Study Of The Acute Hypobaric Hypoxia Induced Injury In Retina Ganglion Cells And The Neuroprotective Effects Of Taurine Pretreatment

Hypobaric hypoxia, often presented in high altitudes (mountains, airplanes, space shuttles), is characterized by low barometric pressure with a consequent fall in the partial pressure of oxygen. As retina has high metabolic demands, it is susceptible to hypoxia. While confronted with hypobaric hypoxia, visual dysfunction such as prolonged dark adaptation and decreased visual acuity, fundus changes such as engorgement of retinal veins with asymptomatic intraretinal hemorrhages and occasional papilloedema were observed even in well-acclimatised climbers. Such syndromes had been correlated with inadequate autoregulatory response of retina vascular system, however, alterations of retinal neurons, the visual signal editor and transducer, had received scant attention. Reduced oscillatory potentials (OPs) and swollen inner layers of retina were reported in previous studies, which indicated acute hypobaric hypoxia exposure leads to injury in the inner retina. Retinal ganglion cells (RGCs), located in the inner retina, are solely responsible for the relay of visual signals from the eye to the brain, and therefore are of critical importance to the visual system. Thus, it is urgent to clarify alterations of RGCs exposed to acute hypobaric hypoxia and develop neuroprotective strategies for RGCs.Although the exact mechanisms of hypoxia-induced neuron damage have not been completely elucidated, the following theories were extensively accepted: (1) Mitochondrial dysfunction have been shown to play centrol roles in hypoxic injuries to neurons. On one hand, hypoxia leas to bioenergetic, biosynthetic, and redox crisis in neurons, which can directly depress the Na~+/K~+ channel and the membrane receptor and impair electrophysiological function. On the other hand, during hypoxia, intracellular Ca2~+ levels are elevated and the presence of excessive free radicals, acidosis, and low adenine nucleotide levels all trigger the opening of mitochondrial permeability transition pore (MPTP), which initiates the mitochondrial permeability transition (MPT). Initiation of the MPT can lead to the functional impairment of mitochondria through depolarization of mitochondrial membrane potential (?ψm) and a reversal of ATP synthase function to accelerate cellular energy depletion. Induction of the mPT can also release proteins normally localized to mitochondria into the cytosol, including cytochrome c (cyt c) and apoptosis-inducing factor (AIF) which can activate caspase and caspase-independent effectors of apoptosis. (2) Calpains has been suggested to be a key proteases in hypoxia-induced neuron damage. Calpains are a famliy of calcium-activated, nonlysosomal neutral cysteine proteases. During hypoxia, the elevated intracellular Ca2~+ levels facilitates calpains activation. The proteolytic activity of calpain was shown to both induce cytoskeletal degradation such as synapsin, tau,α-spectrin and promote activation of pro-apoptotic factors such as caspase-12, caspse-3 and bcl-xL.Taurine is a sulfur-containing amino acid that is enriched in many excitable tissues including the retina, brain, skeletal and cardiac muscles. Physiological actions of taurine are widely involved in membrane stabilization, osmoregulation, neuromodulation, regulation of calcium homeostasis, and antioxidation. Since Hayes et al. reviewed in 1975 that retinal degeneration of cats was associated with taurine deficiency, increasing evidence has accumulated to indicate the pivotal role of taurine in the development and survival of retinal neurons. In addition to the protective effects of taurine demonstrated in diabetic retinopathy and photochemical stress, taurine has also been shown to render neurons and cardiomyocytes resistant to an array of detrimental stimuli such as hypoxia and ischemia. Although our laboratory have found that taurine supplementation (2.4%) can amelimorate acute hypobaric hypoxia induced taurine loss and Müller cell injury in rat retina, the effect of taurine on RGCs is still undetermined.The purpose of this study was to clarify RGCs changes during acute hypobaric hypoxia and further determine whether taurine could protect RGCs from hypoxic injuries and the underlying mechanisms.The sprague-Dawley rat model of simulated 5 000 meters high altitude acute hypobaric hypoxia was used in vivo study and rats were feeded with 2.4% taurine forage for 14d. The immortalized RGC line RGC-5 cultured in hypoxia (5%) condition was used in in vitro study and RGC-5 cells were pretreated with 0.1mM taurine for 4h. multi-focal electroretinogram (mfERG), Fluorogold retrograde-tracing, Immunofluorechemistry, laser confocal analytical assay, flow cytometry, real time-PCR and western blotting method were used to study the structural and functional changes of RGCs induced by acute hypobaric hypoxia and the protective effects of taurine. Furthermore, the probable protective mechanisms of taurine treatment were explored.The main results and conclusions were summarized as follows:1. Retina funcion was detected by mfERG. Acute hypobaric hypoxia (24h) exposure caused on average a 58.6%, 56.5% or 48.3% reduction in the a-, b- wave and OP2 amplitudes respectively in taurine-untreated rats. In animals supplemented with taurine, the reduction of the a-, b- wave and OP2 amplitudes were approximately 22.9%, 34.1% and 35.0% respectively. The differences in amplitudes of a-, b- wave and OP2 amplitudes between hypoxia treated with or without taurine groups were statiscically significant (p<0.05). After 7 days'recovery, there were no significant difference in a-wave amplitude of taurine-untreated and -treated rats compared with rats of the control group. While the b-wave and OP2 amplitudes of the taurine-treated group turns to be normal, there were still about 22.3% and 18.7% reduction in the taurine-untreated rats than that of the control group. These results indicated acute hypobaric hypoxia impaires the retina function and the electrophysiological activity of RGCs, moreover, taurine supplementation could partially prevent the functional impairment.2. After the 24 hours'hypobaric hypoxia, disorganized inner layer cells, swollen inner plexiform layer (IPL) and ganglion cell layer (GCL), degenerated neurites indicated by Fluoro-Jade C (FJC) in IPL and GCL were observed in rats retina. Meanwhile, ultrastructure changes such as the swollen or vacuolated mitochondria in axon, the dissolved mictotube in dentrites and the disrupted synapses were observed in IPL of retina. In the taurine-treated group, the FJC-labled neurites were fewer than the hypoxia group (p<0.05) and synapses were relatively intact and with more synaptic vesicles. All the findings above demonstrated that acute hypobaric hypoxia compromised the functional organelles including the synapses and neurites in IPL and GCL, and taurine treatment could amelimorate the changes of histopathology and ultrastructure in rat retina.3. A few RGCs with fluorogold (FG ) distributed unevenly and branched-type microglia phygocytized with FG were observed in whole mounted retina with RGCs labed with FG in both hypoxia group and taurine-treated hypoxia group, which implyed the RGCs were damaged. TUNEL staining comfirmed that acute hypobaric hypoxia induced apoptosis of cells in INL and GCL. The apoptotic index of RGCs in taurine-treated group was lower than the hypoxia group (1.3±0.2 % vs. 2.4±0.5 %;p<0.05). in vivo study, with prolonged exposure to hypoxia, an increasing number of RGC-5 cells rounded up, the cell viability of RGC-5 cells reduced and lactate dehydrogenase (LDH) release increased. The apoptotic RGC-5 cells increased in a time-dependant manner. After 24h or 48h hypoxia, the apoptotic rate in hypoxia group were increased to (17.75±3.95)% and (47.59±6.70)% respectively. However, serial concentrations of taurine (0.01mM,0.1mM, 1.0mM) pretreatment could promote the RGC-5 cell survival and taurine in 0.1 mM concentration appeared most effective as the 24h hypoxic-induced apoptotic cells was only (17.14±5.14)%.The above indicated acute hypobaric hypoxia induced RGCs apoptosis and loss, taurine treatment could reduce hypoxia-induced RGCs apoptosis.4. Generally, Calpain-2 distributed in retina with lower activity. After 24 hours acute hypobaric hypoxia, the enhanced Calpain-2 activity and expression was observed in the whole retina especially in the IPL and RGCs in both hypoxia group and taurine-treated hypoxia group. However, the comparatively lower content and proteolytic activity were detected in taurine-treated group (p<0.05). As the calpain-2 specific substrates, synapsin,α-spectrin and protein tau were found to have been degraded after 24h hypoxic exposure and taurine partially prevented the degradation. In vivo studies also found that taurine pretreatment could reduce hypoxia-induced calpain-2 transcription and activation, and proteolysis ofα-spectrin and protein tau in RGC-5 cells. These findings showed that the neuroprotective effects of taurine was correlated with its suppression to calpain-2 mediated cytoskeletal proteins disruption.5. in vivo study, during hypoxia, the calcium concentration in RGC-5 cells increased and GSH content decreased gradually. After 24h hypoxia exposure, amount of MPTPs opened, mitochondrial membrane potential (Δψm) and celluar ATP content reduced, moreover, cytoplasm cytochorome c (Cyt C) and the active subunit (17kDa) of caspase-3 and AIF in nucleus elevated. Meanwhile, taurine treatment of RGC-5 cells suppressed the induction of the MPT by reducing intracellular calcium levels and GSH loss, and inhibiting the opening of MPTPs. Moreover, the loss ofΔψm, a decline in cellular ATP levels, a reduction in the amount of Cyt C translocated to the cytoplasm and caspase-3 activation were observed in taurine-treated cultures. In vitro stuy, These results demonstrate the potential for taurine to protect RGCs against hypoxic damage by preventing mitochondrial dysfunction.In brief, our studies revealed that RGCs changes suffered from acute hypobaric hypoxia inculding the reduction of electrophysiological activity, the disruption of synapses and neurites, and cell apoptosis and degeneration. Taurine treatment could amelimorate hypoxic injuries to RGCs, which were associated with its suppression to calpain-2 mediated neuroskeletal proteins degradation and its downregulation the opening of MPTPs and further inhibit the mitochondrial dysfunction and mitochondrial apoptosis signal transduction.