Signed informed dogs’ owners consent about methods and purposes of this study were obtained. The protocol was approved by the Animal Ethics Board of the Department of Veterinary Sciences, University of Messina (protocol number: 13/2017). All treatments, housing and animal care followed the EU Directive 2010/63/EU on the protection of animals used for scientific purposes.
Fifteen mixed-breed dogs, 8 males and 7 females, were enrolled in this study. All dogs were presented at the Veterinary Teaching Hospital of the University of Messina between October 2017 and February 2019 for the assessment of renal perfusion through CEUS, due to a mildly increase of blood urea and creatinine. Age of the enrolled dogs ranged from 1 to 7 years and the mean body weight was 30.9 ± 3.6 (SD) kg.
Dogs were included in the study only if the spleen was homogenous at ultrasonography. Dogs with focal or multifocal large lesions were excluded and fifteen dogs have been selected. The enrolled dogs, submitted to a clinical examination, had normal heart rate, blood pressure, capillary refill time, respiratory rate and hydration status.
All the dogs were subjected to B-mode ultrasonography, Doppler ultrasonography and CEUS. Ultrasound and Doppler examination were performed by the same investigator (FM) using a scanner Mindray M9 (Shenzhen, China), equipped with a linear probe (10-12-MHz). The dogs were not sedated and were manually restrained in the right lateral recumbency, the hair was clipped, alcohol (70%) and coupling gel were applied to the skin.
Spleen tissue was considered normal if the margins were regular and smooth and the parenchyma showed a finely textured and homogeneous pattern, more echogenic than liver and the cortex of left kidney. Colour Doppler was performed to evaluate the splenic vascularization and rule out the presence of splenic intravascular thrombotic structures or ischemic lesions.
CEUS examination was performed, using a linear transducer (10-12-MHz) probe with contrast agent capability. The contrast agent, INN-sulphur hexafluoride (SonoVue®, Bracco International, Milan, Italy), was prepared following the manufacturer’s recommendations and was rapidly injected (0.05 mL/kg of body weight) via a three-way stopcock and 18-gauge catheter placed in a cephalic vein, in accordance with a methodology previously reported [28,29]. Each dog received two bolus injections of contrast agent, which were standardised and administered by the same investigator (SDP). The first bolus was administered to assess the kidney while the second bolus, injected approximately 5 to 10 minutes after the first bolus, was used for the assessment of the spleen. Injection of the contrast agent and activation of a timer were started simultaneously and video clips were recorded for 2 minutes. As animal were not sedated, care was taken to keep the probe in the same position for at least 2 minutes. The spleen was examined with a mechanical index set at a low value (0.09). Standardised settings were adopted for time gain compensation, overall gain and focal zone, as previously reported 
Raw data (good-quality video clips) obtained during CEUS were stored digitally on a hard disk. A trained investigator (LL) analysed all the functional data. A qualified operator (SM) drowned a total of 13 quadrangular or rectangular ROIs. The smallest ROIs (0.05 cm2) were numbered in sequential numerical order from 1 to 9. They were drawn in groups of three, at a distance of one millimetre, on three different depth levels: proximal, middle and distal. Around each group of three ROIs, a larger ROI (0.3 cm2) was drawn, indicated with the letters A, B and C. The total of the 12 ROIs were grouped into a higher ROI (1 cm2), named as ROI MAX (Figure 1). The use of image-analysis software produced post-processing analysis (Qontrast®, Bracco Imaging, Milan, Italy). This software processed the raw data allowing measurement of tissue perfusion in ROIs and determining variables automatically. A time-intensity curve, which is a parametric curve of time versus signal intensity (SI), was also generated for each ROI. The maximal SI was defined as a white band in the grey scale bar (8 bit). The software generated the following parameters: peak enhancement, time to peak (TTP), mean transit time (MTT),regional blood volume (RBV) and regional blood flow (RBF) for each ROI. These parameters were defined as follows:
- peak enhancement as the percentage increase in SI (from 0 to 100 as maximal intensity) reached during transit of the contrast agent at a specific time point;
- TTP as the interval until the maximum SI of the contrast agent;
- MTT as the circulation time of the contrast agent in the examined tissue;
- RBF as the ratio between RBV and MTT.
Descriptive statistical analysis of the quantitative CEUS-derived parameters revealed a normal distribution for each value. To evaluate statistical differences among the bodyweight of the enrolled dogs a paired Student t-test was used.
To define the influence of the location and size of the ROI on perfusion parameters, the coefficient of variation was calculated for each of them. If a parameter showed the lowest coefficient of variation, it was considered acceptable and reliable.
Data obtained from the quantitative CEUS-derived parameters were analysed by ANOVA with the GLM procedure (SAS V.9.4, SAS Institute, 2001), using ROI size and location as variables. All data were expressed as mean ± standard deviation (SD). Statistical significance was assessed at the P < 0.05 level.