Uso da termografia e ultrassonografia power dopplerpara avaliação da resposta tecidual em falhas ósseas preenchidas com biomaterial em ovinos / Termography and power doppler sonography assessment for tissue response evaluation in sheep bone defects filled with biomaterial

Biomateriaistem diversas indicações como auxiliares no processo de reparação óssea, além de terem função de substituto ósseo em perdas extensas. Diversas são as vantagens de sua utilização, como por exemplo, auxílio na osteocondutividade, estímulo de neovascularização, potencial antimicrobiano, entre outros. Falhas ósseas foram realizadas nas tíbias de seis ovinos da raça Santa Inês e preenchidas com biomaterial à base de quitosana, colágeno e hidroxiapatita. Um membro foi considerado controle e outro membro tratado segundo estudo randomizado. Foram realizadas avaliações termográficas e por ultrassonografia Power Dopplerem todos os animais do estudo, semanalmente, nos dias D0, D7, D14, D21, D28, D35, D42 e D56. Não houve diferenças significativas com relação à temperatura mínima, máxima e média entre os grupos com biomaterial e controle nas imagens termográficas. Houveram variações com relação ao tempo dentro de ambos os grupos. Com relação à presença de vasos na ultrassonografia Power Dopplernão houve diferenças estatísticas entre os grupos, exceto no dia 21 (P=0,031). Dentro das possibilidades de avaliação que os exames de imagem fornecem, a termografia e a ultrassonografia Power Dopplermostraram-se ferramentas não invasivas de avaliação pós-operatória de processo inflamatório e neovascularização, sendo realizadas semanalmente, permitindo acompanhamento fidedigno e detalhado ao longo do experimento, sem gerar desconforto ou estresse aos animais. Não houve indícios de complicações relacionadas ao biomaterial.(AU)

Biomaterials have several indications supporting the bone repair process, besides having bone substitute function in extensive losses. There are several advantages of its use, such as contribution in osteoconductivity, stimulation of neovascularization, antimicrobial potential, among others.Tibial bone defects was performed in six Santa Inês breed ewes and implanted with chitosan, collagen and hydroxyapatite biomaterial. One limb was considered the control limb and the other one the biomateriallimb, chosenrandomly. Thermographic and Power Dopplersonography was performed in all animals, weekly for 56 days. The thermography showed no significant differences related to low, high and mean temperature between the control and the biomaterial groups. Statisticalvariations were found between time in both groups. The visualization of neovascularization with the Power Dopplersonography did not present statistical differences, except for day 21 (P=0,031). Within the possibilities provided by imaging exams, thermography and Power Doppler sonography demonstrate to be non-invasive methods for post-operative evaluation of inflammatory process and neovascularization. The weekly management allowed reliable and detailed monitoring throughout the experiment, without causingdiscomfort or stress to the animals. There was no evidence of biomaterial complications.(AU)

Bisacurone gel ameliorated burn wounds in experimental rats via its anti-inflammatory, antioxidant, and angiogenic properties

Purpose: To investigate putative mechanism of wound healing for chitosan-based bisacurone gel against secondary burn wounds in rats. Methods: A second-degree burn wound with an open flame using mixed fuel (2 mL, 20 seconds) was induced in Sprague Dawley rats (male, 180-220 g, n = 15, each) followed by topical treatments with either vehicle control (white petroleum gel, 1%), silver sulfadiazine (1%) or bisacurone gel (2.5, 5, or 10%) for 20 days. Wound contraction rate and paw withdrawal threshold were monitored on various days. Oxidative stress (superoxide dismutase, glutathione, malondialdehyde, and nitric oxide), pro-inflammatory cytokines (tumour necrosis factor-alpha, interleukins by enzyme-linked immunosorbent assay), growth factors (transforming growth factor-ß, vascular endothelial growth factor C using real time polymerase chain reaction and Western blot assay) levels, and histology of wound skin were assessed at the end. Results: Bisacurone gel showed 98.72% drug release with a 420.90­442.70 cps viscosity. Bisacurone gel (5 and 10%) significantly (p < 0.05) improved wound contraction rate and paw withdrawal threshold. Bisacurone gel attenuated oxidative stress, pro-inflammatory cytokines, and water content. It also enhanced angiogenesis (hydroxyproline and growth factor) and granulation in wound tissue than vehicle control. Conclusions: These findings suggested that bisacurone gel can be a potential candidate to treat burn wounds via its anti-inflammatory, antioxidant, and angiogenic properties.

Carcinoma de células escamosas em gato - tratamento com quimioterapia metronômica / Squamous cell carcinoma in a cat - treatment with metronomic chemotherapy

Background: Squamous cell carcinoma (SCC) is a malignant neoplasm associated with prolonged exposure to ultraviolet light and lack of skin pigment in white-coated animals. The disease diagnosis is based on the animal's history, clinical signs and histopathology of the samples collected. The therapeutic alternatives are diverse. Among them, one of the most used is the metronomic chemotherapy which is based on the use of cytostatics drugs, applied in low and frequent doses for a longer period of time. One of the mechanisms of action of this type of therapy is the decrease in angiogenesis. Non-steroidal anti-inflammatory drugs (NSAIDs) are simultaneously used to maximize the angiogenic effect. The objective of this study is to report the results of a treatment accomplised with metronomic chemotherapy (cyclophosphamide with meloxicam) in a domestic feline which presented a squamous cell tumor. Case: A 7-year-old female cat, mixed breed, not neutered, weighing 3.5 kg, was assisted at the Veterinary Specialties Teaching Clinic (CEEV) of the Technical University of Machala (UTMACH), presenting bilateral ulcerative lesion in the nasal plane for 5 months. Chronic exposure to sunlight is referred to in the patient's anamnesis. Aspiration cytology performed in the patient revealed an inflammatory process with a predominance of neutrophils and macrophages, and showed absence of neoplastic cells. A treatment based on antibiotics and corticosteroids was adopted. As there were no favorable results, an excisional biopsy procedure was chosen to collect the affected tissues. The result of the histopathological analysis was a level 2 squamous cell carcinoma (SCC). The initial treatment consisted of surgical removal of the tumor and later the use of metronomic chemotherapy based on Cyclophosphamide, Meloxicam, DHA (docosahexaenoic acid) and Sucralfate for 5 months was adopted. At the beginning of the second phase of treatment, the patient developed a macula that progressively diminished. Currently, the patient does not have any recurrence of the diagnosed neoplasm. Discussion: The diagnosis of squamous cell carcinoma was based on clinical and histopathological findings. The macroscopic lesions were crusted ulcers that do not heal in the nasal plane and auricular pavilion, as reported in the literature. SCC is characterized by the infiltration of squamous cells in the dermal layer of the skin, forming epithelial islands with keratin pearls. The histopathological findings of this case present the same characteristics. The climatic conditions of Ecuador, high temperatures with greater incidence of sunlight, hypopigmented areas of the animal's body and chronic exposure to solar radiation are risk factors present in this case that foster the development of this malignant neoplasm. There are multiple treatments to be introduced in this sort of pathology. In this case, surgical removal of the tumor was adopted, followed by metronomic chemotherapy based on cyclophosphamide at 10 mg/m2 and meloxicam at a 0.05 mg/kg per day for a period of time of five months, obtaining favorable results and absence of recurrence. Despite metronomic chemotherapy being easy to administer, with fewer side effects, which includes a better acceptance of care takers who are reluctant to administer conventional chemotherapy, further studies are still necessary in what concerns certain variables such as age, carcinoma level, appropriate dosages and drugs according to each neoplasm.

Early maturation of corpus luteum in rabbits - effect of sildenafil citrate on luteolytic capacity in the early luteal period

Background: Prostaglandin F2 alpha (PGF2 α) binds to the specific receptor (PTGFR) on the corpus luteum (CL) in mammals, inducing regression of the CL structure (luteolysis) and initiating a new cycle. While PGF2 α is effective only on mature CL, the immature CL structure (early luteal phase) resists PGF2 α. In this study, sildenafil citrate, which is used to increase blood flow in the genital organs for treating specific pregnancy issues in women, was administered during the early luteal phase in a rabbit model to test the hypothesis of enhancing blood flow to the CL, thereby promoting earlier maturation and enabling a response to PGF2 α. Materials, Methods & Results: The study was conducted in 2 sub-studies: clinical and molecular. A large number of rabbits were initially included in the sub-studies to ensure a sufficient number of pseudo-pregnant rabbits. Ovulation in rabbits was induced with buserelin acetate and was considered as day 0 of the study. The sub-studies were continued with rabbits whose pseudo-pregnancies were confirmed according to progesterone (P4 ) results. As a result, the studies were continued with a total of 41 pseudo-pregnant New Zealand female rabbits, 21 of which were included in the clinical sub-study and 20 in the molecular sub-study. In both sub-studies, on day 3 of the luteal period, rabbits in the treatment group received 5 mg/kg sildenafil citrate and all rabbits received a single dose of exogenous PGF2 α on day 4 to induce luteolysis. In the clinical sub-study, echotexture and intraovarian blood flow changes in the ovaries were determined by ultrasonography (USG) examination. In the molecular sub-study, the expression changes of Hypoxia Inducible Factor 1 Alpha (HIF1A) and Vascular Endothelial Growth Factor (VEGF) related to angiogenesis, Steroidogenic Acute Regulatory Protein (StAR) related to P4 metabolism, Prostaglandin-Endoperoxide Synthase 2 (PTGS2) related to prostaglandin (PG) mechanism and 15-Hydroxyprostaglandin Dehydrogenase (HPGD) genes at mRNA level were determined using Real Time Polymerase Chain Reaction (RT-PCR) in CL tissues obtained with ovariohysterectomy (OVH) at 1 and 12 h after PGF2 α injection. In addition, blood samples were collected for determine P4 levels from all rabbits. In the clinical sub-study; there was no difference between the groups in mean gray values (MGV), whereas there was a significant decrease in both pulsatile index (PI) and resistance index (RI) values at 40 min after PGF2 α injection (P < 0.05). In the molecular sub-study, it was determined that sildenafil citrate had no significant effect (P > 0.05) on the expression levels 1 and 12 h after PGF2 α injection. According to the results of the molecular sub-study, no significant effect of sildenafil citrate on the mRNA expression levels in the investigated genes was detected (P > 0.05). However, within each group, differences were found according to OVH time after PGF2 α injection. It was observed that PTGS2 and HPGD mRNA expressions decreased at the 12th h compared to the 1st h, while HIF1A expression increased (P < 0.05). Discussion: According to the results obtained from clinical and molecular sub-studies, it was determined that a single dose of sildenafil citrate (5 mg/kg) applied on the 3rd day of the luteal period did not contribute to the maturation process of the CL, did not increase blood flow, and was insufficient to break the resistance of the CL against PGF2 α applied on the 4th day of the luteal period. However, a significant decrease in the PI value at the 40th min after PGF2 α injection suggests that sildenafil citrate has a supportive effect, and that this decrease is also seen in the RI value, suggesting that its effect is insufficient against the vasoconstrictive effect of PGF2 α.

Canine mammary neoplasms - evaluation of tumor microenvironment

Background: The tumor microenvironment is an important target of studies in different types of neoplasms. Understanding the role of general components such as immune, vascular and fibroblastic cells has the objective of contributing to prognosis and treatment. The aim of this study was to evaluate the relationship between mast cells and angiogenesis in benign and malignant mammary neoplasms by investigating the role of degranulation and microlocation of mast cells and neoformed vessels in canine mammary neoplasms. Materials, Methods & Results: Mammary glands (n = 122) from 50 female dogs submitted to mastectomy without chemotherapy were evaluated and categorized into 3 groups: control group (n = 46); malignant group (n = 57) and benign group (n = 19). Lymph nodes without changes (n = 59) and with metastases (n = 6) were also evaluated. To evaluate the MCD (mast cell density) and angiogenesis, Toluidine Blue (0.1%) and Gomori’s Trichrome techniques were performed and adapted from previous studies. Photomicrographs of 10 hotspot areas on a 40x objective lens of the mammary glands and lymph nodes were captured to assess MCD and angiogenesis. In the absence of these areas, random fields were captured. For the mammary glands of the malignant and benign groups, 20 fields were analyzed, as the analysis considered the microlocation (peritumoral and intratumoral). Counting was performed manually using ImageJ software version 1.42q by 2 observers. The statistical analysis were performed using SPSS software version 19.0. The most frequent histological type in the malignant group was carcinoma in mixed tumor (68.42%; 39/57) and in the benign group was benign mixed tumor (57.89%; 11/19). Female dogs without breed pattern were more frequently affected represented 70% of the animals and the mean age was 9 years and 8 months ± 3 years and 1 month...

Canine mammary neoplasms - evaluation of tumor microenvironment

Background: The tumor microenvironment is an important target of studies in different types of neoplasms. Understanding the role of general components such as immune, vascular and fibroblastic cells has the objective of contributing to prognosis and treatment. The aim of this study was to evaluate the relationship between mast cells and angiogenesis in benign and malignant mammary neoplasms by investigating the role of degranulation and microlocation of mast cells and neoformed vessels in canine mammary neoplasms. Materials, Methods & Results: Mammary glands (n = 122) from 50 female dogs submitted to mastectomy without chemotherapy were evaluated and categorized into 3 groups: control group (n = 46); malignant group (n = 57) and benign group (n = 19). Lymph nodes without changes (n = 59) and with metastases (n = 6) were also evaluated. To evaluate the MCD (mast cell density) and angiogenesis, Toluidine Blue (0.1%) and Gomoris Trichrome techniques were performed and adapted from previous studies. Photomicrographs of 10 hotspot areas on a 40x objective lens of the mammary glands and lymph nodes were captured to assess MCD and angiogenesis. In the absence of these areas, random fields were captured. For the mammary glands of the malignant and benign groups, 20 fields were analyzed, as the analysis considered the microlocation (peritumoral and intratumoral). Counting was performed manually using ImageJ software version 1.42q by 2 observers. The statistical analysis were performed using SPSS software version 19.0. The most frequent histological type in the malignant group was carcinoma in mixed tumor (68.42%; 39/57) and in the benign group was benign mixed tumor (57.89%; 11/19). Female dogs without breed pattern were more frequently affected represented 70% of the animals and the mean age was 9 years and 8 months ± 3 years and 1 month...(AU)

Free omental graft without vascular microanastomosis in the cutaneous wound healing of rabbits / [Enxerto omental livre sem microanastomose vascular na cicatrização de feridas cutâneas em coelhos]

This study aims to evaluate the influence of free omental graft without vascular microanastomosis (FOGWVA) on experimental skin healing in rabbits. Through celiotomy, a 9cm2 free omental fragment was collected in 36 rabbits, with subsequent production of a deep linear cutaneous wound in the dorsal midline measuring 3cm. In 18 animals from the omental group (OG), the omental fragment collected was fixed subcutaneously with six simple interrupted stitches using a 4-0 polyamide thread. In both treated and control (CG) groups, intradermal dermorrhaphy was performed with 4-0 polyamide thread. Experimental wounds were clinically evaluated every day. Each of the groups was divided into three subgroups of 6 animals each for histopathological evaluation on the 7th, 14th, and 28th days of postoperative. In the OG wounds, the increase in volume (omentum activation) stood out after the second postoperative day. Macroscopy showed an organic reaction to the graft on day 7, with a progressive reduction in addition to neovascularization towards the omental graft. The intense presence of mononuclear cells and collagen deposition on day 7 demonstrated an accelerated process of tissue remodeling and repair. The FOGWVA omental graft remained viable and positively influenced the cutaneous healing of the experimental wounds in rabbits.

Neste estudo, objetiva-se avaliar a influência do enxerto omental livre sem microanastomose vascular (FOGWVA) na cicatrização cutânea experimental em coelhos. Por meio de celiotomia, foi coletado fragmento omental livre de 9cm2 em 36 coelhos, com posterior produção de ferida cutânea profunda linear na linha média dorsal medindo 3cm. Apenas em 18 animais, do grupo omento (GO), o fragmento omental coletado foi fixado no subcutâneo com seis pontos simples interrompidos utilizando fio poliamida 4-0. Em ambos os grupos, tratado e controle (GC), efetuou-se dermorrafia intradérmica com fio poliamida 4-0. As feridas experimentais foram avaliadas clinicamente todos os dias. Cada um dos grupos foi dividido em três subgrupos, com seis animais cada, para avaliação anatomopatológica no sétimo, 14º e 28o dias de pós-operatório. Nas feridas do GO, destacou-se aumento de volume (ativação do omento) a partir do segundo dia pós-operatório. A macroscopia evidenciou reação orgânica ao enxerto no dia sete, com redução progressiva, além de neovascularização em direção ao enxerto omental. Intensa presença de células mononucleares e deposição de colágeno no dia sete demonstraram acelerado processo de remodelamento e reparo tecidual. O FOGWVA manteve-se viável e influenciou positivamente na cicatrização cutânea de feridas experimentais em coelhos.

Use of omentum flap for induction of free skin graft healing / [Utilização de flap de omento para indução da cicatrização de enxertos cutâneos]

The objective of this study was to assess healing induction of free skin grafts following transposition of omental flap through a subcutaneous tunnel to the recipient bed. Macroscopic and microscopic evaluations were performed. Nineteen piglets were used. Two surgical wounds were created of each subject. The graft removed from the left side (LS) was placed on the right side (RS) without the omental flap in the graft-bed (control group-CG). On the LS, an omental flap was placed between the graft removed from the RS and the recipient bed (omentum group-OG). Macroscopic evaluations showed edema, which gradually decreased on both groups. Suture dehiscence was highest at day 10 compared to other days in both groups. The CG had a higher incidence of unvitalized tissue compared to OG, although no difference was found among days of postoperative evaluation. The presence of unvitalized tissue was seen on 32% on OG and 53% on CG. Microscopic evaluations revealed higher collagenization, reepithelization, keratinization and less swelling in the OG compared to CG. In conclusion, mesh skin grafts evolved satisfactorily in swine even in newly created bedding without granulation tissue, but with appropriate vascularization. The omentum flap provided better macroscopic and microscopic outcomes regarding graft integration.

O objetivo deste estudo foi verificar a indução da cicatrização de enxertos cutâneos em malha após a utilização de flap de omento transposto através de túnel no subcutâneo até o leito receptor. Avaliações macroscópica e microscópica foram realizadas. Foram utilizados 19 suínos. Duas feridas cirúrgicas foram criadas em cada animal. O enxerto removido do lado esquerdo (LE) foi fixado do lado direito (LD), sem a presença do flap de omento entre o enxerto e o leito receptor (grupo controle - GC). No LE, foi fixado um flap de omento entre o enxerto cutâneo removido do LD e o leito receptor (grupo omento - GO). As avaliações macroscópicas mostraram que o edema diminuiu gradativamente em ambos os grupos. A deiscência foi maior no dia 10 em comparação aos demais dias, em ambos os grupos, entre os diferentes dias de avaliação. Foi verificada a presença de tecido desvitalizado em 32% do GO e em 53% no GC. Foi observada, na avaliação microscópica, maior colagenização, reepitelização, queratinização e menor edema no GO, quando comparado ao GC. Concluiu-se, com este estudo, que enxertos cutâneos em malha evoluíram satisfatoriamente em suínos, mesmo em leito receptor recém-criado e sem presença de tecido de granulação, desde que vascularizado, e que o flap de omento propiciou melhores resultados macro e microscópicos relativos à integração do enxerto.

Treatment of experimentally induced partial-thickness burns in rats with different silver-impregnated dressings

Purpose: To evaluate the morphometric, macroscopic and microscopic aspects of experimentally induced partial-thickness burns in rats treated with different silver-based dressings. Methods: Wistar rats were used, divided into six treatments: saline (NaCl 0.9%); silver sulfadiazine 1%; Silvercel; Mepilex Ag; Aquacel Ag and Acticoat. The animals were monitored daily and euthanized at 7, 14 and 30 days after injury induction (DAI). Results: At 7 DAI, necrosis/crust was greater in control, silver sulfadiazine and Mepilex Ag treatments, granulation tissue was induced by Aquacel Ag, polymorphonuclear infiltrate (PMN) infiltration was intensified by Mepilex Ag; mononuclear infiltrate (MN) infiltration and angiogenesis were increased by Silvercel. At 14 DAI, hemorrhage was decreased by Silvercel and Mepilex Ag, PMN infiltration increased by Acticoat. At 30 DAI, angiogenesis was greater in the Acticoat treatment and fibroblasts were increased by Acticoat and Mepilex Ag. Collagen was induced at 14 DAI by silver sulfadiazine and Aquacel Ag and, at 30 DAI, by silver sulfadiazine and Silvercel treatments. Conclusions: Silvercel and Acticoat presented better results than the other products. However, all the dressings were better than the control at some point during the process, and may contribute to the healing of partial thickness burns. Silvercel and Aquacel Ag treatments induced better cosmetic outcomes regarding wound closure and scarring.

Pouteria ramiflora leaf extract on emulgel in wound healing activity in diabetic rats

Abstract The genus Pouteria has been studied because it presents various activities, among which is its anti-inflammatory potential. The effects of Pouteria ramiflora Carbopol gel on the healing of skin wounds in diabetic rats were evaluated by microscopic imaging. Streptozotocin was administered intraperitoneally in animals that had fasted for 12 hours, a situation confirmed by the glycemic index ( 240 mg dL-1). An excision on the back of the animals was performed and three groups were formed: Control (Gel), Ethanolic extract (Ext) and Gel + extract 2% (Ext+gel); the histopathological evaluation occurred on the 7th, 14th, 21st and 30th days after the post-operative period. The results of the phytochemical prospecting of P. ramiflora extract demonstrated the major presence of phenolic compounds and flavonoids; the assessment of the inflammatory infiltrate on the 7th day was higher on group Ext and Ext+gel when compared to group Control; on the 14th day control and Ext (p 0.05). The quantification of fibroblasts was higher on the 7th day among the three treatments, control and Ext (p 0.05), on the 21st day. Angiogenesis showed a higher number of vessels in Ext+gel group (p 0.05) on the 7th day; in Control, Ext and Ext+gel (p 0.05) on the 14th day; and Control and Ext (p 0.05)on the 21st day. The histopathological results showed that the formulation Ext+gel was efficient in tissue reparation and decrease in inflammatory cells on the diabetics animals.

Resumo O gênero Pouteria apresenta várias aplicações terapêuticas e, dentre elas, grande potencial antiflamatório. Os efeitos do gel de Pouteria ramiflora sobre a cicatrização de feridas na pele de ratos diabéticos foram avaliados pela histomorfometria. A estreptozotocina foi administrada por via intraperitoneal em animais após jejum de 12 horas, a confirmação de indução da diabetes foi confirmada pelo índice glicêmico ( 240 mg dL-1). Foi realizada uma incisão no dorso do animal e foram criados 3 grupos de tratamento: controle (gel carbopol), extrato etanólico (Ext) e Gel + extrato etanólico à 2% (Ext+gel); a avaliação histopatológica foi realizada no 7º, 14º, 21º e 30º dias após o período pós operatório. Os resultados da prospecção fitoquímica dos extratos de P. ramiflora demonstraram majoritariamente a presença de compostos fenólicos e flavonóides; o infiltrado inflamatório avaliado no 7º dia foi maior para animais do grupo controle em relação aos grupos Ext (p 0.05) e Ext+gel 2% (p 0.05); no 14º dia o controle e Exp (p 0.05) apresentaram aumento significativo dos infiltrados inflamatórios. A presença de fibroblastos foi elevada no 7º dia em todos os tratamentos. O processo da angiogênese mostrou um maior número de vasos sanguíneos entre os grupos Ext e Ext+gel (p 0.05) no 7º dia; no 14º dia o grupo controle, Ext (p 0.05), Control e Ext+gel (p 0.05) apresentaram aumento de vascularização, e no 21º dia apenas os grupos controle e Ext (p 0.05). Os resultados histopatológicos mostraram que a formulação gel carbopol + extrato etanólico a 2% foi eficiente na reparação de tecidos e na diminuição de células inflamatórias nos animais diabéticos.

The use of occlusive dressings: influence on excisional wound healing in animal model

Purpose: To analyze the influence of occlusive dressing on the healing of excisional skin wounds in mice. Methods: Pre-clinical, comparative, and translational study. Mice were divided into three experimental groups: wounds occluded with hydrocolloid (HD) dressings, transparent polyurethane film (TF) dressings, and without occlusion (WO), monitored at three, six and 14 days, with eight animals each. Closure rate, infiltration of neutrophils and macrophages, measurement of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) and, histologically, angiogenesis were evaluated. Results: Wound closure was accelerated in the occlusive groups. There was a decrease in TNF-α levels in the HD group when compared to the WO and TF groups. Neutrophils accumulation decreased in the HD group. Increased dosages of macrophages were evidenced in the HD group, compared to the WO and TF groups. Levels of VEGF were increased in the TF and HD groups. Conclusions: It is suggested that the occlusion of wounds modulates the inflammatory response.

The effects of valproic acid on skin healing: experimental study in rats

Purpose: To recognize the effects of valproic acid (VPA), an epigenetic drug, on the skin healing process. Methods: Sixty male Wistar rats were divided into two groups: the experiment treated with VPA (100 mg/kg/day); and the control, with 0.9% sodium chloride by gavage. Skin healing was studied in three moments (the third, the seventh, and the 14th day), evaluating the parameters: inflammatory reaction and its intensity (anti-LCA), angiogenesis (anti-CD34), collagen I and III (anti-collagen I, anti-collagen III and Picrosirius-red F3BA) and myofibroblasts (anti-alpha-AMS). Results: The inflammatory reaction was acute or sub-acute in both groups on the third day. On the seventh and the 14th day, chronic predominated in the control (p=0.006), and sub-acute in the experiment (p=0.020). There was a greater number of leukocytes in the group treated only on the third day (p=0.036). The number of vessels was lower in the treated group at the three times (p3=0.002, p7<0.001, and p14=0.027). Myofibroblasts were rare in the third day and moderate quantity in the remaining periods. Collagen I density was higher in the control at the three times (p<0.001) and collagen III in the treated group (p<0.001). Conclusions: VPA led to a more intense inflammatory reaction, decreased angiogenesis and collagen deposition, especially type I collagen.

Tissue healing changes on wounds in rats after treatment with Hancornia speciosa latex in cream-gel formulation

Purpose: Hancornia speciosa latex has shown pharmacological potential in wound healing processes due to its angiogenic, osteogenic, and anti-inflammatory activities. The aims of this study were to carry out a cream-gel formulation with 5, 10 and 25% of H. speciosa serum latex and to evaluate its potential to stimulate the skin regeneration in rats' wounds. Methods: One hundred and twenty rats were divided into five groups: neutral control with saline (G1), cream-gel based on H. speciosa latex serum at 5% m/v (G2), cream-gel at 15% m/v (G3), cream-gel at 25% m/v (G4), and cream-gel (G5). The animals were euthanized at three, seven, 14 and 21 days after the injury induction, and some parameters were analyzed: wound contraction, necrosis, fibrin, polymorphonuclear and mononuclear infiltrates, fibroblast, angiogenesis, hemorrhage, and collagen. Results:The therapeutic treatment with cream-gel at 15 and 25% is beneficial in the inflammatory phase of healing processes since it increased the angiogenesis and proliferation of mononuclear infiltrations in wounds. Regarding wound contraction, the treatment with cream-gel (5 and 15%) induced a higher rate of contraction in the proliferative phase. The 15% cream-gel formulation stimulated a greater production of collagen in the injured tissues. Conclusions: H. speciosa cream-gel is a low-cost herbal medicine which can aid in tissue repair.

Scrotal hemangiosarcoma in a Large White boar / Hemangiossarcoma na bolsa escrotal de um reprodutor Large White

Tumors are rarely diagnosed in swine specie because of the short lifespan of production animals. Normally, these tumors do not present any clinical signs and are often detected at the time of slaughter. A 2-year-old Large White boar, used in the reproductive management of a farm and without a history of pre-existing problems, was examined for skin lesions on the scrotum. Samples were collected from skin segments containing lesions for histopathological and immunohistochemical diagnosis. Microscopically, the nodes in the scrotum pouch consisted of poorly demarcated, highly cellular, expansile, and multifocally invasive neoplasms, composed of immature endotheliocytes organized into neovascular formations. The tumor cells were pleomorphic, slightly oval to spindle-shaped, with eosinophilic cytoplasm and hyperchromatic nuclei with one to three nucleoli. All the nodules analyzed were compatible with hemangiosarcoma. After immunohistochemical evaluation, for the quantification of tissue angiogenesis the neoplastic cells immunoexpressed the CD31 monoclonal antibodies and factor VIII, through the identification of proteins expressed on the surface of endothelial cells. The Ki67 cell proliferation marker was positive in approximately 10% of the neoplastic cells, demonstrating a high degree of malignancy. Hemangiosarcoma in swine species has already been identified in several organs and tissues; however, to date, no study has demonstrated the diagnosis of this condition on the skin of the scrotum, as reported in this study. Therefore, it is expected that this report will contribute to the knowledge of the frequency of neoplasms in swine species.

Na espécie suína os tumores são pouco diagnosticados devido a curta vida útil dos animais de produção. Normalmente não causam sinais clínicos, sendo, muitas vezes, encontrados no momento do abate. Um reprodutor com dois anos de idade, da raça Large White, utilizado no manejo reprodutivo de uma granja, sem histórico de problemas pré-existentes, foi examinado em função de lesões na pele da bolsa escrotal. Foram coletadas amostras de segmentos de pele contendo lesões para o diagnóstico histopatológico e imunohistoquímico. Microscopicamente os nodos em bolsa escrotal consistiam em neoplasias mal demarcadas, altamente celulares, expansivas e multifocalmente invasivas composta por endoteliócitos imaturos organizados em formações neovasculares. As células tumorais eram pleomórficas, levemente ovais a fusiformes com citoplasma eosinofílico e núcleos hipercromáticos e com um a três nucléolos. Todos os nódulos analisados foram compatíveis com hemangiossarcoma. Para a quantificação da angiogênese tecidual, após a avaliação imunohistoquímica, as células neoplásicas imunoexpressaram os anticorpos monoclonais CD31 e Fator VIII, através da identificação de proteínas expressas na superfície das células endoteliais. Observou-se o marcador de proliferação celular Ki67 positivo em aproximadamente 10% das células neoplásicas, demonstrando um alto grau de malignidade. O hemangiossarcoma na espécie suína já foi identificado em vários órgãos e tecidos, mas, até o momento, nenhum trabalho demonstrou o diagnóstico na pele da bolsa escrotal, como reportado, ineditamente, neste estudo. Face o exposto, espera-se que este relato contribua para a frequência de neoplasias encontradas na espécie suína.

Biocompatibility in vivo of elastic cartilage treated in alkaline solutions / [Biocompatibilidade in vivo de cartilagem elástica tratada em soluções alcalinas]

This study verified the in vivo biocompatibility of bovine elastic cartilage decellularized with alkaline solution in relation to the non-decellularized cartilage implanted in rats. Fifty Wistar rats were divided into two groups, with the experimental group (EG) receiving subcutaneous implants of cartilage treated in alkaline solution and the control group (CG) receiving subcutaneous implants of untreated cartilage. In both groups, the implants were removed on days 3, 7, 14, 21 and 42 with the preparation of histological slides stained with Hematoxylin and Eosin for the quantification of inflammatory cells, fibroblasts, angiogenesis, and cartilage degradation. The results showed that EG presented a less intense inflammatory infiltrate and better organization of collagen fibers compared to CG. It was concluded that the alkaline treatment provided better biocompatibility for elastic cartilage when implanted subcutaneously in rats.

Este estudo verificou a biocompatibilidade in vivo de cartilagem elástica bovina descelularizada com solução alcalina em relação à cartilagem não descelularizada implantada em ratos. Cinquenta ratos Wistar foram divididos em dois grupos; o grupo experimental (GE) recebeu implantes subcutâneos de cartilagem tratada em solução alcalina, e o grupo controle (GC) recebeu implantes subcutâneos de cartilagem não tratada. Em ambos os grupos, os implantes foram retirados nos dias três, sete, 14, 21 e 42, com a preparação de lâminas histológicas coradas com hematoxilina e eosina para a quantificação de células inflamatórias, de fibroblastos, de angiogênese e para a degradação da cartilagem. Os resultados mostraram que o GE apresentou infiltrado inflamatório menos intenso e melhor organização das fibras de colágeno em relação ao GC. Concluiu-se que o tratamento alcalino proporcionou melhor biocompatibilidade para a cartilagem elástica quando implantado por via subcutânea em ratos.

New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Sclerosing encapsulating peritonitis in a dog

Sclerosing encapsulating peritonitis (SEP) is a rare condition which consists of reactive fibrous tissue proliferation with mixed inflammatory infiltration within the abdominal cavity. The present report describes an additional case of SEP affecting a mixed-breed immature female dog presented with persistent vomiting, progressive weight loss, and ascites. Abdominal radiographic and ultrasonographic findings suggested abdominal neoformation resulting in gastric displacement, in addition ascitic fluid was evaluated and cytology showed large numbers of inflammatory effusion. An exploratory laparotomy was performed and were detected multiple thick peritoneal adhesions which restricted mobility of abdominal viscera. Biopsy specimens of these lesions were submitted for histopathological examination. Microscopically, the external serous surfaces of the abdominal organs were covered with dense fibrous connective tissue characterized by intense mature collagen deposition and moderate angiogenesis. The animal was euthanized due to poor prognosis and sent for necropsy. The clinical, imaging, gross and microscopic findings were compatible with SEP.(AU)

Inflammation reduction potential of nanostructured lipid carriers encapsulated with rat's bone marrow cells' lysate / Potencial de redução da inflamação de carreadores lipídicos nanoestruturados encapsulados com lisado de células de medula óssea de ratos

Bone marrow-derived mesenchymal stromal cells (BMSCs) have been used for treating inflammatory disorders. Due to the large size of BMSCs compared to nanoparticles, BMSCs cannot be loaded into the nanoparticles. It is hypothesized that BMSCs lysate loading into the nanocarriers will effectively deliver cellular contents and regulatory elements of BMSCs at the injury site. This study aimed to investigate nanostructured lipid carriers (NLC) loading with BMSCs lysate through basic characterization and morphological analysis. Moreover, this study was mainly designed to investigate the role of NLC loaded BMSCs lysate in reducing inflammation via in-vitro and in-vivoassays. The in-vitro study involves cell viability assays, p53, annexin V and VEGF expression through ELISA and immunocytochemistry, real-time BAX, caspase-3, IL-6, IL-8, TOP2A, PCNA, and Ki-67 gene expression analysis. Additionally, to evaluate in-vivo anti-inflammatory activity, the carrageenan-induced rat paw oedema model was used. In-vitro results showed that NLC loaded BMSCs lysate increased cell viability, decreased apoptosis and pro-inflammatory genes expression and up-regulated angiogenesis and proliferation in H2O2 pre-stimulated cells. Findings of the in-vivo assay also indicated a reduction in rat's paw oedema volume in NLC-loaded BMSCs lysate, and downregulation of BAX, Caspase-3, IL-6, and IL-8 was observed. Enhanced expressions of TOP2A, PCNA, and Ki-67 were obtained. Concluding the results of this study, NLC-loaded BMSCs lysate could reduce inflammation and possibly regenerate damaged tissue mainly via increasing cell viability, angiogenesis and proliferation, and reducing apoptosis and pro-inflammatory cytokines.

Células estromais mesenquimais derivadas da medula óssea (BMSCs) têm sido utilizadas para o tratamento de distúrbios inflamatórios. Devido ao grande tamanho das BMSCs em comparação com as nanopartículas, as BMSCs não podem ser carregadas nas nanopartículas. Supõe-se que o carregamento de lisado de BMSCs no nanocarriers será eficaz na entrega de conteúdos celulares e elementos reguladores de BMSCs no local da lesão. Este estudo teve como objetivo investigar a carga de carreador lipídico nanoestruturado (NLC) com lisado de BMSCs através de caracterização básica e análise morfológica. Além disso, este trabalho foi projetado, principalmente, para investigar o papel do lisado de BMSCs carregado com NLC na redução da inflamação por meio de ensaios anti-inflamatórios in vitro e in vivo. O estudo in vitro envolve ensaios de viabilidade celular, expressão de p53, anexina V e VEGF por ELISA e imunocitoquímica e expressão gênica em tempo real de BAX, caspase-3, IL-6, IL-8, TOP2A, PCNA e Ki-67 . Além disso, para avaliar a atividade anti-inflamatória in vivo,o modelo de edema de pata de rato induzido por carragenina foi utilizado. Os resultados in vitro mostraram que o lisado de BMSCs carregadas com NLC aumentou a viabilidade celular, diminuiu a apoptose e a expressão de genes pró-inflamatórios e aumentou a angiogênese e proliferação em células pré-estimuladas com H2O2. Os achados do ensaio in vivo também indicaram uma redução no volume do edema da pata de rato no lisado de BMSCs carregado com NLC, entretando, foi observada a regulação negativa de BAX, Caspase-3, IL-6 e IL-8. Expressões aumentadas de TOP2A, PCNA e Ki-67 foram obtidas. Assim, concluindo os resultados do estudo, é possível afirmar que o lisado de BMSCs carregado com NLC pode reduzir a inflamação e possivelmente regenerar o tecido danificado principalmente por meio do aumento da viabilidade celular, angiogênese e proliferação e redução da apoptose e citocinas pró-inflamatórias.

Closure of defects in a geometric figure pattern associated with tumescent anesthesia with lidocaine in rabbits (Oryctolagus cuniculus) / Fechamento de defeitos em padrão de figura geométrica associado ao emprego de anestesia por tumescência com lidocaína em coelhos (Oryctolagus cuniculus)

The use of tumescent anesthesia with lidocaine can provide better intra- and postoperative analgesia that would benefit extensive reconstructive surgery. However, lidocaine can interfere with the healing process. Therefore, this study aimed to assess the local interference of the healing of induced and closed skin defects in a geometric pattern associated with the use of tumescent anesthesia with lidocaine in rabbits. Furthermore, we assessed its influence on cardiorespiratory parameters and postoperative analgesia. This study included 27 rabbits divided into three groups: GC (without the use of tumescence), GS (use of tumescence with 0.9% NaCl solution), and GL (use of tumescent anesthesia with lidocaine). There was no statistically significant intergroup difference in any stage of the wound healing process on macroscopic evaluations, in the angiogenesis process, or in the process of collagenization and fibroblast deposition. There were significant differences in heart rate (lower in GL), respiratory rate (higher in GC), mean arterial pressure (higher in GL), and expired concentration of isoflurane (lower in GL). There was no significant intergroup difference in the von Frey filament test or the visual analog scale score used to evaluate postoperative analgesia. We concluded that tumescent anesthesia with lidocaine does not impair postoperative tissue repair. Its use features benefits such as reducing the volume of inhaled anesthetic, maintaining the anesthesia plan, stable heart and respiratory rates, and lower hypotension during the surgical procedure.

O uso da anestesia por tumescência com lidocaína pode proporcionar melhor analgesia transoperatória e pós-operatória, o que seria um benefício em cirurgias reconstrutivas extensas. Entretanto, a lidocaína pode interferir na cicatrização. Diante disso, delineou-se um estudo a fim de avaliar a interferência local da cicatrização de defeitos cutâneos induzidos e fechados em padrão de figura geométrica, associado ao emprego de anestesia por tumescência com lidocaína em coelhos. Ademais, avaliar sua influência em parâmetros cardiorrespiratórios e na analgesia pós-operatória. Foi realizado um estudo com 27 coelhos, separados em três grupos, compreendidos em GC (sem o emprego de tumescência), GS (emprego de tumescência com solução NaCl 0.9%) e GL (emprego de anestesia por tumescência com lidocaína). Não houve diferença estatística em nenhuma etapa do processo cicatricial da ferida entre os grupos, tampouco nas avaliações macroscópicas, e no processo de angiogênese, ou ainda no processo de colagenização e deposição de fibroblastos. Houve diferença significativa na frequência cardíaca (menor no GL) e na respiratória (maior no GC), na pressão arterial média (maior no GL) e na concentração expirada de isoflurano (menor no GL). Não houve diferença significativa entre os grupos no teste de filamentos de von Frey e na Escala Visual Analógica, utilizados para avaliar a analgesia pós-operatória. Conclui-se que anestesia por tumescência com lidocaína não prejudica a regeneração tecidual após a cirurgia. O seu uso oferece benefícios como a diminuição do volume de anestésico inalatório, manutenção do plano anestésico, estabilidade das frequências cardíaca e respiratória, além de menor hipotensão durante o procedimento cirúrgico.

Congenital cutaneous hemangioma in a newborn lamb

Background: Hemangioma is a benign dermal or subcutaneous endothelial cell tumor composed of vascular spaces of varying sizes filled with erythrocytes and lined with a single layer of uniform endothelial cells. Although the pathogenesis is not well defined, these tumors are considered to result from an imbalance in angiogenesis, leading to uncontrolled proliferation of vascular elements. To the best of our knowledge, there are no reports of congenital cutaneous hemangioma in sheep. This report describes the clinical, laboratory, and pathological findings of a case of congenital hemangioma affecting a newborn lamb. Case: A 5-day-old crossbred (Dorper x Santa Inês) lamb presenting with an ear nodule that expanded in the right ear was necropsied. An expansive subcutaneous nodule was observed macroscopically; it occupied approximately 90% of the right ear and had a crusty, irregular surface. The cut surface had multiple cavitations delimited by firm fibrous tissue and a light yellow-to-translucent content. Microscopically, it showed focally extensive subcutaneous neoplastic proliferation and moderate cellularity; it was formed of vascular beds of varied sizes and supported by moderate fibrocollagenous stroma. Suppurative inflammation was observed in the neoplastic vascular beds with large amounts of free basophilic coccoid bacteria inside macrophages. Immunohistochemistry analysis was performed to confirm the diagnosis. Strong cytoplasmic labeling was observed in neoplastic endothelial cells for CD31 and factor VIII. The Ki67 proliferation marker was positive in approximately 5% of neoplastic cells. The cells did not express smooth muscle actin (1A4) or pan-cytokeratin (AE1AE3). Histological characteristics and immunohistochemistry findings were consistent with those of congenital cutaneous hemangioma, a rare neoplasm in sheep. Discussion: The association of clinical, anatomopathological, and immunohistochemical data enabled the diagnosis of congenital cutaneous hemangioma in the 5-day-old lamb. Reports of vascular tumors in sheep are not frequent in literature and usually involve adult animals with no anatomical site predilection. In sheep, the occurrence of nasotracheal hemangioma in a 2-year-old ewe and gingival hemangioma in a 5-year-old sheep have already been described. A cutaneous extra-neural hemangioblastoma was diagnosed in the ear of a 1-month-old lamb. IHC was also used to confirm the diagnosis of hemangioblastoma. Macroscopically, hemangiomas can present as well-delimited and encapsulated masses that when cut, show a reticulated pattern similar to honeycombs that separate the blood-filled cavities. The present case showed a similar conformation but without enough erythrocytes to result in a bloody appearance. Tumor drainage and the predominance of blood serum in the content possibly made it macroscopically translucent. Microscopically, the hemangioma was classified as cavernous. This morphological variation forms large channels separated by fibrous connective tissue stroma, which may contain inflammatory cells. IHC confirmed the endothelial lining of the cystic cavities and was crucial in excluding differential diagnoses. Thus, factor VIII-related antigen was used as a marker for normal and neoplastic cells, as well as for tumoral and reactive neovascularization, in which neoplastic cells were immuno-expressed for CD31 and Factor VIII. In domestic animals, the association between CD31 and Factor VIII is considered more specific for vascular endothelial cells, differentiating them from cells of lymphatic origin. Congenital cutaneous hemangioma occurs in sheep, and its diagnosis and differentiation can be based on histopathology associated with conventional immunohistochemical panels for vascular neoformation.