Molecular Vision 2020; 26:494-504
Received 31 October 2019 | Accepted 25 June 2020 | Published 27 June 2020
1Department of Ophthalmology, Shanghai General Hospital of Nanjing Medical University, Shanghai, China; 2Department of Ophthalmology, Baoshan Hospital of Integrated Traditional Chinese Medicine and Western Medicine, Shanghai, China
Correspondence to: Xingwei Wu, Department of Ophthalmology, Shanghai General Hospital of Nanjing Medical University, Shanghai, China Phone: +86-13918757688; email: firstname.lastname@example.org
Purpose: To observe the effects of tetramethylpyrazine (TMP) on mice retinas injured by sodium iodate (NaIO3).
Methods: Male mice (n = 45) were randomly divided into three groups: the control group (Group C), the NaIO3-degenerated group (Group I), and the TMP-treated group (TMP group). The Group I mice were intraperitoneally injected with 35 mg/kg NaIO3. The Group C mice were injected with similar volumes of PBS. The TMP group mice were intraperitoneally injected with 80 mg/kg TMP starting 24 h after NaIO3 administration once a day for 14 days. Fundus photography, optical coherence tomography (OCT), electroretinography (ERG), hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and western blotting were used to assess the effects of TMP on mice retinas at day 3, 7, and 14 after NaIO3 administration.
Results: TMP effectively prevented the decrease in the thicknesses of the retinas and the outer nuclear layer (ONL), and effectively alleviated the functional decline in the retinas after NaIO3 administration. TMP significantly decreased the number of TUNEL-positive cells in retinas. In addition, TMP rapidly increased the expression of Nrf2 and HO-1 and decreased BAX expression in mice retinas after NaIO3 injection.
Conclusions: TMP alleviates morphological and functional retinal damage in mice exposed to NaIO3 and reduces retinal apoptosis.
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in individuals over 65 years old in developed countries . AMD is characterized by the progressive loss of central vision and is classified into two forms: dry AMD and wet AMD. Dry AMD involves the accumulation of deposits in the RPE and Bruch's membrane and is characterized by drusen and geographic atrophy (GA). Wet AMD is characterized by choroidal neovascularization invading the RPE layer accompanied by bleeding and exudation. Both types eventually show degeneration of the RPE and photoreceptor cells. In recent years, anti-vascular endothelial factor (anti-VEGF) injections have revolutionized the treatment of wet AMD . There are a few promising therapies under investigation, including stem cell transplantation . For dry AMD, no definitive treatments are currently available.
Although the pathogenesis of AMD remains unclear, one of the risk factors is oxidative stress injury of the RPE . Sodium iodate (NaIO3), an oxidizing agent, effectively induces retinal degeneration associated with regional loss of RPE, recapitulating some of the morphological features of geographic atrophy . NaIO3 selectively injures the RPE with secondary effects on photoreceptors which makes it possible to establish a reproducible model of AMD [6,7]. NaIO3–induced retinal structural and functional changes are time and dose dependent . Treatment with >20 mg/kg NaIO3 induces visual dysfunction associated with rapid suppression of phototransduction genes and induces oxidative stress in photoreceptors. NaIO3 can directly alter photoreceptor function and survival . Intravenous injection of 35 mg/kg NaIO3 induces necrosis in RPE cells and apoptosis in photoreceptor cells .
Tetramethylpyrazine (TMP) is extracted from an active ingredient of traditional Chinese herbal medicine Ligusticum wallichii Franchat (Chuanxiong). It has been widely used for treatment of neurovascular disorders, such as ischemic stroke, in China for hundreds of years [10,11]. TMP efficiently protects retinal cells against hydrogen peroxide–induced oxidative stress in mixed rat retinal cell cultures . In addition, TMP protects retinal capillary endothelial cells (TR-iBRB2) against IL-1β-induced nitrative and oxidative stress . In recent years, TMP has been suggested for the treatment of various retinal diseases due to its antioxidant and anti-inflammatory effects.
In this study, we performed in vivo experiments to examine the protective effects of TMP on NaIO3-induced retinal damage. Morphological and functional changes were assessed.
Forty-five 6- to 8-week-old male C57BL/6J mice (19–24 g) from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences were randomly divided into three groups (the control group, Group C; the NaIO3-degenerated group, Group I; and the TMP-treated group, TMP group), and housed in a standard laboratory environment under a 12-h:12-h light-dark cycle at 25 °C. All experimental procedures involving animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and approved by the Institutional Animal Care and Use Committee of Shanghai General Hospital’s Ophthalmic Research Center.
A sterile 7 mg/ml NaIO3 (S4007; Sigma St. Louis, MO) solution was freshly prepared with solid NaIO3 diluted in PBS (1X; 120 mM NaCl, 20 mM KCl, 10 mM Na2HPO4, 5 mM KH2PO4, pH 7.4). Group I (the NaIO3 group) was intraperitoneally injected with 35 mg/kg NaIO3 solution. Group C (the control group) was injected with similar volumes of PBS. The TMP (95,162; Sigma) solution was freshly prepared as 10 mg/ml in dimethyl sulfoxide (DMSO): PBS 1/1,000 (v/v). The TMP group was intraperitoneally administered 80 mg/kg TMP solution beginning 24 h after treatment with 35 mg/kg NaIO3 once a day for 14 days.
The mice were injected intraperitoneally with 4.3% chloral hydrate. Optical coherence tomography (OCT) images and fundus photography centered on the optic nerve head were acquired in live anesthetized animals using an OCT system (Phoenix Research Laboratories, Pleasanton, CA). Fundus images were obtained using the Micron III retinal imaging system in the same machine.
Scotopic and photopic electroretinograms (ERGs) were recorded at day 3, 7, and 14 after NaIO3 administration for four randomly selected mice (eight eyes) in each group. Following 6 h of dark adaptation, the mice were anesthetized with an intraperitoneal injection of 50 mg/kg ketamine plus 10 mg/kg xylazine and prepared under red light. The eyes were dilated with compound Tropicamide eye drops (SANTEN OY, Osaka, Japan). Ground and reference electrodes were placed subdermally on the tail and connected with the corneas, respectively. Scotopic and phototopic ERGs were collected on a Diagnosys Celeris instrument (Diagnosys, Boxborough, MA). Scotopic responses were elicited using eight flash intensities from 0.01 to 10 cd × s/m2, with increasing interflash intervals at each step. Before the photopic ERG recordings, the mice were adapted to light via exposure to 5 min of bright white flashes (adapted with 30 cd × s/m2 light). Photopic responses were elicited using a graded series of flashes with 3 cd × s/m2 light, with the background presented on a 30 cd × s/m2 background. All ERG data were collected at the same time of day. The a-wave amplitude was measured from the baseline to the trough of the first negative wave. The b-wave amplitude was measured from the trough of the a-wave to the peak of the first positive wave or from the baseline to the peak of the first positive wave in case of no a-wave.
Whole eyes were enucleated, fixed with 10% formalin, paraffin embedded, and sectioned at 5 μm. The thicknesses of the retinas and the outer nuclear layer (ONL) of the retina’s posterior pole were measured on hematoxylin and eosin (H&E)-stained slides. Five sections were measured for one eye of each mouse (one eye randomly selected from every mouse). The measurements were taken three times per section and averaged. For each section, measurements were taken at a distance of approximately 300 μm from the optic nerve.
The slides were washed with fresh xylene twice (15 min each time), rehydrated in a graded series of ethanol (absolute ethanol twice for 5 min, 85% and 75% ethanol each for 5 min), and rinsed in PBS for 5 min. The samples were then incubated in proteinase K for 15 min at room temperature and rinsed again three times in PBS for 5 min. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) In situ Cell Death Detection Kit (Roche, Indianapolis, IN) was employed for the analysis according to the manufacturer’s instructions. The slides were counterstained with 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR), incubated for another 10 min, rinsed in PBS three times for 5 min, and then sealed. A fluorescence scanning microscope (Olympus, Tokyo, Japan) was used for assessment.
Proteins were separated and transferred onto polyvinylidene fluoride (PVDF) membranes, which were incubated overnight at 4 °C with primary antibodies, including rabbit anti-mouse Nrf2 monoclonal antibodies (12,721; Cell Signaling Technology, Beverly, MA), rabbit anti-mouse HO-1 polyclonal antibodies (70,081; Cell Signaling Technology), rabbit anti-mouse Bax polyclonal antibodies (2772; Cell Signaling Technology), and rabbit anti-mouse β-actin monoclonal antibodies (4970; Cell Signaling Technology). Then, the membranes were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibodies (7047; Cell Signaling Technology) for 1 h at room temperature. The Chemi Doc XRS gel documentation system (Bio-Rad Laboratories Inc., Hercules, CA) was used to quantify immunoreactive proteins.
Data were presented as mean ± standard error of the mean (SEM). A two-tailed t test was used for statistical analysis of two groups. To compare three groups, two-way analysis of variance (ANOVA) was used. All statistical analyses were performed using Graph Pad Prism 7.0 software (San Diego, CA). A p value of less than 0.05 was considered statistically significant .
To evaluate the effects of TMP in NaIO3-induced injury in the mouse retinas, OCT and colorful fundus photographs of C57 mice in the three groups at day 3, 7, and 14 after administration of NaIO3 were assessed (Figure 1). Three days after NaIO3 treatment, the mouse retinas in Group I and the TMP group showed edemas (Figure 1B,C). Then the retinas were dark, and the pigments were disordered. Yellow-white deposits appeared at day 7 in Group I and the TMP group (Figure 1E,F). By day 14, the colors of the retinas were dirty, and the optic nerves were pale in the two groups (Figure 1H,I). OCT scans showed the retinal laminations in Group I and the TMP group became less clear compared with those in the control group at day 3 (Figure 2B,C). The RPE layers were disordered, and the RPE migrated into the inner and outer segments (IS/OS) at day 7 (Figure 2E,F). Progressive retinal degeneration was observed with RPE disruption and thinning of the entire retina, especially the ONL, in the following days. We could not identify laminations in Group I because of retinal atrophy at 14 days, and they were also blurry in the TMP group (Figure 2H,I).
H&E staining (Figure 3A) of the specimens confirmed the results from the fundus and OCT studies. The structure of each retinal layer in Group C was neatly arranged with an average thickness of 216 ± 3.00 μm; the ONL thickness was 49 ± 2.0 μm. After NaIO3 treatment for 3 days, the ONLs in Group I and the TMP group were wave-like (Figure 3A); the normal RPE structure was destroyed, with swelling and bundling of RPE cells migrated. Black round sediments were observed in the IS/OS (Figure 3A). The entire retinas, especially the ONL, were thin in subsequent days in Group I and the TMP group, compared with Group C. The average retinal thicknesses in Group I were 147 ± 4 μm with an ONL thickness of 26 ± 2.0 μm; in the TMP group, values of 160 ± 5.00 μm and 25 ± 0.7 μm were obtained, respectively, for the retinal and ONL thicknesses. At day 7 after NaIO3 injection, the thicknesses of the ONL were reduced more than 50%, with an average retinal thickness in Group I of 128 ± 4.00 μm, and an ONL thickness of 19 ± 5.0 μm. The average retinal and ONL thicknesses in the TMP group were 146 ± 13.0 μm and 25 ± 1.0 μm. On day 14, the average retinal thicknesses in Group I were 108 ± 4.00 μm, with an ONL thickness of 18 ± 3.0 μm. The average retinal and ONL thicknesses in the TMP group were 131 ± 6.00 μm and 26 ± 5.0 μm, respectively. The central retinal thicknesses of three groups statistically showed significant changes across time (**p<0.01). Retinal thicknesses reduction were less pronounced in the TMP group compared with Group I (**p<0.01; Figure 3B). ONL thicknesses of three groups also showed an apparent difference (**p<0.01), but they did not decrease statistically significantly across the time. The ONL thicknesses were reduced in Group I at day 7 and 14 compared with the TMP group (*p<0.05; Figure 3C). It appeared that TMP could prevent the decrease of the central retina and the ONL after NaIO3 treatment.
Retinal function changes were examined with scotopic and photopic ERGs at day 3, 7, and 14 after NaIO3 injection. Statistically significant reductions in all ERG responses were observed in Group I and the TMP group at three time points. The scotopic and photopic b-wave amplitudes strongly decreased from day 3 post NaIO3 injection. In addition, visual function decreased by day 7, and the scotopic (b-wave) and photopic (b-wave) responses were almost diminished in Group I. These rapid ERG changes also appeared in the TMP group, but the reduction was less than in Group I. Surprisingly, the scotopic b-wave amplitudes in the TMP group were nearly 50% of those in Group C. The ERG examinations in the mice showed that TMP could effectively reduce the decline of the b-wave in the scotopic responses at three time points (Figure 4A). The a- and b-wave amplitudes of the mixed rod-cone responses in the TMP group declined slightly which compared with Group I at three time points; the a- and b-wave latencies were shorter than Group I at day 3 and 7 (Figure 4B). The results for the photopic responses in the mice were similar to those scotopic responses. The ERG examinations showed that TMP could effectively reduce the decline in the b-wave in the photopic responses at day 3 and 14 and the b-wave latencies of the photopic responses in the TMP group were shorter than those in Group I at three time points (Figure 4C).
TUNEL assays showed that the level of apoptosis in the retinas peaked on day 3 after NaIO3 administration, then gradually decreased in the following days, and was not obvious at day 14 in Group I and the TMP group (Figure 5). TUNEL-positive cells (green) were restricted in the ONL, in which photoreceptor nuclei were located. The number of TUNEL-positive cells was statistically significantly increased in Group I compared with the TMP group at day 3 and 7 (**p<0.01; Figure 6).
Western blotting of retinal samples (n = 5) of Nrf2, HO-1 and Bax protein expression at day 3, 7, and 14 after NaIO3 injection (Figure 7A). Western blotting showed that the amount of Nrf2 protein in Group I and the TMP group was statistically significantly higher than that in Group C after NaIO3 injection at day 3, with a more obvious increase in the TMP group compared with that in Group I. These variations were not observed at day 14 (Figure 7B). The HO-1 levels changed with the amount of Nrf2, statistically significantly increasing at day 3 and decreasing gradually with time in Group I and the TMP group. However, there were statistically significant differences among the three groups at day 14 post-NaIO3 treatment, and the TMP group showed higher amounts than Group I (Figure 7C). Western blotting also revealed that the Bax expression levels in Group I and the TMP group were much higher than those in Group C at day 3 post-NaIO3 treatment. The Bax protein levels decreased gradually in the TMP group in the following days, while remaining high in Group I for 14 days. Bax expression was statistically significantly higher in Group I compared with the TMP group (Figure 7D).
AMD is the leading blinding eye diseases in individuals over 65 years old in developed countries . The occurrence of dry AMD is related to oxidative stress, autophagy, inflammation, and other factors . Large amounts of unsaturated fatty acids accumulate in the photoreceptor layer, which is likely to cause oxidative stress damage under continuous visible light irradiation. The retina is sensitive to oxidative stress which damages the retina aggravated by lipofuscin. We used NaIO3 to make an animal model of dry AMD. TMP is an active ingredient of the traditional Chinese herbal medicine Chuanxiong. Its main pharmacological effects include antioxidative stress, antagonism for calcium, and suppression of proinflammatory factors . It was reported that TMP could inhibit free radical production, had antioxidative and neuroprotective effects, and was widely used in neurologic ischemic diseases .
A mouse disease model similar to dry AMD was prepared by intraperitoneal injection of NaIO3. In this study, we selected TMP to intervene mice retinas against NaIO3-induced damage and observed morphological and functional retinal changes. Fundus photography and OCT scan showed that the mice retinas presented yellow-white deposits at day 7, accompanied by RPE layer disorder and reduction of photoreceptor cells in the ONL after NaIO3 injection. H&E staining of the retinal sections showed that the retinal thickness was statistically significantly decreased after NaIO3 treatment; however, the thickness of the retinal thickness in the TMP-treated group declined less than that in the NaIO3-degenerated group. In conclusion, TMP could alleviate NaIO3-induced retinal damage, especially protecting the ONL from injury. Retinal apoptosis peaked on day 3 after NaIO3 injection, gradually decreased, and was no longer obvious on day 14. The ratios of apoptotic cells in the ONL in the NaIO3-degenerated group and the TMP-treated group at day 3 and 7 were statistically significantly different. This suggested that TMP could reduce NaIO3-induced apoptosis in photoreceptor cells. Therefore, TMP could alleviate retina and ONL thickness reductions caused by NaIO3.
We observed a rapid reduction in the b-wave amplitude after NaIO3 treatment, in agreement with a defective synaptic transmission to the second-order neurons. The decrease in the b-wave in the TMP-treated group was nearly half that of the control group, while responses were almost disappeared in the NaIO3-degenerated group. The differences were statistically significant. It was suggested that TMP could effectively protect the function of bipolar cells. The reductions in the a- and b-wave amplitudes of the mixed rod-cone responses in the TMP-treated group were less than those in the NaIO3-degenerated group, suggesting that TMP can effectively protect the function of the photoreceptors. In conclusion, TMP could protect retinal function from NaIO3 damage.
The Keap1-Nrf2/ARE signaling pathway confers resistance to oxidative stress due to internal or external oxidation and chemical substances, and constitutes an important protective pathway. As an antioxidative transcription factor, Nrf2 binds to the ARE antioxidant reaction and induces the expression of multiple antioxidants, anti-inflammatory proteins, and detoxifying enzymes downstream to form the Nrf2/ARE pathway. In combination with ARE, Nrf2 activates the expression of the target genes, which regulates the transcriptional activities of phase II metabolic enzymes and antioxidant enzymes, thus exerting antioxidant effects and restoring intracellular homeostasis . Damage to the Nrf2 pathway leads to oxidative damage to the RPE or triggers an intrinsic immune response that induces apoptosis in RPE leading to pathological changes in AMD . The impaired Nrf2 signaling pathway causes RPE oxidative damage . HO-1 is a downstream product of Nrf2 activation. It not only plays a role in the body’s physiologic state but also has an important function in antioxidation, antiapoptosis, and immune regulation under stress conditions.
This study showed that the amount of Nrf2 protein in the retinas after NaIO3 treatment was statistically significantly higher than that in the control group. However, the increase in the TMP-treated group was more obvious at day 3 and 7. The downstream protein HO-1 changed consistently with Nrf2, and it increased statistically significantly at three time points after NaIO3 treatment. Then the amount of HO-1 in two groups fell, but the amount remained higher than the control values. It indicated that treatment of NaIO3 temporarily activated the expression of Nrf2 and HO-1 in the retinas against oxidation stress, and TMP could increase the expression levels. Bax is the most important apoptotic gene in the human body. It belongs to the Bcl-2 family of genes and forms a heterodimer with Bcl-2, which inhibits Bcl-2. Bax is a key component of apoptosis caused by mitochondrial stress . Upon apoptotic stimulation, Bax forms oligomers and is transferred from the cytosol to the mitochondrial membrane. BAX interacts with the voltage-dependent anion channel of the mitochondrial outer membrane to promote mitochondrial membrane permeability with transient pore opening . These channels promote the release of mitochondrial membrane gap proteins and cause apoptosis. In this study, BAX amounts in retinas were statistically significantly higher than those of the control group after NaIO3 injection at day 3. The amount of BAX in the TMP-treated group began to decline, but the NaIO3-degenerated group retained high levels throughout 14 days. These findings indicate that treatment of NaIO3 leads to BAX upregulation and causes apoptosis that peaks within 3 days, resulting in decreased retinal and ONL thicknesses and significantly impaired retinal function. Meanwhile, TMP intervention upregulates retinal Nrf2 protein and downstream HO-1, decreases BAX elevation, inhibits mitochondrial pathway activation, suppresses apoptosis, reduces retinas and ONL thinning, and decreases retinal function damage induced by NaIO3. The result suggests that the protective effects of TMP against NaIO3-induced injury in retinas may be mediated by upregulation of Nrf2 and HO-1 expression. Therefore, using TMP may provide a novel approach in the treatment of dry AMD.
This study was supported by National Nature Science Foundation of China (NO. 81,674,027), National Key R&D Program of China (2016YFC0904800) and Baoshan District Shanghai Youth Medical Talents Training Program (bswsyq2015A02), Baoshan District Ophthalmology Specialty of Integrated TCM and Western Medicine Program (BSZK2018B01).