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Classical two-dimensional (2D) cell culture as a drug or nanoparticle test system only poorly recapitulates in vivo conditions. Animal studies are costly, ethically controversial, and preclude large-scale testing.We established a three-dimensional (3D) tissue slice air-liquid interface (ALI) culture model for nanoparticle testing. We developed an optimized procedure for the reproducible generation of large sets of tissue slices from tumor xenografts that retain their tissue architecture. When used for the analysis of nanoparticles based on chemically modified polyethylenimines (PEIs) to deliver siRNA or DNA, differences in transfection efficacy and cytotoxicity between nanoparticles were observed more clearly than in 2D cell culture. While nanoparticle efficacies between cell culture and the tissue slice model overall correlated, the tissue slice model also identified particularly suitable candidates whose efficacy was underestimated in 2D cell culture and had already been shown in previous in vivo studies.The ex vivo 3D tissue slice ALI culture model is a powerful system that allows the effective evaluation of biological nanoparticle efficacy and biocompatibility in an intact tissue environment. It is comparably inexpensive, time-saving, and follows the 3R principle, while allowing the identification of critical nanoparticle properties and optimal candidates for in vivo applications.

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice. Supplementary Information The online version contains supplementary material available at 10.1007/s11302-021-09792-9.

Diagnosing traumatic brain injury (TBI) from body fluids in cases where there are no obvious external signs of impact would be useful for emergency physicians and forensic pathologists alike. None of the previous attempts has so far succeeded in establishing a single biomarker to reliably detect TBI with regards to the sensitivity: specificity ratio in a post mortem setting. This study investigated a combination of body fluid biomarkers (obtained post mortem), which may be a step towards increasing the accuracy of biochemical TBI detection. In this study, serum and cerebrospinal fluid (CSF) samples from 30 acute lethal TBI cases and 70 controls without a TBI-related cause of death were evaluated for the following eight TBI-related biomarkers: brain-derived neurotrophic factor (BDNF), ferritin, glial fibrillary acidic protein (GFAP), interleukin 6 (IL-6), lactate dehydrogenase, neutrophil gelatinase-associated lipocalin (NGAL), neuron-specific enolase and S100 calcium-binding protein B. Correlations among the individual TBI biomarkers were assessed, and a specificity-accentuated threshold value analysis was conducted for all biomarkers. Based on these values, a decision tree modelling approach was performed to assess the most accurate biomarker combination to detect acute lethal TBIs. The results showed that 92.45% of acute lethal TBIs were able to be diagnosed using a combination of IL-6 and GFAP in CSF. The probability of detecting an acute lethal TBI was moderately increased by GFAP alone and considerably increased by the remaining biomarkers. BDNF and NGAL were almost perfectly correlated (p = 0.002, R2 = 0.944). This study provides evidence that acute lethal TBIs can be detected to a high degree of statistical accuracy using forensic biochemistry. The high inter-individual correlations of biomarkers may help to estimate the CSF concentration of an unknown biomarker, using extrapolation techniques.

Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growth-promoting effects of Montelukast (MTK, GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration.

Traumatic brain injury (TBI) is a major cause of death and its accurate diagnosis is an important concern of daily forensic practice. However, it can be challenging to diagnose TBI in cases where macroscopic signs of the traumatic head impact are lacking and little is known about the circumstances of death. In recent years, several post-mortem studies investigated the possible use of biomarkers for providing objective evidence for TBIs as the cause of death or to estimate the survival time and time since death of the deceased. This work systematically reviewed the available scientific literature on TBI-related biomarkers to be used for forensic purposes. Post-mortem TBI-related biomarkers are an emerging and promising resource to provide objective evidence for cause of death determinations as well as survival time and potentially even time since death estimations. This literature review of forensically used TBI-biomarkers revealed that current markers have low specificity for TBIs and only provide limited information with regards to survival time estimations and time since death estimations. Overall, TBI fatality-related biomarkers are largely unexplored in compartments that are easily accessible during autopsies such as urine and vitreous humor. Future research on forensic biomarkers requires a strict distinction of TBI fatalities from control groups, sufficient sample sizes, combinations of currently established biomarkers, and novel approaches such as metabolomics and mi-RNAs.

Aufgrund der dezentralen und uneinheitlichen Dokumentation der Vergiftungsfalle in Deutschland besteht im Vergleich zu anderen Landern (USA, Schweiz, Schweden, Niederlande usw.) eine wesentliche nationale Datenlucke, insbesondere in Hinblick auf die Risikofruherkennung und Risikobewertung im Bundesinstitut fur Risikobewertung (BfR). In Deutschland existieren viele Datenquellen, die Auskunft zu Vergiftungsfallen geben. Deren Erfassung basiert jedoch auf verschiedenen Grundlagen und zeigt ein zugleich breites, redundantes und luckenhaftes Spektrum auf. Die Errichtung eines nationalen Monitorings von Vergiftungen in Deutschland ist erklartes Ziel und ein notwendiger Schritt aus Sicht des BfR und der deutschen Giftinformationszentren (GIZ). Durch einen schnelleren und effizienteren technischen Zugang zu akkumulierten Daten zum nationalen Vergiftungsgeschehen ware ein umfangliches Bild zur Situation in Deutschland verfugbar. Daruber hinaus konnte eine zeitnahe Erfassung der Vergiftungsfalle ein schnelles Erkennen und Bestatigen von akuten und bisher nicht erkannten Vergiftungsgefahren ermoglichen. Relevant sind hierbei auch die Sicherheit von Produkten und der damit verbundene Schutz, insbesondere von Kindern, vor Vergiftungsunfallen. Die fortlaufende Uberwachung und Berichterstattung mit einem solchen System erfordern eine erhebliche Arbeitsanstrengung, zusatzlichen Arbeitsaufwand, eine geeignete personelle und IT-Infrastruktur und ein jahrliches Budget, welches zukunftig in der Finanzplanung des Bundes berucksichtigt werden muss.

Glial fibrillary acidic protein (GFAP) is a well-established astrocytic biomarker for the diagnosis, monitoring and outcome prediction of traumatic brain injury (TBI). Few studies stated an accumulation of neuronal GFAP that was observed in various brain pathologies, including traumatic brain injuries. As the neuronal immunopositivity for GFAP in Alzheimer patients was shown to cross-react with non-GFAP epitopes, the neuronal immunopositivity for GFAP in TBI patients should be challenged. In this study, cerebral and cerebellar tissues of 52 TBI fatalities and 17 controls were screened for immunopositivity for GFAP in neurons by means of immunohistochemistry and immunofluorescence. The results revealed that neuronal immunopositivity for GFAP is most likely a staining artefact as negative controls also revealed neuronal GFAP staining. However, the phenomenon was twice as frequent for TBI fatalities compared to non-TBI control cases (12 vs. 6%). Neuronal GFAP staining was observed in the pericontusional zone and the ipsilateral hippocampus, but was absent in the contralateral cortex of TBI cases. Immunopositivity for GFAP was significantly correlated with the survival time (r = 0.306, P = 0.015), but no correlations were found with age at death, sex nor the post-mortem interval in TBI fatalities. This study provides evidence that the TBI-associated neuronal immunopositivity for GFAP is indeed a staining artefact. However, an absence post-traumatic neuronal GFAP cannot readily be assumed. Regardless of the particular mechanism, this study revealed that the artefact/potential neuronal immunopositivity for GFAP is a global, rather than a regional brain phenomenon and might be useful for minimum TBI survival time determinations, if certain exclusion criteria are strictly respected.

The Special AT-rich sequence binding protein 1 (SATB1) is a genome organizer protein that controls gene expression of numerous genes by regulating chromatin architecture and targeting chromatin-remodeling/-modifying enzymes onto specific chromatin regions. SATB1 is overexpressed in various tumors. In head and neck squamous cell carcinoma (HNSCC), SATB1 upregulation is correlated with TNM classification, metastasis, poor prognosis and reduced overall survival. In this paper, we comprehensively analyze cellular and molecular effects of SATB1 in a large set of primary cell lines from primary HNSCC or metastases, using RNAi-mediated knockdown in vitro and, therapeutically, in tumor xenograft mouse models in vivo. In a series of 15 cell lines, major differences in SATB1 levels are observed. In various 2-D and 3-D assays, growth inhibition upon efficient siRNA-mediated SATB1 knockdown depends on the cell line rather than initial SATB1 levels. Inhibitory effects are found to be based on cell cycle deceleration, apoptosis induction, decreased HER3 and Heregulin A&B expression, and effects on EMT genes. In vivo, systemic treatment of tumor xenograft-bearing mice with siRNAs formulated in polymeric nanoparticles inhibits tumor growth of two HNSCC xenograft models, resulting from therapeutic SATB1 reduction and concomitant decrease of proliferation and induction of apoptosis. In conclusion, SATB1 represents a promising target in HNSCC, affecting crucial cellular processes and molecular pathways.

The aim of the given study was to test the in situ stability of biochemical markers of cerebral damage and acute phase response in the early post-mortem interval to assess their usability for forensic pathology. A monocentric, prospective study investigated post-mortem femoral venous blood samples at four time points obtained within 48 h post-mortem starting at the death of 20 deceased, using commercial immunoassays for the ten parameters: S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), C-reactive protein (CRP), procalcitonin (PCT), ferritin, soluble tumor necrosis factor receptor type 1 (sTNFR1), and lactate dehydrogenase (LDH). Significant changes in serum levels were observed only later than 2 h after death for all markers. Inter-laboratory comparability was high, and intra-assay precision was sufficient for most markers. Most of the biomarker levels depended on the severity of hemolysis and lipemia but were robust against freeze-thaw cycles. Serum levels increased with longer post-mortem intervals for S100B, NSE, ferritin, sTNFR1, and LDH (for all p

Organotypic hippocampal slice cultures were used to model the effects of neuroinflammatory conditions following an epileptic state on functional P2X7 receptors (Rs) of subgranular zone (SGZ) neural progenitor cells (NPCs). The compound, 4-aminopyridine (4-AP), is known to cause pathological firing of neurons, consequently facilitating the release of various transmitter substances including ATP. Lipopolysaccharide (LPS) and interleukin-1&beta, (IL-1&beta, ) both potentiated the dibenzoyl-ATP (Bz-ATP)-induced current amplitudes in NPCs, although via different mechanisms. Whereas LPS acted via promoting ATP release, IL-1&beta, acted via its own receptor to directly influence P2X7Rs. Thus, the effect of LPS was inhibited by the ecto-ATPase inhibitor, apyrase, but not by the IL-1&beta, antagonist, interleukin-1RA (IL-1RA), by contrast, the effect of IL-1&beta, was inhibited by IL-1RA, but not by apyrase. Eventually, incubation with 4-AP upregulated the number of nestin/glial fibrillary acidic protein/P2X7R immunoreactive cells and their appropriate staining intensity, suggesting increased synthesis of P2X7Rs at NPCs. In conclusion, inflammatory cytokines accumulating after epilepsy-like neuronal firing may facilitate the effect of endogenous ATP at P2X7Rs of NPCs, thereby probably promoting necrosis/apoptosis and subsequent cell death.

An inflammatory response occurring after fatal traumatic brain injury (TBI) initiates time-dependent cascades of acute phase response. This may offer the potential to monitor postmortem biomarker levels of several pro-inflammatory cytokines to gain information about the cause of death and the trauma survival time. Cerebrospinal fluid (CSF) and serum samples were collected from forensic autopsies of 95 adult cadavers after postmortem intervals up to 6 days. The cases were divided according to their cause of death into fatal TBI (n = 46) with different survival times and age- and gender-matching non-TBI fatalities as controls (n = 49). Quantitative marker levels of interleukin-6 (IL-6), ferritin, soluble tumor necrosis factor receptor type 1, C-reactive protein, and lactate dehydrogenase were analyzed using immunoassays. Standardized statistical tests were performed to differentiate causes of death and survival time of TBI cases. The CSF IL-6, ferritin, and LDH levels after TBI were significantly higher than those in the controls (p

Purinergic signalling 17(1), 139-140 (2021). doi:10.1007/s11302-021-09768-9 special issue: "A Tribute to Professor Geoff Burnstock / edited by Charles Kennedy", Published by Springer Science + Business Media B.V., Dordrecht

Therapeutic gene silencing by RNA interference relies on the safe and efficient in vivo delivery of small interfering RNAs (siRNAs). Polyethylenimines are among the most studied cationic polymers for gene delivery. For several reasons including superior tolerability, small linear PEIs would be preferable over branched PEIs, but they show poor siRNA complexation. Their chemical modification for siRNA formulation has not been extensively explored so far. We generated a set of small linear PEIs bearing tyrosine modifications (LPxY), leading to substantially enhanced siRNA delivery and knockdown efficacy in vitro in various cell lines, including hard-to-transfect cells. The tyrosine-modified linear 10 kDa PEI (LP10Y) is particularly powerful, associated with favorable physicochemical properties and very high biocompatibility. Systemically administered LP10Y/siRNA complexes reveal antitumor effects in mouse xenograft and patient-derived xenograft (PDX) models, and their direct application into the brain achieves therapeutic inhibition of orthotopic glioma xenografts. LP10Y is particularly interesting for therapeutic siRNA delivery.

The whole-cell patch-clamp technique was used to record current responses to AMPA, N-methyl-d-aspartate (NMDA), muscimol and dibenzoyl-ATP (Bz-ATP) in superficial (reactive/gliotic) substantia gelatinosa (SG) astrocytes and neurons of spinal cord slices kept for different periods of time in organotypic culture. Currents induced by AMPA, NMDA and muscimol confirmed the existence of their specific receptors in 2-week-old neurons; astrocytes cultured for the same period of time responded to AMPA and muscimol, but not to NMDA. AMPA had a larger effect on 2-week-old astrocytes than on the 1-week-old ones, in spite of a similar sensitivity of the age-matched neurons to this amino acid. The effect of the prototypic P2X7 receptor agonist Bz-ATP on superficial astrocytes and neurons depended on the drug concentration applied and increased in parallel with the lengthening of the culture period. The amplitudes of Bz-ATP currents of deep (resting) astrocytes were age-independent. Neurons located in deep layers exhibited after 1week of culturing much larger Bz-ATP currents than the superficial ones of the same age. In conclusion, whereas resting astrocytes had culture period-independent P2X7 receptor-sensitivity, reactive/gliotic astrocytes exhibited P2X7 receptor-sensitivity increasing in parallel with the prolongation of the time spent in culture. The results with Bz-ATP agree with the facilitation of AMPA-induced currents in reactive astrocytes during development, and with the hypothesis that extracellular ATP is an ontogenetically early transmitter/signaling molecule in the CNS.

The success of therapeutic nanoparticles depends, among others, on their ability to penetrate a tissue for actually reaching the target cells, and their efficient cellular uptake in the context of intact tissue and stroma. Various nanoparticle modifications have been implemented for altering physicochemical and biological properties. Their analysis, however, so far mainly relies on cell culture experiments which only poorly reflect the in vivo situation, or is based on in vivo experiments that are often complicated by whole-body pharmacokinetics and are rather tedious especially when analyzing larger nanoparticle sets. For the more precise analysis of nanoparticle properties at their desired site of action, efficient ex vivo systems closely mimicking in vivo tissue properties are needed. In this paper, we describe the setup of organotypic tumor tissue slice cultures for the analysis of tissue-penetrating properties and biological activities of nanoparticles. As a model system, we employ 350μm thick slice cultures from different tumor xenograft tissues, and analyze modified or non-modified polyethylenimine (PEI) complexes as well as their lipopolyplex derivatives for siRNA delivery. The described conditions for tissue slice preparation and culture ensure excellent tissue preservation for at least 14days, thus allowing for prolonged experimentation and analysis. When using fluorescently labeled siRNA for complex visualization, fluorescence microscopy of cryo-sectioned tissue slices reveals different degrees of nanoparticle tissue penetration, dependent on their surface charge. More importantly, the determination of siRNA-mediated knockdown efficacies of an endogenous target gene, the oncogenic survival factor Survivin, reveals the possibility to accurately assess biological nanoparticle activities in situ, i.e. in living cells in their original environment. Taken together, we establish tumor (xenograft) tissue slices for the accurate and facile ex vivo assessment of important biological nanoparticle properties. Beyond the quantitative analysis of nanoparticle tissue-penetration, the excellent tissue preservation and cell viability also allows for the evaluation of biological activities.

While probably originating from Africa, the plant Ricinus communis is found nowadays around the world, grown for industrial use as a source of castor oil production, wildly sprouting in many regions, or used as ornamental plant. As regards its pharmacological utility, a variety of medical purposes of selected parts of the plant, e.g., as a laxative, an anti-infective, or an anti-inflammatory drug, have been described already in the sixteenth century BC in the famous Papyrus Ebers (treasured in the Library of the University of Leipzig). Quite in contrast, on the toxicological side, the native plant has become the "poisonous plant 2018" in Germany. As of today, a number of isolated components of the plant/seeds have been characterized, including, e.g., castor oil, ricin, Ricinus communis agglutinin, ricinin, nudiflorin, and several allergenic compounds. This review mainly focuses on the most toxic protein, ricin D, classified as a type 2 ribosome-inactivating protein (RIP2). Ricin is one of the most potent and lethal substances known. It has been considered as an important bioweapon (categorized as a Category B agent (second-highest priority)) and an attractive agent for bioterroristic activities. On the other hand, ricin presents great potential, e.g., as an anti-cancer agent or in cell-based research, and is even explored in the context of nanoparticle formulations in tumor therapy. This review provides a comprehensive overview of the pharmacology and toxicology-related body of knowledge on ricin. Toxicokinetic/toxicodynamic aspects of ricin poisoning and possibilities for analytical detection and therapeutic use are summarized as well.

As a result of decentralized and non-harmonized documentation of poisoning cases within Germany, in comparison to other countries (e.g. the USA, Switzerland, Sweden, and the Netherlands), there is a substantial lack of harmonized national data, in particular for the early detection and risk assessment at the German Federal Institute for Risk Assessment (BfR).The establishment of a national monitoring system for poisonings in Germany is therefore an important step towards enabling faster and more efficient technical access to consolidated accumulated national poisoning data. Additionally, the monitoring would consolidate the publication of annual reports (modelled on the USA National Poison Data System reports) of the German Poison Information Centres and the BfR. Timely assessment of poisoning cases enables fast recognition and confirmation of acute as well as previously unrecognized poisoning hazards. Also relevant here is the safety of products and the associated protection, especially of children, against poisoning accidents.The continuous monitoring and reporting requirements of such a system necessitate additional and challenging work, suitable human resources and IT infrastructure and an annual budget that must be taken into consideration for overall as well as financial planning.

The non-viral delivery of small RNA molecules like siRNAs still poses a major bottleneck for their successful application in vivo. This is particularly true with regard to crossing physiological barriers upon systemic administration. We have previously established polyethylenimine (PEI)-based complexes for therapeutic RNA formulation. These nanoplexes mediate full RNA protection against nucleolytic degradation, delivery to target tissues as well as cellular uptake, intracellular release and therapeutic efficacy in preclinical in vivo models. We herein present data on different polyplex modifications for the defined improvement of physicochemical and biological nanoparticle properties and for targeted delivery. (i) By non-covalent modifications of PEI polyplexes with phospholipid liposomes, ternary complexes ("lipopolyplexes") are obtained that combine the favorable features of PEI and lipid systems. Decreased cytotoxicity and highly efficient delivery of siRNA is achieved. Some lipopolyplexes also allow prolonged storage, thus providing formulations with higher stability. (ii) Novel tyrosine modifications of low molecular weight PEI offer further improvement of stability, biocompatibility, and knockdown efficacy of resulting nanoparticles. (iii) For ligand-mediated uptake, the shielding of surface charges is a critical requirement. This is achieved by PEI grafting with polyethylene glycol (PEG), prior to covalent coupling of anti-HER1 antibodies (Erbitux®) as ligand for targeted delivery and uptake. Beyond tumor cell culture, analyses are extended towards tumor slice cultures from tumor xenograft tissues which reflect more realistically the in vivo situation. The determination of siRNA-mediated knockdown of endogenous target genes, i.e., the oncogenic survival factor survivin and the oncogenic receptor tyrosine kinase HER2, reveals nanoparticle penetration and biological efficacy also under intact tissue and stroma conditions.

The mammalian nervous system is a complex, functional network of neurons, consisting of local and long-range connections. Neuronal growth is highly coordinated by a variety of extracellular and intracellular signaling molecules. Purines turned out to be an essential component of these processes. Here, we review the current knowledge about the involvement of purinergic signaling in the regulation of neuronal development. We particularly focus on its role in neuritogenesis: the formation and extension of neurites. In the course of maturation mammals generally lose their ability to regenerate the central nervous system (CNS) e.g. after traumatic brain injury; although, spontaneous regeneration still occurs in the peripheral nervous system (PNS). Thus, it is crucial to translate the knowledge about CNS development and PNS regeneration into novel approaches to enable neurons of the mature CNS to regenerate. In this context we give a general overview of growth-inhibitory and growth-stimulatory factors and mechanisms involved in neurite growth. With regard to neuronal growth, astrocytes are an important cell population. They provide structural and metabolic support to neurons and actively participate in brain signaling. Astrocytes respond to injury with beneficial or detrimental reactions with regard to axonal growth. In this review we present the current knowledge of purines in these glial functions. Moreover, we discuss organotypic brain slice co-cultures as a model which retains neuron-glia interactions, and further presents at once a model for CNS development and regeneration. In summary, the purinergic system is a pivotal factor in neuronal development and in the response to injury. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Background Adult neurogenesis has been shown to occur throughout life and different brain pathologies were demonstrated to be associated with altered neurogenesis. Here, an impact of heroin addiction on neurogenesis in humans is hypothesised. Methods Post mortem hippocampal specimens of drug addicts with known heroin abuse and a group of non-addictive control subjects were analysed, using antibodies indicating different stages of neurogenesis. The subgranular zone of the dentate gyrus was examined qualitatively and quantitatively. Results The data indicate (i) a decreased number of neural precursor cells, (ii) accompanied by low rates of proliferation and (iii) a marked loss of dendritic trees in targeting cells in heroin fatalities. (iv) The age-dependent increase of differentiating cells in the healthy controls was not observed in the addicts. Additionally, double immunofluorescence labelling indicated the precursor nature of Musashi-1 positive cells in the human subgranular zone of the dentate gyrus. Conclusions Present data firstly demonstrate the influence of drug addiction with known heroin abuse on different developmental stages of progenitors in the dentate gyrus. The patterns of antibody staining suggest a distinct inhibition of neurogenesis at the stage of neural precursor cells and revealed morphological changes in targeting cells in cases of heroin addicts as compared to healthy controls. These alterations could be considerable for memory and cognitive deficits as well as addictive behaviour in chronic drug abusers and may give rise to specific pro-neurogenic therapies.

Two subclasses of acid-sensing ion channels (ASIC3) and of ATP-sensitive P2X receptors (P2X3Rs) show a partially overlapping expression in sensory neurons. Here we report that both recombinant and native receptors interact with each other in multiple ways. Current measurements with the patch-clamp technique prove that ASIC3 stimulation strongly inhibits the P2X3R current partly by a Ca2+-dependent mechanism. The proton-binding site is critical for this effect and the two receptor channels appear to switch their ionic permeabilities during activation. Co-immunoprecipation proves the close association of the two protein structures. BN-PAGE and SDS-PAGE analysis is also best reconciled with the view that ASIC3 and P2X3Rs form a multiprotein structure. Finally, in vivo measurements in rats reveal the summation of pH and purinergically induced pain. In conclusion, the receptor subunits do not appear to form a heteromeric channel, but tightly associate with each other to form a protein complex, mediating unidirectional inhibition., Two subclasses of ligand-gated ion channels (ASIC3 and P2X3) are both present at sensory neurons and might be therefore subject to receptor crosstalk. Here authors use electrophysiology, biochemistry and co-immunoprecipitation to show that the two ion channels interact and affect P2X3 currents.

Until now, it is impossible to identify a fatal traumatic brain injury (TBI) before post-mortem radiological investigations or an autopsy take place. It would be preferable to have an additional diagnostic tool such as post-mortem biochemistry to get greater insight into the pathological pathways and survival times after sustaining TBI. Cerebrospinal fluid (CSF) and serum samples of 84 autopsy cases were collected from forensic autopsies with post-mortem intervals (PMI) of up to 148 h. The cases were categorized into a fatal TBI case group (n = 42) and non-TBI controls (n = 42). The values of glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF), and neutrophil gelatinase-associated lipocalin (NGAL) were analyzed by means of quantitative chemiluminescent multiplex immunoassays. The main results indicate that the usage of liquid samples with good macroscopic quality is more relevant for meaningful biomarker analyses than the length of the PMI. All three proteins were shown to differentiate TBI fatalities from the controls in CSF. In serum, only GFAP could be shown to be able to identify TBI cases. This study is the first approach to measure the three proteins together in CSF and serum in autopsy cases. Determined threshold values may differentiate between fatal TBI and control cases. The presented results emphasize the possible use of post-mortem biochemistry as a supplemental tool in everyday forensic routine.

Background Atrial fibrillation (AF) is the most common chronic arrhythmia in elderly people and is accompanied by remodeling processes. While much is known about changes in ionic channels and in extracellular matrix, less is known about possible changes of intracellular structures. Objective We wanted to investigate, whether AF may also affect the structure of the Golgi apparatus and the microtubular network. Methods One-hundred fifty-three cardiac surgery patients were investigated [n = 24 in sinus rhythm (SR) and n = 129 with chronic AF of >1 year duration]. Tissue samples of the left atrial free wall were examined immunohistochemically. Golgi apparatus was detected by GM130 and its phosphorylated isoform. Furthermore, we investigated the length of the microtubules by α-tubulin staining. We also measured stathmin (phospho-S37), which is known to induce microtubule depolymerization. In addition, we investigated the cyclin-dependent kinase cdk5-activation, a typical stimulus for Golgi fragmentation, by measuring membrane-associated cdk5. Results We found significant fragmentation of the Golgi apparatus in AF together with a reduced fragment size. Significant more fragments of the Golgi were found lateral to the nucleus in AF, while the Golgi in SR was located more to the polar side of the nucleus, that is, in the longitudinal axis of the cell. This was accompanied by a significant reduction of the number of tubulin strands longer than 10 µm. These changes did not go along with an activation of stathmin, but with an increase in membrane association of cdk5. Conclusions The present data may show that AF associated remodeling also involves intracellular remodeling of the Golgi-microtubular apparatus.

P2X7 receptors (Rs) mediate apoptosis/necrosis in neuronal and non-neuronal systems. Patch-clamp recordings from dentate gyrus (DG) granule cells in acutely prepared hippocampal slices of mice showed that incubation with 4-aminopyridine (4-AP) causes an excitability increase. This led to an enhanced sensitivity of P2X7Rs of the underlying subgranular zone neural progenitor cells (NPCs) towards dibenzoyl-ATP (Bz-ATP). The glutamatergic agonists NMDA and AMPA, as well as the purinergic agonist ATP also increased the Bz-ATP-induced current amplitudes (IBzATP). Tetrodotoxin as well as the standard antiepileptic drugs phenytoin, valproic acid and gabapentin counteracted the effect of 4-AP, most likely by decreasing the firing rate and/or action potential duration of DG granule cells and in consequence the release of ATP/glutamate onto NPCs. Experiments with organotypic hippocampal slice cultures confirmed these results also under conditions when 4-AP was applied for longer time periods and at much lower concentrations than used in acute slices. It was concluded that pathological firing modelled by 4-AP might trigger a sensitivity increase of P2X7Rs leading to necrosis/apoptosis of NPCs with the subsequent decrease of NPC, and in consequence, granule cell number. Hence, supersensitive P2X7Rs may exert a beneficial counter-regulatory effect by reducing the chances for the evolution of chronic temporal lobe epilepsy by ectopically located granule cells.

Traumatic brain injury (TBI) is a very common entity that leads to numerous fatalities all over the world. Therefore, forensic pathologists are in desperate need of supplemental methodological tools for the diagnosis of TBI in everyday practice besides the standard autopsy. The present study determined post-mortem neuron specific enolase (NSE) and S100 calcium-binding protein B (S100B) levels as biological markers of an underlying TBI in autopsy cases.Paired serum and CSF samples of 92 fatalities were collected throughout routine autopsies. Afterwards, the marker levels were assessed using commercially available immunoassays (ECLIA, Roche Diagnostics). For statistical analysis, we compared the TBI cases to three control groups (sudden natural death by acute myocardial infarction, traumatic death without impact on the head, cerebral hypoxia). Moreover, the TBI cases were subdivided according to their survival time of the trauma. Brain specimens have been collected and stained immunohistochemically against the aforementioned proteins to illustrate their typical cellular staining patterns with an underlying TBI compared to non-TBI fatalities.CSF NSE and S100B levels were elevated after TBI compared to all control groups (p 0.001). Although this finding can already be investigated among the TBI cases dying immediately subsequent to the trauma, the marker levels in CSF increase with longer survival times until a peak level within the first three days after trauma. There is a strong correlation between both marker levels in CSF (r = 0.67). The presence or absence of cerebral tissue contusion following the initial trauma does not seem to affect the CSF levels of both proteins (p 0.05). Post-mortem serum levels of both proteins were not elevated in TBI cases compared to controls (p 0.05). Former elaborated cut-off values in CSF were confirmed and were only exceeded when a TBI survival time of at least 30 min was reached.The present results report that post-mortem NSE and S100B CSF levels are significantly elevated subsequent to a fatal TBI.

Die Diagnostik von Schadel-Hirn-Traumen (SHT) ist wegen ihrer statistischen Haufigkeit im rechtsmedizinischen Obduktionsgut von herausragender Bedeutung. Bis heute konnte jedoch keine alleinig zuverlassige Methode etabliert werden, mit deren Hilfe das Verletzungsalter eines SHT exakt zu definieren ist. Im Rahmen der vorliegenden Studie sollten die Proteinmengen der 2 Biomarker neuronenspezifische Enolase (NSE) und saures Gliafaserprotein (GFAP) an definierten Lokalisationen im humanen Hirngewebe nach einem todlich verlaufenden SHT bestimmt und hinsichtlich zeitabhangiger Expressionsunterschiede evaluiert werden. Wahrend 42 gerichtlicher Obduktionen mit todesursachlichem SHT und Uberlebenszeiten (ULZ) zwischen wenigen Minuten und 37 Tagen wurden Gewebeproben aus Gros- und Kleinhirn der Verstorbenen entnommen. Als Kontrollen wurden 13 Falle mit kardiovaskularen Todesursachen verwendet. Der Proteinnachweis erfolgte mithilfe des Western-Blots. Grundsatzlich wurde der hochste NSE-Gehalt im Kleinhirn bestimmt, die GFAP-Expression war bis 4 Tage nach der traumatischen Schadigung im Hippocampus am grosten. Auser in der Perikontusionszone kommt es posttraumatisch nach mehr als 2-h-ULZ zu einer NSE-Expressionszunahme. Eine solche fand fur GFAP erst nach ULZ von mehr als 4 Tagen statt. Die Ergebnisse zeigen, dass Western-Blot-Analysen von NSE und GFAP als Erganzung zu Obduktion und konventioneller histologischer Untersuchung mit dem Ziel der Wundalterdiagnostik nach SHT geeignet sind.

The availability of markers able to provide insight into protein changes in the central nervous system after fatal traumatic brain injury (TBI) is limited. The present study reports on the semi-quantitative assessments of the immunopositive neuroglial cells (both astrocytes and oligodendrocytes) and neurons for S100 protein (S100), as well as neuronal specific enolase (NSE), in the cerebral cortex, hippocampus, and cerebellum with regard to survival time and cause of death. Brain tissues of 47 autopsy cases with TBI (survival times ranged between several minutes and 34 d) and 10 age- and gender-matched controls (natural deaths) were examined. TBI cases were grouped according to their survival time in acute death after brain injury (ABI, n = 25), subacute death after brain injury (SBI, n = 18) and delayed death after brain injury (DBI, n = 4). There were no significant changes in the percentages of S100-stained astrocytes between TBI and control cases. The percentages of S100-positive oligodendrocytes in the pericontusional zone (PCZ) in cases with SBI were significantly lower than in controls (p0.05) and in the ABI group (p0.05). In the hippocampus, S100-positive oligodendrocytes were significantly lower in cases with ABI and SBI (both, p0.05), compared with controls. It is of particular interest that there were also S100-positive neurons in the PCZ and hippocampus in TBI cases after more than 2 h survival but not in ABI cases or controls. The percentages of NSE-positive neurons in the hippocampus were likewise significantly lower in cases with ABI, compared with controls (p0.05) but increased in cases with SBI in PCZ (p0.05). In conclusion, the present findings emphasize that S100 and NSE-immunopositivity might be useful for detecting the cause and process of death due to TBI. Further, S100-positivity in neurons may be helpful to estimate the survival time of fatal injuries in legal medicine.

Injury to the peripheral axons of sensory neurons strongly enhances the regeneration of their central axons in the spinal cord. It remains unclear on what molecules that initiate such conditioning effect. Because ATP is released extracellularly by nerve and other tissue injury, we hypothesize that injection of ATP into a peripheral nerve might mimic the stimulatory effect of nerve injury on the regenerative state of the primary sensory neurons. We found that a single injection of 6 μl of 150 μm ATP into female rat sciatic nerve quadrupled the number of axons growing into a lesion epicenter in spinal cord after a concomitant dorsal column transection. A second boost ATP injection 1 week after the first one markedly reinforced the stimulatory effect of a single injection. Single ATP injection increased expression of phospho-STAT3 and GAP43, two markers of regenerative activity, in sensory neurons. Double ATP injections sustained the activation of phospho-STAT3 and GAP43, which may account for the marked axonal growth across the lesion epicenter. Similar studies performed on P2X7 or P2Y2 receptor knock-out mice indicate P2Y2 receptors are involved in the activation of STAT3 after ATP injection or conditioning lesion, whereas P2X7 receptors are not. Injection of ATP at 150 μm caused little Wallerian degeneration and behavioral tests showed no significant long-term adverse effects on sciatic nerve functions. The results in this study reveal possible mechanisms underlying the stimulation of regenerative programs and suggest a practical strategy for stimulating axonal regeneration following spinal cord injury. SIGNIFICANCE STATEMENT Injury of peripheral axons of sensory neurons has been known to strongly enhance the regeneration of their central axons in the spinal cord. In this study, we found that injection of ATP into a peripheral nerve can mimic the effect of peripheral nerve injury and significantly increase the number of sensory axons growing across lesion epicenter in the spinal cord. ATP injection increased expression of several markers for regenerative activity in sensory neurons, including phospho-STAT3 and GAP43. ATP injection did not cause significant long-term adverse effects on the functions of the injected nerve. These results may lead to clinically applicable strategies for enhancing neuronal responses that support regeneration of injured axons.

Objective: The aim was to test the reliability of former reported results of post-mortem laboratory investigations using cerebrospinal fluid (CSF) and serum levels of the biomarkers S100B and neuron specific enolase (NSE) to further evaluate the brain damage after fatal traumatic brain injuries. Methods: CSF and serum samples of 92 cases were collected during routine autopsies. The cases were divided into four groups regarding the causes of death in traumatic brain injury (TBI), isolated torso trauma, diffuse cerebral hypoxia and acute myocardial infarction. The samples were analysed using chemiluminescent immunoassays and the haemolytic index was quantified as internal control per sample (Roche Diagnostics). Results: There were significant differences in CSF values between TBI and all control cases (p