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In vitro-based new approach methodologies (NAMs) provide a pragmatic solution to animal testing of petroleum substances and their constituents. A previous study exposed an in vitro model (A549 cells) at the air–liquid interface (ALI) to assess inhalation toxicity of a single compound, ethylbenzene. Experimental conditions using VITROCELLⓇ 24/48 exposure system were optimized to achieve a deposition efficiency that resulted in dose-dependent biological changes. The feasibility of this set-up was evaluated for testing the complex substance gasoline, which, at only high concentrations, can induce mild respiratory irritation in animals and cough in humans. • Results showed that perpendicular ALI exposure flow systems (VITROCELL® 6/4 and 24/48) may not be appropriate for testing gasoline because it was not possible to achieve enough deposition onto the cells and in the culture medium to measure dose and to determine dose-dependent biological changes (more information can be found in ‘Supplementary material and/or Additional information’ section). • Structural features (e.g. aromatic or saturated hydrocarbon structure) and high hydrophobicity, together with the low concentrations of individual components in gasoline, may have caused the low deposition. • To achieve a higher deposition on the cells, A549 cells were exposed to gasoline at the ALI by passive dosing.The results demonstrate that the presented methodology is a promising NAM for inhalation toxicity testing of (semi-)volatile complex substances with low aqueous solubility.

Standard information reporting helps to ensure that assay conditions and data are consistently reported and to facilitate inter-laboratory comparisons. Here, we present recommendations on minimum information for reporting on the TEER (trans-epithelial/endothelial electrical resistance) assay (MIRTA). The TEER assay is extensively used to evaluate the health of an epithelial/endothelial cell culture model and as an indicator of the potential toxicity of a test substance. This publication is the result of an international collaboration─called the RespTox (Respiratory Toxicity) Collaborative─through which twelve laboratories shared their protocols for assessing the barrier function of respiratory epithelial cells using the TEER assay following exposure to substances. The protocols from each laboratory were reviewed to identify general steps for performing the TEER assay, interlaboratory differences between steps, the rationale for differences, whether these differences impact results or cross-laboratory comparisons between TEER measurements. While the MIRTA recommendations are focused on respiratory epithelial cell systems, these recommendations can be adapted for other cell systems that form barriers. The use of these recommendations will support data transparency and reproducibility, reduce challenges in data interpretation, enable cross-laboratory comparisons, help assess study quality, and facilitate the incorporation of the TEER assay into national and international testing guidance., (© 2024. The Author(s).)

Aging affects the scalp-to-cortex distance (SCD) and the comprising tissues. This is crucial for noninvasive neuroimaging and brain stimulation modalities as they rely on traversing from the scalp to the cortex or vice versa. The specific relationship between aging and these tissues has not been comprehensively investigated. We conducted a study on 250 younger and older adults to examine age-related differences in SCD and its constituent tissues. We identified region-specific differences in tissue thicknesses related to age and sex. Older adults exhibit larger SCD in the frontocentral regions compared to younger adults. Men exhibit greater SCD in the inferior scalp regions, while women show similar-to-greater SCD values in regions closer to the vertex compared to men. Younger adults and men have thicker soft tissue layers, whereas women and older adults exhibit thicker compact bone layers. CSF is considerably thicker in older adults, particularly in men. These findings emphasize the need to consider age, sex, and regional differences when interpreting SCD and its implications for noninvasive neuroimaging and brain stimulation., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze-thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log 10 TCID 50 /ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze-thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities., (© 2024. The Author(s).)

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

In indoor air the reaction of ozone (O 3 ) with terpenes may lead to the formation of irritating gas-phase products which may induce acute airway effects (i.e. sudden, short-term changes or symptoms related to the respiratory system). We aimed to perform an in vitro study on possible health effects of products from the O 3 -initiated reaction of limonene with printer exhaust, representing real-life mixtures in offices. Human bronchial epithelial cells were exposed for 1 hour (h) to limonene and O 3 , combined with printer exhaust. The resulting concentrations represented 34% and 6% of the generated initial concentrations of limonene (400 µg/m³) and O 3 (417 µg/cm³), respectively, which were in range of high end realistic indoor concentrations. We observed that the reaction of limonene with O 3 generated an increase of ultrafine particles within 1 h, with a significant increase of secondary reaction products 4-oxopentanal and 3-isopropenyl-6-oxo-heptanal at high end indoor air levels. Simultaneous printing activity caused the additional release of micron-sized particles and a further increase in reaction products. Relevant cellular endpoints to evaluate the possible induction of acute airway effects were measured. However, none of the test atmospheres representing office air was observed to induce these effects.

Background: Electric field (E-field) modeling is a potent tool to estimate the amount of transcranial magnetic and electrical stimulation (TMS and tES, respectively) that reaches the cortex and to address the variable behavioral effects observed in the field. However, outcome measures used to quantify E-fields vary considerably and a thorough comparison is missing., Objectives: This two-part study aimed to examine the different outcome measures used to report on tES and TMS induced E-fields, including volume- and surface-level gray matter, region of interest (ROI), whole brain, geometrical, structural, and percentile-based approaches. The study aimed to guide future research in informed selection of appropriate outcome measures., Methods: Three electronic databases were searched for tES and/or TMS studies quantifying E-fields. The identified outcome measures were compared across volume- and surface-level E-field data in ten tES and TMS modalities targeting two common targets in 100 healthy individuals., Results: In the systematic review, we extracted 308 outcome measures from 202 studies that adopted either a gray matter volume-level (n = 197) or surface-level (n = 111) approach. Volume-level results focused on E-field magnitude, while surface-level data encompassed E-field magnitude (n = 64) and normal/tangential E-field components (n = 47). E-fields were extracted in ROIs, such as brain structures and shapes (spheres, hexahedra and cylinders), or the whole brain. Percentiles or mean values were mostly used to quantify E-fields. Our modeling study, which involved 1,000 E-field models and > 1,000,000 extracted E-field values, revealed that different outcome measures yielded distinct E-field values, analyzed different brain regions, and did not always exhibit strong correlations in the same within-subject E-field model., Conclusions: Outcome measure selection significantly impacts the locations and intensities of extracted E-field data in both tES and TMS E-field models. The suitability of different outcome measures depends on the target region, TMS/tES modality, individual anatomy, the analyzed E-field component and the research question. To enhance the quality, rigor, and reproducibility in the E-field modeling domain, we suggest standard reporting practices across studies and provide four recommendations., Competing Interests: Declaration of Competing Interest none., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inhalation is a major route by which human exposure to substances can occur. Resources have therefore been dedicated to optimize human-relevant in vitro approaches that can accurately and efficiently predict the toxicity of inhaled chemicals for robust risk assessment and management. In this study-the IN vitro Systems to PredIct REspiratory toxicity Initiative-2 cell-based systems were used to predict the ability of chemicals to cause portal-of-entry effects on the human respiratory tract. A human bronchial epithelial cell line (BEAS-2B) and a reconstructed human tissue model (MucilAir, Epithelix) were exposed to triethoxysilane (TES) and trimethoxysilane (TMS) as vapor (mixed with N2 gas) at the air-liquid interface. Cell viability, cytotoxicity, and secretion of inflammatory markers were assessed in both cell systems and, for MucilAir tissues, morphology, barrier integrity, cilia beating frequency, and recovery after 7 days were also examined. The results show that both cell systems provide valuable information; the BEAS-2B cells were more sensitive in terms of cell viability and inflammatory markers, whereas MucilAir tissues allowed for the assessment of additional cellular effects and time points. As a proof of concept, the data were also used to calculate human equivalent concentrations. As expected, based on chemical properties and existing data, the silanes demonstrated toxicity in both systems with TMS being generally more toxic than TES. Overall, the results demonstrate that these in vitro test systems can provide valuable information relevant to predicting the likelihood of toxicity following inhalation exposure to chemicals in humans., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology.)

Motor control, a ubiquitous part of driving, requires increased cognitive controlled processing in older adults relative to younger adults. However, the influence of aging on motor-related neural mechanisms in the context of driving has rarely been studied. The present study aimed to identify age-related changes in cognitive control and attention allocation during a simulated steering task, using electroencephalography. Midfrontal theta, a marker for cognitive control, and posterior alpha power, a marker for attention allocation, were measured in a total of 26 young, 25 middle-aged, and 28 older adults. By adapting driving speed, the difficulty level of this steering task was individualized for each participant. Results show age-related changes in midfrontal theta power, but not in posterior alpha power, despite similar steering accuracy across age groups. Specifically, only younger and, to a lesser extent, middle-aged adults exhibited increased theta power while driving through more demanding curved segments relative to straight segments. In contrast, theta power upregulation was absent in older adults, suggesting a saturation of cognitive resources while driving, possibly due to a limitation in resource capacity, or less automatic motor-related neural processing., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Interlimb coordination is required for adequate execution of most daily life activities. Yet, aging negatively affects interlimb coordination, impacting the quality of life in older people. Therefore, disentangling the underlying age-related neural mechanisms is of utmost importance. Here, we investigated neurophysiological processes of an interlimb reaction time task, including both simple and complex coordination modes. Midfrontal theta power, measured using electroencephalography (EEG), was analyzed as a marker for cognitive control. In total, 82 healthy adults participated, with 27 younger, 26 middle-aged, and 29 older adults. On a behavioral level, reaction time increased across the adult lifespan, and error rate was higher in older adults. Notably, aging disproportionately affected reaction times in the complex coordination modes, with larger reaction time increases from simple to complex movements than in younger adults, starting already at middle age. On the neurophysiological level, EEG showed that only younger adults had significantly increased levels of midfrontal theta power during complex relative to simple coordination modes, while no significant differences were found between simple and complex movements in middle-aged and older adults. The absence of this theta power upregulation with regard to movement complexity with increasing age might reflect a premature saturation of the available mental resources.

Bimanual coordination is an important part of everyday life and recruits a large neural network, including the cerebellum. The specific role of the cerebellum in bimanual coordination has not yet been studied in depth, although several studies indicate a differential role of the anterior and posterior cerebellum depending on the complexity of the coordination. An activation likelihood estimation (ALE) meta-analysis was used combining the data of several functional MRI studies involving bimanual coordination tasks with varying complexities to unravel the involvement of the different areas of the cerebellum in simple and complex bimanual coordination. This study confirms the general bimanual network as found by Puttemans et al. (Puttemans et al. in J Neurosci 25:4270-4278, 2005) and highlights the differences between preferred in-phase (simultaneous movements of homologous muscle groups) and anti-phase movement conditions (alternating movements of homologous muscle groups), and more complex, non-preferred bimanual movements (e.g., out-of-phase movements). Our results show a differential role for the anterior and posterior vermis in bimanual coordination, with a role for the anterior vermis in anti-phase and complex bimanual coordination, and an exclusive role for the posterior vermis in complex bimanual movements. In addition, the way complexity was manipulated also seems to play a role in the involvement of the anterior and posterior vermis. We hypothesize that the anterior vermis is involved in sequential/spatial control, while the posterior vermis is involved in temporal control of (bimanual) coordination, though other factors such as (visual) feedback and continuity of the movement also seem to have an impact. More studies are needed to unravel the specific role of the cerebellar vermis in bimanual coordination., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

With aging comes degradation of bimanual movement performance. A hallmark feature of bimanual movements is movement-related beta desynchronization (MRBD), an attenuation in the amplitude of beta oscillations associated with sensorimotor activation. Here, we investigated MRBD in 39 healthy adults (20 younger and 19 older adults) in frontal, central, and parietal regions across both hemispheres, during the planning and execution of a bimanual tracking task. Task accuracy decreased with age and during more difficult conditions when both hands had to move at different relative speeds. MRBD was mostly situated in the central region, and increased in older versus younger adults during movement execution but not planning. Irrespective of age, motor planning and execution were associated with increased MRBD in the left and right hemispheres, respectively. Notably, right central MRBD during motor planning was associated with bimanual task performance, particularly in older adults. Specifically, persons who demonstrated high MRBD during motor planning performed better on the bimanual tracking task. Our results highlight the importance of lateralized MRBD during motor planning, thereby shining new light on previous research and providing a promising avenue for future interventions., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Dual-site transcranial magnetic stimulation (ds-TMS) is well suited to investigate the causal effect of distant brain regions on the primary motor cortex, both at rest and during motor performance and learning. However, given the broad set of stimulation parameters, clarity about which parameters are most effective for identifying particular interactions is lacking. Here, evidence describing inter- and intra-hemispheric interactions during rest and in the context of motor tasks is reviewed. Our aims are threefold: (1) provide a detailed overview of ds-TMS literature regarding inter- and intra-hemispheric connectivity; (2) describe the applicability and contributions of these interactions to motor control, and; (3) discuss the practical implications and future directions. Of the 3659 studies screened, 109 were included and discussed. Overall, there is remarkable variability in the experimental context for assessing ds-TMS interactions, as well as in the use and reporting of stimulation parameters, hindering a quantitative comparison of results across studies. Further studies examining ds-TMS interactions in a systematic manner, and in which all critical parameters are carefully reported, are needed., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

Interlimb coordination deteriorates as a result of aging. Due to its ubiquity in daily life, a greater understanding of the underlying neurophysiological changes is required. Here, we combined electroencephalography time-frequency spectral power and functional connectivity analyses to provide a comprehensive overview of the neural dynamics underlying the age-related deterioration of interlimb coordination involving all four limbs. Theta, alpha and beta oscillations in the frontal, central and parietal regions were analyzed in twenty younger (18-30 years) and nineteen older adults (65-78 years) during a complex interlimb reaction time task. Reaction time was significantly higher in older adults across all conditions, and the discrepancy between both age groups was largest in the most complex movement condition. Older adults demonstrated enhanced beta event-related desynchronization (i.e., the attenuation of beta power), which further increased along with task complexity and was positively linked to behavioral performance. Theta functional connectivity between frontal, central and parietal regions generally increased with movement complexity, irrespective of age group. In general, frontoparietal alpha band functional connectivity tended to be reduced in older versus younger adults, although these contrasts did not survive multiple comparison corrections. Overall, spectral results suggest that enhanced beta desynchronization in older adults reflects a successful compensatory mechanism to cope with increased difficulty during complex interlimb coordination. Functional connectivity results suggest that theta and alpha band connectivity are prone to respectively task- and age-related modulations. Future work could target these spectral and functional connectivity dynamics through noninvasive brain stimulation to potentially improve interlimb coordination in older adults., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

The use of animals in neuroscience and biomedical research remains controversial. Policy is built around the "3R" principle of "Refining, Reducing and Replacing" animal experiments, and across the globe, different initiatives stimulate the use of animal-free methods. Based on an extensive literature screen to map the development and adoption of animal-free methods in Alzheimer's and Parkinson's disease research, we find that at least two in three examined studies rely on animals or on animal-derived models. Among the animal-free studies, the relative contribution of innovative models that may replace animal experiments is limited. We argue that the distinction between animal research and alternative models presents a false dichotomy, as the role and scientific value of both animal and animal-free approaches are intertwined. Calls to halt all animal experiments appear premature, as insufficient non-animal-based alternatives are available and their development lags behind. In light of this, we highlight the need for objective, unprejudiced monitoring, and more robust performance indicators of animal-free approaches., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

It has been argued that age-related changes in the neurochemical and neurophysiological properties of the GABAergic system may underlie increases in reaction time (RT) in older adults. However, the role of GABA levels within the sensorimotor cortices (SMC) in mediating interhemispheric interactions (IHi) during the processing stage of a fast motor response, as well as how both properties explain interindividual differences in RT, are not yet fully understood. In this study, edited magnetic resonance spectroscopy (MRS) was combined with dual-site transcranial magnetic stimulation (dsTMS) for probing GABA+ levels in bilateral SMC and task-related neurophysiological modulations in corticospinal excitability (CSE), and primary motor cortex (M1)-M1 and dorsal premotor cortex (PMd)-M1 IHi, respectively. Both CSE and IHi were assessed during the preparatory and premotor period of a delayed choice RT task. Data were collected from 25 young (aged 18-33 years) and 28 older (aged 60-74 years) healthy adults. Our results demonstrated that older as compared to younger adults exhibited a reduced bilateral CSE suppression, as well as a reduced magnitude of long latency M1-M1 and PMd-M1 disinhibition during the preparatory period, irrespective of the direction of the IHi. Importantly, in older adults, the GABA+ levels in bilateral SMC partially accounted for task-related neurophysiological modulations as well as individual differences in RT. In contrast, in young adults, neither task-related neurophysiological modulations, nor individual differences in RT were associated with SMC GABA+ levels. In conclusion, this study contributes to a comprehensive initial understanding of how age-related differences in neurochemical properties and neurophysiological processes are related to increases in RT., (Copyright © 2021. Published by Elsevier Inc.)

Objective: To systematically review how patient characteristics and/or transcranial direct current stimulation (tDCS) parameters influence tDCS effectiveness in respect to upper limb function post-stroke., Methods: Three electronic databases were searched for sham-controlled randomised trials using the Fugl-Meyer Assessment for upper extremity as outcome measure. A meta-analysis and nine subgroup-analyses were performed to identify which tDCS parameters yielded the greatest impact on upper limb function recovery in stroke patients., Results: Eighteen high-quality studies (507 patients) were included. tDCS applied in a chronic stage yields greater results than tDCS applied in a (sub)acute stage. Additionally, patients with low baseline upper limb impairments seem to benefit more from tDCS than those with high baseline impairments. Regarding tDCS configuration, all stimulation types led to a significant improvement, but only tDCS applied during therapy, and not before therapy, yielded significant results. A positive dose-response relationship was identified for current/charge density and stimulation duration, but not for number of sessions., Conclusion: Our results demonstrate that tDCS improves upper limb function post-stroke. However, its effectiveness depends on numerous factors. Especially chronic stroke patients improved, which is promising as they are typically least amenable to recovery., Significance: The current work highlights the importance of several patient-related and protocol-related factors regarding tDCS effectiveness., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

Few studies have identified the intrahemispheric functional connectivity between the ipsilateral dorsal premotor cortex (PMd) and the primary motor hand area (M1 hand ) due to technical limitations. In this proof-of-concept study, a novel neuronavigated dsTMS set-up was employed, combining stimulation over left PMd and left M1 hand using the edge of a butterfly coil and a small cooled-coil. This arrangement was warranted because coil (over)heating and inter coil distance are limiting factors when investigating connectivity between stimulation targets in close proximity and over a longer duration. The proposed set-up was designed to deal with these limitations. Specifically, the effect of four dual-site transcranial magnetic stimulation (dsTMS) protocols on twenty-eight right-handed participants (12 males) was evaluated. These protocols differed in stimulus order, interstimulus interval and current direction induced in PMd. A structural scan with electric (E-)field modeling was obtained from seven participants prior to dsTMS, demonstrating that PMd and M1 hand were effectively stimulated. Results indicate that one protocol, in which a latero-medial current was induced in PMd 2.8 ms prior to stimulation over M1 hand , induced a sex-mediated effect. In males, significant inhibition of motor-evoked potentials was identified, whereas females demonstrated a facilitatory effect that did not survive correction for multiple comparisons. E-field simulations revealed that the E-field induced by the coil targeting PMd was maximal in PMd, with weaker E-field strengths extending to regions beyond PMd. Summarizing, the current dsTMS set-up enabled stimulating at an inter-target distance of 35 mm without any indications of coil-overheating., (Copyright © 2021 IBRO. Published by Elsevier Ltd. All rights reserved.)