Inactivation of the pOFC did not affect conditioned responses to either proximal threat or reward and basal cardiovascular activity was unaffected by manipulations of activity in either subregion. The results highlight the contribution of aOFC and pOFC to regulation of responses to more distal uncertain but not proximal, certain threat and reveal their opposing contribution to that of the immediately adjacent medial OFC, area 14.Structural and functional abnormalities of the orbitofrontal cortex (OFC) have been implicated in affective disorders that manifest anxiety-related symptoms. However, research into the functions of primate OFC has predominantly focused on reward-oriented rather than threat-oriented responses. To redress this imbalance, the present study performed a comprehensive analysis of the independent role of 2 distinct subregions of the central OFC  in the processing of distal and proximal threat. Temporary inactivation of both aOFC and pOFC heightened responses to distal threat in the form of an unknown human, but not to proximal threat assessed in a discriminative Pavlovian conditioning task. Inactivation of the aOFC, however, did unexpectedly blunt conditioned threat responses, although the effect was not valence-specific, as conditioned appetitive responses were similarly blunted and appeared restricted to a discriminative version of the task (when both CS The orbitofrontal cortex (OFC) has been implicated in a number of affective disorders, including depression  and anxiRevealing the contribution of the OFC as distinct from other regions of the prefrontal and cingulate cortex to such traits and disorders is an important step forward in determining the neurobiological basis underlying the marked heterogeneity in the etiology and also treatment of affective disorders. Heterogeneity is considered one of the major causes of the current impasse in effective treatments for anxiety and depression and underlies the drive for individualized treatment strategies . A conceAn additional consideration when studying the OFC is its distinct subregions based on dissociable cytoarchitectonic features and connectivity patterns. Such subregions exist in both primates and rodents, but while these are generally comparable across human and nonhuman primates, their analogous/homologous counterparts in rodents are as yet unclear. In primates, the subdivision of the orbital surface of the macaque monkey by Here, we are focusing on central OFC, specifically comparing the anterior and posterior divisions, area 11 and 13, respectively, in a new world primate, the common marmoset. Previously, permanent excitotoxic lesions of the anterior division in marmoset heightened responsivity to more uncertain, distal threat as measured by the human intruder test and, while not affecting the expression of Pavlovian conditioned proximal threat responses, did cause those responses to become inflexible to changes in stimulus-outcome contingencies . HeighteIn the current study, the anterior (a)OFC and posterior (p)OFC of the marmoset were therefore the subject of a systematic comparative analysis of their contribution to reactivity to uncertain and thus more distal threat and certain, proximal threat. It was hypothesized that the central OFC would contribute to distal but not proximal threat processing based on the findings from human neuroimaging studies . MarmoseCallithrix jacchus), bred on site at the University of Cambridge Marmoset Breeding Colony. Thirteen marmosets were experimentally naïve, while the remaining 3 had taken part in experiments not included in this publication  or posterior (area 13) OFC. Eight of the 16 marmosets also underwent a surgery to implant a telemetry device for the transmission and recording of real-time cardiovascular activity.Marmosets were premedicated with ketamine hydrochloride  and received the nonsteroidal anti-inflammatory carprofen . Anesthesia was then induced through intubation and delivery of 2.0–2.5% isoflurane gas in 0.3 L/min of oxygen. Respiration, heart rate (HR), oxygen saturation, and carbon dioxide blood levels were monitored throughout all surgeries using a pulse oximeter capnograph . Body temperature was monitored using a rectal temperature probe  and maintained using a heat mat. Following surgery, marmosets were monitored to ensure they were capable of maintaining oxygen saturation above 95% and placed in a heated incubator for recovery. Having fully recovered from anesthesia, they were returned to the home cage. Postoperative analgesia was administered using meloxicam  on the 3 days following surgery.Surgery was carried out as previously described . FollowiSurgery was carried out as previously described . FollowiA receptor agonist, Sigma-Aldrich) and baclofen  in saline were infused bilaterally into the brain over 2 min (Mus-Bac). The control was a 2-min infusion of 0.5 μL of sterile saline. The injectors were kept in place for a further 1 min, to ensure diffusion of the drugs into the surrounding tissue. The injectors were then removed, and dummies and caps replaced. Marmosets were returned to the home cage for a period of 25 min, before they were retrieved or prepared for behavioral testing in the home cage. Stimulation of activity (overactivation) of the aOFC and pOFC was also examined in an experiment looking at basal cardiovascular activity, as a comparison to effects of overactivation of area 25 , referred to as CGP-LY. 1.0 μL of CGP-LY was infused over a period of 2 min, with a 15-min pretreatment time . Marmosets were initially trained to enter a Perspex carry box in exchange for a small amount of marshmallow reward. The box was brought to the testing room and slotted into a chamber within the testing apparatus. The chamber was fitted with a houselight , 3 cameras attached to the top of the chamber, and a speaker (Behringer) and microphone concealed behind a panel. A telemetry receiver for the relaying of cardiovascular data was located under the floor of the chamber . All data from the receiver were recorded and analyzed using Spike 2 software (Cambridge Electronic Design). Video footage obtained using the cameras in the chamber was recorded using Power Director software (Cyberlink).This experiment was conducted in the home cage, as previously described . BrieflyMarmosets are known to display a range of different behaviors in response to a human intruder . Video rMarmosets were initially habituated to the sound-attenuated testing apparatus (described above). Marmosets were tested once a day, 5 days a week (Monday to Friday). They were placed inside the testing chamber for 5 min, with the duration of time spent inside gradually increased across each subsequent daily session until the maximum time of 20 min was reached. The house light was kept on and no sounds were played, in order to create an affectively neutral setting. Behavior was monitored to ensure that marmosets were not in distress, and the cardiovascular data recorded for each session. Once HR and systolic blood pressure (sysBP) showed stability across at least 3 consecutive sessions, intracerebral drug infusions began. Only 1 drug infusion per week was carried out and was counterbalanced across marmosets with some animals receiving saline, Mus-Bac, and CGP-LY infusions into the aOFC before the pOFC or vice versa. Following the infusion and pretreatment time, marmosets were placed in the testing apparatus for 20 min. HR and sysBP across the entire session were analyzed. Additionally, heart rate variability (HRV) was calculated using both the root mean square of successive differences (RMSSD) and Toichi’s cardiosympathetic and cardiovagal indices . The values were extracted from R-R interval data using Kubios HRV software.+ (30 s of darkness with 10 s of mildly aversive white noise at 80 dB) and the US− (0.5-s 2 kHz beep at 75 dB). The 10-s period of white noise was pseudo-randomized to occur either within the first, middle, or last 10 s of the 30-s period of darkness. The 2 neutral tones were initially tested in a single preconditioning session to assess the subjects’ intrinsic responses to the “to-be conditioned” tones. Whichever tone produced the higher cardiovascular arousal was selected to be the CS− (tone associated with the US−) and the remaining tone, the CS+ (tone associated with the US+), to ensure that any cardiovascular arousal seen during conditioning reflected conditioning per se. The discrimination training consisted of test sessions 7–12 min in length, with different combinations of CS presentations or trials. Each session contained 2–4 trials, with a maximum of 1 CS+-US+ presentation per session and a maximum of 2 CS+-US+ per week. This gave a number of interspersed session types, including 2 CS−, 3 CS−, 2 CS− with 1 CS+, or 3 CS− with 1 CS+ presentations. The CS+ was never presented as the first trial of the session.The Pavlovian conditioning paradigm was introduced in the same apparatus as that used to measure cardiovascular activity in an affectively neutral setting . MarmoseConditioning was assessed by looking at behavioral and cardiovascular arousal. Behavioral arousal was assessed by recording the duration of vigilant scanning, a behavior associated with rapid side to side head movements and a raised, tense body posture of the marmoset, as previously described . The car+-directed, but not CS−-directed, cardiovascular and behavioral arousal over at least 3 sessions. Responses to the threat itself, the US, were also quantified by comparing the US to the preceding CS .The cardiovascular and behavioral arousal was quantified by looking at the 20-s CS period relative to the immediately preceding 20 s of baseline . Successful conditioning was determined on the basis of consistent increases in CS−-US− trial, followed by a CS+-US+ trial, and a final CS−-US− trial.Once successful conditioning was established, intracerebral infusions began. There was a maximum of 1 infusion per week. The order of infusions was counterbalanced between marmosets with some animals receiving saline and Mus-Bac infusions into the aOFC before the pOFC or vice versa. All infusions were followed by a specific session type, which consisted of an initial CS+  and the US− (an empty food box becoming visible for 120 s). The 2 neutral tones were initially tested in a single preconditioning session to assess the subjects’ intrinsic responses to the “to-be conditioned” tones, as described above for the threat conditioning procedure. The discrimination training consisted of test sessions 4–7 min in length, with different combinations of CS presentations or trials. Each session contained only 1 or 2 trials, with a maximum of 1 CS+-US+ presentation per session and a maximum of 5 CS+-US+ every 2 weeks. This gave a number of interspersed session types, including a single CS−, 2 CS−, a single CS+, or a CS− followed by a CS+ presentation. The CS+ was never presented before a CS−, as the test session was terminated immediately after the CS+-US+. This was due to the fact that the food box door could not be closed again as these risked marmosets trapping their hands in the mechanism while reaching inside.The Pavlovian conditioning paradigm was introduced in a distinct apparatus from the one used for discriminative conditioning to threat. The testing apparatus was fitted with a special slot for the transparent carry box. From the carry box, the marmosets had access to 2 food boxes on either side of the chamber. The food boxes were gated with revolving doors, which could be set to closed, closed but transparent (inside of food box visible), or open (marmoset can reach into the food box) positions. Marmosets were tested once a day, 5 days a week (Monday to Friday). Marmosets were initially trained to retrieve reward from the food box. They were then trained to discriminate between 2 neutral tones , which predicted 2 different outcomes or US: the US+-directed, but not CS−-directed, cardiovascular and behavioral arousal over at least 3 sessions. Responses to the reward itself, the US, were also quantified by comparing the US to the preceding CS . Additionally, consumption of reward was monitored by weighing the amount of marshmallow consumed by the marmoset in the session.Conditioning was assessed by looking at behavioral and cardiovascular arousal. Behavioral arousal was assessed by a count of head jerks, a rapid side to side head flicking motion, as previously described in marmosets during appetitive conditioning . The car−-US− trial followed by the CS+-US+ trial, after which the session was immediately terminated.Once successful conditioning was established, intracerebral infusions began. There was a maximum of 1 infusion per week. The order of infusions was counterbalanced between marmosets with some animals receiving saline and Mus-Bac infusions into the aOFC before the pOFC or vice versa. All infusions were followed by a session which consisted of a CSAt the end of the study, marmosets were sedated with ketamine hydrochloride  and euthanized with pentobarbital sodium . A transcardial perfusion was performed . The brain was carefully removed and kept in 10% formalin for a further 24 h and later transferred to phosphate-buffered saline azide solution (0.01 M) for storage. When ready for sectioning, the brain was transferred to 30% w/v sucrose solution for at least 48 h . The tissue was sectioned into 40–60 μm coronal slices on a freezing microtome. The tissues were mounted onto gelatin-coated glass slides and stained with cresyl violet. The placement of cannulae was determined on the basis of tissue damage by examining the mounted brain sections. The placements were schematized by drawing them on coronal sections of the marmoset brain based on the  marmosetAll data were subject to tests of sphericity (Mauchly’s test in SPSS) and normality (Shapiro–Wilk test in SPSS). In the event that the condition of sphericity was not met, the Greenhouse-Geisser correction was applied. In the event that the condition of normality was not met, the following steps were taken: All pairwise comparisons were conducted using the Wilcoxon test for nonparametric data; all 1-way analyses involving more than 2 samples were conducted using the Friedman test for nonparametric data; all 2- or 3-way analyses were conducted using the aligned rank transform with the ARTool package in R . WhereveThe analyses were separately conducted for the EFA score and all of the component behaviors. The behavioral measures were analyzed using a 2-way linear mixed-effects model analysis  to account for partially overlapping subjects within the aOFC and pOFC samples.All measures of cardiovascular activity were analyzed separately. The data were subject to a 2-way linear mixed-effects model analysis  to account for partially overlapping subjects within the aOFC and pOFC samples.+-directed cardiovascular responses were analyzed using a 2-way repeated measures ANOVA (area × treatment). The US− was only 0.5 s in duration and therefore did not provide sufficient cardiovascular data for comparative analysis.The analyses for CS-directed HR and vigilant scanning behavior were conducted separately. Data for each measure were subjected to a 3-way repeated measures ANOVA . Additionally, US− responses were subtracted from CS+ responses, as previously .The analyses for CS-directed sysBP and head jerks were conducted separately. For the main analysis, CSeviously . The datFor all the key behavioral measures from videos used in the experiments, an inter-rater reliability analysis was conducted on a subset of 6 videos, 2 from each of 3 marmosets. The subset of videos was analyzed by a second rater blind to the conditions. The results were compared using an inter-rater reliability analysis using a 2-way mixed effects model in SPSS, with all results summarized in F = 8.76, P = 0.010), with no other effects . Pairwise comparisons comparing inactivation to control for both aOFC and pOFC confirmed that the effect of inactivation was present for both areas . Inactivation of the pOFC was also associated with a consistent increase in average height .Inactivation of the aOFC and pOFC on the human intruder test A both cae height . There wF = 3.17, P = 0.059; area: F < 1; area × treatment: F = 2.33, P = 0.118). There were no significant effects for RMSSD  = 2.10, P = 0.148; area: F < 1; area × treatment: F = 1.76, P = 0.198), CSI  = 1.23, P = 0.309), or CVI  = 1.26, P = 0.301). A ratio of CSI to CVI was also analyzed but did not reveal significant effects  = 1.70, P = 0.207). For sysBP, there was a significant area by treatment interaction  = 4.23, P = 0.026; area: F = 1.16, P = 0.291; treatment: F = 3.17, P = 0.055). However, post hoc pairwise comparisons did not reveal any significant effects of either treatment when compared with saline , only between CGP-LY and Mus-Bac for aOFC . All of the above data were collected after marmosets showed evidence of successful habituation and stable cardiovascular activity  revealed no significant effects of treatment (F < 1) or treatment by area interaction  = 2.15, P = 0.238).A 2-way ANOVA (area × treatment) on the cardiovascular responses to the USF = 24.27, P = 0.001; area × treatment: F = 8.19, P = 0.064; treatment × CS: F = 1.69, P = 0.262; treatment: F = 7.02, P = 0.077; CS: F = 47.61, P < 0.001). Post hoc pairwise comparisons revealed a significant reduction of CS+-directed responses following aOFC inactivation compared with saline (P = 0.033), with no effect on the CS− . Despite this, CS+-directed responses were still significantly higher than those to the CS− , although blunted compared with saline . They were also significantly above their preceding baseline . In contrast, there were no significant effects of inactivation of pOFC .A 3-way ANOVA (area × treatment × CS) on CS-directed behavioral responses revealed a significant 3-way interaction  = 3.02, P = 0.124), with only a significant effect of CS  = 21.16, P = 0.002; treatment × CS:F = 1.01, P = 0.418; area × treatment: F = 2.75, P = 0.196; treatment: F < 1). However, it can be seen that following aOFC inactivation there was a consistent reduction in HR that mirrored that seen in behavior. When the effects on the CS+-directed HR were considered separately using a 2-way repeated measures ANOVA (area × treatment), there was a significant interaction  = 29.06, P = 0.013; area: F < 1; treatment: F = 1.94, P = 0.258), with pairwise comparisons confirming a significant effect of aOFC inactivation compared with the control (P = 0.037), but no effect with pOFC inactivation (P = 0.814).For HR, there was no significant 3-way interaction on the ANOVA , based on the order of occurrence rather than target area, did not reveal a significant effect of order  = 11.26, P = 0.009, session order by CS type: F < 1; US+-directed HR responses, saline 1 vs. saline 2 paired t-test—t = 1.50, P = 0.231).There was no evidence of significant habituation in performance, based on the US+ in the absence of the US+  revealed that HR responses showed a consistent decline following CSsessions   Fig. 3.F = 8.65, P = 0.060) and no treatment by phase interaction  = 3.80, P = 0.145) and no treatment effect (F < 1).Effects of pOFC inactivation on HR recovery were analyzed using a 2-way repeated measures ANOVA (treatment × phase), where the 60-s recovery period was split into three 20-s recovery bins  and normalized to the CS-directed response. The analysis revealed a trend toward an effect of phase , to identify any effects on the response which could affect subsequent recovery. The analysis revealed a significant effect of area  = 10.93, P = 0.010), a trend toward an area by treatment interaction  = 4.11, P = 0.081) and no main effect of treatment  = 2.22, P = 0.179). Post hoc pairwise comparisons revealed no effect of pOFC inactivation on CS-directed responses , but a clear trend toward a reduction of responses with aOFC inactivation, similar to that seen on the standard task . A single-sample t-test against 0 revealed that the HR increase above baseline was significant for both the pOFC control (P = 0.018) and inactivation (P = 0.018).CS− predicted 2 min of a visible empty food box and the CS+ predicted 2 min of access to a food box filled with marshmallow .On the test day, marmosets were presented with a CS the CS− B, as wel the CS− . Inactivificance C. Despitificance D and theificance E. Inactiificance F–I.+-CS− blood pressure responses revealed a significant area by treatment interaction  = 6.75, P = 0.023). Post hoc pairwise comparisons revealed a significant reduction following aOFC inactivation relative to its control , with no effect of pOFC inactivation . The same analysis on head jerk behavior revealed only a trend toward an effect of treatment  = 0.093; area: F = 4.24, P = 0.060). In addition, there were no significant effects with respect to the US for either blood pressure  = 2.19, P = 0.167; treatment × area: F < 1) or marshmallow consumption . The responses to the CS− and CS+ individually were also analyzed, revealing equivalent effects  of the CS effects .+-directed and CS+-directed sysBP responses across time. The analysis of the 2 saline sessions , based on the order of occurrence rather than target area, did not reveal a significant effect of order  = 93.27, P = 0.002; session order × CS type: F < 1; 2-way repeated measures ANOVA on US-directed responses across saline sessions [order × US type]—order F < 1; US type F = 31.25, P = 0.011; order × US type F = 7.35, P = 0.073).There was no evidence of significant habituation in performance across the course of the Pavlovian appetitive discrimination study, based on the US+ omission (CS+ presented without the expected outcome). This involved presentation of a single CS+ trial. Unexpectedly, inactivation of the aOFC did not produce the expected blunting of CS+-directed responses previously observed in sessions with both trial types  was conducted using a 3-way linear mixed-effects model analysis . The analysis revealed a significant 3-way interaction between area, treatment, and session type  = 4.58, P = 0.041; area × treatment: F = 2.97, P = 0.096; treatment × session type: F = 1.33, P = 0.260; treatment: F < 1; session type: F < 1). Importantly, the analysis revealed that there was a significant difference in the effects of inactivation when the CS+ was presented following a CS− trial and when it was presented alone . Additional post hoc pairwise comparisons confirmed the significant difference between CS+-directed responses following aOFC inactivation compared with the aOFC control in discriminative test sessions (P = 0.016), but showed no effect of inactivation in sessions with the CS+ alone (P = 0.284). In contrast, consistent with no effects of pOFC inactivation on the standard task, there were no effects of pOFC inactivation on the CS+-directed responses compared with the pOFC control in either session type .The comparison of CS+-directed responses were not affected in the single trial sessions and were equivalent following saline and inactivation infusions allowed for the comparison of both aOFC and pOFC inactivation effects on the recovery of appetitive conditioned responses with US+ omission. Inactivation of the aOFC appeared to accelerate the recovery of CS+-directed sysBP responses  = 9.16, P = 0.004), area  = 28.93, P < 0.001), recovery phase , and no other effects  = 3.40, P = 0.072; area × phase: F = 1.22, P = 0.305; treatment × phase: F < 1; area × treatment × phase: F < 1). Post hoc pairwise comparisons revealed that the effect of treatment was primarily attributable to the aOFC, with no effects in the pOFC. There were significant effects of aOFC inactivation on recovery in the later phases  but not the pOFC .A 3-way linear mixed-effects model analysis (area × treatment × recovery phase) on sysBP recovery revealed significant effects of treatment  rather than certain .A major component of affective disorders is increased morbidity and mortality resulting from cardiovascular disease , and recTogether, these experiments into OFC function have presented a unique profile of effects on threat- and reward-elicited behaviors. The effects of aOFC and pOFC inactivation, primarily encompassing areas 11 and 13, respectively, set them apart from the neighboring medial OFC area 14. While aOFC and pOFC inactivation heighten responses to a distal threat, the same effect is observed following overactivation of area 14 . In all In summary, this study has begun to separate out the contribution of the aOFC and pOFC from neighboring regions in the regulation of distal and proximal threat responses. In particular, the finding that inactivation of aOFC and pOFC, but overactivation of areas 14 and 25, induce an apparently similar high anxiety-like phenotype in response to distal threat highlights the varied neurobiological mechanisms that can underlie anxiety symptoms in humans and which may contribute to the individual variation in responsivity to treatments. We propose that a comparison of distinct classes of anxiolytics on heightened threat reactivity induced by opposing interventions in these different prefrontal and cingulate brain regions will provide important new insights into individualized treatment strategies.ZStawicka_Supplementary_Final_bhab240Click here for additional data file.


Retrieval practice improves retention of information in long-term memory more than restudy, but the underlying neural mechanisms of this “retrieval practice effect” (RPE) remain poorly understood. Therefore, we investigated the behavioral and neural differences between previously retrieved versus restudied items at final retrieval. Thirty younger (20–30 years old) and twenty-five older (50+ years old) adults learned familiar and new picture stimuli either through retrieval or restudy. At final recognition, hemodynamic activity was measured using functional magnetic resonance imaging (fMRI). Behaviorally, younger and older adults showed similar benefits of retrieval practice, with higher recollection, but unchanged familiarity rates. In a univariate analysis of the fMRI data, activation in medial prefrontal cortex and left temporal regions correlated with an individual’s amount of behavioral benefit from retrieval practice, irrespective of age. Compatible with this observation, in a multivariate representational similarity analysis (RSA), retrieval practice led to an increase in pattern similarity for retested items in a priori defined regions of interest, including the medial temporal lobe, as well as prefrontal and parietal cortex. Our findings demonstrate that retrieval practice leads to enhanced long-term memories in younger and older adults alike, and this effect may be driven by fast consolidation processes. Compared to repeated study, retrieval practice, or testing of information, is a more beneficial approach to learning and retaining information . DespiteIn terms of mechanistic explanations of the RPE, the fast route to consolidation (FRC) hypothesis  suggestsDirect empirical evidence in humans in favor of the FRC hypothesis is limited, with some studies finding increased activity in cortical areas , but onlTo address these questions, we used a previously established RP paradigm , 2020, iOur sample comprised a total of 64 participants, 31 younger (20–30 years of age) and 33 older (51–77 years of age). They were recruited through the Online Recruitment System for Economic Experiments . InclusiThe experimental paradigm was almost identical to the paradigm described in In Phase 2, participants had to perform two different, randomly alternating tasks, while viewing 160 new stimuli randomly intermixed with the 160 familiarized stimuli. The tasks were designed to induce a restudy, and a retrieval context for half of the new and familiarized stimuli each. In the study task (STU), participants gave simple indoor/outdoor categorization judgments, using keyboard button presses. In the retrieval practice task (RET), participants gave an old/new recognition judgment, again through button presses. The combination of the two factors Task (STU/RET) and stimulus Novelty (OLD/NEW) resulted in a 2 × 2 repeated measures design with 80 stimuli per condition. Task blocks were 8 trials long, each block containing 4 OLD and 4 NEW stimuli in random order. Task (STU/RET) was also randomized across blocks. An instruction on the screen informed participants about the upcoming task prior to the start of each block. Images were presented for 1 s with an interstimulus interval of 3 s (fixation cross). Participants gave their response within 2.8 s using their right index and middle finger. Slower responses were coded as “no button press.” Response-button mappings were counterbalanced across participants. Participants could make a self-paced pause every 64 trials.and could recollect specific thoughts or associations linked to the study episode (recollection). They were asked to choose “Know” when they recognized the picture but were not able to recall specific associations related to the study episode (familiarity). “Unsure” was to be pressed when they did not know whether a picture was old or new, and “New” when they were sure they had not seen the picture before. The jittered interstimulus interval was 3 s  during which participants could still give their response using the scanner button boxes. Participants could make a self-paced pause every 60 trials. Phases 1 and 2 were performed consecutively, with small breaks (5–10 min) between them. Phase 3 was conducted in four functional runs, of circa 10 min each. Participants could do self-paced break between runs.For Phase 3, participants were brought into the scanner and viewed the stimuli via a mirror reflecting a screen positioned at the back of the scanner. In the scanner, participants performed a surprise recognition task roughly 15–20 min after the end of the second phase . The 320 previously encountered stimuli, counterbalanced for location (indoor/outdoor), stimulus Novelty, and Task in Phase 2, were intermixed with 160 unseen distractor images . Each image was presented for 1 s with four response options in German below the image: Remember—Know—Unsure—New . Participants were instructed, orally and in writing, about the meaning of each response option. They were asked to choose “Remember” when they recognized a picture Images were randomly assigned to the different phases and conditions for each subject. To control for effects of illumination, mean luminance on each color channel  was set to 127  and images were presented on a gray background of equal luminance. Prior to each phase of the experiment, participants completed a brief training session, introducing the participant to the upcoming task while using a training set of visual stimuli . For Phase 3, participants practiced twice, once outside and once inside of the scanner. Images used during the training phase were not shown again.d′ (d-prime) was calculated by subtracting the inverse phi  of the hit rate from the inverse phi of the false alarm rate for each subject and condition. As the inverse phi of 0 and 1 is -∞ and ∞ respectively, 0.5 was added to the number of hits and false alarms and 1 was added to the number of signal and no signal trials . Calculation of d′ and hit rates was performed in MATLAB 2019a , while the Bayesian analysis of behavioral data was performed with JASP  × 2  × 2 (stimulus Novelty: old vs new) × 2  repeated measures (Bayesian) ANOVA, and post-hoc tests were conducted on interactions using ith JASP . Barplotith JASP .3; 80 × 80 matrix; 60 transversal slices; GRAPPA factor 2 and simultaneous multislice factor 2). We recorded 4 runs with 345 volumes each (plus 6 dummy scans). Structural images of the whole brain using a 3D T1-weighted MP-RAGE sequence were acquired . Additionally, we acquired resting-state runs of each participant ; however, those data will not be presented in this article. Data were preprocessed using SPM 12  and MATLAB. EPIs were slice time corrected and realigned to correct for head motion. T1 images were coregistered onto mean EPIs. All images were normalized to MNI space based on normalization parameters derived from a segmentation of T1-weighted images into white matter, gray matter, and CSF using default tissue probability maps. Smoothing was performed with a 6 mm full width at half maximum (FWHM) Gaussian kernel. As mentioned above, one participant was excluded due to excessive head motion (exceeding 4 mm in each run). For the RSA, preprocessing followed a similar pipeline but neither normalization nor smoothing was performed on the functional data. In addition, T1 images (mapped onto mean EPIs) were segmented using FreeSurfer  sequence sensitive to blood oxygen level dependent (BOLD) contrast  and custom MATLAB scripts, in case of the RSA. For the univariate analysis in SPM, first- and second-level analyses were performed. On the first level, we included regressors for the levels of Task  and Novelty , resulting in the conditions study-old, study-new, retrieval-old, and retrieval-new. Correct rejections, false alarms, unsure responses, and errors were also modeled on the first level, but not included in the analysis on the second level. The factor memory type (remember vs know responses) could not be included due to insufficient trial numbers in each condition . We selected this cluster extent based on our primary focus of cortical areas, not warranting smaller cluster sizes, which in turn can lead to reporting of spurious effects  and novelty (old vs new), and the between-subjects factor age group (young vs older) see . The unc effects .RSA—multivariate mixed model) were defined with FreeSurfer segmented maps, and data from each ROI were extracted from each trial’s image. Furthermore, we conducted an analysis subdividing the frontal cortex into dorsolateral prefrontal cortex (dlPFC), inferior frontal gyrus (IFG), orbitofrontal cortex (OFC), and caudal anterior cingulate , to further investigate the role of the frontal cortex in retrieval practice. As we were interested in the effects of retrieval practice on cortical representations and, more specifically, whether similarity between stimuli changed, we correlated voxel patterns of every trial with voxel patterns of every other trial across all voxels of a given ROI, for every participant separately. We only included between-run correlations. Then, the correlations from different conditions were averaged across trials. Data were subsequently analyzed across participants in a linear mixed multilevel model in RStudio, Version 1.1.463 . Systematic effects of motion were regressed out (using the glmfit function in MATLAB). The preidentified regions of interest  and reaction times (RTs) in the target detection task between young and older participants in a two-sample T-test . On average, accuracy was high in both age groups, and there was no significant difference in performance between young and older adults (P > 0.4), d′young = 5.43 ± 0.3; d′older = 5.36 ± 0.38 (mean d′ ± SD). Similarly, there was no significant difference in RT between both groups, only a trend for older participants to be slower, t(53) = −1.96, P = 0.055, RTyoung = 556 ± 71 ms, RTolder = 594 ± 71 ms (mean RT ± SD).In Phase 1, we compared accuracy  as within-subjects factor and age (young vs old participants) as between-subjects factor. Note that the factor novelty cannot be analyzed here . For RTs, we conducted a 2 × 2 × 2 ANOVA including the within-subjects factors task and novelty (old vs new stimuli), and the between-subjects factor age. We tested assumptions of normality of residuals with Kolmogorov–Smirnov tests and homogeneity of variance with the Levene test. As some assumptions  were violated for some variables, we conducted Bayesian repeated measures ANOVAs alongside the traditional frequentist analysis. In terms of memory accuracy  = 270.89, P < 0.001, η2 = 0.84), but no main effect of age and no interaction of age × task (P > 0.5). A post-hoc T-test revealed that accuracy was lower in the retrieval task as compared to the study task (t(54) = 16.55, P < 0.001). The Bayesian ANOVA confirms this result, suggesting that the best model includes the factor task (BF10 = 4.84e+28).For Phase 2, we analyzed racy see , the freF = 144.68, P < 0.001, η2 = 0.73) and novelty  = 16.86, P < 0.001, η2 = 0.24), and an interaction of task x novelty  = 5.81, P = 0.019, η2 = 0.1) in the frequentist ANOVA. All other effects were not significant (P > 0.1). Post-hoc T-tests revealed that participants were faster in the study  task (T(54) = −12.07, P < 0.001), and faster for old (compared with novel) stimuli (T(54) = −4.17, P < 0.001). In the Bayesian ANOVA, the best model included both factors task and novelty (BF10 = 2.74e+37), but not the interaction of task x novelty (BFinclusion task x nov = 0.58). Similarly as in the frequentist results, descriptively, participants were slower in the retrieval task, as well as for new items.For reaction times see , there wd′ in a 2 × 2 × 2 × 2 ANOVA, with memory (remember vs know), task , novelty (old vs new in Phase 2), and age (young vs older participants) as factors. Homogeneity of variance (Levene test) was present in all but one variable (retrieved new items that were rated as “known”), and normality of residuals (Kolmogorov–Smirnov tests) was present in all but three variables . Because of the large sample size of n = 55 subjects in total, we still conducted frequentist ANOVAs, but we also conducted a Bayesian repeated measures ANOVA. In the Bayesian analysis, the best model (BF10 = 3.3e+100) mirrored the main effects found in the frequentist ANOVA described below, as well as the interaction of memory by novelty. A model that also included the interaction of memory by task also performed very well (BF10 = 2.55e+100).In Phase 3, we analyzed F = 161.83, P < 0.001, η2 = 0.75), task  = 34.37, P < 0.001, η2 = 0.39), novelty  = 131.4, P < 0.001, η2 = 0.71), and age  = 6.59, P = 0.013, η2 = 0.11). Furthermore, post-hoc T-tests revealed higher recollection than familiarity rates (T(54) = 12.9, P < 0.001), higher memory scores for old items (T(54) = 12.05, P < 0.001), and, importantly, higher memory scores for items previously encountered in the retrieval task  = −7.25, P < 0.001, see T(53) = 1.49, P = 0.071); thus, despite the significant main effect of age in the ANOVA, the effect in the post-hoc T-test was only at trend level  = 17.58, P < 0.001, η2 = 0.25), which was driven by a significant difference between study and retrieval for remembered items, but not for known items (see T(54) = 7.0, P < 0.001, and T(54) = 0.72, P > 0.4, respectively). There was a disordinal interaction between memory and novelty  = 106.23, P < 0.001, η2 = 0.67); old items were associated with more recollection and fewer familiarity responses (see T(53) = −10.0, P < 0.001, and T(48) = −4.27, P < 0.001, respectively). There were no other significant interactions, including task by age .There was a significant interaction of memory by task , and novelty (old vs new in Phase 2), and the between-subjects factor age (young vs old) revealed main effects of age and novelty. The age effect (contrast older > younger) was associated with widely distributed higher activity for older adults in the bilateral precentral/postcentral gyrus, medial and lateral PFC, temporal cortex, basal ganglia, bilateral thalamus, temporal cortex, and supramarginal gyrus see . For thed′Retrieval—d′Study in Phase 3) as a predictor for the difference in brain activation of retrieval versus study . In this analysis, we pooled young and older participants (i) since there was no significant interaction between age and task at the behavioral level; (ii) while the behavioral main effect of task was very strong, the main effect of age only reached trend levels in the post-hoc T-test; and (iii) since the focus of this work was on the retrieval practice effect, while age was of secondary importance. We found three significant clusters (see P < 0.025).We performed a multiple regression analysis, with retrieval benefit . The parietal cortex ROI included the supramarginal gyrus and precuneus  the fronSecond, we investigated differences between the hippocampal formation and 12 other ROIs ; see in X2 = 70.5, P < 0.0001, dfs = 1) for pattern similarity . In terms of fixed effects, the model including task had a significantly better fit  for explaining the pattern similarity data. ROI area  also improved the model significantly . Neither stimulus novelty (P > 0.5) nor age (P > 0.4) as fixed effects improved the model significantly in comparison to the more parsimonious random effects model. The model including both Task and ROI as fixed effects  slightly outperformed either model containing only one of the factors . There were no significant 2-, 3-, or 4-way interactions .The mixed-model analyses revealed participant as a significant random effect : the final model thus included participant as random effect, ROI area and Task as fixed effects, and Task as random slope.We tested for random slopes of Task and ROI. Including Task as a random slope significantly improved model fit  was associated with higher mean similarity with no significant differences between both age groups  suggestiX2 = 74, P < 0.0001, dfs = 2). No interactions contributed to the model fit.The results for the subdivided ROIs mirrored the results for the ROI areas. The final model includes participant as a random effect, Task and ROI as fixed effects, and a random slope of Task .To further investigate a possible interaction of Task and ROI area in line with the FRC hypothesis, we performed post-hoc P > 0.4).In analogy to the regression analysis in the univariate approach, we investigated a possible correlation between individual behavioral retrieval practice benefit and individual random slopes of task. There was no significant correlation  as compared to the medial temporal lobe, including the hippocampus. As such, our findings provide novel insights into the underlying mechanisms of RP across the life span and they are compatible with the notion that the beneficial effects of retrieval practice on recognition memory can—at least in part—be explained by fast changes in neocortical representations.As hypothesized, retrieval of information increased recollection-based recognition memory in both younger and older adults . ImportaInterestingly, the RPE was present irrespective of stimulus novelty in Phase 2. That means, not only familiar but also novel stimuli that were shown in the context of retrieval were remembered better as compared to novel items shown in the restudy context. This is compatible with previous work  includinAt the neural level, a multiple regression analysis across all participants showed that the RPE was correlated with activity in the mPFC, anterior cingulate cortex, left superior temporal gyrus, and temporal pole, with stronger activation in these areas relating to larger RP benefits on an individual level. According to traditional models of systems consolidation, the hippocampus initially stores novel information; over time, through postlearning reactivation in resting periods and sleep, this information is being transferred to and stored within the neocortex including the PFC . ImportaWhile our goal was to analyze recollection and familiarity separately, based on remember and know responses, to further investigate contributions from episodic memory, this was not possible due to a large proportion of participants not providing enough responses in at least one of the categories. As individuals show large biases in responding with either “remember” or “know” (see also The multivariate RSA provides some preliminary evidence that RP leads to rapid changes in the neural representation of information. Specifically, items previously encountered in a retrieval context were, after circa 20 min, significantly more similar in their neural representations in a priori defined regions of interest , as compContrary to our hypothesis and own previous work , 2020, tFinally, direct changes in representational similarity from one phase to the next cannot be assessed in an experimental design that does not measure neural activity at each phase of the paradigm. However, as stimuli were randomized for novelty and task across participants, and the only difference between the stimuli in the contrast “restudy vs retrieval practice” was whether they had been retrieved or restudied in Phase 2, it is extremely unlikely that the observed differences between restudy and retrieval stimuli arose from a different source than the task condition itself.In summary, the retrieval practice effect is characterized by rapid and specific increases in recollection-based recognition memory and changes in neural activation in both young and older adults. In particular, activity in the mPFC/anterior cingulate and left anterior temporal lobe predicted the increase in recognition memory through retrieval, suggesting an involvement of brain regions previously associated with semantic processing, fast learning, and memory consolidation. Furthermore, RP increased pattern similarity, and pattern similarity was generally higher within the frontal cortex, parietal cortex, and temporal cortex. As such, our data extend current theoretical views of the RPE and provide empirical evidence that retrieval practice involves fast changes in neocortical representations of information.Guran_etal_CCC_SuppMat_Feb22_tgac009Click here for additional data file.


Lead is a nonessential heavy metal, which can inhibit heme synthesis and has significant cytotoxic effects. Nevertheless, its effect on the electrical properties of red blood cells (RBCs) remains unclear. Consequently, this study aimed to investigate the electrical properties and the electrophysiological mechanism of lead exposure in mouse blood using Electrical Impedance Spectroscopy (EIS) in 0.01–100 MHz frequency range. Data characteristic of the impedance spectrum, Bodes plot, Nyquist plot and Nichols plot, and Constant Phase Element (CPE) equivalent circuit model were used to explicitly analyze the differences in amplitude–frequency, phase–frequency, and the frequency characteristics of blood in electrical impedance properties.f0) were significantly increased; (iv) the electrical impedance of plasma, erythrocyte membrane, and hemoglobin decreased, while the conductivity increased. (v) The pseudo-capacitance of cell membrane (CPE_Tm) and the intracellular pseudo-capacitance (CPE-Ti) were decreased.Compared with the healthy blood in control mice, the changes in blood exposed to lead were as follows: (i) the hematocrit decreased; (ii) the amplitude–frequency and phase–frequency characteristics of electrical impedance decreased; (iii) the characteristic frequencies (Therefore, EIS can be used as an effective method to monitor blood and RBC abnormalities caused by lead exposure. The electrical properties of the cells can be applied as an important observation in the evaluation of the toxic effects of heavy metals. Lead is a heavy metal material that has been widely applied in pottery, lead welding, dyes, and cosmetics . Exposur. measured the membrane properties of rat erythrocytes and erythrocyte membrane ghosts by either chronic or acute Pb2+ treatments and found that 1.8 μM Pb2+ could directly increase cell membrane permeability, thereby increasing hemolysis  decreases with increasing frequency. As current is hindered by the high impedance of RBCs membrane, it consequently flows through the extracellular plasma, which is expected due to the low impedance properties. Overall, the decreasing trend of the real part of impedance  of RBCs [Notably, we introduce here an estimation protocol based on a multiparameter method of RBCs.  in RBCs ; the for in RBCs , 24. Con in RBCs . Similar in RBCs –28 thus  in RBCs  and decr in RBCs , 28, eve of RBCs .Cm [More importantly, given that these feature parameters defined reference value for blood electrophysiology, future research efforts should explicitly analyze the differences in amplitude–frequency, phase–frequency, and the frequency characteristics of blood in electrical impedance properties. Encouragingly, Constant Phase Element (CPE) equivalent circuit model displayed inspiring curve fitting effect on observation data. The results were consistent with glucose-6-phosphate dehydrogenase (G-6-PD)-deficiency anemia . For sicCm . Our resIn summary, this study used the electrical impedance spectrum, Bode plots, Nyquist plots, and Nichols plots data analysis to confirm that lead exposure can reduce the Hct, decrease the impedance and phase angle characteristics, and increase the first and second characteristic frequencies of blood in mice. These results provided data support and new diagnosis and treatment methods for the hematotoxicity and the potential electrophysiological mechanism of lead exposure. Further, the present study focuses on the electrical properties of RBC suspension using Electrical Impedance Spectroscopy, while the electrical characteristics and microstructural alterations of single cells exposed in Pb have not been deeply studied and explained. Single-cell impedance analysis based on model analysis and parameter extraction deserves in-depth study, which will have more profound research value and reference significance.A total of 20 ICR mice, weighing 27.5 ± 5.1 g on average, were provided by the experimental animal center of Ningbo University. All the animals were housed in an environment with a temperature of 22 ± 1 °C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Ningbo University institutional animal care and conducted according to the AAALAC and the IACUC guidelines.n = 10) was gavaged with 0.5 ml PbA (250 mg/kg bw) per 24 h for 4 weeks; at the same time, the control group (n = 10) was treated with normal saline through identical administration. Blood samples were collected by orbital sinus puncture into heparinized microhematocrit tubes from all mice by diethyl ether anesthesia; blood impedance and hematocrit values were directly determined after pretreatment or centrifugation. Lead acetate (PbA) of AR grade and all other reagents were purchased from Merck  unless otherwise stated.The exposure group (Hematocrit (Hct) was measured with microhematocrit capillary tubes  in a bench-top hematocrit centrifuge  for 5 min at 11,000 rpm. The hematocrit was calculated using the following equation : HematocZ| and the phase angle θ of blood were measured by Agilent 4294A Impedance Analyzer (USA), equipped with Agilent 42942A terminal adapter and an Agilent 16192A parallel electrode SMD test fixture; analyzers were controlled by a Lenovo computer. Measurements were made at room temperature. Dispersion characteristic was measured in the frequency range 0.01–100 MHz with 124 frequency points. Each run was taken three times, and the data points were the average of these runs. The sample cell was made of clear Plexiglas tube and consisted of a chamber with two parallel platinum plates embedded on both sides, which was customized according to size as follows [2 well area, and 0.38 ml sample volume. The complex impedance was expressed as The amplitude | follows  7.7 mm eRm, Ri) and pseudo-capacitance , while the plasma is purely resistive (Rp). The impedance in terms of the parallel/series combinations of elements of the CPE-equivalent circuit is given byZk = Rk, for k ∈ ,  l ∈  while 0 < CPE_T < 1. In addition, α = CPEP. Therefore, the impedance of the equivalent circuit model using fractional circuit theory [s = jω. The complex impedance of (https://softadvice.informer.com/Z_View_Software_Free_Download.html).Importantly, the RBCs may not be an “ideal” capacitor, as commonly assumed in the standard electrical elements. The constant phase element (CPE) model provided the best fit in terms of accuracy for the experimental data . Using tt theory  becomes2t test using SPSS 12.0 software. A p value < 0.05 was considered to be statistically significant.All values were expressed as the means ± standard deviation (SD). The significance of the differences between each value presented by the Control and Lead exposure group was evaluated by the Student


To compare data on transperineal template biopsy (TPTB) under general anesthesia (GA) compared with local anesthesia (LA) procedures using the PrecisionPoint™ Transperineal Access System (PPTAS) in relation to tolerability, cancer detection rate, complications, and cost.A prospective pilot cohort study of patients undergoing transperineal biopsy was performed. Patients were excluded if they had concurrent flexible cystoscopy or language barriers. Patients had a choice of GA or LA. A prospective questionnaire on Days 0, 1, 7, and 30 was applied. The primary outcome was patient tolerability. Secondary outcomes were cancer detection rate, complication rate, and theater utilization.p = 0.3790, p = 0.9832, p = 0.444, p = 0.3939, respectively). Higher median prostate imaging‐reporting and data system (PI‐RADS) score of 4 (interquartile range [IQR] 2) versus 3 (IQR 1) was noted in the LA group (p = 0.0326). Pain was higher leaving recovery in the GA group however not significantly (p = 0.0555). Median pain score at LA infiltration was 5/10 (IQR 3), with no difference in pain at Days 1, 7, or 30 . For GA versus LA, the overall cancer detection rate was 55% versus 55% (p = 1.000) with clinically significant cancer in 22.5% versus 35% (p = 0.217). Acute urinary retention (AUR) occurred in 5% of GA and 2.5% of LA patients (p = 1.000). The GA cohort spent longer in theater and in recovery with a median of 93.5 min versus 57 min for the LA group (p = <0.0001).This study included 80 patients (40 GA TPTB and 40 LA PPTAS). Baseline characteristics including age, prostate‐specific antigen (PSA), digital rectal examination (DRE), findings, and prostate volume were comparable between the groups (This study demonstrates that transperineal biopsy is safely performed under LA with no difference between the cohorts in relation cancer detection or AUR. LA biopsy also consumed less theater and recovery resources. A further larger prospective randomized controlled trial is required to confirm the findings of this study. The inclusion criteria were all patients undergoing TPBx at our institution from February 2020 to September 2020. Patients who had concurrent procedures such as flexible cystoscopy were excluded to avoid confounding factors affecting the outcomes of this study. Non‐English‐speaking patients, as defined by the hospital admission information, were excluded. This was in relation to the process of reading and understanding the consent and study participant information as well as feasibility for follow‐up phone calls. There were five patients excluded due to language barriers. Patients who met the inclusion and exclusion criteria were given time to read the patient information sheet and were consented for participation in this study. No patients declined to participate in the study. As this is a cohort study, enrolment in the LA versus GA TPBx arm of the study was based on patient preference after informed consent and pre‐operative discussion with patients regarding the risk versus benefits of prostate biopsy performed under LA versus GA. Patients who initially underwent LA TPBx but needed a conversion to GA were not included in the final data analysis but were captured to determine the rate of conversion to GA. As this was a pilot study, sample size calculations were not performed. The study aimed to enroll 80 patients (40 LA PPTAS and 40 GA TPTB).Prior to the procedure, patients were asked to rate their pain on a numeric rating scale (NRS) numbered 0–10 with 0 being “No pain” and 10 being “Worst pain,” Pain scores were assessed at eight time points: pre‐operatively, LA infiltration, ultrasound probe insertion, biopsy gun firing, leaving recovery, Day 1 , Day 7, and Day 30. All patients were given a day of surgery questionnaire as well as postoperative phone call questionnaires on Days 7 and 30. A copy of this questionnaire is available in the Clinicopathological data were collected from hospital electronic medical records and included demographic information; age, date of biopsy, prostate‐specific antigen (PSA), comorbidities, digital rectal examination (DRE) findings, multiparametric magnetic resonance imaging (mpMRI) prostate imaging‐reporting and data system (PI‐RADS) score, prostate volume, number of cores taken, and International Society of Urological Pathology (ISUP) histopathological findings. Complications and return to work data were recorded at the time of follow‐up phone call on Days 7 and 30. Ethical approval was granted by the Monash Human Research Ethics Committee on January 29, 2020. Governance approval was obtained from Western Health's Institutional Review Board following this. Informed written consent was obtained from all participants.Stata Statistical Software: Release 16 . Descriptive statistics were used to describe the study groups. Mann–Whitney U test was used to compare median values for nonnormally distributed data. Independent t‐test was used to compare mean values for normally distributed data. Fisher's exact test of independence was used to determine associations between categorical variables given the small sample size. A two‐sided p‐value <0.05 was considered statistically significant.Data analysis was performed using 2.1Two transperineal biopsy approaches, LA PPTAS and GA TPTB, were used in this study. All procedures were carried out in the day procedure setting. All patients were prescribed 400‐mcg Tamsulosin for 3 days pre‐operatively and for 7 days postoperatively. All patients were compliant with this pretreatment. Patients received perioperative intravenous (IV) administration of antibiotics: 2‐g cefazolin, 2‐g cefazolin, and 160‐mg gentamicin or 500‐mg ciprofloxacin depending on surgeon preference. Of the 40 LA PPTAS patients, 20 did not receive any antibiotic prophylaxis. All biopsy naïve patients had a prebiopsy mpMRI performed. Tensile tape (Elastoplast) was used to elevate the scrotum away from the perineum. All biopsies had transrectal ultrasound guidance with the BK™ endocavity 8848 ultrasound probe  in the axial and sagittal planes. All patients had a systematic transperineal biopsy regardless of biopsy approach with cognitive‐assisted target sampling if present on prebiopsy mpMRI. The number of biopsies varied depending on prostate size and surgeon preference. All patients were required to void once prior to discharge.LA PPTAS—Patients were placed in the low lithotomy position. LA to the skin of the intended needle entry site was infiltrated with 10 ml of 1% lignocaine with 1:200000 adrenaline. Peri‐prostatic nerve block using up to 15 ml of 1% lignocaine without adrenaline was then performed bilaterally with transrectal ultrasound guidance. LA was given 5 min to take effect. The ultrasound probe with the attached PPTAS device was then reinserted into the rectum and the access needle advanced into the perineum at the previously anesthetized site. The biopsy gun was then introduced through the access needle, and all cores were obtained through the one perineal access point or two perineal access points on each side, depending on prostate size. The threshold for conversion to GA was relatively low to avoid unnecessary patient discomfort. If a patient did not tolerate a DRE on table or did not tolerate the insertion of the local anesthetics needle, they were converted to GA.GA TPTB—Patients were placed in high lithotomy position. Template biopsies utilized the brachytherapy 5‐mm grid/stepper unit . Administration of local anesthetic (approximately 20 ml of 0.75% Ropivocaine) to achieve a pudendal nerve block on completion of the procedure was dependent on surgeon preference.3p = 0.122). The patient characteristics pre‐operatively are summarized in Table p = 0.0326).A total of 40 men underwent GA TPTB and 40 men underwent LA PPTAS during the study period. Comparing the GA TPTB group with the LA PPTAS group, the median age was 63 (interquartile range [IQR] 10.5) versus 63.5 (IQR 10), the mean PSA was 7.60 (±5.02) versus 7.36 (± 4.32), the mean prostate volume was 49.42 (±18.99) versus 51.03 (±17.03), and the median IPSS was 9p = 0.0080). Using the NRS to evaluate pain postoperatively, the score was not different between the groups on Day 1 (p = 0.1783), Day 7 (p = 0.2202), or Day 30 (p = 0.5686). At the time of leaving the recovery area patients in the GA TPTB group tended to have more pain compared with the LA PPTAS group, but this did not reach the threshold for significance (p = 0.0555). When assessing patients return to work following the procedure, there were 38 patients in employment at the time. Of these 81.5% (n = 31) returned to work within 7 days of the procedure. Patients who had an LA procedure returned to work significantly faster than those who had a GA (p = 0.0459). This is summarized in Table At baseline there was a higher median NRS pain score seen in the LA PPTAS  reported they would be happy to have this procedure performed again if necessary. Similarly, 97.5% of patients in the GA TPTB group would have the same procedure again (p = 0.359). There were two patients  excluded from the study who did not tolerate LA infiltration and required conversion to GA biopsy in the study period. Of the 9 patients in the LA group versus the 8 patients in the GA group who had had a previous GA TPTB; 22.2% (n = 2) versus 37.5% (n = 3) preferred the current procedure, 55.6% (n = 5) versus 50% (n = 4) felt it was comparable to their previous procedure, and 22.2% (n = 2) versus 12.5% (n = 1) felt it was worse than their previous procedure.In the LA PPTAS group 90% (n = 22). The rate of clinically significant PCa (csPCa) ISUP≥2 was 22.5% (n = 9). For patients undergoing a primary biopsy (n = 32), the cancer detection rate overall was 53.1% (n = 17) and for csPCa was 21.6% (n = 7).In the GA TPTB group the overall rate of cancer detection was 55% (n = 22). The rate of csPCa ISUP≥2 was 35% (n = 14). For patients undergoing primary biopsy (n = 31), the cancer detection rate overall was 48.3% (n = 15) and for csPCa was 35.5% (n = 11). There was no statistically significant difference between the two groups when assessing overall cancer detection rate (p = 1.000). The median number of cores taken in the GA TPTB cohort was 27 (IQR 8), compared with 33 (IQR 21.5) in the LA PPTAS cohort (p = 0.0084).In the LA PPTAS group the overall rate of cancer detection was 55% (n = 2) of patients in the GA cohort compared with 2.5% (n = 1) of the LA cohort (p = 1.000). The episode of AUR in the LA group occurred Day 9 postoperatively and was associated with urosepsis secondary to gram‐negative bacteraemia requiring catheterization and intravenous antimicrobial therapy. The patient presented with urinary retention followed by fever but remained haemodynamically stable through the entire episode and only required ward‐based management.Acute urinary retention (AUR) requiring catheterization occurred in 5% (n = 2) of the LA group (p = 0.494). One patient in the GA group was treated with oral antibiotics in the community for dysuria postprocedure; however, his urine sample was sterile. Nonelective re‐admission occurred in 2.5% (n = 1) of the GA cohort compared with 5% (n = 2) of the LA cohort (p = 0.494). All complications that occurred were classified as Clavien‐Dindo ≤2.Culture positive symptomatic UTI occurred in 0% of the GA group and in 5% (p = 0.5292). When anesthetic  time was included, the GA TPTB cohort had a significantly longer time in theater (p < 0.0001). The time the patient was in the theater department from the beginning of the procedure to leaving recovery was significantly shorter in the LA PPTAS group (p < 0.0001). Men in the LA PPTAS group spent a significantly shorter period in the recovery area after the procedure (p < 0.0001). A summary of the times can be found in Table There was no difference in procedure specific time between the cohorts . The use of a pudendal nerve block post operatively in the GA TPTB group was at the discretion of the operating surgeon and therefore was not administered to all patients. This may contribute to the pain score postoperatively in the GA TPTB group. The GA TPBx patients also had a higher number of puncture sites due to the nature of the procedure which may have contributed to their pain scores. In the follow‐up questionnaire 90% (n = 36) of LA patients reported, they would be satisfied to have the same procedure again where they were to require a repeat biopsy. This compares to 97.5% (n = 39) of the GA cohort (p = 0.359).Pain scores on leaving the recovery area trended towards being higher in the GA TPTB group compared with the LA PPTAS group (p = 1.000). A recently published study looking at 174 standard grid‐based biopsy patients compared with 304 freehand biopsy patients has shown an equivalent cancer detection rate between both techniques.It has been well documented that the TP route of biopsy has a superior cancer detection rate compared with TRBx.n = 2) and is in keeping with other PPTAS studies.In the first 7 days following the procedure, there were no documented new infections in either group, indicating that there was no introduction of bacteria via the needle biopsy. This is consistent with previous literature and supports the adoption of the TP route rather than the TR route as a safer method of biopsy by avoiding fecal contamination.n = 2) in the GA TPTB group which was not statistically different to the 2.5% (n = 1) in the LA PPTAS group (p = 1.000). In line with current local guidelines, patients were given 400‐mcg Tamsulosin for 3 days pre‐operatively and this was continued for a further 7 days postoperatively. Compliance with this pretreatment was recorded, and all patients were compliant. Compared with the previous literature on PPTAS, our rate is consistent with Meyer et al. (4.7%), Kum et al. (0.6%), and Gorin et al. (1.1%). Urkmez et al. have assessed 304 patients following a freehand biopsy and reported an AUR rate of just 1%.The rate of AUR requiring catheterization in the present study was 5% , the overall time spent by the patient in the theater including administration of anesthesia was significantly longer in the GA TPTB group (p < 0.001). Given the PPTAS device is new to the surgeons involved, it is anticipated that the procedure time will decrease with increasing experience of the device. In further studies, precise and comprehensive costing per patient will be obtained.Consumption of less theater and recovery times allows the theater resources to be more efficiently utilized to care for more patients. This is important, especially during the COVID‐19 pandemic, with a scarcity of theater resources available to manage the ever‐expanding waiting list. Furthermore, utilization of theater and recovery time can be considered a surrogate marker for cost. Although procedure specific time was similar between the groups : noneGrants or contracts from any entity: noneRoyalties of licenses: noneConsulting fees: nonePayment or honoraria for lectures, presentations, speakers' bureaus, manuscript writing, or educational events: nonePayment for expert testimony: noneSupport for attending meetings and/or travel: nonePatents planned, issued, or pending: noneParticipation on a data safety monitoring board or advisory board: noneLeadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: noneStock or stock options: noneReceipt of equipment, materials, drugs, medical writing, gifts, or other services: noneOther financial or nonfinancial interests: noneWe confirm that this paper has not been published or submitted for publication elsewhere and that all authors have contributed significantly and are all in agreement with the content of the manuscript. The following apply to all listed authors:Data S1. Supporting InformationClick here for additional data file.


We recently showed headache to be common in children (aged 7–11 years) and adolescents (aged 12–17) in Lithuania. Here we provide evidence from the same study of the headache-attributable burden.Lifting The Burden’s global schools-based study, this cross-sectional survey administered self-completed structured questionnaires to pupils within classes in 24 nationally representative schools selected from seven regions of the country. Headache diagnostic questions were based on ICHD-3 beta criteria but for the inclusion of undifferentiated headache . Burden enquiry was conducted in multiple domains.Following the generic protocol for Questionnaires were completed by 2505 pupils , of whom 1858 reported headache in the preceding year, with mean frequency (±SD) of 3.7 ± 4.5 days/4 weeks and mean duration of 1.6 ± 1.9 h. Mean proportion of time in ictal state, estimated from these, was 0.9% . Mean intensity on a scale of 1–3 was 1.6 ± 0.6 (mild-to-moderate). Symptomatic medication was consumed on 1.5 ± 2.8 days/4 weeks. Lost school time was 0.5 ± 1.5 days/4 weeks  based on recall, but about 50% higher for migraine according to actual absences recorded in association with reported headache on the preceding day. More days were reported with limited activity  than lost from school. One in 30 parents (3.3%) missed work at least once in 4 weeks because of their son’s or daughter’s headache. Emotional impact and quality-of-life scores generally reflected other measures of burden, with pMOH causing greatest detriments, followed by migraine and tension-type headache, and UdH least. Burdens were greater in adolescents than children as UdH differentiated into adult headache types.Headache in children and adolescents in Lithuania is mostly associated with modest symptom burden. However, the consequential burdens, in particular lost school days, are far from negligible for migraine  and very heavy for pMOH . These findings are of importance to both health and educational policies in Lithuania. In our recently published prevalence study, we showed headache to be common in children aged 7–11 years) and adolescents aged 12–17) in Lithuania  and medication intake in terms of days in the preceding 4 weeks. We expressed headache intensity on a scale of 1–3 . Duration of episodes [D] was reported in hours as < 1, 1–2, 2–4 or > 4; for analysis we expressed these according to their mid-points , judging these approximations to be no less reliable than participants’ estimates of duration. From F and D we calculated proportion of time in ictal state (pTIS) according to the formula (pTIS = F/28*D/24), and expressed it as a percentage. We calculated disability at individual level (using this term in the sense imputed in GBD) as the product of pTIS and disability weight (DW) attributed in GBD 2013  to the iWe expressed proportions as %, with 95% confidence intervals [CIs], and used chi-squared tests for comparisons. We treated all other variables as continuous, and used descriptive statistics (means and standard deviations [SDs]). For comparisons of continuous data, we used ANOVA when these were distributed normally, and otherwise Kruskal-Wallis test with post hoc Dunn test. We calculated relative risk (RR) of consequential outcomes.p < 0.05 to be significant.We considered Of the 3714 potential participants, 2505 completed the questionnaires . Of these, 1169 (46.7%) were male and 1336 (53.3%) female; 1382 (55.2%) were children and 1123 (44.8%) adolescents; overall mean age was 11.5 ± 3.2 years (median 11) , assuming a 5-day week). This, again, showed an upward trend with age (Table p < 0.01) or UdH (p < 0.001), while pMOH cost those affected one quarter (5/20) of their school days.Table ge Table . Lost scFor all headaches, a greater proportion of days were reported with limited activity than lost from school, even allowing for a denominator for the former of 28 rather than 20. While days of limited activity were clearly related to headache frequency, with ratios of the order of 1:3 for the episodic headaches, pMOH again stood out, affecting almost 1 day in every three (8.4/28).p = 0.1163). They were also too few to relate likelihood of having missed school to reported intensity of HY.In Table Table p < 0.0001). These data are in Table p < 0.001) and five-fold, to one in six (16.7%), for pMOH, although numbers for the latter were small. Taking UdH as reference, the RR for a parent missing work ranged from 2.0  for TTH through 6.9  for migraine to 16.7  for pMOH.One in 30 parents (3.3%) missed work at least once in 4 weeks because of their son’s or daughter’s headache, but this was clearly age-related, and in the opposite direction to other variables trending with age, so that parents of children were at much higher risk than those of adolescents , followed by TTH (Table p < 0.001) than TTH.Emotional impact scores, with a median value of 5.0, were not normally distributed but, judged nonetheless by means, UdH had least emotional impact (TH Table . Migrainp < 0.01) and no headache (p < 0.001). Migraine had more impact than TTH (p < 0.001), which itself was associated with a lower QoL score than UdH or no headache (both p < 0.001)  Table .We previously reported that headache was very common among children and adolescents in Lithuania, with a 1-year prevalence of 76.6%, very roughly one third each of migraine (21.4%), TTH (25.6%) and UdH (24.0%) [The symptomatic burden of child and adolescent headache does not, from these findings, generally appear to be heavy, with a mean pTIS of < 1% and only mild-to-moderate headache on average. Estimates of lost health based on pTIS and DWs from GBD 2013  ranged fWe noted that medication days were fewer than headache days, although only negligibly for pMOH. These age groups do not medicate all headaches, a reflection, presumably, of the short duration of most headaches, a well-recognised characteristic of these age groups. Of course, pMOH stands out: highly frequent, longer lasting and more intense, coupled with symptomatic medication intake on more days than not. The prevalence of pMOH was low overall (0.8% ), but ouWe should note that, more than in adults, duration (a factor in pTIS and symptom-based disability estimates) may be underestimated because of intervening sleep – a commonly effective therapy. While this may reduce symptom burden, its consequences are reflected in activity indices. Indeed, although symptom burdens appeared modest, the reported consequential burdens tell a somewhat different story. Particularly among these is lost school time, with its likely impact on education: a mean of 0.5 days/4 weeks (2.5%) is not inconsiderable. For those with migraine it was 3.5%, while for pMOH it was 25%, which could only be highly damaging to education and prospects. Although the small numbers with pMOH call for cautious interpretation, the larger group with other headache on ≥15 days/month lost 1.2 days (6.0%), which is still a serious impact. We note here that estimates of lost school time based on HY, presumed to be free from recall error (but not necessarily from embellishment), generally paint a picture of rather greater severity. For all episodic headaches, actual lost days yesterday were always somewhat higher  than predicted from 4-week recall. That this was not so for headache on ≥15 days/month suggests, as might be expected, that established patterns of frequent headache and absences are more reliably recalled. The point, however, is that, if the HY data are true, the 2.5% based on recall for all headache may underestimate true absences of 3.3%, and the 3.5% for migraine may in reality be 4.7%. Furthermore, school absences were more frequent among adolescents, in whom the consequences would be more adverse, than in children.On the issue of possible embellishment, we noted previously that HY appeared to be over-reported, according to predictions based on headache frequencies . It is oFor all headache types, more days were reported with limited activity than lost from school. This could be anticipated for two reasons. First, a day of limited activity does not imply a whole day lost (as is the case with lost school days). Second, “activity” embraces a wide range, from chores to socialising and play, much of which, unlike school, is essentially optional and may be abandoned without consequence. Even so, the 1.5 days/4 weeks (5.4%) reportedly affected in this way in those with migraine suggests a considerable life diminution, while the 8.4 days/4 weeks  reported by those with pMOH, and the 4.6 days (one in six) reported by the larger group with other headache on ≥15 days/month, are indicative of major life impairments.Parental work was also a casualty, with one in 30 parents (3.3%) of children or adolescents with headache reportedly missing work as a consequence at least once in the preceding 4 weeks. We did not enquire further because the young participants were unlikely to be fully aware of how much time their parents lost, so the range might be wide, from an hour or two to multiple days. Furthermore, while this finding was obviously influenced by how many parents were actually in paid work, which was unknown for the same reason, it remains valid as a measure of burden at population level without this knowledge. Notably, parental work losses were strongly influenced by headache type, as demonstrated by RRs (with reference to UdH) of 2.0 for TTH, 6.9 for migraine and 16.7 for pMOH. Equally notably, despite the greater burdens among adolescents, including more days lost from school, it was parents of children rather than of adolescents who bore the brunt of these losses.These various burdens were reflected in emotional impact scores. The six questions contributing to this score related principally to concentration, mood, fear of headache and coping with it, so the gradient observed – pMOH having greatest impact, followed by other headache on ≥15 days/month, migraine and TTH, and UdH having significantly the least – was as expected. QoL scores showed a similar picture, with the lack of impact attributable to UdH distinguishing it clearly from all other headache types, a finding that supports its recognition as a distinct entity. Incidentally, QoL scores seemed generally low: < 72% of the possible score of 36 even for those with no headache, while the validation of the full KINDL scale suggests “healthy” scores of about 80% . HoweverFinally, we might note that, while UdH is not reported in any of these measures as a burdensome headache, which is unsurprising since, by definition, it is mild and short-lasting < 1 h), it remains important firstly as a common headache in these age groups, accounting for one third of all reported headache (38.0% in children declining to 27.4% in adolescents) [ h, it reThere are no similar data yet available from other countries, but these will be forthcoming for comparison as the Global Campaign completes its programme of schools-based studies around the world , 10.The strengths and limitations of this study are as noted previously : its tesHeadache, common in children and adolescents, is associated with symptom burdens that may not, for most, be onerous, but the consequential burdens, in particular lost school days, are not insubstantial. These findings are of importance not only to health policy but also to educational policy in Lithuania.


To investigate whether the treatment zone size (TZS) and treatment zone decentration (TZD) will affect the axial elongation in myopic children undergoing orthokeratology treatment.A self-controlled retrospective study was conducted on 352 children who met the inclusion criteria. Axial length was measured before and at 12 months after the initial lens wear. Corneal topography was measured at baseline and at each follow-up after lens wear. The Corneal topography obtained from the 12-month visit was used to quantify TZS and TZD for each subject. Cycloplegic refraction was required for all children before fitting the orthokeratology lenses.P < 0.01). In groups with both small and large TZD, axial elongation was significantly decreased with small TZS . Age, SE, and TZD were significantly associated with axial elongation in multiple regression .Axial elongation was significantly associated with age, baseline spherical equivalent (SE), TZS, and TZD with univariate linear regression. In groups with both small and large TZS, axial elongation was significantly decreased with large TZD (both Relatively smaller TZS and larger TZD may be beneficial in slowing myopia progression in children with orthokeratology treatment. Myopia has become a serious public health issue due to its increased prevalence and severity over the past decades , 2. MyopA variety of methods have been used for controlling the progress of myopia, including orthokeratology, which has an effectiveness supported by several studies –11. An oA series of factors have been proposed to be associated with the individual variation in axial elongation with orthokeratology, such as age, baseline spherical equivalent (SE), corneal shape, and pupil size –23.RecenThe mechanism by which TZS and TZD affect myopia control may be associated with the distribution of the corneal power shift or the conditions of the retinal peripheral defocus. To our knowledge, few study to date has combined these two factors in an orthokeratology study. The purpose of this current study was to explore the effect of TZS combined with TZD on slowing myopia progression in children using orthokeratology. This will enhance our understanding of the effects of orthokeratology in slowing myopia progression over a topography profile.This retrospective study was conducted at the Tianjin Medical University Eye Hospital  between May 2018 and July 2019. This study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Tianjin Medical University Eye Hospital. In total, 352 children were deemed suitable for this study and included for analysis according to the inclusion criteria below. The patients were recruited from the children who visited the myopia control clinic. The initial inclusion criteria for orthokeratology lens fitting were: aged between 8 and 14 years; SE of cycloplegic refraction from − 0.75 D to − 6.00 D; corneal astigmatism ≤1.50 D; best-corrected monocular visual acuity no less than 20/20; cycloplegic refraction with compound tropicamide eye drops  before fitting orthokeratology lenses. Exclusion criteria were: strabismus or ocular surface disease; any history of surgery or contact lens use; binocular vision dysfunction.− 11 (cm2/s) (mL O2/mL · mm Hg). Total lens diameter had a range of 10.2–11.0 mm. Lens fitting procedures strictly followed the guidelines provided by the lens manufacturer. Briefly, the first trial alignment curve for the lens was based on the corneal topography , flat-K, corneal eccentricity, and horizontal visible iris diameter. Fitting quality was evaluated by fluorescence staining 1 h after the lens placement. A good fitting was indicated by an optical zone covering the pupil, no apparent decentration of the lens, blink lens-movement less than 1 mm, and a bullseye pattern with fluorescence staining. Over-refraction was performed to determine target power plus 0.75 diopters as the final order. Children received instructions for contact lens wearing and cleaning at fitting. Lenses were required to be worn for more than 8 h per night, for at least 6 days per week. Follow-up visits were scheduled at one day, one week and one month after the initial lens wear, and at least once every 3 months afterward. All children included were continuously worn the lenses and do topographic map examination within four hours after removing the lenses. A total of 352 children meet the above conditions.Children were fitted with spherical four-zone orthokeratology lenses  composed of oprifocon A (Boston EQUALENS II) with an oxygen permeability of 127× 10Corneal topography was first obtained with a Medmont Corneal Topographer at baseline Fig. A. Three Axial length was measured before lens fitting (baseline) and at 12 months after lens treatment using noncontact optical biometry . At each visit, a single examiner measured the axial length three consecutive times, and the mean value was taken for data analysis. All measurements were done by the same experienced technician, and the results meet the quality control requirements of the instrument.http://www.R-project.org/). A P < 0.05 value was defined as statistically significant.For descriptive purposes, the means and standard deviations were calculated for baseline SE, ages, axial elongation and corneal parameters . The normality of the data was tested with a Schapiro-Wilk test. Univariate linear regression was used to analyze the relationships between axial elongation and age, baseline SE, TZS, TZD. Stepwise multiple linear regression was used to analyze the relationships between axial elongation and age, baseline SE, TZS, TZD and corneal parameters. All statistical analyses were performed using R software  was used for statistical analysis. The mean age of the subjects was 10.28 ± 1.88 years (range 8–14 years). At baseline, the mean SE was − 3.25 ± 1.28 D (range − 6 to − 0.75 D), the mean axial length was 24.92 ± 1.36 mm, the mean corneal flat-K was 42.65 ± 3.88 D, the mean corneal steep-K was 43.88 ± 3.96 D. After treatment, the mean axial elongation was 0.16 ± 0.23 mm a year, the mean TZD was 0.52 ± 0.22 mm (range 0.05 to 1.24 mm), and the mean radius of TZS was 1.90 ± 0.12 mm (range 1.59 to 2.31 mm).P < 0.01) and children with greater baseline SE (P < 0.01). Axial elongation was significantly associated with TZS  or a larger TZD (P < 0.01). The corneal flat-K and steep-K were not associated with axial elongation (both p > 0.05).The association between axial elongation and the parameters were first analyzed by univariate linear regression. Axial elongation was significantly associated with age and baseline SE Fig. A, B. AxiTZS Fig. D and TZDTZS Fig. C. Axial r = − 0.05, P = 0.38, Fig. p > 0.05). The TZD data also followed a normal distribution . In groups with either small TZD or large TZD, myopia control was improved with a decrease in TZS . However, there was no significant difference in axial elongation between children who had both small TZS and small TZD and those who had both large TZS and large TZD .In groups with either small TZS or large TZS, the axial elongation was significantly negatively associated with TZD , and our study, therefore, provides evidence to clarify the relationship between TZD and the slowing of myopia progression with orthokeratology treatment for twelve months follow-up. Children increased TZD was beneficial in controlling myopia, with relatively larger TZD associated with smaller axial elongation  which was in line with previous studies. Li et al. reported a mean TZD of 0.68 ± 0.35 mm (range 0.05–1.49 mm) from a study of 106 subjects , and CheP = 0.3212, Fig. TZS and TZD were two previously neglected factors for myopia control effectiveness, compared with the known factors such as initial age and SE at baseline. We found that in subjects with smaller TZS, larger TZD was associated with the smallest axial elongation (0.06 mm a year, Fig. The mechanism of orthokeratology in control of myopia progression is still not clear. We hypothesized that orthokeratology induces myopia defocus in relative peripheral refractive error interfering the axial elongation pattern as “peripheral refraction theory”, which has been recognized by most researchers –48. Cho There are several limitations to the current study. First, we suggest that TZS and TZD benefit the retinal myopic defocus, but we did not directly measure peripheral retinal defocus in this study. Second, we speculate that the myopic control with TZD might shift more PCRP into the pupillary area as a potential mechanism for orthokeratology, but we did not measure the children’s pupil. Third, we speculated that higher-order corneal aberrations were associated with myopia control, but we did not directly measure higher-order aberrations. Fourth, the method to quantification of TZD relative to the corneal center rather than pupil center may have a slight impact on the results.Children with relatively smaller TZS and larger TZD after Orthokeratology experienced slower axial elongation by the end of 12 months. This effect might be mediated by the induction of a greater amount of relative myopic defocus on the peripheral retina. Further studies are needed to assess whether change on lens designs increase efficacy for slowing progression of myopia.