Functional Study of SNCA p.V15A Variant: Further Linking α-Synuclein and Glucocerebrosidase.

BACKGROUND: SNCA p.V15A was reported in five families. In vitro models showed increased aggregation and seeding activity, mitochondrial damage, and apoptosis. Mutant flies had reduced flying ability and survival. OBJECTIVES: To clinically and functionally evaluate SNCA p.V15A in a large Italian family with Parkinson's disease (PD). METHODS: Genetic diagnosis was reached through next-generation sequencing. Pathogenicity was assessed by molecular dynamics simulation and biochemical studies on peripheral blood mononuclear cells (PBMCs). RESULTS: Five siblings carried SNCA p.V15A; three developed bradykinetic-rigid PD in their 50s with rapid motor progression and variable cognitive impairment. A fourth sibling had isolated mood disturbance, whereas the fifth was still unaffected at age 47. The mutant protein showed decreased stability and an unstable folded structure. Proband's PBMCs showed elevated total and phosphorylated α-synuclein (α-syn) levels and significantly reduced glucocerebrosidase activity. CONCLUSION: This study demonstrates accumulation of α-synV15A in PBMCs and strengthens the link between α-syn pathophysiology and glucocerebrosidase dysfunction. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

PSEN1 Compound Heterozygous Mutations Associated with Cerebral Amyloid Angiopathy and Cognitive Decline Phenotype.

Cerebral amyloid angiopathy (CAA) is a cerebrovascular disorder caused by the deposition of amyloid beta-peptide (Aß) aggregates. Aß aggregates lead to vessel rupture and intracerebral hemorrhages, detected by magnetic resonance imaging (MRI). Presenile CAA is usually genetically determined by mutations in the amyloid precursor protein (APP) gene. However, mutations after codon 200 in the presenilin 1 (PSEN1) gene have been reported to facilitate CAA onset. Here, we analyzed the genetic bases in a patient of 55 years old affected by CAA and cognitive decline. DNA was isolated and genetic analysis was performed by Next-Generation Sequencing (NGS). RNA was extracted and retro-transcribed to perform segregation analysis by TOPO-TA cloning. WB analysis was carried out to check the impact of the mutations on protein. Two compound heterozygous mutations in PSEN1 exon 10, such as a novel stop-gain mutation (c.1070C > G) and a pathogenic splice variant (c.1129A > T), were found by NGS. Both mutations altered the presenilin 1 protein, truncating its C-terminal portion. This is the first case of CAA and cognitive decline caused by two compound mutations in PSEN1. With this report, we suggest extending the genetic analysis to PSEN1 when cerebral microbleeds are observed by MRI investigation in a patient affected by presenile cognitive decline.

The spectrum of intermediate SCN8A-related epilepsy.

OBJECTIVE: Pathogenic variants in SCN8A have been associated with a wide spectrum of epilepsy phenotypes, ranging from benign familial infantile seizures (BFIS) to epileptic encephalopathies with variable severity. Furthermore, a few patients with intellectual disability (ID) or movement disorders without epilepsy have been reported. The vast majority of the published SCN8A patients suffer from severe developmental and epileptic encephalopathy (DEE). In this study, we aimed to provide further insight on the spectrum of milder SCN8A-related epilepsies. METHODS: A cohort of 1095 patients were screened using a next generation sequencing panel. Further patients were ascertained from a network of epilepsy genetics clinics. Patients with severe DEE and BFIS were excluded from the study. RESULTS: We found 36 probands who presented with an SCN8A-related epilepsy and normal intellect (33%) or mild (61%) to moderate ID (6%). All patients presented with epilepsy between age 1.5 months and 7 years (mean = 13.6 months), and 58% of these became seizure-free, two-thirds on monotherapy. Neurological disturbances included ataxia (28%) and hypotonia (19%) as the most prominent features. Interictal electroencephalogram was normal in 41%. Several recurrent variants were observed, including Ile763Val, Val891Met, Gly1475Arg, Gly1483Lys, Phe1588Leu, Arg1617Gln, Ala1650Val/Thr, Arg1872Gln, and Asn1877Ser. SIGNIFICANCE: With this study, we explore the electroclinical features of an intermediate SCN8A-related epilepsy with mild cognitive impairment, which is for the majority a treatable epilepsy.

De novo exonic duplication of ATP1A2 in Italian patient with hemiplegic migraine: a case report.

BACKGROUND: Sporadic Hemiplegic Migraine is a rare form of migraine headache. Mutations in three different genes, two ion-channel genes and one encoding an ATP exchanger, CACNA1A, ATP1A2 and SCN1A are all responsible for the FHM phenotype, thus indicating a genetic heterogeneity for this disorder. Here, we described a de novo exonic duplication of ATP1A2 in an Italian patient with Hemiplegic Migraine. CASE PRESENTATION: We describe the case of a young woman (33 year old) who suffered from the age of 8 years of episodic weakness of the limbs, associated to other subjective and objective features. From aged 25, she developed neurological symptoms, like dizziness, blurred vision and an MRI scan revealed aspecific peritrigonal white matter hyperintensities. Aged 32 she suffered of right hemisomatic sudden-onset paresthesias, hypoesthesia and hyposthenia and the patient was genetically investigated for sporadic hemiplegic migraine. CONCLUSIONS: Here we report, for the first time, an exonic duplication in the ATP1A2 associated with hemiplegic migraine. The variation identified involves exon 21 of the ATP1A2 and is expected to alter the function of the alpha(2) subunit of the Na(+)/K(+) pump; the de novo nature of the duplication further supports its pathogenic role. To date, no other CNVs have been described in the ATP1A2 but only point mutations are reported. The novel mutation may result impaired M9 transmembrane domain, in a loss-of-function of the alpha(2) Na(+)/K(+)-ATPase with glutamate accumulation, alteration of synaptic function and neurotransmission.

Analysis of amplicon-based NGS data from neurological disease gene panels: a new method for allele drop-out management.

BACKGROUND: Amplicon-based targeted resequencing is a commonly adopted solution for next-generation sequencing applications focused on specific genomic regions. The reliability of such approaches rests on the high specificity and deep coverage, although sequencing artifacts attributable to PCR-like amplification can be encountered. Between these artifacts, allele drop-out, which is the preferential amplification of one allele, causes an artificial increase in homozygosity when heterozygous mutations fall on a primer pairing region. Here, a procedure to manage such artifacts, based on a pipeline composed of two steps of alignment and variant calling, is proposed. This methodology has been compared to the Illumina Custom Amplicon workflow, available on Illumina MiSeq, on the analysis of data obtained with four newly designed TruSeq Custom Amplicon gene panels. RESULTS: Four gene panels, specific for Parkinson disease, for Intracerebral Hemorrhage Diseases (COL4A1 and COL4A2 genes) and for Familial Hemiplegic Migraine (CACNA1A and ATP1A2 genes) were designed. A total of 119 samples were re-sequenced with Illumina MiSeq sequencer and panel characterization in terms of coverage, number of variants found and allele drop-out potential impact has been carried out. Results show that 14 % of identified variants is potentially affected by allele drop-out artifacts and that both the Custom Amplicon workflow and the procedure proposed here could correctly identify them. Furthermore, a more complex configuration in presence of two mutations was simulated in silico. In this configuration, our proposed methodology outperforms Custom Amplicon workflow, being able to correctly identify two mutations in all the studied configurations. CONCLUSIONS: Allele drop-out plays a crucial role in amplicon-based targeted re-sequencing and specific procedures in data analysis of amplicon data should be adopted. Although a consensus has been established in the elimination of primer sequences from aligned data (e.g., via primer sequence trimming or soft clipping), more complex configurations need to be managed in order to increase the retrieved information from available data. Our method shows how to manage one of these complex configurations, when two mutations occur.

Immunization with pentraxin 3 (PTX3) leads to anti-PTX3 antibody production and delayed lupus-like nephritis in NZB/NZW F1 mice.

BACKGROUND: Anti-pentraxin 3 (PTX3) antibodies were associated with the absence of lupus glomerulonephritis in humans. AIM: To explore the effects of anti-PTX3 antibodies in New Zealand Black/White (NZB/NZW F1) mice and their inherent mechanisms of action. MATERIALS AND METHODS: 30 NZB/NZW F1 mice were subdivided into 3 groups of 10 mice each and subcutaneously injected with PTX3, alum and PBS (group 1), alum and PBS (group 2) or PBS alone (group 3), 3 times 3 weeks apart, before development of renal disease. Mice were followed until natural death. Histological analysis and immunohistochemistry were performed on harvested kidneys. Effects of anti-PTX3 antibodies on C1q binding to immobilized PTX3-anti-PTX3 immune complexes were evaluated in vitro using human SLE sera. Qualitative characterization of human IgG anti-PTX3 was performed. RESULTS: Only group 1 mice developed anti-PTX3 antibodies. Anti-dsDNA and anti-C1q antibodies appeared significantly later and at lower levels in group 1 mice vs. controls (p < 0.0001). Proteinuria-free and overall survival were significantly increased in group 1 mice vs. controls (p < 0.05 and p = 0.03, respectively). Histopathological analysis showed that glomerular and tubular PTX3 staining and renal lesions were increased in controls compared with immunized mice. Addition of human SLE sera positive for anti-PTX3 antibodies to C1q and fixed PTX3 interfered with C1q binding to PTX3-anti-PTX3 immune complexes. Qualitative characterization of human IgG anti-PTX3 showed an increased proportion of IgG4. CONCLUSIONS: Anti-PTX3 antibodies delay lupus-like nephritis and prolong survival of NZB/NZW F1 mice. In vitro observations suggest anti-PTX3 antibodies may dampen complement activation via their Fc fragment, likely hindering renal inflammation.

Potential role of effector memory T cells in chronic T cell-mediated kidney graft rejection.

BACKGROUND: Chronic T cell-mediated rejection (TCMR) in kidney graft is characterized by reduction of the vessel lumen with marked intimal thickening, fibrous hyperplasia of the small renal arteries and leukocyte infiltrates. The aim of this study was to find specific gene expression profiles in chronic TCMR kidney biopsies. METHODS: RNA extracted from archival formalin-fixed, paraffin-embedded renal biopsies was used for gene expression profiling. Our study included 14 patients with chronic TCMR and 10 with acute TCMR. Fifty-two cadaveric donors were used as controls. The results were validated in an independent set of kidney biopsies. RESULTS: We identified 616 and 243 differentially expressed genes with a fold change ≥1.5 and a false discovery rate <0.05 in chronic and acute TCMR, respectively. Pathway analysis revealed upregulation of OX40 signalling. This pathway is involved in the generation of CD8+ effector memory T cells and the upregulation of killer cell lectin-like receptor G1 (KLRG-1), B lymphocyte-induced maturation protein 1 (BLIMP-1) and CD25, which characterize CD8+ effector memory T cells. However, the enhanced OX40 signalling pathway was specific to chronic TCMR; a significant increase of KLRG-1+/CD8+ and BLIMP-1+/CD8+ was only detected in these specimens. CONCLUSIONS: These results suggest the involvement of memory-committed CD8+ effector T cells in chronic TCMR. The generation of effector memory T cells is mediated by the OX40 gene pathway, and could be considered a future target for the specific treatment of chronic TCMR.

Pro-Apoptotic Effects of Plasma from Patients with Cardiorenal Syndrome on Human Tubular Cells.

BACKGROUND: The pathophysiology of Cardiorenal Syndrome Type 1 (CRS1) is widely studied, although the mechanisms by which renal tubular epithelial cells (TECs) cease to proliferate and embark upon terminal differentiation, following the initial insult of heart failure (HF), remain a key target. This study seeks to provide insight into the pathophysiological pathways in CRS1; we evaluated in vitro the effects of CRS1 plasma on TECs. METHODS: We enrolled 40 acute HF patients and 15 controls (CTR) without HF or acute kidney injury (AKI). Ten out of 40 HF patients exhibited AKI at the time of admission for HF or developed AKI during hospitalization and were classified as CRS1. In vitro, cell viability, DNA fragmentation and caspase-3 levels were investigated in TECs incubated with HF, CRS1, and CTR plasma. We assessed inflammatory cytokines and NGAL expression at the gene and protein levels. RESULTS: We observed a marked pro-apoptotic activity and a significantly increased in vitro level of apoptosis in TECs incubated with plasma from CRS1 patients compared to HF and CTR (p < 0.01). In the CRS1 group, the mRNA expression of IL-6, IL-18 and NGAL resulted significantly higher in TECs incubated with CRS1 plasma compared with those incubated with plasma from HF and CTR (p < 0.01). IL-6, IL-18, NGAL, and RANTES levels were significantly higher in TECs supernatant incubated with CRS1 plasma compared with HF patients and CTR plasma (p < 0.01). CONCLUSION: In vitro exposure to plasma from CRS1 patients altered the expression profile of TECs characterized by increases in proinflammatory mediators, release of tubular damage markers, and apoptosis.

Canalicular adenoma immunoprofile: a case report.

OBJECTIVES: To report a case of canalicular adenoma (CA) and discuss the use of immunohistochemistry to better address the diagnosis given some unusual characteristics in this patient. BACKGROUND: CA is an uncommon benign neoplasm that can develop in minor salivary gland duct tissues throughout the oral cavity. At histology, it shares several features with other salivary tumors. Immunohistochemistry can be useful in the differential diagnosis. MATERIALS AND METHODS: The clinical presentation consisted in a nodule on the left upper lip of an 85-year-old man. The patient's main complaint was upper denture instability secondary to soft tissue changes. The nodule was excised under local anesthesia and underwent histological and immunohistochemical examination to rule out any malignancy. RESULTS: Histological findings, cytokeratin positivity and the absence of any reactivity to specific markers of myoepithelial differentiation confirmed the epithelial nature of the lesion. CONCLUSION: The histological diagnosis of benign salivary tumors such as CA can be confirmed by immunohistochemistry.

Electroclinical features and phenotypic differences in adenylosuccinate lyase deficiency: Long-term follow-up of seven patients from four families and appraisal of the literature.

OBJECTIVE: Adenylosuccinate lyase (ADSL) deficiency is a rare inherited metabolic disorder with a wide phenotypic presentation, classically grouped into three types (neonatal, type I, and type II). We aim to better delineate the pathological spectrum, focusing on the electroclinical characteristics and phenotypic differences of patients with ADSL deficiency. PATIENTS AND METHODS: Seven patients, from four different families, underwent serial electroencephalogram (EEG), clinical assessment, and neuroimaging. We also performed a systematic review of the cases published in the literature, summarizing the available clinical, neurophysiological, and genetic data. RESULTS: We report seven previously unreported ADSL deficiency patients with long-term follow-up (10-34 years). From the literature review, we collected 81 previously reported cases. Of the included patient population, 58 % (51/88) were classified as having ADSL deficiency type I, 28% (25/88) as having type II, and 14% (12/88) as having neonatal. The most frequently reported pathogenic variants are p.R426H homozygous (19 patients), p.Y114H in compound heterozygosity (13 patients), and p.D430N homozygous (6 patients). In the majority (89.2%), disease onset was within the first year of life. Epilepsy is present in 81.8% of the patients, with polymorphic and often intractable seizures. EEG features seem to display common patterns and developmental trajectories: (i) poor general background organization with theta-delta activity; (ii) hypsarrhythmia with spasms, usually adrenocorticotropic hormone-responsive; (iii) generalized epileptic discharges with frontal or frontal temporal predominance; and (iv) epileptic discharge activation in sleep with an altered sleep structure. Imaging features present consistent findings of cerebral atrophy with frontal predominance, cerebellar atrophy, and white matter abnormalities among the three types. SIGNIFICANCE: ADSL deficiency presents variable phenotypic expression, whose severity could be partially attributed to residual activity of the mutant protein. Although a precise phenotype-genotype correlation was not yet feasible, we delineated a common pattern of clinical, neuroradiological, and neurophysiological features.

Three-dimensional molecular reconstruction of rat heart with mass spectrometry imaging.

Cardiovascular diseases are the world's number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na(+), K(+), and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging.

Lipid insudation as a cause of structural failure of a stentless pericardial bioprosthesis.

The Sorin Pericarbon Freedom (SPF) valve is a stentless bioprosthesis made from bovine pericardium, with a peculiar design aimed at preventing the mechanical failures observed with old models of stented pericardial bioprostheses. Herein, the case is described of a patient who presented with severe regurgitation of a SPF six years after aortic valve replacement, caused by commissural dehiscence. Both, microradiographic and histologic investigations, revealed mild calcific deposits and massive lipid infiltration, thus confirming that a patient-related mechanism such as 'atheromasia' can account for structural valve deterioration also in recipients of pericardial bioprostheses.

The pathology of TRI-Tech valve leaflet escape.

BACKGROUND AND AIM OF THE STUDY: The study aim was to investigate leaflet escape in the TRI-Tech mechanical valve, as reported in three patients. METHODS: Among the three patients, one patient with a mitral prosthesis from which leaflet escape occurred underwent a successful reoperation. However, two patients with an aortic prosthesis from which leaflet escape occurred died suddenly. In addition, 150 unimplanted TRI-Tech valve prostheses were evaluated. RESULTS: Tab fracture was observed in all of the escaped leaflets, with a height asymmetry of 0.33 mm in the mitral valve, and 0.55 and 0.41 mm, respectively, in the two aortic valves. Height tab asymmetry was present in all unimplanted valves: this was <0.20 mm in 90% of valves, and >0.20 mm in 10% (in one valve the asymmetry was >0.35 mm). Height asymmetry was greatest in valves sized 21 to 25 mm. A review of data provided by the TRI-Tech quality control unit failed to demonstrate any reference to checking for tab symmetry. CONCLUSION: Significant tab malalignment was observed in the TRI-Tech valves in which tab fracture and leaflet escape had occurred. Tab asymmetry was also present in all unimplanted valves; this was >0.20 mm in 10% of units, and in one case was as high as in valves that had undergone leaflet escape. As tolerance for tab asymmetry is unknown and the risk of rupture is unpredictable, an interruption of the implant program with prophylactic replacement of the implanted TRI-Tech valves must be considered judicious.

Overexpression of SERPIN B3 promotes epithelial proliferation and lung fibrosis in mice.

SERPIN B3/B4, members of the serpin superfamily, are fundamental for the control of proteolysis through a known inhibitory function of different proteases. Several studies have documented an important role of SERPIN B3 in the modulation of inflammation, programmed cell death and fibrosis. To confirm the role of SERPIN B3 in lung fibrosis and overall investigate its influence on epithelial dysfunction, a stratified controlled trial randomly assigning bleomycin (BLM) treatment was performed on both SERPIN B3 transgenic (TG) and wild-type (WT) mice. TG and WT animals were killed 48 h (group T48 h) and 20 days (group T20d) after BLM treatment. Lung fibrosis was assessed by histology and hydroxyproline measurement. Architectural remodeling, inflammation, epithelial apoptosis and proliferation were quantified. Moreover, the profibrogenetic cytokine transforming growth factor (TGF)-ß, cathepsin K, L and S were also investigated. No significant differences were observed between TG and WT mice of group T48 h in any parameters. In group T20d, less inflammation and a significant increase in epithelial proliferation were detected in treated TG than WT mice despite a similar apoptotic index, thus resulting in a different apoptosis/proliferation imbalance with a significant gain of epithelial proliferation. Moreover, TG mice showed higher TGF-ß expression and more extended fibrosis. General linear model analysis, applied on morphological data, showed that interaction between SERPIN B3 expression and treatment was mainly significant for fibrosis. This study provides in vivo evidence for a role of SERPIN B3 in inhibiting inflammation and favoring epithelial proliferation with increased TGF-ß secretion and thus the likelihood of consequent fibrogenesis.

SERPINB3 induces epithelial-mesenchymal transition.

Epithelial-mesenchymal transition is believed to facilitate invasion and metastasis formation of epithelial tumour cells. SERPINB3 is a serine protease inhibitor, physiologically found in normal squamous epithelium but over-expressed in epithelial tumours and known to inhibit apoptosis. We tested the hypothesis that SERPINB3 has a role in invasion by modulating the epithelial-mesenchymal transition programme, using morphological, molecular and cell biology techniques on HepG2 cell clones transfected with the human SERPINB3 gene. The paracrine effect of this serpin was determined by the addition of exogenous recombinant SERPINB3 protein to HepG2 and MDCK cell line. SERPINB3 expression leads to changes in transfected cells morphology, characterized by clusters of loosely connected cells with elongated shape. Ultrastructural analysis confirmed the decrease of desmosomal junctions and widening of intercellular spaces. These alterations were associated with a reduction of E-cadherin and an increase of beta-catenin, with a parallel increase of cell proliferation. SERPINB3 clones, untransfected HepG2 and MDCK cells treated with exogenous SERPINB3 expressed vimentin, undetectable in controls. SERPINB3 induced significant cell scattering, migration and invasiveness in untransfected cells. These effects were not dependent on the anti-protease activity of the protein, as documented by the results obtained with an active loop-deleted recombinant SERPINB3 protein. Scatter activity was inhibited by an anti-SERPINB3 antibody in a dose-dependent manner and SERPINB3-transfected cells formed a significantly higher number of colonies on soft agar than controls. In conclusion, the observed results indicate that SERPINB3 induces deregulation of adhesion processes and increases the invasiveness potential supported by features of epithelial-mesenchymal transition, acting at both the autocrine and the paracrine level.

Anticalcification strategies to increase bioprosthetic valve durability.

In recent years, there has been a revival in the use of bioprosthetic valves as aortic substitutes. For example, during 2008 in Germany, among 12,397 patients who underwent isolated aortic valve procedures, 78% received a biological prosthesis and 21% a mechanical valve, while only 1% underwent valve repair. However, whilst this situation is the reverse of that during the 1990s, the question must be asked as to whether this is simply a fluctuating fashion, or a reflection of other factors.

Intra-oral bone harvesting: two methods compared using histological and histomorphometric assessments.

AIM: This study used morphometric analyses to compare two methods for the intra-oral harvesting of particulate bone: Mectron Piezosurgery® and the Meta Micross®. MATERIALS AND METHODS: Twenty patients requiring bilateral germectomy of the lower third molars for orthodontic reasons were selected and a sample was harvested from each patient from a standardised donor site (the cortical bone in the area of the retromolar triangle). Ten samples were obtained for each method. The particulate collected were subjected to a histological examination and the samples were analysed considering the following parameters: the mean surface area of fragments, the mean surface area considered vital and the mean surface area considered non-vital, the mean percentage of area considered vital and the mean percentage of area considered non-vital, the mean number of normal osteocytes and the mean number of osteocytes with morphological changes identified per unit area (600,000 µm(2)). The results were analysed, calculating the mean and the corresponding standard deviations, and testing their significance using Student's t-test, and plotted in graphs. RESULTS: Mectron Piezosurgery® produced significantly larger particles (P<0.05) than the Meta Micross®, with a larger mean surface area considered vital and a significantly larger (P<0.05) surface area considered non-vital. Mectron Piezosurgery® also produced a smaller mean percentage of area considered vital (64.83%) and a larger mean percentage of area considered non-vital (35.17%) by comparison with the Meta Micross® (75.34% and 24.66%, respectively). The data also showed that the two methods produce a similar quantity of empty lacunae, and that the Mectron Piezosurgery® produces a larger quantity of osteocytes. CONCLUSIONS: The analyses conducted demonstrated that the particulate collected with the Meta Micross® had a smaller mean surface area of the fragments and a smaller surface area of bone considered non-vital than in the particulate collected using Mectron Piezosurgery®.

Surgical OFF-LOADING of the diabetic foot.

Diabetic foot ulcer treatment is a challenge for the healthcare world. Widespread infection and the presence of critical ischemia (especially with end-stage renal disease) can lead to major amputation rather than amenable to conservative treatment. Surgical strategies of the diabetic foot have been changing over the past 10 years and are now focused on reconstructive treatment and limb salvage. These goals were achieved, thanks to an evolution of distal revascularization techniques and a distinct approach, which integrates various methods focused on limb salvage. Podoplastic techniques of the diabetic foot are focused on infection clearance, the surgical treatment of corrective deformities, soft tissue coverage and limb ischemia correction along with the management of diabetes and the comorbidities that compromise tissue repair processes. The reconstructive techniques used in diabetic foot treatment owe their effectiveness in part to the results of technological improvements such as the circular external fixator as a tool for stabilization and surgical site protection. In the last decade, many studies have shown that circular external fixation should be considered as the most useful method to protect the reconstructive surgical site in limb salvage of the diabetic foot. The objective of this review is to highlight the role of surgical offloading using circular external fixation as an adjunct to the podoplastic diabetic foot reconstruction procedures.