|Year : 2022 | Volume
| Issue : 3 | Page : 347-352
Mitigation of photoreceptors abnormalities after low-level laser therapy and chia seeds supplementation in experimental diabetic retinopathy
Abdel Rahman Hassan Mahmoud1, Salwa Ahmed Abdelkawi2, Dina Fouad Ghoneim1, Aziza Ahmed Hassan1, Mona Ebrahim Morsy1
1 Department of Medical Application of Laser, Ophthalmic Unit, National Institute of Laser Enhanced Sciences, Cairo University, Giza, Egypt
2 Department of Vision Science, Biophysics and Laser Science Unit, Research Institute of Ophthalmology, Giza, Egypt
|Date of Submission||28-Aug-2021|
|Date of Decision||19-Apr-2022|
|Date of Acceptance||13-Aug-2022|
|Date of Web Publication||02-Nov-2022|
Salwa Ahmed Abdelkawi
Department of Vision Science, Biophysics and Laser Science Unit, Research Institute of Ophthalmology, Giza
Source of Support: None, Conflict of Interest: None
| Abstract|| |
BACKGROUND: This study aimed to assess the effect of low-level laser therapy (LLLT) and chia seeds on the mitigation of photoreceptors abnormalities in experimental diabetic retinopathy (DR).
MATERIALS AND METHODS: A total of 65 female Wistar rats, 5 rats were served as a control group and 60 rats were injected intraperitoneally with one dose of 55 mg/kg of streptozotocin (STZ) to induce DR after 6–8 weeks. The rats were divided into (n = 20 rats each): (a) DR group: did not receive any treatment, (b) DR+ LLLT group was exposed to 670-nm LLLT for 6 weeks (two sessions/week), and (c) DR+ LLLT+ chia seed group, in which rats were exposed to LLLT and administrated with 250 mg/kg/day of chia seeds flour for 2 weeks before STZ injection and continued to the end of the experiment. Blood glucose (BG) levels and retinal histological examination were employed after 1, 2, 4, and 6 weeks.
RESULTS: The BG level in the DR group and the treated groups were significantly higher (P < 0.001) than in the control group after the four-time periods. Chia seeds group exhibited BG levels less than the DR and the DR+ LLLT groups after 6 weeks (P < 0.01). LLLT improved the degeneration of the photoreceptors after 6 weeks of treatment, while LLLT+ chia seeds supplementation showed early photoreceptors improvement after 2 weeks.
CONCLUSION: The early improvement in the photoreceptors after LLLT+ chia seed may be attributed to the potent antioxidant properties of chia seeds. Therefore, the combination between LLLT and chia seeds should be employed to protect the retinal photoreceptors against DR.
Keywords: Chia seeds, diabetic retinopathy, histological examination, low-level laser therapy
|How to cite this article:|
Hassan Mahmoud AR, Abdelkawi SA, Ghoneim DF, Hassan AA, Morsy ME. Mitigation of photoreceptors abnormalities after low-level laser therapy and chia seeds supplementation in experimental diabetic retinopathy. Oman J Ophthalmol 2022;15:347-52
|How to cite this URL:|
Hassan Mahmoud AR, Abdelkawi SA, Ghoneim DF, Hassan AA, Morsy ME. Mitigation of photoreceptors abnormalities after low-level laser therapy and chia seeds supplementation in experimental diabetic retinopathy. Oman J Ophthalmol [serial online] 2022 [cited 2022 Dec 4];15:347-52. Available from: https://www.ojoonline.org/text.asp?2022/15/3/347/360406
| Introduction|| |
Diabetic retinopathy (DR) is the common microvascular complication of diabetes mellitus (DM), affecting around 25%–44% of patients with diabetes. Early diagnosis and treatments can effectively prevent disease progression for most DR patients. Many therapies, such as argon laser photocoagulation, can cause retinal damage by applying heat to the adjacent choroid and the neurosensory retina, leading to thermal damage and alteration in the retina transparency.
The micropulse subthreshold laser has a beneficial effect on the retina might be referred to the reduction in cytokines and vasoactive substances synthesis, leading to fewer capillaries' permeability. Moreover, micropulse laser produces retinal pigmented epithelium migration and proliferation, leading to drying edematous areas.
Fortunately, it might be possible to apply low-level laser therapy (LLLT) or photobiomodulation (PBM) to treat many retinal diseases. The low-level laser was involved in the treatment of DR, optic nerve degeneration, photoreceptors damage, and diabetic macular edema with no damage to the neurosensory retina.,,
Chia seeds (Salvia hispanica L.) are herbaceous plants related to the Lamiaceae or Labiatae family. Chia seeds have rich gluten-free protein supplies, a high amount of dietary fiber, antioxidants, phenolic compounds, polyunsaturated fatty acids, bone minerals, Vitamin B, omega-3 (alpha-linolenic acid), and omega-6, that aid in digestion and controlling of blood glucose (BG) levels in DM.
Recently, chia seeds are endorsed for ocular healthiness, such as tear production in dry eye syndrome, lens transparency and elasticity, treatment of macular degeneration, and anti-inflammatory effects.
The present work aims to consider the effects of DR on the rat's retina and explore the impact of LLLT by focusing on retinal photoreceptors. Moreover, to evaluate whether this LLLT alone or through supplementation with chia seeds, as an antioxidant, will preserve the photoreceptors against DR.
| Materials and Methods|| |
A total of 65 female Wistar rats (200 ± 20 g) were maintained in a standard 12-h light–dark cycle with permitted access to water and a balanced diet at a temperature of 30°C ± 2°C and 50% humidity. Slit-lamp biomicroscopic examination for rats' eyes revealed no signs of any abnormalities. All the experiments were employed in compliance with the Cairo University Institutional Animal Care and Use Committee (CU-IACUC) (Application No. CU/I/F/5/18), Public Health Guide for the Care and Use of experimental animals.
Induction of diabetes and diabetic retinopathy
Rats were once injected intraperitoneally with a dose of 55 mg/kg of streptozotocin (STZ) (in 0.1 mol/L citrate buffer, pH = 4.4) (Sigma-Aldrich, St. Louis, Missouri, USA). The BG level was measured using an electronic BG meter (Accu-Check, Roche Diabetes Care, Inc., Indianapolis, USA). Rats with BG levels of more than 280 ± 50 mg/dl were involved in the study and were followed up for 6–8 weeks until the establishment of DR.
The rats were divided into a control group that did not receive treatment (n = 5 rats) and the DR group (n = 60 rats). The DR group was then divided into three subgroups (n = 20 rats each) as follows: (a) DR group: did not receive any treatment, (b) DR+ LLLT group: exposed to 670 low-level diode laser for 6 weeks, and (c) DR+ LLLT+ chia seeds group: supplemented with a daily dose of 250 mg/kg of chia seed (Planton Company, New Delhi, India). Freshly prepared chia seeds were minced and soaked in water 1 day before administration through a stomach tube. The administration started 2 weeks before the induction of diabetes. It was continued until the establishment of the DR as well as during the 6 weeks of exposure to 670-nm low-level laser.
Low-level diode laser application
A low-level diode-pumped laser (Cobolt Modulated DPSSL-DRIVER II, Solna, Sweden) was calibrated to deliver 670 nm using a noncontact fiber optic with 0.4 mm in diameter at a distance of 7–9 cm from the rat' eye. Indirect ophthalmoscopy with +20 D Convex Volk Lens was used to permit retinal visualizations. The power used was 50 mW for 90 s (two sessions/week, 2 days apart for 6 weeks) so, the weekly radiant exposure was 9000 mJ/cm2/eye. BG level was recorded directly before sacrificing the animal, while histopathological examination for the retina was employed after 1, 2, 4, and 6 weeks.
Eyes were enucleated, injected as well as immersed in 4% glutaraldehyde in 0.1 M phosphate buffer saline (PBS, pH 7.4). The retina was dissected into sections of 1 mm3 and then further fixed for 8 h with a freshly prepared glutaraldehyde-buffered solution (pH 7.4). The sections were washed for 1 h with PBS at 4°C, fixed in 1.33% osmium tetroxide, and dehydrated in cold ethanol grads (50%, 70%, 80%, 90%, and 96%). The samples were embedded in freshly prepared Araldite CY212 mixtures. Semithin sections (1 μm) were cut by ultratome (LKB Produkter, Sweden), fitted on glass slides, and stained with toluidine blue for microscopic examination.
The levels of BG were recorded as the mean ± standard deviation. A one-way ANOVA was employed with Tukey's test for statistical analysis of BG levels between the different experimental groups. P < 0.05 was considered statistically significant.
| Results|| |
Blood glucose level
The mean value of the BG level was significantly higher in the DR group, DR+ LLLT group, and DR+ LLLT+ chia seeds (P < 0.001) after the four-time periods than in the control rats [Table 1]. The DR+ LLLT+ chia seeds group exhibited BG levels significantly higher than the control animals (P < 0.001) but considerably less than those of the DR and DR+ LLLT groups after 6 weeks (P < 0.01). The DR+ LLLT+ chia seeds group exhibited BG levels significantly higher than the control animals (P < 0.001) because chia seeds were responsible for considerable improvement of the diabetic mellitus state without complete recovery. Furthermore, as a result of the antioxidant effect, the improvement in BG level after chia seeds supplementation was noticeable compared with the DR group and the DR+ LLLT.
|Table 1: Blood glucose level for the control, diabetic retinopathy, diabetic retinopathy + low-level laser therapy, and diabetic retinopathy + low-level laser therapy + Chia seeds groups|
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The light micrograph of the control retina [Figure 1]a revealed regular retinal layers without any abnormality. After 1 week of DR, the retina showed edema of the cytoplasm in the pigmented epithelium layer and hollow spaces between photoreceptors [Figure 1]b. After 2 weeks of DR, the retina showed severe disorganization of photoreceptors [Figure 1]c. Moreover, after 4 weeks, the retina displayed edema in-between the outer segments of photoreceptors and severe disorganization of the photoreceptor [Figure 1]d. After 6 weeks, the retina shows edema in the outer segment of photoreceptors and severe degeneration of photoreceptors with spaces between them [Figure 1]e. Moreover, the outer plexiform layer (OPL) showed prominent edema and thickening in the capillary wall.
|Figure 1: (a) Light micrograph of the well-organized control retina. (b) 1WDR: Showed edema in-between the outer segment of photoreceptors (arrow) and slight disorganization of the photoreceptors. (c) 2WDR: Severe disorganization of photoreceptors. (d) 4WDR: Edema in the outer segments (yellow arrow) and severe disorganization in the photoreceptors layer. (e) 6WDR: Edema of the outer segment of photoreceptor (yellow arrows), severe degeneration of photoreceptors with hollow spaces between them (red stars) and edema and thick-wall capillary in the outer plexiform layer (black arrow). RPE: Retinal pigmented epithelium, PRL: Photoreceptor layer, OPL: Outer plexiform layer, INL: Inner nuclear layer, NFL: Neurofilament layer, 1WDR: One-week diabetic retinopathy (Toluidine blue, scale bar: 20 μm)|
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The retina with DR and exposed to LLLT for 1 week revealed a noticeable disorganized photoreceptor layer (PRL), vacuolization in the OPL, and thickened basement membrane of the capillaries in the nerve fiber layer [Figure 2]a. Then, after 2 weeks, the retina layers showed edema of the cytoplasm in the pigment epithelium layer and slight disorganization in PRL, which persisted until the 4th week [Figure 2]b and [Figure 2]c. In contrast, the retinal layer was improved after 6 weeks [Figure 2]d with no deviation from the control.
|Figure 2: Light micrograph of the retina with DR + LLLT. (a) 1 week there was marked disorganization of photoreceptors, vacuolization in the OPL (yellow arrows), thickened basement membrane of the capillaries in the nerve fiber layer (red arrow). (b) 2 weeks showing edema of the cytoplasm of pigment epithelium layer and slight disorganization in PRL. (c) 4 weeks: Slight disorganization in PRL. (d) 6 weeks showed improved retina with no deviation from the control. (Toluidine blue, scale bar: 20 μm). DR: Diabetic retinopathy, LLLT: Low-level laser therapy, OPL: Outer plexiform layer, PRL: Photoreceptor layer, RPE: Retinal pigmented epithelium|
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One week after treatment with LLLT and chia seeds, the histological structure revealed edema of the pigmented epithelium and disorganization in the photoreceptors layer. Moreover, there was a fragmentation of nuclear chromatin of the cell bodies in the inner nuclear layer [Figure 3]a. After 2 weeks of treatment, the retinal layer displayed an improvement in both the pigmented epithelium and the PRL s. Nevertheless, vacuolation in the OPL and spongy architecture in the inner plexiform layer were observed [Figure 3]b. In contrast, undeviating photoreceptors layer from the control after 4 weeks and 6 weeks, as shown in [Figure 3]c and [Figure 3]d. The histological difference between the experimental groups is summarized in [Table 2].
|Figure 3: Light micrograph of the retina with DR + LLLT + Chia seeds. (a) 1 week: Edema of the retinal pigmented epithelium (arrow), disorganization in the photoreceptors layer, and fragmentation of nuclear chromatin of the cell bodies in the inner nuclear layer. (b) 2 weeks: Displaying improvement in pigmented epithelium layer and PRL with the appearance of vacuolation in OPL and spongy shape in IPL. (c) 4 weeks and (d) 6 weeks: Showed unchanged retinal structure. (Toluidine blue, scale bar: 20 μm). DR: Diabetic retinopathy, LLLT: Low-level laser therapy, OPL: Outer plexiform layer, RPE: Retinal pigmented epithelium, PRL: Photoreceptor layer, IPL: Inner plexiform layer|
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|Table 2: Histological changes in the rat's retina for the control, diabetic retinopathy, diabetic retinopathy + low-level laser therapy, and diabetic retinopathy + low-level laser therapy + Chia seeds groups|
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| Discussion|| |
Photoreceptors cells have high metabolic activity and contain about 75% of the total retinal mitochondria. There are many approaches of using mediators that target photoreceptors to inhibit the progression of DR. These include retinylamine, agonists, antagonists of G-protein-coupled receptors, and retinaldehyde combined with an antioxidant.
In normal conditions, the PRL can renew itself by adding cells and removing the old ones. However, in some retinal degenerative diseases such as DR, there is an imbalance between the adding and removing of photoreceptors cells leading to cellular disruption and photoreceptors degeneration.
In the present study, the histological structure in the DR group revealed marked alterations in the photoreceptors layer. This alteration started slightly by photoreceptors disorganization after 1 week and severely progressed to photoreceptors degeneration, and edema after 6 weeks. Our results agree with Park et al., who elucidated that photoreceptors begin to undergo degeneration after 4 weeks from the induction of experimental diabetes and progressively continue to degenerate for more than 12 weeks.
The dysregulation of glucose metabolism in photoreceptors leads to the expression of hypoxia-inducible factor protein-1α, which is responsible for several cellular activities. These cellular activities include angiogenesis, overexpression of vascular endothelium growth factor, new vessel formation, and metabolic energy. Furthermore, hyperglycemia led to the induction of tumor necrosis factor-α and large amounts of cytokines from photoreceptors. These products will contribute to the damage of retinal microvessels and the killing of retinal endothelial cells. Thus, all these data provide strong evidence that the change in the retinal capillaries that observed after 6 weeks of DR [Figure 1]e may be attributed to the alteration of the retinal photoreceptor cells and therefore, played a significant role in the pathogenesis of retinal vessels.
Recently, light has been used in several studies to reverse vascular lesions resulting from retinopathies. The exposure of the eyelid of patients with nonproliferative DR to 505-nm light showed a reduction in retinal thickness, achromatic contrast sensitivity, and significant improvement in microperimetry thresholds. Analogous research has been explored using far-red light (670 nm) for 3–4 min/day to inhibit the progression of early DR.
An alternative experimental study showed that whole-body exposure to a low-intensity far-red light effectively inhibited diabetes-induced retinal capillaries' degeneration. In addition, this low-intensity light decreases oxidative stress, inflammation, accumulation of albumin in the neural retina, and improves the electroretinogram. Furthermore, this low-level light reversed the edema in diabetic patients having noncenter involved retinal edema. All these aforementioned studies explained the contribution of photoreceptors in the development of retinopathies but do not demonstrate their role in those retinopathies.,
In our study, the improvement in the PRL with LLLT was started after 2 weeks and increased gradually to a noticeable recovery after 6 weeks. This improvement may be attributed to the initiation of the low-level energy to cellular and molecular processes which mediated the repairing effect obtained.
The inner segment layer of retinal photoreceptors is the region that contains most retinal mitochondria. Thus, mitochondrial cytochrome c oxidase (COX) is often considered to be a target of PBM therapy. It has been suggested that 670-nm light photons are absorbed by COX, the rate-limiting enzyme in the terminal phosphorylation of the mitochondrial respiratory chain. The 670-nm light could increase COX activity, elevates adenosine triphosphate synthesis and mitochondrial membrane potential, leading to cytoprotective, and accelerating cellular repair and healing.
We also demonstrated here the potential effects of LLLT and chia seeds on the photoreceptors. The histological profiles revealed an early improvement in PRL occurred after 2 weeks of treatment. This early recovery of PRL may be due to the supplementation of chia seeds as an antioxidant. This data agrees with previous reports that show the inhibition of retinal inflammation and vascular lesions of DR after supplementation of antioxidants.
In DR, the generation of reactive oxygen species (ROS) is increased during the early stages of diabetes causing structural damage to the mitochondria in the photoreceptors. Moreover, long-term hyperglycemia leads to the overproduction of ROS, progressive damage to the mitochondria, and an increase in oxidative stress. The increase in oxidative stress in the diabetic retina suggesting a role in the photoreceptor's degeneration. Thus, chia seeds seemed to partially prevent this oxidative stress and accelerate PRL healing, probably due to their antioxidative activity.
| Conclusion|| |
This study suggested that LLLT is a therapeutic approach and has a beneficial rule to mitigate photoreceptors abnormalities that develop after DR. Even though the combination between LLLT and the antioxidant chia seeds effectively accelerated the improvement of retinal photoreceptors. Hence, this combination has raised the possibility that a simple and inexpensive treatment method could be used to protect the retina against diabetes-induced DR and significantly reduce histopathological abnormalities. Future research accompanied by clinical trials is necessary for DR patients to explore the clinical usefulness of LLLT therapy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]