Male MO offspring had impairments in attention and accuracy in the five-choice serial reaction time task, while female MO offspring were less affected. Targeted gene expression analysis at P21 in the PFC identified alterations in microglial-related and TLR4-related genes, while immunohistochemical analysis in adult brains indicated decreased microglial Iba1 and phagocytic CD68 proteins in the PFC and AMG in males, but females had an increase. Further, both male and female MO offspring had increased social preference. Overall, these data demonstrate male vulnerability to executive function deficits in response to perinatal opioid exposure and evidence for disruptions in neuron–microglial signaling.Children exposed prenatally to opioids are at an increased risk for behavioral problems and executive function deficits. The prefrontal cortex (PFC) and amygdala (AMG) regulate executive function and social behavior and are sensitive to opioids prenatally. Opioids can bind to toll-like receptor 4 (TLR4) to activate microglia, which may be developmentally important for synaptic pruning. Therefore, we tested the effects of perinatal morphine exposure on executive function and social behavior in male and female mouse offspring, along with microglial-related and synaptic-related outcomes. Dams were injected once daily subcutaneously with saline ( This study demonstrates (1) construct validity for a mouse model of perinatal morphine exposure that results in executive function deficits and (2) evidence in support of alterations in neuron–microglial signaling that may underlie these behavioral deficits. The importance of this work is emphasized by the continually worsening opioid epidemic and the challenges of studying long-term behavioral health of exposed children. Our study found that male morphine-exposed offspring had delayed learning and reduced motivation in basic cognitive tasks, followed by executive function deficits in attention and accuracy. Females instead had an increase in impulsivity, but both sexes displayed heightened social preference. Transcriptional profiling identified altered neuron–microglial signaling as an important biological pathway affected by perinatal morphine.Prenatal opioid exposure rates are continuing to rise, largely captured by the 82% increase in Neonatal Opioid Withdrawal Syndrome (NOWS) incidence across the United States since 2010 . As of 2Children born to mothers taking opioid drugs during pregnancy have an increased risk for cognitive problems that most frequently manifest as executive function deficits during early childhood, including inattention and impulsivity . These cOpioid maintenance therapy with methadone or buprenorphine is recommended for pregnant people who disclose an opioid use disorder, but major barriers can prevent access to these therapies . Hence, Morphine and other opioids can activate toll-like receptor 4 TLR4; , a patteThe prefrontal cortex (PFC) is the brain region largely responsible for executive functioning, has heavy bidirectional connections with the amygdala (AMG), and opioid receptors are densely expressed in both PFC and AMG . ImportaTherefore, we hypothesized that long-term prenatal morphine exposure causes offspring changes in PFC-mediated and AMG-mediated behaviors that could vary by sex and potentially align with changes in microglia and synaptic markers. To study this, we used a 7 week perinatal morphine model that encompassed the pregestational, gestational, and lactation periods in mice to include exposure that is equivalent to third trimester brain development in humans. We assessed the social behavior of male and female offspring in adolescence using the three-chambered social interaction test and adult executive function in the operant-based touchscreen five-choice serial reaction time task (5CSRTT). We then examined the offspring PFC and AMG for gene expression at postnatal day 21 (P21) and in adulthood, along with quantifying microglial protein expression of Iba1 and lysosomal phagocytic marker CD68 in adult offspring. Finally, we conducted gene enrichment analysis on transcriptional changes in the P21 PFC to identify pathway-related hypotheses for future work.ad libitum (unless otherwise noted for behavior testing). The housing room had a 12 h light/dark cycle . Experimental procedures were all conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the University of Cincinnati Institutional Animal Care and Use Committee.All experimental subjects were B6D2F1/J hybrid mice bred in-house from sexually naive female C57BL/6J and male DBA/2J mice that arrived at 7 weeks of age and acclimated to the temperature-controlled and humidity-controlled vivarium for 1 week before the experiment. This breeding enhances genetic diversity to increase the generalizability of our findings. All mice were housed in standard polycarbonate cages containing corncob bedding with water and food available n = 12) or saline vehicle , injected daily (subcutaneously), 4 h before lights off. Female injections began 1 week before breeding to habituate and minimize stress during pregnancy. Additionally, this models the establishment of opioid use before pregnancy, instead of after conception. Morphine sulfate  was diluted with sterile saline to achieve an injection volume of 0.1–0.2 ml. Mice were injected with doses of 3, 5, and 7 mg/kg on days 1–3 to gradually acclimate the mice to the desired dose of 10 mg/kg on day 4, which was used throughout pregnancy and lactation, until offspring were weaned on P21 . The second cohort was used for all behavioral testing and adult gene expression and housed in cages of three to four, unless otherwise noted . After 1 week acclimation to the facility, female C57BL/6J mice were randomly assigned to receive either MO  or 4 h (n = 3) after injection. For dams from the experimental cohort killed at 20 h postinjection and the smaller cohort of dams killed at 1 and 4 h postinjection, maternal trunk blood was collected and centrifuged at 3500 × g for 15 min at 4°C. Fetal brains and placentas were collected from dams in the smaller cohort at 1 and 4 h (n = 3–4 offspring/dam) and flash frozen in isopentane. Plasma, brains, and placentas were stored at −80°C for mass spectrometry MO quantification. The small sample size used for this quantification was to verify MO transport to maternal plasma and fetal tissue, not for comparative purposes.Dams used to breed the experimental cohort were killed 24 h after weaning offspring , and 10.1523/ENEURO.0238-22.2022.f1-2Figure 1-2Figure 1-2, DOCX file.Gene expression targets for P21 mPFC, AMG, VTA, and NAc. The + and – symbols indicate included (+) or omitted (–) targets for adult operant PFC. The ^ and ᵒ symbols indicate included (^) or omitted (ᵒ) targets for adult operant AMG. Download 10.1523/ENEURO.0238-22.2022.f1-3Figure 1-3Figure 1-3, DOCX file.Targets added for the adult operant mPFC gene expression analysis. The ^ and ᵒ symbols indicate included (^) or omitted (ᵒ) targets for adult operant AMG. Download 10.1523/ENEURO.0238-22.2022.f1-4Figure 1-4Figure 1-4, DOCX file.Targets added for the adult operant AMG gene expression analysis. Download 10.1523/ENEURO.0238-22.2022.f1-5Figure 1-5A, B, Maternal MO treatment did not alter gestational weight (A) or pup birth weight (B). C, Assessed during the first postnatal week, MO injection acutely reduced time spent in the nest at 1 h, but this was recovered by 4 h . D, On postnatal day 2, MO dams returned their pups faster on average than SAL dams . Download Figure 1-5, TIF file.Maternal and birth characteristics. 10.1523/ENEURO.0238-22.2022.f1-6Figure 1-6Figure 1-6, DOCX file.Values for maternal and birthing outcomes that did not reach statistical significance (mean ± SEM) or median (range). Download 10.1523/ENEURO.0238-22.2022.f1-7Figure 1-7Figure 1-7, DOCX file.Values for offspring body weight (mean ± SEM). Download On P2, the pup retrieval test was performed in the home cage during the light phase under baseline (BL) conditions (before daily injection). The dam was removed from the home cage and placed in a separate clean cage. The pups were then moved out of the nest and scattered apart from each other at the most distal portion of the home cage from the nest. The dam was then returned to the nest and video recorded undisturbed for 10 min. A single investigator blinded to the experimental groups used Behavioral Observation Research Interactive Software (BORIS) to quantify latency to retrieve the first pup, average retrieval latency per pup, and the percentage of pups retrieved during the 10 min session.At 5–6 weeks of age, adolescent offspring were tested in the three-chambered social interaction task (3C) during a single day under dim indirect white light. This was performed 1–8 h after lights off, with order balanced between groups. The apparatus  was divided into a central chamber  and two adjacent right and left chambers . Mice were first placed into the central chamber and allowed to explore all three chambers for 5 min (habituation). Next, mice were guided into the center chamber and briefly contained with dividers while a novel conspecific was placed into a wire cup on one of the side chambers. Dividers were then removed so the experimental mouse could explore for 10 min and become familiar with this mouse . Finally, the experimental mouse was brought back to the center chamber while a second novel conspecific was placed in the second cup, with the experimental mouse allowed to explore either mouse for 10 min . The investigator counterbalanced the sides for familiar versus novel mice and whether the familiar mouse changed sides. Conspecific mice were B6D2F2/J hybrid mice from B6D2F1/J dams × DBA/2J males. A single investigator blinded to the experimental groups used BORIS to quantify time spent and bout frequencies investigating each of the cups under the various conditions . Preference and recognition indexes were calculated as percentages = [time spent investigating target/(time spent investigating target + time spent investigating other cup)] × 100.At 8–9 weeks of age, mice were singly housed and underwent preference testing for high-fat diet and sucrose . Mice were provided with either a 60% high-fat diet  or 4% sucrose water, in addition to standard chow diet and water bottles. Consumption of both options were measured daily for 3 d, with bottle/hopper placement switched daily. For both tests, day 1 was considered habituation and not assessed. Intake on days 2–3 was averaged and calculated as a percentage of total fluid or food intake. In the females, there were three SAL and three MO offspring that shredded the high-fat diet to a degree that prevented accurate measurement, resulting in seven SAL and nine MO mice. After completing both tests, mice were rehoused into their cages of three to four for the remainder of the study.At 12–13 weeks of age, adult offspring were food restricted to maintain 85–90% of their free-feeding body weight for 1 week before training and testing in Bussey-Saksida Mouse Touch Screen chambers using Abet II and Whisker software (Lafayette Instrument). Mice also habituated to the reinforcer for 24 h in their home cages (chocolate Yoo-hoo). Mice were tested 1×/d Monday–Friday for 30 min (or 1 h for progressive ratio) 1–8 h after lights off, with order balanced between groups.Mice habituated to the chamber with magazine training for 3 d . The habituation session began with a 5 s feeder pulse, and all remaining trials for the entire experiment were 280 ms. For magazine training, the magazine light illuminated until the mouse entered to collect the reward, followed by a 10 s intertrial interval (ITI). After habituation, the mice began fixed ratio 1 (FR1) training and testing, during which the center pad of five touchscreen pads became active (Center Only–Mouse Must Touch training v2). The center touchscreen illuminated until the mouse made a touch response, after which the magazine light illuminated until the mouse collected the reward, followed by a 1 s ITI. The lateral four touchscreen pads remained unlit, although touch responses to these blank pads were recorded. Achieving ≥70 trials for 2 consecutive days satisfied the FR1 learning criterion. Each male and female cohort remained on FR1 until all mice had passed the criterion. Once all mice had learned FR1 , they underwent progressive ratio (PR) testing for 2 consecutive days. PR had the same stimulus and ITI as FR1, except after every three trials the number of responses needed to turn off the touchscreen and obtain a single reward increased in the following fashion: 1, 2, 4, 7, 11, 16, 22, 29, and 37. The break point was the response number achieved per reward at the end of the 1 h PR session.After PR testing, mice started the 5CSRTT, which began with 2 d of habituation to any one of the five touchscreen pads illuminating for a trial . The pad remained illuminated until the mouse made a correct response, followed by reward and 5 s ITI. All remaining 5CSRTT sessions had a finite stimulus presentation, and, for a successful trial, the mouse had to respond to the correct touch pad during illumination. Responding to an unlit pad during stimulus presentation counted as an incorrect trial, failure to respond at all during stimulus presentation counted as an omission, and any response during the 5 s ITI was a premature response. An incorrect, omitted, or premature response resulted in a 5 s time-out, during which the house light illuminated. All trials resulted in magazine light illumination, but correct responses also came with a 1 s tone and reward. The magazine light remained lit until the mouse entered to start the ITI. We analyzed the percentage of accuracy, the percentage of omissions, the total number of trials, and the total number of premature responses .The mice completed four progressively harder 5CSRTT schedules at their own learning pace. A mouse passed schedule criterion by reaching at least 20 trials and >50% correct for 2 consecutive days. The first three schedules were finite stimulus presentations that remained constant over the 30 min session and lasted for 16, 8, and 4 s . For the 16 s stimulus, we analyzed the first few days of training the mice encountered before any mouse passed criterion. We then analyzed the first day that each mouse passed criterion for the 16, 8, and 4 s stimulus. After passing the 4 s stimulus, the mice underwent titration testing where stimuli durations were titrated to individual performance during the session . Correctg, at 4°C), the supernatant was mixed with 100% ethanol, transferred to RNeasy columns, and washed with buffers RWT and RPE (Qiagen). An Epoch microplate spectrophotometer (BioTek) was used to assess RNA quantity and quality and cDNA was synthesized with a High Capacity Reverse Transcriptase Kit (Thermo Fisher Scientific). The cDNA samples were preamplified for 32 genes of interest, using TaqMan assays and Preamp Master Mix (Thermo Fisher Scientific) following the manufacturer protocol (Fluidigm) and as previously published , +1.2 to 2.2; dorsoventral (DV), −2.2 to 0.0; mediolateral (ML) ±0–0.6; ublished . For quahttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE206032). Directional poly A RNA-seq was performed via previously described protocols . The data discussed in this publication have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus  and are rotocols . BrieflyMus musculus/MM10 (RefSeq) was used to align reads under first-strand setting, with the analysis using STAR for alignment and Salmon for quantification. BAM and Transcriptome.BAM files were obtained from STAR, with Salmon then using the Transcriptome.BAM file to assign transcripts per million to genes and transcripts. The RNA-Seq Differential Expression app then used the alignment result for differential expression analysis of reference genes with DESeq2 to create differential expression reports in BaseSpace Hub.After sequencing, Illumina BaseSpace Sequence Hub automatically produced Fastq files for downstream analysis. BaseSpace app RNA-Seq Alignment version 2.0.2 and RNA-Seq Differential Expression app version 1.0.1 were used for standard bioinformatics analysis to identify differentially expressed genes. Reference genome p < 0.05; and log2FoldChange >1.2 or less than −1.2), to identify biologically relevant pathways underlying the gene lists. Then to determine the robustness of the upregulated/downregulated gene lists in determining perinatal MO versus SAL exposure, unbiased gene × drug hierarchical clustering was performed using Morpheus (Broad Institute). Pearson and Spearman similarity measures were assessed, along with both hierarchical and k-means clustering (k = 2).Gene set enrichment analysis was performed using Enrichr . Enrichmn = 5; male MO, n = 6; female SAL; n = 3; female MO, n = 5). Perfused brains were collected and stored in 4% paraformaldehyde for 4 h, transferred to 30% sucrose overnight, sectioned on a microtome at 35 μm, and stored in cryoprotectant. For immunohistochemistry, sections were washed 5× 5 min in 1× PBS before transfer to blocking solution . After 1 h of incubation in blocking solution, sections were incubated in the following primary antibodies diluted in blocking solution overnight at 4°C: rabbit anti-Iba1 ; and rat anti-CD68 . Following primary antibody incubation, samples were washed with 1× PBS and incubated 1:500 in goat anti-rabbit cyanine3 and 1:500 in goat anti-rat 647 IgG for 1 h. Sections were washed, mounted to slides, and coverslipped with DABCO Mounting Media. Images were captured at 20× and 40× magnification using Axiovision 4.6 software (Zeiss) with Apotome Deconvolution. At 40×, z-stacks were captured, and subsets of five consecutive 1 μm images in the stack were compressed into projection images (LSM Image Browser) as previously described .A subset of adult behaviorally naive SAL and MO offspring from the same pregnancy cohort were transcardially perfused with 1× PBS followed by 4% paraformaldehyde at 23 weeks of age , and then extracted by solid-phase extraction on a Strata-X-C strong cation exchange column (Phenomenex). Morphine was eluted with organic solvent and liquid chromatography tandem mass spectrometry (LC)-MS/MS analysis was performed using a LC20AD HPLC system (Shimadzu) coupled to a mass spectrometer . Chromatographic separation was achieved on a Kinetex 2.6 μm Phenyl-Hexyl LC column . A gradient mobile phase was used with a binary solvent system, which was ramped from 5% mobile phase B (methanol/0.1% formic acid) to 5% mobile phase A (water/10 mm ammonium formate) at a flow rate of 0.7 ml/min. The total run time was 7 min. The optimal signal for the morphine and morphine-D6 were achieved in positive ion mode with the use of the following instrument settings: ion spray voltage, 2500 V; source temperature, 650°C; curtain gas, 35 psi; ion source gas 1, 60 psi; and ion source gas 2, 50 psi. The ion transitions monitored were selected as mass/charge ratio (m/z) of 286.0 to >152.1 (quantifier ion) and m/z of 286.0 to >165.1  for morphine, and m/z of 292.1 to >152.1 for morphine-D6. Data acquisition on the mass spectrometer was controlled by Analyst 1.7.2 software (SCIEX). Data processing and quantification were performed with MultiQuant software version 3.0.3 (SCIEX).Total morphine concentration was measured in mouse plasma, fetal brain, and placenta by tandem mass spectrometry (MS/MS) using a validated clinical assay for the detection of drugs of abuse in human urine and umbilical cord tissue performed according to proprietary assays (standard operating procedures #Path.CMS 1060 and #Path.CMS 1066) in a College of American Pathologists-accredited and Clinical Laboratory Improvement Amendments-accredited laboratory. Briefly, plasma and homogenized brain and placenta were hydrolyzed with a β-glucuronidase enzyme to obtain unconjugated morphine in the presence of the stable isotopic-labeled internal standard,  and main effects of drug  = 11.8; p = 0.0063) and time  = 4.6; p = 0.0087; Extended Data p < 0.0001) but recovered by 4 h postinjection. MO dams returned their pups to the nest faster on average than SAL dams in the pup retrieval test conducted at BL (t(13) = 2.5; p = 0.028c; Extended Data Maternal MO treatment did not affect gestational weight gain or pup birth weight (Extended Data p < 0.05 vs 50%); however, MO offspring displayed a significant increase in preference for the mouse versus empty cup compared with SAL offspringd  = 5.8; p = 0.02; In the three-chambered social interaction test at 5–6 weeks of age, there was a main effect of drug in the social preference portion. All animals demonstrated a social preference  or sucrose preference (B) tests. Download Figure 2-1, TIF file.High-fat and sucrose preference tests. 2 = 4.2; p = 0.04e; t(20) = 3.3; p = 0.0033f; In FR1, animals learn that each correct touch results in a single reward, which is the basic behavior required for subsequent operant tasks. All female mice acquired the FR1 criterion  in 7 d, while the males took 11 d. In FR1, male MO offspring learned FR1 significantly slower compared with male SAL offspring  = 5.1; p = 0.035g; F = 15.9; p = 0.0007h; F = 11.7; p = 0.0027i). Male MO offspring had decreased accuracy, an increased percentage of omissions, and a decreased number of premature responses without a change in the total number of trials achieved  = 6.8; p = 0.017j) and total number of trials  = 8.4; p = 0.009k; In the 5CSRTT, mice begin by learning the task with a 16 s stimulus, where they encounter a finite stimulus presentation for the first time. All mice took a minimum of 3–5 d of training on the 16 s stimulus before any mouse reached criterion . Across the first 5 d of the 16 s stimulus training in males, there were main effects of drug on accuracy, omissions, and premature responses  = 4.7; p = 0.043l) and premature responses  = 20.9; p = 0.0002m) . Male MO offspring were less accurate, had a comparable percentage of omissions, but again had decreased premature responses (t(18) = 2.7; p = 0.013n; We then analyzed the first day that each mouse achieved criterion  for each of the stimulus lengths. This analysis revealed the main effects of drug in the males on accuracy  and the MO group  = 11.3; p = 0.0031s), MYD88  = 4.6; p = 0.044t), DLG4  = 5.02; p = 0.037u), catechol-O-methyltransferase  = 8.16; p = 0.0098v), PNOC  = 8.2; p = 0.0095w), and DNMT3A  = 7.3; p = 0.014x; F = 4.6; p = 0.045y; F = 9.5; p = 0.0065z), with male MO offspring having reduced TLR4 expression and no difference in female offspring  for observed sex differences in gene expression. Download There were significant sex effects in the AMG with females having increased expression of DLG4, DNMT1, ITGAM, MBP, and SETD7. Females also had decreased AMG OPRK1 expression compared with males. In the NAc, females had increased expression of MYD88 and TLR2  = 28; p = 0.033aa) and neurotensin  = 21; p = 0.0083ab) compared with male SAL offspring  = 2.45; p = 0.0496ac; t(6) = 3.2; p = 0.019ad; t(3) = 3.65; p = 0.036ae; t(8) = 2.99; p = 0.017af; t(7) = 4.3; p = 0.0036ag; Images were captured in the m-mPFC, c-mPFC, MeA A–C, and 10.1523/ENEURO.0238-22.2022.f6-1Figure 6-1Figure 6-1, DOCX file.Male m-mPFC values for Iba1 and CD68 reported as the mean ± SEM. Download 10.1523/ENEURO.0238-22.2022.f6-2Figure 6-2A–D, Representative m-mPFC Iba1/CD68 40× images of female SAL (A) and female MO (B) depict increased Iba1 in female MO offspring; male SAL (C) and male MO (D) depict reduced Iba1 in male MO offspring. Representative c-mPFC CD68 40× images of male SAL (L) and male MO (O) depict reduced CD68 in male MO offspring. Download Figure 6-2, TIF file.Representative images for CD68/Iba1 immunohistochemistry. t(8) = 2.7; p = 0.028ah; In the MeA, 20× analysis revealed that male MO offspring had significantly reduced CD68 integrated density normalized to the cell count (p < 0.05 and log2FoldChange > 1.2 or less than −1.2). In male offspring, this produced 18 genes that were upregulated and 15 genes that were downregulated in MO compared with SAL (padjusted < 0.05). This was mediated by upregulation of oxytocin (OXT), AVP, DNMT3B, DNMT3L, and MYB in male MO offspring (highlighted in padjusted < 0.05). This was driven by the upregulation of genes S100A7A, S100A8, S100A9, KRT17, DSP (desmoplakin), HPX (hemopexin), FGL1, FGB (fibrinogen beta chain), and LTF (lactoferrin) in female MO offspring  construct validity for a mouse model of perinatal morphine exposure that results in executive function deficits in males and (2) evidence in support of alterations in neuron–microglial signaling that may underlie these behavioral deficits. We found that male MO offspring had delayed learning in FR1 and reduced motivation in PR, along with impaired accuracy and decreased attention in the 5CSRTT executive function task. At the end of maternal MO exposure on P21, we detected an increase in gene expression in the microglial pruning gene ITGAM (CR3/CD11b) and neuronal postsynaptic marker DLG4 [postsynaptic density-95 (PSD-95)] in the mPFC in both sexes. Simultaneously, there was a reduction in presynaptic glutamatergic marker SLC17A7 (VGluT1) in the AMG of MO offspring at the same age. In adulthood, female MO offspring had increased Iba1 and CD68 immunolabeling in the mPFC, while these markers were reduced in the mPFC and AMG of male MO offspring.We treated dams with MO daily for 7 weeks to model long-term maternal opioid use and to include all stages of early neurodevelopment. As a result, female MO offspring had increased impulsive errors during the initial learning of the easiest version of the task with the longest stimulus length. However, this effect was absent once females had successfully learned the task. In contrast, male MO offspring showed increased omissions (indicating inattention), and decreased accuracy in the early stage of learning, with reduced accuracy persisting even after successfully learning the task at progressively shorter stimulus lengths. Further, male MO offspring had reduced premature/impulsive errors throughout. The reduction in impulsivity in male MO offspring is reminiscent of reduced impulsivity in a rat model of early life stress . While rWe assessed social behavior because PFC–AMG circuitry is important for social preference  and PFC–1 receptor microglial engulfment during both adolescence and adulthood. This is relevant to our work because D1 receptor pruning by microglia in males drives the developmental decrease in social play (We saw an increase in ITGAM (CR3) and MYD88 expression in the mPFC at P21 in MO offspring, which is consistent with the ability of MO to activate the TLR4 pathway  and a reial play , indicatIn addition to the PFC and AMG being affected by perinatal MO, we found a reduction in TLR4 expression in the male VTA at P21. TLR4 signaling in the VTA is important for morphine conditioned place preference , which ide novo DNA methylation. RNA-seq in male MO offspring revealed DNA methylation as a significantly enriched upregulated pathway, mediated by DNMT3B, DNMT3L, and MYB expression. DNA methylation patterns are highly responsive to environmental challenges during the prenatal period. Clinically, infants treated for NOWS had hypermethylation of the μ opioid receptor gene (OPRM1), and the degree of hypermethylation corresponded to NOWS severity (Finally, we detected increased COMT and PNOC expression in the P21 mPFC of MO exposed offspring, which is consistent with clinical literature that has identified single nucleotide polymorphisms in COMT and PNOC as driving individual differences in NOWS outcomes , 2017. Tseverity . IncreasThe 7 week daily maternal MO injection protocol is a model of opioid use before and during pregnancy, including exposure via lactation to model the third trimester brain development in humans . The 10 In conclusion, perinatal MO exposure during pregestation, gestation, and lactation causes sex-dependent behavioral and molecular responses in the brain, with male offspring being behaviorally more affected than females. Executive function outcomes seem to depend on sex and the timing of opioid exposure during gestation, while social preference does not. The heightened social preference and impaired learning, motivation, attention, and accuracy in male MO offspring could relate to lasting impairments in microglial phagocytic capacity. Female MO offspring appear to have evidence of increased microglial phagocytosis, which potentially could dampen a subset of the behavioral effects.


Salivary exosomes have demonstrated vast therapeutic and diagnostic potential in numerous diseases. This study pioneers previously unexplored roles of SE in the context of corneal wound healing by utilizing primary corneal stromal cells from healthy (HCFs), type I diabetes mellitus (T1DMs), type II DM (T2DMs), and keratoconus (HKCs) subjects. Purified, healthy human SEs carrying tetraspanins CD9+, CD63+, and CD81+ were utilized. Scratch and cell migration assays were performed after 0, 6, 12, 24, and 48 h following SE stimulation (5 and 25 µg/mL). Significantly slower wound closure was observed at 6 and 12 h in HCFs with 5 μg/mL SE and T1DMs with 5 and 25 μg/mL SE. All wounds were closed by 24-hour, post-wounding. HKCs, T1DMs, and T2DMs with 25µg/mL SE exhibited a significant upregulation of cleaved vimentin compared to controls. Thrombospondin 1 was significantly upregulated in HCFs, HKCs, and T2DMs with 25 µg/mL SE. Lastly, HKCs, T1DMs, and T2DMs exhibited a significant downregulation of fibronectin with 25 μg/mL SE. Whether SEs can be utilized to clinical settings in restoring corneal defects is unknown. This is the first-ever study exploring the role of SEs in corneal wound healing. While the sample size was small, results are highly novel and provide a strong foundation for future studies. Exosomes are nanovesicles that originate from the inward budding of cell membranes that are released into the extracellular compartment ,2,3. WhiExosomes are found throughout the body in blood , saliva Corneal wound healing is a complex cascade of events that involves epithelial, stromal, and endothelial cells (depending on the type/depth of injury) undergoing orchestrated proliferation, differentiation, and migration responses, ultimately leading to extracellular matrix (ECM) remodeling. The actual response to corneal injury varies among the three major corneal cell types. For instance, epithelial healing results in the differentiation and migration of limbal stem cells ; stromalDiabetes mellitus (DM) is associated with deficiency of the body to use blood glucose; however, complications can lead to delayed/inefficient wound healing compared to healthy populations. The diabetic cornea suffers from delayed wound healing ,33, relaKeratoconus (KC) is characterized by progressive corneal thinning, resulting in a cone-shaped corneal architecture and eventually leading to the loss of visual acuity due to corneal scarring ,42. KC iTo our knowledge, this is the first report of the impact of SEs in the context of wound healing in healthy, DM, and KC corneal tissue. Future studies are warranted in order to delineate the potential therapeutic role of SEs related to corneal disease and trauma.Results of the purified human SEs demonstrated positive CD9(+), CD63(+), and CD81(+) expression. Proportions for overall CD9+ were 15%, CD63+ were 17%, and CD81+ were 3% A. Particx-axis labels CHIP050, CHIP051, and CHIP052 denote n = 3 for the purified human SE.Exosomes’ sizes typically range from ~50 nm to 150 nm in diameter ,48,49. OScratch assays were performed to determine the effect of SEs on wound closure/migratory cell pattern of healthy (HCFs), KCs (HKCs), type I DM (T1DMs), and type II DM (T2DMs) human corneal fibroblasts cells . All fouThe protein expression of alpha-smooth muscle actin (α-SMA), thrombospondin-1 (TSP-1), fibronectin (EDA-Fn), vinculin, and vimentin was examined using Western blots for all cell types and conditions at 48 h. Beta actin (β-actin) expression was used to normalize data, and the mean ± SEM of four replicates (n = 3) was plotted. Expression of the native and cleaved form of vimentin was determined as previously reported ,51. CultExpression of α-SMA  and vincThe expression of TSP-1 was significantly higher for HCFs stimulated with 25 µg/mL SEs when compared to HCF stimulated with 5 µg/mL SEs A. For HKEDA-Fn expression differed significantly for HKCs, T1DMs, and T2DMs . EDA-Fn Native vimentin expression was significantly decreased in T2DMs stimulated with 25 µg/mL SEs when compared to T2DM controls D. In addThe expression of cleaved vimentin showed significant differences for HKCs, T1DMs, and T2DMs . The cleExosomes are extracellular vesicles derived from body fluids and biological tissues/cells, transporting proteins, enzymes, DNA, RNAs, microRNAs, and lipids ,5,6. TheOur current studies have focused on SEs due to their non-invasive collection methods and similar contents compared to other exosome forms . HoweverOur study investigated the SE impact on HCFs, T1DMs, T2DMs, and HKCs. Both the diabetic and keratoconic cornea are known for their fibrotic phenotype, as well as their wound healing imbalances ,66,67. IAdditionally, a tear analysis of KC patients found decreased anti-inflammatory marker IL-10 and increased pro-inflammatory cytokines, including IL-6, tumor necrosis factor-α, and matrix metalloproteinase (MMP)-9 ,46. InflOur study revealed that HKC and T2DMs stimulation with 25 µg/mL SEs promoted the upregulation of TSP-1 compared to their own controls, possibly resulting in ECM repair and corneal wound healing via the activation of transforming growth factor-beta 1 TGF-β1; B,D. In tSE stimulation caused the significant downregulation of EDA-Fn in HKC, T1DM, and T2DM cells but not in HCFs B–D. EDA-Our results showed increases in cleaved vimentin in SE-stimulated HKCs, T1DMs, and T2DMs, suggesting cleavage of the native vimentin into truncated forms promoting wound healing and cell migration . As a cyThis study highlights the potential role of SEs related to corneal trauma/disease. Our study measured fibrotic marker expressions late in the wound healing process after wound closure was completed. Future studies should track the levels of fibrotic phenotypes throughout the cornea wound healing process to confirm if fibrotic markers expression varies in the early stages compared to later stages of healing. Further studies, including larger number of cornea samples, are needed to understand the impact of SE in other cell types, such as epithelial and endothelial, during corneal wound healing. In addition, SE stimulation using an organ culture and/or our established 3D model is needed in order to closer represent the natural corneal wound healing process. Finally, in vivo studies are necessary to understand better the impact SEs have on corneal wound healing. Regardless, our findings are the first to suggest a potential therapeutic role of SE in cornea wound healing and paves the way for future studies.Tissue collection written consent was obtained following institutional and federal guidelines. The study was approved by the North Texas Regional Institutional Review Board . A total of four corneas from four donors were used, including one from each condition: healthy , KC , T1DM , and T2DM . Healthy , T1DM (Oklahoma Lions Eye Bank), Oklahoma City, OK, USA,, and T2DM  are cadaveric tissue donations. KC tissue donations are live penetrating keratoplasty transplants from Dean McGee Eye Institute .® R100 platform .Commercially available SEs  were purchased and fully characterized with ExoView® Tetraspanin kit , which contains microarray chips to capture and characterize CD9, CD63, and CD81. SEs were diluted 100-fold to optimize signal-to-background expression and split in triplicates. Then, 70 µL of the sample was added to three chips, placed on a plate holder, and incubated overnight at room temperature. The plates were then placed into the ExoView® Chip Washer . After the initial wash, 250 µL of blocker/fluorescent solution  was added containing mouse anti-human: CD81 with Alexa488, CD63 with Alexa647, and CD9 with Alexa555, followed by a 2 h incubation. The chips were dried, scanned, and analyzed using the NanoView Scanner software for particle counts, colocalization counts, and size.In brief, SE’s tetraspanin profiles were examined using an ExoView2. The medium was changed every two days, and cells were passaged in new flasks, as needed. All described experiments were executed with cells passage 3 to 6.Primary human corneal fibroblast cells, HCFs, HKCs, T1DMs, and T2DMs were isolated from donor corneas. In brief, the cornea’s epithelial and endothelial layers were scraped off using a razor and rinsed with sterile phosphate buffer saline . The cornea pieces were cut into ~2 × 2 mm and placed into a T-25 flask  with Eagle’s minimum essential media, , 10% fetal bovine serum , and 1% antibiotic-antimycotic  and incubated at 37 °C in 5% CO5 cells per well and grew confluent over 24 h incubation at 37 °C with 5% CO2.All cell types were seeded in clear flat-bottom 6-well plates  at a concentration of 5 × 109 particles in 2 mL media) or 25 μg/mL (1.42 × 1010 particles in 2 mL media). Cultures with no SEs (media only) served as controls (n = 3). The scratch assay was executed on confluent cultures by scratching each well’s center using a 10 μL pipette tip. The scratch was marked using a red sticker to serve as a reference point. To track the progression of cell migration and wound closure, images were taken at 0, 6, 12, 24, and 48 h post-wounding, using an ACCU-SCOPE microscope  equipped with an EXCELIS HDS camera (AU-600-HD) at 4× magnification. Images were analyzed using ImageJ .Cultures (n = 3) were stimulated with two concentrations of purified SEs of 5 μg/mL . In brief, cultures were washed two times with PBS, followed by the addition of 100 µL of RIPA-PI. All cells were then scraped off the wells with cell scrapers  and incubated for 30-min at 4 °C in an ice bucket. Finally, cell supernatants containing protein were collected following 15 min centrifugation at 12,000 RPM at 4 °C.Protein was quantified using Pierce™ BCA Protein Assay Kit , and Pierce™ Bovine Serum Albumin standards , per manufacturer protocols. A BioTek EPOCH2 microplate reader  was used to measure the absorbances at 562 nm. Values were plotted using linear regression with the standards to calculate and normalize protein concentration.TM fluorescent buffer  for 1 h. Following blocking, 1:500 dilution of a primary antibody including EDA-Fn , TSP-1 , and α-SMA , β-actin , vinculin , or vimentin  was added overnight at 4 °C. The NCMs were washed three times with Tris-Buffer Saline with 1% tween (TBST) for 5 min, followed by a 1 h incubation with the secondary antibody (1:2000); donkey anti-rabbit AlexaFluor 568 , at room temperature. The NCMs were imaged with the iBright FL 15,000 imaging system  and analyzed with the iBright analysis software .Western blots were run by SDS-PAGE at 225 V for 39 min in Novex 4–20% Tris-glycine Mini Wedge 12-well gels  and transferred to nitrocellulose membrane (NCM) using iBlot 2 Dry Blotting System . NCMs were then blocked using 1× Blockerp < 0.05.GraphPad Prism was used for graphical representation and statistical analysis using one-way ANOVA . Data were normalized to β-actin expression, and the average (mean ± SEM) of four replicates (n = 3) was plotted. Differences were considered statistically significant when


Chlamydomonas reinhardtii in comparison with RSL3-induced ferroptosis. After RSL3 and heat shock (50 °C) treatments with or without inhibitors, Chlamydomonas cells were evaluated for cell viability and the induction of ferroptotic biomarkers. Both the heat shock and RSL3 treatment were found to trigger ferroptotic cell death, with hallmarks of glutathione–ascorbic acid depletion, GPX5 downregulation, mitochondrial dysfunction, an increase in cytosolic calcium, ROS production, lipid peroxidation, and intracellular iron accumulation via heme oxygenase-1 activation (HO-1). Interestingly, the cells preincubated with ferroptosis inhibitors (ferrostatin-1 and ciclopirox) significantly reduced RSL3- and heat-induced cell death by preventing the accumulation of Fe2+ and lipid ROS. These findings reveal that ferroptotic cell death affects the iron homeostasis and lipid peroxidation metabolism of Chlamydomonas, indicating that cell death pathways are evolutionarily conserved among eukaryotes.The phenomenon of heat stress leading to ferroptosis-like cell death has recently been observed in bacteria as well as plant cells. Despite recent findings, the evidence of ferroptosis, an iron-dependent cell death remains unknown in microalgae. The present study aimed to investigate if heat shock could induce reactive oxygen species (ROS) and iron-dependent ferroptotic cell death in  RAS-selective lethal 3 (RSL3) directly inactivates GPX4 or depletes glutathione to induce ROS production and lipid peroxidation. The regulatory factors of ferroptosis primarily include the mevalonate pathway, sulfur transfer pathways, and the heat shock factor 1 dependent heat shock protein beta 1 (HSF1-HSPB1) system [Cell death is a fundamental biological process that occurs ubiquitously in all living organisms in response to intrinsic and extrinsic stimuli . In rece) system .Arabidopsis thaliana during heat stress. Heat stress triggered an iron-dependent cell death that showed hallmarks similar to those observed in mammalian cells such as glutathione and ascorbic acid depletion, lipid peroxidation, and the accumulation of ROS and Fe ions. Moreover, an exogenous addition of iron triggered ferroptosis-mediated cell death in plants infected with viruses and fungi [Synechocystis sp. PCC6803 to heat shock [Chlamydomonas, firmly regulate iron assimilation through the differential expression of various iron assimilatory genes [Chlamydomonas is well established, its possible role during abiotic stress is unknown. In microalgal species, apoptosis-like cell death in response to stress is either caspase-dependent or -independent, with established cellular processes such as cell shrinkage, DNA fragmentation, chromatin condensation, the loss of membrane potential, the externalization of phosphatidylserine, and ROS accumulation in cell organelles [Chlamydomonas have also been found to exhibit defined characteristics of ferroptotic cell death during heat stress, which includes the depletion of the glutathione–ascorbate redox system [Chlamydomonas reinhardtii as a model system during heat shock (at 50 °C).In plants, Distefano et al.  identifind fungi . Recentlat shock . Like plry genes . Though ganelles . These sx system , ROS accx system . HoweverC. reinhardtii CC-124 was obtained from the Chlamydomonas Research Center, University of Minnesota, Minneapolis, MN, USA. The culture was maintained in a tris-acetate phosphate (TAP) medium at 25 °C with continuous illumination of 50 µE m−2 s−1. For the heat treatment, the exponential-phase cells (2 × 106 cells/mL) were incubated at 50 °C for 10 min in a shaking water bath in the dark. Then, the cells were recovered under continuous light at 25 °C for programmed cell death to occur [The wild-type strain of to occur . The celto occur . Cell de2DCFDA  for the detection of ROS accumulation, 5 µM C11-BODIPY™ 581/591  for the detection of lipid peroxidation, 3 µM JC-1  for staining the mitochondrial membrane potential, 15 µm Calcium Green-1, AM  for staining cytosolic calcium, and 1 µM FerroOrange  for iron staining at room temperature for 20–30 min, followed by two washes with 1X PBS. Fluorescence images were captured using a Leica fluorescence microscope  equipped with a Leica DFC9000 sCMOS camera. The excitation/emission of fluorescent dyes used the following filters: H2DCFDA (Ex/Em: 492/515 nm), C11-BODIPY™ 581/591 (Ex/Em: 460–495/510–550 nm), JC-1 (Ex/Em: 488/538 nm for monomers and 596 nm for oligomers), Calcium Green-1 (Ex/Em: 506/531 nm), and FerroOrange (Ex/Em: 543/580 nm). For fluorescence intensity, 100 µL of stained cells were transferred into a black 96-well plate and fluorescence was measured using a Synergy HTX Multi-mode Plate Reader  with the indicated excitation and emission wavelengths.After the heat shock treatment (50 °C), cells were stained with 5 µM HChlamydomonas G protein ß-subunit-like polypeptide (endogenous control)) using the 2−ΔΔCT method [Total RNA was isolated from control and experimental cultures using Trizol  and reverse-transcribed using a RevertAid First strand cDNA Synthesis Kit  as per the manufacture’s protocol. RT-qPCR was performed with a CFX96 Touch Real-Time PCR Detection System  using iTaq universal SYBR Green Supermix. The list of RT-qPCR primers is given in T method . All theChlamydomonas cells were harvested, weighed, and used for total lipid extraction. The extracted lipids were treated with or without 10 mM triphenyl phosphine (TPP) for 30 min to reduce lipid peroxides. The TPP-treated and untreated lipids were mixed with 0.5 mL of FOX reagent (1:1 ratio of 50 mM xylenol orange and 5 mM iron(II) sulfate heptahydrate) for 30 min at room temperature, and the absorbance was measured at 560 nm [2O2 (0 to 20 µM) were used to create a calibration curve. For LOX activity, Chlamydomonas cells were harvested and homogenized with 5 mL of a 50 mM ice-cold potassium phosphate buffer (1% polyvinylpyrolidone and 0.1% triton x-100). The samples were centrifuged at 10,000× g, and the supernatant was used for LOX activity, which was carried out according to the protocol of Awad et al. [The ferrous oxidation xylenol orange (FOX) assay was performed to determine the levels of lipid hydroperoxide (LOOH). Treated t 560 nm . Standard et al. . The LOXChlamydomonas cultures were harvested and homogenized with a 0.1 M phosphate buffer (pH 7.2). From the samples, 100 µL of homogenates were mixed with 0.1 mL of 98% sulfuric acid and 70% nitric acid. Samples were completely digested at 100 °C for 3 h, following which 50 µL of 60% perchloric acid was added, and the mixture was dried completely at 100 °C. Finally, 0.5 mL of sterile distilled water, 0.25 mL of 1% thioglycolic acid, 1.5 mL of saturated sodium acetate, and 1.0 mL of 0.08% bathophenanthroline in ethanol were added to the ash samples and vortexed well. The samples were centrifuged at 12,000× g, and the supernatant was used to measure the total iron content colorimetrically at 535 nm. The total iron content (ng mg−1 dry weight) was calculated using a standard iron solution .The measurement of the iron content was performed as described in a previous study . ChlamydChlamydomonas cells were harvested and homogenized with an ice-cold 50 mM phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride, 0.2 mM ethylenediaminetetraacetic acid, and 0.25 mM sucrose. The cell homogenate was centrifuged at 10,000× g for 20 min, and the resulting supernatant was assessed for HO-1 activity [−1 bovine serum albumin, 10 µM hemin, 0.025 units mL−1 spinach ferredoxin-NADP+ reductase, and 4.2 µM ferredoxin. The reaction mixture was incubated at 37 °C for 1 h, and the enzyme activity was measured at 650 nm using a UV spectrophotometer .For the HO-1 activity, activity . The act3+ to Fe2+ with ascorbic acid in an acidic medium resulted in the formation of a red chelate between Fe2+, and 2,2′-bipyridyl was read at 525 nm. Dehydroascorbate was determined by subtracting AsA from AsA and DHA. The ascorbate content was calculated using a series of standards of L-ascorbate . GSH and glutathione disulfide (GSSG) were quantified by following the 5,5′-dithiobis (2-nitrobenzoic acid) method using glutathione reductase and 2-vinylpyridine and were measured at 415 nm according to the protocol of Griffith [The estimation of the total ascorbate (AsA and DHA), reduced ascorbate (AsA), and dehydroascorbate (DHA) was carried out using the method of Gossett et al. . In thisGriffith . The GSHg for 5 min and washed with 1X PBS. The DNA extraction was carried out using a DNeasy Plant Mini Kit  according to manufacturer’s instructions. The purified DNA (5 µg) was subjected to electrophoresis in a 1.5% agarose gel and stained with ethidium bromide. The bands were visualized and documented with a Gel Documentation System .After the treatment, the algal cells were collected via centrifugation at 5000× n = 3). GraphPad Prism statistical software v5.0 was used to analyze the experimental data using a one-way ANOVA with Bonferroni’s post hoc test.All experimental procedures were performed using three biological samples , lipid peroxidation, GPX4 downregulation, and GSH and ascorbate depletion [Chlamydomonas, cells were subjected to RSL3 and a heat shock (50 °C) treatment in the presence or absence of ferroptosis inhibitors, and cell death was observed at regular intervals . However, cells preincubated with inhibitors before the heat shock (50 °C) and RSL3 treatment showed a significant decrease in the accumulation of cytosolic and lipid ROS , the JC-1 fluorescence signal shifted from red to green, suggesting a decrease in the MMP. However, cells preincubated with inhibitors significantly restored the MMP compared to the RSL3 and heat shock treatment , and ascorbate oxidases/reductases [Chlamydomonas cells treated with heat shock at 50 °C in the presence or absence of ferroptosis inhibitors were examined, and the results revealed that the levels of GSH and AsA were significantly lowered in cells under heat shock, whereas cells treated with RSL3 showed no significant change when compared to control cells  plays a vital role in ferroptosis and is the key regulator of the inhibition of lipid hydroperoxides. PHGPX converts GSH into GSSG and reduces cytotoxic LOOH . The inhnhardtii ,41. Siminhardtii ,42. In tnhardtii . Howeverductases . As a reol cells B–F. RSL3Chlamydomonas lacks canonical caspases; however, proteases have been shown to be responsible for caspase-like activities in algal cells during cell death [Chlamydomonas during RSL3 and heat shock treatment, we investigated the effect of preincubation with the reversible caspase-3 inhibitor CHO. We observed that preincubation with CHO significantly reduced the percentage of cell death induced by RSL3 and heat shock  was observed only after 14–16 h of treatment but not at different time points after heat treatment [Chlamydomonas cells for 12 h after the heat shock and RSL3 treatment . The occurrence of ferroptosis-mediated cell death in Chlamydomonas provides new insights into the evolutionarily conserved mechanisms of cell survival and programmed cell death in eukaryotic cells under stress conditions. Moreover, understanding the finer aspects of ferroptotic cell death regulation in microalgae could be useful for the effective control and management of several natural and anthropogenically induced phenomena. To give an example, such knowledge could help us effectively manage harmful algal blooms caused by climate change and their effects on ecosystems.The present investigation indicates that heat shock (50 °C) induces iron-dependent cell death with hallmarks of GPX5 inactivation through GSH and AsA depletion, which leads to an accumulation of intracellular free iron that generates ROS and lipid peroxidation . These b


Here, we report 17 metagenome-assembled genomes (MAGs) recovered from microbial consortia of forest and pasture soils in the Brazilian Eastern Amazon. The bacterial MAGs have the potential to act in important ecological processes, including carbohydrate degradation and sulfur and nitrogen cycling. From the microbial enrichment and cultivation in triplicate, we selected one forest and one pasture consortia sample for further analyses. The total consortia DNA was extracted using the PowerLyzer PowerSoil DNA Isolation Kit . The metagenomic libraries were constructed using the Nextera DNA Flex Library Prep Kit  and sequenced on the HiSeq 2500 platform  (2 × 100 bp).Soil sampling was conducted in the Brazilian Eastern Amazon, state of Pará, in a primary forest located in the Tapajós National Forest (2°51′19.6″S 54°57′30.1″W) and a pasture in the adjacent region (3°07′44.9″S 54°57′15.5″W). In each area, soil samples from 0- to 10-cm depth were collected in quintuplicate after removing the litter layer. Then, the samples of each land use were mixed. Microbial consortia were obtained from 10 g of forest and pasture soil samples which were initially triplicate enriched in CHN50, 56,397 bp) and for pasture was 89.07 Mbp .The 38.5 and 42.1 million paired-end reads from the forest and pasture, respectively, were imported into the KBase platform , and defCheckM v2.2.4  was usedProteobacteria (11 MAGs), Actinobacteriota (4 MAGs), and Bacteroidota (2 MAGs)  . Functio(2 MAGs) , indicatPRJNA849167. The SRA accession numbers for the raw reads are SRR19663812 and SRR19663813. The metagenome-assembled genomes are deposited under the GenBank accession numbers listed in https://doi.org/10.25982/116951.133/1878567.All data are deposited at the NCBI under BioProject


The Ca(OH)2-assisted thermal pretreatment process was optimized to enhance the methane production potential of switchgrass. Solid loading (3–7%), Ca(OH)2 concentration (0–2%), reaction temperature (50–100 °C), and reaction time (6–16 h) were selected as independent variables for the optimization. Methane production was obtained as 248.7 mL CH4 gVS−1 under the optimized pretreatment conditions. Specifically, a reaction temperature of 100 °C, a reaction time of 6 h, 0% Ca(OH)2, and 3% solid loading. Compared to raw switchgrass, methane production was enhanced by 14.5%. Additionally, the changes in surface properties and bond structure, along with the kinetic parameters from first order, cone, reaction curve, and modified Gompertz modeling revealed the importance of optimization.Switchgrass earned its place globally as a significant energy crop by possessing essential properties such as being able to control erosion, low cost of production, biomass richness, and appeal for biofuel production. In this study, the impact of a Ca(OH) Electrified transportation is expected to increase in the future; however, implementing its use will not happen everywhere, in every industry, and all at once . TherefoEnergy crops, as a specific crop species, have been grown for advanced cellulosic biofuel production in recent decades. Switchgrass, one of these promising energy crops, belongs to the Poaceae family and is a native North American prairie grass species that can rapidly grow large amounts of carbohydrate-rich biomass. It is a species of C4-type plants (which utilize the Hatch–Slack cycle for carbon fixation), and as such, it can perform this process four times more productively than C3-type plants (which utilize the C3 pathway for carbon fixation). From an environmental point of view, switchgrass-based biofuels are carbon negative due to switchgrass’s high carbon sequestration ability owing to its deep and complex root system. Moreover, switchgrass has the ability to clean soils and waters by removing excess nutrients  originating from fertilization. This plant is also a great growth and living habitat for wildfowl and mammals, especially in ecotone zones between forest and agricultural fields ,3. From 2 pretreatment is the most widely preferred among the various alkaline chemicals  for enhancing the hydrolysis of various lignocellulosic biomasses [Hydrolysis is the rate-limiting step for the anaerobic digestion (AD) process, even for less complex substrates than lignocellulosic biomass. Therefore, to utilize lignocellulosic biomass in AD efficiently, a pretreatment process needs to be applied. Pretreatment makes the crystalline, compact, and solid structures of lignocellulose suitable for chemical and biochemical conversion. A wide range of pretreatment processes, such as physical, chemical, thermal, and biological, and their combination has been investigated for lignocellulosic biomass in the literature for the last few years . The objiomasses ,12,13,14The AD of switchgrass, considering the methane yield for various harvesting seasons ,16,17,182-assisted thermal pretreatment process to enhance methane production from the switchgrass variety, Shawnee. Central composite design (CCD) of response surface methodology (RSM) was selected for the optimization procedure. Solid loading as DM (3–7%), Ca(OH)2 concentration (0–2%), reaction temperature (50–100 °C), and reaction time (6–16 h) were chosen as independent variables. The effects of these independent variables on pH, sSugar, and methane production were investigated, and the Ca(OH)2-assisted thermal pretreatment process was modeled and optimized for maximum methane yield. The impact of the Ca(OH)2-assisted thermal pretreatment process on surface properties, molecular bond characterization of switchgrass, and anaerobic digestion kinetics were also evaluated.Since switchgrass is a complex substrate as lignocellulosic material, it needs pretreatment to break down the structure, open up cellulose and hemicellulose, and remove lignin to produce biogas. Therefore, this study aims to optimize the Ca(OH)Characterization of biomass for biofuel production is the first step for evaluation. A field-dried, milled, and homogenized variety of Shawnee as an energy crop was used to characterize biomass. The general composition of switchgrass Shawnee is presented in 2-assisted thermal pretreatment experiments are shown in Thomas et al.  indicate2 is applied in the range of 0–2% in the pretreatment experiments, it is expected that changes in the pretreatment inlet and outlet pHs depend on the applied amount of Ca(OH)2. As can be seen in 2 at the reaction temperatures of 50, 75, and 100 °C were measured close to each other before and after the pretreatment experiments. While initial pH values were measured as 6.5–6.7, pH values varied between 5.2 and 6.7 depending on solid loading and reaction time after the pretreatment. In the pretreatment experiments assisted with 2% Ca(OH)2, the initial pH values ranged from 10.3 to 10.9. At the end of the reactions, at low temperatures (50 °C), pH values changed in the range of 7.7–8.2 depending on the pretreatment reaction time, while at high temperatures (100 °C), it changed between 6.5 and 6.9 regardless of the reaction time. When 1% Ca(OH)2 was applied in the pretreatments, the initial pH values were 9.5–9.8, while pH values were measured as 6.1–6.8 after the pretreatment. The pH values were observed to be lower at high temperatures.Since the Ca(OH)−1. The amounts of sSugar were measured at 7% solid loading (SL), as DM is higher than 3% SL when 50 °C reaction temperature, 6 h and 16 h reaction times, and without Ca(OH)2 conditions are applied in the pretreatments. It is normal that sugar’s solubilization from high solid loading was higher than from low solid loading. On the other hand, under the pretreatment conditions of 50 °C reaction temperature, 6 h and 16 h reaction times, and 2% Ca(OH)2, the amount of sSugar at 7% solid loading was lower than 3% solid loading. Higher sSugar values were measured from the raw switchgrass samples at all conditions of 75 °C reaction temperature applied. Furthermore, at 100 °C reaction temperature conditions, the sSugar values measured at 7% SL were higher than those measured at 3% solid loading.sSugar analysis found that raw switchgrass has 50 mg sSugar VS4 gVS−1. In the literature, the methane production potential of switchgrass was reported as 169–252 mL CH4 gVS−1 by Masse et al. [−1 by Frigon et al. [4 gVS−1 by El-Mashad et al. [4 gVS−1 by Grigatti et al. [4 gVS−1 by Başar et al. [4 gVS−1 by Başar and Perendeci [Raw Shawnee switchgrass produced 217.1 mL CHe et al.  and 104.d et al. , the rawi et al. , raw Kanr et al. , and rawerendeci . Conside4 gVS−1, specifically under the pretreatment conditions of 100 °C reaction temperature, 16 h of reaction time, 0% Ca(OH)2, and 3% solid loading. Methane generation was enhanced by 6.6% compared to raw switchgrass. In contrast, the lowest methane production was observed as 62.4 mL CH4 gVS−1 when the 75 °C reaction temperature, 11 h reaction time, 2% Ca(OH)2, and 5% solid loading pretreatment conditions.The maximum methane generation was achieved as 231.4 mL CHAs is seen in 2 from 0% to 2% resulted in high BMP values. A similar trend was observed at 100 °C reaction temperature, 6 h reaction time, and 3% solid loading conditions, but low BMP production was obtained when raising the reaction time from 6 h to 16 h. Furthermore, BMP values slightly rose or remained stable while the reaction temperature increased from 50 °C to 100 °C. When a reduction in methane production is observed, it should be kept in mind that process optimization is required for the amount of chemicals used in the pretreatment process, depending on the composition of lignocellulose. Gunerhan et al. [When 50 °C reaction temperature, 6 h and 16 h reaction times, and 3% solid loading conditions are applied in the pretreatments, an increase in Ca(OH)n et al.  indicate2-assisted thermal pretreatment, containing duplicates of 25 varied pretreatment conditions and triplicate of the center point, were loaded into Design Expert software. The model for BMP was developed by RSM. The accuracy of the BMP model was evaluated by the highest determination of coefficient (R2), adjusted-R2, and predicted-R2. In addition, modification (Backward) was applied to the quadratic BMP model to obtain high R2. Backward selection seeks to remove terms from a model that are detrimental to the criterion. p-value criterion was used in the backward selection. The BMP model equation was used to optimize the Ca(OH)2-assisted thermal pretreatment process for maximum methane production with an acceptable process cost. BMP Model equation, statistics, and information are presented in BMP results from the experiments of 51 Ca(OH)R2 of 0.8572, adjusted–R2 of 0.8121, and predicted–R2 of 0.7646 was developed. The coefficient of determination, expressed as R2, shows the model’s predictive power in percent. The developed model could only explain 85.72% of the relation between BMP and independent variables. The quadratic modified BMP model presented a low probability result , indicating the model is in the confidence range of 99%. p-values less than 0.05 indicate that model terms are significant. According to the BMP model statistics, solid loading (B) and Ca(OH)2 concentration (D) were found to be significant terms from the first-degree effect terms, whereas reaction time (C) and reaction temperature (A) were determined insignificant terms in the BMP model of Ca(OH)2-assisted thermal pretreatment process. Furthermore, solid loading * Ca(OH)2 concentration (BD), square terms of solid loading (B2), and reaction time (C2) are effective independent variables in the BMP model. Since the first-degree effect of the reaction temperature (A) does not change BMP directly and to obtain a hierarchical model, insignificant terms from the quadratic BMP model were excluded by the application of backward modification.A BMP model with 2 assisted thermal pretreatment process on BMP and desirability (0.977) are presented in 4 gVS−1 should be produced by the application of 3% solid loading, 6 h reaction time, and 0% Ca(OH)2 concentration conditions in the pretreatment process. As is seen in 4 gVS−1 by increasing solid loading from 3% to 7%. Furthermore, increasing solid loading, reaction time, and Ca(OH)2 concentration resulted in low desirability and BMP values.The individual effects of independent variables of Ca(OH)3D response surface plots for the effects of independent variables on the BMP model are given in 2 concentration positively impacted BMP values. Where solid loading is kept at a constant minimum of 3%, increasing the Ca(OH)2 concentration did not show any significant impact on the methane yield. On the other hand, where Ca(OH)2 concentration was kept at its constant, increasing the solid loading adversely affected the BMP values; specifically, at the maximum solid loading and 0% Ca(OH)2 concentration, BMP resulted in the lowest value. 2 concentration in any direction did not affect the BMP values significantly. Conclusively, solid loading * Ca(OH)2 concentration was the most effective interaction for the maximum methane yield.In 2-assisted thermal pretreatment process was implemented to generate the maximum specific methane yield using the BMP model. In the optimization solid loading (%) (+++++), reaction time (h) (+++++), Ca(OH)2 concentration (%) (+++++) were minimized, whereas reaction temperature (°C) (+++++) and BMP yield (mL CH4 g VS−1) (+++++) was maximized. Under these conditions, optimum Ca(OH)2-assisted thermal pretreatment process conditions were found as 3% solid loading, 100 °C reaction temperature, 0% Ca(OH)2 concentration, and 6 h reaction time with the 0.977 value of highest desirability. The BMP model predicted methane yield as 213.5 mL CH4 gVS−1 under optimized conditions. The Ca(OH)2-assisted thermal pretreatment trial was done under optimized conditions for the validation. Pretreated switchgrass was subjected to the BMP test, and methane yield was acquired as 248.7 mL CH4 gVS−1. Enhancement of methane yield was computed as 14.5%, compared to raw switchgrass under optimized validation conditions.Optimization of a Ca(OH)Most of the work in the literature focused on the production of methane from switchgrass without pretreatment. Pretreatment conditions for switchgrass and results available in the literature are presented in 2, NaOH, KOH), acidic , and oxidizing (H2O2) chemical agents were preferred in pretreatment. As seen in 2-assisted thermal pretreatment, considering all the process variables for methane enhancement. However, this result revealed that Ca(OH)2-assisted thermal pretreatment for switchgrass does not significantly affect methane enhancement.It can be concluded from HCl, H2SO, and oxi2-assisted thermal pretreatment process. SEM images of raw switchgrass sample (a), pretreated switchgrass samples at 3% SL, 50 °C, 0% Ca(OH)2, 6 h, (b), 3% SL, 100 °C, 2% Ca(OH)2, 16 h (c), and 3% SL 100 °C, 0% Ca(OH)2, 6 h (d) are presented in SEM images and the FTIR analysis of the raw and pretreated switchgrass samples were performed to appraise the alterations in surface properties and the structural-chemical modifications that emerged during the Ca(OH)2, 16 h, and 100 °C conditions   d shows t2, 6 h, (b), 3% SL, 100 °C, 2% Ca(OH)2, 16 h (c), and 3% SL 100 °C, 0% Ca(OH)2, 6 h (d) are presented in FTIR spectra of raw switchgrass sample (a), pretreated switchgrass samples at 3% SL, 50 °C, 0% Ca(OH)−1. It is observed that the switchgrass sample pretreated at 3% SL, 100 °C, 2% Ca(OH)2, and 16 h conditions show sectional similarities with the switchgrass sample pretreated at 3% SL, 100 °C, 0% Ca(OH)2, and 6 h conditions. Nevertheless, upon careful examination, differences in chemical bonds were observed in both samples. Between the 400–1800 cm−1 wavelength, 470 cm−1, 560 cm−1, 620 cm−1, 775 cm−1, 895 cm−1, 1050 cm−1, 1180 cm−1, 1245 cm−1, 1440 cm−1, 1465 cm−1, 1580 cm−1 and 1735 cm−1 wavelengths in particular show remarkable differences. FTIR spectrum of raw switchgrass sample shows differences in Si-O-Si and (PO4)3− bonds at 470 cm−1 wavelengths compared to pretreated switchgrass samples. Additionally, a notable resonance in Si-O-Si and (PO4)3− bonds is observed for the switchgrass samples pretreated at 3% SL, 100 °C, 2% Ca(OH)2, and 16 h conditions compared to raw switchgrass, indicating a concentration increase of fragments containing these groups. However, the resonance in C-O and C=O bonds at 620 cm−1 wavelengths remained identical when high temperatures were applied to the switchgrass samples. Thus, this shows how temperature application makes a difference in C-O and C=O bonds. 775 cm−1 wavelength corresponds to resonance in NH2 bonds. Switchgrass samples pretreated at 3% SL, 100 °C, 2% Ca(OH)2, and 16 h conditions have minimum absorbance value compared to raw switchgrass samples. This observation indicates how much deterioration occurred under the effects of Ca(OH)2-assisted temperature pretreatment. Moreover, 895 cm−1 wavelength states the resonance in β-glucosidic bonds directly related to cellulose. Switchgrass sample either pretreated at 3% SL, 50 °C, 0% Ca(OH)2, 6 h, or at 3% SL, 100 °C, 0% Ca(OH)2, 6 h conditions showed similar properties. However, the sample pretreated at 3% SL, 100 °C, 2% Ca(OH)2, and 16 h conditions had a low absorbance value. This is an indication of the deterioration of the β-glycosidic bonds. The 1050 cm−1 wavelength indicates C-O, C=C, C-OH, and C-O-C stretching vibrations in cellulose and hemicellulose compounds. FTIR spectrums of raw and pretreated switchgrass samples at 3% SL, 50 °C, 0% Ca(OH)2, 6 h, and 3% SL, 100 °C, 0% Ca(OH)2, 6 h conditions indicated similar absorbance values. However, a minimum absorbance value was obtained from the switchgrass sample pretreated at 3% SL, 100 °C, 2% Ca(OH)2, and 16 h conditions. This shows the tension in the C-O, C=C, C-OH, and C-O-C bonds of cellulose and hemicellulose. Alteration in the band position and intensity may represent a reduction in the content of the structural ingredients by the application of pretreatment [−1 and 1245 cm−1 wavelengths are related to the C-O-C asymmetrical bending vibration in cellulose and hemicellulose compounds and the C-O bonds of acetyl groups of hemicellulose, respectively. Switchgrass samples pretreated at 3% SL, 100 °C, 2% Ca(OH)2, and 16 h conditions resulted in lower absorbance values than others at these wavelengths. This is evidence of the disintegration of the acetyl groups of hemicellulose. In the fingerprint zone of the spectra, modifications in spectra, especially at 1440, 1465, and 1580 cm−1 wavelengths, can be observed from the pretreated switchgrass samples compared to raw switchgrass. Chemical bonds at 1140, 1465, and 1580 are related to lignin decomposition. Out of the fingerprint region, at 2900 and 3400 cm−1 wavelengths, modifications were monitored that indicate an alteration in C-H and O-H bonds.Spectroscopic results obtained by FTIR analysis indicate that raw and pretreated switchgrass samples show similar binding properties at the wavelength between 400 and 1800 cmR2 and Adjusted-R2, are presented in The BMP test was finalized at 56 days. Since the biogas production reached a plateau after the first 28 days, this period was considered in the kinetic modeling. Predicted methane production and kinetic parameters from the first order, cone, modified Gompertz, and reaction curve modeling, along with the statistical indicators as R2) was achieved for all models. All four models were successfully applied to determine the biogas potential of the switchgrass. The hydrolysis rate constant (k) is an important parameter showing the methane production efficiency in the anaerobic degradation process. The k values estimated with the first order kinetic model for the different pretreatments applied to the switchgrass varied in the range of 0.115–0.159 d−1. The k value varies between 0.136 and 0.210 d−1 from cone modeling. Parallel with the first order modeling, the minimum k value was estimated from the pretreated switchgrass at 3% SL, 50 °C, 0% Ca(OH)2, 6 h and was followed by the pretreated switchgrass at 3% SL, 100 °C, 0% Ca(OH)2, 6 h conditions and the raw sample. The estimated and experimental results show a variance between 2.55% and 6.41%. The high R2 values and minor differences between model results and experimental results for methane production indicate that the cone model can be used successfully.High compatibility 2, 16 h conditions, 15.97 mL CH4 gVS−1 d−1 for raw sample, 14.37 mL CH4 gVS−1 d−1 from pretreated switchgrass at 3% SL, 100 °C, 0% Ca(OH)2, 6 h, and 14.25 mL CH4 gVS−1 d−1 from pretreated switchgrass at 3% SL, 50 °C, 0% Ca(OH)2, 6 h. Low alpha values are consistent with experimental BMP results. BMP production started quickly in all four samples.Modified Gompertz modeling results indicate the maximum Rm value was calculated as 26.33 mL CH4 gVS−1 d−1 for pretreated switchgrass at 3% SL, 100 °C, 2% Ca(OH)2, 16 h conditions, as in the modified Gompertz model, followed by 22.70 mL CH4 gVS−1 d−1 for raw, 20.33 mL CH4 gVS−1 d−1 from pretreated switchgrass at 3% SL, 100 °C, 0% Ca(OH)2, 6 h, and 20.19 mL CH4 gVS−1 d−1 from pretreated switchgrass at 3% SL, 50 °C, 0% Ca(OH)2, 6 h.When examining the results of the reaction curve model, the In conclusion, in evaluating the success of each four models in this study, every model achieves applicable results, while first order, cone, and reaction curve methods proved to have a better prediction capacity than modified Gompertz. For the comparison, the effects of pretreatments on methane production kinetic parameters available in the literature are presented in Rm) and ultimate specific methane production were improved. At the same time, lag phase time (λ) decreased due to the application of NaOH pretreatment , except the lag phase time (λ) for the condition of 8% NaOH, 50 °C and 1 day. Shen et al. [2 to the switchgrass. The λ and maximum methane production rate (μm) values were calculated as 0.8 d and 6.3 mL CH4 gVS−1 d−1, respectively, for 5% Ca(OH)2 pretreatment conditions.Elalami et al.  concluden et al.  applied λ: 3.1 d and μm: 1.9 mLSTP gVS−1 d−1), while λ decreased, the μm value increased by the pretreatment. In this study, parallel with the literature, the Rm value increased, and the λ value decreased obtained from the reaction curve and modified Gompertz models compared to raw by applying 3% SL, 100 °C, 2% Ca(OH)2, 16 h pretreatment condition.Compared to the untreated raw switchgrass . Wu et al. [k values between 0.021–0.075 d−1 for the leaf and 0.021–0.070 d−1 for the stem part of the switchgrass. Obtained k values from this study are slightly higher than the literature values. However, it should be noted that k values were calculated for the first time for Ca(OH)2-assisted thermal pretreatment for switchgrass.Jackowiak et al.  calculatu et al.  also fouk and the shape factor n to predict methane production Unyay et al. [2-assisted thermal pretreatment in this study, k constants increased, and the n values decreased compared to the work of Unyay et al. [The cone model uses the kinetic parameters of the hydrolysis rate constant y et al.  calculaty et al. .In this study, the switchgrass variety of Shawnee is utilized as biomass. Switchgrass was planted and dried on the field in Karapınar . The size of the samples was reduced to 3 mm to achieve textural homogeneity and consistency ,58,59,60® 3000 HPLC  with an Aminex HPX-87P column  for characterization of switchgrass.Total solids (TS), volatile solids (VS) , Van Soe2-assisted thermal pretreatment on switchgrass was applied by Central Composite Design (CCD) and Response Surface Method (RSM). CCD with 3 levels of 4 independent variables as switchgrass solids loading (3–7%) as DM, reaction temperature (50–100 °C), Ca(OH)2 concentration (0–2%), and reaction time (6–16 h) were designated for the process optimization. Biochemical methane potential (BMP) and sSugar results were selected as dependent variables. Design Expert 11 software  was used for experimental design and statistical analysis.The experimental design of Ca(OH)p- and F-Values. Model quality was determined by the coefficient of determination (R2) and an adjusted determination coefficient (adj-R2). Optimization of Ca(OH)2-assisted thermal pretreatment was done by using maximization and minimization criteria for independent and dependent variables [Experimental results were modeled, and the obtained regression model was evaluated by the analysis of variance (ANOVA), regression coefficients, and the ariables ,65 by us2-assisted thermal pretreatment experiments were conducted in 1 L laboratory-scale glass reactors equipped with a reflux condenser immersed in a 2 L oil heating bath. To provide the desired temperature and mixing conditions, magnetic stirrers with heaters  and oil baths were used. Glass reactors were closed with condensers, which were kept at constant temperature by a circulating water bath  to avoid evaporation.Ca(OH)2-assisted thermal pretreatment experiments, reactors were heated to predetermined temperatures after the addition of a calculated amount of switchgrass, Ca(OH)2, and deionized water. The reaction time was started, and the temperature was kept at the determined temperature during the pretreatment experiments. The reactor contents were continuously mixed with magnetic stirrers at 150 rpm. All pretreatment experiments were performed in duplicates. At the end of reaction time, the temperature of the reactor content was rapidly cooled down to room temperature. pH values of reactor content were measured before and after the pretreatment experiments. Reactor contents were centrifuged two times at 4400 rpm for 5 min and 14,500 rpm for 15 min to obtain the liquid phase for soluble sugar (sSugar) analysis. Soluble sugar (sSugar) analysis was performed by the Dinitro Salicylic Acid (DNS) method [In the Ca(OH)) method  to obser2-assisted thermal. The BMP experiment works on the basis of incubating a specific amount of switchgrass samples mixed with anaerobic sludge at 35 °C and detecting the gas composition and volume produced. The BMP test was conducted according to Perendeci et al. [2/CO2 (70%/30%) gas blend for the anaerobic condition after the addition of the switchgrass sample, seed sludge, required nutrients, buffer solution, and deionized water. The substrate-to-seed ratio was selected as 0.5 (gVS gVS−1 for solid samples). BMP reactors were incubated at 35 °C, and experiments were finalized at 56 days. The gas composition of N2, CH4, and CO2 in headspaces of the BMP reactors was quantified by Varian CP-4900 Micro-Gas Chromatography . Details of the measurements of biogas volume and the analysis of gas composition can be found in Basar et al. [The standard BMP test was applied to define the biochemical methane potentials of raw and pretreated switchgrass by Ca(OH)i et al. . BMP exp/CO2 70%/% gas ble2-assisted thermal pretreatment. Samples firstly were lyophilized and coated with gold-palladium for 120 s under 18 mA current . After coating, the samples were monitored at different magnifications, and images were kept with SEM  at 15 kV voltage.The surface structure of raw and pretreated switchgrass samples was evaluated by SEM to determine the changes caused by Ca(OH)2-assisted thermal pretreatment on the molecular bond characteristics of the lignocellulosic structure, Fourier transforms infrared spectroscopy (FTIR) analyzes were performed for both raw and pretreated samples. Samples were lyophilized, and measurements were performed by a Perkin Elmer, Spectrum Two in 400 cm−1 and 4000 cm−1 wavelength ranges.To determine the effects of Ca(OH)2-assisted thermal pretreatment on the anaerobic digestion reaction kinetics, specific cumulative methane production was modeled by using modified Gompertz and reaction curve equations. Furthermore, first order and cone models were implemented for the determination of the hydrolysis rate. Detailed descriptions of the model equations can be found in the work of Yılmaz et al. [To evaluate the effect of Ca(OH)z et al. . All mod2-assisted thermal pretreatment was applied to energy crops to enhance biodegradability and biofuel yield and remove a significant handicap of lignocellulose by unmasking cellulose. Neat optimization revealed that solid loading as DM (3–7%) and Ca(OH)2 concentration (0–2%) were found to be significant independent variables compared to reaction temperature (50–100 °C) and time (6–16 h) within range desing boundary. Essentially, increasing solid loading, reaction time, and Ca(OH)2 concentration resulted in low desirability and methane production yield. A biochemical methane production model was built and validated specific methane yield with 14.5% increases compared to untreated switchgrass. Nevertheless, it is concluded that Ca(OH)2-assisted thermal pretreatment did not significantly affect methane enhancement compared to other pretreatment methods.Energy crops have garnered considerable attention due to their significant potential as biofuel feedstock and agricultural benefits. Most switchgrass varieties are a powerful alternative to produce energy-efficient biofuel. In this study, Ca(OH)


Purpose: Magnetic resonance elastography (MRE) generates quantitative maps of the mechanical properties of biological soft tissues. However, published values obtained by brain MRE vary largely and lack detail resolution, due to either true biological effects or technical challenges. We here introduce cerebral tomoelastography in two and three dimensions for improved data consistency and detail resolution while considering aging, brain parenchymal fraction (BPF), systolic blood pressure, and body mass index (BMI).Methods: Multifrequency MRE with 2D- and 3D-tomoelastography postprocessing was applied to the brains of 31 volunteers (age range: 22—61 years) for analyzing the coefficient of variation (CV) and effects of biological factors. Eleven volunteers were rescanned after 1 day and 1 year to determine intraclass correlation coefficient (ICC) and identify possible long-term changes.Results: White matter shear wave speed (SWS) was slightly higher in 2D-MRE (1.28 ± 0.02 m/s) than 3D-MRE , with less variation after 1 day in 2D (0.33 ± 0.32%) than in 3D , which was also reflected in a slightly lower CV and higher ICC in 2D  than in 3D . Remarkably, 3D-MRE was sensitive to a decrease in white matter SWS within only 1 year, whereas no change in white matter volume was observed during this follow-up period. Across volunteers, stiffness correlated with age and BPF, but not with blood pressure and BMI.Conclusion: Cerebral tomoelastography provides high-resolution viscoelasticity maps with excellent consistency. Brain MRE in 2D shows less variation across volunteers in shorter scan times than 3D-MRE, while 3D-MRE appears to be more sensitive to subtle biological effects such as aging. MRE genechanics . Promineechanics  and Alzhechanics , multiplechanics , Parkinsechanics , and norechanics . Conversechanics , Valsalvechanics , hypercaechanics , perfusiechanics , idiopatechanics , and funechanics .Specifically, aging has been reported to be associated with up to 0.8% brain softening per year in adults . AlzheimHowever, possible changes in smaller multiple sclerosis lesions might be masked by blurry or noisy MRE maps, which cannot display interfaces between small anatomical subregions or cerebrospinal fluid (CSF) . For exain vivo brain stiffness, it remains to be determined if these challenges reflect technical limitations or biological margins of variability of brain viscoelasticity.Taken together, we identified two main challenges for the clinical application of state-of-the-art cerebral MRE: first, limited resolution of detail and, second, large inter-subject variability. Since no ground truth values exist for k)-based multifrequency dual elasto-visco (k-MDEV) inversion in two variants, once as a new development with shear wave separation in 3D using the curl operator and once with 2D bandpass filtering , cortical gray matter (CGM), white matter (WM), and DGM] as well as the reproducibility of values. Reproducibility is addressed by repeated measurement after a day while longitudinal variation due to possible aging effects is studied by repeated examinations after 1 year.Since the ground truth for the mechanical properties of Regarding the sensitivity of brain MRE to biological effects, we study possible biological influences  and geometrical influences  including wave amplitudes on the measured values by correlation analysis.(i) provide reference values for cerebral tomoelastography of the brain,(ii) demonstrate high-resolution viscoelasticity mapping of anatomical detail,(iii) assess the short-term and long-term consistency of the method based on 1-day and 1-year follow-up examinations, and(iv) discuss pros and cons of 2D and 3D wave inversion in MRE of the brain.Collectively, we aim tohttps://bioqic-apps.charite.de/ in this study. A subgroup of eleven volunteers  were examined two additional times, 1 day and 1 year after the baseline examination. All volunteers underwent both standard anatomical MRI and multifrequency MRE.SPM-segment from the neuroimaging data analysis package SPM12  equipped with a 32-channel head coil. Each volunteer’s head was placed in the same position on the vibration bed with precise connection to the driver during all follow-up examinations. All slice blocks were automatically positioned at the center of the brain using the scanner’s auto align function based on the localizer scan. T1-weighted, high-resolution, whole-brain images were acquired using a magnetization-prepared rapid acquisition of gradient echo sequence . WM volume and the brain parenchymal fraction (BPF), which is the ratio of intracranial brain parenchymal volume (GM plus WM) to total intracranial volume (GM plus WM plus CSF) were calculated from MPRAGE images using the segmentation routine ge SPM12 .2, voxel size 1.6 × 1.6 × 2 mm3, acquisition matrix 126 × 126 × 40, echo time 70 m, repetition time 4,700 m. GRAPPA parallel acquisition  sequence . Eight puisition  with an SPM-realign and Hysco2 (based on field maps with inverted readout direction), respectively, in SPM12. Mean MRE magnitude images were calculated by averaging over the frequencies, encoding directions, and time steps and normalized to the Montreal Neurological Institute (MNI) space based on the ICBM152 template (Complex MRE images were corrected in a slice-wise fashion (2D) for stochastic head motion and field distortions using template  using SPk-)MDEV inversion  and penetration rate  maps  were recnversion  with recnversion . The equnversion . k-MDEV k-MDEV decomposes the measured vector fields d), wave component (c) and frequency (f). Wave numbers are then deduced from the phase gradient of Therefore, as a critical step of preprocessing, G* with its real part or storage modulus G′ and its imaginary part or loss modulus G″, with density 3:Compound property maps of SWS and PR are finally obtained by weighted-averaging (weights w) over components, directions, and N frequencies:https://bioqic-apps.charite.de/  and fully 3D using a newly developed processing pipeline. For 2D data processing, wave images were decomposed in eight propagation directions. Smoothing and suppression of compression waves were done using a bandpass Butterworth filter of third order with a highpass threshold of 15 1/m and lowpass threshold of 250 1/m. SWS and PR maps were reconstructed based on 2D phase gradients. The 2D pipeline is publicly available on rite.de/ . For 3D rite.de/ .Due to edge slice artifacts of the 3D inversion, the four outermost slices in each direction were removed, leaving 32 slices for further analysis. Edge slice artifacts from finite difference operators are common for 3D MRE  but wereSWS and PR maps were normalized to the MNI space using the mean MRE magnitude images to generate averaged parameter maps and tissue probability maps . ProbabiIn eleven volunteers, we further analyzed how the number of slices for a fixed slice block thickness potentially affected SWS and PR values in 3D processing. Starting with 3D processing based on 39 slices, equivalent to 62.4 mm block thickness, we averaged SWS and PR within WM visible in the center slice for reference, and, subsequently, removed the two outermost slices from further 3D processing. The error (in %) relative to the central reference slice was averaged over all volunteers. Data were processed in MATLAB 2020a .p-values were corrected for four comparisons using the Holm-Bonferroni method. A multivariable linear regression model with age and BPF as independent variable was calculated for SWS and PR, respectively .Cross-sectional investigations provided inter-subject variability based on the coefficient of variation (CV) in all brain regions we analyzed. Correlations between reconstructed parameters  and region-specific wave amplitude, age, systolic peripheral blood pressure (BP), BMI, and BPF were analyzed in GBT using Pearson’s correlation coefficient. p-values were corrected for multiple comparisons using the Holm-Bonferroni method.Differences between viscoelastic values reconstructed from 2D and 3D inversion and between different brain regions (WM vs. CGM and WM vs. DGM) were analyzed using a paired Student’s t-test and a correlation analysis. p-values were corrected for two comparisons using the Holm-Bonferroni method.Repeated measurements in eleven volunteers after 1 day provided reproducibility indices for 2D and 3D data processing based on mean relative absolute difference (RAD) and intraclass correlation coefficient (ICC) for GBT, WM, CGM, and DGM. RAD between 2D and 3D data processing was compared using a paired Student’s t-test. ICC estimates and their 95% confident intervals were based on a single-rater, absolute-agreement, two-way, mixed-effects model . One-yeap-values below 0.05 were considered statistically significant.All statistical analysis was done in R version 4.0.2 . 3, 544 ± 21 cm3, 380 ± 22 cm3, and 53 ± 5 cm3, respectively. Mean BPF was 0.77 ± 0.04%.The analyzed MRE volumes covered 65% GBT, 77% WM, 53% CGM, and 84% DGM of the MNI volume, resulting in group-averaged volumes of 909 ± 44 cmp < 0.0001). Both reconstruction methods yielded higher SWS values for DGM  and lower SWS values for CGM  compared with WM (p < 0.0001 for each test). Group statistical plots for GBT, WM, CGM, and DGM in 2D and 3D are shown in r = 0.75, p < 0.0001). A corresponding analysis is provided for the viscosity-related PR parameter in the r = −0.54, p = 0.007, 3D: GBT: r = −0.45, p = 0.04) and positively correlated with BPF . The annual change in SWS of GBT was -0.0014 m/s in 2D  and −0.0019 m/s in 3D . Multivariable analysis for SWS in GBT showed a significant effect of BPF on SWS  while no significant effect of age was observed  given an intercept of 0.90 (standard error = 0.11).SWS in GBT was negatively correlated with age  while no such correlation was observed in 3D. SWS was not correlated with BP or BMI. A correlation analysis of PR, presented in the 2D SWS of DGM was correlated with wave amplitudes in DGM . Again, fluid-filled spaces were slightly enlarged by 3D data processing, however, with lower noise than visible in 2D SWS maps.Group-averaged SWS values  measured at three time points in eleven volunteers are presented in n = 31 and n = 11) was similar. CV for 2D processing was markedly lower than for 3D processing with the lowest values measured in WM and highest values in DGM. As shown in p = 0.004 for WM). Specifically, mean RAD was lowest for 2D WM SWS (0.33 ± 0.32%) and increased for GBT (0.43 ± 0.33%), CGM (0.68 ± 0.46%), and DGM (1.31 ± 1.06%). 3D values for GBT, WM, CGM, and DGM were 1.16 ± 0.71%, 0.96 ± 0.66%, 1.47 ± 0.98%, and 1.34 ± 1.17%, respectively. A summary for CV, ICC and RAD is given in p = 0.037) and between day one and 1-year follow-up by −0.021 . This longitudinal decrease in SWS, most likely attributable to aging, was about tenfold higher than obtained from our previously reported cross-sectional analysis in 31 brains. In contrast, no significant change in WM volume was observable after 1 year, whereas BPF was significantly reduced by 1.1 ± 1.0% (p = 0.019).Significant brain softening after 1 year was observed in GBT, WM, CGM, and DGM using 3D reconstruction. In GBT, SWS changed between baseline and 1-year follow-up by −0.011 m/s , which we consider an important indication of the overall consistency of the proposed inversion pipeline in addition to excellent reproducibility scores.|G*| using the elastic model  facilitates a direct comparison with recently reported values obtained at 50 Hz vibration frequency. |G*| of 2.07 ± 0.42 kPa and 2.62 ± 0.21 kPa  . Convert0.21 kPa  while Svterature .Similarly, our observation of stiffer DGM than WM (10%—20%) and softer CGM than WM (5%—10%) is in agreement with prior research , with la|G*|, we found an annual decrease in GBT stiffness of 5 Pa (2D) and 7 Pa (3D), consistent with the average decrease of 8 Pa reported by Age-related brain softening, as previously shown by different research groups , was rep3) and minimal smoothing. It should be pointed out again that the detail resolution of our maps was better than that of most published MRE maps of the brain. Our study has shown that it is not sufficient today to reject methods that provide higher anatomical fidelity than conventional approaches by referring to the lack of ground truth in brain MRE. Ground truth must be defined over many studies, but with consideration of anatomically plausible structures  as well as high consistency of values in follow up examinations, optimally over a year. It is a remarkable result of our study that the consistency of cerebral tomoelastography was similarly good or better as other quantitative MRI techniques  and inter-subject (2% CV) variability is encouraging in comparison with other MRE methods  given ouchniques . For prochniques  and perfoducible . Thus, ck-MDEV inversion, confirming that sample points per wavelength and sampled wavelength fraction influence SWS reconstruction, as previously shown by k-MDEV may be beneficial for other organs and body regions as well if the spatial support across slices is similar to the in-plane resolution. These technical requirements for 3D MRE should be considered whenever subtle mechanical changes are expected in larger tissue regions, similar to our 1-year follow-up study.It is worth mentioning that a reproducible excitation of wave fields inside the brain and a reproducible selection of imaging volumes are important to achieve a high consistency of MRE values. Our actuator setup is position-sensitive and therefore we ensured that the volunteer’s head was placed in a similar position on the driver setup during all follow-up examinations. Nevertheless, any other actuation setup  that achk-MDEV maps contributed most to the variability of our data and how one could further improve the consistency of brain MRE by refinement of the range of vibration frequencies. However, such an analysis would exceed our concise study design. To avoid a lengthy presentation, we confined ourselves to stiffness analysis in the main text while providing the results of viscosity analysis in the Our study has limitations. First, the number of volunteers in our longitudinal study was rather small, which precluded tests for multiple confounders that may affect brain stiffness over 1 year. Therefore, we focused on the effect of aging within 1 year as the most reproduced and best reported physiological confounder of brain MRE. However, a clear separation of aging effects from loss in BPF was not fully possible. Furthermore, we could not avoid that wave amplitudes varied between individuals even though the technical setup including driver amplitude was identical across all experiments. These amplitude variations are likely due to different head geometries, which affect the efficacy of wave induction. Possibly for that reason, 2D-SWS correlated with wave amplitudes in DGM, which contributed to higher CV values. In the future, variability in brain MRE could be further reduced if wave amplitudes inside the brain were actively controlled using an MRI actuator feedback system. Finally, we focused on frequency compound viscoelasticity maps without in-depth analysis of single frequency data. It would be another interesting and important research question which of the included frequencies in our In summary, this study introduced cerebral tomoelastography based on 2D and 3D multifrequency MRE and wavenumber-based multifrequency inversion. We assessed reproducibility, long-term changes, detail resolution, and biological effects on viscoelasticity parameters in the healthy human brain. Our method enabled high-resolution viscoelasticity mapping of anatomical detail as demonstrated by the stiffness-based separation of DGM regions in individual volunteers, which was consistent with group mean values. Stiffness correlated with age and BPF whereas BP and BMI did not correlate with MRE values. Cerebral tomoelastography was highly consistent in terms of CV and ICC, 2D versus 3D, and long-term effects. 2D MRE shows less variation across volunteers and at 1-year follow-up than 3D MRE and supports thin imaging slabs while 3D MRE seems to be more sensitive to subtle individual changes such as aging within only 1 year. Overall, cerebral tomoelastography has shown excellent consistency and detail resolution compared with both classical MRE of the brain and other quantitative MRI techniques. Therefore, it contributes to the quest for reproducible, quantitative, and biophysically significant MRI biomarkers for clinical applications.