In this paper, we investigate the robustness of electrophysiological responses of relatedness to multiple consecutive word stimuli (probes), in relation to an actively recollected target word. Such relatedness information could be used by a Brain Computer Interface to infer the active semantic concept on a user’s mind, by integrating the knowledge of the relationship between the multiple probe words and the ‘unknown’ target. Such a BCI can take advantage of the N400: an event related potential that is sensitive to semantic content of a stimulus in relation to an established semantic context. However, it is unknown whether the N400 is suited for the multiple probing paradigm we propose, as other intervening words might distract from the established context . We perform an experiment in which we present up to ten words after an initial target word, and find no attenuation of the strength of the N400 in grand average ERPs and no decrease in classification accuracy for probes occurring later in the sequences. These results are groundwork for developing a BCI that infers the concept on a user’s mind through repeated probing, however, low single trial decoding accuracy, and high subject variability may limit practical applicability. Others allow users to spell messages, by selecting characters one by one7. Each approach has their strengths and weaknesses, allowing the choice of BCI to vary depending on the needs and preferences of a user.Brain Computer Interfaces (BCIs) use brain activity as a direct input for a computer. Many BCI applications are designed to offer alternative means of communication to people that are no longer able to use conventional input devices . Existing communication BCIs come in a number of paradigms, achieving communication in different ways. Some offer binary choices selections, that, for instance, allow for “Yes”/“No” answers to questions  as an alternative for existing BCI communication approaches, or for tip-of-the-tongue scenarios, in which the word for a to-be-communicated concept cannot be retrieved, which can be a problem for patients with anomic aphasia9. In that study by Geuze et al. (2014), users encoded a probe’s relatedness status using deliberate responses: Users were presented with a stream of probes and asked to press a button when a presented word was related; a task designed to simultaneously induce a movement related de-synchronization (ERD) and elicit a P300 for related probes (due to their explicit task-related nature).Such an approach toward word selection has been outlined in the past10. Over the years it has proven a robust effect that can be elicited in a sentence context, in which the response to a word is measured based on its relation to earlier parts of the sentence, or in a word-pair context, in which a prime is presented followed by a related or unrelated probe. It is not only elicited with text on a screen, but also occurs when stimuli are pictures11 or when presented auditorily, as speech13, though the N400’s scalp distribution can vary. There are also non-semantic factors that affect the N400 amplitude, for instance, the lexical properties of the presented stimulus word 15, or the repetition of a stimulus within in a short timeframe16. These are of less inherent interest for a BCI application, but may need to be accounted for as potential confounds.While using such deliberate signals is one way to approach such a BCI, there already exists a brain response that is inherently sensitive to the semantic content of a stimulus: the N400. It is an Event Related Potential (ERP) characterised as a wave that is more negative when a presented stimulus is unrelated, compared to a related stimulus, in relation to a previously established context18). Using such an “automatic” brain response might be more pleasant for the user, as they have to provide less effort in deliberately evoking the right response. However, if we want to use the N400 for this BCI paradigm, we first have to establish whether this signal lends itself to such an approach.Importantly, it is not necessary that subjects are explicitly tasked to evaluate the semantic content of a stimulus to elicit an N400, though it appears to require that a stimulus’ meaning is processed , with classification rates across individuals ranging from 54% to 67%20.Van Vliet Secondly, to infer the concept on a user’s mind, a system would have to present a number of probe words consecutively, for the same concept, in order to collect sufficient evidence. Such a sequence of probe words could conceivably influence the context on a users mind, and it is therefore possible that results from a prime-single probe paradigm do not generalise to a setup with multiple  probes. In other words, we want to know whether single trial classification of relatedness based on the N400 is still possible when multiple consecutive probes are presented following an initial target word.et al. (2014) study9, that focused on deliberate responses, to also elicit an N400 response. However, this N400 would predict a difference in the same direction as their expected P300: a more positive response for related probes after 300 ms, so it is hard to estimate the potential contribution of the N400 to their ERP findings. Notably, their grand average ERPs do not show the downward deflection (with a minimum near 400 ms) that is characteristic for the N400.It is good to note that, given the described automatic nature of the N400, we might expect that in the Geuze To establish what the effects of multiple consecutive probes are on the decoding of semantic relatedness, we designed an experiment in which we present a target word for participants to actively keep in mind, followed by up to ten probe words that are either related or unrelated to this target. Subjects are tasked to mentally evaluate the relatedness of probes, but without putting emphasis on related over unrelated probes. While presenting ten probes is not necessarily sufficient for decoding purposes, we will use this data to determine if there is any indication of attenuation  of the N400, when comparing first probes and probes late in the sequence. Specifically, we will compare grand average ERPs in response to probes appearing immediately after the target, with those in response to probes at the end of the sequence, combining data across participants to increase the sensitivity for detecting a difference.21. To evaluate this we will analyse the data in the frequency domain to determine if there are any spectral differences that could be used as additional features for decoding semantic relatedness.In addition to this, there is some evidence that tasks designed to elicit N400s can also induce changes in oscillatory brain responsesIn each trial, participants were presented with a target word to keep in mind, followed by up to ten probe words. At the end of each trial, participants indicated with a button-press whether the most recently presented probe had been related or unrelated to the initial target word (left vs. right button-press respectively). We compared the answers of each participant to the label of that probe and calculated the percentage of agreeing answers across all trials. Agreement between the response and label ranged from 70% to 95% across participants (mean 87%). A mismatch between the response and label can reflect a user error, or a disagreement on the relatedness status of the probe, as relatedness judgements can vary between individuals. Average reaction times per participant ranged from 368 ms to 1420 ms (mean 632 ms), with a strong correlation between reaction time and age . On average, reaction times to related probes were somewhat faster than to unrelated probes .prior to stimulus onset, with the ERP for unrelated probes being more negative before, at, and some period after time 0. A difference in ERPs occurring prior to presentation of the stimulus of interest cannot reflect a brain response to that stimulus, suggesting that there is some structural factor  affecting the responses, that did not average out in the ERP. We hypothesise that this is a consequence of our design: while two related stimuli were never presented sequentially, unrelated stimuli could appear after either a related or an unrelated probe. If we split the data into these three categories and compute grand averages we obtain the results in Fig. To determine if there were any differences in brain responses to related compared to unrelated probes, we computed a grand average ERP, averaging across all related and unrelated probes presented in the experiment. This analysis produced an unexpected result: the grand average showed a difference in response between related and unrelated probes related → unrelated category behaves similarly to the unrelated → unrelated, until at least 1 s after stimulus presentation.More specifically, there appears to be a late response that differs in amplitude between related and unrelated probes, which is still present when the next stimulus is presented. This is followed by a stimulus response (around 200 ms) after which the related → unrelated data (unless noted otherwise), to ensure that effects are not an artifact of our experimental design.With responses to probes preceded by an unrelated probe appearing unconfounded , from here on we consider only these probe’s data, excluding the 22 , confirming that there is a significant difference in the response to related and unrelated probes.The resulting grand average ERP can be found in Fig. th and 10th position). Note, that due to the exclusion of unrelated probes that were preceded by a related probe, there are fewer instances available for unrelated probes in the final positions . For both locations , we plot the difference ERP (unrelated-related) in Fig. In order to determine whether or not the magnitude of the ERP decreases after more probes have been presented, we compare the ERP in response to the first probe in each plot, to the ERPs of those in final positions . Unexpectedly, the figure suggests that there is a difference after one second. To look at this in more detail, we plot the individual ERPs for related and unrelated for both the first and final positions in Fig. For both probes in the first and final positions, the response to unrelated probes was more negative than the response to related probes, resulting in a negative difference from around 400 ms to 800 ms. In the cluster permutation applied to the two difference waves , no significant clusters were found /2), rather than the percentage of correctly predicted probes. In the figure, we shade the 99.74% binomial confidence interval of chance performance in red to get an indication of which participant’s results did not differ from chance . For 12 out of 19 participants, the classification accuracy could be distinguished from chance level. On average, the relatedness of probes was predicted correctly for 58.3% of probes . To ensure our crossvalidation approach did not overestimate the classification accuracies, we compare them to the accuracies obtained with a train-test split (80–20%). For this train-test split, the average accuracy across participants was 59.5%.As our interest is ultimately in the decoding of these signals from single probe presentations, we trained classifiers to predict for a given probe response whether it is related or unrelated, separately for each subject, using a leave-one-sequence-out crossvalidation approach. No classifier was trained for subject 19, as they were unable to complete the full experiment. The accuracy per subject can be found in Fig. ERP plots for each individual subject are included in the Supplementary Information .Using the behavioural measure, we can determine whether there is a relation between how well the participant’s behavioural responses agreed with our labels, and how well the relatedness of a probe can be predicted from their brain signals. The correlation between behavioural agreement and classifier accuracy, across participants, was moderately high . We Bonferroni corrected the significance values for performing these two tests, resulting in a significant effect for the beta band (p = 0.001), but with a non-significant cluster (p = 0.015) for the alpha band, at the two-tailed α/2 = 0.0125 level . These results are plotted in Fig. To determine if there was any difference in oscillatory brain activity when contrasting the related or unrelated probes, we also analysed the data in the time-frequency domain. A cluster permutation test applied to the Time Frequency Representations (TFRs) of related and unrelated probes, found a significant difference between the two conditions, identifying a significant negative cluster ). To visualise this data, we obtain the difference between the the TFR for related probes and the TFR for unrelated probes , as a fraction of the total power per frequency band (summed across the time-dimension) and plot this normalised difference, averaged across electrodes, in Fig. To determine if this difference in oscillatory behaviour can be decoded from single probe presentations, we performed a classification analysis on this TFR data, analogously to the ERP data classification, and also created classifiers for both types of data combined. The results of this analysis can be found in Fig. We have now generated predictions for single probes to estimate how well we can classify related from unrelated probes. However, for a BCI, information across multiple probes would need to be integrated to ultimately infer the original target. We can simulate such an analysis with our data, by using the consecutive predictions from a trial to predict which trial sequence they are from.Each trial has a pattern of related and unrelated probes occurring in specific positions. We can try to predict from which trial a set of consecutive probe predictions was obtained, by comparing the similarity between the consecutive predictions and the true relatedness of all presented trials. That is, for each trial, we compute a similarity as the inner product between the probe relatedness scores predicted by the classifier , and the ‘true’ relatedness expected for every possible trial, given the sequence of probe words. We then rank the trials based on this similarity (high to low). We can use this rank as an estimate of how well trials can be predicted from the sequence of probe predictions, by interpreting it as a percentile: a trial that receives a high percentile rank , obtained (among) the highest score on the basis of its predictions.To show how this estimate changes as trials contain more probes, we start by only considering the first probe of each trial, then both the first and second probe, and so on, until all 10 probes are included in this analysis. In Fig. The figure shows that for random predictions it does not matter how many predictions are accumulated for the percentile rank of the correct trial. For actual predictions, however, the (mean) percentile rank in which the correct trial is found increases when more consecutive probes are considered. There are also participants (in the tail of the boxplots) for which, even with 10 consecutive probes, the percentile rank cannot be distinguished from the random prediction data.In Fig. In this study we aimed to determine whether the N400 can be elicited reliably using multiple probes after an initial target. Overall, our results show that there is a difference in brain response to related versus unrelated probes, as evidenced by both the grand average ERPs and the classification results. Specifically, in the grand average ERP we see an early component, more negative for unrelated stimuli, that matches the N400 in timing and location. Furthermore, the amplitude of this N400 effect is similar for probes appearing at the start and end of a trial Fig. . In addi24, aiming to decode word relevance to a category of interest using sequential presentation of words and Fixation Related Potentials . The authors found a more negative ERP for non-relevant words, similar to the N400. Interestingly, the results show markedly different brain responses for the online phase, where longer sequences were used (100 words), compared to the calibration phase (22 words). This may still be an indication that the N400 amplitude reduces for very long sequences . Alternatively, with only a limited number of words used per category, stimuli were likely repeated frequently, which is known to cause a reduction in the N400 amplitude16.This result is consistent with a recent publicationAn unexpected result in our initial analysis was an overlapping late-negative component occurring after related probes see Figs  and 2b. 21. When decomposing our data into the frequency domain, we visually observe higher power for related probes in the alpha and beta band from 600 to 1200 ms post stimulus presentation, though, only the beta band (16–19 Hz) increase was statistically significant. This is in contrast to Wang et al.21 who showed a decrease in beta power for in-congruent sentence endings (that elicit a more negative N400). This inconsistency in response suggests that either the role of beta power in relation to the N400 is different for sentence close-word paradigms, or that another aspect of our task  results in these changes in beta power. These conflicting results, together with the classification analysis which showed no additional benefit from the inclusion of time-frequency data over ERP data only, suggest only limited usability of this response for a BCI.Previous research has indicated that N400-tasks may also induce changes in power in the frequency domainAgreement between the behavioural responses and the probe labels ranged from 70% to 95% across participants (mean 87%). Disagreement can reflect an error, or an inherent disagreement on the relatedness of the most recent probe and the target. Participants with higher percentage agreement, thus, may have been better at keeping the target on mind, or have more conventional relatedness judgements. The significant correlation between classification accuracy and behavioural agreement (r = 0.58), can in fact be explained by both interpretations.μ = 58%) and a large variability across subjects (50–72%). The fact that for about a third of subjects their accuracy could not significantly be distinguished from chance is a concern for using any BCI application on the basis of this paradigm. There is some precedent in difficulty to detect the N400 within individual subjects in other N400 experimental paradigms25, and having a subset of users for whom the intended brain signal cannot be detected well, is a not an uncommon problem for BCIs (see e.g.26 for a discussion).The classification results overall, show relatively low accuracies  for only a small subset of participants (2–5 out of 20). In reality such long sequences present additional challenges, such as subject motivation and attention decrease over the trial, which may reduce performance further. On the other hand, a closed-loop BCI, could use the information accumulated from previous probes to select (the most) informative future probes, which may reduce the required sequence length compared to the non-optimised probe sequences used in these simulations. This remains an area for future research.One approach to overcome low accuracies in a BCI is to aggregate results over multiple stimulus repetitions. However, as the N400 amplitude can be reduced by stimulus repetition28. For this purpose, word-pair priming or sentence congruence paradigms have previously been used, and the presence of the N400 in these tasks has been shown to correlate with recovery28. The sequential probing paradigm used in our experiment, might be able to detect higher level cognitive processing in patients that exhibit a N400, by determining whether or not these patients can hold a target active over multiple consecutive probe words. These approaches may similarly be useful for assessment of patients in a Complete Locked in State29.While practical utility as a BCI that infers the target word may be limited, the consecutive probing paradigm may have alternate applications. For instance, in assessing linguistic processing in patients with Disorders of ConsciousnessIn summary, our results show that we can decode responses of relatedness from EEG, on the basis of the N400, and we find no indication that this response attenuates across multiple consecutive probes. While this paradigm can in theory be adapted to a BCI that infers the target word on a user’s mind, low singe trial accuracies and high variability in accuracy across subjects make this unlikely to offer practical utility in communication applications. However, the paradigm itself may yet be of use for assessing cognitive processing in certain patients populations.Twenty-one participants took part in the experiment , ranging in age from 18 to 61 years old . Participants were only eligible to participate if they were native Dutch speakers and reported to have no reading problems One participant dropped out of the experiment due to excessive sleepiness. Her data are not included in the analysis. Response data are missing for participant 9, block 6, due to a technical problem, and there is no data for participant 19, block 6 .All participants provided informed consent prior to participation, and the study was approved by and conducted in accordance with the guidelines of the Ethical Committee of the Faculty of Social Sciences at the Radboud University.In the experiment participants completed 212 trials, each consisting of a target word, followed by one to ten probe words. For each trial, participants were tasked to remember the target word, and subsequently mentally assess for each probe whether or not it was semantically related to the target. At the end of a trial participants were prompted to specify by button-press the relatedness status of the most recent probe . 50% of trials contained ten probes, while the remaining 50% contained an equal distribution of one to ten probes. Trials with fewer probes were included to ensure that participants would mentally evaluate all probes, and not only those for which they could predict that a question would follow.Participants were instructed to respond as fast as possible when prompted with a question, and received their reaction time as feedback . This feedback did not reflect ‘correctness’ of the choice as relatedness judgements can vary between individuals. To increase salience and facilitate memorisation, participants were asked to pronounce the target word during its presentation.Each target word was presented for 2000 ms, followed by a 1000 ms interstimulus interval (ISI). Each subsequent probe was presented for 350–370 ms (jittered duration) and also followed by a 1000 ms ISI. The question prompt was displayed after the last ISI, and was replaced by feedback on buttonpress. Across the 212 trials a total of 539 related, and 1072 unrelated probes were presented (33.5% related).th and 10th position). Note that we define the ‘final’ category to consist of two probe positions, in order to get an equal number of brain responses, as only 50% of trials consisted of the full 10 probes. With this analysis in mind we designed trials such that there was an equal distribution of related and unrelated probes for the first and final position(s). Finally, these trials were set up such that no two related probes were ever presented in a row, as a brain response signifying relatedness in such a case could be purely from the pairing with the previous stimulus. The percentage of related probes per probe position can be found in Fig. In our analyses we compare probes occurring in the first position with probes occurring in the ‘final’ position  three words that a given cue-word brought to mind. In our design we used Leuven cue-words as targets, and Leuven response-words as (related) probes. We then used the CELEX Dutch Wordform database31 to obtain words unrelated the the targets, to use as unrelated probes. Probes never occurred more than once across all trials, as repetition of concepts has been shown to affect the N400 amplitude16, though some targets also occurred as probes. Unrelated words were selected to approximate the related probes in distribution of both word length and word frequency. Word frequency in particular, is known to affect N400 amplitudes, with words that occur frequently in a language eliciting smaller N400s15. Across all probes, the mean (log) word-frequency was 1.50 (sd = 0.65) for related, and 1.49 (sd = 0.63) for unrelated probes. On average the length of a related probe was 5.68 characters (sd = 1.98), and an unrelated probe 6.13 characters (sd = 1.92).Stimulus words were obtained from the Leuven association datasetStimuli for a few example trials are depicted in Fig. Trial order was randomised per subject.EEG was recorded with 64 sintered Ag/AgCl active electrodes , placed according to the international 10–20 system, at a sampling rate of 512 Hz. The Biosemi ActiveTwo system uses a Common Mode Sense (CMS) and a Driven Right Leg (DRL) electrode, instead of a ground electrode. The recorded signals reflect the voltages between each electrode and the CMS, which can subsequently be re-referenced to any electrode(s) of choice. We placed two electrodes on the mastoids for this purpose and used four further electrodes to measure vertical and horizontal EOG.32, and then sliced into segments , from 1.5 s prior to and 2 s after each probe presentation. Then, data from different blocks were combined and downsampled to 256 Hz. All electrodes were mean-corrected (centered) and re-referenced to the average of the mastoid electrodes. The four EOG channels were then regressed-out of the EEG channels to remove any influence from eye movements or blinks33. The EEG channels were subsequently lowpass filtered at 40 Hz (6th order Butterworth filter).Data were recorded in 6 blocks of 35 trial sequences each. These data blocks were loaded, highpass filtered at 0.1 Hz (4th order Butterworth filter)34, while bad epochs were marked to be ignored during grand average calculation and training of classifiers. Finally, epochs were baselined to a period from −100 ms to 0 ms from stimulus onset.After these preprocessing steps, any epochs or channels with abnormal activity were marked for removal (epochs) or interpolation (channels). A trial or channel was considered abnormal when the variance of the given unit diverged more than 3.5 standard deviations from the median of all units (channels or epochs). This was repeated, excluding previously marked units in the next iteration, until no units were considered abnormal (max 6 iterations). This resulted in between 12 and 156 epochs and up to 4 channels to be marked per participant. Any identified bad channels were replaced with an interpolated’virtual channel’ using a spherical spline interpolationFor brain responses in the time domain, we obtained grand average ERPs by averaging across relevant probes, and then averaging across subjects. The data for the grand average ERPs were low-pass filtered at 20 Hz, prior to averaging, for plotting purposes (smoothing). To analyse brain responses in the frequency domain, the data from each trial was decomposed into frequency bins of 2 Hz, from 2 to 40 Hz. The power in these bins was calculated for each 50 ms, starting from 0.5 s prior to probe onset to 1.35 s after, using a Hanning window and a frequency dependent window length (ranging 3.5 s - to 175 ms respectively). These Time Frequency Representations (TRFs) were then averaged across participants to obtain grand average TRFs.22 (as implemented in Fieldtrip35). Such a test allows for the combination of information across electrodes and timepoints, to increase sensitivity of the statistical test, without having to correct for multiple comparisons with respect to those aspects. These tests were performed using a within-subject design, and a dependent samples t-test as test-statistic. Note that this non-parametric cluster-based permutation test does not rely on assumptions with regard to the distribution of the data, regardless of the chosen test-statistic (the test-statistic is merely used to quantify the difference between datapoints). In all cases, we performed a two-tailed test, , using 1000 permutations, supplying all channels, and timepoints and/or frequency bins as specified.To determine whether brain responses between conditions  were significantly different, we used non-parametric cluster-based permutation tests36, consisting of a spectral filter, a spatial whitening and classification using a regularized classifier. Specifically, the preprocessed data from time 0 to 1 s and all remaining epochs and channels , were spatially whitened, and subsequently classified using a L2-regularized logistic regression. The regularization parameter was optimised using a 5-fold (nested) crossvalidation. A separate classifier was trained for each participant. For classification analysis on data in the time-frequency domain, preprocessed data were again decomposed into frequency bins of 2 Hz (2–40 Hz), but here the bins were calculated for each 100 ms from probe onset (0 s), until the end of the trial (1.35 s), using a Hanning window and a frequency dependent window length (1 s–175 ms respectively). These time-frequency data were then processed using the same classification pipeline.For classification analyses on data in the time domain, the preprocessed data were further downsampled to 64 Hz and low-pass filtered to 15 Hz to reduce the amount and complexity of data passed to the classifier and prevent overfitting. We use a classification pipeline identified as robust for different types of ERPsTo estimate classification performance, the data were separated into crossvalidation folds of training and test sets, for each participant, where all but one trial sequences were used as training data, and the data from probes in the excluded trial sequence were used as the test set. Only trial sequences in which ten probes were presented were used as test sets, to avoid biasing the results toward responses to early probes. Epochs that were marked for removal during preprocessing were excluded when occurring in a training set, but were included when part of the test set .Supplementary Information


The number of women entering medical school and careers in science is increasing; however, women remain the minority of those in senior faculty and leadership positions. Barriers contributing to the shortage of women in academics and academic leadership are numerous, including a shortage of role models and mentors. Thus, achieving equity in a timelier manner will require more than encouraging women to pursue these fields of study or waiting long enough for those in the pipelines to be promoted. Social media provides new ways to connect and augments traditional forms of communication. These alternative avenues may allow women in academic medicine to obtain the support they are otherwise lacking. In this perspective, we reflect on the role of Twitter as a supplemental method for navigating the networks of academic medicine. The discussion includes the use of Twitter to obtain (1) access to role models, (2) peer-to-peer interactions, and continuous education, and (3) connections with those entering the pipeline—students, trainees, and mentees. This perspective also offers suggestions for developing a Twitter network. By participating in the “Twittersphere,” women in academic medicine may enhance personal and academic relationships that will assist in closing the gender divide. The number of women pursuing seats in medical schools continues to increase, and women comprise nearly one-half of applicants and more than half of matriculants ,2. TheseWomen who leave academics early in their careers cite multiple concerns, including inequalities in compensation, lack of role models, difficulties achieving work-life integration, effects of unconscious and implicit bias on experiences, frustrations in research including funding gaps, and a non-collaborative, and biased work environment ,7. In acThe Association of American Medical Colleges (AAMC), whose mission it is to “improve the nation's health through the advancement of academic medicine” , is atteMentorship is important for increasing diversity at the top and identifying a role model who “looks like you” makes it easier to follow suit ,15. SeveEducators have also realized the need to draw on informal learning networks to complement formal supports and institutions and have turned to Twitter and other social media platforms as extensions of the learning environment . RelatioThe value of access to a large pool of same-gender role models, mentors, and sponsors accessible on Twitter may be particularly important in situations where these relationships are not uniformly available to junior faculty -25. For In addition, as men currently hold the majority of leadership positions, it is imperative that women identify and build relationships with men who are gender inclusive in supporting the development of leaders ,29. ProfIntra- and interdisciplinary peer-to-peer connections may develop through participation in Twitter-based movements, chats, journal clubs, and other educational platforms. These allow individuals to create personal learning networks (PLNs), systems of “interpersonal connections and resources that support informal learning” ,21,31. TPhysicians across the country and worldwide have met virtually and in person and garnered support from participation in #ILookLikeASurgeon, #NYerORCoverChallenge, #WomenInMedicine, and #MeTooMedicine ,33. SeveThere are many hashtags devoted to teaching vignettes  and journal clubs like general surgery (#UMichSurgJC), trauma surgery (#EASTjc), Association of Women Surgeons (#AWSchat), nephrology (#nephJC), and pathology (#pathJC). Sharing conference material with the use of a specific hashtag allows interested parties to follow meeting content and participate in the conversation regardless of physical presence at the event. For example, the #EWIMS hashtag facilitated connectivity for the varied communities of conference attendees, faculty, and worldwide followers. In addition to ease of participation, an international group of researchers recently showed that the perceived costs of conference participation via social media were far lower than live attendance while nearly half of respondents felt that they would learn the same or more from the social media-based format . When foTwitter also provides a platform from which to reach and inspire girls and women who are students and trainees, especially those who might not otherwise have access to women role models. Social media can help faculty to connect with talented students, residents, and others for inspiration, promotion of program and institution, and recruitment. Pearls of wisdom and other messages are shared broadly among communities of medical educators and learners using hashtags such as #DearFutureMD, #MedEd, and #SurgEd. Students in other fields, including art and history, have also realized the value of access to multiple educators and peers outside of their classrooms to provide feedback and leading them to explore national organizations, institutions, and publications that they otherwise may not have been aware . Among mOne example of how Twitter can facilitate such a relationship includes an #EWIMS participant who was contacted by a high school student who lived over a thousand miles away. This connection led to a Skype Chat with the student’s all-female medicine interest club, a behind-the-scenes tour of the student’s local academic medical center arranged by the #EWIMS participant, and ongoing mentorship via Twitter, and other social media outlets.Like any tool, there are challenges when utilizing Twitter for the above pursuits. Messages must be concise, despite a recent doubling of the length, as tweets are limited to 280 characters. Most of this work is often performed before or after hours and on personal devices as many organizations block the use of social media sites ,21. ThosWhile popular among many, the reach of Twitter is limited as only 23% of active American internet users (or 20% of the adult population) have a Twitter account . Also, tSome consider Twitter to be “frivolous, superficial, or dangerous” and do not find professional value in social media ,42. OtheWith unprecedented numbers of women entering medical and science careers, there has never been a more important or realistic opportunity to enhance the field’s emphasis on retaining women in academic medicine. Twitter’s ability to connect women faculty in academic medicine with others surviving and thriving in the field can facilitate and promote long-term success in a challenging career environment.. Medical schools should support the use of Twitter among faculty, such as including professional social media activity as one component of the faculty dossier. Profile pages of faculty at schools of medicine should publish Twitter handles, allow faculty to integrate Twitter feed on their profile pages, and encourage faculty Twitter engagement. Schools may also report journal article influence using metrics, such as the Altmetric, that measure the social media impact of research articles. When faculty publish articles, they should be encouraged to share through Twitter and discouraged from believing that it is shameless self-promotion. Second, facilitate connections. Faculty on Twitter should make a point of acknowledging participation as part of routine networking, education, and mentoring activities . In academic presentations, faculty should include Twitter handles (not just email addresses) as contact information. As part of mentoring and leadership development activities, mentors and sponsors should be encouraged to use social media and to support their trainees in doing so responsibly. Training programs should include workshops on how to get the most out of Twitter, facilitated by faculty seasoned in its use.We suggest the following strategies for developing a supportive environment through the use of Twitter. First, promote a culture of Tweeting among academicsClosing the sustained gender gap in hiring, promoting, and retaining women in academic medicine will certainly require a multi-faceted approach. As the authors of this piece have experienced, the use of Twitter as part of this approach can facilitate ties fundamental to the long-term success of women in medicine and science. We are encouraged by the growing flurry of tweets that have become the roar of many academic women in medicine and science advancing through their careers.


Samples were run on a 39-plex Luminex  assay to assess cytokine concentrations. We found that fluid recovery was inadequate in 50% of epochs with conventional PF, whereas Dextran PF overcame this limitation. The AR was higher in the Dextran PF samples for a majority of cytokines, and RR was significantly increased for macrophage colony-stimulating factor and transforming growth factor-alpha. In summary, Dextran PF improved fluid and cytokine recovery as compared to conventional PF and is a suitable alternative to albumin supplemented PF for protein microdialysis.Cerebral microdialysis (CMD) is used in severe traumatic brain injury (TBI) in order to recover metabolites in brain extracellular fluid (ECF). To recover larger proteins and avoid fluid loss, albumin supplemented perfusion fluid (PF) has been utilized, but because of regulatory changes in the European Union, this is no longer practicable. The aim with this study was to see whether fluid, absolute (AR), and relative (RR) recovery for the novel carrier, Dextran 500, was better than conventional PF for a range of cytokines and chemokines. An  The membrane is attached to inlet and outlet tubing through which perfusion fluid (PF) is slowly pumped and collected.3 To measure metabolites in clinical practice, such as glucose, lactate, and pyruvate, a 20-kDa MWCO is adequate and an isotonic solution, mimicking cerebrospinal fluid, is used as a carrier.4 Microdialysis of proteins is limited by both lower absolute concentrations within the brain ECF, as well as the larger molecular weight, necessitating the use of larger MWCO membranes. This causes a number of problems, including non-specific adsorption to the device materials, clogging of membranes, and protein-protein interactions, which all negatively affect recovery.5–7 A further issue with increased MWCO catheters  is loss by convection of fluid within the catheter.8 This is attributed to the hydrostatic pressure differences (with a relatively low osmotic pressure in the PF) and referred to as ultrafiltration. This may impact on both the ability to carry out analysis on the diminished volume of fluid recovered by the catheter as well as potentially impacting on the biology of the extracellular space, such that it is not representative of the underlying processes of interest. In order to mitigate this phenomenon, addition of colloid to the PF to increase the oncotic pressure has been recommended, typically albumin.9 However, a regulatory reclassification of albumin within the European Union as a blood product has made formulation of albumin-supplemented fluid logistically and financially impractical. Further, the theoretical risk of albumin leak and accumulation in the surrounding tissues has been raised, with potential negative consequences.8 Our group and others have shown that cytokines and chemokines are key mediators in the inflammatory processes after TBI,10–12 and in order to advance the study of brain protein recovery and potential therapeutic advances, an accurate estimation of relative recovery is necessary. Thus, it is imperative for the continued clinical use of microdialysis that alternative strategies for improving cytokine recovery which do not require blood products are developed and tested.Cerebral microdialysis (CMD) is a technique enabling sampling from the extracellular fluid (ECF) 13–15 Dextrans are branched glycans of varying molecular sizes (3–2000 kDa), of which ranges between 60 and 500 kDa have been extensively studied in the microdialysis setting.17 In comparison to normal PF, and even albumin PF, studies have shown an improved recovery of macromolecules using different molecular weights and concentrations of Dextrans.16 Recent in vitro studies have suggested that a 3% Dextran 500-kDa solution is the most suitable additive given that it is large enough not to pass through the microdialysis membrane,18 maintains the greatest fluid recovery,14 and does not lead to an inflammatory response in the surrounding tissue.8An alternative colloid to increase the osmotic and hydrostatic pressure of microdialysis PF is Dextran.in vitro, in comparison to normal PF available for clinical use . The two types of PF were tested using an in vitro setup that closely approximates the clinical environment, to ascertain whether Dextran would be worthwhile to use during microdialysis sampling in human patients.Thus, the aim of this study was to determine whether PF supplemented with the recently commercially available 3% Dextran 500 kDa  could improve the fluid, absolute (AR), and relative recovery (RR) of inflammatory markers (39 cytokines and chemokines) during microdialysis sampling 2O) used was of high-performance liquid chromatography grade . All reagents were also of analytical grade, purchased from Sigma-Aldrich , and used as received, unless otherwise stated. Sodium chloride and potassium chloride were purchased from BDH Laboratory Supplies .All high-purity deionized water , microdialysis vials, Perfusion Fluid CNS, Perfusion Fluid CNS Dextran, M Dialysis 106 microdialysis pumps, and corresponding batteries and syringes were purchased from M Dialysis . Both PFs contain 147 mM of NaCl, 2.7 mM of KCl, 1.2 mM of CaClIn vitro microdialysis sampling experiments were performed using a VWR  advanced hotplate magnetic stirrer with temperature probe. Catheters were held in place during in vitro sampling using a triple-lumen cranial access device . Custom Invitrogen eBioscience ProcartaPlexTM human cytokine and chemokine 39-plex bead assays and human cytokine and chemokine standards  were purchased from Thermo Fisher . A complete list of the cytokines and chemokines analyzed is provided in ® software. Wash steps were performed using a ProcartaPlex hand-held magnetic plate holder.In vitro microdialysis sampling was performed using an artificial external solution (ES) representative of the brain extracellular environment. The ES comprised PF with 0.05% (w/v) sodium azide, 1 mg/mL of human serum albumin (HSA), and 39 human cytokines and chemokines, prepared in a 50-mL centrifuge tube  as follows. Microdialysis PF for the external solution was made in-house , to the same specifications as Perfusion Fluid CNS used for CMD in patients. The mixed cytokine and chemokine standards , received as lyophilized powders, were resuspended in accord with the manufacturers' instructions and subsequently diluted to 1:100 in PF with 0.05% (w/v) sodium azide and 1 mg/mL of HSA . The total volume of the ES was 25 mL. The final cytokine and chemokine concentrations are shown in The centrifuge tube (50 mL) containing the ES (25 mL) was suspended using a clamp stand in a thermostatically controlled glycerol bath (to avoid condensation) set to 37°C. Very gentle agitation of the external solution was applied using a magnetic stirrer. Two M Dialysis 71 brain microdialysis catheters were placed into the external solution through a triple bolt cranial access device, which was secured within the centrifuge tube using self-adhesive plastic film. Each catheter was perfused at 0.3 μL/min using M Dialysis 107 pumps, with syringes loaded with approximately 1.5 mL of either normal PF or PF containing 3% Dextran 500. Both the normal PF and Dextran PF were used as received from the manufacturer. The microdialysate samples were collected in microdialysis vials at the end of each catheter. Pumps and collection vials were kept at the same height either side of the glycerol bath, to nullify any hydrostatic pressure differences.in vitro sampling setup is shown in in vitro sampling test was repeated in three independent experiments over the course of 4 weeks . All samples were stored at −80°C before analysis.Sampling was performed for 48 h in total; the microdialysis vials were changed every 6 h, and sample from the ES was drawn at the 0-, 24-, and 48-h time points. A schematic of the TM human 39-plex bead-based immunoassay kits. Samples were thawed and gently mixed before analysing. In total, 25 μL of sample was used per well; all samples were analyzed in duplicate. The assay was performed as per the manufacturers' instructions. Wash steps were carried out using a hand-held magnetic plate holder (ProcartaPlex). All assays were analyzed on a Luminex 200 platform. We established that Dextran 500 did not interfere with the analysis by running a standard curve using the Dextran PF as a diluent and compared it to a normal PF standard curve, and Dextran PF had no discernible effect on the standard response.Quantitative analysis of cytokine and chemokines was performed using custom ProcartaPlex® .19 For each described analysis below, complete case analyses were conducted. A p value ≤0.05 was considered significant in all analyses. Graphical presentation was conducted using the R packages tidyverse,20 cowplot,21 and RColorBrewer,22 unless otherwise stated.All statistical analyses were conducted using the statistical software R, and its graphical interface Rstudio23 and conducted a linear mixed model per cytokine, using cytokine recovery as a dependent variable and time together with flush as independent variables.24 In each model, the independent experiment was considered to be the random intercept. Some cytokines  could not be quantified in the microdialysates, given that the concentrations recovered were below the lower limit of detection (as specified by the kit manufacturer) for the assay. Assumptions were examined graphically with regard to equal variance, linearity, and normal distribution.To assess how flushing the pumps affected cytokine recovery in the remaining fluid collected in that epoch, we used the R package nlme23–26 The dependent variable was the recovered cytokine value. For random intercepts, we used independent experiments. IFN-γ was excluded from analysis because the returned levels were below lowest levels of detection for both PFs. Assumptions were examined graphically as described above.28Similarly, for absolute recovery analysis, time and carrier were used as independent variables in a mixed model.t-test  or  a two-sided Mann-Whitney U test.RR was calculated as the ratio between the recovered cytokine in the microdialysis vial (numerator) and the recovered cytokine in the ES (denominator) obtained concomitantly at 24 and 48 h. For inferential analysis, cytokines exhibiting less than three positive observations  were excluded. For the remainders, the cytokine retrieval capacity of the different carriers  were compared using a two-sided Student's Only vials perfused with the standard PF demonstrated inadequate fluid recovery in certain epochs over the three experiments and hence needed intermittent flushing in the initial phase of the epoch . In totaThroughout the analyzed cytokines, the absolute recovery was systematically higher for the Dextran CNS PF carrier compared to the standard CNS PF . Two exaAt 24 and 48 h, the ES was sampled to enable calculation of the RR, generating a maximum of six RRs per cytokine . Overall, there was a general trend toward a higher RR for the Dextran perfusion fluid as compared to the standard CNS ; Fig. 4.in vitro cytokine/chemokine recovery using this new fluid carrier as compared to the conventional CNS PF. We found that the fluid and absolute recovery were much more robust when the Dextran was used in the carrier fluid. Two cytokines also had a significantly higher RR when Dextran was used in the carrier fluid, suggesting it to be a preferable PF in comparison to conventional CNS PF.To our knowledge, this is the first study using the CE-marked commercially available Perfusion Fluid CNS Dextran 500 from μDialysis, applicable for human use. This work represents a comprehensive overview of 9 though other explanations, such as varying catheter capabilities depending on the surrounding medium (in vivo vs. in vitro), have also been suggested.6 Similarly, in an in vitro recovery study by Dahlin and coworkers, they noticed a 30% fluid recovery decrease in CNS PF compared to an in-house Dextran 500 solution and only if special surface coated catheters were used ,7 similar to Kahl and colleagues using Ringer's solution as PF.16 Our 50% is similar to the previous study from our group comparing 3.5% albumin with normal CNS PF, which revealed that 44% of CNS PF epochs had inadequate fluid recovery compared to none using the albumin colloid.6 There have been no comparisons between Dextran 500 and 3.5% albumin PF, but a study from 2005 analyzed Dextran 60 and observed that the fluid recovery was slightly better for albumin, but this difference was insignificant given that both fluid recoveries were almost 100%.9 It should be noted that the lower fluid volumes we noticed is not as evident for catheters with normal CNS PF placed in brain ECF in vivo, presumably attributed to different pressure gradients and other factors such as endogenous proteins,14 but a phenomenon more commonly observed in in vitro studies.29 That being said, we believe the robust fluid recovery observed for Dextran PF is translatable to the clinical scenario akin to the benefit observed with 3.5% Albumin PF.6 In our experience, colloid-supplemented PF mitigates against catheter failure during clinical use and reduces the need for catheter replacement. Overall, the Dextran 500 PF shows superior fluid recovery as compared to the normal CNS PF, most likely attributed to the opposing oncotic pressure generated by the colloid within the microdialysis perfusate.The catheters perfused with normal CNS PF needed to be “flushed” in 50% of the epochs to reach expected adequate sample volumes, as compared to the Dextran PF, which always reached sufficient volumes of recovery in the collection vials. This is presumably attributed to ultrafiltration causing a fluid loss over the membrane of CNS PF because of low osmotic pressure,7 However, their study was not structured in a similar fashion as ours, making direct comparisons difficult; but researchers from Uppsala have now shifted to an in-house Dextran 500 as a colloid in PF and successfully recovered larger proteins in swine and rat brain injury models.31We found that, overall, the Dextran PF was superior to normal CNS PF in recovery across a range of cytokines. Almost all cytokines had a significantly higher improved AR, and the RRs in the current study were significantly higher for two of the proteins, namely M-CSF and TGF-alpha, using the Dextran PF, compared to CNS PF. Although data on Dextran 500 PF from M Dialysis has never been published before in a similar fashion, Dahlin and coworkers, in Uppsala, Sweden, have escalated Dextran concentrations, and molecular weight of Dextran molecules, and noted an improved recovery of some cytokines for Dextran 500 compared to CNS PF.in vitro analyses comparing a 3.5% albumin PF versus normal CNS PF,6 showing an RR improvement in the colloid in 9 of 12 analyzed cytokines. Further, the RRs in that study reached 30–50%, often double that of CNS PF.6 These RRs were higher than those observed in the current study, which revealed mean RRs of 1–10%, with some higher responders . We cannot easily explain this apparent disparity with our earlier studies, but this might be partly attributable to the different assay used and more extensive protein-protein interaction attributed to more proteins involved in the present study. In the present study, we used continuous stirring of the external solution. Comparing with the literature, some in vitro recovery studies utilized stirring, whereas some did not.33 It is conceivable that stirring may have an effect on recovery, given that the surface chemistry of interaction with a solution is a potentially complex situation in which layers and gradients can form, potentially affected by stirring. However, these lower RRs seem to be more in line with some previously published studies,34–37 where RRs in the range of 1–10% are often noted, although with some reaching higher levels. This distinct heterogeneity in results—depending on different study setups—means that caution must be exercised when comparing absolute RR between different studies.Our group has previously performed similar 38 We compared RRs at 24 versus 48 h, and this was not evident in our data set (data not shown), so even if protein depositions occurred they did not affect the recovery. Instead, a more probable explanation for the decrease in AR is that the ES concentrations decreased for many cytokines over time, which may be attributed to decay of the cytokines in the standard over 48 h at 37°C .12Previous studies have shown that catheters are susceptible to biofouling over time , decreasing the RR.16Dextran 500 and 3.5% albumin have not been compared directly in a similar fashion. However, Khan and coworkers noted that albumin is preferable to hydroxyethyl starch  with superior recovery for some cytokines, whereas HES was better for others, tentatively prompting investigators to a choice of PF depending on which cytokine is to be analyzed.13 and albumin in PF has been shown to have lower recovery of lactate compared to other colloids containing different concentrations of HES.16 Therefore, Dextran is presumably a preferable choice clinically as compared to albumin and normal CNS PF for the common metabolites, especially if 100-kDa catheters are used. However, before widespread clinical use, more-extensive examinations of RR of normally monitored metabolites need to be performed.It is unknown what effect (if any) Dextran PF will have on the recovery of clinically analyzed metabolites  as compared to normal CNS PF. A variant of Dextran 60 in PF was reportedly preferable to saline solutions in PF to recover glucose,n = 6 versus n = 6 measurements. However, given that many cytokines were not recovered in several epochs, many of these samples returned concentrations below the lower limit of detection by the Luminex assay for this analyte. Although it could be considered a limitation to compare fewer “positive” samples, these “zero” levels presumably represent important information as to highlight which cytokines and concentrations are suitable to recover using microdialysis in the current and similar scenarios. Further, many of the recovered cytokines exhibited a higher variability than expected on the basis of our previous studies. We acknowledge that additional experimental runs would have been desirable in the present study to increase the statistical sample sizes (n), as previously stated. Even so, the current results shed light on the individual cytokines' different propensities for variability in recovery and thus identify those cytokine species that behave most consistently with the current microdialysis technique.In the context of RR, the study was planned and designed for 13 membrane coating,39 fluid pressure,14 inclusion of nanoparticles,40 and microdialysis pump speed,7 which could be other ways to improve the recovery depending on the situation. We have specifically focused on the methodological constraints within clinical practice as a prelude to utilizing this perfusion fluid in clinical studies. As mentioned above, we checked that Dextran PF did not interfere with the Luminex assay. If different analytes are to be measured by other assay techniques, it would be important to perform tests to ascertain whether or not Dextran PF interferes with those assays. For example, preliminary tests we performed with ISCUSflex measurement of glucose, lactate, and pyruvate suggested that Dextran 500 did not interfere with such measurements.We only compared the effect of the PF in this study, whereas many other factors have been shown to alter microdialysis recovery, including membrane lengths,In vitro studies are not fully representative of the situation in vivo, perhaps attributed to direct tissue/catheter interactions such that the outer boundary of the microdialysis catheter is in contact in vivo with cells, extracellular matrix, and extracellular fluid in the brain tissue, rather than a simple in vitro fluid solution. This makes microdialysis measures such as FR and RR a necessarily crude estimate of the recovery of a given species in vivo. Nevertheless, the overall benefit of Dextran PF over conventional PF for the recovery of cytokines and chemokines is supported by these in vitro results, and we therefore regard Dextran PF as showing promise as a perfusion fluid for use in clinical microdialysis studies requiring recovery of protein.


In Mpumalanga province, more than 45 000 persons with disability receive a disability grant. Although research regarding social grants in general and disability grants specifically had previously been conducted from various perspectives, none has been carried out in Mpumalanga and none to explore the impact of the disability grant on the lives of the recipients. The objective of the study was to gain an understanding of the impact of the disability grant on the lives of recipients living in Mpumalanga. The study was conducted as a contextual, exploratory and qualitative study. The target population was persons with a disability receiving a disability grant. Data gathering was conducted in October 2010 using a semi-structured interview technique. The data were analysed in terms of the social and economic impact of the disability grant in the life of the participant. A combination of three qualitative data analysis methods was used to analyse the data. The qualitative findings indicate that although it is an individual grant, the disability grant was used to support the whole family and was frequently the family's only income. Food, clothes and electricity was most frequently bought with the disability grant. Food often did not last for a month. The families were living precariously and any crisis, for example lapsing of the grant, would result in hunger and desperation as a result of their complete dependence on the disability grant. Without insight in how people live their lives, registered nurses may give health education to patients that they cannot implement, perpetuating the burden of disease in South Africa. The South African Welfare System is premised on full employment Nattrass :3, implyIt is imperative that health care professionals and social workers have some understanding of the lives of a person who has to survive on a disability grant. Registered nurses or social workers in health care settings or government and municipal service departments respectively, frequently have to care for to persons living on a disability grant as the only income in a household. A person's socio-economic background has a direct influence on their health related needs and ability to access health and follow a treatment plan. Frequently, a health consultation and treatment plan does not have the intended outcome, resulting in chronic disease and unforeseen new health problems such as multidrug or extreme drug resistant tuberculosis or the frequent measles outbreaks. The registered nurse has to take cognisance of the context of the person cared for when giving health education. If the person cannot adhere to advice because of a lack of funds, health care workers are contributing to the burden of disease in South Africa. Although the current study was conducted in Mpumalanga province, the insight gained would be applicable anywhere in South Africa.From a legislative perspective, the Constitution of South Africa , a social grant means a child support grant, a foster care grant, a disability grant, an older person's grant, a war veteran's grant and a grant-in-aid .Social assistance: Social assistance is state-provided basic minimum protection to relieve poverty, essentially subject to qualifying criteria on a non-contributory basis .Impact: Impact, in terms of the study, is defined as the social and economic influence of the disability grant on the life of the recipient from the perspective of the recipient.The study was contextual, exploratory and qualitative, in investigating the impact of the disability grant on the life of the recipient living in Mpumalanga from the perspective of the participant. The design was suitable for the study as the focus was on the participants’ perceptions of how they live their lives.The study was conducted in the three districts of Mpumalanga, namely Gert Sibande, Nkangala and Ehlanzeni. In each district, an urban and rural setting was purposefully chosen to investigate the impact of the grant in a variety of settings. The target population was the recipients of disability grants in Mpumalanga. As there may be a difference in the impact on the lives of the recipients in the various districts, disability grant recipients of all three districts were included and the sample size of each district was saturated independently. The municipalities in each district were classified as predominantly urban or rural and one of each was chosen conveniently. In each of the chosen municipalities, the South African Social Security Agency (SASSA) prepared a list of disability grant recipients in the municipality. People on the SASSA lists were contacted and invited to participate. Because of the logistical challenges of gathering data in such a vast province, specifically in the rural areas, a decision was taken to conduct 30 semi-structured interviews per district, 15 in each municipality and to do the analysis after data gathering. If it was found that the data were not saturated, additional interviews would have been carried out.The sampling method was purposive and convenient. The inclusion criteria were: a recipient of a disability grant from Mpumalanga; willingness to participate in the study; both male or female; and living in the chosen urban or rural municipality in the three districts of Mpumalanga.Saturation was achieved in all six municipalities at the 10th to 12th interview.A semi-structured interview was used to gather data and generate a self-report in October 2010. The data-gathering instrument used was an interview schedule which consisted of two sections, including a demographic profile of the participant which was extensive, containing information regarding:The location of the participant .Type of disability.Socio-demographic information .Grant information .The interviewer completed the demographic data on the prepared form and then audio-taped the interview with permission. Rich data were gathered by direct questioning of people to report on their own experiences. The section exploring the impact of the disability grant included several open-ended questions and focused on the collection of the grant, the use of the grant in the household, and decision-making regarding the spending of the grant.Data analysis commenced after the interviews were completed. A combination of three qualitative data analysis methods was used. A template analysis style . In Pixley ka Seme and Mbombela, more females (n = 11 and n = 10) were included, whereas in Victor Khanye, more males (n = 10) were included in the sample. The modal age group was 41 to 50 years with 19 participants for the combined group, followed by 15 participants in the 31 to 40 years age group. For all three urban municipalities, the majority of the participants (32 of 45) was never married. A third of the combined urban group (n = 16) never had any schooling, followed by 23 participants who achieved less than grade 12. The Pixley ka Seme sample was less literate compared to the Victor Khanye and Mbombela samples. The number of children per household varied between none in 10 households to five in four households: the modal number of children per household being two (15 of 45 households). In each of the urban municipalities, the ages of the majority of the children tended to be younger . The majority of the children (46 of 64) were still in primary school.A total of 90 participants were interviewed. The demographic profile for the urban municipalities is presented first . In Pixln = 12), in Dr JS Moroka emotional disability (n = 7) and in Bushbuckridge, multiple disability (n = 8). In all three municipalities, both males and females were included in the study with more males (n = 24) than females (n = 21). The modal age group was 21 to 30 years (24 of 45) followed by a 51 to 60 years age group, but in Albert Luthuli, all the participants were in the 21 to 30 years age group. As was the case with the urban municipalities, the majority of the rural sample (n = 36) was never married. Seven of the 45 households had no children as part of the family. The modal number of children per household was two (n = 14). Nine households had three to four children to support and only two households supported five children. There were more children in the combined rural sample (n = 98) when compared to the combined urban sample (n = 87). A large proportion (43 of 51) of the children was still in primary school. In terms of the age of the children, more than half of the combined rural children sample (54 of 98) were 10 years of age or younger. The modal age group was 11 to 15 years in 28 children.In terms of the rural municipalities, the type of disability varied within each municipality. In Albert Luthuli, the majority of the sample had a physical disability  but then I was not able to give my children pocket money for school so I left it.’No participant reported being able to save any money.In terms of transport, participants mostly walked to their destination, using a taxi infrequently.In a household where there were more than one recipient of a grant , the grants were used in combination. Each person retained the right to spend their own grant, but if there was a need, resources were combined, for example:‘I do a lot because if they need a shirt like the other one who needed shoes we had to add money and buy the shoes so that he can continue and study.’‘The grant helps me here at home. My mother receives a grant as well, she buys food and when I get mine, I also give her money. Even the children's grant, it helps us a lot.’In most of the six municipalities, more than two-thirds of the participants had school-going children and in all instances, the disability grant was used to support the children's school fees, to buy uniforms or to pay for additional trips. Participants explained as follows:‘My younger sibling goes to school. I pay school fees and uniform. He sometimes tells me that he needs school shoes, even if I buy, it will just be the shoes.’disability grant] is not enough because you will find there is much costs then.’‘I buy clothes for the school going children but it . When the money comes back it has interest its equal R65.’Some participants reported that they would get a loan at an exorbitant interest rate. The repayment would deplete their disability grant to such an extent that they would have to borrow money again the next month: ‘There is this other lady who is a loan shark, I go to her and ask for a 12.5 kg [The most frequent type of disability, for both the urban and rural group, was physical. In some instances the deduction, from the narratives, was that the underlying disease was HIV and AIDS related, but a large group also mentioned arthritis and orthopaedic related skeletal health problems. Support for the non-infectious origin of the disability is the age distribution of the urban group. More than half of the urban group (27 of 45) were older than 41 years of age, but in the rural group, only 14 of the 45 participants were in this age category.The use of transport was infrequent, mostly for health related visits and always budgeted for carefully. The majority of the participants used the local clinic or hospital for health care as it was free of charge. Specialists were only used after a referral from the local health practitioner. There was one exception. In Albert Luthuli, although participants would use the clinic if they felt really ill, they would, although expensive, go to a private doctor, at R200.00 to R250.00 per consultation. The reason for not trusting the local health care system is unclear.In both urban and rural samples, the majority of the participants (39 of the 45) did not achieve grade 12 level of education. The proportion of participants not receiving any schooling was high in the urban sample (16 of 45) compared to 3 of 45 participants in the rural sample. There were also variations in the urban sample with 10 of the 15 participants in Pixley ka Seme not having any schooling compared to 1 of 15 Mbombela participants. The high level of functional illiteracy seems to have an influence on the way information is disseminated to disability grant recipients.It was unclear whether all eligible children received a child grant and there was no specific question to determine if they should. The number of children younger than 10 years old were high in both rural (54 of 98) and urban (51 of 87) households. Additionally, in the rural sample, there were 11 households with 4 or 5 children per household and 8 such households in the urban sample. If the disability grant recipients were as uninformed regarding child grants as they were regarding their own disability grant, it is quite possible that many of the households did not receive the financial support they were legally eligible to.Families frequently used the disability grant to pay school fees and to buy school clothes. Although, being an individual grant and on paper supposed to be used for the disabled individual, in practice this was not the case. Support for this statement is found in the number of persons supported by the disability grant in a household. In total, the disability grant issued to 90 persons supported 185 children, most of who were still in primary school. Research should be conducted to inform policy with regards to using a disability grant to pay school fees, where it is the only income in the family.Saving for a future problem was almost non-existent in the combined urban and rural sample. It was not as though saving was not considered important, but it was mostly impossible. The most frequent method of saving was participating in a stokvel group where a number of women would contribute a monthly amount, usually about R100.00, and use the cash when it was her turn to buy either clothes or a piece of furniture. A large group of the participants, except for the Mbombela sample, paid monthly into a funeral society which was also community based and in some instances would only supply a coffin, not the social occasion accompanying a funeral. The understanding gained was that it was the sensible thing to do as we would all die some day and the person should not be a burden to the family post-mortem.The following limitations of the study are noted:Six different field workers were used to gather the qualitative data. Although they were trained twice, depth of probing was, in some instances, lacking. The lack of depth may, however, be offset by the culturally congruent nature of the fieldworkers in the three districts.Because of the qualitative nature of the study, some aspects were raised but not explored in depth.The data was self-reported and, as such, social desirability bias may have influenced it.There is a lack of literature to support or contrast the findings of the current study.Several recommendations emanated from the evidence generated in the study.Any health care-related prescription should take the context of the lives of this group of people into consideration.In a household where the disability grant is the only income, exemption of paying for essential services such as electricity and water should be considered.The reality of school going children depending on the grant, the majority of whom are still in primary school, should be accepted. Several recommendations regarding school going children emanated from the study:Although some participants reported that their children were eligible and received a child grant, it was unclear whether all eligible children received a grant. An investigation is required.Children living in a household where the disability grant was the only income for a livelihood should be exempted from paying school fees.Many of the children in the household were orphans and it was unclear whether the children were receiving appropriate financial assistance, or whether the disability grant recipients were knowledgeable regarding the foster care grant. Support from the Department of Social Development is required.The disability grant fulfils a life-saving function in the family where a person with a disability lives. The influence is profound and literally a matter of life and death, frequently being the only income in the family. The group most assisted by the disability grant is children and specifically children less than 10 years old. Without an understanding of how people live in their environments, health care professionals may not understand why health education is not followed. A feeling of frustration and desperation could result if the person living on a disability grant cannot comply with the health education as a result of a lack of funds. Health promotion must be considered carefully and contextualised for the individual, because without insight into the lives of our patients, the result may be harming instead of improving health.


Experimental acute lung injury models are often used to increase our knowledge on the acute respiratory distress syndrome (ARDS), however, existing animal models often do not take into account the impact of specific fluid strategies on the development of lung injury. In contrast, the current literature strongly suggests that fluid management strategies have a significant impact on clinical outcome of patients with ARDS. Thus, it is important to characterize the role of fluid management strategies in experimental models of lung injury. In this study we investigated the effect of two different fluid strategies on commonly used outcome variables in a short-term model of acute lung injury, in relation to age. Infant (2–3 weeks) and adult (3–4 months) Wistar rats received intratracheal instillations of lipopolysaccharide and 24 hours later were mechanically ventilated for 6 hours. During mechanical ventilation, rats from both age groups were randomized to either a standard or conservative intravenous fluid strategy. We found that the hemodynamic response in infant and adult rats was similar in both fluid strategies. Lung wet-to-dry ratios were lower in adult, but not in infant rats receiving the conservative fluid strategy as compared to the standard fluid strategy. There were age-related differences in markers of alveolar capillary barrier disruption and alveolar fluid clearance, yet these were unaffected by fluid strategy. Finally, we found significantly higher IL-1β and TNF-α concentrations in the adult rats treated with the conservative as compared to the standard fluid regimen. In conclusion, the choice of fluid strategy in mechanically ventilated rats with experimental LPS-induced acute lung injury has a significant effect on pulmonary extravascular water, an important and well-recognized lung injury marker, and on the local pro-inflammatory cytokine profiles. We advocate the use of a more uniform, conservative, fluid strategy regimen in experimental models of acute lung injury. Over the last decades, accumulating evidence has shown that fluid overload is associated with adverse outcomes in critically ill patients requiring mechanical ventilation (MV). In a large clinical randomized controlled trial in adult ICU patients, a conservative fluid regimen was found to be associated with significantly more ventilator-free days and improved oxygenation . The impThe likely underlying pathophysiological mechanism of the adverse effect of fluid overload is aggravation of extravascular and interstitial edema in the lungs and other organs , but mayThe modelling of human ARDS by experimental induction of acute lung injury in animals has proven to be an important tool to increase our knowledge of this complex syndrome . Given tIn general, experiments of acute lung injury in animals have a relatively short duration (hours) as compared to the clinical setting of human ARDS (days to weeks). Consequently, the impact of fluid overload, which is a relatively slow process, on clinical disease outcomes may be limited. However, we hypothesize that fluid strategy has potential impact on short-term and commonly used outcome variables in models of acute lung injury , such asEscherichia coli, serotype: O127:B8, Sigma Aldrich, St. Louis, MO, USA) with a nebulizer  24 hours before the onset of mechanical ventilation (MV).The animal protocols were approved by the national Ethical Committee for Animal Experiments . Animals were used in compliance with Institutional Standards for Use of Laboratory Animals. The rats were housed in a specific pathogen-free facility on a 12/12h light/dark cycle and 20–24°C ambient temperature and allowed to acclimatize for 7 days prior to start of the experiment. Infant  and adult  specific-pathogen free RccHan Wistar rats  received intratracheal inoculations of LPS, 5mg/kg  subcutaneously and an intraperitoneal injection of pentobarbital sodium . Subsequently, animals received a tail vein cannula for administration of maintenance anesthesia and fluids. A tracheostomy was performed for initiation of MV. During MV, anesthesia was maintained by intravenous infusion of 20–30 mg/kg/hour pentobarbital sodium . A polyethylene catheter was inserted into the right carotid artery and connected to a pressure sensor in order to continuously monitor blood pressure and heart rate. Temperature was maintained at the normal body temperature for a rat (±37.5°C) using a heating pad.2O. The inspired oxygen fraction (FiO2) was set at 30% and the respiratory rate at 60 breaths per minute with an inspiratory/expiratory ratio of 1:1.5. The peak inspiratory pressure (PIP) was adjusted to maintain normocapnia (PaCO2 = 35–45 mmHg) and a normal pH (7.35–7.45). Static lung compliance was determined every hour upon an inspiratory hold.The rats were mechanically ventilated in a pressure-controlled mode  for 6 hours aiming at tidal volumes ranging from 6–8 ml/kg as measured by a pneumotachometer  and recorded by respiration software . Positive end-expiratory pressure (PEEP) was set at 3.5 cmHPrior to the onset of MV, all rats were randomly allocated to one of the two experimental groups; standard fluid strategy or conservative fluid strategy (n = 6–8 for each group). These fluid strategies were based on pilot experiments measuring the blood pressure response to each strategy during 6 hours of MV. Volume associated with maintenance anesthesia was incorporated in the allocated fluid strategies. Ringer’s lactate solution was administered to reach the required volume of the allocated fluid strategy. The standard fluid regimen consisted of 6.5 ml/kg/hour intravenous fluid, based on the normal daily fluid intake of a healthy rat . The cong, 4°C and the supernatant stored at -80°C. The middle lobe of the right lung was flash-frozen for homogenization. The total water content in lung, kidney, liver and heart tissue was measured by calculating wet-to-dry weight ratios.The rats were euthanized after 6 hours of MV by intravenous injection of sodium pentobarbital and exsanguination. Bronchoalveolar lavage fluid (BALF) was obtained from the left lung by three separate aliquots of 0.9% NaCl  and the aliquots were pooled. Immediately after performing the BAL, a fluid aliquot was retrieved for determination of total cell counts. The remainder of the bronchoalveolar lavage fluid (BALF) was centrifuged for 10 min at 1500 x +/K+/ATPase activity, an important ion transport regulator of alveolar fluid clearance marker, was determined in lung homogenate using the Na+/K+/ATPase microplate assay kit . All assays were used according to the manufacturer’s instructions. Haematoxylin and eosin (H&E)-stained lung sections were prepared and evaluated under a standard light microscope by a blinded investigator using the lung injury scoring system as stated by the American Thoracic Society , the whiskers represent 1.5 IQR; n = 6–8 animals per group. **p<0.01.(PDF)Click here for additional data file.S2 FigLiver wet-to-dry weight ratio of each experimental group of LPS-inoculated and mechanically ventilated rats. Data are presented as median + interquartile range [IQR], the whiskers represent 1.5 IQR; n = 6–8 animals per group. **p<0.01.(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Fluid overload is a potential complication of hysteroscopic procedures with the possibility of dangerous electrolyte changes. This prospective randomized controlled trial aimed to compare perioperative outcomes and changes in electrolytes after hysteroscopic procedures between the manual infusion (MI) and the pump infusion (PI) methods for distending media infusion. One hundred consecutive women who had hysteroscopic procedures between December 2013 and February 2017 were recruited and randomly allocated to either the MI or PI group. The PI group was associated with an increased volume of infused fluid and collected fluid compared with the volumes of the MI group. Almost all serum electrolyte levels differed significantly between the baseline and postoperative values in both groups; however, no significant differences were noted between the groups. The change in potassium level was positively correlated with the volume of fluid deficit , whereas the change in calcium level was negatively correlated with the volume of fluid deficit . With no between-group differences in the changes in the other perioperative parameters and electrolytes, the MI method can be a good alternative for delivering distending media for hysteroscopic procedures. Any type of distending media can potentially cause the above complications following rapid systemic absorption.Fluid overload is a life-threatening complication in hysteroscopic procedures. Excessive absorption of distending media, especially hypotonic, electrolyte-free media, may result in dilutional hyponatraemia, potentially causing cerebral oedema, coma and death2. Studies comparing unipolar and bipolar electrodes support the safety profile of bipolar electrodes in hysteroscopic surgeries5. However, in locations where bipolar electrodes are unavailable, various methods have been adopted to reduce the incidence of fluid overload, including automated fluid monitoring, preoperative use of vasopressin or gonadotropin-releasing hormone agonists1.Technological advancements in recent years have resulted in the development of bipolar electrodes6. However, the study results might be biased given its retrospective nature6. Thus, the primary objective of this prospective randomized controlled study is to compare the infused volume of the MI method with the PI method in hysteroscopic procedures. The secondary objective of this study was to compare the other perioperative outcomes and postoperative changes in serum electrolytes and blood osmolarity between the MI and PI groups.In a previous retrospective study, manual infusion (MI) of distending media was associated with a significantly reduced average volume of infused fluid, reduced operative time and lower postoperative abdominal pain scores compared with the pump infusion (PI) methodOne hundred and two consecutive women were enrolled in this study. Two women did not receive allocated interventions for personal reasons. Forty-nine women in the MI group and 51 women in the PI group underwent hysteroscopic procedures  had blood transfusion due to preoperative symptomatic anaemia. In the MI group, one woman with menorrhagia was found to have a grade 3 endometrioid endometrial cancer after hysteroscopic myomectomy. She subsequently underwent a robotic staging operation, and her pathological staging was International Federation of Gynecology and Obstetrics stage 1 A with negative peritoneal washing cytology. She remained alive without evidence of disease.Almost all serum electrolyte levels differed significantly between the baseline and postoperative values in both groups; however, no significant differences were noted between the groups Table . In the 6. The continuous flow based on pressure by an automatic pump in the PI group instead of the intermittent flow based on visibility in the MI group might explain the above finding of higher infused and collected fluid volumes in the PI group. Theoretically, the greater the infused volume, the greater the risk of excessive fluid absorption will be, as concluded in the findings of the previous study6. Wong et al., who evaluated the efficacy of transcervical injection of vasopressin during hysteroscopic myomectomy, found a positive correlation between the infused volume and fluid deficit7. We also found a significant correlation between the infused volume and fluid deficit  in the current study; therefore, it is advised to keep the amount of infused volume as low as possible.In this study, the MI group had a significantly lower volume of infused fluid and collected fluid compared with the PI group Fig. , which w9. Haemolysis can occur either with the use of distilled water, which is extremely hypotonic, or iatrogenically during the collection of blood samples10. Hyperkalaemia, especially a serum potassium level of >6 mEq/L, may result in life-threatening arrhythmia. Gupta et al. reported that the percentage increase in serum potassium may be up to 16.19% if a large amount of infused fluid was used during the transurethral resection of the prostate (TURP) procedure9. Panovska Petrusheva et al. also reported an increase in the potassium level after the TURP procedure that was associated with the amount of infused fluid11. The TURP procedure is similar to the operative hysteroscopic procedure. Thus, the serum potassium level should also be reassessed if a large fluid deficit was noted during the hysteroscopic procedure.The change in potassium level was positively correlated with the volume of fluid deficit in this study, which could be explained as a consequence of haemolysis or intracellular uptake of the infused fluid12. Severe hypocalcaemia may be associated with seizures, hypotension, heart failure, or laryngospasm. Lee et al. reported that hypotension occurred in a woman with TURP syndrome  due to a reduced ionized calcium concentration during hysteroscopy, and her refractory hypotension improved after 40 mL of 3% calcium chloride12. Agarwal et al. also reported a case of TURP syndrome with hypocalcaemia after the TURP procedure13. Thus, our findings support the coexistence of hypocalcaemia in women with large fluid deficits after hysteroscopic or TURP procedures13. Although hyponatraemia rarely develops in women who received a bipolar hysteroscopic procedure, it is prudent to assess the serum calcium level if a large fluid deficit occurred.Our study revealed a negative correlation between the change in calcium level and the fluid deficit volume Table . The dilet al. reported a decrease of 4.77 ± 0.831 mmol/L in serum sodium levels after unipolar hysteroscopic surgeries4. Similarly, Berg et al. reported a reduction in the mean serum sodium to 133.8 mmol/L from 138.8 mmol/L in the unipolar group5. Nonetheless, the operative time was markedly longer in Tammam et al.’s study (31.93 ± 12.92 minutes) compared to that in our study  compared to that in our study , such as the Truclear device  and the MyoSure device 24. Nevertheless, the choice for hormonal endometrial preparation before hysteroscopic surgery requires robust cost-effectiveness analyses23.In addition, preoperative use of dienogest, an orally administrable progestin, has good endometrial thinning effects and tolerable side effects25. Distilled water was the original distending medium for resectoscopic urologic surgery14. Nonetheless, it is worth mentioning that if distilled water is absorbed systemically, one of the risks was haemolysis. The addition of solutes such as sorbitol and glycine increased the medium’s osmolality to a degree that haemolysis could be largely prevented14.In our study, due to the unavailability of 3% sorbitol and 1.5% glycine in our institution, distilled water was used for unipolar hysteroscopic myomectomy28. Nonetheless, intraoperative absorption of these electrolyte-free fluids, including distilled water, 3% sorbitol and 1.5% glycine, can cause hyponatraemia, hypoosmolality, volume overload and pulmonary oedema27. It is important to meticulously monitor fluid balance and reduce operation time to minimize complications.Electrolyte-free low viscosity solutions, such as 3% sorbitol and 1.5% glycine, have been widely used as distending media during hysteroscopic surgeries using unipolar instruments14. The in-flow pressure is approximately 70 to 100 mmHg when the bag with distending medium is 1 to 1.5 m above the uterus15, and the height should be kept at the minimum elevation to allow sufficient uterine distention. It has been demonstrated that systemic absorption is greater with increasing intrauterine pressure, especially if it exceeds mean arterial pressure14. The mean arterial pressure in normal healthy people without known cardiovascular risk factors has been reported to be 88 ± 9 mmHg29. Hasham et al. also reported that there was no absorption of the distending medium into the venous system at an intrauterine pressure of 70 mmHg, while at 150 mmHg, contrast distending medium was clearly seen entering the uterine venous plexus30. Thus, the in-flow pressure of 70–100 mmHg in our PI group seems to be reasonable.Gravity is the simplest method to deliver the distending medium into the intrauterine cavityThe main limitation of this study is the limited number of hysteroscopic myomectomy procedures. However, the randomized study design might compensate for this limitation. In addition, although there were statistically significant between-group differences in the infused and collected fluid volumes, owing to their limited powers Table , the intIn conclusion, the MI method was associated with a reduced infused fluid compared with that of the PI method. With no between-group significant differences in the changes in the other perioperative parameters and electrolytes, the MI method can be a good alternative for delivering distending media for less complex hysteroscopic procedures.This study was performed at the Department of Obstetrics and Gynecology of Far Eastern Memorial Hospital and was approved by the Research Ethics Committee of the institution. All women aged 20 years and older who had indications for hysteroscopic procedures between December 2013 and February 2017 were invited to participate in the study. Before receiving the intervention, all women provided written informed consent. All methods were performed in accordance with the guidelines and regulations specified by Far Eastern Memorial Hospital.6.Women were allocated to the MI or PI group in a randomized order based on computer-generated random numbers at a ratio of 1:1. The surgical procedures were performed by the corresponding author. Under intravenous anaesthesia, cervical dilation was performed with Hegar uterine dilators, followed by insertion of a unipolar resectoscope with an outer diameter of 8 mm  into the uterine cavity. The uterine cavity was then infused with distilled water for inspection, resection, or ablation of tissue. In the MI method, a 60-mL disposable syringe  was connected to the resectoscope via a 90-cm extension tube , and an assistant helped to pump the distilled water manually. Two 60-mL syringes were used at one time to minimize the waiting time required for refilling the syringe. A large collecting bag was tucked beneath the woman’s gluteal region and secured to the surgeon’s gown to capture fluid spilled from the cervix and the resectoscope. The total volumes of infused fluid and outflow fluid were recorded. In the PI method, a continuous-flow fluid infusion pump device  was used to deliver the fluid media at a constant in-flow pressure of 70–100 mmHg, depending on the visibility of the surgical view, and the remaining procedures were performed in a manner similar to that of the MI groupBlood samples were drawn before and after surgery. The blood sample was sent for analysis of complete blood count, blood osmolarity and serum electrolytes, including sodium, potassium, chloride, magnesium, calcium and phosphorus. The women’s characteristics, clinical indications for the hysteroscopic procedure, the volume of distending media used, and perioperative data were recorded. The fluid deficit was calculated by subtracting the total volume of collected fluid media from the total infused volume. The postoperative pain scores (0–10) were recorded twice by self-reporting using a visual analogue scale at baseline when the patient recovered from anaesthesia in the recovery room  and 30 minutes later prior to discharge from the recovery room . Changes in serum electrolytes, blood osmolarity and haemoglobin were determined by subtracting the preoperative measurements from the postoperative measurements.The Wilcoxon rank-sum test and the Chi-square test were employed for statistical analyses using STATA software . A p-value of less than 0.05 was considered statistically significant.6. To detect a difference in the amount of infused fluid, we conducted a test with a significance level of 0.05 and a power of 0.9. We anticipated that two groups of equal size would be required. Thus, we concluded that at least 25 subjects in each group were required to test the above hypothesis.Given that the primary objective of this study is to compare the infused volume between the MI and PI groups, the variable was used to calculate the sample size required. A previous study reported an infused volume of 1117 ± 712 mL in the MI group and 2216 ± 1502 mL in the PI groupwww.clinicaltrials.gov.NCT02012010, registered on 16/12/2013. Available at ClinicalTrials.gov, www.clinicaltrials.gov, NCT02012010, 16/12/2013. Number of IRB and date of approval: 102124-F, 21/10/2013.Clinical Trial Registration Number and date of registration: ClinicalTrials.gov, CONSORT 2010 checklistResearch Protocol