An allergen-fused dendritic cell-binding peptide enhances in vitro proliferation of equine T-cells and cytokine production.

Allergen-specific immunotherapy (AIT) constitutes the only curative approach for allergy treatment. There is need for improvement of AIT in veterinary medicine, such as in horses suffering from insect bite hypersensitivity, an IgE-mediated dermatitis to Culicoides. Dendritic cell (DC)-targeting represents an efficient method to increase antigen immunogenicity. It is studied primarily for its use in improvement of cancer therapy and vaccines, but may also be useful for improving AIT efficacy. Immunomodulators, like the Toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid-A (MPLA) has been shown to enhance the IL-10 response in horses, while CpG-rich oligonucleotides (CpG-ODN), acting as TLR-9 agonists, have been shown to induce Th1 or regulatory responses in horses with equine asthma. Our aim was to evaluate in vitro effects of antigen-targeting to equine DC with an antigen-fused peptide known to target human and mouse DC and investigate whether addition of MPLA or CpG-ODN would further improve the induced immune response with regard to finding optimal conditions for equine AIT. For this purpose, DC-binding peptides were fused to the model antigen ovalbumin (OVA) and to the recombinant Culicoides allergen Cul o3. Effects of DC-binding peptides on cellular antigen uptake and induction of T cell proliferation were assessed. Polarity of the immune response was analysed by quantifying IFN-γ, IL-4, IL-10, IL-17 and IFN-α in supernatants of antigen-stimulated peripheral blood mononuclear cells (PBMC) in presence or absence of adjuvants. Fusion of DC-binding peptides to OVA significantly enhanced antigen-uptake by equine DC. DC primed with DC-binding peptides coupled to OVA or Cul o3 induced a significantly higher T-cell proliferation compared to the corresponding control antigens. PBMC stimulation with DC-binding peptides coupled to Cul o3 elicited a significant increase in the pro-inflammatory cytokines IFN-γ, IL-4, IL-17, as well as the anti-inflammatory IL-10, but not of IFN-α. Adjuvant addition further enhanced the effect of the DC-binding peptides by significantly increasing the production of IFN-γ, IL-4, IL-10 and IFN-α (CpG-ODN) and IL-10 (MPLA), while simultaneously suppressing IFN-γ, IL-4 and IL-17 production (MPLA). Targeting equine DC with allergens fused to DC-binding peptides enhances antigen-uptake and T-cell activation and may be useful in increasing the equine immune response against recombinant antigens. Combination of DC-binding peptide protein fusions with adjuvants is necessary to appropriately skew the resulting immune response, depending on intended use. Combination with MPLA is a promising option for improvement of AIT efficacy in horses, while combination with CpG-ODN increases the effector immune response to recombinant antigens.

Novel immortalization approach defers senescence of cultured canine adipose-derived mesenchymal stromal cells.

In the last decades, the scientific community spared no effort to elucidate the therapeutic potential of mesenchymal stromal cells (MSCs). Unfortunately, in vitro cellular senescence occurring along with a loss of proliferative capacity is a major drawback in view of future therapeutic applications of these cells in the field of regenerative medicine. Even though insight into the mechanisms of replicative senescence in human medicine has evolved dramatically, knowledge about replicative senescence of canine MSCs is still scarce. Thus, we developed a high-content analysis workflow to simultaneously investigate three important characteristics of senescence in canine adipose-derived MSCs (cAD-MSCs): morphological changes, activation of the cell cycle arrest machinery, and increased activity of the senescence-associated ß-galactosidase. We took advantage of this tool to demonstrate that passaging of cAD-MSCs results in the appearance of a senescence phenotype and proliferation arrest. This was partially prevented upon immortalization of these cells using a newly designed PiggyBac™ Transposon System, which allows for the expression of the human polycomb ring finger proto-oncogene BMI1 and the human telomerase reverse transcriptase under the same promotor. Our results indicate that cAD-MSCs immortalized with this new vector maintain their proliferation capacity and differentiation potential for a longer time than untreated cAD-MSCs. This study not only offers a workflow to investigate replicative senescence in eukaryotic cells with a high-content analysis approach but also paves the way for a rapid and effective generation of immortalized MSC lines. This promotes a better understanding of these cells in view of future applications in regenerative medicine.

Increased teat wall thickness in response to machine milking.

Transiently increased teat wall thickness in response to machine milking has been documented by various methods, including ultrasound. However, correlative ultrasonography and histology to detect the origin of this phenomenon is lacking. The first goal of the present study was to evaluate and compare milking-related changes of the teat tissue in 2 breeds of dairy cows (11 Simmental and 3 Holstein) using B-mode ultrasonography. Additionally, the observed changes were compared with ultrasonographic findings in a Holstein cow with periparturient udder edema. Finally, corresponding histological sections of the Simmental teats were analyzed and compared with those from a lactating nonmilked Angus cow. We hypothesized that the mechanical load of both stretching by the vacuum during phases of open teat cup liner and compression by the closed liner during machine milking results in a transient congestion of blood vessels in the teat wall. The barrel of 1 front teat of each cow was scanned immediately before and after machine milking (system vacuum: 42 kPa; pulsation rate: 60 cycles/min; pulsation ratio: 65:35). Shortly after milking (33 ± 6 min), the Simmentals were slaughtered, and their scanned teat was immediately removed and processed for investigation by light microscopy. Ultrasonography after milking revealed anechoic tubular structures mainly in the inner half of the teat wall. Histological examination revealed these structures to be thick-walled veins. The left front and hind teats of the nonmilked lactating cow, collected and prepared identically to those from the Simmental cows, showed the same histological features. Ultrasonographic measurements showed that the diameter of these veins significantly increased after milking compared with matching images before milking. This effect was most pronounced in the Holstein cows. Similarly, these veins were very prominent in the periparturient cow. However, neither the milked cows, including the periparturient cow, nor the lactating nonmilked cow provided any evidence of edematous extravasation on ultrasonography or histology. These findings corroborated our hypothesis that the increase in size of thick-walled veins in the teat tissue is the main reason for the thickening of the teat walls in response to machine milking.

Establishment of caprine airway epithelial cells grown in an air-liquid interface system to study caprine respiratory viruses and bacteria.

Respiratory diseases negatively impact the global goat industry, but are understudied. There is a shortage of established and biological relevant in vitro or ex vivo assays to study caprine respiratory infections. Here, we describe the establishment of an in vitro system based on well-differentiated caprine airway epithelial cell (AEC) cultures grown under air liquid interface conditions as an experimental platform to study caprine respiratory pathogens. The functional differentiation of the AEC cultures was monitored and confirmed by light and immunofluorescence microscopy, scanning electron microscopy and examination of histological sections. We validated the functionality of the platform by studying Influenza D Virus (IDV) infection and Mycoplasma mycoides subsp. capri (Mmc) colonization over 5 days, including monitoring of infectious agents by titration and qPCR as well as colour changing units, respectively. The inoculation of caprine AEC cultures with IDV showed that efficient viral replication takes place, and revealed that IDV has a marked cell tropism for ciliated cells. Furthermore, AEC cultures were successfully infected with Mmc using a multiplicity of infection of 0.1 and colonization was monitored over several days. Altogether, these results demonstrate that our newly-established caprine AEC cultures can be used to investigate host-pathogen interactions of caprine respiratory pathogens.

Herpesvirus-Associated Proliferative Skin Disease in Frogs and Toads: Proposed Pathogenesis.

A comparative study was carried out on common and agile frogs (Rana temporaria and R. dalmatina) naturally infected with ranid herpesvirus 3 (RaHV3) and common toads (Bufo bufo) naturally infected with bufonid herpesvirus 1 (BfHV1) to investigate common pathogenetic pathways and molecular mechanisms based on macroscopic, microscopic, and ultrastructural pathology as well as evaluation of gene expression. Careful examination of the tissue changes, supported by in situ hybridization, at different stages of development in 6 frogs and 14 toads revealed that the skin lesions are likely transient, and part of a tissue cycle necessary for viral replication in the infected hosts. Transcriptomic analysis, carried out on 2 naturally infected and 2 naïve common frogs (Rana temporaria) and 2 naturally infected and 2 naïve common toads (Bufo bufo), revealed altered expression of genes involved in signaling and cell remodeling in diseased animals. Finally, virus transcriptomics revealed that both RaHV3 and BfHV1 had relatively high expression of a putative immunomodulating gene predicted to encode a decoy receptor for tumor necrosis factor in the skin of the infected hosts. Thus, the comparable lesions in infected frogs and toads appear to reflect a concerted epidermal and viral cycle, with presumptive involvement of signaling and gene remodeling host and immunomodulatory viral genes.

Time-Dependent Internalization of Polymer-Coated Silica Nanoparticles in Brain Endothelial Cells and Morphological and Functional Effects on the Blood-Brain Barrier.

Nanoparticle (NP)-assisted procedures including laser tissue soldering (LTS) offer advantages compared to conventional microsuturing, especially in the brain. In this study, effects of polymer-coated silica NPs used in LTS were investigated in human brain endothelial cells (ECs) and blood-brain barrier models. In the co-culture setting with ECs and pericytes, only the cell type directly exposed to NPs displayed a time-dependent internalization. No transfer of NPs between the two cell types was observed. Cell viability was decreased relatively to NP exposure duration and concentration. Protein expression of the nuclear factor ĸ-light-chain-enhancer of activated B cells and various endothelial adhesion molecules indicated no initiation of inflammation or activation of ECs after NP exposure. Differentiation of CD34+ ECs into brain-like ECs co-cultured with pericytes, blood-brain barrier (BBB) characteristics were obtained. The established endothelial layer reduced the passage of integrity tracer molecules. NP exposure did not result in alterations of junctional proteins, BBB formation or its integrity. In a 3-dimensional setup with an endothelial tube formation and tight junctions, barrier formation was not disrupted by the NPs and NPs do not seem to cross the blood-brain barrier. Our findings suggest that these polymer-coated silica NPs do not damage the BBB.

A novel technique for laser-assisted revascularization: an in vitro pilot study.

The common limitation of surgical revascularization procedures for severe tissue ischemia due to cardiovascular diseases is the need to interrupt blood flow during the intervention. We aim to introduce a new technique that allows a sutureless, non-occlusive revascularization. A 3-step technique was developed using rabbit's aorta to simulate a side-to-side anastomosis model. It enables the creation of a bypass circuit for revascularization. The first step was the soldering of 2 vessels in a side-to-side fashion based on the laser-assisted vascular anastomosis (LAVA) principle using a diode laser emitting irradiation at 810 nm with an albumin-based solder patch between them, followed by the creation of a channel within the patch using either a holmium-doped yttrium aluminum garnet laser (Ho:YAG) at λ = 2100 nm or a xenon-chloride excimer laser (XeCl) at λ = 308 nm. Thereby, a bypass circuit was created, thus allowing a non-ischemic revascularization. The system was deemed functional when a flow was observed across the anastomosis. The highest average tensile strength recorded after side-to-side LAVA using a diode laser power of 3.2 W for 60 s was 2278.6 ± 800 mN (n = 20). The Ho:YAG laser created the channels with less tension on the anastomosis than the excimer laser. Histological analysis showed limited thermal damage and good patch-tissue adaptation. The preliminary results of this feasibility study outline the foundations for an entirely sutureless laser-assisted revascularization procedure. The next studies will evaluate the rheological parameters across the bypass circuit to optimize the post-anastomotic flow.

Ex vivo study of vagal branches at risk for iatrogenic injury during laryngoplasty in horses.

OBJECTIVE: To localize vagal branches within the surgical field of laryngoplasty and identify potentially hazardous surgical steps. STUDY DESIGN: Observational cadaveric study. SAMPLE POPULATION: Five equine head-neck specimens and four entire equine cadavers. METHODS: Dissection of the pharyngeal region from a surgical perspective. Neuronal structures were considered at risk if touched or if the distance to instruments was less than 5 mm. RESULTS: The branches of the pharyngeal plexus (PP) supplying the cricopharyngeal muscle (PPcr), the thyropharyngeal muscle (PPth), and the esophagus (PPes) were identified in the surgical field in nine of nine, five of nine, and one of nine specimens, respectively. The internal branch of the cranial laryngeal nerve (ibCLN) was identified within the carotid sheath in six of nine specimens. The external branch of the cranial laryngeal nerve (ebCLN) was identified close to the septum of the caudal constrictors in nine of nine specimens. The blade of the tissue retractor compressed the ibCLN in six of six, the ebCLN in four of six, the PPcr in six of six, the PPth in two of three, and the PPes in two of two specimens in which the respective nerves were identified after further dissection. Surgical exploration of the dorsolateral aspect of the pharynx and the incision of the septum of the caudal constrictors harmed the ebCLN in nine of nine, PPcr in seven of nine, and PPth in four of eight specimens. CONCLUSION: Several vagal branches were located in the surgical field and must be considered at risk because of their location. CLINICAL SIGNIFICANCE: Use of the tissue retractor, dissection over the pharynx, and dissection of the septum of the caudal constrictors involve a risk to damage vagal branches.

Abnormal keratinocyte differentiation in the nasal planum of Labrador Retrievers with hereditary nasal parakeratosis (HNPK).

Hereditary nasal parakeratosis (HNPK) is an inherited disorder described in Labrador Retrievers and Greyhounds. It has been associated with breed-specific variants in the SUV39H2 gene encoding a histone 3 methyltransferase involved in epigenetic silencing. Formalin-fixed biopsies of the nasal planum of Labrador Retrievers were screened by immunofluorescence microscopy for the presence and distribution of epidermal proliferation and differentiation markers. Gene expression of these markers was further analysed using RNA sequencing (RNA-seq) and ultrastructural epidermal differences were investigated by electron microscopy. Differentiation of the nasal planum in the basal and suprabasal epidermal layers of HNPK-affected dogs (n = 6) was similar compared to control dogs (n = 6). In the upper epidermal layers, clear modifications were noticed. Loricrin protein was absent in HNPK-affected nasal planum sections in contrast to sections of the same location of control dogs. However, loricrin was present in the epidermis of paw pads and abdominal skin from HNPK dogs and healthy control dogs. The patterns of keratins K1, K10 and K14, were not markedly altered in the nasal planum of HNPK-affected dogs while the expression of the terminal differentiation marker involucrin appeared less regular. Based on RNA-seq, LOR and IVL expression levels were significantly decreased, while KRT1, KRT10 and KRT14 levels were up-regulated (log2fold-changes of 2.67, 3.19 and 1.71, respectively) in HNPK-affected nasal planum (n = 3) compared to control dogs (n = 3). Electron microscopical analysis revealed structural alterations in keratinocytes and stratum corneum, and disrupted keratinocyte adhesions and distended intercellular spaces in lesional samples (n = 3) compared to a sample of a healthy control dog (n = 1). Our findings demonstrate aberrant keratinocyte terminal differentiation of the nasal planum of HNPK-affected Labrador Retrievers and provide insights into biological consequences of this inactive SUV39H2 gene variant.

Treponema phagedenis (ex Noguchi 1912) Brumpt 1922 sp. nov., nom. rev., isolated from bovine digital dermatitis.

'Treponema phagedenis' was originally described in 1912 by Noguchi but the name was not validly published and no type strain was designated. The taxon was not included in the Approved Lists of Bacterial Names and hence has no standing in nomenclature. Six Treponema strains positive in a 'T. phagedenis' phylogroup-specific PCR test were isolated from digital dermatitis (DD) lesions of cattle and further characterized and compared with the human strain 'T. phagedenis' ATCC 27087. Results of phenotypic and genotypic analyses including API ZYM, VITEK2, MALDI-TOF and electron microscopy, as well as whole genome sequence data, respectively, showed that they form a cluster of species identity. Moreover, this species identity was shared with 'T. phagedenis'-like strains reported in the literature to be regularly isolated from bovine DD. High average nucleotide identity values between the genomes of bovine and human 'T. phagedenis' were observed. Slight genomic as well as phenotypic variations allowed us to differentiate bovine from human isolates, indicating host adaptation. Based on the fact that this species is regularly isolated from bovine DD and that the name is well dispersed in the literature, we propose the species Treponema phagedenis sp. nov., nom. rev. The species can phenotypically and genetically be identified and is clearly separated from other Treponema species. The valid species designation will allow to further explore its role in bovine DD. The type strain for Treponema phagedenis sp. nov., nom. rev. is B43.1T (=DSM 110455T=NCTC 14362T) isolated from a bovine DD lesion in Switzerland.

Ceratohyoidectomy in standing sedated horses.

OBJECTIVE: To investigate the feasibility and complications associated with ceratohyoidectomy (CHE) in standing sedated horses unaffected (experimental horses) and standing sedated horses affected (clinical cases) with temporohyoid osteoarthropathy (THO). STUDY DESIGN: Case series. ANIMALS: Six experimental horses and four clinical cases. METHODS: Standing CHE was performed in six experimental horses euthanized 30 minutes (n = 3) and 7 days (n = 3) postoperatively. The four clinical cases were presented because of central facial nerve paralysis (n = 3), vestibular ataxia (n = 3), auricular hemorrhage (n = 2), quidding (n = 1), and oesophageal impaction (n = 1). Evolution was assessed by clinical examination during hospitalization and later by telephone interviews for the clinical cases. RESULTS: The procedure was successfully performed in all horses. Experimental horses did not show any short-term postoperative complications. Hemorrhage was experienced intraoperatively in one of the clinical cases and was successfully managed with placement of hemostatic forceps. Vestibular ataxia and other symptoms of THO improved within days, but facial nerve paralysis did not improve until 9 days to 6 months after surgery. Follow-up ranged from 9 to 24 months. All clinical cases returned to performance, and client satisfaction was excellent. CONCLUSION: Ceratohyoidectomy was consistently feasible in standing sedated horses. The method did not result in postoperative complications and led to resolution of clinical signs associated with THO. CLINICAL SIGNIFICANCE: Standing CHE should be considered in horses affected with THO, especially when horses present with marked vestibular deficits and ataxia, to reduce risks associated with recovery from general anesthesia.

Silica nanoparticle-exposure during neuronal differentiation modulates dopaminergic and cholinergic phenotypes in SH-SY5Y cells.

BACKGROUND: Silica-ε-polycaprolactone-nanoparticles (SiPCL-NPs) represent a promising tool for laser-tissue soldering in the brain. After release of the SiPCL-NPs in the brain, neuronal differentiation might be modulated. The present study was performed to determine effects of SiPCL-NP-exposure at different stages of neuronal differentiation in neuron-like SH-SY5Y cells. The resulting phenotypes were analyzed quantitatively and signaling pathways involved in neuronal differentiation and degeneration were studied. SH-SY5Y cells were differentiated with all-trans retinoic acid or staurosporine to obtain predominantly cholinergic or dopaminergic neurons. The resulting phenotype was analyzed at the end of differentiation with and without the SiPCL-NPs given at various times during differentiation. RESULTS: Exposure to SiPCL-NPs before and during differentiation led to a decreased cell viability of SH-SY5Y cells depending on the differentiation protocol used. SiPCL-NPs co-localized with the neuronal marker ß-3-tubulin but did not alter the morphology of these cells. A significant decrease in the number of tyrosine hydroxylase (TH) immunoreactive neurons was found in staurosporine-differentiated cells when SiPCL-NPs were added at the end of the differentiation. TH-protein expression was also significantly downregulated when SiPCL-NPs were applied in the middle of differentiation. Protein expression of the marker for the dopamine active transporter (DAT) was not affected by SiPCL-NPs. SiPCL-NP-exposure predominantly decreased the expression of the high-affinity choline transporter 1 (CHT1) when the NPs were given before the differentiation. Pathways involved in neuronal differentiation, namely Akt, MAP-K, MAP-2 and the neurodegeneration-related markers ß-catenin and GSK-3ß were not altered by NP-exposure. CONCLUSIONS: The decrease in the number of dopaminergic and cholinergic cells may implicate neuronal dysfunction, but the data do not provide evidence that pathways relevant for differentiation and related to neurodegeneration are impaired.

Bovine Epithelial in vitro Infection Models for Mycoplasma bovis.

Mycoplasma bovis causes bovine mycoplasmosis. The major clinical manifestations are pneumonia and mastitis. Recently an increase in the severity of mastitis cases was reported in Switzerland. At the molecular level, there is limited understanding of the mechanisms of pathogenicity of M. bovis. Host-pathogen interactions were primarily studied using primary bovine blood cells. Therefore, little is known about the impact of M. bovis on other cell types present in infected tissues. Clear in vitro phenotypes linked to the virulence of M. bovis strains or tissue predilection of specific M. bovis strains have not yet been described. We adapted bovine in vitro systems to investigate infection of epithelial cells with M. bovis using a cell line (MDBK: Madin-Darby bovine kidney cells) and two primary cells (PECT: bovine embryonic turbinate cells and bMec: bovine mammary gland epithelial cells). Two strains isolated before and after the emergence of severe mastitis cases were selected. Strain JF4278 isolated from a cow with mastitis and pneumonia in 2008 and strain L22/93 isolated in 1993 were used to assess the virulence of M. bovis genotypes toward epithelial cells with particular emphasis on mammary gland cells. Our findings indicate that M. bovis is able to adhere to and invade different epithelial cell types. Higher titers of JF4278 than L22/93 were observed in co-cultures with cells. The differences in titers reached between the two strains was more prominent for bMec cells than for MDBK and PECT cells. Moreover, M. bovis strain L22/93 induced apoptosis in MDBK cells and cytotoxicity in PECT cells but not in bMec cells. Dose-dependent variations in proliferation of primary epithelial cells were observed after M. bovis infection. Nevertheless, an indisputable phenotype that could be related to the increased virulence toward mammary gland cells is not obvious.

The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8+ T cells.

T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b-/- CD8+ T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b-/- CD8+ T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b-/- CD8+ T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8+ T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue-resident T cell populations.

Binding of indocyanine green in polycaprolactone fibers using blend electrospinning for in vivo laser-assisted vascular anastomosis.

BACKGROUND AND OBJECTIVE: The clinical application of laser-assisted vascular anastomosis is afflicted by unreliable and low bonding strengths as well as tedious handling during microvascular surgery. The challenge to be met arises from the flow-off of the chromophore during soldering that changes the absorption and stains the surrounding tissue, leading to an uncontrollable thermal damage zone. In this study, we investigated the feasibility to produce an indocyanine green (ICG)-loaded patch by electrospinning and tested its applicability to both in vitro and in vivo microvascular laser soldering. MATERIALS AND METHODS: A blend of polycaprolactone and ICG was electrospun to produce a pliable patch. Prior to soldering, the patch was soaked in 40% wt. bovine serum albumin solution. The solder patch was wrapped in vitro around blood vessel stumps of rabbit aortas. An intraluminal balloon catheter enabled an easy alignment and held the setup in place. The soldering energy was delivered via a diffusor fiber from the vessel lumen using a diode laser at 810 nm. During the procedure, the surface temperature was observed with an infrared camera. Afterward, samples were embedded in methylmethacrylate and epon to study thermal damage. The quality of the fusion was assessed by measuring the tensile strength. After in vitro tests with rabbit aortas, eight large white pigs were subjected to an acute in vivo experiment, and the artery of the latissimus dorsi flap was anastomosed to the distal femoral artery. RESULTS: The ICG-loaded patch, produced by electrospinning, has a thickness of 279 ± 62 µm, a fiber diameter of 1.20 ± 0.19 µm, and an attenuation coefficient of 1,119 ± 183 cm-1 at a wavelength of 790 nm. The patch was pliable and easy to handle during surgery. No leakage of the chromophore was observed. Thermal damage was restricted to the Tunica adventitia and Tunica media and the area of the vessel wall that was covered with the patch. Six pigs were successfully treated, without any bleeding and with a continuous blood flow. The in vivo flap model yielded a similar tensile strength compared to in vitro laser-assisted vascular anastomoses (138 ± 52 vs. 117 ± 30 mN/mm2 ). CONCLUSION: Our study demonstrated the applicability of the ICG-loaded patch for laser-assisted vascular anastomosis. By using electrospinning, ICG could be bound to polymer fibers, avoiding its flow-off and the staining of the surrounding tissue. This patch demonstrated several advantages over liquid solder as it was easier to apply, ensured a high and reliable bonding strength while maintaining a constant concentration of ICG concentration during the surgery. Lasers Surg. Med. 49:928-939, 2017. © 2017 Wiley Periodicals, Inc.

Generation of a molecular clone of an attenuated lentivirus, a first step in understanding cytopathogenicity and virulence.

Small ruminant lentiviruses infect goats and sheep, inducing clinical disease in a minority of infected animals. Following an eradication campaign, clinical cases may disappear in a population. The complete elimination of these lentiviruses is however difficult to achieve and the spreading of less virulent strains often parallels the elimination of their virulent counterparts. Here, we characterized three such strains isolated from a flock in the post-eradication phase. We completely sequenced their genomes, showing that one of the isolates was most probably the product of a recombination event between the other two viruses. By comparing the sequences of these isolates with those of virulent strains, we found evidence that particular LTR mutations may explain their attenuated phenotype. Finally, we constructed an infectious molecular clone representative of these viruses, analyzing its replication characteristics in different target cells. This clone will permit us to explore the molecular correlates of cytopathogenicity and virulence.

Ultrasound-guided proximal paravertebral anaesthesia in cattle.

OBJECTIVE: To develop and evaluate a method for ultrasound-guidance in performing the proximal paravertebral block for flank anaesthesia in cattle through a cadaveric study, followed by clinical application. STUDY DESIGN: prospective experimental cadaveric study and clinical series. ANIMALS: Previously frozen lumbar sections of cows without known spinal abnormalities were used. The clinical case group comprised of ten animals for which a right flank laparotomy was indicated. METHODS: Twenty cow cadavers were used to perform ultrasound-guided bilateral injections of 1.0 mL dye (1.0 mL 1% Toluidine Blue in 1% Borax) at the intervertebral foramen at the level of T13, L1 and L2 spinal nerves. Distance and depth of injection, staining of the dorsal and ventral nerve branches, and deviation from the target were evaluated. The investigator's confidence as to visualisation and expected success at staining the nerve was assessed. Ten clinical cases received the ultrasound-guided proximal paravertebral anaesthesia. Analgesic success was evaluated using a 4-grade scoring system at 10 minutes after the injection and during surgery, respectively. Categorical variables were described using frequencies and proportions. RESULTS: Both dorsal and ventral branches of the spinal nerves T13, L1 or L2 were at least partially stained in 41% of injections, while in 77% of injections one of the branches was stained. Five out of ten clinical cases had a satisfactory anaesthesia. There was no significant association between confidence at injection and either staining or analgesic success. CONCLUSION: Results from the cadaveric and clinical study suggest no significant improvement using ultrasound guidance to perform proximal paravertebral block in cows compared to our previous clinical experience and to references in the literature using the blind method. CLINICAL RELEVANCE: Further research should be conducted to improve the ultrasound-guided technique described in this study.

Ultrasound-guided block of sciatic and femoral nerves: an anatomical study.

The sheep is a popular animal model for human biomechanical research involving invasive surgery on the hind limb. These painful procedures can only be ethically justified with the application of adequate analgesia protocols. Regional anaesthesia as an adjunct to general anaesthesia may markedly improve well-being of these experimental animals during the postoperative period due to a higher analgesic efficacy when compared with systemic drugs, and may therefore reduce stress and consequently the severity of such studies. As a first step 14 sheep cadavers were used to establish a new technique for the peripheral blockade of the sciatic and the femoral nerves under sonographic guidance and to evaluate the success rate by determination of the colorization of both nerves after an injection of 0.5 mL of a 0.1% methylene blue solution. First, both nerves were visualized sonographically. Then, methylene blue solution was injected and subsequently the length of colorization was measured by gross anatomical dissection of the target nerves. Twenty-four sciatic nerves were identified sonographically in 12 out of 13 cadavers. In one animal, the nerve could not be ascertained unequivocally and, consequently, nerve colorization failed. Twenty femoral nerves were located by ultrasound in 10 out of 13 cadavers. In three cadavers, signs of autolysis impeded the scan. This study provides a detailed anatomical description of the localization of the sciatic and the femoral nerves and presents an effective and safe yet simple and rapid technique for performing peripheral nerve blocks with a high success rate.

Development of an ultrasound-guided technique for pudendal nerve block in cat cadavers.

The objective of this prospective experimental cadaveric study was to develop an ultrasound-guided technique to perform an anaesthetic pudendal nerve block in male cats. Fifteen fresh cadavers were used for this trial. A detailed anatomical dissection was performed on one cat in order to scrutinise the pudendal nerve and its ramifications. In a second step, the cadavers of six cats were used to test three different ultrasonographic approaches to the pudendal nerve: the deep dorso-lateral, the superficial dorso-lateral and the median transperineal. Although none of the approaches allowed direct ultrasonographical identification of the pudendal nerve branches, the deep dorso-lateral was found to be the most advantageous one in terms of practicability and ability to identify useful and reliable landmarks. Based on these findings, the deep dorso-lateral approach was selected as technique of choice for tracer injections (0.1 ml 1% methylene blue injected bilaterally) in six cat cadavers distinct from those used for the ultrasonographical study. Anatomical dissection revealed a homogeneous spread of the tracer around the pudendal nerve sensory branches in all six cadavers. Finally, computed tomography was performed in two additional cadavers after injection of 0.3 ml/kg (0.15 ml/kg per each injection sites, left and right) contrast medium through the deep dorso-lateral approach in order to obtain a model of volume distribution applicable to local anaesthetics. Our findings in cat cadavers indicate that ultrasound-guided pudendal nerve block is feasible and could be proposed to provide peri-operative analgesia in clinical patients undergoing perineal urethrostomy.

Sciatic-femoral nerve block with bupivacaine in goats undergoing elective stifle arthrotomy.

The aim of this study was to describe the sciatic-femoral nerve block (SFNB) in goats and to evaluate the peri-operative analgesia when the goats underwent stifle arthrotomy. The animals were randomly assigned to one of four treatment groups: groups 0.25, 0.5 and 0.75 received 0.25%, 0.5% and 0.75% of bupivacaine, respectively, while group C (control group) received 0.9% NaCl. In all groups, the volume administered was 0.2 mL/kg. Intra-operatively, the proportion of animals receiving rescue propofol was significantly lower in groups 0.5 and 0.75, compared to group C. Post-operatively, the visual analogue scale (VAS) and total pain score were significantly higher in group C than in the other groups. Group 0.75 had the highest percentage of animals showing motor blockade. SFNB performed with bupivacaine resulted in better intra- and post-operative analgesia than SFNB performed with saline. Compared to the other concentrations, 0.5% bupivacaine resulted in satisfactory analgesia with acceptable side effects.