An analytical model is presented that allows predicting the progress and the final depth obtained by laser micromachining of grooves in metals with ultrashort laser pulses. The model assumes that micromachined grooves feature a V-shaped geometry and that the fluence absorbed along the walls is distributed with a linear increase from the edge to the tip of the groove. The depth progress of the processed groove is recursively calculated based on the depth increments induced by successive scans of the laser beam along the groove. The experimental validation confirms the model and its assumptions for micromachining of grooves in a Ti-alloy with femtosecond pulses and different pulse energies, repetition rates, scanning speeds and number of scans.  Laser micromachining of grooves with ultrashort laser pulses is a versatile process that can be applied for various applications, such as cutting through thin metal foils , dicing The laser micromachined grooves with depths of a few tens up to several hundreds of micrometers typically feature a V-shaped geometry in metals ,8, semicThe in situ measurement of the current depth during processing has already been demonstrated using optical coherence tomography for percussion drilling of microholes ,17 and fA simplified analytical model for the prediction of the depth progress in laser machining of V-shaped grooves with ultrashort laser pulses is therefore introduced in the following section. The model was experimentally verified for the case of laser micromachining of grooves with a depth of up to y–z-plane perpendicular to the scanning direction x. An analytical, recursive model for the calculation of the depth progress of laser-processed V-shaped grooves in metals can be derived in analogy to the model for percussion drilling of conical microholes as presented in . Figure z-direction). At the surface of the workpiece, the transversal distribution of the incident fluence (red curve) is given byCx − x and y − yC are the distances from the centre of the laser beam located at , w0 is the beam radius and ϕ0 denotes the peak fluence, which is given byEP is the pulse energy. At normal incidence, material removal by ablation on the surface occurs when the locally absorbed fluence A is the material-specific absorptivity at the wavelength of the incident radiation, exceeds the valuelE denotes the effective penetration depth of the absorbed energy density and hV denotes the volume-specific enthalpy required for heating and complete vaporization of the material. The effective penetration depth lE is dominated either by the optical penetration depth or by the electron heat diffusion length, depending on the peak fluence of the incident radiation [lE and thus ϕth decrease with increasing number of pulses applied to the surface [lE and thus the ablation threshold ϕth are assumed to be constant over the entire process for the presented analytical model. The error caused by this simplification during the first 100 pulses is negligible as typically, more than several thousands of pulses are applied to each location for the production of laser machined grooves.The pulses of a Gaussian laser beam are irradiated onto the metal sample (the grey hatched cross section) at normal incidence dG=2⋅znG, after n ∈ 1,2,…N scans can be calculated byzn − 1G, denotes the groove depth after n − 1 scans and znS, denotes the depth ablated by the nth scan ηA(dGgiven by (9)NR absorbed from one single pulse of a Gaussian laser beam during the nth scan along the groove at the location x in a stripe with the width dx inside the groove between the edges at y = ±dG/2 amounts tox-axis at the time at which the jth pulse hits the workpiece and where j ∈ ℤ and yC was set to zero for the beam, which is centred on the groove. The energy Aη only defines the amount of energy dEn,jA,(x) absorbed in the groove but does not specify the transversal distribution of the fluence in the y–z-plane  and end with ϕn,jtip,(x) at the tip of the groove (y = yC = 0). With this assumption, the energy dEn,jA,(x) absorbed at the location x from a single pulse j in a stripe of width dx amounts toy–z-plane  and replacing the integral with ϕn,jtip,(x) with a spatial offset of xδ each absorbed at the tip of the groove around an arbitrary point x0. For the sake of clarity, the figure is divided into two parts showing the pulses with j ≤ 0 in j ≥ 0 in x0 at the moment when the 0th pulse hits the workpiece . Considering this diagram, it becomes evident that from the perspective of a point  located at x0 somewhere along the centre line of the groove, the individual pulses of a scan can only contribute to the ablation of the groove at this point x0 as long as the fluence absorbed at the tip ϕn,jtip,(x0) > ϕth exceeds the ablation threshold ϕth. The fluence that is absorbed at the tip of the groove from each of the pulses j of one scan  at the location x = x0 is given by the intersection of the fluence distribution ϕn,jtip,(x) with the ordinate at x = x0, as indicated by the colored small arrows in j = −2 to j = 2 contribute to material ablation at , as only their fluences ϕn,jtip,(x0) exceed the ablation threshold ϕth, whose value is indicated by the black dotted line. The intersection ϕn,jtip,(x) with the ablation threshold ϕth determines the maximum number of pulses jnabl, contributing to ablation in this direction. Using Equation (12) and solving for jnabl, yieldsIn the example depicted in nth scan znS, as seen by the spot x = x0 located on the centre line of the groove corresponds to the accumulated depth ablated by the pulses zn,jP,(x) denotes the depth ablated by the pulse j during the nth scan. According to the logarithmic ablation law [As a result, the depth ablated by the tion law ,26, the Aη  as given by Equations (8) and (9) is assumed during one scan over the groove. This induces a negligible error since Aη changes very slowly with an increasing number n of scans as long as znS, << dG, which is typically the case in micromachining processes with a reasonable pulse overlap Ωx in the range of 30–95%. In the present model, a constant absorptance znG, can be recursively calculated as a function of the number n of scans. A useful way to proceed is by starting with the calculation of the constant parameters that are not affected by the recursive calculation, such as the spatial offset xδ between the impact locations of two consecutive pulses using Equation (6). Furthermore, the peak fluence ϕ0 and ablation threshold ϕth can be calculated with Equations (2) and (3), respectively, in order to determine the width of the groove dG using Equation (4). With the first scan (n = 1) at the beginning of the recursive calculation, a very small value should be chosen for the initial groove depth, e.g., zG,0 = 1 nm , so as not to divide by 0 in the subsequent calculation of the absorptance Aη  in Equations (8) and (9). Then, the maximum number of pulses jabl,1 contributing to ablation in each direction is calculated using Equation (13), followed by the calculation of the fluence ϕjtip,1,(x) deposited at the tip of the groove with Equation (12) for each contributing pulse j during this first scan. The depth increment zjP,1,(x) ablated by each pulse j is calculated using Equation (15) and accumulated according to Equation (14). Then, the accumulated depth of the first scan zS,1 is added to the initial groove depth zG,0 as given by Equation (7). The calculation of the absorptance Aη  of the groove with increased depth zG,1 starts the second loop of the recursive calculation. This procedure must be repeated n times to receive the groove depth znG, after micromachining with n scans.With the above equations, the progress of the increasing groove depth ϕn,jtip,(x) at the tip of the groove decreases with increasing groove depth due to the increasing length of the sidewalls Aη  due to the increasing number NR of reflections within the groove (cf. Equations (8) and (9)). The maximum attainable groove depth z,∞G obtained after n → ∞ scans is reached when the fluence ϕn,jtip,(x = xC) at the tip of the V-shaped groove converges to the value of the ablation threshold ϕth. The maximum groove depth z,∞G can therefore be found with Equation (12) by setting ϕn,jtip,(x = xC) = ϕth, and solving for z,∞G, which yieldsη∞ = Aη  denotes the absorptance of a groove micromachined with ∞−1 scans. As the absorptance, in turn, depends on the groove depth zn−1G, (cf. Equations (8) and (9)), the maximum groove depth z,∞G cannot be calculated directly but has to be found by a recursive calculation using Equation (7). Assuming a high aspect ratio zG,∞/dG of the final groove, the absorptance can, however, be approximated to be η∞ ≈ 1, and the maximum achievable groove depth zG,∞ obtained with a given parameter set can directly be estimated using Equation (16) by setting ∞η = 1. Equation (16) also shows that the maximum achievable groove depth does neither depend on the repetition rate frep nor on the scanning parameters such as the scanning speed xv and that—for a given beam radius w0 and with the material-specific value of ϕth—it can only by increased by increasing the pulse energy EP.The absorbed fluence EP, the repetition rate frep, the radius w0 of the laser beam, the scanning speed xv, and the number of scans n, as well as the three material parameters, absorptivity A, energy penetration depth lE, and the enthalpy hV for heating and complete vaporization of the material.It is noted that the model for the calculation of the depth progress presented in this section only requires five generally known laser and scanning parameters, namely the pulse energy Pharos from Light Conversion with a wavelength of 1030 nm was used for micromachining. The laser emitted pulses with a pulse duration of 260 fs. The circularly polarized laser beam with a Gaussian intensity distribution had a beam propagation factor of M² < 1.3. The beam was scanned over the surface of the samples by means of a Galvanometer-Scanner  and was focused by an F-Theta lens  with a focal length of 340 mm, resulting in a focal radius of w0 = 55 ± 5 µm. The focus position was always set on the surface of the samples. Grooves with a length of 10–35 mm were micromachined in the Ti-samples with different pulse energies EP, repetition rates frep, scanning speeds xv and number of scans n, as summarized in xδ of the impact locations of two consecutive pulses and the corresponding pulse overlap Ωx were calculated according to Equation (6) and Equation (5), respectively.The model for the prediction of the depth and width of laser micromachined grooves derived in the previous section was compared to experimental results obtained by micromachining samples with a size of 50 × 50 mm² and a thickness of 1 mm made of Ti6Al4V (ASTM Grade 5), a Ti-alloy often used in the aerospace industry and for biomechanical applications due to the high specific strength, corrosion resistance and biocompatibility. The ultrafast laser system y–z-plane), and cross sections were prepared by grinding and polishing in order to investigate the shape of the grooves and measure their depth and width using an optical microscope . After micromachining, the samples were cut perpendicular to the grooves ). The fitted value of lE = 30 nm is consistent with experimentally determined values of the optical penetration depth of 26 nm for Ti6Al4V [The material parameters for titanium published in ,29 were A = 0.51 . The eff Ti6Al4V  and 30 n Ti6Al4V .dG = 126 µm and dG = 100 µm, respectively. The experimentally determined widths of The groove widths are given by Equation (4) for the peak fluences of 3.81 J/cm² (P1) and 1.45 J/cm² (P2) yield zG,n as a function of the number n of scans was recursively calculated as described above. The calculations are compared to the experimental results in The progress of the groove depth zG/dG ≈ 1.5, the measured groove depth increases almost linearly with the number of scans. The progress of the depth is found to slow down for aspect ratios beyond zG/dG > 1.5. At constant repetition rate frep and scanning speed xv, higher depth progress and deeper grooves were achieved for higher pulse energies (cf. P1 and P2). For constant pulse energy EP and constant pulse overlap Ωx, the groove depth as a function of number of scans is similar (cf. P2 and P3). However, the net processing time is divided in half for P3 in comparison to P2 due to double the scanning speed vx at a twofold repetition rate frep. At constant pulse energy EP and constant repetition rate frep, higher depth progress is achieved with lower scanning speeds xv . The relations observed in this work regarding the depth progress in micromachining of grooves in Ti6Al4V confirm the observations made for semiconductors in [EP = 181 µJ and the highest number of scans n = 10,000 for this parameter combination (P1). The maximum measured groove depth for a constant pulse energy EP = 69 µJ and different scanning parameters (from P2 to P5) is in the range of Up to an aspect ratio (depth/width) of ctors in  and for ctors in : The groEP = 69 µJ, but micromachined with different scanning parameters (from P2 to P5) converge to the same maximum groove depth, which agrees well with the theoretical prediction of the model of 326 µm calculated by Equation (16) for ∞η = 1. Deviations from calculation and measurement might result from uncertainties regarding the material parameters used for the calculation, in particular the fitted value for the effective penetration depth lE, or from deviations of the assumed ideal V-shape, as shown before in n = 10,000, with the bending of the tip. Complete vaporization is assumed in the proposed analytical model, whereas additional effects such as melting and spallation can cause a deviating process enthalpy and thus a different ablation rate [The groove depths as calculated by the model  and as measured by the cross sections with the optical microscope  are in very good agreement for the different parameter combinations and for the different number of scans. The depth progress predicted by the model decreases with increasing number of scans and stagnates when the fluence in the tip converges the ablation threshold, which corresponds well with the results from . As a rezG and groove width dG, as calculated by the model derived in the previous section.Nevertheless, for a broad range of laser and scanning parameters, the V-shape and calculated groove dimensions by the model correspond to the shape and groove dimensions as measured by the cross sections shown in EP and frep, the scanning speed xv and beam radius w0, and the three material parameters A, lE and hV, the model allows for the prediction of the groove dimensions as a function of the number of scans n and maximum achievable groove depth.Knowing the laser parameters An analytical model for the prediction of the depth and width of V-shaped grooves in metals micromachined with ultrashort laser pulses was derived. The model predicts the progress of the micromachining depth for V-shaped grooves in a Ti-alloy with ultrashort laser pulses as a function of laser parameters, scanning parameters and material parameters. The corresponding assumptions for the model were experimentally validated for different pulse energies, repetition rates, scanning speeds and number of scans using cross sections and optical microscopy. The analytical model derived in our paper provides a useful tool for the estimation of the groove dimensions, process windows of micromachining with high depth progress and the maximum achievable groove depth.


The use and interpretation of diagnostic cerebrospinal fluid (CSF) biomarkers for neurodegenerative disorders, such as Dementia with Lewy bodies (DLB), represent a clinical challenge. According to the literature, the composition of CSF in DLB patients varies. Some patients present with reduced levels of amyloid, others with full Alzheimer Disease CSF profile (both reduced amyloid and increased phospho-tau) and some with a normal profile. Some patients may present with abnormal levels of a-synuclein. Continuous efforts will be required to establish useful CSF biomarkers for the early diagnosis of DLB. Given the heterogeneity of methods and results between studies, further validation is fundamental before conclusions can be drawn. Dementia with Lewy bodies (DLB) is the second most common cause of neurodegenerative dementia in the elderly, following Alzheimer’s disease, and it is characterized by a progressive cognitive deterioration, accompanied by various combinations of clinical features, including parkinsonism, visual hallucinations, fluctuations  psychiatric/behavioral features, REM sleep behavior disorder and autonomic dysfunction . Further42), a marker of amyloid pathology (plaque formation), total tau protein (τT), a non-specific marker of neurodegeneration and neuronal/axonal loss and phosphorylated tau protein at threonine 181 (τP-181), a marker of tau pathology (neurofibrillary tangle formation) have been proven useful in the  diagnosis of Alzheimer’s disease (AD) and have been incorporated in AD diagnostic criteria and guidelines [42 and high τP-181, based on the so-called AT(N) system [Established  biomarkers of dementia in the cerebrospinal fluid (CSF), namely amyloid beta with 42 amino acids (Aβidelines . The latidelines . The typ) system , which i42, τP-181 and τT may be more useful in determining the concomitant pathology of AD or in predicting cognitive impairment. On the other hand, CSF α-synuclein has not yet been shown to be an established biomarker but appears to be a promising one.The development of widely applicable CSF biomarkers for DLB remains unclear. To date, the three classical biomarkers Aβ42 levels, and this may be more common than previously thought. Aβ is a proteolytic cleavage product of amyloid precursor protein (APP). Decreased Aβ42 in the CSF is associated with neuropathological features in the brain, including amyloid plaques and neuronal loss, findings that have been proposed to be good predictors of cognitive impairment and dementia detection [42 in DLB, especially at the demented stage [42, and Aβ40). At the prodromal stage, contrary to AD patients, DLB patients’ biomarker levels in the CSF were not altered. At the demented stage of DLB, A42 levels were reduced [42 were older, more often females, had a shorter duration of the disease and had severe cognitive impairment [38, Aβ40, and Aβ42, while AD is characterized by a relatively isolated reduction in Aβ42. This study included 72 patients with a diagnosis of probable DLB and matched them for age and sex, with 38 patients with a diagnosis of probable AD and 38 subjects with subjective cognitive decline who served as controls. The abovementioned patients and controls were selected from the Amsterdam Dementia Cohort [42 may be associated with more rapid cognitive decline in DLB patients, as was concluded by a European multicenter study on DLB, in which they selected 100 patients with diagnostic criteria for probable DLB [42 was the only abnormal biomarker [42 could show less prominent or marginal reduction or may even be increased in DLB as compared to AD, as proposed by a study with 1194 patients diagnosed with several neurodegenerative diseases, and another, in which they include CSF samples of 33 patients with probable AD without parkinsonism, 25 patients with all the core features of DLB, and 46 age-matched controls [A typical finding in DLB patients is a decrease in CSF Aβetection . Severaled stage ,8,9,10. a Cohort . The reda Cohort . Reducedable DLB . A retroiomarker . Howevercontrols ,15. Withcontrols . The retcontrols . Some of42 combined with pathological τΤ and/or τP-181. In line with the previous observation, another study, in 2019 [Τ [In a large memory clinic cohort  and in l in 2019 , reveale 2019 [Τ .42 is a rather frequent finding in patients with DLB. However, a significant percentage may show the typical CSF AD profile, indicating mixed pathology, not only with amyloid plaques but also with tangles, compatible with the additional presence of AD. Indeed, there seems to be a spectrum with pure DLB and pure AD at the two ends and mixed cases in between [Thus, a reduction in Aβ between . This co between ,18, one Although α-syn is not a classical biomarker, it is important to study its possible diagnostic role. Intracellular accumulation of α-syn, a protein that is abundantly expressed in the brain, is a feature of several late-onset synucleinopathies, such as DLB ,3. SimilIn a patient with a clinical presentation suggestive of DLB, positive CSF results for AD biomarkers indicate the presence of AD and may be compatible with mixed pathology (both synucleinopathy and AD) ,20,21. TThe form of quantitated α-syn may play some role, since synucleinopathies, such as DLB, may exhibit lower total α-syn and higher ratio of phosphorylated/total α-syn ratiο, as compared to other neurodegenerative diseases . FurtherThe heterogeneity of results on α-syn could have been attributed, at least to some degree, to confounding factors. Initially, the immunoassays used are based on different antibodies that recognize different fragments of the protein and with variable affinity. In addition, patient cohorts show high variability at the time of CSF collection. Further, the absence of the application of strict standardized instructions regarding the collection and storage protocols and the limitation of the presence of blood in the CSF sample are significant hurdles. This suggests that CSF α-syn could serve as a potential marker of synucleinopathy if only a number of the above-mentioned factors have been controlled to reduce the observed variability in results. Furthermore, there should be application of standard instructions on collection and storage protocols and also for blood contamination, since the presence of blood in CSF alters the results. Among the various confounders of CNS α-Syn quantification, blood contamination has been widely recognized, as α-Syn is abundant in the red blood cells. Nevertheless, other methodological differences should be kept in mind, including differences in α-syn species quantified (its type may be different among the synucleinopathies) and, in addition, differences in capture/detection antibodies among the immunoassays used in different studies .Furthermore, worth noting is that recent findings have pointed out the potential role of the two members of the family of synucleins {α-synuclein (140 amino acids) and β-synuclein (134 amino acids) as biomarkers for the diagnostic characterization of patients affected by synucleinopathies as DLB .42, τP-181 and τΤ. The first biomarker in the AT(N) classification system (NIA-AA 2018) [42, with reduced values being abnormal. Reduction in this biomarker alone may indicate  the Alzheimer’s continuum [P-181 is increased, in addition to decreased Aβ42, this is indicative of Alzheimer’s disease. DLB often overlaps with AD in clinical and pathological features, sometimes making it challenging to differentiate between these conditions. Some DLB patients may have abnormal (decreased) Aβ42 alone, while others may have both Aβ42 and τP-181 abnormal. Taken together, the data from the literature described above indicate that decreased levels of CSF Aβ42 are a rather common finding in DLB patients, whereas a simultaneous increase in τP-181 may be observed in a significant percentage of patients as the disease progresses [P-181 may identify additional AD pathology in patients with DLB [Until now, classical (established) CSF biomarkers have included AβAA 2018)  is amyloontinuum . When τPogresses ,11,13. Twith DLB . By avoi42 or a typical AD profile, the presence of another more indicative biomarker is mandatory. This role could be attributed to α-syn. Nevertheless, there are still some methodological issues, currently preventing CSF α-syn to be an established biomarker, although it may become so in the near future. This could be feasible by achieving some control of several confounding parameters, in addition to the standardization of α-syn species measured.Therefore, given the fact that patients with DLB may frequently show a reduced AβFurthermore, given the overlapping of neuropathological, neurochemical and neuropsychiatric profiles of neurodegenerative diseases, larger studies of homogeneous design are required to demonstrate the relationship of the CSF biomarkers and the underlying pathology.Recognizing DLB remains challenging due to the highly variable presentation of clinical symptoms, which include cognitive fluctuations, visual hallucinations, parkinsonism, sleep disorders and autonomic dysfunction, and the fact there is considerable clinical and pathological overlap, primarily with AD. They may occur together, not uncommonly, especially in older patients, but correct clinical identification of the underlying pathology is not always easy. The discovery of validated biomarkers for DLB is essential for the early diagnosis and treatment of patients affected by this prominent cause of neurodegenerative dementia. Currently, the only condition recognized by established CSF biomarkers is the presence or absence of concomitant AD. Diagnosis “by inclusion” has not been achieved yet. Until now, quantitative analysis of CSF α-syn levels has varied widely between studies, probably reflecting (pre)analytical issues, whilst a real biological cause cannot be excluded. Based on the heterogeneity of methods and results between studies, further validation is fundamental before conclusions can be drawn. The recognition of fluid biomarkers reflecting α-syn pathology is urgent  and may Additionally, one fact to be optimistic about is that recent studies support that proteins expressed during the inflammatory changes in synucleinopathies, such asCSF APR proteins, may be proven to be potential biomarkers of clinical diagnostic utility .


A range of neurological pathologies may lead to secondary hydrocephalus. Treatment has largely been limited to surgical cerebrospinal fluid (CSF) diversion, as specific and efficient pharmacological options are lacking, partly due to the elusive molecular nature of the CSF secretion apparatus and its regulatory properties in physiology and pathophysiology.CSF obtained from patients with subarachnoid hemorrhage (SAH) and rats with experimentally inflicted intraventricular hemorrhage (IVH) was analyzed for lysophosphatidic acid (LPA) by alpha-LISA. We employed the in vivo rat model to determine the effect of LPA on ventricular size and brain water content, and to reveal the effect of activation and inhibition of the transient receptor potential vanilloid 4 (TRPV4) ion channel on intracranial pressure and CSF secretion rate. LPA-mediated modulation of TRPV4 was determined with electrophysiology and an ex vivo radio-isotope assay was employed to determine the effect of these modulators on choroid plexus transport.+/K+/2Cl− cotransporter (NKCC1) implicated in CSF secretion.Elevated levels of LPA were observed in CSF obtained from patients with subarachnoid hemorrhage (SAH) and from rats with experimentally-inflicted intraventricular hemorrhage (IVH). Intraventricular administration of LPA caused elevated brain water content and ventriculomegaly in experimental rats, via its action as an agonist of the choroidal transient receptor potential vanilloid 4 (TRPV4) channel. TRPV4 was revealed as a novel regulator of ICP in experimental rats via its ability to modulate the CSF secretion rate through its direct activation of the NaTogether, our data reveal that a serum lipid present in brain pathologies with hemorrhagic events promotes CSF hypersecretion and ensuing brain water accumulation via its direct action on TRPV4 and its downstream regulation of NKCC1. TRPV4 may therefore be a promising future pharmacological target for pathologies involving brain water accumulation.The online version contains supplementary material available at 10.1186/s12987-022-00361-9. The mammalian brain is submerged in cerebrospinal fluid (CSF), the majority of which is continuously produced by the choroid plexus, a monolayered epithelial structure that protrudes from the ventricular wall , 2. The R1-LPAR6) i with BAPTA-AM). The 86Rb+ efflux in the presence of GSK  was reduced in the absence of Ca2+ , Fig. 86Rb+ efflux was thus halved in the absence of Ca2+ , Fig. 86Rb+ efflux , Fig. 86Rb+ efflux was thus more than halved by WNK inhibition and completely abolished by SPAK inhibition. These data demonstrate that NKCC1 is activated by TRPV4 via Ca2+-mediated activation of the WNK/SPAK signalling pathway.Ca kinases , the lat kinases . RNASeq ity Fig. F, H, but86Rb+ efflux rate in the presence of this lipid. LPA elevated the efflux rate  [Here, we reveal a molecular coupling between a brain hemorrhagic event and the ensuing ventricular enlargement signifying PHH. The origin of hydrocephalus formation is generally sought in blockage of the CSF exit routes –59. In c1-LPAR6) –18. Here1-LPAR6) , but in 1-LPAR6) , 28, 64,1-LPAR6) ). Inhibi1-LPAR6) , 27 and 1-LPAR6) .Xenopus laevis oocytes, an elevation that was absent in the presence of a TRPV4 inhibitor and in control oocytes lacking TRPV4 expression. Such a reduced experimental system allows for determination of direct functional interactions and promoted LPA as an agonist of TRPV4. The molecular coupling between TRPV4 activation and elevated activity of a transport mechanism implicated in CSF secretion originated in the TRPV4-mediated Ca2+ influx, which activated the WNK/SPAK signaling pathway as also observed in salivary glands [TRPV4 can be directly activated by various lipid compounds , and we y glands . These ky glands , 66, 67.y glands , 21 as ry glands . A couply glands , 17, andy glands  and it ry glands , 17 wouly glands . Importay glands ), which y glands , the LPAy glands , the NKCy glands , 70 and y glands  inhibitiLimitations to the study include CSF sampling from cisterna magna in the rodent experimentation, from the ventricular compartment in the patients with SAH, and from the basal cisterns in the control patients. The different patient sample sites were dictated by ethical limitations in invasive CSF sampling, but could influence our results if the CSF composition differs between these locations. The control samples may contain blood contamination from the surgical opening. Such contamination is expected to raise the LPA levels in the control CSF. Our results would thus be expected to show even further differences between the two CSF groups, if one could test control CSF samples with no traces of blood. In the rat IVH model, the control rats received a saline injection (instead of the orthologous blood injection) and were therefore not naïve at the point of CSF sampling 24 h later. We cannot rule out that this manipulation could promote an inflammatory response, which in itself could affect the CSF LPA levels. However, the human CSF samples were obtained from patients undergoing preventive surgical clipping of unruptured aneurisms and therefore had to such surgical intervention prior to the sampling event. Of note, with the invasive nature of experimental approaches towards determination of agonist-induced modulation of intracranial pressure, cerebrospinal fluid secretion rates, and choroidal transport mechanisms, these parameters were obtained from the rodent (IVH) animal model. Such findings may not fully capture the human SAH condition. With the expected lipid-plastic interaction during the experimental procedures, most (approximately 97% ) of the In conclusion, we demonstrate that the serum lipid LPA, entering the ventricular system during a hemorrhagic event, acts directly on TRPV4, and thus serves as a novel lipid agonist of this choroidal ion channel. Activation of TRPV4 causes the NKCC1 hyperactivity underlying the TRPV4-mediated elevated CSF secretion rate seemingly contributing to the ensuing ventriculomegaly signifying PHH. Future studies aimed at elucidating the pathophysiological changes in brain fluid dynamics occurring with diverse neuropathologies, ideally, should take into account formation rates of CSF, its path through the ventricular system, as well as its drainage, as most of these pathologies are likely to affect more than one of these aspects.Additional file 1. LPA quantification, choroid plexus viability, and 3D imaging of the rat ventricular system.


CSF MTBR-tau measures are reproducible in repeated lumbar punctures and can be used to distinguish CBD from control (receiver operating characteristic area under the curve (AUC) = 0.889) and other FTLD-tau, such as PSP (AUC = 0.886). CSF MTBR-tau275 and MTBR-tau282 may represent the first affirmative biomarkers to aid in the diagnosis of primary tauopathies and facilitate clinical trial designs.Despite recent advances in fluid biomarker research in Alzheimer’s disease (AD), there are no fluid biomarkers or imaging tracers with utility for diagnosis and/or theragnosis available for other tauopathies. Using immunoprecipitation and mass spectrometry, we show that 4 repeat (4R) isoform-specific tau species from microtubule-binding region (MTBR-tau Cerebrospinal fluid measures of isoform-specific tau species from the microtubule-binding region serve as the first fluid biomarkers of primary tauopathy. Additionally, recent progress in positron emission tomography (PET) imaging now enables the measurement of aggregated Aβ and tau in the brains of living patients with AD12. In contrast, such progress is lacking for other tauopathies classified as FTLD-tau, including CBD, PSP, argyrophilic grain disease (AGD), globular glial tauopathy, chronic traumatic encephalopathy (CTE) and Pick’s disease (PiD). Accurate diagnoses are challenging without fluid biomarkers for the tauopathies because these disorders fall within a spectrum comprising multiple and overlapping clinical phenotypes. Most tauopathies can only be definitively diagnosed by brain autopsy. Antemortem fluid biomarkers for these tauopathies will be required to improve the accuracy of clinical diagnosis and facilitate clinical trials for tauopathy therapeutics.Tauopathies are a heterogeneous group of neurodegenerative diseases that all include aggregated tau proteins. The symptomatic phases of these fatal illnesses involve neurological impairments that typically progress over years to decades, leading to substantial medical, social and financial burden on patients and families. For the most common tauopathy, AD, the CSF biomarkers amyloid-β (Aβ) and total and phosphorylated tau have been used to aid diagnosis13. Tauopathies can be classified into 3R, 4R and 3R/4R mixed tauopathies based on the dominant isoforms found in tau aggregates. Cryogenic electron microscopy (cryo-EM) studies demonstrated that there are distinct tau filament structures in AD (3R/4R)14, PiD (3R)15, CBD (4R)16, PSP (4R)17 and CTE (3R/4R)18. R3 and R4 repeat domains are commonly present in tau aggregates from AD and other tauopathies14. In contrast, a 4R isoform-specific R2 repeat domain in addition to R3 and R4 are found in tau aggregates from CBD and PSP17. Recently, we used biochemical extraction and mass spectrometry (MS) to show that specific tau fragments, such as the residues 243–254 , 299–317  and 354–369 , differentially enriched in AD brains with disease progression19. Furthermore, we showed that the truncated tau containing MTBR could be detected and quantified in CSF and that CSF soluble concentrations of MTBR-tau fragments reflected AD clinical severity and correlated strongly with tau PET measures19. Others also reported a correlation between CSF soluble truncated MTBR-tau and insoluble tau aggregates measured by tau PET measures20, suggesting that measures of MTBR-tau fragments might serve as fluid biomarkers of tau aggregation in AD.Recent structural and biochemical analyses implicate that distinct tau species constitute brain tau aggregates in subtypes of tauopathies. Six tau splicing isoforms are expressed in the adult human brain, including isoforms containing R1, R3 and R4 (3R) and R1, R2, R3 and R4 (4R) repeat domains in the MTBR275) and 282–290 (MTBR-tau282) that are in the R2 region and specific to 4R tau splicing isoforms. We tested differential diagnostic abilities of MTBR-tau275 and MTBR-tau282 normalized to total tau, to distinguish FTLD-tau, FTLD with TAR DNA-binding protein aggregates (FTLD-TDP), control and within different subtypes of tauopathies.In this study, we hypothesized that 4R isoform-specific MTBR-tau species accumulate in the brain of specific subtypes of 4R tauopathies. Then, we investigated if these changes are reflected in the CSF and can distinguish different subtypes of primary tauopathies. We specifically monitored tau fragments 275–280 (MTBR-tau21. MTBR-tau275 (275–280) and MTBR-tau282 (282–290) located within the R2 repeat domain and specific to 4R isoforms were normalized with total tau (t-tau) measured by mid-domain tau peptide 181–190 to account for individual variabilities in t-tau  and threefold in FTLD-MAPT (0.762 ± 0.333) compared to normal control  and non-tauopathy FTLD-TDP , 4R tauopathies, AGD (0.200) and PSP , and 3R/4R mixed tauopathy, AD . MTBR-tau275/t-tau moderately increased in PSP (approximately twofold) compared to FTLD-TDP (P < 0.01). Brain MTBR-tau282/t-tau had a similar increase profile to MTBR-tau275/t-tau , which was not observed in MTBR-tau275/t-tau. These results suggest that 4R-specific MTBR-tau species are enriched in the insoluble fraction of SFG/insular cortex brain tissue in a subset of 4R tauopathies, such as CBD and FTLD-MAPT, and moderately increased in a different PSP (4R tauopathy) and AD (3R/4R mixed tauopathy).First, frozen brain tissues from 59 individuals with autopsy-confirmed AD, FTLD-tau or FTLD-TDP and three normal control individuals without clinical diagnoses of neurodegenerative diseases were processed for biochemical extraction of insoluble tau and underwent MS analyses Table . The suptau Fig. . Brain Mtau Fig.  but modeMAPT and 78 autopsy-confirmed cases of AD, primary tauopathies and FTLD-TDP were analyzed for MTBR-tau275 and MTBR-tau282 , AD (0.00472 ± 0.00085) and FTLD-MAPT (0.00491 ± 0.00207), compared to normal control  and non-tauopathy control, FTLD-TDP  and PSP , and the 3R tauopathy, PiD . CSF MTBR-tau282/t-tau had similar decrease profiles to CSF MTBR-tau275/t-tau  across all disease groups (n = 54) and strongly . This suggests that 4R-specific MTBR-tau species have inverse correlation in the CSF and brain in 4R tauopathies. In CBD, MTBR-tau275/t-tau and MTBR-tau282/t-tau from the CSF and brain correlated moderately but no statistical significance was obtained  = 0) had fewer changes in MTBR-tau275/t-tau and MTBR-tau282/t-tau in both brain and CSF . This may suggest that the changes of these biomarkers depend on the severity of the disease.To assess if the soluble CSF MTBR-tau/t-tau measures reflected brain tau pathology measured by the paired insoluble brain MTBR-tau/t-tau measures, MTBR-tau/t-tau from antemortem CSF and brain from the same individuals were analyzed for correlation  were investigated (Table n = 81) and 4 ± 1 years for CBD-only (n = 18). In CBD, there were negative correlations between duration and CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau  although statistical significance was not observed . This result suggests that participants with longer duration who are at later pathological stages have larger degrees of decrease in the CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau biomarkers.To assess if CSF MTBR-tau275/t-tau in an independent cohort of 25 participants who underwent repeated lumbar punctures (LPs) (3–5 times) within approximately 4 months as a part of an ongoing study examining protein turnover kinetics24 (Extended Data Table n = 7) or corticobasal syndrome , with two participants having autopsy-confirmed CBD. They also included seven MAPT mutation  carriers who were either symptomatic or asymptomatic and two noncarrier family members who are normal controls. The mean coefficient of variation for CSF MTBR-tau275/t-tau in repeated LPs was 12 ± 7%, establishing the high reproducibility and stability of CSF MTBR-tau measures within 4 months.To evaluate reproducibility and stability of the CSF MTBR-tau measurements within the same individual, we examined CSF MTBR-tauMAPT cases analyzed with the pathologically confirmed cohort, CSF MTBR-tau275/t-tau decreased in FTLD-MAPT mutation carriers in the repeated LP cohort. Interestingly, CSF MTBR-tau275/t-tau decreased in two symptomatic FTLD-MAPT P301L mutation carriers  and a symptomatic FTLD-MAPT R406W mutation carrier  compared to normal control . However, CSF MTBR-tau275/t-tau did not change in FTLD-MAPT R406W mutation carriers who were asymptomatic at LPs (numbers 06 and 07). The FTLD-MAPT variant IVS10+16 promotes the splicing of tau exon 10, resulting in greater production of 4R over 3R isoforms. Indeed, symptomatic FTLD-MAPT IVS10+16 mutation carriers  had 1.38-fold higher CSF MTBR-tau275/t-tau compared to normal controls, indicating that an increase in 4R isoforms is reflected in the CSF.Consistent with the FTLD-275/t-tau decreased in the two participants who were clinically diagnosed as PSP-RS but later were autopsy-confirmed with CBD (numbers 10 (0.00396) and 11 (0.00535)), which is consistent with the pathologically confirmed CSF cohort results. However, CSF MTBR-tau275/t-tau did not change in participants clinically diagnosed with PSP-RS (0.00779 ± 0.00052) or CBS (0.00748 ± 0.00187), who are not yet autopsy-confirmed during the repeated measures studies. Average duration across diseases was 5 ± 3 years (n = 21) and 4 ± 2 for CBS only (n = 9), which were similar to the pathologically confirmed cohort.CSF MTBR-tau275/t-tau in an additional independent cohort of 238 primary tauopathies with single LP . However, CSF MTBR-tau275/t-tau did not statistically change in either AD or clinically diagnosed CBS compared to control or other tauopathies.To estimate the CSF MTBR-tau biomarker performance in clinically diagnosed primary tauopathies, we measured CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau in a pathologically confirmed primary tauopathy cohort. First, CSF t-tau (mid-domain peptide 212–221) and phosphorylated tau (pT217/T217) were examined in primary tauopathies for comparison  and can differentiate AD from FTLD-tau  with an AUC of 0.794  and can differentiate AD from FTLD-tau with an AUC of 0.987 , PiD and PSP with AUCs of 0.800–0.889. CBD can be distinguished from FTLD-TDP with AUCs of 0.701–0.770. When AD copathology cases were excluded, CSF MTBR-tau275/t-tau and CSF MTBR-tau282/t-tau can distinguish CBD from PSP with AUCs of 0.859 and 0.886, respectively  had clinical syndromes of either CBS (n = 9), bvFTD (n = 4), nonfluent variant PPA  or PSP-RS (n = 1). Without biomarkers, the diagnostic accuracy of CBD within CBS was 9 out of 15 (60%). Among 15 individuals with CBS, 7 and 10 had lower than cutoff values of CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau and 6 and 7 were CBD, respectively. With these biomarkers, the diagnostic accuracies of CBD within CBS were 6 out of 7 (86%) and 7 out of 10 (70%) for CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau, respectively. Across different clinical syndromes of CBD, 12 out of 18 (67%) and 15 out of 18 (83%) had CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau values lower than cutoff, suggesting that these biomarkers identify CBD regardless of clinical syndromes with up to 83% accuracy. Other applications of these biomarkers include distinguishing CBD from PSP among PSP-RS since 0 out of 11 (0%) and 2 out of 11 (18%) PSP had lower than cutoff for CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau, respectively.Lastly, we retrospectively assessed CSF MTBR-tau31. We recently demonstrated that CSF pT217/T217 increased in FTLD-MAPT R406W compared to control without Aβ pathology25. However, phosphorylated tau changes most substantially in AD and thus these studies may not have captured key pathological changes in primary tauopathies. In this study, we focused on MTBR-tau, which constitutes the core regions of tau aggregates in the brain and also exists in the CSF as a truncated C-terminal tau fragment19. Using biochemical purification and quantitative MS, we showed that 4R isoform-specific MTBR-tau275 and MTBR-tau282 that are normalized to t-tau decrease in the CSF soluble tau as they increase in brain insoluble tau in primary tauopathies, especially CBD and FTLD-MAPT P301L. MTBR-tau/t-tau measures are inversely correlated in the CSF and brain, suggesting that there may be an equilibrium or a unidirectional transfer between soluble CSF MTBR-tau and insoluble brain MTBR-tau in these primary tauopathies. This study provides the possibility of the first fluid biomarker that reflects brain pathology in primary tauopathies.Despite the long quest for antemortem biomarkers of FTLD-tau pathology, to date no fluid biomarkers have been identified that can differentiate subgroups of tauopathies other than AD. Previous studies showed that CSF phosphorylated tau at T181 decreased in PSP and FTLD-TDP relative to controls275 and MTBR-tau282 were only observed in a subset of 4R and 3R/4R mixed tauopathies. As expected, these 4R isoform-specific measures did not change in 3R tauopathy (PiD) or non-tauopathy FTLD (FTLD-TDP). However, CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau specifically decreased in CBD and FTLD-MAPT but not PSP and AGD even though all are categorized as 4R tauopathies. This may be due to higher variability in the degree of neocortical pathology in PSP and AGD cases compared to CBD and most FTLD-MAPT cases. Many cases of PSP have 4R tau aggregates primarily in subcortical regions, such as the thalamus and brain stem32, and AGD pathology is most severe within the medial temporal lobe, even in advanced stages33. In contrast, CBD has more abundant and widespread tau pathology in the cerebrum34. FTLD-MAPT  can lead to a very high deposition of 4R tau aggregates in neurons and glia in multiple brain regions, including the hippocampus, neocortex and substantia nigra35. An alternative explanation is that CBD has wispy and fine filamentous inclusions within neuronal cell bodies, whereas PSP neurons tends to harbor a larger proportion of more compact tau aggregates36. CBD is commonly associated with abundant cortical astrocytic plaque pathology and neuritic tau pathology in both gray and white matter, while PSP neuronal and astrocytic pathology (tufted astrocytes) are often restricted to the motor and premotor cortex and subcortical nuclei. It is possible that tau aggregates in PSP may have different physicochemical property from CBD and the status of equilibrium between insoluble and soluble forms may be different. Together, we speculate that the quantity or total burden of 4R tau pathology in the whole brain may reflect changes in CSF 4R-specific MTBR-tau.One of the interesting findings of this study was that changes in 4R isoform-specific MTBR-tau275/t-tau and MTBR-tau282/t-tau may potentially be used to positively identify a subset of primary tauopathies and may be useful in assisting with antemortem differential diagnosis. We confirmed in our repeated lumbar puncture study that CSF MTBR-tau/t-tau measures are reproducible and stable over 4 months, which will reliably provide biomarker values in the clinic or clinical trial settings. In the clinically diagnosed cohorts without autopsy confirmation, CSF MTBR-tau275/t-tau did not change in CBS and there was a higher overlap between CBS and PSP-RS, which might be attributable to lack of one-to-one relationship between clinical syndromes and neuropathological diagnosis in FTLD. However, from retrospective clinical syndrome analyses in the pathologically confirmed cohort, we showed that CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau biomarkers may be able to identify individuals with CBD regardless of clinical syndromes  with as high as 83% accuracy, which is higher than approximately 25–50% diagnostic accuracies of CBD without these biomarkers.CSF MTBR-tauAdditional limitations of this study include the following: tauopathies with shorter duration or during the asymptomatic stage may not yet show decrease in these biomarkers. Future studies targeting larger cohorts with different severity and longitudinal samples with clinical measures would help address whether CSF MTBR-tau can capture disease at an earlier stage of primary tauopathies; the lack of orthogonal measures to identify brain tau pathology in living patients  limited our ability to assess correlation between CSF and brain MTBR-tau antemortem; the moderate size of the cohorts with small sample sizes for subgroups such as AGD and PiD can limit interpretation; the 77G7 MTBR-tau antibody used for sequential immunoprecipitation in the study may also be targeting specific pools of truncated tau; and there may be future technical advancements and analytical method developments that may unveil additional or new populations of tau species in biofluids that reflect qualitative and quantitative aspects of tau pathology in primary tauopathies. Overall, these findings advance our knowledge of heterogeneous pathophysiology in primary tauopathies and open avenues for the development of therapeutics and clinical trials targeting primary tauopathies.The retrospective study of pathologically confirmed cohort included participants seen at UCSF or participating research sites under the following projects: Hillblom Healthy Aging Study; UCSF Alzheimer’s Disease Research Center Program Project Grant (P30AG062422); ARTFL-LEFFTDS Longitudinal Frontotemporal Lobar Degeneration (ALLFTD) (U19AG063911); and the Four Repeat Tauopathy Neuroimaging Initiative (R01AG038791). Collection and use of biospecimens was approved by the institutional review board at ALLFTD or each research center from which the individual was recruited and this study was approved by the Biospecimen Resource Committee at UCSF. Participants provided written informed consent at the time of recruitment. Participants underwent a standardized clinical evaluation that included collection of demographic data, structured participant/informant interview, functional assessment, neurological examination and neuropsychological testing. CSF draws were performed at the same visit as the clinical evaluation; primary clinical syndrome was determined based on all available data at the time of clinical evaluation by an experienced neurologist or panel of neurologists according to established diagnostic criteria. Consent to undergo autopsy was provided by the patient or their surrogate according to the principles outlined in the Declaration of Helsinki 2013.The repeated LP study (NCT03545126) was approved by the institutional review board at Washington University in St. Louis and University College London. All participants or their delegates consented to the collection and sharing of biofluid samples and brain autopsy and were compensated. Exclusion criteria included any contraindications for LPs or lumbar catheters, including a bleeding disorder, active anticoagulation and active infection.http://www.biobanques.eu)). Authorization to handle personal data was granted by the French Data Protection Authority  under no. 1709743 v0.The study of the clinically diagnosed cohort was approved by the ethics committee of the Montpellier University Hospital , 150 mM sodium chloride, 10 mM EDTA, 10 mM EGTA, phosphatase inhibitor cocktail and protease inhibitor cocktail. The homogenate was clarified by centrifugation for 20 min at 11,000 g at 4 °C and stored at −80 °C as the whole-brain extract. The demographics of the brain donors included in this study are described in Table Neuropathological diagnoses of human brain donations were rendered according to established diagnostic criteria at Washington University and UCSF. The frozen human brain tissue samples selected for this study were processed as described previouslyhttp://www.adni-info.org/). The demographics of participants in the pathologically confirmed cohort is in Table 24. The demographics of participants in the repeat LP study are described in Extended Data Table 25.CSF collection methods were similar across all cohorts examined in this study. The CSF collection method from pathologically confirmed cases was as described previously in the Alzheimer’s Disease Neuroimaging Initiative procedures manual  and internal standards for tau (15N-labeled 2N4R recombinant tau) were prepared in polypropylene tubes before CSF addition. Then, 0.5 ml CSF was added and immunoprecipitated with Tau1, HJ8.5 and HJ8.7 anti-tau antibodies with epitopes residing in the N terminal to mid-domain of tau25. Post-immunoprecipitated samples depleted of the N terminal to mid-domain of tau were sequentially immunoprecipitated with 77G7 anti-tau antibody to the MTBR (residue 316–335) to measure MTBR-tau species. After washing, samples were digested with trypsin, desalted and analyzed by Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific). The MS methods used to measure MTBR-tau were as described previously with some modifications19. To account for individual variability in t-tau concentrations, we used the ratio of MTBR-tau275 or MTBR-tau282 normalized to the mid-domain tau peptides  that are common to all isoforms.CSF MTBR-tau was analyzed as described previouslyP < 0.05 was considered statistically significant and corrected for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR) method with FDR set at 5%38. The P values reported in the tables and figures were corrected using the Benjamini–Hochberg method (FDR = 5%). Spearman correlations were used to assess the associations between CSF tau biomarkers and neuropathological changes.All statistical analyses were performed using Prism v.9.3.1 (GraphPad Software). Differences in biomarker values were assessed with a one-way analysis of variance (ANOVA) unless otherwise specified. A two-sided Further information on research design is available in the Any 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/s41591-022-02075-9.Supplementary InformationSupplementary Figs. 1–6.Reporting Summary


Objective: The objective of this study was to evaluate the efficacy and safety of intranasal midazolam as part of a paediatric sedation and analgesic procedure during the suturing of traumatic lacerations in paediatric emergency departments. Methodology: A systematic review of clinical trials was completed in July 2021. The databases consulted were PUBMED, SCOPUS, WEB OF SCIENCE, NICE and Virtual Health Library. Eligibility criteria: randomised and nonrandomised clinical trials. Two independent, blinded reviewers performed the selection and data extraction. The participants were 746 children, of whom, 377 received intranasal midazolam. All of the children were admitted to an emergency department for traumatic lacerations that required suturing. The quality of the articles was evaluated with the Jadad scale. This systematic review was conducted according to the PRISMA  guidelines. Results: Nine studies were included in the review. The intranasal administration of midazolam in healthy children produces anxiolysis and minimal/moderate sedation without serious side effects. Although there are combinations of parenteral drugs that produce deeper sedation, they also have greater adverse effects. No significant differences in the initiation of sedation and the suture procedure were found between the intranasal route and the parenteral route. Conclusions: The use of intranasal midazolam in healthy children produces sufficiently intense and long-lasting sedation to allow for the suturing of traumatic lacerations that do not present other complications; therefore, this drug can be used effectively in paediatric emergency departments. In children, trauma frequently causes minor lacerations that should be evaluated at paediatric emergency departments. In the United States, lacerations account for approximately 8.2% of annual emergency department visits, which means that between seven and nine million lacerations are treated in the US each year .When suturing is necessary, the fear of pain, separation from parents and refusal to be examined or remain in a certain position manifest as anxiety, crying and aggression, especially in young children. In turn, these experiences increase the suffering of the child and his or her parents, making it difficult for the health professional to perform the suture ,3,4.Local anaesthetics produce analgesia but lack the ability to control the child’s fear and anxiety ,6. HowevIn young children, the parenteral administration of sedative drugs may not be adequate, since it increases anxiety, fear and anguish due to the puncture . In addiThe intranasal route has proven to be a non-invasive, simple-to-use, safe and effective option . The nasAccording to our study, although there are currently no official statistics on the number of traumatic laceration suture procedures in children, this is a common procedure in paediatric emergency departments; therefore, it is necessary for health professionals to be aware of the available alternatives and to know which anxiolytic/sedative drugs offer the best guarantees of safety and efficacy.Therefore, the objective of this systematic review was to synthesise the available scientific evidence based on clinical trials to evaluate the efficacy and safety of intranasal midazolam as an anxiolytic and sedative as part of a paediatric sedation and analgesic (PSA) procedure during the suturing of traumatic lacerations in paediatric emergency departments.A systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . This syThe databases consulted from January to July 2021 were PUBMED, SCOPUS, WEB OF SCIENCE, NICE and Virtual Health Library (VHL).The search strategy was guided by the PICO research questions  shown in We consider that a drug is “effective” if it produces a rapid state of sedation in the child allowing for the successful suturing of a traumatic laceration without complications in the emergency department. Likewise, we consider that a drug is “safe” if we observe that the doses administered do not produce significant adverse effects or require a prolonged recovery of the child until they are discharged from hospital.Two researchers (F.J.G.-M. and J.A.L.-A.) independently and blinded to one another selected the studies according to the predetermined inclusion and exclusion criteria and evaluated all of the references of studies identified in the search. After searching the databases mentioned above, a total of 99 articles were obtained. These potentially relevant articles were imported to the Mendeley reference manager to eliminate duplicates (35 studies), in accordance with the PRISMA Systematic Review Flowchart . Once duQuality was assessed using the Jadad scale . This toAll included studies underwent quality assessment and were independently evaluated by two reviewers (F.J.G.-M. and A.I.C.-C.). The disagreements and discrepancies were discussed until consensus was reached. If consensus was not achieved, another reviewer was consulted (J.M.C.-T.). For data extraction, two authors (F.J.G.-M. and J.M.C.-T.) independently used a standardised data collection form. From each selected study, the following data were collected: (1) the name of the first author and year of publication; (2) country; (3) study design and type of intervention; (4) characteristics of the sample: sample size, selection, type of blinding, age of participants and size of the laceration; (5) characteristics of the intervention: dose, drug, route of administration, intranasal method of administration and vital signs; and (6) main results of the intervention: level of sedation/anxiolysis, time until the start of sedation, time until discharge from the hospital, observed adverse effects and degree of satisfaction of parents/health professionals.A narrative synthesis of each of the studies included in this systematic review was performed. The data were analysed to compare the results for intranasal midazolam with those for different sedative drugs and placebo  based onOf the 99 articles that were obtained, nine clinical trials were finally selected ,20,21,22The studies had a randomised clinical trial (RCT) ,20,21,22The Jadad scale was used to assess the methodological quality of the studies and detect bias . Accordip < 0.003), struggled less (p < 0.04) and had a significantly lower maximum heart rate (p < 0.002) and maximum systolic blood pressure (p < 0.04) than the placebo/control group [Theroux M. et al., 1993) assessed the degree of crying, struggling and movement by children during the suturing procedure [93 assessol group .No significant differences were observed in the level of anxiety reduction ,20,21 orAccording to Klein et al., (2011), the intranasal route presents a faster initiation of action, a higher proportion of adequately sedated children, an optimal score on the activity scale that was used and a greater proportion of parents who would choose this method of sedation in the future .No serious adverse effects were observed with any of the examined routes of administration ,20,21.McGlone R. et al., (1998) did not observe significant differences between intranasal midazolam and intramuscular ketamine and atropine during the suturing procedure in terms of the level of oxygen saturation, the behaviour of the child during recovery, adverse reactions and total time until discharge from the hospital, although, in this case, a somewhat lower median time was recorded in the group that received intranasal midazolam (75 min vs. 82 min) [p < 0.01) [The combination of ketamine and atropine induced deeper sedation, which reduced the need for physical restraint of the children (14% vs. 86% of the midazolam group) ( < 0.01) . This co < 0.01) .Sixty-six percent of children who received intranasal midazolam were resistant to receiving nasal drops .p < 0.001; 95% CI (3.2–7.4 min)), compared to the intranasal midazolam group [The initiation of sedation was faster in the group that received intravenous ketamine and midazolam, at 5.3 min on average ) [The intranasal midazolam group was discharged from the hospital on average 19 min earlier than the group that received intravenous drugs (33 min)) .Parents and health professionals were more satisfied with sedation via the intravenous route .Two-thirds of children who received intravenous drugs experienced random movements of the extremities . No signp = 0.034) [p = 0.011) than intranasal midazolam (26.1 ± 9 min) [According to Everitt I. et al., (2002), the oral route was better tolerated than the intranasal route (= 0.034) . Oral di± 9 min) , a longe± 9 min) . No adveNeville D. et al., (2016) compared two groups of children  and obtained the following results : (a) posDexmedetomidine and midazolam behaved similarly, with no significant differences between the groups of children for all other measures recorded: anxiety at other times during the procedure, anxiety perceived by parents, satisfaction of parents and doctors, success of the suturing procedure, complications/adverse effects and total time to discharge .Tsze D. et al., (2017) observed slight differences  in the iIn clinical trials published before 2002, intranasal midazolam was administered via the instillation of drops in the nostrils ,8,19,20;In the analysed studies, there were no significant differences in the time elapsed from the administration of the drug to the start of sedation and suturing among the intranasal route , the intravenous route (ketamine + midazolam) and the intramuscular route (ketamine + atropine) ,22.For different volumes of intranasal midazolam, the time until the onset of the sedative effect was very similar .Intranasal dexmedetomidine produced a deeper sedative effect during the initial stages of the suture procedure .The combination of intravenous ketamine and midazolam and intramuscular ketamine and atropine showed deeper and more prolonged sedation ,22.No significant differences were found in the level of oxygen saturation, heart rate, respiratory rate or blood pressure between children who received intranasal midazolam and those who received the midazolam via other routes of administration or those who received other sedative drugs ,10,20,21Two-thirds of the children who received intravenous ketamine and midazolam exhibited random movements of their extremities .Three of the analysed studies did not assess parents’ and/or professionals’ satisfaction with the outcomes of sedation ,8,20.Theroux M. et al., 1993) observed a high degree of parental satisfaction with the use of intranasal midazolam compared with no drug or placebo [93 observSeveral studies showed that parents and health professionals prefer deeper sedation ,22. McGlKlein E. et al., (2011) found that parents would request sedation with intranasal midazolam in the future, indicating that they were satisfied with the sedation outcomes .Neville D. et al., (2016) did not observe differences in the satisfaction of parents and health professionals with sedation by dexmedetomidine and midazolam .According to the analysed data, the intranasal administration of 0.2–0.5 mg/kg midazolam in healthy children at a paediatric emergency department achieved minimal/moderate sedation during the suturing procedure for an uncomplicated traumatic laceration.Although intravenous sedation with midazolam is widely used in paediatrics  due to iIn the analysed clinical trials, the time passed from the administration of intranasal midazolam until the children were sedated (conscious sedation) was variable, lasting between 4.3 and 28 min ,20,21,22Regarding the oral and intraoral routes, in the analysed studies, there were no significant differences among the oral, intraoral  and intranasal administration of midazolam in terms of the degree of anxiety reduction ,20,21, tThe oral administration of midazolam requires the child’s cooperation in ingesting the drug and requires a higher dose than the intranasal route to achieve the same level of sedation ,20,26,27On the other hand, according to several of the analysed studies, the intranasal route is least tolerated by children due to local discomfort  ,20,21,22Nasal discomfort was also evidenced in other studies ,11,31; hThe nasal burning produced by midazolam is mild and lasts only a few seconds , but is Nasal mucosa spray devices  convert To determine the necessary dose of a nasal spray, the 0.1 mL dead space of the device must be taken into account ,14. AccoThe presence of nasal secretions caused by respiratory infections can hinder the absorption of the drug. This problem can be solved with a nasal wash with saline solution prior to the administration of the drug . If the The rectal administration of midazolam presents an initiation of action, duration of effect and recovery time similar to the intranasal route, although a moderate effectiveness of 60–75% has been shown . In addiAccording to the analysed results, oral diazepam has a longer duration of sedation and lighter sedation . TherefoAccording to the analysed studies, intranasal dexmedetomidine has a better initiation of sedation during the initial phases of the suture procedure  than intranasal midazolam; however, no significant differences were found for the rest of the procedure, recovery or the time elapsed until discharge from the hospital . HoweverKetamine, which was used intravenously and intramuscularly in two of the studies included in this review ,22, prodIn the analysed clinical trials, doses of intranasal midazolam between 0.2–0.5 mg/kg were considered effective. However, the intensity of sedation depends on the administered dose. Yelay D. et al.  analysedIn Spain, midazolam is marketed as an injectable solution, a buccal solution and oral tablets . CurrentClinical nurses and physicians often perform the suturing of traumatic lacerations in the emergency department. For this procedure, health professionals have different drugs for sedation or analgesia, it being necessary to know the route for their administration. The rapid administration of drugs in the paediatric population in emergency situations is sometimes complicated for many reasons: agitation, problems with the insertion of intravenous catheters, impossibility of communication, etc. To avoid increasing the child’s discomfort by performing invasive techniques, we can use non-invasive routes, such as the intranasal route, to administer analgesic and/or sedative drugs to reduce the state of agitation or anxiety of paediatric patients, if this route is not contraindicated  .Based on our results, the intranasal administration of midazolam may be considered a good choice for children requiring suturing for traumatic lacerations due to its rapid sedative effect, similar to the effect obtained using the parenteral route and without significant adverse effects. However, this technique is easier to perform and has fewer complications. It also avoids traumatic procedures for children, such as needle sticks. It is true that, in older and heavier children, the amount of volume to be administered intranasally is greater, so this fact should be taken into account. Regarding the limitations of the present study, it is worth noting the variability of the methods and scales used to assess the anxiety and sedation levels of the children in the clinical trials included in this review. Another limitation is the variability in the method of intranasal midazolam administration (drop instillation or administration with a spray device). In addition, not all studies included the location and size of the traumatic laceration that had to be sutured. Another limitation was the inclusion of low-quality articles due to the fact that one of the nine articles included showed a score < 3 on the Jadad scale. Additionally, it was not possible to verify the efficacy and safety of intranasal midazolam in comparison with other sedative drugs that are used in paediatrics  Given the absence of recent RCTs comparing the use of these sedatives in suture procedures, it would be wise to conduct more clinical trials in this area.According to the analysed results and the doses of drugs used in the clinical trials included in this systematic review, we consider that the administration of intranasal midazolam in healthy children is a non-invasive method that is simple and effective (depending on the dose), producing a rapid state of minimal/moderate sedation in the child as part of a paediatric sedation and analgesic procedure. This sedative effect is similar to that produced using the intravenous route, which allows for the successful suturing of traumatic lacerations that do not present complications. Likewise, according to the results and the doses analysed, the intranasal midazolam is safe since it does not produce a significant adverse effect or require a lengthy recovery. In the analysed studies, children who received intranasal midazolam recovered rapidly, and the time until hospital discharge was similar to or even shorter than that of other drugs. However, this administration route sometimes requires the child to be restrained during the administration procedure. Health professionals’ prior training for its correct application is also necessary. In general, the intranasal route is a good option for short procedures such as suturing lacerations.Additional good-quality RCTs are needed to compare intranasal midazolam with other sedative drugs used in paediatrics and to clarify the effectiveness and safety of midazolam versus intranasal dexmedetomidine at different doses in children.


However, the mechanisms of acacetin on osteoclast differentiation and type H vessel formation, as well as the effect of preventing bone loss, remain unclear. Here, we firstly used primary bone marrow derived macrophages (BMMs), endothelial progenitor cells (EPCs), and ovariectomized (OVX) mice to explore the function of acacetin on bone remodeling and H type vessel formation. In this study, we found that acacetin inhibits osteoclast formation and bone resorption of BMMs induced by the macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) in a concentration of 20 μM without exerting cytotoxic effects. It was accompanied by downregulation of osteoclast differentiation marker genes  and cell fusion genes . Moreover, acacetin disrupted actin ring formation and extracellular acidification in osteoclasts. Mechanistic analysis revealed that acacetin not only inhibits the expression of the major transcription factor NFATc1 and NF-κB during RANKL-induced osteoclast formation, but also suppresses RANKL-induced the phosphorylation of Akt, GSK3β, IκBα, and p65. Additionally, acacetin enhanced the ability of M-CSF and RANKL-stimulated BMMs to promote angiogenesis and migration of EPCs. We further established that, in vivo, acacetin increased trabecular bone mass, decreased the number of osteoclasts, and showed more type H vessels in OVX mice. These data demonstrate that acacetin prevents OVX-induced bone loss in mice through inhibition of osteoclast function and promotion of type H vessel formation via Akt/GSK3β and NF-κB signalling pathway, suggesting that acacetin may be a novel therapeutic agent for the treatment of osteoporosis.Osteoporosis, characterized by the destruction of bone resorption and bone formation, is a serious disease that endangers human health. Osteoporosis prevention and treatment has become one of the important research contents in the field of medicine. Acacetin, a natural flavonoid compound, could promote osteoblast differentiation, and inhibit osteoclast formation  Osteoporosis is a chronic bone disease characterized by destruction of bone microstructure and low bone mass, leading to increased bone fragility and thus increased fracture susceptibility . Bone reOsteoclasts are multinucleated cells, that differentiate from monocytes/macrophages in the presence of M-CSF and RANKL . When RAin vitro   was purchased from Herbest Bio-Tech Co., Ltd. . Alpha-minimal essential medium (α-MEM) was obtained from Gibco . Foetal bovine serum (FBS) was obtained from HyClone . M-CSF and RANKL were provided by R&D . Sodium carboxymethyl cellulose (CMC-Na), peroxidase conjugate-wheat germ agglutinin (WGA) (L3892), FITC-WGA (L4895), acid phosphatase, leukocyte (TRAP) kit, rhodamine-conjugated phalloidin, acridine orange, and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich . Antibodies against CTSK (sc-48353), ACP5 (sc-376875), MMP9 (sc-13520), NFATc1 (sc-7294), PI3K (sc-376112), p-Akt (sc-293125), Akt (sc-81434), p-GSK3β (sc-373800), GSK3β (sc-53931), p-IκBα (sc-8404), IκBα (sc-1643), p-NF-κB p65 (sc-136548), NF-κB p65 (sc-8008), p-ERK (sc-81492), ERK (sc-514302), p-JNK (sc-6254), JNK (sc-7345), p-p38 (sc-7973), p38 (sc-7972), CD31 (365804), EMCN (sc-65495), PDGF-BB (sc-365805), TRAP (sc-376875), and GAPDH (sc-166574) were obtained from Santa Cruz Biotechnology . ECL chemiluminescence reagents were obtained from Pierce Biotechnology . The DAB Horseradish Peroxidase Colour Development Kit, One Step TUNEL Apoptosis Assay Kit, Calcein AM, 4′,6-diamidino-2-phenylindole (DAPI), paraformaldehyde (PFA), ethylenediaminetetraacetic acid (EDTA), Haematoxylin and Eosin (H&E) Staining Kit, horseradish peroxidase (HRP)-conjugated IgG, RIPA Lysis Buffer, Calcein AM, and Triton X-100 were obtained from Beyotime Biotechnology . Matrigel was obtained from BD . TRIzol reagent and methyl thiazolyl tetrazolium (MTT) were obtained from Invitrogen . A one-step PrimeScript RT reagent kit and SYBR Premix Ex TaqIIkit were provided by TaKaRa . A mouse PDGF-BB ELISA kit was obtained from Jingmei Biotechnology . A glutamic-oxalacetic transaminase (GOT/AST) activity assay kit, glutamic-pyruvic transaminase (GPT/ALT) activity assay kit, urea nitrogen assay kit, and creatinine (CREA) assay kit were obtained from Sangon Biotech .5/well and induced with conditioned medium for 4 days in a 5% CO2 incubator at 37°C.Primary bone marrow derived macrophages (BMMs) were isolated from the femur and tibia of six-week-old C57BL/6J mice by flushing the marrow with a syringe and then cultured in α-MEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/ml streptomycin in the presence of 5 ng/mL M-CSF for 24 h. Then, cells in the supernatant were collected as primary BMMs and seeded for osteoclastogenic differentiation induction. Osteoclastogenesis of BMMs was induced by conditioned medium in α-MEM supplemented with 10% FBS, 100 μg/ml streptomycin and 100 U/mL penicillin in the presence of 10 ng/mL M-CSF and 10 ng/ml RANKL. The cells were seeded into 24-well plates at a density of 2 × 102 incubator.Endothelial progenitor cells (EPCs)  were culture in endothelial growth medium-2 (EGM-2) (Lonza). Endothelial basal medium-2  was used for experiments that do not require growth factors. The cells were incubated at 37°C in a 5% CO2 incubator.The MC3T3-E1 osteoblastic cell line were cultured in α-MEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/ml streptomycin and incubated at 37°C in a 5% CO4/well, and cultured with α-MEM supplied with 5 ng/mL M-CSF and treated with various concentrations of acacetin . After 24 and 48 h of incubation, 0.5 mg/ml MTT was added to each well and incubated for 3 h at 37°C. Then, DMSO was used to dissolve the formazan crystals, and the optical density was measured at a wavelength of 490 nm using a microplate analyser .BMMs were seeded into 96-well plates at a density of 4 × 105/well, cultured with α-MEM supplied with 5 ng/mL M-CSF and treated with various concentrations of acacetin . After 24 and 48 h of incubation, the TUNEL staining assay was performed using a One Step TUNEL Apoptosis Assay Kit. In brief, BMMs were washed once with precooled PBS for 3–5 min. Then, the cells were fixed in 4% PFA for 30 min and washed again with precooled PBS. The cells were permeabilized with 0.3% Triton X-100 for 5 min and washed with PBS twice. After adding 50 μL TUNEL solution/well, the samples were developed in the dark for 1 h at 37°C, washed 3 times with precooled PBS and observed using an inverted fluorescence microscope . The apoptosis rate of in randomly chosen areas was quantified using ImageJ software.BMMs were seeded into 24-well plates at a density of 2 × 105 cells/well) were seeded into 24-well plates, incubated with 10 ng/ml RANKL and 10 ng/mL M-CSF and treated with various concentrations of acacetin  for 4 days. After fixation in 4% PFA for 20 min, the cells were subjected to TRAP staining using the acid phosphatase, leukocyte (TRAP) kit according to the manufacturer’s instructions. The number of TRAP+ osteoclasts containing more than 3 nuclei was counted using an optical microscope .BMMs . The osteoclast number/endocortical surface  was measured using ImageJ.Gapdh was used as an internal control for mRNA detection. All primers were purchased from Sangon Biotech .BMMs were induced with conditioned medium and treated with acacetin  for 4 days. Total RNA was isolated from cells and tibiae using TRIzol reagent according to the manufacturer’s instructions. Reverse transcription was performed using the one-step PrimeScript RT reagent kit for mRNA analysis. Real-time PCR assays for mRNA analysis were performed using a SYBR Premix Ex TaqII kit with a Thermal Cycler C-1000 Touch System . The primers used for real-time PCR are listed in Whole-cell lysates for western blotting were prepared by extracting proteins from the cells using RIPA lysis buffer, and then blotting them on a polyvinylidene fluoride membrane. The membranes were incubated with antibodies against CTSK , ACP5 , MMP9 , NFATc1 , PI3K , p-Akt , Akt , p-GSK3β , GSK3β , p-IκBα , IκBα , p-NF-κB p65 , NF-κB p65 , p-ERK , ERK , p-JNK , JNK , p-p38 , p38 , and GAPDH . Secondary antibodies conjugated with HRP  were used for signal detection. The blots were visualized using ECL chemiluminescence reagents.Bone resorption assays were performed using Osteo Assay surface . BMMs were seeded onto osteo surface and induced with 10 ng/mL M-CSF and 10 ng/ml RANKL for 5 days. Bone resorption pits were sonicated in PBS to remove adherent cells. The bone resorption pits on the osteo surface were visualized using optical microscope .Primary BMMs on bone slices or glass chamber slides were induced with 10 ng/mL M-CSF and 10 ng/ml RANKL and treated with various concentrations of acacetin for 4 days. The cells were washed 2–3 times with PBS, and fixed in 4% PFA for 15 min and washed again with precooled PBS 2–3 times. Then, the cells were permeabilized with 0.5% Triton X-100 for 10 min and washed 2–3 times with precooled PBS for 3–5 min each. The cells were stained with 0.5 μg/ml rhodamine-conjugated phalloidin in the dark for 40 min. After incubation, the cells were washed twice with precooled PBS for 3–5 min each. Finally, the nuclei were counterstained with 1 μg/ml DAPI in the dark for 3 min, washed twice with precooled PBS for 3–5 min and observed under fluorescence microscopy .Primary BMMs on glass chamber slides were induced with 10 ng/mL M-CSF and 10 ng/ml RANKL for 4 days and then treated with acacetin (0 and 20 μM) for another 24 h. The cells were washed 2–3 times with PBS and incubated with 5 μg/ml acridine orange for 15 min at 37°C. Then, the cells were washed with PBS and chased with fresh medium for 10 min, and stained cells were observed under fluorescence microscopy .5 cells/well) were seeded into 24-well plates, incubated with 10 ng/mL M-CSF and 10 ng/ml RANKL, and treated with various concentrations of acacetin  for 4 days. At the end of induction, serum-containing conditioned medium from the mature osteoclasts was collected. Serum-free conditioned medium containing the same concentrations of M-CSF, RANKL, and acacetin was harvested after another day of culture. All the aliquoted conditioned media were centrifuged at 2,500 rpm for 10 min and stored at −80°C.Conditioned media from osteoclasts treated with acacetin  was prepared. BMMs . After 6 h, the cells were stained with calcein AM, and network formation was imaged using an inverted fluorescence microscope . The total tube lengths and numbers of intersections in randomly chosen areas were quantified using ImageJ software.EPCs were seeded into 6-well plates and grown to 90% confluence. An injury was created in the cell monolayer using a sterile 200 ml pipette, and unattached cells were removed by washing twice with PBS. Then, the cells were allowed to migrate into the empty space for 18 h in acacetin or serum-free conditioned medium from osteoclasts cultured with acacetin . The cells were imaged during migration using an inverted optical microscope . The width of the injury was measured using ImageJ software.5 cells/well) were seeded into 24-well plates, incubated with 10 ng/mL M-CSF and 10 ng/ml RANKL, and treated with various concentrations of acacetin  for 4 days. The conditioned medium was subjected to PDGF-BB ELISA analysis using a mouse PDGF-BB ELISA kit according to the manufacturers’ instructions.BMMs (2 × 10n = 6 in each group), including the sham-operated (sham) group, the OVX group, and the OVX with acacetin (OVX + Acacetin) group. The sham group was treated with 0.5% CMC-Na with a sham operation, while both the OVX and OVX + Acacetin groups were ovariectomized, and orally administered 0.5% CMC-Na or 20 mg/kg/d acacetin for 8 weeks, respectively. The choice of dosage was based on previous studies . Mice were randomly divided into three groups  and Rankl/osteoprotegerin (Opg) mRNA expression.Serum was evaluated for AST, ALT, BUN, and CREA content using assay kits according to the manufacturer’s guidelines for each.3), bone surface area/bone volume , bone surface to tissue volume , bone volume to tissue volume , trabecular number , trabecular spacing , and trabecular thickness .The distal metaphysis of the femur and 5th lumbar vertebrae were fixed in 4% PFA and scanned using a Bruker SkyScan 1,276 Micro-CT device . In brief, the scanning parameters were set as following, energy: 70 kV, 114 μA; angle of increment: 0.2°, exposure time: 810 ms/frame, scanning time: 14 min, and the scanning resolution were set as 10 μm. For femur, a region of interest (ROI) that start from the growth plate to 1.5 mm below the growth plate was selected for analysis (threshold = 117.4 mg HA/ccm) using CTvox software , and three-dimensional (3D) reconstruct by NRecon Reconstruction software . For vertebrae, the ROI of trabecular bone of 5th vertebral body was selected for analysis (threshold = 282.15 mg HA/ccm) and 3D reconstruction. The following parameters of trabecular bone and vertebrae were calculated using the direct three-dimensional measurement method: bone mineral density , EMCN (1:100), PDGF-BB (1:100), and TRAP (1:100) overnight at 4°C. Then, the sections were washed and incubated with secondary antibodies conjugated with fluorescence in the dark for 1 h. Finally, the nuclei were counterstained with 1 μg/ml DAPI in the dark for 3 min and washed twice with precooled PBS for 3–5 min. CD31hiEMCNhi vessels were quantitatively analysed as previously described (−2) of bone marrow area in the trabecular bone. For immunohistochemistry analyses  were added to the upper compartment of the transwell system in α-MEM complete medium, and BMMs were added into lower chamber in α-MEM complete medium without RANKL or M-CSF. After 5 days co-culture, the cells in both upper and lower chamber were selected for further study.A transwell assay with polycarbonate membranes (0.4 μm pore size)  was used for osteoblast-osteoclast co-culture. MC3T3-E1 cells , and the effect was most obvious at 20 μM . Interesd groups . Additiod groups . Taken tc-Fos and Pu.1 were reduced by 20 μM acacetin after 4 days of induction . Real-time PCR results showed that expression of nduction , while tnd 20 μM . Furthernd 20 μM . These dTo further study the role of acacetin in osteoclast function, we performed a bone resorption assay. Equal numbers of BMMs were cultured on the osteo assay surface and treated with 10 ng/mL M-CSF, 10 ng/ml RANKL for 5 days, and treated with acacetin at concentrations of 0, 5, 10, and 20 μM for another 2 days. Bone resorption assays indicate that the total resorption area was significantly reduced in the acacetin treatment groups (10 and 20 μM) .CD9, CD47, Atp6v0d2, Dc-stamp, and Oc-stamp was reduced in response to 20 μM acacetin treatment after 4 days of induction  treated osteoclasts was measured. As evidenced by ELISA, the production of PDGF-BB in conditioned medium was increased by acacetin treatment at a concentration of 20 μM . FurtherRunx2, Alp and Ocn were upregulated by acacetin, while the expression of Rankl/Opg was not changed  or vehicle (CMC-Na) for 2 months. Serum analysis revealed that acacetin did not exert liver or kidney toxicity in mice because there was no significant change in AST, ALT, BUN, or CREA content . Micro-C changed . Furtheracacetin . These rin vivo, we analysed sections of the femora by TRAP staining. The results showed that TRAP+ osteoclasts on the trabecular bone of distal femurs and cortical bone were increased, while acacetin reduced the number of osteoclasts in OVX mice (Acp5 and Rankl/Opg in the femurs of OVX mice was upregulated in response to acacetin treatment (To investigate osteoclast development OVX mice . ImmunohOVX mice . Moreovereatment .hiEmcnhi vessels (hiEMCNhi vessels adjacent to the growth plate (GP) of OVX mice, which was similar to the sham-operated mice (+ cells that were positive for PDGF-BB than sham-operated mice, while acacetin rescued the proportion of PDGF-BB+/TRAP+ cells in OVX mice (+/TRAP+ preosteoclasts that stimulate angiogenesis and bone formation in OVX mice.An increase in the number of preosteoclasts induces the formation of CD31 vessels . Immunofted mice . Preosteted mice . TherefoOVX mice . CollectOsteoporosis is a common bone disease characterized by excessive bone resorption mediated by osteoclasts and resulting in bone loss . It is ein vitro (Acp5, Ctsk, and Mmp9, also demonstrated the inhibitory effects of acacetin on osteoclastogenesis. Next, we tested the role of acacetin in osteoclast function. The bone resorption assay, actin-ring staining, and acridine orange staining confirmed that acacetin inhibited the bone resorption and extracellular acidification of osteoclasts. These results indicate that acacetin suppresses RANKL-induced osteoclast formation and function in vitro.To prevent the development of osteoporosis, a great deal of work has been done to identify effective treatments. Overactivation of osteoclasts plays a major role in bone destruction, therefore, osteoclast differentiation is considered as a major therapeutic target for developing new drugs . Clinicain vitro . HoweverThe binding of RANKL to RANK during osteoclast differentiation activates key downstream signalling pathways, such as Akt and NF-κB . Previouin vitro and in vivo (Type H vessels play an important role in maintaining normal bone structure . Previou in vivo . HoweverAcp5 and Rankl/Opg in bone were significantly reduced by acacetin treatment. Type H vessels play an important role in maintaining normal bone structure (+PDGF-BB+ cells were induced by acacetin in OVX mice. Taken together, these observations demonstrated that acacetin exerts a protective effect on bone loss in vivo.Finally, a preclinical study using an OVX mouse model of osteoporosis confirmed the role of acacetin in reducing bone loss. The micro-CT results showed that treatment with acacetin restored OVX-induced damage to the trabecular bone architecture of the femur and vertebrae via increases in BMD, BS/TV, BV/TV, Tb.N, and Tb.Th, and a decrease in the BS/BV and Tb. Sp parameters. Moreover, acacetin increased the number of osteoprogenitor cells in the femurs. Our results demonstrated that OVX-induced increases in the number of osteoclasts and enhanced expression of tructure . Previoutructure . The numtructure . TherefoIn summary, we demonstrated that acacetin prevents OVX-induced bone loss by regulating bone resorption and type H vessel formation. Notably, our results reveal that acacetin treatment inhibits osteoclastogenesis and bone resorption through suppression of the Akt/GSK3β and NF-κB signalling pathways. Therefore, this study provides potential application prospects for the pharmacological countermeasures of acacetin in osteoporosis treatment.