Neuron-specific Protein Research Articles

Mechanism of mild hypothermia promoting nerve regeneration after traumatic brain injury in rats

Objective To investigate whether mild hypothermia can promote neurogenesis in the dentate gyrus of hippocampus and cognitive function recovery after traumatic brain injury (TBI) through inhibiting apoptosis of hippocampal neurons. Methods A total of 66 healthy adult Sprague-Dawley rats were randomly divided into sham group, TBI group and TBI+ hypothermia group, with 22 rats in each group. The rat TBI model was established using the fluid percussion device. The rats in TBI+ hypothermia group received 4-hour hypothermia therapy immediately after injury, with the target temperature of 33.5℃. Bromodeoxyuridine (BrdU) was injected into the rats' abdominal cavity to label the mitotic cells. The test of Morris water maze was used to evaluate the rats' spatial learning and memory capabilities. Immunofluorescence staining was used to observe the expression levels of BrdU, doublecortin (DCX), neuron specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3) and cleaved caspase-3 expressions in dentate gyrus of hippocampus at 7 days and 28 days after injury. Expressions apoptosis-related proteins including the factor associated suicide (FAS)/factor associated suicide ligand (FASL), B-cell lymphoma-2 (Bcl-2), caspase-3 and cleaved caspase-3 expressions were detected by Western blot assay. Results The water maze tests at 28 days after injury showed that compared with TBI group, the escape latency in TBI+ hypothermia group was significantly shorter [(24.2±5.9)s∶(18±4.1)s], and both the time in the target quadrant and the number of platform crossing were increased significantly [(24.9±6.5)s∶(31.7±5.2)s; (1.9±0.8) times∶(3.5±1.2)times](P<0.05). Compared with the sham group, in TBI group and TBI+ hypothermia group, the BrdU+ new-born cells in the dentate gyrus of hippocampus were significantly increased at 7 days after injury [(9.4±4.1)∶(33.4±3.8); (9.4±4.1)∶(45.8±5.6)], the BrdU+ /DCX+ new-born neurons were increased at 7 days after injury [(2.0±0.6)∶(9.6±1.6); (2.0±0.6)∶(19.2±3.7)], and the BrdU+ /NeuN+ mature neurons were increased at 28 days after injury [(2.6±1.0)∶(17.2±3.9); (2.6±1.0)∶(33.6±9.1)] (P<0.01). TBI group showed more obvious increase than the TBI+ hypothermia group (P<0.01). Moreover, compared with 7 days after injury, the number of BrdU+ cells at 28 days after injury was further increased in TBI+ hypothermia group but decreased in TBI group [(45.8±5.6)∶(58.8±9.2); (33.4±3.8)∶(22.0±3.5)](P<0.05 or <0.01). Compared with the sham group, the caspase-3+ NeuN+ and caspase-3+ NeuN+ apoptotic neurons were significantly increased at 7 days after injury in TBI group [(2.0±0.9)∶(11.6±2.6); (2.6±1.0)∶(10.2±2.9)] (P<0.05). Compared with the TBI group, the cleaved caspase-3+ NeuN+ apoptotic neurons were decreased in TBI+ hypothermia group [(6.6±2.0)∶(11.6±2.6)](P<0.05). Furthermore, compared with the TBI group, mild hypothermia might down-regulate the expression of FAS, FASL, cleaved caspase-3 and caspase-3 and up-regulate the expression of Bcl-2 in the hippocampus [(1.54±0.15) ∶(1.14±0.12); (1.06±0.04)∶(0.80±0.09); (0.84±0.03)∶(0.62±0.08); (0.93±0.06)∶(0.86±0.09); (0.71±0.01)∶(1.58±0.18)](P<0.05). Conclusions Mild hypothermia might inhibit apoptosis of hippocampal neurons through cleaved caspase-3, FAS/FASL and Bcl-2 pathways, thus improving the neurogenesis and maturation of neurons in the dentate gyrus of hippocampus and facilitating cognitive function recovery in rats. It indicates that the function of hypothermia in anti-apoptosis and neurogenesis and maturity of hippocampal neurons may have a potential role in predicting the prognosis of TBI patients. Key words: Hypothermia; Brain injuries; Hippocampus; Neuve regeneration; Apoptosis

A novel transgenic zebrafish line allows for in vivo quantification of autophagic activity in neurons

ABSTRACT The pathophysiology of most neurodegenerative diseases includes aberrant accumulation of protein aggregates. Recent evidence highlights the role of protein degradation pathways in neurodegeneration. Concurrently, genetic tools have been generated to enable zebrafish, Danio rerio, to be used as an animal model to study neurodegenerative processes. In addition to optical clarity and fast ex utero development, the zebrafish brain is relatively small and has conserved structures with its mammalian counterparts. To take advantage of this model organism and to aid further studies on autophagy and neurodegeneration, we created a stable transgenic zebrafish line that expresses eGFP-Map1lc3b specifically in post-mitotic neurons under the elavl3 promoter. This line is useful for indirectly monitoring autophagic activity in neurons in vivo and screening for macroautophagy/autophagy-modulating compounds. We determined the applicability of this transgenic line by modulating and quantifying the number of autophagosomes via treatment with a known autophagy inducer (rapamycin) and inhibitors (3-methyladenine, protease inhibitors). Additionally, we proposed an in vivo method for quantifying rates of autophagosome accumulation, which can be used to infer occurrence of autophagic flux. Last, we tested two FDA-approved drugs currently undergoing clinical studies for Parkinson disease, isradipine and nilotinib, and found that isradipine did not modulate autophagy, whereas nilotinib induced both autophagosome number and autophagic flux. It is hoped that others will find this line useful as an in vivo vertebrate model to find or validate autophagy modulators that might be used to halt the progression of neurodegenerative diseases. Abbreviations 3MA: 3-methyladenine; BafA: bafilomycin A1; dd: dorsal diencephalon; dpf: days post fertilization; e: eye; eGFP: enhanced green fluorescent protein; Elavl3: ELAV like neuron-specific RNA binding protein 3; FDA: Food and Drug Administration; hb: habenula; hpt, hours post treatment; Map1lc3b: microtubule-associated protein 1 light chain 3 beta; nt: neural tube; ot, optic tectum; P/E: pepstatin A and E64d; PD: Parkinson disease; PMTs: photomultiplier tubes; PTU: 1-phenyl-2-thiourea; Ta: annealing temperature; Tel, telencephalon

Dynamics of blood brain barrier permeability and tissue microstructure following controlled cortical impact injury in rat: A dynamic contrast-enhanced magnetic resonance imaging and diffusion kurtosis imaging study

ObjectiveThe blood-brain barrier (BBB) and cerebral tissue microstructure can be impaired following traumatic brain injury (TBI). However, the spatiotemporal changes of BBB leakage and tissue microstructure are not completely understood. In this study, we evaluated the spatiotemporal changes of BBB leakage and tissue microstructure using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and diffusion kurtosis imaging (DKI) in controlled cortical impact (CCI) rats. Materials and methodsThe DCE-MRI parameters volume transfer coefficient (Ktrans) and DKI parameters were longitudinally measured in bilateral cortex, hippocampus, thalamus and corpus callosum (CC) at baseline (D0), acute stage (D1, D3), and subacute stage (D7, D14 and D28) post-injury. Immunohistochemistry analysis was performed at D28 after MRI scanning. Repeated-measures ANOVA was used to assess the temporal changes of MRI parameters. ResultsKtrans abnormality was only localized to ipsilateral perilesional cortex with a significant temporal change (F = 144.2, p < 0.0001). Compared to baseline, increased mean kurtosis (MK) was observed in ipsilateral regions of cortex and hippocampus and CC for all the time points (p < 0.05 for all). Increased MK was also observed in ipsilateral thalamus (p = 0.005) at subacute stage but not at acute stage while no change was observed with MD and FA (p > 0.05 for both). In ipsilateral cortex, the overall Ktrans value of D0, D1, D3, D7, D14, and D28 post-injury were significantly correlated with MK value (r = 0.84, p < 0.0001). The CCI group showed higher staining of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba-1) and lower staining of neuron-specific nuclear protein (NeuN) and myelin basic protein (MBP) in ipsilateral regions of cortex, hippocampus, thalamus and CC (p < 0.05 for all) as compared to control group. There were no significant differences in the contralateral regions by immunohistochemistry. ConclusionThe BBB disruption reflected by Ktrans correlated well with MK value in ipsilateral cortex. In addition, MK could detect the delayed microstructural changes in thalamus. DCE-MRI and DKI could be used to assess the BBB breakdown and cerebral microstructural changes of TBI.

Regulation effects of Ngn2 gene transfection on retinal neuron differentiation in three-dimensional optic vesicle of mice

Objective To investigate the regulation effects of Ngn2 gene transfection on retinal neuron differnetion in three-dimentional optic vesicle (OV) of mice. Methods OV was cultured in vitro using mouse induced pluripotent stem cells (iPSC) under specific conditions.During OV culture, it was transfected multiple times by lentivirus-mediated Ngn2 gene and then it was induced after maturation.The cells were specificly differentiated toward retinal nerve cells in OV.Using the green fluorescent protein (EGFP) gene as control, the differentiation of retinal nerve cells in OV was detected by immunohistochemistry.Reverse transcription PCR and Western blot were used to quantitatively detect the expressions of retinal neuron-specific proteins Pax6, Islet1 and Brn3b. Results The mouse iPS-derived OV was successfully cultured.The number of neural cells in the OV transfected with the Ngn2 gene was increased by the lentiviral-mediated lentivirus.The expressions of PAX6, Islet1 and Brn3b in the Ngn2 transfection group were significantly higher at the gene and protein levels than those in the control group, with significant differences between the two groups (P<0.05). Conclusions The Ngn2 gene can effectively increase the number of retinal neuron differentiation in OV and make in vitro cultured OV more mature and form a more perfect retinal cell neural circuit. Key words: Neuroretina; Ngn2 genes; Optic vesicle; differentiation; Gene transfection

Chapter 13 - Biomarkers in neonatal hypoxic–ischemic encephalopathy—Review of the literature to date and future directions for research

The widespread introduction of therapeutic hypothermia as a standard of care in hypoxic-ischemic encephalopathy (HIE) has brought increasing pressure on clinicians to make an early and accurate assessment of the degree of hypoxic injury (HI) that has occurred and the severity of the encephalopathy that will ensue. No single blood-based marker is currently robust enough to detect significant HI or predict outcome. However, research in the field has been active in the last 10 years and we know that HIE is associated with predictable alterations in the expression of a number of inflammatory proteins, neuron-specific proteins, metabolite pathways, and microRNA. These alterations evolve quickly over the first hours and days of life. Predictive power varies depending on the timing of measurement of the biomarker, the sample type, and the case mix of the cohort examined. Combining clinical data with biochemical measurements is currently the most likely path toward improved detection and prediction of outcome in neonatal HIE.

Adiponectin Attenuates the Inflammation in Atopic Dermatitis-Like Reconstructed Human Epidermis.

BackgroundAtopic dermatitis (AD) is a chronic disorder, with a vicious cycle of repetitive inflammation and deterioration of the epidermal barrier function. Adiponectin, an adipokine, has anti-inflammatory effects on various metabolic and inflammatory disorders. Recently, its level was found to be reduced in serum and tissue samples from AD patients.ObjectiveWe aimed to investigate the effects of adiponectin on epidermal inflammation and barrier structures in AD skin.MethodsA three-dimensional in vitro epidermal equivalent model mimicking AD was obtained by adding an inflammatory substance cocktail to normal human epidermal equivalents (HEEs). The expression of epidermal differentiation markers, primary inflammatory mediators, and lipid biosynthetic enzymes was compared between adiponectintreated AD-HEEs, untreated control AD-HEEs, and normal HEEs.ResultsAdiponectin co-treatment 1) inhibited the increase in mRNA expression of major inflammatory mediators (carbonic anhydrase II, neuron-specific NEL-like protein 2, thymic stromal lymphopoietin, interleukin-8, tumor necrosis factor-alpha, and human beta-defensin-2) from keratinocytes in AD-inflammatory HEEs, 2) enhanced the expression of lipid biosynthetic enzymes (fatty acid synthase, HMG CoA reductase, and serine-palmitoyl transferase), and 3) promoted the expression of differentiation factors, especially filaggrin. We also found that the expression of adiponectin receptor-1 and -2 decreased in the epidermis of chronic AD lesion.ConclusionActivation of the adiponectin pathway is expected to enhance epidermal differentiation and barrier function as well as attenuate inflammatory response to AD as a therapeutic approach.

Multiplexed electrochemical and SERS dual-mode detection of stroke biomarkers: rapid screening with high sensitivity

In this work, a real-time assay for a highly sensitive, label-free, multiplexed electrochemical and surface-enhanced Raman spectroscopic (SERS) detection of stroke biomarkers by neuron-specific enolase (NSE) and S100-β protein was developed using lateral flow devices.

The neuron-specific interleukin-1 receptor accessory protein alters emergent network state properties in Vitro

Small in vitro neuronal/glial networks exhibit sleep-like states. Sleep regulatory substance interleukin-1β (IL1) signals via its type I receptor and a receptor accessory protein (AcP). AcP has a neuron-specific isoform called AcPb. After sleep deprivation, AcPb, but not AcP, upregulates in brain, and mice lacking AcPb lack sleep rebound. Herein we used action potentials (APs), AP burstiness, synchronization of electrical activity (SYN), and delta wave (0.5–3.75 Hz) power to characterize cortical culture network state. Homologous parameters are used in vivo to characterize sleep. Cortical cells from 1–2-day-old pups from AcP knockout (KO, lacking both AcP and AcPb), AcPb KO (lacking only AcPb), and wild type (WT) mice were cultured separately on multi-electrode arrays. Recordings of spontaneous activity were taken each day during days 4–14 in vitro. In addition, cultures were treated with IL1, or in separate experiments, stimulated electrically to determine evoked response potentials (ERPs). In AcP KO cells, the maturation of network properties accelerated compared to those from cells lacking only AcPb. In contrast, the lack of AcPb delayed spontaneous network emergence of sleep-linked properties. The addition of IL1 enhanced delta wave power in WT cells but not in AcP KO or AcPb KO cells. The ontology of electrically-induced ERPs was delayed in AcP KO cells. We conclude IL1 signaling has a critical role in the emergence of sleep-linked network behavior with AcP playing a dominant role in the slowing of development while AcPb enhances development rates of sleep-linked emergent network properties.

Are signaling pathways inversely regulated in Alzheimer's disease and glioblastoma multiforme?

Background: Epidemiological studies suggest an inverse association between cancer and neurodegenerative disorders, including Alzheimer’s disease (AD). AD and cancers, such as glioblastoma multiforme (GBM), are characterized by abnormal but opposing cellular behavior. AD is characterized by accumulation of the processing products of amyloid β (Aβ) and its metabolizing enzymes amyloid precursor protein (APP), β-secretase (or BACE1), and γ-secretase. Our rationale is unraveling cell signaling pathways linking AD and GBM. We hypothesized low-grade gliomas (LGG) and high-grade gliomas (HGG) would have differential expression of neuronal and synaptic markers. Furthermore, protein expression profiles of these markers, APP metabolites, and BACE1 would be different among LGG, HGG, and AD cases. &#x0D; Experimental Design or project methods: Specific neuronal protein markers (e.g., NSE), presynaptic proteins (e.g., synaptophysin and SNAP25), and post-synaptic proteins (e.g., PSD-95) have been measured in glioma samples. Characterization are done by Western immunoblotting and ELISA. Protein biomarkers will be analyzed in LGG and HGG of biopsy samples, and the results will be compared with brain samples from AD cases. &#x0D; Results: Using specific primary and secondary antibodies and optimal protein range, we have standardized an immunoblotting procedure to detect our desired proteins in blinded LGG and HGG samples. After unblinding and analyzing results, expression signals will be compared between GBM and AD brain samples. &#x0D; Conclusion and potential impact: Our results would shed light on diverging and/or shared cell signaling pathways between AD and GBM. In addition, potential impact would be utilizing GBM-derived cultures to test and develop therapeutics for both AD and GBM.

Diagnostic value of NT-proCNP compared to NSE and S100B in cerebrospinal fluid and plasma of patients with sepsis-associated encephalopathy

Sepsis-associated encephalopathy (SAE) has significant impact on the neurocognitive outcome of sepsis survivors. This study was conducted to analyze the amino-terminal propeptide of the C-type natriuretic peptide (NT-proCNP) as a biomarker for SAE in comparison to neuron-specific enolase (NSE) and S100B protein. Cerebrospinal fluid (CSF) and plasma samples from twelve septic patients with SAE and nine non-septic controls without encephalopathy were analyzed. The assessment of SAE comprised a neuropsychiatric examination, delirium screening using the confusion assessment method in the ICU (CAM-ICU) and magnetic resonance imaging (MRI) in all participants. NSE, S100B and NT-proCNP were measured in plasma at study days 1, 3 and 7 in sepsis patients, once in controls and once in the CSF of both groups. The long-term outcome was assessed using the validated Barthel index (BI). Plasma NT-proCNP levels were significantly higher in the sepsis cohort compared to controls with peak concentrations at study day 1 (10.1 ± 6.6 pmol/l vs. 3.3 ± 0.9 pmol/l; p < 0.01) and a decrease over time. Plasma NT-proCNP levels at day 7 correlated with NT-proCNP in CSF (r = 0.700, p < 0.05). A comparable decrease of significantly higher plasma S100B values in sepsis patients compared to controls was observed. Plasma NSE levels were not significantly different between both groups. CSF NT-proCNP levels just tended to be higher in sepsis patients compared to controls and tended to be higher in patients with septic brain lesions seen on MRI. In the sepsis cohort CSF NT-proCNP levels correlated with CSF Interleukin-6 (IL-6) levels (r = 0.616, p < 0.05) and systemic inflammation represented by high plasma procalcitonin (PCT) levels at day 3 (r = 0.727, p < 0.05).The high peak concentration of plasma NT-proCNP in the early phase of sepsis might help to predict the emergence of SAE during the further course of disease. NT-proCNP in plasma might, in contrast to CSF, indicate neurological impairment in patients with SAE.

Isolation and characterization of neural stem cells from ovine foetal cerebral cortex

Neural stem cells are a potential source in cell based therapy for neurodegenerative diseases and traumatic brain injuries. The aim of this study is to isolate the ovine (Ovis aries) fetal neural stem cells and to analyze their neurogenic characteristics in vitro. Cerebral cortex pieces harvested from ovine fetal brain tissue were triturated for dispersal into single cells, cultured in neuronal stem cells culture medium and expanded in vitro in adherent culture. The morphological changes and the expression of neuron-specific gene markers and proteins were evaluated. These isolated NSCs were plastic adherent, self-renewing cells maintained the capacity to differentiate into brain specific cell types and they were found positive for neuronal stem cell markers, Nestin and â III tubulin by Reverse Transcription-PCR. Immunocytochemistry of the isolated NSCs also showed positive for Nestin, an established marker for neural and glial precursors as well as MSC marker CD90 and negative for hematopoietic CD34 markers.

Specific translocation of protein kinase Cγ to lipid rafts in primary somatosensory cortex is involved in remifentanil-induced hyperalgesia

Objective To determine the involvement of neuron-specific protein kinase C γ isoform(PKCγ) translocation to lipid rafts in primary somatosensory cortex(S1) in remifentanil-induced hyperalgesia. Methods Fifty adult male SD rats weighing 240-260 g (at the age of 8-10 weeks) were randomly assigned to 3 groups: control, the propofol (group P) and the remifentanil group (group R). Intravenous anesthesia with either propofol (group P) or propofol combined with remifentanil(group R) was performed through a 24 G catheter inserted in tail vein for 3 h. The total doses of propofol were 36.0 mg and 26.0 mg in group P and R, respectively. The dose of remifentanil was 19.5 g in group R. Mechanical allodynia in the rats was evaluated by the von Frey filament test, and PKCγ levels in the whole-cell lysates and lipid rafts in the S1 neurons were determined by Western blot. Results ① The threshold of paw-withdrawal response to von Frey filament stimulation in group R at 2 h after stopping drug infusion was significantly lower than that in group P(P<0.05). ② Two hours after stopping drug infusion, the PKCγ protein levels in the lipid rafts of S1 in group R were significantly higher than those in group R(P<0.05). Conclusions These results suggest that increased translocation of PKCγ to lipid raft of S1 neurons may contribute to postoperative pain in rats after propofol-remifentanil-based anesthesia. Key words: Remifentanil; Hyperalgesia; Protein kinase C γisoform; Lipid rafts; Primary somatosensory cortex

Prognostic values of serum neuronal biomarkers NSE and protein S-100 in acute period of severe hypoxic-ischemic encephalopathy in term infants

It remains unclear if the serum concentrations of neuron-specific enolase (NSE) and protein S-100 correlate with severity and possible complications of hypoxic ischemic encephalopathy (HIE) as well as monitoring parameters of cerebral perfusion and central hemodynamics in term newborns.The aim of research. To determine diagnostic and predictable values of NSE and protein S-100 in acute period of severe hypoxic-ischemic encephalopathy in neonates.Materials and methods. Data of 89 term infants with Apgar score at birth of seven or less and Sarnat stage II-III was collected during ≤72 hours of life. The correlation between NSE, S-100 and Glasgow Coma Scale, seizures, confirmed by the aEEG, the unwanted development of neurological complications such as cerebral leukomalacia, and the short-term clinical results were analyzed.Result. In acute period of HIE the concentration of serum neuronal biomarkers S-100 and NSE proteins substantially exceeds the reference values for the first hours after labor and exceeds the normal range until the 5th day of life. The dynamics of these data significantly changes during this period. Mostly diagnostically and prognostically significant the measurement of biomarkers was on the 3rd day of treatment, immediately after the end of the therapeutic hypothermia, at the beginning of rewarming and brain reperfusion, when both indicators were most closely correlated with the assessment of the GCS: NSE (r=-0.3; p=0.002 ) and S-100 (r=-0.3; p=0.003). The value of S-100 on day 1 correlating with the following unwanted development of leukomalacia is above 1.8 μg/L (p=0.002), which is more than 3 times higher from the reference level of 0.51 μg/L given by Simon-Pimmel J. (2017) for term newborns.Conclusion. The evaluation of the serum concentrations of protein S-100 and neuron-specific enolase in the acute period of hypoxic ischemic encephalopathy in the term newborns has a significant diagnostic and predictable value and correlates with the severity and short-term clinical results of HIE

Visualization of Tau⁻Tubulin Interaction in a Living Cell Using Bifluorescence Complementation Technique.

Tau is a neuron-specific microtubule-binding protein that stabilizes microtubules. It is generally thought that highly phosphorylated tau dissociates from microtubules and becomes insoluble aggregates, leading to neuronal degeneration. Due to the implication of tau aggregation in neurodegenerative disorders, including Alzheimer’s disease, great efforts have been made to identify the tau aggregation process. However, tau interaction with tubulin during the aggregation process remains largely unknown. To scrutinize the tau-tubulin interaction, we generated a cell model that enables visualization of the tau-tubulin interaction in a living cell using the Bifluorescence Complementation (BiFC) Technique. Upon diverse chemical stimulation that induced tau pathology, tau-tubulin BiFC cells showed significantly increased levels of BiFC fluorescence, indicating that tau aggregates together with tubulin. Our results suggest that tubulin should be considered as a key component in the tau aggregation process.

Diffusion Kurtosis Imaging Characterizes Brain Microstructural Changes Associated with Cognitive Impairment in a Rat Model of Chronic Traumatic Brain Injury

This study aims to investigate the value of diffusion kurtosis imaging (DKI) in assessing microstructural changes associated with cognitive impairment in chronic traumatic brain injury (TBI). At 7 months, six TBI rats and six control rats underwent Morris water maze (MWM) tests, followed by DKI examinations. DKI parameters were measured in bilateral cortex, hippocampus, and callosum. Brain immunohistochemistry (IHC) analysis of neuron [neuron-specific nuclear protein (NeuN)], astroglia [glial fibrillary acidic protein (GFAP)], microglia [ionized calcium binding adaptor molecule 1 (Iba-1)], and myelin [myelin basic protein (MBP)] was performed in the same area as DKI parameter. The DKI parameters, IHC results, and MWM results were compared between TBI and control groups. Correlation analysis was performed to analyze the relationship between DKI parameters and IHC and MWM results. TBI group had worse performance in MWM test. DKI showed higher mean diffusion (MD) in all ipsilateral regions of interest (ROIs), and lower mean kurtosis (MK) in ipsilateral cortex and callosum in TBI group (P < 0.05). TBI group also showed lower IHC staining of NeuN, and higher staining of Iba-1 and MBP in all ipsilateral ROIs (P < 0.05). Further correlational study showed a positive relationship between MK and NeuN, MD and MBP in ipsilateral cortex, and a negative relationship between MK and Iba-1, MBP in ipsilateral cortex and hippocampus (P < 0.05). The MK in ipsilateral cortex and hippocampus were also correlated with MWM test results (P < 0.05). Our study suggests that DKI could be used to assess the microstructural changes associated with cognitive impairment in chronic TBI.

Proteomic identification of hippocalcin and its protective role in heatstroke-induced hypothalamic injury in mice.

Heatstroke is a devastating condition that is characterized by severe hyperthermia and central nervous system dysfunction. However, the mechanism of thermoregulatory center dysfunction of the hypothalamus in heatstroke is unclear. In this study, we established a heatstroke mouse model and a heat-stressed neuronal cellular model on the pheochromocytoma-12 (PC12) cell line. These models revealed that HS promoted obvious neuronal injury in the hypothalamus, with high pathological scores. In addition, PC12 cell apoptosis was evident by decreased cell viability, increased caspase-3 activity, and high apoptosis rates. Furthermore, 14 differentially expressed proteins in the hypothalamus were analyzed by fluorescence two-dimensional difference gel electrophoresis and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Expression changes in hippocalcin (HPAC), a downregulated neuron-specific calcium-binding protein, were confirmed in the hypothalamus of the heatstroke mice and heat-stressed PC12 cells by immunochemistry and western blot. Moreover, HPAC overexpression and HPAC-targeted small interfering RNA experiments revealed that HPAC functioned as an antiapoptotic protein in heat-stressed PC12 cells and hypothalamic injury. Lastly, ulinastatin (UTI), a cell-protective drug that is clinically used to treat patients with heatstroke, was used in vitro and in vivo to confirm the role of HPAC; UTI inhibited heat stress (HS)-induced downregulation of HPAC expression, protected hypothalamic neurons and PC12 cells from HS-induced apoptosis and increased heat tolerance in the heatstroke animals. In summary, our study has uncovered and demonstrated the protective role of HPAC in heatstroke-induced hypothalamic injury in mice.

Neuron-specific enolase and protein S100β are not affected by different target temperature management protocols in a clinically relevant model

Neuron-specific enolase and protein S100β are not affected by different target temperature management protocols in a clinically relevant model

Cardiac Surgery is Associated with Neuronal Damage

Background: Post-operative cognitive (POCD) decline is common in cardiac surgery and may be caused by neuronal damage. Plasma concentrations of the neuronal proteins tau and neurofilament light (NFL) are established biomarkers for acute neuronal injury. Our objective was to investigate if cardiac surgery is associated with neuronal injury reflected by changes in these plasma biomarkers. Methods: We performed a single centre case-control study of 25 patients undergoing cardiac surgery and 26 patients undergoing otolaryngeal surgery. The cohorts were sampled at several time-points before, during and after surgery and plasma concentrations of tau, NFL, and betaamyloid (Aβ40 and Aβ42) were measured. Findings: s Tau levels increased greatly by 1700% during cardiac surgery compared to baseline and normalized seven days post-surgery. NFL levels rose sharply seven days post-surgery (1100%). No changes were observed in patients undergoing otolaryngeal surgery. Furthermore, cardiac surgery patients on extracorporeal circulation (on-pump) had higher levels of tau (up to 2200%) and NFL (up to 430%) compared to cardiac surgery patients off-pump. However, the levels of tau and NFL were still significantly elevated in the cardiac surgery patients off-pump compared to the patients undergoing otolaryngeal surgery. Only minor fluctuations were observed for Aβ40 and Aβ42. Interpretation: Cardiac surgery, but not otolaryngeal surgery, is associated with massive release of neuronal-specific proteins indicating neuronal injury, which is aggravated by extracorporeal circulation. Thus, neuronal damage may contribute to the increased frequency of POCD in cardiac surgery. Analyses of NFL and tau may help to improve surgical procedures to minimize neuronal damage and possibly POCD. Funding Statement: This study was funded by grants no. N/ST/ZB/14/001/1128, 143-28594L, 133-28785L, N/ST/ZB/16/001/1128 from Medical University of Bialystok Clinical Hospital, Poland, the Swedish Research Council, Swedish State Support for Clinical Research, Alzheimer’s Association, the Knut and Alice Wallenberg Foundation, the Torsten Soderberg Foundation, the Alzheimer Foundation (Sweden), the European Research Council (grant number 681712), the Innovative Medicines Initiative Joint Undertaking under EMIF grant agreement n° 115372, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies. Declaration of Interests: PL received consultation and/or lecture honoraria from IBL International, Fujirebio Europe, AJ Roboscreen, and Roche. KB has served as a consultant or at advisory boards for Alzheon, BioArctic, Biogen, Eli Lilly, Fujirebio Europe, IBL International, Merck, Novartis, Pfizer and Roche Diagnostics. HZ has served on scientific advisory boards for Eli Lilly, Roche 17 Diagnostics, Wave, Samumed and CogRx, and has received travel support from Teva. None of the other authors declares competing interests. Ethics Approval Statement: The study was approved by the Ethical Committee, Medical University of Bialystok Clinical Hospital, Poland. All patients gave written informed consent.

Quantum dot-based immunofluorescent imaging and quantitative detection of DNER and prognostic value in prostate cancer.

DNER, Delta/Notch-like epidermal growth factor (EGF)-related receptor, is a neuron-specific transmembrane protein carrying extracellular EGF-like repeats. The prognostic value of DNER in prostate cancer has not been evaluated. Here we showed that the up-regulation of DNER protein was observed in prostate cancer detected by immunohistochemistry (IHC) and quantum dot-based immunofluorescent imaging and quantitative analytical system (QD-IIQAS). However, a higher accuracy of measurements of DNER expression in prostate cancer was found by QD-IIQAS than by IHC (AUC = 0.817 and 0.617, respectively). DNER was significantly higher in patients undergoing bone metastasis (P = 0.045, RR = 3.624). In addition, DNER overexpression was associated with poor overall survival (OS) (P = 0.028, adjusted HR = 8.564) and recurrence-free survival (RFS) (P = 0.042, adjusted HR = 3.474) in patients suffering prostate cancer. Thus, QD-IIQAS is an easy and accurate method for assessing DNER and the DNER expression was an independent prognostic factor in prostate cancer.

Involvement of advillin in somatosensory neuron subtype-specific axon regeneration and neuropathic pain

Advillin is a sensory neuron-specific actin-binding protein expressed at high levels in all types of somatosensory neurons in early development. However, the precise role of advillin in adulthood is largely unknown. Here we reveal advillin expression restricted to isolectin B4-positive (IB4+) neurons in the adult dorsal root ganglia (DRG). Advillin knockout (KO) specifically impaired axonal regeneration in adult IB4+ DRG neurons. During axon regeneration, advillin was expressed at the very tips of filopodia and modulated growth cone formation by interacting with and regulating focal-adhesion-related proteins. The advillin-containing focal-adhesion protein complex was shed from neurite tips during neurite retraction and was detectable in cerebrospinal fluid in experimental autoimmune encephalomyelitis, oxaliplatin-induced peripheral neuropathy, and chronic constriction injury of the sciatic nerve. In addition, advillin KO disturbed experimental autoimmune encephalomyelitis-induced neural plasticity in the spinal-cord dorsal horn and aggravated neuropathic pain. Our study highlights a role for advillin in growth cone formation, axon regeneration, and neuropathic pain associated with IB4+ DRG neurons in adulthood.