LIM kinase inhibitor T56-LIMKi protects mouse brain from photothrombotic stroke.

Primary Objective: In an ischemic stroke, the damage spreads from the infarction core to surrounding tissues. The present work was aimed at the search of effective neuroprotectors that restrict injury propagation. Research Design: We studied possible protective effects of inhibitors of protein kinases LIMK2 (T56-LIMKi), DYRK1A (harmine), and tryptophan hydroxylase (4-chlorophenylalanine) on infarction size and morphology of peri-infarct area after photothrombotic stroke (a model of ischemic stroke) in mouse brain. Methods and Procedures: Photothrombotic stroke was induced by laser irradiation of mouse cortex after administration of photosensitizer Bengal Rose, which does not penetrate cells and remains in blood vessels. Under light exposure, it induces vessel occlusion. Infarct volume and histological changes in the cerebral cortex were evaluated 3, 7 and 14 days after photothrombotic impact. Main Outcomes and Results: Harmine and 4-chlorophenylalanine did not influence infarct volume and morphology of peri-infarct area in the mouse brain cortex after photothrombotic stroke. However, LIMK2 inhibitor T56-LIMKi significantly reduced infarct volume 7 and 14 days after photothrombotic stroke. It also increased the percent of normochromic neurons and decreased the fraction of altered cortical cells (hypochromic, hyperchromic and pyknotic neurons). Conclusions: T56-LIMK2i may be considered as a promising anti-stroke agent.

HDAC1 Expression, Histone Deacetylation, and Protective Role of Sodium Valproate in the Rat Dorsal Root Ganglia After Sciatic Nerve Transection.

Nerve injury is an important reason of human disability and death. We studied the role of histone deacetylation in the response of the dorsal root ganglion (DRG) cells to sciatic nerve transection. Sciatic nerve transection in the rat thigh induced overexpression of histone deacetylase 1 (HDAC1) in the ipsilateral DRG at 1-4 h after axotomy. In the DRG neurons, HDAC1 initially upregulated at 1 h but then redistributed from the nuclei to the cytoplasm at 4 h after axotomy. Histone H3 was deacetylated at 24 h after axotomy. Deacetylation of histone H4, accumulation of amyloid precursor protein, a nerve injury marker, and GAP-43, an axon regeneration marker, were observed in the axotomized DRG on day 7. Neuronal injury occurred on day 7 after axotomy along with apoptosis of DRG cells, which were mostly the satellite glial cells remote from the site of sciatic nerve transection. Administration of sodium valproate significantly reduced apoptosis not only in the injured ipsilateral DRG but also in the contralateral ganglion. It also reduced the deacetylation of histones H3 and H4, prevented axotomy-induced accumulation of amyloid precursor protein, which indicated nerve injury, and overexpressed GAP-43, a nerve regeneration marker, in the axotomized DRG. Therefore, HDAC1 was involved in the axotomy-induced injury of DRG neurons and glial cells. HDAC inhibitor sodium valproate demonstrated the neuroprotective activity in the axotomized DRG.

The Expression of E2F1, p53, and Caspase 3 in the Rat Dorsal Root Ganglia After Sciatic Nerve Transection.

Neurotrauma is among the main causes of human disability and mortality. Nerve injury impairs not only neurons but also causes death of satellite glial cells remote from the injury site. We studied the dynamics of expression of different proapoptotic proteins (E2F1, p53, caspase 3) in the dorsal root ganglia (DRG) of a rat after sciatic nerve transection. TUNEL staining and immunoblotting were used for analysis of cell apoptosis and axotomy-induced biochemical changes. Apoptosis of glial cells was observed at 24 h after sciatic nerve transection and increased on day 7, when apoptosis of some neurons only started. The earliest proapoptotic event in the injured DRG was overexpression of transcription factor E2F1 at 4 h after sciatic nerve transection. This preceded the induction of p53 and cleavage of caspase 3 at 24-h post-axotomy. The nerve injury marker amyloid precursor protein and the nerve regeneration marker GAP-43 were overexpressed in DRG on day 7 after sciatic nerve transection. We also developed a novel fluorescence method for differential visualization of the rat DRG and nerves by means of double staining with propidium iodide and Hoechst 33342 that impart red and blue-green fluorescence, respectively. The present experiments showed that glial cells remote from the nerve transection site were more vulnerable to axotomy than DRG neurons. E2F1 and p53 may be considered promising molecular targets for development of potential neuroprotective agents.

The Localization of p53 in the Crayfish Mechanoreceptor Neurons and Its Role in Axotomy-Induced Death of Satellite Glial Cells Remote from the Axon Transection Site.

Neuron and glia death after axon transection is regulated by various signaling proteins. Protein p53 is a key regulator of diverse cell functions including stress response, DNA repair, proliferation, and apoptosis. We showed that p53 was overexpressed in crayfish ganglia after bilateral axotomy. In the isolated crayfish stretch receptor, a simple natural neuroglial preparation, which consists of a single mechanoreceptor neuron (MRN) enveloped by glial cells, p53 regulated axotomy-induced death of glial cells remote from the axon transection site. In MRN, p53 immunofluorescence was highest in the nucleolus and in the narrow cytoplasmic ring around the nucleus; its levels in the nucleus and cytoplasm were lower. After axotomy, p53 accumulated in the neuronal perikaryon. Its immunofluorescence also increased in the neuronal and glial nuclei. However, p53 immunofluorescence in the most of neuronal nucleoli disappeared. Axotomy-induced apoptosis of remote glial cells increased in the presence of p53 activators WR-1065 and nutlin-3 but reduced by pifithrin-α that inhibits transcriptional activity of p53. Pifithrin-µ that inhibits p53 effect on mitochondria increased axotomy-induced apoptosis of remote glial cells but reduced their necrosis. Therefore, axotomy-induced apoptosis of remote glial cells was associated with p53 effect on transcription processes, whereas glial necrosis was rather associated with transcription-independent p53 effect on mitochondria. Apparently, the fate of remote glial cells in the axotomized crayfish stretch receptor is determined by the balance between different modalities of p53 activity.

Са2+- and NF-κB-dependent generation of NO in the photosensitized neurons and satellite glial cells.

Photodynamic therapy (PDT) is used for killing of malignant cells in tumors including brain cancer. It can also damage normal neurons and glial cells. Nitric oxide (NO) is known to control PDT-induced cell death. To study the mechanisms that regulate NO generation in photosensitized neurons and glial cells, we used a simple model object - isolated crayfish mechanoreceptor that consists of a single sensory neuron surrounded by glial cells. PDT induced NO generation in glial cells, neuronal dendrites, and, less, in soma and axon. Using modulators of the cytosolic Ca2+ level and nuclear factor-kappa B (NF-κB) activity, we showed that Ca2+ and NF-κB regulate NO generation in the photosensitized neurons and glia. Actually, NO production was stimulated by 4-fold cadmium chloride (CdCl2) concentration in the saline, Ca2+ ionophore ionomycine, or inhibition of Ca2+-ATPase in the endoplasmic reticulum by 2,5-ditert-butylbenzene-1,4-diol (tBuBHQ). Oppositely, CdCl2 or nifedipine, blockers of Ca2+ channels in the plasma membrane, decreased NO generation. NO production was also inhibited by S-methylthiouronium sulfate (SMT), inhibitor of Ca2+-independent inducible NO synthase. SMT also prevented the stimulation of PDT-induced NO generation by NF-κB activator prostratin. This suggests the involvement of both Ca2+-dependent neuronal NO synthase and Ca2+-independent inducible NO synthase, which is regulated by NF-κB, in NO production in the crayfish neurons and glia.

Apoptosis regulation in the penumbra after ischemic stroke: expression of pro- and antiapoptotic proteins.

Ischemic stroke is the leading cause of human disability and mortality in the world. The main problem in stroke therapy is the search of efficient neuroprotector capable to rescue neurons in the potentially salvageable transition zone (penumbra), which is expanding after brain damage. The data on molecular mechanisms of penumbra formation and expression of diverse signaling proteins in the penumbra during first 24 h after ischemic stroke are discussed. Two basic features of cell death regulation in the ischemic penumbra were observed: (1) both apoptotic and anti-apoptotic proteins are simultaneously over-expressed in the penumbra, so that the fate of individual cells is determined by the balance between these opposite tendencies. (2) Similtaneous and concerted up-regulation in the ischemic penumbra of proteins that execute apoptosis (caspases 3, 6, 7; Bcl-10, SMAC/DIABLO, AIF, PSR), signaling proteins that regulate different apoptosis pathways (p38, JNK, DYRK1A, neurotrophin receptor p75); transcription factors that control expression of various apoptosis regulation proteins (E2F1, p53, c-Myc, GADD153); and proteins, which are normally involved in diverse cellular functions, but stimulate apoptosis in specific situations (NMDAR2a, Par4, GAD65/67, caspase 11). Hence, diverse apoptosis initiation and regulation pathways are induced simultaneously in penumbra from very different initial positions. Similarly, various anti-apoptotic proteins (Bcl-x, p21/WAF-1, MDM2, p63, PKBα, ERK1, RAF1, ERK5, MAKAPK2, protein phosphatases 1α and MKP-1, estrogen and EGF receptors, calmodulin, CaMKII, CaMKIV) are upregulated. These data provide an integral view of neurodegeneration and neuroprotection in penumbra. Some discussed proteins may serve as potential targets for anti-stroke therapy.

Epigenetic Alterations Induced by Photothrombotic Stroke in the Rat Cerebral Cortex: Deacetylation of Histone h3, Upregulation of Histone Deacetylases and Histone Acetyltransferases.

Ischemic penumbra that surrounds a stroke-induced infarction core is potentially salvageable; however, mechanisms of its formation are not well known. Covalent modifications of histones control chromatin conformation, gene expression and protein synthesis. To study epigenetic processes in ischemic penumbra, we used photothrombotic stroke (PTS), a stroke model in which laser irradiation of the rat brain cortex photosensitized by Rose Bengal induces local vessel occlusion. Immunoblotting and immunofluorescence microscopy showed decrease in acetylation of lysine 9 in histone H3 in penumbra at 1, 4 or 24 h after PTS. This was associated with upregulation of histone deacetylases HDAC1 and HDAC2, but not HDAC4, which did not localize in the nuclei. HDAC2 was found in cell nuclei, HDAC4 in the cytoplasm and HDAC1 both in nuclei and cytoplasm. Histone acetyltransferases HAT1 and PCAF (p300/CBP associated factor) that acetylated histone H3 synthesis were also upregulated, but lesser and later. PTS increased localization of HDAC2 and HAT1 in astroglia. Thus, the cell fate in PTS-induced penumbra is determined by the balance between opposite tendencies leading either to histone acetylation and stimulation of gene expression, or to deacetylation and suppression of transcriptional processes and protein biosynthesis. These epigenetic proteins may be the potential targets for anti-stroke therapy.

Axotomy-Induced Changes of the Protein Profile in the Crayfish Ventral Cord Ganglia.

We suggest novel experimental model of nerve injury-bilaterally axotomized ganglia of the crayfish ventral nerve cord (VNC). Using proteomic antibody microarrays, we showed upregulation of apoptosis execution proteins (Bcl-10, caspases 3, 6, and 7, SMAC/DIABLO, AIF), proapoptotic signaling proteins and transcription factors (c-Myc, p38, E2F1, p53, GADD153), and multifunctional proteins capable of initiating apoptosis in specific situations (p75, NMDAR2a) in the axotomized VNC ganglia. Simultaneously, anti-apoptotic proteins (p21WAF-1, MDM2, Bcl-x, Mcl-1, MKP1, MAKAPK2, ERK5, APP, calmodulin, estrogen receptor) were overexpressed. Some proteins associated with actin cytoskeleton (α-catenin, catenin p120CTN, cofilin, p35, myosin Vα) were upregulated, whereas other actin-associated proteins (ezrin, distrophin, tropomyosin, spectrin (α + ß), phosphorylated Pyk2) were downregulated. Various cytokeratins and ßIV-tubulin, components of intermediate filament and microtubule cytoskeletons, were also downregulated that could be the result of tissue destruction. Downregulation of proteins involved in clathrin vesicle formation (AP2α and AP2γ, adaptin (ß1 + ß2), and syntaxin) indicated impairment of vesicular transport and synaptic processes. The levels of L-DOPA decarboxylase, tyrosine, and tryptophan hydroxylases that mediate synthesis of serotonin, dopamine, norepinephrine, and epinephrine decreased. Overexpression of histone deacetylases HDAC1, HDAC2, and HDAC4 contributed to suppression of transcription and protein synthesis. So, the balance of multidirectional processes aimed either at cell death, or to repair and recovery, determines the cell fate. Present data provide integral, albeit incomplete, view on the nervous tissue response to axotomy. Some of these proteins can be probably potential markers of nerve injury and targets for neuroprotective therapy.

Photothrombotic Stroke as a Model of Ischemic Stroke.

The search of effective anti-stroke neuroprotectors requires various stroke models adequate for different aspects of the ischemic processes. The photothrombotic stroke model is particularly suitable for the study of cellular and molecular mechanisms underlying neurodegeneration, neuroprotection, and neuroregeneration. It is a model of occlusion of small cerebral vessels, which provides detailed study of molecular mechanisms of ischemic cell death and useful for search of potential anti-stroke agents. Its advantages include well-defined location and size of ischemic lesion that are determined by the aiming of the laser beam at the predetermined brain region; easy impact dosing by changing light intensity and duration; low invasiveness and minimal surgical intervention without craniotomy and mechanical manipulations with blood vessel, which carry the risk of brain trauma; low animal mortality and prolonged sensorimotor impairment that provide long-term study of stroke consequences including behavior impairment and recovery; independence on genetic variations of blood pressure and vascular architecture; and high reproducibility. This review describes the current application of the photothrombotic stroke model for the study of cellular and molecular mechanisms of stroke development and ischemic penumbra formation, as well as for the search of anti-stroke drugs.

Ca2+ mediates axotomy-induced necrosis and apoptosis of satellite glial cells remote from the transection site in the isolated crayfish mechanoreceptor.

Severe nerve injury such as axotomy induces neuron degeneration and death of surrounding glial cells. Using a crayfish stretch receptor that consists of a single mechanoreceptor neuron enveloped by satellite glia, we showed that axotomy not only mechanically injures glial cells at the transection location, but also induces necrosis or apoptosis of satellite glial cells remote from the transection site. We studied Ca2+role in spontaneous or axotomy-induced death of remote glial cells. Stretch receptors were isolated using the original technique that kept the neuron connected to the ventral cord ganglion (control preparations). Using Ca2+-sensitive fluorescence probe fluo-4, we showed Ca2+ accumulation in neuronal perikarion and glial envelope. Ca2+ gradually accumulated in glial cells after axotomy. In saline with triple Ca2+ concentration the axotomy-induced apoptosis of glial cells increased, but spontaneous or axotomy-induced necrosis was unexpectedly reduced. Saline with 1/3[Ca2+], oppositely, enhanced glial necrosis. Application of ionomycin, CdCl2, thapsigargin, and ryanodine showed the involvement of Ca2+ influx through ionic channels in the plasma membrane, inhibition of endoplasmic reticulum Ca2+-ATPase, and Ca2+ release from endoplasmic reticulum through ryanodine receptors in axotomy-induced glial necrosis. Apoptosis of glial cells surrounding axotomized neurons was promoted by ionomycin and thapsigargin. Possibly, other Ca2+ sources such as penetration through the plasma membrane contributed to axotomy-induced apoptosis and necrosis of remote glial cells. Thus, modulating different pathways that maintain calcium homeostasis, one can modulate axotomy-induced death of glial cells remote from the transection site.

The Focal-Focal Preconditioning Effect of Photothrombotic Impact on the Signaling Protein Profile in the Penumbra Surrounding the Ischemic Core Induced by Another Photothrombotic Impact.

Ischemic tolerance is the establishment of brain resistance to severe ischemic damage by a mild preconditioning stimulus, insufficient to irreversible tissue damage, but capable of initiating a defense response. We developed the model of focal-focal ischemic tolerance, in which the first local photothrombotic infarct (PTI) in the rat brain cortex reduced the infarct caused by second PTI applied to the contralateral cortex of the same rat 7 days later. Using antibody microarrays, we compared protein profiles in the penumbra surrounding the PTI core after single and double PTI. We observed up- or downregulation of several dozens of proteins that are aimed at neurodegeneration or neuroprotection. Both single and double PTI induced damaging processes in the rat cerebral cortex that included over-expression of various pro-apoptotic and signaling proteins and downregulation of other signaling proteins and regulators of proliferation, some components of actin, intermediate fiber and microtubular cytoskeletons, and proteins involved in vesicle transport and synaptic transmission. The simultaneous protective processes included the upregulation of different signaling and anti-apoptotic proteins, stimulators of proliferation, and proteins involved in remodeling of actin cytoskeleton. The elevated expression of some signaling proteins, such as calcium-dependent PLCγ1, PKVα1, CaMKIIα, calnexin, and calreticulin was preserved after double PTI. Less pro-survival proteins were downregulated in the penumbra after double than single impact.

Photo-Induced Oxidative Stress Impairs Mitochondrial Metabolism in Neurons and Astrocytes.

Photodynamic therapy is selective destruction of cells stained with a photosensitizer upon irradiation with light at a specific wavelength in the presence of oxygen. Cell death upon photodynamic treatment is known to occur mainly due to free radical production and subsequent development of oxidative stress. During photodynamic therapy of brain tumors, healthy cells are also damaged; considering this, it is important to investigate the effect of the treatment on normal neurons and glia. We employed live-cell imaging technique to investigate the cellular mechanism of photodynamic action of radachlorin (200 nM) on neurons and astrocytes in primary rat cell culture. We found that the photodynamic effect of radachlorin increases production of reactive oxygen species measured by dihydroethidium and significantly decrease mitochondrial membrane potential. Mitochondrial depolarization was independent of opening of mitochondrial permeability transition pore and was insensitive to blocker of this pore cyclosporine A. However, irradiation of cells with radachlorin dramatically decreased NADH autofluorescence and also reduced mitochondrial NADH pool suggesting inhibition of mitochondrial respiration by limitation of substrate. This effect could be prevented by inhibition of poly (ADP-ribose) polymerase (PARP) with DPQ. Thus, irradiation of neurons and astrocytes in the presence of radachlorin leads to activation of PARP and decrease in NADH that leads to mitochondrial dysfunction.

Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes.

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200 nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).

Protein Profile and Morphological Alterations in Penumbra after Focal Photothrombotic Infarction in the Rat Cerebral Cortex.

After ischemic stroke, cell damage propagates from infarct core to surrounding tissues (penumbra). To reveal proteins involved in neurodegeneration and neuroprotection in penumbra, we studied protein expression changes in 2-mm ring around the core of photothrombotic infarct induced in the rat brain cortex by local laser irradiation after administration of Bengal Rose. The ultrastructural study showed edema and degeneration of neurons, glia, and capillaries. Morphological changes gradually decreased across the penumbra. Using the antibody microarrays, we studied changes in expression of >200 neuronal proteins in penumbra 4 or 24 h after focal photothrombotic infarct. Diverse cellular subsystems were involved in the penumbra tissue response: signal transduction pathways such as protein kinase Bα/GSK-3, protein kinase C and its ß1 and ß2 isoforms, Wnt/ß-catenin (axin1, GSK-3, FRAT1), Notch/NUMB, DYRK1A, TDP43; mitochondria quality control (Pink1, parkin, HtrA2); ubiquitin-mediated proteolysis (ubiquilin-1, UCHL1); axon outgrowth and guidance (NAV-3, CRMP2, PKCß2); vesicular trafficking (syntaxin-8, TMP21, Munc-18-3, synip, ALS2, VILIP1, syntaxin, synaptophysin, synaptotagmin); biosynthesis of neuromediators (tryptophan hydroxylase, monoamine oxidase B, glutamate decarboxylase, tyrosine hydroxylase, DOPA decarboxylase, dopamine transporter); intercellular interactions (N-cadherin, PMP22); cytoskeleton (neurofilament 68, neurofilament-M, doublecortin); and other proteins (LRP1, prion protein, ß-amyloid). These proteins are involved in neurodegeneration or neuroprotection. Such changes were most expressed 4 h after photothrombotic impact. Immunohistochemical and Western blot studies of expression of monoamine oxidase B, UCHL1, DYRK1A, and Munc-18-3 confirmed the proteomic data. These data provide the integral view on the penumbra response to photothrombotic infarct. Some of these proteins can be potential targets for ischemic stroke therapy.

Profiling of Signaling Proteins in Penumbra After Focal Photothrombotic Infarct in the Rat Brain Cortex.

In ischemic stroke, cell damage propagates from infarct core to surrounding tissue. To reveal proteins involved in neurodegeneration and neuroprotection, we explored the protein profile in penumbra surrounding the photothrombotic infarct core induced in rat cerebral cortex by local laser irradiation after Bengal Rose administration. Using antibody microarrays, we studied changes in expression of 224 signaling proteins 1, 4, or 24 h after photothrombotic infarct compared with untreated contralateral cortex. Changes in protein expression were greatest at 4 h after photothrombotic impact. These included over-expression of proteins initiating, regulating, or executing various apoptosis stages (caspases, SMAC/DIABLO, Bcl-10, phosphatidylserine receptor (PSR), prostate apoptosis response 4 (Par4), E2F1, p75, p38, JNK, p53, growth arrest and DNA damage inducible protein 153 (GADD153), glutamate decarboxylases (GAD65/67), NMDAR2a, c-myc) and antiapoptotic proteins (Bcl-x, p63, MDM2, p21WAF-1, ERK1/2, ERK5, MAP kinase-activated protein kinase-2 (MAKAPK2), PKCα, PKCß, PKCµ, RAF1, protein phosphatases 1α and MAP kinase phosphatase-1 (MKP-1), neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), estrogen and EGF receptors, calmodulin, CaMKIIα, CaMKIV, amyloid precursor protein (APP), nicastrin). Phospholipase Cγ1, S-100, and S-100ß were down-regulated. Bidirectional changes in levels of adhesion and cytoskeleton proteins were related to destruction and/or remodeling of penumbra. Following proteins regulating actin cytoskeleton were over-expressed: cofilin, actopaxin, p120CTN, α-catenin, p35, myosin Va, and pFAK were up-regulated, whereas ezrin, tropomyosin, spectrin (α + ß), ßIV-tubulin and polyglutamated ß-tubulin, and cytokeratins 7 and 19 were down-regulated. Down-regulation of syntaxin, AP2ß/γ, and adaptin ß1/2 indicated impairment of vesicular transport and synaptic processes. Down-regulation of cyclin-dependent kinase 6 (Cdk6), cell division cycle 7-related protein kinase (Cdc7 kinase), telomeric repeat-binding factor 1 (Trf1), and topoisomerase-1 showed proliferation suppression. Cytoprotection proteins AOP-1 and chaperons Hsp70 and Hsp90 were down-regulated. These data provide the integral view on penumbra response to photothrombotic infarct. Some of these proteins may be potential targets for antistroke therapy.

Photodynamic therapy-induced nitric oxide production in neuronal and glial cells.

Nitric oxide (NO) has been recently demonstrated to enhance apoptosis of glial cells induced by photodynamic therapy (PDT), but to protect glial cells from PDT-induced necrosis in the crayfish stretch receptor, a simple neuroglial preparation that consists of a single mechanosensory neuron enveloped by satellite glial cells. We used the NO-sensitive fluorescent probe 4,5-diaminofluorescein diacetate to study the distribution and dynamics of PDT-induced NO production in the mechanosensory neuron and surrounding glial cells. The NO production in the glial envelope was higher than in the neuronal soma axon and dendrites both in control and in experimental conditions. In dark NO generator, DEA NONOate or NO synthase substrate L-arginine hydrochloride significantly increased the NO level in glial cells, whereas NO scavenger 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) or inhibitors of NO synthase L-NG-nitro arginine methyl ester and N?-nitro-L-arginine decreased it. PDT induced the transient increase in NO production with a maximum at 4 to 7 min after the irradiation start followed by its inhibition at 10 to 40 min. We suggested that PDT stimulated neuronal rather than inducible NO synthase isoform in glial cells, and the produced NO could mediate PDT-induced apoptosis.

On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells.

Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.