High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

Inner branched complex aortic repair outcomes from a national multicenter registry using the E-xtra design platform.

BACKGROUND: Complex aortic pathology still represents an open issue in contemporary endovascular management, with continuous technological advancement being introduced in practice over time aiming to improve outcomes. Thus far, the dualism between the fenestrated and branched configuration for visceral artery revascularization is yet unsolved, with each approach having its own pros and cons. The inner branched technology for endovascular aneurysm repair (iBEVAR) aims to take the best out of both strategies, offering wide applicability and stable bridging stent sealing. The objective of this study was to evaluate the early outcomes obtained with a single manufacturer custom-made inner-branched endograft in a multicenter Italian experience. METHODS: All patients consecutively treated with E-xtra design devices in three Italian facilities were enrolled. Anatomic characteristics and perioperative data were analyzed. The main objective was to asses technical and clinical success after iBEVAR. Secondary end points were overall survival, aortic-related mortality, target visceral vessel (TVV) patency, and freedom from target vessel instability during follow-up. RESULTS: From 2016 to 2021, 45 patients were treated with an E-xtra design device revascularizing at least one visceral vessel through an inner branch. The mean age at the time of the procedure was 71.1 ± 9.3 years and 77.8% were males. The total number of target visceral arteries to be bridged with an inner branch was 159. The extent of aortic repair was thoracoabdominal in 91.1% of the cases. Technical success was achieved in 93.3% of the procedures (42/45) with all failures owing to a type I endoleak at final angiography. Each TVV was successfully connected to the graft's main body as planned without complications. Following their intervention, five patients developed spinal cord ischemia and in three of these cases symptoms persisted after discharge (6.7%). At 30 days clinical success was 93.3% (42/45). No death as well as no TVV thrombosis occurred within 30 days from the primary procedures. The mean follow-up was 22.8 ± 14.2 months. The Kaplan-Meier estimate of overall survival and TVV patency at 36 months were 83.9% and 95.9%, respectively. CONCLUSIONS: Inner branches seem to be a promising technology in the complex aortic repair landscape, with an applicability ranging from type II thoracoabdominal aneurysm to type I endoleak repair after infrarenal endografting. Whether iBEVAR could offer results comparable with those provided by fenestrated/branched endovascular aneurysm repair in terms of target vessel patency and stent stability is yet to be established and further studies are, therefore, needed.

Balloon Endoclamping for Postdissection Infrarenal Aorto-Iliac Aneurysm Repair.

BACKGROUND: Isolated post dissection infrarenal and iliac aneurysm is a rare condition that often requires surgical treatment. Surgical repair should involve the replacement of the aneurysmal segments and a wide fenestration in the residual proximal untreated abdominal aorta. However, in these patients proximal aortic clamping may be challenging. Indeed, infrarenal clamping may hamper an appropriate fenestration in the proximal dissecting lamella, and suprarenal or supraceliac clamping can be dangerous and highly demanding, especially in acute and subacute patients. Here we report our initial experience with a balloon endoclamping technique. MATERIAL AND METHODS: Our technique includes 1) direct aortic true lumen catheterization, 2) balloon endoclamping of the proximal thoracic aorta, 3) wide fenestration of the infrarenal aorta followed by external clamp positioning, 4) infrarenal aorta and iliac artery reconstruction. RESULTS: Between October 2018 and November 2019, 4 patients (male n = 4, median age 57 years) underwent postdissection iliac aneurysm repair in our institution. All patients had previously undergone emergent thoracic aorta repair. Postoperative courses were uneventful in all cases. At a median FU of 13 months, all patients remain well, with stable diameters in visceral aorta. CONCLUSIONS: In our initial experience, proximal aortic endoclamping appeared to be a safe technique associated with promising results. This approach may facilitate proximal aortic clamping and allow for a wide aortic fenestration. Further larger clinical trials are needed to validate our preliminary observations.

Bell-bottom technique in iliac branch era: mid-term single stent graft performance.

BACKGROUND: Endovascular abdominal aortic aneurysm repair (EVAR) is considered the primary option for abdominal aortic aneurysm but the management of concomitant wide or aneurysmal iliac arteries (CIAs) is still controversial. METHODS: We retrospectively evaluated mid-term results of patients receiving standard EVAR combined with bell-bottom technique (BBT) using Medtronic Endurant endograft between January 2009 and December 2018. Patients were followed up by CT scan performed 1 month after the procedure and by duplex ultrasound annually (with or without contrast medium) followed by CT scan in case of evolution. RESULTS: Seventy-one patients (67 males; mean age of 77,1 years) with abdominal aortic aneurysm and wide or aneurysmal common iliac artery (distal landing zone diameter up to 25 mm and length more than 20 mm) were treated with standard EVAR and BBT (107 limbs) using Endurant stent graft. The median aortic diameter was 56,1 mm (31.0-85.0). Technical success was obtained in 100%. Mean procedural time was of 100.1 min. No 30 days' mortality, renal failure or limb ischaemia occurred. The median follow-up was of 36.56 months (1-136). 5-year aneurysm related mortality was not found. At 5 years, the number of all-cause deaths was seven. The freedom from secondary intervention was 91.6% at 5 years. Three patients (4.4%) were treated for iliac related complications at 5 years: internal iliac artery aneurysm, iliac obstruction, type 1b endoleak, all successfully treated by endovascular technique. CONCLUSIONS: According with this study BBT using Endurant stent graft is effective and safe with good mid-term results, with low rate of iliac related complications and no aneurysm related mortality.

Chimney technique with endoanchors in treatment of late type 1a endoleak after endovascular aortic repair.

The late type Ia endoleak after endovascular aortic repair could be a challenging issue. Over the last years, in case of short or enlarged neck, fenestrated and branched stent grafts have been increasingly employed with improving results. However, these devices have limited use in urgent/emergent cases as custom graft manufacturing takes long time, and may not be fit in patients with particular anatomic features. In this setting, chimney and relining remain an alternative but sometimes may not be adequate. According to literature, the use of the endoanchors associated to chimney technique can improve the procedure results in primary endovascular aortic repair. We treated two patients with a late type Ia endoleak after endovascular aortic repair with a simultaneous relining, single renal chimney, and endoanchors implant. These patients were valuated unfit for open repair with neck configuration unadapt for a simple relining, ballooning, or stenting. The patient conditions were unfavorable for an endovascular repair with branched endovascular aortic repair-fenestrated endovascular aortic repair. The same procedure was performed in both patients. Postoperative angio-computed tomographic scan demonstrated the resolution of the endoleak with patency of renal graft. Our preliminary experience, in these selected cases, demonstrate the feasibility of the technique in late type Ia endoleak.

Pararenal aortic aneurysm in situs inversus totalis: open repair with right retroperitoneal approach.

Situs inversus totalis (SIT) refers to a mirror-image reversal of the internal organ position. The coexistence of abdominal aortic aneurysm and SIT is extremely rare; very short series have been reported; therefore its prevalence has never been estimated. The presence of anatomical anomalies could increase operative risk. The authors describe a case of successfully repaired pararenal abdominal aortic aneurysm with a right retroperitoneal approach in situs inversus totalis.

Emergency Endovascular Treatment of Coral Reef Aorta with an Unconventional Technique.

Coral reef aorta (CRA) is a rare condition featured by rock-hard calcifications that grow into the lumen of the thoracoabdominal aorta. Patients suffering from CRA may present severe downstream ischemic and embolic events involving the viscera and the lower limbs. In these patients, open surgical repair is the first choice of treatment. We present a case of a 70-year-old woman with acute presentation of bilateral limb ischemia and abdominal pain. An angio-computed tomography (CT) scan showed the subocclusion of the distal thoracic aorta due to a severe calcified coarctation with intraluminal thrombosis, a chronic occlusion of the superior mesenteric and celiac trunk arteries, a hypertrophic inferior mesenteric artery associated with signs of partial left kidney ischemia. The patient underwent urgent endovascular repair. A soft dilatation of the coarctation using a 7-mm noncompliant balloon was initially performed; a 21-mm conformable thoracic stent graft was subsequently deployed and increasingly dilated using progressively larger angioplasty balloons. The procedure was uneventful and the aortic gradient was reduced to zero. A postoperative CT scan revealed the proper deployment of the graft with no residual stenosis. Endovascular approach with the covered thoracic stent graft could be an accurate technique to treat CRA in the emergency setting. It allows for a primary protected balloon angioplasty and, in case of aortic-graft recoiling, a subsequent placement of covered or uncovered balloon-expandable stent graft can be safely and easily performed.

Results of a multicenter universal newborn screening program for sickle cell disease in Italy: A call to action.

BACKGROUND: Sickle cell disease (SCD) is a chronic multisystem disorder requiring comprehensive care that includes newborn screening (NBS) as the first step of care. Italy still lacks a national SCD NBS program and policy on blood disorders. Pilot single-center screening programs and a regional targeted screening have been implemented so far, but more evidence is needed in order to impact health policies. POPULATION AND METHODS: NBS was offered to parents of newborns in gynecology clinics in Padova and Monza, tertiary care university hospitals in northern Italy. High-performance liquid chromatography (HPLC) was performed as the first test on samples collected on Guthrie cards. Molecular analysis of the beta-globin gene was performed on positive samples. RESULTS: A total of 5466 newborns were enrolled; for 5439, informed consents were obtained. A similar family origin was seen in the two centers (65% Italians, 9% mixed couples, 26% immigrants). Compared with SCD NBS programs in the United States and Europe, our results show a similar incidence of SCD patients and carriers. All SCD patients had a Sub-Saharan family background; HbS carriers were 15% Caucasians (Italian, Albanians) and 10% from other areas (North Africa-India-South America); carriers of other hemoglobin variants were mainly (47%) from other areas. CONCLUSIONS: Our results demonstrate the feasibility of a multicentric NBS program for SCD, give information on HbS epidemiology in two Northern Italian Areas, and support previous European recommendation for a universal NBS program for SCD in Italy: a high incidence of patients and carriers has been detected, with a high percentage of Caucasian carriers, impossible to identify in a targeted NBS.

Evaluation of Technical Issues in a Pilot Multicenter Newborn Screening Program for Sickle Cell Disease.

A multicenter pilot program for universal newborn screening of Sickle cell disease (SCD) was conducted in two centres of Northern Italy (Padova and Monza). High Performance Liquid Chromatography (HPLC) was performed as the first test on samples collected on Guthrie cards and molecular analysis of the ß-globin gene (HBB) was the confirmatory test performed on the HPLC-positive or indeterminate samples. 5466 samples of newborns were evaluated. Of these, 5439/5466 were submitted to HPLC analysis and the molecular analysis always confirmed in all the alteration detected in HPLC (62/5439 newborns); 4/5439 (0.07%) were SCD affected, 37/5439 (0.68%) were HbAS carriers and 21/5439 (0.40%) showed other hemoglobinopathies. Stored dried blood spots were adequate for HPLC and ß-globin gene molecular analysis. Samples were suitable for analysis until sixteen months old. A cut-off of A1 percentage, in order to avoid false negative or unnecessary confirmation tests, was identified. Our experience showed that several technical issues need to be addressed and resolved while developing a multicenter NBS program for SCD in a country where there is no national neonatal screening (NBS) program for SCD and NBS programs occur on a regional basis.