Acquisition of chromosome 1q duplication in parental and genome-edited human-induced pluripotent stem cell-derived neural stem cells results in their higher proliferation rate in vitro and in vivo.

OBJECTIVES: Genetic engineering of human-induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC) may increase the risk of genomic aberrations. Therefore, we asked whether genetic modification of hiPSC-NSCs exacerbates chromosomal abnormalities that may occur during passaging and whether they may cause any functional perturbations in NSCs in vitro and in vivo. MATERIALS AND METHODS: The transgenic cassette was inserted into the AAVS1 locus, and the genetic integrity of zinc-finger nuclease (ZFN)-modified hiPSC-NSCs was assessed by the SNP-based karyotyping. The hiPSC-NSC proliferation was assessed in vitro by the EdU incorporation assay and in vivo by staining of brain slices with Ki-67 antibody at 2 and 8 weeks after transplantation of ZFN-NSCs with and without chromosomal aberration into the striatum of immunodeficient rats. RESULTS: During early passages, no chromosomal abnormalities were detected in unmodified or ZFN-modified hiPSC-NSCs. However, at higher passages both cell populations acquired duplication of the entire long arm of chromosome 1, dup(1)q. ZNF-NSCs carrying dup(1)q exhibited higher proliferation rate than karyotypically intact cells, which was partly mediated by increased expression of AKT3 located on Chr1q. Compared to karyotypically normal ZNF-NSCs, cells with dup(1)q also exhibited increased proliferation in vivo 2 weeks, but not 2 months, after transplantation. CONCLUSIONS: These results demonstrate that, independently of ZFN-editing, hiPSC-NSCs have a propensity for acquiring dup(1)q and this aberration results in increased proliferation which might compromise downstream hiPSC-NSC applications.

Perforator Dissection Simulation: A High-Fidelity Five-Flap Porcine Training Model.

INTRODUCTION: Perforator dissection can be technically demanding with a steep learning curve. Inadvertent perforator damage during dissection can be minimized with practice and familiarity with tissue-handling techniques unique to perforator dissection. There currently lacks a simulation model that mimics the human perforator in size and course. We present a porcine training model with five consistent perforator flaps per side that can be readily harvested and is a reproducible simulation model. MATERIALS AND METHODS: Five fresh cadaveric pigs were used in this study to evaluate the feasibility and location of the perforators. Ten perforators were dissected out in each pig (five per side) by the same surgeon. The length of perforator was measured and intramuscular route was noted. The ease of dissection was graded, and its similarity to actual surgery was graded as well. RESULTS: Five consistent perforators were identified across each side of five fresh cadaveric pigs. The perforators were located, namely in the neck, anterior flank, posterior flank, rectus and hindlimb. They were fasciocutaneous and had an intramuscular course of each (average 2.5 cm length). The perforators were found to be on each side of the pig, giving ten perforators for dissection practice in total. DISCUSSION: The five perforators named in this porcine model are easily replicated and can be performed for perforator dissection simulation and practice.

Dexamethasone and levetiracetam reduce hetero-cellular gap-junctional coupling between F98 glioma cells and glial cells in vitro.

Gap junctions (GJs) in astrocytes and glioma cells are important channels for cell-to-cell communication that contribute to homo- and heterocellular coupling. According to recent studies, heterocellular gap-junctional communication (H-GJC) between glioma cells and their surrounding environment enhances glioma progression. Therefore, we developed a new in vitro model to examine H-GJC between glioma cells, astrocytes and microglia. Consequently, F98 rat glioma cells were double-labeled with GJ-impermeable (CM-DiI) and GJ-permeable dye (calcein AM) and were seeded on unlabeled astrocyte-microglia co-cultures. Dual whole cell voltage clamp recordings were carried out on selected cell pairs to characterize the functional properties of H-GJC in vitro. The expression of four types of connexins (Cxs), including Cx32, Cx36, Cx43 and Cx45, and microglial phenotypes were analyzed by immunocytochemistry. The H-GJC between glioma cells and astrocytes/microglia increased after a longer incubation period with a higher number of glioma cells. We provided evidence for the direct GJ coupling of microglia and glioma cells under native in vitro conditions. In addition, we exploited this model to evaluate H-GJC after incubation with levetiracetam (LEV) and/or dexamethasone (DEX). Previous in vitro studies suggest that LEV and DEX are frequently used to control seizure and edema in glioma. Our findings showed that LEV and/or DEX decrease the number of heterocellular coupled cells significantly. In conclusion, our newly developed model demonstrated H-GJC between glioma cells and both astrocytes and microglia. The reduced H-GJC by LEV and DEX suggests a potential effect of both drugs on glioma progression.

Low-flow lower body perfusion for spinal protection in a frozen elephant trunk simulation model.

OBJECTIVES: The frozen elephant trunk (FET) procedure using isolated selective cerebral perfusion (SCP) at moderate hypothermia is associated with an increased risk for spinal cord ischaemia. The aim of this study was to evaluate the benefit of a combined selective cerebral and low-flow lower body perfusion (CLBP) in a porcine model. METHODS: Twenty pigs (46 ± 5 kg) were cooled on cardiopulmonary bypass (CPB) to 28°C. After aortic clamping and occlusion of the thoracic segmental arteries (TSAT4-T13), a pressure-controlled SCP (50 mmHg) was established for 90 min. Randomly, in n = 10 animals, an additional lower body perfusion (LBP) was performed with 15 ml/kg/min (CLBP). Regional spinal cord blood flow (SCBF), cerebrospinal fluid pressure (CSFP) and motor-evoked potentials (MEPs) were registered at six time points. The animals were sacrificed after 120 min of weaning from CPB, and the spinal cord was analysed histologically using a schematic scoring system (0 = normal, 8 = total necrosis). RESULTS: Isolated SCP led to an SCBF decrease from 18.5 ± 9.4 to 0.9 ± 1.4 ml/min/100 g in the L1-L5 region (P = 0.005). CLBP preserved an almost physiological lumbar SCBF of 11.3 ± 5.3 ml/min/100 g. CSFP decreased in both groups during cooling and SCP/CLBP to 70-80% and increased during reperfusion to 150%, without showing significant differences between groups. The MEP amplitude decreased in both groups, with certain regional differences: T7-T11. MEP recording revealed a more pronounced amplitude decrease in the CLBP group (52.5 ± 2.0 vs 71.3 ± 0.9%), but MEP amplitudes recovered in both groups (SCP: 73.7 ± 0.5 vs CLBP: 82.6 ± 0.1%). During selective hypothermic perfusion, SCP-treated animals showed significant lower MEP amplitudes, when compared with CLBP-treated animals: 60 ± 9 vs 90 ± 3% (P < 0.001). After weaning, CLBP animals showed a better MEP recovery, especially in the L1-L5 region (99 ± 7 vs 70 ± 13%; P < 0.001). The histological analysis did not show significant differences in the necrosis extension in the thoracic spinal cord. A different situation was seen in the L1-L5 area: all animals with isolated SCP, but only 50% of the CLBP animals presented a score of >5. A higher grade of lumbar ischaemia could be seen after isolated SCP (score: 5.9 ± 0.6 vs 3.6 ± 2.9). CONCLUSION: The prolonged SCP provides an insufficient lumbar spinal cord protection during the FET procedure at 28°C. The use of a low-flow LBP in addition to SCP may reduce functional and structural spinal damage.

Multiple Sclerosis Patient-Specific Primary Neurons Differentiated from Urinary Renal Epithelial Cells via Induced Pluripotent Stem Cells.

As multiple sclerosis research progresses, it is pertinent to continue to develop suitable paradigms to allow for ever more sophisticated investigations. Animal models of multiple sclerosis, despite their continuing contributions to the field, may not be the most prudent for every experiment. Indeed, such may be either insufficient to reflect the functional impact of human genetic variations or unsuitable for drug screenings. Thus, we have established a cell- and patient-specific paradigm to provide an in vitro model within which to perform future genetic investigations. Renal proximal tubule epithelial cells were isolated from multiple sclerosis patients' urine and transfected with pluripotency-inducing episomal factors. Subsequent induced pluripotent stem cells were formed into embryoid bodies selective for ectodermal lineage, resulting in neural tube-like rosettes and eventually neural progenitor cells. Differentiation of these precursors into primary neurons was achieved through a regimen of neurotrophic and other factors. These patient-specific primary neurons displayed typical morphology and functionality, also staining positive for mature neuronal markers. The development of such a non-invasive procedure devoid of permanent genetic manipulation during the course of differentiation, in the context of multiple sclerosis, provides an avenue for studies with a greater cell- and human-specific focus, specifically in the context of genetic contributions to neurodegeneration and drug discovery.

Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

INTRODUCTION: Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43) and estrogen receptors (ERs). Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2) on Cx43 expression in two glioma cell lines with variable native expression of Cx43. MATERIALS AND METHODS: F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERß and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique. RESULTS: E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERß was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERß in C6 cultures, while it decreased ERα expression in F98 glioma cells. DISCUSSION: These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

Spinal cord ischemia after selective cerebral perfusion in a porcine "frozen elephant trunk" simulation model.

BACKGROUND: The "frozen elephant trunk" procedure (FET) represents the therapy of choice for extended aortic diseases. The aim of our study was to analyze whether 90 minutes of selective cerebral perfusion (SCP) at 28 °C followed by permanent occlusion of the thoracic segmental arteries (TSA) would cause spinal cord ischemia in a porcine model. METHODS: 14 pigs (41 ± 3 kg) were cooled on CPB to 28 °C. After aortic clamping, SCP was established for 90 minutes. Randomly, in 7 animals the TSA were clipped (T4-T13); the TSA of 7 animals remained untouched. After the animals were weaned from CPB, hemodynamic data were registered for 120 minutes. Regional spinal cord blood flow (SCBF) was calculated, and motor-evoked potentials (MEP) were assessed at 6 time points. After sacrifice of the animals, the spinal cord was analyzed histologically by use of a schematic grading system (0 = normal; 8 = total necrosis). RESULTS: During SCP the SCBF was maintained at baseline (5.9 ± 2.4 mL/min/100 g) in the T4-T13 region but showed a decrease (from 8.4 ± 4.3 to 1.3 ± 1.5 mL/min/100 g) in the L1-L5 region. During reperfusion it increased, with two to three times higher values in the nonclipped animals. After 90 minutes of SCP, the MEP reached lower levels in the L1-L5 region of the TSA-clipped animals: 59% ± 7% vs 84 ± 15% (vastus medialis muscle) and 48% ± 6% vs 82% ± 26% (tibialis anterior muscle). The MEP recovered only in the nonclipped group. Higher ischemia rates were seen in the L1-L5 region of the TSA-clipped animals (score: 6.0 ± 0.6 vs 2.5 ± 2.3). CONCLUSIONS: 90 minutes of SCP provided sufficient spinal cord protection during arch replacement at 28 °C. In combination with permanent TSA occlusion, the lumbar spinal cord perfusion may be altered, which causes functional and structural damage.

Bisphosphonates and connexin 43: a critical review of evidence.

Bisphosphonates (BPs) are drugs commonly used in the treatment of various disease arising or affecting bone tissue. There is a standard use in bone neoplasia and metastasis, hormonal and developmental disorders as well as for compensation of adverse effects in several medical therapies. Many in-vivo and in-vitro studies have assessed the efficacy of this drug and its function in cellular scale. In this concern, BPs are described to inhibit the resorptive function of osteoclasts and to prevent apoptosis of osteoblasts and osteocytes. They can preserve the osteocytic network, reduce fracture rate, and increase the bone mineral content, which is therapeutically used. Connexin 43 (Cx43) is a crucial molecule for basal regulation of bone homeostasis, development, and differentiation. It is described for signal transduction in many physiological and pathological stimuli and recently to be involved in BP action.

Internal ribosomal entry site (IRES) activity generates endogenous carboxyl-terminal domains of Cx43 and is responsive to hypoxic conditions.

Connexin43 (Cx43) is the most abundant gap junction protein in higher vertebrate organisms and has been shown to be involved in junctional and non-junctional functions. In addition to the expression of full-length Cx43, endogenously produced carboxyl-terminal segments of Cx43 have been described and have been suggested to be involved in manifold biological functions, such as hypoxic preconditioning and neuronal migration. Molecular aspects, however, behind the separate generation of carboxyl-terminal segments of Cx43 have remained elusive. Here we report on a mechanism that may play a key role in the separate production of these domains. First, stringent evidence derived from siRNA treatment and specific knockouts revealed significant loss of the low molecular weight fragments of Cx43. By applying a dicistronic vector strategy on transfected cell lines, we were able to identify putative IRES activity (nucleotides 442­637) in the coding region of Cx43, which resides upstream from the nucleotide sequence encoding the carboxyl terminus (nucleotides 637­1149). Functional responsiveness of the endogenous expression of Cx43 fragments to hypoxic/ischemic treatment was evaluated in in vitro and in vivo models, which led to a significant increase of the fastest migrating form (20 kDa) under conditions of metabolic deprivation. By nano-MS spectrometry, we achieved stringent evidence of the identity of the 20-kDa segment as part of the carboxyl-terminal domain of full-length Cx43. Our data prove the existence of endogenously expressed carboxyl-terminal domains, which may serve as valuable tools for further translational application in ischemic disorders.

Relevance of gap junctions and large pore channels in traumatic brain injury.

In case of traumatic brain injury (TBI), occurrence of central nervous tissue damage is frequently aligned with local modulations of neuronal and glial gap junction channel expression levels. The degree of gap junctional protein expression and intercellular coupling efficiency, as well as hemichannel function has substantially impact on the course of trauma recovery and outcome. During TBI, gap junctions are especially involved in the intercellular molecule trafficking on repair of blood vessels and the regulation of vasomotor tone. Furthermore, gliosis and astrocytic swelling due to mechanical strain injury point out the consequences of derailed gap junction communication. This review addresses the outstanding role of gap junction channels in TBI pathophysiology and links the current state of results to applied clinical procedures as well as perspectives in acute and long-term treatment options.

Glia and epilepsy: experimental investigation of antiepileptic drugs in an astroglia/microglia co-culture model of inflammation.

PURPOSE: The contribution of glial cells, mainly astrocytes and microglia, to the pathophysiology of epilepsy is increasingly appreciated. Glia play a pivotal role in the initiation and maintenance of the central nervous system (CNS) immune response and neuronal metabolic and trophic supply. Recent clinical and experimental evidence suggests a direct relationship between epileptic activity and CNS inflammation, which is characterized by accumulation, activation, and proliferation of microglia and astrocytes. Concomitant glia-mediated mechanisms of action of several antiepileptic drugs (AEDs) have been proposed. However, their direct effects on glial cells have been rarely investigated. We aimed to investigate the effect of commonly used AEDs on glial viability, the gap junctional network, the microglial activation, and cytokine expression in an in vitro astroglia/microglia co-culture model. METHODS: Primary astrocytic cultures were prepared from brains of postnatal (P0-P2) Wistar rats and co-cultured with a physiologic amount of 5%, as well as 30% microglia in order to mimic inflammatory conditions. Co-cultures were treated with valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHE), and gabapentin (GBT). Viability and proliferation were measured using the tetrazolium (MTT) assay. The microglial activation state was determined by immunocytochemical labeling. The astroglial connexin 43 (Cx43) expression was measured by Western blot analysis. The transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) cytokine levels were measured by the quantitative sandwich enzyme immunosorbent assay (ELISA). KEY FINDINGS: Astrocytes, co-cultured with 5% microglia (M5 co-cultures), showed a dose-dependent, significant reduction in glial viability after incubation with PHE and CBZ. Furthermore, VPA led to highly significant microglial activation at all doses examined. The antiinflammatory cytokine TGF-ß1 release was induced by high doses of GBT and PHE. Astrocytes co-cultured with 30% microglia (M30 co-cultures) revealed a dose-dependent significant reduction in glial viability after incubation with PHE, accompanied by increased TGF-ß1 and TNF-α levels. However, CBZ significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia. Finally, CBZ resulted in reduced viability at all doses examined. SIGNIFICANCE: CNS inflammation is characterized by a disturbance of glial cell functions. Strong microglial activation, a typical hallmark of inflammation, was induced by VPA in M5 and continued in M30 co-cultures. With regard to the direct relation between CNS inflammation and seizures, VPA seems to be unsuitable for reducing inflammatory conditions. The reverse effect was achieved after CBZ. We noticed significant microglial inactivation, after incubation of the M30 co-cultures. In conclusion, we suggest that AEDs with antiinflammatory glial features are beneficial for seizures caused by persistent brain inflammation.

Pannexin1 channel proteins in the zebrafish retina have shared and unique properties.

In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.

An electrophysiologic approach to quantify impaired synaptic transmission and plasticity in experimental autoimmune encephalomyelitis.

Despite its various limitations, for many decades the experimental autoimmune encephalomyelitis (EAE) has been indispensable for understanding the pathology of multiple sclerosis (MS) and for establishing widely used MS therapeutics. We tested whether synaptic plasticity is a suitable measure for EAE and whether it can detect detrimental effects on supra-spinal structures that are too subtle to be captured by the motor score. Our data show functional synaptic deficits in the EAE that were beyond the measurable EAE score: long-term depression responses were strongly weakened in superior colliculus and cerebellum resulting from impaired postsynaptic transmission. In addition to further insight into neuronal deficits associated with the autoimmune disease, quantification of synaptic transmission may serve as a complementary method of EAE evaluation.

Material attrition and bone micromorphology after conventional and ultrasonic implant site preparation.

OBJECTIVES: Little is known about the recently introduced ultrasonic implant site preparation. The purpose of this study was to compare material attrition and micromorphological changes after ultrasonic and conventional implant site preparations. MATERIAL AND METHODS: Implant site preparations were performed on fresh bovine ribs using one conventional (Straumann, Freiburg, Germany) and two ultrasonic (Piezosurgery; Mectron Medical Technology, Carasco, Italy and Variosurg; NSK, Tochigi, Japan) systems with sufficient saline irrigation. Sections were examined by environmental scanning electron microscopy (ESEM). Energy-dispersive X-ray spectroscopy (EDX) was performed to evaluate the metal attrition within the bone and the irrigation fluid. RESULTS ESEM: After conventional osteotomy, partially destroyed trabecular structures of the cancellous bone that were loaded with debris were observed, whereas after ultrasonic implant site preparations, the anatomic structures were preserved. EDX: None of the implant site preparation methods resulted in metal deposits in the adjacent bone structures. However, within the irrigation liquid, there was significantly higher metal attrition with ultrasonic osteotomy (P < 0.0001 and P < 0.0001 for Mectron and NSK, respectively). Whereas for Straumann system used, 15.5% of the SEM/EDX findings were drill-origin metals, this percentage increased to 37.3% and 37.9% with the application of Mectron and NSK, respectively. CONCLUSIONS: Ultrasonic implant site preparation is associated with the preservation of bone microarchitecture and with the increased attrition of metal particles. Therefore, copious irrigation seems to be even more essential for ultrasonic implant site preparation than for the conventional method.

Functions of connexins and large pore channels on microglial cells: the gates to environment.

Microglial cells are not only sensitive indicators for pathology of the central nervous system (CNS), they are a key factor for neurotoxicity and degeneration in many diseases. Neuronal damage leads to reactive gliosis and to activation of microglia including cytoarchitectonic changes accompanied by alterations in surface receptor and channel expression. In this context, the release of neuroactive soluble factors like pro-inflammatory cytokines can result in increased cellular motility and a higher grade of phagocytotic activity. Ligands including glutamate, tumor necrosis factor alpha (TNF-α), cytokines, superoxide radicals and neurotrophins released by microglia have in turn effects on neuronal function and cell death. The current review focuses on large pore and hemichannel function in microglial cells under different conditions of activation and elucidates the role of these channels in cytokine release, as well as putative targets for clinical intervention in case of inflammatory processes. This article is part of a Special Issue entitled Electrical Synapses.

Pannexin1 stabilizes synaptic plasticity and is needed for learning.

Pannexin 1 (Panx1) represents a class of vertebrate membrane channels, bearing significant sequence homology with the invertebrate gap junction proteins, the innexins and more distant similarities in the membrane topologies and pharmacological sensitivities with gap junction proteins of the connexin family. In the nervous system, cooperation among pannexin channels, adenosine receptors, and K(ATP) channels modulating neuronal excitability via ATP and adenosine has been recognized, but little is known about the significance in vivo. However, the localization of Panx1 at postsynaptic sites in hippocampal neurons and astrocytes in close proximity together with the fundamental role of ATP and adenosine for CNS metabolism and cell signaling underscore the potential relevance of this channel to synaptic plasticity and higher brain functions. Here, we report increased excitability and potently enhanced early and persistent LTP responses in the CA1 region of acute slice preparations from adult Panx1(-/-) mice. Adenosine application and N-methyl-D-aspartate receptor (NMDAR)-blocking normalized this phenotype, suggesting that absence of Panx1 causes chronic extracellular ATP/adenosine depletion, thus facilitating postsynaptic NMDAR activation. Compensatory transcriptional up-regulation of metabotropic glutamate receptor 4 (grm4) accompanies these adaptive changes. The physiological modification, promoted by loss of Panx1, led to distinct behavioral alterations, enhancing anxiety and impairing object recognition and spatial learning in Panx1(-/-) mice. We conclude that ATP release through Panx1 channels plays a critical role in maintaining synaptic strength and plasticity in CA1 neurons of the adult hippocampus. This result provides the rationale for in-depth analysis of Panx1 function and adenosine based therapies in CNS disorders.

Micromorphometrical analyses of five different ultrasonic osteotomy devices at the rabbit skull.

OBJECTIVES: The recently introduced ultrasonic osteotome procedure is an alternative to conventional methods of osteotomy. The aim of the present study was to establish the differences between five recently introduced ultrasonic osteotomes and to perform micromorphological and quantitative roughness analyses of osteotomized bone surfaces. MATERIALS AND METHODS: Fresh, standard-sized bony samples were taken from a rabbit skull using the following ultrasonic osteotomes: the Piezosurgery 3 with insert tip OT7, Piezosurgery Medical with insert tip MT1-10, Piezon Master Surgery with insert tip SL1, VarioSurg with inert tip SG1, and Piezotome 2 with insert tip BS1 II. The required duration of time for each osteotomy was recorded. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), and confocal laser scanning microscopy (CLSM). RESULTS: All of the investigated piezoelectric osteotomes preserved the anatomical structure of bone. The mean roughness values of the osteotomized bone edge obtained using the investigated piezoelectric osteotomes were as follows: 2.47 µm (Piezosurgery 3), 9.79 µm (Piezosurgery Medical), 4.66 µm (Piezon Master Surgery), 6.38 µm (VarioSurg), and 6.06 µm (Piezotome 2). Significantly higher roughness values were observed when using the Piezosurgery Medical in comparison with those achieved by the Piezosurgery 3 (P<0.0001) and Piezon Master Surgery (P=0.002). Different osteotomy durations were achieved using the different piezoelectric osteotomes: 144 s (Piezosurgery 3), 126 s (Piezosurgery Medical), 142 s (Piezon Master Surgery), 149 s (VarioSurg), and 137 s (Piezotome 2). CONCLUSIONS: In the present study, micromorphological differences following the use of various ultrasonic devices were clearly identified. According to this study, it can be concluded that the power and the composition of the teeth of the insert tip might impact procedure duration and cutting qualities.

Electromagnetic field effect or simply stress? Effects of UMTS exposure on hippocampal longterm plasticity in the context of procedure related hormone release.

Harmful effects of electromagnetic fields (EMF) on cognitive and behavioural features of humans and rodents have been controversially discussed and raised persistent concern about adverse effects of EMF on general brain functions. In the present study we applied radio-frequency (RF) signals of the Universal Mobile Telecommunications System (UMTS) to full brain exposed male Wistar rats in order to elaborate putative influences on stress hormone release (corticosteron; CORT and adrenocorticotropic hormone; ACTH) and on hippocampal derived synaptic long-term plasticity (LTP) and depression (LTD) as electrophysiological hallmarks for memory storage and memory consolidation. Exposure was computer controlled providing blind conditions. Nominal brain-averaged specific absorption rates (SAR) as a measure of applied mass-related dissipated RF power were 0, 2, and 10 W/kg over a period of 120 min. Comparison of cage exposed animals revealed, regardless of EMF exposure, significantly increased CORT and ACTH levels which corresponded with generally decreased field potential slopes and amplitudes in hippocampal LTP and LTD. Animals following SAR exposure of 2 W/kg (averaged over the whole brain of 2.3 g tissue mass) did not differ from the sham-exposed group in LTP and LTD experiments. In contrast, a significant reduction in LTP and LTD was observed at the high power rate of SAR (10 W/kg). The results demonstrate that a rate of 2 W/kg displays no adverse impact on LTP and LTD, while 10 W/kg leads to significant effects on the electrophysiological parameters, which can be clearly distinguished from the stress derived background. Our findings suggest that UMTS exposure with SAR in the range of 2 W/kg is not harmful to critical markers for memory storage and memory consolidation, however, an influence of UMTS at high energy absorption rates (10 W/kg) cannot be excluded.

Unified patch clamp protocol for the characterization of Pannexin 1 channels in isolated cells and acute brain slices.

In the central nervous system, Pannexin 1 (Panx1) channels are implicated in a variety of physiological and pathological conditions. One of the prerequisites to enlighten the role of Panx1 is the development and standardization of reliable methods. Here, we address the applicability of voltage clamp protocols to identify Panx1 channel mediated currents in neurons of acutely dissected brain slices. We improved an established protocol and report on a modified paradigm that robustly evokes Panx1 channel currents. Crucial advances are the use of physiologic ion gradient conditions and a preconditioning step of depolarizing membrane potential ramps of long duration. This new paradigm provides significant impact on membrane current generation at hypo- and depolarized holding potential steps post voltage ramp preconditioning in heterologous expression systems and primary hippocampal CA1 neurons of mouse brain slices in vitro. Finally, we demonstrate that under these conditions the analysis of tail currents elicited by repolarization of the cells from preconditioning holding potential depolarization permits an independent method to isolate Panx1 mediated channel activity. In summary, this study provides a comprehensive methodological improvement in the biophysical analysis of Panx1 channels with a particular focus on investigations under physiological conditions in complex tissues.