MIC-MAC: An automated pipeline for high-throughput characterization and classification of three-dimensional microglia morphologies in mouse and human postmortem brain samples.

The phenotypic changes of microglia in brain diseases are particularly diverse and their role in disease progression, beneficial, or detrimental, is still elusive. High-throughput molecular approaches such as single-cell RNA-sequencing can now resolve the high heterogeneity in microglia population for a specific physiological condition, however, the relation between the different microglial signatures and their surrounding brain microenvironment is barely understood. Thus, better tools to characterize the phenotypic variations of microglia in situ are needed, particularly for human brain postmortem samples analysis. To address this challenge, we developed MIC-MAC, a Microglia and Immune Cells Morphologies Analyser and Classifier pipeline that semiautomatically segments, extracts, and classifies all microglia and immune cells labeled in large three-dimensional (3D) confocal image stacks of mouse and human brain samples. Our imaging-based approach enables automatic 3D-morphology characterization and classification of thousands of individual microglia in situ and revealed species- and disease-specific morphological phenotypes in mouse aging, human Alzheimer's disease, and dementia with Lewy Bodie's samples. MIC-MAC is a precision diagnostic tool that allows a rapid, unbiased, and large-scale analysis of microglia morphological states in mouse models and patient brain samples.

Two novel POC1A mutations in the primordial dwarfism, SOFT syndrome: Clinical homogeneity but also unreported malformations.

Primordial dwarfism encompasses rare conditions characterized by severe intrauterine growth retardation and growth deficiency throughout life. Recently, three POC1A mutations have been reported in six families with the primordial dwarfism, SOFT syndrome (Short stature, Onychodysplasia, Facial dysmorphism, and hypoTrichosis). Using a custom-designed Next-generation sequencing skeletal dysplasia panel, we have identified two novel homozygous POC1A mutations in two individuals with primordial dwarfism. The severe growth retardation and the facial profiles are strikingly similar between our patients and those described previously. However, one of our patients was diagnosed with severe foramen magnum stenosis and subglottic tracheal stenosis, malformations not previously associated with this syndrome. Our findings confirm that POC1A mutations cause SOFT syndrome and that mutations in this gene should be considered in patients with severe pre- and postnatal short stature, symmetric shortening of long bones, triangular facies, sparse hair and short, thickened distal phalanges.

Microglia phenotypes are associated with subregional patterns of concomitant tau, amyloid-ß and α-synuclein pathologies in the hippocampus of patients with Alzheimer's disease and dementia with Lewy bodies.

The cellular alterations of the hippocampus lead to memory decline, a shared symptom between Alzheimer's disease (AD) and dementia with Lewy Bodies (DLB) patients. However, the subregional deterioration pattern of the hippocampus differs between AD and DLB with the CA1 subfield being more severely affected in AD. The activation of microglia, the brain immune cells, could play a role in its selective volume loss. How subregional microglia populations vary within AD or DLB and across these conditions remains poorly understood. Furthermore, how the nature of the hippocampal local pathological imprint is associated with microglia responses needs to be elucidated. To this purpose, we employed an automated pipeline for analysis of 3D confocal microscopy images to assess CA1, CA3 and DG/CA4 subfields microglia responses in post-mortem hippocampal samples from late-onset AD (n = 10), DLB (n = 8) and age-matched control (CTL) (n = 11) individuals. In parallel, we performed volumetric analyses of hyperphosphorylated tau (pTau), amyloid-ß (Aß) and phosphorylated α-synuclein (pSyn) loads. For each of the 32,447 extracted microglia, 16 morphological features were measured to classify them into seven distinct morphological clusters. Our results show similar alterations of microglial morphological features and clusters in AD and DLB, but with more prominent changes in AD. We identified two distinct microglia clusters enriched in disease conditions and particularly increased in CA1 and DG/CA4 of AD and CA3 of DLB. Our study confirms frequent concomitance of pTau, Aß and pSyn loads across AD and DLB but reveals a specific subregional pattern for each type of pathology, along with a generally increased severity in AD. Furthermore, pTau and pSyn loads were highly correlated across subregions and conditions. We uncovered tight associations between microglial changes and the subfield pathological imprint. Our findings suggest that combinations and severity of subregional pTau, Aß and pSyn pathologies transform local microglia phenotypic composition in the hippocampus. The high burdens of pTau and pSyn associated with increased microglial alterations could be a factor in CA1 vulnerability in AD.

The Parkinson's-disease-associated mutation LRRK2-G2019S alters dopaminergic differentiation dynamics via NR2F1.

Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental abnormalities in dopaminergic neurons in a model of Parkinson's disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1.

3D Cultures of Parkinson's Disease-Specific Dopaminergic Neurons for High Content Phenotyping and Drug Testing.

Parkinson's disease (PD)-specific neurons, grown in standard 2D cultures, typically only display weak endophenotypes. The cultivation of PD patient-specific neurons, derived from induced pluripotent stem cells carrying the LRRK2-G2019S mutation, is optimized in 3D microfluidics. The automated image analysis algorithms are implemented to enable pharmacophenomics in disease-relevant conditions. In contrast to 2D cultures, this 3D approach reveals robust endophenotypes. High-content imaging data show decreased dopaminergic differentiation and branching complexity, altered mitochondrial morphology, and increased cell death in LRRK2-G2019S neurons compared to isogenic lines without using stressor agents. Treatment with the LRRK2 inhibitor 2 (Inh2) rescues LRRK2-G2019S-dependent dopaminergic phenotypes. Strikingly, a holistic analysis of all studied features shows that the genetic background of the PD patients, and not the LRRK2-G2019S mutation, constitutes the strongest contribution to the phenotypes. These data support the use of advanced in vitro models for future patient stratification and personalized drug development.

Neural Stem Cells of Parkinson's Disease Patients Exhibit Aberrant Mitochondrial Morphology and Functionality.

Emerging evidence suggests that Parkinson's disease (PD), besides being an age-associated disorder, might also have a neurodevelopment component. Disruption of mitochondrial homeostasis has been highlighted as a crucial cofactor in its etiology. Here, we show that PD patient-specific human neuroepithelial stem cells (NESCs), carrying the LRRK2-G2019S mutation, recapitulate key mitochondrial defects previously described only in differentiated dopaminergic neurons. By combining high-content imaging approaches, 3D image analysis, and functional mitochondrial readouts we show that LRRK2-G2019S mutation causes aberrations in mitochondrial morphology and functionality compared with isogenic controls. LRRK2-G2019S NESCs display an increased number of mitochondria compared with isogenic control lines. However, these mitochondria are more fragmented and exhibit decreased membrane potential. Functional alterations in LRRK2-G2019S cultures are also accompanied by a reduced mitophagic clearance via lysosomes. These findings support the hypothesis that preceding mitochondrial developmental defects contribute to the manifestation of the PD pathology later in life.

Shape-aware surface reconstruction from sparse 3D point-clouds.

The reconstruction of an object's shape or surface from a set of 3D points plays an important role in medical image analysis, e.g. in anatomy reconstruction from tomographic measurements or in the process of aligning intra-operative navigation and preoperative planning data. In such scenarios, one usually has to deal with sparse data, which significantly aggravates the problem of reconstruction. However, medical applications often provide contextual information about the 3D point data that allow to incorporate prior knowledge about the shape that is to be reconstructed. To this end, we propose the use of a statistical shape model (SSM) as a prior for surface reconstruction. The SSM is represented by a point distribution model (PDM), which is associated with a surface mesh. Using the shape distribution that is modelled by the PDM, we formulate the problem of surface reconstruction from a probabilistic perspective based on a Gaussian Mixture Model (GMM). In order to do so, the given points are interpreted as samples of the GMM. By using mixture components with anisotropic covariances that are "oriented" according to the surface normals at the PDM points, a surface-based fitting is accomplished. Estimating the parameters of the GMM in a maximum a posteriori manner yields the reconstruction of the surface from the given data points. We compare our method to the extensively used Iterative Closest Points method on several different anatomical datasets/SSMs (brain, femur, tibia, hip, liver) and demonstrate superior accuracy and robustness on sparse data.

Multiple stressors in estuarine waters: Effects of arsenic and salinity on Ruditapes philippinarum.

Marine organisms are constantly exposed to multiple stressors creating a range of associated environmental and ecotoxicological risks. Several stressors have been identified as key drivers of environmental change that may significantly influence marine near-shore systems. These include increased frequency and duration of extreme rainy events and drought periods, arising from climate change, and the constant discharge of contaminants into aquatic systems. A growing body of evidence demonstrates that climate change can have direct and indirect impacts on marine organisms although the combined effects with other stressors, namely with metals and metalloids, have received very little attention to date. The present study evaluated the biochemical alterations induced in the clam Ruditapes philippinarum, also known as Manila clam, when simultaneously exposed (96 h) to different arsenic concentrations (0, 4 and 17 mg/L) and a range of salinities (14, 21, 28, 35 and 42 g/L). Results obtained revealed that, when acting alone, both stressors induced oxidative stress in clams, with higher LPO levels and lower GSTs activity induced by As contamination, and a stronger inhibition of the antioxidant defenses induced by salinity increase. Furthermore, when exposed to the combination of both stressors, clams experienced stronger biochemical alterations, presenting higher LPO increases and greater decreases of antioxidant enzymes, especially noticed at higher salinities. The present findings may indicate that climate change, including predicted drought periods that will increase salinities in aquatic systems, will seriously affect the clam R. philippinarum, especially those inhabiting contaminated ecosystems.

Realineamiento Proximal del Mecanismo Extensor en la Luxación Congénita de la Rótula en Niños y Adolescentes / Proximal Realignment of the Extensor Mechanism in Congenital Dislocation of the Knee in Children And Adolescents

Introducción La luxación congénita de la rótula (LCR) es una entidad poco frecuente. El diagnóstico precoz suele ser difícil porque la rótula en el recién nacido es pequeña y de difícil palpación. El tratamiento no quirúrgico ha demostrado ser ineficaz. El enfoque terapéutico de esta patología requiere de la liberación externa y extensa del cuádriceps, el avance del vasto interno oblicuo y de la transferencia o hemitransferencia del aparato extensor. Materiales y métodos se busca evaluar la funcionalidad de los pacientes tratados quirúrgicamente mediante realineamiento proximal del mecanismo extensor por la técnica descrita por Green y modificada por los autores. Desde el año 2000 hasta el año 2015 se recolectaron 15 pacientes (20 rodillas) con diagnóstico de LCR tratados quirúrgicamente mediante la técnica descrita. Resultados Los resultados postoperatorios fueron evaluados mediante el cuestionario Kujala: 93.8/100 en promedio. Discusión El tratamiento de la LCR es un desafío y más aún si se encuentra asociada a retracción idiopática del recto anterior. Los resultados clínicos y funcionales obtenidos sugieren que la recolocación anatómica y estable de la rótula dentro de la tróclea femoral permite una satisfactoria alineación extensora del cuádriceps, desapareciendo su acción flexora inicial, lo que permite la elongación progresiva del mecanismo extensor durante las fases postoperatorias de rehabilitación. La técnica quirúrgica descrita ofrece una buena alternativa para el tratamiento de esta infrecuente patología congénita de la rodilla.

Background Congenital dislocation of the patella (CDP) is a rare condition. The early diagnosis is difficult, as the patella in the newborns is small and also difficult to recognise. The therapeutic approach of this disorder requires the extended and external release of the quadriceps, the forward movement of the internal oblique vastus, and the transfer or hemi-transfer of the extensor compartment. Methods To review the functionality of those patients who have received surgical treatment with proximal realignment of the extensor mechanism using Green's surgical technique and the one modified by the author. Between the years 2000 and 2015, a total of 15 patients with 20 knees found to have had congenital dislocation of the patella, and had received the abovementioned surgical treatment. Results The post-operative results were evaluated using the Kujala questionnaire, which gave a mean result of 93.8 / 100. Discussion The CDP treatment is a challenge, and even more so if it is associated with an idiopathic retraction of the anterior rectus. The clinical and functional results obtained suggest that an anatomic and stable realignment of the patella in the femoral trochlea allows a satisfactory extensor alignment of the quadriceps. This leads to the disappearance its initial flexor action, which allows the gradual elongation of the extensor mechanism during the postoperative phases of rehabilitation. The surgical technique described offers a good alternative for the treatment of this rare congenital disorder of the knee.

Luxación congénita de rodilla bilateral. Reporte de caso / Bilateral congenital dislocation of the knee (BDK). Case report

Introducción La luxación congénita de rodilla (LCR) es una patología muy poco habitual, pues se estima una incidencia de 0,017 por cada mil nacidos vivos o, aproximadamente, del 1% de la incidencia de la displasia de la cadera en desarrollo (DCD). Debido a su baja incidencia existe controversia respecto al tratamiento definitivo. Presentamos el caso de un paciente con un cuadro sindrómico, con luxación congénita bilateral de rodillas, tratadas satisfactoriamente mediante manejo quirúrgico. El propósito de la publicación es describir el caso clínico de un paciente con LCR bilateral asociado con un cuadro sindrómico no clasificado genéticamente, sin la existencia de otras luxaciones articulares. Materiales y métodos Se describe el caso clínico de un niño de 2 años y 6 meses, a quien se le realizó tratamiento quirúrgico de LCR bilateral mediante reducción abierta y asociada con cuadriceplastia selectiva (distal en una de las rodillas y proximal en la otra) en Colombia. Se intervino primero la rodilla derecha y luego la rodilla izquierda con un intervalo de tiempo de 1 mes entre una cirugía y la otra. El manejo postoperatorio consistió en inmovilización con yesos cerrados inguinopédicos con rodilla a 90° de flexión durante 2 meses, luego tubo de yeso con rodilla en flexión de 45° y posteriormente férulas anteriores de yeso con flexión de 60° en cada rodilla de uso intermitente. Se realizó valoración funcional de los resultados postoperatorios mediante arcos de movilidad de las rodillas (activa y pasiva), posición cuadrúpeda, posición de rodillas, gateo y extensión de rodillas contra gravedad ya que el paciente aún no camina. Resultados En el seguimiento, los padres del paciente reportaron estar «muy satisfechos¼ con los resultados obtenidos. Los arcos pasivos de movilidad de ambas rodillas fueron: flexión de 125° (promedio de las dos rodillas) y extensión de -5°. No se reportó inestabilidad ni recurrencia de la luxación. Los controles fueron realizados después de 5 meses del tratamiento quirúrgico para la rodilla derecha y después de 4 meses para la rodilla izquierda. Discusión Se indicó manejo quirúrgico de las deformidades por edad del paciente, falla en los tratamientos ortopédicos no quirúrgicos previos en otras instituciones y por tratarse de una asociación con cuadro sindrómico, mediante la realización de cuadriceplastia selectiva bilateral, en la cual se realizó solo un abordaje anterior, suprarrotuliano e infrarrotuliano medial, cuadriceplastia en Z proximal para la rodilla izquierda y cuadriceplastia distal para la rodilla derecha, lo cual disminuyo las comorbilidades por número de abordajes y permitió una recuperación más rápida. El tratamiento quirúrgico de la LCR mediante la técnica de cuadriceplastia selectiva en pacientes mayores, cuya patología se asocia con síndromes genéticos que empobrecen los resultados de manejo y dificultan los tratamientos no quirúrgicos, hasta la fecha ha ofrecido buenos resultados en términos de funcionalidad, satisfacción de los padres y ausencia de recurrencia de la luxación a los 5 meses de seguimiento en el caso descrito. Nivel de evidencia clínica Nivel IV.

Background Congenital dislocation of the knee (CDK) is a very rare condition, with an incidence of 0.017 per thousand live births and approximately 1 percent incidence in patients diagnosed with developing hip dysplasia (DHD). Due to its low incidence, there is controversy regarding definitive treatment. We present the case of a syndromic patient with bilateral congenital dislocation of the knees, satisfactorily treated by surgical management. The purpose of the publication is to describe the clinical case of a patient with bilateral CDK associated with non-genetically classified syndromic syndrome, without the presence of other joint dislocations. Materials and methods We describe the clinical case of a 2-year and 6-month-old child who underwent bilateral CDK surgical treatment through open reduction and associated selective quadriceplasty (Distal in one knee and Proximal in the other knee). The right knee was first intervened and then the left knee with a time interval of one month between one surgery and another. Postoperative management consisted of immobilization with closed inguinopedic casts with knee at 90° flexion for two months, then plaster tube with knee at 45° flexion and then anterior gypsum ferrules with 60° flexion at each knee of intermittent use. Functional assessment of the postoperative results was performed by knee mobility arches (active and passive), quadruped position, knee position, crawling and knee extension against gravity, because the patient still does not walk. Results At follow-up, the patient's parents reported being "very satisfied" with the obtained results. The passive arches of mobility of both knees were: flexion of 125° (on average for the two knees), extension of - 5°. No instability or recurrence of dislocation was reported. Controls were performed after 5 months of surgical treatment for the right knee and 4 months after for the left knee. Discussion Due to age, failure in previous non-surgical orthopedic treatments in other institutions, and a syndromic association, surgical management of the patient's deformities were indicated, performing bilateral selective quadriceplasty. A single restricted anterior medial suprapatellar and infrapatellar approach was performed accordingly to each knee, proximal "Z" quadriceplasty for the left knee and distal quadriceplasty for the right knee were also performed, a decreased in co-morbidities was observed by avoiding several approaches as well as a faster postsurgical recovery for the patient. After 5 months follow-up, this approach has provided good results in terms of functionality, satisfaction of the parents and absence of recurrence of dislocation. Evidence level IV.