Evaluation of lead toxicity on the retina of pregnant rats and their pups: the possible ameliorative role of pomegranate juice

Abd El-Fattah B. M. El-Beltagy; Karoline K. Abdelaziz; Amira M. B. Saleh; Hassan I. H. Elsayyad; Reham A. Gahnem

doi:10.12688/f1000research.110028.1

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Research Article

Evaluation of lead toxicity on the retina of pregnant rats and their pups: the possible ameliorative role of pomegranate juice

[version 1; peer review: 1 approved with reservations, 1 not approved]

Abd El-Fattah B. M. El-Beltagy ¹, Karoline K. Abdelaziz¹, Amira M. B. Saleh¹, Hassan I. H. Elsayyad²⁺, Reham A. Gahnem³

Abd El-Fattah B. M. El-Beltagy ¹, Karoline K. Abdelaziz¹, [...] Amira M. B. Saleh¹, Hassan I. H. Elsayyad²⁺, Reham A. Gahnem³

⁺ Deceased author

PUBLISHED 26 Apr 2022

Author details Author details

¹ Zoology, Faculty of Science, Damanhour University, Damanhour, Damanhour, Egypt
² Zoology, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt
³ Oral Biology Department, Faculty of Oral and Dental Medicine, Delta University for Science and Technology, Gamasa, Egypt Abstract:, Mansoura, Egypt

Abd El-Fattah B. M. El-Beltagy
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration

Karoline K. Abdelaziz
Roles: Project Administration, Resources, Software, Validation, Writing – Review & Editing

Amira M. B. Saleh
Roles: Data Curation, Formal Analysis, Investigation, Validation, Visualization

Hassan I. H. Elsayyad
Roles: Data Curation, Resources, Supervision, Visualization, Writing – Review & Editing

Reham A. Gahnem
Roles: Data Curation, Investigation, Visualization, Writing – Original Draft Preparation

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Agriculture, Food and Nutrition gateway.

Abstract

Background: Lead (Pb) exposure even at a low dose can induce functional and structural impairments in both humans and experimental animals. The present study evaluated the potential ameliorative role of pomegranate juice (PJ), as a powerful antioxidant fruit against histopathological and ultrastructural changes caused by Pb in the retina of pregnant rats and their neonates.
Methods: 24 pregnant female rats were selected and randomly divided into four groups (n=6): control, PJ supplemented (100 μL PJ), Pb treated (18.5 mg / kg B.Wt), and Pb co-supplemented alternatively with PJ group. After birth, the mother rats, as well as their neonates at different ages (7, 14, and 21 days old), were dissected and the eyes were removed for histological, ultrastructural, and immunohistochemical investigation of the retina.
Results: The obtained results revealed deleterious histological and ultrastructural lesions in all retinal cell layers of Pb-treated female rats and their offspring. Such lesions included hypertrophied cells of retinal pigmented epithelium (RPE) with pronounced vacuolated mitochondria and fragmented Bruch's membrane. The outer and inner segments of photoreceptors appeared fragmented and detached from the RPE. Additionally, the outer nuclear layer (ONL) and inner nuclear layer (INL) appeared disorganized with vacuolated cytoplasm and pyknotic nuclei. The immunohistochemical results displayed glial fibrillary acidic protein (GFAP) weak expression and P53 strong expression in the retinal sections of the Pb-treated group of female rats and their neonates if compared with control.
Conclusions: PJ successfully alleviated the deleterious histological and ultrastructural as well as immunohistochemical changes induced by lead.

Keywords

Lead, Pomegranate, Retina, Histopathology, TEM

Corresponding author: Abd El-Fattah B. M. El-Beltagy

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2022 El-Beltagy AEFBM et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The author(s) is/are employees of the US Government and therefore domestic copyright protection in USA does not apply to this work. The work may be protected under the copyright laws of other jurisdictions when used in those jurisdictions.

How to cite: El-Beltagy AEFBM, Abdelaziz KK, Saleh AMB et al. Evaluation of lead toxicity on the retina of pregnant rats and their pups: the possible ameliorative role of pomegranate juice [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2022, 11:461 (https://doi.org/10.12688/f1000research.110028.1) First published: 26 Apr 2022, 11:461 (https://doi.org/10.12688/f1000research.110028.1) Latest published: 26 Apr 2022, 11:461 (https://doi.org/10.12688/f1000research.110028.1)

Introduction

Lead (Pb) is a prominent environmental toxic heavy metal that has adverse effects on human health. Pb and its compounds can easily accumulate in air, water, and soil¹. After absorption, Pb is transported to most body organs especially the liver, kidney, brain, and finally accumulates in bone throughout life.²^,³ It had been reported that there is no safe dose of Pb exposure and even very low doses have toxic effects on both humans and animals.¹ Moreover, prolonged exposure to Pb can cause neurological, hematological, cardiovascular, gastrointestinal, immunological, and fertility disorders.⁴ During gestation, the maternal stored Pb can cross the placenta by diffusion resulting in adverse effects on the developing fetuses.⁵ Another study revealed that there is a positive correlation between maternal and umbilical cord blood Pb levels, indicating the transfer of Pb from mother to fetus.⁶ Also, Pb can transfer to neonates during lactation via the breast milk.⁷^,⁸ It had been claimed that neonates are more sensitive than adults to Pb.⁹ The toxic response for Pb depends on several factors, like the dose, the age, the life stage of a woman, duration of exposure, and health and nutritional status of the individual.¹⁰ Accordingly, Pb constitutes a persistent public health problem.¹¹ Previous studies claimed that the primary site of action of Pb is the central nervous system (CNS).¹²^,¹³ Due to the lineal correlation between eyes and the CNS, there is no doubt that the ability of Pb to resist the development of the CNS will also affect eye development in fetuses. Pb exposure leads to reduced sensitivity of photoreceptors, blurred vision and cataracts, and optic neuritis.¹⁴

Certain fruits and vegetables contain high levels of phenolic compounds that typically act as natural antioxidants.¹⁵ Pomegranate (Punica granatum) is a sweet fruit that have an ancient history and is mentioned in many Holy Scriptures such as the Torah, the Bible, and the Holy Quran.¹⁶ The pomegranate fruit has essential polyphenolic compounds in different parts of the fruit that include the peel, seeds, and arils. Other compounds such as organic acids, sugars, minerals and vitamins have been reported in pomegranates by many instigators.¹⁷^,¹⁸ Moreover, the peel part of pomegranate contains bioactive phenolic compounds such as ellagitannins, flavonoids, and proanthocyanidin as well as minerals and complex polysaccharides.¹⁹^,²⁰ The arils part contains 85% H₂O, 10% total sugars, especially fructose and glucose, pectin, organic acids such as malic acid, ascorbic acid, and citric acid as well as phenolic compounds and flavonoids like anthocyanins.¹⁸ The pomegranate seeds are rich with total lipids such as linoleic, stearic acid, punicic acid, palmitic acid and oleic acid.²¹^,²² Also, the seeds are rich with protein, minerals, vitamins, pectin, crude fibers, sugars, isoflavones and polyphenols.²³

Various medicinal uses have been documented for pomegranate extracts.²⁴^,²⁵ The pomegranate is widely used in various medicinal practices such as wound-healing,²⁶ as well as having anti-tumor properties,²³^,²⁷ anti-atherosclerotic capacities,²⁸ antihepatotoxic agents,²⁹ and in the amelioration of cardiovascular diseases.³⁰ Moreover, it has been documented to have anti-diabetic,²⁵^,³¹ anti-inflammatory,³² and antimicrobial³³ activities as well as ameliorative properties for general metabolic health.³⁴^,³⁵ Accordingly, the present study was mainly designed to evaluate the potential ameliorative role of pomegranate juice against Pb-induced retinal cell damage in pregnant rats and their neonates.

Methods

- Ethical considerations: The ARRIVE guidelines were followed. All efforts were made to ameliorate the suffering of animals. This study was approved by the Bioethics Committee of Damanhur University, no. EA 23122, 2020. All experiments inclusive of animal handling and sacrifice were conducted as per the guidelines of the Bioethics Committee of Damanhur University.
- Equipment: Light microscope fitted with digital canon camera, electron microscope, flow cytometry system (FL2-H), blender, microwave rotatory vacuum.
- Chemicals: Lead acetate (C₄H₆O₄Pb₃H₂O) in the form of white crystals, GFAP and P53 antibodies were purchased from Sigma Chemical Company, Cairo, Egypt.

Preparation of pomegranate juice (PJ)

Fresh pomegranates fruits were washed, crushed, squeezed using a blender and then treated with pectinase to obtain PJ and by-products. The PJ was filtered, pasteurized, concentrated using microwave rotatory vacuum and atmospheric heating process respectively, and stored at -20°C. 20 mL of concentrated PJ was diluted in 500 mL of distilled water and kept in refrigerator until use. A total of 2.5 mL diluted PJ includes 100 μL PJ, which is equivalent to 2.8 μmol total polyphenols was supplemented to rats each other day.³⁶^,³⁷

Experimental animals

For this study, 32 Wistar albino rats (24 females and 8 males) were obtained from the Holding Company for Biological Products and Vaccines (VACSERA, Cairo, Egypt). They were 2-3 months old and had an average weight of 180 g. Animals had the same housing conditions of a clean house, regular dark-light cycle, normal ventilation and an adequate, stable, balanced diet with water ad libitum. After an acclimatization period of two weeks, the animals were mated in the special matting cages (1 male: 3 females) overnight. After 3-4 days and ensuring of pregnancy via observation of vaginal plug and using vaginal smear method, pregnant females were separated from males. The pregnant rats were randomly divided into four groups (n=6): group 1, control; group 2, pomegranate juice (2.5ml of diluted PJ, every other day); group 3, lead (oral dose, 18.5 mg/kg B. Wt, each other day);³⁸ and group 4, Pb & PJ (Pb alternatively with PJ). Researchers were aware of the group allocations. Groups 2, 3 and 4 were exposed to the appropriate dose of treatment from the 4^th day of pregnancy till the end of weaning. The offspring (six from each group) were weighed postnatal and examined at the ages of one week, two weeks and three weeks. The mothers were also examined and processed at the end of weaning period. The study took place from August 2020 – August 2021. No rats were excluded from the final analysis.

Experimental procedure

Investigated parameters

Statistical analysis

Data were expressed as mean + standard error, (n=6 per group). Statistical analyses were one-way ANOVA followed by post hoc test, performed in Excel using the data analysis – ANOVA function. Means in the same row with different superscript (*) are significantly different (p>0.05), *significant at p-value>0.05, ** significant at p-value>0.01 and ***significant at p-value>0.001.

Histological and immunohistochemical techniques

Histological sections by hematoxylin and eosin

The animals were euthanized using di-ethyl ether and the eyes of mothers (at the end of weaning) and their neonates (7, 14 and 21 days old, 3 offspring for each age from each group) were removed immediately, washed in normal saline and fixed in 10% neutral buffered formalin.³⁹ After fixation, the eye specimens were dehydrated with ascending grades of ethanol, cleared with xylene, and embedded in paraffin. A 5 μm circular cut sections was made up vertical to the corneal margin and parallel with the optic nerve. The sections of the eye were stained in Mayer’s haematoxylin and eosin and processed for investigation of the retina under bright field light microscope and photographed.

Immunohistochemical labeling of glial fibrillary acidic protein (GFAP) and P53

5 μm thick paraffin sections were cut, mounted onto positively charged slides, de-paraffinized and rehydrated in descending series of alcohol. Endogenous peroxidase activity was inhibited using 3% H₂O₂ in methanol for 40 min at room temp. The retinal sections were retained at 25°C and processed for antigen retrieval by digestion in 0.05 % trypsin. After thorough washing in Tris buffered saline (TBS), pH 7.6, the sections were treated with normal swine serum and divided into two groups for further incubation with the appropriate primary antibody. For demonstration of GFAP activity, the sections were incubated for 30 minutes with polyclonal rabbit anti- GFAP antibody (1:300 dilutions, DAKO Corporation, California, and USA). After three washes in TBS the sections were incubated for 30 minutes with anti-rabbit antibody raised in swine. After thorough washing with TBS the sections were incubated with horseradish peroxidase-rabbit anti-horseradish peroxidase complex (PAP) for 30 minutes and washed for 30 min. Other sections were processed for demonstration of P53 activity by incubation for 45 min with diluted 1:10 monoclonal primary antibody (Anti-p53; clone DO-7 Dako). Slides were then rinsed in PBS and subsequently incubated in the presence of the secondary antibody for 20 min. For all sections, the complex sites were shown brown using 3, 3 diaminobenzidine tetrahydrochloride with fresh hydrogen peroxide substrate. The sections were counterstained with Mayer’s haematoxylin, mounted and photographed by phase-contrast Olympus light microscope fitted with digital canon camera. Incidences of cellular accumulations of GFAP and p53 protein were determined for each group.

Transmission electron microscope investigation (TEM)

The ultrastructural investigation was mainly focused on the retina of the mother rats and their neonates at 21 days old. Immediately after dissection of the selected animals, small pieces about 1 mm² of retina were placed in freshly prepared 2.5% cold glutraldehyde as a fixative for about 5 hours. Samples were then washed in two changes of cold phosphate buffer, PH 7.3 for 1 hour. The specimens were post-fixed for 1-2 hours in buffered 1% Osmium tetraoxide, followed by washing in buffer then dehydrated in ascending series of cold ethyl alcohol, cleared in propylene oxide and mounted in epoxy resin. Semi-thin sections of 0.7μm thickness were cut with glass knives on the 6000 MT RMC ultramicrotome. The sections were mounted on glass slides and stained with 0.25% toluidine blue. For the electron microscope preparations, thin sections were cut from a preselected area and mounted in copper grids and stained with lead citrate and uranyl acetate according to.⁴⁰ The sections were then examined and photographed by a JEOL 1200 EX11 transmission electron microscope in the EM Unit at Mansoura University.

Flow cytometry of cell cycle

Slices from retinas of all studied groups were taken, and cell suspension was prepared with Tris-EDTA buffer (pH7.4) (Sigma-Aldrich Co.). Cell suspension was fixed in ice-cold 96-100% ethanol (Sigma) at 4 °C overnight, centrifuged at 1,500 rpm for 10 min, and then resuspended in PBS containing 50 μg/mL propidium iodide (PI) (Sigma-Aldrich Co.). For each sample, since analysis was based on measurement of 10000 cells. Single cell suspensions were prepared from retinas from at least six rats of each of the age of the animals, and 1.5-3×10⁶ cells were stained for expression of the designated lineage markers. Cells were harvested by centrifugation, washed in ice-cold PBS, and fixed in 80% ethanol that had been prechilled to 20°C. They were then re-pelleted and re-suspended at a concentration of 0.1-0.3 106/ml in PBS containing 18 mg/ml propidium iodide (PI; Sigma) and 8 mg/ml RNase A (Sigma; PI solution). After incubation in the dark for at least 1 h, cell cycle profile analysis was performed on 10,000-20,000 cells on a FAC Sort flow cytometer using the Cell quest analysis program (Becton Dickinson, Sunnyvale, CA, USA).

Results

Changes of body weight

The mean body weights of Pb-exposed mothers were significantly lower (p<0.001) than control. On the other hand, the mean body weights of mother rats of group 4 (Pb and PJ) did not appear to be significantly different (p>0.05) compared with the control group. Also, for all studied neonates maternally induced with Pb (7, 14, and 21 days old), the mean body weights were significantly lower (p<0.001) than their corresponding ages of the control. The mean body weights of 7 and 14 day-old neonates of group 4 appeared to be non-significant (p>0.05) in comparison with those of control group, however a significant (p<0.05) decrease in body weight was still recorded in 21 days neonates (Table 1).

Table 1. Illustrating the mean body weight (g) among the different studied groups of mother rats and their neonates.

Group (n=6)	Control (G1)	PJ (G2)	Pb (G3)	Pb & PJ (G4)
7days	17.09±0.19	16.56±0.26	10.56±0.28^***	16.33±0.27
14 days	27.69±0.34	26.98±0.31	16.74±0.28^***	26.60±0.25
21 days	35.88±0.38	34.71±0.32	25.46±0.32^***	33.78±0.94^*
Mother rats	234±1.68	227±3.81	189±2.87^***	225±4.36

* Significant at p-value≤0.05,

** Significant at p-value≤0.01 and

*** Significant at p-value≤0.001. PJ: pomegranate juice; Pb: lead.

Histopathological observations (Figure 1)

Figure 1. Photomicrograph of histological sections through the retina among the different studied group of mother's rats and their neonates. A-A3: Control; B-B3: PJ; C-C3: Pb; D-D3: Pb+PJ group (H&E).

(The software used to edit the images and labels: Adobe photoshopCS8me).

Abbreviations: Inner limiting membrane (ILM), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), outer limiting membrane (OLM), photoreceptors cell layer (PCL), pigment epithelium (PE) and choroid (Ch).

In control and PJ supplemented groups of mother rats, the retinal sections appeared with normal histological architecture. On the other hand, the retinal layers of Pb-treated group showed obvious deleterious histological changes especially in the pigmented epithelium (RPE), photoreceptors cell layer (PCL) and ganglion cell layer, however mild changes were recorded in outer nuclear layer (ONL) and the outer plexiform layer (OPL). The RPE cells appeared hypertrophied with bulged peripheral nuclei. The PCL appeared detached from the RPE with fragmented and vacuolated outer segments. The nuclei of the ONL were aggregated in clusters and separated from the bases of photoreceptors. The OPL appeared relatively thin and disintegrated if compared with control. Moreover, the cells of GCL appeared hypertrophied, highly vacuolated and lost its normal striation pattern.

In Pb group of mother rats co-supplemented with PJ, the retina showed marked amelioration in its histological architecture. Such ameliorations were represented by restoration of retinal detachment and normal arrangement of photoreceptors without vacuoles. The RPE appeared normal with centrally located nuclei. Also, the ONL appeared homogenous and adjacent to the bases of photoreceptors inner segments. The OPL layer appeared compact and in contact with the ONL. The retinal ganglion cells become highly organized in one row and their axons formed a continuous nerve fiber layer.

In the different ages of neonates (7, 14, 21 days old) maternally induced with Pb, the RPE illustrated pronounced wavy and hypertrophied cells, detached outer segments of PCL. On the other hand, marked histological improvement was recorded in the retina of neonates of group 4 (Pb plus PJ) where the outer nuclear layer and the RPE were tight.

Ultrastructural observations by TEM

Changes in RPE (Figure 2)

Figure 2. Transmission electron micrograph (TEM) through the RPE of the mothers rats (A1-F1) and their neonates (A-F) among the different studied groups. A-B1: Control; C-D1: Pb and E-F1; Pb+PJ group.

(The software used to edit the images and labels: Adobe photoshopCS8me). Abbreviations: BI: basal in folding; BM: Bruch's membrane; CC: choriocapillaris; CP: cytoplasmic processes; DBI: descending basal infolding; DBM: disintegrated Bruch's membrane; GB: Golgi bodies; L: lysosomes; M: mitochondria; N: nucleus; OS: outer segments; Ph: phagosomes; RER: rough endoplasmic reticulum; SER: Smooth endoplasmic reticulum; V: vacuoles; VM: vacuolated mitochondria.

In control and PJ groups of mother rats, the TEM investigation revealed that RPE consists of a single layer of low cuboidal cells which have numerous basal (scleral) infoldings and apical (vitreal) processes enclosing the tips of outer segments of photoreceptors. The cells of RPE appeared rich with smooth endoplasmic reticulum (SER), lysosomes (L), and rough endoplasmic reticulum (RER). In addition, the phagosomes of photoreceptor outer segment are observed.

In Pb-exposed mothers and their neonates, the cells of RPE displayed multiple ultrastructural changes like fragmentized Bruch’s membrane, cytoplasmic vacuolation, vacuolated mitochondria, disintegrated basal infoldings and apical processes as well as loss of RER. Moreover, the nucleus appeared pyknotic with irregular nuclear membranes. On the other hand, the RPE cells of Pb-exposed rats co-supplemented with PJ displayed remarkable improvement in their subcellular architecture. Such improvements were represented by rebuilding of Bruch’s membrane and extension of the cytoplasmic processes in between the photoreceptors’ outer segments. The mitochondria appeared normal with obvious regular cristae. The SER and RER appeared regularly scattered in the cytoplasm especially near the nucleus. Further observation revealed that the nucleus becomes vesicular with their intact membrane.

Changes in Photoreceptors and ONL (Figures 3 and 4)

Figure 3. TEM through the photoreceptors (outer and inner segments) of the mothers rats (A1-F1) and their neonates (A-F) of the different studied groups. A&A1: Control; B&B1: PJ; C-D1: Pb and E-F1; Pb+PJ group.

(The software used to edit the images and labels: Adobe photoshopCS8me). Abbreviations: BM: Bruch's membrane; CP: cytoplasmic processes; DIS: damaged inner segment; FOS: fragmented outer segment; IS: inner segment L: lysosomes; M: mitochondria; MD: membranous disc; N: nucleus; OLM: outer limiting membrane; ONL: outer nuclear layer; OS: outer segments; V: vacuoles.

Figure 4. TEM through the outer nuclear layer (ONL) of the mothers rats (A1-D1) and their neonates (A-D) among the different studied groups. A&A1 control; B&B1: PJ; C&C1: Pb and D&D1: Pb+PJ group.

(The software used to edit the images and labels: Adobe photoshopCS8me). Abbreviations: ONL: outer nuclear layer; PN: pyknotic nuclei; V: vacuoles; VM: vacuolated mitochondria.

Our results by TEM revealed that the photoreceptors of control and PJ supplemented mother rats and their offspring appeared regular, and their outer segments interdigitate with the cytoplasmic processes of the RPE cells. The outer segments (OS) of photoreceptors consist of organized membranous discs, however the inner segments displayed rounded to oval nuclei. The outer limiting membrane appeared intact. In Pb treated mother rats, the OS of photoreceptors appeared fragmented and separated from the IS. Furthermore, the IS of photoreceptors were dislocated from the ONL and became aggregated in clusters with pronounced loss of their mitochondria. The neonates of Pb-exposed rats didn’t show any changes in their photoreceptors.

Co-supplementation of PJ to Pb-exposed rats successfully restored the deleterious ultrastructural changes induced by Pb, whereas the OS of photoreceptors displayed a regular pattern of membranous discs and the IS appeared in direct contact with the ONL. In Pb treated mother rats and their neonates, the ONL appeared with multiple pyknotic nuclei and fragmented cell membranes with wide intercellular spaces. Furthermore, the ONL of mother rats and their neonates of group 4 appeared normal with less intercellular spaces.

Changes in INL of retina (Figure 5)

Figure 5. TEM through the inner nuclear layer (INL) of the mother rats (A1-D1) and their neonates (A-D) among the different studied groups. A&A1 control; B&B1: PJ; C&C1: Pb and D&D1: Pb+PJ group.

(The software used to edit the images and labels: Adobe photoshopCS8me). Abbreviations: AC: amacrine cells; BC: bipolar cells; HC: horizontal cells; IPL: inner plexiform layer; MC: Muller cells; RER: rough endoplasmic reticulum; V: vacuoles.

Generally, among mammals, the inner nuclear layer is made up of four categories of neuronal cells: bipolar cells (BC), horizontal cells (HC), amacrine cells (AC) and Muller cells (MC). The BCs are numerous, rounded or oval in shape and each is prolonged into an inner and an outer process. The HCs lie in the outer part of the INL and possess somewhat flattened cell bodies. Their dendrites divide into numerous branches in the OPL, while their axons run horizontally for some distance and finally ramify in the same layer. The ACs are localized in the inner part of the INL, they have not yet been shown to possess axis-cylinder processes. Their dendrites undergo extensive ramification in the inner plexiform layer. The MCs display dark, elongated nuclei. The Muller cells form an almost continuous layer separating the sclerally located BC and HC from AC somata are mostly positioned at the vitreal side of the inner nuclear layer.

In lead acetate treated mother rats and their neonates, the INL exhibited marked degeneration of horizontal cells, bipolar cells and amacrine cells. Such cellular degeneration was represented by disintegration of most cytoplasmic organelles, nuclear pyknosis and damage of cell membranes. Furthermore, the cells of the INL of Pb-exposed rats post-supplemented with PJ (group 4) appeared normal with less vacuolation still found in the cytoplasm in some cells.

Immunohistochemical observations (Figure 6)

Figure 6. Photomicrograph of paraffin embedded sections through the retina of mothers' rats and their offspring stained with anti-GFAP antibody (A-C1) and with anti-P53 antibody (A2-C3).

(The software used to edit the images and labels: Adobe photoshopCS8me).

The arrows heads indicate the degree of immunoreactivity of GFAP and P53.

Immunohistochemical reaction for GFAP

The immunoreactivity of GFAP appeared strong in the retinal sections of control neonates of rats if compared with their mothers. However, the retinal sections of Pb-exposed mother rats and their neonates displayed weak to moderate expression of GFAP if compared with control. Further observations revealed that the retinal sections of group 4 (Pb plus PJ) expressed a moderate to strong immunoreactivity for GFAP that was approximately similar to that of the control. For all studied groups the immunoreactivity of GFAP appeared prominent in ONL and INL rather than the other layers (Figure 6A-C1).

Immunohistochemical reaction for P53

The retinal sections of control mother rats and their offspring showed negative to weak immune expression for P53 protein. In contrast, an intense reaction for P53 was observed in all retinal cell layers of Pb-exposed mother rats and their neonates. In Pb-treated mother rats post-supplemented with PJ and their offspring, the retinal sections showed pronounced decrease in the degree of P53 immuno-reactivity (Figure 6A2-C3).

Flow cytometric analysis for apoptosis (Figure 7)

Figure 7. A flow cytometric chart of cell cycle analysis illustrating the mean % value of apoptotic cells in the retinal tissues of the different studied groups of mother's rats and their offspring (A&B: Control; A1&B1:PJ; A2&B2: Pb and A3&B3: Pb+PJ).

Note: highly % value of apoptotic cells in Pb-exposed groups of mothers rats and their offspring (83.12%, 50.12%) in comparing with control value (48.59%, 36.53%) respectively. Pomegranate juice successfully restored the mean % values of apoptotic cells near to the normal values.

The obtained flow cytometric results revealed that the mean percentage value of cellular apoptosis in the retina of Pb-treated mothers (83.12% versus 48.59%) and their offspring (54.84% versus 36.53%) was significantly different than that of control. Post-supplementation of PJ to Pb-treated group, the mean % values of apoptotic retinal cells were significantly decreased compared to the control values (50.12% for mother rats and 38.52% for offspring).

Discussion

Lead exposure is known to disrupt most of body processes due to its toxicity to our vital organs, particularly our bones, heart, kidneys and nervous system.¹^,²^,⁴¹ However, there has been little research into its direct effects on eye, a fundamentally cognitive process. Due to the direct correlation between the development of eyes and the central nervous system (CNS), there is no doubt that the potency of lead can prohibit the development of the CNS will inevitably cause effects on the eye. These effects are collectively termed “optic atrophy” or “blurred vision”, appearing only in cases of lead poisoning severe enough to cause brain damage.¹⁴^,⁴² Pomegranate has a protective role as a phytotherapeutic agent due to its active ingredients like tannins,⁴³ phenolic acids⁴⁴ estrogenic flavonoids and conjugated fatty acids.⁴⁵^,⁴⁶ Accordingly, this work was done for the first time to evaluate the possible ameliorative effects of pomegranate juice against lead acetate-induced retinal toxicity in pregnant rats and their neonates. Previous studies revealed that Pb compound can induce toxic effects on the mothers and their pups due to its transfer from the mothers to their pups during gestation and suckling periods.⁸

The result of the present work revealed that the mean body weights of Pb-exposed mothers and their offspring were significantly lowered than the control group. However, post supplementation of PJ to Pb-induced rats the body weight was successfully restored near to that of control. The obtained results are in agreement with previous researchers.⁴⁷^,⁴⁸ Aprioku and Siminialayi⁴⁹ reported that the decreased body weights of embryos maternally induced with Pb compound may be attributed to oxidative stress-mediated deleterious alteration in the ovarian and/or placental functions which interfere with fetal nutrition and O₂ utilization. Additionally, placental disorders may also result in inhibition of transport of essential compounds to the fetuses, and consequently inhibit the normal growth rate of embryos. Another study explained that the toxic ions are one of the factors which decrease the body weight via the mechanism involving disturbance in metabolic enzymes.⁵⁰ Restoration of body weight after supplementation of PJ to Pb exposed group of mother rats and their offspring may be attributed to the antioxidant constituents of PJ which allow easy assimilation of all the nutrients found in the blood stream by the body.⁵¹

Several histological lesions were recorded in the retinal cell layers of Pb-treated mothers’ rats and their offspring. Such alterations were represented by hypertrophied RPE, detached photoreceptors with fragmented outer segments and cytoplasmic vacuolation as well as the nuclei of the ONL and INL were aggregated in irregular clusters. Additionally, the GCL was highly vacuolated and lost its normal striation pattern. Fox and Chu⁵² reported that adult rats’ exposure to low-level Pb results in long-term selective photoreceptor deficits. Another study revealed that lead acetate or its derivatives could induce deleterious histological changes on the embryonic development of RPE and photoreceptors resulting in retinal detachment.⁵³ Raafat et al.³⁸ found that lead toxicity in mother rats could induce folding of pigment epithelium and fragmentation of photoreceptors outer segments. It had been postulated that the fragmentation of photoreceptors segments as well as appearing of cytoplasmic vacuolation under lead neurotoxicity is mainly due to rupture of bi-membranous discs and mitochondria.⁵⁴ Elgohary et al.⁵⁵ elucidated that aggregation of nuclei is an evident sign for pyknosis. Raafat et al.³⁸ proved that animals that received lead acetate have swollen ganglion cell layer. The histopathological signs caused by lead acetate were more pronounced in the mothers and their offspring of 21 days old in comparing with the other two ages. Such variation in the degree of effects may be attributed to long period of exposure to lead acetate.

The results of the TEM study revealed that Pb led to the creation of deleterious ultrastructural changes all-over the retinal cell layers especially in the RPE and photoreceptor cell layers. In lead acetate treated mother rats and their offspring of 21 days old, the RPE cells displayed pyknotic nuclei, swollen and vacuolated mitochondria, vacuolated cytoplasm and remarkable disassembled Bruch’s membrane. Similar observations were recorded in the RPE of rabbit⁵⁶ and rat neonates.⁵⁷ Also similar alterations were noticed in the RPE of monkeys that were exposed to mercury⁵⁴ and in humans exposed to cadmium.⁵⁸^,⁵⁹ Vacuolation of the RPE cells post lead acetate-treatment may represent a type of cellular defense against its toxicities as well as a source of accumulating toxic agents interfering with its biological interactions in cell metabolism.⁶⁰ Schlötzer-Schrehardt et al.⁶¹ reported that retinal detachment is mainly attributed to fragmentation of Bruch’s membrane.

Further observations by the TEM study revealed that the photoreceptors of lead acetate treated mothers and their offspring at 21 days old exhibited severe deleterious changes especially in mothers. Such changes were apparently represented by fragmented outer and inner segments with obvious vacuolation among them. Similar ultrastructural findings were recorded in retina of experimental animals after exposure to heavy metals.⁶²^,⁶³ Furthermore, the previous experimental studies revealed that the low or moderate Pb dose could induce disorganization of photoreceptors in the mothers and children.⁶⁴ Sanders et al.⁶⁵ suggested that degeneration of photoreceptors under the influence of Pb is mainly due to the binding of lead and calcium to the internal metal-binding site of the mitochondrial transition pore, subsequently open the transition pore, and stimulate the cytochrome C-caspase activation cascade leading to death of photoreceptors. Moreover, the ONL and INL of lead acetate treated mothers and their offspring at 21 days old showed severe pyknotic nuclei, cytoplasmic lysis and vacuolation as well as remarkable wide intracellular spaces. Similar ultrastructural alterations were recorded in the two nuclear layers of adult rats⁶⁶ and mice⁶⁷ after exposure to low level of lead during gestation period.

GFAP is an intermediate filament protein that is expressed by numerous cell types of the central nervous system (CNS) including astrocytes⁶⁸ and ependymal cells.⁶⁹ GFAP is involved in many important CNS processes, including cell communication and the functioning of the blood brain barrier.⁷⁰ Also, GFAP has been shown to play a role in mitosis by adjusting the filament network present in the cell.⁷¹ Our results revealed that the retinal sections from Pb treated mothers and their offspring exhibited weak expression for GFAP compared with the control group. The reactivity for such protein was more confined to the nerve cell fibers of ganglion layer, Muller cells and ONL. Similar observations were recorded in the brain of rat offspring⁷² and adult male rats post-treated with lead acetate.⁷³ Chen et al.⁷⁴ reported that depletion of GFAP activity plays a crucial role in activation of caspase-3 which would lead to increased apoptosis.

P53 is a tumor suppressor gene that in an inactivated form tends to be associated with a high risk of certain cancers and inhibition of apoptosis.⁷⁵ Moreover, most evidence suggests that the key contribution of P53 to apoptosis is primarily dependent on transcriptional activity. P53 has the ability to activate transcription of various proapoptotic genes.⁷⁶ Therefore, increased P53 activity can also trigger apoptosis by repression of antiapoptotic genes, such as surviving, thus promoting caspase activation.⁷⁷ In compared with control, strong immunohistochemical reactivity for P53 protein was markedly observed all-over the retinal cell layers of Pb treated mothers and their offspring. According to the previous discussion, the decreased GFAP activity and overexpression of P53 reactivity in the retina of lead acetate groups confirmed an elevated risk of cellular apoptosis and that lead is the strongest neurodegenerative agent. Further examination to confirm the apoptosis was carried out by flow cytometric analysis to detect DNA damage. The results of the present work showed a significant increase in the percentage of cellular apoptosis in Pb treated groups in relative to control and pomegranate groups. Jia et al.⁷⁸ revealed that lead acetate could induce a progressive loss in human mesangial cells viability together with a significant increase in the number of apoptotic cells using the assay of flow cytometry. Another study explained that Pb could induce oxidative stress, DNA damage and alteration of P53, Bax and Bcl-2 expressions in mice.⁷⁹ The main mechanism of DNA damage caused by heavy metal ions, which contributed to genotoxicity, is the excess liberation of free radicals. These free radicals attack DNA double chains and break them. If the broken DNA strands cannot be repaired timely, it will affect the function of DNA and result in genotoxicity.⁸⁰

In the present work, the pomegranate juice exerts ameliorative effects against the retinal histopathology as well as the immunohistochemical alterations induced by lead acetate. The pomegranate has been regarded as a “healing food” that plays a role in regulation of metabolism.⁸⁰ It was commonly used as an anti-inflammatory, and in the treatment of ulcers, diarrhea, hemorrhages, microbial infections and respiratory diseases.³²^,⁸¹^,⁸² Improvement of pomegranate juice against lead acetate treated retina may explain the reduction of inflammation, re-differentiation of retina and regulation of apoptosis as well as maintain normal pattern of GFAP and P53. All of these beneficial effects could be attributed to the polyphenols inclusion of pomegranate which has antioxidant capacity.

Detailed studies are required to understand the mechanism by which pomegranate juice can exert beneficial effects on the retina.

Conclusion

On the basis of our findings, the oral administration of PJ to lead acetate-treated rats during gestation and lactation could ameliorate the damage to retinal layers and restore their structure as well as enhance the antioxidative defense system due to presence of bioactive polyphenolic compounds which play a role in scavenging free radicals, and also prevent DNA damage.

Data availability

Underlying data

figshare: Raw data for body weight. https://doi.org/10.6084/m9.figshare.19276061.v2⁸³

This project contains the raw data on rats’ body weighs.

figshare: Data of Flow cytometry. https://doi.org/10.6084/m9.figshare.19294352.v2⁸⁴

This project contains the flow cytometry data.

figshare: Underlying data.pdf. https://doi.org/10.6084/m9.figshare.19237473.v4⁸⁵

This project contains the images used in the manuscript.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

The underlying image data for this study are too large to share openly. The image files are predominately JPG files, and approximately 200 MB (210,739,647 bytes) in size. Considering the large size and multiple images (n=280), the image files will be shared on request to readers. Please contact the corresponding author (beltagyaaa@yahoo.com) if you would like to request access to the image files. Representative images are shown in the figures and can be found in the Figshare repository.

Reporting guidelines

figshare: ARRIVE checklist for ‘Evaluation of lead toxicity on the retina of pregnant rats and their pups: the possible ameliorative role of pomegranate juice’. https://doi.org/10.6084/m9.figshare.19237293.v3

Acknowledgments

The authors would like to thank the Light and Electron microscope unit of Mansoura University, for providing facilities to bring to this work.

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¹ Zoology, Faculty of Science, Damanhour University, Damanhour, Damanhour, Egypt
² Zoology, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt
³ Oral Biology Department, Faculty of Oral and Dental Medicine, Delta University for Science and Technology, Gamasa, Egypt Abstract:, Mansoura, Egypt

Abd El-Fattah B. M. El-Beltagy
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration

Karoline K. Abdelaziz
Roles: Project Administration, Resources, Software, Validation, Writing – Review & Editing

Amira M. B. Saleh
Roles: Data Curation, Formal Analysis, Investigation, Validation, Visualization

Hassan I. H. Elsayyad
Roles: Data Curation, Resources, Supervision, Visualization, Writing – Review & Editing

Reham A. Gahnem
Roles: Data Curation, Investigation, Visualization, Writing – Original Draft Preparation

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No competing interests were disclosed.

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El-Beltagy AEFBM, Abdelaziz KK, Saleh AMB et al. Evaluation of lead toxicity on the retina of pregnant rats and their pups: the possible ameliorative role of pomegranate juice [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2022, 11:461 (https://doi.org/10.12688/f1000research.110028.1)

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Reviewer Report 05 Sep 2022

Hidehiro Oku, Osaka Medical College, Takatsuki, Japan

Approved with Reservations

https://doi.org/10.5256/f1000research.121598.r148791

This reviewer has some concerns about histological findings using light microscope.

1. Figure 1. It seems very hard to segment each retinal layer in rats 7 days after birth. In addition, some vacuolar changes exist in the ... Continue reading

This reviewer has some concerns about histological findings using light microscope.

1. Figure 1. It seems very hard to segment each retinal layer in rats 7 days after birth. In addition, some vacuolar changes exist in the INL even in the control rats of 14 and 21 days after birth.

2. Muellar cells are primary glial cells in the retina which run transversely throughout whole retinal layers. Please correct the following description: “the inner nuclear layer is made up of four categories of neuronal cells: bipolar cells (BC), horizontal cells (HC), amacrine cells (AC) and Muller cells (MC)”.

3. In addition, please discuss less immunoreactivities to GFAP in rats treated with Pb exposure. Retinal injuries almost always accompany activation of Muellar cells and increase immunoreactivities to GFAP.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Neuroprotection.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 30 Aug 2022

Matt Rutar, The University of Canberra, Canberra, Australia

Not Approved

https://doi.org/10.5256/f1000research.121598.r146540

The study by El-beltagy and colleagues sought to determine a retinal neuroprotective effect of pomegranate juice in rats exposed to lead toxicity. The authors investigated this by treating lead-affected mice with pomegranate juice and then performed comparative histological analysis on ... Continue reading

The study by El-beltagy and colleagues sought to determine a retinal neuroprotective effect of pomegranate juice in rats exposed to lead toxicity. The authors investigated this by treating lead-affected mice with pomegranate juice and then performed comparative histological analysis on retinal tissue sections, as well as rudimentary cytometry and immunostaining. Though the overall premise of the study is reasonable, I have major concerns with methodology and analysis of the data throughout the manuscript. In general, there is a severe lack of (1) adequate quantification and presentation of the data, (2) appropriate description in the methods, and (3) mechanistic insight. The most glaring of these are detailed below:

The figures documenting histological changes in retinal structure are entirely qualitative, making the authors assertion of a protective effect of PJ questionable. Moreover, the representative retinal tissue sections presented are not clear and are of variable quality (Figure 1 in particular). At a minimum, the cell layer thickness, hypertrophy, or other parameters must be measured and quantified with respect to each group, and with appropriate statistical analysis included. Ideally, ERG recordings should also be included to determine whether PJ confers a protective effort with respect to vision function.
The GFAP and P53 staining figures are inadequate to draw a meaningful conclusion (Figure 6). The staining presented is difficult to resolve clearly, as is any change with regard to PJ treatment, and there is also no quantification of the data. GFAP staining should be apparent within GCL astrocytes and muller cell processes, though the data presented appears to show staining throughout the retina (particularly in Figure 6 C). The authors should also include in any negative or positive controls in their analysis (either as part of the figure or a supplementary).
The cytometry data are confusing (Figure 7). It is not abundantly clear (1) what they are actually analysing, (2) what the overall data show, and (2) how this was undertaken (methods for this are threadbare at best). I assume that it would be apoptotic cells, though it is not described how the data presented in Figure 7 relate to this. The figure appears to show merely the raw data taken from the cytometer, with no indication of what retinal cell type they assessing, and no apparent statistical correlation between groups. The figure also does not mesh with the text description – the data show ‘counts’, though the text refers to ‘percentages’.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: I am a retinal cell biologist with particular interest in retinal degenerations and neuroimmunology. Techniques include cellular (histology, cytometry) and molecular (RNA profiling) methods for interrogating changes in retinal pathophysiology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Matt Rutar, The University of Canberra, Canberra, Australia
Hidehiro Oku, Osaka Medical College, Takatsuki, Japan

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Reviewer Report

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05 Sep 2022 | for Version 1

Hidehiro Oku, Osaka Medical College, Takatsuki, Japan

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Approved With Reservations

This reviewer has some concerns about histological findings using light microscope.

1. Figure 1. It seems very hard to segment each retinal layer in rats 7 days after birth. In addition, some vacuolar changes exist in the INL even in the control rats of 14 and 21 days after birth.

2. Muellar cells are primary glial cells in the retina which run transversely throughout whole retinal layers. Please correct the following description: “the inner nuclear layer is made up of four categories of neuronal cells: bipolar cells (BC), horizontal cells (HC), amacrine cells (AC) and Muller cells (MC)”.

3. In addition, please discuss less immunoreactivities to GFAP in rats treated with Pb exposure. Retinal injuries almost always accompany activation of Muellar cells and increase immunoreactivities to GFAP.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neuroprotection.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report

13 Views

30 Aug 2022 | for Version 1

Matt Rutar, The University of Canberra, Canberra, Australia

13 Views Cite this report Responses(0)

Not Approved

The study by El-beltagy and colleagues sought to determine a retinal neuroprotective effect of pomegranate juice in rats exposed to lead toxicity. The authors investigated this by treating lead-affected mice with pomegranate juice and then performed comparative histological analysis on retinal tissue sections, as well as rudimentary cytometry and immunostaining. Though the overall premise of the study is reasonable, I have major concerns with methodology and analysis of the data throughout the manuscript. In general, there is a severe lack of (1) adequate quantification and presentation of the data, (2) appropriate description in the methods, and (3) mechanistic insight. The most glaring of these are detailed below:

The figures documenting histological changes in retinal structure are entirely qualitative, making the authors assertion of a protective effect of PJ questionable. Moreover, the representative retinal tissue sections presented are not clear and are of variable quality (Figure 1 in particular). At a minimum, the cell layer thickness, hypertrophy, or other parameters must be measured and quantified with respect to each group, and with appropriate statistical analysis included. Ideally, ERG recordings should also be included to determine whether PJ confers a protective effort with respect to vision function.
The GFAP and P53 staining figures are inadequate to draw a meaningful conclusion (Figure 6). The staining presented is difficult to resolve clearly, as is any change with regard to PJ treatment, and there is also no quantification of the data. GFAP staining should be apparent within GCL astrocytes and muller cell processes, though the data presented appears to show staining throughout the retina (particularly in Figure 6 C). The authors should also include in any negative or positive controls in their analysis (either as part of the figure or a supplementary).
The cytometry data are confusing (Figure 7). It is not abundantly clear (1) what they are actually analysing, (2) what the overall data show, and (2) how this was undertaken (methods for this are threadbare at best). I assume that it would be apoptotic cells, though it is not described how the data presented in Figure 7 relate to this. The figure appears to show merely the raw data taken from the cytometer, with no indication of what retinal cell type they assessing, and no apparent statistical correlation between groups. The figure also does not mesh with the text description – the data show ‘counts’, though the text refers to ‘percentages’.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

I am a retinal cell biologist with particular interest in retinal degenerations and neuroimmunology. Techniques include cellular (histology, cytometry) and molecular (RNA profiling) methods for interrogating changes in retinal pathophysiology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

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Evaluation of lead toxicity on the retina of pregnant rats and their pups: the possible ameliorative role of pomegranate juice

Abstract

Keywords

Introduction

Methods

Preparation of pomegranate juice (PJ)

Experimental animals

Statistical analysis

Histological and immunohistochemical techniques

Transmission electron microscope investigation (TEM)

Flow cytometry of cell cycle

Results

Changes of body weight

Table 1. Illustrating the mean body weight (g) among the different studied groups of mother rats and their neonates.

Histopathological observations (Figure 1)

Figure 1. Photomicrograph of histological sections through the retina among the different studied group of mother's rats and their neonates. A-A3: Control; B-B3: PJ; C-C3: Pb; D-D3: Pb+PJ group (H&E).

Ultrastructural observations by TEM

Figure 2. Transmission electron micrograph (TEM) through the RPE of the mothers rats (A1-F1) and their neonates (A-F) among the different studied groups. A-B1: Control; C-D1: Pb and E-F1; Pb+PJ group.

Figure 3. TEM through the photoreceptors (outer and inner segments) of the mothers rats (A1-F1) and their neonates (A-F) of the different studied groups. A&A1: Control; B&B1: PJ; C-D1: Pb and E-F1; Pb+PJ group.

Figure 4. TEM through the outer nuclear layer (ONL) of the mothers rats (A1-D1) and their neonates (A-D) among the different studied groups. A&A1 control; B&B1: PJ; C&C1: Pb and D&D1: Pb+PJ group.

Figure 5. TEM through the inner nuclear layer (INL) of the mother rats (A1-D1) and their neonates (A-D) among the different studied groups. A&A1 control; B&B1: PJ; C&C1: Pb and D&D1: Pb+PJ group.

Immunohistochemical observations (Figure 6)

Figure 6. Photomicrograph of paraffin embedded sections through the retina of mothers' rats and their offspring stained with anti-GFAP antibody (A-C1) and with anti-P53 antibody (A2-C3).

Flow cytometric analysis for apoptosis (Figure 7)

Figure 7. A flow cytometric chart of cell cycle analysis illustrating the mean % value of apoptotic cells in the retinal tissues of the different studied groups of mother's rats and their offspring (A&B: Control; A1&B1:PJ; A2&B2: Pb and A3&B3: Pb+PJ).

Discussion

Conclusion

Data availability

Underlying data

Reporting guidelines

Acknowledgments

References

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