Nerve Regeneration after a Nerve Graft in a Rat Model: The Effectiveness of Fibrin Glue.

BACKGROUND: Simulating the post-traumatic continuity defect of small human peripheral nerves, we compared the effectiveness of fibrin glue with neurorrhaphy for nerve gap restoration. METHODS: In twenty-four male Wistar rats, a fifteen mm defect in one sciatic nerve only was made and immediately repaired with an inverted polarity autograft. According to the used technique, rats were divided into Group A (Control), using traditional neurorrhaphy, and Group B (Study), using fibrine glue sealing; in total, 50% of rats were sacrificed at 16 weeks and 50% at 21 weeks. Before sacrifice, an assessment of motor function was done through Walking Track Analysis and an electroneurophysiological evaluation. After sacrifice, selected muscle mass indexes and the histology of the regenerated nerves were assessed. All data were evaluated by Student's t test for unpaired data. RESULTS: No significant differences were found between the two groups, with only the exception of a relative improvement in the tibialis anterior muscle's number of motor units in the study group. CONCLUSION: Despite the fact that the use of fibrin glue as a nerve sealant is not superior in terms of functional recovery, its effectiveness is comparable to that of microsurgical repair. Hence, the faster and technically easier glueing technique could deserve broader clinical application.

Antibiotic Resistance Genes Carried by Commensal Escherichia coli from Shelter Cats in Italy.

Antimicrobial resistance is a widespread global health problem. The presence of resistant bacteria and antibiotic resistance genes has been demonstrated not only in humans but also in animals, including pets. Stray cats share the urban environment with people and pets. This may facilitate transmission of resistant bacteria and resistance genes between stray animals, people and domestic animals. Several studies have investigated the role of stray cats as a fecal carrier of ESBL-producing bacteria. However, there are many genes and resistance mechanisms that can be detected in commensal E. coli, which, because of its genetic plasticity, is considered an indicator for monitoring antibiotic resistance. In this study, rectal swabs were collected from stray cats from colonies and shelters in the city of Monza (Monza Brianza, Italy) to isolate commensal E. coli. Phenotypic tests, such as the minimum inhibitory concentration (MIC) and the double disc test (DDST), and molecular analyses to detect antimicrobial resistance genes (ARGs) were used to study the resistance of these isolates. The results obtained confirm that stray cats can carry ESBL-producing E. coli (6.7%) and genes conferring resistance to other important antibiotic classes such as tetracyclines and sulfonamides.

Peripheral Nerve Regeneration at 1 Year: Biodegradable Polybutylene Succinate Artificial Scaffold vs. Conventional Epineurial Sutures.

The utilization of a planar poly(1,4-butylene succinate) (PBS) scaffold has been demonstrated as an effective approach for preserving nerve continuity and facilitating nerve regeneration. In this study, we assessed the characteristics of a microfibrous tubular scaffold specifically designed and fabricated through electrospinning, utilizing PBS as a biocompatible and biodegradable material. These scaffolds were evaluated as nerve guide conduits in a rat model of sciatic nerve neurotmesis, demonstrating both their biodegradability and efficacy in enhancing the reconstruction process over a long-term period (1-year follow-up). Histological assay and electrophysiological evaluation were performed to compare the long-term outcomes following sutureless repair with the microfibrillar wrap to outcomes obtained using traditional suture repair.

Inflammatory-like status and acute stress response in horses after road transport.

This study aimed to evaluate the change of white blood cell count, serum concentration of cortisol, C-reactive protein, albumin and globulin fractions in horse after road transport, and to assess the linkage among hypothalamic-pituitary-adrenal axis (HPA) and inflammatory reaction. From 10 horses blood samples were collected at rest, before road transport (218 km) (BT); after unloaded (AT), 30 and 60 min after unloaded (AT30 and AT60) in order to assess white blood cell count (WBC), serum cortisol, C-reactive protein (CRP), total proteins, albumin, α1-, α-2, ß1-, ß2- and γ-globulins. WBC, cortisol, CRP, α1-, α-2 and ß2-globulins values increased after road transport than rest condition (p < 0.001). Albumin and A/G ratio showed lower values after road transport than rest (p < 0.001). Pearson's test showed a negative correlation between cortisol and the values of WBC, CRP, α1-, α2-, ß1-, ß2- globulins, and a positive correlation between WBC and serum concentration of CRP, α1- and α2-, ß1-, ß2-globulins at AT and AT30. The results showed that road transport evokes an inflammatory like-status in horses. Moreover, the activation of HPA and the onset of acute phase reaction in response to road transport seem to be interconnected with effects on horse's immune status.

Tentacle Mesh for Fixation-Free Spigelian Hernia Repair: Mini-Invasive Approach Granting Broad Defect Overlap.

BACKGROUND: Compared to other types of abdominal protrusions, Spigelian hernias are not very common. In prosthetic repair of abdominal protrusions, mesh fixation and defect overlap are an open issue, as they are a source of complications. A newly developed tentacle-shaped mesh has been used to ensure a fixation-free repair with a broader defect overlap in the repair of abdominal hernias. This study describes the long-term results of a fixation-free repair of Spigelian hernias carried out with a tentacle mesh. METHODS: A proprietary mesh composed of a central body with integrated radiating arms was used for repairing Spigelian hernias in 54 patients. The implant was positioned in preperitoneal sublay, and the straps were delivered across the abdominal musculature with a needle passer, and then, after fascia closure, cut short in the subcutaneous layer. RESULTS: The friction of the straps passing through the abdominal wall served to hold the mesh in place, guaranteeing a wide overlap over the defect without fixation. In a long-term follow-up of 6 to 84 months (mean 64 months), a very low rate of complications occurred, but no recurrence was reported. CONCLUSIONS: The tentacle strap system of the prosthesis allowed for an easy, fast and safe fixation-free placement granting a wide overlap, avoiding intraoperative complications. Greatly reduced pain and a negligible amount of postoperative complications characterized the postoperative outcome.

Physiologic Cyclical Load on Inguinal Hernia Scaffold ProFlor Turns Biological Response into Tissue Regeneration.

Surgical repair of groin protrusions is one of the most frequently performed procedures. Currently, open or laparoscopic repair of inguinal hernias with flat meshes deployed over the hernial defect is considered the gold standard. However, fixation of the implant, poor quality biologic response to meshes and defective management of the defect represent sources of continuous debates. To overcome these issues, a different treatment concept has recently been proposed. It is based on a 3D scaffold named ProFlor, a flower shaped multilamellar device compressible on all planes. This 3D device is introduced into the hernial opening and, thanks to its inherent centrifugal expansion, permanently obliterates the defect in fixation-free fashion. While being made of the same polypropylene material as conventional hernia implants, the 3D design of ProFlor confers a proprietary dynamic responsivity, which unlike the foreign body reaction of flat/static meshes, promotes a true regenerative response. A long series of scientific evidence confirms that, moving in compliance with the physiologic cyclical load of the groin, ProFlor attracts tissue growth factors inducing the development of newly formed muscular, vascular and nervous structures, thus re-establishing the inguinal barrier formerly wasted by hernia disease. The development up to complete maturation of these highly specialized tissue elements was followed thanks to biopsies excised from ProFlor from the short-term up to years post implantation. Immunohistochemistry made it possible to document the concurrence of specific growth factors in the regenerative phenomena. The results achieved with ProFlor likely demonstrate that modifying the two-dimensional design of hernia meshes into a 3D outline and arranging the device to respond to kinetic stresses turns a conventional regressive foreign body response into advanced probiotic tissue regeneration.

Primary Ovarian Leiomyoma in a White Tiger (Panthera tigris).

Ovarian leiomyomas are very rare in domestic cats and occasionally mentioned in studies reporting general pathological findings and neoplasm occurrence in non-domestic large felids. This report describes a case of ovarian leiomyoma in a 22-year-old white tiger (Panthera tiger), treated with deslorelin implants, detailing pathological and immunohistochemical characteristics. Gross examination revealed a markedly enlarged, firm, whitish right ovary with a multinodular appearance. On a cut surface, multiple brown-fluid-filled cysts interspersed with solid grey-to-white areas were observed. On histopathological examination, the ovary was enlarged and replaced by a densely cellular neoplasm composed of spindle cells arranged in fascicles, or occasionally in a herringbone pattern, embedded in a large stroma of collagenous connective tissue. Neoplastic cells showed mild nuclear atypia and pleomorphism and low mitotic rate. Immunohistochemistry confirmed smooth muscle origin of the neoplasm, and cells were positive for vimentin, alpha-smooth muscle actin, desmin, and caldesmon. A low rate (<1%) of Ki-67-positive cells was observed. Although rare, ovarian leiomyoma should be considered when a mass is present in the ovary of a tiger with reproductive failure. Because cancer of the reproductive system impacts on species conservation by affecting reproduction, regular health monitoring is warranted to support wildlife conservation. Finally, the adverse effects associated with long-term treatment with synthetic GnRH analogues as contraceptives in non-domestic felids are worthy of future investigation.

Dynamic Responsive Inguinal Scaffold Activates Myogenic Growth Factors Finalizing the Regeneration of the Herniated Groin.

BACKGROUND: Postoperative chronic pain caused by fixation and/or fibrotic incorporation of hernia meshes are the main concerns in inguinal herniorrhaphy. As inguinal hernia is a degenerative disease, logically the treatment should aim at stopping degeneration and activating regeneration. Unfortunately, in conventional prosthetic herniorrhaphy no relationship exists between pathogenesis and treatment. To overcome these incongruences, a 3D dynamic responsive multilamellar scaffold has been developed for fixation-free inguinal hernia repair. Made of polypropylene like conventional flat meshes, the dynamic behavior of the scaffold allows for the regeneration of all typical inguinal components: connective tissue, vessels, nerves, and myocytes. This investigation aims to demonstrate that, moving in tune with the groin, the 3D scaffold attracts myogenic growth factors activating the development of mature myocytes and, thus, re-establishing the herniated inguinal barrier. METHODS: Biopsy samples excised from the 3D scaffold at different postoperative stages were stained with H&E and Azan-Mallory; immunohistochemistry for NGF and NGFR p75 was performed to verify the degree of involvement of muscular growth factors in the neomyogenesis. RESULTS: Histological evidence of progressive muscle development and immunohistochemical proof of NFG and NFGRp75 contribution in neomyogenesis within the 3D scaffold was documented and statistically validated. CONCLUSION: The investigation appears to confirm that a 3D polypropylene scaffold designed to confer dynamic responsivity, unlike the fibrotic scar plate of static meshes, attracts myogenic growth factors turning the biological response into tissue regeneration. Newly developed muscles allow the scaffold to restore the integrity of the inguinal barrier.

Nontraumatic Paraureteral Urinoma in a Cat with Urolithiasis.

Urinoma is an encapsulated collection of urine due to a disruption in the collection system of the urinary tract. This condition is rarely reported in veterinary medicine. The aim of this study is to describe the clinical and diagnostic findings of a well-encapsulated paraureteral urinoma associated with urinary tract stones in a critical feline patient. The ultrasound examination of the abdomen revealed a well-defined collection of anechoic fluid containing the left kidney in the retroperitoneal space, while the radiographic examination revealed a loss of soft tissue contrast in the lumbar region. Both techniques confirmed the presence of stones in the bladder. Following the ultrasound-guided drainage of the lesion, the nature of the fluid was also confirmed to be urine. Due to the worsening of the cat's health, to better define the extent and genesis of the lesion, a positive contrast radiographic study was performed by means of an ultrasound-guided percutaneous injection of a contrast medium into the lesion, which highlighted a wide and homogeneous radiopaque area in the left retroperitoneal space. These findings were confirmed with a postmortem examination, corroborating the diagnostic suspicion of urinoma. The percutaneous contrast inoculation performed in this critically ill patient plays a role in the diagnostic process to reach a final diagnosis in cases in which the patient's clinical condition does not allow for performing an excretory positive contrast study.

In vivo efficacy of verteporfin loaded gold nanorods for combined photothermal/photodynamic colon cancer therapy.

The high incidence of cancer recurrences and the frequent occurrence of multidrug resistance often stem from a poorly selective and inefficient antineoplastic therapy, responsible for the onset of undesired side effects as well. A combination of minimal-invasive approaches could thus be a useful strategy to surmount these shortcomings, achieving a safe and solid cancer therapy. Herein, a multi-therapeutic nanotool was designed by merging the photothermal properties of gold nanorods (AuNRs) with the photodynamic activity of the photosensitizer verteporfin. AuNRs were coated with the natural materials lipoic acid and gellan gum (AuNRs_LA,GG) and subsequently loaded with verteporfin (AuNRs_LA,GG/Vert) producing stable colloidal dispersions. AuNRs_LA,GG/Vert were characterized in terms of stability, size and morphology. The hyperthermia exhibited after NIR excitation (810 nm) was also evaluated to highlight the effect on increasing the drug released profile in intra-tumoral mimicking media, as well as cytotoxicity on human colon cancer cell line (HCT116). In vivo studies on HCT116 murine xenograft models were carried out to prove the ability of AuNRs_LA,GG to arrest the tumor growth via NIR laser-triggered hyperthermia. Furthermore, the complete xenograft depletion was demonstrated upon AuNRs_LA,GG/Vert administration by combined photothermal (PTT) and photodynamic (PDT) effects.

Oxidant and Antioxidant Parameters' Assessment Together with Homocysteine and Muscle Enzymes in Racehorses: Evaluation of Positive Effects of Exercise.

This study aimed to evaluate the changes in serum oxidant and antioxidant parameters together with the serum values of homocysteine (Hcy) and muscle enzymes including creatine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in regularly trained athletic horses during official races of 1200, 1600 and 2000 m. Thirty Thoroughbred horses were divided into three groups of 10 subjects each according to the race distance: Group 1, 1200 m race; Group 2, 1600 m race; Group 3, 2000 m race. Blood samples were collected from horses 1 week prior to the race (1WB), on the day of the race at rest (TREST), immediately after the race (TPOST), and after 30 (TPOST30) and 120 (TPOST120) minutes. Serum total proteins, reactive oxygen metabolites (dROMs), thiol antioxidant barrier (SHp), antioxidant barrier (Oxy-ads), Hcy, CK, AST and LDH values were assessed. A two-way repeated measures ANOVA did not show differences referable to the race distance (Group effect) on all investigated parameters (p > 0.05). An exercise effect on oxidative stress markers, Hcy and muscle enzymes herein investigated was found in all groups (p < 0.001). A Pearson's test showed dROMs positively correlated with SHp, Oxy-ads and Hcy after exercise (p < 0.05). This study suggests that, though well-trained racing horses are subjected to oxidative stress during a race, a proper antioxidant capacity may improve their ability to cope with exercise-induced oxidative stress.

Postmortem Electrical Conductivity Changes of Dicentrarchus labrax Skeletal Muscle: Root Mean Square (RMS) Parameter in Estimating Time since Death.

Electric impedance spectroscopy techniques have been widely employed to study basic biological processes, and recently explored to estimate postmortem interval (PMI). However, the most-relevant parameter to approximate PMI has not been recognized so far. This study investigated electrical conductivity changes in muscle of 18 sea bass specimens, maintained at different room temperatures (15.0 °C; 20.0 °C; 25.0 °C), during a 24 h postmortem period using an oscilloscope coupled with a signal generator, as innovative technology. The root mean square (RMS) was selected among all measured parameters, and recorded every 15 min for 24 h after death. The RMS(t) time series for each animal were collected and statistically analyzed using MATLAB®. A similar trend in RMS values was observed in all animals over the 24 h study period. After a short period, during which the RMS signal decreased, an increasing trend of the signal was recorded for all fish until it reached a peak. Subsequently, the RMS value gradually decreased over time. A strong linear correlation was observed among the time series, confirming that the above time-behaviour holds for all animals. The time at which maximum value is reached strongly depended on the room temperature during the experiments, ranging from 6 h in fish kept at 25.0 °C to 14 h in animals kept at 15.0 °C. The use of the oscilloscope has proven to be a promising technology in the study of electrical muscle properties during the early postmortem interval, with the advantage of being a fast, non-destructive, and inexpensive method, although more studies will be needed to validate this technology before moving to real-time field investigations.

Preliminary study for the application of Raman spectroscopy for the identification of Leishmania infected dogs.

Raman spectroscopy is a rapid qualitative and quantitative technique that allows the simultaneous determination of several components in biological fluids. This methodology concerns an alternative technique to distinguish between non-healthy and healthy subjects. Leishmaniasis is a zoonosis of world interest, the most important agent is L. infantum. Dogs are the principal reservoirs affected by a broad spectrum of clinical features. During a clinical exam, blood samples were collected in tubes without anticoagulants, from twenty two dogs. One aliquot was used for serological test for Leishmaniasis, one aliquot was subjected to the Raman spectroscopic analysis. Animals were divided into two groups of equal subjects, Leishmania group (LG) constituted by infected dogs, and control group (CG) constituted by healthy dogs. The acquired spectra were different in the region 1200-1370 cm-1, in which it is possible to distinguish the amide III vibration (~ 1300 cm-1). In LG, an evident shift to the shortwave region is observed in spectral frequencies of the band centered at ~ 1250 cm-1. Our results distinguished between LD group and CG. Further studies are necessary to exclude the effect of metabolic modification due to disease on the recorded spectra changes and to consolidate the achievability of Raman spectroscopy as rapid and less expensive diagnosis of Leishmaniasis.

Polybutylene succinate artificial scaffold for peripheral nerve regeneration.

Regeneration and recovery of nerve tissues are a great challenge for medicine, and positively affect the quality of life of patients. The development of tissue engineering offers a new approach to the problem with the creation of multifunctional artificial scaffolds that act on various levels in the damaged tissue, providing physical and biochemical support for the growth of nerve cells. In this study, the effects of the use of a tubular scaffold made of polybutylene succinate (PBS), surgically positioned at the level of a sciatic nerve injured in rat, between the proximal stump and the distal one, was investigated. Scaffolds characterization was carried out by scanning electron microscopy and X-ray microcomputed tomography and magnetic resonance imaging, in vivo. The demonstration of the nerve regeneration was based on the evaluation of electroneurography, measuring the weight of gastrocnemius and tibialis anterior muscles, histological examination of regenerated nerves and observing the recovery of the locomotor activity of animals. The PBS tubular scaffold minimized iatrogenic trauma on the nerve, acting as a directional guide for the regenerating fibers by conveying them toward the distal stump. In this context, neurotrophic and neurotropic factors may accumulate and perform their functions, while invasion by macrophages and scar tissue is hampered.

Improved Bone Regeneration Using Biodegradable Polybutylene Succinate Artificial Scaffold in a Rabbit Model.

The treatment of extensive bone loss represents a great challenge for orthopaedic and reconstructive surgery. Most of the time, those treatments consist of multiple-stage surgeries over a prolonged period, pose significant infectious risks and carry the possibility of rejection. In this study, we investigated if the use of a polybutylene succinate (PBS) micro-fibrillar scaffold may improve bone regeneration in these procedures. In an in vivo rabbit model, the healing of two calvarial bone defects was studied. One defect was left to heal spontaneously while the other was treated with a PBS scaffold. Computed tomography (CT) scans, histological and immunohistochemical analyses were performed at 4, 12 and 24 weeks. CT examination showed a significantly larger area of mineralised tissue in the treated defect. Histological examination confirmed a greater presence of active osteoblasts and mineralised tissue in the scaffold-treated defect, with no evidence of inflammatory infiltrates around it. Immunohistochemical analysis was positive for CD56 at the transition point between healthy bone and the fracture zone. This study demonstrates that the use of a PBS microfibrillar scaffold in critical bone defects on a rabbit model is a potentially effective technique to improve bone regeneration.

A regenerative 3D scaffold for inguinal hernia repair. MR imaging and histological cross evidence. Qualitative study.

BACKGROUND: Inguinal hernia is a degenerative disease occurring in a high motile surround. Stopping degeneration and promoting tissue regeneration should be the treatment goal. Groin hernias are conventionally managed with static flat meshes, mostly fixated to the delicate inguinal environment. Far from a regenerative effect, the biologic response of conventional hernia meshes is characterized by a foreign body reaction leading to a stiff/shrunken scar plate, which is often the source of unpleasant complications. Recently, a newly engineered 3D device for inguinal hernia repair - ProFlor-has been developed to produce a regenerative biological response. Unlike conventional hernia meshes, this regenerative 3D hernia scaffold seems to demonstrate suitable features for a pathogenetical and physiological coherent treatment of the disease. The aim of this manuscript is to cross evidence these features through magnetic resonance imaging (MRI) and histology. STUDY DESIGN: The biological response of ProFlor at three defined post-implantation stages has been evaluated through MRI signal intensity and compared to neighbouring muscles and fat. As additional proof, histology of tissue specimens excised at the same post-implantation periods from porcine models during an experimental attempt were also evaluated. RESULTS: MRI of newly ingrown tissue in ProFlor demonstrated similar signal intensity of muscles while fat tissue showed remarkably higher values. These data matched with the histology of ProFlor biopsies excised from pigs. CONCLUSIONS: The motile compliance to groin movements of ProFlor appears to induce a probiotic biologic response comparable to a regenerative scaffold, allowing to physiologically resolve the degenerative source of inguinal hernia disease.

A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity.

The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (51Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate 51Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release 51Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells' killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.

Evaluation of Tear Production as Measured by Schirmer Test I in Dogs after Acepromazine and Acepromazine-Methadone Premedication.

The purpose of the current study was to investigate the effects of two commonly used sedation protocols in dogs, acepromazine (ACP) and acepromazine-methadone (ACP-MET) combination, on tear production measured by the Schirmer Tear Test (STT) 1. We hypothesized that both sedation protocols cause a reduction in canine tear production for a variable time. Fifteen client-owned dogs were recruited for the study. Each dog was subjected to sedation twice, 2-3 weeks apart, and they were randomly allocated to one of two groups receiving ACP (0.015 mg kg-1) or ACP-MET (0.010 mg kg-1 and 0.2 mg kg-1) intramuscularly. In both eyes, tear production was measured 15 min before sedation (T0) and 20 min (T20 m), 40 min (T40 m), 1 h (T1), 2 h (T2), 4 h (T4) and 8 h (T8), after drug administration. Two-way repeated measures ANOVA, followed by the Bonferroni post hoc test (p < 0.05), showed a significant effect of time (p < 0.0001) and treatment (p < 0.0001). A significant decrease in tear production at T20 m, T40 m, T1 and T2 compared to T0 was observed in the ACP experimental protocol, while in the ACP + MET protocol, this reduction persisted until T8. Comparing the two experimental protocols, no statistically significant differences were observed at T0 or T20 m, and STT 1 values were statistically lower in the ACP + MET than the ACP protocol at the other data points. In the ACP + MET group, at T40 m, 100% of dogs showed STT 1 readings lower than 15 mm/min. This finding is clinically relevant as it can predispose dogs to corneal injuries. The major reduction in tear production due to the ACP + MET protocol proves the need for adequate corneal hydration, particularly to discourage its use in animals with altered tear production. The data obtained provide important information helping clinicians to better manage the drug's effects on tear production.

Impact of region-of-interest size and location on quantitative contrast-enhanced ultrasound of canine splenic perfusion.

BACKGROUND: During contrast enhanced ultrasound (CEUS), the features of the regions of interest (ROI) can affect the value of the perfusion-related parameters obtained from a time intensity curve (TIC). In veterinary medicine, conflicting have been reported on the influence of ROI size and location on renal CEUS. There are some disagreeing evidences regarding the optimal method for selecting ROI in quantitative analysis of renal perfusion using CEUS. The aim of this study was to evaluate the effect of the size and location of ROIs in the spleen of conscious dogs on perfusion variables determined using sulphur hexafluoride contrast-enhanced ultrasounds. RESULTS: A prospective observational study on 15 client-owned mixed-breed adult dogs was performed using a system equipped with contrast-tuned imaging technology. Qualitative and quantitative assessments of the spleen enhancement pattern were carried out. Three square ROIs (0.05 cm2) were manually drawn in a row and spaced 1 mm apart, placing adjacent ROIs at three different depths. Three medium rectangular ROIs (0.3 cm2) include the 3 smallest ROIs in each row, indicated by the letters A, B and C, and a single large square ROI (1 cm2) was drawn containing all previous ROIs. Software analysis of time-intensity curves generated within each ROI allowed us to calculate the perfusion-related parameters: peak enhancement, time to peak, regional blood flow, mean transit time and regional blood volume. The coefficient of variation for all blood-related parameters was always lower in the larger ROI than in the other smaller ROIs. ROI A and B, positioned proximally and medially, levels respectively, showed similar coefficients of variation to the largest ROI. The analysis of variance model exhibited a significant effect of location and size of the ROIs in the quantitative analysis of canine spleen perfusion, with a reduction of perfusion-related parameters in the distal ROI. CONCLUSIONS: The recommendation for a quantitative CEUS examination of a dog's spleen is to analyze splenic perfusion by drawing a sufficiently large ROI proximal to the ultrasound beam on the splenic parenchyma. This may be of clinical relevance in the diagnosis of splenic diseases.

Anti-Atherogenic Effect of 10% Supplementation of Anchovy (Engraulis encrasicolus) Waste Protein Hydrolysates in ApoE-Deficient Mice.

Fish protein consumption exerts beneficial metabolic effects on human health, also correlating with a decreased risk for cardiovascular disease. Fish waste contains high amount of proteins and utilization may offer the opportunity for generating compounds advantageous for human health. Especially, fish waste protein hydrolysates beneficially influence pathways involved in body composition, exerting anti-inflammatory and antioxidant activities, making their potential supplementation in human disorders of increased interest. This study assessed the effect of a 10% (w/w) anchovy waste protein hydrolysate (APH) diet for 12 weeks in reducing atherosclerosis in ApoE-/- mice, through histological and immunohistochemical methods. In addition, monitoring of plaque development was performed, using high-frequency ultrasound and magnetic resonance imaging. Overall, the APH diet attenuated atherosclerotic plaque development, producing a regression of arterial lesions over time (p < 0.05). Twelve weeks on an APH diet had an anti-obesity effect, improving lipid metabolism and reducing hepatic enzyme activity. A significant reduction in plaque size and lipid content was observed in the aortic sinus of APH-fed mice, compared to the control (p < 0.001), whereas no differences in the extracellular matrix and macrophage recruitment were observed. Supplementation of APH significantly attenuates atherosclerosis in ApoE-/- mice, exerting a lipid-lowering activity. The opportunity to use fish waste protein hydrolysates as a nutraceutical in atherosclerosis is worthy of future investigations, representing a low cost, sustainable, and nutritional strategy with minimal environmental impact.