The current standard treatment for patients with Duchenne muscular dystrophy (DMD) involves corticosteroids. Granulocyte colony-stimulating factor (G-CSF) induces the proliferation of satellite cells and myoblasts and, in turn, muscle regeneration. Beneficial effects of G-CSF were also described for skeletal muscle disorders.We assessed the safety and effects of using G-CSF to promote muscle strength in patients with DMD.Inclusion criteria were as follows: patients aged 5–15 years with diagnosed with DMD confirmed by genetic test or biopsy. Fourteen patients were treated with steroids, and their use was not changed in this study. Diagnoses were confirmed by genetic tests: deletions were detected in 11 patients and duplications in 5 patients. Nineteen 5- to 15-year-old patients diagnosed with DMD—9 were in wheelchairs, whereas 10 were mobile and independent—completed an open study. Participants received a clinical examination and performed physiotherapeutic and laboratory tests to gage their manual muscle strength, their isometric force using a hand dynamometer, and aerobic capacity  before and after therapy. Each participant received G-CSF (5 µg/kg/body/day) subcutaneously for five consecutive days during the 1st, 2nd, 3rd, 6th, and 12th month. Laboratory investigations that included full blood count and biochemistry were performed. Side effects of G-CSF treatment were assessed during each visit. During each cycle of G-CSF administration in the hospital, rehabilitation was also applied. All patients received regular ambulatory rehabilitation.The subcutaneous administration of G-CSF improved muscle strength in participants. We recorded a significant increase in the distance covered in the 6MWT, either on foot or in a wheelchair, increased muscle force in isometric force, and a statistically significant decrease in the activity of the muscle enzyme creatine kinase after nearly every cycle of treatment. We observed no side effects of treatment with G-CSF.Our findings suggest that G-CSF increases muscle strength in patients with DMD, who demonstrated that G-CSF therapy is safe and easily tolerable. Muscular dystrophies comprise about 30 disease entities , 2. CurrDuchenne muscular dystrophy is a hereditary X-linked neuromuscular disorder caused by mutations in the dystrophin gene, with a prevalence of 1:5,000 male newborns . It is cResearchers are searching for an effective therapeutic approach, including gene and stem cell-based therapies. Beneficial effects of granulocyte colony-stimulating factor (G-CSF) were also described for skeletal muscle disorders , 11. StrGranulocyte colony-stimulating factor is a hematopoietic cytokine, widely used for mobilization of hematopoietic stem cells from bone marrow and to treat neutropenia after chemotherapy . In 2014Granulocyte colony-stimulating factor induces (directly or through the increase in circulating stem cells) the production of many growth factors  and may have other methods of action on the system of musculature, vessels, and nerves yet to be described . The mecTo date and to the best of our knowledge, there have not been any published studies reporting the use or effects of G-CSF in children with muscular dystrophies.The purpose of this open trial is to evaluate the efficacy, and we assessed the safety and effects of G-CSF on muscle strength in patients with DMD.A prospective, non-randomized clinical trial assessed the efficacy and safety of G-CSF treatment in patients with muscular dystrophy.We enrolled 26 patients with muscular dystrophies under care of our department. Details are shown in Figure Inclusion criteria were as follows: patients aged 5–15 years with diagnosed with DMD confirmed by genetic test or biopsy. Fourteen patients were treated with steroids, and their use was not changed in this study. Diagnoses were confirmed by genetic tests: deletions were detected in 11 patients and duplications in 5 patients.Nine children (47.4%) were wheelchair-bound, and the others (52.6%) were mobile and self-independent. Details are shown in Table We expected that G-CSF (5 μg/kg/body/day) administration subcutaneously in patients with muscular dystrophies during the 1st, 2nd, 3rd, 6th, and 12th month increase in the passed distance in the 6-min walk test (6MWT) by feet or wheelchair, and an increase in muscle force compared with baseline would be observed.Disease course was evaluated clinically by neurological assessments . Manual Laboratory investigations that included full blood count, biochemistry  were performed. Blood was collected before G-CSF administration and on the fifth day of each treatment cycle. The assessment of hematopoietic stem/progenitor cells (CD34+), endothelial progenitor cells (CD34+ CD133+ CD309), monocyte subsets  was performed using flow cytometry in 11 patients in previous study .Abdominal ultrasonography with a spleen measurement was done before and after G-CSF administration. Electrocardiographic records were also performed. Side effects of G-CSF treatment were assessed during each visit.Granulocyte colony-stimulating factor (5 μg/kg/body/day) was administrated subcutaneously for five consecutive days during the 1st, 2nd, 3rd, 6th, and 12th month . We decided to apply half of G-CSF dose using for oncology treatment in children because of safety requirements. During each cycle of G-CSF administration in the hospital, rehabilitation was also applied. All patients received regular ambulatory rehabilitation. The treatment protocol schema is shown on Figure Interview and physical examinationPhysiotherapeutic examination: 6MT, Lovett Test, Hand Dynamometer TestLaboratory testsUltrasound of abdominal cavity with liver and spleen measurementECGSpirometryEthical approval for the study was obtained from the ethic committee of the Medical University of Bialystok (R-I-002/375/2013). Written informed consent was obtained from the patients and parents before participation.Clinicaltrials.gov to increase significance of the trial. The study Clinicaltrials.gov Identifier: NCT02814110.We have started our study in March 1, 2013, and we recruited patients from this time. We have registered  our clinical trial at website of t-test was applied to compare difference in time. The critical level for all tests of significance was p < 00.05.All statistical analysis was performed with SPSS package 15.0. Results are presented as mean values ± SD. Parametric paired Of the 19 patients, 16 (84.2%) completed the study. Patients were 5–15 years old, with a mean age (9.4 ± 2.6). Between March 2013 and February 2017, a total of 19 patients were treated with G-CSF. Sixteen patients completed five courses of the G-CSF treatment, and three boys ended earlier—after the fourth course .No side effects after G-CSF administration were reported by patients. White blood cell count increased at fifth day after each G-CSF administration as a reaction to drug application. Red blood count, platelets, CRP, creatinine, glucose, electrolytes—Na, K, Cl, Ca, Mg, fibrinogen, partial thromboplastin time, and prothrombin time were in the normal range. We observed a statistically significant decrease of the activity of muscle enzyme—CK after each cycle of treatment Table .In an ultrasound examination, the spleen size was normal during treatment. Electrocardiographic records did not differ significantly. We evaluated the effect of G-CSF treatment on the muscle strength and physical activity of the patients by performing these tests: (i) 6MWT to evaluate walking distance or moving wheelchair distance during 6 min, (ii) isometric force with hand dynamometer, and (iii) manual muscle testing (Lovett Test).We found significant increase of distance in 6MWT between baseline and first, second, third, and fourth cycle in ambulant patients with DMD. We observed also significant increase of distance between baseline first and third cycle in wheelchair-dependent patients with DMD. Details are shown in Table We also found significant increase of muscle strength in right hand in ambulant patients with DMD after the first, second, third, fourth, and fifth cycle of G-CSF treatment in comparison with the baseline. Only significant increase of muscle strength in left hand was noted after the fifth cycle.Significant increase of muscle strength in right and left hand was noted in wheelchair-dependent patients with DMD only after the fourth cycle of G-CSF treatment in comparison with the baseline. See Table The minimal increase of muscle force was found (Lovett test), but not statistically significant. Details are not shown.p = 0.001) increase of forced vital capacity in patients with DMD after fifth cycle of G-CSF treatment (1.69 ± 0.44 L) in comparison with the baseline (1.54 ± 0.43 L). We also noted significant (p = 0.032) increase of forced expiratory volume in 1 s (FEV1) in patients with DMD after fifth cycle of G-CSF treatment (1.53 ± 0.40 L) in comparison with the baseline (1.42 ± 0.37 L).We found significant , whereas the first administrations were 1 month apart. The increase of muscle strength testing by hand dynamometer was less spectacular but also detected. In laboratory tests, we observed a decrease in the activity of muscular enzymes.Although stem-cells therapy is still in its beginning stages, there are more and more observations of the positive effects of it and G-CSF treatment in patients with neurological diseases . G-CSF iIt has been shown that G-CSF decreases inflammatory processes and acts positively on peripheral nerve regeneration during the course of muscular dystrophy. This effect was observed in Simões’s study on mdx mice . The autThere is a growing body of evidence that monocytes/macrophages play an important role in muscle regeneration. Macrophages MI (pro-inflammatory cells) are involved in immune activation, phagocytosis, and muscle cell lysis. Macrophages MII exert anti-inflammatory properties and participate in the vascularization process. This population is able to support muscle cell regeneration by including satellite cell proliferation and tissue revascularization . To findAs to the administration of G-CSF in clinical trials, researchers have found neurological improvement in motor and sensory functions in adult patients with worsening symptoms of compression myelopathy . These fIn this study, we found significant decrease in the activity of muscle enzymes CK after each cycle of treatment and a stability of this level until 6 months of observation. After 12 months, a decrease of this enzyme activity was also observed, and it was statistically significant. In patients with muscular dystrophy the loss of muscle mass is observed, and, after an increase, a decrease of CK activity in the blood is noted. In our investigation, the situation seems to have been different. The abovementioned changes in laboratory parameters were accompanied by an augmentation of passed distance—by foot or on wheelchair and an increase in muscle force detected by dynamometry measurement.Furthermore, it is known that with G-CSF-induced mobilization of many trophic cells—endothelial progenitor cells, monocyte subsets, and cells with proangiogenic potential—there is the suggestion of possible anti-inflammatory effect, mobilization of existing satellite cells, improvement of local vascularity, and cytoprotection. We think that in DMD patients, the effect of G-CSF treatment comes from its positive impact on the regeneration and maintenance of the muscle fibers.Because the effects of therapy diminished after a 6-month break from G-CSF application, it seems to be more beneficial to administer a drug each 4 months. Our report has several limitations and strengths that are shown in Table The major limitation is that this was an open study. Patients in different stages of disease, with or without steroid therapy were treated. In a qualitative evaluation, both medical staff and parents observed that children were more active, they had better mood, appetite, their state of balance and precise movements improved.New genetic-based therapies in patients with DMD are promising. Exon skipping is a therapeutic approach for DMD. This method uses antisense oligonucleotides (AONs) to modulate pre-mRNA splicing of dystrophin transcripts to restore the disrupted DMD reading frame . RecentlOur study shows that G-CSF therapy is safe and well tolerated by the patients. We showed statistically significant increase in 6MWT and muscle strength, due to G-CSF administration. Significant decrease of CK after each of G-CSF treatment cycle was seen. Our data suggest that G-CSF increases muscle strength in children and adolescents with muscular dystrophy. We recommend further studies to address this proposition, as well as the mechanism of action identified by the Canadian research, in that the effects could be both to increase the number of viable  stem cells and growth factors, and also to increase the number of successful divisions that occur. The collective effect of these two features may well explain the significant collective improvements reflected in our data.Ethical approval for the study was obtained from the ethic committee of the Medical University of Bialystok (R-I-002/375/2013). Informed consent was obtained from the patient and parents before participation.DS, WK, BO-Z, and GP-P contributed equally to this work, study conception, data analysis, data collection, and paper writing; JW, KS, AK, and KO made physiotherapeutic tests and data collection; JS made statistics; EM revised the paper and made remarks.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RJ and handling editor declared their shared affiliation.


Donor liver function in transplantation is defined by mitochondrial function and the ability of mitochondria to recover from the sequence of warm and/or cold ischemia. Mitochondrial resilience maybe related to assembly and- subunit composition of Complex 1. The aim of this study was to determine if Complex 1 subunit composition was different in donor livers of varying quality and whether oxygen exposure had any effect.Five human livers not suitable for transplant were split. One half placed in cold static storage and the other half exposed to 40% oxygen for 2 h. Protein was extracted for western blot. Membranes were probed with antibodies against β-actin and the following subunits of Complex 1: MTND1, NDUFA10, NDUFB6 and NDUFV2. No difference in steady state Complex 1 subunit composition was demonstrated between donor livers of varying quality, in terms of steatosis or mode of donation. Neither did exposure to oxygen influence Complex 1 subunit composition. This small observational study on subunit levels suggest that Complex 1 is fully assembled as no degradation of subunits associated with the different parts of the enzyme was seen. Liver transplantation is limited by the lack of organs and combined with lengthening waiting lists results in a waiting list mortality of up to 20%. To address this disparity the marginal liver in terms of donor age, liver steatosis or donation after cardiac death (DCD) is increasingly used. But there is a risk to the recipient in terms of graft dysfunction, graft loss or death.Previous work has demonstrated that mitochondrial recovery and ability to generate adenosine triphosphate (ATP) effectively is a determinant of survival after liver transplantation. Mitochondria are central to cellular health through the production of ATP by oxidative phosphorylation (OXPHOS) and reactive oxygen species (ROS) that both drive and define cell function . ConsequThe largest complex of the respiratory chain is Complex 1 (> 900 kDa). It is an L-shaped multimeric protein complex, composed of 45 subunits and is the electron entry point of the respiratory chain. Disruption in the organization of Complex 1 can lead to an energy switch from OXPHOS to glycolysis . The facThe aim of this work was to determine if Complex 1 subunit composition in discarded human donor livers of varying quality was different, and whether composition could be modulated by oxygen exposure.Donor livers (n = 5) utilized in this study had been declined for both solid organ and cell transplantation. Table 2 exposed/A-PSF) section of donor liver was kept on ice in UW throughout, while the non-PSF or control section of donor liver was rebagged for standard cold static storage in UW on ice.To assess the response of the donor liver to A-PSF an experimental split model was developed. This involved performing a surgical left lateral segment (LLS) split. Producing two sections of liver for study from one donor liver with an inflow portal vein (PV) and outflow hepatic vein. A-PSF was via the PV with gaseous venting through the hepatic veins. The experimental  for formalin fixation and snap freezing in liquid nitrogen for storage at − 80 °C.Oxygen enriched atmospheric air was supplied with a portable electrochemical oxygen concentrator . For this study atmospheric air was enriched to 40% oxygen. The PV was cannulated and A-PSF was performed with 40% On-dodecyl β-d-maltoside  on ice. All reagents were from Sigma-Aldrich unless otherwise stipulated. After extraction, samples were centrifuged at 13,000g for 10 min at 4 °C. The cytoplasmic fraction (supernatant) was transferred into pre-cooled new tubes. For quantitation of total protein the Pierce™ BCA Protein Assay Kit  was used.Frozen liver biopsies were homogenized on ice using a micropestle in a 1.5 ml microcentrifuge tube. Homogenization buffer composition is as follows: 10 mM Hepes NaOH pH 7.4, 1 mM sodium EDTA, 250 mM sucrose, 1 μg/ml pepstatin A, 1 μg/ml leupeptin and 1 mmol phenylmethanesulfonyl fluoride. After homogenization the samples were stored at − 80 °C for later use. Homogenates were further extracted in homogenization buffer containing 1.5% lauryl ® Tetra Vertical Electrophoresis Cell, 4-15% mini-PROTEAN® TGX Stain-Free™ Precast Gels and Trans-Blot® Turbo™ Transfer System  were used. Reference ladder was Precision Plus Protein™ Dual Color Standards . 20 μg of protein was loaded per lane. The polyvinylidene difluoride (PVDF) membrane was dried overnight before blocking and probing. In brief, membranes were blocked in 5% skimmed milk/phosphate buffered saline, 0.3% Tween (PBS-T) for 1 h followed by rinsing in PBS-T. The membrane was then probed with the primary antibody in PBS-T for 2 h at room temperature unless otherwise stated.For protein electrophoresis and blotting the mini-PROTEAN® ab6276); NDUFB6 , 17 kDa, 1:1500 overnight incubation at 4 °C (abcam® ab110244); NDUFV2 , 24 kDa, 1:1000 overnight incubation at 4 °C (proteintech™ 15301-1-AP); NDUFA10 (middle part of membrane arm), 42 kDa (abcam® ab174829); and MTND1 , 36 kDa (proteintech™ 19703-1-AP).Primary antibodies used and dilutions are as follows: β-actin, 42 kDa, 1:120,000  were used, diluted 1:3000 in PBS-T and incubated with the membrane for 1 h. Clarity™ Western ECL Substrate  was used to develop the image. Image capture, background subtraction and quantification of band signal intensity was performed using Image Lab™ Software v5.1 . The protein of interest was normalized to β-actin. Human nomenclature for Complex 1 subunits has been used.Where appropriate, data is expressed as a mean and standard deviation. For comparison of groups the unpaired two tailed t test was used, significance value was set as p < 0.05. Statistical analysis was performed with GraphPad Prism v7.0b .2 via the hepatic vein.Five human donor livers declined for transplantation were split, considering graft type, 2 were DBD and 3 DCD, producing 10 sections of liver for study. The mean donor age was 54 ± 5.8 years, cold ischemic time 10.4 ± 1.7 h and warm ischemic time, where applicable, was 19.7 ± 4.5 min. On Hematoxylin and Eosin histology, steatosis was not demonstrated in 2 livers and in the remaining 3, steatosis ranged from 20 to 50%. The main documented reason for donor liver decline for transplantation was pathology in the donor, identified histologically as steatosis in 2 and cancer risk in another 2. In the remaining declined liver, there were concerns about preservation perfusion (Table 2 exposed (n = 5) to non O2 (n = 5) exposed liver sections  had any bearing on Complex 1 subunit composition.Complex 1 is a L-shaped structure composed of 45 subunits. One limb is a hydrophobic membrane arm sitting in the mitochondrial inner membrane and the other arm is hydrophilic, projecting into the mitochondrial matrix . ComplexSupercomplex assembly is thought to provide structural and functional advantages to the enzymes of the respiratory chain , therebyLow energy charge and purine quantity in donor liver parenchyma determines poor outcome in transplantation, which is related to mitochondrial function. The application of persufflation, gaseous perfusion with oxygen, to ‘resuscitate’ organs for transplantation has been explored in a number of solid organs . The mitThe factors that determine supercomplex assembly and function are still being resolved but it is recognized that mitochondrial metabolic needs, as defined by cell type and physiological status are a determinant . PutativIn conclusion, the presented data on subunit composition of Complex 1 suggest it is fully assembled, as no degradation of subunits associated with the different parts of the enzyme was seen. However, more work is needed to characterize, the factors that define the active/de-active state of Complex 1 and whether Complex 1 assembly, is a critical determinant of donor liver mitochondrial function and transplant outcome.The number of samples available for analysis and comparison are small. No functional assays have been performed to assess Complex 1 activity. Neither have any co-immunoprecipitation experiments been undertaken to demonstrate protein associations. Ideally, Blue Native polyacrylamide gel electrophoresis (BN-PAGE) to study assembly of Complex 1 combined with in gel measurement of enzyme activity on a larger number of transplanted and non transplanted livers needs to be undertaken.


Hydrocephalus is a multifactorial condition, whose aetiology is not fully understood. Congenital hydrocephalus frequently occurs in small and brachycephalic dog breeds. Although it is widely accepted that the cribriform plate located in the rostral cranial fossa (RCF) is a site of cerebrospinal fluid (CSF) drainage, the RCF has not been studied extensively. Literature reports indicate that a decreased caudal cranial fossa (CCF) volume in the course of the Chiari-like malformation may obstruct CSF circulation. We hypothesised that morphological diversity among different breeds in the volume of the RCF may affect CSF circulation. The aim of the study was to carry out a volumetric analysis of the RCF and the cranial cavity and to determine the ratio between them in dog breeds of different size and morphotype. We performed computed tomography (CT) morphometric analysis of the RCF compartment by obtaining volume measurements from the transverse and reformatted sagittal and dorsal planes.The rostral cranial fossa percentage – volume of the rostral cranial fossa/volume of cranial cavity × 100 (volRCF/volCC × 100) was lower in small and brachycephalic dog breeds than in the other dogs.A reduced RCF volume was detected in small and brachycephalic dog breeds, some of which are predisposed to congenital hydrocephalus. This may lead to overcrowding of brain parenchyma in the RCF and may impede CSF circulation. Our observations may be useful for future studies focusing on the causes and new therapies to treat conditions such as hydrocephalus and syringomyelia.The online version of this article (10.1186/s12917-018-1483-3) contains supplementary material, which is available to authorized users. Hydrocephalus is a pathological condition caused by an imbalance between cerebrospinal fluid (CSF) production, flow and drainage. This results in an accumulation of CSF in the intracranial space, most commonly within the ventricular system . HydroceAs opposed to the caudal cranial fossa (CCF), the RCF has not been studied volumetrically in dogs. The CCF structures have been studied extensively due to the occurrence of the Chiari-like malformation (CM) in some breeds of small dogs –12. CM hWe assumed that similar relationships may occur in the RCF. We hypothesised that in small and brachycephalic breeds predisposed to hydrocephalus the volume ratio of the RCF to the cranial cavity (CC) is smaller than in the others. That may potentially cause “overcrowding” of brain structures and disrupt CSF flow through the cribriform plate to the NL. The aim of the study was to carry out a volumetric measurement of the RCF and CC and to assess the relationship between those parameters in dog breeds of various size and morphotype.p = 0.0002) and brachycephalic and mesaticephalic 1 breed group (p = 0.0024). There were no statistically significant differences among the remaining groups.Individuals were divided into four groups based on skull index. Dolichocephalic breed group (skull index ≤50.00) consisted of 13 individuals . Mesaticephalic 1 breed group (skull index 50.01–65.00) contained 33 individuals . There were 12 animals in mesaticephalic 2 breed group (skull index 65.01–80.00) . Brachycephalic breed group (skull index ≥80.01) comprised 13 animals . The different dog breeds in each group are presented in Table p = 0.0002), small and large breed group (p = 0.0002) and small and giant breed group (p = 0.0002). There were no statistically significant differences among the remaining groups. Rostral cranial fossa percentage values of different breeds in small breed group are presented in Table Individuals were divided into four groups based on the breed size. Small breed group contained 25 individuals . Medium breed group comprised 17 individuals . There were 20 animals  in large breed group and nine in giant breed group . The number of dogs of each breed is presented in Table The cranial cavity can be divided into three regions called the RCF, middle cranial fossa and the CCF . CSF, whImpaired CSF drainage may result in CNS disorders. A block to CSF flow in the region of the basal cisterns in the middle cranial fossa resulted in hydrocephalus in the rat. Interestingly, the degree of cerebral ventricular enlargement was proportionate to the degree of impaired CSF flow through the cribriform plate . In humaHydrocephalus can be congenital or acquired. Toy and brachycephalic breeds, such as the Pekingese, Yorkshire Terrier, Maltese, Miniature Poodle, Pomeranian, Chihuahua, Pug, Boston Terrier and English Bulldog are predisposed to congenital hydrocephalus , 3. ThisThe concept of the CSF flow being disrupted by the crowded brain structures is not novel. CCF pathologies may also cause disturbances in CSF flow. Some breeds of dogs suffer from CM , 24, 25,The determination of volume ratios between different compartments of the cranial cavity is essential to understand certain CNS disorders associated with disrupted CSF circulation. Our results indicate that the ratio of RCF volume to CC volume is lower in small and brachycephalic breeds of dogs. A small rostral cranial fossa percentage is found in dogs predisposed to congenital hydrocephalus. We are aware that our findings do not prove impaired CSF outflow through the cribriform plate. However, they could form the basis for future research on this area of the cranial cavity. The ratios of the brain parenchyma within the RCF to the volume of the RCF should be calculated. The flow of CSF through the cribriform plate to the NL should be thoroughly studied in dogs with hydrocephalus.Seventy-one skulls obtained from adult dogs of both genders  from 2 to 16 years old were analyzed. Mixed-breed dogs and mongrel dogs were not included in the study. The skulls were divided based on the breed size into small, medium, large and giant breed group. Based on the skull index individuals were divided into the dolichocephalic, mesaticephalic 1, mesaticephalic 2 and brachycephalic breed group .The skulls were obtained from animals euthanised by veterinary doctors at the Small Animal Clinic of the Department of Clinical Sciences, Faculty of Veterinary Medicine, of the Warsaw University of Life Sciences. The animals were not euthanized for the purpose of this study. The owners consented to the euthanasia and to the use of the animal tissue for scientific purposes. None of the animals had any neurological symptoms prior to euthanasia. Examined animals were adults, with closed skull sutures and without dome-shaped skull. According to the Polish law, the post mortem use of tissue does not require the approval from the Ethics Committee . The corpses were subjected to a process of maceration. Soft tissue were removed from cranial cavity prior to CT examination.Computed tomography images of the skulls placed in ventral recumbency were acquired with a helical CT scanner (Siemens SOMATOM Emotion 16 CT Scanner) and a slice thickness of 0.75 mm to provide detailed image of the cranial cavity. Images were reconstructed with bone filter and displayed using a bone window (window width 2000 HU and window level 350 HU). In some cases the window was adjusted manually to better outline specific bony structures. CT data were transferred as a DICOM file to an image analysis workstation with Osirix® software in order to perform the image analysis. The originally obtained transverse slices were reformatted into the sagittal and dorsal planes. The volCC  and volRCF  were calculated. The volCC was obtained using the slice by slice method. The region of interest (ROI) was selected on each slice in the sagittal plane  software was used to assess the results. The distribution of the variables in the examined population was evaluated using the W Shapiro-Wilk test. The obtained data had a normal distribution, and they were analysed using the one-way analysis of variance. The effect of the breed size and the morphotype of the animals on the rostral cranial fossa percentage were assessed separately. Statistical significance between the groups was calculated using Tukey’s honestly significant difference (HSD) test.Additional file 1:Individuals value of AP, ZyZy, volRCF, volCC and volRCF/volCC ratio (RCF/CC). (XLS 20 kb)


Chemical and mechanical cues from the cerebrospinal fluid (CSF) can affect the development and function of the central nervous system (CNS). How such cues are detected and relayed to the CNS remains elusive. Cerebrospinal fluid-contacting neurons (CSF-cNs) situated at the interface between the CSF and the CNS are ideally located to convey such information to local networks. In the spinal cord, these GABAergic neurons expressing the PKD2L1 channel extend an apical extension into the CSF and an ascending axon in the spinal cord. In zebrafish and mouse spinal CSF-cNs originate from two distinct progenitor domains characterized by distinct cascades of transcription factors. Here we ask whether these neurons with different developmental origins differentiate into cells types with different functional properties. We show in zebrafish larva that the expression of specific markers, the morphology of the apical extension and axonal projections, as well as the neuronal targets contacted by CSF-cN axons, distinguish the two CSF-cN subtypes. Altogether our study demonstrates that the developmental origins of spinal CSF-cNs give rise to two distinct functional populations of sensory neurons. This work opens novel avenues to understand how these subtypes may carry distinct functions related to development of the spinal cord, locomotion and posture. Multiple studies indicate that the CSF also conveys signals affecting the development and output functions of the CNS, such as feeding, sleep, and locomotion6. These observations suggest that chemical or mechanical cues from the CSF may act on neurons in the brain and spinal cord. Cerebrospinal fluid-contacting neurons (CSF-cNs) are precisely located at the interface between the CSF and the neuronal circuits8. In the vertebrate spinal cord, CSF-cNs reside around the central canal15 and project an apical extension in contact with the CSF18 and have a locally-projecting axon20 that contacts other spinal neurons21.The cerebrospinal fluid (CSF) is a complex solution circulating around the central nervous system (CNS). It has classically been assumed that the CSF ensures the homeostasis of the CNS22, which belongs to the Transient Potential Receptor (TRP) family, appears as a robust candidate to label CSF-cNs24. PKD2L1, originally identified as the sour taste receptor in the taste buds, has been found in the spinal CSF-cNs of mouse26, zebrafish and macaques13. The opening of the PKD2L1 channel is modulated by variations in pH27 and osmolarity28. Although the physiological variations of pH and osmolarity in the CSF are not well known, these observations suggest that CSF-cNs could be interoceptors modulated by chemical and/or mechanical cues from the CSF23.One essential step to understand spinal CSF-cN functions lies in identifying specific markers for these cells. In this regard, the polycystic kidney disease 2 like 1 (PKD2L1) channel also called TRPP332. In zebrafish, CSF-cNs are subdivided into the ventral population, referred to as KA”, originating from the progenitor domain p3 and a dorsal population, referred to as KA’, originating from pMN30. In mouse, spinal CSF-cNs were recently shown to originate from the p3 and p2 progenitor domains26. In addition, some secreted compounds have been previously reported in a restricted number of spinal CSF-cNs, such as the somatostatin34 or serotonin36.There is evidence that spinal CSF-cNs do not constitute a homogeneous population of neurons. In particular, these cells originate from distinct progenitor domains and are specified differentially by several cascades of transcription factors30, the morphology and physiological roles of CSF-cNs in vivo due to its transparency at early stages37. Yet, few functional markers of CSF-cNs related to their sensory or secretory functions have been identified in this species. Here, we investigate whether the two types of spinal CSF-cNs defined by distinct developmental origins can be discriminated by morphology, projection on neuronal targets and expression of secreted compounds. Using a quantitative measure of cell shape, we show that ventral and dorsal CSF-cNs have differently shaped apical extensions as well as different axonal projections and consequently project onto distinct neuronal targets within the spinal cord. By electron microscopy (EM), we found that both populations of CSF-cNs exhibit large granular vesicles in accordance with secretory properties. We demonstrate that these two cell types express distinct modulators and peptides: while ventral CSF-cNs transiently express serotonin, dorsal CSF-cNs express the sst1.1 somatostatin paralog. We observed that the Pkd2l1 channel is not required for the differentiation of CSF-cN axonal projections, for the expression of serotonin or somatostatin. Altogether, our results show that spinal CSF-cNs constitute two distinct functional cell types that differ in apical and axonal morphology, neuronal targets within the spinal cord as well as in the transient expression of secreted compounds.The zebrafish has emerged as an ideal model organism to study the developmentpkd2l1 promoter  staining  zebrafish spinal cord by transmission electron microscopy (TEM). Without specific markers, we observed cells around the central canal that bore an apical extension with microvilli icm22 larvae or F-actin itself in Tgicm28 larvae or larvae transiently expressing  and . Using these different approaches, we found that the shape of the apical extension differs between ventral and dorsal CSF-cNs at 3 and 6 dpf  DNA construct to label single cells 44), V0-v (Tg(vglut2a:lox:DsRed:lox:GFP)45) or CoPA (Tg(tbx16-GFP)46). We observed that only ventral CSF-cNs form the stereotypical basket structure around the soma of CaP motor neurons  motor neurons and commissural primary ascending (CoPA) sensory interneurons within the escape circuit34 and serotonin36. By taking advantage of pkd2l1 expression in CSF-cNs13, we used the Tg(pkd2l1:GCaMP5G)icm07 transgenic line to selectively target CSF-cNs37. We demonstrated the specificity of this line using fluorescent in situ hybridization (FISH) for pkd2l1 mRNA coupled to GFP immunohistochemistry (IHC) to amplify the endogenous GCaMP5G signal from 24 hours post fertilization (hpf) to 5 dpf 51. Testing the expression of all of them, we only detected expression of sst1.1 in CSF-cNs. Expression of sst1.1 was observed only in dorsal CSF-cNs . Since sst1.1 had been reported as expressed transiently in motor neurons from 19 hpf until 55 hpf52, we showed using FISH for sst1.1 with GFP IHC in 24 hpf Tg(mnx1:GFP) embryos53 that sst1.1 expression was excluded from motor neurons icm07 embryos and larvae from 24 hpf to 72 hpf. At 24 hpf, no 5-HT immunostaining was detected in the zebrafish spinal cord (data not shown). At 48 hpf, we detected 5-HT in ventral CSF-cNs . At 3 dpf, although 5-HT was still expressed along the entire spinal cord in other cells, the staining was absent in CSF-cNs in the rostral spinal cord  within CSF-cNs in our EM data, combined with expression of sst1.1 and 5-HT for dorsal and ventral CSF-cNs respectively, reinforces this hypothesis.Due to their peculiar apical extension contacting the CSF, it had been hypothesized that CSF-cNs may release compounds into the CSF57 as well as mechanosensory cells58. In zebrafish larvae, we previously showed that dorsal CSF-cNs respond to lateral bending of the spinal cord37 while ventral CSF-cNs respond to longitudinal contractions43. The difference in shape of the apical extension between dorsal and ventral CSF-cNs may reflect a structural difference relevant for either their mechanosensory functions (direction of maximum sensitivity to bending of the tissue or CSF flow) or their secretory functions .Previous works have shown that CSF-cNs are chemosensory59. Four morphological types of CSF-cNs were then described based on the shape of their soma, their axonal projection, and on the expression of the peptide Met-Enk-Arg-Gly-Leu60. These indications together with our data reveal a high level of heterogeneity among spinal CSF-cNs. Future studies will investigate the role of CSF-cN structural differences in sensory and secretory functions.There are indications that spinal CSF-cNs harbor different morphologies in other species as well. In rat, spinal GABAergic CSF-cNs were first classified into three subtypes according to the shape of their soma15 as well as CaP primary motor neurons and CoPA sensory interneurons43. Here we show that only ventral CSF-cNs form the characteristic basket-like contact onto CaP soma while we only found dorsal CSF-cNs contacting ventrolateral glutamatergic neurons, most likely V0-v interneurons49 previously shown to receive inputs from CSF-cNs15. Interestingly CoPA sensory interneurons received innervation from both dorsal and ventral CSF-cNs. Our results suggest that targets of the slow swimming circuits receive innervation from dorsal CSF-cNs while targets of the fast swimming circuit receive innervation from ventral CSF-cNs, and sensory interneurons involved in processing feedback receive innervation from both ventral and dorsal CSF-sNs. Further investigations will be necessary to establish whether other targets of the slow and fast swimming circuits follow the same dichotomy, receiving inputs from dorsal and ventral CSF-cNs respectively.Using a single cell labelling approach, we show that ventral CSF-cNs have on average a longer and broader axonal arborization and cover a higher dorso-ventral spinal cord range with more axonal branches than dorsal CSF-cNs. These differences in axonal projections suggest the two CSF-cN populations might have distinct targets within the spinal cord. We had shown that CSF-cNs form active GABAergic synapses onto glutamatergic descending V0-v interneurons34, dopamine64 and serotonin65. The expression of most markers was reported in a restricted fraction of CSF-cNs. Here we tested whether some of these markers showed restricted expression patterns among CSF-cNs. We found that ventral CSF-cNs were at 48 hpf transiently serotoninergic as previously described in other species65 in accordance with findings from Montgomery et al.66, in zebrafish. In addition, we demonstrated that, even at 48 hpf, not all ventral CSF-cNs expressed 5-HT. This suggests that there may be different subtypes of ventral CSF-cNs based on 5-HT expression. Moreover, Montgomery et al.66, also showed that the rate-limiting enzyme involved in 5-HT synthesis tryptophan hydroxylase 1 a (tph1a)68 is expressed in the ventral spinal cord at 24 and 48 hpf suggesting that ventral CSF-cNs could constitute a transient source of serotonin at early stages. One possible explanation for the disappearance of 5-HT in ventral CSF-cNs at later stages could be the emergence of descending fibers from 5-HT+ neurons originating from the brainstem raphe nuclei from 48 hpf onwards67. 5-HT from descending fibers has been previously shown in mammals to suppress the monoaminergic expression of CSF-cNs71. The physiological relevance of the transient 5-HT expression among this ventral population of CSF-cNs remains to be elucidated.Multiple markers have been found in subsets of CSF-cNs across vertebrate species: somatostatinsst1.1 was the only one expressed. We demonstrated that the expression of this gene is specific and restricted to the dorsal population of CSF-cNs, identifying for the first time a specific marker of dorsal CSF-cNs. We also demonstrated that this peptide is excluded from motor neurons contrary to what previous studies suggested52. The physiological relevance of sst1.1 expression by dorsal CSF-cNs is not known. In mammals and lamprey, it has been shown that somatostatin can reduce locomotor frequency73. The release of SST by dorsal CSF-cNs could impact the frequency of locomotor events along with the release of neuropeptides of the UII family, urp1 and urp2 by ventral CSF-cNs only74 as previously suggested by the presence in coho salmon of a UII-like immunoreactive ventral population of CSF-cNs and a distinct somatostatinergic one75. Taken together, our results confirm that ventral and dorsal CSF-cNs express distinct peptides and neuromodulators. The transient nature of the expression of 5-HT suggest that it could have developmental roles to be further investigated.Regarding the origin of somatostatin in zebrafish CSF-cNs, we found that among the six SST paralogs, Danio rerio) adults and larvae were maintained and raised on a 14/10 hour light cycle. Fish lines used in this study are referenced in Table pkd2l1icm02 mutants was described in ref. et al. 2016). Zebrafish icm22 line, the TagRFP-CAAX sequence was amplified using a TagRFP-forward (5′-CCCGGGATCCACATGGTGTCTAAGGGCGAAG-3′) and reverse primer (5′-GATCGCGGCCGCTCAGGAGAGCACACACTTGCAGCTCATGCAGCCGGGGCCACTCTCATCAGGAGGGTTCAGCTTATTAAGTTTGTGCCC-3′) and inserted into the pME-MCS vector76 via BamHI/NotI restriction digestion. The resulting pME-TagRFP-CAAX vector was recombined via a Gateway reaction (MultiSite Gateway Three-Fragment Vector Construction Kit) with p5E-4nrUAS77, p3E-pA and pDest-Tol2; cmlc2:eGFP76 resulting in . Using a similar approach, the pME_LifeAct-GFP plasmid was generated from the plasmid pCS2_LifeAct-GFP (kind gift from Nicolas David) using the SP6 promoter sequencing primer (5′-ATTTAGGTGACACTATAG-3′) and inserted into the pME_MCS vector via a BamHI/NotI restriction digestion. The final three-way gateway reaction used the pME_LifeAct-GFP, pDest_cryaa:V and p5′E_polyA plasmids to generate the  construct. Microinjection of these plasmids was performed with Tol2 mRNA (25 ng/µl) following standard protocols. Transgenic founder fish Tgicm22 where screened based on GFP expression in the heart. These two lines were also screened based on transactivation of their respective transgene when crossed with various Gal4 lines.In order to generate the 78. We generated a three-fragment Gateway recombining reaction  to design the (UAS:APEX2-TagRFP) construct. This plasmid was injected into single-cell stage Tgicm10 embryos at 60 ng/µl or co-injected with the icm10 construct into Tg(pkd2l1:GaMP5G)icm07 singe-cell stage embryo. To generate the  DNA construct, we extracted the coding sequence of the tagged protein LifeAct-TagRFP from the plasmid (mTagRFP-T- LifeAct-7)  by PCR using a forward (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTAGATCTCTGCCACCATGGGCGTGGCCGACTTGATC-3') and a reverse (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTACTAGTTTACTTGTACAGCTCGTCCATGCC-3') primer. LifeAct-TagRFP was then inserted into the plasmid pDONR221 by a BP reaction to produce the pME_LifeAct-TagRFP plasmid and a three-way gateway reaction was performed using pME_LifeAct-TagRFP, pDest_cryaa:C, p5′E_10XUAS and p3′E_polyA plasmids to produce the  construct.In order to investigate the CSF-cNs ultrastructure, we took advantage of the engineered peroxidase APEX22 was added for 45 min. Whole embryos were then rinsed in the same buffer 5 × 2 min each. Functional aldehyde excess was blocked by treating embryos for 5 min with 20 mM glycine in the sodium cacodylate buffer. Other rinses in buffer (5 × 2 min) were then carried out. The APEX2 peroxidase was further revealed in diaminobenzidine  to which 10 mM hydrogen peroxide (30%) was added , in 100 mM cacodylate buffer. After 5 min, the reaction was stopped by rinsing embryos twice in cold buffer before post fixation in 2% osmium tetroxide (OsO4) in the same buffer for 45 min. Embryos were then rinsed in cold distilled water, stained in 2% uranyl acetate in distilled water overnight in a cold chamber. Animals were returned to room temperature, rinsed in distilled water, then dehydrated in a graded series of ethanol, cleared in acetone, and embedded in epoxy resin . Embryos were oriented coronally in resin molds cured at 60 °C for 48 hr. Whole embryos were imaged using a Leica DMRB microscope to assess the presence of DAB positive CSF-cNs and determine their location within the animals. Embryos were first cut in 1 µm semi-thin sections with an ultramicrotome . Sections were picked up every µm, stained with toluidine blue. Semi-thin sectioning was performed until the first level of DAB+ CSF-cN cell appears. Then, serial ultra-thin sections (~70 nm thick) were collected onto copper grids (about 8 sections per grid). 12 ventral CSF-cNs, and 3 dorsal CSF-cNs were analyzed. They were contrasted in urany-less solution  for 1–2 min, rinsed in distilled water, dried for at least 1 hr. Observations were made with an Hitachi HT 7700 electron microscope operating at 70 kV. Electron micrographs from DAB+ CSF-cNs were taken at low (×6200), medium  and high  magnifications, using an integrated AMT XR41-B camera (2048 × 2048 pixels). In all the images displayed, dorsal is up and rostral is left.To label CSF-cNs for electron microscopy, we followed the procedure described in refs Tg transgenic line with membrane TagRFP and the transient expression of 15 and  DNA constructs injected into Tg(pkd2l1:GCaMP5)icm07 eggs at the one cell stage to label F-actin with LifeAct79. At 6 dpf, we took advantage of the stable Tgicm28 line. 3 and 6 dpf larvae were screened for expression in CSF-cNs and fixed using 4% paraformaldehyde (PFA) during 4 hours at 4 °C and immunostained as described below. We sliced 50 µm-thick transverse sections on larvae mounted in 3% low melting point agarose using a vibratome . For each cell, Z stacks (step size 0.25 µm) were acquired to image the entire apical extension and maximum projection was performed in Fiji80. The shape of the apical extension (S) was calculated as the ratio of the width  over the height . W and H were extracted by drawing a polygon around the apical extension using the polygon tool in Fiji and estimated by the parallel and perpendicular axis, respectively, of the best fitting ellipse  construct generated with a three-fragment Gateway recombineering reaction into single-cell stage embryos from pkd2l1icm02/+ incrosses (see ref. pkd2l1-TagRFP) construct has been injected in the Tg(pargmnet2-GFP)44 and Tg(tbx16-GFP)46lines and the  with (UAS:synaptophysin-GFP)81 constructs in the Tg(vglut2a.lox-DsRed-lox-GFP)line45. Some of these animals have been imaged live.To assess whether see ref. . 3 dpf lTo label and trace individual CSF-cNs, we followed the same procedure as described in ref. pkd2l1 ISH probe was generated as previously described13. The sst1.1 plasmid originates from the Argenton lab, Padova, Italy82. pkd2l1 and sst1.1 plasmids were respectively linearized with NotI and SalI. Digoxigenin (DIG)- and fluorescein (Fluo)-labeled probes were synthesized using SP6 RNA polymerase with the RNA Labeling Kit  to generate both pkd2l1 and sst1.1 antisense probes. All probes were purified using the mini Quick Spin RNA Column . Whole-mount ISH were performed as previously described83 on embryos or larvae fixed in 4% PFA in phosphate buffered saline (PBS) overnight at 4 °C.The Procedures for IHC were described in Supplemental Information.pkd2l1 and sst1.1 FISH were performed prior to IHC of green fluorescent protein (GFP): embryos and larvae were washed and immunostained with the chicken anti-GFP antibody  overnight at 4 °C, and then incubated with the corresponding Alexa-conjugated secondary antibodies IgG  combined with DAPI .Procedures were described in ref. To compare the density of markers investigated, we systematically imaged three regions along the rostrocaudal axis of the fish: segments 3–6 , 10–13  and 23–26 .Tg(pkd2l1:GCaMP5G)icm07 transgenic embryos and larvae with pkd2l1, sst1.1 FISH or GABA and 5-HT IHC, fish were mounted laterally in 1, 5% agarose covered of Vectashield Mounting Medium . To analyze apical extensions, slices were transferred into Vectashield mounting medium as well .Images were acquired using an Olympus FV1000 confocal microscope equipped with a 20 and 40x water and 60X oil immersion objectives using the 405, 473 and 543 nm laser lines or using an upright microscope  equipped with a spinning disk head  and a modular laser light source . To determine the overlap of GFP in the Procedures for optogenetic stimulation of CSF-cNs and electrophysiological recordings of CaP motor neurons are described in Supplemental Information and Supplemental Fig. pkd2l1icm02/icm02 mutants and two-way ANOVAs to test the interaction between the genotypes and the cells investigated. The level of significance was p < 0.05 for all datasets. p values are represented as the following: (*)p < 0.05; (***)p < 0.001; (****)p < 0.0001.We used Student’s t-tests for the morphological comparison of ventral versus dorsal CSF-cNs in WT and Supplementary Info File #1


In this pilot study, we examined the trends of S-100β and antioxidant capacity in the CSF during and after TEVAR. Thoracic Endovascular Aortic Repair (TEVAR) has substantially decreased the mortality and major complications from aortic surgery. However, neurological complications such as spinal cord ischemia may still occur after TEVAR. S-100T0) and immediately (T1) and 24 (T2) and 48 hours (T3) after the deployment of the aortic stent. S-100β and CSF antioxidant capacity were analyzed with the use of commercially available kits. We recruited 10 patients who underwent elective TEVAR. CSF samples were collected through a lumbar catheter at the following time points: before the start of surgery  increased. However, the levels of S-100β at T1 and T3 were comparable to the baseline value. The antioxidant capacity remained unchanged. No patient had a clinical neurologic complication. We observed that the level of S-100 Our observations may indicate biochemical/subclinical central nervous system injury attributable to the deployment of the aortic stent.  The development of postoperative paraplegia and stroke are major complications of open surgery (with use of either cardiopulmonary bypass or aortic cross-clamping) for repair of thoracic aortic aneurysms . In receβ has been shown to be a sensitive marker to detect spinal cord injury  (ABTS) with ferryl myoglobin, produced through the reaction of the ABTS•+ radical, which can be monitored by reading absorbance at 750 nm or 405 nm. In the presence of antioxidants, the absorbance of this radical cation is quenched to an extent directly related to the concentration of antioxidants present in CSF sample. The extent to which the antioxidants in the sample solution inhibited the reaction was calibrated with use of a standard antioxidant solution.Three ml of CSF fluid was withdrawn from the lumbar drain before the start of surgery (t-tests (with a Bonferroni correction due to multiple testing) to test for a statistically significant difference in S-100B and Trolox levels postoperatively, at 24 hours, and 48 hours, compared to preoperative levels. We then used an analysis of variance (ANOVA) to test for an overall difference in S-100B levels between time points. A statistical result of p < 0.017  was considered significant for the paired t-test analyses, while a level p < 0.05 was considered significant for the ANOVA analysis. All analyses were conducted using STATA 13.0 .Data are expressed as proportions (percent) and median (interquartile range). We used paired Our cohort consisted of ten patients with a median age of 67 (63–74) years; among this cohort, 6 (60%) were male. The demographic data of the patients is summarized in β was significantly higher at 24 hours after the deployment of the aortic stent (T2), compared to preoperative levels (p = 0.007). S-100β levels were increased at 24 hours in all 10 patients. However, the S-100β levels postoperatively (p = 0.04) and at 48 hours (p = 0.10) after the deployment of the stent did not differ significantly  compared to baseline, although the postoperative level trended toward significance (β biomarkers levels over the entire time period (p = 0.023).The CSF level of S-100ificance . Using ap = 0.79), at 24 hours (p = 0.40) or at 48 hours (p = 0.20) (Antioxidant capacity in the CSF (expressed as Trolox) did not change significantly after stent deployment compared to baseline ( = 0.20) .No patients had neurologic complications after surgery, and the postoperative neurological examinations remained unchanged compared to the preoperative status.β levels in the CSF increased significantly at 24 hours after stent deployment but returned to baseline after 48 hours. In addition, the antioxidant Trolox levels did not change during the 48 hours after TEVAR. This data, coupled with clinical observations in the postoperative period, indicate that most patients experience mild central nervous ischemia following stent deployment, but it generally resolves and usually does not lead to clinically relevant neurologic insults. By following the dynamics of S-100β and antioxidant capacity, our study adds to the knowledge of when the spinal cord is exposed to ischemia after TEVAR. This may help guide protocols for CSF monitoring and drainage during and after TEVAR.In summary, our results show that, in patients undergoing TEVAR, the S-100β is a calcium binding protein which is located selectively in glial and Schwann cells and is therefore unique to the central nervous system. S-100β in CSF has previously been identified as a specific and reliable biomarker for central nervous system injury [β in the CSF of the patients whom they considered higher risk for postoperative neurological dysfunction; however, S-100β in CSF did not significantly increase throughout their sampling points [S-100m injury . Brunnekg points . This coβ tends to increase over 48 hours after stent deployment but only reached statistical significance at 24 hours. However, the clinical significance of this increase of S-100β is unclear because even the maximum level of CSF S-100β in this study was below 1.0 microgram/L, the previously identified upper limit of CSF S-100β levels in subjects without any neurological symptoms [β with MRI findings. Importantly, CSF was drained in all patients and hemodynamics were managed to maintain the spinal perfusion pressure >80 mmHg and this might have prevented the development of persistent spinal ischemia/neurological deficits.Our result indicates that S-100symptoms . Additiosymptoms . As we dThe central nervous system is very vulnerable to ischemia, due to its nature of high metabolic requirement for oxygen. CNS ischemia initiates a complicated cascade of events that culminate in oxidant generation. This response is normally balanced by an increase in the production of endogenous antioxidants including enzymes such as superoxide dismutase and glutathione peroxidase. Therefore, antioxidant production has been considered a logical surrogate for ischemic injury –17. Whilβ with MRI findings of ischemia, rather than relying on the clinical exam alone. Second, our small sample size makes it difficult to have statistical estimates of a high precision. Lastly, variations in patient demographics, anesthetic techniques, and surgical care may have contributed to the results that we observed.There are several limitations to our study. First, the lack of neuroimaging may have precluded the ability to study the association of S-100β increased significantly 24 hours after stent deployment for thoracic aorta repair, but it did not reach a clinically significant value. Endogenous antioxidant capacity as measured by Trolox levels was unchanged throughout the study period. Therefore, our study adds to the mechanistic data of the development of SCI after TEVAR, but further studies are needed to elucidate the clinical relevance of our findings. Future research should employ larger samples sizes with more frequent sampling points to evaluate clinical outcomes and study the association of CSF biomarkers with neuroimaging abnormalities.This pilot study demonstrates that CSF levels of S-100


What’s more, biochemical indices in plasma and tissue histology were measured to further study therapeutic effects on the damages of oMWCNTs in mice. Isotope tracing and improved CT imaging results showed that low dosages of DOPC and TD didn’t affect the distribution of oMWCNTs in mice; conversely, the distribution and metabolism of DOPC and TD were influenced by oMWCNTs. Moreover, DOPC and/or TD improved the biocompatibility of oMWCNTs in erythrocyte suspension in vitro. Biochemical index and histopathological results indicated that DOPC and TD didn’t prevent injuries caused by oMWCNTs effectively. But TD showed a good therapeutic effect for damages. This study is the first to investigate prevention and treatment effects of drugs on damages caused by oMWCNTs and provides new insights and breakthroughs for management of nanoparticles on health hazards.With the development and application of nanotechnology, large amounts of nanoparticles will be potentially released to the environment and possibly cause many severe health problems. Although the toxicity of nanoparticles has been investigated, prevention and treatment of damages caused by nanoparticles have been rarely studied. Therefore, isotope tracing and improved CT imaging techniques were used to investigate the biodistribution influence between oMWCNTs and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/or simvastatin (TD)  Particularly, the potential application of carbon nanotubes in drug delivery, tumour probing and imaging has attracted considerable research attention7. Therefore, carbon nanotubes have been used broadly in various areas such as industrial application, consumer products and so on, moreover, it’s reported that the global production of carbon nanotubes sum up to 100–1000 t/a8. The widespread usage of carbon nanotubes will increase their concentration in environment so as to cause diverse ecological problems10.Carbon nanotubes are new types of materials that have been widely studied and applied in electronic engineering, catalytic materials, environmental engineering, biological medicine and other related fields13. The biological toxicity of nanomaterials has been evaluated to determine their safety in various environments, the toxicity of nanomaterials is related to their structure, size, functional groups and mechanism of exposure18. In addition, different functional groups or structure of nanoparticles have strong interaction each other after entering into body, then leading to more complicated damages or effects on biological organisms. Several examples as follows: Li et al. intravenously studied bio-distribution in mice co-exposed to oxidised multi-walled carbon nanotubes (oMWCNTs) and oxidised graphene through isotope tracer technique, and found that the bio-distribution of oxidised graphene was considerably affected by oMWCNTs after co-exposure; both of these compounds cause damages to liver and lung19. Wang et al. reported that single-walled carbon nanotubes (SWCNTs) accumulated mainly in the liver and kidney when mice were exposed intraperitoneally to hydroxylated SWCNTs; SWCNTs also slightly accumulated in the spleen and lung but eventually excreted mainly from urine within 18 days20. Singh et al. also reported that ammonium-functionalised water-soluble SWCNTs linked with DTPA-111In were excreted mainly via renal route without uptake in the liver and spleen after being intravenously injected to mice21. These findings indicate that the nature of functionalization on nanotubes is the most influential factor on the behaviour and toxicity of carbon nanotubes in mice. In addition, several studies about the toxicity of nanoparticles to sensitive groups  found that oMWCNTs could cross placental barrier into the placenta and cause damages to matrix, and the complication reduces the ability of the placenta to provide nutrition for the offspring and affects foetal development, which could cause a series of pregnancy disorders22. But more importantly than all of above, there are many researches about the toxicity and interaction of protein, cell and microorganism basing on carbon nanotubes27, and find that carbon nanotubes could enter into and destroy cell or microorganism, further induced cell apoptosis or function disorder of microorganism, and revealing that the toxicity mechanism of carbon nanotubes may relate to the interaction of protein and carbon nanotubes. However, it can be seen that almost of all reports are about the toxicity and damage of carbon nanotubes in biology body or others, but there are rare or even not researches about the treatment or prevention effects of drugs on the toxicity or damages caused by nanoparticles.Carbon nanotubes are foreign particulate matter in biological organisms; exposure to nanotubes can cause tissue damage or toxicityin vivo through small-molecular drugs were designed and carried out in here. DOPC in cell membranes are first damaged when carbon nanomaterials (such as graphene) enter into the biological body. It was found that graphene strongly adsorbs DOPC on the surface in vitro to form a compound coated with DOPC28. It can be imagined that when nanoparticles enter into biology body, the supplementary extra DOPC will compete with the DOPC of cell membrane to be adsorbed by nanoparticles, and so as to decrease the toxicity of nanoparticles; on the other side, many reports prove that the biocompatibility and toxicity of nanoparticles can be improved after coated with DOPC. It can be imagined that nanoparticles adsorb the free extra DOPC in blood vessel after enter into body, and then the biocompatibility of nanoparticles is improved to make them eliminate from body easily. As well know, statins have obvious effects on regulating blood lipid, delaying the extent of atherosclerosis, anti-inflammation and anti-thrombosis, and thus are commonly used to cardiovascular and cerebrovascular diseases or anti-inflammatory in clinical31. In addition, one of statins pharmacological effects is widening myocardial tissue clearance according to the view of doctors. Thus, statins may promote the excretion and anti-inflammation of nanoparticles that accumulated in the body to a certain degree, thereby reducing or eliminating tissue inflammation35. In the present work, therefore, prevention and treatment capability of simvastatin (TD) and DOPC on the toxicity of oMWCNTs in mice was studied firstly. The reliability of isotope tracer technique on the biodistribution of nanoparticles in vivo was investigated to further study the biodistribution of oMWCNTs in vivo. The improved CT imaging was then used to investigate the distribution of oMWCNTs in mice in vivo. For single oMWCNTs no development under CT imaging, Ag/oMWCNTs nanocompounds were synthesised as reported in the literature36; this technique improved the CT imaging ability of oMWCNTs to further study their biodistribution in mice. This study provides new breakthrough and reference data for prevention and treatment effects of drugs on damages and toxicity caused by nanoparticles to the biological body.It is a great severe effect on health after exposure to nanoparticles environment. It’s also widely believed that carbon nanotubes can accumulate in tissues for a long time to cause inflammation or function disorders. Therefore, it can be speculated that the toxicity or damage of carbon nanotubes might be decreased or cured through urgent elimination of the accumulated nanoparticles from the biological body as well as treatment of inflammatory disorders or others. Basing on this speculation, the experiment of curing the toxicity of nanoparticles in mice The TEM characterisation results showed that oMWCNTs had 1–10 μm length and 10–30 nm diameter. In addition, the TEM images of Ag/oMWCNTs showed that almost all Ag ions were connected to the surface of oMWCNTs, and the Ag/oMWCNTs nanocompounds were synthesised Figure . Figure 131I isotope tracer technique , indicating successful establishment of oMWCNTs-model mice. After exposing the oMWCNTs-model mice to DOPC/or TD, the AST, ALT and TB levels in the plasma of DOPC-exposed mice were lower than those of the oMWCNTs-model group mice and approximate to those of the control group of mice. However, for the TD-exposed group of mice, the BUN, ALT, AST, Cys-C, TB and CRP contents in the plasma were considerably different from those of the oMWCNTs-model group of mice. Most of the measured indices reverted to the normal levels after the oMWCNTs-model group mice was exposed to TD. In addition, the histology of tissues showed the same results . However, the biochemical index contents, except for ALT and AST content, did not change in the exposed groups of DOPC + oMWCNTs and TD + oMWCNTs . However, only a part of the biochemical indices of the DOPC/or TD-model mice exposed to oMWCNTs differed from those of mice exposed to oMWCNTs alone. Therefore, analysis of the biochemical indices indicated that the lack of prevention effect of DOPC/or TD on damages caused by oMWCNTs in mice. After exposing the DOPC-model mice to oMWCNTs, the pathological section showed that ruptured and bleeding pulmonary alveoli and obvious browning of the nanoparticles. The liver and kidney were nearly normal, the spleen nearly underwent fibrosis, and the cardiac muscle fibres became thin because of haemorrhage. The TD-model mice exposed to oMWCNTs possessed severely ruptured and bleeding of the pulmonary alveoli, destroyed central vein of liver and no obvious changes in the spleen. However, the kidney showed a slight haemorrhage and the gap of the cardiac muscle fibres increased without lesions. The pathological results further confirmed that the DOPC/or TD showed no preventive effects on the damages of oMWCNTs in mice . The contents of Cys-C, ALT, AST and TB in the plasma gradually increased to the normal levels as the oMWCNTs-model mice were exposed to TD . As a result, high accumulation of 131I-oMWCNTs was observed in the spleen and liver  were calcined at 450 °C for 24 h to remove the amorphous carbon. The purified oMWCNTs were characterised by TEM, FTIR spectroscopy and Raman spectroscopy analyses and were used to prepare 5 g/L suspension. 1,2-Dioleoyl-sn-glycero-3-phosphocholine  was bought from Xi’an Ruixi Biological Technology Co., Ltd, and TD (purity over 99%) was purchased from Nanjing Ailikaide Chemistry Co., Ltd. All other reagents were analytical pure.MWCNTs were prepared through chemical vaporization deposition and were commercially obtained from Shenzhen Nanotech Port Co. Ltd., Guangdong, China. According to the product specification, the as-received MWCNTs were subjected to transmission electron microscopy (TEM) analyses. The particles had 10 μm length and 10 to 30 nm diameter, and purity of >96% (wt.%); the particles contained <3% amorphous carbon and <0.2% ash. The pristine MWCNTs (untreated MWCNTs) were added to 3 mol/L HNO131I (about 1 mCi) was provided by the People’s Hospital of Gansu Province. According to the literature38, chloramine-T method was used to label oMWCNTs, DOPC and TD with Na 131I. The labelled mixtures were centrifuged at 12000 rpm for 10 min to purify 131I-oMWCNTs, and the supernatant was discarded to remove free Na 131I. The labelled mixtures were placed in a separation column to remove free Na 131Ito purify 131I-DOPC and 131I-TD  The yields of the labelled compounds were measured using a paper chromatogram with chromatographic solutions containing normal saline and acetone. If the labelling yield was over 60%, the labelled compounds could effectively reflect their distribution and metabolism in vivo  and approved by Institutional Animal Care and Use Committees of Gansu Province Medical Animal Center and Lanzhou University Animal Committees Guideline (China).The mice were grouped randomly (five mice/group) before the experiment. 131I-oMWCNTs + DOPC, oMWCNTs + 131I-DOPC, 131I-oMWCNTs + TD and oMWCNTs + 131I-TD (dose (1:1) 12.4 mg/kg.bw) solutions were injected intravenously into different groups of mice. The animals were sacrificed at 1, 6, 16 and 24 h post injection; heart, lung, liver, spleen and kidney tissues were immediately dissected. About 1–2 mL of blood was also collected. Each tissue was wrapped in foil, weighed and counted for 131I. The interactions between oMWCNTs and DOPC /or TD in mice were also investigated.Kunming mice , with initial weights of 15–18 g, were provided by the Laboratory Centre for Medical Science, Lanzhou University, Gansu, China. All animals were housed in individual cages with controlled environment at 21 °C to 22 °C and lights on from 08:00 to 20:00 h. The mice were fed with food and tap water 36. Briefly, AgNO3 solution  was added to the oMWCNTs solution  under magnetic stirring for 30 min. An alkaline solution of sodium borohydride  was added to the oMWCNTs solution under vigorous stirring at room temperature for 2 h. A black dispersion of Ag/oMWCNTs was formed, and Ag/oMWCNTs heterostructures were injected into the mice for CT imaging . In this section, the mice were injected to Ag/oMWCNTs (about 12.4 mg/kg.bw) with different dosages of DOPC/or TD . Mice in the control group were exposed to normal saline. After 2 h, all mice were subjected to CT imaging. The effects of varying dosages of DOPC/or TD on the biodistribution of oMWCNTs in mice were further investigated through improved CT imaging.Ag/oMWCNTs heterostructures were prepared through the reduction of Ag ions in the oMWCNTs dispersion solution by using a previously reported method43. If there are damages or toxicity in tissues, the relative biochemical indices (such as C-reaction protein (CRP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TB)) will change in blood compared with normal group mice45. The kidney plays an important role in drug excretion and detoxification. The most common used biochemical indices or proteins in blood are creatinine (CREA), blood urea nitrogen (BUN) or cystatin C (Cys-C), both assessing the functional status of the kidney and detecting specific damage46. And so, biochemical indices in the plasma were detected using ELISA Kit , reflecting the toxic effects of oMWCNTs on mice.The mice were randomly divided into four groups (five mice/group). Three groups of mice were injected intravenously with 6.2, 18.6 and 31 mg/kg.bw of the oMWCNTs solution. The control group was treated with normal saline. Finally, the experimental mice were sacrificed at 24 h after exposure to oMWCNTs. Blood was collected, stored for 10 min at room temperature and was centrifuged at 4000 rpm to obtain the plasma. As well know, the liver is the main tissue for metabolism and detoxification of drugs in body, the induced hepatotoxicity is one of important and most common cases after exposure oMWCNTs to miceThe following experimental processes were designed to study the prevention and treatment effects of DOPC and/or TD on the toxicity of oMWNCTs in mice:1. Treatment effects: The oMWCNTs-model mice were obtained by intravenously injecting mice with 12.4 mg/kg.bw oMWNCTs on the first day. The oMWCNTs-model mice were randomly divided into three groups (five mice/group). The grouped mice were intravenously injected with 12.4 mg/kg.bw DOPC/or TD and with normal saline solution on the second and the third days, respectively. All mice were sacrificed on the fourth day, and blood was collected and stored for about 10 min at room temperature. The collected blood was centrifuged at 4000 rpm to obtain the plasma, which was stored in the refrigerator at about −20 °C.2. Prevention effects: The normal mice were randomly categorised into three groups (about five mice/group). The mice were intravenously injected with 12.4 mg/kg.bw DOPC/or TD and normal saline on the first and second days, respectively. The mice were then exposed to 12.4 mg/kg.bw oMWCNTs on the third day. All mice were sacrificed on the fourth day. Blood was collected and stored for about 10 min at room temperature. The blood was then centrifuged at 4000 rpm to obtain the plasma, which was stored in the refrigerator at about −20 °C.3. Synergistic effects: Normal mice were randomly categorised into three groups (five mice/group) and were intravenously injected with DOPC + oMWCNTs [(12.4 + 12.4) mg/kg.bw], TD + oMWCNTs [(12.4 + 12.4) mg/kg.bw] and normal saline. All mice were sacrificed at one day after the injection. Blood was collected and stored for about 10 min at room temperature. The blood was centrifuged at 4000 rpm to obtain the plasma, which was stored in a refrigerator at about −20 °C.49, and the results were showed in Figure The concentrations of BUN, CREA, Cys-C, ALT, AST, TB and CRP in the plasma were obtained through the abovementioned processes and measured using an ELISA Kit. Mice from each group were dissected to collect heart, liver, spleen, lung and kidney tissues were collected. These tissues were fixed in 10% buffered formalin and processed for routine histology with haematoxylin and eosin staining by Shanghai Shunbai Biological Co., Ltd. The tissues were microscopically observed with an Olympus Microscope-CX41  coupled with a digital camera. The extent of tissue injury was estimated semi-quantitatively and lesions scored as multi-focal fibrosis/or necrosis according to relative reportsOn the first day, the mice were developed by intravenously injecting with 12.4 mg/kg.bw oMWCNTs as model. Then the mice were randomly categorised into five groups (five mice/group), and exposed intravenously to normal saline (as single oMWCNTs control group), 3.1, 6.2, 9.3 and 18.6 mg/kg.bw TD on the second and third days. Mice in the control group were treated with normal saline. All mice were sacrificed on the fourth day. Blood was collected, stored for about 10 min at room temperature and then centrifuged at 4000 rpm to obtain the plasma. The same biochemical indices of the plasma were determined using the above methods. In order to further prove the treatment effect of TD on the toxicity of oMWCNTs in mice, the serum cytokines levels of interleukin 6 (IL6), interleukin 10 (IL10) and tumor necrosis factor α (TNF-α) were carried out here. Briefly, two group mice (five to six mice/group) were exposed to 12.4 mg/kg.bw oMWCNTs solution intravenously at first day, then one group mice were exposed to 12.4 mg/kg.bw TD another one group mice were treated with normal saline at second and third days. In addition, the control group mice (five mice) were treated with normal saline at all the time. All mice were scarified on the fourth day. Blood was collected, stored for 24 h at 4 °C and then centrifuged at 4000 rpm to obtain serum. Then the cytokines of IL6, IL10 and TNF-α levels in serum were measured through ELISA Kit .51. The plasma and buffy coat were removed by aspiration. The separated erythrocytes were washed three times by centrifugation  in 10 volumes of saline solution. The supernatant and buffy coats of white cells were carefully removed with each wash. The washed erythrocytes were finally re-suspended to the desired hematocrit level using the same solution and stored at 4 °C. The effects of oMWCNTs, DOPC, TD, DOPC + oMWCNTs and TD + oMWCNTs (mass 1:1) on the morphology of erythrocytes were investigated by optical microscopy and scanning electron microscopy (SEM).The effects of DOPC/or TD on the toxicity of oMWCNTs to erythrocyte suspension were also studied. Fresh blood was obtained from normal mice and anticoagulated with heparin. Anticoagulated blood was centrifuged at 4000 rpm for 5 minThe erythrocyte suspension (10% hematocrit) was incubated with the solutions of oMWCNTs, DOPC, TD, DOPC + oMWCNTs and TD + oMWCNTs, respectively, and kept the content of exposure drugs about 0.5 g/L in the mixture solution. After 2 h, the reaction mixtures were dropped onto a glass slide and directly observed the morphology of erythrocytes affected under the presence of oMWCNTs, DOPC, TD, DOPC + oMWCNTs and TD + oMWCNTs through an optical microscopy. For SEM observations, the treated erythrocyte suspension was fixed with a glutaraldehyde solution (3%), centrifuged and washed with gradient ethanol. Finally, the samples were vacuum dried for use.In order to study further the effect of DOPC/or TD on the damage of oMWCNTs in mice, the four groups mice were exposed to 12.4 mg/kg.bw oMWCNTs with different dosages of 0, 6.2, 12.4 and 18.6 mg/kg.bw DOPC/or TD intravenously as experimental groups, and the control groups mice were injected same volume saline. All mice were sacrificed at 24 h after various treatments, and blood was collected to measure the levels of white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), red blood cell specific volume (HCT), blood platelet (PLT) and plateletcrit (PCT) through blood routine examination.Statistical significance in the difference was carried out by analysis of variance (ANOVA) or Kruskal-Wails-test method after One-sample Kplmogorov-Smirnov Npar-test and Homogeneity of Variances test, and all statistical calculations were evaluated by SPSS software, and data were showed as the mean values + SD of multiple determinations.Supplementary Information


Tribulus terrestris L. (TT) is an annual plant of the family Zygophyllaceae that has been used for generations to energize, vitalize, and improve sexual function and physical performance in men. The fruits and roots of TT have been used as a folk medicine for thousands of years in China, India, Sudan, and Pakistan. Numerous bioactive phytochemicals, such as saponins and flavonoids, have been isolated and identified from TT that are responsible alone or in combination for various pharmacological activities. This review provides a comprehensive overview of the traditional applications, phytochemistry, pharmacology and overuse of TT and provides evidence for better medicinal usage of TT. Tribulus, Hard thorns, and goat head in China. It is mainly planted in the Mediterranean and in sub-tropical regions such as India, China, South America, Mexico, Spain, Bulgaria, and Pakistan. It is a small, prostrate, 10–60 cm high, hirsute or silky hairy shrub. The leaves are opposite, often unequal, paripinnate, pinnate from 5 to 8 pairs and elliptical or an oblong lanceolate. The fruits from the five mericarps are ax-shaped, 3–6 mm long, and arranged radially and have a diameter of 7–12 mm and a hard texture. The root is slender, fibrous, cylindrical and frequently branched, bears a number of small rootlets and is light brown in colour , mating ability and fertility [Another animal study describes the protective role of TT against AlClertility .−1 day−1) restored metronidazole (MTZ)-induced spermatogenic inhibition and reduced the epididymal sperm count. The restoring potential of TT against MTZ-induced alterations in the spermatogenesis appears to be due to the presence of antioxidative flavonoids rather than steroidal saponins [TT was reported to cause reproductive system enhancement and possess antioxidant activity, which may assist in the choice of drugs for longer durations that can be prescribed safely without affecting the fertility potential in males. A high dose of the fruit extract of TT . Its probable mechanism of action involves an increase in the serum levels of free and bioavailable testosterone [HSDD is defined in Diagnostic and Statistical Manual of Fourth Edition as persistent or recurrent deficiency (or absence) of sexual fantasies/thoughts, and/or desire for or receptivity to sexual activity, which causes personal distress . TT was osterone .Other clinical research established that regarding the treatment in the domains of desire and sexual interest of 74 postmenopausal women with sexual dysfunction, the TT treatment  was considered to be effective in treating sexual problems among menopausal women .n-butanol fraction, due to higher contents of quercetin, diosgenin and tannic acid, has a protective capacity rather than a curative property against urolithiasis [The fruits of TT have long been used in traditional systems of medicine for the treatment of various urinary diseases including urolithiasis. Calcium oxalate is a major type of crystal found in kidney stones. Calcium oxalate is classified into two types: calcium oxalate monohydrate stones (COM) and calcium oxalate dihydrate (COD). Many medicinal plants have been used for centuries for the treatment of urinary stones in spite of the lack of rationale behind their use. The aqueous extract of TT fruits and its fractions were studied to evaluate its antiurolithiatic potential using different models. The inhibitory potency of the plant was tested on the nucleation and growth of the most commonly occurring kidney stones and COM. The results showed that the bioactive ithiasis .7) of Arabidopsis thaliana after matching peptide mass fingerprints with the MASCOT search engine. CCD7 belong to a family of dioxygenases, which possess five characteristic conserved histidines spread throughout their primary protein sequence. Histidine is said to induce the conversion of oxalate to formic acid and carbon dioxide (CO2). The purified protein decreased cell injury induced by oxalate in a concentration dependent manner and showed the ability to inhibit calcium oxalate (CaOx) crystallization in vitro [A protein (60 kDa) purified from TT showed the highest similarity with carotenoid cleavage dioxygenase 7  showed inhibitory activity against α-glucosidase. In addition, it showed the inhibition activities of a postprandial increase in blood glucose and improvement in insulin dependent diabetes symptoms [T. terrestris (WETT) has an antidiabetic activity. The fasting blood glucose, 2-h postprandial glucose, glycosylated haemoglobin and lipid profile of diabetic women treated with TT extract (1000 mg day−1) for three months were lowered compared to those of the placebo group [Diabetes mellitus is a metabolic disorder with chronic hyperglycaemia, which results from a defect in insulin secretion, insulin action, or both . The grosymptoms . Animal symptoms . Clinicabo group .α and interleukin (IL)-6, increased SOD and the rate of apoptosis, and improved the structure of cardiomyocytes in rats [T. terrestris (METT) fruits, which mainly contains ferulic acid, phloridzin and diosgenin, had an effect on mitochondrial dysfunction in a cell-based (H9c2) myocardial ischaemia model. The extract guards the mitochondria via its antioxidant potential [2O2) induced by angiotensin II, significantly inhibits the increase in intracellular free Ca2+ induced by H2O2, significantly inhibits the increase in phospho-ERK1/2 induced by angiotensin II, and significantly inhibits the increase in the mRNA expressions of c-fos, c-jun and pkc-α induced by angiotensin II [Presently, the clinical treatments are thrombolysis and nerve protection. Thrombolysis has a significant effect. However, it is limited by a narrow therapeutic time window. Therefore, the development of neuroprotective agents is of great significance. Studies have shown that GSTT has a neuroprotective effect on cerebral ischaemia injury, and these saponins have been commercially available as active compounds in traditional Chinese medicine formulations, such as “Xin-nao-shutong”, which has been used for the treatment of cardiovascular disease . Meanwhi in rats . Moreove in rats . The metotential . The celensin II .α and Socs3. It also decreased the mRNA expressions of Akt1, AMPKα1, JAK2, LepR and STAT3 induced by ox-LDL. The most notable changes were for JAK2, LepR, PI3Kα, Socs3 and STAT3. It is thought that the JAK2/STAT3 and/or PI3K/AKT pathway might be a very important pathway that is involved in the mechanism of TT as a vascular protective agent [TT significantly suppressed the proliferation of ox-LDL-induced human umbilical vein endothelial cells (HUVECs) and the apoptosis rate. It also prolonged the HUVEC survival time and postponed the cells’ decaying stage (from the 69 h to over 100 h). TT normalized the increased mRNA expressions of PI3Kve agent .In addition, TT functions as a protector of the myocardium. It supported cardioprotective properties against myocardial ischaemia, protected myocardial cells and reduced the apoptosis rate induced by oxidative stress damage . Inhibitα and IL-1β. It plays a neuroprotective role in rat cerebral ischaemia reperfusion injury by inhibiting the inflammatory response and PPARγ protein expression [2O2. The membrane potential of mitochondria and Bcl-2 protein expression in PC12 cells of the GSTT group was significantly increased in a dosage-dependent manner [TT has a protective effect for neuron injury mainly via its anti-inflammatory and antioxidant effects. GSTT has a neuroprotective effect on cerebral ischaemia–reperfusion injury in rats by suppressing NF-κB, TNF-pression . GSTT det manner . After ct manner .The apoptosis of retinal ganglion cells (RGCs) is an important cause of glaucoma. TT can block the optic nerve injury pathway and enhance the survival of the optic nerve to protect the optic nerve , 83. It Athletic fatigue is generally measured by the levels of testosterone and corticosterone, and the testosterone and corticosterone (T/C) ratio. Herbs and herbal combinations have been used to improve athletic ability through several ways that mimic epinephrine effects, mimic testosterone effects, and increase the productions of corticotropin and cortisol. TT contains gitonin, protodioscin, and tribulosaponins A and B, which are believed to mimic testosterone-like effects in humans because of the similarities of their chemical structures , 86. The−1) can prolong the time to exhaustion and increase body mass, relative mass, and protein levels of gastrocnemius in overtrained rats. The level of testosterone can directly affect the motor ability of the body and its restoration. Corticosterone can accelerate the decomposition of proteins in the body [The administration of GSTT  assay [−1) was rich in saponin and had notable quenching of nitric oxide (90.30%), hydroxyl radicals (90.02%), and hydrogen peroxide radicals (89%) [T. terrestris was found to have great antioxidant activity [TT exhibited effective antioxidant activity in a concentration-dependent manner by 2,2-di-(4-tert-octylphenol)-1-picrylhydrazyl (DPPH), Hr) assay . The expr) assay . Comparels (89%) . Diosgenactivity .N-trans-ρ-caffeoyl tyramine isolated from TT had marked anti-inflammatory activities [N-trans-ρ-caffeoyl tyramine inhibited the productions of nitric oxide (NO), TNF-α, IL-6 and IL-10 in lipopolysaccharide (LPS) stimulated RAW264.7 cells in a dose dependent manner. In addition, N-trans-ρ-caffeoyl tyramine markedly suppressed the expression of cycloxygense (COX)-2 and the production of prostaglandinE2 (PGE2) through decreasing p-JNK expression.The EETT and tivities . EETT an−1) showed a dose-dependent inhibition of rat paw volume in a carrageenan-induced rat paw edema model. The TT extract and diclofenac sodium (a COX-inhibitor) were injected 30 min prior to carrageenan. The results showed that both drugs can reduce the paw volume 1–4 h after injection of carrageenan by inhibiting the releases of histamine, serotonin and kinins in the early phase. Furthermore, the anti-inflammatory effect of 400 mg kg−1 of TT extract is equivalent to that of 20 mg kg−1 of Diclofenac sodium [METT and causing death with lower doses. The effects were comparable with that of Albendazole . In addie leaves .Streptococcus mutans is an important oral pathogen that causes dental caries. The anticarious effect of TT was evaluated for inhibiting S. mutans bacteria. In vitro studies showed that the extract exhibited antibacterial activity for inhibiting S. mutans growth in a dose dependent manner. Meanwhile, TT extract suppressed the adherence of S. mutans to saliva-coated hydroxyapatite (S-HA), which simulated teeth, and inhibited the formation of water-insoluble glucans [TT was certified to have an anticarious effect.  glucans .d-galactose-induced senile mice. Compared with the ageing model group, the GSTT group showed a thicker dermis and more compactly arranged fibre content. The skin morphology of the GSTT group was close to that of the normal group [GSTT can effectively increase SOD activity, decrease MDA and hydroxyproline (Hyp) in the skin and increase the activities of CAT and GPx in the whole blood of al group .Ageing is accompanied by a decline in memory, but GSTT can improve memory impairment. A study showed that GSTT significantly improved obtained memory disorder, consolidated memory disorder and recovered memory disorder . The effTT promotes absorption. The biopharmaceutics classification system (BCS) is a scientific classification method based on solubility in vitro and permeability of drugs in the intestine. Metformin hydrochloride (HCl) is a BCS class III drug with a high solubility and poor absorption characteristics. Therefore, it is necessary to increase the intestinal permeability of drugs to improve their bioavailability. The experiment indicated that TT can enhance the absorption of Metformin HCl in a goat intestine . The abs−1, given orally to 5 mice for 14 days). The methanol extracts mainly consisted of flavonoids, anthraquinones, phenols/tannins, and steroids/triterpenes. As a result, there were no toxic symptoms or mortality observed in any animals and no obvious differences between the treated and control animals regarding behavioural changes and toxicological signs  [An animal study investigated the acute toxicity of METT (2 g kgcrement) .Salmonella typhimurium strains, was evaluated [−1 METT, tDNA%: 11.43) and cytotoxic activities (IC50 = 160 mg mL−1) than WETT and the chloroform extracts of T. terrestris (CETT) but did not damage the deoxyribonucleic acid (DNA), whereas the 300 mg mL−1 WETT might induce frame shift mutations when metabolically activated. WETT showed oestrogenic activity at concentrations higher than 27 mg mL−1 (2.6-fold), and none of the extracts had androgenic activity.The genotoxic potential of TT extracts, as assessed by a Comet assay in a rat kidney cell line and by an Ames assay in valuated . The METThe traditional pharmacological activities of TT focused on improving sexual function and cardiotonic properties. Modern investigation showed that steroidal saponins and flavonoids with the prominent antiaging and anti-inflammatory activities were the main contributors to the traditional pharmacological activities. While the clinical trials with TT are scarce, and randomized placebo controlled clinical trials should be done in future. In addition, we should give more attention to the traditional curative effect of skin pruritus. TT maybe have more utilization as cosmetic plant materials on skin. A critical assessment of the results presented in this review may provide scientific evidence for the reasonable utilization of TT and promote further investigation for the development of new herbal medicine and health products.