Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

Alzheimer's disease (AD) has a characteristic hallmark of amyloid-β (Aβ) accumulation in the brain. This accumulation of Aβ has been related to its faulty cerebral clearance. Indeed, preclinical studies that used mice to investigate Aβ clearance showed that efflux across blood-brain barrier (BBB) and brain degradation mediate efficient Aβ clearance. However, the contribution of each process to Aβ clearance remains unclear. Moreover, it is still uncertain how species differences between mouse and human could affect Aβ clearance. Here, a modified form of the brain efflux index method was used to estimate the contribution of BBB and brain degradation to Aβ clearance from the brain of wild type mice. We estimated that 62% of intracerebrally injected (125)I-Aβ40 is cleared across BBB while 38% is cleared by brain degradation. Furthermore, in vitro and in silico studies were performed to compare Aβ clearance between mouse and human BBB models. Kinetic studies for Aβ40 disposition in bEnd3 and hCMEC/D3 cells, representative in vitro mouse and human BBB models, respectively, demonstrated 30-fold higher rate of (125)I-Aβ40 uptake and 15-fold higher rate of degradation by bEnd3 compared to hCMEC/D3 cells. Expression studies showed both cells to express different levels of P-glycoprotein and RAGE, while LRP1 levels were comparable. Finally, we established a mechanistic model, which could successfully predict cellular levels of (125)I-Aβ40 and the rate of each process. Established mechanistic model suggested significantly higher rates of Aβ uptake and degradation in bEnd3 cells as rationale for the observed differences in (125)I-Aβ40 disposition between mouse and human BBB models. In conclusion, current study demonstrates the important role of BBB in the clearance of Aβ from the brain. Moreover, it provides insight into the differences between mouse and human BBB with regards to Aβ clearance and offer, for the first time, a mathematical model that describes Aβ clearance across BBB.

Arginine-vasopressin (AVP) is a peptide hormone normally secreted from neuroendocrine cells via the regulated secretory pathway. In Familial Neurohypophyseal Diabetes Insipidus (FNDI), an autosomal dominant form of central diabetes insipidus, mutations of pro-vasopressin appear to accumulate in the endoplasmic reticulum (ER) causing a lack of biologically active AVP in the blood. To investigate the effect of pro-vasopressin mutations regarding intracellular functions of protein targeting and secretion, we created two FNDI-associated amino acid substitution mutants, e.g., G14R, and G17V in frame with green fluorescent protein (GFP) and pro-vasopressin (VP) in frame with red fluorescent protein (VP-RFP). Fluorescence microscopy of Neuro-2a cells expressing these constructs revealed co-localization of VP-GFP and VP-RFP to punctate granules along the length and accumulating at the tips of neurites, characteristic of regulated secretory granules. In contrast, the two FNDI-associated amino acid substitution mutants, e.g., G14R-GFP, and G17VGFP, were localized to a perinuclear region of the Neuro-2a cells characteristic of the endoplasmic reticulum. Co-expression of these mutants with VP-RFP showed VP-RFP was retained in the ER, co-localized with the mutants suggesting the formation of heterodimers as found in FNDI. Stimulated secretion experiments indicated that VP-GFP was secreted in an inducible manner whereas, G14R-GFP and G17V-GFP were retained to nearly 100% within the cells. Analysis by western blotting and semi-quantitative RT-PCR indicated an increased protein and mRNA expression for an ER resident molecular chaperone, BiP. Further analysis of ER-storage disease-associated proteins such as caspase 12 and CHOP showed an increase in these as well. The results suggest that G14R-GFP and G17V-GFP are retained in the ER of Neuro-2a cells, resulting in up-regulation of the molecular chaperone BiP, and activation of the ER-storage disease-associated caspase cascade system.

Alzheimer's disease (AD) is characterized by accumulation and deposition of Abeta peptides in the brain. Abeta deposition in cerebral vessels occurs in many AD patients and results in cerebral amyloid angiopathy (AD/CAA). Abeta deposits evoke neuro- and neurovascular inflammation contributing to neurodegeneration. In this study, we found that exposure of cultured human brain endothelial cells (HBEC) to Abeta(1-40) elicited expression of inflammatory genes MCP-1, GRO, IL-1beta and IL-6. Up-regulation of these genes was confirmed in AD and AD/CAA brains by qRT-PCR. Profiling of 54 transcription factors indicated that AP-1 was strongly activated not only in Abeta-treated HBEC but also in AD and AD/CAA brains. AP-1 complex in nuclear extracts from Abeta-treated HBEC bound to AP-1 DNA-binding sequence and activated the reporter gene of a luciferase vector carrying AP-1-binding site from human MCP-1 gene. AP-1 is a dimeric protein complex and supershift assay identified c-Jun as a component of the activated AP-1 complex. Western blot analyses showed that c-Jun was activated via JNK-mediated phosphorylation, suggesting that as a result of c-Jun phosphorylation, AP-1 was activated and thus up-regulated MCP-1 expression. A JNK inhibitor SP600125 strongly inhibited Abeta-induced c-Jun phosphorylation, AP-1 activation, AP-1 reporter gene activity and MCP-1 expression in cells stimulated with Abeta peptides. The results suggested that JNK-AP1 signaling pathway is responsible for Abeta-induced neuroinflammation in HBEC and Alzheimer's brain and that this signaling pathway may serve as a therapeutic target for relieving Abeta-induced inflammation.

The prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming alpha-subunit of BKCa channels in breast cancer metastasis and invasion.

Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages-a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in vitro differentiation capacity of isolated MSCs. Bone marrow was obtained from the femoral cavity of twelve donors during total hip arthroplasty in the absence or presence of heparin. No coagulation was observed in the absence of heparin. The number of mononuclear cells was independent of heparin addition. Isolated MSCs were characterized by morphology, population doubling times, expression of cell surface antigens and in vitro differentiation. Results of these analyses were independent of the amount of heparin. Transcriptome analyses of cells from three randomly chosen donors and quantitative realtime PCR (qRT-PCR) analysis from cells of all donors demonstrated no clear effect of heparin on the transcriptome of the cells. This excludes heparin as a potential source of disparate results.

The hydrogen sulfide (H2S) and the oxytocin/oxytocin receptor (OT/OTR) systems interact in trauma and are implicated in vascular protection and regulation of fluid homeostasis. Acute brain injury is associated with pressure-induced edema formation, blood brain barrier disruption, and neuro-inflammation. The similarities in brain anatomy: size, gyrencephalic organization, skull structure, may render the pig a highly relevant model for translational medicine. Cerebral biomarkers for pigs for pathophysiological changes and neuro-inflammation are limited. The current study aims to characterize the localization of OT/OTR and the endogenous H2S producing enzymes together with relevant neuro-inflammatory markers on available porcine brain tissue from an acute subdural hematoma (ASDH) model. In a recent pilot study, anesthetized pigs underwent ASDH by injection of 20 mL of autologous blood above the left parietal cortex and were resuscitated with neuro-intensive care measures. After 54 h of intensive care, the animals were sacrificed, the brain was removed and analyzed via immunohistochemistry. The endogenous H2S producing enzymes cystathionine-ɤ-lyase (CSE) and cystathionine-β-synthase (CBS), the OTR, and OT were localized in neurons, vasculature and parenchyma at the base of sulci, where pressure-induced injury leads to maximal stress in the gyrencephalic brain. The pathophysiological changes in response to brain injury in humans and pigs, we show here, are comparable. We additionally identified modulators of brain injury to further characterize the pathophysiology of ASDH and which may indicate future therapeutic approaches.

Background: The success of a cochlear implant (CI), which is the standard therapy for patients suffering from severe to profound sensorineural hearing loss, depends on the number and excitability of spiral ganglion neurons (SGNs). Brain-derived neurotrophic factor (BDNF) has a protective effect on SGNs but should be applied chronically to guarantee their lifelong survival. Long-term administration of BDNF could be achieved using genetically modified mesenchymal stem cells (MSCs), but these cells should be protected - by ultra-high viscous (UHV-) alginate ('alginate-MSCs') - from the recipient immune system and from uncontrolled migration. Methods: Brain-derived neurotrophic factor-producing MSCs were encapsulated in UHV-alginate. Four experimental groups were investigated using guinea pigs as an animal model. Three of them were systemically deafened and (unilaterally) received one of the following: (I) a CI; (II) an alginate-MSC-coated CI; (III) an injection of alginate-embedded MSCs into the scala tympani followed by CI insertion and alginate polymerization. Group IV was normal hearing, with CI insertion in both ears and a unilateral injection of alginate-MSCs. Using acoustically evoked auditory brainstem response measurements, hearing thresholds were determined before implantation and before sacrificing the animals. Electrode impedance was measured weekly. Four weeks after implantation, the animals were sacrificed and the SGN density and degree of fibrosis were evaluated. Results: The MSCs survived being implanted for 4 weeks in vivo. Neither the alginate-MSC injection nor the coating affected electrode impedance or fibrosis. CI insertion with and without previous alginate injection in normal-hearing animals resulted in increased hearing thresholds within the high-frequency range. Low-frequency hearing loss was additionally observed in the alginate-injected and implanted cochleae, but not in those treated only with a CI. In deafened animals, the alginate-MSC coating of the CI significantly prevented SGN from degeneration, but the injection of alginate-MSCs did not. Conclusion: Brain-derived neurotrophic factor-producing MSCs encapsulated in UHV-alginate prevent SGNs from degeneration in the form of coating on the CI surface, but not in the form of an injection. No increase in fibrosis or impedance was detected. Further research and development aimed at verifying long-term mechanical and biological properties of coated electrodes in vitro and in vivo, in combination with chronic electrical stimulation, is needed before the current concept can be tested in clinical trials.

Rotator cuff tear is the most frequent tendon injury in the adult population. Despite current improvements in surgical techniques and the development of grafts, failure rates following tendon reconstruction remain high. New therapies, which aim to restore the topology and functionality of the interface between muscle, tendon and bone, are essentially required. One of the key factors for a successful incorporation of tissue engineered constructs is a rapid ingrowth of cells and tissues, which is dependent on a fast vascularization. The dorsal skinfold chamber model in female BALB/cJZtm mice allows the observation of microhemodynamic parameters in repeated measurements in vivo and therefore the description of the vascularization of different implant materials. In order to promote vascularization of implant material, we compared a porous polymer patch (a commercially available porous polyurethane based scaffold from Biomerix™) with electrospun polycaprolactone (PCL) fiber mats and chitosan-graft-PCL coated electrospun PCL (CS-g-PCL) fiber mats in vivo. Using intravital fluorescence microscopy microcirculatory parameters were analyzed repetitively over 14 days. Vascularization was significantly increased in CS-g-PCL fiber mats at day 14 compared to the porous polymer patch and uncoated PCL fiber mats. Furthermore CS-g-PCL fiber mats showed also a reduced activation of immune cells. Clinically, these are important findings as they indicate that the CS-g-PCL improves the formation of vascularized tissue and the ingrowth of cells into electrospun PCL scaffolds. Especially the combination of enhanced vascularization and the reduction in immune cell activation at the later time points of our study points to an improved clinical outcome after rotator cuff tear repair.

We previously showed that attenuated glucocorticoid receptor (GR) function in mice (GRdim/dim) aggravates systemic hypotension and impairs organ function during endotoxic shock. Hemorrhagic shock (HS) causes impaired organ perfusion, which leads to tissue hypoxia and inflammation with risk of organ failure. Lung co-morbidities like chronic obstructive pulmonary disease (COPD) can aggravate tissue hypoxia via alveolar hypoxia. The most common cause for COPD is cigarette smoke (CS) exposure. Therefore, we hypothesized that affecting GR function in mice (GRdim/dim) and pre-traumatic CS exposure would further impair hemodynamic stability and organ function after HS.

Cerebral cavernous malformations (CCMs) are vascular malformations that can be the result of the deficiency of one of the CCM genes. Their only present treatment is surgical removal, which is not always possible, and an alternative pharmacological strategy to eliminate them is actively sought. We have studied the effect of the lack of one of the CCM genes, CCM3, in endothelial and non-endothelial cells. By comparing protein expression in control and CCM3-silenced cells, we found that the levels of the Epidermal Growth Factor Receptor (EGFR) are higher in CCM3-deficient cells, which adds to the known upregulation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in these cells. Whereas VEGFR2 is upregulated at the mRNA level, EGFR has a prolonged half-life. Inhibition of EGFR family members in CCM3-deficient cells does not revert the known cellular effects of lack of CCM genes, but it induces significantly more apoptosis in CCM3-deficient cells than in control cells. We propose that the susceptibility to tyrosine kinase inhibitors of CCM3-deficient cells can be harnessed to kill the abnormal cells of these lesions and thus treat CCMs pharmacologically.

Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-β (TGF-β) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and β-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

As a response to pro-inflammatory signals mesenchymal stem cells (MSCs) secrete agents and factors leading to lymphocyte recruitment, counteracting inflammation, and stimulating immunosuppression. On a molecular level, the signalling mediator TGF-β-activated kinase 1 (TAK1) is activated by many pro-inflammatory signals, plays a critical role in inflammation and regulates innate and adaptive immune responses as well. While the role of TAK1 as a signalling factor promoting inflammation is well documented, we also considered a role for TAK1 in anti-inflammatory actions exerted by activated MSCs. We, therefore, investigated the capacity of lipopolysaccharide (LPS)-treated murine MSCs with lentivirally modulated TAK1 expression levels to recruit lymphocytes. TAK1 downregulated by lentiviral vectors expressing TAK1 shRNA in murine MSCs interfered with the capacity of murine MSCs to chemoattract lymphocytes, indeed. Analysing a pool of 84 secreted factors we found that among 26 secreted cytokines/factors TAK1 regulated expression of one cytokine in LPS-activated murine MSCs in particular: interleukin-6 (IL-6). IL-6 in LPS-treated MSCs was responsible for lymphocyte recruitment as substantiated by neutralizing antibodies. Our studies, therefore, suggest that in LPS-treated murine MSCs the inflammatory signalling mediator TAK1 may exert anti-inflammatory properties via IL-6.

The design of implants for tissue transitions remains a major scientific challenge. This is due to gradients in characteristics that need to be restored. The rotator cuff in the shoulder, with its direct osteo-tendinous junction (enthesis), is a prime example of such a transition. Our approach towards an optimized implant for entheses is based on electrospun fiber mats of poly(ε-caprolactone) (PCL) as biodegradable scaffold material, loaded with biologically active factors. Chitosan/tripolyphosphate (CS/TPP) nanoparticles were used to load transforming growth factor-β3 (TGF-β3) with increasing loading concentrations for the regeneration of the cartilage zone within direct entheses. Release experiments were performed, and the concentration of TGF-β3 in the release medium was determined by ELISA. Chondrogenic differentiation of human mesenchymal stromal cells (MSCs) was analyzed in the presence of released TGF-β3. The amount of released TGF-β3 increased with the use of higher loading concentrations. This correlated with larger cell pellets and an increase in chondrogenic marker genes (SOX9, COL2A1, COMP). These data were further supported by an increase in the glycosaminoglycan (GAG)-to-DNA ratio of the cell pellets. The results demonstrate an increase in the total release of TGF-β3 by loading higher concentrations to the implant, which led to the desired biological effect.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of β-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost- and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.

Human bone marrow-derived stromal cells (hBMSCs) derived from the adult organism hold great promise for diverse settings in regenerative medicine. Therefore a more complete understanding of hBMSC biology to fully exploit the cells' potential for clinical settings is important. The protein CD24 has been reported to be involved in a diverse range of processes such as cancer, adaptive immunity, inflammation, and autoimmune diseases in other cell types. Its expression in hBMSCs, which has not yet been analyzed, may add an important aspect in the understanding of hBMSC biology. The present study therefore analyzes the expression, regulation, and functional implication of the surface protein CD24 in hBMSCs. Methods used are stimulation studies with TGF beta as well as shRNA-mediated knockdown and overexpression of CD24 followed by microarray, immunocytochemistry, and flow cytometric analyses. To our knowledge, we demonstrate for the first time that the expression of CD24 is an inherent property of hBMSCs. Importantly, the data links the upregulation of CD24 to the adoption of a myofibroblast-like gene expression pattern in hBMSCs. We demonstrate that CD24 is an important modulator in transforming growth factor beta 3 (TGFβ3) signaling with a reciprocal regulatory relationship between these two proteins.

Mesenchymal stem cells (MSCs) are regarded as an immune privileged cell type with numerous regeneration-promoting effects. The in vivo behavior of MSC and underlying mechanisms leading to their regenerative effects are largely unknown. The aims of this study were to comparatively investigate the in vivo behavior of canine (cMSC), human (hMSC), and murine MSC (mMSC) following intra-cerebroventricular transplantation. At 7 days post transplantation (dpt), clusters of cMSC, hMSC, and mMSC were detected within the ventricular system. At 49 dpt, cMSC-transplanted mice showed clusters mostly consisting of extracellular matrix lacking transplanted MSC. Similarly, hMSC-transplanted mice lacked MSC clusters at 49 dpt. Xenogeneic MSC transplantation was associated with a local T lymphocyte-dominated immune reaction at both time points. Interestingly, no associated inflammation was observed following syngeneic mMSC transplantation. In conclusion, transplanted MSC formed intraventricular cell clusters and exhibited a short life span in vivo. Xenogeneically in contrast to syngeneically transplanted MSC triggered a T cell-mediated graft rejection indicating that MSCs are not as immune privileged as previously assumed. However, MSC may mediate their effects by a "hit and run" mechanism and future studies will show whether syngeneically or xenogeneically transplanted MSCs exert better therapeutic effects in animals with CNS disease.

The hydrogen sulfide (H2S) and the oxytocin/oxytocin receptor (OT/OTR) systems interact in the central nervous and cardiovascular system. As a consequence of osmotic balance stress, H2S stimulates OT release from the paraventricular nuclei (PVN) in the hypothalamic regulation of blood volume and pressure. Hemorrhagic shock (HS) represents one of the most pronounced acute changes in blood volume, which, moreover, may cause at least transient brain tissue hypoxia. Atherosclerosis is associated with reduced vascular expression of the main endogenous H2S producing enzyme cystathionine-γ-lyase (CSE), and, hence, exogenous H2S administration could be beneficial in these patients, in particular after HS. However, so far cerebral effects of systemic H2S administration are poorly understood. Having previously shown lung-protective effects of therapeutic Na2S2O3 administration in a clinically relevant, long-term, porcine model of HS and resuscitation we evaluated if these protective effects were extended to the brain.

The cochlear implant outcome is possibly improved by brain-derived neurotrophic factor treatment protecting spiral ganglion neurons. Implantation of genetically modified mesenchymal stem cells may enable the required long-term brain-derived neurotrophic factor administration. Encapsulation of mesenchymal stem cells in ultra-high viscous alginate may protect the mesenchymal stem cells from the recipient's immune system and prevent their uncontrolled migration. Alginate stability and survival of mesenchymal stem cells in alginate were evaluated. Brain-derived neurotrophic factor production was measured and its protective effect was analyzed in dissociated rat spiral ganglion neuron co-culture. Since the cochlear implant is an active electrode, alginate-mesenchymal stem cell samples were electrically stimulated and alginate stability and mesenchymal stem cell survival were investigated. Stability of ultra-high viscous-alginate and alginate-mesenchymal stem cells was proven. Brain-derived neurotrophic factor production was detectable and spiral ganglion neuron survival, bipolar morphology, and neurite outgrowth were increased. Moderate electrical stimulation did not affect the mesenchymal stem cell survival and their viability was good within the investigated time frame. Local drug delivery by ultra-high viscous-alginate-encapsulated brain-derived neurotrophic factor-overexpressing mesenchymal stem cells is a promising strategy to improve the cochlear implant outcome.