Tetrandrine protects mouse retinal ganglion cells from ischemic injury

This study aimed to determine the protective effects of tetrandrine (Tet) on murine ischemia-injured retinal ganglion cells (RGCs). For this, we used serum deprivation cell model, glutamate and hydrogen peroxide (H2O2)-induced RGC-5 cell death models, and staurosporine-differentiated neuron-like RGC-5 in vitro. We also investigated cell survival of purified primary-cultured RGCs treated with Tet. An in vivo retinal ischemia/reperfusion model was used to examine RGC survival after Tet administration 1 day before ischemia. We found that Tet affected RGC-5 survival in a dose- and time-dependent manner. Compared to dimethyl sulfoxide treatment, Tet increased the numbers of RGC-5 cells by 30% at 72 hours. After 48 hours, Tet protected staurosporine-induced RGC-5 cells from serum deprivation-induced cell death and significantly increased the relative number of cells cultured with 1 mM H2O2 (P<0.01). Several concentrations of Tet significantly prevented 25-mM-glutamate-induced cell death in a dose-dependent manner. Tet also increased primary RGC survival after 72 and 96 hours. Tet administration (10 μM, 2 μL) 1 day before retinal ischemia showed RGC layer loss (greater survival), which was less than those in groups with phosphate-buffered saline intravitreal injection plus ischemia in the central (P=0.005, n=6), middle (P=0.018, n=6), and peripheral (P=0.017, n=6) parts of the retina. Thus, Tet conferred protective effects on serum deprivation models of staurosporine-differentiated neuron-like RGC-5 cells and primary cultured murine RGCs. Furthermore, Tet showed greater in vivo protective effects on RGCs 1 day after ischemia. Tet and ciliary neurotrophic factor maintained the mitochondrial transmembrane potential (ΔΨm) of primary cultured RGCs and inhibited the expression of activated caspase-3 and bcl-2 in ischemia/reperfusion-insult retinas.


Introduction
Tetrandrine (Tet) is a bisbenzylisoquinoline alkaloid extracted from the root of the Chinese herb creeper Stephania tetrandra. 1,2 It is a calcium channel blocker 3,4 that inhibits lipid peroxidation 5 and the generation of reactive oxygen species (ROS) 6 and suppresses the production of cytokines and inflammatory mediators in the brain after ischemia reperfusion injury, 6,7 anoxia, 8 or Alzheimer's disease. 9 Tet ameliorates amyloid-β(1-42)-induced spatial learning and memory impairment, and the beneficial effect of tetrandrine treatment may be linked to the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity and downregulation of interleukin-1β and tumor necrosis factor-α. 9 Furthermore, it has been found that Tet can increase neuroprotective brain-derived neurotrophic factor (BDNF) protein levels in the rat hippocampus and regulate the central monoaminergic neurotransmitter system. 10 Cytosolic ionized calcium often increases within seconds or minutes after an injury or other cell damage, long before cell death, and the increased calcium concentration activates various signaling processes that include phospholipases, endonucleases, proteases, and protein kinases, [11][12][13] followed by both apoptotic and necrotic cell death of neurons, 14,15 including retinal neurons. 16,17 For example, excitotoxic activation of calpain, a calciumdependent protease, may facilitate necrosis and apoptosis after cerebral hypoxia-induced ischemia. 17,18 There have been no studies to indicate a neuroprotective action of Tet on retinal ganglion cells (RGCs), although neuroprotective effects on neurons of the brain have been reported. 9,10 To determine the neuroprotective effects of Tet on RGCs, we utilized a series of in vitro models of cell death, namely serum deprivation as well as glutamate and hydrogen peroxide (H 2 O 2 ) treatment of RGC-5 cells, staurosporine (SSP)-induced RGC-5 cells, and purified RGCs in culture. We also used an in vivo model of transient retinal ischemia to determine whether Tet would protect RGCs from retinal ischemia reperfusion injury.

Materials and methods animals
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. 19 The protocol was approved by the IACUC of the Penn State University College of Medicine (Permit Number, 2006-080) and the Peking University Third Hospital (Permit Number, 11161). All surgeries were performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering. All animals were housed in temperature-controlled rooms with a 12-hour light/dark cycle and free access to food and water.
Unless noted, all other reagents were obtained from Sigma-Aldrich.

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Tetrandrine protects mouse retinal ganglion cells from ischemic injury rgc-5 cell line Cultures of the rat retinal ganglion cell line (RGC-5) (ATCC; Manassas, VA, USA) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 mg/L glucose and 2 mM L-glutamine, supplemented with 10% FBS, and 10 µg/mL gentamicin in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. For the experiments, the RGC-5 cells were plated on 96-well culture plates in DMEM with or without 10% FBS at a density of 5 × 10 4 cells/mL.

ssP-induced rgc-5 line
A protocol developed by Yang et al, 20 with modification, was used to induce RGC-5 cells, which led to their differentiation into highly branched, nonmitotic neuron-like cells. RGC-5 cells were cultured in DMEM with 10% FBS and plated onto 96-well plates, coated with a poly-D-lysine and laminin, at a density of 1 × 10 5 cells/mL. Cells were allowed to adhere to the substrate for 1 hour at 37°C and were then treated with the general kinase inhibitor SSP (1 µM) for 1 hour. Following differentiation, the medium was changed to a Neurobasal ® medium (Life Technologies, Carlsbad, CA, USA) with Tet and the number of cells were counted 24 hours and 48 hours after treatment.
glutamate-and h 2 O 2 -mediated cell death L-glutamic acid (glutamate) and H 2 O 2 were used to induce cell death in RGC-5 cells. Following treatment with various concentrations of Tet for 6 hours, RGC-5 cells were exposed to glutamate (25 mM) or H 2 O 2 (1 µM) for 12 hours to 72 hours depending on the experiment. Cell numbers were determined with a WST-8 assay (CCK-8) described below. The number of living cells in each well was expressed as a value relative to the control value.

WsT-8 assay
The relative numbers of the living RGC-5 and SSP-induced RGC-5 cells were determined with a WST-8 assay kit. WST-8 -2H-tetrazolium, monosodium salt) produces a water-soluble formazan dye on bioreduction in the presence of an electron carrier, 1-methoxy-5-methylphenazinium methyl sulfate. After exposure to Tet under the different conditions, the medium was replaced by a 10% WST-8 solution in DMEM. The number of living cells in each well was determined by measuring the optical density at 460 nm with a microplate reader (680XR; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and was expressed as a value relative to the control value.
Retinas from 24 0-2-day-old C57Bl/6 pups of either sex were dissected on ice and incubated at 37°C for 30 minutes in 33 U/mL papain, 70 U/mL collagenase, 0.004% deoxyribonuclease, and 0.2 mg/mL L-cysteine in D-PBS with Ca 2+ and Mg 2+ containing 0.2 mg/mL bovine serum albumin (BSA). To yield a suspension of single cells, the tube was gently agitated ten times until the suspension was cloudy. Then, 2 mL 1% trypsin inhibitor BSA solution was added to the tube and the cells were collected by centrifugation at 80 × gravity for 10 minutes. The cell pellet was resuspended in 2 mL 0.02% BSA in D-PBS containing integrin αM (Mac-1) antibody.
Sterile petri dishes (100 and 60 mm in diameter; BD Biosciences) were prepared for immunopanning. The 100 mm petri dishes were coated with 10 µg/mL affinity-purified goat anti-mouse IgG (H+L) antibody, and the 60 mm petri dishes were coated with 10 µg/mL affinity-purified rat anti-mouse IgG (H+L) antibody in 50 mM Tris (pH 9.5) at 4°C overnight. After washing, the surface of the 100 mm petri dishes was blocked with 0.2 mg/mL BSA in D-PBS for 1 hour. The 60 mm petri dishes were incubated with mouse anti-rat CD90.2 antibody for 1 hour at room temperature. The retinal cells in 0.02% BSA were placed in a 100 mm petri dish precoated with affinity-purified goat anti-mouse IgG (H+L) antibody for 20 minutes at 37°C. Nonadherent cells were transferred to the second 100 mm petri dish for 20 minutes at 37°C. The nonadherent cells were passed through a mesh filter (40 µm pore size; BD Biosciences) to obtain single cells, which were then transferred to the 60 mm dish precoated with rat anti-mouse CD90.2 antibody and incubated for 30 minutes at 37°C. After removal of nonadherent cells (collected for non-RGC testing), the dish was washed ten times with D-PBS. Adherent cells were mechanically scraped off in Neurobasal ® medium containing 1% BSA, selenium (6.7 ng/mL), transferrin (5.5 µg/mL), putrescine (60 µM), 3,5,3-triiodothyronine (T3, 100 nM), progesterone (20 nM), B27 (1%), sodium pyruvate (1 mM), glutamine (2 mM), BDNF (50 ng/mL), CNTF (10 ng/mL), forskolin (5 µM), and gentamicin (10 µg/mL). The cells were plated at a low density of approximately 1,000 cells/cm 2 on a surface precoated with poly-D-lysine and laminin. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO 2 and 95% air. Mitochondrial membrane potential in primary cultured rgcs (∆Ψm) by JC-10 fluorescence and flow cytometry The ∆Ψm of primary cultured RGCs was determined using the fluorescent probe JC-10. Briefly, primary cultured RGCs were exposed to CNTF (12.5 ng/mL), Tet (10 nM), or the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (5 µM) for 1 hour and then incubated in culture medium containing 3 µM JC-10 for 1 hour at room temperature. The cells were washed with PBS and analyzed by flow cytometry. Photomultiplier settings were adjusted to detect JC-10 monomer and aggregate fluorescence on the FL1 (525 nm) and FL2 (595 nm) detectors. The fluorescence ratio at these wavelengths was used to monitor changes in mitochondrial membrane potential. 26

intravitreal injection of tetrandrine
The protocol of performing an intravitreal injection has been previously described ( Figure 1B). 27 For the intravitreal injection of Tet into a transient retina ischemic model, 20 BABL/c mice (24 eyes) of either sex (4-week-old mice, 18-24 g) were used. Animals were anesthetized with a cocktail of intraperitoneal ketamine (75 mg/kg) and xylazine (5 mg/kg). Atropine ointment was applied to achieve mydriasis. Twenty-four hours before ischemic insult, the mice received intravitreal injections of Tet in the experimental group or of PBS in the control group. All injections were performed under visual control using an ophthalmic microscope. Mice in both experimental and control groups received 2 µL intravitreal injections. Intravitreal injections were performed with a 34-gauge blunt needle attached to a 5 µL syringe (Hamilton Company; Reno, NV, USA) after 10-0 needle puncture through the sclera, approximately 1 mm behind the limbus. The needle remained in the vitreous cavity for 1 minute after the injection and was then gently withdrawn. Vetropolycin ointment was used on the injected eyes after the operation. The injected animals were allowed to recover for 24 hours before being subjected to ischemic insults.

Transient retina ischemic model
The transient retina ischemic model was induced according to a previously reported protocol, 27-29 with a modification. Transient retinal ischemia was induced in the right eyes, and the contralateral eyes served as nonischemic control. Briefly, animals were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and xylazine hydrochloride (5 mg/kg). Atropine ointment was applied to achieve mydriasis. The anterior chamber of the right eye was then cannulated with a 30-gauge needle connected to a sterile saline-filled bottle. Intraocular pressure (IOP) was maintained at 110 mmHg for 60 minutes by lifting the bottle to a height of 1,496 mm above the eyes. Vetropolycin ophthalmic ointment was applied to treated eyes after the operation.

cell counting of rgcs in retinal cross sections
RGCs were counted using published procedures 30 with little modification. Briefly, each retinal section was divided into central, middle, and peripheral regions (1, 2, and 3 mm from the optic disc, respectively). Microscopic fields measuring 210 × 210 µm 2 were chosen to count DAPIlabeled RGCs. Six microscopic fields were used in each retina for counting ( Figure 1B)

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Tetrandrine protects mouse retinal ganglion cells from ischemic injury from each retina of the experimental and control groups were used for counting.

statistical analysis
Statistical analyses of the means for more than two groups were performed using one-way analysis of variance. The Student's t-test was applied for samples with an insignificant difference in variance. The null hypothesis was rejected at the 0.05 level. All statistical computations were carried out using SPSS 13.0 (IBM Corporation, Armonk, NY, USA). The relative cell numbers are expressed as the mean ± standard deviation.

Results
Protective influence of Tet on RGC-5 and ssP-induced neuron-like cell death resulting from serum deprivation Serum deprivation models were used to evaluate the protective effects of Tet on RGC-5 cell survival. The relative cell numbers from control, 0.01% DMSO, and Tet-treated groups of RGC-5 cells are shown in Figure 2. All cell numbers were measured at 24 hours, 72 hours, and 96 hours after treatment in both serum deprived ( Figure 2A) and 10% FBS growth conditions ( Figure 2B).
Under conditions of serum deprivation, as shown in Figure 2A, the RGC-5 cell numbers increased 30% at 72 hours using concentrations of Tet from 10 nM (1.32±0.060; P=1x10 -11 ) to 0.1 µM (1.31±0.080; P=1×10 -11 ) as compared to the cells treated with DMSO alone. At 96 hours after Tet treatment, the increase was less pronounced but still significant ( Figure 2B). The absolute numbers of RGC-5 cells in 10% FBS ( Figure 2C) were significantly higher than those under the serum deprivation condition at 48 hours (P=1.5×10 -9 ) and 72 hours (P=1.9×10 -7 ). These results suggest that Tet has little effect on cells that are growing rapidly in 10% FBS, but can protect cells under stressed conditions. To eliminate confounding factors related to cell division, we examined Tet function in postmitotic RGC-5-derived neuron-like cells. SSP (1 µM) optimally induced and maintained neuron-like morphology in RGC-5 cells from 2 hours to 72 hours (data not shown). We measured the protection of SSP-induced neuron-like cells at 48 hours after treatment with 5 nM (P=9.67×10 -5 ), 10 nM (P=9.67×10 -5 ), and 50 nM (P=2.58x10 -3 ) Tet ( Figure 2D). The numbers of SSP-induced neuron-like cells increased approximately 16% to 26% after Tet pretreatment, as compared to DMSO alone ( Figure 2D). This suggests that Tet promotes survival of postmitotic cells, which more closely represent mature neurons.

Tet protected cells from oxidative stress and glutamate-induced neurotoxicity
To determine whether Tet protects cells from oxidative stress and glutamate neurotoxicity, insults closely related to glaucoma, we examined cell survival in response to H 2 O 2 and glutamate stresses using a WST-8 cell proliferation assay ( Figure  3). As shown in Figure 3A, 100 nM Tet, but not 1 nM or 10 nM (data not shown), was able to significantly retard the reduction in cell numbers caused by 1 mM H 2 O 2 (P=4.2x10 -4 ), although they did not return to control levels.
Similarly, a range of Tet concentrations from 10 nM to 0.5 µM significantly prevented the cell death induced by 25 mM glutamate-induced neurotoxicity, in a dose-dependent manner ( Figure 3B), with cell numbers almost reaching control levels after Tet treatment with the highest concentration (0.5 µM). Together, these results indicate that Tet can promote cell survival in the context of oxidative stress or glutamate-induced neurotoxicity.

Tet slows down the loss of primary cultured rgcs in vitro
To confirm that Tet can exert protective effects on primary cultures, we examined the effects of Tet on the survival of murine primary RGCs. The immunopanning method, [21][22][23][24][25] with modifications, was used to isolate RGCs from neonatal mouse retinas. Primary RGCs were grown at a low density (approximately 1,000 cells/cm 2 ) for 4 days and were characterized with a neuron-specific antibody ( Figure 4A). The cells extended long processes from variably sized cell bodies ( Figure 4B), and exhibited a gradual loss of viability with a more stable period of approximately 40%-60% survival between 3 days and 9 days after isolation ( Figure 4C). The primary RGCs from this period were used to examine Tet function.
Two concentrations of Tet, 1 nM and 10 nM, were investigated with regard to their relative capacity to protect RGCs from death after isolation, as compared to

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Tetrandrine protects mouse retinal ganglion cells from ischemic injury DMSO alone ( Figure 5). Where the cell count immediately after isolation was deemed to be 100%, 72 hours after Tet treatment, significantly increased RGC survival was observed in both the 1 nM (35.7%±10.4%, P=0.009) and 10 nM (56%±10.5%, P=3.8×10 -4 ) Tet groups as compared to DMSO alone. At 96 hours after Tet treatment, a significant increase in RGC survival rate was still detected in both the 1 nM (36.6%±15.5%, P=0.011) and 10 nM (40.6%±8.5%, P=0.006) Tet groups. There were no differences between 1 nM Tet, 10 nM Tet, and DMSO groups at 120 hours after treatment (P.0.05). These results confirmed that Tet increased the survival rates of primary cultured RGCs, but Tet could not extend the survival times for primary cultured RGCs under the culture conditions tested.

Tet preserves ischemic rgcs in vivo
To  We examined the effects of Tet on RGCs 3 days after I/R and observed no significant differences in GCL cell survival in the Tet-I/R and CNTF-I/R groups versus the PBS-I/R group throughout the retinal regions ( Figure 7B and C; P.0.05). These results suggest a single dose of Tet protects retinal neurons immediately after I/R injury, but only for a limited time, comparable to the protection provided by CNTF.

effects of Tet and cnTF on mitochondrial transmembrane potential (∆Ψm)
The staining of primary cultured RGCs with the lipophilic cation JC-10 permits measurement of ∆Ψm within intact cells. JC-10 has the unique property of forming J-aggregates locally and spontaneously under high mitochondrial ∆Ψm; the aggregates fluoresce red and the monomeric form fluoresces green. Figure 8 shows that about 65% of PBS-, 56% of FCCP-, 86% of Tet-, and 80% of CNTF-treated RGCs ( Figure 8A-D) exhibited red and green fluorescence. The percentage of cells with polarized mitochondria in RGCs exposed to PBS, FCCP, Tet, and CNTF for up to 60 minutes were about 22%, 29%, 4%, and 5%, respectively ( Figure 8A-D). Figure 8E-H show the survival of RGCs 24 hours after exposure to PBS, FCCP, Tet, and CNTF, respectively, for up to 60 minutes.

effects of Tet and cnTF on activated caspase-3 and Bcl-2 expression in i/r insult retinas
Bcl-2 and cleaved caspase-3 (Asp175) were detected in the retinas one day after I/R. Expression was lower in the GCL and outer nuclear layer cells of retinas in the

Discussion
Our previous experiments suggested that 0.3% Tet exhibits an ocular antihypertensive effect that is comparable to 0.5% timolol in a laser-induced glaucoma model, 31 suggesting that Tet may be a potentially useful IOP-lowering agent in the treatment of glaucoma. Herein, we provide evidence that pretreatment with Tet can also promote the survival of cells in a wide variety of conditions. We measured protection of RGC-5 cells in the contexts of serum deprivation (10-500 nM of Tet), glutamate neurotoxicity (10-500 nM of Tet), H 2 O 2 (100 nM of Tet), protection of 1 µM SSP-differentiated RGC-5 cells (5-100 nM of Tet), and purified RGCs (1 nM and 10 nM of Tet) in vitro, as well as RGCs in vivo following retinal ischemic injury (10 µM of Tet). Together, the results showed a direct neuroprotective action of Tet on RGCs, which is consistent with a previous study on neuronal apoptosis in the hippocampus. 9 The RGC-5 cell line, derived by transforming postnatal day 1 rat retinal cells with ψ2EA1 virus, 32 was used to evaluate the protective roles of Tet in the context of glaucomatous insults such as ischemic serum deprivation, glutamate, The numbers of rgcs in the PBs-i/r, Tet-i/r, and cnTF-i/r retinas 1 day and 3 days after ischemia were much lower than the corresponding numbers in the PBs, Tet, and cnTF groups, respectively (P,0.05). a 2 µl volume of Tet, cnTF, or PBs was intravitreally injected 24 hours prior to ischemia. results are expressed as means ± sD. *P,0.05 as compared to control, one-way anOVa. # P,0.05, one-way anOVa (n$3

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Tetrandrine protects mouse retinal ganglion cells from ischemic injury stress-induced apoptosis. Glutamate excitotoxicity is another important molecular mechanism in RGC degeneration during glaucoma and other retinal diseases with ischemic involvement. 42 Tet reportedly prevents glutamate excitatory toxicity in cultured rat cerebellar granule cells, 43 and our present study extends this observation to L-glutamate-treated RGC-5 cells. The Tet-induced reduction in intracellular Ca 2+ levels, as has been described in cultured neurons of fetal mice, 3,44 could limit the toxic Ca 2+ overload resulting from prolonged activation of the Ca 2+ -permeable N-methyl-Daspartate receptors, which trigger calcium influx, organelle stress, and activation of proapoptotic pathways. 42 In addition to protection against ischemic insult, Tet exhibited multiple functions with regard to the promotion of cell survival. 41,43 In this study, we also used primary cultured RGCs and a transient retinal ischemia animal model to confirm the neuroprotective function of Tet on RGCs. Tet caused a significant increase in primary RGC survival, in agreement with the effect of CNTF on primary RGC numbers 45 and the effect of Tet on neuronal apoptosis in the hippocampus, 46 indicating that Tet protects RGCs directly. Furthermore, Tet treatment resulted in a significant increase in RGC survival  Tet could involve multiple pharmacologic  actions such as blocking the calcium influx, 3,4 inhibiting lipid peroxidation, 5 reducing the generation of ROS, 6 suppressing the production of cytokines and inflammatory mediators, and reducing neuronal apoptosis. 9,47 After induction of high IOP, topical administration of 0.1%-0.3% (1.6-4.8 µM) Tet, twice daily, for 1 week led to a significant decline in IOP in adult Sprague Dawley rats. 31 Although this involved a different route of drug application, the concentrations of Tet used in our previous study were equal to or less than the concentration achieved by an intravitreal injection of 2 µL of 10 µM Tet that markedly protected RGCs against ischemic reperfusion.
We also discovered that Tet maintains ∆Ψm in primary cultured RGCs and reduces I/R-induced RGC apoptosis by inhibiting expression of cleaved caspase-3 and Bcl-2 one day after I/R. The mechanism of this effect remains unknown.
Although a protective effect of Tet on RGCs was observed 1 day after ischemia, a single injection did not prevent damage to RGCs 3 days after that. The most likely explanation for this is that the metabolism or clearance of Tet caused the concentration to drop below a therapeutic range, although reperfusion might result in greater loss of tissue with secondary injury than that caused by the initial ischemia. 17,48 Overall, our results support the suggestion that Tet may be a novel medication that imparts effective neuroprotection.