The aims of the study were to evaluate the external and internal work load of trained handcyclists during a mountain time trial, to compare the results with a world-class handcyclist, and to identify time trial performance determinants.Ten trained and one world-class handcyclists performed a graded exercise test to determine power output and heart rate at the (first and second) ventilatory thresholds and exhaustion. Power output and heart rate were continuously measured during the race.P < 0.001) was lower in the trained handcyclists compared with the world-class handcyclist. The absolute and relative heart rate during the race  and relative power output during the race  were similar. Trained handcyclists cycled significantly less time at a power output between first and second ventilatory thresholds  and more at a power output greater than second ventilatory threshold . Power output at the second ventilatory threshold showed the strongest correlation with finish time (r = −0.78) and peak power output with mean power output of the race (r = 0.90).The mean absolute power output during the race (119 ± 21 vs. 203 W, The laboratory outcome peak power output and power output at the second ventilatory threshold are important performance determinants for longer time trials in handcyclists, and it is, therefore, important to improve these outcomes with training. Because the trained handcyclists cycled most of the race in intensity zones 2 and 3, it is recommended to incorporate these zones also in the training. What Is KnownThe HandbikeBattle is mountain time trial in Austria in which former patients of rehabilitation centers participate. Although there are studies on handcycling in the rehabilitation setting and competitive sport domain, the physical demands of climbing a mountain in a handcycle are yet unclear.What Is NewThis study shows that power output at the second ventilatory threshold and at exhaustion are important performance determinants for mountain time trials in handcyclists. Because the trained handcyclists cycled most of the race in intensity zones 2 and 3, it is recommended to incorporate these zones also in the training.1 These former patients are selected by their rehabilitation centers because they might need this challenge to become mentally and/or physically fit. Some of the HandbikeBattle participants discover that they are really talented in handcycling and start to participate in competitive handcycling events and eventually in the Para-cycling World Championships and Paralympic Games.The HandbikeBattle is a yearly organized mountain time trial  in Austria in which former patients of Dutch rehabilitation centers participate.2 and in the competitive sport domain,3 the mechanical and physiological demands of climbing a mountain in a handcycle are yet unclear. A previous study1 investigated the exercise intensity distribution of 17 handcyclists with spinal cord injury (SCI) during this HandbikeBattle time trial. Results showed that the HandbikeBattle participants exercised most of the time at a vigorous intensity (73%),1 which is in contrast to the low to moderate intensities for level ground marathons and/or ultra-long handcycling races.5Although there is an emerging body of evidence regarding handcycling in the rehabilitation setting6 HR can be influenced by environmental conditions, cardiovascular drift, the VO2 slow component,7 and, among people with SCI, also by impairment of the autonomic nervous system.8 While PO is often an explicit outcome in individual laboratory-based peak handcycle performance tests, both absolute and relative indicators of PO and HR allow a description of the external and internal load during race events. Power output is often derived from specific sensor devices and is deemed to be the best objective indicator of external load because it takes both velocity and mechanical resistance forces  into account. The PO distribution has been studied during races in professional cycling.11 From these studies, it is clear that PO distribution is different in time trials (mostly in high-intensity PO zone) compared with mass start stages (mostly in low-intensity PO zone)9 and that the absolute PO is similar between flat and semimountainous terrain but higher in mountainous terrains.11 Furthermore, it was shown that compared with PO, HR underestimated the time spent at the low- and high-intensity zones and overestimated time spent in the moderate intensity zone.10 While the power profile has been investigated extensively in road cycling, current research on the PO demands of handcycling outside the laboratory is limited to level ground handcycling,12 which leaves a gap regarding preparation for uphill handcycling races. Last, not much is known about the PO determinants of a handcycling time trial performance. Previous studies found that peak aerobic PO,14 aerobic lactate threshold,14 and PO at 4 mmol/l14 are important determinants of a 15- to 16-km handcycling time trial performance in a laboratory14 or on a cycling racing circuit.13Despite the linear relationship between internal load measures such as heart rate (HR) and external load measures such as power output (PO) in handcycling,To get more insight in the mechanical and physiological demands of uphill handcycling, the HR and PO of trained handcyclists during the HandbikeBattle mountain time trial were investigated. For a good interpretation, these results were compared with a world-class (WC) handcyclist who was familiar with the HandbikeBattle route. The aims of this study were, therefore, (1) to investigate the external and internal work load of trained handcyclists and compare their results with a WC handcyclist (the “criterion standard”); (2) to investigate the external and internal work load during three sections  of the HandbikeBattle; and (3) to identify the most important PO determinants of this time trial performance.n = 36) was selected to participate in the present study. This subgroup was selected based on having a peak handcycling PO (POpeak) greater than 100 W in the GXT at the medical screening, trained relatively often, and were motivated to handcycle with a power meter during all training sessions and the race. Exclusion criteria for the study were: not being able to come to the rehabilitation center in the month before the race for an additional GXT, not owning a handcycle in which a wheel with power meter could be fitted, and not understanding the Dutch language.People with lower-limb disabilities are allowed to compete in the HandbikeBattle if they have no contraindications for physical exercise. This was checked by a mandatory medical screening including a graded exercise test (GXT). Because of a limited availability of the expensive power meters, a device on a (hand)cycle that measures the PO of the rider in real life, a subgroup  was an international medalist at major global championships,16All HandbikeBattle participants were classified by official classifiers before the HandbikeBattle and following the classification rules from the Union Cycliste Internationale.This study was approved by the local ethics committee of the Centre for Human Movement Sciences, University Medical Centre Groningen, the Netherlands (ECB/2012_12.04_l_rev/Ml). All participants signed an informed consent before participating in this study.1Power output and HR were measured during a laboratory-based GXT and during the HandbikeBattle race. Data were collected in the month before (laboratory) and during five editions (2015–2019) of the HandbikeBattle. The race was held each year in June on the Kaunertaler Gletscherstrasse in Austria, starting at the toll gate and ending at the Ochsenalm.In the month before the HandbikeBattle, the selected handcyclists conducted the GXT. The GXT was performed in the participant’s own handcycle, which was attached to a Cyclus2 ergometer . During the GXT, breath-by-breath data were collected using a COSMED K4B2  in 2015 and 2016, an Oxycon Mobile  in 2017 and a Cortex  in 2018 and 2019. Heart rate was measured continuously using a Polar HR sensor chest strap .The participant started the GXT with a 5-min warm up at a self-selected cadence and PO. Thereafter, the participant had 2-min rest, followed by incremental 3-min stages at a self-selected cadence greater than 60 revolutions per minute (rpm). With every 3-min block, the PO increased with 20 W until volitional exhaustion. The starting PO was determined as the achieved POpeak during the first GXT, performed during the medical screening, minus 120 W. If the participant had a POpeak of 120 W or lower at the first GXT, the PO started at 30 W and increased with 10 W every 3 mins.The GXT of the WC athlete was performed in the athlete’s own handcycle using a Cyclus2 ergometer. Breath-by-breath and HR data were collected using a COSMED Quark CPET . The starting PO was set at 100 W with an increase of 15 W per 3 mins at a cadence approximately 85 rpm. When the second ventilatory threshold (VT2) was reached, the PO increased with 15 W every minute until volitional exhaustion.17 Breath-by-breath data were filtered with a moving average filter over 15 breaths before the analysis. Three plots were presented to an experienced rater to determine VT1 and VT2: (1) VCO2 versus VO2, (2) the ventilatory equivalents of oxygen (Ve/VO2) and carbon dioxide (Ve/VCO2) versus time, and (3) respiratory exchange ratio versus time.From the GXT, the first ventilatory threshold (VT1) and VT2, POpeak, and HRpeak were determined as described in detail by Kouwijzer et al.2 at the time of VT1 and VT2 were determined as the 15-breaths-average value at the VT.The PO accompanied with this time that VT1 and VT2 occurred was determined as the highest PO maintained for 3 mins with an addition of 3.3 W for every 30 secs maintained in the block in which the VT occurred (1.7 W for the 10-W protocol). The HR and VO18 VO2peak (in liters per minute) and HRpeak (in beats per minute [bpm]) were defined as the highest 15-breath average of VO2 and HR, respectively.The POpeak (in watts) was defined as the highest PO maintained for 3 mins during the GXT with an addition of 3.3 W for every 30 secs maintained in the final block (1.7 W for the 10-W protocol).The PO during the race was measured with a PowerTap P1 Hub system . The PowerTap (sample frequency: 1 Hz) and a Garmin HR monitoring chest strap were connected to a Garmin Edge 500 GPS system .The WC handcyclist used a Quarq Dzero crank powermeter , a Garmin 1000 Edge GPS system , and a Tacx HR monitoring chest strap . Together, this equipment enabled the registration of PO, speed, traveled distance, HR, and cadence at every second throughout the HandbikeBattle.Before race data analysis, outlier values of PO above 2*POpeak and HR below the HR at rest were replaced by the average of the values before and after the data point, because they are thought to be unrealistic, that is, erroneous values. The total time the participant spent on stops during the race (%time stopped) was calculated as the time that both speed and cadence were 0 , which was divided by the total race time. Furthermore, data points where the speed was 0 m/sec but cadence greater than 0 rpm were considered errors and not used for further analysis. When, as a result, a data set had too many missing data (>10% of the data), this data set/participant was excluded from the study.The valid race data were used to determine the mean and peak values for HR (in bpm), PO (in watts), cadence (in rpm), and velocity (v in meters per second). The mean and peak HR and PO were also expressed as percentage of the peak value achieved during the GXT.10 In zone 1, the PO or HR during the race is below the PO or HR at VT1 (low intensity). In zone 2, the PO or HR during the race lies in between the PO or HR at VT1 and VT2 (moderate intensity), that is, lactate begins to accumulate in the blood and the breathing rate begins to increase. In zone 3, the HR or PO during the race is above the PO or HR at VT2 (high intensity), that is, lactate quickly accumulates in the blood and the person needs to breathe heavily. The amount of time spend in every HR and PO zone during the race was calculated as a percentage of the total race time.The VT1 and VT2 determined in the GXT were used to calculate three PO and HR zones.Furthermore, the race data were cut into three sections based on the race trajectory, using the GPS data recorded by the Garmin device. Figure t test.First, potential differences in personal characteristics and GXT outcomes between the group with complete PO data (study participants) and the group with incomplete PO and/or HR data (nonstudy participants) during the time trial were tested with an independent t test was used to test for differences between the trained handcyclists and the WC handcyclist.Descriptives were calculated for the outcomes of the GXT, the total race, and the three sections of the race. The one-sample A repeated measures analysis of variance (within-subject factor: section) was used to determine whether there was a difference between sections in PO and HR outcomes within the trained handcyclists. Bonferroni tests were performed as post hoc analyses.Pearson correlation coefficients were calculated between finish time and the PO outcomes of the laboratory test and during the time trial to identify the most important PO determinants of the time trial performance.P < 0.05. Effect sizes (Cohen d) were calculated and classified as small when 0.20, medium when 0.50, and large when 0.80.19All statistical analyses were performed using SPSS . Values were considered significant at P < 0.001), VO2peak (P = 0.001), and slower finish time  compared with the study participants ; and five handcyclists had too many missing PO data >10%). Therefore, data of 11 handcyclists (1 WC and 10 trained handcyclists) were analyzed. One of the trained handcyclists only collected PO but no HR data during the race. Descriptives of the 10 trained handcyclists, all male, are shown in Table 0%. There2peak and POpeak as well as the VO2 and PO at the VTs were significantly lower for the trained handcyclists.Table The exercise profile of the total race is shown in Table Table r = −0.775). The correlations between PO during (sections of) the race and finish time showed slightly higher values (Table r = −0.841) with the finish time. The outcomes of the laboratory test were correlated strongest with the POmean during the mountainous part with POpeak showing the highest correlation (r = 0.901).Moderate to strong associations were found between the PO laboratory outcomes and the finish time Table , with thes Table , with thIn summary, the trained athletes seem to pace the race differently compared with the WC athlete, that is, the WC handcyclist was able to cycle a larger percentage of the time in a less intensive PO zone (zone 2 instead of 3). In addition, PO at VT2 showed the strongest correlation with the finish time while POpeak showed a very strong correlation with POmean during the mountainous part of the race.20 Thus, the longer race duration itself might also have an effect on the mean PO that can be maintained and subsequently on the distributions in PO zones.Although the absolute POmean during the race was lower in the trained athletes, the relative PO (as %POpeak) was comparable with the WC athlete. The WC athlete was able to produce more PO at a similar HR when compared with the trained handcyclists, indicating that the WC athlete’s body is operating more efficiently because of central and peripheral adaptations. The physical capacity of the WC handcyclist is, of course, related to the higher race PO and shorter race time. However, the longer race time in the trained handcyclists also has an effect on the power-duration curve. A previous study indicated that the mean PO over the last 30-sec of a 3-min all-out test decreased by 8% after 2 hrs of heavy-intensity exercise.11 This could have different causes but is, as far as known, not well studied. For example, the angle of attack on the cranks is different between a grade and level ground, and on a climb, you cannot have pauses in power production in contrast to flat sections. Furthermore, air resistance and gravitational resistances will be different during flat or mountainous sections. Of course, the mass of the athlete and handbike has a more important role in the (semi)mountainous sections, that is, more mass will require more external PO to produce the same velocity. Furthermore, a decrease in cadence during the steeper sections was also found before in cycling.11 The trained handcyclists had a lower cadence to climb the mountain compared with the WC handcyclists, with the largest difference during the flat section (57 vs. 91 rpm). A previous cycling study21 showed similar results, that is, that less-trained noncyclists chose lower cadences (65–80 rpm) at all standardized POs than trained cyclists (90–100 rpm). The authors21 speculated that less-trained noncyclists, who cycled at a higher percentage of VO2max, may have selected lower preferred cadence to reduce aerobic demand. This could also be the explanation for the lower cadence in our trained handcyclists.The POmean was significantly lower on the flat terrain compared with the other two sections, which was also found in elite cyclists.9 However, when cycling a time trial, the elite cyclists perform mainly in zone 3 (approximately 65%).9 Although our race was a time trial, our handcyclists were cycling most of the race in PO zone 2. This might be explained by the mountainous character and/or the duration of the HandbikeBattle time trial, which was much longer than the cycling time trial (38 mins) in Sanders et al.9During all three sections, the trained handcyclists spent more time in PO zone 3 (18%–38%) compared with the WC handcyclist (6%–18%). During the flat part, the trained handyclists cycled mainly in PO zone 1, while in the other sections, they cycled mainly in PO zone 2. A similar pattern was seen for the WC handcyclist. During flat cycling stages, up to 80% is spent in PO zone 1 and this percentage decreases slightly in semimountainous (76%) and mountain stages (69%).10 One of the reasons for this might be that the regulation of the cardiovascular system is slower to adapt to the quick changes in high and low PO.10 During descents, for instance, HR can still be in zone 3 while PO is already in zone 1. However, this cannot fully explain our results because the mountain time trial, especially the (semi)mountainous sections 1 and 3, did not fluctuate much, that is, it was mainly uphill. Cardiovascular drift might be another explanation.22 Heart rate increases up to 15% from 5 to 60 mins of exercise have been reported.22 Furthermore, cardiovascular drift is accentuated by factors such as dehydration and heat stress,22 which can play a role during a mountain time trial. Nevertheless, HR remains a useful marker to estimate exercise intensity because it displays the body’s response to a given performance.11The percentage time in the exercise intensity zones is quite different within the handcyclists when it is based on PO or HR Fig. . Some of14 which took 29 mins on average. The main determinants of the finish time of the simulated race were POpeak and PO at 4 mmol/l, the latter is comparable with our VT223  and aerobic lactate threshold, which is comparable with our VT123 . Other handcycling studies showed even stronger correlations between PO at 4 mmol/l (r = 0.927)13 and 15-km time trial velocity and between POpeak and 22-km time trial velocity (r = 0.85).24 These results indicate that both PO at VT2 and POpeak are important indicators of the race time during handcycling races of different durations.The PO values measured in the laboratory, especially PO at VT2 and POpeak, were strongly associated with finish time as well as with POmean during the (different sections) of the race. Our results were almost similar to the determinants of a 16-km simulated handcycling time trial,r = 0.901). Interestingly, the POmean during the flat part of the race showed the strongest correlation with the finish time. Of course, to finish in a good time, it is also important to speed up during the flat parts of a race, which seemed to be the strategy of the WC handcyclist.Peak power output showed slightly stronger correlations with POmean during the different race sections than the PO at VT2. The correlation of POpeak with the POmean during the mountainous part was extremely high . Unfortunately, it was, because of the low sample size, not possible to correct for potential confounders, such as disability or weather conditions.Because of the high dropout rate, the results cannot be generalized to the total group of HandbikeBattle participants. The high dropout rate might be due to the low velocities observed during the race in the nonstudy group. A previous cycling study showed that the PowerTap can validly and reliably measure PO even at the lower levels of PO and speed.The WC handcyclist had a different GXT protocol with shorter stage duration above VT2. This could have led to a higher POpeak compared with the trained handcyclists. Last, we unfortunately did not collect information about the weather condition over the race years, neither did we collect information about the handcycles. Both could have impacted the climbing performance compared with the laboratory-based data.The increasing number of people with a disability who are active in recreational and competitive handcycling necessitates scientific knowledge to guide the safe preparation for training and competition. Information about the PO profile during handcycling could be used to develop training programs and to assist in pacing mountainous time trials. Power meters are becoming less expensive and, therefore, will hopefully be more accessible for trained handcyclists in the near future. When power meters become more affordable, future studies might be able to collect PO and HR data during training and competition in a large sample. That would make it feasible to also investigate mechanical and physiological responses in subgroups  in different conditions .26 described the training intensity distribution in three zones based on the rating of perceived exertion (RPE) during the training period. The intensity of the training was, on average over the group, equally distributed over the three zones, that is, 35% in RPE 1 to 4, 31% in RPE 5 to 6 and 34% in RPE 7 to 10.26 This indicates that most of the HandbikeBattle participants also train in the higher intensity zones. Although RPE is, like HR, an internal training load measure, it correlates very well with PO measures during training in most handcyclists.6Because the handcyclists cycled most of the race in intensity zones 2 and 3, it is recommended to incorporate these zones also in the training when participating in longer time trials (>20 mins). Especially, training at an intensity around the VT2 might be important because the PO at VT2 is an important determinant of the finish time and is associated strongly with the POmean during the mountain time trial. A previous HandbikeBattle studyLaboratory outcomes POpeak and PO at VT2 are important performance determinants for longer time trials in handcyclists, and it is, therefore, important to improve these outcomes with training. Because the trained handcyclists cycled most of the race in intensity zones 2 and 3, it is recommended to incorporate these zones also in the training.


Plate motion is a key diagnostic to distinguish between plate and stagnant-lid tectonics, but palaeomagnetic tests have been thwarted because the planet’s oldest extant rocks have been metamorphosed and/or deformed4. Herein, we report palaeointensity data from Hadaean-age to Mesoarchaean-age single detrital zircons bearing primary magnetite inclusions from the Barberton Greenstone Belt of South Africa5. These reveal a pattern of palaeointensities from the Eoarchaean (about 3.9 billion years ago (Ga)) to Mesoarchaean (about 3.3 Ga) eras that is nearly identical to that defined by primary magnetizations from the Jack Hills 7, further demonstrating the recording fidelity of select detrital zircons. Moreover, palaeofield values are nearly constant between about 3.9 Ga and about 3.4 Ga. This indicates unvarying latitudes, an observation distinct from plate tectonics of the past 600 million years (Myr) but predicted by stagnant-lid convection. If life originated by the Eoarchaean8, and persisted to the occurrence of stromatolites half a billion years later9, it did so when Earth was in a stagnant-lid regime, without plate-tectonics-driven geochemical cycling.Plate tectonics is a fundamental factor in the sustained habitability of Earth, but its time of onset is unknown, with ages ranging from the Hadaean to Proterozoic eons Magnetic palaeointensity data from the Barberton Greenstone Belt (South Africa) as well as the Jack Hills  show nearly constant palaeofield values between 3.9 Ga and 3.4 Ga, providing evidence for stagnant-lid mantle convection. This method has been applied to document the past strength of the geomagnetic field using progressively older Archaean rocks13. The results are consistent with the few available palaeointensity studies of whole rocks of low metamorphic grade15 and/or those having signals dominated by silicate-hosted magnetizations17. Extant rocks older than approximately 3.45 billion years (Gyr) have generally been metamorphosed to amphibolite or higher grades, compromising any primary magnetic signals, but detrital single crystals found in younger sedimentary units have the potential to record even older geomagnetic fields4. The orientation of these detrital crystals at the time of their magnetization is unknown and, therefore, their magnetization direction does not constrain site latitude. The acquisition of palaeointensity data does not require knowledge of this orientation. SCP data can place bounds on motion because of the relationship between field strength and latitude, which is—in turn—a function of field morphology , whereby single mineral crystals that contain magnetic inclusions capable of recording the ancient field are studied, provides an approach to see through this metamorphismrphology . Field-g4. However, the magnetic measurement of zircons and interrogations of their magnetizations to determine whether they preserve primary signals are formidable technical challenges7. The first SCP measurements of zircons were from the JH Discovery site of Western Australia6. Exhaustive tests led to selection of only approximately 2% of the zircons separated from their host rock, and these yielded palaeointensity data suggesting the presence of a geodynamo in the Hadaean eon, approximately 4.2 Ga (ref. 6). This report was followed by a study showing how relatively young zircons successfully recorded the field21 but also attempts to disprove the original findings23. Critiques have been systematically addressed28 and, in so doing, further evidence on the preservation of primary magnetizations in zircons has been discovered. In particular, the presence of primary magnetite inclusions in select JH Discovery site zircons has been documented7, consistent with magnetic unblocking temperature data presented in the first report on zircon magnetizations6.The only known detrital crystals that can be accurately dated, and that are able to provide constraints on lithospheric mobility spanning the multi-hundred-million-year timescales that typify plate-tectonic cycles, are detrital zircons bearing primary magnetic inclusions7 is one of several lines of evidence indicating primary magnetizations rather than magnetic resetting (‘Evidence for primary magnetite inclusions and magnetizations in JH zircons’ section in 29). The BGS is less deformed and of lower metamorphic grade than the JH metaconglomerate zircon host rocks, with peak temperatures of ≤350 °C (ref. 30). Palaeomagnetic conglomerate tests from the Barberton Greenstone Belt indicate the preservation of a high unblocking temperature primary magnetization15.The distinctive time sequence defined by the JH palaeointensity values7. These include: (1) relatively large (>1 μm) particles  isoles Fig.  and (3) les Fig. . We alsoles Fig. . For potles Fig. , reflectles Fig. . The Fe-les Fig.  include 2 laser for demagnetization, allowing heating durations at least an order of magnitude less than those typical of conventional palaeomagnetic ovens, limiting laboratory alteration, and measurement with a WSGI ultrasensitive three-component direct-current superconducting quantum interference device (SQUID) magnetometer at the University of Rochester  and Mann–Whitney U tests  and allow the plates to traverse 90° of latitude, one backtracking south, the other north  subduction7 and, therefore, separations were performed at the University of Rochester using non-magnetic gravimetric methods. Methylene iodide was the heavy liquid used and zircons were handpicked from the separate. Zircon selection follows the methods of Tarduno et al.7, with some modifications. The BGS zircons isolated are generally slightly smaller than those studied from the JH Discovery site and, therefore, we focus on zircons larger than approximately 70 μm (versus 150 μm). The NRM intensity selection cutoff for analysis  was slightly lower for the BGS zircons (approximately 9 × 10−13 A m2 versus approximately 1 × 10−12 A m2).Zircons are more dispersed in the BGS relative to zircons in the JH Discovery site samples studied by Tarduno et al.−13 A m2. Sixty-five of these crystals without visible fractures or large inclusions (that might be multidomain oxides) were used for the 565 °C palaeointensity experiments. Seven zircons were rejected before experiments because of non-reproducibility of initial NRM intensity measurements; the initial value was likely contaminated by a viscous remanent magnetization. A further five zircons failed because of a large intensity drop after the first thermal treatment, again suggesting a viscous remanent magnetization. Six zircons failed palaeointensity checks (they did not lose NRM on heating or did not gain thermoremanent magnetization (TRM) or failed the multidomain-tail check). Forty-seven zircons meeting palaeointensity criteria as established by Tarduno et al.6 were subject to SEM and cathodoluminescence analyses and 12 were rejected because of metamictization or other evidence of internal grain disturbance. The 35 selected zircons represent a success rate of at least approximately 3.5% from those isolated from the methylene iodide heavy liquid separate. Although techniques differ, this success rate is higher than that of the study of JH zircons , consistent with a better preservation of the BGS zircons.Viable zircons were collected at three sites . The separations yielded >1,000 zircons, but only about 500 met the size requirements. Of these, approximately 300 crystals could be isolated that were single zircons without attached non-zircon grains and that were clear of obvious surface alteration. One hundred and thirty zircons had NRM values ≥9 × 10Optical microscopy studies used a Nikon Eclipse LV100POL microscope with both transmitted and reflected-light capabilities, a maximum of 1,000× magnification and a SPOT Insight 4-MP CCD colour digital camera assembly. SEM analyses were also conducted at the University of Rochester using a Zeiss Auriga SEM. For subsurface inclusions, we estimated depth using Electron Flight Simulator Version 3.1E software.7 and are outlined here. All experiments were conducted in the shielded room (ambient field < 200 nT) of the Paleomagnetism Laboratory at the University of Rochester. The 565 °C palaeointensity method6 was used to derive palaeofield strength estimates while limiting the effects of laboratory-induced alteration by reducing the number of heatings. A temperature of 565 °C is chosen for comparison with the JH palaeointensity dataset, which also used this temperature, and to represent blocking temperatures best reflecting single-domain-like magnetite inclusion carriers, while also retaining a measurable demagnetized value. The demonstration that 565 °C falls within the interval of sharp magnetic unblocking :l and m are spherical harmonic degree and order, respectively, re is the radius of Earth and the Gauss coefficients 10, the geocentric axial dipole  by:The geomagnetic field can be as described by the scalar potential 40and procedure for U-Pb calibration by reference materials41 were followed. Two 1-inch epoxy mounts (IP985 and IP987) were built containing the JH and BGS zircons as well as zircon reference materials z6266 with an accepted 206Pb/238U age of 559 Myr41 and z1242 with an accepted 207Pb/206Pb age of 2,679.6 ± 0.2 Myr42. Polishing with diamond suspension exposed the internal structure of the zircon grains. After coating with 10 nA of gold, the grains were imaged in cathodoluminescence (CL) and backscattered electron detector (BSD) mode using a MIRA3 TESCAN field emission scanning electron microscope. In order to target pristine areas of zircon grains and avoid cracks and alteration, a 16O- ion beam 13 μm in diameter and with an average beam current of 1 nA was used. The analytical runtable consisted of 11 masses including background with species of Hf, Yb, Zr, Pb, Th, U analysed over six scans. Data reduction used SQUID3 (ref. 43) . Steiger and Jäger44 decay constants were used. Two analytical sessions were carried out on each of the two epoxy mounts. The 1σ external error for the 206Pb/238U calibration relative to reference material z6266 for sessions IP985_1 and IP985_2 was ±0.88% or ±1.01, respectively. The 1σ external error for the 206Pb/238U calibration for sessions IP987_2 and IP987_3 was ±0.75% or 1.07%, respectively. These errors are also specified in the footnote to the data table (Supplementary Table 1). The requirement for a correction due to instrumental mass fractionation of the Pb isotopes was assessed through replicate analyses of reference material z1242. The measured weighted mean 207Pb/206Pb age of those analyses are reported for each session in the footnotes of Supplementary Table 1. No fractionation correction was applied to the Pb- isotope data except for session IP987_2, in which a correction of −0.64% was applied. Concordia plots were generated, and weighted means calculated using Isoplot v. 4.15 (ref. 45). The uncertainties for the weighted mean ages reported in the text and Supplementary Materials are at the 95% confidence level.Standard SHRIMP U-Pb instrumental setup7. Specifically, zircon microconglomerate tests and the identification of distinct secondary components of magnetization separate from the primary characteristic magnetization exclude magnetic resetting after incorporation of the zircons into the JH host metasediment (figure 2 of ref. 7). 7Li profiling data  argues against thermal resetting of Hadaean and Eoarchaean data, whereas reflected-light microscopy, microtectonic analysis, SEM/EDS, focused ion beam and NanoMOKE investigations document the presence of primary magnetite inclusions (figures 3–5 of ref. 7). Pb-Pb screening of data, documented in refs. 7 (figure 7 of ref. 7) argues against a magnetic resetting age older than the age of incorporation into the host sediment in the selected zircons. This is further enforced by the distinct change in palaeointensity data, with values from late Hadaean zircons being higher than those of Palaeoarchaean to Eoarchaean age, inconsistent with Palaeoarchaean magnetic resetting. Reference 6 notes that iron oxyhydroxides can be found in JH zircons that are likely related to weathering, but these cannot be the carrier of the magnetic remanence used for palaeointensity determination. Furthermore, an assemblage of magnetite inclusions is needed to account for the magnetizations observed from the zircon, satisfying Maxwell–Boltzmann statistical limits on magnetic recording7. Reference 7 also describes why remagnetization scenarios calling on the neoformation of magnetic grains represent particles too small or too few in number to record stable magnetizations. Reference 7 also notes that the association of magnetic particles with dislocations does not mean that the particles are secondary22; instead, this is expected given the deformation history of the JH conglomerate.Palaeomagnetic, reflected-light microscope, electron microscope, microtectonic, geochemical and palaeointensity data indicate the presence of primary magnetite inclusions in select JH zircons and that these have primary magnetic signals46 data on a JH zircon illustrate differences in the high unblocking temperature magnetic primary remanences isolated in refs. 7 and other potential Fe-bearing minerals. Reference 46 interprets quantum diamond microscope data from ref. 22 on a single zircon as indicating Fe-bearing zones and links those to the carriers of natural magnetic remanence. Atom probe tomography data from one zone within that zircon is further interpreted to contain Fe nanoclusters  and a maximum age of approximately 1.4 Gyr is assigned; this is interpreted as a natural remanent magnetization age. A quantum diamond microscope does not have the sensitivity to measure the natural magnetic remanence of JH zircons. Reference 7 explains that the authors instead measured the magnetization of a laboratory-induced isothermal remanence magnetization ; this strong field can enhance the magnetization of Fe oxides/oxyhydroxides that do not contribute to the primary remanence. The zircon microconglomerate tests7 discussed above supersede older tests and document that the high unblocking temperature magnetization in JH zircons must be older than the approximately 3-Gyr depositional age of the JH conglomerate. Therefore, the hypothetical remanent magnetization inferred in ref. 46 with an assigned age of approximately 1.4 Gyr cannot be the key high unblocking temperature component of JH magnetization7.Although the zircon studied would not pass our selection criteria, atom probe tomography47. In thin section, our BGS samples used for zircon separation have a greenschist-grade anastomosing foliation in the generally fine-grained matrix. The foliation is cut by several conjugate through-going fractures. The competency difference between the competent zircon grains and the weaker surrounding matrix could result in stress buildups at the grain boundaries that might generate cracks within the zircon grains. Below, reference is made to specific zircons studied by reflected-light and scanning electron microscopy.Samples for this study were collected from the lower approximately 1 m of the approximately 3–5-m-thick BGS. Our samples appear distinct from those of the lowest 20 cm of the BGS, which show extensive shear zones in thin sectionSEM sample BGS2-z51. The fractures appear to be in three main systems . The ‘east-dipping’ and ‘west-dipping’ sets appear to be part of the same (112) crystallographic system as close-packed planes . The melt inclusions , which would suggest an E–W compression direction when those fractures formed. The E–W fracture on either side of the ‘football-shaped’ inclusion probably formed during the same compression. The fractures coming off the tapered ends of the ‘football-shaped’ inclusion are likely guided by the inclusion itself. The tapered ends of an inclusion have the smallest radius of curvature and, therefore, generate the highest stress concentrations, causing the fracture to propagate out from the tapered tip. Those fractures do not appear to be guided by the crystal structure of the zircon—if the fractures had propagated farther, they might have reoriented to follow a crystallographic close-packed plane. There is a possible fracture within the inclusion , a set is perpendicular to the c axis  and there is a pair of conjugate sets (perhaps the (112) set) indicating compression approximately in the E–W direction. The globular form of the quartz inclusion highlighted in Extended Data Fig. c axis.SEM sample BGS2-z36, Fig. 10); trace Fe signals could reflect such inclusions within the zircons, but further work is needed to distinguish these from Fe intrinsic to the crystals in the case of feldspar. When viewed under reflected light, we note that isolated inclusions often show extinction after rotation from 0° to 90° polarization angles. For those shallow enough in the crystal to be sampled by SEM EDS analysis, the presence of Fe together with extinction provides evidence for magnetite7.Magnetic inclusions on the tens to hundreds of nanometres scale are well established within silicates , the two-sample KS test51 and the Mann–Whitney U test52. The Student t-test differs from the KS and Mann–Whitney U tests in the assumption that each sample is normally distributed with equal variance. Given the small sample sizes for some comparisons and unequal variances in each population (see below), the two-tailed Welch’s version of the t-test is appropriate50, which compares each sample mean and variance to the t distribution. The non-parametric two-sample KS test compares two samples and measures the maximum distance between their empirical cumulative distributions (DKS) and rejects the null hypothesis if pKS exceeds some critical threshold. The Mann–Whitney U test is another non-parametric test that examines two samples , with the null hypothesis being that a random value from sample X has an equal probability of being greater or less than a random value from population Y; this is the expected outcome if the two samples share the same underlying distribution. All three tests compare the hypotheses H0 and H1 and return a P value. If the P value is greater than a defined significance threshold (α), typically α = 0.05, then the H0 hypothesis cannot be rejected; if the P value is less than the defined significance, then H0 can be rejected at (1 − α) confidence in favour of H1. For our hypotheses, if P > 0.05, then it is reasonable to infer that the JH and BGS zircon palaeointensity distributions cannot be distinguished at the 95% confidence threshold.To compare the JH and BGS zircon palaeointensity data, we first examine the following hypotheses: t-test, non-parametric two-sample KS test and Mann–Whitney U test all yield P values > 0.05, indicating that we cannot reject the null hypothesis H0  for both the JH and BGS datasets, centred on 3,408 Ma and 3,508 Ma. The Welch’s t-test for each interval yields P values of 0.17 and 0.09, respectively, and thus again the null hypothesis H0 that the JH and BGS data record the same field intensity cannot be rejected. Non-parametric tests yield similar results, with P values exceeding 0.05 for both intervals . When applied to the new BGS, the P value is 0.32, which supports the hypothesis that the JH model can predict the Barberton zircon results. Although the P value is above the 95% threshold, the distributions nevertheless merit some discussion. The JH palaeointensities are clearly more scattered than the BGS results. This could signal one, or both, of the following:(1) the BGS zircons are, in general, better preserved, having less amorphous iron oxides in cracks than the JH and, therefore, they might be better recorders; (2) the JH might record the field on shorter timescales than the BGS, averaging higher-frequency secular variations of the past geomagnetic field. Palaeointensities from the SCP analyses of Nondweni dacite samples are within the BGS values of the same age, but SCP of Barberton dacites at 3.45 Ga are higher. Both the Nondweni and Barberton SCP values are plotted as individual results from two relatively shallow intrusions, and these might be expected to sample higher-frequency variations of the geomagnetic field than the zircons.The null hypothesis is that the two samples  are drawn from the same distribution. In the case in which the new data are well predicted by the model, the KS test should return a 53, a continuous full-plate reconstruction model spanning the past 1 Gyr, is used. A set of sites are defined for the present day and the site palaeolocations are reconstructed back from the present day to 600 Ma using GPlates54. The distribution of palaeolocations is described and comparisons between sites located on different plates is made. From this empirical dataset, a set of statistical tests is constructed.Here we consider the probability of observing two sites, located on different plates, experiencing both little to no latitudinal motion as well as little to no relative latitudinal distance separation during an interval of 600 Myr. To construct this test, the plate-reconstruction model of Merdith et al.55, which yields a uniform distribution of locations distributed globally with a median separation of approximately 6°. From this grid, 1,000 sites are assigned to plates using a built-in GPlates function. Sites that do not fall within the boundaries of a plate are removed before the analysis. Using the plate-motion model of Merdith et al.53, site palaeolocations are reconstructed in 1-Myr steps from the present day to 600 Ma. From the initial set of sites, only those assigned to plates that existed 600 Ma are preserved, resulting in a set of 228 locations ;, however, sites with Δλ falling in the lower 5% of the distribution  have ages ranging from 150 to 480 Ma. We note that Δλ estimates can be biased downward by as much as approximately 10° when sites are downsampled at 100-Myr versus 1-Myr intervals.From the distribution of ΔWith these general trends in mind, we next consider whether any sites experience little to no latitudinal motion. No sites could be identified with near-zero latitudinal motion irrespective of the sampling level . The bound on relative distance between the plate pair is the combination of this backtracked southward motion for one plate and the maximum backtracked northward motion of another plate consistent with the palaeointensity data (5°), for a total of 53°.Given the rarity, or absence, of plates showing no motion over 600 Myr, the likelihood of observing two sites on separate plates with little to no latitudinal motion is very low. We next consider the bounds of Δata Fig. . The casλ and relative distance characteristics, a hypergeometric distribution is appropriate. A hypergeometric distribution models a sampling without replacement, in contrast to the more common binomial distribution, which samples trials with replacement56. Sampling without replacement is appropriate for this scenario because the same site (plate) cannot be selected twice. The probability mass function (p(k)) for a hypergeometric function is defined as:N = 66 is the number of plates, K is the number of plates in the 600-Myr dataset known to record latitudinal motion and relative distance less than or equal to a specified threshold, n = 2 represents the number of unique plates selected in a single random sampling and k is the number of plates in that sampling showing limited latitudinal motion. The resulting probability of identifying a single pair of plates (k = 2) that both show limited latitudinal motion, here defined as Δλ ≤ 48° and relative latitudinal distance ≤53° at 100-Myr downsampling, is <1% , we normalize longitudinal motion by analysing the fraction of maximum angular travel from the initial position that is not explained by latitudinal change. We note that plates having dominant longitudinal motion  are rare (about 5%). In these cases, there is at least 40° of latitudinal motion.58 but challenged by others59. Arguably the strongest geologic correlations are between the approximately 2.7–2.8-Ga volcanism on these cratons . Palaeomagnetic data for the Kaapvaal Craton, applying a tilting interpretation59, imply emplacement at a very high latitude (75.4°). There is an approximately 700-Myr-long age gap between this volcanic association and the time when the BGS and JH zircon records start to diverge, which might hint at a start of horizontal motions characteristic of plate tectonics. This duration is 100 Myr longer than that over which we used to assess the latitudinal-motion characteristics of plate tectonics. Nevertheless, our analysis can be used as a conservative measure to assess whether plate-motion rates typical of plate tectonics are compatible with a high-latitude Kaapvaal Craton in the Neoarchaean and a low-latitude position in the Palaeoarchaean. Given that the median maximum latitudinal displacement from 0–600 Ma is 76°, it is clear that plate-motion rates are compatible. Assuming a location of the Kaapvaal craton solely in one (N or S) hemisphere yields an average latitudinal component of motion of 8 mm year−1. Assuming one equatorial crossing results in an average latitudinal component of motion of 16 mm year−1.A ‘Vaalbara’ supercraton involving the Pilbara and Kaapvaal cratons between about 3.1 and 2.7 Ga has been proposed by some authors61. Palaeointensity variations of superchron versus reversing intervals65 have also been interpreted as reflecting these variations, and this is supported by some numerical geodynamo models66. More recently, it has been suggested that such changes may extend to the Devonian67 and/or might have started in the Ediacaran Period19. If palaeointensity variations similar to those observed in the Phanerozoic had been present in our 3.9–3.4-Ga data, we might not have been able to separate changes in dynamo efficiency from palaeolatitude changes. But because the palaeointensity record is constant, we infer that palaeolatitudes were constant and mantle processes were not creating the latitudinal core–mantle boundary heat-flux patterns necessary to greatly affect geodynamo efficiency.Palaeomagnetic variations, principally the occurrence of reversing and seemingly non-reversing periods (superchrons), but also inferences on secular variation, have long been used to argue for changes in core–mantle boundary structure influencing the efficiency of the geodynamoAny methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-06024-5.Supplementary InformationThis file contains Supplementary Text and Supplementary ReferencesPeer Review FileSupplementary Table 1SHRIMP U-Pb geochronology data


However, DCD hearts suffer from serious ischemia/reperfusion injury (IRI). Recent studies found that the activation of NLRP3 inflammasome could play a significant role in organ IRI. Mcc950, which is a novel inhibitor of the NLRP3 inflammasome, can be applied to treat various kinds of cardiovascular diseases. Therefore, we hypothesized that the treatment of mcc950 could protect DCD hearts preserved with normothermic Donor-heart rats were randomly divided into four groups: Control group; Vehicle group; MP-mcc950 group; and MP + PO-mcc950 group. Mcc950 was added into the perfusate of normothermic EVHP in the MP-mcc950 and MP + PO-mcc950 groups, and was injected into the left external jugular vein after heart transplantation in the MP + PO-mcc950 group. Cardiac functional assessment was performed. The level of oxidative stress, inflammatory response, apoptosis, and NLRP3 inflammasome-associated protein of donor hearts were evaluated.max, and dP/dtmin of the left ventricular of DCD hearts at 90 min after heart transplantation in both MP-mcc950 and MP + PO-mcc950 groups. Furthermore, mcc950 added into perfusate and injected after transplantation in both MP-mcc950 and MP + PO-mcc950 groups significantly attenuated the level of oxidative stress, inflammatory response, apoptosis, and NLRP3 inflammasome compared with the vehicle group.The treatment with mcc950 significantly increased the developed pressure (DP), dP/dtvia inhibiting NLRP3 inflammasome.Normothermic EVHP combined with mcc950 treatment can be a promising and novel DCD heart preservation strategy, which can alleviate myocardial IRI  HoweverNevertheless, the inevitable warm ischemia time, which is from systolic blood pressure lower than 50 mmHg after the withdrawal of life-sustaining therapy to reperfusion or cardioplegia, results in more serious myocardial ischemia/reperfusion injury (IRI) in the DCD hearts, thereby leading to higher incidence of primary graft dysfunction (PGD) for the patients undergoing DCD heart transplantation , 5. TherRecent reports found that nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome exerts a significant effect on increasing myocardial IRI , 7. Foll20H23N2NaO5S; Molecular weight: 426.46), as a novel and selective inhibitor of NLRP3-inflammasome amino]carbonyl]-4-(1-hydroxy-1-methylethyl)-2-furansulfonamide sodium; Shorthand name: Cammasome , interruammasome , 12. Mccammasome –15. Howeex vivo heart perfusion (EVHP) is becoming a novel and promising preservation strategy for donor hearts  used in this study received care in compliance with the Guide for the Care and Use of Laboratory Animals . All animal experiments were submitted, checked, and approved by the Ethical Committee of the Laboratory Animal Research Center of Southern Medical University Nanfang Hospital . The rats were housed in temperature-controlled (22 ± 2°C) rooms with a 12-h light-dark cycle, given food and sterilized water, and acclimatized for 1 week.2.2.31 male Lewis rats  were introduced as donor-heart rats and also blood donors for blood-based perfusate of the normothermic EVHP system. Another 31 male Lewis rats  were regarded as recipient rats undergoing abdominal heterotopic heart transplantation.In addition, another 6 male Lewis rats  were introduced as donor-heart rats in the control group. In this control group, 6 male Lewis rats were directly sacrificed and their hearts tissue sample was collected.via the abdominal aorta to procure blood for EVHP. Subsequently, the donor-heart rats were subjected to the DCD procedure by suffering 15-min warm ischemia injury. Next, the hearts were procured and preserved by the normothermic EVHP system for 90 min. Cardiac function of the heart was assessed at the end of the normothermic EVHP period. Abdominal heterotopic heart transplantation was carried out after 90-min normothermic EVHP. At 90 min after heart transplantation, cardiac function of the donor heart was measured. Donor heart tissue and recipients' blood were collected to analyze the level of inflammation, apoptosis, oxidative stress, and NLRP3 inflammasome  Control group: 6 non-DCD hearts were procured from heart-beating rats and heart tissue was immediately collected. (2) Vehicle group (11 rats): 100 µl saline was mixed into 12 ml blood-based perfusate before the initiation of normothermic EVHP and saline(1 ml/kg body weight) was injected into the left external jugular vein after heart transplantation; (3) MP (Machine perfusion)-mcc950 group (10 rats): 100 µl saline with mcc950  was mixed into 12 ml blood-based perfusate before the initiation of normothermic EVHP and saline (1 ml/kg body weight) was injected into the left external jugular vein after heart transplantation; (4) MP + PO (Post-operation)-mcc950 group (10 rats): 100 µl saline with mcc950  was added into 12 ml blood-based perfusate before the initiation of normothermic EVHP and mcc950  was injected into the left external jugular vein after heart transplantation to achieve the dosage of 5 mg/kg body weight.Two of the recipients were dead because of an overdose of anesthesia and another two recipients failed because of anastomotic leak. Finally, totally 31 pairs of rats underwent heart transplantation successfully and all of their donor hearts re-beat.2.3.Details of the operative procedure, including anesthesia, procuring of blood and donor heart from the donor rat, the operation of EVHP, heterotopic abdominal heart transplantation, and sample collection, are described in the 2.4.2.4.1.Immunohistochemistry was performed to analyze the expression of 4-hydroxynonenal (HNE), which is an indicator of oxidative stress in donor hearts. The immunoreactivity to HNE  was measured. The antigen-antibody reaction was visualized by the diaminobenzidine reaction. The fields of each slice were selected and recorded randomly in a blinded manner under a conventional light microscope. Image-Pro Plus software  was applied to perform image analysis. Four random and non-overlapping fields of the heart tissue were selected for evaluation, and the average value of each rat was calculated. The expression of HNE was measured by counting integrated optical density (IOD).2.4.2.κB p65) in myocardial tissue were measured by western blotting as described below.The expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nuclear factor kappa-B p65  staining  was performed to detect DNA-strand breaks of the donor hearts as previously described . TUNEL-p2.4.4.With regard to the immunofluorescence method, myocardial tissue slices were incubated with the following primary antibodies: Rabbit anti-NLRP3 , anti-Caspase 1 , anti-IL-1β , anti-IL-18 , anti-ASC .The slices were then washed and detected with appropriate Fluor dye, DyLight 488 , as secondary antibodies followed by counterstaining with DAPI in the dark. Afterward, immunofluorescent signaling was observed with a fluorescence microscope (IX73 Olympus). Digital images and data were recorded and analyzed by applying ImageJ (NIH).2.4.5.κB , and TNF-α  were evaluated. Except for Caspase-3 and ASC, the other reagents were all purchased from Proteintech Group, Inc, USA.Myocardial protein expression of donor hearts was assessed as previously described by western blotting . The exp2.5.p < 0.05 was considered statistically significant.The results were expressed as mean ± standard error of the mean (SEM). GraphPad Prism 8.3 software  was used to perform statistical analysis. Shapiro-Wilk test was performed to test the normality of data before statistical tests were applied. One-way ANOVA followed by Tukey's post-hoc-test was performed for multiple comparisons between three experimental groups. If the data failed the normality test, the non-parametric Kruskal–Wallis test followed by Dunn's post-hoc-test was used. Intraventricular pressure measurement recordings among vehicle, MP-mcc950, and MP + PO-mcc950 groups were compared by two-way ANOVA according to different time points. A value of 3.3.1.To test a hypothesis that NLRP3 inflammasome could be presented in the DCD hearts which had suffered myocardial IRI, the expression of NLRP3, ASC, Caspase-1, IL-1β and IL-18 in the donor hearts was evaluated by immunofluorescence and western blotting analysis.In –5A,B, co3.2.As shown in max, and dP/dtmin, in the MP-mcc950 and MP + PO-mcc950 groups. However, no significant difference could be observed among the three groups concerning heart rate. Furthermore, there was no significant difference between the MP + PO-mcc950 and MP-mcc950 groups in terms of post-transplant cardiac function of donor hearts.We measured the cardiac function of donor hearts 90 min after heart transplantation. As indicated in 3.3.As an indicator of oxidative stress, the expression of HNE in the donor hearts among the four groups was determined by immunohistochemical analysis. Compared with the control group, warm ischemia-reperfusion injury significantly increased the expression of HNE in the vehicle group. However, the addition of mcc950 into the circuit of EVHP could lead to a significantly decreased expression of HNE in the DCD hearts 90 min after heart transplantation. Furthermore, the additional administration of mcc950 after heart transplantation in the MP +PO-mcc950 group could significantly reduce the expression of HNE in the donor hearts compared with the MP-mcc950 group .3.4.A significantly increased number of TUNEL-positive nuclei in the heart graft could be observed in the vehicle group compared with the control group. However, the treatment with mcc950 during normothermic EVHP in the MP-mcc950 group significantly decreased DNA strand breaks in the DCD hearts after heart transplantation compared with the vehicle group. Finally, the level of myocardial apoptosis in the DCD hearts was significantly lowered in the MP + PO-mcc950 group compared with the MP-mcc950 group .In addition, western blot assay showed that the expression of pro-apoptosis protein markers Bax and Caspase-3 significantly decreased, while anti-apoptosis protein Bcl-2 increased in the MP + PO-mcc950 groups compared with the vehicle group .3.5.κB in heart tissue was measured by western blotting to evaluate the inflammatory response in the myocardium. As shown in κB in the DCD heart submitted to EVHP, while the treatment of mcc950 significantly attenuated inflammatory response in the DCD heart in MP-mcc950 and MP + PO-mcc950 groups.The expression of IL-6, TNF-α, and NF-3.6.The expression of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the donor hearts, which demonstrates the generation of the NLRP3 inflammasome, was evaluated by immunofluorescence analysis and western blotting.In the immunofluorescence analysis, compared with the vehicle group, the level of the NLRP3 inflammasome significantly reduced in the post-transplant donor hearts in the MP-mcc950 and MP + PO-mcc950 groups –5. In ad4.Ex vivo heart perfusion with mcc950 treatment could protect the DCD hearts against myocardial IRI by attenuating the level of NLRP3 inflammasome.In our present study, we demonstrated the protective effect of the selective NLRP3 inflammasome inhibitor mcc950 on transplantation outcomes in a rat DCD heart transplantation model. Increasing evidence showed that NLRP3 inflammasome was a potential therapeutic target for IRI in myocardial infarction , 23. SheIn line with these previous studies, we also observed that the level of NLRP3 inflammasome  in the vehicle group significantly increased compared with the control group. Therefore, inhibiting the activation of NLRP3 inflammasome could be a significant therapeutic target in reducing myocardial IRI in DCD hearts. In line with this novel theory, our previous study , demonstin vivo and in vitro by inhibiting NLRP3-mediated pyroptosis. Consequently, we hypothesized that normothermic EVHP and mcc950 treatment could be a novel and effective organ preservation strategy, which might reduce myocardial IRI in the DCD hearts and increase the number of transplantable hearts. In the present study, we introduced the heterotopic heart transplantation model to fully present the cardioprotective potential of normothermic EVHP combined with mcc950 treatment. Cardiac function, as reflected by developed pressure, dP/dtmax, and dP/dtmin, significantly improved in the DCD hearts in the MP-mcc950 and MP + PO-mcc950 group at 90 min after heart transplantation. However, no significant difference could be found in the cardiac function of DCD hearts between the MP + PO-mcc950 and MP-mcc950 groups. We supposed that the treatment of mcc950 after transplantation might influence the intracellular metabolism of myocardial tissue, including the generation of ROS and apoptosis , but not the cardiac function of DCD hearts. As Anantha et al. (Mcc950, as a selective inhibitor, has been shown to inhibit the activation of the NLRP3 inflammasome and expression of Caspase-1, IL-18, and IL-1β. Yu et al.  reporteda et al.  showed, κB, MAPKs and PI3K/Akt signaling pathway (Apoptosis, as one kind of programmed cell death, was reported to be suppressed when NLRP3 inflammasome was inhibited , 29. The pathway . In our  pathway , we also pathway .In the present study, no significant difference was observed among vehicle and MP-mcc950 groups with regard to the cardiac function of DCD hearts measured at the end of 90-min normothermic EVHP. Furthermore, there was no significant difference between MP + PO-mcc950 and MP-mcc950 groups in terms of post-transplant cardiac function of donor hearts measured at the end of 90-min reperfusion. We supposed that 90-min reperfusion, either EVHP or heterotopic abdominal heart transplantation, might not be long enough for mcc950 to significantly exert its cardioprotective effect on DCD hearts. However, mcc950 preconditioning during 90-min EVHP could significantly improve the post-transplant cardiac function of DCD hearts, which indicated that 180-min reperfusion, including 90-min EVHP and 90-min reperfusion after heterotopic heart transplantation, could be an optimal timepoint for us to observe the profoundly improved cardiac function as a result of mcc950 treatment.There are several limitations of this study that need to be discussed. Firstly, the induction of circulatory death in the rats was exsanguination, which might not closely mimic a clinical DCD setting. Secondly, the dosage of mcc950 in the perfusate for normothermic EVHP might not be ideal. Compared with the previous study  in which5.via inhibiting NLRP3 inflammasome in a rat heart transplantation model of DCD. Therefore, normothermic EVHP combined with mcc950 treatment could be a promising and novel DCD heart preservation strategy, thereby increasing the number of transplantable hearts in heart transplantation.The present study reveals that normothermic EVHP combined with mcc950 treatment can alleviate myocardial IRI


Mycobacterium leprae is the etiological agent of leprosy. Macrophages (Mφs) are key players involved in the pathogenesis of leprosy. In this study, immunohistochemical analysis was performed to examine the phenotype of Mφ subpopulations, namely M1, M2, and M4, in the skin lesions of patients diagnosed with leprosy. Based on the database of treatment-naïve patients treated between 2015 and 2019 at the Department of Dermatology of the University of the State of Pará, Belém, routine clinical screening samples were identified. The monolabeling protocol was used for M1 macrophages  and M2 macrophages , and the double-labeling protocol was used for M4 macrophages . To confirm the M4 macrophage lineage, double labeling of the monoclonal antibodies CD68 and MRP8 was also performed. Our results demonstrated a statistically significant difference for the M1 phenotype among the Virchowian (VV) , Borderline , and tuberculoid (TT)  clinical forms of leprosy. Additionally, the M2 phenotype showed a statistically significant difference among the VV , Borderline , and TT  forms. For the M4 phenotype, a statistically significant difference was observed in the VV , Borderline , and TT  forms. A significant correlation was observed between the VV M1 and M4  and between the VV M2 × TT M1  phenotypes. The M1 Mφs constituted the predominant Mφ subpopulation in the TT and Borderline forms of leprosy, whereas the M2 Mφs showed increased immunoexpression and M4 was the predominant Mφ phenotype in VV leprosy. These results confirm the relationship of the Mφ profile with chronic pathological processes of the inflammatory response in leprosy. Mycobacterium leprae [Leprosy is an infectious disease with low morbidity owing to the population’s resistance to m leprae . It has m leprae . Howeverm leprae .Mφs are capable of undergoing phenotypic modification and expressing receptors and co-stimulatory molecules such as cytokines that induce the development of appropriate immunological responses . StudiesClassical activation of Mφs occurs via IFN-γ stimulation, which generates MφM1 with high pathogen-killing potential and upregulates the secretion of pro-inflammatory cytokines such as IL-6, IL-12, inducible nitric oxide synthase (iNOS), and TNF-α. The expressed molecules or factors are Janus kinase 1 (JAK1), JAK2, signal transducer and transcription activator 1 (STAT1), and STAT2 . In the MφM2 play a key role in the resolution of inflammation, promoting the removal of debris and enabling an increase in the contractility of the smooth muscle, thereby contributing to the expulsion of pathogens . ActivatMφM1 and MφM2 mainly differ with respect to their receptors, effector functions, and cytokine production. Upon induction by lipopolysaccharides (LPSs) or IFN-γ, activated MφM1 of the Th1 lineage express high levels of iNOS, which metabolizes arginine to nitric oxide and citrulline. In contrast, MφM2 of the Th2 lineage are characterized by their expression of arginase, which hydrolyzes arginine to ornithine and polyamines. Although M1/M2 polarization leads to opposing outcomes of inflammatory reactions, the balance between cytotoxicity (MφM1) and immunosuppression (MφM2) is vital for the homeostasis of the immune system . A growiThe advancement of knowledge on immunology in recent years has contributed to a deeper understanding of the mechanisms involved in the pathogenesis of leprosy. Due to the fact that macrophages are the main cells involved in the immune response to M. leprae, and due to the few comparative studies published in the scientific literature on the role of M1, M2, and M4 macrophage subpopulations in the pathogenesis of cutaneous lesions in the different clinical forms of leprosy, the objective of this work was to comparatively evaluate the presence and possible role of these cells using the immunohistochemistry technique.n = 14), TT (n = 14), or VV (n = 14) leprosy, according to Ridley and Jopling’s classification. Samples from underage patients, co-infected with HIV, who had presented reactions or who underwent a biopsy of the lesion after starting multidrug therapy, according to information obtained from the medical records, were excluded. Samples from patients diagnosed with borderline leprosy were also excluded, due to the immunological instability characteristic of this clinical form, which would compromise the analysis of the data obtained. Statistical analysis was performed using the BioStat 5.0 program. In the univariate analysis, frequencies and measures of central tendency and dispersion were obtained, together with ANOVA. A limiting significance level of 5% (p = 0.05) was adopted for all tests.This is a cross-sectional study, in which we analyzed 42 samples of skin lesions obtained from patients treated at the Dermatology Service of the State University of Pará between 2015 and 2019, available on file, and therefore a sample of convenience. All patients were treatment-naïve and diagnosed with borderline .The monolabeling protocol was used for M1 macrophages and M2 macrophages and the double-labeling protocol for M4 macrophages. M1 macrophages were characterized by immunostaining for monoclonal antibodies to iNOS, IL-6, and TNF-α. M2 macrophages were characterized by immunostaining for monoclonal antibodies to IL-10, IL-13, CD163, Arginase 1, TGF-β, and FGFb. M4 macrophages were characterized by immunostaining for monoclonal antibodies to IL-6, Metalloproteinase 7, MRP8, TNF-α, and CD68. To confirm the M4 macrophage lineage, double labeling of the monoclonal antibodies CD68 and MRP8 was also performed, following the protocol of Quaresma et al. . We perfThe study was approved by the Research Ethics Committee of the Federal University of Pará, under the approval number 2.338.865.The pattern of immunostaining for the macrophage subpopulations studied was characterized by the identification of brownish areas deposited in the cytoplasm or cell nucleus on the immunostained slides on a blue background stained with hematoxylin .Quantitative analysis of the Mφ subpopulations  was performed based on three clinical forms of leprosy, namely VV, Borderline and TT. The numbers represent the average number of positive cells stained for each antibody, with the standard deviation. A statistically significant difference was observed between the three groups, as shown in p < 0.0001), Borderline , and TT  leprosy. Based on the MφM2 profile, a statistically significant difference was observed between VV , Borderline , and TT  leprosy. Based on the MφM4 profile, a statistically significant difference was observed between VV , Borderline , and TT  leprosy.The mean activity of MφM1 showed a statistically significant difference between VV  and in the M1 profile in Borderline (p = 0.0001) and TT (p = 0.0001) leprosy.To improve characterization of Mφ levels in the three clinical forms, intra-lesion group as well as between-lesion comparisons were performed. In the intra-lesion group comparison, a greater number of cells were observed in the M2 profile in VV (p = 0.0001) as well as MφM2 and MφM4 in the VV form (p = 0.0001 for both).In addition, for the comparison of Mφ levels between lesions, statistical significance was observed for MφM1 in the TT form .Elucidation of the complex interaction between the tubercle bacillus and host remains a challenge for scientists, as it depends on the characteristics of virulence, evasion, and phenotype of the pathogen as well as factors related to the host’s immune response mechanisms ,11,12,13p < 0.0001) compared to those with other clinical forms of the disease.Our findings revealed that patients diagnosed with TT were positive for the MφM1 phenotype , indicating a strong anti-inflammatory response triggered by this subpopulation of Mφs. It was suggested that the high bacterial load in the cells not only enhances the chronicity of the VV form but also increases the presence of MφM2.Our findings further showed that the M2 phenotype was significantly different in the VV clinical form of leprosy  can facilitate the ingestion and spread of intracellular pathogens via opsonization ,43. Infe leprosy .In leishmaniasis, oligosaccharides such as mannose and galactose are integrated into lipophosphoglycan structures , and plap = 0.0001), indicating the behavior of IDO in the Virchowian pole, whereby the presence of CD68 in this pole could be associated with the migration of activated Mφs to the infected host cells [Our findings showed a positive correlation between the M2 phenotype of the VV form and the M1 phenotype of TT form . These data confirm the similarity between MφM4 that affect treatment response in leprosy and the immunosuppressive behavior of the host [Results of our study revealed a significant increase in the M4 subpopulation in VV leprosy  [The phenotypic similarity of Mφs was validated in a study on Mφ phenotype modulation using CXCL4 (M4) and M-CSF (M2), whereby similar mRNA expression levels, leukocyte counts, and myeloid marker protein levels were observed. Transcriptome analysis was performed to confirm the strong correlation between phenotypes of Mφs  .p = 0.0001) in the VV form, indicating that in the absence of the CD163 receptor, the MφM4 exhibit a cytokine secretion profile similar to that of the MφM1. In a study on Mφ polarization associated with atheromatous intraplaque hemorrhages, hemoglobin-induced Mφ polarization led to increased expression of IL-10, high levels of the hemoglobin receptor inhibitor CD163, and low expression of HLA-DR [Furthermore, we found an association between MφM1 and MφM4 .The presence of specific biomarkers for Mφ activation in leprosy indicates the dynamic and transient nature of this immune cell type. Regulatory Mφs upregulate expression of several biologically important proteins, including MMP  and DC-SThe increased expression of DC-STAMP following the stimulation via the presence of FcγR crosslinking is associated with increased phagocytosis and reduced antigen presentation and cytokine production ,80, therp < 0.0001) when compared to other clinical forms of the disease.Simultaneous immunostaining of skin samples from patients diagnosed with the Borderline form of leprosy showed a predominant M1 phenotype  and TT (p = 0.0001). MφM1 played a pro-inflammatory role, evidenced by the immunoexpression of the TNF-α, IL-6, and iNOS markers. Based on the immunoexpression pattern of MφM1 in the three clinical forms of leprosy, our data showed predominance of MφM1 in the TT form (p < 0.0001). Statistically significant differences were observed between the means of the clinical forms VV, Borderline, and TT with the subpopulation of MφM1 (p < 0.0001).First, MφM1 predominated in the clinical forms of Borderline (p = 0.0001) and characterized by their immunoexpression pattern with the markers IL-10, IL-13, TGF-β, FGFb, CD163, and arginase-1. The immunoexpression pattern of the M2 phenotype was significantly different in the VV clinical form (p = 0.0001). There were statistically significant differences between the means of the clinical forms  and the subpopulation of MφM2 (p = 0.0001).Second, MφM2 were the predominant Mφs in the VV clinical form (p = 0.0001), indicating the association between MφM4 and chronic pathological processes in leprosy. The pro-inflammatory function of MφM4 was indicated in their positive immunoexpression pattern for TNF-α, IL-6, CD68, MMP7, and MRP8. The immunoexpression pattern of the M4 phenotype was significantly correlated to the VV form of leprosy (p = 0.0001). ANOVA revealed statistically significant differences between the means of the three clinical forms  and the subpopulation of MφM4 (p = 0.0001).Third, MφM4 predominated the Mφ subpopulations in VV leprosy (p = 0.0000), as well as between VV M2 × TT M1 .Fourth, our data also revealed a highly significant positive linear correlation between VV M1 × VV M4 (r = 0.8712; Our findings revealed the relationship of macrophage subpopulations and their profile according to the respective clinical forms in persistent pathological processes of the inflammatory response during the disease. This makes it possible for new studies to advance in the diagnosis and personalized treatment of leprosy.


Purkinje cytoplasmic autoantibody type 1 (PCA-1)/anti-Yo autoimmunity is a common high-risk paraneoplastic neurological disorder, traditionally attributed antigenically to cerebellar degeneration–related protein 2 (CDR2), predominantly affecting women with gynecologic or breast adenocarcinoma. Single-modality CDR2 testing may produce false-positive results. We assessed the performance characteristics of the more recently purported major PCA-1/Yo antigen, CDR2-like (CDR2L), side by side with CDR2, in a line blot format.CDR2 and CDR2L were tested in six specimen groups ). Group 1, PCA-1/Yo mouse brain indirect immunofluorescence assay (IFA) positives; Group 2, PCA-1/Yo IFA mimics; Group 3, suspected CDR2 line blot false positives; Group 4, consecutive patient samples tested for neural antibodies over 1 year; Group 5, healthy subject serums; and Group 6, polyclonal (non-specific) immunoglobulin G (IgG)-positive serums.Group 1: Of 64 samples tested, all but two were CDR2 positive (both CSF samples) and all were CDR2L positive. In individual patients, CDR2L values were always higher than CDR2. The two “CDR2L-only” positives were CSF samples with low titer PCA-1/Yo by IFA with serum negativity but with typical clinical phenotype. Group 2: All 51 PCA-1/Yo mimics were CDR2/CDR2L negative. Group 3: Nine samples [six of 1289 (0.47%) serums and three of 700 CSF samples (0.43%) were PCA-1/Yo IFA negative/CDR2 positive; two of the six available (serums from the same patient) were also CDR2L positive; the other four CDR2L negative had low CDR2 values (17–22). Group 4: Twenty-two patients had unexpected CDR2 or CDR2L positivity; none had tissue IFA positivity. Eleven of the 2,132 serum (0.5%) and three of the 677 CSF (0.4%) samples were CDR2 positive; median value was 19 . Seven of the 2,132 serum (0.3%) and three of the 677 CSF (0.4%) samples were CDR2L positive; median value was 18 . Group 5: All 151 healthy serum samples were negative. Group 6: One of the 46 polyclonal serum samples was CDR2L positive. Optimum overall performance was accomplished by requiring both CDR2 and CDR2L positivity in serum  and positivity for CDR2L in CSF .CDR2L provides additional PCA-1/anti-Yo sensitivity in CSF, and dual positivity with CDR2 provides additional specificity assurance in serum. Combining antigen-specific and tissue-based assays optimizes PCA-1/anti-Yo testing. Purkinje cytoplasmic autoantibody type 1  is a biomarker of paraneoplastic neurological autoimmunity, usually manifesting as cerebellar ataxia (paraneoplastic cerebellar degeneration) in women with gynecologic or breast adenocarcinoma . The diaThis retrospective study was approved by the Mayo Clinic Institutional Review Board . Medical records of patients who consented to research review were included.Group 1 (PCA-1/Yo IFA positives): Samples (64) were serum (39) or CSF (25) from 48 patients identified in our clinical service laboratory . All samples yielded PCA-1/Yo by tissue IFA (January 2019 to December 2022). Fifteen patients had serum/CSF pairs available for testing. Group 2 (PCA-1/Yo IFA mimics): There were 53 samples (30 serum and 23 CSF samples) with immunohistochemical staining mimicking but not fulfilling criteria for PCA-1/Yo (January 2019 to December 2022). Group 3 : There were nine samples  reflexed from IFA (27 February 2022 to 14 March 2023) without PCA-1/Yo by IFA, in which CDR2 was incidentally detected during confirmation testing for another antibody . Six (five serum and one CSF samples) were available for CDR2L testing. Group 4 : There were 2,809 consecutive available specimens , from patients all neurologically evaluated at Mayo Clinic, and referred for neural antibody testing in the Mayo Clinic Neuroimmunology Laboratory (19 September 2016 to 25 October 2017). Group 5 : There were 151 serum samples from healthy adult donors (EUROIMMUN). Group 6 : There were 46 serum samples from patients without neurological disease but known to have polyclonal hypergammaglobulinemia .The following groups of patient samples were evaluated. Groups 1–4 were samples derived from clinical laboratory service, Neuroimmunology Laboratory, Mayo Clinic. Groups 5 and 6 were additional control samples tested. Neurological and cancer histories were obtained where possible for samples with unexpected results. IFA utilizing a composite of murine brain, gut, and kidney was performed as previously described . CriteriCorrelation of CDR2 and CDR2L values was evaluated using logistic regression analyses. Serum and CSF values were compared using Mann–Whitney U-test.Anonymized data used for this study are available upon request.All 48 patients were positive for PCA-1/Yo by tissue IFA (All 53 samples were CDR2 and CDR2L negative (2 = 0.970)  and the three CSF samples from 19 to 30 . Six of the samples were available for CDR2L testing (five serum and one CSF samples). The solitary CDR2-positive CSF specimen tested was CDR2L negative. Two of the five PCA-1 IFA-negative/CDR2-positive serums were also CDR2L positive  . Both had paraneoplastic disorders in the setting of adenocarcinoma . Neither had samples residual for CDR2L testing. An additional 22 patients had unexpected CDR2 or CDR2L positivity, where tissue IFA was PCA-1/Yo negative. Thirteen patients were CDR2-only positive, eight patients were CDR2L-only positiv, and one patient was positive for both CDR2 and CDR2L  who had None of the 151 serum samples were CDR2 or CDR2L positive  was CDR2L positive   and used the presence or absence of tissue IFA criteria as the gold standard for PCA-1 autoimmunity. We did not include Group 3 data because CDR2L testing was only performed on the available nine false-positive CDR2-positive cases from that cohort of 1,989 samples tested. Sensitivity data were as follows: CDR2 in serum, 100%; CDR2L in serum, 100%; CDR2 in CSF, 92%; and CDR2L in CSF, 100%. Specificity data were as follows: CDR2 in serum, 99.5%; CDR2L in serum, 99.7%; CDR2 in CSF 99.6%; and CDR2L in CSF, 99.6%. Combining CDR2 and CDR2L with a requirement for detection of both analytes above the cutoff to yield a positive result, sensitivity was 100% in serum and 92% in CSF, and specificity was 99.9% for both specimen types. Combining CDR2 and CDR2L with a requirement for one analyte above the cutoff to yield a positive result, sensitivity was 100% in both serum and CSF samples, and specificity was 99.2% for serum and 99.6% for CSF.PCA-1 (anti-Yo) autoimmunity is the second most common high-risk paraneoplastic antibody after ANNA-1 (anti-Hu) detected in clinical laboratory practice . AlthougWe addressed sensitivity of the line blot by comparing CDR2 and CDR2L as reflex confirmatory tests among samples meeting IFA criteria for PCA-1/Yo. The available histories from patients assessed neurologically at the Mayo Clinic indicated that this group was representative of PCA-1/Yo autoimmunity . Both antigens had equivalent sensitivity to confirm PCA-1/Yo IFA results in serum (100%). However, CDR2L values were universally higher than CDR2 in PCA-1/Yo IFA-positive cases. This finding is consistent with those from a study using a peptide-based phage-immunoprecipitation platform . In partWe addressed the specificity of the line blot by testing for CDR2 and CDR2L among samples with IFA mimicking PCA-1/Yo, sequential Mayo Clinic patients in whom neurological autoimmune testing had been requested, and healthy control serums and serums with the potential to produce interference in an immunoassay . Although the non-detection of CDR2/CDR2L among samples producing IFA features mimicking PCA-1/Yo and healthy controls was reassuring, some false positives were encountered for both analytes among consecutive Mayo Clinic patient samples generally referred for neurological autoimmune testing. These false positives were more commonly detected in serum than in CSF, usually for one analyte only and occurred more frequently for CDR2 than CDR2L. Overall, requiring positivity for both CDR2 and CDR2L optimized specificity for both serum and CSF samples, but there was some loss of sensitivity for CSF. Thus, optimum overall performance might be accomplished by requiring positivity for both CDR2 and CDR2L in serum and positivity for either in CSF. In the Mayo Clinic experience to date, the values for CDR2 false-positive results (highest being 59 for serum and 19 for CSF) have been lower than that for true positives (lowest being 70 for serum and 39 for CSF). It should be borne in mind that some laboratories screen by line blot and reflex to tissue-based assay for confirmation, the opposite of the approach described here.The two patients encountered with immune-mediated neurological disorders in whom both CDR2 and CDR2L were detected at intermediate or high values, but without PCA-1/Yo observed by tissue IFA, did not have PCA-1/Yo typical phenotypes. The immunological and clinical significance of these findings are unknown. One patient was a man with steroid-responsive meningoencephalitis without cancer with dual positivity in CSF but not in serum. The other was a woman with cerebellar degeneration but in the context of ANNA-1 (anti-Hu) by tissue IFA, with dual-CDR2/CDR2L positivity in serum (CSF not available) and had the ANNA-1–associated neoplasm  rather than PCA-1/Yo–associated breast or gynecologic neoplasia. Consistent with the immune response against multiple neural antigens encountered in small cell carcinoma, she was also positive for Hu, SOX-1, and ZIC4 by line blot , 16. It Limitations of our study include missing clinical data for some subjects, and the focus on line blots currently used commonly in clinical practice. Future studies will need to examine promising CDR2L cell–based assays in a similarly high-throughput laboratory practice . In addiWe conclude that, for PCA-1/Yo autoimmunity, CDR2L in the line blot format improves sensitivity in CSF and is optimized for specificity in serum when combined with CDR2. Our observations give emphasis to the importance of pairing tissue-based and antigen-specific assays for the diagnosis of paraneoplastic neurological disorders . AlthougThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Mayo Clinic Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.NV: Data curation, Investigation, Methodology, Writing – review & editing. MS: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. RM: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. ST: Formal Analysis, Writing – review & editing. DD: Data curation, Formal Analysis, Writing – review & editing. JM: Data curation, Formal Analysis, Writing – review & editing. SP: Data curation, Formal Analysis, Writing – review & editing. AZ: Data curation, Formal Analysis, Writing – review & editing. AO: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. AM: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing.


In patients with SAA rapid CSF drainage while performing durotomy must be avoided by utilizing cotton pads and lowering the head level to avoid catastrophic complications. In patients with SAA rapid CSF drainage while performing durotomy must be avoided by utilizing cotton pads and lowering the head level to avoid catastrophic complications. She suffered from spinal adhesive arachnoiditis in craniovertebral junction leading that underwent posterior fossa decompression and developed subsequent supratentorial subdural hematoma formation requiring surgical evacuation.Spinal adhesive arachnoiditis (SAA) is characterized by collagen deposition in pia matter of arachnoid space due to inflammation caused by various etiologies.Intracranial hematoma, remote from surgical site, has been reported in literature.22.1A 37‐year‐old single woman from southern Iran was referred to our clinic due to progressive spastic gate and apraxia along with progressive thoracolumbar scoliosis for the past 6 months prior to presentation. She had no significant past medical, family or drug history except a falling object head trauma 5 years earlier. In physical examination, she had scoliotic thoracic spine and lower extremity proximal weakness (3/5). She also had right upper extremity radicular pain and proximal weakness (4/5). Plantar reflexes were upward bilaterally along, the Hoffmann's sign was positive and the deep tendon reflexes (DTR) were exaggerated (3/4). She could walk only with assistance utilizing a walker. Fundoscopic examination revealed normal optic disc borders without papilledema.2.2Whole spine magnetic resonance imaging (MRI) was conducted revealing a large syrinx extending from C2 to lower thoracic levels Figure , a large2.3In standard prone position with her head fixed in Mayfield head‐holder and slight neck flexion, the patients underwent bilateral suboccipital craniotomy. About 3 × 3 cm midline suboccipital craniectomy, including dorsal rim of Foramen Magnum, in addition to C1 partial laminectomy was done. Dura was opened in linear fashion, 5‐mm from upper edge of craniectomy down to C1 lamina. There was a collection at dorsal aspect of CVJ, covering with numerous thick arachnoid webs, distorting normal anatomy of neurovascular structures in the area, without normal pulsation of arachnoid membranes. Then, microscopic arachnolysis with preservation of tethered arteries and nerves in arachnoid membranes was performed until floor of fourth ventricle was seen. The cisterna magna was also opened and the CSF normal flow was restored. The arachnolysis was continued caudally to reach normal dorsal spinal SAS. After microscopic coagulation of arachnoid webs with preservation of neurovascular structures, direct passage of CSF from fourth ventricle to spinal SAS was confirmed. The Duroplasty with autologous fascial graft was performed and the wound was closed.2.4We had no major bleeding or hemodynamic catastrophic events during surgery. A postoperative brain computed tomography (CT) scan was performed before transferring to intensive care unit (ICU) revealing right frontoparietal (FP) acute SDH with nearly 5‐mm right to left midline shift Figure . The pat2.5The patient retained his normal consciousness a day after surgery and pupillary reflexes were bilateral and equal. She developed CSF leakage from CVJ surgical site at the fourth postoperative day, that was managed with insertion of lumbar drainage tube and medications. After 3 weeks of postoperative care, neurological examination improved significantly and she could walk without assistance. Her right upper extremity radicular pain and weakness were also resolved. She was discharged with both surgical wounds healed, having no sign of infection, no CSF leakage or subcutaneous collection at the 23rd postoperative day.2.6Serial neurologic examination during 6‐months postoperative period was acceptable and no new neurological deficits were observed. We obtained serial OPD visit and brain CT scans with two episodes of brain and spine MRIs which revealed no hydrocephalus or hematoma and partial resolution of syringomyelia. The patient myelopathy and spastic gate improved significantly.33.1Cerebrospinal fluid (CSF), as the protector of mammalian CNS, is mainly (75–90%) produced by choroid plexus of lateral ventricles and other structures such as brain parenchyma, ependymal cells, and interstitial fluid. CSF flows from lateral ventricles to third ventricle though foramina of Monro. Then, passing through cerebral aqueduct to the fourth ventricle and leaves it from  foramina of Lushka to cerebral cisterns and SAS; and  foramen of Magendie to spinal SAS. The CSF is absorbed at Superior Sagittal Sinus (SSS) arachnoid granulations and Virchow–Robin space of peripheral SAS.3.2Syringomyelia is classified into communicating and non‐communicating types.3.3Spinal adhesive arachnoiditis (SAA) is referred to formation of multiple arachnoid membranes in SAS due to various pathologies, mainly caused by inflammation.3.4Herein we presented our case of foramen magnum SAA due to previous traumatic brain injury, forming numerous arachnoid membranes in CVJ, altering CSF circulation in CVJbeside local accumulation of CSF in dorsal aspect of FM with compression effect on brainstem and fourth ventricle. It obviously caused resistance in CSF flow and higher‐pressure gradient between cranial and spinal regions, documented in CSF dynamic study. Decompression of FM and release of arachnoid adhesions, are mainstay of SAA of the SVJ being associated with improved clinical outcome and resolution of the syrinx.We assumed that CSF dynamics in these patients adapt with a higher‐pressure gradient; explaining that CSF flow resistance in CVJ, causes higher cranial and lower spinal CSF pressure. CSF outflow and absorption adaptations, might cause higher cranial CSF pressure without ventriculomegally or other imaging findings of intracranial hypertension similar to normal pressure hydrocephalus (NPH) or idiopathic intracranial hypertension (IIH).4Spinal adhesive arachnoiditis (SAA), caused by various etiologies leads to alterations in CSF circulation and neural structure tethering or compression. In CVJ, beside brainstem and cerebellum compression, spinal syrinx formation is a common sequela which is associated with scoliosis and neurologic deficits. Foramen magnum decompression and arachnolysis with direct observation of CSF flow from fourth ventricle to spinal SAS, is considered the standard of care. Posterior fossa decompression with abrupt CSF drainage is associated with cerebral cortical vein sagging and subsequent supratentorial SDH formation. We recommend in similar cases, to avoid rapid CSF drainage while performing durotomy by utilizing cotton pads and lowering the head level to avoid such catastrophic complications.Keyvan Eghbal: Conceptualization; methodology; project administration; writing – review and editing. Majid Reza Farrokhi: Conceptualization; resources; supervision; writing – review and editing. Seyed Reza Mousavi: Data curation; investigation; supervision; writing – review and editing. Mohammadhadi Amir Shahpari Motlagh: Investigation; software; validation; writing – review and editing. Ali Kazeminezhad: Data curation; formal analysis; software; visualization; writing – review and editing. Fariborz Ghaffarpasand: Data curation; formal analysis; investigation; validation; writing – original draft; writing – review and editing.None of the authors have any conflict of interest to declare regarding the manuscript.All procedures performed were under the institutional and/or national research committee's ethical standards and the 1964 Helsinki Declaration and later amendments or comparable ethical standards. Shiraz University neurosurgery department board members supervised and approved this report on behalf of the Ethical Committee of Shiraz University of medical sciences (SUMS).The patient and their legal guardians provided their written consent for publication of the manuscript and the case for research purposes.