Under static loading for the [(0(IMS60))/(0(E-glass))]S, [(0(E-glass))/(0(IMS60))]S, [(0(E-glass))/(0(IMS60))2]S, [(0(E-glass))/(0(IMS60))3]S, and [(0(E-glass))/(0(IMS60))5]S HFRP specimens, the stress applied to the specimen was almost linearly proportional to the strain until failure. However, the tensile stress–strain curves of the [(0(E-glass))2/(0(IMS60))]S, [(0(E-glass))3/(0(IMS60))]S, and [(0(E-glass))5/(0(IMS60))]S HFRP specimens had a complicated shape (jagged trace). The Weibull statistical distributions of the tensile strength values were also examined. The Weibull moduli for the [(0(E-glass))/(0(IMS60))]S, [(0(E-glass))/(0(IMS60))2]S, [(0(E-glass))/(0(IMS60))3]S, [(0(E-glass))/(0(IMS60))5]S, [(0(E-glass))2/(0(IMS60))]S, [(0(E-glass))3/(0(IMS60))]S, and [(0(E-glass))5/(0(IMS60))]S HFRP specimens were higher than those for the mono carbon fiber-reinforced epoxy (CFRP) and glass fiber-reinforced epoxy (GFRP) specimens. Under fatigue loading, the fatigue properties of the HFRP specimens showed CFRP-dominant behaviour at high stress levels and GFRP-dominant behaviour at low stress levels. The fatigue properties of the HFRP specimens increased with increasing volume fraction of CFRP in the following order: ([(0(E-glass))/(0(IMS60))5]S > [(0(E-glass))/(0(IMS60))3]S > [(0(E-glass))/(0(IMS60))2]S > [(0(IMS60))/(0(E-glass))]S > [(0(E-glass))/(0(IMS60))]S > [(0(E-glass))2/(0(IMS60))]S > [(0(E-glass))3/(0(IMS60))]S > [(0(E-glass))5/(0(IMS60))]S).The static and fatigue tensile properties of high-strength polyacrylonitrile (PAN)-based carbon (IMS60) and electronic (E)-class glass (E-glass) hybrid fiber-reinforced epoxy matrix composites (HFRPs) were investigated. The fiber orientations of the HFRP specimens were set to unidirectional with [(0 By mixing two or more types of fibers in a common matrix to form a hybrid composite, it may be possible to create a material possessing the combined advantages of the individual composite.Fiber-reinforced polymer matrix composites (FRPs) have become a dominant material in the aerospace, high-performance automotive, and sporting goods industries5. The tensile stress–strain curves of the hybrid CFRP specimens showed a complicated shape (jagged trace). The hybrid composite can be considered one example of a material that prevents instantaneous failure.Naito et al. characterized the tensile properties and fracture behavior of high-strength polyacrylonitrile (PAN)-based and high-modulus pitch-based hybrid carbon fiber-reinforced epoxy and polyimide matrix composites (CFRPs)10. This interest stems from a more cost-effective utilization of expensive fiber if it is used in hybrid form11. The development of fiber-hybrid composites is a logical evolution toward even more design freedom and hence more possibility for optimization and cost reduction12. Although fatigue behavior is an important property for many applications, the effects of hybridization on this property have not been extensively studied13. Wu et al.14 reported the fatigue properties of hybrid composites. The addition of CFRP to a basalt fiber-reinforced polymer matrix composite increased the number of cycles to rupture of the hybrid composites. On the other hand, the addition of CFRP to a glass fiber-reinforced polymer matrix composite (GFRP) did not have the same effect. However, measuring the static and fatigue tensile failure of the same hybrid composites remains a challenging issue. Demonstrating the static and fatigue tensile failure of the same hybrid composites are a major original contribution of this work.A number of papers were written approximately 1970–1980 on the advantages and applications of hybrid composites, such as carbon/glass hybrid composites, under static loadingIn the present work, static and fatigue tensile tests of high-strength PAN-based carbon (IMS60) and electronic (E)-class glass (E-glass) hybrid fiber-reinforced epoxy matrix composite (HFRP) specimens were performed to evaluate their potential. The Weibull statistical distributions of the static tensile strength and stiffness reduction during fatigue loading of the HFRP specimens were also evaluated.2, resin content (RC): 35%) and 0.137 mm  were used.HFRP laminates were produced using an epoxy matrix-based unidirectional (UD) FRP prepreg material QC133-149A  and E-glass-UD/epoxy . The IMS60 carbon fiber was a high-strength PAN-based carbon fiber, and the E-class glass fiber was an alumino-borosilicate glass fiber with less than 1% w/w alkali oxides. IMS60 (QC133-149A) prepreg was supplied by Toho Tenax Co., Ltd., and E-glass (E-glass-UD/epoxy) prepreg was supplied by Arisawa Mfg. Co., Ltd. All sheets were manufactured using conventional prepreg technology. FRP prepregs with nominal thicknesses of 0.142 mm : 145 g/m(IMS60))4 (subscript 4 means four layers and (0(IMS60)/0(IMS60)/0(IMS60)/0(IMS60))), (0(E-glass))4, [(0(IMS60))/(0(E-glass))]S (subscript S means symmetry and [(0(IMS60))/(0(E-glass))/(0(E-glass))/(0(IMS60))]), [(0(E-glass))/(0(IMS60))]S, [(0(E-glass))/(0(IMS60))2]S, [(0(E-glass))/(0(IMS60))3]S, [(0(E-glass))/(0(IMS60))5]S, [(0(E-glass))2/(0(IMS60))]S, [(0(E-glass))3/(0(IMS60))]S, and [(0(E-glass))5/(0(IMS60))]S, respectively. The mono CFRP ((0(IMS60))4), GFRP ((0(E-glass))4), and the HFRP specimens ([(0(IMS60))/(0(E-glass))]S, [(0(E-glass))/(0(IMS60))]S, [(0(E-glass))/(0(IMS60))2]S, [(0(E-glass))/(0(IMS60))3]S, [(0(E-glass))/(0(IMS60))5]S, [(0(E-glass))2/(0(IMS60))]S, [(0(E-glass))3/(0(IMS60))]S, and [(0(E-glass))5/(0(IMS60))]S) were described as MC, MG, HA, HB, HC, HD, HE, HF, HG, and HH, respectively.The prepreg sheets were cut into the appropriate size and fiber orientation. The sheets were placed on a vacuum molding board. HFRP laminates were made using a hand lay-up and vacuum bagging technique (no bleeder). The fibre orientations of the mono CFRP and GFRP specimens and the HFRP specimens were set to unidirectional with  and 10 mm in width. The fiber axis in the specimen was oriented in line with the length of the tensile test specimen (0° direction specimen). To remove the effect of stress concentrations caused by surface roughness from the edges, the edges of the tensile test specimens were polished to remove scratches. Thinner plain-woven fabric glass fiber-reinforced plastic  tapered tabs were affixed to the tensile test specimen to minimize damage from the grips on the tensile testing machine. Similar specimen preparation procedures of other hybrid composites have been observed in the reported literature5.The laminates were cut into rectangular straight-side tensile test specimens with dimensions of 200 mm in length  with a load cell of 50 kN. The specimen was set up in the testing machine. A crosshead speed of 5.0 mm/min was applied, and all tests were conducted under the laboratory environment at room temperature (at 23 °C ± 3 °C and 50% ± 5% relative humidity). Strain gauges were used to measure longitudinal strains. Similar static test procedures of other hybrid composites have been observed in the reported literatureR, of the minimum stress to the maximum stress was 0.1. The fatigue tests were terminated after 1 × 107 cycles. All tests were conducted in the laboratory environment at room temperature (at 23 °C ± 3 °C and 50% ± 5% relative humidity). Strain gauges were used to measure longitudinal strains. Similar fatigue test procedures of other composites have been observed in the reported literature16.Fatigue tests of HFRP specimens were conducted using a servo-hydraulic testing machine  with a 50-kN load cell at a frequency of 10 Hz under cyclic loading with a constant amplitude. The waveform of the cyclic loads was sinusoidal. The stress ratio, σ–ε) curves for the HFRP specimens, as well as for the MC and MG specimens. For the MC and MG specimens, and HA, HB, HC, HD, and HE specimens, the stress–strain response was linearly proportional until failure. However, the HF, HG, and HH specimens showed a complicated shape (jagged trace)5. For the IMS60 layers, the HFRP specimens showed an intermediate modulus in the initial stage of loading, which was taken as the tensile modulus, E, after which the load reached a maximum point taken as the tensile strength, σf, and corresponding initial failure strain, εf. Subsequently, when the IMS60 layers began to fail, the high-ductility E-glass layers held the load without instantaneous failure, exhibiting a so-called secondary tensile modulus, E* which was calculated for a constant strain range using a least square method. Finally, the load reached its secondary maximum, and fracture of the HFRP specimen occurred at the secondary fracture strength, σ*f, and corresponding secondary failure strain, ε*f. Because higher ductility E-glass fibers bear the load for a certain time after initial failure occurred, HFRP specimens with jagged traces could be considered one example of a material able to prevent instantaneous failure5. The average tensile modulus (E), tensile strength (σf), failure strain (εf), secondary tensile modulus (E*), strength (σ*f), and failure strain (ε*f) are shown in Table 5.Figure σmax, and the number of cycles to failure, Nf, also defined as the S–N curves for the HFRP specimens. The S–N curves for the MC and MG specimens are also shown in this figure. For the MC and MG specimens, the fatigue properties of the MC were ~ 2–4 times higher than those of the MG. The fatigue properties of the HFRP specimens showed CFRP-dominant behaviour at high stress levels and GFRP-dominant behaviour at low stress levels. The fatigue properties of the HFRP specimens increased with increasing volume fraction of CFRP (HE > HD > HC > HA > HB > HF > HG > HH).Figure EHFRP, and secondary tensile modulus, E*HFRP, of the HFRP specimens were calculated using a simple rule of mixtures:EF(IMS60), EF(E-glass), EM, and EGFRP are the tensile moduli of the IMS60 fiber, E-glass fiber, matrix, and E-glass GFRP, respectively. VGFRP is the volume fraction of E-glass GFRP. The volume fraction of each element is already known. The tensile modulus of the matrix is assumed to be EM = 3.5 GPa. The tensile moduli of the CFRP and GFRP are obtained from the static tensile tests for the MC and MG specimens, and ECFRP = 159 GPa and EGFRP = 38 GPa, respectively. EF(IMS60) and EF(E-glass) are estimated from Eq. (EF(IMS60) = 277 GPa and EF(E-glass) = 75 GPa.The tensile modulus, Ecal) and secondary tensile modulus  are shown in Table The estimated tensile modulus , and secondary fracture strength, σ*f(HFRP), of the HCFRP specimens were also calculated using the rule of mixtures:σfF(IMS60), σfF(E-glass), and σf(GFRP) are the tensile strength values of the IMS60 fiber, E-glass fiber, and E-glass GFRP, respectively. The volume fraction and tensile modulus of each element are already known. The tensile strength values of the CFRP and GFRP are obtained from the static tensile tests for the MC and MG specimens, are σf(CFRP) = 3.023 GPa and σf(GFRP) = 1.109 GPa, respectively. σfF(IMS60) and σfF(E-glass) are estimated from Eqs. (σfF(IMS60) = 5.279 GPa and σfF(E-glass) = 2.169 GPa.The tensile strength, εf(HFRP) and secondary failure strain ε*f(HFRP) of the HCFRP specimens were calculated using the following equations:The failure strain σf.cal), secondary fracture strength , failure strain , and secondary failure strain  are shown in Table The estimated tensile strength  of the composite, and σ0 is a Weibull scale parameter (characteristic stress). The cumulative probability of failure, PF, under a particular stress is given byi is the number of composite specimens that have broken at or below a stress level and n is the total number of composite specimens tested.There is an appreciable scattering of tensile strength for these composites. The statistical distribution of strength values is usually described by the Weibull equationm, for the MC and MG specimens were calculated to be 17.60 for the MC specimen and 12.15 for the MG specimen. m values for the HFRP specimens are shown in Table m for the HA specimen was similar to that for the MC specimens, and m for the HB, HC, HD, HE, HF, HG, and HH specimens was higher than that for the MC and MG specimens. In particular, m for the HF, HG, and HH specimens showed higher values.Figure m can be attributed to the nature and distribution of the flaws present in the specimens. It is well known that many defects, including voids, fiber breakage, and fiber misalignment29, are known to be introduced into these types of laminates during manufacturing and subsequent treatment. Outer high-ductility E-glass GFRP hybridization reduced the effects of the strength-limiting defects of IMS60 CFRP, which in turn, improved the Weibull moduli of HB, HC, HD, HE, HF, HG, and HH specimens. In contrast, the failures of the inner high-ductility E-glass GFRP hybridized HFRP specimen were predominantly initiated by outer IMS60 CFRP defects. Hybridization effects are less likely to appear. Consequently, the HA specimen did not improve the Weibull modulus of the IMS60 CFRP specimens.The results clearly show that the HFRP specimens, except for the HA specimen, improved the specimen Weibull moduli of tensile strength. The differences in m of the HFRP specimens as a function of the hybrid ratio and tensile properties (modulus and strength). There is a clear Weibull modulus transition value in the hybrid ratio, tensile modulus, and strength.Figure 30. A similar fracture morphology  was observed for the MC, MG, and the HFRP specimens. The HFRP specimens failed by extensive longitudinal splitting, resulting in a brush-like fracture surface and suggesting that the higher-strength IMS60 fiber dominant the fracture behavior and increased fiber efficiency. A distinct difference in morphology between the HA, HB, HC, HD, and HE specimens and the HF, HG, and HH specimens was observed. The HF, HG, and HH specimens were covered with a large amount of E-glass GFRP layers. The principal transverse crack related to fiber fracture ran across the whole width and thickness of the IMS60 CFRP layers. A delamination crack was produced at the intersection of the transverse crack and propagated in the length direction near the interface between the IMS60 CFRP layers and the E-glass GFRP layers. The fractured surfaces of the HF, HG, and HH specimens showed large splitting surfaces with delaminated E-glass GFRP layers.Axial tension failure in unidirectional CFRP and GFRP specimens led to fracture in the transverse direction at several points and was associated with longitudinal splitting of the compositeS–N curves for the MC and MG specimens can be described by a power law model. The power law model31 is given bya and b are experimental constants. The least squares fitting of the fatigue trends with the power law model is illustrated in Fig. a, and slope, b, are calculated to be 3.292 and − 0.0356 for the MC specimen and 1.884 and − 0.0899 for the MG specimen, respectively.The entclass1pt{minimaS–N curves of the CFRP- and GFRP-dominant behaviors in the HFRP specimens were calculated using the power law model of the CFRP and GFRP specimens and a simple rule of mixtures. The S–N curves of the CFRP-dominant HFRP behaviors were estimated to add the load acted on GFRP (different in the ratio of CFRP/GFRP) to the load of CFRP using the S–N curve of the MC specimen. The S–N curves of the GFRP-dominant HFRP behaviors were estimated to add the load acted on CFRP (different in the ratio of CFRP/GFRP) to the load of GFRP using the S–N curve of the MG specimen. The estimated results are also shown in Fig. The Xexp − Xcal)/Xcal, and X is the maximum applied stress for the same cycles) as a function of the hybrid ratio.Figure The CFRP-dominant behavior of the fatigue properties for the HA and HB specimens was higher than that of the estimated results. The GFRP-dominant behavior of the fatigue properties for the HA and HB specimens was higher and lower, respectively, than that of the estimated results. The fatigue properties of the HA specimen were approximately 5% higher than those of the HB specimen. For the HB, HC, HD, and HE specimens, the CFRP-dominant behavior of the fatigue properties decreased with increasing volume fraction of CFRP, and the GFRP-dominant behavior, which was lower than that of the estimated results, decreased with increasing volume fraction of CFRP. The CFRP-dominant behavior of the fatigue properties for the HE specimen was lower than that of the estimated results. On the other hand, for the HB, HF, HG, and HH specimens, the CFRP-dominant behavior of fatigue properties, which was higher than the estimated results, decreased with increasing volume fraction of CFRP. For the same specimens, the GFRP-dominant behavior, which was lower than the estimated results, increased with increasing volume fraction of CFRP.36. Figure Fatigue damage, such as matrix cracking and delamination, often results in a significant reduction in the modulus of composite laminates. Hence, it is crucial to develop an analytical model to describe the cumulative damage of composites due to fatigue based on apparent stiffness reductionMost of the stiffness reduction occurred in the earlier stages of fatigue life, whereas the damage density increased steeply. The rate of stiffness degradation became very low as soon as the damage density reached a saturated value. The stiffness reduction trends of the HA, HB, HC, HD, and HE specimens were similar to those of the MC specimen. The stiffness reduction trends of the HF, HG, and HH specimens were similar to those of the MG specimen.34 for the MC, MG, and HFRP specimens, Di, is defined asE0 and Ei represent the apparent stiffness at the first cycle and the i-th cycle, respectively.Stiffness reduction reflects the damaged state under fatigue cycles after the distribution of damage for the MC, MG, and HFRP specimens. The cumulative fatigue damageNi/Nf (Ni represents the i-th cycle), which is widely used in the literature36.Figure Di, for the MC, MG, and HFRP specimens increased with increasing Ni/Nf. For the MC, MG, and HFRP specimens, there exists a relationship between Di and Ni/Nf, given byC, n, m1, and m2 are experimental constants. Dth and DC are the threshold and critical cumulative fatigue damages, respectively, and are assumed to be Dth = 0 and DC = 1. The estimated relationship between Di and Ni/Nf is also shown in Fig. The cumulative fatigue damage, mentclasspt{minimaThe static and fatigue tensile properties of high-strength PAN-based (IMS60) and E-glass hybrid fiber-reinforced epoxy matrix composites (HFRP) were examined.Under static loading, for the HA, HB, HC, HD, and HE specimens, the stress applied to the specimen was almost linearly proportional to the strain until failure. However, the tensile stress–strain curves of the HF, HG, and HH specimens showed a complicated shape (jagged trace). The tensile modulus and secondary tensile modulus for the HFRP specimens could be estimated from the rule of mixtures. The tensile strength values of the HB, HC, HF, HG, and HH specimens are higher than the values predicted by the rule of mixtures. The secondary fracture strength values of the HG and HH specimens are higher than those of the predicted values. The failure strains of the HB, HC, HF, HG, and HH specimens are higher than that of the MC specimen. The Weibull statistical distributions of the tensile strength were also examined. The Weibull moduli for the HB, HC, HD, HE, HF, HG, and HH specimens are higher than those for the MC and MG specimens. The Weibull modulus of the HA specimen is almost similar to that for the MC specimen.Under fatigue loading, the fatigue properties of the HFRP specimens show CFRP-dominant behavior at high stress levels and GFRP-dominant behavior at low stress levels. The fatigue properties of the HFRP specimens increase with increasing volume fraction of CFRP (HE > HD > HC > HA > HB > HF > HG > HH). The fatigue properties of the HA specimen are higher than those of the HB specimen. For the HB, HC, HD, and HE specimens, the CFRP-dominant behavior of the fatigue properties decreases with increasing volume fraction of CFRP and the GFRP-dominant behavior decreases with increasing volume fraction of CFRP. On the other hand, for the HB, HF, HG, and HH specimens, the CFRP-dominant behavior of the fatigue properties decreases with increasing volume fraction of CFRP and the GFRP-dominant behavior increases with increasing volume fraction of CFRP. The stiffness reduction trends of the HA, HB, HC, HD, and HE specimens are similar to those of the MC specimen. The stiffness reduction trends of the HF, HG, and HH specimens are similar to those of the MG specimen.


Altered physiology caused by critical illness may change midazolam pharmacokinetics and thereby result in adverse reactions and outcomes in this vulnerable patient population. This study set out to determine which critical illness‐related factors impact midazolam pharmacokinetics in children using population modeling. This was an observational, prospective, controlled study of children receiving IV midazolam as part of routine care. Children recruited into the study were either critically‐ill receiving continuous infusions of midazolam or otherwise well, admitted for elective day‐case surgery (control) who received a single IV bolus dose of midazolam. The primary outcome was to determine the population pharmacokinetics and identify covariates that influence midazolam disposition during critical illness. Thirty‐five patients were recruited into the critically ill arm of the study, and 54 children into the control arm. Blood samples for assessing midazolam and 1‐OH‐midazolam concentrations were collected opportunistically  and in pre‐set time windows (control arm). Pharmacokinetic modeling demonstrated a significant change in midazolam clearance with acute inflammation (measured using C‐Reactive Protein), cardio‐vascular status, and weight. Simulations predict that elevated C‐Reactive Protein and compromised cardiovascular function in critically ill children result in midazolam concentrations up to 10‐fold higher than in healthy children. The extremely high concentrations of midazolam observed in some critically‐ill children indicate that the current therapeutic dosing regimen for midazolam can lead to over‐dosing. Clinicians should be aware of this risk and intensify monitoring for oversedation in such patients. Pharmacokinetic modelling revealed significantly reduced midazolam clearance in critically ill children with acute inflammation and cardio‐vascular compromise, resulting in up to 10‐fold higher plasma concentrations compared to healthy children. An intravenous bolus dose of the drug alleviates symptoms in a matter of minutes and, when administered prior to a short‐lived unpleasant procedure, prevents significant patient distress.Continuous IV infusion midazolam is commonly used in pediatric critical care to provide sustained patient sedation.A receptors.A signal transduction, diminished hepatic blood flow, and altered liver enzyme metabolizing capacity. The latter two factors are potential modifiers of midazolam clearance in the critically ill.Midazolam acts by potentiating the effects of gamma‐aminobutyric acid (GABA) on GABAThis prospective, observational, pharmacokinetic (PK) study explored midazolam disposition in otherwise healthy children undergoing elective surgery (control group) and critically ill patients requiring mechanical ventilation in intensive care. Contemporaneous recruitment of a control group should enable critical illness‐related covariates influencing PK parameters to be identified with greater certainty.22.1This was a single‐center, observational, prospective study of IV midazolam pharmacokinetics in two groups of children; clinically well children (control group) receiving IV midazolam prior to elective day case surgery and ill, intubated, and ventilated children receiving IV midazolam in intensive care . Children were aged between 1 month  and less than 16 years and admitted either for planned surgical procedures requiring general anesthesia or to the pediatric intensive care unit (PICU). Although there was no sample size calculation, a minimum of 50 children were to be recruited with at least 150 PK samples to enable a robust population PK model to be developed.The study was conducted between January 2015 and September 2016 and complied with the principles of the 1964 Declaration of Helsinki and its later amendments. The study was approved by the East Midlands‐ Derby Research Ethics Committee in England (14/EM/1261) and registered on the EUDRACT database (2014‐004958‐34).−1) as part of general anesthetic induction. In the critically ill group, infants and children received an initial 25–50 mcg kg−1 bolus dose and were then initiated on a continuous infusion of 50–200 mcg kg−1 h−1. The target sedation level was assessed and reviewed at least twice daily. The continuous infusion rate was altered according to the unit's algorithm for dose adjustment and titrated to the desired sedation score. Similarly, additional bolus doses were occasionally administered as necessary to achieve and maintain the desired sedation level.In the control group, midazolam was administered as a single IV ‘bolus’ dose  were taken at various time points, starting from the pre‐dose sample before surgery, during surgery, and after surgery was complete, and surgical drapes covering the child were removed. Subsequent samples  were taken either in the recovery suite or on the wards. PK blood samples were thus obtained for up to 6 h post‐dose.In the critically ill group, blood samples for PK were either scavenged (from laboratory samples obtained for monitoring patients) or opportunistic . PK blood samples were obtained for the duration of the period the child was on midazolam infusion and up to 96 h after treatment had stopped.CRP, liver, and kidney function assessments were determined at least once daily. To measure cardio‐vascular status, a scoring system was developed for this study utilizing data available in the PICU cohort Table . Each vaA secondary objective of the study was to validate the volumetric absorptive microsampling system of PK sampling as an alternative to the collection of conventional blood samples in tubes. This volumetric system involved using Mitra® microsampling devices based on VAMS® technology to collect blood for analysis as a dried sample. In both groups of patients, each PK sample was collected as a duplicate: a whole blood wet sample (for centrifugation and processing) and a 10 μl dry sample using the Mitra device with a VAMS system. The validation data for the VAMS system met internationally accepted guideline criteria.A population pharmacokinetic model of midazolam and 1‐hydroxy‐midazolam plasma concentrations was developed using the nonlinear mixed effects modeling program NONMEM  (Table The median (range) age was 22 months (1 month to 15 years). In the control group, 80% of children were over 2 years of age, whereas in the critically ill group the age was skewed toward the younger age group with 67% less than 2 years of age. The body weight ranged from 2.9 to 78.4 kg  and the BMI ranged from 9.8 to 21.8 kg mControl group children were mainly admitted for ENT [19 (53%)] and urological [13 (36%)] day case procedures. In the critically ill group, cardiac [23(42%)], respiratory [19 (34%)] and neurological [7 (13%)] disorders were the commonest primary systems causing critical illness.−1. The mean (range) duration and rate of midazolam infusion in the critically ill group was 11.8 h (0.25–372) hours and 129 (8.33–760) mcg kg−1 h−1, respectively.The mean (range) midazolam bolus dose in the control group was 34.6 (34.6–41.7) mcg kg−1. Mean (range) 1‐OH Midazolam concentrations in the control group were 9 (5–64) ng ml−1 and in the critically ill group was 56 (5–1507) ng ml−1. Scatter plots of dose‐corrected midazolam and 1‐hydroxy midazolam concentrations are presented in Figure In total, 626 plasma midazolam and 628 1‐hydroxy midazolam PK samples were included in the modeling dataset, of which 119 (19%) and 318 (51%) were below LLOQ, respectively. Mean (range) midazolam concentrations in the control group were 28 (5–356) ng/ml and in the critically ill group was 332 (5–1987) ng ml−1. This suggests a significant proportion of patients were septic. CRP was assumed to be normal (≤3 mg L−1) in the control group. The mean (range) CV score was 7 (4–12) in the critically ill group. Control group patients did not have cardiovascular dysfunction or any requirement for inotropic support and therefore a fixed score of 4 was imputed during model development.Mean (range) CRP in the critically ill group was 375.5 (3–306) mg L3.2Vc). Separate residual error models were applied for the two cohorts and the use of the third variance of residual error was utilized for 1‐hydroxy‐midazolam observations across both cohorts. Due to the wide range of body weights across the two cohorts, body weight‐based allometry was tested upfront during the development of the base model, and was found to be highly significant on CLmid (with a fixed exponent of 0.75) but not on Vc. Testing of the incorporation of a published maturation function for midazolam clearance  could only be optimally quantified for midazolam clearance (CLmid), 1‐hydroxy midazolam clearance (CLHmid), and volume of distribution of the central compartment Vc. SeparaFollowing the selection of the base PK model, exploratory plots of the IIV versus patient covariates revealed four visually compelling relationships, all involving CLmid: CRP, CV score, serum albumin, and total bilirubin in the blood , a rise >200 mg L−1 results in a 3‐fold increase in steady state concentrations. A similar fold increase is seen when the CV score increases from baseline (=4) to 12. A combination of high CRP > 200 mg L−1 and significant cardiovascular compromise (CV score = 12) results in a massive 7–10 fold increase in steady state concentrations.The simulations predict a significant increase in steady state midazolam concentrations with rising CRP and CV score. Compared to baseline CRP  and those with a CRP > 200 ng ml−1 Clmid reduces around 70% compared to otherwise healthy children with a CRP <3 ng ml−1.C‐reactive protein (CRP) is an acute phase reactant that increases during periods of inflammation, e.g., due to sepsis or trauma. Its concentration in blood correlates with concentrations of interleukin‐6 (IL‐6) a pro‐inflammatory cytokine that is known to induce CRP. The PK model developed herein estimates that critically ill children with a CRP > 100 ng ml−1 and CV score = 12 will result in median midazolam concentrations between 1000 and 3000 ng ml−1 (higher in children than infants) when administered continuous IV midazolam infusion at recommended dosing rates. Although no pharmacodynamics assessments were included in our analysis and therefore the clinical impact of increasing midazolam exposure cannot be quantified, previous studies have suggested that the therapeutic range is in the region of 200–800 ng ml−1, but concentrations as high as 2000 ng ml−1 were recorded in several of the critically ill children in this study.Reduced urine output, treatment to support blood pressure (inotropic drugs such as dopamine and boluses of fluid), and increased acid (negative base excess) production are all features of depressed cardiovascular function. In this study, these variables were used to develop a score to provide an integrated measure  investigated the effect of inflammation and disease severity on midazolam pharmacokinetics in 21 critically ill children.In healthy individuals, midazolam has a low/intermediate hepatic extraction ratio. Consequently, midazolam clearance in health is largely dependent on liver CYP3A4 activity and less influenced by changes in liver blood flow.The clinical implication here is that intensivists need to be cognizant of the impact of rising CRP and poor cardiovascular status on the PK of midazolam and other CYP3A4 substrates too. Inflammation and cardiovascular status are dynamic in a critically ill patient and therefore may account for the large intra‐individual variability. Such pathophysiological changes in critically ill patients can modify exposure to a previously stable drug regimen, possibly resulting in either an increased incidence of adverse reactions or a lack of efficacy. Extrapolating these results to other CYP3A4 substrates needs to be confirmed through clinical studies, but the impact of inflammation, for example, needs to be considered when unexpected variations in the blood concentrations of a CYP3A4 substrate occur in the absence of direct drug–drug interactions.Despite clearly associating two pathophysiological variables with midazolam clearance in critically ill children, in this study, nearly 60% of the variance in clearance was assigned to inter‐individual variability, i.e. remains unexplained. This suggests a need for further studies to investigate and model the influence of genetic and other factors on midazolam. However, it also suggests that clinical outcomes may be significantly improved through a more personalized approach to midazolam dosing in pediatric intensive care units.Some limitations of our study should be acknowledged. Only a single bolus dose was administered to the healthy cohort whereas the critically ill child received prolonged, continuous infusions as well as multiple bolus doses. Thus, the pooled data from the two populations were unbalanced with respect to the dosing profile. However, single‐dose midazolam PK is predictive of steady state concentrations following continuous infusion and therefore the pooled analysis is unlikely to be biased. Although the inclusion of healthy children in the study adds weight to the identification of influential covariates, it does not in itself definitively establish a causal relationship between inflammation (rise in CRP) and CYP3A4 suppression. Future proof of mechanism study could perhaps utilize IL‐6 blocking antibodies to provide definitive proof. Finally, our cardiovascular status scoring tool was a bespoke development for this study and not a validated tool. Nevertheless, the inclusion of inotropes, fluid resuscitation, base excess, and urine output in the scoring calculation are considered standard clinical indicators and taken together, sensitive to changing cardiovascular status.5In this population PK study of midazolam, we have shown that acute systemic inflammation and cardiovascular status significantly influence midazolam clearance in children. This finding has implications for other drugs metabolized by CYP3A4 too. This confirms the findings of previous studies, but the inclusion of a control group of healthy children gives greater confidence in the identification of these two influential pathophysiological effects. Even with guideline‐recommended dosing regimens, significantly reduced midazolam clearance will result in supra‐therapeutic systemic concentrations and substantially increase the risk of adverse reactions that can impact short and long‐term clinical outcomes. Intensive care clinicians need to be mindful of this; intensify monitoring of such patients with a frequent assessment of sedation scores, arousal scales, daily interruption of sedatives, and monitoring for delirium to avoid overdosing.BN, HP, and HM had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: BN, HP, NS, JR, and HM. Analysis and interpretation of data: BN, HP, TP, RA, and HM. Drafting of the manuscript: BN, TP, HP, RA, and HM. Critical revision of the manuscript for important intellectual content: NS and JR.The authors declare no conflicts of interest.The study was approved by the East Midlands‐ Derby Research Ethics Committee in England (14/EM/1261).EUDRACT: 2014‐004958‐34. Date of registration: 30th/Jan/2015Written informed consent (and assent where appropriate) was obtained from each patient's parent(s)/legal guardian before any procedures or assessments were performed.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


Regardless of the degree of stenosis, vulnerable plaque is an important cause of ischemic stroke and thrombotic complications. The changes of the immune microenvironment within plaques seem to be an important factor affecting the characteristics of the plaque. However, the differences of immune microenvironment between stable and vulnerable plaques were remained unknown.In this study, RNA-sequencing was performed on superficial temporal arteries from 5 traumatic patients and plaques from 3 atherosclerotic patients to preliminary identify the key immune response processes in plaques. Mass cytometry (CyTOF) technology was used to explore differences in immune composition between 9 vulnerable plaques and 12 stable plaques. Finally, immunofluorescence technique was used to validate our findings in the previous analysis.Our results showed that more CD86+CD68+ M1 pro-inflammatory macrophages were found in vulnerable plaques, while CD4+T memory cells were mainly found in stable plaques. In addition, a CD11c+ subset of CD4+T cells with higher IFN-r secretion was found within the vulnerable plaque. In two subsets of B cells, CD19+CD20-B cells in vulnerable plaques secreted more TNF-a and IL-6, while CD19-CD20+B cells expressed more PD-1 molecules.In conclusion, our study suggested that M1-like macrophages are the major cell subset affecting plaque stability, while functional B cells may also contribute to plaque stability. The latest Global Burden of Disease report showed that ischemic heart disease and stroke had become the leading causes of mortality. The common pathological basis was atherosclerosis, a disease of vascular stenosis, in which subendothelial resident LDL undergoes a series of oxidations to produce ox-LDL with immune effects –3. On thThe bifurcation of the carotid artery is the most common site of atherosclerosis. About 10% to 15% of patients with carotid artery stenosis will experience an ischemic stroke, mainly related to local thrombosis caused by plaque rupture –9. UsualIn this study, we used Mass Cytometry , a precise, high-dimensional approach, to identify the differences in immune microenvironment between stable and vulnerable plaques. The results showed that more M1 pro-inflammatory macrophages were found in vulnerable plaques, while CD4+T memory cells were mainly found in stable plaques. In contrast, B-cell subsets and T-cells within vulnerable plaques showed higher activity. These clusters may be important for plaque vulnerability. Our study revealed the surface characteristics of these clusters in detail, which may help identify vulnerable plaque cells and understand the relevant mechanisms of rupture.From August 2021 to November 2021, 21 atherosclerotic patients undergoing carotid atherectomy at Beijing Tiantan Hospital were enrolled in this study after informed consent provided. The detailed clinical data of the enrolled patients are shown in Peripheral blood and plaque tissues from 3 patients were used for RNA-seq analysis. In addition, superficial temporal artery samples from 5 patients undergoing craniocerebral trauma surgery were used as blank controls. The plaque tissues and artery samples were washed with DPBS  within 1 hour after surgical resection. Then, TRIzol method was also adopted. Sequencing libraries were generated using rRNA-depleted RNA with an NEBNext Ultra Directional RNA Library Prep Kit for Illumina  following the manufacturer’s recommendations. We then performed the paired-end sequencing on illumina NovaSeq 6000as recommended by the supplier. After cluster generation, the libraries were sequenced on the Illumina HiSeq platform, and 150-bp paired-end reads were generated.http://genome.ucsc.edu). The clean reads were aligned with both genome hg19 and transcript reference using STAR v2.2.1, and gene expression was calculated by RSEM v1.3.0 using FPKM (fragments per kilobase of exon per million fragments mapped). We then compared RNA-seq in peripheral blood between healthy individuals and atherosclerotic patients. P<0.05 indicated statistically significant difference in expression. R was used for analysis of the gene expression data.In order to obtain clean data, raw data in fastq format were first processed through in-house Perl scripts. Reference genome and gene model annotation files were downloaded directly from the genome website. The index of the reference genome was built using bowtie2 v2.2.8, and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Hg19 RefSeq  was downloaded from the UCSC Genome Browser  within 1h after surgery. Each specimen was digested at 37°C for 1h using miscible liquids that contain collagenase type IV , DNase , hyaluronidase , collagenase type XI  and collagenase type II . The mixture was filtered through a 70μm cell strainers with DPBS and washed with red blood cell (RBC) lysis buffer . The dissociated cell suspension was then washed once with DPBS and resuspended in 1 mL of staining buffer .6 cells/mL in deionized water containing a 1:20 dilution of EQ Four Element Beads . The samples were then examined by CyTOF2 mass cytometry .A panel of 39 antibodies designed to distinguish a broad range of immune cells was used. Antibodies were either purchased in a preconjugated form from Fluidigm  or purchased from Biolegend  in a purified form and conjugated inhouse using the Maxpar X8 Multimetal Labeling Kit  according to the manufacturer’s instructions. The antibodies and reporter isotopes are included in CyTOF data were acquired in the form of.fcs files from the CyTOF2 system. The addition of EQ Four Element Beads allowed us to use the MATLAB-based normalization technique. The obtained data were uploaded to Cytobank. Firstly, beads are filtered and active cells were selected from the specific gate. Then, CD45+ cells were gated (see Plaques from 9 patients (4 vulnerable plaques and 5 stable plaques) were fixed overnight in 4% formalin (4°C) and embedded in paraffin blocks for paraffin sections. Three paraffin sections (4 um) were cut from each specimen. Hematoxylin and eosin (H&E) staining was performed. For immunofluorescence, paraffin sections were washed twice 15 min in PBS , permeabilized in 0.2%-0.5% Triton X-100  and blocked in 5% normal donkey serum  for 1 h and stained with primary antibody overnight. Primary antibody was detected using fluorescent-conjected second antibodies . Primary antibodies were: anti-CD68 , anti-CD86 , and anti-HLA_DR . Sections were mounted with fluorescence mounting medium . Fluorescent images were acquired on a Zeiss LSM880 NLO microscope and Zeiss Axio Scope Al was used to obtain H&E images. Three fields were randomly selected from each staining and the number of fusion particles of CD68, CD86 and HLA_DR in each field was counted. The Wilcoxon rank test was used and P<0.05 indicated statistically significant difference.In order to explore the composition of immune microenvironment in plaques, RNA sequencing data from plaques of 3 atherosclerotic patients and data from superficial temporal artery of 5 patients undergoing craniocerebral trauma surgery were analyzed. A total of 16036 differentially expressed genes (defined as |log2FoldChange|>1 and FDR <0.05) were identified. Among them, 3656 genes were upregulated in the atherosclerosis plaques 3.Finally, based on these RNA-Seq data, we used xCell  to infer the type and proportion of cells contained in atherosclerotic plaques. The results showed that endothelial cells, smooth muscle cells and fibrous cells were composed of superficial temporal artery (According to the imaging (CTA/MRA/UTA) results, we divided the enrolled specimens into 12 stable plaques and 9 vulnerable plaques  , 16. TheTwo types of B cells existed in samples. These B cells have different surface molecular patterns. Cluster9 expressed CD19+, whereas Cluster8 expressed CD20 but not CD19 , 18. TheMacrophages were the most abundant cluster in plaques and contained multiple cell subsets . In addiStroke has become a severe disease endangering human health, and its high mortality and disability rate have increased the global burden of disease , 23. ManAs an important cell type in plaques, T lymphocytes are widely involved in the development of atherosclerotic lesions , 26. Comvia these proinflammatory cytokines. The other CD19-CD20+ B cells from vulnerable plaques expressed higher level of PD-1. Studies have found that high expression of PD-1 by innate-type B cells after their activation by antigens and PD-1 molecule on the surface of B cells facilitate the adaptive responses through longer-lived plasma cells and memory cells generated  and Ethics Committee of Beijing Tiantan Hospital. The patients/participants provided their written informed consent to participate in this study.PG conducted the experiment and wrote this article. HL designed the experiment and collected the data. XY provided the surgical specimen and analysis data. YX, QH, LM helped organize some of the data. QZ and XY performed the atherectomy. WW provided the guidance for this experiment. DZ and JZ supervised this experiment. All authors contributed to the article and approved the submitted version.


Laboratory analysis of basic cerebrospinal fluid (CSF) parameters is considered as essential for any CSF evaluation. It can provide rapidly very valuable information about the status of the central nervous system (CNS). Our retrospective study evaluated parameters of basic CSF analysis in cases of either infectious or non-infectious CNS involvement. Neutrophils are effector cells of innate immunity. Predominance of neutrophils was found in 98.2% of patients with purulent inflammation in CNS. Lymphocytes are cellular substrate of adaptive immunity. We found their predominance in 94.8% of patients with multiple sclerosis (MS), 66.7% of patients with tick-borne encephalitis (TBE), 92.2% of patients with neuroborreliosis, 83.3% of patients with inflammatory response with oxidative burst of macrophages in CNS and 75.0% of patients with malignant infiltration of meninges (MIM). The simultaneous assessment of aerobic and anaerobic metabolism in CSF using the coefficient of energy balance (KEB) allows us to specify the type of inflammation in CNS. We found predominantly aerobic metabolism (KEB > 28.0) in 100.0% CSF of patients with normal CSF findings and in 92.8% CSF of patients with MS. Predominant faintly anaerobic metabolism (28.0 > KEB > 20.0) in CSF was found in 71.8% patients with TBE and in 64.7% patients with neuroborreliosis. Strong anaerobic metabolism (KEB < 10.0) was found in the CSF of 99.1% patients with purulent inflammation, 100.0% patients with inflammatory response with oxidative burst of macrophages and in 80.6% patients with MIM. Joint evaluation of basic CSF parameters provides sufficient information about the immune response in the CSF compartment for rapid and reliable diagnosis of CNS involvement. Basic cerebrospinal fluid (CSF) analysis is a very important approach to quickly assess the current state of the central nervous system (CNS). Despite this fact, basic CSF examination is often underestimated. The aim of this study is to present our scheme of basic CSF examination, to show the interpretation of results in several subgroups of patients with different CNS involvement and to stimulate interest in this important part of CSF analysis.Our basic CSF examination consists of a simultaneous assessment of the blood-cerebrospinal fluid barrier (BCB) permeability (see 1.1.), cytological composition (see 1.2.), energy ratios (see 1.2.) and detection of CNS tissue damage (see 1.3.). Measurement of total CSF protein concentration or albumin quotient is used to assess BCB permeability. Cytological analysis provides the essential information, especially addressing the presence of immunocompetent cells in CSF, possibly also the presence of tumor cells, signs of tissue damage, bleeding, presence of microbial pathogens, etc. Energy parameters, i.e., the simultaneous assessment of immunocompetent cells and KEB values in CSF is called cytological-energy analysis and allows us to determine intensity and the type of local inflammatory response in the CNS. CNS tissue damage at the level of baseline CSF examination is evidenced by aspartate aminotransferase (AST) catalytic activity determination.Approximately 80% of cerebrospinal fluid (CSF) is produced by ultrafiltration of blood plasma through the endothelium of the choroid plexus vessels. This structure is called the blood-cerebrospinal fluid barrier (BCB) and regulates the flow of immune system components into the CSF. The absence of pathological processes in the CSF is accompanied by the influx of a limited number of immunocompetent cells and a low concentration of proteins. In contrast, pathological processes in the CSF are associated with an increased number of cells and changes in the concentration of humoral components in the CSF. This condition is usually referred to as “increased BCB permeability”. It is evidenced as elevated concentration of total protein in CSF or albumin quotient   1,2,3,4,43,4,5.The brain parenchyma is highly vascularized. The endothelial cells of the brain capillaries are one from the key components of blood-brain barrier (BBB), which significantly influences the composition of the extracellular fluid in the brain. After crossing the ventricular wall, the fluid replenishes the remaining approximately 20% volume of the CSF. Therefore, CSF is thus an important source of information about the physiology or pathophysiology of the brain parenchyma  2,5,6,7,6,75,6,7The evaluation of cytological and energy parameters in the CSF compartment is performed in two steps. The first is to determine the number and composition of immunocompetent cells in the CSF. The second is to determine the level of their activation by examining energy parameters in the CSF. To determine energy parameters, we have recently proposed so-called coefficient of energy balance . KEB is calculated using the molar concentrations of glucose and lactate in CSF and calculate as followed:KEB is defined as the theoretical average number of adenosine triphosphate (ATP) molecules that are produced from one glucose molecule under the appropriate energy conditions in the CSF compartment. Activation of immunocompetent cells correlates with an increase in glucose and oxygen consumption and the development of anaerobic metabolism in CSF. The decrease in ATP production is reflected by a decrease in KEB values ,15,16,1728.0 to 38.0: aerobic metabolism in the CSF.20.0 to 28.0: slight anaerobic metabolism in the CSF.10.0 to 20.0: moderate anaerobic metabolism in the CSF.<10.0: strong anaerobic metabolism in the CSF.Based on the KEB values, we stratified our cases as follows:We consider aspartate aminotransferase (AST) catalytic activity in the CSF to be a readily available, easily measurable and inexpensive parameter for reliable assessment of CNS injury. This enzyme is present in all nucleated cells. Cellular damage is characterized by release of AST with subsequent elevation of AST activity in body fluids including CSF ,18.The absence of a pathological process in the CNS is characterized by no significant immune response in the CSF. There is the only basal immune surveillance there. The cellular and humoral components of the immune system in the CSF are at a basic level. The maximum leukocyte count in CSF is 4 cells/1 µL with predominance of resting lymphocytes (about 70%) and a minority of resting monocytes (about 30%). The low concentration of total protein in the CSF (<430.0 mg/L) indicates normal permeability of the BCB or the absence of any disturbance in the CSF circulation. Energy metabolism in CSF is aerobic with KEB values above 28.0. AST catalytic activity below 18.0 IU/L is not indicating tissue destruction in the CNS. No signs of hemorrhage are detected by CSF cytology ,18,19,20The inflammatory response in the CSF compartment is followed by mobilization of cellular and humoral components into the CSF. The consequence of increased BCB permeability is increase in total protein concentration in CSF. The number of cells in the CSF is increased and their composition is reflecting the type of inflammatory response. Immunocompetent cells are activated, and more energy is required. Glucose and oxygen consumption is increased in this way. The concentration of glucose in the CSF is decreased. There is the switch from aerobic glucose metabolism to anaerobic one. Energy production in the form of adenosine triphosphate (ATP) molecules is decreased. This process is revealed as decrease in KEB values. In addition, local inflammation in the CNS may be associated with tissue destruction resulting in increased AST catalytic activity in the CSF ,18,19,20Precise determination of the nature of the inflammatory reaction in the CSF can define the spectrum of causes of CNS pathologies.Neutrophils represent the major population of phagocytic cells and are the final effector cells of innate immunity, with a primary role to clear extracellular pathogens . The proIncreased BCB permeability, pleiocytosis with lymphocytes predominance, and aerobic or faintly anaerobic metabolism in the CSF compartment (28.0 > KEB > 20.0) usually represent the presence of serous inflammatory response in the CSF induced by either virus or spirochetes present in the CNS ,39,40,41Very similar findings in CSF corresponding to mononuclear or lymphocytic pleiocytosis, hyperproteinorhachia, hypoglycorhachia and hyperlactatorhachia have been described in patients with neurotuberculosis, neurolisteriosis and cryptococcal meningitis ,47,48,49There are also numerous non-infectious causes of inflammatory response in CSF, such as autoimmunity, injury, hemorrhage, ischemia, tumors and neurodegenerative disorders ,53,54,55Multiple sclerosis (MS) is an autoimmune immunopathological disease affecting primarily white matter of the brain and spinal cord. Immunopathological inflammation targets myeline sheets of neurons thus impairing nerve signal transduction with ultimate axonal loss. Other CNS structures, including oligodendrocytes, are also targeted. Blood-brain barrier (BBB) contributes significantly to the pathogenesis of multiple sclerosis. It is a gateway for autoreactive lymphocytes entry into brain parenchyma. Basic analysis of CSF of patients with multiple sclerosis usually shows only subtle nonspecific changes. Total protein concentrations are often within normal limits, leukocyte counts are low or only slightly elevated, CSF energy ratios are usually insignificant, and CNS tissue destruction is not apparent. Lymphocytic oligocytosis or slight lymphocytic pleiocytosis with predominance of activated lymphocytes and the presence of plasmocytes in the CSF are frequently found. The gold standard of laboratory analyses in multiple sclerosis is still detection of intrathecal oligoclonal immunoglobulin synthesis by isoelectric focusing of CSF and blood ,56,57,58Cytological analysis of CSF plays a key role in the detection of malignant infiltration of the brain meninges (MIM). Deep analysis of inflammatory parameters can identify the presence of malignancy in CNS even if tumor cells in the CSF cytology are absent. Elevated proteins, pleiocytosis with predominance of lymphocytes and hypoglycorrhachia are characteristic in CSF patients with malignant infiltration of meninges ,59,60,61This retrospective study was approved by the local Ethics Committee of the Masaryk Hospital Usti nad Labem, Czech Republic (reference number: 305/19). No informed consent was required for this study as this work did not involve any human experiment. All patient records and information were anonymized and deidentified.We performed a basic analysis of 524 cerebrospinal fluid samples evaluated as normal serving as controls for this study . In total, 304 CSF samples from patients with multiple sclerosis (MS), 39 CSF samples from patients with tick-borne encephalitis (TBE), 51 CSF samples from patients with central neuroborreliosis (NB), 113 CSF samples from patients with purulent inflammation (P) in the CNS induced by extracellular bacteria, and 31 CSF samples from patients with malignant infiltration of meninges (MIM) were enrolled to this study. We separately evaluated 6 CSF samples taken from 1 patient with cryptococcal meningitis, 1 patient with neurotuberculosis, 1 patient with neurolisteriosis and 3 patients with neuroborreliosis. These CSF samples were evaluated as “serous” inflammation in terms of cytological analysis and “purulent” in terms of energy analysis .BCB permeability was assessed using only cerebrospinal fluid total protein concentrations. We did not evaluate the albumin quotient because in some emergency cases cerebrospinal fluid samples only without blood samples were analyzed.Cerebrospinal fluid samples were centrifuged, and the mass concentration of total protein was determined by the turbidimetry method with bensetonium chloride on a Cobas 6000 analyzer .The samples of CSF were collected into tubes without anticoagulants and immediately transported to our clinical laboratory. The total number of elements in these samples was enumerated using a Fuchs-Rosenthal chamber under the optical microscope. Cytological smear using cytocentrifuge method was prepared immediately after receiving the sample in all cases. Permanent cytological smears were stained using Hemacolor . Microscopic analyses to determine cellular composition of CSF were performed by trained laboratory personal using Olympus BX40 microscope .Another aliquot of the samples was centrifuged and the molar concentrations of glucose using the hexokinase method and lactate using the lactate-oxidase and peroxidase method on a Cobas 6000 analyzer  were determined.KEB values were calculated for all samples, including rare cases with very low glucose concentrations below the measurement limit (=0.11 mmol/L). Glucose concentration of 0.11 mmol/L was used to calculate KEB values in all these anaerobic cases.The cerebrospinal fluid samples were centrifuged, and the catalytic activities of aspartate aminotransferase (AST) were determined by the IFCC method on a Cobas 6000 analyzer . Catalytic activities of AST in CSF exceeding 18.0 IU/L were identified as evidence of CNS tissue damage .Concentrations of total protein, numbers of leukocytes, the percentages of lymphocytes, neutrophils and monocytes and AST catalytic activities in the CSF are in box plots expressed as a median, the 1st and 3rd interquartile range and non-outlier range of values. KEB values are divided into subgroups with aerobic metabolism (>28.0), slight anaerobic metabolism (20.0 to 28.0), moderate anaerobic metabolism (10.0 to 20.0), and strong anaerobic metabolism (<10.0) in CSF in the bar graph. The nonparametric Mann-Whitney two sample tests were performed to compare each patients group with our control group. The variables were age-adjusted before testing. The 5% level was the criterion of significance.All statistical tests were carried out using Statistica 14.0 software .Using the Mann-Whitney two-sample test, we compared the CSF findings of our patients with CNS involvement to normal CSF findings.p < 0.001), tick-borne encephalitis , central neuroborreliosis , purulent inflammation , intensive inflammation with oxidative burst of macrophages , and malignant infiltration of meninges  , we found significantly higher total protein concentrations and leukocyte counts in the CSF of patients with multiple sclerosis  .p < 0.001) and NB (p < 0.001), and a significantly lower percentage of lymphocytes was found in patients with purulent inflammation (P) in the CNS (p < 0.001 *)  .p < 0.001), NB (p = 0.003), P (p < 0.001) and MIM (p > 0.001). The absolute highest neutrophil count is typical for patients with purulent inflammation in the CNS (P)  .p < 0.001), TBE (p < 0.001), NB (p < 0.001) and P (p < 0.001)  .p = 0.005), P (p < 0.001), MF (p = 0.012) and MIM (p < 0.001) compared to normal findings   .We consider basic CSF examination to be a very important part of complex CSF analysis. Its results provide key information about the current status of the CSF compartment and CNS. Many CNS impairments can be reliably detected by basic CSF analysis.Lymphocytes are immunocompetent cells of adaptive immunity. Consistent with observations of many authors, we found predominance of these cells in the CSF of patients with multiple sclerosis, thick-borne encephalitis, central neuroborreliosis, malignant infiltration of meninges and a very small group of several patients with cytologically proven “serous inflammation” and energy proven “purulent inflammation” in the CSF  10,19,2,37,40,41Energy parameters, especially KEB values, allows us to distinguish multiple sclerosis patients with predominantly aerobic metabolism in the CSF KEB > 28.0) of patients with tick-borne encephalitis and central neuroborreliosis with predominantly slightly anaerobic metabolism in the CSF (28.0 > KEB > 20.0), and in patients with malignant infiltration of meninges or in a very small group of several patients with cytologically proven “serous inflammation” and energy proven “purulent inflammation” in the CSF with strongly anaerobic metabolism in the CSF (KEB < 10.0) . The sam.0 of patWe are convinced that important cause for moderate expression of inflammatory response in the CSF of patients with multiple sclerosis is the lower contribution of BCB in the pathogenesis of this disease. Autoreactive lymphocytes are migrating from the blood into the brain parenchyma across the blood-brain barrier (BBB) . AutoimmOn the other hand, inflammation induced by invasion of pathogens usually manifests itself directly in the CSF. Therefore, we can observe increased BCB permeability with a higher influx of immunocompetent cells and proteins into the CSF . There iLymphocytic pleiocytosis and hyperproteinorhachia are typical in patients with thick-borne encephalitis and central neuroborreliosis . We founAlmost all samples of CSF of our patients with purulent inflammation in the CNS were characterized with high BCB permeability, large number of neutrophils and strong anaerobic metabolism in CSF . These fMycobacterium tuberculosis and Listeria monocytogenes [We separately evaluated a small group of cases with cytologically proven “serous” inflammation and energy proven “purulent inflammation”. These included one patient with cryptococcal meningitis, one patient with neurotuberculosis, one patient with neurolisteriosis, and three patients with neuroborreliosis with an atypical CSF laboratory picture. These cases are very similar with regards to mononuclear pleiocytosis, strong anaerobic metabolism in the CSF compartment (KEB < 10.0) and very high BCB permeability . Bicanicytogenes ,63,64. OFor this reason, we excluded these three cases from our group of patients with central neuroborreliosis.Some authors described hyperproteinorhachia, pleiocytosis with lymphocyte predominance, and hypoglycorrhachia in the CSF of patients with malignant infiltration of meninges ,59,60,61We evidenced the catalytic activity of AST in CSF as a reliable parameter to assess CNS parenchyma damage in our recent studies ,9,18. InWe have recently published the significance of AST catalytic activity evaluation in CSF of patients after CNS hemorrhage . Very prWe consider basic CSF analysis as a solid framework for both rapid differentiation of the type of local inflammatory response in the CNS and for optimization of subsequent CSF investigation.Inflammatory reactions in the cerebrospinal fluid are classified according to the predominant involvement of components of innate or adaptive immunity. Innate immunity activation is revealed by purulent inflammation usually induced by the presence extracellular bacteria in CNS. Reliable signs of this inflammation are extremely high numbers of neutrophils and strong anaerobic metabolism in CSF (KEB < 10.0).Adaptive immunity activation is characterized by the presence of lymphocytes in the CSF. The significant presence of these immunocompetent cells is evident in other subgroups of our patients. Whereas aerobic (KEB > 28.0) and slight anaerobic metabolism (28.0 > KEB > 20.0) are significantly predominant in the CSF of patients with multiple sclerosis, tick-born encephalitis and central neuroborreliosis, CSF of patients with inflammation with oxidative burst of macrophages in the CNS induced by intracellular bacteria, yeasts and tumor is characterized by a predominantly strong anaerobic metabolism (KEB < 10.0).Inflammatory response with a predominance of lymphocytes and a predominance of aerobic (multiple sclerosis) or slightly anaerobic metabolism  in the CSF is called serous inflammation. This type of inflammation is usually associated with low or slightly increased BCB permeability and absence of CNS tissue destruction. In contrast, intense inflammation with oxidative burst of neutrophils (purulent inflammation) or macrophages is associated with increased BCB permeability and destruction of CNS tissue is proven by increased catalytic activity of AST in the CSF (>18.0 IU/L).


Compartment Syndrome (CS) has been recognized as a potential factor that worsens testicular viability after detorsion, especially in borderline cases of prolonged ischemia. Fasciotomy of the testicular tunica albuginea to relieve the pressure associated with CS has been proposed to accommodate edema after detorsion, embracing the raw fasciotomy area with tunica vaginalis flap (TVF) or graft. Fashioning the TVF can be tedious in cases of severe scrotal edema. Herein we present a technique that facilitates and expedites the procedure, maintaining the fasciotomy area decompressed. In testicular torsion, where the testis remains with dark coloration and questionable viability after detorsion a longitudinal releasing incision is made in the tunica albuginea (fasciotomy) to decrease compartmental pressure. If signs of parenchymal recovery  are seen an orchio-septopexy is performed, suturing the incised albuginea’s edges to the septum with a running suture, avoiding CS as well as re-torsion. Orchio-septopexy was performed in 11 cases with a mean age of 11.9 years (3-17). All cases had clinic follow-up and testicular Doppler US with a mean of 9.5 months (6-24). 6/11 cases (54%) were salvaged, with good vascularity in the Doppler US and maintained more than 50% testicular volume compared to the contralateral side. Orchio-septopexy after testicular fasciotomy is a simple and fast technique that can be utilized in cases of prolonged testicular ischemia and questionable viability. More than half of the testes recovered, encouraging us to propose its utilization as well as its validation by other surgeons. Testicular torsion (TT) is a common emergency condition encountered by Pediatric urologist with annual incidence of 3.8 per 100,000 pediatric patients   . The teWe performed a retrospective review of all cases with testicular torsion that underwent scrotal exploration between January 2018 and 2020. Patients with unilateral intravaginal TT and a minimum of one documented follow up visit have been included. Perinatal TT, torsion of undescended testis, TT in patients with single gonad and documented contra-lateral testicular abnormality were excluded. Patient’s demographics as well as time of evolution was noted.https://intbrazjurol.com.br/videos/20220128_Pippi-Salle_et_al.mp4 >The scrotum is explored via midline incision, identifying and preparing the testicular septum as well as exposing the affected side first in order to confirm the diagnosis of TT. The testicle is untwisted and kept covered with gauzes soaked with warm saline. Attention is then driven to the contralateral testicle that is pexed to avoid future torsion. Then the affected side is re-assessed. If it recovers normal colour it is then pexed with monofilamentar non absorbable 5/0 sutures. If it remains darker after detorsion a generous incision in TA is made, aiming decompression and improvement of vascularization. If parenchyma remains dark and with bleeding points it is assumed that irreversible necrosis have ocurred and orchiectomy is warranted. If color improves and bleeding points appear in the parenchyma an orchio-septopexy is performed via running suture of polydioxanone 5.0 sutures , starting at the lower edges of the fasciotomy   . For adThe summary of intra-operative approach of TT as shown in Post-operative physical examination as well as Doppler US were performed at least 6 months after surgery. The definition of post-operative testicular salvageability is having adequate blood flow on Doppler US, maintaining at least of 50% testicular volume compared to the contralateral side.Between January 2018 and 2020, 21 patients underwent scrotal exploration for TT. The age of patients ranged from 3 to 17 years old. Left sided was slightly more affected than right . Five cases (23.8%) improved with detorsion alone and underwent routine bilateral orchiopexy. In 16 patients the color did not improve after detorsion and underwent fasciotomy. Dark coloration with necrotic looking parenchyma remained in 5 cases and these patients underwent orchiectomy. Eleven testicles improved the color, having points of parenchyma bleeding and underwent orchio-septopexy. All patients who underwent orchio-septopexy after fasciotomy had clinic follow-up and testicular Doppler US with a mean of 9.5 months   .Compartment syndrome (CS) is a well-known and important initiator of post ischemic insult to various organs constrained by natural envelops, including but not limited to orthopedics and trauma, being well documented in the literature   . The inAlthough we did not measure the time spent to perform orchio-septopexy or compare with other techniques, it seems faster and easier than the TVF technique, done previously by the senior author. This modification, with a follow-up of 9.5 months, confirms the results published in previous paper from Toronto, a similar experience with 11 patients who had TVF after fasciotomy, obtaining 54% of testicular viability on Doppler US at 7.9 months mean follow-up   .Orchio-septopexy seems to be particularly useful in patients with prolonged clinical evolution. Our study confirms the impression of Chu, et al. who retrospectively reviewed a cohort of 182 patients who 49, 36, and 97 underwent orchiectomy, TVF and orchiopexy alone, respectively   . In theOur study has limitations: it is retrospective and reviews a small number of patients with relatively short follow-up. In addition, in order to confirm our impression that the proposed technique is faster and easier to perform, it should be ideally done in a randomized fashion with the established utilization of TVF to cover the fasciotomy area, measuring the duration of the procedures with both techniques as well as the outcomes. However, its simplicity and the encouraging early results stimulate us to propose its utilization and possible validation by other surgeons.In conclusion, orchio-septopexy after fasciotomy of tunica albuginea is a simple and fast technique that can be utilized in cases of prolonged testicular ischemia with questionable viability. More than half of the testis recovered, encouraging us to propose it as alternative to treat testis jeopardized by compartment syndrome after detorsion.


To evaluate the efficacy and adverse effects of hypoglossal nerve stimulation in adolescents with down syndrome and obstructive sleep apnea.A systematic search was conducted using PubMed, Web of Science, Embase, and Scopus databases. The systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. A comprehensive search strategy used a combination of Medical Subject Headings and free words with “OR” and “AND.” Articles were screened to extract data reporting apnea-hypopnea index, quality of life, voltage, follow-up duration, and complications. All included participants were adolescents with down syndrome and obstructive sleep apnea.P < 0.001) after 2 case reports were excluded. The pooled OSA-18 were significantly decreased in 88 patients after treatment  after excluding 5 studies. Four investigations examined the necessity to optimize stimulation voltage for arousal during treatment. The most common complication was pain or discomfort in the tongue or mouth. Most studies had relatively short patient follow-up periods, with the most extended follow-up being 44–58 months.A total of 92 articles were identified, of which 9 articles met the inclusion criteria. A total of 106 patients were included. All the studies showed that patients receiving hypoglossal nerve stimulation experienced a significant decrease in apnea-hypopnea index (at least 50%). The pooled AHI was significantly lower in patients following treatment (mean AHI reduction 17.43 events/h, 95% confidence interval 13.98–20.88 events/h, Hypoglossal nerve stimulation significantly reduces apnea-hypopnea index and improves the quality of life; and thus, could be a potential alternative therapy for obstructive sleep apnea in adolescents with down syndrome. The adolescent's age, potential complications, adverse events, long-term efficacy, and comfort, needs to be considered while performing hypoglossal nerve stimulation. Obstructive sleep apnea (OSA) is a sleep-related breathing disorder that causes hypoxia and fragmented sleep, because of repeated airway obstruction or collapse . OSA affDown syndrome (DS), trisomy 21, or redundancy of chromosome 21, is one of the most complex human congenital diseases . AdolescUntreated OSA can affect a child's development, including reduced learning abilities, speech and language delays, and impaired cognitive flexibility and memory . CurrentSince 2014, hypoglossal nerve stimulation (HNS) has been approved by the US Food and Drug Administration (FDA) for treating OSA in adults . HNS impHowever, there is no consensus about reducing OSA in adolescents with DS using HNS. In 2016, the first case of HNS for OSA in adolescents with DS was reported . AlthougTo the best of our knowledge, this is the first review of HNS for treating OSA in adolescents with DS based on the existing research. We sought to evaluate the efficacy and adverse effects of HNS in adolescents with DS and OSA and to clarify the underlying processes of HNS for treating OSA.This systematic review was conducted following the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines . EthicalA systematic search was carried out through PubMed, Web of Science, Embase, and Scopus databases. The search strategy used a combination of Medical Subject Headings (MeSH) and free words with “OR” and “AND.” Retrieval words included “DS,” “Trisomy 21,” “OSA,” “sleep apnea syndromes,” “HNS,” and “upper airway stimulation.” The detailed retrieval strategy is available in The requirements for studies to be included were as follows: (I) Types of participants – adolescents (the age ranges from 10 to 21 years)  with DS Two reviewers (WK and KZ) extracted data using Excel  spreadsheet. The data included were as follows: authors, year of publication, study design, voltage titration, sample size, age, treatment assessment, intervention time, follow-up duration, main results, adverse events, and conclusions.Two reviewers (WK and KZ) independently evaluated the selected articles. The quality of the articles included in this review was assessed using the National Institutes of Health quality assessment tools  . The quaP value was <0.05 and 95% confidence intervals (CIs) were given. The Q statistic, with a significance level of P < 0.10, was used to investigate the heterogeneity of Mean differences. The study was divided into two groups according to whether the number of cases was >10, and a subgroup analysis was conducted. The random-effects model was employed throughout the analysis.Mean differences (MD) were calculated to create forest plots of continuous data to analyze the variations in the apnea-hypopnea index (AHI) and A validated, disease-specific quality of life instrument for OSA (OSA-18) between HNS and non-HNS. We merged data using Review Manager 5.3 software. The test was regarded as statistically significant when the A total of 92 articles were obtained from the 4 databases, out of which 41 articles remained after excluding the duplicate ones. The literature was then further screened using the article titles and abstracts and 20 irrelevant articles were excluded. Next, two abstracts without full text, one surgical procedure study, one with cellular experimental study, four review articles, one no result article, and three non-adolescent studies, were also excluded by full-text literature identification. Ultimately, 9 studies met the inclusion criteria for this systematic review.A total of 106 patients were included in all investigations; three articles had a sample size with more than 10, while 6 articles had a sample size of no more than 10 cases. The first related publication was published in 2016. Further, an article was published each, in 2017, 2019, and 2020 years. Because of the rising interest in this topic, four relevant articles were published in 2021. P < 0.001); however, there was moderate heterogeneity between the studies . The forest plot of studies investigating AHI is shown in All the studies showed that patients receiving HNS experienced a significant decrease in AHI, based on the Polysomnography (PSG) results. Diercks et al. reported the first case of HNS treatment for a 14-year-old boy, whose AHI dropped from 48.5 to 3.4 events/h . Yu et aP < 0.001), and there was no evidence of study heterogeneity . The forest plot of studies investigating OSA-18 is shown in Five studies used the OSA-18  and Epworth Sleepiness Scale (ESS) questionnaires, to examine the improvements in treatment durations , 25, 26.Four investigations examined the necessity to optimize stimulation voltage for arousal during treatment , 20, 23.Most researchers followed patients with DS and OSA for a short duration. Only one study had a follow-up duration of 44 to 58 months , two stu2 (P = 0.96)  factors , 23–26, 2 , 23, 26. = 0.96) . Age- anPain or discomfort in the tongue or oral cavity, were the most common complications. Notably, three studies documented the occurrence of serious adverse events, such as the incidences of reading and reoperations , 25, 26.In this review, we investigated the therapeutic effects of HNS for OSA in adolescents with DS, the enhancement of participants' quality of life, and the benefits of the intervention, based on the current literature. In addition, we discussed its potential therapeutic mechanism. After receiving HNS therapy, all participants experienced a significant reduction in AHI. Participant's OSA-18 and ESS scores also indicated noticeable improvements in some studies. Therefore, HNS can be considered as an effective treatment for OSA in adolescents with DS and is a better tolerated option than CPAP or equally well tolerated option than CPAP.OSA is a complex condition that demands a multimodal treatment strategy, particularly for adolescents with DS, due to the abnormalities in their airway structure . NowadayWith the advancement of medical technology, electrical stimulation devices have shown numerous benefits in treating OSA. According to research, the loss of genioglossal muscle tone is strongly correlated with airway collapse . A hypogThe degree of AHI reduction and quality of life improvement, are the essential measures to monitor the effectiveness of therapeutic modalities for treating OSA. All the adolescents included in this systematic review had used CPAP before receiving HNS therapy, but none of them was able to tolerate it. The PSG of all the participants showed a significantly lower AHI score during the HNS therapy, than that before receiving it. This result is comparable to that obtained using typical pediatric CPAP treatment. According to King et al., CPAP therapy decreased the AHI of pediatric patients from 9.8 (5.7–46.0) to 3.3 (0.4–2.2) . InteresAlthough there are positive findings on the efficacy of HNS in adults, the data are inconsistent. Zhu et al. followed 82 patients with moderate-to-severe OSA for 4 years and found that the stimulation threshold of the hypoglossal nerve remained constant . FurtherThe voltage threshold of HNS is also an important factor to consider and to investigate. In adolescents, the voltage threshold for HNS may differ from that of adults. Since adolescents with DS are going through a particular stage of rapid physical growth, the efficacy and safety of HNS treatment should be focused on. While four studies have been examined at titrating stimulation voltage, none have precisely investigated how variations in voltage stimulation intensity affect the efficacy of the HNS treatment. Diercks et al. observed that increasing stimulation voltage during HNS treatment significantly reduced the AHI of patients . The thrSo far, it is unknown whether higher voltage stimulation is necessary during HNS therapy to obtain improved efficacy. According to certain studies, HNS can effectively control OSA in adolescents with DS, but the underlying OSA is likely to continue until adulthood . TherefoIt is well recognized that adolescents with DS suffer from cognitive impairment and that prolonged sleep hypopnea might worsen this impairment . Further2, and no circumferential airway collapse at the level of the velopharynx (BMI may have a significant impact on HNS treatment outcomes. The Food and Drug Administration (FDA) has recommended HNS for treating OSA in neurotypical adults with an AHI <50 events/h, a BMI <32 kg/mopharynx , 39. Adoopharynx . Accordiopharynx . CurrentAdolescents with DS who were treated with HNS may experience complications or adverse events. Therefore, understanding the reasons behind adverse occurrences and their consequences can help improve therapeutic outcomes. Three studies reported adverse events, such as tongue or mouth pain, rash, tissue inflammation, cheek swelling, irritation-related discomfort, insomnia, pneumothorax, and swallowing or speech-related problems. These adverse events were primarily caused by device displacement, infection, device migration, and poor postoperative pain control. If the patient has a small chest, the stimulator could become squeezed by the beating heart . As the This review has some limitations. First, the sample sizes of the included studies were limited, and several were case reports. Also, none of these studies had a control group. Thus, these investigations might be affected by research bias. Second, the safety of HNS therapy and the reasons behind some adverse events, including some severe ones, weren't fully understood by these investigations. Additionally, there was no record of adolescent tolerance to electrical stimulation in these investigations. It is important to consider the safety of HNS therapy and guard against adverse outcomes. Third, the follow-up duration of these participants was typically limited, and the long-term consequences of HNS on adolescents with DS and OSA remains unknown. Therefore, large-scale, prospective, randomized controlled, multicenter studies, are required in the future.In conclusion, HNS can significantly reduce the AHI and improve the quality of life of adolescents with DS, and can be considered as a potential alternative treatment for OSA. As adolescents get older, more studies are required to fully demonstrate the effectiveness of HNS, with a greater focus on potential complications, adverse events, long-term efficacy, comfort, and cost-effectiveness throughout HNS treatment. Comprehensive therapy protocols incorporating two or more therapeutic techniques, including CPAP, upper airway surgery, and HNS, are also worthy of investigation. Currently, HNS has not yet received FDA approval for pediatric patients, which has restricted its widespread clinical application.The original contributions presented in the study are included in the article/PL: conceptualization, methodology, writing, review and editing, and approval for final version. WK and KZ: investigation, data curation, formal analysis, and approval for final version. CF: investigation, data curation, software, and approval for final version. XD: methodology, writing—review and editing, and approval for final version. XM: conceptualization, writing, review and editing, and approval for final version. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


There is no consensus on anatomic landmarks or reference axes with which to accurately align rotational position of tibial component. Using the tibial tubercle, commonly referring to the Akagi line and the Insall line, for anatomic reference was widely accepted. However, it is unknown about the predictors that may affect the reliability of using the tibial tubercle for aligning tibial component rotation. The aims of our study were (1) to investigate the reproducibility and accuracy of using the tibial tubercle for aligning tibial component rotation and (2) to determine predictors resulting in discrepancies of the tibial component rotation when referring to the tibial tubercle.α formed by the tibial anteroposterior (AP) axis and the Akagi line and the angle β formed by the tibial AP axis and the Insall line were measured to quantify the discrepancies of the Akagi line and the Insall line. Independent variables, including the tibial tubercle-to-trochlear groove distance (TT-TG), tibial tubercle to posterior cruciate ligament (TT-PCL), and knee rotation angle (KRA), hip–knee–ankle angle (HKA), medial proximal tibial angle (MPTA), and tibial bowing (TB), were measured. Pearson’s product moment correlation coefficients and multivariable linear regression analysis were calculated to assess relationships between independent variables and the two defined angles.A total of 160 patients with osteoarthritis were recruited before total knee arthroplasty. The angle α and the angle β. In terms of a cutoff of TT-TG = 9 mm, 100% cases and 97% cases for using the Akagi line and Insall line, respectively, were located in the defined safe zone (− 5° to 10°).All defined measurement were available for 140 patients. The Akagi line rotated internally with 1.03° ± 4.25° in regard to the tibial AP axis. The Insall line rotated externally in regard to the tibial AP axis with 7.93° ± 5.36°. Three variables, including TT-TG, TT-PCL, and KRA, tended to be positively correlated with the angle The tibial tubercle  is found to be a useful and promising anatomic landmark for aligning the tibial component rotation. The TT-TG, with a cutoff value of 9 mm, is helpful to choose the Akagi line or Insall line, alternatively. Total knee arthroplasty (TKA) is one of the most common elective orthopedic surgeries performed worldwide and has been recognized as the highly cost-effective and curative method to relieve pain and improve function for patients with end-stage knee osteoarthritis when conservative treatment is unsuccessful , 36. OveBesides restoration of the coronal and sagittal alignment of the affected lower extremity, proper femoral and tibial component rotational positioning is also essential for successful TKA. The consequences of component malrotation are considered to produce abnormal patellofemoral kinematics , 35, 47,Unlike the femoral side, there is no consensus on anatomic landmarks or reference axes with which to accurately align rotational position of tibial baseplate. High rates of malalignment greater than 3° were reported to occur in rotational position of tibial component , 32. To The two basic objectives in this study were (1) to investigate the reproducibility and accuracy of using the tibial tubercle for aligning tibial component rotation and (2) to determine predictors resulting in discrepancies of the tibial component rotation when referring to the tibial tubercle.The study was approved by the Huabeiyiliao Jiankangjituan Fengfeng Zongyiyuan (ID: 2018-049-11). Informed consent was obtained from all patients before enrolled in the study.This team successively reviewed, from January 2018 to October 2019, medical records and imaging documents of 160 patients diagnosed with severe knee osteoarthritis and prior to primary TKA in our institute. Patients were excluded if any of these following items were met: (a) rheumatoid arthritis or inflammatory arthritis else; (b) surgical history of the affected lower extremities; (c) unavailable imaging modalities or the scheduled anatomic landmarks could not be distinctly identified; and (d) instability of knee joints or knee flexion contractures ≥ 10°.All patients had finished CT scans of the affected knee and standard standing AP radiographs of the entire lower extremities. The patients were placed in the supine position on the scanning table the knee fully extended with neutral or slight external rotation as needed for comfort. Straps were wrapped around the thigh and lower leg to minimize motion. A 16-detector row CT scanner  was used. These CT scans were acquired using the following parameters: 512 × 512 matrix, 120 kV, 100 mAs, 1 s rotation time, 1 mm slice thickness, 0 mm slice skip, a 14 cm field of view, and bone kernel. The CT images obtained were then imported into a personal computer to carry out our measurements using RadiAnt DICOM software  with an accuracy of 0.1° and 0.1 mm. This system allows linear and angular measurements to be made on images and marked while scrolling through successive axial CT images. The AP radiographs were performed in standing position.To analyze risk factors that might affect the accuracy and reliability of the tibial tubercle used as a landmark for rotationally positioning the tibial component, this investigation reviewed axial measurements, including the tibial tubercle-to-trochlear groove distance (TT-TG), tibial tubercle to posterior cruciate ligament (TT-PCL), and knee rotation angle (KRA), and coronal measurements, including hip–knee–ankle angle (HKA), medial proximal tibial angle (MPTA), and tibial bowing (TB). Two authors, one well-trained orthopedist and one specialist in musculoskeletal radiology, independently performed all measurements in a blinded and randomized fashion. A subset of 30 patients was randomly selected to conduct all measurements again after four weeks.α formed by the tibial AP axis and the Akagi line and the angle β formed by the tibial AP axis and the Insall line were used to quantify the discrepancies of the Akagi line and the Insall line  axis was determined as the vertical line of the TEA, passing through the center of the PCL. Two points, locating at the medial border and the medial third of the patellar tendon, were labeled on the level of the most cephalad image on which the patellar tendon was completely in contact with the tibial tubercle, and then were cast on the plane of the tibial AP axis. The Akagi line and the Insall line were determined connecting the center of the PCL and the medial border and the medial third of the patellar tendon. The angle ine Fig. . A positThe TT-TG distance was measured according to the technique described by Schoettle et al. . The posTwo aforementioned levels, containing the tibial tubercle and the PCL, were selected to calculate the TT-PCL distance. The TT-PCL distance was determined as the mediolateral distance between one line, passing the midpoint of the patellar tendon attached at the tibial tubercle and perpendicular to the tibial posterior condylar line, and the other line, passing the medial border of the PCL and perpendicular to the tibial posterior condylar line  Fig. 3)3).Fig. 3The KRA represented relative rotation of the femur and tibia in relation to each other and was defined as the angle between the posterior condylar line of the femur and a tibial posterior condylar line Fig. .Fig. 4KRThe HKA was formed by the mechanical axis of the femur  and the mechanical axis of the tibia (connecting the center of the ankle and the center of the knee). A positive value indicates a genu varum Fig. a. The MPp value < 0.05 denoted a statistically significant difference. To detect an expected Pearson correlation magnitude of 0.25 , a sample size of 112 subjects was necessary.Descriptive analyses were performed using frequencies with percentages for discrete or dichotomous variables as well as means with standard deviations (SDs) for continuous variables. The Kolmogorov–Smirnov normality test was performed to test whether our data fit a Gaussian distribution and all data passed the test. Pearson’s product moment correlation coefficients were initially calculated to assess the relationship between the discrepancies of the Akagi line and predictors, including sex, BMI, affected sides, age at surgery, and the defined imaging parameters. Subsequently, variables with a P value below 0.2 or a correlation coefficient of ≥ 0.25 were entered into a stepwise multivariable linear regression analysis. Model fit of the final selected model was assessed via normal quantile–quantile plots of the standardized residuals. The intra-observer and interobserver reliability were determined by calculating the interclass correlation coefficient (ICC), and an agreement of > 0.75 was considered excellent. All analyses were conducted using the IBM SPSS Statistics version 22.0 software package . All statistical tests were two-tailed, and a α or angle β. All defined measurements were showed in Table Ultimately, baseline demographics and defined measurements were available for 140 patients (Table α = − 1.03 ± 4.25). Three variables, including TT-TG, TT-PCL, and KRA, tended to be positively correlated with the angle α , without multicollinearity . The regression coefficient were -10.83 of the constant, 0.54 for the TT-TG, 0.13 for the TT-PCL, and 0.57 for the KRA, and the R2 values and adjusted R2 values were 0.716 and 0.709, respectively. For the prediction of the angle α, a formula could be expressed as: Angle α (degrees) = − 10.83 + 0.54 × TT-TG + 0.13 × TT-PCL + 0.57 × KRA , without multicollinearity . The regression coefficient were − 3.22 of the constant, 0.72 for the TT-TG, 0.12 for the TT-PCL, and 0.56 for the KRA, and the R2 values and adjusted R2 values were 0.626 and 0.618, respectively. For the prediction of the angle β, a formula could be expressed as: Angle β (degrees) = − 3.22 + 0.72 × TT-TG + 0.12 × TT-PCL + 0.56 × KRA , there were no outliers to use the Akagi line when the TT-TG > 9 mm, and were two outliers to use the Insall line when the TT-TG < 9 mm  It is advisable to determine the tibial component rotation according to the combination of the Akagi line and Insall line with a cutoff of TT-TG = 9 mm .The present study demonstrated several main interesting findings: (1) The tibial tubercle, referring the Akagi line and Insall line, could be a reliable anatomic landmark for aligning the tibial component rotation with promising accuracy and excellent reproducibility. (2) Independent variables, including TT-TG, TT-PCL, and KRA, were found to be risk contributors for the angle The axial rotation alignment, namely the reciprocal internal–external rotation alignment between the femoral and tibial components on their respective bony articular surface, deserves as a crucial requirement for successful TKA. High rates of discrepancies have been reported to be associated with the postoperative rotational alignment of tibial baseplate , 32. Rotα values indicate that using the Akagi line as rotational reference makes all cases within the defined safe zone when TT-TG distance ≥ 9 mm. On the other hand, using the Insall line as rotational reference makes 97% (75 in 77) cases within the defined safe zone when TT-TG distance < 9 mm. The effect, reported by Howell et al. [α and angle β. There were else literature demonstrating that the Akagi line rotated mildly externally relative to the tibial AP axis [According to the results, the Akagi line is approximately parallel to the tibial AP axis with slight internal rotation. The distributions of the angle l et al. , of the l et al. , 4. Notel et al.  and Kim l et al.  who also AP axis , 27. The AP axis , 27. A s AP axis . Lützner AP axis . In contRecently, several novel references have been put forward and been suggested to be used to determine the rotation alignment of the tibial component , 23. TheTo avoid depending on anatomic landmarks, the ROM (range of motion) technique, which allowed the tibial baseplate to float into the orientation with respect to the femoral component while the knee was placed through a full arc of motion, was preferred , 16, 17.