Idiopathic normal pressure hydrocephalus diagnosis: Quantitative and qualitative score predicting outcome of extended lumbar drainage.

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Minimally Invasive Surgery of Deep-Seated Brain Lesions Using Tubular Retractors and Navigated Transcranial Magnetic Stimulation-Based Diffusion Tensor Imaging Tractography Guidance: The Minefield Paradigm.

BACKGROUND: Surgical treatment of deep-seated brain lesions is a major challenge for neurosurgeons. Recently, tubular retractors have been used to help neurosurgeons in achieving the targeting and resection of deep lesions. OBJECTIVE: To describe a novel surgical approach based on the combination of tubular retractors and preoperative mapping by navigated transcranial magnetic stimulation (nTMS) and nTMS-based diffusion tensor imaging (DTI) tractography for the safe resection of deep-seated lesions. METHODS: Ten consecutive patients affected by deep-seated brain lesions close to eloquent motor/language/visual pathways underwent preoperative nTMS mapping of motor/language cortical areas and nTMS-based DTI tractography of adjacent eloquent white matter tracts, including optic radiations. The nTMS-based information was used to plan the optimal surgical trajectory and to guide the insertion of tubular retractors within the brain parenchyma without causing injury to the eloquent cortical and subcortical structures. After surgery, all patients underwent a new nTMS-based DTI tractography of fascicles close to the tumor to verify their structural integrity. RESULTS: Gross total resection was achieved in 8 cases, subtotal resection in 1 case, and a biopsy in 1 case. No new postoperative deficits were observed, except in 1 case where a visual field defect due to injury to the optic radiations occurred. Postoperative nTMS-based DTI tractography showed the integrity of the subcortical fascicles crossed by tubular retractors trajectory in 9 cases. CONCLUSION: The novel strategy combining tubular retractors with functional nTMS-based preoperative mapping enables a safe microsurgical resection of deep-seated lesions through the preservation of eloquent cortical areas and subcortical fascicles, thus reducing the risk of new permanent deficits.

Cortical Excitability and Connectivity in Patients With Brain Tumors.

Background: Brain tumors can cause different changes in excitation and inhibition at the neuronal network level. These changes can be generated from mechanical and cellular alterations, often manifesting clinically as seizures. Objective/Hypothesis: The effects of brain tumors on cortical excitability (CE) have not yet been well-evaluated. The aim of the current study was to further investigate cortical-cortical and cortical-spinal excitability in patients with brain tumors using a more extensive transcranial magnetic stimulation protocol. Methods: We evaluated CE on 12 consecutive patients with lesions within or close to the precentral gyrus, as well as in the subcortical white matter motor pathways. We assessed resting and active motor threshold, short-latency intracortical inhibition (SICI), intracortical facilitation (ICF), short-latency afferent inhibition (SAI), long-latency afferent inhibition, cortical silent period, and interhemispheric inhibition. Results: CE was reduced in patients with brain tumors than in healthy controls. In addition, SICI, ICF, and SAI were lower in the affected hemisphere compared to the unaffected and healthy controls. Conclusions: CE is abnormal in hemispheres affected by brain tumors. Further studies are needed to determine if CE is related with motor impairment.

Mapping and Preserving the Visuospatial Network by repetitive nTMS and DTI Tractography in Patients With Right Parietal Lobe Tumors.

INTRODUCTION: The goal of brain tumor surgery is the maximal resection of neoplastic tissue, while preserving the adjacent functional brain tissues. The identification of functional networks involved in complex brain functions, including visuospatial abilities (VSAs), is usually difficult. We report our preliminary experience using a preoperative planning based on the combination of navigated transcranial magnetic stimulation (nTMS) and DTI tractography to provide the preoperative 3D reconstruction of the visuospatial (VS) cortico-subcortical network in patients with right parietal lobe tumors. MATERIAL AND METHODS: Patients affected by right parietal lobe tumors underwent mapping of both hemispheres using an nTMS-implemented version of the Hooper Visual Organization Test (HVOT) to identify cortical areas involved in the VS network. DTI tractography was used to compute the subcortical component of the network, consisting of the three branches of the superior longitudinal fasciculus (SLF). The 3D reconstruction of the VS network was used to plan and guide the safest surgical approach to resect the tumor and avoid damage to the network. We retrospectively analyzed the cortical distribution of nTMS-induced errors, and assessed the impact of the planning on surgery by analyzing the extent of tumor resection (EOR) and the occurrence of postoperative VSAs deficits in comparison with a matched historical control group of patients operated without using the nTMS-based preoperative reconstruction of the VS network. RESULTS: Twenty patients were enrolled in the study (Group A). The error rate (ER) induced by nTMS was higher in the right vs. the left hemisphere (p=0.02). In the right hemisphere, the ER was higher in the anterior supramarginal gyrus (aSMG) (1.7%), angular gyrus (1.4%) superior parietal lobule (SPL) (1.3%), and dorsal lateral occipital gyrus (dLoG) (1.2%). The reconstruction of the cortico-subcortical VS network was successfully used to plan and guide tumor resection. A gross total resection (GTR) was achieved in 85% of cases. After surgery no new VSAs deficits were observed and a slightly significant improvement of the HVOT score (p=0.02) was documented. The historical control group (Group B) included 20 patients matched for main clinical characteristics with patients in Group A, operated without the support of the nTMS-based planning. A GTR was achieved in 90% of cases, but the postoperative HVOT score resulted to be worsened as compared to the preoperative period (p=0.03). The comparison between groups showed a significantly improved postoperative HVOT score in Group A vs. Group B (p=0.03). CONCLUSIONS: The nTMS-implemented HVOT is a feasible approach to map cortical areas involved in VSAs. It can be combined with DTI tractography, thus providing a reconstruction of the VS network that could guide neurosurgeons to preserve the VS network during tumor resection, thus reducing the occurrence of postoperative VSAs deficits as compared to standard asleep surgery.

Effects of low-dose X-ray medical diagnostics on female gonads: Insights from large animal oocytes and human ovaries as complementary models.

Diagnostic imaging has significantly grown over the last thirty years as indispensable support for diagnostic, prognostic, therapeutic and monitoring procedures of human diseases. This study explored the effects of low-dose X-ray medical diagnostics exposure on female fertility. To aim this, cumulus-oocyte complexes (COCs) recovered from the ovaries of juvenile sheep and human ovaries were used as complementary models for in vitro studies. In the sheep model, the effects of low-dose X-rays on oocyte viability and developmental competence were evaluated. In human ovaries originated from two age group (21-25 and 33-36 years old) subjects with gender dysphoria, X-rays effects on tissue morphology, follicular density and expression of apoptosis-related (NOXA, PUMA, Bcl2, Bak, γH2AX) and cell cycle-related genes (p21 and ki67) were investigated. It was noted that in sheep, the minimum dose of 10 mGy did not influence most of examined parameters at oocyte and embryo levels, whereas 50 and 100 mGy X-ray exposure reduced oocyte bioenergetic/oxidative activity but without any visible effects on oocyte and embryo development. In addition, blastocyst bioenergetic/oxidative status was reduced with all used doses. Overall data on human ovaries showed that low-dose X-rays, similarly as in sheep, did not alter any of examined parameters. However, in women belonging to the 33-36 year group, significantly reduced follicular density was observed after exposure to 50 and 100 mGy, and increased NOXA and Bax expression after exposure at 50 mGy. In conclusion, used low-doses of X-ray exposure, which resemble doses used in medical diagnostics, produce weak damaging effects on female fertility with increased susceptibility in advanced age.

Ochratoxin A affects oocyte maturation and subsequent embryo developmental dynamics in the juvenile sheep model.

The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 µmol/L-0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels.

Meagre Argyrosomus regius (Asso, 1801) Stem Spermatogonia: Histological Characterization, Immunostaining, In Vitro Proliferation, and Cryopreservation.

The meagre, Argyrosomus regius, is a valued fish species of which aquaculture production might be supported by the development of a stem germ cell xenotransplantation technology. Meagre males were sampled at a fish farm in the Ionian Sea (Italy) at the beginning and end of the reproductive season. Small and large Type A undifferentiated spermatogonia were histologically identified in the germinal epithelium. Among the tested stemness markers, anti-oct4 and anti-vasa antibodies labeled cells likely corresponding to the small single Type A spermatogonia; no labeling was obtained with anti-GFRA1 and anti-Nanos2 antibodies. Two types of single A spermatogonia were purified via density gradient centrifugation of enzymatically digested testes. Testes from fish in active spermatogenesis resulted in a more efficient spermatogonial stem cell (SSC) yield. After cell seeding, meagre SSCs showed active proliferation from Day 7 to Day 21 and were cultured up to Day 41. After cryopreservation in dimethyl-sulfoxide-based medium, cell viability was 28.5%. In conclusion, these results indicated that meagre SSCs could be isolated, characterized, cultured in vitro, successfully cryopreserved, and used after thawing. This is a first step towards the development of a xenotransplantation technology that might facilitate the reproduction of this valuable species in captivity.

Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm.

Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300 ×g for 5', 300 ×g for 10', 1500 ×g for 5' and 1500 ×g for 10' vs no centrifugation) on motility and oxidative status in cryopreserved stallion sperm, were analyzed. After centrifugation, almost all motility parameters were significantly altered, as observed by computer-assisted sperm analysis. A polarographic assay of oxygen consumption showed a progressive decrease in mitochondria respiration from the gentlest to the strongest protocol. By laser scanning confocal microscopy, significant reduction of mitochondrial membrane potential, at any tested protocol, and time-dependent effects, at the same centrifugal force, were found. Increased DNA fragmentation index at any tested protocol and time-dependent effects at the same centrifugal force were found, whereas increased protein carbonylation was observed only at the strongest centrifugal force. These results provide more comprehensive understandings on centrifugation-induced effects on cryopreserved stallion sperm and suggest that, even at a weak force for a short time, centrifugation impairs different aspects of equine sperm metabolism and functionality.

Sperm selection in assisted reproduction: A review of established methods and cutting-edge possibilities.

Male infertility often involves idiopathic or unknown causes, leading to an increasing demand for assisted reproduction technologies (ART). Conventional sperm sorting techniques rely on centrifugation steps that are known to cause oxidative stress and consequently damage cells. Alternative novel techniques have been introduced but offer disadvantages that need to be overcome. These techniques are also employed to increase the number and the quality of subjects in the animal breeding industry, to obtain purebred subjects or to preserve endangered animal species. Microfluidics deals with the manipulation of small amounts of volume within a microdevice known as lab-on-a-chip (LOC), which offers rapid analyses, ease of use, small reagent sample volumes, high-throughput processing and wide reproducibility owing to automation and standardization. As the LOC allows gamete handling within a microenvironment that strictly mimics physiological in vivo conditions and avoids centrifugation steps and long processing time, the use of microfluidics for sperm sorting and selection have been proposed during the last 15 years and is currently under investigation. Moreover, LOC technologies to sort, identify and analyse other kinds of cells could be transferred to sperm selection and analysis, thus opening the way to a novel approach to the sperm cell selection and manipulation. This review describes the techniques routinely performed in human and animal clinical practice for sorting good-quality sperm for in vitro fertilization procedures, and focuses on the positive and negative aspects of each method. Emerging microfluidic devices, recently proposed for sperm selection, are also described and, when possible, compared with standard methods.

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep.

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus-oocyte-complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase-mediated dUTP nick-End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short-term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long-term carry-over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late-stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3-0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.

Modern Treatment of Brain Arteriovenous Malformations Using Preoperative Planning Based on Navigated Transcranial Magnetic Stimulation: A Revisitation of the Concept of Eloquence.

BACKGROUND: Navigated transcranial magnetic stimulation (nTMS) provides a reliable identification of "eloquent" cortical brain areas. Moreover, it can be used for diffusion tensor imaging fiber tracking of eloquent subcortical tracts. We describe the use of nTMS-based cortical mapping and diffusion tensor imaging fiber tracking for defining the "eloquence" of areas surrounding brain arteriovenous malformations (BAVMs), aiming to improve patient stratification and treatment. METHODS: We collected data of BAVMs suspected to be in eloquent areas treated between 2017 and 2019, and submitted to nTMS-based reconstruction of motor, language, and visual pathways for the definition of the eloquence of the surrounding brain areas. We describe the nTMS-based approach and analyze its impact on patient stratification and allocation to treatment in comparison with the standard assessment of eloquence based on anatomical landmarks. RESULTS: Ten patients were included in the study. Preliminarily, 9 BAVMs were suspected to be located in an eloquent area. After nTMS-based mapping, only 5 BAVMs were confirmed to be close to eloquent structures, thus leading to a change of the score for eloquence and of the final BAVMs grading in 60% of patients. Treatment was customized according to nTMS information, and no cases of neurological worsening were observed. Radiological obliteration was complete in 7 cases microsurgically treated, and accounted for about 70% in the remaining 3 patients 1 year after radiosurgical treatment. CONCLUSIONS: The nTMS-based information allows an accurate stratification and allocation of patients with BAVMs to the most effective treatment according to a modern, customized, neurophysiological identification of the adjacent eloquent brain networks.

Multimodal Surgical Treatment of High-Grade Gliomas in the Motor Area: The Impact of the Combination of Navigated Transcranial Magnetic Stimulation and Fluorescein-Guided Resection.

BACKGROUND: Fluorescein-guided surgery of high-grade gliomas (HGGs) increases the extent of tumor resection but its efficacy has been questioned, especially for tumors located close to functional networks. In these cases, navigated transcranial magnetic stimulation (nTMS) may be used to plan and guide a safe resection. The aim of this study was to assess the impact of these techniques combined with intraoperative neurophysiologic mapping (IONM) to achieve the maximal safe resection of tumors located in the motor area. METHODS: We collected data of patients operated using a multimodal combination of sodium fluorescein-guided resection, nTMS motor planning, and IONM for HGGs in the motor area. The nTMS planning accuracy, extent of resection, and postoperative motor and functional status were compared with a matched control group of patients with HGG operated on only by IONM-guided resection. RESULTS: Forty-one patients treated by multimodal approach (group A) and 41 controls (group B) were included. The nTMS-based planning reliably identified the tumor/motor pathway spatial relationship (accuracy, 92.68%). We obtained in group A versus controls a higher gross total resection rate (73.17% vs. 51.22%; P = 0.04), and a reduction of cases with new permanent motor deficits (9.75% vs. 29.27%; P = 0.04) or Karnofsky Performance Status worsening (12.19% vs. 31.71%; P = 0.03). CONCLUSIONS: This study supports the role of the combination of sodium fluorescein-guided resection and nTMS-based planning for surgery of HGGs close to the motor pathway. This multimodal approach in combination with IONM may lead to customized preoperative planning, increased extent of resection, and improved functional outcome, compared with standard IONM-guided surgery.

Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP-induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP-exposed oocytes. VB addition during IVM limited DEHP-induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.

Supplementation with nanomolar concentrations of verbascoside during in vitro maturation improves embryo development by protecting the oocyte against oxidative stress: a large animal model study.

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress.