Comparison of the efficiencies of intrathecal and intraganglionic injections in mouse dorsal root ganglion

Background/aim: Dorsal root ganglia (DRG) are structures containing primary sensory neurons. Intraganglionic (IG) and intrathecal (IT) applications are the most common methods used for viral vector transfer to DRG. We aim to compare the efficiencies and pathological effects of IT and IG viral vector delivery methods to DRG, through in vivo imaging. Materials and methods: Mice were divided into four groups of six each: IT, IG, IT-vehicle, and IG-vehicle. Adeno-associated virus (AAV) injection was performed for EGFP expression in IT/IG groups. DRGs were made visible through vertebral window surgery and visualized with multiphoton microscopy. After imaging, spinal cords and DRGs were removed and cleared, then imaged with light sheet microscopy. Results: No neuronal death was observed after IT injection, while the death rate was 17% 24 h after IG injection. EGFP expression efficiencies were 90%–95% of neurons in both groups. EGFP expression was only observed in targeted L2 DRG after IG injection, while it was observed in DRGs located between L1-L5 levels after IT injection. Conclusion: IT injection is a more suitable method for labeling DRG neurons in neurodegenerative injury models. However, when the innervation of DRG needs to be specifically studied, IT injection reduces this specificity due to its spread. In these studies, IG injection is the most suitable method for labeling single DRG neurons

Development of a chemogenetic approach to manipulate intracellular pH

Chemogenetic Operation of iNTRacellular prOton Levels(pH-Control)is a novel substrate-based enzymatic method that enables precise spatiotemporalcontrol of ultralocal acidification in cultured cell lines and primaryneurons. The genetically encoded biosensor SypHer3s showed that pH-Controleffectively acidifies cytosolic, mitochondrial, and nuclear pH exclusivelyin the presence of beta-chloro-d-alanine in living cellsin a concentration-dependent manner. The pH-Control approach is promisingfor investigating the ultralocal pH imbalance associated with manydiseases.CE254SWXHI ; NN254SWPZX ; CP254SWT2

Active shrinkage protects neurons following axonal transection

Trauma, vascular events, or neurodegenerative processes can lead to axonal injury and eventual transection (axotomy). Neurons can survive axotomy, yet the underlying mechanisms are not fully understood. Excessive water entry into injured neurons poses a particular risk due to swelling and subsequent death. Using in vitro and in vivo neurotrauma model systems based on laser transection and surgical nerve cut, we demonstrated that axotomy triggers actomyosin contraction coupled with calpain activity. As a consequence, neurons shrink acutely to force water out through aquaporin channels preventing swelling and bursting. Inhibiting shrinkage increased the probability of neuronal cell death by about 3-fold. These studies reveal a previously unrecognized cytoprotective response mechanism to neurotrauma and offer a fresh perspective on pathophysiological processes in the nervous system.Yüzüncü Yıl Universit

Investigating transneuronal degeneration in cell culture with live cell imaging

Sinir sisteminde kısıtlı bir alanda meydana gelen bir hasar öncelikle hasar bölgesindeki hücreleri etkiler ve sonrasında çevre bölgelerde ikincil hasar diye de adlandırılan patolojik bir sürecin başlamasına sebep olur. İkincil hasar, çevredeki hücrelerin ölüm ve dejenerasyonuna sebep olduğunda transnöronal dejenerasyon olarak da adlandırılır. Çalışmamızda bu süreçte rol oynadığı düşünülen P2X7 reseptörü ve hasar sonrası yayılan kalsiyum sinyali, femtosaniye kızılötesi lazer kullanılarak oluşturduğumuz in-vitro hasar modeli ile araştırılmıştır. Hücre kültürü ortamında sınırlı sayıda sinir hücresinde aksotomi hasarı oluşturulduktan 24 saat sonra yapılan canlılık testlerinde ölüm oranının kontrol grubuna kıyasla arttığı ve yalnızca glia hücrelerinde bulunan P2X7 reseptörü bloklandığında ölüm oranın anlamlı bir şekilde kontrol seviyelerine düştüğü gözlenmiştir. Boya alım analizlerinde de hasarın hemen sonrasında çevredeki glia hücrelerinde boya alımının arttığı ve bu artışın hasar bölgesine olan mesafe ile negatif korelasyon gösterdiği gözlenmiştir. P2X7 reseptörü bloklandığında kontrol seviyelerine düşmesi glia hücrelerindeki P2X7 reseptörünün aktivasyonunun hücre zarında büyük bir por açılmasını sağladığını düşündürmüştür. Panneksin1 ve P2X7 boyamaları da aksotomi sonrası komşu hücrelerde bu proteinlerin hücre zarındaki miktarının da zamana bağlı olarak değiştiğini göstermiştir. Kalsiyumun hasar sinyalinin yayılmasındaki etkisinin araştırıldığı deneylerde ise hasarlanan nöronun etrafındaki hücrelerin %4,5 inde kalsiyum artışı olduğu, bu oranın NMDA reseptörü ve oluklu bağlantılar bloklandığında değişmediği fakat NMDA reseptörlerinin bloklanmasının aksotomi sonrası kalsiyum cevabı görülen hücrelerdeki ölüm oranının azalttığı görülmüştür. Tüm bu bulgular transnöronal dejenerasyon sinyalinin yayılmasında gliaların etkin bir rol oynayabileceğini ve P2X7, panneksin1 ve NMDA reseptörlerinin bu süreçte yer aldıklarını ortaya koymuştur.An injury occurred in limited region of nervous system primarily affects cells at injury region later induces a pathological process in neighboring cells called secondary injury. If secondary injury causes death and degeneration of neighboring cells it's also called transneuronal degeneration. In our study, P2X7 receptor and spreading calcium signal are considered as possible actors in this process and researched using a precise femtosecond infrared laser induced in-vitro injury model. In cell cultures, after limited number of neurons were axotomized, viability tests after 24-hour indicated that total death ratio were increased compared to control and blocking of P2X7 receptors which present only in glial cells, significantly decreased cell death. In dye uptake assays, shortly after axotomies, glial cells showed increased dye uptake level and this increase was negatively correlated with distance to injury region. Blocking of glial P2X7 receptors decreased dye uptake of glial cells to control levels and this make us thought that P2X7 receptors may open a large pore on the cell membrane. Immunocytochemistry indicated that the amount of P2X7 and Pannexin1 channels in cell membranes of neighboring cells were also changed time dependently after injury. Experiments testing the effect of the spreading calcium signal in response to injury indicated that 4.5% of neighboring neurons were showed calcium increase as a response to axotomy. Inhibition of NMDA receptors and gap junctions didn't alter this ratio but inhibition of NMDA receptors decreased death ratio of the neighboring neurons which showed increased levels of calcium in response to axotomy. All of these findings indicated that glial cells may have an active role in spreading of transneuronal degeneration signal and P2X7, Pannexin1 and NMDA receptors are involved in the process

Transfer and integration of breast milk stem cells to the brain of suckling pups

WOS: 000445336600057PubMed ID: 30250150Beside its unique nutritional content breast milk also contains live cells from the mother. Fate of these cells in the offspring has not been adequately described. In this study, we aimed to detect and identify maternal cells in the suckling's blood and the brain. Green fluorescent protein expressing transgenic female mice (GFP+) were used as foster mothers to breastfeed wildtype newborn pups. One week and two months after the birth, blood samples and brains of the sucklings were analyzed to detect presence of GFP+ cells by fluorescence activated cell sorting, polymerase chain reaction and immunohistochemistry on the brain sections and optically cleared brains. The tests confirmed that maternal cells were detectable in the blood and the brain of the pups and that they differentiated into both neuronal and glial cell types in the brain. This phenomenon represents breastfeeding - induced microchimerism in the brain with functional implications remain to be understood.The Scientific and Technological Research Council of Turkey (TUBITAK) [114R078]This project was supported by "The Scientific and Technological Research Council of Turkey" (TUBITAK) with a grant number 114R078

Adult mouse dorsal root ganglia neurons form aberrant glutamatergic connections in dissociated cultures

Cultured sensory neurons can exhibit complex activity patterns following stimulation in terms of increased excitability and interconnected responses of multiple neurons. Although these complex activity patterns suggest a network-like configuration, research so far had little interest in synaptic network formation ability of the sensory neurons. To identify interaction profiles of Dorsal Root Ganglia (DRG) neurons and explore their putative connectivity, we developed an in vitro experimental approach. A double transgenic mouse model, expressing genetically encoded calcium indicator (GECI) in their glutamatergic neurons, was produced. Dissociated DRG cultures from adult mice were prepared with a serum-free protocol and no additional growth factors or cytokines were utilized for neuronal sensitization. DRG neurons were grown on microelectrode arrays (MEA) to induce stimulus-evoked activity with a modality-free stimulation strategy. With an almost single-cell level electrical stimulation, spontaneous and evoked activity of GCaMP6s expressing neurons were detected under confocal microscope. Typical responses were analyzed, and correlated calcium events were detected across individual DRG neurons. Next, correlated responses were successfully blocked by glutamatergic receptor antagonists, which indicated functional synaptic coupling. Immunostaining confirmed the presence of synapses mainly in the axonal terminals, axon-soma junctions and axon-axon intersection sites. Concisely, the results presented here illustrate a new type of neuron-to-neuron interaction in cultured DRG neurons conducted through synapses. The developed assay can be a valuable tool to analyze individual and collective responses of the cultured sensory neurons.This work is funded by Bogazici University Research Fund to author AG under the Project Code 8080D. During the experiments some facilities of REMER (Istanbul Medipol University, Regenerative and Restorative Medicine Center) were used. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Bogazici Universit

A transcriptome based approach to predict candidate drug targets and drugs for Parkinson's disease using an in vitro 6-OHDA model

The most common treatment strategies for Parkinson's disease (PD) aim to slow down the neurodegeneration process or control the symptoms. In this study, using an in vitro PD model we carried out a transcriptome-based drug target prediction strategy. We identified novel drug target candidates by mapping genes upregulated in 6-OHDA-treated cells on a human protein-protein interaction network. Among the predicted targets, we show that AKR1C3 and CEBPB are promising in validating our bioinformatics approach since their known ligands, rutin and quercetin, respectively, act as neuroprotective drugs that effectively decrease cell death, and restore the expression profiles of key genes upregulated in 6-OHDA-treated cells. We also show that these two genes upregulated in our in vitro PD model are downregulated to basal levels upon drug administration. As a further validation of our methodology, we further confirm that the potential target genes identified with our bioinformatics approach are also upregulated in post-mortem transcriptome samples of PD patients from the literature. Therefore, we propose that this methodology predicts novel drug targets AKR1C3 and CEBPB, which are relevant to future clinical applications as potential drug repurposing targets for PD. Our systems-based computational approach to predict candidate drug targets can be employed in identifying novel drug targets in other diseases without a priori assumption

Validation of an In-Vitro Parkinson’s Disease Model for the Study of Neuroprotection

Parkinson’s disease (PD) is the second most common neurodegenerative disease with an estimation of 10 million people living with the disease and it is increasing in prevalence every year. Familial cases of PD are source of valuable information to determine genetical risk factors yet sporadic cases can emerge from distinct mechanisms so, identifying common pathways of familial and sporadic cases of PD may provide worthwhile insights to determine underlying mechanisms through the progression. LRRK2 mutations are the most common indicators of both sporadic and familial cases of PD and α-synuclein aggregation is one of the hallmarks of both cases of PD as well as in other synucleinopathies. As in the case of most neurological diseases, human studies addressing the molecular basis of pathology are generally restricted to post-mortem materials. For this reason, cell culture systems and animal models are widely used. There are two main approaches for modelling PD: genetically constructed PD models and neuroxin-based models. In this study, we aim to construct and compare both approaches by overexpressing wild-type (WT) and A53T mutant α-synuclein and treating cells with well-known neurotoxin 6-hydroxidopamine (6-OHDA) using dopaminergic human neuroblastoma SH-SY5Y cell line. Our findings suggest that WT or A53T α-synuclein overexpression by itself is not sufficient to cause significant toxicity in SH-SY5Y cells in the presented time scale. As expected, 6-OHDA treatment caused toxicity with an IC50 value of ~100 µM. In addition, 6-OHDA treatment causes 3- and 2.5-fold increase in SNCA and LRRK2 expression respectively

The protective effect of platelet-rich plasma administrated on ovarian function in female rats with Cy-induced ovarian damage

Purpose We evaluated the protective effect of PRP on ovarian function in female rats with cyclophosphamide (Cy)-induced ovarian damage. Methods Thirty-two adult female Sprague-Dawley rats were randomly divided into four groups. Group 1 (control-sodium chloride 0.9%; 1 mL/kg, single-dose ip injection), group 2 (Cy); 75 mg/kg, single-dose ip injection and sodium chloride 0.9% (1 mL/kg, single-dose ip injection), group 3 Cy plus PRP, Cy (75 mg/kg, single-dose and PRP (200 mu l, single-dose) ip injection), group 4 (PRP, 200 mu l, single-dose ip injection). Primordial, antral, and atretic follicle counts; serum anti-Mullerian hormone (AMH) levels; AMH-positive granulosa cells; and gene expression analysis of Ddx4 were assessed. Results Serum AMH levels were significantly lower in group 2 compared to groups 1, 3, and 4 (p < 0.01, p < 0.01, and p = 0.04, respectively). A significant difference was found in the primordial, primary, secondary, antral, and atretic follicle counts between all groups (p < 0.01). There was a statistically significant difference in AMH-positive staining primary, secondary, and antral follicles count between the groups (p < 0.01). There was a statistically significant difference in primary, secondary, and antral AMH positive staining follicle intensity score between the groups (p < 0.01). Ddx4 expression in group 4 was highest compared to other groups. Conclusion Our study may provide evidence that PRP could protect ovarian function against ovarian damage induced by Cy. It could lead to improved primordial, primary, secondary, and antral follicle numbers