Alzheimer’s disease is a common neurodegenerative disease. Microglia induces oxidative stress in the brain for engulfing bacteria and viruses. The accumulating data indicate that oxidative stress and apoptosis are two main actors for the induction of microglia activation-induced Alzheimer’s Disease. Oxidative stress is one of many triggers that activate the transient receptor potential melastatin 2 (TRPM2) channel. Glutathione (GSH) is a main cytosolic antioxidant in the mammalian cells. The GSH depletion via the activation of TRPM2 induces oxidative stress and apoptosis in neuronal cells. It has not yet been researched how GSH depletion via activation of TRPM2 affects oxidative stress and apoptosis in microglial cells with the Alzheimer's disease model. The BV2 cells divided into 5 groups as control, buthionine sulphoximine (BSO and 0.5 mM for 6 h), amyloid beta (1 uM for 72 h), amyloid beta+BSO, and amyloid beta+BSO+GSH (10 mM for 2 h). In the BSO group, the levels of apoptosis, mitochondrial membrane potential, cytosolic free oxygen reactive species (cyROS), caspase (Casps) -3, Casps -8, and Casps -9 were increased as compared to the control group, although cell viability level was decreased. The expression levels of TRPM2, Casps -3, Casps -9, Bax, Bcl-2, and PARP-1 were also increased in the BSO group. In addition, their levels were further increased in the amyloid beta and BSO+amyloid beta groups as compared to the BSO group. However, the changes were modulated in the BSO+amyloid beta+GSH group by the incubation of GSH. In conclusion, the depletion of GSH increased apoptosis and cyROS levels via activation of caspases and TRPM2 in the amyloid beta-induced microglia cells. The treatment of GSH may be a potential target on the apoptosis and oxidative stress in the amyloid beta-induced microglia cells.

Epilepsy is the fourth most common neurological disorder and affects people of all ages. Medication for epilepsy is often life-long and has a major impact on the quality of life - mostly being related to substantial adverse effects. Therefore, over 30% of people with epilepsy do not achieve sufficient seizure control whilst effective medication being available.  Ion channels are often primary targets of anticonvulsant drugs. They can either act as blockers for voltage gated sodium and calcium channels or as activators for potassium or chloride channels. Additionally, modulators of ligand gated ion channels (GABA or Glutamate receptors) are frequently used to treat epilepsy.  Employing a panel of functional electrophysiological assays using fluorescence based methods and patch-clamping on a broad range of voltage and ligand gated ion channels, we were able to successfully screen for drugs with a beneficial action profile. In successful leads we found drugs that selectively interacted with TTX sensitive, neuronal voltage gated sodium channels. Activation and fast inactivation were unchanged, while an increased affinity in the slow inactivated state was observed. This profile is in contrast to traditional anticonvulsant drugs which show their major effects on the fast inactivated state of voltage gated sodium channels. One drug showed substantial shifts of the voltage dependence of the slow inactivation only for NaV1.2 and 1.6. This favours this drug for treating patients with diseases with compromised NaV1.1 function in interneurons, such as Alzheimer's disease.

Microglial cell is a highly plastic cell in which it retracts its branched processes upon activation by structurally diverse molecules. Elevation of these molecules in the brain has been implicated in a diversity of diseases conditions in the CNS, where these molecules promote production of toxicity mediators, such as ROS. Microglial cell activation in response to ROS has been of particular interest. Emerging evidence supports a role for the TRPM2 channel in ROS-induced neuroinflammation. Thus, the current study aims to examine the role of the TRPM2 channel in mediating H2O2-induced microglial activation. A multidisciplinary approach was adopted, including primary microglial isolation, single cell calcium imaging, immunocytochemistry, confocal microscopy and computer-aided analysis of cell morphology. H2O2-induced microglial activation were observed in WT microglial cells but were ablated by genetic or pharmacological inhibition of the TRPM2 channel. Exposure to H2O2 raised the [Ca2+]i via promoting Ca2+ influx, which was prevented by TRPM2-KO. H2O2 induced ROS production and PARP-1 activation.  H2O2induced ROS production and PARP-1 activation as well as an increase in the [Ca2+]i and microglial activation, were suppressed by inhibiting PKC and NOX. Furthermore, H2O2-induced PARP-1 activation, increase in the [Ca2+]i and microglial activation were attenuated by inhibiting the Ca2+-sensitive PYK2 and downstream MEK/ERK kinases. The findings provide strong evidence to support that the TRPM2 channel is functionally expressed and plays a major role in ROS-induced Ca2+ signalling as well as cell activation in microglia. Such information is useful for a better understanding of microglial cells in oxidative stress-related pathologies. 

Chemotherapeutic agents such as cisplatin and 5fluorouracil are very effective and commonly used chemotherapeutic agents in treatment of several cancers including breast, testicular, ovarian and lung cancers. However, they have adverse effects and apoptosis in normal cells and neurons including optic nerve (Cardellicchio et al. 2014). Oxidative stress occurs during the several physiological functions such as mitochondria and phagocytosis. If the products of oxidative stress such as superoxide radical and hydrogen peroxide will be controlled by antioxidants the cell injury in normal tissue will not be occur. Results of recent reports indicated that optic nerve injury was induced through excessive production of reactive oxygen species (ROS) in rats by chemotherapeutic agents, although ROS were scavenged by antioxidants such as pycnogenol and rutin (Icel et al. 2018; Taşlı et al. 2018). It seems that the chemotherapeutic agentsinduced excessive ROS production results in increased levels of lipid peroxidation as malondialdehyde and inflammation markers such as TNF-α and NF-κB levels, but decrease of glutathione and total antioxidant levels. Apoptosis in the optic nerve was induced in ARPE19 eye cells by activation of intrinsic apoptosis pathway and death receptor signaling (Güçlü et al. 2018). In the presentation, we discussed novel effects of oxidative stress and apoptosis on the optic nerve injury in rodentsand human.  The results of current data suggest that oxidative stress has a main role in chemotherapeutic agentsinduced optic nerve injury in rodents, although the injury was attenuated by the antioxidant treatment. 

Abnormalities of intracellular free Ca+2 concentration is caused through activation of mitochondrial membrane depolarization by excessive levels of reactive oxygen species (ROS). In etiology of cerebral ischemia, the abnormalities of intracellular free Ca+2 concentration and excessive productions of ROS play an important role in the pathophysiology of cerebral ischemia (Chinopoulos and Adam-Vizi, 2006). Ca2+ influx occurs through activation of different cation channels. Well-known cations channels in cell membrane are chemical and voltage gated channels. Apart from the well-known cation channels, there is transient receptor potential (TRP) superfamily. The TRP superfamily is containing 28 members in 7 subfamilies in mammalian. Activation and inhibition mechanisms of the TRP channels are very different from the wellknown calcium channels. TRPM2 channel is activated by ADP-ribose NAD+. Another member of TRP superfamily is TRPV1 channel and it is activated several stimuli, including capsaicin, heat (≥43 °C) and acidic pH (≤ 6) (Chinopoulos and Adam-Vizi, 2006; Toda et al, 2019). Both channels are also activated by oxidative stress. Recent data indicated protective roles of some drugs on cerebral ischemia in rodents. One of the drug is duloxetine (DULOX) and it reduced the effects of Ca2+ entry and ROS through inhibition of TRPM2 channel (Toda et al. 2019). Another drug is dexmedetomidine (DEX) and it is an important drug for long-term sedation in intensive care patients, because it induces a rapid response. In addition to the intensive care patients, it has been started to use for sedation and analgesia in emergency medicine patients (McMorrow and Abramo, 2012). Recently, the protective role of DEX through inhibition of TRPM2 and TRPV1 channels on experimental cerebral ischemia in rats was reported (Akpınar et al. 2016). In the oral presentation, I discussed novel effects of TRPM2, TRPV1 and oxidative stress on the cerebral ischemia in rodents and human.  I concluded that the results of current data suggest that antioxidant drugs such as DEX and DULOX treatments reduce cerebral ischemia-induced oxidative stress and intracellular Ca2+ signaling through inhibition of TRPM2 and TRPV1 channels. It seems to that the exact relationship between TRP channel activation and the drugs in cerebral ischemia still remains to be determined. 

Glia constitutes a heterogeneous cell population that makes up half of the cells in the central nervous system (CNS). Glial cells include macroglia, astrocytes and oligodendrocytes, and microglia. Their roles are very diverse but overall they orchestrate CNS formation and function by providing neurons with essential support. Although glia-derived immortalized cell lines are now available, primary cultures of glial cells still constitute the most reliable method to study glial functions as the primary cultures retain important characteristics and markers of glia from their normal brain environment. Isolation and culturing of glia from postnatal rodent brain is well-characterized and give higher yield than from adult brain. Therefore, isolation of glial cells from postnatal mouse brains, with an emphasis on microglia, will be described. It will include a protocol describing the steps of isolation and necessary equipments and reagents, as well as the subsequent cell culture monitoring and potential applications.

Our ability to perceive nociceptive pain is crucial to respond to harmful stimuli: it warns us about noxiously hot or cold objects. Sensory nerve endings that respond to pain (nociceptors) initiate an electrical signal in the periphery and transport it to the dorsal horn and eventually to higher brain centers. The detection of noxious stimuli involves the expression of nociceptive ion channels in sensory nerve endings, such as transient receptor potential (TRP) channels. Recently we found that the acute response to noxious heat relies on the functional expression of TRPV1, TRPM3 and TRPA1 in sensory nerve endings. Calcium imaging on isolated dorsal root ganglia (DRG) is a widely accepted model to study the involvement of TRP channels in acute pain responses. However, DRGs are clusters of nerve cell bodies, present in the dorsal root of spinal nerves, far away from the skin where the physiological stimulus detection take place. We use two different optical measurement protocols to study ion channel activity in sensory neurons: an in vivo-protocol to image DRGs and an in tissue-protocol to visualize intact skin measurements. A cre-dependent GCaMP3 mouse line is used. These mice express the genetically encoded calcium indicator GCaMP3 in specific somatosensory neurons. Using this technique, we study the role of TRP channels in different pain models, such as inflammation. 

Epilepsy is one of the most frequent and heterogeneous neurological disorders and it is characterized by several disabilities. Epilepsy is affecting about 3% of people worldwide. Current antiepileptic drugs are only effective in 60% of individuals and many drugs can induce several unwanted side effects in patients. Etiology of epilepsy has not been clarified fully. However, increased intracellular calcium ion (Ca2+) concentration has main role in etiology of epilepsy. Ca2+ passes the cell membrane through different cell membrane channels. One of the channels is TRP superfamily. The family is containing six subfamilies. TRPV1 channel is a member of TRPV subfamily. Capsaicin is a component of hot chili pepper. The TRPV1 channels is activated by different stimuli such as acidic pH, high temperature (≥ 42° C) and capsaicin, causing pain, inflammation and hyperalgesia in peripheral nervous system (Caterina et al. 1997). Is has been well known that hippocampus is main area in the brain for induction of epilepsy. Expression levels of TRPV1 channels in different areas of hippocampus are high (Gonzalez-Reyes et al. 2013). Results of recent studies indicated involvement of TRPV1 channels in epilepsy (Nazıroğlu and Övey, 2015; Cho et al. 2018). In the oral presentation, I discussed novel roles of TRPV1 on the epilepsy induction by the capsaicin. Results of a recent study indicated increased levels of intracellular Ca2+ concentration in hippocampus of epilepsy induced rats (Nazıroğlu and Övey, 2015). They also observed increased levels of intracellular mitochondrial oxidative stress and apoptosis levels in the neurons by the capsaicin stimulation. However, their levels were decreased by inhibition of TRPV1 channel blocker, capsazepine. I concluded that the results of recent studies suggest that TRPV1 stimulation through capsaicin causes oxidative stress and intracellular Ca2+ signaling in epileptic rats. It seems to that the certain role of TRPV1 channel activation in in the epilepsy still remains to be determined.

Fibromyalgia (FM) is a common chronic pain syndrome affecting up to 2% of the adult population.Several factors such as excessive oxidative stress and overload calcium ion (Ca2+) influx play main roles in the etiology of FM. Several pharmaceutical drugs such as antidepressants and voltage-gated calcium channel blockers are recommended for the treatment of FM; however, they fail to produce a satisfactory response in patients with FM because of the unclear etiology of the disease. Transient receptor potential (TRP) channels have six subfamilies and 27 members in human. Most of these channels are responsible in dorsal root ganglia (DRG) neurons for the Ca2+ permeation especially in neuronal cells. Expression level of the TRPM2 and TRPV1 channels are high in the DRG neurons and they show oxidative stress dependent activation (Tan and McNaughton 2016; Santos et al. 2018). The TRPM2 and TRPV1 channel expression levels in the DRG increased in different types of pain. Selenium as an antioxidant trace element is implicated as a neuroprotective agent in peripheral pain through the inhibition of apoptosis and regulation of the TRPM2 and TRPV1 channels (Kahya et al. 2017). Since a decade, a recent theory have argued that both supporting of intracellular antioxidant system and extracellular antioxidant administration may helpful in fibromyalgia for the inhibition of TRP channels mediated Ca2+ influx (Yüksel et al. 2017). In the oral presentation, I discussed novel effects of selenium on the treatment of irregular oxidative status and fibromyalgia by the regulation of TRPM2 and TRPV1 channels in rats. In conclusion, present literature information indicated that protective effects of selenium on TRPM2 and TRPV1 channels may novel approach to treat FM induced pain and mitochondrial oxidative stress. However, the subject should be clarified by further studies.

Traumatic brain injury (TBI) is induced in the brain by external forces such as traffic accidents and heat trauma. Death and disability are induced by the TBI. Indeed, worldwide, about 10 million people are annually deaths or hospitalizations annually by the TBI exposures. In addition, about 57 million exposed to brain injury after TBI annually (Xiong et al. 2013). There is no direct treatment method for the TBI. After the TBI, different pathological processes such as oxidative stress, inflammation and apoptosis are induced by the brain injury. Hence, investigations of new treatment methods in rodent models have important role for inhibition of the pathological processes of human. Marmarou method has been used to make a diffuse head trauma (Marmarou et al. 1994) and it is popular for induction of TBI in rats. Before induction of TBI, the animals should anesthetized by anesthetics such as ketamine and xylazine combination. The animals are placed in prone position on the trauma table under the anesthesia. After skin incision, a steel disc (10 mm X 3 mm) is placed midline between coronal and lambdoid sutures on the animal’s skull, and a 250-300 g weight is freely dropped through a cylindrical tube, with 19 mm inner diameter, from 2 m height onto the head of the animal (Marmarou et al. 1994). In the presentation, a selection of the principal models is described and the model was compared