Hydrostatic pressure does not cause detectable changes to survival of human retinal ganglion

Purpose: Elevated intraocular pressure (IOP) is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP). The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC) survival in the human retina was investigated. Methods: A chamber was designed to expose cells to increased HP (constant and fluctuating). Accurate pressure control (10-100mmHg) was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs) from donor eyes (<24h post mortem) were cultured in serum-free DMEM/HamF12. Increased HP was compared to simulated ischemia (oxygen glucose deprivation, OGD). Cell death and apoptosis were measured by LDH and TUNEL assays, RGC marker expression by qRT-PCR (THY-1) and RGC number by immunohistochemistry (NeuN). Activated p38 and JNK were detected by Western blot. Results: Exposure of HORCs to constant (60mmHg) or fluctuating (10-100mmHg; 1 cycle/min) pressure for 24 or 48h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100mmHg; 1 cycle/min) for 15, 30, 60 and 90min durations, whereas OGD (3h) increased activation of p38 and JNK, remaining elevated for 90min post-OGD. Conclusions: Directly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina

Inhibition of G Protein-Activated Inwardly Rectifying K+ Channels by Different Classes of Antidepressants

Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K+ (GIRK, Kir3) channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects

Akt (Protein Kinase B) subtypes in retinal ischemic postconditioning

Topic: Experimental NeurosciencesINTRODUCTION: We have previously described enhancement of recovery in a rat model of retinal ischemia by ischemic post-conditioning using transient elevation of intraocular pressure after ischemia. We previously demonstrated involvement of protein kinase B (Akt) in this form of neuroprotection. In this study, we examined the hypothesis that specific Akt subtypes underlie this phenomenon. METHODS: Ischemia was produced by elevation of intraocular pressure above systolic arterial blood pressure for 55 min in anesthetized adult Wistar rats. Post-conditioning was produced by 8 min elevation of intraocular pressure to the same level at 5 min after reperfusion following ischemia. Interfering RNA (siRNA) to Akt subtypes 1, 2, or 3, or non-silencing siRNA was injected into the vitreous 6 h prior to ischemia. Recovery was assessed using electroretinography (ERG) and histological examination of 5-micron thick retinal paraffin embedded sections. RESULTS: Injection of interfering RNA to Akt subtype 1 significantly decreased the post-conditioning ischemia protective effect as reflected by decreased recovery of the electroretinogram b wave and oscillatory potentials (OPs) (Figure).[figure1]CONCLUSIONS: The results show the involvement of specific Akt subtypes in the intriguing and clinically relevant phenomenon of post-ischemic conditioning. Enhancing expression of these subtypes may be a viable means to decrease neuronal injury after ischemia.link_to_OA_fulltex

Dual specificity phosphatase MKP1 and retinal ischemic preconditioning

Topic: Experimental NeurosciencesINTRODUCTION: We previously described the phemenon of retinal ischemic preconditioning (1) and showed significant increases in the dual-specificity phosphatase MKP-1 with preconditioning (2). In this study we examined the role of MKP-1 in ischemic preconditioning and specifically, its role in regulating MAPK p38. METHODS: Ischemia was produced by elevation of intraocular pressure above systolic arterial blood pressure for 55 min in anesthetized adult Wistar rats. Pre-conditioning was produced by ligating the central retinal artery for 5 min using suture occlusion, 24 h prior to ischemia. Interfering RNA (siRNA) to MKP-1 or, non-silencing siRNA, was injected into the vitreous 6 h prior to ischemia. Recovery was assessed using electroretinography (ERG) and histological examination of 5-micron thick retinal paraffin embedded sections. The a and b waves, and oscillatory potentials (OPs) were measured before and 1 week after ischemia and normalized relative to pre-ischemic baseline and corrected for diurnal variation in the normal non-ischemic eye. The P2 (which reflects function of the rod bipolar cells in the inner retina) was derived using the Hood-Birch model (3). Levels of phosphorylated MAPKs p38, ERK, and JNK were measured relative to total levels of protein at 24 h after ischemic preconditioning +/- siRNA to MPK-1 or non-silencing siRNA. RESULTS: Injection of interfering RNA to to MKP-1 significantly (P < 0.05) attenuated the protective effect of ischemic preconditoning as reflected by decreased recovery of the electroretinogram a, b wave and oscillatory potentials (OPs) and the P2 (Figure).[figure1]The blockade of MKP-1 by siRNA resulted in an increase in activation of p38 at 24 h following preconditioning to 146 +/- 7 % vs that with non-silencing siRNA (108 +/ 13%, P < 0.05). MKP-1 siRNA did not alter the levels of phosphorylated JNK or ERK after preconditioning. CONCLUSIONS: The results show the involvement of dual-specificity phosphatase MKP1 in ischemic preconditioning and suggest that MKP-1 is involved in ischemic preconditioning by regulating levels of activated MAPK p38.link_to_OA_fulltex

Late post-conditioning to enhance recovery after retinal ischemia in rats

Poster Session: Experimental Neurosciences: abstract no. A102

Post-ischemic conditioning decreases apoptosis after retinal ischemia

Topic: Experimental NeurosciencesIschemic preconditioning (IPC) protects the rat retina against the deleterious effects of severe ischemia. Ischemic post-conditioning (Post-C) involves a brief ischemic stimulus delivered after ischemia. We studied the impact of Post-C on apoptosis following ischemia and the impact of combining IPC and Post-C. A second purpose of the study was analyze the effect of Post-C on inflammation following ischemia. METHODS: IPC and ischemia were induced in rats as previously described (1). IPC was 8 min ligation of the central retinal artery ([start_en]201C;ligation PC”) 24 h prior to ischemia, while post conditioning ([start_en]201C;Post-C') was increased intraocular pressure (IOP) for 5 min, beginning 10 min after the end of 55 min ischemia. Ketamine-xylazine anesthetized rats breathed spontaneously with temperature held constant at 36-37 C, whilst monitoring systemic blood pressure, and pulse oximetry. At 24 h after ischemia, when apoptotic injury peaks, retinas were removed and 7 micron frozen sections prepared, and stained using TUNEL. % TUNEL retinal ganglion cells (RGCs) was calculated using simultaneous staining with the nuclear dye, DAPI. Identity of the TUNEL-positive RGCs was confirmed by double labeling with RGC-specific anti-thy1 antibody. Inflammation was assessed on frozen retinal sections using antibodies specific for infiltrating macrophages (anti-MIP1), monocytes (anti-MCP), and leukocytes (anti-CD45). Data were quantitated using NIH Image. Data were analyzed as previously described (1). RESULTS: Figure 1 shows that TUNEL cells were thy-1 positive, identifying them as RGCs.[figure1]There was a significant reduction in TUNEL positivity with Post-C; previous application of IPC blocked the anti-apoptotic effect of Post-C (Table 1).[table1]There was no significant leukocyte infiltration, but both macrophages and monocytes infiltrated retinas after ischemia, with Post-C decreasing the macrophage infiltation (Figure 2).[figure2]CONCLUSIONS: Post-C and IPC are not additive in decreasing apoptosis, suggesting that IPC alters the post-ischemic environment that prevents the post-C protection effect. Post-ischemic inflammation as reflected by inflammatory cell infiltration was decreased by Post-C.link_to_OA_fulltex

The Role of Akt/Protein Kinase B and P38-alpha in Retinal Ischemic Post-Conditioning

Poster Session 5: Cerebrovascular Disease: Basic ScienceThe 62nd Annual Meeting of the American Academy of Neurology, Toronto, Ontario, Canada, 10-17 April 2010, Poster Session V: Cerebrovascular Disease: Basic Scienc

Protein kinase B (Akt) and mitogen-activated protein kinase p38α in retinal ischemic post-conditioning

In previous studies, it was shown that post-conditioning, a transient period of brief ischemia following prolonged severe ischemia in the retina, could provide significant improvement in post-ischemic recovery, attenuation of cell loss, and decreased apoptosis. However, the mechanisms of post-conditioning in the retina have not been elucidated. We hypothesized that two kinases, mitogen-activated protein kinase p38α and protein kinase B (Akt), were involved in the mechanism of post-conditioning. Ischemia was induced in rat retina in vivo. Recovery after ischemia followed by 8 min of post-conditioning early in the reperfusion period after prolonged ischemia was assessed functionally (electroretinography) and histologically at 7 days after ischemia. We examined the role of p38α and Akt subtypes 1-3 in post-conditioning by intravitreal injection of interfering RNA 6 h prior to ischemia and post-conditioning and compared the results to injection of non-silencing interfering RNA sequence. The blockade of p38α significantly decreased the recovery after ischemia and post-conditioning, and enhanced cell loss and disorganization of the retina. Blockade of Akt1, and to a lesser degree, Akt2, significantly decreased the recovery after ischemia and enhanced cell loss and disorganization. These differences in the effects of blockade of Akt subtypes were not explainable by distribution of Akt subtypes in the retina, which were similar. In conclusion, both p38 and Akt are essential components of the neuroprotection induced by post-ischemic conditioning in the retina. © 2011 Springer Science+Business Media, LLC.link_to_subscribed_fulltex

Mechanisms of bone-marrow stem cell-mediated delayed ischemic neuroprotection in rat retina

The abstract can be viewed at http://www.asaabstracts.com/strands/asaabstracts/abstract.htm;jsessionid=8B0404AE3FDBF0AB9EAE20152CF76D98?year=2011&index=10&absnum=5635Poster Session: Experimental Neurosciences: abstract no. A130