Introduction: The reduction of drag is a crucial concern within the shipping industry as it directly influences energy consumption. This study addresses this issue by proposing a novel approach inspired by the unique ridge structure found on killer whale skin. The objective is to develop a non-smooth surface drag reduction method that can effectively decrease drag and improve energy efficiency for ships.Methods: The study introduces a technique involving the creation of transverse bionic groove surfaces modeled after the killer whale skin’s ridge structure. These grooves are aligned perpendicular to the flow direction and are intended to modify the behavior of turbulent boundary layer flows that form around the ship’s hull. Numerical simulations are employed using the Shear Stress Transport k-ω model to analyze the effects of the proposed groove surface across a wide range of flow conditions. The research investigates the impact of various parameters, such as the width-to-depth ratio (λ/A), groove depth, and inlet velocity, on the drag reduction performance of the bionic groove surface.Results: The study reveals several key findings. Optimal shape parameters for the bionic groove surface are determined, enabling the most effective drag reduction. The numerical simulations demonstrate that the proposed groove surface yields notable drag reduction benefits within the velocity range of 2∼12 m/s. Specifically, the friction drag reduction ratio is measured at 26.91%, and the total drag reduction ratio at 9.63%. These reductions signify a substantial decrease in the forces opposing the ship’s movement through water, leading to enhanced energy efficiency.Discussion: Comparative analysis is conducted between the performance of the bionic groove surface and that of a smooth surface. This investigation involves the examination of velocity gradient, streamwise mean velocity, and turbulent intensity. The results indicate that the bionic groove structure effectively mitigates viscous stress and Reynolds stress, which in turn reduces friction drag. This reduction in drag is attributed to the alteration in flow behavior induced by the non-smooth surface.Conclusion: The study proposes a novel approach for drag reduction in the shipping industry by emulating the ridge structure of killer whale skin. The transverse bionic groove surface, aligned perpendicular to flow direction, demonstrates promising drag reduction outcomes across diverse flow conditions. Through systematic numerical simulations and analysis of key parameters, the research provides insights into the drag reduction mechanism and identifies optimal design parameters for the groove surface. The potential for significant energy savings and improved fuel efficiency in maritime transportation underscores the practical significance of this research. Reducing energy consumption has always been an important objective in engineering. Due to the drag force, ships that undertake maritime traffic and transport functions consume a large amount of fuel. For underwater vehicles such as submarines, reducing the total drag by 10% can increase their cruising speed and range by approximately 3.75% under the same conditions . FrictioInspired by the unique non-smooth surface structures of organisms in nature, bionic non-smooth surface drag reduction methods, such as the pit structures of dung beetle skin and the denticle structures of shark skin, are a new feasible means of applying bionic theory to reduce the drag force of underwater vehicles. This method involves changing the surface structure of an object to change the flow field . By inteRecent research has shown that bionic non-smooth surfaces can effectively reduce surface frictional resistance. For instance, Besides, However, the disadvantages of microstructure such as difficulty in fabrication, poor sustainability, and expensive maintenance cannot be ignored. Furthermore, surface fouling caused by the adhesion of marine organisms makes it easy for the microstructure on the surface of an underwater vehicle to lose its drag reduction effect . And theOrcinus orca have high swimming speeds, which have been attributed to the drag-reducing properties of their unique skin structure  was used to generate the bionic groove curve by the cubic spline interpolation method. The interpolation points were chosen to ensure that the shape of the bionic groove approximates the ridge of killer whale. The bionic groove curve is shown in L (L = 2m), and the transverse bionic grooves were arranged along the perpendicular to the flow direction (x-direction) with coordinates y = 0 and y = −A corresponding to the peaks and troughs of the bionic grooves, respectively. The total length of the bionic groove area is 1.2 m, and the length of the front and rear smooth surfaces is the same.Novel bionic grooves were arranged on a plate to investigate their ability and mechanism to reduce friction drag. The arrangement of the bionic grooves is shown in A) should be less than the thickness of the boundary layer (δ)  is 998.2 kg/m3, dynamic viscosity (μ) is 0.001003, and the flow velocity range is 2–12 m/s. The Reynolds number range in the bionic groove region is 7.96 × 105 to 1.91 × 107, ensuring that its flow state is turbulent. The thickness of the smooth surface boundary layer at the corresponding location in the bionic groove region is shown in A ≤ 6.8 mm.The influence of non-smooth structure on the boundary layer fluid is an important way to reduce frictional resistance, so the amplitude of the bionic groove (ayer (δ) . The thiOrcinus orca satisfy the scaling requirements for non-smooth surface drag reduction, they have the same microstructural drawbacks. Therefore, the ridges of Orcinus orca were appropriately scaled up according to the bionic principle to ensure the drag reduction performance of the new bionic grooves while making them possible for practical application. To study the effect of the variation of the geometric parameters of the bionic grooves on the drag reduction, 9 types of grooves were designed with the geometric parameters shown in Although the ridges of SST k-ω model is a deformation of the standard k-ω model, which incorporates cross-diffusion originating from the ω equation, and the turbulent viscosity takes into account the propagation of turbulent shear stresses. Moreover, it has higher accuracy for simulating near-wall flow, inverse pressure gradient flow, and turning. The equations of the SST k-ω model are as follows:k is the turbulent kinetic energy, ω is the specific dissipation rate, μt is the turbulent kinematic viscosity coefficient, Gk and Gω are the turbulent kinetic energy generated by the mean velocity gradient and ω equations, respectively, σk and σω represent the turbulent Planter constants for k and ω, respectively, Sk and Sω are the correlation source terms, Yk and Yω represent the divergence terms for k and ω, respectively, and Dω are the turbulent cross terms.In the present study, the numerical simulations are carried out using the SST (shear stress transport) turbulence model, with the SIMPLE (Semi Implicit Method for Pressure Linked Equations) algorithm. The discrete format used for the numerical simulation is the second-order upwind scheme. And the physical parameters of incompressible liquid water are kept constant during the calculation. The SST k-ω turbulence model are as follows:The expressions of the turbulent eddy viscosity coefficient for the a1 is Bradshaw’s constant with a value of 0.31; Ω is the absolute value of the vortex; F2 is a switching function, and in the SST k-ω turbulence model, F2 takes a value of 1 in wall turbulent and F2 takes 0 in free shear flow. The expression F2 is:In the above equation, x, y, and z directions are denoted by u, v, and w, respectively, and the total length of the flow domain along the flow direction is denoted by L(L = 2m). The width and height of the flow domain is always 0.2 m.A 3D geometric model of the flow domain used in numerical simulation was established according to the bionic groove arrangement in x = 0), the flow enters the flow domain with a uniform velocity. The groove surface that can be replaced with the smooth surface is a no-slip wall at the bottom of the flow domain, the upper surface has a zero pressure and no velocity gradient across it and the outlet (at the plane x = L) has zero velocity gradient across it and zero pressure. The two side walls are symmetric boundary conditions.The boundary conditions of flow domain are shown schematically in y+ close to 1, τw is the wall shear stress. The grid of the entire flow domain was refined to verify that the computational results were independent of the number of grids. As a result, the friction drag of the smooth surface was only reduced by 0.1%, when the number of grids increased from 1 × 106 to 1.2 × 106. To improve the computational efficiency, the number of grids for subsequent calculations was set to 1 × 106.The near-wall grids of the groove surface and smooth surface are shown in Moreover, under the simulation setting, the simulation reliability verification was carried out. Rf is the friction drag reduction ratio, Rt is the total drag reduction ratio, Nf is the friction drag of the smooth surface, Nfn is the friction drag of the groove surface, Nt is the total drag of the smooth surface, Ntn is the total drag of the groove surface. The smooth surface is selected as a reference. Rf > 0 indicates that the goal of reducing friction drag is achieved, whereas Rf < 0 means that the friction drag is increased, Rt is also like that.In this study, the friction drag reduction ratio and total drag reduction ratio of the bionic groove surface were analyzed, and they can be calculated by the following equations:The drag reduction ratio of all bionic groove surfaces at different velocities are shown in The trend of the friction drag reduction ratio is the same for all groove surfaces, and only numerical differences exist. The trend exhibited is that the friction drag reduction ratio gradually decreases as the flow velocity increases, but the groove surface still obtained a friction drag reduction rate of 24.16% at the velocity of 12 m/s. In addition, the peak friction drag reduction ratio is achieved at the velocity of 2 m/s for all groove surfaces.For high Reynolds number turbulent flow fields, the wall shear stress of plate is influenced by both viscous stress and turbulent Reynolds stress. In the near-wall region, the viscous stress plays a major role, and away from the wall, the Reynolds stress becomes the dominant force due to the pulsation of a large number of fluid micro clusters. Since groove type 8 has a better friction drag reduction performance, the changes of viscous stress and turbulent Reynolds stress in the flow field are analyzed in terms of boundary layer thickness, velocity gradient and turbulent intensity to explore the drag reduction mechanism.6 (u = 2 m/s) and 2.39 × 107 (u = 12 m/s) reveals periodic variations in velocity and pressure in the near-wall region. u) contour in one groove range, when x = 0.5, Streamwise velocity contour of the middle cross-section at (a) 2 m/s and (c) 12 m/s have minimum values of −0.0016 and −0.001, respectively; and λ. From Periodic bionic groove surfaces cause changes in turbulent flow. Analysis of the flow field in the middle region of groove surface 8 at Reynolds number of 3.98 × 10y-direction) locations over a groove range is shown in The pressure contour over a groove range is shown in In summary, the change of pressure gradient induces the change of streamwise velocity, from the peak to the trough, due to the flow of the fluid against the pressure gradient, the fluid particles in the boundary layer are affected by viscosity and the retardation of the inverse pressure gradient, then the kinetic energy is rapidly lost. So that the turbulent stress decreases and the fluid accumulates in the trough, the viscous shear stress gradually decreases. From trough to peak, boundary layer fluid flows at an accelerated rate along the pressure gradient, which increases the kinetic energy of the fluid particle and makes the viscous effect between the fluids more obvious, so the turbulent stress increases, resulting in the increase of viscous shear stress.x-direction) velocity vector over the range of a groove is shown in The variation of the cross-sectional (y+<30), there is almost no difference between the groove surface and the smooth surface, and in the region where y+<5, u+ is linearly distributed for both surfaces, satisfying u+ = y+. And in the region where y+>30, the distribution of u+ along the normal direction of the groove surface differs significantly from that of the smooth surface, but still satisfies the law of logarithmic distribution. Moreover, when the value of y+ is the same, the u+ of the groove surface is significantly higher than that of the smooth surface, and there is a significant upward shift in the log-law region and the outer region of the dimensionless streamwise mean velocity distribution curve on the groove surface.To study the streamwise velocity variation caused by a single bionic groove structure as a whole, the streamwise velocities at the same normal position in the range of adjacent peaks are averaged. The distribution of the dimensionless streamwise mean velocity  the Reynolds shear stress on the groove surface is smaller than that on the smooth surface when it is closer to the wall, which indicates that the velocity pulsation of the boundary laminar flow field is suppressed by the groove surface, and the effect on the near-wall flow field is more obvious. The Reynolds shear stress of smooth surface and groove surface reaches the peak at y+≈100, and begins to close at y+≈200. When approaching the second peak, the Reynolds shear stress on the groove surface and the smooth surface gradually decreases as it moves away from the wall, indicating that the suppression effect of the groove surface on turbulent pulsation gradually decreases.The y+ corresponding to the maximum turbulent intensity is similar. When y+ is the same, the turbulent intensity at the peak of the groove surface is lower than that of the corresponding smooth surface, indicating that the turbulent pulsation intensity at the peak of the groove surface is lower than that of the corresponding smooth surface.The maximum turbulent intensity at the trough of the groove surface is close to that of the smooth surface. Meanwhile, the turbulent intensity at the trough of the groove surface first reaches the peak value and then decreases rapidly, appearing quite different compared with the smooth surface, which should be caused by the presence of a high turbulent intensity region on the windward side of the bionic groove.When far from the surface, the turbulence statistics of the flow field on the bionic groove surface and the smooth surface are not different, and the turbulent motion away from the surface is almost unaffected by the surface conditions, which is consistent with the findings of The wall shear stress contours and curves of the groove and smooth surface are given in To sum up, the flow field analysis on the bionic groove surface demonstrates that the streamwise velocity, pressure, and velocity gradient change periodically, such that the fluid flows under adverse pressure gradient from the peak to the trough. As a result, the streamwise velocity and velocity gradient gradually decrease, leading to a reduction in the viscous stress in the near-wall region, while the opposite trend is observed from the trough to the peak.y+<30), but the groove structure leads to an upward shift in the log-law and outer regions of the mean velocity profile. This observation suggests that the fully developed turbulent flow region is relatively far from the wall. The comparison of the turbulent intensity of the bionic groove surface with that of the smooth surface reveals that the bionic groove structure reduces the turbulent intensity of the flow field and suppresses the velocity pulsation, thus reducing the Reynolds stress.The study investigates the influence of individual bionic groove as a whole on the dimensionless streamwise mean velocity, and it is found that there is little variability between the groove surface and the smooth surface in the near-wall region (It can be concluded that compared with the smooth surface, the viscous stress and Reynolds stress of the groove surface are significantly reduced, resulting in the overall shear stress is significantly less than that of the smooth surface. Consequently, this corresponds to the drag reduction effect observed on the groove surface.Orcinus orca possess a distinctive skin structure characterized by regular ridges that cover most of their bodies. This paper presents a design for nine new transverse bionic grooves with larger geometric scales that are inspired by the killer whale skin ridges. In comparison with the traditional groove structure, the new bionic groove has a greater width-to-depth ratio and hence larger geometric scale at the same depth. The numerical simulation results reveal that the drag reduction effect improves as the depth of the bionic groove surface decreases, with the optimal width-to-depth ratio being 25. Over the flow velocity range of 2–12 m/s, the friction drag reduction ratio decreases gradually as the flow velocity increases, but the trend tends to level off, with the optimal friction drag reduction ratio reaching 26.91% and the total drag reduction ratio reaching 9.63%.Therefore, the transverse bionic grooves designed in this paper has a broad engineering application prospect. The next step is to explore the drag reduction effect of applying the new bionic groove to a full-size model at high Reynolds number for an aircraft or submarine model. This will provide a theoretical basis for the practical application of transverse bionic grooves.


Endoscopic endonasal surgery (EES) is commonly performed for resection of lesions of the anterior/middle cranial fossa region. Cerebrospinal fluid (CSF) leakage is a major complication. Skull base reconstruction after EES is challenging. We describe our reconstruction strategy and technique and analyze its outcomes.We retrospectively analyzed 703 patients with pituitary adenoma who underwent EES in our center from January 2020 to August 2022. Clinical, imaging, operative, and pathologic data were recorded from the medical records and analyzed. Skull base reconstruction was performed to achieve the following three goals: seal the original leak, eliminate dead space, provide blood supply, and early ambulation. Reconstruction was tailored to individual patients based on grade of CSF leakage encountered during surgery.The number of patients with a grade 0, 1, 2, and 3 intraoperative CSF leak was 487, 101, 86, and 29, respectively. Overall incidence of postoperative CSF leakage was 0.14% (1/703). Fascia sutured and vascularized nasoseptal flap were selected for all grade 3 CSF leaks. One patient who experienced postoperative CSF leakage developed intracranial infection and were treated with lumbar CSF drainage that failed; eventually re-exploration surgery for repair was required. Other patients did not have complications such as CSF leak and infection. 29 patients with grade 3 CSF leakage did not complain of severe nasal complications after operation. No perioperative complications related to the strategy  occurred. Incidence of postoperative CSF leak according to intraoperative leak grade was as follows: grade 0, zero; grade 1, zero; grade 2, 1.16% (1/86); and grade 3, zero.The principles of sealing the original leak, eliminating dead space, providing blood supply, and early ambulation are key in skull base reconstruction after EES. Individualization of these principles can significantly reduce the incidence of postoperative CSF leakage and intracranial infection and reduce the use of lumbar CSF drainage. Skull base suture technique is safe and effective in patients with high-flow cerebrospinal fluid leaks. Endoscopic endonasal surgery (EES) is widely used for resection of basicranial tumors –3. CerebIn 2006, Hadad et al. first described using a vascularized nasoseptal flap for skull base reconstruction, which substantially reduced the incidence of postoperative CSF leakage . A systeDespite these improvements, postoperative CSF leakage may still occur. To further decrease the incidence of CSF leakage in our center, we implemented a new skull base reconstruction strategy in 2020 based on three principles: sealing the original leak, eliminating dead space, and providing blood supply. In this report, we describe our reconstruction strategy and technique and analyze its outcomes.The inclusion criteria were endoscopic surgery and pathological diagnosis of pituitary adenoma. We retrospectively analyzed 703 patients in our center from January 2020 to August 2022. Clinical, imaging, operative, and pathologic data were recorded from the medical records and analyzed. Postoperative CSF leakage, intracranial infection, and nasal quality of life were the indicators we focused on. All patients were followed up by telephone at 3 and 6 months after surgery to ask whether the above complications occurred. The study was approved by the ethics committee of Beijing Tiantan Hospital, Capital Medical University. All patients provided informed consent.The goal of sealing the original leak is to achieve the same cranial physiological state as before surgery where possible and prevent CSF leakage. We believe that this principle is key when reconstructing the skull base.Filling any dead space with repair material can protect the dura of the diaphragma sellae and prevent empty sella syndrome.The vascularized nasoseptal flap, contralateral rescue flap, and pedicled middle turbinate mucosal flap provide blood supply to the reconstructed area. Considering the impairment of olfactory function, we prepared mucosal flaps only for patients deemed likely to experience high-flow (grade 3) CSF leakage based on preoperative assessment. These included patients with a pituitary adenoma classified as Hardy–Wilson grade B, C, or D .After surgery, patients were encouraged to stand and walk as much as possible to shorten the recovery time.With grade 0 CSF leakage, the diaphragma sellae is intact and CSF leakage is not observed. The general principle is to restore the normal structure of the saddle area using fibrin glue to strengthen the diaphragma sellae, then packing gelatin sponge within the sella.Although no diaphragma sellae damage is seen in grade 1 CSF leaks, a small amount of CSF leakage is seen. The principles of repair are reinforcement of the original leak and elimination of dead space by packing fat within the sella, which can achieve both. The artificial dura is then attached to the outer layer of fat, fixed with a thin layer of fibrin glue to strengthen the sellar dura, and the sphenoid sinuses are then filled with absorbent cotton .Grade 2 CSF leakage is defined as a significant leak at the diaphragma sellae. The principles of repair are sealing the original leak and eliminating dead space by filling the damaged area of the diaphragma sellae with an appropriately sized autologous fat graft and keeping the diaphragma sellae intact with a “bathtub plug.” Then, the sella is filled with autologous fat to eliminate dead space. Then fibrin glue was used to fix an artificial dural on the outside of the fat. A vascularized nasoseptal flap is not used. After repair, the nasal passage is filled with iodoform gauze that is removed 1 week after surgery .With a grade 3 CSF leak, a large defect in the diaphragma sellae and/or anterior skull base dura has been created as part of the surgical approach and the floor of the third ventricle is open. Therefore, the degree of CSF leakage is large. We classify grade 3 CSF leaks into two subtypes. In the first, the planum sphenoidale dura has been opened and the sellar region structure has been completely destroyed. In the second, “fishnet” CSF leakage from the diaphragma sellae occurs while the structure of the sellar region has remained intact. For repair of the first type, autologous fascia is continuously sutured with the dura of the anterior skull base. For the second, the first step is filling the sella with autologous fat to eliminate dead space followed by continuous suturing of autologous fascia with saddle bottom dura mater to seal the leak. Then, a vascularized nasoseptal flap is placed. After repair, the nasal passage is filled with iodoform gauze that is removed after 7–10 days .continuous suturing of the dura with the fascia lata grafts, was performed using 5–0 resorbable sutures . The key step of the skull base suture technique is how to end the operation as soon as possible while ensuring the watertightness. We will introduce our surgical techniques. First, we will cut the appropriate size of autogenous fascia and place it at the “3 o'clock position” of the sellar dura mater defect as the first suture stitch. The direction of the fascia is from outside to inside. The injection direction of the dura mater is from the inside to the outside, and the knot of the first needle must be absolutely firm. Suture continuously counterclockwise, every 2–3 stitches, tighten the suture to prevent the knot from loosening. After sewing a circle, the last stitch is tied with the first stitch. The needle distance is about 2–3 mm, and care should be taken not to damage the bilateral internal carotid arteries when suturing the dura mater on both sides. We believe that the advantage of continuous suture is that it can shorten the operation time and reduce the difficulty of operation caused by multiple knots. But its disadvantage is that it is necessary to keep the suture in a tight state to prevent the knot from slipping, which undoubtedly increases the difficulty of cooperation between the surgeon and the assistant. Another key operation of skull base suture is knotting, the knot was made outside the nasal cavity and then slid into the operative region.We did not routinely use lumbar CSF drainage. Patients were kept in bed on the day of surgery. On the day after, posture and diet were not restricted. Patients were instructed to avoid actions that increase intracranial pressure. The nasal cavity was cleaned approximately 1 week after surgery to prevent nasal mucosal adhesions that can result in olfactory dysfunction.Intravenous antibiotics (ceftriaxone 2 g daily or cefuroxime 1.5 g twice daily) were administered for 48 h after surgery. Patients with signs of infection were treated according to results of biochemical CSF testing. For patients with a postoperative CSF leak, surgical exploration was performed if an intraoperative repair had not been performed. In those whom intraoperative repair had been performed, a lumbar drain was placed; if leakage persisted more than 3 days, surgical repair was performed.No after operative complications related to the strategy  occurred. Patient characteristics are shown in Overall, one patient experienced postoperative CSF leakage and infection (0.14%). In each, lumbar CSF drainage was ineffective and additional exploratory repair was required. The occurrence of CSF leakage according to pathological tumor type is shown in In our opinion, for patients with po-CSF leakage, we reviewed the procedure of the second and third operations. At the second operation, we found an inflammatory discharge covering the dural at the base of the saddle and evidence of local infection. Therefore, we believe that the main causes of CSF leakage after the patient's first operation are poor local healing and local infection. At the same time, we also believe that the patient's emotional instability will lead to intracranial elevation, which is also the cause of po-CSF leakage. For patients with grade 2 CSF leakage during the operation, the size of the fat mass is the key to determine the success of the repair of CSF leakage. If the fat mass is too small, it is easy to cause po-CSF leakage. If the fat block is too large, it is prone to the occupying effect. After the appropriate size of the fat blocked the leak, the fat block could be seen pulsating with the pulse of the CSF. During the third operation, we found no obvious signs of infection locally, but there was a gap between the fat mass and the leakage. In order to prevent the recurrence of CSF leakage completely, we chose continuous suture as the repair method. Now, our retrospective analysis suggests that higher levels of repair should be selected when patients first experience CSF leakage after surgery.EES plays an important role in the treatment of basicranial tumors. However, it requires skull base reconstruction, which can be challenging. The most serious complication of EES is CSF leakage. Risk factors include intraoperative CSF leakage and skull base defect location and size. The proposed reconstruction strategies described above, which are based on the grade of intraoperative CSF leak encountered, are expected to further decrease the incidence of CSF leakage and use of lumbar drainage.In recent years, the incidence of CSF leak after EES has decreased significantly in conjunction with advances in skull base reconstruction technology and techniques . HoweverGrade 1: packing fat within the sella; Grade 2: fat “bathtub” plug; Grade 3: autologous fascia suturing). The fat plays a very important role in the “3F” reconstruction strategy proposed by Solari et al., which is consistent with our chosen repair strategy in grade 1–2 CSF leakage, that fat can seal the leak like a “cork”. More importantly, the fat can be plastic in the surgical cavity, thus filling some irregular cavities. Different from the “3F” strategy, we chose a continuous suture method for the repair of grade 3 CSF leakage. In our center, we previously conducted a retrospective analysis on patients with high-flow CSF leakage during surgery to discuss the role of continuous dural suture in endoscopic surgery. A total of 79 adult patients, all of whom had grade 3 CSF leakage during surgery, were compared between patients who underwent and did not undergo endoscopic dural suture after tumor resection. 79 adult patients had Esposito Grade 3 high-flow CSF leakage during surgery. Po-CSF leakage occurred in 10 patients (12.7%). Po-CSF leakage occurred in 1 of 36 patients who underwent intraoperative dural suture, while po-CSF leakage occurred in 9 of 43 patients who did not undergo intraoperative dural suture (p = 0.016). Regression analysis showed that dural suture significantly reduced the incidence of po-CSF leakage . Fascia Lata was chosen as the repair material because it is more plastic than other dural alternatives and can reduce financial costs for patients. We also examined the learning curve for dural suturing under EETSS. When this procedure was first implemented, it took more than 90 min to complete closure with dural suturing. After gaining experience with 15–20 cases,2 veteran surgeons could finish the suturing procedure within 30 min , which is suitable because it can adhere to the surrounding tissue. In addition, fat is hydrophobic, light in weight, and will not compress local tissue. However, it takes several days or weeks for adherence to occur; therefore, patients with a grade 3 CSF leak may need additional fixation. We suture dura for fixation, ensuring close contact between the dura and fat to prevent CSF leakage. This appears to ensure rapid and tight adhesion. Previously reported repair methods emphasize the principle of filling the sella , 10. ForProviding blood supply is our third principle of skull base reconstruction. Vascularized nasoseptal flaps were first described in 2006  and haveIn our center, lumbar drainage was not routinely used in all patients after surgery, and patients were asked to rest in a semi-sitting position on the postoperative day to reduce the pressure on the skull base defect. On the first postoperative day, patients were encouraged to get out of bed. This is consistent with “Flash” in the “3F” intermediate strategy, and we believe that early ambulation is an important measure to accelerate postoperative recovery.Retrospective analysis is the main limitation of this study. Moreover, the manuscript was designed to clarify the philosophical issue of strategy and specific reconstruction methods for skull base reconstruction, and there was no control group, so statistical analysis was not applicable to this study. Short follow-up time was another limitation. Future prospective studies should be selected to further clarify the effectiveness of this reconstruction strategy.The principles of sealing the original leak, eliminating dead space, providing blood supply and early ambulation are key in skull base reconstruction after EES. Individualization of these principles can significantly reduce the incidence of postoperative CSF leakage and intracranial infection and reduce the use of lumbar CSF drainage. Skull base suture technique is safe and effective in patients with high-flow cerebrospinal fluid leaks.


Clearance of neurotoxic brain proteins via cerebrospinal fluid (CSF) to blood has recently emerged to be crucial, and plasma biomarkers of neurodegeneration were newly introduced to predict neurological disease. This study examines in 106 individuals with neurological disorders associations between plasma biomarkers  and magnetic resonance imaging measures of CSF-mediated clearance from brain via extra-vascular pathways (proxy of glymphatic function) and CSF-to-blood clearance variables from pharmacokinetic modeling . We also examine how biomarkers vary during daytime and associate with subjective sleep quality. Plasma concentrations of neurodegeneration markers associate with indices of glymphatic and meningeal lymphatic functions in individual- and disease-specific manners, vary during daytime, but are unaffected by sleep quality. The results suggest that plasma concentrations of neurodegeneration biomarkers associate with measures of glymphatic and meningeal lymphatic function. Plasma neurodegeneration biomarkers are increasingly utilized to predict neurological disease. Here, authors show in different neurological disorders associations between plasma neurodegeneration biomarker concentrations and various measures of glymphatic and meningeal lymphatic functions. Clearance of brain proteins occurs along different transport routes, such as passage across the blood–brain-barrier (BBB) to the systemic circulation, cellular degradation of metabolites in the brain by macrophages and glia cells, and cerebrospinal fluid (CSF)-mediated clearance2. Independent of whether clearance occurs directly from the brain to blood or via lymph as an intermediate step, all metabolites eventually reach the blood circulation. Therefore, measurements of plasma concentrations of brain metabolites, such as 40 and 42 amino acid-long Aβ peptides (Aβ40 and −42), tau protein variants, glial fibrillary acidic protein (GFAP), and neurofilament light (NfL), have emerged as diagnostic biomarkers of neurodegenerative diseases, currently in particular for Alzheimer’s disease (AD) and post-traumatic brain injuries4. At present, biomarkers like Aβ40, Aβ42, phosphorylated tau (P-tau), GFAP, and NfL make a huge impact on the diagnostics of neurodegenerative diseases4.Abnormal aggregations in the brain of toxic proteins such as amyloid-β (Aβ) and phosphorylated tau (P-tau), the main constituents of amyloid plaques and neurofibrillary tangles, are key features in neurodegeneration diseases. Several different mechanisms have been proposed as important for promoting aggregation, such as increased production or decreased clearance, sequence variants increasing hydrophobicity, protein truncations or post-translational modifications, and chaperone proteins5, and of functional meningeal lymphatic vessels  in 20157; for review see also2. An increasing body of evidence points to the role of the glymphatic5 and meningeal lymphatic systems8 in the clearance of Aβ and tau. Importantly, the glymphatic and meningeal lymphatic systems seem to be under circadian control9. It is presently assumed that the glymphatic system is a waste clearance system mostly active during sleep10, while the meningeal lymphatic system is active during the awake state11. Hence, under physiological circumstances, brain metabolites may be mobilized from the brain to CSF and lymphatic structures during nighttime, while clearance via the meningeal lymphatic system is enhanced during daytime. From this, it might be expected that plasma concentrations of CNS-derived metabolites and biomarkers for neurodegenerative disease processes are impacted in concert by the glymphatic and meningeal lymphatic systems. However, it remains unclear how alterations in CSF-mediated clearance of CNS-derived molecules via the glymphatic and meningeal lymphatic systems relate to their plasma concentrations. These mechanisms are important to understand, not least because potentially modifiable clearance routes may represent a target for treating disease processes underlying changes in plasma biomarker concentrations.The role of CSF-mediated clearance of brain metabolites has gained renewed interest during the last decade, not least facilitated by the discoveries of CSF-mediated molecular clearance from the brain via paravascular pathways (denoted the glymphatic system) in 201212. The meningeal lymphatic function was examined by the CSF-to-blood clearance recently described by our group13. The MRI contrast agent gadobutrol is hydrophilic and distributes freely in CSF, and enriches the extravascular spaces of the brain12. We aimed to address the following hypotheses: (1) Whether plasma concentrations of CNS-derived biomarkers correlate with indices of glymphatic clearance, assessed by extravascular brain enrichment of an intrathecal CSF tracer. The tracer does not cross the intact BBB and can here thus serve as a surrogate marker of clearance along extravascular pathways. Recent research suggests that impaired extravascular clearance (glymphatic function) is a major cause of deteriorated clearance of metabolites from the brain to CSF and further to meningeal lymphatic transport routes12. A negative correlation might be expected, provided that increased tracer levels in the brain during the clearance phase (24 and 48 h) reflects impaired glymphatic function. (2) Whether plasma biomarker concentrations correlate with indices of meningeal lymphatic function, assessed by variables of CSF-to-blood clearance. We recently reported a population pharmacokinetic model for CSF-to-blood clearance of the presently used CSF tracer (gadobutrol)14. The hypothesis is that meningeal lymphatic clearance represents a final common pathway for the egress of metabolites from CSF15. Hypothetically, in subjects with intact BBB and no abnormal CSF leakage, the CSF-to-blood clearance may reflect the total meningeal lymphatic clearance capacity. In line with this hypothesis, we previously demonstrated enrichment in the parasagittal dura of this intrathecal CSF tracer16. Furthermore, the CSF-to-blood clearance variables of the pharmacokinetic model showed large variation between individuals and across disease catgeories14. Therefore, the direction of the correlation might be expected to vary. (3) Whether plasma biomarker concentrations vary during daytime and relate to chronically impaired sleep quality. This could be expected given the proposed role of sleep and circadian rhythm on both glymphatic and meningeal lymphatic clearance function18.This study was undertaken to examine in humans how plasma concentrations of neurodegeneration biomarkers associate with tentative indices of glymphatic and meningeal lymphatic functions. The glymphatic function was examined as the brain-wide distribution of a magnetic resonance imaging (MRI) contrast agent, gadobutrol, administered intrathecal, and utilized as a CSF tracer19. The other cohorts consisted of patients who were diagnosed with communicating hydrocephalus (cHC), arachnoid cyst (AC), idiopathic intracranial hypertension (IIH), or spontaneous intracranial hypotension (SIH), and who underwent surgery for these diseases. Twelve individuals were denoted reference (REF) subjects in which no apparent CSF disturbance was diagnosed. As compared with the REF subjects, the patient cohorts with iNPH and arachnoid cysts (ACs) were older and with different sex distributions.The present study included 106 patients who underwent intrathecal contrast-enhanced MRI as part of a work-up for tentative CSF disturbances , time to maximum concentration (Tmax), and lag-time (Tlag) , and plasma biomarker concentrations , GFAP, and NfL, while Aβ40 and Aβ42 did not show this pattern. (2) There were significant correlations between CSF-to-blood clearance variables  and plasma biomarker concentrations, but the relationships were biomarker- and disease-specific. This may be related to the extensive inter-individual variation in CSF-to-blood clearance capacity. (3) In the total cohort, plasma T-tau, GFAP, and NfL concentrations changed significantly during the day, while we found no daytime variability in plasma concentrations of Aβ40 and Aβ42. In this cohort, subjective sleep quality was not associated with plasma biomarker concentrations. (4) Plasma GFAP, NfL and T-tau concentrations were increased in iNPH compared with REF subjects, but the difference could be caused by age differences.This study provides several lines of evidence suggesting that plasma concentrations of neurodegeneration biomarkers associate with indices of glymphatic and meningeal lymphatic functions: (1) There was a significant correlation between levels of an intrathecal CSF tracer in the brain during the clearance phase  and plasma biomarker concentrations. Hence, for the entire cohort, impaired tracer clearance from the brain correlated significantly with lower plasma concentrations of T-tau  have been introduced to assess tau pathology25. Available plasma tau assays address phosphorylation at different amino acids, such as 181 (P-tau-181), 217 (P-tau-217), and 231 (P-tau 231)26. Increased plasma P-tau isoform concentration relates to the degree of cerebral Aβ pathology, predicts the density of tau tangles, and differentiates AD from other dementias28. At present, P-tau is primarily considered a marker of AD, and not for other types of dementia. The T-tau presented in the present study does not refer to any particular type of phosphorylation and is not specific to AD. Notably, the cerebral aggregations of Aβ and P-tau associate with inflammatory responses and neuronal and glial damage. Here we focused on GFAP and NfL. GFAP is a marker of astrocyte activation, not specific for AD, but as well for traumatic brain injury and a variety of other brain diseases29. In the context of AD pathology, GFAP is considered to reflect an astrocyte response to Aβ aggregation in the brain31, but not tau pathology31. Notably, the association between GFAP and Aβ pathology is stronger for plasma than CSF pathology31. NfL is a non-specific marker of neuroaxonal injury and is found to increase in a variety of neurodegenerative disorders32, with increasing levels during aging33. The CSF and blood concentrations of NfL correlate34. One study reported increased CSF and plasma concentrations of NfL 7 years before symptom onset in autosomal dominant AD subjects who were cognitively intact35.We here examined the association between plasma concentrations of neurodegeneration biomarkers and indices of glymphatic and meningeal functions in patient groups that may not necessarily be categorized as neurodegenerative diseases. In this regard, it should be noted that the current knowledge about the plasma biomarkers referred to here, primarily derives from studies of Alzheimer’s disease, frontotemporal dementia, atypical parkinsonian disorders, and post-traumatic brain injury2. To this end, the passage route for Aβ has been most studied; for which passage via BBB is important, as well as cellular degradation within the brain by macrophages and microglia, in addition to egress via CSF2. The latter may perhaps be more important than previously considered, given recent data on an important clearance route of Aβ via meningeal lymphatic vessels and the beneficial effect of lymphatic enhancement8. The recent observations that Aβ-labeled cells were far more abundant in cervical than inguinal lymph nodes support this view36. Perhaps enhancement of meningeal lymphatic function could be one way to modify CSF-mediated Aβ clearance?. The transport route for the tau protein, on the other hand, is less characterized; no dedicated BBB transport has been identified2, but passage across the BBB was observed in mice20. Moreover, experimental data indicate that efflux via dural meningeal pathways may be crucial37. The efflux routes for GFAP and NfL are largely unknown. In this regard, it should be emphasized that neurodegenerative diseases involve BBB damage to a variable degree38, which may facilitate the passage of CNS-derived biomarkers directly to the blood. With regard to the present observations, we do not expect BBB disruption to be a significant factor, though some impairment of BBB cannot be excluded. It is generally accepted that BBB becomes impaired with increasing age, which is relevant for the iNPH cohort with a median age of 72.5 ± 5.7 years. In addition, previous histopathological studies have shown some degree of BBB disruption in iNPH39 and IIH40 patients, though the effect on BBB is not major in these cases.While the present observations suggest an association between concentrations of plasma biomarkers of neurodegeneration disease and indices of glymphatic and meningeal lymphatic clearance function, we remind that the specific efflux routes from the brain and CSF to the blood of the present biomarkers are not fully understood. Additional pathways than via CSF are involved12. However, given the low resolution of MRI (1 mm), the exact transport route of the CSF tracer at the microscopic level  cannot be defined, though it may be concluded that tracer transport occurs extravascular. With the term “glymphatic”, we refer to CSF-mediated tracer enrichment and clearance from the brain along extravascular pathways, well aware that this represents only one aspect of the glymphatic concept presented in 20125. This concept as a whole is still heavily debated41; the controversy particularly concerns mechanisms behind the solute movement (role of convective forces versus diffusion), the role of aquaporin-4 (AQP4) located at perivascular astrocytic endfeet, the role of directionality of CSF movement, and lack of evidence for efflux along perivenous spaces. Despite this ongoing debate, we here use the term glymphatic with an awareness of these yet unresolved issues. In this regard, an obvious question is how glymphatic function assessed by a non-endogenous CSF tracer (gadobutrol) may be relevant for excretion from the brain and CSF of endogenous compounds such as Aβ40, Aβ42, T-tau, GFAP, and NfL? In soluble form, these proteins are expected to be transported paravascular in the human brain42. The CSF tracer used here, gadobutrol, has a molecular weight (MW) of 604 Da, which is many times smaller than the plasma metabolites: Aβ40 (MW about 4.3 kDa), Aβ42 (MW about 4.5 kDa), tau (MW up to 80 kDa) GFAP (MW about 50 kDa), NfL (MW about 70 kDa). However, these large proteins may as well be transported paravascular. For example, one study demonstrated in pig brain glymphatic paravascular tracer enrichment of a CSF tracer [AlexaFluor647-conjugated bovine serum albumin (BSA-647)] with MW about 66 kDa43. The tracer enrichment in the pig brain utilizing this large MW compound was comparable to the CSF tracer (gadobutrol) enrichment seen in the human brain after intrathecal injection43. Second, gadobutrol is a hydrophilic substance that does not pass the intact BBB, nor does it permeate the cell membrane or interact with other molecules of the brain. Third, the transport of plasma biomarkers and CSF tracer in the subarachnoid compartments may differ due to the membranes compartmentalizing the subarachnoid space45. In this regard, ref. 45 recently described a subarachnoid lymphatic-like membrane (SLYM) compartmentalizing the subarachnoid space, where this mesothelial membrane was impermeable to substances >3 kDa.Here, we applied a surrogate marker for in vivo human assessment of glymphatic and meningeal lymphatic functions, as direct measurements cannot be done in humans. In vivo visualization of extra-BBB clearance pathways in humans can be done by multiphase and standardized MRI utilizing a CSF tracer46 used technetium-99 m (99mTc-) diethylenetriaminepentaacetic acid (DTPA) imaging to estimate clearance from CSF to the periphery after intrathecal injection. This method as well provides for visualization of tracer. It may be considered a limitation that the radiolabel half-life of this compound is 6 h (time to maximum concentration in the present study was 8.1 ± 4.0 h). However, radiotracers may be used for dynamic scans that are three to four times longer than their half-life. Moreover, a recent study suggested the useful role of radiotracers for in vivo imaging of glymphatic function47. We recently introduced another method to assess CSF-to-blood clearance capacity13, incorporating an intrathecal injection of a CSF tracer , followed by repeated plasma samples. A population pharmacokinetic model based on consecutive blood samples (n = 1140) from 161 subjects demonstrated marked inter-individual variation and even marked variation across disease categories14. It is important to note that this method measures total CSF-to- blood clearance capacity, independent of the clearance route. For substances also passing the BBB, direct passage to blood may contribute. In subjects with impaired BBB integrity, which must be anticipated in older subjects48, the passage of substances directly to blood may be expected to some degree. In other rare diseases, e.g. spontaneous intracranial hypotension due to CSF-to-venous-fistula49, there is an abnormal direct passage to blood. However, for substances primarily being excreted via CSF, we tentatively suggest that the CSF-to-blood clearance capacity can be considered a surrogate marker of meningeal lymphatic clearance capacity, and that brain clearance may indirectly be dependent on meningeal lymphatic CSF clearance capacity. We previously reported that the intrathecal CSF tracer enriched extensively in parasagittal dura, but identified a few arachnoid granulations also being enriched by tracer16. The volume fraction of efflux via arachnoid granulations could not be determined, and requires further studies. However, knowledge of the exact clearance route is not critical, as the CSF-to-blood clearance aims to measure total clearance capacity, independent of the clearance pathway. For compounds largely excreted via CSF, the CSF-to-blood clearance capacity may largely reflect the total meningeal lymphatic clearance capacity.With regard to meningeal lymphatic clearance capacity, no specific methods for assessments have been established. Verma et al.51. Failed CSF clearance has been shown to represent a feature of AD, related to Aβ deposition and to the pathology of AD24. Most importantly, CSF clearance failure is expected to occur in the preclinical stages of dementia disease52, and impaired CSF clearance due to failure of meningeal lymphatic function may be involved in other diseases, such as malignant glioma53 and ischemic stroke54. Given the prospects of interventions that may enhance meningeal lymphatic function56, direct estimation of CSF-to-blood clearance capacity, as described here, might be useful to monitor treatment regimens. Therefore, defining CSF-to-blood clearance changes in other diseases than dementias such as AD seems warranted.It remains to be determined how CSF-to-blood clearance variables from pharmacokinetic modeling provide information about the underlying biological processes preceding neurodegenerative diseases. It may be of particular interest, however, that emerging evidence suggests that CSF-to-blood clearance by impaired meningeal function to be a risk factor for abnormal aggregation of toxic proteins 31 might perhaps suggest direct release into the blood at the glia-neuro-vascular interface. The presently reported significant positive correlation between CSF-to-blood clearance and plasma GFAP, indicative of higher blood levels with impaired CSF-to-blood clearance, could support this assumption. If the correlation had been negative, it might rather indicate higher plasma levels due to more efficient CSF-to-blood clearance. The weaker association of CSF GFAP with Aβ pathology may, however, also reflect the recently discovered instability of the protein in CSF, which is much less of a problem in the plasma matrix57. Overall, the present results point to the need for individualized assessments concerning causes for altered plasma concentrations of neurodegeneration biomarkers.The variable profiles of the presently reported plasma metabolites may reflect their different clearance routes from the brain. On the one hand, GFAP showed significant daytime variation, correlation with glymphatic function, and correlation with CSF-to-blood clearance, indicative of meningeal lymphatic function. On the other hand, Aβ40 and Aβ42 showed no daytime variation in the total cohort (though varied during the day in disease cohorts such as iNPH and IIH), no correlation with glymphatic function and no correlation with CSF-to-blood clearance for the entire cohort (though for certain disease categories). Moreover, both T-tau and NfL showed daytime variation in plasma concentrations and correlated with glymphatic function, while CSF-to-blood clearance was disease-specific. Tentatively, these observations may be interpreted as CSF clearance being more dominant for T-tau, GFAP, and NfL than for Aβ being excreted by different routes . In this regard, the observations of a closer association between Aβ pathology and plasma GFAP than what has been observed for CSF GFAP58; for example, accumulation in the brain of Aβ and/or tau is seen in a significant proportion of iNPH subjects59. Moreover, previous studies showed a comparable reduction of CSF production and turnover in AD and iNPH patients60, though CSF Aβ40, Aβ42, and T-/P-tau also were able to differentiate iNPH from AD subjects61. iNPH involves impaired glymphatic function62, which was proposed as a common mechanism behind dementias and AD63. The iNPH disease is also characterized by impaired CSF-to-blood clearance capacity14. In this study, impaired clearance of tracer from CSF, cerebral cortex, and subcortical white matter, indicative of impaired glymphatic function, was accompanied by reduced plasma T-tau concentration. This finding is of interest, considering the increased tau deposition within the brain of this patient group59. In line with this observation, iNPH patients not responding to shunt surgery had increased CSF T-tau concentration65, and also increased CSF NfL concentration65. Cognitively impaired iNPH patients also had higher P-tau concentration than cognitively intact iNPH subjects61. They also presented a lower Aβ42/Aβ40 ratio61. Here, we found that impaired clearance of tracer from CSF was associated with a significantly lower plasma Aβ42/Aβ40 ratio in iNPH patients at 24 h and within the total cohort at 48 h after tracer injection. The present results also disclosed higher plasma concentrations of Aβ40, T-tau, GFAP, and NFL in the iNPH cohort, indicative of ongoing neurodegeneration. In comparison, immunohistochemistry of brain tissue specimens of iNPH patients demonstrates astrogliosis with increased GFAP immunostaining66. It remains to be determined whether the reduced plasma concentrations of Aβ40 and Aβ42 and increased plasma levels of tau, GFAP, and NfL towards the evening reflect the clearance pathways and clearance rates of these proteins from the brain. A significant negative correlation in iNPH between plasma Aβ42 and lag-time could indicate that clearance via CSF and meningeal lymphatics has a major role in this isoform.Here, we addressed patient categories that has been less studied concerning plasma biomarkers of neurodegeneration disease. The existing knowledge about plasma biomarkers largely relates to AD, while their role in other brain disorders remains less understood. The patient category closest to AD is the iNPH cohort. The iNPH disease is a neurodegenerative disease and subtype of dementia with close histopathological overlap towards AD67. In IIH, plasma Aβ40 and Aβ42 concentrations declined during the day, as did GFAP, perhaps due to more efficient CSF-to-blood clearance, which we previously provided evidence for14. This is supported by the present observation that plasma Aβ40 and Aβ42 concentrations increased with increasing time-to-maximum, indicative of slower CSF-to-blood clearance capacity. Furthermore, the different profiles of the various disease categories concerning the association between plasma biomarker concentrations and CSF-to-blood clearance variables suggest variation across individuals and disease types. These results point to the need for individualized assessments, i.e. individual precision diagnostics. This point was illustrated when comparing plasma biomarker concentrations between disease cohorts. As such, the iNPH and REF cohorts demonstrated differences in plasma concentrations of Aβ40, T-tau, GFAP, and NfL, which remained significant for GFAP and NfL after correction for kidney function (GFR). We cannot exclude that age difference is a confounding factor for differences between REF and iNPH subjects, but iNPH is a disease of the aged population.iNPH differed from the IIH disease, which is characterized by high intracranial pressure, impaired glymphatic function, and enhanced CSF-to-blood clearance18. Despite these negative results, there is accumulating evidence for the role of sleep on biomarker concentrations in CSF and plasma. In subjects with mild-moderate AD, lack of deep sleep was associated with higher NfL concentration in CSF68. Furthermore, impaired sleep quality was also correlated with reduced Aβ42/Aβ40 and increased T-tau/Aβ42 ratios69.In this study, we found no correlation between subjective sleep quality (assessed by the PSQI) and plasma biomarker concentrations, which was somewhat unexpected given the previous observations of different glymphatic enrichment of tracer in poor and good sleepersIt might be considered a limitation of this study that lumbar CSF concentrations were not measured. On the other hand, multi-hour repeated sampling of lumbar CSF would not be feasible, as it would require a lumbar drainage system, and heavily interfere with the interpretation of the CSF tracer studies. We also decided not to retrieve lumbar CSF prior to the intrathecal administration of gadobutrol in order not to affect the normal CSF circulation. In future dedicated experimental setups, simultaneous sampling of CSF and plasma over time should nevertheless be considered. Another limitation is that the intrathecal use of gadobutrol is currently off-label. For this reason, we will explore in future studies the utility of a computer tomography (CT) contrast agent, which is approved for intrathecal use. As MRI and CT contrast agents share many of the same properties, such as similar molecular size, being hydrophilic and inert, we hypothesize that these groups of contrast agents also share the same clearance pathways and clearance rates.9. Overall, solute clearance from CSF may be a significant contributor to the plasma concentrations of neurodegeneration biomarkers, and assessment of CSF clearance capacity by administration of an exogenous tracer substance provides dynamic information that goes beyond what can be retrieved by point measurements of endogenous solutes in CSF and blood. The finding that CSF-to-blood clearance variables derived from pharmacokinetic modeling14 associated with plasma biomarker concentrations in a biomarker- and disease-specific manner, may suggest the need for individualized assessment of CSF-to-blood clearance at an individual level. In particular, this may be utilized in the preclinical phase and as a tool to monitor treatments and interventions initiated to modify CSF-mediated solute clearance.In conclusion, plasma concentrations of neurodegeneration biomarkers are associated with indices of glymphatic and meningeal lymphatic functions. The plasma concentrations also varied during the daytime, supporting previous data that CSF-mediated molecular clearance is under circadian controlThe research study was approved by The Institutional Review Board (2015/1868), Regional Ethics Committee (2015/96), and the National Medicines Agency (15/04932-7), and registered in Oslo University Hospital Research Registry (ePhorte 2015/1868). Patients were included after written and oral informed consent.The study design was prospective and observational: Randomization of patients was not relevant; neither was a priori sample size calculation needed.Consecutive patients referred to the Department of Neurosurgery, Oslo University Hospital—Rikshospitalet, Oslo, Norway, for various tentative CSF disturbances were included. The indication for intrathecal contrast-enhanced MRI was made for clinical reasons.19 or subjects with communicating hydrocephalus (cHC) not defined as iNPH. The subjects with arachnoid cysts (AC) underwent surgery followed by post-operative clinical improvement. The idiopathic intracranial hypertension (IIH) subjects were shunted with clinical improvement thereafter. The patients with spontaneous intracranial hypotension (SIH) had an identified CSF leakage, requiring surgery to close the leakage.Concerning the categorization of CSF disorders, reference subjects (REF) are individuals in whom no apparent evidence of CSF disturbance was identified and no indication for surgery. The hydrocephalus category was either subjects with idiopathic normal pressure hydrocephalus (iNPH) who underwent shunting with a demonstrated clinical improvement, thereby qualifying for the diagnosis of Definite iNPH according to the Japanese guidelinestm, Philips Medical Systems, Best, The Netherlands) for every time point. Gadobutrol increases the T1 relaxation of water, which provides a higher T1 signal intensity at the image gray scale, thereby providing a semi-quantitative measure of the tracer level. The images were post-processed using FreeSurfer software (version 6.0) to determine the percentage change in normalized T1 signal units, indicative of tracer enrichment. For this, FreeSurfer software (version 6.0) (http://surfer.nmr.mgh.harvard.edu/) was used for segmentation, parcellation, and registration/alignment of the longitudinal data, and to determine the increase in T1 signal caused by the CSF tracer, as previously reported70. We further adjusted for changes in the gray scale between MRI scans, by dividing the T1 signal unit for each time point by the T1 signal unit of a reference region of interest (ROI) for the respective time point. The reference ROI was placed within the posterior part of the orbit, as previously described17. The ratio is the normalized T1 signal units, which corrects for baseline changes of image gray scale due to automatic image scaling. Tracer enrichment was semi-quantified as the percentage change in normalized T1 signal at different time points, relative to the pre-contrast injection.The MRI contrast agent gadobutrol was used as a CSF tracer and was administered intrathecally in doses of either 0.25 or 0.5 mmol . Standardized T1-weighted MRI scanning was performed before and at defined time points after administration of gadobutrol, utilizing a 3 Tesla Philips Ingenia MRI scanner  was used for MRI. Equal imaging protocol settings were applied at all time points to acquire sagittal 3D T1-weighted volume scans, with the following imaging parameters: repetition time = “shortest” , echo time = “shortest” , Flip angle = 8 degrees, a field of view = 256 × 256 cm and matrix = 256 × 256 pixels (reconstructed 512 × 512). Hundred and eighty-four over-contiguous (overlapping) slices with 1 mm thickness were sampled, which were automatically reconstructed to 368 slices with 0.5 mm thickness. The duration of each image acquisition was 6 min and 29 s. To secure consistency and reproducibility of the MRI slice placement and orientation, slice orientation of image stacks were defined using an automated anatomy recognition protocol based on landmark detection in MRI data (SmartExamoC) for a few hours before centrifuge and thereafter storage in an ultra-freezer (−80 oC). Aβ40, Aβ42, GFAP, and NfL concentrations were measured using the Single molecule array (Simoa) Human Neurology 4-Plex E (N4PE) assay, whilst T-tau was measured using the Simoa Tau Advantage kit according to instructions from the kit manufacturer . All measurements were performed on an HD-X instrument  in one round of experiments using one batch of reagents by board-certified laboratory technicians who were blinded to the clinical data. Intra-assay coefficients of variation were below 10%.Venous blood samples were obtained at empirically determined regular time points up to about 48 h after intrathecal administration of gadobutrol at the time of the MRI acquisitions and stored in the refrigerator  The absorption half-life , which is the time for half the amount of gadobutrol in the CSF to be cleared to blood, serving as one surrogate marker for CSF-to-blood clearance capacity of gadobutrol. (ii) Time to maximum concentration (Tmax) in plasma, which was obtained directly from the individual predictions. (iii) Lag-time of absorption to blood (Tlag) is the model-estimated time for the tracer to reach the site of clearance in CSF, implying that longer Tlag means that the tracer stays longer within CSF or that it takes longer before the clearance process to blood starts. (iv) Time to maximum concentration (Cmax) in plasma were obtained directly from the individual predictions. (v) Area under the concentration-time curve from zero to infinity (AUC0-∞) is a measure of systemic exposure to gadobutrol. The Cmax and AUC0-∞ were normalized with respect to dose in order to compare parameters across multiple doses. We have previously demonstrated dose linearity in the relevant range14.The CSF-to-blood clearance capacity was estimated by quantifying the intrathecally administered gadolinium in plasma according to a previously described method71, referring to the patient’s sleep over the past months, i.e. how their sleep is in general, not exclusively at the time of the MRI/blood sampling. A global PSQI score ≤5 is considered indicative of good sleep quality71.We assessed subjective sleep quality utilizing the Pittsburgh Sleep Quality Index (PSQI) questionnairet-test assessed the mean difference between groups. Due to skewed T-tau values, we used the logarithm of T- tau values (log10T-tau). A few unusually high values of GFAP above 3000 pg/ml and NfL above 400 pg/ml were assumed to be outliers and removed from the dataset.Statistical analyses were performed using Stata/SE 17.0 . Statistical significance was accepted at the 0.05 level (two-tailed). Daytime variation in biomarker concentrations was analyzed using a nonlinear model; a fractional polynomial linear regression with a maximum of one degree of the fractional polynomial and robust standard error for repeated measurements of the same subject. The plots were presented with the linear prediction (estimated mean from the regression model) and 95% confidence interval. A general linear model or a two-sample Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


To investigate the potential role of keratometry on whole globes in situ of deceased patients by assessing its repeatability and comparing it with sterile donor tomography after excision and preservation in organ culture.A sequence of 5 measurements was taken from 40 eyes in situ of deceased patients < 24 h after death using the portable Retinomax K-plus 3 . Keratometry of whole globes in situ, from which sclerocorneal discs were taken for organ culture, was compared to those obtained after measuring these sclerocorneal disks through their cell culture flask in medium I after 5 ± 4 days using the anterior segment optical coherence tomograph Casia 2 , and to 964 different donor corneas in medium II.P). The steepest (44.5D) and flattest (41.1D) P as well as the astigmatism (3.4D) of in situ corneas remained unchanged after preserving sclerocorneal discs in medium I . The comparison of the in situ values with the 964 measured different donor corneas in medium II showed significantly (p < 0.001) higher P at the steep (45.4D) and flat (43.9D) meridian and smaller astigmatism (1.4D) for sterile donor tomography.Cronbach’s alpha of the in situ keratometry was 0.891 and 0.942 for the steepest and flattest corneal power (Measuring deceased patients’ eyes in situ with the portable Retinomax K-plus 3 represents a feasible and reliably repeatable screening method in the eye bank. In comparison to donor tomography in medium I, it measures a similar power and astigmatism. Any corneal microsurgeon who performs high-volume penetrating keratoplasties (PKP) may have inadvertently transplanted donor corneas with  keratoconus or with a history of keratorefractive surgery, which typically entails significant postoperative refractive disadvantages for the patient –4. Such In this context, many authors have focused on the potential role of donor tomography in the eye bank. Initial studies on this subject were already performed in 2003 by Priglinger et al.  on a timDespite the scientific advances, sterile measurement of sclerocorneal disks in a sterile container has been a serious challenge for a long time due to the flat interfaces of this latter. Nevertheless, our group –16 succeIn addition to the development of donor tomoraphy, other methods have been proposed throughout the years as refractive screening devices in the eye bank. Stoiber et al.  presenteThe purpose of this study was to examine a potential new screening technique to avoid postoperative refractive surprises after corneal transplantation, as an alternative for sterile donor tomography for eye banks lacking an AS-OCT. In this context, the potential role of keratometry on whole globes in situ of deceased patients was investigated by assessing its repeatability and comparing it with AS-OCT parameters of sclerocorneal discs after excision and preservation in organ culture.Firstly, consent for corneal harvesting was obtained on the basis of an organ donor card  or from relatives (according to TPG § 4). Subsequently, a detailed medical and surgical history was collected from the donor’s medical records to exclude contraindications based on the guidelines of the European Eye Bank Association (EEBA) , whereafPrior to corneal explantation, 44 eyes from deceased patients were measured in situ using the portable Retinomax K-plus 3  within an interval of 1 day after death. First, a Mellinger eye speculum  was used to keep the eyelids apart. Then, a drop of sodium hyaluronate (0.2%) was instillated to ensure a more regular corneal surface before keratometric reading. In situ measurements with the portable Retinomax K-plus 3 were repated 5 times consecutively.2 Canted-Neck Cell Culture Flask, Corning Inc., Corning, NY, USA) that were initially filled with an isotonic (307 mOsmol/kg) nutrient medium (medium I) . This standard organ culture medium I  is composed of 10% Minimum Essential Medium (MEM) with Earle’s balanced salts supplemented with 1% penicillin/streptomycin, 1% amphotericin B (250 μg/mL), 1% l-glutamine (200 mM), 1.25% Hepes buffer (1 M), 2.93% NaHCO3, and 2% fetal calf serum. Due to the absence of dehydrating agents, the donor corneas swell considerably in this medium. The tissues were stored in this organ culture at 34 °C for a maximum of 34 days. Donor corneas designated for penetrating keratoplasty based on tissue evaluation  were transferred 1–3 days prior to surgery into another culture medium (medium II)  in the eye bank’s laminar flow unit. This hypertonic medium (353 mOsmol/kg) contains 6% dextran T500, a hydrophilic macromolecule that induces colloid osmotic pressure and thus deswells the corneas in order to obtain a corneal graft thickness comparable to that of the recipient cornea at the time of surgery.Donor corneal tissues were harvested in a sterile manner as sclerocorneal disks with a diameter of 15 mm. After harvesting, the donor corneas were preserved in our Klaus Faber Centre for corneal diseases incl. LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz in Homburg/Saar. The sclerocorneal disks were stored in cell culture flasks . These preoperative donor tomographies were carried out at least 12 h after transfer of the corneal tissues into medium II in order to enable a reliable measurement of the donor corneal tissue after deswelling . This OCn = 44) of the repeated in situ keratometric readings were compared to the routinely measured sterile tomography of 964 donor corneas using a Mann–Whitney U test in regards to the dioptric power (P) at the steep and flat meridian of the cornea, as well as the corneal astigmatism. The respective keratometric values of the whole globes, from which sclerocorneal discs were harvested for organ culture in the eye bank, were also compared to those obtained after measuring the same sclerocorneal discs in medium I (n = 25) after 5 ± 4 days, using a Wilcoxon test.All the means  version 20. Result values are expressed as mean x̄ ± standard deviation (SD) (minimum–maximum) unless otherwise stated. A n = 40) was 1.3 D and 0.9 D for the power (P) at the steep and flat meridian of the cornea, respectively, and 1.4 D for keratometric astigmatism. The corresponding Cronbach’s alphas were 0.891, 0.942, and 0.794, respectively.The mean standard deviation of the five in situ measurements (P (44.5 ± 2.3 D) of in situ corneas remained unchanged (p = 0.09) after preserving the same sclerocorneal discs (n = 25) in medium I (sterile tomography 44.7 D ± 2.1 D) after 5 ± 4 days. Again, the flattest P (41.1 ± 1.8 D) of in situ corneas remained unchanged as well (p = 0.17) compared to sterile tomography of the same sclerocorneal discs after explantation (41.4 ± 2.0 D). The keratometric astigmatism (3.4 ± 1.8 D) also remained unchanged (p = 0.09) in comparison to tomographic values in medium I (3.3 ± 1.8 D)  of deceased patients with the routinely measured sterile tomography of 964 different donor corneas in medium II showed a statistically significantly different (p < 0.001) value for all examined parameters. Indeed, P at the steep meridian was measured significantly (p < 0.001) smaller  with in situ keratometry (44.2 ± 2.5 D) in comparison to sterile tomography (45.4 ± 1.8 D). Again, P at the flat corneal meridian was measured significantly (p < 0.001) smaller  with in situ keratometry (41.0 ± 1.8 D) in comparison to sterile tomography (43.9 ± 1.3 D). The keratometric astigmatism (3.2 ± 2.0 D) was significantly (p < 0.001) higher than the sterile tomographic astigmatism (1.4 ± 1.7 D)  . On the morphological  as well as curvature anomalies  [However, in contrast to donor tomography, in situ donor keratometry does not allow further analysis of the posterior curvature nor corneal thickness. In this context, the sterile donor tomography allows a more detailed analysis of donor corneas in the eye bank. Moreover, donor tomography allows the detection of corneal tissues with surgery) –17, wherIn contrast to in situ donor keratometry, uneven epithelial coating between corneas in organ culture could lead to differences in donor tomographic measurements. Indeed, Neubauer et al.  showed tDuring in situ donor keratometry, the corneas were examined in their natural environment. However, pressure applied by the eye speculum (eyelid retractor) could still have biased the measured radii of curvature by distortion following mechanical stress. During sterile donor tomography, sclerocorneal disks are examined in their cell culture flask. The storage and attachment of these corneas to the cell culture flask holder may cause slight deformation of the donor corneal tissue, rendering the measured geometry inconsistent with in situ conditions , 22, 23.Independent of its clinical relevance, in situ keratometry seems to represent a highly repeatable measurement method with regard to the calculated Cronbach’s alpha . SterileWith regard to the aforementioned results presented in this study and regardless of the above-described (dis)advantages of in situ keratometry, this technique on whole globes of deceased patients seems to represent a suitable screening method for eye banking when compared to sterile donor tomography, as the keratometric values  remained statistically unchanged after preserving the same sclerocorneal discs in medium I. Nevertheless, those values differed statistically when being compared to the routinely in organ culture medium II measured sterile tomography of 964 different donor corneas (much larger sample size). However, it has contradictorily also been shown that the (hourly measured) corneal curvature did not change within 24 h after deswelling in medium II , which cMoreover, the validity of the donor tomographic measurements should be examined by means of available tomographic measurements during the donor’s lifetime or alternatively using histological reprocessing of unused corneal tissues to detect corneal pathologies or situations with a history of LASIK or keratokonus in respectively remarkably flat and steep donor corneas. Only then, the significance of donor tomography could be proven and be seen as a reference method with which other techniques may be compared. These projects are currently being researched in our eye bank.Given the results and information presented in this study, the question of the clinical role of in situ keratometry arises. Much more than an alternative for eye banks lacking an AS-OCT, in situ keratometry can be considered as an “addition to” rather than as a “replacement for” sterile donor tomography. Indeed, sterile donor tomography provides a more complete screening of donor corneas, but in situ donor keratometry allows a pre-harvesting screening, avoiding the collection of extremely curved/flat corneas und thus possibly screening out likely unsuitable corneas before harvesting.To avoid postoperative refractive surprises, donor corneas with an out of range refractive power should be discarded from penetrating (PKP) or deep anterior lamellar keratoplasty (DALK), but may potentially be suitable for posterior lamellar keratoplasty such as Descemet membrane endothelial keratoplasty  or DescIn situ donor keratometry represent a suitable, sterile, and reliably repeatable method to help prevent refractive surprises after keratoplasty, thus further optimizing tissue selection in the eye bank. Further studies about the quality of in situ keratometry should determine if this latter may represent a valid alternative for sterile donor tomography for routine screening of donor corneas in daily practice. Positive controls of donor corneas with ectasia or prior keratorefractive surgery still need to be screened prior harvesting in order to demonstrate the sensitivity of the technique in such detection.


Cryptococcus neoformans. Biopsy studies show that the drug exhibits adequate brain penetration although levels of cerebral spinal fluid (CSF) are highly variable. Considering that CSF is one of the main surrogates for CNS exposure, the present work proposed the building of a population pharmacokinetic modeling (popPK) model able to describing the exposure achieved by voriconazole in the plasma, interstitial cerebral fluid and CSF of healthy and infected rats. The developed popPK model was described by four compartments, including total plasma, free brain and total CSF concentrations. The following PK parameters were determined: Km = 4.76 mg/L, Vmax = 1.06 mg/h, Q1 = 2.69 L, Qin = 0.81 h−1 and Qout = 0.63 h−1. Infection was a covariate in the Michaelis–Menten constant (Km) and intercompartmental clearance from the brain tissue compartment to central compartment (Qout). Simulations performed with the popPK model to determine the probability of reaching the therapeutic target of fAUC > MIC showed that VRC has sufficient tissue exposure in the interstitial fluid and in the CSF for the treatment of fungal infections in these tissues at prevalent minimum inhibitory concentrations.Voriconazole is a triazole antifungal used empirically for the treatment of complicated meningitis associated with  Cryptococcus [Cryptococcal meningitis is one of the most common fungal infections that affect the central nervous system (CNS), accounting for a large number of deaths annually ,2,3. Thetococcus ; howevertococcus . Regarditococcus ,9. An altococcus ,9,10.Recently, case reports have shown that voriconazole (VRC) could be used as an alternative treatment for patients with cryptococcal meningitis whose standard treatment has failed ,12,13. VC. neoformans caused an increase in the free brain penetration of VRC in the infected animals, with the brain exposure in terms of the ratio of the area under the concentration curve (AUC) in the brain related to plasma being 1.86 ± 1.05 for the infected group and 0.85 ± 0.22 for the healthy group [VRC presents good penetration into human brain tissue, reaching concentrations similar to or even higher than those observed in plasma. However, VRC concentrations that are observed in human cerebrospinal fluid (CSF) tend to be reduced, with CSF/plasma concentration ratios of 0.22 to 1.0 in adults ,19,20 anIn the clinical scenario, concentrations of drugs in the brain are measured in the CSF, which is the most accessible site to assess the drug extension effect. However, CSF concentrations cannot be assumed to be equal to interstitial fluid (ISF) concentrations without first investigating the distribution and concentration in these compartments due to the inherent characteristics of the barriers present in the CNS, drug-related factors and the presence of infection ,24.With the aim of building a single model by grouping plasma data and data from two CNS regions, this work built a POP-PK model based on plasma, ISF and CSF to understand the differences that occur in the penetration of VRC into the CNS and to allow for a better evaluation of clinical scenarios and understanding whether isolated CSF concentrations, which are used in the clinic, are sufficient predictors of drug availability in the CNS.Cryptococcus neoformans-infected male Wistar rats, who received a 5 mg/kg dose of VRC i.v. [The popPK model was developed using data from three preclinical studies. The study design and methodologies used to quantify VRC concentrations in the collected samples have been extensively described in those publications ,25,26. BVRC i.v. . Lelièvr® v.3.0.0  were used to keep track of run records and results. For graphical analysis, R program, version 4.1.1, RStudio, version 1.4.1717  and the packages xpose4 and ggplot2 were used.Concentration–time profiles were analyzed using non-linear mixed-effects modeling in NONMEM v.7.4.3 , applying the first-order condition estimation method with interaction (FOCE-I) and ADVAN13 subroutine. PsN software version 4.9.0  and PIRANAThe data were modeled as log-transformed and free plasma concentrations were calculated using plasma protein binding value of 66% . MicrodiStructural model building was performed sequentially. First, the observed VRC plasma concentrations obtained from healthy and infected rats were fitted to a two-compartment model. The model was then expanded to include the free brain and CSF concentrations. Thus, inter-individual variability (IIV) was modeled exponentially, and residual variability was described separately for plasma, microdialysate and CSF data with a log-additive error model.p ˂ 0.05) and backward elimination (p ˂ 0.01) procedure. The infection and body weight were analyzed as covariates. Model selection was guided by significant changes in the value of the objective function (OFV), measured by at least a decrease of 3.84 or 6.64 points in OFV (p < 0.05 or p < 0.01), visual exploration of goodness-of-fit (GOF) plots and the relative standard error (RSE), informing the precision of model parameters.Once the base structural model had been determined, the contributions of covariates to the population parameters variability were assessed by applying a stepwise forward inclusion ) to describe the difference between the weights of rats of different lineages from the data used ,30.(1)PiP corresponds the individual parameter, max) and Michaelis–Menten constant (Km), and k = 1 for all the other parameters.In this equation, Model evaluation was performed according to GOF plots, %RSE and conditional number and through prediction-corrected visual predictive checks (pcVPCs) for each group . The pcVPC generated 1000 simulated profiles with its respective 10th, 50th and 90th percentiles calculated and visualized with the experimental data. A non-parametric bootstrap resampling analysis, stratified on concentration measurement site and study group with 1000 replicates, was performed to obtain the medians and confidence intervals (CIs) of the 5th and 95th quartiles, checking the model stability.p,uu,CSF) for rats by the simulation of 1000 individuals of the dataset randomly distributed. We calculated the Kp,uu,CSF (Equation (2)) of rats using the free plasma and free CSF exposure described by the area under the concentration–time curve (AUC) of the profiles. To calculate Kp,uu,CSF for humans, we corrected the value from rats with the albumin concentration in humans and rats and free plasma fraction [u,CSF represents the unbound fraction of CSF, which was calculated using Equation (3):alb is the ratio of albumin concentrations of CSF to plasma and was set to 0.005 for humans and 0.003 for rats.Using the popPK model developed, we performed simulations to predict the human exposure to VCR in CSF and to evaluate if this exposure will be effective in treatment. To perform these simulations, we used VRC dosing regimens indicated for the treatment of fungal infections , allometfraction .(2)Kp,upuu,CSF value calculated for rats and humans was used to estimate free concentrations in the CSF by the simulation of a model with 1000 individuals. The exposure obtained in the CSF of rats using human doses was used to predict the probability of target attainment (PTA) of treatment using a pharmacokinetic/pharmacodynamic (PK/PD) index free AUC over MIC (fAUC/MIC) higher than 25 [C. neoformans MIC (from 0.002 to 512 mg/L) . A PTA > 90% was assumed as adequate clinical outcome. So far, a PK/PD index value has not been established for voriconazole against C. neoformans. Therefore, we used the PK/PD ratio described for Candida and Aspergillus [The K than 25 ,34 for Vergillus ,34, assuergillus .Cryptococcus neoformans-infected rats), we added 4 more healthy rats to the initial group for data enrichment, and 284 microdialysate concentrations in brain tissue  and 18 CSF concentrations were included in the population analysis. A summary table of individuals/groups and the respective number of observations is provided in After a total of 287 plasma concentrations , peripheral volume of distribution (V2) and intercompartmental clearance (Q1). The final structural model was expanded to four compartments to accommodate unbound brain concentrations and CSF concentrations. Compartments three and four describe the brain. The brain is represented as the third compartment, where microdialysate samples were collected. The volume of this compartment (V3) was fixed as 0.00041 L, the interstitial physiological value [in and Qout, respectively). The fourth compartment, linked to the brain interstitial fluid, is represented by CSF concentrations, where we have the CSF volume of distribution (V4) and intercompartmental clearance (Q4) (Plasma concentrations were best described by a two-compartment model with Michaelis–Menten (MM) elimination, parameterized in terms of Val value . This conce (Q4) .1, A2, A3 and A4 are plasma, peripheral plasma, brain and CSF unbound amounts of VRC, respectively; Vmax is the maximum rate of metabolism; Km is the Michaelis–Menten constant; V1 is the central volume of distribution; V2 is the peripheral volume of distribution; Q1 is the intercompartmental clearance from the central compartment to peripheral compartment; Qin is the intercompartmental clearance from the central compartment to brain tissue compartment; Qout is the intercompartmental clearance from the brain tissue compartment to central compartment; V3 is the volume of the brain tissue compartment; Q4 is the intercompartmental clearance from the central compartment to CSF compartment; and V4 is the volume of the CSF tissue compartment. To compare observed plasma concentrations with the free concentrations estimated by the model, the estimates were divided by 0.35, the unbound fraction of the drug [Equations used to estimate free concentrations at the different compartments are described below (Equations (4)–(7)):the drug .m, V1, V2 and Qout. An additive error model for each plasma, brain and CSF concentration was sufficient to describe the residual unexplained variability. The infection was included as a covariate in Km and Qout. The parameters Km and Qout were calculated separately for the infected group. The calculated Km was higher in infected animals ) compared to the healthy animals (Km = 4.76 (mg/L)) and the calculated Qout was lower in infected animals ) compared to the healthy animals (Qout = 0.634 (mg/L)).IIV was described by an exponential model and was then estimated for Km and for Qout, respectively; m and for Qout, respectively; and the infected status is defined as a categorical covariate with 0 and 1 for healthy and infected animals, respectively. The final individual parameters, including the covariates, are expressed as follows:Model parameters were estimated with good precision and diagnostic plots showed a good agreement between the observed and predicted data . The pc-C. neoformans for free plasma, free brain and free CSF concentrations. The target PK/PD index used was a VRC fAUC/MIC greater than 25 [To assess the PTA of dosing regimens used in clinical practice, simulations of 1000 profiles using the final popPK model were conducted to evaluate different allometrically scaled VRC dose regimens . Figure  than 25 ,34 with C. neoformans MICs, both in plasma and tissues, when using an initial dose of 6 mg/kg/12 h of VRC on the first day and a maintenance dose of 4 mg/kg/12 h on each subsequent day [The PTA for the healthy and infected groups was more than 90% for the most prevalent uent day ,32. HoweWhen we look at treatment with an initial dose of 3 mg/kg/12 h of VRC on the first day and a maintenance dose of 2 mg/kg/12 h on each subsequent day, which represent a 50% reduction compared to standard treatment ,32, we oVRC is used for the treatment of systemic fungal infections, it being indicated for the treatment of cryptococcal meningitis in cases of resistance to other drugs, such as 5-FH, FLU and AmB. When used in monotherapy, VRC presents good activity, which can be attributed to its penetration into the CNS, including the CSF .C. neoformans cryptococcal meningitis was investigated using the PK/PD index ƒAUC/MIC > 25.In this work, we investigated the penetration of VRC in the CNS, specifically in the ISF and CSF, developing a preclinical popPK model with data obtained from total plasma concentrations, free brain concentrations and total CSF sampling in rats. The developed model was simulated to predict free brain and free CSF exposure after VRC doses allometrically scaled from humans to rats. The PTA of free concentration–time profiles generated by model simulation for different dosing regimens to treat C. neoformans infection in brain [in and Qout) and the compartment CSF was linked to the brain compartment. This developed model showed alterations in the VRC distribution during the infection caused by C. neoformans through modifications in the pharmacokinetic parameters, such as the Michaelis–Menten constant (Km) and intercompartmental clearance from the brain tissue compartment to central compartment (Qout). The literature describes that the VRC presents non-linear pharmacokinetics due to the saturation of its metabolism from doses more than 5 mg/kg [The final popPK model was an extension of that previously reported for in brain , with an 5 mg/kg ,22,25,26m was higher in infected animals ) compared to the healthy animals ) and the value of Qout was lower in infected animals ) compared to the observed value in healthy animals ). One of the factors most associated with infection that can affect drug exposure in the CNS is inflammation of the meninges. This inflammation makes the BBB more permeable, in addition to causing a decrease in the return clearance for venous blood. This factor helps to explain a reduction in the Qout of infected animals in relation to the Qout of healthy animals. It should be noted that, during infection, concentrations are increased in the CNS; however, during treatment, they may change as patients respond, and thus inflammation may decrease, thereby lowering target site concentrations necessary for disease eradication [The calculated Kdication .0–t) was performed with predicted data obtained from the model to compare with the AUC0–t of the works from which the data were taken. The results can be seen in Post hoc analysis of the area under the concentration–time curve , calculated by the ratio between free AUC in plasma and in the brain, which was 0.82 for healthy mice. For infected mice, the fTPLASMA/BRAIN was 1.50 in ISF, i.e., the infected group presented an increased VRC in brain. These results are similar to those found by Alves et al. (2017) who observed a fTPLASMA/BRAIN of 0.85 ± 0.22 for the healthy group and 1.86 ± 1.05 for the infected group [fTPLASMA/CSF of 2.57, indicating greater penetration into the CSF, agreeing with the findings by Lelièvre and collaborators, where they obtained a fTPLASMA/CSF of 2.10 [The AUCed group . However of 2.10 . In addi of 2.10 .CSF concentrations are used clinically as the same as or as substitutes for ISF concentrations due to the greater ease of collecting these samples in relation to ISF ,24,39. AC. neoformans and that the PK/PD target used for the analysis was the one described for Candida and Aspergillus, considering that these fungi share some similarities with C. neoformans [fAUC/MIC > 25 as a target showed that, for the most prevalent MICs (0.06 and 0.125 mg/L) of C. neoformans plasma, ISF and CSF concentrations are efficient for both healthy and infected groups. From the MIC of 4.0 mg/L for both doses, none of the observed concentrations reach PTA > 90%.Using the developed model, we calculated the PTA in different treatments used in clinical practice to assess the probability of these treatments reaching a pharmacological target. It is necessary to take into account that, so far, there is no established PK/PD target for VRC and oformans ,34. PTA fAUC > MIC shows that voriconazole has sufficient tissue exposure in the cerebrospinal fluid for the treatment of fungal infections in these tissues in the most prevalent MICs. The use of plasma concentrations can lead to the use of higher doses than necessary because, as we demonstrate, the CSF concentrations are higher than plasma. Furthermore, the concentrations of voriconazole observed in the CSF are shown to be predictors of the concentrations observed in the ISF; however, this information should be analyzed with caution since there is no established PK/PD index for VRC against C. neoformans, even though the literature describes that we can assume that the PK/PD ratio is consistent within a drug class and between pathogens.The probability of reaching the therapeutic target of C. neoformans in relation to the healthy group, and the concentrations of free CSF showed a greater penetration in healthy animals. The PTA analysis demonstrated that the CSF concentrations obtained after an allometric dose on a scale from humans to rats are sufficient to eradicate the infection and that the CSF concentrations can be used as a predictor of the concentrations observed in the ISF in the case of voriconazole since both concentrations showed the same result in terms of reaching the PTA (>90%) in both conditions and doses tested.In conclusion, we present the results of a popPK model of VRC concentrations in plasma, ISF and CSF. The penetration of VRC was greater in the tissue of rats infected by