Splenic Segments Research Articles

Ultrasonographic findings of a rare case of splenic rupture and torsion in a dog

AbstractA 7‐year‐old dog presented with acute lethargy, anorexia and vomiting. Ultrasonographic examination revealed a hypoechoic mass lesion in the left abdomen, characterised by a hilum‐like structure and intraparenchymal gas. A mild amount of hyperechoic peritoneal effusion was present. An apparently normal spleen was visualised, which was not associated with the described mass lesion. Surgery revealed a presumed previously ruptured and separated spleen. The caudal portion of the spleen was acutely torsed and there was a mild amount of peritoneal fluid present. Histopathology confirmed splenic torsion of the previously ruptured and separated caudal splenic segment. Bacterial culture of the peritoneal effusion after enrichment indicated Staphylococcus aureus. The patient recovered successfully and maintained excellent clinical conditions at 14‐month follow‐up. Splenic torsion of a ruptured splenic segment should be considered as a differential diagnosis for an emphysematous mass containing a hilum‐like structure in the abdomen in dogs.

Spontaneous Splenic Rupture Case Report: an Approach to Anatomy Importance for Radiological Correlation

Spontaneous splenic rupture (SSR) is a rare entity 1, usually associated with hematologic malignancies, infectious processes or inflammatory diseases. Timely diagnosis by ultrasound and/or computed tomography 1 allows early clinical and/or surgical management 2, reducing the mortality 1.Our aim is to expose, through a clinical case of SSR, the importance of anatomy knowledge and its radiological correlation in medical education.A 26‐year‐old man, diagnosed with chronic myeloid leukemia (CML) without chemotherapy treatment, attends to the emergency department for 10/10 (VAS) colic pain in the right inferior abdominal quadrant, irradiated to the ipsilateral inguinal region, associated with non‐emetic nauseas, abdominal distention and dysuria. At presentation, heart rate was 80 beats/minute, respiratory rate 18 breaths/minute, blood pressure 123/71 mmHg, temperature 36.0 ° C and SatO2 87%.Initial workup positive findings were leukocytosis (270.000/mm3), low hemoglobin (11.2 g/dL) and hematocrit (33.3%), thrombocytosis (925.000 platelets), and Hypokalemia (2,96 mEq/L). Abdominal computed tomography showed hepato‐splenomegaly and a poorly defined lesion with irregular borders, infiltration and vascular tracts on the spleen inferior segment. Considerable accumulation of free liquid in the abdominal cavity was present.After 2 hours the patient reported an increase in pain, associated to abdominal guarding and rebound tenderness. Heart rate of 134 beats/minute, respiratory rate 30 breaths/minute, blood pressure 150/70 mmHg, SatO2 84% and metabolic acidosis (pH 7.0).Based on the suspicion of hypovolemic shock, an emergency xipho‐pubic laparotomy was performed, finding a 1500cc hemoperitoneum, active bleeding, clots and splenic segments. Complete splenectomy was selected obtaining a surgical specimen (spleen) of 2 Kg, 32 cm in cephalo‐caudal length, and a lower pole rupture. Patient’s postoperative evolution was satisfactory, specific treatment for CML was initiated and he was discharged 14 days after the surgical procedure.An early diagnosis is crucial for the reduction of morbidity and mortality in patients suffering a SSR. This is carried out through the proper interpretation of diagnostic images. This clinical approach requires a broad body anatomy knowledge, and the capacity to extrapolate it to a bidimensional representation. Several researchers confirm that the inclusion of diagnostic‐images in the anatomy curriculum leads to an early exposure of medical student to these tools, which improves their imaging interpretation and diagnostical skills 2. Finally, the implementation of these diagnostic tools in the anatomy program could lead to better medical practice and, as seen in this case, to a reduction on mortality.

A comparison of the anatomical structure of the pancreas in experimental animals.

As basic knowledge for evaluation of pancreatic toxicity, anatomical structures were compared among experimental animal species, including rats, dogs, monkeys, and minipigs. In terms of gross anatomy, the pancreases of dogs, monkeys, and minipigs are compact and similar to that of humans. The rat pancreas is relatively compact at the splenic segment, but the duodenal segment is dispersed within the mesentery. In terms of histology, the islet of each animal is characterized by a topographic distribution pattern of α- versus β-cells. β-cells occupy the large central part of the rat islet, and α-cells are located in the periphery and occasionally exhibit cuffing. In dog islets, β-cells are distributed in all parts and α-cells are scattered in the center or periphery of the islet (at body and left lobe); whereas β-cells occupy all parts of the islet and no α-cells are present in the islet (at right lobe). Monkey islets show two distinct patterns, that is, α-cell-rich or β-cell-rich islets, and the former represent peripheral β-cells forming an irregular ring. Minipig islets show an irregular outline, and both α- and β-cells are present in all parts of the islet, intermingling with each other. According to morphometry, the endocrine tissue accounts for <2% of the pancreas roughly in rats and minipigs, and that of monkeys accounts for >7% of the pancreas (at tail). The endocrine tissue proportion tends to increase as the position changes from right to left in the pancreas in each species.

Acoustic radiation force impulse elastography of the spleen in healthy dogs of different ages.

To determine the elastographic characteristics of splenic parenchyma in clinically healthy dogs of various ages in order to establish preliminary qualitative and quantitative standards/reference intervals for this technique. Thirty three healthy dogs categorized as young, adult and elderly were used. Splenic echotexture, echogenicity, size and ages were assessed with B-mode ultrasonography. Using qualitative elastography, the spleen (head, body and tail) was examined for homogeneity and presence of deformities. Shear velocities in different splenic segments were then quantitatively evaluated. All splenic segments visualised with the B-mode ultrasonography appeared normal. Different splenic segments examined with qualitative elastography were free of any detectable malformations and the images appeared as homogeneous dark areas. The mean shear velocity values were 2 · 32 m/s for head, 2 · 16 m/s for body and 2 · 25 m/s for tail of the spleen (P = 0 · 40), and they did not vary between the different age groups (P > 0 · 05). Quantitative and qualitative ARFI elastography of the spleen in clinically healthy dogs differing in age could be easily performed, and it may aid in the diagnosis and evaluation of splenic abnormalities routinely assessed in veterinary practice with B-mode ultrasonography.

Temperature Measurement Methods During Direct Heat Arterial Tissue Fusion

Fusion of biological tissues through direct and indirect heating is a growing area of medical research, yet there are still major gaps in understanding this procedure. Several companies have developed devices which fuse blood vessels, but little is known about the tissue's response to the stimuli. The need for accurate measurements of tissue behavior during tissue fusion is essential for the continued development and improvement of energy delivery devices. An experimental study was performed to measure the temperatures experienced during tissue fusion and the resulting burst pressure of the fused arteries. An array of thermocouples was placed in the lumen of a porcine splenic artery segment and sealed using a ConMed Altrus thermal fusion device. The temperatures within the tissue, in the device, and at the tissue-device interface were recorded. These measurements were then analyzed to calculate the temperature profile in the lumen of the artery. The temperature in the artery at the site of tissue fusion was measured to range from 142 to 163 °C using the ConMed Altrus. The corresponding burst pressure for arteries fused at this temperature was measured as 416 ± 79 mmHg. This study represents the first known experimental measurement of temperature at the site of vessel sealing found in the literature.

Experimental study on the anticancer effect of curcumin on mouse of S180 in vivo

Objective To study curcumin's growth inhibitory effects and morphology changes on sarcoma graft's of S180 mouse, with further, inquiry into the possible mechanisms. Methods Thirty S180 mouse were randomly assigned into 3 groups: saline group (blank control group). Cytoxan group (positive control group) and curcumin group. The tumor inhibitory rates, the index of thymus and spleen, the growth of tumor and the change of pathology-morph, the index of apoptosis cells and morphology changes of apoptosis cells in the different groups were observed. Results (1) Tumor's inhibitory rate in curcumin group and cytoxan group was 68.32% and 70. 43%. respectively. Compared to blank control group, the 2 groups had significant elevated tumor inhibitory rate ( P<0.01). (2) Thymus index of curcumin group did not have significant decrease compared to blank control group ( P>0.05). (3) Under electroscope, curcumin group and positive control group had significant decrease in terms of growth of tumor, degree of infiltration of tumor, the number of nucleus fission, and blood vessels number compared to saline group ( P<0.05). However, the degree of cell necrosis, the number of splenic segments and macrophage are increased significantly compared to negative group. (4) Accumulative score of apoptosis cell in curcumin group was significantly higher than other two groups ( P<0.05). Conclusion Internal organ study and cell morphology observation show curcumin can effectively inhibit the growth and cause the death of sarcoma graft of S180 mouse without interference with thymus.

Surgical anatomy of the human spleen.

Post-splenectomy sequelae are now well recognized, and conservative splenic surgery is widely advocated. However, controversies exist regarding splenic surgical anatomy. We studied 127 human spleens using anatomical dissection and a sequential injection method involving both radiology and corrosion casting, with the primary aim of examining segmental splenic anatomy and blood supply. The existence of well-defined splenic segments was confirmed and these ranged in number from 3 to 7 with a mean of 4.3. Each segment had its own arterial supply and venous drainage. The segments were separated from each other by avascular planes. Subsegments with independent blood supply were also identified. The splenic artery was found to divide into two branches; in all cases these further divided into segmental arteries supplying the central segments of the spleen. The polar segments were supplied by segmental vessels of highly variable origin and size. This study aims to highlight the importance of identifying these segmental vessels at operation when splenic conservation is considered.

Avaliação da função fagocitária em remanescentes de esplenectomia subtotal e auto-implante esplênico autógeno

Para diminuir os efeitos adversos da esplenectomia total, abordagens conservadoras devem ser tentadas, quando for possível. Esplenectomia subtotal e auto-implantes esplênicos são boas alternativas nessa situação. O objetivo do presente estudo foi comparar as funções fagocitárias do pólo superior remanescente de esplenectomia subtotal e de tecido esplênico implantado no omento maior. Esta investigação foi conduzida em 35 ratos Holtzman adultos de ambos os sexos. Todos os animais foram submetidos a esplenectomia subtotal preservando o pólo superior suprido pelos vasos esplenogástricos e a auto-implante do segmento esplênico removido, no omento maior. A função fagocitária foi verificada por dois métodos diferentes: cintilografia com enxofre coloidal marcado com 99mTc e contagem, ao microscópio, de macrófagos que continham carbono coloidal. Durante os três primeiros meses, a fagocitose foi maior no pólo superior remanescente. Após esse período, não houve diferença entre o pólo superior e o auto-implante. Concluindo, o remanescente de esplenectomia subtotal e o auto-implante esplênico mantêm a função de filtração do baço mediante remoção de partículas coloidais do sangue.

Non-invasive single cell pH measurements in the isolated perfused pancreas.

1. A new method was developed for non-invasive investigations of intracellular pH (pHi) regulation in different cell types of the isolated perfused pancreas using a confocal laser scanning technique. 2. After removal of the rat pancreas the coeliac artery was cannulated and the splenic segment of the pancreas was perfused with dextran (5%)-Ringer solution at a constant flow rate of 2 mL/min. In a temperature-controlled (37 degrees C) chamber, pH regulation was studied using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein (BCECF) with a confocal microscope (MRC-600; Bio-Rad, Hercules, CA, USA). 3. Image analysis permitted the identification and comparison of different cell types with a pHi of 7.26+/-0.1 in acinar cells and of 7.02+/-0.1 in endothelial cells. Increasing PCO2 from 5 to 20% resulted in a rapid decrease in pHi. Omission of sodium from the perfusate resulted in a smooth decline in pHi. Both decreases were found to be fully reversible. Increasing PCO2 under sodium-free conditions also resulted in a drop of pHi that was, however, not fully reversible, suggesting involvement of the Na+/H+ exchanger in the regulation of pHi in the intact organ. 4. The above method completely preserves tissue integrity and, therefore, allows the study of pH regulation in different cell types of the pancreas simultaneously and without interference with their functional arrangement. The technique should be of specific value to investigate experimental disease states of the pancreas.

Anatomie und Physiologie bei milzerhaltenden Eingriffen

Anatomy and Physiology in Spleen-Preserving Surgery The basis for a successful partial splenectomy is the blood supply of each splenic segment and a high frequency of upper and lower polar arteries. The compartments of the spleen are the red pulp, the white pulp, and the marginal zone. Recent functional and immunohistological studies have documented major species differences. Furthermore, the spleen only attains its typical structure at a certain age. The development of this organ and its functional relevance for pediatric surgery are largely unknown. In splenic remnants the function of the red pulp might be sufficient to prevent Howell-Jolly bodies in the blood, but the white pulp and the marginal zone are inadequate in septicemia. The spleen plays a major role in lymphocyte recirculation, but the adhesion molecules involved are largely unknown. The immune function of the spleen is also regulated by nerves that might be involved in the regeneration of autotransplanted splenic tissue. The extrapolation from the structure and function of the spleen in adults to that in young children or from animal experiments to the human situation should only be undertaken with great caution.

Anatomical and surgical aspects of splenic segmentectomies

Based upon the anatomicosurgical segments of the spleen, suggested by DiDio and demonstrated in cadavers, classified and named by Neder (1958) and Zappalá (1958, 1959, 1963), the normal segmental organization was anatomically and radiologically confirmed in 51 human spleens, after studying corrosion casts and radiograms of intraparenchymal vessels (Christo, 1959 a, b, 1960, 1962, 1963, 1993). From 1958 to 1965, pioneer segmental resections were performed successfully in 34 dogs and in 9 patients to safely remove traumatic injured splenic segments. At the same time, the overwhelming postsplenectomy infection (OPSI) became well identified. Consequently, to save normally functioning splenic parenchyma became the most important issue in the management of splenic injuries. The anatomical basis for partial splenectomy and splenic segmentectomy is discussed. The term "splenorrhaphy" was employed to designate all conservative or parenchyma saving operations of spleen based upon its vascular supply: from topical packings to splenic sutures including "cappings" and partial splenectomies. From analysis of 38 consecutive reports in 20 years, covering 4,076 patients, it was concluded that "splenorrhaphies" had been electively employed in 46% of the injuries and partial splenectomies were identified in 8.6% of these surgical interventions. However, the critical minimal mass of splenic tissue to be preserved after partial splenectomies is still to be defined. Postoperative complications directly related to "splenorrhaphies" are rare. Uncommonly performed after splenectomies, the heterotopical splenic autotransplantation has presented dubious results. Trials with nonoperative management of splenic blunt trauma injuries have been safer among children, whose spleens are predominantly transversally disrupted and have a higher relationship "capsular resistance/parenchymal bulk". Splenectomies have been most frequently the ultimate result of delayed laparotomy and underlying risks of growing blood requirements may surpass the advantages of preventing OPSI.

Proliferation and differentiation of pancreatic ?-cells: ultrastructural analysis of the pancreas in diabetic mice induced by selective alloxan perfusion

To clarify the mechanism of regenerative processes of pancreatic β-cells, we constructed a new diabetic model of mice and investigated their pancreatic endocrine cells by electron microscopy. Male ICR mice (8 weeks old) were partially and chemically depancreatized by perfusing alloxan (100 mg/kg body weight) via the caudal vein after clamping the cranial mesenteric artery. By this method, we could render the mice diabetic by partial reduction of β-cells localized in the splenic, gastric, and parabiliary segment. Glucose intolerance gradually ameliorated without any treatment. In the perfused segments, pancreatic β-cells showed pyknosis and the mitochondria were swollen 6h after the treatment, while non-β-cells including α-cells remained intact. At 5 days, β-cells were few and the islets became smaller in size. At 20 weeks, small islet cell clusters (ICCs) were observed budding from interlobular and intralobular ductal epithelial cells. β-cells scattering in the exocrine pancreas were also frequently observed. In the alloxan-nonperfused segment, β-cells with thin rough endoplasmic reticulum and immature secretory granules without an electron-opaque halo were observed, and the number of mitochondria increased in some β-cells at 1 day and 5 days after the treatment. At 20 weeks, β-cells that contained only mature granules were observed in hypertrophic islets. In this model, both proliferation of residual β-cells and differentiation of pancreatic endocrine cells from the ductal epithelial cells were recognized.

Overnight storage of the porcine isolated splenic artery enhances endothelium-dependent contractions to NG-nitro-L-arginine methyl ester without impairing endothelium-dependent dilator function.

This study examined the influence of overnight storage on endothelium-independent contractions to 5-hydroxytryptamine (5-HT), endothelium-dependent contractions to NG-nitro-L-arginine methyl ester (L-NAME), and endothelium-dependent relaxations to substance P (SP) and L-arginine, using the porcine isolated splenic artery. In endothelium-intact (E+) segments from fresh porcine isolated splenic arteries or segments from the same vessels stored overnight at 4 degrees C, either in Krebs-Henseleit saline or in Krebs-Henseleit saline containing 1 mM L-arginine, 5-HT caused concentration-related contractions that were similar under all three conditions. Overnight storage enhanced contractions of the splenic artery to L-NAME, an effect not observed if the vessels were co-stored with 1 mM L-arginine. L-NAME failed to contract endothelium-denuded (E-) segments from fresh tissues or tissues stored overnight, indicating that its constrictor effects were endothelium-dependent. SP caused concentration-related, endothelium-dependent relaxations of the splenic artery that were inhibited by 100 microM L-NAME, indicating that the relaxations could be attributed to the stimulated release of NO from endothelial cells. Established contractions to 100 microM L-NAME in E+ segments from fresh tissues, or segments from the same tissues stored overnight at 4 degrees C, either in Krebs-Henseleit saline or in Krebs-Henseleit saline containing 1 mM L-arginine, were all reversed by 1 mM L-arginine to similar extents, indicating that overnight storage did not affect endothelium-dependent dilator responses to L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatal hemorrhage caused by disease progression after partial splenectomy for type III Gaucher's disease

An 18-month-old boy with Gaucher's disease, massive hepatosplenomegaly, and hypersplenism underwent partial (85%) splenectomy and recovered without complication. During the next 4 months, his residual splenic segment showed progressive enlargement from 2 to 6 cm below the left costal margin. By this time, genetic typing had indicated central nervous system involvement (type III). A compatible donor was not available for bone marrow transplantation. Complete splenectomy was not recommended despite continued enlargement of the organ into the pelvis and across the abdominal midline by 7 1 2 months postoperatively. The next week he presented to the emergency room in shock with an hematocrit of 6% and a platelet count of 18,000/mm 3. During emergency surgery, massive intraabdominal hemorrhage was noted from a splenic rupture on the inferior pole that was opposite the site of prior division. He died in the operating room during attempted splenectomy. This is a report of fatal outcome after partial splenectomy for type III Gaucher's disease. Patients with subacute neuronopathic disease may not be candidates for subtotal splenectomy.

Anatomic basis for conservative surgery of the spleen

100 pancreatico-splenic blocks obtained at autopsy were treated by the injection-corrosion method. The splenic artery ended in the lienorenal ligament in 64% of cases, in the pancreatic segment in 32% and in the hilar segment in 4%. In 84% of cases, the artery divided into two lobar arteries, upper and lower. In 16% the artery trifurcated. The pedicle was 20 to 60 mm long (mean 35 mm). The branches of the splenic artery were in three successive stages: the primary or lobar arteries were each associated with a lobe; the secondary or segmental arteries each supplied one of the constituent segments of a lobe; the tertiary or subsegmental arteries supplied the various areas which made up a splenic segment. Nine cases of extraparenchymal anastomoses were found. There were 32 intraparenchymal anastomoses, being either interlobar or intersegmental. There were a few interlobar anastomoses. The findings support the notion of upper and lower intersegmental and interlobar planes. The latter is almost avascular, while the former has limited vascularity. These planes make conservative surgery of the spleen possible.

Pancreatic growth after partial resection of normal and enlarged pancreas in rats fed soya flour

Pancreatic growth was studied after partial resection of the normal-sized pancreas in rats fed heated soya flour (HSF) or the enlarged gland in rats fed raw soya flour (RSF). Resection involved the removal of the splenic and gastric segments and in both the normal and enlarged gland this represents a loss of approximately 55% of total pancreatic mass. After partial resection animals were either continued on these preresected diets or changed to the alternative diets. For at least the first 8 days after resection, in all conditions studied, there was a significant increase in DNA synthesis in the pancreas, involving both parenchymal and nonparenchymal cells as shown by autoradiography. The increased cell turnover was not associated with any increase in total DNA content of the gland, indicating that the increase paralleled cell loss in response to injury caused by the surgery. By 160 days after resection of the normal pancreas, RSF feeding caused both hypertrophy and hyperplasia of the remnant, but after partial resection of the enlarged gland, growth was limited to hypertrophy. These results suggest that the pancreas has a limited capacity for additional growth after that initially caused by RSF.

Segmental splenectomy using the ultrasonic surgical aspirator

The technique described seems to significantly decrease blood loss in segmental splenectomy. Resection of the damaged splenic segments offers the potential advantage of reducing rebleeding and secondary hemorrhage from sites of repaired lacerations. Also, the viable splenic fragment that is left behind may afford protection from postsplenectomy sepsis. The ultrasonic surgical aspirator seems well suited for the purpose of segmental splenectomy in both the trauma and staging laparotomy setting.

Intrinsic Nerves of the Pancreas After Celiac and Superior Mesenteric Ganglionectomy in Rats

The effect of surgical celiac and superior mesenteric ganglionectomy (GGX) on intrinsic pancreatic innervation was studied in male Wistar rats. Neurons were stained in the pancreas of control and ganglionectomized rats with histochemical methods evidencing the acetylcholinesterase (AChE) activity and the induced fluorescence of catecholamines. Two segments of the head of the pancreas ("duodenal" and "splenic" segments) were studied. The surface density of neuronal cell bodies was larger in the splenic segment than in the duodenal segment of the head of the pancreas (p less than 0.01), both in control and in ganglionectomized rats. The surface density of the neuronal cell bodies and the intensity of AChE staining were not significantly different in control and in ganglionectomized rats. Most AChE-positive fibers survived after ganglionectomy; only some of them disappeared. These disappearing fibers corresponded to the thinnest fiber bundles found in the pancreatic tissue of control rats. Only 10% of catecholamine fibers remained after ganglionectomy. Some of the remaining fibers may run through direct duodenopancreatic pathways.

Different characteristics of postjunctional alpha-adrenoceptors on arterial and venous smooth muscle.

The postsynaptic alpha-adrenoceptors of rabbit ear artery, dog splenic artery and dog splenic vein were characterized in vitro by the use of the selective antagonists prazosin (alpha 1) and rauwolscine (alpha 2). In isolated segments of rabbit ear artery, prazosin was a potent antagonist of norepinephrine-induced contraction, with a receptor dissociation constant (KB) of 6.4 nM. In contrast, rauwolscine was effective only at very high concentrations (KB = 4,000 nM), suggesting that the alpha-receptor of the ear artery has conventional alpha 1-characteristics. The opposite order of potency was observed in a presynaptic alpha-receptor model, the isolated guinea pig atrium, where rauwolscine was potent (KB = 4.5 nM) and prazosin essentially inactive. Rauwolscine had an intermediate potency in the dog splenic vasculature, with a KB of 125 nM in splenic arterial segments and 5-15 nM in segments or helical strips of splenic vein. In contrast to the results with rauwolscine, prazosin was essentially equipotent in dog and rabbit tissues as an antagonist of norepinephrine-induced contraction (KB = 3-9 nM). Schild analysis showed competitive antagonism for both rauwolscine and prazosin in all tissues. These results are consistent with the presence of a homogeneous population of alpha-receptors in dog vasculature having characteristics of both the alpha 1 and alpha 2 receptor subtypes.

Comparative Study of the Arterial and Venous Segmental Circulation in the Human Spleen

The authors have studied the vascular organization of the human spleen with particular regard to the relationship between the arterial and venous segmental circulation. Sixty fresh adult human spleen obtained at post-mortem were stud ied. After anatomical dissection of the splenic vessels at the hilus, in a group of thirty spleens (group I) the injection of radioopaque material (Lipiodol Extraf luid) was performed and angiograms were taken. In a second group of thirty spleens (group 2) epoxy resins of different colours (Biodur E 20) were injected in the splenic vessels and corrosion-casts obtained. By means of these methods the presence of a segmental vascular organization in the human spleen was evi denced ; every main splenic vascular segment, both arterial and venous, was independent and no visible anastomoses took place. In addition, there were complete correspondence of the arterial and venous segments, which were su perimposable since each arterial segment had the same limits as the correspon dent venous one. A parallel distribution of the arterial and venous segmental branches provided a further demonstration that each main splenic vascular seg ment may be considered as a real independent morphological unit. These ana tomical findings support the fact that partial splenectomies can be considered in a variety of pathological conditions, as an alternative to total splenectomy, par ticularly in young patients.