant difference between tissues (p = 0.0009), with significantly lower values in the smooth muscle in relation to plasma (p<0.001) and higher in striated muscle in relation to smooth muscle (p<0.05) was observed. CONCLUSION: There was no change in the analysis of oxidative stress in mice which were undergone a single session of aerobic exercise.(AU)

OBJETIVO: Avaliar a influência do exercício físico aeróbico sobre o estresse oxidativo em camundongos. MÉTODOS: Participaram do estudo 20 camundongos (Swiss), distribuídos em dois grupos: controle-sedentário (GA) e exercício (GB) cada um contendo dez animais. Todos os animais passaram por um período de adaptação de sete dias. Após os animais do grupo (GB) receberam treinamento em roda giratória angulada montada sobre eixo articulado por cinco minutos durante três dias consecutivos. No quarto dia foram submetidos à única sessão de exercício por 45 minutos. A avaliação do estresse oxidativo foi realizada por meio dos níveis de malondiadeído pelo método do TBA. As amostras foram lidas em espectrofotômetro a 535nm. RESULTADOS: Não houve diferença significativa na comparação intergrupos nos tecidos avaliados. Diferença significativa foi observada na comparação intragrupo para o GA (p=0,0201). O post hoc test de Tukey apontou valores significantemente inferiores no músculo liso em relação ao plasma. A análise de variância do GB apontou diferença significativa entre os tecidos (p=0,0009), com valores menores no músculo liso em relação ao plasma (p<0,001), e maiores no músculo estriado em relação ao músculo liso (p<0,05). CONCLUSÃO: Não houve alteração nas análises de malondialdeído tecidual entre os grupos avaliados.(AU)