We augmented these with two additional cases of secondary CSF escape related to neuroborreliosis and HSV-2 encephalitis and analyzed these two non-HZ cases for factors contributing to CSF HIV-1 RNA concentrations. HIV-1 CSF escape and discordance were associated with higher CSF white blood cell (WBC) counts than their non-escape (P = 0.0087) and non-discordant (P = 0.0017) counterparts, and the CSF WBC counts correlated with the CSF HIV-1 RNA levels in both the treated (P = 0.0047) and untreated (P = 0.002) group pairs. Moreover, the CSF WBC counts correlated with the CSF:plasma HIV-1 RNA ratios of the entire group of 27 subjects (P = <0.0001) indicating a strong effect of the CSF WBC count on the relation of the CSF to plasma HIV-1 RNA concentrations across the entire sample set. The inflammatory response to HZ and its augmenting effect on CSF HIV-1 RNA was found up to 5 months after the HZ outbreak in the cross-sectional sample and, was present for one year after HZ in one individual followed longitudinally. We suggest that HZ provides a ‘model’ of secondary CSF escape and discordance. Likely, the inflammatory response to HZ pathology provoked local HIV-1 production by enhanced trafficking or activation of HIV-1-infected CD4+ T lymphocytes. Whereas treatment and other systemic factors determined the plasma HIV-1 RNA concentrations, in this setting the CSF WBC counts established the relation of the CSF HIV-1 RNA levels to this plasma set-point.HIV cerebrospinal fluid (CSF) escape is defined by a concentration of HIV-1 RNA in CSF above the lower limit of quantification of the employed assay and equal to or greater than the plasma HIV-1 RNA level in the presence of treatment-related plasma viral suppression, while CSF discordance is similarly defined by equal or higher CSF than plasma HIV-1 RNA in untreated individuals. During secondary CSF escape or discordance, disproportionate CSF HIV-1 RNA develops in relation to another infection in addition to HIV-1. We performed a retrospective review of people living with HIV receiving clinical care at Sahlgrenska Infectious Diseases Clinic in Gothenburg, Sweden who developed uncomplicated herpes zoster (HZ) and underwent a research lumbar puncture (LP) within the ensuing 150 days. Based on treatment status and the relationship between CSF and plasma HIV-1 RNA concentrations, they were divided into 4 groups:  Hiv infects various compartments in the body including the central nervous system (CNS). Thus it is important to be certain that antiretroviral treatment is CNS effective to avoid risk for cognitive disorders.HIV-1 RNA is detected in the cerebrospinal fluid (CSF) throughout the course of untreated systemic infection, beginning early after initial exposure  and contneurosymptomatic CSF escape based on the clinical presentation of neurological deficits in the presence of detectable CSF HIV-1 RNA despite systemic viral suppression[However, there are notable exceptions to this effective and proportionate effect of ART on CSF HIV-1 infection that have been collectively designated CSF escape in which the concentration of HIV-1 in CSF remains above the LLoQ and equal to or greater than that in plasma in the face of therapeutic plasma virus suppression –17. Thisppression, 17, 30.ppression. It at lppression, 32, andppression, 34, neuppression. In thesppression, 20, 29.ppression, 20, 27.asymptomatic CSF escape, has also been documented in which CSF HIV-1 RNA is increased despite plasma suppression, but without clinical evidence of neurological injury. Asymptomatic CSF escape is more common than the neurosymptomatic form and seemingly largely benign [A second type of CSF escape, designated y benign . Its repy benign , 37. In y benign , 36. Unuy benign . Since tsecondary CSF escape, in which another nervous system infection appears to provoke, or at least associate with, elevation of the CSF HIV-1 RNA concentration despite plasma viral suppression, presumably as a consequence of the host local inflammatory responses to the inciting non-HIV-1 pathogen[The current report deals with a third type of CSF HIV-1 escape, termed  pathogen.https://aidsinfo.nih.gov/guidelines/html/4/adult-and-adolescent-opportunistic-infection/392/whats-new-in-the-guidelines).While it occurs in those without HIV-1 infection, herpes zoster ((HZ) is more common in PLWH in which it is more frequent in those with depressed blood CD4+ counts , but alsi.e., in individuals with CSF escape and CSF discordance.Having noted some cases of secondary escape in individuals with HZ in our HIV-1 cohort study and in the course of HIV-1 clinical care in Gothenburg, we undertook a review of all the cases of HZ in HIV-1-infected individuals who underwent lumbar puncture (LP) in the longitudinal Gothenburg HIV CSF Study , 49 withThe prominent inflammatory aspect of HZ provides a relatively common setting to examine the effects of focal inflammation within the neuraxis on CSF HIV-1 concentrations, and we therefore undertook a review of our experience with HZ in PLWH who underwent LP and CSF analysis.This was a retrospective study drawn from the records between 1996–2018 of the Gothenburg Infectious Diseases Clinic, searched for HIV-1 infection, HZ rash and LP. All records were reviewed by one of us (LH), and those with an LP within 150 days of the onset of the dermatomal rash were included. All LPs were performed within the Gothenburg HIV CSF Study Cohort protocol, approved by the institutional IRB and with oral and written informed consent . The identified individuals with HZ were classified by treatment status and the relation of CSF to plasma HIV-1 RNA concentrations into four groups using the following definitions:Secondary CSF escape. a) on chronic stable and consistent ART treatment (at least 8 months of therapy); b) plasma HIV RNA <500 copies/mL; c) CSF HIV-1 RNA concentrations above the clinical laboratory LLoQ of 20 copies/mL; and d) CSF ≥ plasma HIV-1 RNA.On therapy no escape. as in a) above; but b) CSF HIV-1 RNA below the laboratory LLoQ, or CSF < plasma HIV-1 RNA.CSF discordance. a) untreated (naïve or off ART >8 months); and b) CSF ≥ plasma HIV-1 RNA.No CSF discordance. untreated as in a) above; but b) with CSF < plasma HIV-1 RNA.10 values: log10CSF HIV-1 RNA–log10plasma HIV-1 RNA.Cross-sectional analysis included data from the first LP after the onset of HZ. CSF and plasma HIV-1 RNA were measured in all subjects using the Roche Cobas Taqman assay with the LLoQ of 20 copies/mL; a value of 19 copies/mL was assigned as the default value for any levels below 20 in calculations and descriptive statistics. The CSF:plasma ratio was calculated using log1 for lumbosacral dermatome, 2 for thoracic, 3 for cervical and 4 for cranial nerve. Selected cases in which CSF HIV-1 measurements were available before and after the HZ episode were reviewed for longitudinal findings.ART regimen at the times of HZ outbreak and CSF sampling were available for all subjects. CSF white blood cell (WBC) counts, concentrations of CSF and blood neopterin and CSF neurofilament light chain protein (NfL) were measured for most using previously reported methods , 51. TheFor perspective on the findings in the HZ experience, We also examined a sample of neuroasymptomatic PLWH without a history of recent HZ from our Gothenburg HIV cohort. These were randomly chosenmatched according whether on or off antiretroviral therapy, including two subjects in each category for each one in the HZ groups, incjluding: 32 neuroasymtomatic PLWH naïve to treatment were compared with the 18 HZ PLWH off of antiviral therapy and 18 PWLH on therapy without HZ were compared to the 9 HZ co-infected PLWH on therapy.Additionally, to supplement the experience with secondary escape, in selected analyses we included two additional individuals with secondary CSF escape related to other infections in some of the analyses: one with neuroborreliosis  and an uncommon case of HSV-2 encephalitis documented by CSF PCR. These two individuals were the only non-HZ PLWH with secondary escape identified in the data base.10 transformed; in the case of CSF WBCs, when the initial laboratory value was zero, we substituted a value of one for analysis and graphing of the transformed data.Descriptive statistics, comparisons of subgroups and analysis of correlations among variables generally used nonparametric methods, with linear regressions and t-test or Fisher’s exact tests performed in selected analyses. These along with graphic presentations were all performed using Prism 8.3 (GraphPad Software). For some analyses, data were logWe identified 25 HIV-1-infected individuals who underwent LP within 150 days after the onset of HZ rash: 9 on and 16 off ART . These w10 copies per mL, a modest but distinct level of CSF escape from therapy, particularly in comparison to the fully suppressed CSF levels in the no escape group. As expected, the plasma HIV-1 RNA concentrations were higher in the two untreated groups (mean of 4.69 log10 copies per mL in the discordant and 5.14 log10 copies per mL in nondiscordant) but with the CSF distinctly higher than its companion plasma in the discordant samples while the plasma HIV-1 RNA was higher than that of the CSF in the nondiscordant group. This figure not only shows the relationships between CSF and plasma HIV-1 RNA that conform to their group definitions but emphasizes the distinct differences among these four groups that serves as the basis for comparing their other characteristics.In order to better understand the reversal of CSF:plasma HIV-1 RNA ratios by HZ in some, but not all, of the identified individuals, we examined a number of other variables, including the CSF WBC count, CSF neopterin concentration, blood CD4+ T-lymphocyte counts, CSF NfL concentration, time between the HZ rash outbreak and CSF sampling, and distance between the HZ outbreak and the site of LP .The CSF WBC counts differed in the escape (including the two non-HZ cases) and discordant groups with CSF ≥ plasma HIV-1 RNA concentrations compared to their non-escape and nondiscordant counterparts , so that10 transformations  and untreated (discordant and non-discordant) groups using linear regression after logrmations . This sh10 difference between CSF and plasma HIV-1 RNA) across the entire sample set as shown in i.e., ≥1) at a mean CSF WBC count of about 16 cells per μL where the regression crossed the line of no difference between CSF and plasma HIV-1 RNA depicted by the horizontal dotted line in Because of this separation between the two sample pairs, we next examined the correlation of the CSF WBC counts with the ratios of CSF to plasma HIV-1 RNA (expressed as the log2 = 0.231). Thus, CSF neopterin measurements supported the association of inflammation with the CSF relation to plasma HIV-1 RNA, but were not as clear a predictor as the CSF WBC count, though the CSF neopterin and WBC counts, themselves, were strongly correlated .The Gothenburg cohort study included measurement of CSF neopterin at most LP visits. THis is a sensitive measure of enhanced immune activation-inflammation in the CNS during HIV-1 infection . In thisThe blood CD4+ T-cell counts of the HZ patients were mostly above the levels predisposing to opportunistic infections, with a median near 400 cells/μL in the treated groups and 350 cells/μL in the discordant group off ART . They weThe Gothenburg cohort study also measured CSF NfL at most visits. Because HZ can elevate the CSF NfL presumably as a result of axonal injury of nerve roots and perhaps at times spinal cord , we reasCSF and blood samples from one of the discordant subjects with HZ (SID #5348 in the Table) were obtained at several study visits before and after the index HZ LP included in the cross-sectional analysis . He deve6/L, and the comparison group (n = 50) 44,7 years and 412 cellsx106/L. The HZ patients on treatment had significantly higher number of individuals with HIV CSF virus detected and number of CSF cells > 5x106/l (p<0.01 Fischer exact test) than controls on treatment which confirms that HZ is associated to secondary escape. Furthermore, the HZ patients off treatment had significantly higher CSF viral count (log 4.55) than controls (log 3.35) (p<0.01 t-test) but did not differ significantly in plasma viral load (log 4.92 copies compared with log 4.55 copies) (p = 0.3 t-test). When comparing the discordant group with the non-discordant group statistical analyses showed that the CSF virus load was the cause to drive the quotient to discordance (p<0.01 t-test).The mean age and CD4 cells of the HZ study population (n = 25) was 43.9 years and 343 cellsx10This retrospective analysis indicates that HZ was associated with an increase in local HIV-1 production within the CSF compartment and strongly suggests that it was mediated through the local inflammatory response to VZV reactivation, and particularly by the lymphocytic pleocytosis.–This secondary CSF escape and CSF discordance during HZ infection, may be an important mechanistic component, and have implications for other types of CSF escape. Both CSF escape and discordance are characterized by a ‘disproportionate’ CSF HIV-1 RNA concentration compared to that of plasma in the treatment and non-treatment settings, respectively. These relationships of CSF to plasma HIV-1 are unusual as indicated by the comparison with the comparison group with lower CSF WBC counts and CSF RNA levels than their counterparts with HZ infection. In the absence of treatment CSF HIV-1 RNA levels are usually about 10-fold or more lower than those in plasma, while ART that effectively suppresses systemic viremia is usually similarly effective in suppressing CSF HIV-1 RNA , 13; eveOur findings establish secondary escape as a third type of CSF escape joining the previously characterized neurosymptomatic and asymptomatic clinical forms of CSF escape discussed earlier , 20, 36.Our case series was small, but overall more than two-thirds of our PLWH and HZ exhibited pleocytosis (defined as a CSF WBC counts >5 cells/μL), a higher frequency than the nearly 50% reported in non-HIV patients . In our Common to both the treated and untreated groups, there was a relationship of the CSF WBC count to the CSF viral load and to the ratio of CSF to plasma HIV-1 RNA concentrations. Both treated and untreated groups showed a threshold between 10–30 cells/μL at which this ratio transitioned to higher CSF than plasma HIV-1 RNA concentrations that defined CSF escape or discordance depending on treatment status. The impact of the CSF WBC count on CSF HIV-1 infection in both treated and untreated individuals was most evident in relation to the CSF:plasma HIV-1 RNA ratio in the entire group of samples as depicted in HZ is an inflammatory disorder in those without and with HIV-1 infection that begins in the dorsal root ganglion, spreads centrifugally along the peripheral nerve, and seeds the skin of the related dermatome, leading to the characteristic rash. Infection also spreads centripetally to the nerve root and, to a variable degree, beyond, into the spinal cord or brain stem –60. It aOur leading hypothesis in explaining the development of CSF escape and discordance in this setting is that HZ provokes local inflammation along this pathway that includes CD4+ T-cells, an important component in VZV defense –63. SomeOur efforts to define the influence of the timing of the HZ rash on the CSF HIV-1 RNA concentration and its relation to that of plasma did not provide a clear definition of this factor, though the numbers were small and the timing of LPs inconsistent. However, the aggregate cross-sectional experience with escape and discordance together with the longitudinal observations show that the effect of HZ on CSF HIV-1 RNA, in parallel with the inflammatory reaction, is protracted, lasting up to the 5 month limit of our study definition and beyond one year in the longitudinal example in The two non-HZ escape cases included in this analysis exhibited features that align with the four individuals with HZ-related secondary escape, but also included some differences. Most notably, they shared the association of the CSF HIV-1 RNA concentrations with the WBC counts, enough to fit into the linear correlation between these two key variables . HoweverHZ provides a relatively common and useful model of secondary escape. Even in the presence of suppressive ART its incidence is higher in PLWH than in the general population, providing an opportunity to extend these retrospective observations. Secondary escape in HZ at least partially resembles asymptomatic escape and raises the question of whether HZ itself, with or without rash , or othea priori design to carefully define the frequency or time course of the effect of HZ on local HIV-1 infection, but rather was determined mainly by the protocol-specified schedule and non-protocol clinical decisions. This almost random timing in relation to the HZ episode had the advantage of showing the prolonged period of pleocytosis and CSF escape in some subjects but did not allow consistent assessment of the time course of this escape as would a study specifically focused on this issue. The longitudinal observations before and after HZ in some individuals were useful in providing interesting background to the impact of ART and its disruption by HZ, but only afforded a preliminary view of the time course of HZ effects. The two non-HZ cases appear to support the findings in HZ but may involve some different mechanisms despite their seeming similarity, so their integration into more unifying conclusions needs to be cautious. In the untreated individuals the zoster episode led to initiation of ART, and consequently follow-up and natural history without HIV-1 treatment was not available nor was follow-up information on CSF after treatment . On the other hand, these untreated individuals exhibiting parallel relations of CSF WBCs to CSF HIV-1 RNA provide an important and provocative perspective on secondary escape related to their likely shared mechanisms.Our study had several limitations. The number of individuals in each of the four HZ groups was relatively small, particularly in the two treated groups. It was also a retrospective study of a single cohort, though this particular cohort study had the good fortune to include protocol LPs and CSF analyses of HIV-1 RNA, and therefore afforded a valuable opportunity to examine this issue. The timing of LPs after HZ was not set by This study defines secondary CSF escape as a coherent entity and HZ as a relatively common and useful model of it. The study also shows some of the parallel features of CSF escape and discordance in treated and untreated individuals, respectively, that likely indicate similar pathogenetic mechanisms, particularly the interaction of reactive inflammation and CSF HIV-1 RNA levels. The results suggest that the robust local or regional inflammatory response to VZV gives rise to secondary HIV-1 production that is reflected in CSF. They also demonstrate that secondary escape is an entity worthy of further study, including virological and immunological dissection that may contribute to a broader understanding of some of the features of a contributing source of CSF HIV-1 infection.


In heart transplantation, donor hearts inevitably suffer from ischemia/reperfusion (I/R) injury, which leads to primary graft dysfunction and affects patients’ survival rate. Bone marrow mesenchymal stem cells (BMSCs) have been reported to attenuate myocardial I/R injury via their paracrine effects, which can be enhanced by hypoxic preconditioning. We hypothesized that the donor heart preservation with hypoxic conditioned medium (CdM) derived from BMSCs would improve post-transplant graft function.2) or hypoxic (1% O2) condition. Donor hearts were explanted; stored in cardioplegic solution supplemented with either a medium (vehicle), normoxic CdM (N-CdM), or hypoxic CdM (H-CdM); and then heterotopically transplanted. Antibody arrays were performed to compare the differences between hypoxic and normoxic CdM.Normoxic or hypoxic CdM were isolated from rat BMSCs cultured under normoxic  and cardiac troponin I in the N-CdM group compared with the vehicle group. These therapeutic effects can be further enhanced by hypoxic preconditioning. Antibody arrays revealed that nine proteins were significantly increased in hypoxic CdM compared with normoxic CdM. Furthermore, compared with vehicle and N-CdM groups, the protein levels of PI3K and p-Akt/Akt ratio in the transplanted hearts significantly increased in the H-CdM group. However, no significant difference was found in the phosphorylation of Smad2 and Smad3 for the donor hearts among the three groups.Our results indicate that the cardioplegic solution-enriched with hypoxic CdM can be a novel and promising preservation solution for donor hearts. Heart transplantation remains a well-established therapy of choice for patients with refractory heart failure . DespitePrevious studies have shown that ischemia/reperfusion (I/R) injury plays a significant role in PGD , therebyThe administration of bone marrow-derived mesenchymal stem cells (BMSCs) is emerging as a promising approach for I/R injury-induced myocardial infarction due to its multiple differentiation potential , 8. Howe2 BMSCs) added to a preservation solution could improve in vivo left ventricular (LV) graft dysfunction after heart transplantation and that these cardioprotective effects could be enhanced by hypoxic preconditioning (1% O2 BMSCs) in the well-established heterotopic heart transplantation model [Therefore, in the current study, we hypothesized that CdM-BMSCs under normoxic conditions  received care in compliance with the Guide for the Care and Use of Laboratory Animals . All procedures and handling of animals during the investigations were reviewed and approved by the Ethical Committee of Laboratory Animal Research Center of Southern Medical University Nanfang Hospital. The animals were housed at constant ambient temperature (22 ± 2 °C) in light-controlled rooms (12–12-h light-dark cycles), were given food and water access ad libitum, and acclimatized for 1 week.Rats were randomly assigned to three groups. The donor hearts were arrested and stored for 1 h in either cold Custodiol  supplemented with a serum-free medium vehicle (α-MEM) , or Custodiol supplemented with normoxic CdM-BMSCs , or Custodiol supplemented with hypoxic CdM-BMSCs . Then, the donor hearts were heterotopically transplanted.2 on culture flasks. The primary culture was subcultured at a ratio of 1:2 when 80% confluency was reached. Only the third passage was used in subsequent experiments.As previously reported , BMSCs w2 and 20% O2. The hypoxic group was cultured at 37 °C in 1% O2, 5% CO2, and 94% N2 in a hypoxic incubator . The primary CdM was collected after incubation for 24 h, and the cell debris was removed using a syringe filter . The primary CdM-BMSCs was then sequentially concentrated to 15-fold by the centrifugation through Amicon Ultra Centrifugal Filter  according to the manufacturer’s protocol. Finally, the novel donor heart preservation solution was produced by diluting 300 ul α-MEM (vehicle group), concentrated normoxic (N-CdM group), or hypoxic CdM-BMSCs (H-CdM group) from the filtrate tube of the top unit in 2700-ul Custodiol cardioplegic solution as indicated in Fig. Normoxic and hypoxic CdM-BMSCs were acquired as described previously with slight modifications . A simplorg.Hs.eg.db” and “clusterProfiler”) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (R package “clusterProfiler”) were performed using the open-source program R (version 3.5.1) to investigate the biological process categories and pathways of proteins that were highly expressed between hypoxic and normoxic CdM-BMSCs, as previously described [The cytokines/chemokines of isolated CdM-BMSCs were measured by using a G-series rat cytokine array 67  according to the manufacturer’s instructions. The Gene Ontology (GO) analysis  and embedded in paraffin. Five-micron-thick slices of myocardium were stained with hematoxylin and eosin. The pathology changes of each heart slice were evaluated by scoring according to the grades 0–4 : (0) NilTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to detect DNA-strand breaks of the donor hearts after functional measurements was performed as previously described . The numBlood samples from the recipient rats were obtained at 1.5 h after reperfusion just before the sacrifice of rats. Blood samples were drawn from the abdominal aorta. Then, plasma samples were obtained after centrifugation . The levels of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6) and cardiac troponin I (cTnI) were measured via Rat ELISA kit .Myocardial protein expression was assessed by Western blot as previously described . The ratt test was applied to analyze the difference between N-CdM and H-CdM groups. If the normality test failed, a nonparametric Mann-Whitney test was used. One-way ANOVA followed by Tukey’s post hoc test was performed for multiple comparisons between three experimental groups. If the data failed the normality test, the nonparametric Kruskal-Wallis test followed by Dunn’s post hoc test was used. A value of p < 0.05 was considered statistically significant.The results were expressed as mean ± standard error of the mean (SEM). GraphPad Prism 7.02 software  was used to perform statistical analysis. Shapiro-Wilk test was performed to test the normality of data before statistical tests were applied. For data with normal distribution, a two-sample Student’s Time to return of spontaneous contraction of the donor hearts immediately after successful heart transplantation was significantly decreased in the N-CdM and H-CdM groups compared to the vehicle group Fig. . BesidesOne transplanted graft in the vehicle and H-CdM group could not be included in the heart function analysis since part of the intestine covered the transplanted heart, making the evaluation of heart function by echocardiography very difficult and challenging. After heart transplantation, significantly increased left ventricular EF and FS were observed in N-CdM and H-CdM groups compared to the vehicle group Fig. a, b. MorSignificant improved myocardial recovery in N-CdM and H-CdM groups compared with the vehicle group was noted with regards to the increased LVIDs Fig. c. FurtheMoreover, significantly increased SV and CO were observed in the H-CdM group compared to N-CdM and vehicle groups Fig. e, f. WhiIn general, the preservation of the donor hearts with Custodiol solution supplemented with normoxic CdM-BMSCs appeared to protect cardiac contractility in the donor hearts subjected to acute I/R injury, and hypoxic CdM-BMSCs added in the preservation solution could further enhance this cardioprotective effect for grafts Fig. h.After heart transplantation, histological evaluation of hematoxylin and eosin-stained myocardial tissue demonstrated more inflammatory cell infiltration, irregularly arranged, and disordered-structure myocardial fibers in the vehicle group when compared with N-CdM and H-CdM groups Fig. a, b. HypAfter heart transplantation, a significantly increased number of TUNEL-positive nuclei in the graft were observed in the vehicle group compared with N-CdM and H-CdM groups Fig. . BesidesThe levels of proinflammatory cytokine plasma IL-1β, IL-6, and TNF-α measured at 1.5 h after reperfusion were significantly lower in the N-CdM and H-CdM groups compared with the vehicle group Fig. a–c. The p = 0.041), cytokine-induced neutrophil chemoattractant-2 (CINC-2) , platelet-derived growth factor-AA (PDGF-AA) , neuropilin-2 , Activin A , and beta-nerve growth factor (b-NGF)  showed the largest differences in expression.An antibody-based protein array (67 rat cytokines) analysis was performed to investigate the component of BMSC secretome see the . The scrp value = 0.00032)  Fig. b.Fig. 9Our KEGG analysis demonstrated that PI3K/Akt signaling might participate in the protective effect of hypoxic CdM-BMSCs against graft dysfunction associated with I/R injury in heart transplantation Fig. b. TherefSince Decorin  and Activin A (a member of TGF-β superfamily), which were involved in the Smad2/3 activation (phosphorylation) , 32, sigIn the present study, we investigated the role of Custodiol-supplemented with normoxic and hypoxic CdM-BMSCs as a novel preservation solution to attenuate I/R injury-induced graft dysfunction in a rat heart transplantation model. The novelty of our work is the comparison of the cardioprotective effect of normoxic and hypoxic CdM-BMSCs on the donor heart preservation. In the present study, the application of Custodiol-supplemented with normoxic CdM-BMSCs in the cold storage of the donor hearts was associated with a shorter time to return of spontaneous contraction, higher post-transplant cardiac contractility, and lower histopathological scores of the transplanted hearts. The cardioprotective effects might be associated with the inhibition of apoptosis and inflammation, as reflected by less TUNEL-positive cells and lower levels of plasma proinflammatory cytokines  and cardiac troponin I in the N-CdM group compared with the vehicle group. Additionally, these beneficial cardioprotective effects are significantly enhanced after hypoxic preconditioning.In heart transplantation, the donor hearts inevitably suffer from cold ischemia and warm reperfusion injury, which can result in PGD for transplanted hearts. I/R injury is a critical pathological process influencing graft clinical outcomes  due to iRecently, an increasing number of studies have demonstrated that BMSCs can secrete a vast array of paracrine factors, including cytokines, microRNA, growth factors, antioxidants, proteasomes, and exosomes , which aRecent studies have shown that hypoxic preconditioning can intensify the paracrine effects of BMSCs, thereby promoting BMSCs to secrete more nutritional bioactive factors, such as VEGF, HGF, and Activin A , 18. UntIn the present study, the cardioprotective effect of the donor hearts from CdM-BMSCs could be enhanced by hypoxic preconditioning. Compared with normoxic CdM-BMSCs, the application of Custodiol-supplemented with hypoxic CdM-BMSCs in the cold storage of the donor hearts before heart transplantation was associated with a shorter time to return of spontaneous contraction, lower histopathological scores, higher post-transplant cardiac contractility, a lower rate of DNA-breaks and DNA fragmentation in the myocardium of donor hearts, and a lower level of plasma proinflammatory cytokines  and indices for myocardial injury (cTnI). In line with the above findings, recent studies have shown hypoxic CdM-BMSCs can be used to treat retinal ischemia  and ischThe highly secreted soluble factors in hypoxic CdM-BMSCs may account for improved post-transplant cardiac function through the inhibition of apoptosis and inflammation. The antibody array in the present study indicated that hypoxic preconditioning resulted in an increased expression of nine cytokines. Among these identified cytokines, VEGF  and ActiIn the present study, GO term enrichment analysis showed that the biological process of increased proteins in hypoxic CdM-BMSCs included the positive regulation of ERK1/2 cascade, positive regulation of vascular endothelial growth factor signaling pathway, and positive regulation of protein autophosphorylation. Interestingly, previous studies reported that the activation of ERK1/2 cascade  and vascInterestingly, no significant difference was found in the phosphorylation of Smad2 and Smad3 for the donor hearts among the three groups. Although our antibody array showed that H-CdM contained a higher level of Decorin and Activin A compared with N-CdM, Decorin  and ActiThere are several limitations to our present studies. Firstly, although our rat model of heterotopic heart transplantation has been introduced as a suitable model to study global myocardial I/R injury, the LV unloading can lead to donor heart atrophy, thrombus formation in LV cavities, and faster myocardial recovery after I/R injury, which may not fully represent the real world of transplanted hearts. Secondly, the dose-response relationship between both normoxic and hypoxic CdM-BMSCs and beneficial effects for the donor heart preservation remains unclear. Thirdly, the present study was unable to identify precisely the specific cytokines or signaling pathways that account for the therapeutic effects of hypoxic CdM-BMSCs for cold storage of the donor hearts. To fully understand the precise mechanisms, future investigations can be performed by overexpressing or inhibiting some of the specific cytokines in hypoxic CdM-BMSCs. Fourthly, the impact of longer cold ischemia time for the donor hearts and the effect of CdM-BMSCs on right ventricle structure and function were not evaluated.Despite these limitations, the current study demonstrated promising potential for the hypoxic CdM-BMSC-based therapeutic strategy against myocardial I/R injury in heart transplantation. From a clinical perspective, preconditioning heart preservation solution to mitigate myocardial I/R injury during heart transplantation can reduce the incidence of PGD for donor hearts, thereby improving short- and long-term graft function and recipient’s survival. Thus, one of the most promising and novel alternatives may be the supplementation of heart preservation solution with secretome derived from BMSCs under either normoxic or hypoxic conditions. Compared with living BMSCs, CdM-BMSCs can provide some advantages, including fewer complications for the donor hearts , convenience for storage, and transportation. However, before its successful clinical translation, some key issues should be solved, including the verification of optimal concentration and cardioprotective effect of hypoxic CdM-BMSCs on the donor hearts preservation in a big animal model, the standardization of BMSC cultivation and hypoxic preconditioning BMSCs, isolation, storage, and transportation of CdM-BMSCs in the clinical setting.Taken together, the present in vivo study reveals that the preservation of the donor hearts with cardioplegic solution supplemented with hypoxic CdM-BMSCs significantly improves post-transplant cardiac graft contractility compared with normoxic CdM-BMSCs in a rat model of heterotopic heart transplantation via the inhibition of apoptosis and inflammation. Furthermore, hypoxic-preconditioning BMSCs confers the ability to upregulate some cytokines which are associated with various potential molecular mechanisms underlying hypoxic CdM-BMSC-mediated protection against myocardial I/R injury for the donor hearts. In summary, our results indicate that the cardioplegic solution-enriched with hypoxic CdM-BMSCs can be a novel and promising preservation solution for the donor hearts.Additional file 1. : Table 1. The secreted cytokine levels between hypoxic and normoxic CdM-BMSCs measured by antibody array


P < 0.01). Through histological observation at different phases, the distinctive pathological changes of the spinal cord included early inflammatory reaction, adhesion and blockage in the subarachnoid space and the central canal, perivascular space enlargement, central canal expansion, which suggested the CSF flow being blocked by multiple ways after kaolin-induction. In conclusion, experimental scoliosis can be successfully induced by intraspinal kaolin injection. In this model, continuous hypodynamic change of CSF flow was correlated to the formation of scoliosis, which could be an important factor of scoliotic pathogenesis being explored furtherly.Scoliosis is often associated with syringomyelia (SM). As an important role in SM formation, the influence from abnormal cerebrospinal fluid (CSF) flow is still unclear to scoliosis. The aim of this experimental work is to explore the connection between CSF flow and scoliosis through imaging and histological analysis on the basis of a kaolin-induced scoliotic rabbit model. For imaging observation, in 40 kaolin-induced rabbits by C7 spinal cord injection, through pre- and postoperative MRI and radiography, CSF flow and scoliosis formation were detected at consecutive phases. According to the final formation of scoliosis until postoperative week 12, the kaolin-induced rabbits were divided into 2 groups. Through comparing the 2 groups, the relationship between the changes of CSF flow velocity and scoliosis formation were reviewed and analyzed. For histological observation, another 20 kaolin-induced rabbits were used for consecutive histological observations of spinal cord at postoperative 3-day, 2-week, 4-week and 6-week. After kaolin-induction, abnormal spinal coronal curve was observed from postoperative week 6 in the 37 survived rabbits. At postoperative week 12, scoliosis formation was detected in 73.0% kaolin-induced rabbits and the mean Cobb angle was 27.4°. From the comparison between scoliotic and non-scoliotic groups, the difference of the velocities of CSF flow was more obviously from postoperative week 4 to 12, especially after week 6. In the scoliotic group, the peak velocity of CSF flow was diseased gradually following scoliosis formation after induction. Moreover, the decrease of the peak velocities of CSF flow from preoperation to postoperative 12 weeks (ΔVmax), including up-flow (ΔVUmax) and down-flow (ΔVDmax), were positively correlated to the final scoliotic Cobb angle ( Meanwhile, SM also can be detected by magnetic resonance imaging (MRI) in 19.5% infantile, 26% juvenile and 20% adolescent patients ever being diagnosed as idiopathic scoliosis (IS)2. Moreover, in the patients with severe scoliosis (Cobb angle more than 90°), the rate of associated-SM is even up to 60.4%3. However, the relationship between scoliosis and SM is still obscure, which has been explained as a causal link through the “paraspinal muscle denervation” theory: the pressure from an asymmetrically expanded syrinx may cause the disturbances in innervation of the trunk musculature and thus causing the imbalance that leads to resultant scoliosis7. However, this hypothesis could not explain several clinical phenomena reasonably, such as the inconsistence between the magnitude of scoliosis and the size of syrinx, the direction of curve and the eccentricity of syrinx in the spinal cord, the site of syrinx and the apex of scoliosis, etc.10. Additionally, primary neurosurgical decompression can reduce syrinx and improve neurological function, but could fail to stabilize the associated scoliosis13. Therefore, the relationship between scoliosis and SM should not be believed as a simple causal link.Scoliosis is often associated with syringomyelia (SM) in clinical, which can be found in 25–85% of the patients being diagnosed as SM16. As a crucial reason of SM, the hypodynamic change of cerebrospinal fluid (CSF) flow has been studied furtherly20. In the pathological process of SM-associated scoliosis, CSF flow hypodynamic change plays an important role in SM formation, but makes unclear effects to associated scoliosis.SM is a pathological state of the spinal cord as a result of trauma, malformation etc.Based on that consideration, in this study, on the basis of a rabbit scoliotic model being induced by injecting kaolin into spinal cord at the level of seventh cervical vertebra (C7), the following hydrodynamic changes of CSF flow in the process of coronal curve development and formation were recorded and analyzed consecutively by Phase Contrast–Cine MRI (PC-cine MRI) scanning. According to the final formation of scoliosis until postoperative week 12, the kaolin-induced rabbits were divided into scoliosis (group S) and non-scoliosis (group Non-S). Then in order to further understand the role of CSF flow in scoliosis formation, through comparison between the 2 groups, the relationship between the changes of CSF flow velocity and scoliosis formation were analyzed. Additionally, combined with histological observation at several consecutive phases after kaolin-induction, the postoperative extramedullary and intramendullary pathological changes of the spinal cord were observed for understanding how the modeling method to affect the CSF flow.In the 40 kaolin-inducted rabbits for imaging detections, 37 survived more than 12 weeks after operation and were included in the final experimental analysis. In the 3 died rabbits: 1 was related to the surgical process and possibly from mass blood loss, 2 was from the postanesthetic hypothermia. None occurred incision infection. Most kaolin-induced rabbits exhibited signs of lethargy and anorexia. None was dead in the shame animals.In the 37 survived kaolin-induced rabbits, before the week 6 after surgery, even minor curve of spine was found in none. But from the week 6 to 12, scoliosis formation was detected in 73.0% (27/37) of the experimental rabbits and the final Cobb angle was up to 27.4 ± 10.0° (15–59°) .Meanwhile, syrinx also started to be observed at postoperative week 6, and MRI scanning showed syrinx occurrence mainly in the cervical-thoracic segments. Syrinx was found in 70.3% 26/37) of the experimental rabbits at postoperative week 12 . Meanwhile, at C7 level, similar but more obvious differences were detected through the comparison between groups S and Non-S   and C7 levels were increased more obviously from week 4 to 12 after kaolin-induction, especially at postoperative week 6. The hydrodynamic changes of CSF flow at C0 and C7 levels were similar. At C0 level, to postoperative 12 weeks, the peak velocities of CSF flow (Vmax), including up-flow (VUmax) and down-flow (VDmax) of group S were much slower than group Non-S  and 4.2 ± 1.0 cm/sec (P ≤ 0.001) obviously, and the VUmaxs were also decreased to 4.4 ± 1.2 cm/sec (P ≤ 0.001) and 4.6 ± 0.9 cm/sec (P ≤ 0.001). Meanwhile, the spinal curve was observed initially from week 6, then the cobb angle of scoliosis was increased to 27.4 ± 10° at week 12   and ∆VUmax (the decrease of VUmax from preoperative status to postoperative 12 weeks). At C0 and C7 levels, both ∆VDmax and ∆VUmax were positively correlated to the final scoliotic Cobb angle (01) Fig. .Figure 5In the 25 rabbits for histological observation, 3 rabbits died: 2 was related to the mass blood loss in surgical process, 1 was to unclear reason.Through the hematoxylin-eosin (HE) staining of spinal cord tissue being harvested at postoperative 3-day, 2-week, 4-week and 6-week after kaolin-induction, a series consecutive pathological changes of the spinal cord were observed, which was characterized by early inflammatory reaction, adhesion and blockage in the subarachnoid space and the central canal, perivascular space (Virchow-Robin space) enlargement, ependymal cell shedding, central canal expansion Figs. . Moreove21. Then a series of experimental SM models induced by kaolin injection into spinal cord and subarachnoid space have been reported29. Inflammatory reaction, apoptosis and secondary obstruction of the channels of CSF circulation are the main sources of using kaolin to induce SM models, which are also the important pathological basis of clinical SM occurrence. Especially, the formation of SM is critically related to the hypodynamic changes of CSF caused by several reasons.Kaolin-induction is widely used to build SM animal model in different experimental researches. McLaurin and associates firstly described using kaolin injection into the cisterna magna consistently resulted in the development of chronic obstructive hydrocephalus in experimental animals30. After intramedullary kaolin injection, whether or not SM occurred, scoliosis would occur in a significant proportion of experimental animals. This was an interesting and meaningful phenomenon, which hinted that there seemed to be a connection between scoliosis and SM, but not a simple causal relationship. In order to further study the specific relationship between the change of CSF hydrodynamics and scoliosis formation, a modified kaolin injection process was used to induce experimental scoliosis in present study for improving the rate of scoliotic formation. After kaolin being injected into subarachnoid space and spinal cord layer by layer, at postoperative 6 weeks, spinal curve occurrence on the coronal plane was observed in 32.4% rabbits. To 12 weeks after kaolin-induction, the rate of scoliosis formation was up to 73.0%. Moreover, through MRI scanning, syrinx also started to be observed at postoperative week 6, which was mainly in the cervical-thoracic segments. Therefore, postoperative 6-week was an important phase, which represented the start point of scoliosis and syrinx after kaolin-induction. The period in the postoperative 6 weeks can be believed as the pre-scoliosis and pre-syrinx states, which would be very helpful in researches of this pathological process. Additionally, the final rate of syrinx formation was 70.3% at postoperative week 12. As an important part of pathological outcomes in the cord, syrinx observation outcomes by MR imaging was used to show there was no different of syrinx formation rates between the animals with and without scoliosis , which demonstrated there were similar influences to syrinx formation from kaolin-induced modeling approach no matter spinal coronal curve formation or not, which did not support there was a potential causal connection between scoliosis formation and SM.In our early exploration of inducing SM by intramedullary injecting kaolin into spinal cord, we had ever noticed thoracic scoliosis occurrence in some experimental rabbits even without any spinal hydromyelia or syrinx formation31. For the scoliotic patients with syringomyelia, neurosurgical intervention such as posterior fossa decompression may prevent curve progression. The aim of neurosurgical decompression in these patients were to restore hydrodynamics of CSF flow, and the stop of scoliotic progression or even improved also hints there is some link between CSF hydrodynamics and scoliosis formation33. Recently, results from a study from Grimes et al., which was based on ptk7 mutant zebrafish with defects in ependymal cell cilia development and CSF flow, indicated the irregularities in CSF flow could be an underlying biological cause of idiopathic scoliosis34. In this study, through the comparison between the groups S and Non-S, the differences of the velocities of CSF flow became more obvious from postoperative week 4 to 12 after kaolin-induction, including at C0 and C7 levels, especially after postoperative 6 weeks. The hypodynamic changes of CSF flow at C0 and C7 levels were similar. In the group S, at the week 12, being compared with the preoperative state, the VDmax and VUmax at the level of C0 and C7 were decreased obviously. Meanwhile, the spinal curve was observed initially from the postoperative week 6 with obvious increase to the week 12. Moreover, from preoperation to postoperative 12 weeks, the changes of VDmax and VUmax positively correlated to the final scoliotic Cobb angle. Our results indicated there was a close relationship between the slow-down of CSF flow and scoliosis formation in the kaolin-induced rabbit model. The hypodynamic changes of CSF flow could play an important role in the process of scoliosis formation.In the previous clinical study, we had reported the use of electrocardiogram-synchronized cardiac-gated PC-cine MRI to study the differences between the patients of SM & CM-I malformation with and without scoliosis and found the worse dynamics of CSF flow in the patients with scoliosisMoreover, to focus on the postoperative 6-week after kaolin-induction as an important period including pre-scoliosis and pre-syrinx states, consecutive histological observations were also performed in this study. The histological changes was characterized by early inflammatory reaction, adhesion and blockage in the subarachnoid space and the central canal, perivascular space (Virchow-Robin space) enlargement, ependymal cell shedding, central canal expansion, etc. Combining with the significant changes of CSF flow at 4–6 weeks after kaolin induction in the previous part of this study, the histological changes indicated that the hydrodynamic change to CSF flow could not only be derived from the subarachnoid inflammatory adhesion and blockage by kaolin crystal itself, but also from intramedullary lesions. The involved pathological processes could be speculated as follows: kaolin-induced subarachnoid inflammation caused the normal CSF flow to be blocked, which in turn caused increased subarachnoid pressure; meanwhile, the intramedullary inflammatory response could lead to the opening of the perivascular space as well as the CSF with increased pressure entering into the central canal through it. Additionally, the kaolin crystals and intramedullary inflammatory reaction also could cause the central canal to be blocked, which would increase the accumulation of CSF in the central canal and the final formation of syrinx. Therefore, this animal scoliotic modeling method intervened the normal CSF flow by multiple ways, especially the obstruction of the subarachnoid space and the central canal. Moreover, the following hypodynamic change of CSF flow is prior to the formation of scoliosis, which suppled a powerful evidence of the consecutive hypodynamic change of CSF flow could be an important potential cause of scoliosis formation.There are some limitations of the current study. In this study, we could not find a reliable approach to quantitatively analyze whether there were histological differences in the cord and subarachnoid space between the animals with and without scoliosis. Although this study primarily established the correlation between hypodynamic change of CSF flow and scoliosis formation, the specific relationship between them was still obscure. The different degree of cord damage and arachnoiditis could result in lower CSF flow velocities and scoliosis development. In that case, the possibility of lower CSF flow velocities and scoliosis development would be two symptoms of a more severe cord damage and/or arachnoiditis, which could not be excluded in this study. The aim of this study was to introduce hypodynamic CSF flow as a possible factor of soloistic pathogenesis, more evidences and researches are needed to explain how the hydrodynamic change of CSF flow affects the structure of spine as well as the following scoliosis occurrence and progression. The process maybe through intervertebral discs, paravertebral muscles, intraspinal structures or other factors, which still needs further researches in future. And these researches would be helpful to reveal the potential mechanisms of scoliosis formation and to explore future novel treatments.In summary, scoliosis can be induced in most of the rabbits by kaolin injection into cervical spinal cord and subarachnoid space. This kaolin-induced scoliosis model could be a good choice for the pathogenesis exploration of idiopathic scoliosis. In this animal model, postoperative 6-week is a start of scoliosis after kaolin-induction, and continuous hypodynamic change of CSF flow was correlated to the formation of scoliosis, which could be an important factor of scoliotic pathogenesis being explored furtherly.This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, following ethical approval from the Animal Care and Ethics Committee of the Kunming Medical University (# SYDW20080125001). A total of 75 Japanese white rabbits  were included in this study, aged 2–3 months old and weighting 1.8–2.5 kg. Each animal was kept in an individual cage and had free access to food and water. The room temperature range was 20–28 °C, with a relative humidity of 35–60% and a 12 hours light-dark cycle. In the 75 rabbits, 25 were used for histological observation and 50 were for imaging observation . The skin was shaved and prepared with povidone iodine. Then animals were placed prone on a self-made U-shape frame to raise the cervicothoracic junction for avoiding the interrupt from the scapula to facilitate next steps. A posterior median longitudinal incision (about 2 cm length) was made from C7 to the first thoracic vertebra (T1). Then the C7 spinous process was cut off, and at its basis, a tiny part of the lamina and ligamentum flavum were resected for exposing the dura. An 100 microliter glass microinjector with a 28-gauge needle  was held by a stereotaxic apparatus  to be used for the dura puncture. The site of puncture was near the middle line of the dura but avoiding the dorsal vessels, the depth was 1.0–2.0 mm under the dura. Then 0.1 ml of 25% kaolin  was injected from the subarachnoid space to the center of the spinal cord layer by layer  using cervical and spinal coils. PC-cine MRI scans were performed preoperatively and 4, 6, 8, 10, 12 weeks postoperatively.The methodology of PC-cine MRI observation was based on our previous studyTen minutes before MRI scanning, the general anesthesia was induced by intramuscular injection of 3% pentobarbital sodium  through the gluteus maximus at a dose of 30 mg/kg. MRI scanning was started at a prone position with steady heart rate and respiration. Sagittal and axial T1-weighted, T2-weighted imaging sequences of the brain and cervical spinal cord were collected. Synchronized cardiac gating was used to divide the cardiac cycle into 23 images. CSF flow studies were performed at the levels of C0 and C7 in the axial plane with following sequence: TR 1800 msec/TE 93 msec, flip angle 180°, slice thickness 4 mm, matrix 256 × 196, and encoding velocity 20 cm/sec.The acquired phase-contrast images were transferred to Argus software  for CSF flow analysis. Regions of interest (ROIs) were selected in the axial plane: 4 ROIs were evenly placed in the anterior and posterior subarachnoid spaces at C0 and C7 levels. The peak velocities of CSF flow, including VUmax and VDmax were measured and averaging values at the 4 different ROIs were recorded at different phases Fig. .Figure 9According to the occurrence of scoliosis at the end of week 12 after kaolin-induction, the kaolin-induced rabbits were divided into two groups: scoliosis (group S) and non-scoliosis groups (group Non-S). On the basis of the grouping, the changes of CSF flow velocity were reviewed and analyzed being combined with/without scoliosis formation: 1. what was the role of the CSF flow velocity in curve formation? 2. what the relationship between the change of CSF flow velocity and the curve progression?30. Therefore, in order to study structural changes in the spinal cord after kaolin induction, the rest 20 kaolin-inducted rabbits and 5 normal ones were sacrificed at different phases in postoperative 6 weeks, including postoperative 3-day, 2-week, 4-week and 6-week .All numerical data are presented as expressed as mean ± standard error (S.E.M). Independent sample t test was used to test the differences of the velocities of CSF flow between groups S and Non-S, and the changes of the central canal area at postoperative different phases. Correlation studies were done using Pearson Correlation test when appropriate. Differences between two groups were assessed by independent The experimental protocol for this study was approved by the Animal Care and Ethics Committee of the Kunming Medical University (# SYDW20080125001). The approval was obtained before the start of the experiments. A licensed breeder had bred all animals for the sole purpose of being used in animal experiments. All efforts were made to minimalize animal suffering.


Approval of nusinersen, an intrathecally administered antisense oligonucleotide, for the treatment of 5q-spinal muscular atrophy (SMA) marked the beginning of a new therapeutic era in neurological diseases. Changes in routine cerebrospinal fluid (CSF) parameters under nusinersen have only recently been described in adult SMA patients. We aimed to explore these findings in a real-world setting and to identify clinical and procedure-associated features that might impact CSF parameters. Routinely collected CSF parameters , oligoclonal bands) of 28 adult SMA patients were examined for up to 22 months of nusinersen treatment. Total protein and QAlbumin values significantly increased in the first 10 months, independent of the administration procedure. By month 14, no further increases were detected. Two patients developed transient pleocytosis. In two cases, positive oligoclonal bands were found in the beginning and in four patients throughout the whole observation period. No clinical signs of inflammatory central nervous system disease were apparent. Our data confirm elevated CSF total protein and QAlbumin during nusinersen treatment. These alterations may be caused by both repeated lumbar punctures and the interval between procedures rather than by the medication itself. Generally, there were no severe alterations of CSF routine parameters. These results further underline the safety of nusinersen therapy. SMN)1 gene, resulting in degeneration of anterior horn cells in the spinal cord and motor nuclei of the lower brainstem. The almost identical SMN2 gene, which is commonly present in several copies, differs only in a single nucleotide. This change causes the disruption of an exonic splice enhancer in exon 7, and therefore, the gene encodes only low levels of the functional SMN protein [SMN2 at the pre-mRNA level and thereby increase the production of the functional, full-length SMN protein [With U.S. Food and Drug Administration (FDA) approval of the antisense oligonucleotide (ASO) nusinersen, the first disease-modifying treatment for 5q-spinal muscular atrophy (SMA) has become available . SMA is  protein . ASOs ar protein . Nusiner protein . A major protein . In a co protein . In a la protein . Besides protein . A poten protein . A slowd protein .This study was approved by the local ethics committee of the Hannover Medical School . All participants gave written informed consent to participate in the study and publication of pseudonymous cohort data before study enrollment.SMN2 copy number were collected at baseline. The route of nusinersen administration was either by conventional LP or computed tomography (CT)-guided LP. In this study, only CT-guided LP instead of fluoroscopy-guided LP was performed due to better tolerability and feasibility [In this prospective, single-center, observational study, CSF and serum samples of patients with confirmed 5q-associated SMA treated with nusinersen at the Department of Neurology at the Hannover Medical School were collected between November 2017 and December 2019. Clinical parameters and demographics such as age, disease duration, history of scoliosis and spondylodesis, SMA type (2–4), and sibility . The motsibility  and the sibility . The RUL−3). Intrathecal immunoglobulin synthesis for IgG, IgA, and IgM was measured regarding Reiber’s revised hyperbolic function referring IgG, IgA, and IgM quotients to QAlbumin [All CSF and serum samples were collected before nusinersen administration and were further analyzed within one hour after LP. Determination of the CSF leukocyte count and protein analysis of CSF and corresponding serum samples were performed, as previously described . BrieflyQAlbumin . OCB werQAlbumin . OCB typQAlbumin .n = 1, S16) and SMA patients with CSF–blood barrier dysfunction at baseline  and compared CSF total protein and QAlbumin values by using the Wilcoxon signed-rank test. We used the Mann–Whitney U-test to examine the influence of CT-guided LP at every time point. We analyzed associations between CSF routine parameters and clinical parameters  by using Spearman’s correlation coefficient (rho). For investigating the associations between SMA type, LP method, SMN2 copy number, side effects, and CSF routine parameters, the point-biserial correlation coefficient (rPB) was used. To investigate the association between sex, LP technique, OCB status, and procedure-related side effects, the chi-square test was used. The phi-coefficient (phi) was used to describe the effect size. We applied a linear mixed model with restricted maximum likelihood analysis, with treatment time points d0, d14, d63, m6, m10, m14, and m18 as repeated measurements. With either protein concentration or QAlbumin as a dependable variable, we controlled other factors such as age, gender, or LP method, which may be confounding in univariate analysis. Additionally, estimated marginal means were calculated for the treatment time point, gender, and LP method. For post hoc multiple comparisons, p-values were corrected by Bonferroni’s procedure. For all statistical tests, a two-tailed significance level of p < 0.05 was considered statistically significant. Because of the explorative character of this study, all statistical test results were interpreted as hypothesis generating and not as confirmatory. No adjustment for multiple testing was done. Missing data of CSF routine parameters or incomplete analysis of any participant’s CSF were not taken into account for this calculation.All statistical analyses were performed using either IBM SPSS Statistics version 26  or GraphPad Prism 7.0 . According to the Shapiro–Wilk test, values for the CSF cell count, CSF total protein, QAlbumin, and OCB were not normally distributed. Therefore, the Wilcoxon signed-rank test was used for comparison of CSF values within the SMA patient group and at every treatment time point. Additionally, we stepwise excluded SMA patients with outlying CSF protein parameters  with a median age of 36 years (range 19–65 years) were enrolled in this study. The one SMA type 4 patient was further analyzed together with SMA type 3 patients. The majority of patients were male (64.3%). Almost every second patient (46.4%) underwent CT-guided LP. The reason for CT-guided LP in the majority of cases was scoliosis (84.6%). In two patients, CT-guided LP was performed after conventional LP had failed beforehand. In seven SMA patients who underwent CT-guided LP for nusinersen treatment, transforaminal injections were necessary at least once during the treatment course. The median scores for the HFMSE and RULM at d0 were 9.5 (range 0–63) and 18.5 (range 0–37), respectively. Ambulatory patients reached 390 meters in the 6MWT in the median (range 0–512 m). It took on average 32.5 years (range 2–62.5 years) from symptom onset to initiation of nusinersen therapy. Two patients discontinued the therapy after 6 and 14 months, respectively. No side effects were reported in 58% of all conducted LP (n = 179). Reports on side effects were missing for three LPs. Reported side effects were mainly (80.2%) classified as procedure related.Demographic data of participants are summarized in n = 28), d14 (n = 28), d28 (n = 26), d63 (n = 27), m6 (n = 24), m10 (n = 22), m14 (n = 15), m18 (n = 7), and m22 (n = 2). In total, 179 CSF samples of 28 patients were collected. Data were missing for leukocyte count (n = 3 samples), lactate (n = 33), total protein (n = 33 samples), and QAlbumin (n = 34 samples) due to various reasons . After blood correction by excluding all CSF samples with an erythrocyte count above 2500 cells/µL, 161 CSF leukocyte count values, 130 CSF lactate values, 136 CSF total protein values, and 135 QAlbumin values were analyzed. Since data for m22 were only available for two patients, this time point was not included in the calculation. CSF/serum quotients of IgG, IgA, and IgM were not analyzed in this study, because intrathecal immunoglobulin synthesis was found only in one SMA patient. In CSF routine parameters of SMA patients were available for baseline  was found in a 36-year-old SMA type 2 female patient (S11) at d14, i.e., two weeks after the first nusinersen administration. The LP was conducted conventionally. No intrathecal immunoglobulin synthesis according to the revised hyperbolic function by Reiber or CSF-specific OCB was found. Subsequently, the leukocyte count was <5/µL during further treatment (d28 to m22). In the CSF of a 32-year-old SMA type 3 male patient (S22), an elevated cell count was found at d14 (33/µL) and d28 (12/µL). The LP was conducted via CT guidance by the standard interlaminar approach. At d0, the CSF leukocyte count was normal (1/µL). At m10, CSF pleocytosis was no longer detectable. However, intrathecal IgA synthesis was found at m10. At every time point (d0 to m10), weakly positive OCB were found in this patient.In the second of the two patients with positive OCB at baseline, a 51-year-old SMA type 2 female patient (S02), OCB were also positive at both d0 and d14 to m6 as well as m14 to m18. Only at m10, no positive OCB were found. No intrathecal immunoglobulin synthesis  according to Reiber graphs and increased leukocyte count were present at any time point.In two patients without CSF-specific OCB at baseline, a 45-year-old SMA type 3 male patient (S04) and a 39-year-old SMA type 3 female patient (S09), positive OCB were found at a single time point during further treatment  but no longer in subsequent CSF samples.p = 0.004), d0 and d28 (p = 0.024), d0 and d63 (p = 0.015), and d0 and m14 (p = 0.038). Summarizing OCB results, in 2 of 24 tested SMA patients, positive OCB were found at baseline. Throughout therapy, four SMA patients tested positive for OCB.In general, the leukocyte count in the CSF ranged from 1 to 5/µL in 24 of 26 patients. Despite mainly being within the normal range, an increase in the leukocyte count was observed between d0 and the following time points, with the exception of m18, reaching statistical significance between d0 and d14 . Other CSF parameters  were within the normal range. The remaining 18 patients had CSF protein levels within the reference range (211–477 mg/L). While there was no significant difference in total protein after the first injection of nusinersen, from d28 onward, significant changes were found . After m14, the increase persisted but was no longer significant. Fifteen patients (53.6%) were at least once above the upper reference level of 500 mg/L during therapy. After the first and second administration of nusinersen, no significant changes in QAlbumin were detected. We found significant changes in QAlbumin between d0 and d63 , between d0 and m6 , and between d0 and m10 . Similar to changes in total protein, no further significant changes in QAlbumin were found from m14 onward. According to age-dependent adjustments for QAlbumin, 12 patients (42.9%) displayed CSF–blood barrier dysfunction at least once throughout therapy. Because of the very high CSF total protein values and corresponding QAlbumin values, we repeated the statistical analysis after excluding the above-mentioned SMA type 3 patient (S16). We then still found a significant difference in total protein values between d0 and d28 (p = 0.033). QAlbumin values first differed significantly between d0 and d63 (p = 0.001). A significant difference in the total protein and QAlbumin values was found until m6 . After excluding all SMA patients with a baseline dysfunctional CSF–blood barrier according to age-adjusted QAlbumin values , we still detected a significant difference in both total protein (p = 0.008) and QAlbumin (p = 0.026) between d0 and d28. This significant difference remained present until m10  and became insignificant thereafter. All 22 available lactate values at baseline were within the normal range between 1.26 and 1.96 mmol/L. During the treatment course, lactate levels were mainly stable but showed significant differences between d0 and m6 (p = 0.024), d0 and m10 (p = 0.034), and d0 and m18 (p = 0.028). At baseline, seven SMA patients (28%) showed total protein values in the CSF above the upper reference level of 500 mg/L (range 502–588 mg/L), and six (25%) were found with a disturbed blood–CSF barrier function, five of those with an elevation of total protein. The patient with the highest total CSF protein levels in our study was a 57-year-old SMA type 3 male patient (S16). Total protein levels in this patient ranged from 583 mg/L (d0) to 907 mg/L (m14), rising from baseline to 817 mg/L at d63 and decreasing slightly to 742 mg/L at m6 and 624 mg/L at m10. From baseline on, QAlbumin values of this patient indicated CSF–blood barrier dysfunction at all time points (d0: 8.74 × 10p = 0.016) and m14 . Lactate levels ranged below the upper level of normal in both groups at both time points .In 13 patients, CT-guided lumbar puncture was necessary due to scoliosis and previous spinal surgery. Lactate, CSF total protein, and QAlbumin values did not differ significantly in patients undergoing CT-guided LP compared to those undergoing conventional LP at baseline. At all following injections, there was no significant difference between the CSF leukocyte count, CSF total protein levels, and QAlbumin values in patients who underwent CT-guided lumbar puncture . Only lactate levels significantly differed between patients with conventional versus CT-guided LP at m10  as well as between total protein levels and age . During the observation period (d0 to m18), there was no significant correlation between QAlbumin or total protein levels and the HFMSE, RULM, and 6MWT but a correlation of age with QAlbumin  and total protein levels . We found a significant association between QAlbumin values and total protein levels and SMA type , sex , SMN2 copy number , and modality of LP . Significantly higher total protein levels and higher QAlbumin values were found in SMA type 3 patients, in male patients, in patients with SMN2 copy number ≥3, and in patients who underwent conventional lumbar puncture. QAlbumin values and total protein levels significantly correlated with procedure-related side effects . Patients with lower CSF total protein levels or QAlbumin values experienced more procedure-related side effects. Overall, procedure-related side effects did not correlate with age, OCB status, and LP technique. However, a weak but significant association between sex and side effects was found: women reported procedure-related side effects in 50% of conducted LPs compared to 25.3% in men . The linear mixed model analysis confirmed our former approach of correlating QAlbumin values with the LP method  and age , where we observed an additive effect . All post hoc evaluations were abandoned due to the lack of sufficient data density to perform the estimated marginal means.No significant correlations between QAlbumin values at baseline (d0) and HFMSE, RULM, and 6MWT results were found. CSF total protein levels at baseline (d0) did not correlate with the HFMSE, RULM, and 6MWT. There was a positive correlation between QAlbumin at baseline (d0) and age  to confirm recently published changes in CSF routine parameters in a representative cohort of adult nusinersen-treated SMA patients, (2) to assess a potential procedure-based effect caused by repeated LPs on CSF routine parameter changes under nusinersen treatment, and (3) to determine whether nusinersen is well tolerated according to CSF routine parameters in SMA patients with borderline-pathogenic CSF metrics at baseline. Overviewing a treatment period of a maximum of 18 months, we observed a significant elevation in CSF total protein levels and respective QAlbumin values in 38.9% of cases during the first 10 months of treatment, mainly without significant changes in lactate levels, leukocyte count, and OCB status, which is consistent with previously published data . ElevateSMN2 copy number, sex, and LP method were identified. An explanation is that CSF total protein levels and QAlbumin levels are age dependent and increase over time [SMN2 copies [SMN2 copy number consequently displayed higher total protein levels and QAlbumin values. Patients who underwent conventional LP (higher number of SMA 3 patients) were older than patients who underwent CT-guided LP . Therefore, we suppose that the patients’ age mainly explains different findings for total protein levels and QAlbumin values, as previously described by Wurster et al. [No clinical parameters influencing CSF protein parameters in SMA patients during the treatment course were identified in a previous study . Consistver time ,29. SMA 2 copies , patientr et al. . Multivar et al. . Since rr et al. . Therefor et al. , but no Our results contain important information for the new field of gene-specific therapies. Ongoing studies on ASO treatment for genetic variants of ALS ,34,35 wiAt baseline, in 28.6% of SMA patients, at least one abnormal CSF parameter was detected . In a previously published study, positive OCB were found in 3.9% and CSF–blood barrier disorder in 24.6% of nusinersen-treated SMA patients at baseline . In our This study confirms the previous findings on mild changes in CSF routine parameters in adult SMA patients under nusinersen treatment. Our results confirm the notion that intrathecal administration of nusinersen in adult SMA patients is generally safe and well tolerated. Conspicuous pre-treatment CSF findings such as OCB had no impact on the tolerability of nusinersen. Considering ongoing studies assessing intrathecal administration of ASOs in other neurological diseases, it is important to further investigate and monitor CSF routine parameters.


R2 ≥ 0.9942, and 0.4–7 µM for CSF, R2 ≥ 0.9949). Moreover, MEPS significantly reduced the matrix effect of serum compared to liquid–liquid extraction (LLE), which was revealed in the example of reducing the amount of cholesterol and its relative compounds.Indole-containing acids—tryptophan metabolites—found in serum and cerebrospinal fluid (CSF) samples of patients with diseases of the central nervous system (CNS) were determined with the use of microextraction by packed sorbent (MEPS) followed by silylation and gas chromatography–mass spectrometry (GC–MS) analysis. MEPS with the following silylation led to the reproducible formation of derivatives with an unsubstituted hydrogen ion in the indole ring, the chromatographic peaks of which are symmetric and can be used for GC–MS analysis without additional derivatization. The recoveries of analytes at the limit of quantitation (LOQ) levels were 40–80% for pooled CSF and 40–60% for serum. The limit of detection (LOD) and LOQ values were 0.2–0.4 and 0.4–0.5 µM, respectively, for both CSF and serum. The precision  value of less than 20% and the accuracy  value of less than ±20% at the LOQ concentrations meet the Food and Drug Administration (FDA) recommendations. Linear correlations for all analytes were determined over a potentially clinically significant range of concentrations (0.4–10 µM for serum,  The study of the metabolism of aromatic α-amino acids in the human body has been of scientific interest since the middle of the last century. Transformations of tyrosine, phenylalanine and tryptophan have both normal pathways in a healthy body and alternative pathways associated with various pathologies ,2,3.p-hydroxyphenyllactic acids) are ubiquitous among healthy people and are detected in their serum in low concentrations. The concentrations of these compounds, as well as the appearance of other metabolites , became diagnostically important, indicating the possible development of severe infectious complications + and [M-CH2COOSi(CH3)3]+ ions, respectively. Peaks with m/z = 261, m/z = 143 and m/z = 144 in the mass spectrum of the mono-TMS derivative of 3IPA correspond to the molecular radical cation, [M-HCOOSi(CH3)3]+ radical cation and the [M-COOSi(CH3)3]+ ion, respectively. Peaks with m/z = 233 and m/z = 218 in mass spectrum of mono-TMS derivative of 3ICA correspond to the molecular radical cation and [M-CH3]+ ion, respectively 3]+ ions are characteristic; for 3ICA—[M-CH3]+ ions, for 3IPA—[M-CH2COOSi(CH3)3]+ ions, for 3ILA—[M-CH(OSi(CH3)3)-COOSi(CH3)3]+. It confirms that the recorded mass spectra absent from the NIST mass spectral library correspond to the incompletely substituted TMS derivatives of 3ICA, 3IPA and 3ILA. The peak forms of the incompletely substituted derivatives of indolic acids are symmetric and can be used in quantitative analysis.Fragmentations of both completely and incompletely substituted derivatives proceed according to similar schemes . For exaThe selection of derivatization conditions allowed us to obtain only incompletely substituted derivatives of indolic acids using BSTFA . The results were reproducible and the relative signals  of the TMS derivatives of indolic acids were the following: 3IAA 0.7 ± 0.2, 3ICA 0.35 ± 0.03, 3IPA 1.3 ± 0.1, 3ILA 0.8 ± 0.2, 5HIAA 0.5 ± 0.1. Subsequently they were used to calculate the recoveries of indolic acids from model solutions and biological samples in tert-butyldimethylsilyl (TBDMS) derivatives as an alternative silylating reagent was studied. The Rt values of obtained TBDMS derivatives were 2–4 min higher than for TMS derivatives (m/z = 218 in the mass spectrum of the mono-TBDMS derivative of 3ICA and the peak with m/z = 249 in the mass spectrum of di-TBDMS derivative of 3IPA correspond to [M-C(CH3)3]+ ions, which are also formed during fragmentation of the TBDMS derivatives of 3IAA, 3ILA and 5HIAA. The peak with m/z = 130 in mass spectrum of 3IPA is also characteristic for 3IAA and 3ILA mass spectra; the peak with m/z = 117 corresponds to indole [C8H7N]+ radical cation. Thus, the recorded mass spectra that are not present in the NIST mass spectral library correspond to incompletely substituted TBDMS derivatives of 3ICA and 3IPA. The peak forms of the incompletely substituted TBDMS derivatives of indolic acids are symmetric and can be used for quantitative analysis as the results were reproducible. Therelative signals of TBDMS derivatives of indolic acids were the following: 3IAA 0.3 ± 0.1, 3ICA 0.012 ± 0.001, 3IPA 0.6 ± 0.2, 3ILA 0.90 ± 0.04, 5HIAA 0.17 ± 0.06. Subsequently, they were used to calculate the recoveries of indolic acids from model solutions in Derivatization using MTBSTFA with the formation of ivatives . All TBDivatives e,f, but ivatives . The masm/z and Rt values, as described in We have developed the conditions based on LLE with diethyl ether, derivatization with BSTFA and GC–MS analysis for the determination of aromatic acids in serum samples of both healthy people and critically ill patients . While in = 288) of critically ill patients with stroke, myocardial infarction, postoperative surgical complications, and sepsis. The derivatives of 3IAA, 3IPA, and 3ICA were found; the frequency of appearance was 91, 9, and 6%, respectively. Notably, 3ICA was not detected in serum samples according to HMDB. 3IPA was detected in di-TMS form (n = 25) and in mono-TMS form (n = 2). 3ICA was detected in di-TMS form (n = 16) and in mono-TMS form (n = 1).The search for the chromatographic peaks of target compounds was made on the mass chromatograms of serum samples (A1 ˃ A2 or A1 ˂ A2).3IAA  was founn = 138) of neurosurgical patients with various intracranial diseases or injuries. The derivatives of 3IAA, 3ILA, and 5HIAA were found; the frequency of appearance was 26, 2, and 0.7%, respectively. Notably, 3ILA was not detected in CSF samples according to HMDB. 3IAA was detected in di-TMS form (n = 36) and in mono-TMS form (n = 2). 3ILA was detected in tri-TMS form (n = 3); 5HIAA was detected in di-TMS form (n = 1).The retrospective analysis of the chromatographic peaks of target compounds was also made on the mass chromatograms of the CSF samples  using the conditions of LLE developed for aromatic acids is inappropriate.MEPS was chosen as an alternative sample preparation method to LLE. The conditions for the extraction of the indolic acids from serum and CSF samples were based on our previous study  and theyRt values for TMS derivatives were less than for TBDMS ones.Preliminary experiments with model solutions showed that the application of MEPS with C18 sorbent and derivatization with BSTFA allows for detecting only incompletely substituted forms of indolic acids. Recoveries of the indolic acids from model solutions (concentration of addition was 100 µg/L/0.4–0.5 µM) using MEPS with BSTFA and MTBSTFA were comparable: 50–80% for BSTFA and 40–80% for MTBSTFA . BSTFA wThe recoveries from pooled CSF samples (40–80%) were comparable to those obtained from model solutions (50–80%) and higher than those from serum (40–60%). Recoveries at higher concentrations are also demonstrated in R2 ≥ 0.9960) and serum samples (R2 ≥ 0.9942) and over the 0.4–7 µM range of concentrations for pooled CSF samples (R2 ≥ 0.9949). The LOD values were 0.1–0.2 µM for model solutions, and 0.2–0.4 µM for pooled CSF and serum. The LOQ values were 0.4–0.5 µM for all matrices. The reproducibility (RSD) and relative error (RE) values for the LOQ concentration were less than 20% and ±20%, respectively  and CSF (n = 3) samples of different patients with CNS diseases like cerebral infraction, meningitis and transient ischemic attack. Mono-TMS derivative of 3IAA was detected in all samples :CSF samples: #1–(0.42 ± 0.08) µM; #2–(0.6 ± 0.1) µM; #3–(0.43 ± 0.03) µM.Serum samples: #A–(0.47 ± 0.09) µM; #B–(0.43 ± 0.06) µM; #C–(0.6 ± 0.1) µM.MEPS was used for the extraction of indolic compounds from serum  described in Rt values of the indolic derivatives are close to those of the compounds with higher boiling points. These matrix compounds are usually coextracted with target compounds when using LLE, leading to the contamination of chromatography and mass spectrometry systems, long GC analysis times, and high final temperatures [Rt = 23.71 min corresponds to the TMS derivative of cholesterol; Rt = 25.43, 26.50 and 26.96 min correspond to the derivatives of related sterol compounds. MEPS with C18 sorbent demonstrated perfect results in reducing the content of these interfering matrix components even in the serum sample of a critically ill patient.The eratures . The uti5-benzoic acid , N,O-bis(trimethylsilyl)trifluoroacetamide , N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide , formic acid (≥95%), hexane (≥97.0%), methanol (≥99.9%) were purchased from Sigma-Aldrich ; sulfuric acid, acetone, diethyl ether were Laboratory Reagent grade and obtained from Khimreactiv .Indole-3-carboxylic acid , indole-3-acetic acid , indole-3-propionic acid , indole-3-lactic acid , 5-hydroxyindole-3-acetic acid , 2,3,4,5,6-DA mixture of aqueous stock solutions of each indolic acid (0.8 mg/L) and internal standard (1.0 mg/L) were prepared. Then, the aliquot of this mixture was added to the serum or pooled CSF solution to prepare solutions with concentrations of 100 (0.4–0.5 µM), 500 (2.0–2.5 µM), 1000 (4–5 µM) and 2000 (8–10 µM) µg/L to create respective calibration curves in the 0.4–10 µM range of concentrations for serum samples; 100 (0.4–0.5 µM), 500 (2.0–2.5 µM) and 1500 (6–7 µM) µg/L to create respective calibration curves in the 0.4–7 µM range of concentrations for pooled CSF samples.n = 3), CSF (n = 3) samples of different patients with CNS diseases such as cerebral infraction, meningitis and transient ischemic attack and a healthy donor were collected in the Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology . The approval of the local ethics committee was obtained (N 26/2019). Serum and CSF samples were stored at −30 °C for not more than 3 months and were defrosted at room temperature prior to use. Pooled CSF samples were used due to the absence of those of healthy donors and were prepared by mixing CSF samples from several patients with unconfirmed bacterial or viral infection of the CNS.Serum . 2,3,4,5,6-DMEPS was performed using a 50 µL volume syringe coupled with barrel insert and needle assemblies packed with ~4 mg of C18 . Prior to each sample preparation, the C18 sorbent was activated by consistent conditioning with methanol, distilled water and 0.3 mM formic acid. A total of 50 µL of sample was passed through the C18 sorbent 20 times. Subsequently, the solid phase was washed with 20 µL of 0.3 mM formic acid to remove interferences. The elution of analytes using diethyl ether was conducted after the sorbent drying, which was achieved by the passing of the air through the sorbent for 12 times. The analytes were derivatized using BSTFA after the complete drying of the organic eluate.The recoveries, linearity, LOD, LOQ, accuracy, and precision were evaluated according to the guidelines of the FDA  .The recoveries of indolic acids were calculated at three different concentration levels  as follows:A—peak area of the TMS derivative of the indolic acid;StA—peak area of the TMS derivative of the internal standard;StA/A)—relative signal obtained after sample preparation using MEPS;(StA/A)100%—relative signal obtained from organic solution without any kind of sample preparation (except derivatization). were calculated according to the following formula:SD—the standard deviation;b—the slope.LOQs (µM) were defined as the lowest point of the calibration curve (100 µg/L / 0.4–0.5 µM).The precision was defined as RSD (%). The accuracy was defined as RE (%):The values of RSD not deviating more than 20% and RE not deviating more than ±20% from the nominal concentration at the LOQ were considered acceptable as well as RSD not deviating 15% and RE not deviating ±15% from the nominal concentration for standards other than LOQ.Selectivity for all analytes and internal standards was also evaluated for CSF by three parallel analysis of pooled CSF samples from three different patients and for serum by three parallel analysis of a healthy donor serum. After this, serum and pooled CSF samples were used in the preparation of calibration standards and were subsequently subjected to MEPS sample preparation and GC–MS analysis. Response of the interfering compounds did not exceed 20% of the LOQ values.The instability of aqueous solutions of indolic acids was observed and fresh stock solutions were prepared on a daily basis.The MEPS carryover effect was evaluated as follows: needle, which had been used for all previous extraction procedures with calibration standards, was used for extraction of pooled CSF or serum sample without the addition of indolic acids; the extract was derivatized and introduced into the chromatographic system. We did not observe an increase in the areas of chromatographic peaks during the elution of the target analytes, so the carryover effect of MEPS was considered to be insignificant.The blank sample of the eluting solvent was introduced into the chromatographic system immediately after the analysis of all calibration standards to evaluate the carryover effect of the chromatographic system, which did not exceed 0.1% of the areas of chromatographic peaks of indolic acids obtained during the previous analysis.n = 3, p = 0.95). All data were analyzed using Microsoft Excel 2016.The statistical processing of experimental data was performed based on the results of three parallel experiments and represented the mean with a confidence interval  and CSF (n = 138) samples of different groups of patients using LLE revealed interesting facts. The derivatives of 3IAA were found both in serum and CSF ; 3IPA and 3ICA were found in serum ; 3ILA and 5HIAA were found in CSF . The facts that 3ICA was found in serum samples and 3ILA was found in CSF samples are not described in HMDB. However, quantitative analysis of indolic acids using LLE is inappropriate because of the irreproducible formation of different silyl derivatives of analytes.The qualitative retrospective analysis of tryptophan metabolites on the chromatograms of serum . Some analytical characteristics of the methodology were calculated, and their values met the requirements of the FDA recommendations. The applicability of the developed conditions was demonstrated by analyzing real samples, where 3IAA was reproducibly determined in the CSF and serum samples of patients with CNS diseases at the 0.4–0.6 µM level. Other acids were not detected at this stage of research and the analysis of biological samples of the extended group of patients is required. A comparison of the chromatograms of the same serum sample obtained after MEPS and LLE demonstrated that the utilization of MEPS significantly reduced the amount of sterol matrix components of serum compared to LLE. All of the mentioned advantages of MEPS with the C18 sorbent suggests that this approach can be an excellent alternative to traditional sample preparation techniques for the analysis of small target analytes in complex matrices such as blood serum and CSF.