Low level sarin nerve gas and other anti‐cholinesterase agents have been implicated in Gulf War illness (GWI), a chronic multi‐symptom disorder characterized by cognitive, pain and fatigue symptoms that continues to afflict roughly 32% of veterans from the 1990–1991 Gulf War. How disrupting cholinergic synaptic transmission could produce chronic illness is unclear, but recent research indicates that acetylcholine also mediates communication between axons and oligodendrocytes. Here we investigated the hypothesis that oligodendrocyte development is disrupted by Gulf War agents, by experiments using the sarin‐surrogate acetylcholinesterase inhibitor, diisopropyl fluorophosphate (DFP). The effects of corticosterone, which is used in some GWI animal models, were also investigated. The data show that DFP decreased both the number of mature and dividing oligodendrocytes in the rat prefrontal cortex (PFC), but differences were found between PFC and corpus callosum. The differences seen between the PFC and corpus callosum likely reflect the higher percentage of proliferating oligodendroglia in the adult PFC. In cell culture, DFP also decreased oligodendrocyte survival through a non‐cholinergic mechanism. Corticosterone promoted maturation of oligodendrocytes, and when used in combination with DFP it had protective effects by increasing the pool of mature oligodendrocytes and decreasing proliferation. Cell culture studies indicate direct effects of both DFP and corticosterone on OPCs, and by comparison with in vivo results, we conclude that in addition to direct effects, systemic effects and interruption of neuron–glia interactions contribute to the detrimental effects of GW agents on oligodendrocytes. Our results demonstrate that oligodendrocytes are an important component of the pathophysiology of GWI. Oligodendrocytes are important contributors to the pathophysiology of Gulf War illness, a chronic condition with wide‐ranging neurological symptoms. Media was collected from shaken flasks and plated onto two 6 cm tissue culture dishes per flask, for 15 min, to separate OPCs from heavier endothelial and astrocyte cells. Supernatant from 6 cm dishes was collected and centrifuged for 10 min at 1200 rpm. Cells were then plated onto 25 mm glass coverslips coated with 0.1 mg/mL poly‐L‐lysine (PLL)  and 0.1 mg/mL poly‐L‐ornithine (PLO) . Coverslips were used for calcium imaging 1–3 days post‐plating. 80–90% of the cells on coverslips used for experiments were oligodendrocytes as confirmed by immunocytochemistry with the pan‐oligodendrocyte marker, Olig2. Purified OPCs were grown in DMEM+10% FBS (described above) without additional growth factors.2.32, 2 mM MgCl2. One mililiter of the diluted Fura‐2 was added to a 35 mm dish containing the 25 mm coverslip containing OPCs for 15 min in the dark at 37°C. After 15 mins, 1 mL of HEPES buffer was added and the cells were then incubated a second time for 15 min in the dark at room temperature. Coverslip was washed one time for 10 min with HEPES buffer before use. MetaFluor Software (Molecular Devices) was used to image and measure fluorescence emission at 340 and 380 nm excitation wavelengths. Acetylcholine was diluted in HEPES buffer to 1 and 50 μM concentrations. Intracellular Fura‐2 levels were calibrated using 10 μM A23187 with and without EGTA or Ca2+ in buffer. Calcium concentrations were calculated from fluorescence levels using the equation derived by Grynkiewicz, Poenie, and Tsien . Fifty microgram Fura‐2 AM was added to 50 μL Pluronic Acid . Fifteen microliter of the Fura‐2 Pluronic acid solution was added to HEPES buffer and brought to a total volume of 1.5 mL. HEPES buffer (pH 7.4) consisted of 8 g/L Sucrose, 1 g/L D‐glucose, 20 mM HEPES stock , 2 mM CaClnd Tsien . While s2.4OPCs were differentiated in N1 media with 0.2% FBS. OPCs were plated from flasks, as described above, in DMEM + 10% FBS for 24 hr and then switched to N1 media + 0.2% FBS for the remainder of the experiment.2.5At 3–4 weeks post dissection, flasks were shaken overnight at 37°C and 1200 rpm to remove microglia and dead cells. Media was removed, and flasks were washed twice with sterile Earle's Balanced Salt Solution (EBSS). Trypsin, warmed to 37°C, was added to the flasks and trypsinization was stopped 10 min later with 1:1 addition of DMEM + 10% FBS. Cells were collected and centrifuged at 1200 rpm and then plated on PLL/PLO coated 25 mm glass coverslips.2.6OPCs isolated from postnatal day 2 (P2) cerebral cortex were treated 24 hr after plating. Cells were fixed with 4% paraformaldehyde and immunocytochemistry was performed 72 hr following treatment.2.7Coverslips were fixed with 4% paraformaldehyde  in Phosphate Buffered Saline  for 20 min followed by the addition of 0.1% Triton X‐100 (Sigma‐Aldrich) in PBS for 5 min. The coverslips were washed three times with PBS and blocked for 1 hr in 5% Goat Serum in PBS (Fisher Scientific). The coverslips were then incubated overnight at 4°C with the primary antibody. The coverslips were then washed three times with PBS and incubated with secondary antibody for 2 hr at room temperature. Coverslips were washed three times and plated on microscope slides with Vectorshield Antifade Mounting Medium with DAPI  for imaging. For immunocytochemistry, the primary antibodies used were: Olig2  at 1:500, Olig2  at 1:500, Myelin Basic Protein  at 1:200, AChE  at 1:200, Cleaved Caspase‐3 (Asp175)  at 1:200. The secondary antibodies used were: Alexa Fluor 488  at 1:1000, Alexa Fluor 488  at 1:1000, Alexa Fluor 568  at 1:1000, Alexa Fluor 568  at 1:1000, Alexa Fluor 555  at 1:1000.2.8Acetylcholine (Sigma), DFP (Sigma), Corticosterone (Sigma) and Calcium Ionophore A23187 (Sigma) were used in the study. Ethanol (0.6%) was used as the corticosterone vehicle for in vivo and in vitro experiments. Doses used for each experiment are described in the results section.2.9Adult male Sprague Dawley rats aged 6–8 weeks received Corticosterone  in drinking water for days 1–7, followed by a single subcutaneous (s.c.) injection of DFP (1.5 mg/kg) on the morning of day 8. Animals were sacrificed 12, 24, 72 hr, or 21 days after DFP exposure.2.10.Rats were sacrificed by decapitation and brains were rapidly removed. One hemisphere was frozen for protein analysis and the other was post‐fixed in 4% paraformaldehyde overnight and cryopreserved in 30% sucrose for up to 4 days. Following adequate cryopreservation, brains were embedded in optimal cutting temperature (OCT) embedding media . Embedded tissue was cyrosectioned into 14 μm thick sagittal sections2.11Tissue sections were brought to room temperature and then rinsed one time with PBS to dissolve the embedding medium OCT. The sections were then incubated in Citrate buffer, pH 6, at 94°C for 10 min to promote epitope retrieval. The tissues sections were then rinsed three times with PBS containing 0.1% TritonX‐100 and blocked for 1 hr in blocking buffer . Sections were incubated overnight at 4°C with the primary antibodies. Primary antibodies used include: APC/CC1  at 1:500; Olig2  at 1:500; Ki67  at 1:500; Olig2,  at 1:100. The following day, the tissue sections were rinsed three times with PBS containing 0.3% TritonX‐100 and then incubated for 2 hr at room temperature with secondary antibodies (listed in the Immunocytochemistry methods section) at 1:200 dilution. Tissue sections were then rinsed three times with PBS and mounted on coverslips using mounting medium (Vectashield Antifade Mounting Medium with DAPI). Ki67 staining was performed on animals fixed 24 hr after treatment. CC1 staining was performed on animals fixed 21 days after treatment. A fluorescent light microscope with AxioCam MRm was used to acquire 10 images per region per animal at 40 X magnification.2.12Cells were plated onto coverslips as described above and incubated with 150 μL buffer or DFP at 37°C. Conditioned buffer was collected, and flash frozen after 4 hr. Buffer was analyzed with a Choline/Acetylcholine fluorometric assay kit (Abcam). To measure total levels of acetylcholine and choline ([Ach + Ch]), AChE enzyme was used in the buffer. To measure choline levels excluding acetylcholine, ([ACh]), DFP was added to the buffer with no AChE.2.13To extract proteins from tissue and cell culture, samples were lysed in RIPA buffer (Sigma Aldrich) with protease inhibitor cocktails . Lysate was mixed with LDS sample buffer (Thermo Fisher Scientific) and electrophoresed in a 4–12% Bis‐Tris Gel (Invitrogen) for 2 hr at 150 V in MOPS‐SDS running buffer (Thermo Fisher Scientific). The samples were transferred to PVDF membrane  overnight at 4°C in Tris‐Glycine transfer buffer (Thermo Fisher Scientific). Membranes were blocked in blocking buffer, containing TBS , 0.1% (vol/vol) Triton X‐100 and 5% (wt/vol) bovine serum albumin  or 5% nonfat dry milk (American Bio), for 1 hr at room temperature (RT). The appropriate primary antibody was diluted in blocking buffer and incubated overnight with the PVDF membrane at 4°C. Primary antibodies used were: GAPDH  used at 1:4000; NSE  used at 1:2000; GAPDH  used at 1:4000 dilution; MBP  used at 1:1000; GFAP  used at 1:500, GAP43  used at 1:1000, NG2  used at 1:1000. The PVDF membrane was washed four times, 15 min each, in washing buffer, TBS  and 0.1% (vol/vol) Triton X‐100. The corresponding secondary antibody, diluted in blocking buffer, was then incubated with the PVDF membrane for 2 hours at RT. Secondary antibodies that were used include ECL Anti‐Mouse IgG Horseradish Peroxidase‐linked F(ab’)2 fragment or ECL Anti‐Rabbit IgG Horseradish Peroxidase‐linked F(ab’)2 fragment. Chemiluminescent substrate was applied for 10 min . Membranes were quantified with densitometry using Image J software and normalized to NSE loading control. In reported bar graphs of data, each treatment condition was normalized to saline control levels.2.14Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH). All animals for in vivo and in vitro studies were approved through the Department of Defense Animal Care and Use Review Office (ACURO).All animal studies for in vivo experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of the Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, and the animal facility was certified by AAALAC International. All animal studies for in vitro experiments were performed at the Section on Nervous System Development and Plasticity, Bethesda, MD, according to Animal Study Protocol #15–007, and were approved by the Institutional Animal Care and Use Committee (IACUC), 2.15N was defined as an animal and n was defined as a microscopic field of view. For in vitro imaging, N was defined as biological experimental replicates and n was defined as a microscopic field of view. For calcium imaging, N was defined as a coverslip and n was defined as an individual cell. Grouped data are mean ± SEM unless stated otherwise.Statistics were performed with Minitab 18  and GraphPad Prism 7 . Immunoblot data were analyzed with One‐Way ANOVA  followed by Dunnett's multiple comparison post‐test. Cell counting data from histology and cell‐culture experiments were analyzed using the Chi‐Squared statistical test. Figures were made in SigmaPlot 14.0 . While sampling for immunoblotting, N was defined as an animal. For histology, 33.1To understand the etiology of white matter disruption present in veterans with GWI, we investigated how Cort exposure and disrupting cholinergic signaling through AChE inhibition may interfere with oligodendrocyte biology. To do so, we first determined the capacity for OPCs to engage in cholinergic signaling across development. Immunocytochemistry indicated that all cells of the oligodendrocyte lineage express muscarinic acetylcholine receptors (mAChRs) M1–M5 in vitro and expression of these receptors varied in abundance and localization across oligodendrocyte development from immature progenitors to mature myelinating cells Figure , but sar2+, but M2 and M4 receptors signal through cAMP. Live‐cell calcium imaging of OPC monoculture demonstrated that OPCs respond to 50 μM ACh  and to concentrations as low as 1 μM ACh,  with robust and heterogenous calcium kinetics indicating mAChRs were functional on OPCs. ACh doses of 50 and 1 μM were chosen because similar ACh concentrations have been used previously in studies of the cholinergic neuromuscular synapse . The dynamics of intracellular calcium responses varied in different cells in response to ACh (1 μM). The responses included prolonged oscillations, dampened oscillations, and a sharp rise to peak which plateaued and partially recovered  or Cort alone  had no effect on intracellular calcium levels measured over a 300 s treatment . The lack of measurable ACh in OPC and astrocyte monocultures suggests that ACh is not secreted from either OPCs or astrocytes. This supports the hypothesis that release of ACh by neurons signal to oligodendrocytes and that disruption of this neuro‐glial signaling could disrupt oligodendroglial development and function.The calcium imaging experiments were performed in HEPES Buffer , which does not contain ACh. DFP treatment alone . This suggests that inhibition of AChE on OPC cell membrane by DFP increased the concentration of ACh in the extracellular environment, thus eliciting responses from more OPCs. A larger percentage of cells also responded to 50 μM ACh compared with the 1 μM treatment . We conclude that AChE expressed on OPCs is functional and that its inhibition in the presence of ACh leads to increased ACh‐dependent Ca2+ signaling, which could influence OPC development and function.We therefore applied low concentrations of ACh to OPC cultures, together with DFP, to test whether inhibiting AChE activity on oligodendrocytes would alter their Ca3.2Any effect on oligodendrocyte development and function in the GWI animal model or in GW veterans could be due to direct effects on biology of oligodendrocyte lineage cells, or due to interruption of cholinergic neuron‐oligodendrocyte communication or caused by indirect systemic effects resulting from exposure to Cort and DFP. Cholinergic signaling and stress hormone signaling have diverse biological effects in the CNS, PNS, the cardiovascular, and the immune system. Therefore, to test for direct, non‐systemic effects of the GW agents on OPCs, we exposed OPCs in vitro to the AChE inhibitor, DFP, and to Cort. We performed a dose response study with DFP Figure a and Cor2 = 26.604, p < 0.001). DFP alone had no effect on OPC proliferation . Cort+DFP co‐treatment, as used in the GWI animal model, also decreased OPC proliferation due to the effect of Cort . Cell counts used to determine Ki67+ Olig2+ frequency are reported in Figure The data showed that Cort treatment significantly decreased OPC proliferation (Ki67+ Olig2+ cells)  and by Cort+DFP  treatment due to the effect of DFP. The vehicle used to dissolve Cort, (0.00004% ethanol) caused a small, but statistically significant increase in apoptosis when used alone , but in combination with Cort had no measurable effect . Cell counts used to determine CC3 + Olig2+ frequency are reported in Figure Apoptosis, identified by cleaved caspase 3 (CC3) expression, was increased by DFP treatment , DFP , and Cort+DFP  conditions increased the number of Olig2+ cells that were post‐mitotic and pre‐myelinating (CC1+). Cell counts used to determine CC1 + Olig2+ frequency are reported in Figure 2 = 8.419, p = 0.004). DFP  and Cort+DFP  had no significant effect on the number of MBP+ mature oligodendrocytes. Cell counts used to determine MBP+ Olig2+ frequency are reported in Figure To determine the effects of AChE inhibition and Cort on oligodendrocyte lineage cell maturation, OPCs were differentiated into oligodendrocytes using N1 differentiation media for 3 days in the presence of Cort alone, DFP alone, and in the presence of both Cort and DFP. The data show that Cort .To test if ACh alone could directly affect MBP expression, we treated oligodendrocytes with ACh in vitro. We found that elevated ACh was not sufficient to increase MBP expression in cell‐culture  and Cort having no significant effect . Paradoxically, in combination, DFP + Cort increased the number of proliferating oligodendrocytes . Cell counts of frequencies of proliferating oligodendrocytes (Ki67+ Olig2+) are reported in Figure 2 = 27.611, p < 0.001) and Cort with DFP  had a similar effect, but Cort treatment alone did not affect maturation of oligodendrocytes to the CC1+ stage . Representative immunohistochemistry images from the PFCare shown in Figure Cell culture studies indicate that Cort and DFP have complex effects on OPC proliferation, survival, and maturation. Oligodendrocytes in vivo are likely affected by these agents, making it important to determine how oligodendrocytes may be affected in the GWI animal model. Any differences found in the animal model from the results in cell culture will indicate systemic effects and neuron–glia interactions that are not modeled in culture. In addition, the exposures in vivo could be different due to metabolic breakdown of the compounds. We examined the expression of CC1, a nuclear marker for postmitotic but pre‐myelinating oligodendrocytes , but not DFP  decreased OPC proliferation and this effect persisted when Cort was delivered together with DFP . Cell counts for corpus callosum oligodendrocyte proliferation frequencies are reported in Figure 2 = 225.445, p < 0.001) and Cort + DFP , but DFP alone decreased maturation . Thus, Cort has opposite effects in the PFC and corpus callosum on proliferation and maturation. DFP decreased proliferation and differentiation in both regions, but the effects in the corpus callosum are either small or not statistically significant.The PFC is implicated in GWI and it is still undergoing myelinating in young adults. The major white matter tract in rodents is the corpus callosum. We therefore analyzed the effects of GW agents on oligodendrocyte lineage cells residing in grey matter PFC versus white matter corpus callosum. The effects of DFP and Cort on proliferation and maturation of OPCs differed in the corpus callosum compared with the PFC. This is not unexpected, given the differences in the cellular composition and environment in these two regions. In the corpus callosum, 19.9% of Olig2+ cells were proliferating, immature OPCs, but in the PFC more cells were in this state 25.6%), as this grey matter region is still undergoing active myelination. In the corpus callosum, Cort  and this effect persists at 24 hr , 72 hr  and 21 days . Subcortical white matter was the ideal medium for the immunoblot experiments because it is enriched in white matter, including the corpus callosum, which allowed for robust testing of the hypothesis. For the histology experiments, we chose a specific area, the corpus callosum, to eliminate heterogeneity of each subcortical region.Changes in the integrity of white matter tracts has been found to underlie the key cognitive and sensory impairments in GW veterans . Histological analysis showed that at later time points, Cort and Cort + DFP increased the number of mature oligodendrocytes in the corpus callosum . At 1 day postexposure GW agents significantly altered NG2 levels  and post‐hoc analysis reveals DFP treatment significantly increased NG2 levels compared to saline . This could imply an early spurt in proliferation due to increased availability of ACh, which promotes proliferation, via DFP mediated inhibition of AChE, however NG2 is expressed by perivascular and other cells , there were no significant differences between the treatment conditions, suggesting an increase in the proliferative pool of OPCs was likely not the sole source of the persistently increased MBP across early and later time‐points. Abundance of transcription factor Olig2 at 12 hr  and 24 hr  was not significantly different among any treatment condition at these early timepoints, consistent with the transient changes in NG2 levels resulting from factors other than the number of oligodendrocytes.Interestingly, DFP treatment alone produced a significant increase in MBP expression at 12 hours , 24 hr , or 72 hr  postexposure to DFP and Cort, either alone or in combination. Therefore, the increase in MBP is not explained by myelination of new axons.Alternatively, neuroinflammation and injury can alter axonal sprouting , 24 hr  and 72 hr  posttreatment. This is consistent with studies showing GFAP expression is not altered up to 3 days posttreatment with GW agents . GFAP expression was significantly reduced with Cort alone  and DFP alone  treatment conditions. These observations are consistent with decrease in GFAP expression with chronic exposure to Cort  and by an increase in the number of mature oligodendrocytes in the corpus callosum at later timepoints (21 days). Further, the finding that exogenous ACh on an in vitro monoculture of OPCs is insufficient to change MBP protein levels Figure  suggestsTogether these results show effects on OPC proliferation, survival, maturation, and increased myelin basic protein expression in the subcortical white matter resulting from treatment with Cort and DFP in the GWI animal model. These effects differ in different brain regions and in cell culture because of the differential contribution of cholinergic and non‐cholinergic effects of GW agents and differences in cellular environments of grey and white matter regions where these oligodendroglial populations reside Table .4In this study we have investigated the plasticity of myelinating glia in the context of white matter abnormalities in GWI and demonstrated that biology and development of oligodendrocyte lineage cells are significantly affected by exposure to AChE inhibiting agents and the stress hormone, corticosterone. We conclude that DFP, a sarin nerve gas surrogate, decreases maturation of OPCs when acetylcholine signaling is present. DFP also decreases proliferation of oligodendrocytes in the PFC, a region with a higher percentage of proliferating oligodendrocytes than in the corpus callosum.Our finding that DFP decreases maturation of oligodendrocytes is consistent with the current literature about the role of ACh signaling on oligodendrocyte development  Total Olig2 counts (Olig2 + DAPI+) summed over 10 fields of view. DFP doses used were: 0, 1, 5, 10, 50, 100 μM. . (b) Total Olig2 counts (Olig2 + DAPI+) summed over 10 fields of view. Corticosterone doses used were: 0, 0.1, 0.5, 1, 5, 10, 50 μM. . Bars are average counts per field ± SEM.Click here for additional data file.Supplemental Figure 2 ACh treatment alone is not sufficient to induce MBP changes in OPCs (a) Immunoblots of MBP and GAPDH from differentiating oligodendrocytes with ACh (1 μM in N1 differentiation media) and without ACh . (b) Immunoblots were quantified with Image J as the ratio MBP/GAPDH  = 0.816, p = 0.55). Individual MBP isoform bands were summed to give a single MBP value for each sample.Click here for additional data file.Supplemental Figure 3 No evidence for an early increase in OPC population in the GWI animal model. Immunoblot of whole brain homogenate for NG2 and NSE loading control at (a) 12 hours, (b) 24 hours, and (c) 72 hours post DFP treatment. (d) All treatment conditions were compared to the control condition, Saline. NG2 levels were significantly increased at 24 hours in DFP condition . No significant difference in NG2 at 12 hours  or 72 hours  = 2.825, p = 0.1068). NG2 band occurs at 300 kDa and NSE occurs at 47 kDa. Immunoblot of subcortical brain homogenate for Olig2 protein and NSE loading control at (e) 12 hours, and (f) 24 hours post DFP treatment. Olig2 band occurs at 32 kDa, NSE band occurs at 47 kDa. (g) No changes in Olig2 protein expression was observed  = 1.48, p = 0.3472; 24 hours: ANOVA F = 2.618, p = 0.1877). All treatment conditions were normalized to the control condition, Saline. * indicates p < 0.05.Click here for additional data file.Supplemental Figure 4 No change in GAP43 expression in GWI animal model. Immunoblots of subcortical brain homogenate showing GAP43 expression in Saline, Cort, DFP, and Cort+DFP conditions at (a) 12 hours, (b) 24 hours, (c) 72 hours. GAP43 band occurs at 43 kDa; NSE band occurs at 47 kDa. (d) GAP43 expression levels were not affected by the treatment conditions  = 2.179, p = 0.1684; ANOVA 24 hours: F = 3.638, p = 0.0640; ANOVA 72 hours: F = 1.534, p = 0.2790). Protein expression levels were normalized to NSE at each time point and all treatment conditions were compared to the control condition, Saline.Click here for additional data file.Supplemental Figure 5 No change in GFAP expression in GWI animal model. Immunoblots of subcortical brain homogenate showing GFAP expression with Saline, Cort, DFP, and Cort+DFP treatment at (a) 12 hours, (b) 24 hours, (c) 72 hours, and (d) 21 days post treatment. (e) GFAP levels were not affected by any treatment condition at 12, 24 or 72 hours  = 1.039, p = 0.4263); 24 hours: ANOVA F = 2.826, p = 0.1067; 72 hours: ANOVA F = 2.543, p = 0.1295; 21 days: ANOVA F = 6.638, p = 0.0040) post treatment. Protein expression levels were normalized to NSE at each time point and all treatment conditions were compared to the control condition, Saline.Click here for additional data file.


Laparoscopic surgery typically requires deep neuromuscular blockade (NMB), but whether deep or moderate NMB is superior for thoracoscopic surgery remains controversial.Patients scheduled for thoracoscopic lobectomy under intravenous anesthesia were randomly assigned to receive moderate [train of four (TOF) 1–2] or deep NMB . Depth of anesthesia was controlled at a Narcotrend rating of 30 ± 5 in both groups. The primary outcome was the need to use an additional muscle relaxant (cisatracurium) during surgery. Secondary outcomes included surgeon satisfaction, recovery time of each stage after drug withdrawal , blood gas data, VAS pain grade after extubation, the time it takes for patients to begin walking after surgery, postoperative complications and hospitalization time. Results were analyzed on an intention-to-treat basis.p < 0.001). Surgeons reported significantly higher satisfaction for patients undergoing deep NMB . The mean difference between the two groups in the time from withdrawal until TOF recovery of 25% or 90% was 10 min (p < 0.001). The two groups were similar in other recovery data, blood gas analysis, VAS pain grade, days for beginning to walk and mean hospitalization time.Thirty patients were enrolled per arm, and all but one patient in each arm was included in the final analysis. Among patients undergoing moderate NMB, surgeons applied additional cisatracurium in 8 patients because of body movement and 5 because of coughing . Additional cisatracurium was not applied to any of the patients undergoing deep NMB (Deep NMB can reduce the use of additional muscle relaxant and increase surgeon satisfaction during thoracoscopic lobectomy.ChiCTR-IOR-15007117, 22 September 2015.Chinese Clinical Trial Registry,  Sufficient muscle relaxation is mandatory for most surgical procedures, and particularly for minimally invasive surgery –3. For lWhether deep NMB can similarly benefit patients undergoing thoracoscopic surgery is unclear. On one hand, it may seem unnecessary because the ribcage provides thoracic support and one-lung ventilation usually provides a satisfactory surgical field. On the other hand, thoracoscopic surgeries involve areas adjacent to major blood vessels and can trigger intraoperative body movement, cough, and diaphragm movement , the diaHere we compared deep and moderate NMB for their ability to reduce requirement of additional muscle relaxant and improve surgeon’s assessment in patients undergoing thoracoscopic lobectomy. An anaesthesiologist blinded to patient allocation was responsible for collecting perioperative data. We expected that deep NMB would be superior because of its demonstrated ability to reduce peak pressure and plateau pressure in the airway as well as improve lung compliance and peripheral oxygen saturation during one-lung ventilation .This is a single-center, randomized, controlled trial approved by the Ethics Committee of research institution  and written informed consent was obtained from all subjects participating in the trial. Trial registration: Chinese Clinical Trial Registry, ChiCTR-IOR-15007117, 22 September 2015.2. Patients were excluded if they had history of diabetes, viral hepatitis, asthma, glaucoma, neuromuscular disease, or if airway difficulties were anticipated. Patients were also excluded if they had hepatic or renal dysfunction, defined as one of the following: ALT > 40 U/L, AST > 40 U/L, SCr > 133 μmol/L, or total bilirubin > 30 μmol/L .The trial was carried out between October 2015 and July 2016 in the Department of Anesthesiology. Patients had to satisfy the following inclusion criteria: (1) age of 18–65 years, (2) elective thoracoscopic lobectomy, (3) American Society of Anesthesiologists (ASA) classification of I or II, and (4) body mass index (BMI) of 18–25 kg/mPatients were allocated to deep or moderate NMB groups using random numbers generated by computer  and concealed in opaque envelopes. An anaesthesiologist aware of patient allocation monitored depth of anesthesia and NMB. A different anaesthesiologist blinded to patient allocation was responsible for airway management, intubation, arteriovenous puncture, monitoring of vital signs and temperature and blood gas analysis. Patients and surgeons were blinded to group assignment. The same group of surgeons performed all procedures for both groups of patients.Patients underwent lobectomy involving standard thoracoscopy as described . Lymph nSurgery was conducted under total intravenous anesthesia without preoperative medication. Anesthesia was conducted using propofol TCI (4 μg/ml) and sufentanil (0.7 μg/kg). NMB calibration was carried out under ventilation with a face mask after patients lost consciousness. Cisatracurium  was given after TOF stabilization. When TOF reached 0, patients were intubated with a double-lumen endotracheal tube for single lung ventilation under bronchofiberscope guidance. Ventilation was carried out with pure oxygen and adjusted to maintain end-tidal carbon dioxide pressure at 35–40 mmHg.Anesthesia was maintained with propofol TCI (2–3 μg/ml) and dexmedetomidine (0.4 μg/kg/min) to maintain the Narcotrend (Germany) rating at 30 ± 5. Sufentanil was continued at 5–10 μg/h. Dezocine (5 mg) and ramosetron (0.6 mg) were given 15 min before the end of the operation. After the last skin suture, propofol and dexmedetomidine were discontinued.2 = 40%), observed closely for 10 min and transferred to the post-anesthesia care unit. When blood gas results were normal, patients were transferred to the ward or the intensive care unit.Given the need for continuous muscle relaxant monitoring and in light of the stability of patient baseline parameters, we did not move patients until TOF recovered to 90%. The double-lumen tube was removed in the operating room and patients were given mask oxygen . Electrodes were placed over the ulnar nerve proximal to the wrist at a distance of 3–6 cm. The TOF-Watch applies an electrical stimulus to the ulnar nerve and measures contractions of the adductor pollicis muscle through a sensor attached to the tip of the thumb. Deep NMB was defined as 1 ≤ PTC ≤ 5 .Tetanic stimulation was applied to the ulnar nerve (50 Hz for 5 s) once the patient was unconscious, then the TOF-Watch was calibrated. After TOF stimulation remained stable for at least 3 min, stimulation was switched to the TOF stimulation pattern . If the TOF ratio differed by 10%, the TOF-Watch was recalibrated. TOF was monitored every 15 s, and PTC every 3 min.Muscle relaxant infusion started when PTC reached 1 in the deep NMB group or 8 in the moderate NMB group. Initial rate of cisatracurium infusion was 0.1 mg/kg/h. The infusion rate was adjusted in increments of 0.01 mg/kg/h in order to maintain PTC at 1–5 in the deep NMB group or TOF at 1–2 in the moderate NMB group. Core temperature was controlled at 36–37 °C; skin surface temperature was maintained above 32 °C using a warm air blower. Additional cisatracurium (0.05 mg/kg) was given when requested by the surgeon, regardless of the reasons.Infusion of muscle relaxants was stopped upon placement of the chest drainage tube. When TOF recovered to 20%, neostigmine (0.02 mg/kg) and atropine (0.02 mg/kg) were injected. In all patients, neuromuscular monitoring continued until the TOF ratio reached 90%.The primary outcome was the use of additional cisatracurium during surgery. One secondary outcome was the surgeon’s assessment with how well he was able to perform the procedure without interference from coughing, bucking, muscle contractions or lung movement. The four-point scale  was (1) We calculated  a minimum requirement of 20 subjects in each arm of the study based on the idea that 40% of patients in the moderate NMB group and none of the patients in the deep NMB group would receive additional muscle relaxant  and baset test when the data were normally distributed or using the Wilcoxon rank sum test when the data were skewed. Results associated with a two-sided P < 0.05 were considered statistically significant.Statistical analyses were performed using SAS 9.2  on an intention-to-treat basis. Numbers of surgeon requests for additional muscle relaxant, incidence of intraoperative events and postoperative complications were compared between the two groups using Fisher’s exact probability method. Surgical characteristics and VAS pain grade were compared between the two groups using the Wilcoxon rank sum test. Differences in baseline and recovery characteristics were compared using Student’s n = 2), history of diabetes (7), viral hepatitis (2), asthma (1), and anticipated airway difficulties (2). Of the 60 patients finally enrolled, one was excluded from the deep NMB group because the battery ran out during muscle relaxant monitoring, and one was excluded from the moderate NMB group because of intraoperative bleeding and sent to ICU intubated after surgery. Thus 58 patients were included in the intention-to-treat analysis  of patients during intubation. The average interval between cisatracurium administration during anesthesia induction and recovery of PTC = 1 was 43.33 ± 5.53 min. Among patients in the moderate NMB group, additional muscle relaxant was requested by the operating surgeon for 8 patients due to body movement and 5 due to coughing. Additional relaxant was not requested for any patients in the deep NMB group . The mean difference between the two groups in the time from withdrawal until TOF recovery to 25%  and recovery of TOF = 90% Fig. c, as wel25% Fig. a or 90% 25% Fig. b was 10 25% Fig. e-h.Fig. P = 0.529; Fig. P = 0.818; Fig. P = 0.455; Fig. The VAS pain grade in two groups were less than or equal to 3  or hypoxemia (pO2 < 80 mmHg) occurred. All patients were able to get out of bed and walk within three days after surgery, the patients in both groups were hospitalized for an average of 5 days, and no pulmonary complications occurred. These results suggest that deep NMB does not substantially affect the prognosis of patients undergoing thoracoscopic lobectomy.In both groups, patients showed normal pH, pCO2.We found that in the deep NMB group, the average time for PTC to return to 1 after induction with 0.2 mg/kg cisatracurium was 43.33 ± 5.53 min, and the average time from pump stoppage (when PTC = 1–5) to recovery of TOF = 25% was 35.86 ± 6.50 min. These results indicate that an induction dosage of 0.2 mg/kg cisatracurium is reliably sufficient for 32 min (mean - 2SD) in ASA1–2 patients with BMI 18–25 kg/mWhile we used cisatracurium in our study, because compared to other non-depolarizing muscle relaxants, cisatracurium released less histamine, its impact on the cardiovascular system is small and suitable for elderly patients. Rocuronium is also widely used and it behaves substantially differently. One study  reportedOur study is one of the few to report continuous NMB monitoring. We ensured that the relative position of the palm and finger would not change when the patient was turned over . As a result, we were able to ensure continuity in the calibration and comparability between patients. This may provide a superior approach to that of a previous study in which continuous pumping of muscle relaxants during thoracolaparoscopic esophagectomy was able to provide relatively stable surgical conditions but did not allow continuous monitoring of NMB .Despite its strengths, our study has limitations, which should be taken into account when interpreting our results. We used the 4 point scale to evaluate surgical conditions, while this scale was adapted from a 5 point scale from Martini et al. and it seemingly had only been used in one study before and had not been validated, much more studies are needed to explore the validated scale for thoracic surgery. In addition, we used relatively wide range of 1 ≤ PTC ≤ 5 as the definition of deep NMB. Further study should compare surgical conditions under the two depths of 1 ≤ PTC ≤ 2 and 3 ≤ PTC ≤ 5, which can minimize the dosage range of muscle relaxants to meet surgical requirements. Since our study involved a small sample from a single center, our findings should be verified and extended in larger studies, preferably from multiple centers.Our study shows that deep NMB provides better surgical conditions for thoracoscopic lobectomy. It can reduce the need for additional muscle relaxant by surgeon and improve surgical conditions during thoracoscopic lobectomy. Deep NMB provide these benefits while prolonging recovery time.


To determine if the rate of weight loss (WL) is associated with metabolic changes independent of the absolute WL.  WL and health changes were assessed in 11,281 patients attending a publicly funded clinical weight management program over a treatment period of 12.7 months. Early weight loss rate (WLR) in the first 3–6 months and overall WLR were categorized as Fast WLR (≥1 kg/wk), Recommended WLR (0.5 to 0.9 kg/wk), or Slow WLR (<0.5 kg/wk). P > 0.05). Conversely, the absolute WL sustained is significantly associated with changes in metabolic health independent of WLR (P < 0.001). Similar results were observed with WLR over the entire treatment period.  On average, patients attained a 6.6 ± 7.3 kg (5.8 ± 5.7%) WL over 12.8 ± 13.1 months. Prior to adjusting for covariates, patients with Fast WLR (−24.7 ± 13.4 kg) at 3–6 months had a greater overall WL as compared to those with Recommended WLR (−13.3 ± 8.7 kg) and Slow WLR (−5.0 ± 5.4 kg). Fast WLR also had greater improvements in the overall waist circumference and blood pressure than patients with Slow or Recommended WLR. However, after adjustment for absolute WL, Early and overall Recommended and Fast WLR did not differ in the changes in any of the health markers ( Faster rates of WL are associated with a greater absolute WL and larger improvements in waist circumference and blood pressure. However, after adjusting for the larger absolute WL sustained, early and overall faster WLR do not appear to have advantages for improving metabolic health markers. Thus, the absolute WL attained may be the most important factor for improving metabolic health. Current weight management guidelines emphasize the importance of attaining a 5% weight loss (WL) to achieve health benefits , 2. HoweInterestingly, the recommended 1-2 lb/wk (∼0.5 to −1 kg/wk) WLR (Rec WLR) in the 1998 guidelines  was baseThus, the objective of the current study is to determine whether the rate of WL is associated with changes in health factors independent of the absolute WL.n=25). All participants gave their written consent for the use of their medical data for research purposes and were informed that their decision to participate would not influence their medical treatment at WMC. Participants did not receive any form of stipends. All methods were approved by the York University Research Ethics Board .The study population included 11,283 patients who attended the Wharton Medical Clinic (WMC) Weight Management Program between July 2008 and July 2017. Data up to 5 years of treatment were obtained from electronic medical files. To ensure a fairer comparison among WLR groups, patients were included only if they attended the clinic for at least 3 months and lost at least 0.1 kg of weight  and had repeated metabolic variable measurements. Patients who had bariatric surgery were excluded from the analysis ; thus, the sample size for the metabolic data analyses varied depending on the availability of data.Patients undergo a standard battery of clinical tests including waist circumference, blood pressure (BP), fasting glucose, triglycerides (TG), and high-density lipoprotein (HDL) assessed using standard clinical methods. Participants were included if they had change data for at least one health measure (Body weight (BW) was measured by staff at each patient visit on a calibrated MedWeight, MS-2510 Digital High Capacity Platform Scales . Weight change was calculated as final observed BW-initial BW. For each health measure, the final weight that most closely corresponded to the health measure timeframe was used. The rate of weight loss was calculated as the weight change/treatment time during the first 3–6 months and over the entire treatment period that exceed, met, or was slower than the 1-2 lb/wk (∼0.45–0.91 kg/wk) recommendation: Fast WLR (≥0.91 kg/wk), Rec WLR (0.45 to 0.90 kg/wk), or Slow WLR (<0.45 kg/wk).P < 0.05.Patient characteristics and baseline metabolic variables were presented as means ± SD stratified by early WLR. Group differences in participant characteristics were assessed using an analysis of variance for continuous variables and chi-square tests for categorical variables. The independent associations of overall and early WL and the rate of WL  on changes in health markers were adjusted for age, sex, baseline values, relevant medication (yes/no), and use of weight loss medications (yes/no). Results were similar when only baseline metabolic value and absolute WL were adjusted for, and thus, only the final model is presented. Bonferroni post hoc tests were used. All statistical analyses were conducted using SAS 9.4 . Statistical significance was established at P < 0.05 for all). These differences remained true with adjustment for age, sex, baseline body weight, treatment time, and WL medication use (results not shown).Participant characteristics stratified by early WLR is shown in P < 0.05), but modest to no changes in the lipids or glucose (P > 0.05), whereas absolute WL remained independently associated with changes in all of the health markers (P < 0.0001). When early WLR during the first 3 to 6 months was examined as a continuous variable, the WLR was associated with greater reductions in glucose independent of absolute weight loss , but there was no association between early WLR and the other metabolic risk factors (P > 0.05).In a model adjusting for absolute WL, patients with early Slow WLR, Rec WLR, and Fast WLR had similar improvements in waist and BP . Conversely, the overall Slow WLR had significantly better improvements in HDL as compared with Rec WLR and Fast WLR (P < 0.05). As with early WLR, absolute WL remained significantly associated with all health changes independent of the overall WLR (P < 0.0001). When the WLR over the entire intervention was examined as a continuous variable, the WLR was associated with greater improvements in SBP , but smaller improvements in HDL .Similarly, when examining the rate of WL independent of absolute WL over the entire intervention, the overall Rec WLR and Fast WLR did not differ in changes in any of the health markers  with a 5% WL target overall to achieve health benefits , 8. HoweWhether the WLR results in differential effects on health independent of the absolute WL is unclear. Three studies in smaller cohorts report that there are no differences in health changes between fast and slow WL –15. UsinStrengths and limitations of the current study warrant mention. Our study sample consisted of patients from a publicly funded weight management clinic that was predominately middle-aged women, and the applicability of these findings to other demographics is unclear. The individuals included in this analysis were generally at the clinic for less than 2 years, and thus, we are unsure of the long-term sustainability of their WL, weight regain, or health changes. Because of the nature of care provided at the weight management clinic, reporting of gall stones is inconsistent, as care would be provided at the hospital and follow-up treatment by their primary care physician. Nevertheless, the risk of gallstones is well documented and important to consider. Furthermore, as only two time points were used, we cannot speak to the influence of weight loss patterns on the health changes; however, it is suggested that the weight regain after fast and slow WL is similar , 14, 19.In summary, we demonstrate that patients with rates of WL faster than the commonly recommended 1-2 lb/wk (0.45–0.91 kg/wk) tended to have greater obesity reduction and superior health improvements than slower WL. However, the superior improvements in health associated with faster WL are abolished after adjusting for absolute WL. Faster WLRs either early in the intervention or over the entire treatment were associated with similar changes in metabolic health at the commonly recommended rate in patients attending a publicly funded clinical weight management program. Thus, future WL interventions aimed at improving metabolic health should focus on the absolute weight loss attained and long-term weight management.


MAPT) gene polymorphisms could be associated with increased risk for AD.Alzheimer's disease (AD) is the world leading cause of dementia. Early detection of AD is essential for faster and more efficacious usage of therapeutics and preventive measures. Even though it is well known that one ε4 allele of apolipoprotein E gene increases the risk for sporadic AD five times, and that two ε4 alleles increase the risk 20 times, reliable genetic markers for AD are not yet available. Previous studies have shown that microtubule‐associated protein tau  and MAPT haplotypes are associated with AD pathology, as measured by cerebrospinal fluid (CSF) AD biomarkers amyloid β1–42 (Aβ1–42), total tau (t‐tau), tau phosphorylated at epitopes 181 (p‐tau181), 199 (p‐tau199), and 231 (p‐tau231), and visinin‐like protein 1 (VILIP‐1).The present study included 113 AD patients and 53 patients with mild cognitive impairment (MCI), as well as nine healthy controls (HC) and 53 patients with other primary causes of dementia. The study assessed whether six MAPT rs1467967 genotype, CC MAPT rs2471738 genotype and in patients with H1H2 or H2H2 MAPT haplotype.Significant increases in t‐tau and p‐tau CSF levels were found in patients with AG and AA MAPT haplotype‐tagging polymorphisms and MAPT haplotypes should be further tested as potential genetic biomarkers of AD.These results indicate that  Aβ1–42 indicates the presence of senile plaques in the brain  were previously used in genome‐wide association studies (GWAS) as endophenotypes for detection of AD risk genes  gene were associated to their pathological levels in CSF. Although AD is not caused by mutations in the MAPT gene, previous studies demonstrated that CSF biomarkers of AD differ among patients with different MAPT genotypes , the most common primary cause of dementia, is a complex disease with poorly understood etiology. The characteristic amyloid plaques and neurofibrillary changes seen in AD brains are frequently observed in other neurodegenerative diseases. As a consequence, many AD cases are misdiagnosed. One such autopsy‐confirmed series showed sensitivity of AD diagnosis to range from 70.9% to 87.3%, while specificity ranged from 44.3% to 70.8% , more than 20 common loci have been associated with risk for sporadic AD, age at onset, and progression of cognitive decline, but reported genome‐wide significant loci do not account for all the estimated heritability and provide little information about underlying biological mechanisms  and 112 male (M) subjects: 113 AD patients (60F/53M) and 53 mild cognitive impairment (MCI) patients (27F/26M), nine healthy controls  and 53 patients with other causes of dementia , all recruited at the University Hospital Center, Zagreb , and thyroid function test, and had a negative serology for syphilis or Lyme’s disease. All procedures were approved by the Ethical Committee of the Clinical Hospital Center Zagreb and by the Central Ethical Committee of the University of Zagreb Medical School , and were in accord with the Helsinki Declaration , Montreal Cognitive Assessment (MoCA), and Alzheimer’s Disease Assessment Scale‐cognitive subscale (ADAS‐Cog), underwent neurological examination and complete blood tests including levels of vitamin BFor AD, we used diagnostic criteria of McKhann et al. , while M2.2g for 10 min, CSF samples were aliquoted and stored in polypropylene tubes at −80°C. Levels of Aβ1–42, t‐tau, p‐tau231, p‐tau199, p‐tau181, and VILIP‐1 were determined using the following enzyme‐linked immunosorbent assays (ELISA): Aβ1–42 , t‐tau , p‐tau231 , p‐tau199 , p‐tau181 (Innotest Phospho‐Tau (181P), Fujirebio), and VILIP‐1 , respectively. Each CSF sample was analyzed in duplicate. Concentrations ranges of each biomarker are listed in Table Cerebrospinal fluid was taken by lumbar puncture between intervertebral spaces L3/L4 or L4/L5. After centrifugation at 2,000 2.3MAPT gene polymorphisms  were determined using primers and probes purchased from Applied Biosystems as TaqMan® SNP Genotyping Assay by ABI Prism 7300 Real Time PCR System apparatus . The del‐In9 deletion in MAPT Intron 9 defines the H1/H2 MAPT haplotype division caused by the inversion. H1 and H2 MAPT subhaplotypes were determined using haplotype tagging SNPs in following order: rs1467967, rs242557, rs3785883, rs2471738, del–In9, and rs7521 (Table Venous blood samples (4 ml) were collected into plastic syringes with 1 ml of acid citrate dextrose as an anticoagulant. Genomic DNA was extracted from peripheral blood using the salting‐out method  were used for comparison of the biomarkers’ levels between groups of subjects with different MAPT genotypes and haplotypes. After Kruskal–Wallis test for pairwise comparisons of independent samples, we performed the post hoc analysis. Since this post hoc testing option in SPSS incorporates calculation of the corrected p‐value, there was no need for additional corrections due to multiple comparisons. Nevertheless, due to the relatively small sample size, we performed statistical analysis with and without outliers. Only one statistically significant difference was lost after exclusion of outliers. Only results of the statistical analysis without outliers are presented here.All statistical analyses were performed in SPSS 19.0.1  with the statistical significance level set at 2.5The present study was conducted according to the 6th revised Declaration of Helsinki  and Good Clinical Practice Guidelines and was approved by the local ethics committee of the Clinical Hospital Centre Zagreb. All the patients signed informed consent form for lumbar puncture, genetic research and for participation in this study. The consent form was explained in details to the patients and their legal guardians/caregivers. All procedures were approved by the Central Ethical Committee of the University of Zagreb Medical School .33.11–42, t‐tau, p‐tau181, p‐tau199, p‐tau231, and VILIP‐1) in the analyzed subjects are presented in Table MAPT rs242557, MAPT rs3785883, and MAPT rs7521 genotypes.The concentrations of CSF protein biomarkers . T‐tau levels were significantly higher in patients with AG compared to GG MAPT rs1467967 genotype (p = 0.002) and AA compared to GG MAPT rs1467967 genotype (p = 0.022) when all patients were analyzed together . P‐tau181 levels were significantly higher in patients with AG in comparison to GG MAPT rs1467967 genotype when combining AD and MCI patients . More precisely, t‐tau levels were significantly higher in subjects with CC in comparison to TC MAPT rs2471738 genotype , our analysis of MAPT polymorphisms was conducted in all patients including the HC group , as such, the small number of HC is unlikely to have influenced the outcome of the analysis. The levels of t‐tau and p‐tau181 were significantly higher in patients with AG compared to GG MAPT rs1467967 genotype. Levels of t‐tau were significantly higher in patients with AA compared to GG MAPT rs1467967 genotype. Levels of t‐tau were significantly higher in patients with CC compared to TC MAPT rs2471738 genotype. Additionally, levels of t‐tau, p‐tau181 and p‐tau231 were significantly higher in patients with CC compared to patients with TT or TC MAPT rs2471738 genotypes. Also, levels of t‐tau and p‐tau231 were significantly higher in patients with H1H2 or H2H2 haplotypes compared to patients with the H1H1 haplotype.This preliminary study of a Croatian cohort investigated whether certain variants of MAPT rs242557 . Although Compta et al. compared the levels of t‐tau and p‐tau181 in PD patients with different MAPT rs1880753, rs1880756, rs1800547, rs1467067, rs242557, rs2471738, and rs7521 genotypes, they found no significant differences in t‐tau and p‐tau181 levels in contrast to the present study that reveals t‐tau and p‐tau levels to be altered in patients with different MAPT rs1467967 and rs2471738 genotypes. Elias‐Sonnenschein et al.  could occur through MAPT expression. In other words, individuals carrying risk MAPT alleles have a higher MAPT brain expression and consequently an increased neurodegeneration and leakage of tau protein in CSF  differs from the risk allele detected in the study of Myers et al. , while previous studies testing the association of MAPT polymorphisms with AD used only core CSF biomarkers as endophenotypes . The p‐tau231 endophenotype showed significant difference between groups of patients with different MAPT genotypes. Finally, we detected 23 H1 and 5 H2 MAPT subhaplotypes in this Croatian cohort, revealing that MAPT haplotype‐tagging polymorphisms and MAPT haplotypes should be further tested as potential genetic biomarkers of AD.In conclusion, the present study resulted in several notable findings. The association of s et al.  and Vázqs et al.  (T‐allels et al. , and thiThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.None declared.MAPT haplotypes. MNP and NP determined MAPT genotypes. MBL and GŠ determined levels of CSF biomarkers. MBL, ZS and GŠ completed statistical analysis. PRH substantially contributed to the interpretation of data and to manuscript preparation. All authors contributed to revising and editing the manuscript critically for important intellectual content. All authors read and approved the final version of the manuscript. All authors met the criteria for authorship, as defined by the International Committee of Medical Journal Editors.GŠ conceived and directed the study. NK and FB performed the clinical assessments and lumbar puncture. NW and RdS determined


The neuroactive mycotoxin lolitrem B causes a neurological syndrome in grazing livestock resulting in hyperexcitability, muscle tremors, ataxia and, in severe cases, clonic seizures and death. To define the effects of the major toxin lolitrem B in the brain, a functional metabolomic study was undertaken in which motor coordination and tremor were quantified and metabolomic profiling undertaken to determine relative abundance of both toxin and key neurotransmitters in various brain regions in male mice. Marked differences were observed in the duration of tremor and coordination between lolitrem B pathway members, with some showing protracted effects and others none at all. Lolitrem B was identified in liver, kidney, cerebral cortex and thalamus but not in brainstem or cerebellum which were hypothesised previously to be the primary site of action. Metabolomic profiling showed significant variation in specific neurotransmitter and amino acid profiles over time. This study demonstrates accumulation of lolitrem B in the brain, with non-detectable levels of toxin in the brainstem and cerebellum, inducing alterations in metabolites such as tyrosine, suggesting a dynamic catecholaminergic response over time. Temporal characterisation of key pathways in the pathophysiological response of lolitrem B in the brain were also identified. Lolium perenne L.) pastures containing the indole diterpenoid toxin lolitrem B1. The toxin is produced by an endophytic fungus, Epichloë festucae var. lolii, that lives symbiotically within its perennial ryegrass host. The syndrome has been well documented in livestock and other herbivores4 and isolated lolitrem B has been shown to cause a sustained tremor response in both sheep and mouse models of the disease7. Pastures associated with toxic outbreaks contain a number of potentially neuroactive metabolites of the lolitrem biosynthetic pathway8 and currently their role in the presentation, severity and duration of the clinical signs, their location, and mode of action in the brain has not been well defined.‘Perennial ryegrass staggers’ is associated with ingestion of perennial ryegrass (E. festucae var. lolii) produces a number of important biochemical intermediates which also have biological functions, including protection from herbivory and drought tolerance11. The non-tremorgenic compounds paspaline and terpendole E12 represent a key divergence of two biosynthetic pathways, one of which will terminate with the tremorgenic diterpenoid toxins penitrem D13, janthitrem C15, aflatrem5 and the other which produces lolitrem B and terpendole K16 as endpoints 17. BK channel inhibitors have many pharmaceutical applications, which have resulted in possible development of indole-diterpenes in drug design and discovery21. BK channels are found ubiquitously throughout the brain but are highly expressed in both the cerebellum and cerebral hemispheres23 and are suggested to constitute a primary target for these compounds. The cerebral cortex comprises a number of regions that regulate emotion, cognition, language, memory, homeostasis as well as auditory and visual areas24. BK channels are involved in the regulation of neurotransmitter release and neuronal excitability23 thus a blockade of these channels would cause downstream biological effects which have not yet been characterised in relation to lolitrems.The sustained tremors elicited by lolitrem B and its reversible response is considered to be a phenomena that has pharmacological significance17. Also, the differences displayed by the in vitro interaction are in correlation with the in vivo response of these compounds, such as the duration of tremor exhibited and the affinity of the compounds to the receptor. This suggests that motor function deficits induced by lolitrems are mediated by BK channels17. However, lolitrem E and paspalicine although showing potent BK channel activity, elicit a non-tremorgenic effect on animals. This could be related to structural changes occurring in vivo, rendering it less active25. Knaus et al., suggested that although some pharmacological properties could be explained by BK channel inhibition, tremorgenicity may not be directly related to channel blockade26. Thus, it is possible that the relationship between tremor and BK channel activity is more of a complex interaction. This is further supported by the wide range of neurological observations in animals suffering from lolitrem B toxicosis, which are not restricted to movement, but include changes in behavior4. Thus, the hypothesis that lolitrem B affects, in part, neurotransmitter receptors leading to interference with neurotransmitter release in the central and peripheral nervous system needs to be examined.The inhibition of BK channel currents by the indole-diterpene molecules suggest that these receptors are most likely the major molecular target for these compounds. It has been reported that mice deficient in BK ion channels are unaffected by these neurotoxins at concentrations that are lethal to wild-type mice29. Together these data suggest region specific effects for this toxin in the brain, the effects of which have not been previously investigated.Also, early work investigating the mode of action of the mycotoxins penitrem A and verruculogen shows that these toxins interfere with amino acid neurotransmitter release mechanisms in the central nervous system31. Quantitation of concentration of lolitrem B in the brain was determined and neurotransmitter and amino acid expression compared in four key regions of the brain: cerebral cortex, thalamus, cerebellum and brainstem at the two time points. Principle components analysis (PCA) was undertaken to identify key determinants of effect.To determine the effects of lolitrem B, and members of the indole diterpenoid biosynthesis pathway, on neurotransmitter and amino acid levels in the brain, a functional metabolomics study was undertaken. Adult mice were exposed to purified lolitrem B, and biosynthetic pathway members terpendole B, C and E, at two doses. Time to peak tremor was determined with tissue samples collected for analysis at the onset of peak tremor and 24 h later. Motor coordination was measured using the accelerating rotarod testin vivo suggest a primary mode of action of the toxin in catecholaminergic neurotransmitter pathway production, recycling or synthesis, a correlation that has not been previously demonstrated. This study identifies both region and metabolic specific action of the lolitrem B toxin in the brain, effects that may contribute to the clinical signs observed in animals affected by ingestion of toxic pastures or in controlled animal models.This study showed that lolitrem B exerted region specific effects on neurotransmitter and amino acid levels in the brain of intoxicated mice with the cerebral cortex showing greatest changes in response to toxic exposure. These data suggest the cerebrum, a region that is involved in cognition, emotion and mental activity, to be an important site of action of lolitrem B toxin in affected animals. This hypothesis was further supported by the high levels of toxin found in this region. Furthermore, alteration in neurotransmitter levels observed in the lolitrem B-exposed brain To determine the neurotoxic activity of lolitrem B pathway components, motor coordination and tremor were tested in response to treatment with four selected indole-diterpenoids, lolitrem B, terpendoles B, C and E Figs –3.FigureHIGH and LolBLOW mice showed increased motion power percentage (MPP) indicative of tremor  = 53.99; p < 0.0001) and of time  = 4.15; p < 0.0001). However, the interaction between time and treatment was not significant  = 1.125; p = 0.25) reflecting prolonged and sustained tremors detected over the 24 h period. Peak tremor occurred in LolBHIGH mice at 6 h post treatment with animals exhibiting an MPP of 80% ± 0.04% compared to ControlVEH . LolBHIGH mice showed tremors for the duration of the study and tremors remained significant at 24 h (MPP = 77 ± 0.04%) compared to ControlVEH . LolBLOW mice showed peak tremor at 4 h post treatment (MPP of 76% ± 0.03%) compared to ControlVEH . Tremors for LolBLOW mice also lasted for 24 h post treatment (MPP = 71% ± 0.01) compared to ControlVEH . A dose-dependent response was observed with LolBHIGH mice showing significantly greater tremor response than LolBLOW counterparts. Control and ControlVEH mice showed no tremor response at any time point tested.Both LolBmor Fig. , compareF = 54.33; p < 0.0001) but no significant effect of time  = 1.33; p = 0.16) or interaction between time and treatment  = 1.16; p = 0.21). LolBHIGH mice exhibited a significantly reduced latency to falling for the first eight time points of the testing period with peak effects observed at 2.7 to 5 h post treatment compared to ControlVEH    suggesting that DMSO is not an inert carrier  Fig. . LolBLOW06) Fig. . There w06) Fig. . Controlier Fig. .32. Analysis by two-way ANOVA revealed a significant effect of treatment  = 7.444 p < 0.0001), time  and interaction between time and treatment  = 4.265; p < 0.0001). Peak tremor occurred in TerpCHIGH mice at 24 min post-treatment with mice exhibiting an MPP of 80% ± 0.06% compared to ControlVEH . Significant tremor was recorded until 36 min post injection . Although MPP% was similar in TerpCLOW and TerpCHIGH mice at 12 min post treatment, low dose mice rapidly returned to control levels by 24 min.Mice treated with terpendole C exhibited tremor but the profile of effect was markedly different to lolitrem B. Terpendole C has previously been identified to be a fast-acting tremorgenion Fig.  after whF = 59.84; p < 0.0001), time  = 11.13; p < 0.0001) and interaction between time and treatment  = 2.657; p < 0.0001). TerpCHIGH showed the greatest motor deficits from 12 to 36 min compared to ControlVEH  which is consistent with the tremor profile of this toxin. TerpCLOW treated mice also exhibited significantly reduced latency to falling compared to ControlVEH with peak effects also observed in the first 36 min   for TerpCHIGH and 72 min (p = 0.019) for TerpCLOW compared to ControlVEH.Similar to lolitrem B treated mice, loss of motor coordination was also observed in a dose-dependent manner for terpendole C treated mice Fig. . Analysi42) Fig. . However33 and is a biosynthetic precursor to terpendole B. Terpendole B has been previously isolated, however; its tremorgenic activity has not been previously investigated. Our data confirm that terpendole E is non-tremorgenic and reveal that terpendole B showed no significant tremorgenic activity at any dose tested  = 32.65 p < 0.0001), time  = 55.01; p < 0.0001) and interaction between time and treatment  = 9.779; p < 0.0001). A similar tremor profile was observed in mice used for metabolomics profiling as compared to their counterparts in the behavioural study.To confirm the tremor intensities ascertained for lolitrem B in the behavioural study Fig. , tremor Although lolitrem B has been extensively postulated to exert its effect via interaction with BK channel receptors in the brain, presence of the parent compound or its bioactive metabolites, has not been confirmed in this tissue. To address this, quantitation of lolitrem B was undertaken in dissected brain tissue: cerebral cortex, thalamus, cerebellum and brain stem, and the distribution of toxin examined in comparison to levels found in the liver and kidney. Bioaccumulation, or elimination, were investigated over time in these tissues.HIGH mice at 6 h post treatment, lolitrem B was also identified in the cerebral cortex (13.2 ± 2.2 pg/g) and thalamus (8.8 ± 1.4 pg/g)  Table . No signg) Table .Figure 5HIGH and ControlVEH at 6 h. None of the samples fell outside the 95% confidence limit (blue dashed line). Loadings plots were generated to identify differentially expressed metabolites contributing to the variation. Statistical significance of these data were determined by Student’s t-test.To determine the metabolic effects of lolitrem B, brain tissues were analysed using targeted LCMS. Metabolic variation with respect to amino acid and neurotransmitter expression was analysed using PCA score plots Fig.  of the cThe amino acid metabolites that exerted the greatest influence across all the brain regions include isoleucine, leucine and valine whose concentrations were increased compared to controls; tyrosine was decreased compared to controls. Also, with the exception of the brainstem, all other regions of the brain show a pronounced decrease in concentration of methionine.p = 0.0001), leucine (p = 0.0008) and isoleucine (p = 0.020) and the major catecholamine precursor, tyrosine (p = 0.039). Methionine (p = 0.022) also contributed to the separation illustrated in the PCA score plot. Compared to the ControlVEH the branched chain amino acid valine in the thalamus  and cerebellum  region was also significantly different. The discriminating metabolites in the thalamus region were tyrosine (p = 0.035) and methionine (p = 0.021). Branched chain amino acids  contributed to the separation in the brainstem region.The strong separation in the cerebral cortex could be attributed to the branched chain amino acids, valine . Together these data demonstrate the impact of lolitrem B intoxication on brain metabolism at peak tremor (6 h post injection), with lolitrem B exerting the greatest effect on the cerebral cortex.Temporal metabolic variation suggests metabolites return to a pre-treated state, except for threonine which maintained similar levels at LolBons Fig. . This isLOW, PCA scores plots were generated . The low dose treated mice exhibit weaker variation compared to the high dose mice, however notable effects in the brain are clearly observed. Loadings plots , tyrosine (p = 0.038) and tryptophan (p = 0.049). The thalamus region also showed significant changes in methionine (p = 0.036) and tyrosine (p = 0.018). Other metabolites in the thalamus region that showed pronounced variation are phenylalanine (p = 0.008), tryptophan (p = 0.028), histidine (p = 0.041), glutamine (p = 0.042) and GABA (p = 0.048). The brainstem showed significant changes in methionine (p = 0.039) and tyrosine (p = 0.030). Other metabolites also showed notable changes in the brainstem including threonine (p = 0.008), lysine (p = 0.028), tryptophan (p = 0.049) and histamine (p = 0.004). The significantly affected metabolite in the cerebellum was found to be tyrosine (p = 0.035). Temporal variation suggests metabolites return to a pre-treated state  and tyrosine and methionine strongly influence separation between vehicle control and treatment groups. Thus ROC (receiver-operating characteristic) curves were used to evaluate the specificity and sensitivity; sensitivity and 1-specificity corresponded to the true positive rate and false positive rate, respectivelyLOW brain regions. The SVM model predicted the brainstem region with 75% accuracy (6 out of 8 replicates of LolBLOW 6 h); cerebral cortex and thalamus with 71.4% accuracy (5 out of 7 replicates of LolBLOW 6 h) and cerebellum with 42.8% accuracy (3 out of 7 replicates of LolBLOW 6 h). These results indicate that the SVM model based on the combinatorial biomarkers could effectively predict mild to severe cases of lolitrem B intoxication across most brain regions.To evaluate the predictive performance of the combination of biomarkers, Linear SVM was applied. Based on KM Clustering 4 out of the 5 top biomarkers were selected to build a classification model. The ROC curve generated from the model resulted in an AUC of 0.95 and 0.859–0.991 at 95% confidence interval Fig. . The croin-vivo models. The indole diterpene class of compounds is known to display biological activities based on very specific conformational, structural and stereo-specific features33. The indole diterpenes studied exhibited biological activities consistent with previous reports39. Terpendole B had not been previously tested and, like terpendole E, showed no tremorgenic activity and did not affect coordination or movement in exposed mice. Our results show that rotarod, motor performance and tremor tests provide an effective and sensitive way of measuring indole diterpenoid toxicity in mice in a dose-dependent manner.This study investigated movement and coordination using sensitive screening platforms that enable accurate and effective measures of tremorgenicity and motor coordination deficits in To elucidate the mode and site of action of the indole diterpenoid toxins examined in this study, tremor and rotarod performance was coupled to metabolomics profiling using ESI + LCMS quantitation analysis to determine the effect of lolitrem B exposure on brain metabolism in C57Bl/6J mice at two time points, 6 h and 24 h, post treatment. Our results show that lolitrem B caused a profound disruption in metabolic profile across all regions of the brain, but particularly the cerebral cortex at 6 h and were coincident with high levels of lolitrem B in this brain region.7. Our findings also support a hypothesis that the movement deficits observed in these animals are upstream of the cerebellum, with the cerebellar lesions being secondary via super-excitation of efferent fibres entering the cerebellum from the cranial nerve nuclei or via subcortical movement centres. The highly lipophilic nature of lolitrem B allows for strong association and accumulation of the toxin in fatty tissues42. Strong bioaccumulation, and slow removal of lolitrem B from the brain explains the prolonged tremors observed in animals subsequent to ingestion of toxic pastures. Dysregulation of neurotransmitters and amino acids was observed at 6 h in high and low dose lolitrem B treated mice correlating with this bioaccumulation profile.Recently, the clinical signs presenting in ruminants exposed to lolitrem B toxin have been defined as those of a central rather than cerebellar tremorEffects of toxicity were also observed in a dose-dependent manner with low doses of lolitrem B exerting a lower tremorgenic effect than high doses in mice in this study. Conversely, concentrations of lolitrem B in all brain regions examined at both doses, and both timepoints analysed did not show significant differences, indicating that both low and high levels of toxic exposure cause significant bioaccumulation of toxin in the brain over a short period of time. This bioaccumulation was not, however, associated with significant changes in metabolic profile except in the cerebral cortices.43 similar to those observed in lolitrem B intoxicated animals. The branched-chain amino acids leucine, isoleucine and valine have important biochemical functions in the brain. Particularly, the synthesis of serotonin, dopamine and norepinephrine, which are derived from the aromatic amino acids tryptophan, tyrosine and phenylalanine respectively50. These have wide-ranging effects across multiple neurological systems, including those associated with emotion and behaviour, alterations that are observed in lolitrem B intoxicated animals7.Metabolomic profiling identified that the metabolites exerting the majority of the effect at 6 h were isoleucine, leucine, valine, tyrosine and methionine. Isoleucine, leucine, and valine were increased compared to controls, with tyrosine and methionine decreased compared to controls. Decreased levels of tyrosine, the precursor of dopamine, may influence catecholamine synthesis. Low levels of dopamine in Parkinson’s disease and similar central neurodegenerative disorders are associated with symptoms of hypokinesia, tremor, rigidity, and abnormal posture50. Increased levels of branched-chain amino acids found in metabolic diseases such as maple syrup urine disease results in serious neurological disorders such as ataxia and lethargy52.Branched-chain amino acids compete for transport across the blood-brain barrier with aromatic amino acids. It has been reported that a rise in plasma branched-chain amino acid concentrations are known to have a direct effect on the synthesis and the release of biogenic amines derived from these aromatic amino acids, notably serotonin (from tryptophan) and catecholamines, dopamine and norepinephrine , in the brain54. The kidney, heavily intoxicated with lolitrem B at 6 h, could have had impaired capacity to catabolise branched-chain amino acids which may then accumulate. This may be the cause of the increase in these amino acids in the brain as observed in this study and be directly affecting catecholamine synthesis pathways in the brain.Metabolism of complex compounds and amino acids occurs primarily in the liver. However, many branched-chain amino acids are catabolised in non-hepatic tissues such as cardiac muscle and kidney50. In this study we observed a strong reduction in levels of tyrosine and mild decreases in tryptophan and phenylalanine with variable expression of serotonin and dopamine with region and time-dependent differences. Cellular differences are, however, not measurable by this analysis and only changes in the overall metabolomic pool can be detected. Since neurotransmitters are present in such low abundances, small changes at localised sites are not reliably detected56. Further experiments would be required to understand the effect of lolitrem B at a cellular level.A summary of the major metabolic pathways perturbed in the cerebral cortex, identified in our analysis, resulting from lolitrem B exposure is shown in Fig. 57. Methionine has been reported to modify dopamine levels and alleviate symptoms of Parkinson’s disease58. However, excess levels of S-adenosylmethionine (SAM), a precursor of methionine, has also been reported to deplete dopamine levels, inducing symptoms characteristic of Parkinson’s disease43. Therefore alteration in this metabolic pathway could be underlying the tremor phenotype observed in the mice in this study. In a pathological state, such as infection and cancer59, amino acids as substrates are in high demand for energy production59. Mice in this study exhibited an overall increase in amino acids, particularly in the cerebral cortex. The strong metabolic variation induced by lolitrem B is either implicated in the catecholamine pathway or neuromodulatory reaction, suggesting increased excitatory activity in the brain.Decreased levels of methionine across all brain regions and treatments were observed in this studyThe temporal variation in the metabolome observed in this study shows that whilst peak tremor (6 h) triggers a clear perturbation in multiple pathways, the majority of those key metabolites show a clear return to a pre-treated state at 24 h. This correlates with the tremor profile in these mice where tremor was visibly reduced at 24 h compared to 6 h post exposure, suggesting a moderation of neurotransmitter production or recycling, despite no observable difference in toxin concentrations. Study of the binding kinetics of lolitrem B and its pathway intermediates to BK channel receptors would further elucidate the mode of action of this toxin and its potential uses for pharmacological purposes.In conclusion, this study is the first functional metabolomics analysis of any naturally occurring toxin in a rodent model. Our data identified lolitrem B synthetic pathway members that had both tremorgenic and non-tremorgenic activity. Key differences were identified in concentration of the toxin in different parts of the brain, with highest concentrations observed in the cerebral cortex and levels below the limit of detection in the cerebellum. Metabolomics profiling identified key differences in neurotransmitter and amino acid profiles between different brain regions, and over time, with greatest differences observed at 6 h post exposure, the point of greatest tremor intensity. Dysregulation of catecholaminergic neurotransmitter pathways appear to be delivering the majority of effect in the forebrain with both up and down dysregulation of key biosynthetic precursors identified. Future studies should be undertaken to further understand the bioaccumulation of lolitrem B toxin in the brain and its receptor binding kinetics in order to determine potential pharmacological uses for this potent family of neuroactive toxins.60 and were shown to be at least 98% pure by NMR spectroscopy. Terpendole C (98% pure) was purchased from Jomar Life Research (sc-391042) and terpendole E (98% pure) sourced from Sigma Aldrich (SML1127). All toxins were administered by intraperitoneal (i.p.) injection in the vehicle carrier 9:1 (v/v) dimethyl sulfoxide (DMSO: H2O)33.Lolitrem B and terpendole B were isolated and purified as previously describedLOW) and 2.0 mg/kg (LolBHIGH). These doses correspond to hypothesized subclinical (low) doses and known disease-inducing (high) concentrations subsequent to ingestion in pasture-fed herbivores61. Terpendole C was administered at 2.0 (TerpCLOW) and 4.0 mg/kg (TerpCHIGH) and terpendole E at 8 (TerpELOW) and 16 mg/kg (TerpEHIGH). Terpendole B was only available in very small quantities and a single dose was therefore used: 0.5 mg/kg (TerpB). Two negative control groups were used for comparison of tremor and behaviour: (1) an untreated control (Control), and (2) a vehicle control (ControlVEH) which had been treated with the intraperitoneal (i.p.) injection vehicle 9:1 (v/v) DMSO: H2O only.Lolitrem B was administered at 0.5 (LolBad libitum. Ambient temperature of housing and testing rooms was 21 ± 2 °C and mice were housed under a 12 h light–dark cycle (lights on at 7 am). A total of 184 male 8–9 week old C57Bl/6J mice were sourced from a breeding colony at the Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria. Mice were allowed to acclimatise to the facility conditions for a period of 1 week prior to behavioural testing. All mice underwent a series of behavioural tests to examine effects of intoxication on tremor and movement.All mouse studies were approved by the La Trobe University Animal Ethics Committee (Protocol number 15–87) and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes set out by the National Health and Medical Research Council of Australia. The mice were housed in groups of two to four during the experimental period in individually-ventilated cages  with standard pellet food and water available 5, tremor testing and rotarod tests for lolitrem B treated mice were conducted in 40 min intervals for 12 h, then again at 24 h post treatment. For terpendole C, measurements were conducted at 12 min intervals for 2 h post treatment. For terpendole B and E, tremor and rotarod activity were undertaken at 40 min intervals for 8 h post treatment before final testing and euthanasia at 30 h.To capture peak neurotoxic response and measure return to baseline for both tremor analysis and coordination62. Data from the sensor were converted through an AC amplifier  with a bandpass of 1–50 Hz applied and a sampling rate of 100 samples/s. Motion power output was recorded digitally for 3 min and analysed for change of frequency and power using LabChart™ 7.0 .To measure frequency and amplitude of tremor associated with intoxication, mice were placed individually into a closed container mounted on a piezo-electric pulse transducer sensor  to convert force applied to the surface of the transducer into an analog signal2)) between 0 and 45 Hz by Fast-Fourier-Transformation (FFT). Control mice showed peak activity defined by a 0 and 10 Hz bandwidth, representing normal movement. All tremorgenic mycotoxins tested induced a prominent peak in the motion power spectrum between 10 and 30 Hz, with reduction in power output at other frequencies  for each 3 min epoch were converted to a power output spectrum (Frequency (Hz) vs. Power . Mice were first trained to use the apparatus over 8–10 trials, with 20 min between each trial, or until 3 stable baseline performances had been achieved. For each test, acceleration was applied from 4 to 40 rpm over 120 s then held constant for a further 60 s and latency to falling recorded. Mice that rotated passively were deemed to have fallen.2 before storage at −80 °C for metabolomics analysis.Brain tissues of mice exposed to low (0.5 mg/kg) or high (2.0 mg/kg) doses of lolitrem B were collected at either 6 h or 24 h post-treatment and compared to untreated controls or vehicle injected controls (n = 8 at each time point) by metabolomic analysis. To confirm the intoxication phenotype was consistent to previous analyses, all mice were subjected to tremor testing 5 min prior to euthanasia and tremor data compared to data collected from the behavioural analysis described above. Mice were euthanized by cervical dislocation and tissues were collected, including liver, kidney, and the brain . Samples were weighed and snap-frozen in liquid N63. Briefly, methanol:H2O  was added to tissue powder and vortex-mixed prior to addition of dichloromethane (DCM) . Samples were then sonicated on ice before addition of 1:1 v/v DCM:H2O. Samples were centrifuged for 10 min at 13,000 rpm and the upper aqueous layer transferred into HPLC vials ready for LCMS analysis of polar metabolites. An aliquot (10 μl) of each sample was combined to generate a pooled biological quality control (PBQC) sample, which was used to monitor analytical reproducibility. Two PBQC samples were prepared for each tissue type . The solvent layer was then transferred to a separate tube and evaporated under a stream of N2. Solvent extracts were also analysed to determine lolitrem B concentration by LCMS.Frozen samples of kidney, liver, thalamus and cerebral cortex were transferred into 4 mL polycarbonate tubes with 3/8″ stainless steel grinding balls and kept frozen in liquid nitrogen. Sample tubes were placed into pre-frozen 24 well cryo-blocks on the Geno/Grinder 2010  and the tissues were homogenised at 1,750 rpm for 1 min. The fine powder was stored at −80 °C until ready to be weighed. Kidney and liver samples (50–52 mg) as well as cerebral cortex and thalamus (20–22 mg) samples were each weighed in 2 mL Eppendorf tubes. Liver, kidney, cerebral cortex and thalamus samples of known weight were extracted using a modified Bligh-Dyer extraction method2O was added to each sample and placed in a Precellys 24-tissue homogeniser  fitted to a Cryolys N2 Cooling System. When the temperature reached −20 °C, samples were homogenized at a speed of 5,500 rpm over 3 consecutive 20 s cycles with 30 s intervals. Cerebellum and brainstem were subsequently extracted with 4:1 v/v methanol: H2O (40 µL/mg sample) and the aqueous phase transferred into HPLC vials with inserts for LCMS analysis. Quality controls were prepared as before. In addition, an aliquot of the remaining extract was evaporated under N2 before freeze drying for NMR analysis.The sample sizes of the cerebellum and brainstem were smaller (30–70 mg) than other brain regions collected so a modified method was used. Cerebellum and brainstem were placed directly in pre-weighed Lysing matrix D tubes  prefilled with 1.4 mm ceramic beads. For homogenization, 1 mL of cold 4:1 v/v methanol: HLCMS data were analysed in Thermo Xcalibur Qual Browser v.2.3.26 (Thermo Fisher Scientific™). Assessment of peak retention time and ion extraction window (m/z) confirmed the presence of the targeted polar compounds within the standard mixes.All extracts were analysed on a Vanquish Ultra-High Performance Liquid Chromatography (UHPLC) system  with a binary pump, autosampler and temperature-controlled column compartment coupled with a QExactive (QE) Plus mass spectrometer  detector. The Thermo Fisher QExactive Plus mass spectrometer was set at FT switching positive and negative mode over a mass range of 70–1,200 amu with resolution set at 35,000. Nitrogen was used as the sheath, auxillary and sweep gas at a flow rate of 28, 15 and 4 L/min, respectively and spray voltage was set at 3,600 V (positive) and 3,300 V (negative). Samples were randomized, and blanks were run every 5 samples. A PBQC was run every 10 samples. Prior to data acquisition, the system was calibrated with Pierce® LTQ Velos ESI Positive and Negative Ion Calibration Solution (Thermo Fisher Scientific™). Mass spectrometry data was acquired using Thermo Xcalibur V. 2.1 . Quantitative analysis was conducted using LCQUAN™ Quantitative Software (Thermo Fisher Scientific™).2O), and B (0.1% formic acid: acetonitrile) at 0.5 mL/min with 98% A to 0% A over 11 min with a linear gradient. All amino acids and neurotransmitter standards were purchased from Sigma-Aldrich. For quantitative analysis, standard curves were prepared in H2O for alanine (Ala), asparagine (Asn), aspartic acid (Asp), arginine (Arg), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine  and 50% acetonitrile/water for 10 biogenic amines: dopamine (DA), epinephrine, GABA, histamine (HA), N-methylphenethylamine, norepinephrine, octopamine, phenethylamine, serotonin (5-HT) and tryptamine in a range of concentrations from 0.0001 to 30 µg/mL.Polar metabolites were eluted from the column (Phenomenex 250 × 4.6 mm 4 µm Synergi HPLC column) using a gradient mobile phase, A (0.1% formic acid: H2O) and B (0.1% formic acid in 55% isopropanol (IPA) in acetonitrile) at 0.5 mL/min with 98% to 0% A over 11 min. Two standard curves were prepared for lolitrem B, a high concentration range of 6 to 2400 ng/mL and low concentration range from 0.5 to 60 ng/mL. Assessment of peak retention time and ion extraction window (m/z) on Thermo Xcalibur Qual Browser v.2.3.26 (Thermo Fisher Scientific™) confirmed the presence of lolitrem B. Spike recovery analysis was performed for 300 and 600 ng/mL standards of lolitrem B for each tissue type in duplicate. Limit of detection (LoD) of lolitrem B by this method was 0.8 ng/mL.To determine concentrations of lolitrem B, reconstituted tissue extracts were eluted from the column  using a gradient mobile phase, A . Data were analyzed by two-way ANOVA followed by uncorrected Fisher’s LSD test for multigroup comparisons against vehicle control. Data are expressed as the mean ± SEM. The threshold of P values were calculated using unpaired Student’s t-test data using the MetaboAnalyst 3.064. The threshold of p < 0.05 was designated as statistically significant for all tests. A multivariate statistical approach was also employed to assess differentially expressed LCMS targeted metabolites on the normalized data. An unsupervised principal component analysis (PCA) was carried out on the quantitation data in MatLab using PLStoolbox_731. The Hotelling T2 statistic and the Q residuals were used to indicate how well each sample conformed to the PCA model as well as detect residual outliers in the datasets. The optimum values of the Q residuals and the Hotelling T2 value are 0% and 100%, respectively65. The Q residual is a measure of the difference, or residual, between a sample and its projection into the k principal components used to build the model66. The Hotelling T2 statistic is a measure of the variation in each sample within the PCA model, or otherwise defined as a measure of the distance from the multivariate mean to the projection of the sample onto the k principal components67.For metabolomics analysis, data were normalised by log transformation (log 10) and mean centering pre-processing. t tests (p < 0.05) for metabolite ratios for each tissue were ranked based on AUC values. All specific biomarker candidates were subjected to linear SVM (support vector machine) to establish a statistical prediction model.To discriminate effects between control and treatment groups a Univariate Receiver Operating Characteristic (ROC) Curve analysis was applied using MetaboAnalyst 3.0. A prediction model was developed, and data were log2 transformed/normalised with no scaling applied. ROC curve model evaluation was selected, and the top four features were identified for each treatment (Table Supplementary information