Allodynia is a state in which pain is elicited by innocuous stimuli. Capsaicin applied to the skin results in an allodynia that extends to a broad region beyond the application site. This sensitization is thought to be mediated by spinal networks; however, we do not have a clear picture of which spinal neurons mediate this phenomenon. To address this gap, we used two-photon calcium imaging of excitatory interneurons and spinal projection neurons in the mouse spinal dorsal horn. To distinguish among neuronal subtypes, we developed CICADA, a cell profiling approach to identify cell types during calcium imaging. We then identified capsaicin-responsive and capsaicin-sensitized neuronal populations. Capsaicin-sensitized neurons showed emergent responses to innocuous input and increased receptive field sizes consistent with psychophysical reports. Finally, we identified spinal output neurons that showed enhanced responses from innocuous input. These experiments provide a population-level view of central sensitization and a framework with which to model somatosensory integration in the dorsal horn. Altered mechanosensation by application of capsaicin to the skin is thought to be spinally mediated. Here, the authors use Ca2+ imaging in spinal neurons and develop a cell profiling approach to identify populations involved in central sensitization. This central sensitization can be protective in the context of acute injury because it engenders hypervigilance during recovery. In the context of chronic pain, however, central sensitization is maladaptive, leading to suffering. Given the link between central sensitization and the chronification of pain, it is important to understand activity-induced changes in the central processing of nociception.Persistent noxious input drives changes in central circuitry that alters the integration of somatosensory stimuli, resulting in abnormally amplified pain2 and transmit nociceptive signals to a variety of supraspinal locations by way of spinal output neurons, particularly those in lamina I, most of which collateralize within the lateral parabrachial nucleus6. The large diversity of cell types within the superficial laminae have been defined with a variety of methods including morphology, location, intrinsic firing properties, expression profiling, and neurochemical markers7 but we have yet to reach a consensus on the number and identity of bona fide cell types. Numerous studies have investigated the role of a given population for normal sensation and in the context of allodynia17. A common thread in many of these studies is that any disruption in the balance between inhibition and excitation can drive allodynia via loss of inhibitory tone25 or sensitization of excitatory pathways30 including spinoparabrachial (SPB) neurons35.The spinal dorsal horn processes heterogeneous primary afferent input through complex local interneuron circuits. These neural circuits contribute to spinally mediated reflexes37 and electrophysiological studies40. In this model, cutaneous application of capsaicin activates TRPV1+ C-fibers resulting in burning pain that lasts minutes, and central sensitization in the dorsal horn that lasts hours. Two salient features of this central sensitization are that low threshold mechanical stimuli, which are normally innocuous, are perceived as painful , and that the area of allodynia (the secondary zone) is much larger than the area of capsaicin-treated skin. In light of these findings, capsaicin-induced allodynia is thought to be a central phenomenon, likely due to plasticity at the level of the spinal cord42. Consistent with this idea, electrophysiological studies have revealed that there are synaptic changes in spinal projection neurons after capsaicin treatment43. However, the identity of which spinal populations are involved in generating this plasticity have yet to be examined.Capsaicin-induced hypersensitivity is a model of central sensitization that is well characterized in psychophysical2+ imaging is a powerful approach to visualize neural activity at a population level, and several groups have applied this approach to the spinal dorsal horn50. However, imaging studies in this region are challenging to perform due to respiratory movement and optical access46 and difficult to interpret due to neuronal diversity51. In the ex vivo somatosensory preparation, the skin, nerves, dorsal root ganglia, and spinal cord are dissected out in continuum, allowing the application of quantitative sensory stimuli to the skin. This reduced preparation is advantageous because it provides superior image quality and stability, eliminates the confound of anesthesia, and facilitates extensive pharmacological manipulation. Additionally, rather than imaging a mixed population of unidentified neurons, we focused on excitatory neurons, and further distinguished interneurons from projection neurons by virtue of retrograde labeling from the lateral parabrachial nucleus. To further differentiate the excitatory populations, we leveraged the heterogeneous expression of Gq-coupled G-protein coupled receptors (GPCRs). Because activation of Gq-coupled GPCRs gives rise to the release of Ca2+ from internal stores, the application of GPCR ligands allows us to visualize which cells express which receptors. We have called this approach CICADA for Cell-type Identification by Ca2 + -coupled Activity through Drug Activation.Ca2+ imaging in the superficial dorsal horn to define the response properties of both spinoparabrachial neurons and excitatory spinal interneurons to quantitative mechanical and thermal stimuli. Using CICADA, we defined seven excitatory interneuron populations based on their responses to Gq-coupled GPCR agonists. We then determined how central sensitization alters their response properties and identified distinct CICADA-defined populations that comprise capsaicin-responders and capsaicin-sensitized populations. Within identified capsaicin-sensitized populations, some show selectively amplified responses to low threshold input suggestive of allodynia, whereas others show increases in receptive field size consistent with a secondary zone of allodynia expanding beyond the capsaicin injection site. Finally, we show that capsaicin-induced central sensitization alters the response properties of SPB neurons, which show a shift in tuning towards low threshold input. Together, these data represent a population view of central sensitization in the superficial dorsal horn.Here, we used 2-photon Ca2+ imaging of excitatory neurons in the superficial dorsal horn of the spinal cord in response to natural cutaneous stimulation  or 0.16 g for low threshold (LT). For dynamic mechanical stimulation, we applied a wide brush or an air puff. Thermal stimuli consisted of fast and slow ramps to warm (40 °C), hot (52 °C), cool (18 °C), or cold (4 °C) temperatures using a Peltier thermode. Examples of the Caown Fig. , illustr2+ transient with a peak amplitude that was at least 125% ΔF/F and more than 6 standard deviations above baseline activity  had significantly larger receptive fields than neurons that were deeper   Fig. .Fig. 3Re2+ transients in many neurons simultaneously, it is difficult to determine the specific identity of these neurons, thereby hampering the degree to which the data can be interpreted. To overcome this limitation, we developed a cell profiling strategy that leverages the fact that different subtypes of neurons express different combinations of GPCRs. We reasoned that pharmacological profiling could be used to visualize which cells express which receptors because activation of Gq-coupled GPCRs gives rise to IP3-mediated release of Ca2+ from internal stores. To isolate the direct effects (cell autonomous) from indirect effects (network responses) we included the Na+ channel blocker tetrodotoxin (TTX) in the bath solution, which prevents action potential propagation of neuronal activity. In the presence of TTX, the cells that show a Ca2+ transient in response to a given agonist are only those that express its receptor, allowing us to unambiguously define cell types based on their responses to GPCR agonists applied in series at the end of each experiment , an agonist for the muscarinic acetylcholine receptor M3 (CHRM3); gastrin-releasing peptide (GRP), an agonist of the gastrin-releasing peptide receptor (GRPR); taltirelin, an agonist of the thyrotropin-releasing hormone receptor (TRHR); neuromedin-B (NMB), an agonist of the neuromedin-B receptor (NMBR); cholecystokinin (CCK), an agonist of the cholecystokinin type B receptor (CCKBR); Substance-P (SP), an agonist at the Substance-P receptor (TACR1); oxytocin (OXT), an agonist at the oxytocin receptor (OXTR); and Neurokinin-B (NKB), an agonist at the tachykinin receptor 3 (TACR3)   Fig. . BecauseR3) Fig. , we nextons Fig. . The clunds Fig. . Moreove5%) Fig. . Notably5%) Fig. , with po5%) Fig.  than dee5%) Fig. . This su37. The mechanical allodynia in this model is understood to be the result of central, rather than peripheral, sensitization because the primary afferents that innervate the secondary zone are unchanged after the capsaicin injection42. However, the specific spinal neurons and circuits involved in mediating the response has not yet been visualized at a population level.Previous human psychophysical experiments have shown that intradermal injection of capsaicin causes an immediate response consisting of brief burning pain (<5 min) that is restricted to the capsaicin injection site, followed by a period of sensitization which manifests as long-lasting mechanical allodynia  extending over a large region outside the capsaicin injection site Fig. 37. The m2+ imaging coupled with post-hoc cell identification through CICADA  even though Ex1TACR3/1 makes up <10% of excitatory neurons in lamina I. Thus, intradermal capsaicin drives activity in excitatory neurons in the superficial dorsal horn, and this activity is significantly enriched within Ex1TACR3/1, a population of relatively untuned  of excitatory superficial cells without significantly affecting the responses to other modalities , it is unlikely that we imaged many, if any, PKCγ or CCK cells. These deeper populations could be providing low-threshold input to the superficial populations we examined here through disinhibition via deep dorsal horn populations40 and parvalbumin inhibitory neurons22 although it was not specifically examined in this work.Current models of neural circuitry in the dorsal horn suggest that after injury there are a variety of different mechanisms, many of which depend on the type of injury, that could lead to an allodynia driven by central sensitization. One of the most established concepts is that allodynia is caused by disinhibition involving a loss of inhibitory neuron activity, which is especially common in the context of neuropathic injuriesIn psychophysical studies of capsaicin-induced central sensitization, participants report two salient phenomena: touch stimuli evoke pain , and the region of sensitivity extends beyond the initial site of injury (extended secondary zone). To help visualize the neural basis of allodynia, we developed a vector-based method to represent neuronal tuning to somatosensory stimuli. This way of visualizing the data is advantageous because it emphasizes the relative tuning of neurons to distinct types of stimuli. Because neurons in the superficial dorsal horn are rarely unimodal, we speculate that the sensory tuning may be the salient feature that is relevant for neural coding Fig. . Althoug37. A limitation of our study is that we did not capture the corresponding neural representations of thermal hyperalgesia or dynamic allodynia, likely because the Peltier thermode and brush stimulus used in this study were applied to the entire receptive field, thereby limiting our ability to see localized changes in the dorsal horn. However, using this large Peltier ensures we have covered the entirety of the skin’s receptive zone, which makes the decreased response to heat after capsaicin induced sensitization within Ex2TACR1  facilitated the development of CICADA, 2) yielded hundreds of neurons per animal, which is required for population-scale analysis, (3) provided higher quality recordings due to fewer movement artifacts, (4) facilitated a more quantitative application of cutaneous stimuli, and (5) enabled recordings in the absence of anesthesia. Although this ex vivo approach has a number of appealing benefits, we acknowledge that it is a reduced preparation that lacks numerous components, notably descending modulation which has been well established to inhibit or facilitate nociceptive signaling. In addition, our ability to perform receptive field mapping is limited by the number of nerves and the size of skin attached,  yielded dal Fig. , a findiTACR3/1, Ex2TACR1, and the WDR SPB neuron subtype) express the receptor for SP. Whether any of these populations also express receptors for CGRP remains unclear. The receptor for CGRP is Gs-coupled (rather than Gq-coupled) and, as a result, the activation of this receptor is not visible using the current CICADA strategy. The future development of tools and strategies that allow us to visualize Gs- and Gi-coupled signaling events would not only aid in our understanding of neuromodulatory mechanisms, but also permit a much more complete analysis of neuronal subtypes through pharmacological profiling.Because prolonged activity in TRPV1-positive afferents gives rise to the release of SP and CGRP in the dorsal horn, it is tempting to speculate that these neuropeptides may be involved capsaicin-mediated sensitization. In this regard, it is noteworthy that many of the neurons that showed altered activity following capsaicin  the Ai96 allele  were used for all experiments. Mice were housed on a 12-hour light cycle with ad-libitum water and food in accordance with the United States National Institutes of Health guidelines for the care and use of laboratory animals. Relative humidity was kept between 30% and 70% and temperature was kept between 20 and 26 °C. In total 10–15 fresh air changes per hour was provided for adequate ventilation. Daily observation of all animals was required, and bedding was changed at least weekly depending on the number of animals. Social housing was utilized with no more than 4 adult mice per cage was permitted. Single housing was not used except for cases of animal welfare, e.g., aggressive males being separated. Enrichment was provided in the form of soft bedding that can be burrowed within, plastic housing domes, and running wheels. Ca2+ imaging was performed on mice of both sexes ranging from 5 to 10 weeks of age ranging from 20 to 25 grams depending on sex and age.All experiments were performed with approval of the University of Pittsburgh’s Institutional Animal Care and Use Committee . Mice, from a C57/BL6 background, that were heterozygous for both the Mice ranging from 4 to 5-weeks old were placed under isoflurane anesthesia and mounted in a Kopf stereotaxic headframe. After exposing and leveling the skull a burr hole was made at  and a glass micro-pipette filled with Fast DiI oil  was used to inject 250 nl into the left parabrachial nucleus over the course of 5 min. The pipette was left in place for 10 min before retracting the pipette and closing the wound. Injected mice were then used for imaging a minimum of 4–5 days after injection. DiI-labeled SPB neurons were validated by taking fresh transverse spinal cord slices and coronal brain sections and checking for fluorescence in the appropriate regions as mapped in the Allen Mouse Brain Reference Atlas.2/5%CO2 saturated, chilled, sucrose-based artificial cerebrospinal fluid (ACSF). The sucrose-based ACSF contained (in mM): 234 sucrose, 2.5 KCl, 0.5 CaCl2, 10 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 11 Glucose. A complete dorsal laminectomy was performed followed by excision of the spinal column (taking care not to remove tissue between the right leg and column), right ribs, and right leg. The tissue was transferred into a dissection dish and the lateral femoral cutaneous and saphenous nerves were dissected out in continuum from the skin to the DRG. The thoracolumbar spinal cord was then removed from the bone and the roots were carefully pulled away from the cord to allow movement of the spinal cord. The spinal cord was pinned at an angle such that the grey matter of the spinal cord was parallel with the ground plane. The dura mater was removed among the entire spinal cord and the pia was only removed along the L1 - L2 recording area. The roots and nerves were loosened from the tissues as much as possible and the skin was pinned out as far away from the spinal cord as possible. This ensured sufficient separation from the spinal cord as to allow a 3D printed light shield to block the visible light used to localize stimulation on the skin from reaching the high sensitivity detectors on the microscope. Once the skin and spinal cord were securely pinned in place using Minutien pins (FST Cat: 26002-20) the dish was transferred to the microscope and washed with standard normal ACSF solution containing (in mM): 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, 11 glucose), which was slowly washed in over 15 min. Next, the temperature of the wash solution was raised from room temperature to 28 °C in 2 °C steps every 5–10 min. Once the tissue had equilibrated to the standard ACSF, a manual wash was performed to fully remove any leftover detritus and dissection solution using a 50 ml syringe filled with ACSF. Imaging began 30–45 min after the final wash in order to allow time for thermal expansion and settling of the tissue. Once ~1 L of solution was washed through the dish, the remaining 4 L of bubbled ACSF was then recirculated for the duration of the physiology experiments.Mice were deeply anesthetized with a ketamine/xylazine mixture (1.75 mg ketamine and 0.25 mg xylazine per 20 g body weight). The right hind paw, leg, and back were shaved with an electric clipper leaving 2–3 mm of hair in place. Next, mice were transcardially perfused with 95%O2+ imaging was performed on a Leica SP5 MP microscope using a Leica 20x NA 1.0 water immersion lens with a Coherent Chameleon Ultra II Ti:Si femtosecond laser set to 940 nm for Ca2+ imaging and 1040 nm for imaging of DiI within the SPBPNs. Fluorescence was captured with HyD detectors and standard FITC/TRITC emission filter sets with a 570 nm dichroic beam splitter. Three different planes of the superficial grey matter were simultaneously imaged with a spacing of 14 µm between planes, which yielded a range of depths from 0 to 60 µm below the spinal cord surface. For each plane, scanning was performed at 1 Hz with a 1.4x optical zoom at 512 × 214 pixel resolution and a field size of 528.2 × 220.8 µm size yielding a pixel resolution of 1.03 µm/pixel. This volumetric scanning allowed sampling from multiple lamina simultaneous and typically yielded ~300 excitatory neurons in a given experiment.CaAs the location of afferent input for each animal is determined by relative somatotopy, the region of the grey matter imaged thus needed to be functionally identified for each animal. The exact field of view imaged in the SDH was determined by using a low magnification image and a search stimulus within the dissected skin. A wide brush which stimulated the entire length of the skin was used to activate as many afferent inputs as possible. The imaging field of view was iteratively refined, centering the image on the region that had the most change in fluorescence and then zooming to the final 1.4x optical zoom. Subsequently, a 2.0 g von Frey (VF) filament was used to delineate a 15 × 15 mm region of skin that showed the most input into the final field of view in the SDH. The region was marked with a surgical felt tip pen. Imaging, stimuli controllers, feedback sensors , and event times were synchronized using a Power1401 and Signal (CED).After the final round of sensory testing post capsaicin was completed, 500 nM tetrodotoxin (TTX) was applied and allowed to circulate for 15–20 min before proceeding. CICADA agonists were then applied in a randomized order for 2 min followed by 5 min wash in the continued presence of 500 nM TTX. CICADA agonists were kept frozen in single use aliquots until application when they were thawed and diluted in TTX recording solution. Post CICADA, a brief pulse of 5 ml of ACSF containing 30 mM KCl ACSF solution was injected into the perfusion lines in order to confirm cell viability and assist in ROI generation and refinement. All drugs were purchased from Tocris and Sigma.2+ imaging protocol but encompassing the entire recorded volume of the grey matter at high Z resolution taken from ~10 µm above the surface to ~60 µm below, at a Z resolution of 1 µm. During this time, 30 mM KCl ACSF solution was applied in order to visualize all the GCaMP6s expressing cells and provide a high contrast image for volumetric reconstruction. Post-hoc, the structural reference was aligned with the functional Ca2+ imaging reference image, smoothed with a 3D median filter, contrast enhanced using Contrast Limited Adaptive Histogram Equalization, and then XY down sampled into voxels representing a 10 × 10 × 1 XYZ area. The mean value of the voxels was then used to find the surface of the dorsal horn grey matter for each 10 ×10 XY coordinate. The calculated Z coordinate of the surface was based on identifying the asymptotic increase in fluorescence as the surface of the dorsal horn comes into focus. Each of the 3 recording planes was then aligned to a Z-plane in the structural image and each ROI was then offset in the Z-dimension by the amount indicated by their XY location. This analysis provided the final dorso-ventral location of each cell.In order to accurately calculate the depth of cells in the dorsal horn, the curved surface of the dorsal horn needed to be taken into account. Briefly, after all experimental manipulations had finished, a volumetric time-lapse (XYZT) imaging protocol was loaded at the same field of view and XY resolution as the CaThe presentation order of natural stimuli was randomized within each testing block (i.e. baseline or post capsaicin testing periods). All natural stimuli were applied to an identical 15×15 mm area of skin using stimulators that were specifically designed to fit in the same footprint. A dim red light was focused on the skin in order to provide sufficient illumination for the experimenter and reduce the amount of light contamination reaching the sensor. For manually applied stimuli, a program was written in Signal (CED) to produce a synchronized output to an audio monitor that provided a 3,2,1 countdown for each stimulus, a 1 s tone for duration of application, and ensured both even spacing and duration of stimuli application.2, we estimated the thickness of the brush when compressed during a stroke was ~2 mm resulting in an effective area closer to 30 mm2 when actively applying pressure. The brushing was repeated 3 times, 60 s apart.Airpuff was applied at a constant supply pressure of 60 PSI for 1 s duration and repeated 3 times 60 s apart and controlled by a PicoSpritzer III (Parker Hannifin). The airpuff applicator device was a custom designed module with a 6 ×6 grid of evenly spaced 18-gauge stainless steel nozzles with an inner diameter of 0.8 mm to fit within the delineated region (15 × 15 mm) and provide equal pressure across the skin. The device held in place 1 cm above the skin with a micromanipulator. Brushing was done manually with a quantitative sensory testing brush . Average force applied was ~20 g for brushing and ~5 g for airpuff and was calibrated by use of a load sensor coupled to the imaging chamber and controlled by an Arduino. As brushing was manual, we did observe some variance in the force applied but it typically stayed within the range of 15 to 25 g. The applied pressure for each mechanical stimulus was estimated as follows: air puff, 0.2 kPa; brush, 6.5 kPa; 0.16 g VF (LT), 78 kPa; 2.0 g VF (HT), 270 kPa. While the Somedic brush is a 15 × 5 mm brush with an area of 75 mmReceptive field mapping was done with a LT (0.16 g) or HT (2.0 g) von Frey filament. A custom cut flexible plastic grid was placed upon the delineated region of skin in order to assist with consistent targeting of the filaments to each of the 16 sites to be mapped in a 4 ×4 grid arrangement. Each round of mapping consisted of applying the filament for 1 s to each location spaced 15 s apart. For example, in Fig. A custom-made water-cooled Peltier was placed on the delineated region of skin and held at a thermoneutral 30 °C between stimulations. The Peltier was then driven by a custom-designed Peltier controller to the indicated temperatures at specified rates and set point. Measured surface temperatures were recorded in Signal 7 (CED) with a CED 1401. For fast rate heat ramps the Peltier was driven at 4 °C/s from 30 °C to 52 °C and held for 5 s at 52 °C before returning to neutral. Fast cold ramps were similarly run at 4 °C/s but the effective cooling rate slows as it approaches 0 °C  was used for all animals.2+ dysregulation which would signal signs of apoptosis. Then ΔF/F was calculated in Excel (Microsoft) for each cell using a rolling ball baseline: for each frame (Fi) the 30th percentile value of the surrounding 5 minutes of frames was used as a baseline (Fb) to perform the ΔF/F calculation ((Fi- Fb)/Fb). Rolling ball normalization was preferred here as the effects of slow photobleaching of the indicator and/or small changes in location were minimized in these long recordings.For image processing, we utilized a Suite2p  pipeline empirically tuned for superficial dorsal horn cells for image registration and initial segmentation. The masks were transferred to FIJI  and additional ROIs were added by hand as the heterogeneity in morphology and size in the superficial layers of the spinal cord was difficult for most segmentation programs to accurately capture. Masks were then spot checked using a maximum activity (ΔF/F or the first derivative of fluorescence across time) reference image generated from 5-min bins along the entire recording. This maximum activity reference image resulted in ~50 frames that represented 5-min bins of activity and could be visually scrolled through to quickly check the 4- to 5-h long recording for physical deformations, drift, blebbing, or other pathologic changes. Any cells that showed significant deviation from their XY location, signs of Z-drift , blebbing, or cell death in the latter portions of the recording were manually deleted. Once the masks were finalized, the mean fluorescence was calculated and any cell that has significant drift (>200%) in their 5-min binned median fluorescence value was deleted because such a change was indicative of either physical drift or Cai.e. taking the 8 data points for amplitudes of response to CICADA, the formula is as follows: (CellResponse - Cellmin)/Cellmax. For cells in Fig. 65.Once ΔF/F values had been generated, traces were analyzed for binary responder analysis (yes/no) as determined by a peak value in ΔF/F that was both >125% of baseline in the 5 s following stimulation and also > 6σ (standard deviations) of basal trace noise in the 30 s prior. These criteria were selected based on a receiver operator characteristic (ROC) analysis of σ and amplitude using a manually annotated data set of 500 cells as the ‘ground truth’ against which we optimized the AUC from the ROC analysis. For the manual annotation, we generated image files for each cell with stimulation event markers. The 500 traces were then manually marked as a responder or not based on whether the amplitude of response was clearly distinguishable over the background recording noise. This ‘ground truth’ data was then used to calibrate against multiple objective measures of quantification  and the static LT and HT stimulation each stimulate a distinct region of skin so each of the stimulations were not equal. To calculate the size of the receptive field, the total sum of responses at the 16 tested fields (4 × 4 grid) were averaged across 2 trials providing a possible receptive field size of 0 to 16. As each of the 16 tested sites were spread across a 225 mm2 region spaced apart by ~3.75 mm, each response likely represents ~3-4 mm2 of skin, i.e. a cell which responded to 4 points has an estimated receptive field size of 15 mm2. For clarity, the conversion to square mm2 was not performed and was left as number of receptive fields. Of note, while changes in LT receptive field size post capsaicin are largest in Lamina II cells , mechanical/LT Static +210) and cold/HT static (+330). These angles (e.g. +90) are relative to the positive X-axis moving in a counter-clockwise direction. Using these angles and the normalized amplitudes, 3 separate vectors were calculated, summed, and then graphed in Matlab  where the length of the vector was the normalized amplitude of response, and the direction of each vector was as described for each modality. For examples in interpreting these vectors we can examine Cells #1-3 from 66.K-Means clustering was performed in the statistical package OrangeXi-Xavg)/Xmax.To determine whether dorso-ventral location  or CICADA population best predict cardinal polymodality, we performed a multiple logistic regression in GraphPad Prism 9 software where the outcome was cardinal polymodality and we included depth and CICADA population as variables. For Supplementary Fig. Vglut2 cre x GCaMP6s animals were anesthetized with isoflurane and decapitated. Within 5 min of euthanasia, the L3/L4 spinal segments were removed and placed in OCT, flash frozen, and kept on dry ice until cryosectioning. Spinal cord was then sectioned in the transverse plane at 15 μm thickness, mounted on slides, and were probed for GCaMP6s and Vglut2 using RNAscope Multiplex Fluorescent Assay  using: Mm-Slc17a6 (Cat No. 319171) for Vglut2 and Mm-GCaMP6s-O1 (Cat No. 557091) for GCaMP6s. After hybridization and amplification slides were mounted with Prolong Gold with DAPI. Sections were then imaged using an upright epifluorescence microscope (Leica THUNDER Imager Tissue) using a 20x/0.80 NA air lens at 3883×3886 resolution (0.32 μm per pixel) and analyzed offline with FIJI. A positive cell required a clearly defined nucleus and fluorescent signal forming a ring around the nucleus, as shown before67. 60-76 GCaMP6s+ nuclei were quantified within the superficial dorsal horn per mouse.t test (paired where appropriate e.g. before and after capsaicin) for comparison of exactly 2 groups, ordinary 1-way analysis of variance (ANOVA) with multiple comparisons post hoc test if indicated by the main effect (comparison of >2 groups), 2-way repeated measures ANOVA (restricted maximum likelihood (REML) was used where balanced repeated measures data was unavailable) with multiple comparisons post hoc test if indicated by the main effect (time course with comparison of 2 or more groups). All tests were 2 tailed and a value of P < 0.05 was considered statistically significant in all cases. For cases where multiple post hoc comparisons were indicated by the main effect, a correction for multiple comparisons was implemented using a False Discovery Rate of 0.05 via the two-stage step-up method of Benjamini, Krieger and Yekutieli. Reported Q-values are the FDR corrected P-values, and in all cases Q < 0.05 was considered significant. For all data plotted as a function of depth . We estimated our sample size with G*Power after 2 experiments were completed. All other statistical analyses were performed using GraphPad Prism 9 software.Data are expressed as the mean ± SEM unless otherwise indicated in figure legend. The following tests were used for statistical analyses: Student Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Medical trainees frequently note that cardiac anatomy is difficult to conceive within a two dimensional framework. The specific anatomic defects and the subsequent pathophysiology in flow dynamics may become more apparent when framed in three dimensional models. Given the evidence of improved comprehension using such modeling, this study aimed to contribute further to that understanding by comparing Virtual Reality (VR) and 3D printed models (3DP) in medical education.We sought to systematically compare the perceived subjective effectiveness of Virtual Reality (VR) and 3D printed models (3DP) in the educational experience of residents and nurse practitioners.Trainees and practitioners underwent individual 15-minute teaching sessions in which features of a developmentally typical heart as well as a congenitally diseased heart were demonstrated using both Virtual Reality (VR) and 3D printed models (3DP). Participants then briefly explored each modality before filling out a short survey in which they identified which model (3DP or VR) they felt was more effective in enhancing their understanding of cardiac anatomy and associated pathophysiology. The survey included a binary summative assessment and a series of Likert scale questions addressing usefulness of each model type and degree of comfort with each modality.p < 0.001. Most participants felt comfortable with modern technology (7.6 ± 2.1). 87% of participants preferred VR over 3DP.Twenty-seven pediatric residents and 3 nurse practitioners explored models of a developmentally typical heart and tetralogy of Fallot pathology. Most participants had minimal prior exposure to VR (1.1 ± 0.4) or 3D printed models (2.1 ± 1.5). Participants endorsed a greater degree of understanding with VR models (8.5 ± 1) compared with 3D Printed models (6.3 ± 1.8) or traditional models of instruction (5.5 ± 1.5) Our study shows that, overall, VR was preferred over 3DP models by pediatric residents and nurse practitioners for understanding cardiac anatomy and pathophysiology.The online version contains supplementary material available at 10.1186/s41205-022-00164-6. Medical trainees frequently note that cardiac anatomy and pathophysiology is difficult to fully conceive in a two dimensional 2D) framework , 2. WhilD framewo3DP has found multiple applications in pediatric cardiology education. Several studies have incorporated 3DP into curricula to compare learner response compared to traditional teaching modalities , 7–9. ThWhile 3DP has found several landing points in medical education, surgical preparation, and clinical reinforcement, 3DP has important limitations including cost and limited cutting planes. Significant interest has therefore developed in alternative 3D dimensional representations including Virtual Reality (VR), Augmented Reality (AR) and Mixed Reality (MR) , 10. SacA comparative study in the field of neurosurgery found VR was more effective than 3DP and traditional 2D representations in enhancing understanding of craniovertebral junction deformities . SimilarAs an initial step to create the 3D models, cardiac CT/MRI data for a developmentally typical heart (15 year old patient) and tetralogy of Fallot (15 year old patient) were identified from our institutional cross sectional imaging database. Raw DICOM data from either MRI or CT was loaded into MIMICS  and segmented to label the blood pool and myocardium. Objects were generated and exported to 3-MATIC  for the following steps: wrapping, island removal, smoothing, exterior hollowing, Boolean union (blood pool’s derived shell with the myocardium), vessel trimming to provide a visually and ambiguous heart and slicing into parts to ensure that the goal features of anatomy would be easily visualized  with cyanoacrylate infiltration. As thin sections such as valve component or vessels branches remained fragile, models were reprinted in multi-jet fusion . Then, magnets were placed, the parts were selectively dyed with conventional fabric dye, and employed for the described work. Segmentation and post-processing were performed by a trained and experienced pediatric cardiologist (SB) and imaging scientist (SDF) Figs. The VR models were derived from the same models created from the CT/MRI data used to form the 3D printed models. These models were viewed using the VR/AR interface of Z-Space software  The zSpace® system consists of a central processing unit (CPU), a 23.6 in, 1080-p high definition liquid crystal 3D stereoscopic display screen, a 3 button stylus with integrated haptic technology, and a set of polarized eye glasses with reflective sensors for tracking cameras Fig. a-b. The To achieve randomization, participants signed up for time slots unrestricted and ungrouped by any participant characteristics. Each consecutive participant was then assigned to be taught using either the VR models or the 3D models in an alternating pattern. Following consent, trainees and practitioners underwent individual 15-minute teaching sessions with us where we demonstrated features of a developmentally typical heart as well as a congenitally diseased heart  using both VR and 3DP. The participants were trained in the same way regardless of whether they were being shown a developmentally typical or pathological heart. The 3DP demonstration included identification of key structures along the pre-set slicing planes. The VR demonstration included a brief demonstration on how to manipulate the VR model as well as how to use the slicing tool to obtain multiple cuts through each model. Participants then briefly explored each modality individually before filling out a short survey . Statistical significance was defined as n = 30). The pediatric residents consisted of 7 interns, 12 second year residents and 8 third year residents. Overall, participants endorsed a greater degree of understanding with VR models (8.5 ± 1) compared with 3DP (6.3 ± 1.8) or traditional models of instruction (5.5 ± 1.5) p < 0.001  or 3-D printed models 2.1 ± 1.5). Based on broad minimal exposure and sample size, no adjustment was made for previous exposure to VR or 3DP. Most participants expressed comfort with the use of modern technology at baseline (7.6 ± 2.1). “Modern technology” was not explicitly defined but implicitly alluded to the digital tools that form part of modern daily lived experience. By level of training, 4/7 (57%) interns preferred VR while 11/12 (92%) of second year residents and 8/8 (100%) of third year residents held a similar preference for VR  expressed a preference for Virtual Reality models compared to 13% (n = 4) for 3DP. In addition, participants offered narrative elaboration on their preferences . We hypothesized that there would be a significant number of participants for whom the tactile and haptic qualities of 3DP models would make them preferable to VR models. For the few who in fact had this preference, these factors were mentioned. Also noted, however, was the limitation of the pre-determined cutting planes. In contrast, VR, had a slicing tool which offered multiple planes in virtually any orientation and was repeatedly cited as an appealing factor in narrative comments Table . This abAn additional consideration in comparing these modalities is the effect of prior exposure, whether direct as in VR/3DP in CHD education or indirect as in other settings such as video game usage. Related to this exposure question is Roger and Cohen’s 2020) discussion of generational learning where he advocates for a framework that is best suited to the current generation of learners to include technology [20 discusDigging deeper into what it means to “like” or “prefer” a modality raises additional questions: did intrinsic parts of the educational process such as learning to use the VR wand offer an internal reward that made preference more likely? Less ambiguous is that experienced raters were essentially unanimous in their VR preference. This preference could reflect less of a need or desire on the part of early learners for dimensional data than expert learners.The educational potential of Virtual Reality is certainly being explored in a number of other fields as well with potentially transferable principles. In nursing, for example, an intervention group who were taught a procedure using VR were able to perform more of these procedures in an hour compared to the control group . HoweverOf critical importance in the ongoing evaluation of these modalities is to consider both objective effectiveness and feasibility. In a recent study examining the impact of VR on participant understanding of atrioventricular canals, no difference was found in post-test scores between the control group (desk-top computer) and intervention group (VR). However, the VR group did report a better learning experience and engagement level. Almost counterintuitively, the VR group also had a stronger correlation between their perceived strength of knowledge and their actual performance suggesting that this modality may have role in bridging the gap between perceived knowledge and actual knowledge . In a coHaving demonstrated effectiveness, the final hurdle for the wider use of such modalities is feasibility. 3DP models are expensive and time consuming to prepare . VR, depThis was a single center study with a modest number of participants. Results were self-reported and such perceptions in learning impact are by nature subjective. A comprehensive, objective post-test would more rigorously support improvements in learning and would be an important consideration for subsequent comparative studies. As the participants were primarily residents, these results are not necessarily generalizable to higher level trainees such as fellows. In addition, our group was primarily composed of medical residents; surgical residents may have derived a differential benefit from each representation. Again, a comparative study, perhaps with more complex lesions would be a promising future line of inquiry in this direction. With the global phrasing of our questions in the questionnaire, our conclusions were limited regarded perceived understanding of specific features of the tetralogy of Fallot heart for example. Along with including more complex lesions in the future, it will be important to ask more specific anatomic questions to assess understanding of pathology more precisely. Finally, it was challenging to fully account for the effect of prior “VR like”experience. While most participants endorsed minimal prior exposure to VR, VR like experiences in non-educational settings such as video games and mobile applications may have influenced their preference for this representation. Future studies can more specifically ask about these experiences as well as track if VR preference tracks along any demographic lines.Our study shows that, overall, VR was preferred over 3DP by pediatric residents and nurse practitioners for understanding cardiac anatomy and pathophysiology. Future comparative studies with objective assessments as well as explorations into questions of feasibility such as cost and portability will help to illuminate the full pedagogical value of these modalities.Additional file 1.


Chronic kidney disease (CKD) is a worldwide health problem that its incidence increases nowadays with the increase in the risk of environmental pollution. CKD can progress to end-stage renal disease (ESRD) which usually ends fatally. This study aimed to examine the therapeutic potential of Camel Wharton jelly-mesenchymal stem cells (CWJ-MSCs) in chronic kidney disease model induced in dogs.CWJ-MSCs were injected directed to the kidney with ultrasonographic guidance in dogs with 5/6 nephrectomy to evaluate its therapeutic potency in such cases. Analysis of variance was applied in normally distributed quantitative variables while a non-parametric Mann–Whitney test was used for non-normally distributed quantitative variables.The serum urea and creatinine in the treated group were significantly decreased transferring dogs in the treated group from stage 3 to stage 2 CKD according to the IRIS staging system. Histopathology of renal tissue revealed improving CKD lesions by increasing regeneration of degenerated tubules, maintaining the integrity of glomeruli. New vascularization with blood vessels remodeling were common findings. Periodic acid Schiff stain of renal tissue showed the integrity of renal tubules and thickness of the glomerular basement membrane. Fibrosis of cortex and medulla was lower in the treated group than in the CKD model as monitored by Mallory’s trichrome stain (MTC). NGAL and KIM-1 genes expression were decreased while VEGF and EGF genes expression were increased indicating renal tissue repair.CWJ-MSCs have a therapeutic potential in the CKD model induced in dogs. Chronic kidney disease (CKD) is an irreversible process with progressive loss of renal function . The kidIn preclinical studies, the 5/6 nephrectomy was considered a good model for chronic kidney diseases. It was applied successfully in rats , 15 and Wharton’s jelly mesenchymal stem cells (WJ-MSCs) are more primitive and efficient MSCs than those derived from adult fat or bone marrow , 19. NevA total number of (6) male Mongrel dogs  were purchased from a local breeder. They were housed in kennels of Surgery, Anesthesiology and Radiology Department, Faculty of Veterinary Medicine, Cairo University, and maintained at a constant temperature. Animals had free access to a standard diet and drinking water. The experimental animals were divided into 2 equal groups.Umbilical cord specimens were obtained fresh from a healthy pregnant camel and placed in sterile saline at 4 °C. The cord was washed several times with sterile saline and cut into 2–4 cm lengths. The cord was opened after stripping the vessels manually and the tissue was digested for 60 min in (3 mg/ml) collagenase II enzyme  at 37 °C. Tissue debris was removed from strained digested tissue and then centrifuged at 250 × g for 5 min at room temperature. A complete culture medium formed of Dulbecco's modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) was used for suspension of strained cells. Cells were incubated for 12–14 days at 37 °C in 5% humidified CO2 and were considered as passage 0 . IsolateEach experimental group was subjected to laboratory screening including a complete blood cell count (CBC), urinalysis (UA), and serum biochemistry, to evaluate the general health conditions and normal kidney function of the animals under the experiment and to detect subclinical disease such as hypoproteinemia and anemia that may affect the animal response to anesthesia . CompletG (1): CKD was induced in three dogs by excision of two poles (5/6 nephrectomy) through surgical removal of the upper and lower pole of the right kidney and contralateral nephrectomy after two weeks from the first surgical operation according to Ismail et al. .6 cells) directly in the renal cortex guided by ultrasonography. The time interval between the two injections was two weeks [G (2): CKD was also induced in the three dogs as in G1 and injected twice with undifferentiated CWJ-MSC at a dose . Ultrasound-guided renal injection of CWJ- MSCs were directly injected into one site of the renal cortex at 3rd week after the first surgery and the second dose after two weeks from the first dose . CWJ MSCBefore the first operation, blood samples were taken from each group to estimate the serum urea and creatinine level, it was considered the zero time. Every week blood samples were collected from the jugular vein till receiving the second dose of CWJ MSCs then the blood samples were taken every two weeks. Serum Urea and creatinine were measured using commercial kits  according to manufacture protocol.After 9 weeks from the first stem cells injection, dogs were humanely euthanized by intravenous injection of a fully saturated solution of potassium chloride, and 20% solution of pentobarbital sodium . Tissue Semiquantitative scoring of the severity of glomerular sclerosis (GS) and tubular injury (TI) was performed based on a previous scoring system . BrieflyImmunohistochemistry of vascular endothelial growth factor (VEGF) was performed on paraffin-embedded renal tissue using a primary antibody against VEGF , anti-species secondary antibody (biotinylated goat anti-rabbit), and an avidin–biotin-peroxidase complex kit according to manufacturer protocol  . Image aTotal RNA was extracted from homogenized tissues with Direct-zol RNA Miniprep Plus  and then quantified by Beckman dual spectrophotometer (USA).Reverse transcription of extracted RNA was performed followed by PCR using SuperScript IV One-Step realtime-PCR (RT-PCR) kit . The thermal profile employed in 48-well plate StepOne instrument  was as follows: 10 min at 45 ºC for RT, 2 min at 98 ºC for RT inactivation and initial denaturation by 40 cycles of 10 s at 98 ºC, 10 s at 55 ºC and 30 s at 72 ºC for the amplification step. The data were expressed in Cycle threshold (Ct) for the target genes and housekeeping gene after the RT-PCR run. Normalization for variation in the expression of each target gene; Neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule (KIM-1), and Epidermal growth factor receptor (EGFR) was carried out related to the mean critical threshold (CT) expression values of the GAPDH housekeeping gene by the ΔΔCt method. The relative quantitation (RQ) of each target gene is quantified following the calculation of the 2-∆∆Ct method. Primers sequence for NGAL gene was; forward 5′-GGTACGTCATCGGCATAGCA-3′ and reverse 5′-CCTGAGTAGGGTGGAGGTGA-3′, for KIM-1 gene, was; forward 5′‐ATGAATCAGATTCAAGTCTTC‐3′ and reverse 5′‐TCTGGTTTGTGAGTCCATGTG‐3′ for EGFR gene was; forward 5′‐GAAAGCTTGACCAAGCAAGCAC‐3′ and reverse 5′‐ACGGGACAGTACGTTAAGATGAACA‐3′, and GAPDH housekeeping gene was; forward 5'-TGTGTCCGTCGTGGATCTGA-3' and reverse 5'-TTGATGTTGAAGTCGCAGGAG -3'.P values less than 0.05 were considered statistically significant.Data were analyzed using the statistical package SPSS version 17. Data were presented as mean ± standard error. Analysis of variance (ANOVA) followed by post hoc tests was applied in normally distributed quantitative variables while non-parametric Kruskal–Wallis test and Mann–Whitney test were used for non-normally distributed quantitative variables . P valueCamel Wharton’s Jelly-derived MSCs were isolated, propagated in culture, and identified by their morphological fibroblast spindle-shaped cells Fig. a. AnalysIn G1, the serum urea level increased gradually till the end of the experimental period reaching (71 ± 3.78 mg/dL). In G2, there was a marked increase in serum urea level till the 3rd week (55 ± 2.88 mg/dL) before CMSCs injection. The serum urea level begins to decrease from the 4th week till the end of the experiment reaching (26.6 ± 1.76* mg/dL) and recording a significant decrease compared to the values in G1.  showed great improvement of glomerular lesions and tubular injury recorded in the CKD model (G1). The cortical and medullary fibrosis in G1 Fig. a, b becaThe number of glomeruli that showed lesions were less than that observed in G1. The thickening of the glomerular basement membrane, mesangial hyperplasia, and bowman’s space exudation was less observed in the glomeruli as demonstrated by the lesion score of glomerular lesions. The lesion score of cortical and medullary glomerular lesions and fibrosis were significantly lower than the CKD group but was still significantly higher than the normal group Fig. b. The stThe stained sections of the kidneys by PAS showed the improvement and the integrity of kidney structure in G2 compared to the other groups, as the brush border of convoluted tubules was well distinct Fig. a, b, c. VEGF expression was mainly observed in the renal tubular epithelium. The area percent of its expression was significantly low in G1 compared to the control group. On the other hand, the area percent of VEGF expression in G2 was significantly high compared to control and G1 Fig. .Fig. 8thThe continuously asked question is; Can stem cells, especially the mesenchymal type, regenerate kidney tissue? The answer originates from the fact that the nephrons have a mesenchymal origin, so, MSCs can repair the injury in renal tissue and differentiate into collecting ducts and nephrons . In the The dogs treated with CWJ MSCs showed improved kidney function tests after one week of stem cells injection and by the end of the experiment, they got classified as stage 2 of CKD according to the staging system developed by IRIS . A similThe improvement of renal lesions in the treated group was accompanied by a decrease in the expression of KIM-1 and NGAL genes compared to the CKD group indicating renal tissue repair as these genes expression increases with increased inflammation, renal fibrosis, and chronically damaged tubular cells . The renThe most important study limitation was the inability to collect urine from dogs for albumin analysis. This was mainly due to the difficulty of catheterization in dogs for a long time and the vulnerability to infection.In Conclusion: the transplantation of CWJ-MSCS in the CKD model induced in dogs can improve kidney function tests, have the power to regenerate damaged renal tubules, maintain the integrity of renal corpuscles, and decrease fibrosis.


Deviations from mirror symmetry in the development of bilateral organisms are common but the mechanisms of initial symmetry breaking are insufficiently understood. The actin cytoskeleton of individual cells self-organises in a chiral manner, but the molecular players involved remain essentially unidentified and the relationship between chirality of an individual cell and cell collectives is unclear. Here, we analysed self-organisation of the chiral actin cytoskeleton in individual cells on circular or elliptical patterns, and collective cell alignment in confined microcultures. Screening based on deep-learning analysis of actin patterns identified actin polymerisation regulators, depletion of which suppresses chirality (mDia1) or reverses chirality direction (profilin1 and CapZβ). The reversed chirality  is mDia1-independent but requires the function of actin-crosslinker α−actinin1. A robust correlation between the effects of a variety of actin assembly regulators on chirality of individual cells and cell collectives is revealed. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry. In this work, the authors identify regulators of actin filament assembly involved in chiral organisation of the actin cytoskeleton in single cells and chiral alignment of cells in groups. This provides insights into how actin-driven chirality underlies tissue and organ asymmetry. Several examples of left-right asymmetry emerging have been discovered. At the level of entire organisms, emergence of left-right asymmetry has been thoroughly described for visceral organs in vertebrates2 and formation of asymmetric body in snails3. The processes of asymmetric organogenesis include heart-looping in vertebrates4, chiral shaping of hindgut and genitalia6, and asymmetric tilting of wing bristles in Drosophila7. Cell groups confined to micropatterned adhesive substrates (in the form of stripes or rings) demonstrated chiral cell alignment and movement10. Finally, on the single cell level, the processes of intracellular swirling, cortical flow, and cell migration can demonstrate left-right asymmetry15.While many animals demonstrate approximate bilateral symmetry, many important features of the body layout such as position of visceral organs, as well as the shape of organs themselves are usually asymmetric. These asymmetries are tightly programmed and aberrations in such a program can lead to severe defects in embryonic development16, and in particular the chiral cytoskeletal fibres17. Indeed, several classes of cytoskeletal proteins were shown to be involved in the processes of left-right asymmetry development listed above. While the asymmetric positioning of visceral organs depends on numerous cilia-related proteins18 and attributed to cilia function in specialised cells located in the embryonic node (left-right organiser), actin cytoskeleton-related proteins are involved in other examples of asymmetry. In particular, non-conventional myosins 1d and 1c are needed for asymmetric hindgut and male genitalia development in Drosophila6, and myosin 1d is sufficient to induce chirality in other Drosophila organs19. We previously proposed the role of formin family proteins in the development of actin cytoskeleton chirality11 and several recent publications established the role of diaphanous formin in dextral snail chirality21, Daam formin in Drosophila hindgut and genitalia chirality22, and Caenorhabditis elegans CYK-1 formin in chiral cortical flow of C.elegans zygote23. Finally, some data indicated the involvement of the actin filament crosslinking protein α−actinin1 in emergence of chirality in cultured individual cells and multi-cell spheroids24. However, how intrinsic chirality of the actin filaments25 is translated in vivo into chirality of cells, and whether this is sufficient to explain the emerging of chirality in multicellular groups such as tissue and organs remains obscure.It is commonly believed that mechanisms underlying emergence of left-right asymmetry in diverse biological systems are based on the function of special chiral molecules19 (perhaps different proteins in different systems) or is mediated by coordinated activities of a group of proteins with complementary functions. Here, we addressed this question by systematic investigation of the involvement of major actin-associated proteins in the regulation of left-right asymmetry of the actin cytoskeleton in individual cells. In the course of this analysis, we examined the effects of depletion of chirality regulators found in previous studies, namely formins, myosin 1c and 1d, and α−actinin1. We show that, even in this simple system, the chiral swirling of actin depends on the functions of several groups of actin regulators. We reveal several types of such regulators: depletion of some of them reduced or abolished chirality, while depletion of others reversed the direction of chirality.In particular, it is unknown whether left-right asymmetry emerges as a result of the activity of a single chiral determinant such as myosin 1dThe discovery of numerous regulators of chiral morphogenesis in individual cells allowed us to perform detailed comparison between factors affecting individual and collective cell chirality. It was previously unclear whether chiral asymmetry of the actin cytoskeleton in individual cells is related to emergence of collective chirality in cell groups. Here, we found that the majority of treatments affecting the asymmetric self-organisation of the actin cytoskeleton in individual cells also affected the asymmetric alignment of cell groups. In particular, all factors that reversed direction of cytoskeletal chirality in individual cells also reversed the direction of chiral cell alignment in cell groups. Altogether our findings provide the background for the future understanding of the processes of emerging left-right asymmetry in tissues and organs.2 formed a chiral pattern of organisation of actin filament bundles. Radial actin fibres originating from focal adhesions at the cell edge eventually tilted to the right from the axis connecting the focal adhesion with the cell centre. This coincided with the development of chiral swirling – an anti-clockwise bias in centripetal movement of transverse fibres along the tilted radial actin fibres11 (Supplementary Movie We have previously shown that human fibroblasts (HFF) plated on fibronectin-coated circular islands with an area of 1800 μmWe evaluated the effects of the knockdowns of 10 of 15 formin family members expressed in fibroblasts on chiral organisation of radial fibres . At 48 h following plating, the cells approached confluency and aligned mainly along the diagonal of the rectangles as seen from the orientation of nuclei or local average cell orientation characterised by nematic directors on phase-contrast images26 Fig. . In comp26 Fig. . In cont26 Fig. . In otheWe found that knockdown of mDia1 converted the distribution of the angles characterising individual microcultures into bimodal distribution Fig. , meaning27. The suppression of the actin-nucleating Arp2/3 complex via the knockdown of its major component ARPC2 , effectively reversed the direction of chirality – inducing sinistral actin pattern  and sinistral (rare) coiling. Since formin mDia1 is shown to be required for dextral anti-clockwise chirality Figs. , we exam11    Fig. . Ranking11. During spreading on elliptical micropatterns, the growth of radial fibres and focal adhesions were stronger at the vertex regions characterised by higher curvature than at the sides of the ellipses  seem to be important players. This role of formin family proteins might be related to their rotation at the tip of actin filaments during polymerisation due to helical organization of actin filament37.In contrast to earlier studies that focused on a single gene as the main regulator of left-right asymmetry, our study revealed that multiple actin-associated proteins are involved in the control of chiral morphogenesis in individual cells and multicellular microcultures. Among the proteins involved in actin assembly, formins, Arp2/3 complex, cofilins, capping protein and profilin appeared to be potent regulators of left-right asymmetry development. Some of these identified actin regulators were also reported to influence actomyosin-powered cortical flowsitus inversus in vertebrates, reversed chirality of hindgut in flies, and sinistral chirality in snails3), the reversion of chirality in isolated individual cells was not sufficiently explored. In our studies, we found that the actin cytoskeleton of individual cells can also demonstrate a pattern of organisation that looks like a mirrored reflection of the normal chiral pattern. The most striking examples are knockdowns of profilin 1 (but not profilin 2) and CapZβ subunit of capping protein CapZ, which both led to negative average tilting of the radial fibres in individual cells and clockwise (rather than anti-clockwise) cytoskeleton swirling. Another group of treatments that efficiently reversed chirality direction was treatment with low concentrations of actin polymerisation inhibitors, latrunculin A and swinholide A.A remarkable type of response observed in our study was switching from dextral to sinistral chirality. While mutations reversing the direction of chirality at the organismal level have often been observed (11 and present study), prevented the reversal of direction of individual cells chirality. α−Actinin1 is a major crosslinking protein in radial fibres and its function in chirality determination may depend on its possible role in restricting individual filament rotation and regulation of radial fibre twisting. In our previous paper, we speculated that the torque induced by formin-driven rotation of trapped filament can occasionally be released (when elastic energy of the system approaches some threshold), leading to the rotation of filament in the opposite direction38. Such consideration could explain the reversal of cell chirality in α−actinin1 overexpressing cells11. In recently published papers40, this model was elaborated and applied to the explanation of the phenomenon of chirality reversion. However, which factor induces filament rotation in cells with sinistral chirality in the absence of mDia1 remains to be elucidated.Surprisingly, mDia1, the formin critical for the dextral cell chirality appeared to be dispensable for sinistral chirality induced by the aforementioned treatments. At the same time, knockdown of α−actinin1, which did not interfere with normal dextral chirality in individual cells , then the clockwise-rotation of the bundles  results in anti-clockwise swirling in the cell. However, if the circumferential structures are ‘below’ the radial actin bundles, then the clockwise-rotation of the bundles should produce clockwise swirling. It is possible that some perturbations of the actin dynamics and/or crosslinking could change the mutual position of radial and transverse fibres, thereby changing the direction of cell chirality. Future structural information is needed to evaluate this hypothesis.Another model connects the direction of cell chirality with the structural organisation of the interactions between radial and transverse actin fibres. We posited previously that the initial breaking of the left-right symmetry starts when the radial actin bundle rotating unidirectionally around its long axis begins to ‘roll’ on circumferential transverse actomyosin structures in a ‘rack-and-pinion’ mechanism10. Our study revealed a remarkable correlation between responses of individual cells and cell collectives in microcultures. With only few exceptions, the treatments which affected formation of asymmetric actin pattern in individual cells also affected asymmetric alignment of cell groups. Treatments that reversed actin chirality direction in individual cells always resulted in a change of the direction of average cell alignment in cell groups. These data, in line with42, provide strong experimental support to the hypothesis that the development of the chiral organisation in multicellular cultures, tissues, and organs is determined by the chirality of the actin cytoskeleton in the individual cell.Our experimental systems allowed us to perform systematic quantitative comparison between effects of diverse genetic and pharmacological treatments on development of left-right asymmetry in individual cells and multicellular microcultures confined to rectangular patterns. Asymmetric alignment of cells in our microcultures resembles chiral behaviour seen in cells confined to stripes or ring-shaped patterns43. We postulate that cells could preserve intrinsic actin cytoskeletal chirality when aligned with the boundaries of the adhesive pattern, in such a way that aligned cell migrates preferentially anti-clockwise around the pattern’s boundary. In this scenario, individual cell anti-clockwise bias in collectives might causes an anticlockwise biased streaming of cells around the boundary of the rectangular micropattern because of the combination of contact inhibition of locomotion, lateral interactions and leader-follower behaviours ultimately aligning cells’ polarisation axes with each other to collectively evolve into the И-orientation. This biased streaming of cells along the boundaries was also observed in other independent studies on collective cell chirality on stripes and rings patterns10. The future goal is to elucidate how chiral actin organisation drives chiral cell movement along the boundary of adhesive islands.Our data on chiral deviation of the average direction of stress fibres from the long axis of elongated cells confined to an elliptical pattern could shed some light on the relationship between individual and collective cell chirality. Previous study suggests that the orientation of an individual cell starts with orientation of the systems of focal adhesions and stress fibres, followed by orientation of the cell as a whole21 and in Drosophila22, and probably for asymmetric heart looping in vertebrate development4. The formation of asymmetry of heart and visceral organs positioning in vertebrate deserves special discussion. It is well-established that in many species the key asymmetric factor triggering the signalling cascade determining left-right body asymmetry is an asymmetric flow generated by ciliated cells in the node (left-right organiser of the embryo)2. Recent studies have shown, however, that in birds and reptilia the nodal asymmetry does not depend on cilia1. Thus, the cilia may function as an amplifier, but not the primary source of asymmetry. Since the position and orientation of basal bodies can in principle be regulated by the actin cytoskeleton44, this suggests that the primary asymmetric factor in nodal cilia-dependent systems could still be the intrinsic asymmetry of the actin cytoskeleton. Extensive future studies are necessary to explore this possibility. In conclusion, our study revealed an actin polymerisation-dependent mechanism of establishment of left-right asymmetry in individual cells and cell groups which could be involved in the development of left-right asymmetry in organs and organisms.The hypothesis of the role of asymmetric self-organisation of the actin cytoskeleton in the emergence of chirality in tissues, organs, and even in whole organism could unify the existing data on the development of left-right asymmetry in snails2 at 37 °C. Enucleated cells were generated as described in our earlier work in ref. 12. Briefly, non-transfected or LifeAct-transfected cells were seeded onto plasma-treated plastic coverslips (ibidi GmbH) and left overnight to ~70% confluency. Next, cells were treated with 5 ml of 10 μg ml−1 cytochalasin B (Sigma) in a 50 ml falcon tube and centrifuged at 10,864 g (Beckman centrifuge X30R) for 1 h at 37 °C to enucleate cells. Cells were washed three times with complete medium and allowed to recover for at least 2 h in complete medium following which cells were trypsinised for seeding onto the micropatterned substrate for the experiment. Presence of nuclei were verified by labelling with Hoechst 33342 (10 μg ml−1 for 10 min) and live imaging of the nucleus. Cells were transfected with DNA plasmids via electroporation  following manufacturer’s instructions. Electroporation condition consists of two pulses of 1150 V for 30 milliseconds. Expression vectors encoding the following fluorescent fusion proteins were used: LifeAct-GFP11, mRuby-LifeAct12, mEmerald-mDia1-C-14 (Addgene plasmid # 54156), pEGFP-C1 (Clontech), GFP-α−actinin111, GFP-α−actinin4 , GFP-ABDdel-α−actinin111 , EGFP-Filamin A , mCherry-Cofilin 1 , mCherry-Profilin1 (Addgene plasmid #55121), Pfn1-P2A-eGFP  and GFP-VASP11. Fluorescence-activated cell sorting (FACS) were performed to select for upper 30% of cells expressing high level of fluorescent fusion proteins of pEGFP-C1, GFP-α−actinin1, GFP-α−actinin4 or GFP-ABDdel-α−actinin1. All cell culture and transfection reagents were obtained from Invitrogen. Other chemicals and reagents were obtained from Sigma, unless otherwise stated.Human foreskin fibroblasts (HFF) from American Type Culture Collection  were cultured in Dulbecco’s modified Eagle’s medium high glucose supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate and antibiotics (penicillin and streptomycin) at 5% COCells were seeded into a 35 mm dish on day 0 and transfected with 100 μM of siRNA using Lipofectamine RNAiMAX on days 1 and 2. For experiment involving individual cells, siRNA-transfected cells were trypsinised on day 4 and replated onto circular micropatterns. For experiment involving cell microcultures, siRNA-transfected cells were trypsinised on day 3 and replated onto rectangular micropatterns. As needed, transfection of plasmids via electroporation into siRNA-treated cells were performed on day 3 and cells were replated on day 4. siRNA transfected cells had their proteins or RNAs extracted on day 4 for immunoblotting or RNA sequencing respectively. siRNAs used in this study are listed in Supplementary Table 2 circular or elliptical micropatterns of different aspect ratio , or 300 × 600 µm rectangles (multicellular microculture experiment). Each micropatterned substrate was fabricated by stencil patterning as previously described in our earlier work12. Briefly, polydimethylsiloxane (PDMS)  was cast on the photoresist mould, containing micropattern designs of interest, using a 10:1 ratio (w/w) of elastomer to crosslinker and cured for 2 h at 80 °C. The crosslinked PDMS layer was peeled off and stamps were cut out manually. The PDMS stamp was then inverted and placed onto a hydrophobic uncoated 35 mm μ-dish (ibidi GmbH). Norland Optical Adhesive 73  was deposited along an edge of the stamp and allowed to flow through the gaps between the PDMS stamp and dish by capillary action, upon which the stamp was sealed on all sides using NOA-73. The NOA-73 stencil was cured under ultraviolet illumination for 15 s. After peeling off the PDMS stamp, the stencil and dish were incubated with fibronectin  at a concentration of 50 μg ml−1 in 1 × PBS at 4 °C overnight after a brief degassing at 10 mbar. At the end of the incubation, the fibronectin solution was aspirated, and the stencil was removed. The printed dish bottom was passivated with 0.2% Pluronic acid-H2O for 10 min. Finally, the passivated dishes were washed thrice with 1 × PBS before cell seeding.Cells were seeded on substrates containing either 1,800 µm4 cells ml−1 for 10 min. The medium containing unattached cells was then replaced with fresh DMEM. After 6 h incubation, the cells were fixed using 4% paraformaldehyde  in PBS for 10 min, followed by three 1 × PBS washes. Cells were permeabilised using 0.1% Triton-X-100 in PBS, and then blocked with 2% bovine serum albumin (BSA)-PBS for 1 h at room temperature (RT) before incubation with appropriate labelling reagents. Actin and nucleus staining were performed using phalloidin (Molecular Probes) and Hoechst 33342 (Invitrogen), respectively. For live cell imaging experiment, cells were seeded on circular micropatterns at a density of 5 × 104 cells ml−1 for 10 min. The medium containing unattached cells was then replaced with Leibovitz’s L-15 containing 10% FBS. Cells were left for at least 2 h before imaging at 37 °C with 5% CO2. Time-lapse images for 12–16 h at 10–20 min intervals and Z-stacks of step-size 0.35 μm with total height of 10–15 µm were acquired with a spinning disc confocal microscope (PerkinElmer Ultraview VoX) attached to an Olympus IX81 inverted microscope, equipped with a 100× oil immersion objective , an EMCCD camera  for image acquisition, and Volocity software (PerkinElmer) to control the set-up. Fixed samples were also imaged with the same step-up. Maximum projection of the Z-stack images was performed with Volocity software or with Fiji software and exported as 16-bit TIFF files (512 × 512 pixel and 0.138502 μm pixel−1). Each image contained a single cell and these images were subsequently used for deep learning-based identification of radial fibres.Cells were seeded on printed dishes containing circular micropatterns at a density of 5 × 104 cells ml−1 for 10 min. The medium containing unattached cells was then replaced with fresh DMEM. After 6 h incubation, the cells were fixed using 4% paraformaldehyde  in PBS for 10 min, followed by three 1×PBS washes. Cells were permeabilised using 0.5% Triton-X-100 in PBS, and then blocked with 5% bovine serum albumin (BSA)-PBS for 1 h at RT before overnight incubation at 4 °C with appropriate primary antibodies. Cells were then incubated for 45 min at RT with appropriate labelling reagents: AlexaFluor-conjugated secondary antibodies  and Phalloidin (Molecular Probes). For live cell imaging experiment, cell seeding was as above except that the medium containing unattached cells was then replaced with Leibovitz’s L-15 containing 10% FBS. Time-lapse images at 3 min intervals and Z-stacks of step-size 0.3 μm with a total height of 11 µm were acquired using a 60× oil immersion objective  with the spinning disc confocal microscope (PerkinElmer Ultraview VoX). Fixed samples were also imaged with the above step-up using a 100× oil immersion objective , or on the spinning disc confocal microscopy coupled with the live super-resolution (SR) module (Roper Scientific) attached to a Nikon Eclipse Ti-E inverted microscope with Perfect Focus System, equipped with 100× oil immersion objective , a sCMOS camera (Photometrics Prime 95B) for image acquisition, and MetaMorph software (Molecular Devices) to control the set-up. Maximum projection of the Z-stack images was performed with Volocity software or with Fiji software and exported as 16-bit TIFF files. Angle (°) of stress fibre tilt was measured using images of actin cytoskeleton in cells on elliptical micropattern labelled either by phalloidin or LifeAct with the OrientationJ plugin in Fiji software. As needed, image was rotated using transform tool with bicubic interpolation to align the long axis of the elliptical cell to the vertical orientation. An elliptical mask was then applied to the image and the angle of stress fibre tilt of each elliptical cell, single or in a time-lapse series, was measured using the OrientationJ Measure plugin.Cells were seeded on printed dishes containing elliptical micropatterns with an aspect ratio of 1:2 (34:68 μm) or 1:3 (27.5:84 μm) at a density of 5 × 105 cells ml−1 for 20 min. The medium containing unattached cells was then replaced with fresh DMEM and cell microcultures were incubated for a total of 48 h before cell fixation using 4% paraformaldehyde-PBS for 10 min. Just prior to fixation, cell nuclei were stained with 1 µg ml−1 Hoechst 33342 for 10 min. Phase-contrast and fluorescence images of cell microcultures were taken using a 20× air objective  on an Olympus IX81 inverted microscope, equipped with Andor Neo 5.5 sCMOS camera and light source (Lumencor SOLA SE Light Engine). Single plane images of phase contrast and DAPI channels were taken. Each image contained a single rectangular cell microculture and these images were subsequently used for measurement of average nematic directors angle and nuclei orientation angles in rectangular microcultures.Cells were seeded on rectangular micropatterns at a density of 1 × 10For drug inhibition studies, 10 min following cell seeding on micropatterns, the medium containing unattached cells was replaced with fresh medium containing either 20 nM latrunculin A  or 5 nM swinholide A (SCB Inc.). For experiments that lasted more than 24 h, fresh drugs were added every 24 h until the end of the observation period. All inhibitors remained in the medium during the entire period of observation, except in drug washout experiments.45, implemented in Python, was trained to identify actin radial fibres in cells confined on circular micropattern. Briefly, the following steps were taken. The model was trained using 32 images of actin cytoskeleton labelled by fluorescent protein tagged-LifeAct. These training images comprise of cells with their actin cytoskeleton in a radial or chiral organisation, and images of varied intensities were selected. Data augmentation was done using the Albumentations library46. The ground-truths  were prepared by manual demarcation of actin radial fibres in Fiji. The code and complete list of parameters of the trained deep learning model is available via https://github.com/gohweijia/Cell-Chirality-Analysis. This trained model was used to identify radial fibres in both phalloidin- and LifeAct-labelled cells, returning a 32-bit image of identified radial fibres. Segmentation of these identified radial fibres was performed using a custom MATLAB script, in which background subtraction  and then Niblack local thresholding47, with window size of 15 × 15, k = −0.3 and offset = −0.01, were applied. The resulting binary image was then skeletonised using the MATLAB built-in function, bwmorph. Intersecting radial fibres were separated by branch point removal, and fibre segments with similar orientation (angle difference ≤ 30°) and at nearby position (distance ≤ 30 pixels) were connected as a single fibre.Images of the actin cytoskeleton were first converted to 8-bit and the ‘Enhance brightness/contrast’ function in Fiji software was used with the ‘saturated pixels’ parameter set to the default of 0.35. A Unet-ResNet50 deep learning model2) was estimated to be 63.353 µm2 using a dataset of ~100 cells. This constant was subtracted from the cell area before the computation of cell radius R. Only cells with area between 1700 and 2000 µm2 were analysed.The following procedures were performed using a custom MATLAB script unless otherwise stated. Cell segmentation was performed by thresholding the Gaussian-smoothed  actin image using Otsu binarization , followed by a series of mathematical morphological operations . Cell centroid and cell spread area were calculated based on this cell mask. The cell mask was also used for generating concentric ring masks of 4 µm in width starting from the cell edge, with 2 µm increments, for masking the segmented radial fibres. In each ring, the angle of each radial fibre segment was measured relative to the cell edge. See also Supplementary Fig. 1c. The angle at the cell edge The following procedures were performed using a custom MATLAB script unless otherwise stated. First, identification and segmentation of individual rectangular microculture using phase contrast images was done by performing a Wiener filter with a neighbourhood size of 20 × 20 pixels to remove image noise. This was followed by an entropy filter with a 3 × 3 pixels structural element and morphological opening with a 9 × 9 pixels structural element. This results in an image that differentiates between areas with and without cells. Otsu binarization was then performed to segment the image, the segmented area at the centre of the image was selected as the segmentation mask. This serves as an indicator of the area covered with cells. The bounding box enclosing this segmented area serves to represent the dimensions of the microculture. Only microcultures with bounding box width of 225 to 375 µm and height of more than 550 µm, and with a segmentation mask that covered more than 80% of the bounding box area were analysed. For each bounding box, the centre 200 × 500 µm region of interest was used for subsequent steps in the measurements of average nuclei orientation and average nematic director orientation.49. A Wiener filter using a neighbourhood size of 9 × 9 pixels was performed prior to segmentation. The concaved-point based splitting algorithm50 was used to separate any overlapping nuclei. Segmented objects above the size of 10000 pixels were removed as these corresponded to the background regions, while objects smaller than 500 pixels were also removed as these were either fragmented nuclei or noise regions that were segmented by chance. In addition, only nucleus that had a centroid position that laid within the bounding box of the segmented phase contrast image was selected for further analysis. The number of nuclei in the bounding box was also counted. Microcultures that had less than 50 nuclei were removed as these microcultures often had too few cells to cover the entire rectangular micropattern. The orientation of these nuclei was then calculated based on the angle of the long axis of a fitted ellipse with respect to the long axis of the rectangular micropattern. Alignment of the cell group was determined based on the mean resultant length51 of the nuclei orientation. A cutoff value of 0.35 was selected and any rectangle with a mean resultant length greater than that was classified as aligned. The mean nuclei orientation per aligned microculture was determined by calculating the mean of all the orientations of the individual nuclei within a single microculture.Segmentation of the nuclei was achieved using NICK adaptive binarization26. Briefly the orientation tensor was obtained using OrientationJ implemented in Fiji and the nematic director was obtained using a window size of 60 × 60 µm2 and 70% overlap. The orientation of each directors was measured as the angle relative to the long axis of the rectangular micropattern. The orientation of the directors within the region of interest was then used to determine the alignment of the microculture in a similar manner as that for the nuclei orientation. A higher cutoff value of 0.5 for alignment was set due to more coherent nature of the nematic directors. The mean nematic director angle per aligned microculture was determined by calculating the mean of all the orientations of the directors within a single microculture.Local cell orientation in the phase contrast image was calculated by obtaining the nematic director field as described in ref. −1 protease inhibitors cocktail , and were then mechanically lysed by syringing through a 27.25 G needle on ice. Protein concentration was quantified using the Micro BCA Protein Assay Kit (Thermo Scientific) according to manufacturer’s instructions. 20 µg of protein lysate was dissolved in 1×Laemmli sample buffer supplemented with 10% 2-mercaptoethanol, and separated by 4–20% SDS-polyacrylamide gel (GenScript USA Inc) electrophoresis at 100 V for 1 h and then transferred to a 0.4 μm pore size PVDF membrane  at 100 V for 2 h for formin proteins and 1 h for other proteins in an ice bath. The PVDF membrane was blocked using Intercept® (TBS) Blocking Buffer  or 5% nonfat milk (Bio-Rad) in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h at RT before incubation at 4 °C overnight with appropriate primary antibodies. Primary antibodies were diluted in Intercept® (TBS) Blocking Buffer containing 0.1% Tween-20 at their respective concentrations summarised in Supplementary Table Cell pellets were collected in RIPA buffer (SCB Inc.) supplemented with 2 μL mlRNAs were extracted using RNeasy® Plus Universal Kits (Qiagen) according to manufacturer’s instructions. Library was prepared using TruSeq Stranded mRNA LT Sample Prep Kit and sequenced using NovaSeq6000 Illumina platform. Alignment was performed  and trimmed reads were mapped to GRCh38 reference genome (BioProject: PRJNA312570) with HISAT2, splice-aware aligner. Gene expression was presented using transcript per million (TPM) reads.P < 0.05. Mann-Whitney test was two-tailed. Kruskal-Wallis test was implemented with Dunn’s multiple comparisons test and report multiplicity adjusted P value for each comparison. Wilcoxon signed-rank test was implemented against a hypothetical value of zero. For each histogram, we compared the fit of Gaussian versus Sum of two Gaussians with default software recommended parameters, including least squares regression, asymmetrical confidence intervals (CI) using 95% confidence level and plot 95% confidence bands. There was neither special handling of outliers nor weighting. Bimodal distribution is defined by the following parameters: preferred fit by the Sum of two Gaussians and represented by 1 negative (Mean1) and 1 positive (Mean2) means demonstrating chiral sign in opposite directions. Reliability of bimodality is checked by plotting the fitting curve with 95% confidence bands over the histogram  of individual cells and microcultures analysed for all of the quantitative data are specified in the figure legends and summarised in Supplementary Table Further information on research design is available in the Supplementary InformationPeer Review FileDescription for Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Reporting Summary