Aiming at the problems of low fertilization efficiency, mainly the process operation and inconsistent fertilization depth of domestic tea garden fertilizer machines, a single-spiral fixed depth ditching and fertilizing machine is appropriately designed. This machine is capable of performing the integrated operation of ditching, fertilization, and covering soil at the same time through the operation mode of single-spiral ditching and fertilization. The theoretical analysis and design of the structure of the main components are properly carried out. The fertilization depth can be adjusted through the established depth control system. The performance test reveals that the single-spiral ditching and fertilizing machine exhibits a maximum stability coefficient of 96.17% and a minimum of 94.29% in terms of trenching depth and a maximum of 94.23% and a minimum of 93.58% in terms of fertilization uniformity, meeting the production requirements of tea plantations. The main method of ditching and fertilizing in China is to build a trench with a rotary tiller, then utilize a fertilizer spreader or spread the fertilizer manually, and finally do soil tillage by hand7. This fertilization approach tends to have relatively low fertilizer use efficiency and residual fertilizer left on the surface, which could easily pollute the environment9. A suitable fertilization situation is able to improve the fertilizer utilization ratio11. A fertilization position that is too far from the roots could easily lead to poor crop absorption and a low fertilizer ratio, and a fertilization position that is too close to the roots could easily result in root burning fertilizer problems. Research by agronomists has revealed that a reasonable fertilization position ensures maximum absorption of fertilizer by crop roots, improves fertilizer utilization ratio and is crucial for lessening the fertilizer rate14.Fertilizer application is one of the crucial aspects of crop production, the quality of fertilizer application directly influences the growth of crops, and reasonable fertilizer application is commonly regarded as one of the important measures to ensure high quality and high yield of crops17. The start of ditching and fertilizing machines was relatively late in the country, with rotary furrow fertilizing machines being mainly used18. Xiao et al.19 developed a double spiral ditch and fertilizer machine for gardens, which is used in conjunction with a tractor to apply fertilizer evenly and can meet the agronomic needs of garden fertilization. Gaomi Yifeng Machinery Co., Ltd. produced a self-contained multi-purpose fertilization device, which passes through a screw fertilizer distributor to adjust the amount of fertilizer planting and a crawler chassis with good passing performance. Shen Congju et al.20 developed a self-propelled gas deep loosening fertilization device for gardens, which solved the technical problems of fertilizer punching and dosing in hard ground, and a theoretical basis and technical support for the development of a gas explosion deep loosening fertilizing machine for orchard21.Foreign developed countries have the early start of research on fertilizing machines, and the development of their ditching machines has led to the rapid development of ditching and fertilizing devices, experiencing several stages of share plows, rotary ditchers, and chain ditchersFrom the results of research works inside and outside the country, it is clear that domestic fertility machines are mainly one-process operations and lack automatic depth adjustment devices. Foreign ditching and fertilizing machines are more advanced, but they are expensive and do not meet the operational needs of our country. In the present paper, a spiral fixed-depth ditching and fertilizing machine is designed, which could appropriately perform the integrated operation of ditching, fertilization, and covering soil, and can automatically adjust the fertilization depth to achieve constant depth fertilization, in order to enhance the operational efficiency and fertilization effect of the ditching and fertilizing machine. This article is divided into four parts, where the first part gives an introduction to the current state of research on fertilizing machines. The second part is aimed to design the mechanical structure and control system of the fertilizing machine. The third part deals with the ditch test, fertilization test, and field test. The fourth part summarizes the whole article and the main obtained results.In order to improve the fertilization efficiency, along with the agricultural needs of tea planting, a single-spiral fixed depth ditching and fertilizing machine is appropriately designed, where the corresponding whole structure is schematically illustrated in Fig. The depth of the ditching is predetermined according to the agronomic requirements of fertilization before working the ditching and fertilizing machine operates. During ditching and fertilization, the tractor pulls the ditching and fertilizing device forward, the ditching and fertilizing machine cuts into the soil, the fertilizer is discharged through the screw fertilizer distributor and transported to the ditching and fertilizing mechanism through the fertilizer delivery pipe, and finally falls into the furrow cut by the ditching and fertilizing mechanism. At the same time, the soil falls back to complete the covering soil, realizing the integrated action of ditching, fertilizing, and covering the soil.The fertilizer apparatus adopts the screw fertilizer distributor, which essentially consists of a fertilizer box, fertilizer outlet, fertilizer inlet, rotating shaft, and fertilizer spiral blade, as demonstrated in Fig. 22. Since the amount and speed of fertilizer planting are not taken into account, the formula for evaluating single-ring sowing-fertilizer takes the following form:D represents the outer diameter of the screw fertilizer distributor (mm), d denotes the inner diameter of the screw fertilizer distributor (mm), s is the pitch of the screw fertilizer distributor (mm), b signifies the average thickness of the screw tooth of the screw fertilizer distributor (mm), h represents the depth of the screw tooth of the screw fertilizer distributor (mm), L is the average length of the screw tooth of the screw fertilizer distributor (mm), ρ denotes the fertilizer volume weight (g/mm3), and φ stands for the filling coefficient of the screw fertilizer distributor.The diameter of the screw fertilizer distributor is one of the crucial factors of screw fertilizer distributors, which directly affects the amount of fertilizer planted by the screw fertilizer distributor and the uniform variation coefficient of fertilizer distributionq) depends on various factors, including D, d, S, ρ, and φ. The single-ring fertilizer sowing amount (q) changes by altering the values of D, d, and S. The relationship between the outer diameter of the screw fertilizer distributor and the fertilizer sowing amount in the entire fertilizer sowing operation is given by: Q represents the fertilizer sowing amount of screw fertilizer distributor (t/h), A is the material comprehensive characteristic coefficient, K denotes the material comprehensive coefficient, c is the proportional coefficient of pitch to diameter, λ represents the mass of the material unit volume (t/m3), and ε denotes the transfer coefficient.By virtue of Eq. , it can Qs represents the fertilizer rate (t/h), g is the fertilizer rate to travel a distance (t), and s stands for the distance traveled (m).When the machine is in continuous operation, the fertilizer rate can be expressed by:Qs) can be obtained as 0.6t/h and the fertilizer sowing amount (Q) is also predicted to be 0.6t/h on the basis of Eq. (φ) is 0.25, the comprehensive characteristic coefficient of the fertilizer (A) is 28, the unit mass of the material of the fertilizer is 1.2 T/m3, the proportional coefficient of the pitch to diameter is 0.9, and the conveying coefficient is 0.9, which are substituted into Eqs. .23, and the pitch of the screw fertilizer distributor is evaluated per the following relation:K1 signifies the proportional coefficient of the pitch and diameter, usually in the range of 0.8–1.0, which is mostly related to the flow ability of the fertilizer.The pitch of the screw fertilizer distributor determines the angle of ascent of the screw and the speed of fertilizer progress, which exhibits a crucial impact on the amount and uniformity of fertilizer from the screw fertilizer distributorK1=0.8, so the pitch is 70.4 mm, rounded to 70 mm.According to the existing design experience, comprehensive analysis to take 24. When the rotational speed is too high, the fertilizer will stick to the inner wall of the screw fertilizer distributor under the action of centrifugal force; therefore, the centrifugal force of the fertilizer and gravity should satisfy the following requirements:In order to maximize the fertilization uniformity while meeting the fertilizer requirements, the suitable rotational speed of the rotating shaft can ensure the stability of the fertilizer rate. It should be noticed that the rotational speed of the screw fertilizer distributor can be determined based on the fertilizer rate, the diameter of the screw fertilizer distributor, and the relevant material parameters of the fertilizer to determine the better rotational speedA represents the fertilizer comprehensive coefficient, K0 denotes the fertilizer comprehensive characteristic coefficient, nmax is the maximum speed of the screw fertilizer distributor (r/min), and n stands for the rotational speed of the crew fertilizer distributor (r/min). The fertilizer sowing amount of the crew fertilizer distributor can be calculated as:Considering the effect of various fertilizers,n ≤ 200 (r/min).According to Eqs.  and 16)16), the The spiral ditching device of the single-spiral ditching and fertilizing machine uses single-spiral ditching, as demonstrated in Fig. D1 represents the diameter of the spiral blade (mm), and D2 denotes the ditch width of the fertilizing machine (mm).The single-spiral ditching and fertilizing machine is driven in tea fields, relying on the spiral blade for ditching operation, and together with the relevant agricultural needs, the ditch width is determined to be not less than 250 mm. The diameter of the spiral blade is:The diameter of the spiral blade is selected as 250 mm since the single-spiral and fertilization machine requires a maximum ditch depth of 450 mm. Further, the height of the spiral blade is set as 350 mm, and the height of the tool bit is adjusted to 100 mm because the spiral blade should accomplish the cutting and slanting operation to the soil. At the same time, it performs the straight line ditching operation, indicating the high necessity for high strength and hardness of the material; hence, CR12MOV with a thickness of 10 mm is chosen as the material.25,26. The single-spiral ditching and fertilizing machine employs a cylindrical variable spiral ditching mechanism, and the variable pitch spiral curve consists of numerous tangents , n denotes the number of spiral turns, γ signifies the spiral angle (°), n1 is the number of spiral turns at B, β represents the spiral angle at C (°), and h denotes the height of point F (mm).Consider a point A on the generatrix along the The efficiency of the spiral ditching and fertilizing mechanism relies on the action of the spiral blade to complete the cutting and lifting operation on the soil. According to the agricultural needs of tea fertilization, the width of ditching is set in the range of 200–300 mm and the corresponding depth is considered to be 300–400 mm. Taking into account the power consumption, as well as the depth and width of fertilization, the main parameters of the spiral ditching and fertilizing mechanism are designed by combining Eqs.  and 18)18), as pThe single-screw ditching mechanism cuts to the bottom of the ditch and its automatic adjustment process has been illustrated in Fig. H) is obtained by setting the height of the fixed depth (H1) according to the growth of tea trees and the agronomic requirements of fertilization. The ditching cutter arbor rotates and cuts into the soil, as the fixed-depth ground wheel touches the ground. At this time, the depth sensor of the fixed depth ground wheel transmits the signal to the single-chip microcomputer in real-time, which is responsible for controlling the expansion and contraction of the hydraulic cylinder. To this end, the relay and solenoid valve are mainly employed to realize the real-time adjustment of the ditching and fertilizing depth and keep that constant . The adjustment principle diagram is provided in Fig. During the ditching operation, the depth of the ditch and fertilization  and the Measuring Methods for Agricultural Machinery Testing Conditions-General Rules (GB/T 5262-2008).For the ditching test, the single-spiral ditching and fertilizing machine was operated at a uniform speed of 1.5 m/s to make three operational strokes, each 80 m long, with 10 m before and after the two stroke’s sections remaining as its reserved area, and to test 5 points equally between 20 and 70 m. The average ditching depth and the stability coefficient of each stroke are separately evaluated, and the test results are given in Table The ditching test results indicate that the maximum difference in depth is 17 mm subjected to various strokes and the minimum value of the coefficient of stability of the ditching depth in the case of three strokes is 94.81%, which is in accordance with the evaluation index for the ditching machine operation.In the test tea garden, the single-spiral ditching and fertilizing machine performed under 6 strokes of fertilizing operation. For this purpose, the length of each operational stroke and the length of the protected area before and after was set equal to that of the ditching test, from a stroke of 10 m to a stroke of 40 m. In this interval, based on considering each 10 cm as a test interval, each stroke was divided into 30 test intervals, resulting in a total of 180 measurements. During the test, the single-spiral ditching and fertilizing machine moved at a uniform speed, and in the preparation stage, the spiral ditching and fertilizing mechanism raised and traveled through the test area at a constant speed. The fertilization in each area was then collected separately, and its weight was assessed by an electronic scale. After the completion of the individual travel operation, its fertilization uniformity was calculated in turn, and its appropriate test results are provided in Table Th Fertilization test results reveal that the fertilization uniformity for each individual stroke is higher than 93.15%, which meets the operational requirements of the evaluation index for the fertilizing machine.Test site: The test was conducted in April 2022 at the tea garden of Pingshan Tea Factory in Liuhe District, Nanjing, China, and the field trial has been illustrated in Fig. Test conditions: Sunny weather, temperature 4–22 °C, breeze, plant spacing of 290 mm and row spacing of 1600 mm in the tea garden, flat land in the test tea garden, the soil is sandy loam, and soil moisture content of 13.2%.Test materials: Granular organic fertilizer produced by Hebei Dewaldo Fertilizer Company.Repeated tests were performed with three strokes and the test results are presented in Table In the current investigation, a single-spiral fixed depth ditching and fertilizing machine is designed, which can perform the integrated operation of ditching, fertilization, and covering soil, and can automatically adjust the fertilization depth to achieve a constant fertilizing depth. Through the theoretical analysis, the screw fertilizer distributor, the spiral ditching and fertilizing mechanism, and the corresponding control system are designed, and the ditching depth is automatically adjusted by the fixed depth control system, so that the operation efficiency and fertilization effect of the ditching and fertilizing machine were appropriately improved.The fertilizing machine designed in this paper can effectively solve the problem that the fertilization position of the traditional fertilizing machine is unreasonable, which readily makes the fertilizer use efficiency relatively low, and the residual fertilizer on the surface can easily pollute the environment. The fixed depth fertilization can be realized through the constant depth control system, so as to ensure the reasonable position of fertilization, which enhances the utilization rate of fertilizer. The fertilizing machine designed is able to effectively solve the problem of unreasonable fertilization position, improves the utilization rate of fertilizer, and exhibits significant impacts to reduce the fertilizer rate.The field test results revealed that the coefficient of stability of the ditch depth of the single-spiral ditching and fertilizing machine was at the maximum of 96.17% and at the minimum of 94.29%. The maximum fertilization uniformity value was also reported to be 23.94% and 93.58% at the minimum, meeting the requirements of tea plantation production.Supplementary Information.


Hoplobatrachus rugulosus (Anura: Dicroglossidae) is distributed in China and Thailand and the former can survive substantially lower temperatures than the latter. The mitochondrial genomes of the two subspecies also differ: Chinese tiger frogs (CT frogs) display two identical ND5 genes whereas Thai tiger frogs (TT frogs) have two different ND5 genes. Metabolism of ectotherms is very sensitive to temperature change and different organs have different demands on energy metabolism at low temperatures. Therefore, we conducted studies to understand: (1) the differences in mitochondrial gene expression of tiger frogs from China (CT frogs) versus Thailand (TT frogs); (2) the differences in mitochondrial gene expression of tiger frogs (CT and TT frogs) under short term 24 h hypothermia exposure at 25 °C and 8 °C; (3) the differences in mitochondrial gene expression in three organs  of CT and TT frogs.RT-qPCR and comparing control groups at 25 °C with low temperature groups at 8 °C, we came to the following results. (1) At the same temperature, mitochondrial gene expression was significantly different in two subspecies. The transcript levels of two identical ND5 of CT frogs were observed to decrease significantly at low temperatures (P < 0.05) whereas the two different copies of ND5 in TT frogs were not. (2) Under low temperature stress, most of the genes in the brain, liver and kidney were down-regulated (except for COI and ATP6 measured in brain and COI measured in liver of CT frogs). (3) For both CT and TT frogs, the changes in overall pattern of mitochondrial gene expression in different organs under low temperature and normal temperature was brain > liver > kidney.Utilizing ND5 gene between CT and TT frogs could result in the different tolerances to low temperature stress. (2) At low temperatures, the transcript levels of most of mitochondrial protein-encoding genes were down-regulated, which could have a significant effect in reducing metabolic rate and supporting long term survival at low temperatures. (3) The expression pattern of mitochondrial genes in different organs was related to mitochondrial activity and mtDNA replication in different organs.We mainly drew the following conclusions: (1) The differences in the structure and expression of the  FR10 and other related antifreeze proteins are synthesized to avoid damage to the organism under low-temperature stress . Th. ThND5 gaptation . Hence,  disease . Studiesry chain . In addiactivity , and is activity . Thus, moduction , 44. 288-helices . The mitironment –48.COIII, ND1, ND2, ND3, ND4L and Cytb (P < 0.05) were found in the cold of CT and TT frogs. Gene transcript levels of ND1, ND2 and ND4L in liver were significantly reduced in both CT and TT frogs in response to cold. In kidney, most genes showed significant downregulation in CT frogs, but in TT frogs, only downward trend was detected, but no significant difference was found. Several previous studies have also demonstrated similar relationships between mitochondria and low-temperature exposure. For example, Colinet et al. [Drosophila melanogaster would temporarily decrease under exposure to low temperature. Cold exposure also led to an extreme reduction of the resting metabolic rate of Bufo marinus, which showed an adaptation of mitochondrial bioenergetics [COI transcript level was decreased with exposure to freezing in D. versicolor, as part of the modulation of mitochondrial protein-coding gene expression in the molecular response to freezing [In this study, we sought to determine if environmental temperature might influence the transcript levels of mitochondrial protein-coding genes in three organs  of CT and TT frogs. Our results showed that low-temperature stress decreased the transcript levels of mitochondrial protein-coding genes in all three tissues of tiger frogs examined Fig. . In brait et al.  found thergetics . Similarfreezing . Mitochofreezing . Oxidatifreezing –31, 52. freezing , low temP < 0.05), but no significantly down-regulated genes were found in TT frogs. Studies have suggested that gene expression in the brain is more complex than in other parts of an organism to reflect the diverse functions of neurons and glia [In addition, under low temperature stress, the number of down-regulated genes (11 genes) in brain of TT frogs was higher than that in CT frogs (7 genes), whereas in kidney, eight genes were significantly down-regulated in CT frogs  versus Thai (TT frogs) populations showed significant differences in mitochondrial gene expression at different temperatures (25 °C versus 8 °C), which reflects prolonged adaptation of mitochondrial bioenergetics in these two subspecies to the prevailing environmental temperature conditions. Under low temperature stress, especially in the liver, the H. rugulosus including CT and TT frogs were collected from a farm in Jinhua, Zhejiang province, China on 20 June 2018. All tiger frogs are adult females for one year with similar size. All animals were washed in a tetracycline bath and were fed in a plastic incubator at 25 °C for one week. Four CT frogs and four TT frogs from the 25 °C temperature groups were randomly selected and placed in a plastic box under a wet towel for 24 h at 25 °C as the control group. At the same time, four CT and TT frogs were exposed to low-temperature stress for 24 h at 8 °C as the low-temperature stress group. Subsequently, tiger frogs from both control and low-temperature groups were euthanized by pithing followed by rapid dissection of liver, brain and kidney and freezing in liquid nitrogen. Thereafter, organ samples were stored in an ultra-low temperature freezer at − 80 °C until use.28 S and 18 S ribosomal RNA [Frozen samples (50–100 mg) of brain, liver and kidney from CT and TT frogs were used for RNA extraction using a TaKaRa MiniBEST Universal RNA Extraction Kit , according to manufacturer’s instructions. Samples were then subjected to electrophoresis on a 1% agarose gel and run at 135 V and 120 mA for 15 min followed by staining with Goldview. RNA integrity was verified by the presence of sharp bands for omal RNA . RNA wasRT-qPCR) were designed according to the gene sequences of H. rugulosus [Specific primers for reverse transcription-quantitative polymerase chain reaction  [β-actin primers are shown in Table RT-qPCR, each sample was combined with 10 µL SYBR Premix Ex Taq II (2×), 0.4 µL ROX Reference Dye (50×), 0.8 µL each of forward and reverse primers (10 µM), 6 µL ddH2O and 2 µL RT reactants (cDNA). Three technical replicates were run for each gene corresponding to each primer. Reactions were carried out under the following conditions: initial denaturation at 95 °C for 30 s, and then 40 cycles of 95 °C for 5 s, 60 °C for 30 s. Relative mRNA quantification was done by calculating the ratio of the starting quantity of the target gene to the starting quantity of the reference gene for each sample.Transcript levels of the 13 mitochondrial protein-coding genes from TT and CT frogs were quantified in a StepOnePlus™ Real-Time PCR System (Life Technologies). For real-time fluorescence quantification, CAGA-3′) , 44. Qua−ΔΔCt method and standardized to the β-actin gene. All data were analyzed using Statistical Program for Social Sciences 21.0 software . Gene transcript levels in frog tissues from the control and low-temperature groups were compared using the Student’s t-TEST function, and P < 0.05 was considered statistically significant [Data are expressed as mean expression ± SE for each experimental condition, and four independent biological replicates for each gene were analyzed for the two experimental groups. The relative transcript levels of mRNA in organs at specific stages were analyzed by the 2nificant . Data ar


Objectives: The use of handwashing sinks for activities other than hand hygiene (HH) is associated with higher rates of β-lactamase–producing Enterobacteriaceae. However, little has been published about the handwashing sink activities in Singapore hospitals. We explored the handwashing sink activities in a tertiary-care hospital in Singapore. Methods: Five trained shadow observers conducted this observational study between December 18 and 21, 2018  in acute-care general wards. We divided the handwashing sink activities by healthcare workers (HCWs) and non-HCWs  and by HH- and non–HH-related activities. We used Stata version 15 software for the analysis. The study was approved by the Institutional Review Board of the National Healthcare Group, Singapore (DSRB no. 2020/01257). Results: In total, 657 handwashing sink activities were recorded . Of the 475 HCW handwashing sink activities, 451 (94.9%) were HH-related, 10 (2.1%) were for patient nutrition, 7 (1.5%) were for environmental care, 6 (1.3%) were for medical equipment cleaning, and 1 (0.2%) was patient personal-item cleaning. Of the 182 handwashing sink activities by non-HCWs, 117 (64.3%) were HH related, 30 (16.5%) were for patient nutrition, 21 (11.5%) were for personal hygiene, 14 (7.7%) were patient personal-item cleaning. The distribution of handwashing sink activities differed significantly (P < .01) between HCWs and non-HCWs. The odds of non–HH-related handwashing sink activities among non-HCWs was 10× higher than among HCWs . Conclusions: Handwashing sinks use for non–HH-related activities is higher among non-HCWs than HCWs. Further studies are needed to understand the impact of non-HH handwashing sink activities on nosocomial infections and ways to reduce them.


PCSF) from lumbar region, a spinal needle and a spinal manometer are used. PCSF measurement via LP with the use of a spinal manometer may not yield correct PCSF results due to prolonged times required to obtain an accurate pressure value. Equilibrium pressure may be underestimated in circumstances where spinal manometry procedure is terminated prematurely, with the wrong assumption that equilibrium pressure is reached. Elevated PCSF levels can lead to visual loss and brain damage when go undiagnosed. In this study, the spinal needle-spinal manometer combination was modelled with a first-order differential equation and a time constant (τ) was defined as the product of the resistance to flow of the needle with the bore area of the manometer divided by the dynamic viscosity of CSF, i.e. τ= RA/ρCSF. Each needle/manometer combination had a unique constant as a predictor of the equilibrium pressure. The fluid pressure in the manometer rose in an exponential manner which was tested in a simulated environment using 22G spinal needles namely Braun-Spinocan, Pajunk-Sprotte and M.Schilling. Curve fitting of the manometer readings were obtained with regression coefficients of R2 ≥ 0.99 to determine measurement time constants. The residual differences between predicted and true values were less than 1.18 cmH2O. For a given needle/manometer combination, time required to reach equilibrium pressure was identical for all pressure levels. PCSF measured at reduced times can easily be interpolated to their equilibrium level allowing clinicians to obtain PCSF values with high accuracy within seconds. This method can be used as an indirect estimation of ICP in routine clinical practice.Intracranial pressure (ICP) is an important parameter in clinical management and diagnosis of several neurological diseases which is indirectly measured via lumbar puncture (LP). In routine measurements of cerebrospinal fluid pressure (The online version contains supplementary material available at 10.1186/s12868-023-00805-4. PCSF) from the lumbar region is classically measured with a spinal manometer [PCSF measurement, prolonged times are required to obtain an accurate pressure value. Two critical factors have impact on PCSF measurements: the flow rate of CSF through the spinal needle and the needle response to transduce the 90% of PCSF with high speed and accuracy. A quick and accurate assessment of PCSF is essential for the health and the comfort of the patients. Equilibrium pressure may be underestimated in circumstances where spinal manometry procedure is terminated prematurely, with the wrong assumption that equilibrium pressure is reached. This assumption is due to very slow upward movement of the CSF in the manometer in the case where smaller-diameter spinal needles, that is ≥ 20G are used. Needles with larger inner diameter can transduce correct PCSF value less than a minute yet the use of large-diameter needles have been reported as a risk factor for Post-dural Puncture Headache (PDPH) occurrence [PCSF. Equilibration time of a given PCSF is dependent on the inner diameter of the spinal needle and the bore diameter of the manometer that is the spinal needle-spinal manometer assembly which has not been investigated according to our knowledge.Intracranial pressure (ICP) is an important parameter in clinical management and diagnosis of several neurological diseases including hydrocephalus, idiopathic intracranial hypertension (IIH) and demyelinating disorders . The curanometer , 5–8. DuPCSF values are 10–18 cm H2O in lateral position [2O [2O is a risk of mortality. Under various pathological conditions, the relationships between volumes of parenchyma, blood and CSF changes. An average adult produces approximately 500 ml of CSF daily which is replaced about four times a day indicating that approximately 20 ml of CSF is replenished every hour. CSF is mainly produced by the choroid plexus residing in the ventricles. CSF flow is determined by fine tuning of production and absorption of CSF. The measurement of flow rate Q of CSF through the spinal needle can accurately predict the intracranial pressure. Disruption of this delicate balance impairs the CSF flow rate resulting in diseases such as hydrocephalus. The pulsatility of the CSF flow have been demonstrated approximately four decades ago via magnetic resonance imaging (MRI) studies [Normal tion [2O . Elevate studies , 13. The studies .PCSF are independent of the final equilibrium level and waiting for CSF to reach the equilibrium height is not necessary. This method has the potential to revolutionize routine diagnostic LP procedures allowing clinicians to obtain PCSF values with high accuracy within seconds.In this study, we hypothesized that spinal needle-spinal manometer assembly could be represented with a first-order differential equation. We have identified that each needle/manometer combination has a unique time constant. To our knowledge, this is the first time where spinal needle-spinal manometer assembly was defined with a first-order model revealing the fact that measurement times required for a given percentage of CSF, the reservoir tube diameter must be at least ten times the manometer diameter (D* ≥ 10D), in order to compensate for a loss of fluid volume in the reservoir. Reservoir with a diameter of at least ten times will result in a pressure drop of less than 1% in the manometer at equilibrium. [The CSF measurement model we proposed, consisted of a large diameter CSF reservoir made to communicate with a spinal needle and a spinal manometer, as illustrated in Fig. librium. –17.Fig. h(t) in the manometer equals the cerebrospinal fluid pressure PCSF, and then the flow stops. Capillary action of the manometer is omitted, since according to Jurin’s law of capillary action, a 3 mm bore tube will result in a negligibly small height increase of 5 mm only [PCSF and the pressure exerted by the CSF height h(t) in the manometer determines the CSF flow rate:ρ the density of CSF. The flow rate can also be expressed as Q = dV(t)/dt, V(t) being the instantaneous CSF volume accumulated in the manometer:The model behavior is governed by Pascal’s law of communicating vessels. As the spinal needle and the manometer connection is made, CSF runs up the manometer tubing, where the height of the fluid slowly but continuously fills the manometer until the pressure exerted by the fluid column  mm only . ClinicaA is the inner cross-sectional area of the manometer tube. Combining Eqs. Here, Rearranging,This is a first-order differential equation and the standard form for the homogeneous solution is τ) xpressed as the product of the resistance to flow and the manometer cross sectional area, assuming the density ρCSF is approximately 1 (= 1.0006 g/ml). The general solution to the system has then the form:This implies that, for each spinal needle-spinal manometer combination, there will be a unique characteristic time constant (r = h(t)/PCSF of the equilibrium pressure height is independent of PCSF. The ratio r is 50%, 63%, 86%, 95% and 99% when measurement times are 0.693τ, τ, 2τ, 3τ and 5τ respectively.This Equation implies that time taken for the manometer to reach a specified ratio R by using one of the following method.The resistance to flow in plastic tubings is a well-known concept and never considered by the spinal needle manufacturers. Therefore, prior to pressure measurements, it is necessary to determine L and internal diameter d) was linearly related to the driving pressure ∆P:µCSF is the dynamic viscosity of CSF and equals 1.002 mPa.s at 20 °C. The expression µCSF (cmH2O s), the diameter d and the length L of the spinal needle.By opening one end of the needle to atmosphere via the 3-way stopcock in Fig. Q, under a given pressure PCSF, can be calculated by measuring the total number and total mass mCSF of CSF drops (of constant rate) from the end of the spinal needle, over a fixed time period ∆t [ρCSF is the density of CSF (from 1.005 g/ml to 1.006 g/ml) [The flow rate eriod ∆t , 19:8\do06 g/ml) . We empl*) and 30 cm height, filled with Ringer lactate solution, was used. The condition D* ≥ 10 D guaranteed a pressure drop of less than 1% at system equilibrium. The reservoir was made to directly communicate with the spinal needle and the spinal manometer using a tubing, as illustrated in Fig. Q under the given pressure of 13 cmH2O  until a final height of 13 cm was reached in the manometer  (20 breaths per minute) and cardiovascular (> 1 Hz) activities [The model behaves as a low-pass filter with a 3 dB cut-off frequency at tivities .10\documWe previously compared pulsatile and non-pulsatile CSF models for CSF pressure measurement via LP where measurements were indifferent . The carFor monitoring the dynamic behavior of CSF, the spinal manometer was replaced with an electronic pressure transducer to detect negligibly small pressure changes (few mm) due to physiological effects , 23. TheSimple nonlinear regression analysis  was used as a tool to quantify the relationship between pressure and time. GraphPad Prism 8.3.0. version was used in all analysis.2 ≥ 0.99, when determining the measurement time constants.Curve fitting of the manometer readings were performed with regression coefficient RR was then directly determined from the formula R = P/Q . The time constants for 22G Pajunk Sprotte, the 22G Braun Spinocan and the 22G M. Schilling spinal needles were τ = 72 s; τ = 152 s and τ = 111 s, respectively.Using the R from the Table CSF in the reservoir was set to 13cmH2O as shown in Fig. τ = 72 s), as expected had the lowest equilibration time. Statistics for the data presented in Table First-order differential equation of the form given by the Eq. 2O in the manometer , to simulate a patient lying in the lateral position [Q for each needle by using the measurements from Carson et al.; i.e. by inserting the total mass mCSF of fluid drops collected over a fixed period of 10 min [R by making use of Eq. To further verify that each needle/manometer combination had a unique time constant, data from other researchers were used. In the reference paper by Carson position . A largef 10 min . Using tf 10 min  Table 2)2 were equal or better than 0.99, in all cases.Referring to the same paper Table . We testτ against the resistances to flow R, with the assumption of µCSF = 1.10–5 (cm CSF s). The viscosity is unlikely to play a role in this case; even with high CSF protein and a pleocytosis. The viscosity of CSF and saline are similar and do not have a significant impact on CSF flow [R-21.01, with the slope of 0.067 representing the cross-sectional area of a manometer of 3 mm diameter. Although the bore size of the manometer was not specified in the reference paper, a personal communication with Rocket® Medical revealed that internal diameter of their manometer was 3.05 mm ± 0.08 mm.For the verification of Eq. CSF flow , 17. ThePCSF results due to prolonged times required to obtain an accurate pressure value. Equilibrium pressure may be underestimated in circumstances where spinal manometry procedure is terminated prematurely, with the wrong assumption that equilibrium pressure is reached. PCSF is an important parameter in clinical management and diagnosis of several neurological diseases including hydrocephalus, IIH and demyelinating disorders. These diseases may present with overlapping symptoms such as headache and visual defects [PCSF is essential. Our novel technique overcomes the caveats of spinal manometry allowing rapid and accurate estimation of PCSF.Indirect measurement of ICP via lumbar puncture with the use of a spinal manometer may not yield correct  defects –27. TherPCSF to facilitate collection of at least 2 mL/min implying a small R; and the needle response to transduce the 90% of PCSF accurately and quickly in less than 1 min. The length and the bore, hence the gauge of the needle, determine the resistance to flow. We have shown that time to respond to any CSF pressure level, can be computed by a first-order modelling of the measurement system, with the time constant calculated as the product of the needle R and the spinal manometer bore area.In CSF pressure measurements via lumbar puncture, two parameters are of most importance: the needle flow rate of CSF at normal τ, a lower PCSF could be targeted to reduce measurement time, the improvement in measurement time would be in a ratio of approximately 7 to 1, when the measured pressure is reduced from 99% of PCSF to 50% of PCSF. The viscosity is unlikely to play a role here; even with high CSF protein and a pleocytosis, viscosity of CSF and saline were not very different and do not impact appreciably on CSF flow [The validity of the first-order model was tested 22G needles of different brands and data from the literature with a range of needle types from 24 to 22G. It is reported that spinal needles with a size of 20G and 22G can take from 1 to 5 min to reach equilibrium pressure and smaller needles such as 25G can take up to half an hour . DiffereCSF flow . The modRA/ρCSF. Hence, each needle/manometer combination was expected to have a unique time constant. The time constant concept was tested on spinal needles with the same gauge (22G) from different brands. Spinal needles used in the published data was also considered for verification of the model. Curve fitting of the manometer readings were accomplished with regression coefficient R2 ≥ 0.99, when determining the measurement time constants. Measurement times required for a given percentage of PCSF were independent of the final equilibrium level, in accordance with the experimental findings. CSF pressure values measured at reduced times could easily be interpolated to their equilibrium level; because the speed of measurement is very important for the maximum comfort of the patient. CSF pressure measurement times can be greatly minimized by selecting lower percentages of the equilibrium level by determining the time constant before the measurement. Therefore, we strongly recommend that spinal needle producers declare the resistance to flow R parameter in their data sheet. Similarly, spinal manometer manufacturers must always specify their bore diameters. This is a major limitation for our study since our spinal needle-spinal manometer assembly presented with a first-order differential equation requires precise inner diameter measurements of the manometer and the needle to provide accurate PCSF values. It is not possible for a clinician to measure inner diameters of the needle and the manometer prior to performing an LP. Another limitation is that although this technique provides fast and accurate PCSF measurement, the upward movement of the CSF might be too fast in certain spinal needle-spinal manometer combinations rendering the observation more difficult. Recent comparisons with electronic manometers and intravenous giving sets used in infusion therapy had shown that spinal column manometer is still the best alternative for pressure measurements during lumbar puncture [PCSF measurement via LP which is used as a reference to compare non-invasive parameters.The CSF measurement system of spinal needle and spinal manometer was compatible with the first-order model to predict the time required to reach the equilibrium CSF pressure in the manometer. A time constant τ was defined as the product of the resistance to flow of the needle with the bore area of the manometer divided by the dynamic viscosity of CSF: τ= puncture , 22, 23.puncture , 28. OnePCSF are independent of the final equilibrium level and waiting for CSF to reach the equilibrium height is not necessary. With this novel technique, it is possible to obtain PCSF values with high accuracy within seconds. Given the importance of ICP in clinical management and diagnosis of neurological diseases, improvement of PCSF measurement with our novel technique hold the potential to revolutionize the ICP estimation. As a future work, the model will be further tested for validation in the clinical settings.In this study, the spinal needle-spinal manometer combination was modeled with a first-order differential equation and a time constant was determined for each needle/manometer combination representing a unique constant as a predictor of the equilibrium pressure. Development and validation of our novel method was conducted in a simulated environment using a CSF model filled with an artificial fluid resembling CSF characteristics which served as an alternative to laboratory animals. We have found that measurement times required for a given percentage of Additional file 1: Table S1: Non-linear least square fitting .


The Myodural Bridge (MDB) is a physiological structure that is highly conserved in mammals and many of other tetrapods. It connects the suboccipital muscles to the cervical spinal dura mater (SDM) and transmits the tensile forces generated by the suboccipital muscles to the SDM. Consequently, the MDB has broader physiological potentials than just fixing the SDM. It has been proposed that MDB significantly contributes to the dynamics of cerebrospinal fluid (CSF) movements. Animal models of suboccipital muscle atrophy and hyperplasia were established utilizing local injection of BTX-A and ACE-031. In contrast, animal models with surgical severance of suboccipital muscles, and without any surgical operation were set as two types of negative control groups. CSF secretion and reabsorption rates were then measured for subsequent analysis. Our findings demonstrated a significant increase in CSF secretion rate in rats with the hyperplasia model, while there was a significant decrease in rats with the atrophy and severance groups. We observed an increase in CSF reabsorption rate in both the atrophy and hyperplasia groups, but no significant change was observed in the severance group. Additionally, our immunohistochemistry results revealed no significant change in the protein level of six selected choroid plexus-CSF-related proteins among all these groups. Therefore, it was indicated that alteration of MDB-transmitted tensile force resulted in changes of CSF secretion and reabsorption rates, suggesting the potential role that MDB may play during CSF circulation. This provides a unique research insight into CSF dynamics. CSF plays an important role in a number of physiological processes. As a buoyant medium, CSF provides mechanical support and reduces the effective weight of the brain by allowing it to float3. CSF also helps to cushion the brain and spinal cord from pressure, especially during trauma3. Additionally, CSF is responsible for nourishing neural tissue and removing metabolic waste products from the CNS3. Together with intracranial vessels, CSF also helps to maintain homeostasis in the intracranial environment4. Thus, the regulation of CSF circulation is critical for CNS health5. CSF circulation involves the production, drainage, and reabsorption of fluid within the CNS5. Compare to the CSF in spinal tube, ventricular CSF features more clinical implications. The cellular lining of the ventricle both can produce and is responsive to CSF. Anomalies of the CSF/ventricular system serve as diagnostics and may cause CNS disorders, further highlighting their importance6. While the flow direction, velocity, and quantity of CSF have been studied, the precise mechanism of CSF circulation remains unclear10. However, it is thought to be influenced by factors such as arterial pulsations, respiratory movements, body position, intracranial blood circulation, and the myodural bridge (MDB)14.Cerebrospinal fluid (CSF) is a clear liquid that is found in the ventricles and subarachnoid space of the central nervous system (CNS) and is the major extracellular fluid of the CNS16. Comparative anatomical studies have revealed that the MDB is an evolutionarily conserved structure found in both terrestrial and marine mammals, reptiles, and birds20. The MDB's shape, location, fiber properties, and bridging characteristic have led to several speculated functions, including preventing in-folding of the SDM during head extension, transmitting proprioception, maintaining the integrity of the subarachnoid space, and affecting CSF circulation23. To better understand the MDB's functions, Zheng et al24 proposed the concept of the MDB complex (MDBC), an integrated functional unit involving several muscles, ligaments, and MDB fibers, in this study, the authors suggested that the CSF circulation could be changed by cervical body motion after the status of MDBC has been changed.The fibrous structure known as the MDB connects the suboccipital musculature to the cervical spinal dura mater (SDM) via the atlantooccipital and atlantoaxial interspace25. And for humans, the aqueduct is at more of a vertical position, and thus the humans may require more powers to equilibrate CSF against hydrostatic forces; even so, up to date, the direct experimental evidence showing hydrostatic force is the key factor for CSF circulation remains missing.However, it has been argued that CSF dynamics is influenced in quadrupeds with a minor exposition to hydrostatic forces by their more horizontally oriented aqueduct, and thus body motions in quadrupeds may be more critical regulators to CSF dynamics compare to humans26. Furthermore, experiments conducted on dogs have suggested that in humans, MDB and body motions have strong potentials to play a role in CSF circulation through controlling the suboccipital muscles23. Moreover, our previous study revealed that hyperplasia of suboccipital muscles lead to increased intracranial pressure in rats27, suggesting the body motion, MDBC dysfunction, and pressure changes inside the CSF circulation pathways are correlated to each other, together form a dynamic equilibrium. More to the point, patients with Chiary I malformation exhibited hindered CSF dynamics28, suggested that without changes in upright position or hydrostatic forces of human CSF, a local malformation or dysfunction at cervical level could be enough to unbalance the CSF dynamics or circulation in humans. These collectively suggested that not only the hydrostatic forces, MDBC is highly possible to be one of the key regulatory factors in CSF dynamics of humans as well. As this result, although these studies only examined or implied the transient effects of MDB on CSF circulation, it is indicative that a quadrupeds animal model with MDBC dysfunction is capable to be a human mimicry.However, previous studies have demonstrated that in humans, head rotation and nodding can alter the volume, velocity, and pressure of CSF flowIt is why the power system of human CSF is still under debate. We in all time believe that humans need more against hydrostatic forces, but at the same time we do not believe the hydrostatic forces plays the key role in CSF dynamics. It must be regulated by multiple factors.29. BTX-A injections have been used for aesthetic treatments such as wrinkle reduction, as well as for treating muscle spasticity and hypertrophy30. Animals treated with BTX-A exhibited significantly reduced muscle mass and corresponding decreases in muscle strength31. ACE-031 is a specific inhibitor of myostatin, also known as GDF-8, a secreted protein that regulates muscle size and function by suppressing myocyte proliferation and differentiation33. Natural or artificially induced myostatin mutations have been shown to cause marked muscle hypertrophy, resulting from both muscle cell hyperplasia and hypertrophy35. ACE-031 binds competitively to myostatin proteins, antagonizes the normal myostatin receptor ACTVIIb, and blocks the downstream signaling pathway of myostatin, resulting in a physiological dysfunction of myostatin proteins33. A previous study has verified that myostatin local blockage with ACE-031 can increase muscle strength27.BTX-A is a bacterial exotoxin that targets the neuromuscular junction, inhibiting the release of acetylcholine from nerve terminals, which can cause muscle atrophy37. The rate of CSF secretion was taken as the rate of CSF formation (Vf)38. The researchers also selected six CP-CSF-related proteins to study the secretion mechanism of CSF44. Over last two decades, studies showed that the olfactory lymphatic pathway seemed the main way of CSF absorption, rather than arachnoid villus47. To evaluate the rate of CSF reabsorption (Va), the researchers used SPECT/CT to detect the radioactive intensity of the turbinate area after lateral cerebral ventricle injection of radionuclide. By analyzing both Vf and Va, the researchers explored the mechanism of how muscle strength alteration could lead to different pulling effects of the MDB and changes in CSF dynamics. The results of the study may support the theory that MDBC could be one of the factors affecting the dynamic circulation of CSF and provide unique insights into the mechanism of CSF dynamics.In this study, the researchers injected BTX-A and ACE-031 into rats' suboccipital muscles to create suboccipital muscle atrophy (BTX-A group) and hyperplasia (MSTN group) animal models, respectively. For the negative control, the researchers established a suboccipital muscles severed group (SEV group) to surgically eliminate the physiological functions of the suboccipital muscles. Meanwhile, another group without any surgical operation was established as a wild-type control (CTL group). The researchers then measured the secretion rate of CSF in live rats by blocking the aqueduct of Sylvius and inserting a glass capillary into the lateral ventricleThis study adhered strictly to the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" as presented by the National Institutes of Health. The experimental protocol was approved by the Committee on the Ethics of Animal Experiments at Dalian Medical University. All surgical procedures were conducted using Avertin anesthesia, and all possible measures were taken to minimize any potential discomfort or pain experienced by the animals involved. This study was carried out in compliance with the ARRIVE guidelines.Male rats were utilized in this study, and all experimental rats were obtained from different female rats and raised from 8 weeks of age for subsequent experiments. The animals were housed in a controlled environment with a 12-h light cycle and ad libitum access to standard rodent diet  and water. Four experimental groups were established for this study, consisting of 46 animals randomly divided into MSTN group (n = 13), CTL group (n = 13), BTX-A group (n = 10), and SEV group (n = 10). Prior to and 2 weeks after model establishment, measurements of weight, food intake, and water intake were obtained to evaluate the baseline physiological conditions among the groups. Ethical guidelines were strictly followed in the care and use of the laboratory animals, and the study protocol was approved by the Committee on the Ethics of Animal Experiments of the Dalian Medical University.To control the surgical interference, a group of animals without any surgical operation was firstly established as a wild-type control in contrast .For the other experimental groups of the present study, prior to the surgery and injection procedure, all rats were fasted overnight. Anaesthesia was induced using a 2.5% Avertin  solution administered through intraperitoneal injection. All experimental animals were set on stereotaxic apparatus for the subsequent surgical procedures . A 1.5–2 cm incision was made on the dorsal side of the rat's neck to expose the superficial muscle layer until the midline was visible . The superficial muscle was then incised along the midline to expose the RCDma . In the suboccipital muscles severed group of rats , the RCDma and RCDmi were cut along the posterior border of the occipital bone with a scalpel until the dura was visible . The RCDma and RCDmi were then separated and taken out along the lateral border of the muscles, and the severed ends of the muscles were cauterized with an alternating current-operated bipolar coagulation device to stop the bleeding and prevent re-connecting to the occiput . The superficial muscles and skin were sutured separately to complete the procedure . For the BTX-A and ACE-031 local injection group of rats , the entry point was 2 mm below the inferior margin of the occiput, and 5 mm to the left and right of the midline. A 25 μl micro syringe with a calibre of 0.22 mm (32G) was used for all the injections. The angle of the injections was made perpendicular to the inferior surface of the occiput . Once the pinhead touched the posterior edge of the occiput, it was withdrawn by 0.5 mm to ensure it was located in the RCDmi, while it was withdrawn by 5 mm to be in the RCDma. Each rat was injected on both sides of the RCDmi and RCDma. Every injection was performed at a constant speed at 1μ/sec during the entire process. In the BTX-A group, 5 μl BTX-A  was injected at a concentration of 0.05U/μl into each muscle, totalling 20 μl per rat. Similarly, in the MSTN group, a total of 20 μl ACE-031  was injected at a concentration of 50 ng/μl into each muscle. Following the injection procedure, the superficial muscles and skin were sutured separately to complete the operation .36 and Liu et al37. Rats were anesthetized with avertin and then fixed onto a stereotaxic apparatus . A small hole, approximately 0.6 mm in diameter, was drilled above the right lateral ventricle using a dental drill. The coordinates for the hole (insertion point), relative to bregma, were x = 1.5 mm and y = 0.5 mm  = π·r2·d, where r is the radius of the capillary (0.25 mm) and d is the distance that CSF moves in the capillary. The rate of CSF formation (µl/min) was then calculated from the slope of the volume-time relationship.Two weeks after model establishment, the CSF secretion rate was quantified in accordance with the modified method described by Karimy et alMoreover, to assess the efficacy of mineral oil in obstructing the aqueduct of Sylvius, and to confirm the insertion of the catheter reaches the fourth ventricle, before we actually started to test the CSF secretion rates, a blockage test was conducted. Similar to the procedure which was described above, right after the oil injection into the fourth ventricle, the rat's head was positioned horizontally , and 20 μl of eosin was slowly injected at a rate of 5 μl/min using a micro syringe. To ensure proper staining, the syringe was removed after 10 min of injection. The rat's brain was extracted following a standard perfusion process, and sliced horizontally to visualize all ventricles at the same level. The staining of each ventricular wall was observed under real-time conditions, no staining or damage could be seen on any parts of brain tissues; thereby confirmed the efficacy of the oil blockage as well as the positioning of the fourth and the lateral ventricle insertion. All images were captured using a Canon EOS 7D Mark II digital SLR camera  Fig. C.Two weeks after the establishment of the animal models, the CSF reabsorption rate was measured using standard procedures. Specifically, rats were prepared as described previously, up to the point of skull hole drilling and with their heads placed in a horizontal position. A micro syringe containing 15 μl Technetium 99-labeled diethylene-triamine-pentaacetate (99mTc-DTPA), a radioactive tracer, was inserted 0.6 mm deep into the right-side lateral ventricle and injected at a rate of 3 μl/min . After 25 min, the rat was euthanized, decapitated, and subjected to a median sagittal incision to examine the turbinate tissues under direct vision , and 20 μg of protein were loaded into each lane of the western blot (protein immunoblot) for analysis of myostatin expression levels. Specifically, to assess myostatin expression, equal amounts of protein extracted from the RCDma and the RCDmi, the semispinalis capitis muscle and the gastrocnemius muscles obtained from the MSTN group (n = 3) and the CTL group (n = 3) were subjected to western blot. The rats were sacrificed two weeks after local injections, and their RCDma, RCDmi, and gastrocnemius muscles were extracted and frozen in liquid nitrogen. The anti-myostatin (GDF-8) was produced in goats , and the anti-α-tubulin was produced in rabbits  and used as the primary antibodies. Furthermore, the secondary antibodies, HRP-conjugated anti-Goat IgG  and HRP-conjugated anti-Rabbit IgG , were diluted in TBS with Tween-20 (TBST) in 5% skim milk. The bands were detected using ECL prime , and the experimental protocol was performed according to the manufacturer's instructions.2EDTA•2H2O). Thereafter, regular paraffin embedding was performed, and 8-μm thick sections were obtained using a rotary microtome . To verify the result of model building, Masson trichrome (aniline blue) stained sections were photographed using a Nikon NIS image system . Additionally, Masson trichrome (light green) staining was also performed on RCDma and RCDmi to measure myocyte cross-section area and evaluate muscle condition after local injection. The results of the staining were quantified using Image-J software . At least 100 myocytes were measured per animal to assess myocyte condition.Following conventional perfusion and fixation, tissue samples comprising suboccipital muscle tissue, occipital bone, atlas, axis, and semispinalis capitis muscle were decalcified with ethylenediaminetetraacetic acid disodium salt  after regular paraffin embedding. The primary antibodies used were AQP-1 mouse monoclonal antibody from Santa Cruz Biotechnology (1:500), AQP-4 mouse monoclonal antibody from Bioss (1:200), OTX-2 mouse monoclonal antibody from Santa Cruz Biotechnology (1:500), TTR sheep polyclonal antibody from Santa Cruz Biotechnology (1:400), KCNE-2 rabbit monoclonal antibody from Bioss (1:200), and Na + -k + -ATPase rabbit monoclonal antibody from Signalway Antibody (1:400). The incubation with the primary antibodies occurred for 18 h at 4 ℃ according to the product manual, and the negative control group used PBS instead of the primary antibody. Visualization was performed with HRP-conjugated secondary antibodies (Biotin-labeled goat anti-rabbit IgG (ZSGB-BIO); Biotin-labeled goat anti-mouse IgG (ZSGB-BIO); Biotin-labeled rabbit anti-sheep IgG  and DAB. All stained sections were photographed using a Nikon NIS image system , and the optical density was semi-quantified using Image-J software (N.I.H.)Statistical analysis was carried out using Prism 8.0 (Graphpad Software) and Microsoft Excel Statistics Toolkit (Microsoft). Paired t-tests were applied. Results shown are mean + SEM, a p-value of < 0.05 was statistically significant.The study measured weight, food intake, and water intake before and 2 weeks after model establishment for each group of rats Fig. A. There The study established suboccipital muscle hyperplasia and atrophy models after a single local injection of ACE-031 and BTX-A for 2 weeks. Western blot analysis was utilized to measure the levels of Myostatin (GDF-8) of RCDma and RCDmi since ACE-031 targets Myostatin (GDF-8). Gastrocnemius and splenius capitis muscle were used as controls  staining and analysis of muscle-atlas area ratio showed increased and decreased suboccipital muscle area in BTX-A group and MSTN group, respectively. Meanwhile, hardly any suboccipital muscles were observed in the SEV group Fig. A,B. To sp < 0.05), while it was significantly decreased in both the BTX-A group (p < 0.001) and the SEV group (p < 0.001)  Fig. E.Figure To further understand the mechanism of CSF secretion rate change, six CP-CSF-related proteins were selected for IHC semi-quantitative analysis. However, no significant difference in the protein levels of these modeled animals was observed in any group Fig. A,B. Thesp < 0.05) and the MSTN group  showed a significant increase in CSF reabsorption rate, while the SEV group  did not show a significant change . The suboccipital region is one of the most complex regions in humans, containing unique bony structures, several pairs of short muscles, abundant nerves, and vasculature54. These rich vascular communications correlate the movement of the suboccipital muscles with the regulation of intracranial blood flow55. Considering about the kinetoterapy of lower limbs varicosity, constant muscular relaxation and contraction circles may act as a pump to the local veins, propels the compression of the intramuscular and deep veins, which increase the venous pressure and promote the blood in the deep vein back to the heart56. Therefore, predictively, changes in the contraction force of suboccipital musculature alter the pressure on the wall of blood vessels, resulting in changes in the diameter of blood vessels that can affect the secretion and reabsorption of CSF. Quantification of these changes may be represented by changes in the volume of CSF and CBF. However, this predictive implication lacks of experimental support in the present study.The contraction force of suboccipital muscles can transmit to the dura mater, causing a displacement of the dura mater and leading to a regional negative pressure of the subarachnoid space, resulting in alterations of CSF flow. This effect may initially be considered local, but with constant stimulation, it could become global29. Intramuscular injections of BTX-A can relieve focal spasticity and dystonia, and long-term injections of BTX-A can cause disuse atrophy of muscle31. ACE-031 is a specific inhibitor of myostatin (GDF-8) that can upregulate muscle size, mass, and strength34. Additionally, we severed the target muscles as a functional negative control and set another group of wild-type control with no operation.To further study the role of MDBC in CSF dynamics, we applied local injections of BTX-A and ACE-031 to functionally downregulate and upregulate the target muscles, respectively. BTX-A is a neurotoxin that binds to receptors on presynaptic cholinergic nerve terminals, blocking the release of acetylcholinef, in vivo and calculated CSF reabsorption rate, referred to as Va, by detecting the radioactive intensity of the turbinate area, which is known to be the main site of CSF reabsorption in rodents. We then examined changes in Vf and Va under different conditions of suboccipital musculature.The present study aimed to investigate the effects of suboccipital musculature on CSF dynamics in living rats. To this end, we measured the rate of CSF secretion, referred to as V27, whereas botulinum toxin reduced the muscle strength57; in the present study, rats in the MSTN group exhibited increased muscle mass in the right and left cranial dorsal muscles (RCDma and RCDmi), along with increased Vf and Va. Conversely, rats in the BTX-A group displayed decreased muscle mass in RCDma and RCDmi, as well as decreased Vf but increased Va. As a functional negative control, rats in the SEV group exhibited rare RCDma and RCDmi muscles, and showed a significant decrease in Vf but only a slight decrease in Va with no statistical significance. These findings suggest that changes in suboccipital musculature can significantly affect CSF dynamics, as measured by Vf and Va.According to previous studies, rats with ACE-031 blockage showed increased muscle strength27 and the present studies collectively indicated that muscle hyperplasia brought increased contraction force which MDB transmitted; Moreover, since MDB directly link to the SDM and has been acknowledged as a fixing device of SDM22, it is highly predictive that the suboccipital hyperplasia could increase the displacement of SDM pulled by MDB. Stated another word, there is a great potential that the function of the MDBC unit was enhanced. In this case, we at the same time measured an increase in both Vf and Va. According to these results, we speculated that enhanced MDBC function may lead to increased Vf. Assuming no other change occurred, CSF volume would increase for the formation speed exceeded the reabsorption speed. As a result, the hydrostatic pressure gradient between CSF formation sites and CSF reabsorption sites increased, which offered a greater driving force for CSF reabsorption. Thus, Va increased as well to reach, or tend to reach, an intracranial homeostasis. Therefore, to keep a relatively stable intracranial environment and maintain the CSF volume, an increased Va was observed after the increased Vf . Additionally, considering the MDB universally exist in mammals20, these collectively suggested that there may be strong potential between MDBC and clinical CSF disorders, no matter in human or other mammals. During the present study, the experimental animals shared highly similar patterns of life and daily routines, which includes similar food and water intakes, similar sleep regulations, similar exercise intensities, etc. In addition, considering the animals with or without surgical treatments exhibited similar food/water intake, body weight. These in all indicated that these animals shared similar normal physiologies, which includes non-hindered body movements. Consequently, although these animals exhibited Va and Vf changes, the inductive factors were easy to track.The MDB is recognized as a direct connective linkage of suboccipital muscles and the cervical dura mater. In middle 2023, a novel anatomical definition of occipito-atlantal cistern (OAC) has been introduced, OAC is a subarachnoid space independent from the cisterna magna. More to the point, the dura mater which in the posterior of OAC is just where the MDB connects on humansHowever, humans feature different life regulations and schedules, different dietary habits, and different enthusiasm to exercise; not to mention the humans spend more than half their lives in upright position. These collectively result in different homeostasis of circulation, respiratory, as well as endocrine system, which are all important affecters of CSF dynamics. Furthermore, in human the aqueduct is compared to quadrupeds oriented in a more vertical position and the requirement to equilibrate CSF against hydrostatic forces is much higher than in quadrupeds.Nevertheless, humans spend nearly halfway of their lives in laying down or sitting positions, and these are very similar to the conditions of the modeling animals in the present study. The MDBC could be a key regulator while in sleeping or resting conditions for humans. As this result, long-time improper postures and excessive burden to the neck have potentials leading to CSF disorders; conversely, it is also inferred that a proper exercise which aim to keep or rebuild the functions of neck muscle  could be an effective way for the prevention and therapy to the diseases which related to CSF dynamics disorders. On the other hand, it also provided a mechanical support for the efficacy of cervical massage and manipulative therapy.16. This study investigated the impact of changes in MDBC on the fundamental components of CSF dynamics, namely Vf and Va, and their potential influence on intracranial homeostasis. Our results provide direct experimental evidence supporting the idea that MDBC can affect CSF circulation, as we observed changes in Vf and Va following intervention in suboccipital muscles. We used a 2-week animal model to mimic the course of the clinical patient, which is more relevant than immediate effects assessed in previous experiments.The presence of MDB in suboccipital muscles establishes a unique anatomical connection between these muscles and the dura materf and Va values59. Fluid  may affect the real-time physiological CSF dynamics. However so far, due to the technical limitations, there is no perfect non-invasive measuring method of CSF production/absorption rates60. A novel non-invasive method was firstly described in 2020 to assess human the water transition of the blood-cerebrospinal fluid barrier61, this study certainly showed us a novel perspective to assess the CSF production rate non-invasively, and yet not 100% reliable because of the different study focus. In the present study, the possible alterations of CSF dynamics caused by fluid injections had been reduced as much as possible by controlling the experimental background as consistent as possible. Nevertheless, our corresponding speculations based on these results remain valid. Secondly, the present study simplified the model of CSF dynamics by observing only Vf and Va and speculating the possible changes in intracranial homeostasis. We did not investigate diseases or symptoms associated with altered suboccipital muscle status, such as headache, which is closely linked to CSF and intracranial pressure65. Third, limited to the small volume of the suboccipital muscles, we did not find any direct way to test the contractive forces of the RCDma and RCDmi, although our previous study and publication of other researchers evidently demonstrated the correlations of contraction force changes with muscular atrophy and hyperplasia, a direct experimental proof remains unpresented. Fourth, for the muscular atrophy/hyperplasia models, the implications on related blood vessels have not yet been test, and thus the experimental results regarding Va and VF changes have not yet been fully interpreted. Moreover, the 2-week modelling duration used in this study may not fully correspond to the clinical course of cervicogenic headaches, considering most of headaches present chronicy65.However, there are some limitations to our study. Firstly, the measurement method chosen for this experiment differs from previous perfusion methods and may not fully represent actual Vf and Va. Future studies should focus on the relationship between MDBC and CSF dynamics in greater depth, investigating potential clinical outcomes of altered suboccipital muscle status, including headache, and accounting for the complexity introduced by the perivascular spaces and the glymphatic system.In conclusion, our study provides evidence supporting the hypothesis that MDBC can affect CSF circulation and intracranial homeostasis through changes in VSupplementary Information.


A novel corona virus called SARS-CoV-2 was identified at the end of December 2019, and the illness induced by it was designated as coronavirus disease 2019 (COVID-19). Severity of the disease could vary significantly since most of the infected individuals experience mild to moderate respiratory symptoms and recover without specialized care. Genetic polymorphisms have implications in influencing the varying degrees of COVID-19 severity. This study aims to assess the potential association between the CXCL12 rs2839693 polymorphism and the severity of COVID-19 in Assiut University Quarantine Hospital during the period from May 2022 to August 2022.The present study is a cross-sectional study and is applied to 300 COVID-19 patients confirmed by RT-PCR admitted to Assiut University Quarantine Hospital from May 2022 to August 2022. Based on the clinical symptoms, the recruited participants had been divided into two groups. Group I involved mild or moderate cases; Group II involved severe or critical conditions. The rs2839693 polymorphism was detected by real time PCR using TaqMan assay probe.p value < 0.001). C-reactive protein (CRP) and D-dimers are significantly elevated in the combined variants (CT + TT) and the TT compared with the CC  and the CC,CT genotypes . The combined variants (CT + TT) of CXCL12 were found to be independent predictors to severe or critical COVID-19 risk with P value =  < 0.001, OR = 3.034& 95% CI = 1.805–5.098.The frequency of the T allele and the TT genotype was significantly higher in the severe or critical group compared with the mild or moderate group (Our findings revealed that CXCL12 rs2839693 had a role in the development and seriousness of COVID-19. Patients with the TT genotype or the T allele at increased risk developed severe or critical rather than mild or moderate disease. A novel corona virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified at the end of December 2019, and the illness induced by it was designated as coronavirus disease 2019 (COVID-19) : GAAGGGGACGACAGGATGCTCTAGG[C/T]ACCTGGGGAGGGGAGAATGGAGAGC was utilised using the TaqMan ready-made SNP assay . A 20 μL PCR mixture was comprised of 3μL extracted DNA, 0.5μL SNP assay, 5μL Master Mix, and 11.5μLdistilled water was carried out. The Applied Biosystems 7500 real-time polymerase chain reaction  apparatus was used.With the use of the IBM SPSS software package version 20.0, data were inputted into the computer and analyzed . NumbersTable Our data showed that lymphocyte count in most cases was within the normal values, while lymphopenia was detected in 84 28.0%) of COVID-19 patients. Ferritin, cell reactive protein (CRP), and D-dimers were elevated in most cases. Regarding CT findings, our results revealed that bilateral ground glass opacity (GGO) was the most frequent CT abnormality . The frequency of the TT genotype and the T allele were higher in the severe or critical group than in the mild or moderate group. Regarding genotypes, patients with the CT + TT genotype had 3.08 higher risks to develop severe or critical COVID-19 than the CC genotype. Regarding alleles, patients with the T allele had 2.91 higher risks to develop severe or critical COVID-19 than the C allele.Table P value =  < 0.001, OR = 3.034& 95% CI = 1.805–5.098  of CXCL12 were found to be independent predictors to severe or critical COVID-19 risk with P value < 0.001, 0.015, and < 0.001 respectively). Dyspnea was more prevalent in the combined CT + TT and the TT genotypes compared with the CC and the CT, CC genotypes, respectively. Vomiting and Dysarthria were significantly prevalent in the TT genotype compared with the CT, CC genotypes. Regarding clinical course of the disease, we found that severe or critical illness was associated with the combined CT + TT and the TT genotypes where it was more prevalent in the combined CT + TT, and the TT genotypes was compared with the CC and the CT, CC genotypes, respectively.Subsequently, we compared demographic data, comorbidity, and symptoms between CXCL12 genotypes (CC vs. CT + TT and CC vs. CT vs. TT) as presented in Table P value 0.006 and 0.017 respectively) and the CC, CT genotypes ; while Ferritin was significantly elevated in the TT compared with the CC, CT genotypes .Table P value 0.022). Clexane was more prevalent in the TT genotype than the CC and CT genotype. The number of patients without any demand for oxygen therapy was significantly different between the genotypes  where it was more prevalent in the CC genotype compared with the combined CT + CC and the TT genotypes. Nasal cannula and invasive mechanical ventilation were more prevalent in the TT and the combined CT + TT genotypes compared with the combined CC, CT and the CC genotypes, respectively, . Mask oxygen was used more prevalently in patients of the CT + TT genotypes than those of the CC genotype .As indicated in Table P value 0.002 and 0.006 respectively). The need for ICU stay was significantly higher in patients with the combined CT + TT and the TT genotype compared with CC and CC, CT, respectively, . Regarding mortality rates, our results revealed that they were significantly higher in the combined CT + TT and the TT genotype compared with the CC and the CC, CT, respectively .Patients with the TT genotype had a longer duration of in-hospital stay than patients with the CC genotype .The number of patients needed home management was significantly higher in the CC genotype compared with the combined CT + TT and the TT genotypes  and non-smokers (60.7% compared with 39.3% smokers). Hypertension and DM were the most common co-morbid diseases found in 35.7% and 26.0% patients respectively; while renal disease, heart disease, and thyroid disease were less frequent. These findings agree with a study by Petrakis et al., who reported that the most frequent comorbid diseases were hypertension in 38.4% patients and DM in 20.9% patients .Additionally, regarding clinical manifestations of the studied groups, The principal COVID-19's clinical complaints were fatigue (55.0%), dry cough (53.7%), and dyspnea (48.0%).These findings are similar to those by Rodriguez-Morales et al., who reported that the principal COVID-19's clinical complaints were cough(57.6), and dyspnea (45.6); in contrast to our results fatigue (29.4%) .9/L), while lymphopenia was observed in 84 (28.0%) patient. Ferritin and CRP were elevated in most cases 200 (66.7%) and 250 (83.3%), respectively. Moreover, D-dimers were elevated in 180 (60.0%) of our cases. Our results in agree with Kadhim et al., and Smail et al., who observed lymphopenia, elevated CRP, ferritin, and D-dimers among COVID-19 patients [Regarding laboratory assessment, lymphocytes values in most cases 185 (61.7%) were typically within the normal values between . Pneumonic consolidation was another prevalent CT characteristic in COVID-19 patients 107 (35.7%). These findings are similar to a meta-analysis research conducted on 13 previous studies and concluded that GGO was the principal CT findings followed by consolidation in COVID-19 patients .Most of our participants received antibiotic as a treatment 288 (96.0%) patients; while 323 (77.3%) and 176 (58.7) patients received antiviral and Glucocorticoids, respectively. Oxygen therapy was needed in 227 (75.7%) patients: nasal cannula in 78 (26.0%), Mask oxygen in 150 (50.0%), and Invasive mechanical ventilation in 18 (6.0%). A meta-analysis study by Langford et al., on the use of antibiotic therapy on COVID-19 found that the frequency of antibiotic therapy was 74.6% .The average length of hospitalization was 7.0 days. These findings agree with Alwafi et al., who indicated that the average length of hospitalization was 6.0 days . While tThe median duration of recovery was 15.0 days. Our data agree with SeyedAlinaghi et al., who found that the median length of recovery was 13.5 days . RegardiThe frequency of the TT genotype and the T allele of CXCL12 rs2839693 was significantly different between mild or moderate and severe or critical patients. The frequency of the TT genotype and the T allele was higher in the severe or critical group than in the mild or moderate which may indicate the role of CXCL12 rs2839693 in the pathogenesis and severity of COVID-19. Patients with the TT genotype or the T allele at increased risk developed severe or critical rather than mild or moderate one. Our result agree with Mohamed et al., who reported no differences in the frequency of the TT genotype or the T allele of CXCL12 rs2839693 among ITP patients and controls, and that there is no correlation between this SNP and disease severity .Normal CXCL12 is the only ligand that could bind to CXCR4 receptor. SARS-CoV-2 acts as a competitor for CXCL12 on CXCR4 receptor. The reason for why patients with TT genotype possess a severe form of the disease may be due to the impact of the polymorphisms on CXCL12 that makes it works dysfunctionaly. So, it keeps the way clear for SARS-CoV-2 to bind CXCR4 as a co-receptor leading to increase viral load and disease severity.CXCL12 has a function in inflammation resolution, for example, by increasing angiogenesis and tissue repair. Prolonged CXCL12 activity may not only improve leukocyte chemotaxis, which is advantageous, but it may also worsen the chronic inflammation identified in COVID-19 . CXCL12 The expression of CXCL12 is elevated in the Fibroblasts from severe COVID-19 patients which may help to attract immune cells with the CXCR4 receptor, such as macrophages, T cells, and NK cells . PreviouCXCL12 gene polymorphisms might influence the regulation of ACE2, the receptor used by the SARS-CoV-2 virus to enter human cells. Altered ACE2 expression or function could impact viral entry and disease progression. Patients with severe COVID-19 have much higher levels of ACE2 expression, which allows more viral invasiveness .Gene polymorphisms in CXCL12 might lead to differences in immune cell recruitment and activation, which could affect the immune response to the virus and the development of severe COVID-19. Effective hematopoiesis, homing of T and memory B cells to lymph nodes, and monocyte recruitment depend on CXCL12, the CXCR4 ligand. Several viruses employ the inhibition of this axis to reduce the number of circulating immune cells and raise their own proliferative rate .CXCL12 polymorphisms could potentially impact the ability to clear the virus from the body, affecting the duration and severity of COVID-19. Cellular clearance of the virus depends on the production of virus-specific antibodies, which block the entry of free virions into uninfected cells, opsonize the virus for inactivation by complementing proteins or elimination by phagocytic immune cells that contain CXCL12 receptor like macrophages and neutrophils. They also inactivate or start the killing of infected cells by activating the complement cascade and through antibody-mediated cytotoxicity processes, essential for .Elimination of SARS-CoV-2 results in either an earlier time of recovery or a decrease in the severity of disease. According to Zheng, F. et al., the removal of viral shedding after viral clearance in patients would also aid to lessen viral transmission .In agreement of our data Wang et al., found that patients with CXCL12 rs2839693 were associated with increased susceptibility to sepsis . Zhang eThe multivariate logistic regression analysis of the genotypes against clinical COVID19 revealed that the combined genotype CT + TT acts as independent predictor for the severe or critical clinical condition of COVID 19.According to CXCL12 rs2839693 genotypes, our results revealed a non-significant difference among genotypes regarding age, gender, vaccination, and Co-morbid diseases. On the other side, regarding symptoms, our findings showed significant associations of genotypic distributions with dyspnea, vomiting, and dysarthria of COVID-19 patients. Dyspnea was more prevalent in the combined CT + TT and the TT genotypes compared with the CC and the CT, CC genotypes, respectively. Vomiting and dysarthria were significantly prevalent in the TT genotypes compared with the CT, CC genotypes. Regarding clinical course of the disease, we found that severe or critical illness associated with the combined CT + TT and the TT genotypes was more prevalent in the combined CT + TT and the TT genotypes compared with the CC and the CT, CC genotypes respectively.According to laboratory evaluation, our results showed significant differences between genotypes as regard to CRP, D-dimers, and ferritin. CRP and D-dimers are significantly elevated in the combined CT + TT and the TT compared with the CC and the CC, CT genotypes; while ferritin was significantly elevated in the TT compared with the CC, CT genotypes. Additionally, no significant differences were found between the 3 genotypes as regarding lymphocyte count, leucocyte count, and CT findings.Regarding treatment, a significant difference between genotypes as regarding clexane as a treatment was detected; Clexane was more prevalent in the TT genotype than the CC and the CT genotype. The number of patients without any demand for oxygen therapy was significantly different between the genotypes where it was more prevalent in the CC genotype compared with the combined CT + CC and the TT genotypes. Nasal cannula and invasive mechanical ventilation were more prevalent in the TT and the combined CT + TT genotypes compared with the combined CC, CT and the CC genotypes, respectively. Mask oxygen was used more prevalently in the patients of the CT + TT genotypes than those of the CC genotype.The number of patients needed home management was significantly higher in the CC genotype compared with the combined CT + TT and the TT genotype. The need for ICU stay was significantly higher in patients with the combined CT + TT and the TT genotype compared with the CC and the CC, CT, respectively. Regarding the mortality rates, our results revealed that they were significantly higher in the combined CT + TT and the TT genotype compared with the CC and the CC, CT, respectively. Patients with the TT genotype had a longer duration of in-hospital stay than patients with the CC genotype.Our findings may provide new insights on understanding the different factors affecting disease severity and mechanisms of cytokine storm syndrome which could affect COVID-19 outcomes and treatment strategies.There are two limitations that we encountered while conducting this study. First, the number of patients was limited due to the difficulty in obtaining samples from critical cases, cases without symptoms, or cases that had recovered from the disease. Secondly, some cases were removed from the study due to negative smears or some comorbidities that are not suitable for the study.The present study recommends studying other SNPs in the CXCL12 gene and their relationship with COVID-19 pathogenicity and severity. It also recommends expanding the study patient population to better understand the effect of CXCL12 gene variations on COVID-19 patients.Our data indicated the signature of CXCL12 rs2839693 in the pathogenesis and severity of COVID-19. Patients with the TT genotype or the T allele at increased risk developed severe or critical rather than mild or moderate disease. Also, patients' admission circumstances, such as vaccination, comorbidities, and symptoms may indicate disease severity. These variables require additional exploration and should be taken into account for risk categorization.


Hydrocephalus is a devastating condition characterized by excess cerebrospinal fluid (CSF) in the brain. Currently, the only effective treatment is surgical intervention, usually involving shunt placement, a procedure prone to malfunction, blockage, and infection that requires additional, often repetitive, surgeries. There are no long-term pharmaceutical treatments for hydrocephalus. To initiate an intelligent drug design, it is necessary to understand the biochemical changes underlying the pathology of this chronic condition. One potential commonality in the various forms of hydrocephalus is an imbalance in fluid–electrolyte homeostasis. The choroid plexus, a complex tissue found in the brain ventricles, is one of the most secretory tissues in the body, producing approximately 500 mL of CSF per day in an adult human. In this manuscript, two key transport proteins of the choroid plexus epithelial cells, transient receptor potential vanilloid 4 and sodium, potassium, 2 chloride co-transporter 1, will be considered. Both appear to play key roles in CSF production, and their inhibition or genetic manipulation has been shown to affect CSF volume. As with most transporters, these proteins are regulated by kinases. Therefore, specific kinase inhibitors are also potential targets for the development of pharmaceuticals to treat hydrocephalus. Hydrocephalus is a devastating condition that is considered a spectrum of diseases all characterized by excess cerebrospinal fluid (CSF) in the brain. There are several methods of classification within this spectrum. A functional classification is based on whether the cause of the hydrocephalus results in an obstruction to the flow of CSF within the ventricles , resulting in “noncommunicating” hydrocephalus, or whether there is an unobstructed ventricular flow, termed “communicating” hydrocephalus. The latter can be caused by either the overproduction or underabsorption of CSF. Alternatively, hydrocephalus can be classified as “secondary” (acquired) due to an exogenous precipitating cause or “primary”  . It has The most visible, and therefore the most well-known, form of hydrocephalus occurs in children where the patients’ heads are enlarged because the skull is not ossified and the buildup of CSF causes a cranial distension. Pediatric hydrocephalus is observed in approximately 1 in 1000 live births in high-income countries, such as the U.S., where it is due primarily to intraventricular hemorrhage in premature infants ,9. The iPosthemorrhagic hydrocephalus (PHH) is not just a disease of premature infants. PHH can occur at any age, especially in the older population, who are more prone to strokes. Post-traumatic hydrocephalus (PTH) is a secondary consequence of traumatic brain injury (TBI). The Centers for Disease Control reports approximately 1.5 million cases of TBI annually in the United States. The reported prevalence of PTH varies considerably, but some estimates suggest that as high as 80% of TBI patients deal with post-traumatic hydrocephalus of varying severity. Estimates specific to current and former military service personnel report some degree of hydrocephalus in as many as two-thirds of members who suffered moderate to severe TBI ,13,14,15Long-standing overt ventriculomegaly in adults (LOVA) is a heterogenous disease thought to arise in childhood, followed by an asymptomatic, possibly compensated, phase and then an adult-onset symptomatic phase with a progressive course . The etiElderly patients may suffer from a poorly understood and underdiagnosed disease called “idiopathic normal pressure hydrocephalus” (iNPH). Often misdiagnosed as Parkinson’s, this age-related ventriculomegaly is characterized by urinary incontinence, gait instability, and cognitive impairment that progresses to dementia if not treated. This condition has been estimated to occur in up to 6% of patients over 80 and, if surgically treated, is one of the few reversible causes of cognitive impairment in the elderly ,20,21,22Currently, the only effective treatment for any form of hydrocephalus is surgical intervention. The most common surgery is the placement of a shunt to redirect CSF from the brain to another body cavity for reabsorption. Shunts are prone to malfunction and infection and require additional, often repetitive, surgeries due to blockage, mechanical malfunction, or infection ,25,26,27Endoscopic third ventriculostomy (ETV) is a surgical alternative to shunting in some patients, such as those with noncommunicating hydrocephalus. This procedure involves the creation of a small opening in the third ventricle toward the subarachnoid space to create a bypass for CSF circulation from the ventricles to the subarachnoid space. A third type of intervention is ETV in conjunction with irreversible cauterization of the choroid plexus in the lateral ventricles in order to limit the amount of CSF produced ,25,26.There are no long-term pharmaceutical treatments for hydrocephalus, and the potential candidates that have entered clinical trials were either ineffective or fraught with unacceptable side effects . To initOne potential commonality in the various forms of hydrocephalus is an imbalance in fluid–electrolyte homeostasis. This can be due to the overproduction or underabsorption of CSF. Alternatively, there may be a change in the ionic composition of the CSF, leading to fluid accumulation via osmotic forces. Regardless of the initiating cause, treating the changes in fluid–electrolyte balance may provide an effective treatment. Therefore, good targets for drug development are the electrolyte and water transport proteins of the choroid plexus epithelium (CPe) that are responsible for the production and unique composition of the CSF. Given the dynamic changes in the characteristics of the CSF according to circadian rhythms and the need for immediate homeostatic changes in normal physiological responses, key transporters are regulated on an acute basis. This type of regulation most typically involves intracellular kinase-mediated signaling pathways.The CP is a complex tissue found in the brain ventricles. This small structure produces the majority of the CSF and is one of the most secretory tissues in the body, producing approximately 500 mL of CSF per day in an adult human . The CP In this manuscript two transport proteins, transient receptor potential vanilloid 4 (TRPV4) and sodium, potassium, 2 chloride co-transporter 1 (NKCC1), will be considered. Both have been postulated to play key roles in CSF production, and their inhibition or genetic manipulation has been shown to affect CSF volume. As with most transporters, these important proteins are regulated by kinases. Therefore, specific kinase inhibitors are also potential targets for the development of pharmaceuticals to treat hydrocephalus.2+ and Na+ into cells, which, in turn, stimulates intracellular Ca2+ signaling processes, including the activation of Ca2+-sensitive channels , and inflammatory mediators, such as arachidonic acid metabolites ,38,39,40channels . TRPV4 a regions ,43,44,45 regions ,43,44,45Human genetic diseases involving TRPV4 have been identified, particularly as skeletal dysplasias and arthropathies Thus, it appears from a whole-body perspective that the pharmacological activation of TRPV4 is physiologically deleterious, while the inhibition of the channel activity is relatively innocuous. These findings support the contention that TRPV4 is activated as a stress response. This role is enhanced by the ability of TRPV4 to act as a hub protein capable of integrating exogenous mechanical and chemical signals.Tmem67 model, treatment with TRPV4 antagonists ameliorated hydrocephalic development ..2+ that The signaling elements listed above are not exhaustive but serve to indicate that the kinase-regulated pathways controlling TRPV4 and NKCC1 have multiple points of potential interaction. It is not unexpected that a tissue like the CPe will have a complex regulatory network in order to maintain secretory function in the face of a dynamic extracellular milieu.Hydrocephalus in a multispectrum disease that strikes people of all ages. Although there are multiple causes, most forms of the disease are treated with the same surgical interventions. The efficacy of surgical intervention, combined with similarities in the symptomatic presentation in the multiple disease entities, suggests that the biochemical pathways underlying the pathology may be similar and may result, in part, from electrolyte and fluid imbalances. Preclinical studies have shown that systemic treatment with TRPV4 antagonists  or intra