Video-assisted ovarian sterilization by occluding the vascular supply with a modified Miller's knot and performing bilateral partial excision of the ovaries in the red-eared slider turtle (Trachemys scripta elegans).

Invasive alien species pose a serious threat to native biodiversity and the ecosystem structure through predation or competition, and this threat is especially severe in China. Red-eared sliders (Trachemys scripta elegans) were introduced to mainland China to meet demands for food, traditional Chinese medicine, and the pet trade. However, there has been a lack of field surveys to assess the population, behavior and ecological impacts of introduced red-eared sliders in urban aquatic ecosystems in northern China. We conducted such research in 33 urban parks in Beijing from April to November 2016. The alien sliders were recorded on 514 occasions at 19 parks, and 45 individuals were captured from 4 parks to evaluate their diet composition and reproductive status. The observations showed that red-eared sliders accounted for 95% of the recorded turtles in the parks. The male to female ratio was 1.0:3.5, and the ratio of adults to juveniles was 1.0:3.1. According to stomach content analysis, the sliders fed mainly on animals: the dominant prey were Gastropoda, Osteichthyes, Crustacea, Hexapoda, Bivalvia, Reptilia and Aves in descending order. The high frequency (83%) of follicles and oviduct development in adult females suggested a strong reproductive capacity, which was further supported by one case of early female maturity. Bacteriological examinations demonstrated that the carrying rate of Salmonella bacteria was up to 17.6%. This study demonstrated that red-eared sliders have successfully colonized the waters of urban parks in Beijing and potentially threaten native aquatic biodiversity.

Cutaneous wounds are common in chelonians. The clinical and histological features of wound healing in these species are not well described and this prevents evaluation of new therapies. To describe clinical and histopathological features of cutaneous wound healing in the red-eared slider turtle (Trachemys scripta elegans). Twenty four healthy adult females housed in outdoor facilities with free access to water and exposed to daily variations in temperature. Full thickness 6 mm skin biopsy punch wounds were created in the rear limbs. The turtles were assigned to Group 1 (n = 12 for clinical evaluation) and Group 2 (n = 12 for microscopic study). Group 1 was photographed on Day 1 and weekly, until 28 days post wounding. Wound retraction was expressed as the percentage of perimeter reduction. For Group 2, three skin wounds were sampled at 2, 7, 14, 21, 28, 42, 60 and 135 days post wounding for histological study. The avidin-biotin-peroxidase (ABC) staining method was used to evaluate five commercial antibodies. Wound contraction was limited; crust persisted at least 28 days. Re-epithelialization was complete by Day 14 in many animals; active inflammation persisted until 28 days; connective tissue re-constitution and remodelling was achieved from 42 to 135 days. Antibodies AE1/AE3, Factor VIII, MAC 387, CD3 and NCL-MSA showed cross reactivity with the cell counterpart in turtle tissues. Second intention wound healing progressed slowly and with an indolent behaviour. Microscopically there was marked overlapping of the inflammatory and proliferative phases over a long time period.

Background:Coeliotomy in chelonian species is performed to gain access to the abdominal cavity and requires general anesthesia. In reptile medicine, alfaxalone and propofol are often preferred, although the usage of these drugs is limited by cost and/or vascular access. Furthermore, according to a recent study, propofol produces significant oxidative stress in red-eared sliders (Trachemys scripta elegans), and in such cases, a ketamine and medetomidine combination may provide a better choice. Case description:Three owned red-eared sliders were separately presented with a history of recent lethargy, inappetence, dyspnea, and restlessness. Findings/treatment and outcome:In these three cases of chronic dystocia, a ketamine and medetomidine combination was used to achieve anesthesia induction for surgical treatment. Additionally, intrathecal administration of lidocaine was performed to achieve a potential analgesic effect in deeper intracoelomic reproductive organs. Preoperative changes in blood parameters were observed in one animal, suggesting abnormal kidney function. The same animal showed signs of resedation postoperatively, prolonged recovery, and had a fatal outcome 48 h after the surgery with signs of kidney damage in histopathological evaluation. In the recovery period, none of the surviving animals (n=2) showed neurological or respiratory complications that could be associated with subcarapacial sinus anesthesia administration or intrathecal local anesthesia. Conclusion:According to our observations, intrathecal lidocaine administration provided sufficient analgesia during prefemoral coeliotomy and reproductive organ manipulation. These modes of anesthesia in chelonians require further investigation.

Traumatic lesions of the shell are common problems in chelonians. Lesions of the shell vary in severity from a simple superficial abrasion (with a loss of the external skin layer and exposure of the bone) to complicated fractures of the carapace with exposure of internal organs of the coelom and injury of the soft tissues (Barten, 2006). In most shell or skeletal injuries in the turtle, plain radiographic data do not reveal the extent of complex fractures due to superimposed bone structures (Abou-Madi et al., 2002). In Comparison with conventional radiography, computed tomography (CT) allows better distinction of specific tissue densities and discrete changes in organ size, shape, margin, contour and position (Gaudron et al., 2001). Computed tomography is a non-invasive, cross-sectional diagnostic imaging technique that offers significant advantages for detection of pathologies in chelonians, and is ideal for diagnosing skeletal and soft tissue abnormalities (Gumpenberger and Henninger, 2001). The aim of the present work was to evaluate the three dimensional computed tomography (CT) for clinical examination of shell lesions in a red eared slider turtle.

BackgroundThe tissue growth necessary to achieve a complete or partial restitution ad integrum as a result of injury to soft tissue and/or hard times in reptiles is variable and often needs long time in relation to the species, to the habitat and to their intrinsic physiological characteristics. The purpose of this work was to see if the tissue optimization (TO) treatment with radio electric asymmetric conveyer (REAC) provided good results in these animals and whether its use translates into reduced time of tissue repair. This paper describes preliminary results with in promoting the tissue repair in reptiles. Cases presentationA 5 year old male Testudo graeca (Leo) and Trachemys scripta scripta (Mir) and a 15 year old female Testudo hermanni (Juta) were evaluated because of soft tissue injuries. A female 25 year old Trachemys scripta elegans (Ice), a female 2.5 year old Trachemys scripta scripta (Penelope) as well as a 50 year old male Testudo graeca (Margherito) were evaluated because of wounds of the carapace. Following debridement and traditional therapies, Leo, Penelope and Margherito were exposed to the radio electric asymmetric conveyer (REAC) device, with a specific treatment protocol, named tissue optimization-basic (TO-B). Also Ice and Mir were subjected to REAC treatment after wounds debridement. Juta was treated only with REAC treatment. Complete wound healing was evident after 17 days for Leo, 7 days for Penelope, 27 days for Mir, 78 days for Ice and after 14 days for Margherito. Juta showed a considerable tissue activation in 2 days and complete wound healing in 5 days. ConclusionOur findings suggest that REAC TO-B treatment may provide advantages over other traditional methods after complete wound healing in Leo, and also suitable healing in the other patients. Then REAC device with its specific treatment TO-B protocol, which induces tissue repair without causing severe stress to the patient, could be a potential therapy for tissue damage healing in reptiles. Further studies still need to be conducted to support our observations.

The myofibroblast, a major component of granulation tissue, is a key cell during wound healing, tissue repair and connective tissue remodelling. Persistence of myofibroblasts within a fibrotic lesion leads to excessive scarring impairing function and aesthetics. Various wound-healing cytokines can be modulated by topical application of active agents to promote optimal wound healing and improve scar quality. Thus, the myofibroblast may represent an important target for wound-healing modulation to improve the evolution of conditions such as hypertrophic scars. The purpose of this work is to study the modulation of myofibroblasts and integrin alphavbeta3 in a full thickness wound performed on rabbits treated with different topical agents using: (1) saline, (2) Tegaderm occlusive dressing (3) silver sulfadiazine and (4) moist exposed burn ointment (MEBO). The reepithelialisation was 4 days faster in the MEBO group compared with the other therapies with less oedema formation, delayed contraction, less inflammatory cells and the lowest transepidermal water loss (TEWL) resulting in a soft scar. Although alpha-smooth muscle actin (alpha-SMA) was the highest around day 12 in the MEBO group, wound contraction and myofibroblast's activity were the least for the same period probably because of a downregulation of the integrin alphavbeta3. It seems that the effect of MEBO could be more pronounced on force transmission rather then on force generation. Greater insight into the pathology of scars may translate into non surgical treatments in the future and further work in myofibroblast biology will eventually result in efficient pharmacological tools, improving the evolution of healing and scar formation.

Detection of connexin (Cx) proteins has been used as an indicator of the presence of structural and functional gap junctions in tissues. To examine the role of gap junctions during follicular growth and atresia, the presence of three major connexins, Cx43, Cx32, and Cx26, was evaluated in bovine ovaries by using immunohistochemistry and Western immunoblot analysis. Cx43 was not present in primordial follicles, but was present in granulosa cells of primary/secondary and antral follicles. Cx43 also was present on the borders between granulosa cells and the oocyte. Expression of Cx43 increased in healthy developing antral follicles, but decreased during follicular atresia. Cx32 was not present in healthy follicles but was present in granulosa cells of atretic antral, and especially small antral follicles. Cx26 was present in the oocyte of primordial and primary/secondary follicles, and in the granulosa and/or thecal cell layers of healthy antral follicles. The percentage of healthy antral follicles that expressed Cx26 also increased during follicular development, but decreased during atresia. Cx32 and Cx26 also were detected in ovarian blood vessels and in stromal tissues adjacent to the tunica albuginea in some ovaries. The pattern of expression of these Cx indicates that gap junctional proteins may be involved in the control of follicular growth and atresia in cows.

Chapter 3 - Connective tissue inflammation, repair and remodelling

Mast cells (MCs) are active participants in blood coagulation and innate and acquired immunity. This review focuses on the development of mouse and human MCs, as well as the involvement of their granule serine proteases in inflammation and the connective tissue remodeling that occurs during the different phases of the healing process of wounded skin and other organs. The accumulated data suggest that MCs, their tryptases, and their chymases play important roles in tissue repair. While MCs initially promote healing, they can be detrimental if they are chronically stimulated or if too many MCs become activated at the same time. The possibility that MCs and their granule serine proteases contribute to the formation of keloid and hypertrophic scars makes them potential targets for therapeutic intervention in the repair of damaged skin.

The vascular changes of the ovary were studied by scanning electron microscopy of vascular corrosion casts in estrous, pseudopregnant (stimulated with human chorionic gonadotropin - hCG) and pregnant rabbits. The results demonstrated that ovarian cycle and pregnancy may induce both structural and functional changes in the ovarian vessels. In fact the ovarian blood vessels adapted their structure to the temporary functional needs of the recruited follicles or corpora lutea. These changes involved both larger ovarian blood vessels (supplying the hilus and the medulla) and cortical blood vessels (supplying the luteo-follicular complexes). Hilar and medullary blood vessels also displayed morphological devices for the continuous control of the blood flow (spiral arteries) and for the local recirculation of endocrine products (artero-venous contacts). Such contacts, may likely sustain a countercurrent mechanism which was also shown in ovaries of other species. Scanning electron microscopy of vascular corrosion casts even demonstrated that cortical blood vessels are subjected to continuous remodeling. SEM showed different morphological types of vascular plexuses which supplied antral follicles (Types 1-2), atretic follicles (Types 3-4), peri-ovulatory follicles (Type 5), growing pseudopregnant corpora lutea (Type 6) regressing pseudopregnant corpora lutea (Type 7) and pregnant corpora lutea (Type 8). In estrous rabbit, growing to mature follicles (Types 1-2) showed a gradual enlargement and proliferation of the theca capillaries. These changes, associated with capillary hyper-permeabilization were observed in ovulatory and post-ovulatory follicles (Type 5), after hCG stimulation. The corpus luteum formation (Type 6 and 8) was accompanied by additional capillary dilation, diffuse angiogenetic sprouting and organization of conspicuous venous drainage which appeared more enhanced in pregnant (type 8) than in pseudopregnant (type 6) corpora lutea. The regression of the corpus luteum (Type 7) was characterized by the appearance of avascular areas within the gland and by regression of vascular dilatation. Finally, in estrous rabbit the atretic follicles (Types 3-4) wall showed large interruptions (avascular areas) and focal invasion of the central cavity by newly formed capillaries arranged randomly. The hCG stimulation did not affect consistently the interstitial microvasculature. It is concluded that the gradual increase of ovarian blood flow occuring during follicle growth and corpora lutea formation is provided by a functional adaptation of major ovarian vessels and by capillary functional (vasodilation) and structural (angiogenesis) support as well.

Surgical wound closure is critical in oncologic surgeries and directly influences healing, postoperative complications, and recovery time. In canine mastectomies, conventional skin closure with nylon sutures remains the standard technique; however, surgical staples have emerged as a potential alternative that can potentially optimize surgical efficiency and outcomes. This study evaluated the efficacy of surgical staples as an alternative to routine standard suture (nylon) in female dogs undergoing mastectomies. Fifty female dogs were divided into two groups: one was treated with nylon suture (NS group, n = 25) and the other with surgical staples (SS group, n = 25). Surgical time, local wound temperature, and wound healing characteristics were evaluated. Thermographic analysis of the surgical scar was performed on a subsample. Logistic regression and multivariate linear analysis were used to compare differences between the techniques, and an independent samples t test was applied to compare surgical and suture times. No significant differences were found between the groups regarding age, weight, incision size, body size, and reproductive status. Total surgical time was significantly shorter in the SS group (p = 0.011). The closure time was 9.8 times shorter in the SS group versus the NS group (p < 0.001). The average wound temperature was higher in the NS group (p < 0.001). The SS group showed fewer occurrences of wound alterations (p = 0.029) and a lower frequency of focal inflammatory exudate (p = 0.013). The thermographic analysis showed more white pixels and higher temperatures in the surgical wound in the NS group versus the SS group (p = 0.032 and p < 0.001, respectively). Surgical staples were found to be safe and effective for wound closure in mastectomies in female dogs, offering a viable alternative to traditional nylon suture techniques. Although the cost of staples is slightly higher, the benefits of shorter surgical time and reduced complications can make this technique a favorable choice, especially for oncologic patients with additional health concerns or surgical time restrictions.

In the present study, we have localized desmin and alpha-smooth-muscle actin in the ovaries of the zebrafish, the axolotl, and the red-eared turtle, using the unlabelled antibody peroxidase-antiperoxidase technique. In the axolotl ovary both smooth-muscle markers were demonstrated in cord-like structures, extended along ovarian blood vessels, and in some inner ovarian epithelium cells. In the ovaries of the teleost, smooth-muscle-like cells are detected in a suspensory apparatus formed by venous cords, the tunica albuginea, and the coat around the ovarian artery. Also, in the turtle ovary, smooth-muscle-like cells were found in a suspensory apparatus formed by chordae, the tunica albuginea, and the theca externa of the ovarian follicles. At the present time, the prevailing hypothesis is that, in addition to a role in the mechanical support, the smooth-muscle-like cells in the ovaries of these vertebrates seem to be important with respect to ovarian contractile processes.

Background: Matrix metalloproteinases (MMPs) are major contributors to tumor invasion, remodeling of connective tissue and infiltration of inflammatory cells and may be important mediators in developing allergic inflammation. Overexpression of MMP-9 mRNA by eosinophils in the asthmatic airways has been reported. To clarify the relative significance of MMP as an inflammatory mediator from eosinophils, we determined the content of MMP-9 in the peripheral blood eosinophils and compared it with the other leukocyte fractions. Methods: Peripheral blood eosinophils, neutrophils, and mononuclear cells were purified from normal and allergic donors with Percoll gradient centrifugation and CD16 negative selection. Cell lysate and culture supernatants stimulated with IL-5, PAF, and PMA were tested for MMP-9 with gelatin zymography and ELISA. Results: The amount of MMP-9 in highly purified eosinophils, neutrophils, and mononuclear cells was 2.5 ± 0.9, 4,073 ± 581, and 7.6 ± 1.4 ng/5 × 10⁶ cells, respectively. There was no difference in MMP-9 content of eosinophils between normal donors and patients with asthma. Culture of peripheral blood eosinophils with IL-5 for 4 days did not induce MMP-9 production. The stimulation of eosinophils with PMA and other secretogogues caused only small amounts of MMP-9 secretion as compared with neutrophils. Conclusions: These findings suggest that circulating eosinophils normally have only small amounts of MMP-9 and that eosinophils may need complex activation signals to produce significant amounts of MMP as seen in tissues of allergic inflammation.

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68+ and HmAIF-1+ macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

Transforming growth factor beta (TGFbeta) plays a critical role in connective tissue remodeling by fibroblasts during development, tissue repair, and fibrosis. We investigated the molecular pathways in the transmission of TGFbeta signals that lead to features of connective tissue remodeling, namely formation of an alpha-smooth muscle actin (alpha-SMA) cytoskeleton, matrix contraction, and expression of profibrotic genes. TGFbeta causes the activation of focal adhesion kinase (FAK), leading to JNK phosphorylation. TGFbeta induces JNK-dependent actin stress fiber formation, matrix contraction, and expression of profibrotic genes in fak+/+, but not fak-/-, fibroblasts. Overexpression of MEKK1, a kinase acting upstream of JNK, rescues TGFbeta responsiveness of JNK-dependent transcripts and actin stress fiber formation in FAK-deficient fibroblasts. Thus we propose a FAK-MEKK1-JNK pathway in the transmission of TGFbeta signals leading to the control of alpha-SMA cytoskeleton reorganization, matrix contraction, and profibrotic gene expression and hence to the physiological and pathological effects of TGFbeta on connective tissue remodeling by fibroblasts.