Protective Effects of Vaccinium Uliginosum Extracts on Rat Retina Against Microwave- induced Damage

doi:10.21203/rs.3.rs-1606324/v1

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Protective Effects of Vaccinium Uliginosum Extracts on Rat Retina Against Microwave- induced Damage

https://doi.org/10.21203/rs.3.rs-1606324/v1

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Objective

To study the protective effect of Vaccinium Uliginosum (VU) extracts on rat retina against microwave radiation induced injury.

Methods

The rats were randomized into 3 groups (12 in each) and treated as follows: group 1: no irradiation; group 2: irradiation without administration; group 3: irradiation with administration in advance. After administration to group 3 with VU extracts for 1 week, group 2 and 3 were irradiated. 24 hours later, all the rats were recorded with electroretinography (ERG). Half of each group were performed with pathological examination, and the rest were performed with biochemical detection [superoxide dismutase (SOD) and glutathione peroxidase (GPx)].

Results

1.Compared with group 2, both of group 1 and group 3 showed a significant increase in a-wave ERG amplitudes in scotopic and photopic conditions, and the b-wave amplitude of group 3 was significantly higher than group 2 but lower than group 1 in photopic condition. 2. The thickness of outer nuclear layer (ONL) in group 2 was thicker than group 1 and group 3; The cell density of inner nuclear layer (INL) and the ganglion cells layer (GCL) both showed that group 3 was more than group 2 but less than group 1. 3. The SOD activitiy in group 2 was lower than group 1 and 3. The GPx activitiy in group 3 was lower than group 1, but higher than group 2.

Conclusions

VU extracts can protect rat retina against microwave-induced damage. And these results may provide a base for exploring the possible preventive and therapeutic measures.

Vaccinium Uliginosum

anthocyanins

retinal injury

microwave

antioxidant

Microwave is widely used in military fields, such as radar troops, ships, aircraft, and microwave itself is even put into the battlefield as a new technical weapon. Besides, microwave is used in ordinary life and work, including mobile phone, GPS navigator, Bluetooth, Wi-Fi, microwave oven and so on ^[1]. But with the wide application of microwave, more and more focus is attracted on human health, especially by the high power microwave (HPM) whose wavelength is 10cm in S band level and the peak power is more than 100MW^[2–3]. Microwave radiation can cause retinal edema, vision decreased, contrast sensitivity reduction, color perception threshold increased and so on ^[4–5].

All countries have formulated relevant protective measures against microwave radiation, including Brooks Unit of the U.S. Naval Health Research Center, the Experimental Institute of the U.S. Naval Medical Research Center and the Radio Frequency Radiation Research Unit of the U.S. Air Force Laboratory. They only recommend avoiding long-term contact and wearing protective clothing. However, due to the particularity of troops, it is inevitable to get long-term contact with the radiation. Moreover, because of the unpredictability of microwave damage in wartime, equipment protection has obvious limitations. So, drug protection based on human body should be considered seriously. Despite a lot of exploration, there is no exactly effective drug at home and abroad.

Studies have shown that free radicals induced lipid peroxidation ^[6–7] is the main mechanism of biological damage by microwave radiation. Anthocyanins has good antioxidant and anti-apoptotic properties ^[8]. However, anthocyanins has been proved protective against microwave-induced damage to 661W cells (retinal photoreceptors) in vitro in the early stage by our team ^[9]. In this study, the effect on retina would be studied by animal experiment.

2.1 Experimental Animals

Thirty-six Brown Norway rats, half male and half female, weighing 195 ± 16g, were provided by Vital River Laboratory [Certification Number: SCXK (Jing) 2021-0006]. Studies were conducted in adherence with Association for Research in Vision and Ophthalmology (ARVO) and followed guidance and principles of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Each rat was examined by slit lamp and ophthalmoscopy before the experiment and showed no abnormalities.

2.2Drugs and Reagents

Anthocyanins was extracted from Vaccinium Uliginosum (VU), which was provided by School of Food Science and Nutrition Engineering of Agricultural University of China. By Ultrasonic assisted-solvent extraction method, the exact high performance liquid chromatography and total ion flow diagram of the extracts has been made clear^[9]. Malonaldehyde (MDA) test kit was provided by Abcam Co. Ltd.(Britain); GPx test kits were provided by Shanghai biyuntian Biotechnology Co. Ltd.

2.3 Main Instrument

The microwave radiation source was from the WKZ-II microwave transmitter. Its frequency was 2450 MHz, and the emittance was 100mW/cm². Each rat was put into a plexiglass cage and the electromagnetic field was uniform distributed.

2.4 Animal Grouping and Intervention

The rats were randomized into 3 groups (12 in each) and treated as follows: Group 1 (sham): no irradiation; Group 2: irradiation without VU extracts administration; Group 3: irradiation with VU extracts administration in advance.

The rats were maintained on a 12-hour light/dark cycle at 22℃, and standard rat chow and water were provided ad libitum. Group 1 and 2 were administrated with normal saline intragastrically once a day. Group 3 was administrated with extracted VU extracts intragastrically (250mg/kg) once a day. One week later, group 3 was irradiated for 15 minutes.

2.5 Electroretinography

The rats were pre-adapted overnight and all the procedures were performed in dim red light. After dark adaption, rats were anesthetized using a combination of ketamine and xylazine (16 mg and 80 mg/kg respectively), pupils were dilated with 0.5% tropicamide. The response to a light flash stimulation (3.0 candela seconds/m²) was amplified. Cone-related ERGs were recorded 10minutes after complete light adaption. The baseline to maximum a-peak was recorded as a-wave amplitude. The b-wave amplitude was recorded as maximum a-peak to maximum b-peak.

2.6 Ocular Section Histology

Half of each group (6 rats) were selected randomly. Eyes were enucleated and fixed with 10% formaldehyde. Afterward, samples were embedded in paraffin and sagittal sections were cut at 5 micrometer. Sagittal plane sections containing the optic nerve were selected to be stained with hematoxylin and eosin (HE) and photographed under the microscope. The thickness of ONL and the cell density of INL and GCL were measured by image analysis system software.

2.7 Biochemical Analysis

The retina tissue of the other half of each group (6 rats) was harvested and rinsed in PBS. The tissue homogenates were resuspended in the suitable buffers provided with the SOD and GPx kits, and the assays were performed according to the manufacturer’s instructions. SOD activity was measured in terms of the absorbance at 450 nm. The GPx activity content was measured via microplate reader. Each experiment was performed in triplicates.

2.8 Statistical Analysis

All data were expressed as means ± standard deviation, and analyzed with SPSS21.0 software. ANOVA is applied for data with normal distribution and homogeneity of variance. One-way ANOVA was applied for comparison of measurement data among groups. In case the difference was significant, the Dunnett test was used to test the significance of intergroup differences. P < 0.05 was considered statistically significant.

3.1 Protective Effect on Retinal Function

Retinal function was assessed by ERG and a- and b-waves in both scotopic and photopic conditions were recorded. At the flash intensities, there were significant differences among the groups. 1. There was no significant between group 1 and group 3 in a-wave ERG amplitude whether in scotopic or in photopic conditions, but the a-wave amplitudes of group 1 and 3 were both higher than group 2. There was no significant difference between group 2 and group 3 in b-wave amplitude in scotopic condition, and both of them were lower than group 1. 3. The b-wave amplitude of group 3 was significantly higher than group 2 but lower than group 1 in photopic condition. Figure 1 shows the comparison in details.

3.2 Protective Effect on tissue structure

The retinal tissue morphology was observed under light microscope. No obvious abnormalities were found in the sham irradiation group, the retinal tissue structure was complete, and the cells in each layer were arranged orderly (A in Fig. 2); One day after irradiation, obvious morphological damage of retina could be observed in group 2 (B in Fig. 2), which showed retinal edema, inner segment/outer segment photoreceptor (IS/OS) getting disordered, and both of pyknosis and vacuolar degeneration in INL and GCL. In the VU extracts pretreatment protection group (C in Fig. 2), the damage to retinal tissue structure was totally less serious. The tissue structure of each layer was basically clear and in order, and fewer cells showed pyknosis and vacuolar degeneration.

For the thickness of ONL, there were significant difference among the three groups. Group 2 was thicker than group 1 and group 3, and there was no significant difference between group 1 and 3. The cell density of INL and GCL were measured and both of them showed that group 3 was more than group 2 but less than group 1. A full list of statistical comparisons is available in Fig. 3 (A to C). The above results showed that VU extracts had a good protective effect on the pathological injury of retina caused by irradiation.

Retinal morphology showed the difference among the three groups. Figure 2A, 2B and 2C stand for group 1, 2 and 3 respectively. Figure 2A The retinal tissue structure was complete and arranged neatly. Figure 2B The retinal tissue structure was obviously loose and deformed, and the cells were arranged disorderly. Pyknotic nuclei (green arrow) and cells vacuole (red arrow) could be seen in INL. ONL was swelling and IS/OS got disordered. Cells density in INL and GCL decreased. Figure 2C The change of tissue structure in group C was similar to that in group B, but which was less serious.

3.3 Oxidative Stress Markers

The SOD activity of group 2 was significantly lower than group 1 and group3, but there was no difference between group 1 and group 3 (Fig. 4A). The GPx activity of group 3 was significantly lower than group 1 but higher than group 2 (all P < 0.05; Fig. 4B).

Because of special water-rich circulation and relatively poor heat dissipation in eyes, it is a sensitive target organ under microwave action. Radiation could lead to increased apoptosis and decreased cell proliferation ^[10–11]. And injuries to the retina is the researching focus ^[12]. Modern medical research has found the pathogenesis of microwave injury to the retinal is oxidative damage ^[13].

VU is mainly distributed in north China, Korea, Japan, northern Europe and North America. Its berry is nearly spherical or oval. It was named Wenpu (Shengjing Tongzhi) and xiongguo (Chinese Bencao) in ancient Chinese books. It’s commonly known as red beans, tooth pimples and Wenpu black rice tree. They are warm in nature and bitter in taste.

Light damage is called "light poison" in traditional medicine. Its nature is hot and dirty. Many ophthalmology monographs such as "the secret biography ophthalmology Longmu Lun · Volume 7" and "syndrome and treatment guidelines · qiqiaomen" called it "dark eyes". Just because of its diuretic and detoxifying characteristic, VU was used for curing "light poison" and "dark eyes" by detoxification and supplementing the deficiency of liver and kidney. The main active component of VU is anthocyanin, whose antioxidant effect has been widely proved in modern medicine ^[14–15].While, in modern medical research, the pathogenesis of light injury is oxidative damage ^[16]. Based on the consistency of traditional and modern medicine in the treatment mechanism of antioxidantion, the anthocyanins extracted from VU could be used to study the prevention and treatment of microwave damage. The school of food science and nutrition engineering of China Agricultural University was responsible for purification of VU which was applied in this study. And the high performance liquid chromatography (HPLC) and total ion flow diagram of anthocyanins from VU extracts were provided, which can not only maximize the natural antioxidant capacity of plants, but also ensure the uniqueness and scientificity of the research object.

When assessing retinal function through ERG, no significant difference was found between group 1 and group 3 in a-wave ERG amplitude, but both of them were higher than group 2. Besides, the b-wave amplitude of group 3 was significantly higher than group 2 but lower than group 1 in photopic condition. Generally speaking, a-wave originates in rod and cone cells and represents the electrical activity of photoreceptor, and b-wave represents the electrical activity of bipolar cells and inner nuclear layers ^[17]. So these results indicate the retina is injured by microwave from the inner layer to the outer layer, and the VU extracts could prevent and alleviate the injury.

Histopathologic findings of this experiment confirm such results. Retinal edema, IS/OS getting disordered, pyknosis and vacuolar degeneration in INL and GCL could be seen in group 2, which could be obtained the same findings in Wang’s study ^[18], but all of those presented minor changes in group 3. In quantitative terms, the thickness of ONL of group 2 was thicker than group 1 and group 3, and there was no significant difference between group 1 and 3. The cell density of INL and GCL showed that group 3 was more than group 2 but less than group 1.

To confirm such protective effect by VU extracts is for the reason of antioxidative function, classic oxidative stress markers were tested in this study. Similarly, the SOD activity of group 2 was found significantly lower than group 1 and group3, but there was no difference between group 1 and group 3. The GPx activity of group 3 was significantly lower than group 1 but higher than group 2.

SOD plays an important role in the main antioxidant defense system in the body ^[19]. GPx widely exists in the body as a peroxidase which could clear the peroxides in living cells from free radical damage. When cells encounter harmful conditions caused by reactive oxygen species (ROS), as antioxidant enzymes, SOD and GPx could ensure the integrity of the cells as a compensation mechanism ^[20]. While it has been demonstrated that ROS is an initiating factor in the injury induced by microwave irradiation ^[21].

Previous studies have found anthocyanins extracted from VU could reduce apoptosis and alleviate oxidant injury of mouse retina by activating Nrf2/HO-1 signal pathway and inducing HO-1 translation ^[9]. Combined with the functional and tissue structure performance, the changes of these oxidative stress markers further showed that anthocyanins extracted by VU were protective to rat retina against microwave-induced damage through antioxidation.

Author contributions

Both of the authors participated in the manuscript. Lan Yin and Si-jun Fan conducted the experiment and wrote the manuscript; Fan SJ provided the ideas.

Conflict of interest

The authors declare no competing interests. The authors did not receive any financial AND non-financial support from any organization for the submitted work.

Reproduced images were authorized to Radiation and Environmental Biophysics by both authors.

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No competing interests reported.

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Version 1

posted

You are reading this latest preprint version

Protective Effects of Vaccinium Uliginosum Extracts on Rat Retina Against Microwave- induced Damage

Status:

Version 1

Abstract

Objective

Methods

Results

Conclusions

Figures

1. Introduction

2. Methods

2.1 Experimental Animals

2.3 Main Instrument

2.4 Animal Grouping and Intervention

2.5 Electroretinography

2.6 Ocular Section Histology

2.7 Biochemical Analysis

2.8 Statistical Analysis

3. Results

3.1 Protective Effect on Retinal Function

3.2 Protective Effect on tissue structure

3.3 Oxidative Stress Markers

4. Dicussion

Declarations

References

Additional Declarations

Status:

Version 1