Acute radiation-induced changes in the submandibular salivary gland and possible radioprotective role of silver nanoparticles: a histological, immunohistochemical, and ultrastructural study

Abstract

Gamma radiation is widely used in the treatment of head and neck malignancies, it can cause irreversible damage to the submandibular salivary glands, leading to xerostomia and oral infections. This study evaluated the acute radiation-induced changes in the submandibular gland and the prophylactic and therapeutic effects of silver nanoparticles (AgNPs) in adult male albino rats. Forty-five adult male albino rats (150–200 g) were divided into five groups. Twenty rats served as normal and sham controls, while 25 rats received a single 15 Gy dose of gamma radiation and were assigned to irradiated (n=10), prophylactic AgNPs (n=10), and therapeutic AgNPs (n=5) groups. Right submandibular glands were processed for histological and immunohistochemical analyses, and left glands for ultrastructural examination. Irradiated glands showed acinar atrophy, cytoplasmic vacuolation, pyknotic nuclei, ductal dilatation, reduced PAS optical density, and significantly increased CD3 and BAX immunoreactivity compared with controls. Ultrastructural examination revealed clumped chromatin, swollen mitochondria, and disrupted rough endoplasmic reticulum. Prophylactic AgNP administration largely preserved glandular architecture, maintained acinar area and duct diameter, reduced CD3 and BAX expression, and restored normal ultrastructural features. In contrast, therapeutic AgNP administration produced only minimal improvement, with persistent structural abnormalities. Prophylactic AgNP administration effectively attenuated acute gamma radiation-induced submandibular gland injury, whereas post-irradiation treatment provided only limited therapeutic benefit.

Introduction

Radiation is a form of energy that propagates to the surrounding medium in the form of electromagnetic waves [1]. Radiotherapy is used to inhibit and control growth, metastasis and proliferation of malignant tumor cells using various types of ionizing radiation [2]. It is delivered either by external beam emission with X-rays, γ rays, or photonsor internally by implantation of radioactive sources into or near the tumor [3]. The effects of ionizing radiation are directly related not only to the type of the tissue receiving the radiation, but also to the absorbed radiation dose [4].

Ionizing radiations are capable of damaging DNA and regulatory proteins. By splitting chemical bonds on the helical backbone, they can create single and double-strand breaks with any amount of exposure [5]. Indirect DNA affection occurs when the high-energy particles of ionizing radiation penetrate the body and liberate electrons from atoms and molecules. Reactive oxygen species (ROS) have been proposed to account for the majority of radiation-induced cellular genetic damage [6, 7].

Most patients with head and neck tumors are treated with radiotherapy alone or in combination with chemotherapy and/or surgery. This often results in co-irradiation of salivary glands. Radiation-induced side effects of salivary glands are either acute or chronic. Acute effects occur during radiotherapy while chronic complications arise at the completion of it. Radiation-induced damage to salivary glands is seen soon after the initiation of treatment and at relatively low doses. The sensitivity of salivary glands in humans has been recognized since the beginning of the era of radiotherapy for head and neck tumors [8].

Patients receiving radiotherapy over head and neck will experience moderate or severe xerostomia, secondary to hypo-salivation, alterations in speech and taste, difficulties with mastication and deglutition, oral infections and dental caries occur. These sequelae severely hamper the quality of life of affected patients [5, 9]. The severity of glandular damage is dependent on the irradiated gland volume, the cumulative radiation dose and ability of surviving stem cells to re-populate [8].

Silver nanoparticles (AgNPs) can be radioprotective and antioxidant at low doses [10]. AgNps have antibacterial, antifungal, antiviral, anti-inflammatory and anti-oxidant activities [10]. AgNps can attenuate inflammation by modulating pro-inflammatory signaling pathways and cytokine production. They inhibit nuclear factor kappa protein (NF-kB) activation which is a key transcription factor in inflammatory responses. Previous studies reported that nanosilver decreases the expression of COX-2 protein, leading to decreased prostaglandins production. In addition, they play a role in macrophage polarization [11]. Nanosilver induces oxidative stress in cancer cells causing mitochondrial disruption. They can enhance the radiotherapy anti-cancer effect by increasing radio-sensitivity of the tumor cells [12].

AgNPs draw attention as a potent cellular radio-protector. In 2012, Chandrasekharan and Nair [13], elicited the radioprotective properties of AgNPs in rodents exposed to gamma radiation. Previous workers suggested that AgNps protect DNA integrity from radiation-induced damage, they prevent strand breaks and interact with proteins involved in cellular repair [14]. Radiation often causes apoptosis through the activation of p53 and caspase pathways. AgNPs can inhibit apoptosis by modulating these pathways thereby increasing cell survival after radiation exposure. AgNPs have been shown to reduce the incidence of apoptosis and promote cellular proliferation after exposure to radiation [15, 16]. Silver nanomaterial have been proved to be radioprotective on rat bone marrow, lungs, skin, salivary glands and several in vitro cell lines [17, 18].

Materials and Methods

Chemicals

AgNps: were purchased in the form of colloidal liquid-suspension (50 ppm), average particle size 50 nm for particle, the nanoparticles were mostly spherical in shape, with a relatively narrow size distribution. The surface charge of the AgNps was characterized by a negative zeta potential, and were stabilized by a biocompatible coating layer of citrate. They were purchased from Nanotech Company for Photo-electronics, Cairo, Egypt.

Procedure

Induction of radiation

The process of irradiation was conducted in the National Center of Radiation Research and Technology, Nasr City, Cairo, Egypt. Rats were transported to and from the radiation center in suitable cages (40×40 cm). The Irradiated and stress control groups received 2.5 mg/kg bw of midazolam as intraperitoneal injection prior to irradiation to induce sedation and prevent their movement during the radiation beam emission period. Irradiation of the head and neck region was carried on after placement of a lead cone to shield the path of radiation. Regarding the irradiated groups only, rats were irradiated with ionizing gamma radiation emitted from a cobalt 60 gamma cell unit, positioned at a 20 cm distance from the skin surface, over a time period of 108 sec [19].

Animals

Animal selection

The study was conducted on 45 adult male albino rats weighing 150–200 g. The animals were obtained from the Animal House of Kasr Al-ainy, Faculty of Medicine, Cairo University. They were acclimatized for a period of two weeks before proceeding into the experiment. The rats were housed in metal cages, 5 rats/cage under standard laboratory and environmental conditions. The animals were given standard rodent food pellets and water ad libitum. The approval of the Institutional Animal Care and Use Committee, Cairo University was obtained before proceeding into the study, approval number is CU/III/F/25/26.

Animal grouping

The animals were divided into five groups. The day of induction of radiation was considered as day zero of the experiment.

- Group I (normal control): 10 animals received no manipulations, then were subdivided into two equal subgroups:

Group Ia: was sacrificed on day one.

Group Ib: was sacrificed on day seven.

- Group II (stress control): 10 animals were subjected to the steps of irradiation procedure apart from exposure to radiation. The group was divided into two equal subgroups:

Group IIa: were sacrificed on day one.

Group IIb: were sacrificed on day seven.

- Group III (irradiated): 10 animals was irradiated with a single dose of 15 Gy of gamma rays over the head and neck region [19]. The group were divided into two equal subgroups:

Group IIIa (acute effect): were sacrificed on day one.

Group IIIb (recovery): were sacrificed on day seven.

- Group IV (prophylactic AgNPs–irradiated): 10 animals received a single dose of 150 µg/kg bw of AgNps through intra-peritoneal injection one day before irradiation [20]. The rats were divided into two equal subgroups:

Group IVa: were sacrificed on day one.

Group IVb: were sacrificed on day seven.

- Group V (irradiated-therapeutic AgNPs): 5 rats, received a single dose of 150 µg/kg bw of AgNps through intraperitoneal injection one day after irradiation then were sacrificed on day seven [20].

Scarification

Each animal was sacrificed by intra peritoneal injection of phenobarbitone sodium (100 mg/kg bw) [21]. Cervical dislocation was avoided to protect the submandibular glands from any damage. The right glands were fixed in 10% formaldehyde solution for histological and immunohistochemical studies. The left glands were preserved in 2.5% glutaraldehyde solution for ultrastructural study.

Histological study

The right submandibular gland specimens were subjected to:

- H&E-stain: for standard histological examination of the submandibular gland.

- Periodic acid sciff (PAS) stain: to visualize the glycogen content within the cellular granules.

Immunohistochemical (IHC) study

The following immune markers were assessed using specific immunoassay kits for detection of antigen antibody reaction:

- Cluster of differentiation 3 (CD3): to assess inflammation and lymphocytic infiltration.

- Bcl-2 associated protein X (BAX): as a marker for cellular apoptosis.

Histomorphometric study

Histo-morphometric parameters were examined within the standard measuring frame of a known area equal to 11,694.91 µm square, using Image J analyser software and data were expressed as mean values. For each specimen, three non-overlapping fields were randomly selected, field selection was carried out using a systematic randomization to avoid selection bias. To ensure objectivity, all image acquisition and analysis were conducted in a blinded manner; where the examiner was unaware of the experimental group allocation.

The following parameters were measured:

1. Acinar area (µm³): was measured at a magnification ×100.

2. Striated duct diameter (µm): was measured at a magnification ×200.

3. Optical density of PAS staining in PAS stained section: at a magnification ×100.

4. CD3 positive cell count: at a magnification ×100.

5. Mean area % of BAX protein expression: at a magnification ×100.

Electron microscopic study

The left submandibular glands were fixed with modified Karnovsky solution containing 2.5% glutaraldehyde and 2% formaline in 0.1 M sodium phosphate buffer at pH 7.4. Tissue was rinsed in the buffer solution for 15 min at 4°C. Post fixation was performed in 1% osmium tetroxide in the same buffer for 2 h, then dehydrated in graded series of alcohol and embedded in Spurr resin. Semi thin sections were taken firstly, stained with toludine blue and examined by light microscopy, to ensure the accuracy of the tissue section in the resin capsule. Then, ultrathin sections (90 nm) thickness sections were obtained using gold knife in a Reichert Ultra Cut microtome. The Ultra-thin sections were mounted on copper grids of 200 meshes. The grids were stained by 4% uranyl acetate and 0.4% lead citrate solutions and examined in a ThermoFisher Talos 12 transmission electron microscope adjusted to 80 kV.

Statistical analysis

Statistical analysis was performed using a statistical package for the social sciences (SPSS) version 27 (IBM Co.). Quantitative data were presented by mean±SD and inter quartile range. Tests of significance was done (using two way ANOVA test). Significance was considered when the P-value is ≤0.05 throughout the study [22].

Results

Light microscopic study

H&E-stained sections

Normal and Stress Control Groups: submandibular gland sections obtained from groups Ia and Ib revealed normal histological architecture of the gland. Each lobule was made of serous acini and intralobular ducts (intercalated, striated and granular convoluted ducts). The acini were mostly spherical, closely packed with narrow interacinar space. The acini were lined by pyramidal cells with eosinophilic cytoplasm and basophilic rounded nuclei. The granulated convoluted tubules were scattered within the lobules. The striated ducts were more abundant and easily identified. They exhibited regular rounded lumen and were lined by simple columnar epithelium. Blood vessels lined by flat endothelial cells were seen around the striated ducts (Figs. 1A, 2A). H&E-stained sections obtained from stress control groups (IIa and IIb) elicited similar histological findings to normal control group. Most specimens showed the normal glandular structure; including intact serous acini lined by active pyramidal cells with intralobular ducts in between them (Fig. 1B).

Fig. 1.

(A) A photomicrograph of normal control group sacrificed on day 1 showing intact excretory ducts (ED) with regular lumen and columnar epithelial cell lining, surrounded by spherical eosinophilic serous acini (SA) closely packed together with minimal interacinar spaces and intervening blood vessels (BV). (B) A photomicrograph of stress control group (day 1) illustrating abundant eosinophilic SA, scattered striated ducts (SD) with an ED (arrow). (C) A photomicrograph of irradiated group (day 1) eliciting wide inter acinar spaces (stars), SA are irregular in shape (arrows), granular convoluted ducts appear disrupted (arrowheads). (D) A photomicrograph of AgNps protected group (day 1): showing closely packed SA, intact granular convoluted tubule (GCT) and SD. Interacinar spaces are minimal (stars) (H&E, ×200).

Fig. 2.

(A) A photomicrograph of control group sacrificed on day 1 showing spherical shaped serous acini (SA) lined by pyramidal cells with vesicular nuclei, with an intervening intercalated duct (ID). (B) A photomicrograph of irradiated group (day 1) showing cytoplasmic vacuoles (arrows) and karryolysis (arrowheads) in the cells lining SA. (C) A photomicrograph of AgNps- protected group (day 1) showing spherical shaped SA with less cytoplasmic vacuoles, some cells show dark pyknotic nuclei (red arrows), striated duct (SD) appears lined by low columnar epithelial cells with vesicular nuclei (H&E, ×400).

Irradiated group III: (1) Subgroup IIIa: H&E sections of subgroup IIIa elicited disrupted lobular architecture. The serous acini showed shrinkage, losing their spherical shape and arrangement, with increased inter acinar spaces in between them. The acinar cells encountered generalized cytoplasmic vacuolation and pyknotic nuclei were seen at higher magnification. The intralobular ducts; granulated tubules and straited ducts were dilated and the cells lining them exhibited apoptotic changes; in the form of cytoplasmic vacuoles dark pyknotic nuclei. The intercalated ducts were difficult to be recognized in most sections even at high magnifications. The excretory ducts were dilated with shedding of their lining into their lumen. Blood vessels were congested and areas of cellular infiltrations were encountered (Figs. 1C, 2B). (2) Subgroup IIIb: H&E-stained sections obtained from subgroup IIIb showed widely separated and irregularly arranged serous acini. Some of them exhibited cytoplasmic vacuoles and darkly stained pyknotic nuclei. The granulated convoluted tubules and striated ducts showed dilated lumens, irregular outlines with disrupted epithelial lining. The excretory ducts had irregular outlines and dilated lumen with compressed lining epithelium. Congested blood vessels were encountered within the connective tissue septa (Figs. 3C, 4A).

Fig. 3.

(A) A photomicrograph of normal control group sacrificed on day 7 showing intact striated ducts (SD) with regular lumen, spherical eosinophilic serous acini (SA) closely packed together with minimal interacinar spaces and intervening blood vessels (BV). (B) A photomicrograph of stress control group (day 7) illustrating eosinophilic serous acini (SA) and GCT (arrow) with some intercalated ducts (arrowheads). (C) A photomicrograph of irradiated group (day 7) eliciting widely spaced gland acini (square), that are irregular in shape (arrow), acini cells show vacuolated cytoplasm (arrowheads). (D) A photomicrograph of AgNps protected group (day 7): showing irregular serous acini (SA), granular convoluted tubule (GCT) are lined with low columnar epithelial cell lining (thick arrow), SD (thin arrows) and moderate inter-acinar spaces with congested BV. (E) A photomicrograph from AgNps treated group (day 7) showing disrupted granular convoluted tubules (thick arrows) (H&E, ×200).

Fig. 4.

(A) A photomicrograph from irradiated group (day 7) showing irregular shaped serous acini with vacuolated cytoplasm (red arrows) and pyknotic nuclei (arrowheads). (B) A photomicrograph from AgNps protected group eliciting acini pyramidal shaped cells with vesicular rounded nuclei (red arrows) with considerable interacinar spaces in the section (stars). (C) A photomicrograph from AgNps-treated group illustrating acini widely spaced from each other (stars) with vacuolated cytoplasm (red arrows) (H&E, ×400).

Group IV: (1) Subgroup IVa: Glandular sections from subgroup IVa encountered integrated serous acini structure. They appeared spherical in shape, close to each other and with minimal cellular vacuolization. Striated ducts elicited regular rounded lumen and intact cell lining. Areas of hemorrhage and ductal destructions with retained secretions were recognized in some sections (Figs. 1D, 2C). (2) Subgroup IVb: Specimens obtained from subgroup IVb exhibited rounded regularly arranged, closely packed serous acini with eosinophilic pyramidal cell lining. Inter acinar spaces were minimal with connective tissue septa in between the gland lobules, encompassing wide lumen excretory ducts and congested blood vessels (Figs. 3D, 4B).

Group V: H&E-stained sections of the submandibular gland in this group showed partial regained structure of the gland; serous acini were small in size with considerable spaces in between them. Intralobular ducts were disrupted and widened in many sections with areas of inflammatory cellular infiltration (Figs. 3E, 4C).

PAS staining

Groups I and II: Submandibular gland sections stained with PAS stain in subgroups Ia and Ib revealed strong positive staining of the acinar and duct cell membranes. The acinar cells exhibited focal stained cytoplasmic areas with magenta red color (Figs. 5A, 6A). Similarly sections obtained from subgroups IIa and IIb elicited similar findings to normal control group, where acinar and ductal cell membranes showed strong positive staining (Figs. 5B, 6B).

Fig. 5.

(A) A photomicrograph of normal control group (day 1) eliciting strong positive periodic acid sciff (PAS) reaction in submandibular gland parenchymal cells (arrow). (B) A photomicrograph of stress control group (day 1) showing strong positive PAS staining in parenchymal cells cytoplasm (arrow). (C) A photomicrograph of irradiated group (day 1) illustrating less PAS reactivity in serous acini and duct lining cellular cytoplasm (arrow). (D) A photomicrograph of AgNps – protected group (day 1) indicating moderate PAS staining in gland parenchymal cells (PAS, ×200).

Fig. 6.

(A) A photomicrograph of normal control group (day 7) eliciting strong positive periodic acid sciff (PAS) reaction in submandibular gland parenchymal cells (arrow). (B) A photomicrograph of stress control group (day 7) showing moderate positive PAS staining in parenchymal cells cytoplasm (arrow). (C) A photomicrograph of irradiated group (day 7) illustrating less PAS reactivity in gland parenchyma and duct lining cellular cytoplasm (arrow). (D) A photomicrograph of AgNps–protected group (day 7) indicating moderate PAS staining in gland parenchymal cells. (E) A photomicrograph representing group V (AgNps-treated group) showing weak PAS reaction (PAS, ×200).

Group III: PAS stained sections of group IIIa exhibited a weak reaction to the Schiff reagent indicating depletion of glycoprotein content of both the acini and the duct system. Also the color density of the endothelial cells basement membrane was apparently decreased. Group IIIb also elicited weak PAS staining among acinar and duct cells in most examined sections (Figs. 5C, 6C).

Group IV: PAS stained sections of group IVa (Fig. 5D) and IVb (Fig. 6D) encountered indifferent findings; most sections showed moderate positive reaction to the Schiff reagent in the serous acini and ducts.

Group V: PAS stained sections of this group revealed moderate positive reaction to the Schiff reagent (Fig. 6E).

Immunohistochemical study

CD-3 immune-staining

Groups I and II: Light microscopic interpretation of CD-3 stained sections obtained from subgroups (Ia, Ib) elicited weak positive immune reaction in few stromal cells around the gland acini and ducts (Figs. 7A, 8A). Submandibular gland sections from subgroups (IIa and IIb) revealed weak reaction to CD-3 antibodies in few scattered stromal cells (Figs. 7B, 8B).

Fig. 7.

(A) A photomicrograph of normal control group (day 1) eliciting negative reaction to CD3 antibodies in submandibular gland parenchymal cells (arrow). (B) A photomicrograph of stress control group (day 1) showing negative CD3 antigen-antibody reaction in parenchymal cells cytoplasm (arrow). (C) A photomicrograph of irradiated group (day 1) illustrating strong cytoplasmic reactivity in serous acini (arrows). (D) A photomicrograph of AgNps – protected group (day 1) indicating positive brown coloration in acini-lining cells cytoplasm (arrow) (CD-3, ×200).

Fig. 8.

(A) A photomicrograph of normal control group (day 7) eliciting weak reaction to CD3 antibodies in submandibular gland parenchymal cells. (B) A photomicrograph of stress control group (day 7) showing negative CD3 antigen-antibody reaction in parenchymal cells cytoplasm (arrow). (C) A photomicrograph of irradiated group (day 7) illustrating strong cytoplasmic reactivity in serous acini (arrow). (D) A photomicrograph of AgNps – protected group (day 7) indicating weak reaction in acini-lining cells cytoplasm (arrow). (E) A photomicrograph of AgNps-treated group (day 7) showing strong Ag-Ab reaction in the cellular cytoplasm (arrows) (CD3, ×200).

Group III: CD-3 stained sections of group IIIa presented strong and extensive positive cytoplasmic reaction in acinar and ductal cells, whereas group IIIb revealed indifferent findings; cells lining serous acini and ducts exhibited strong Ag-Ab reaction to CD-3 antibodies (Figs. 7C, 8C).

Group IV: Specimens obtained from subgroup IVa exhibited weak to moderate CD3 reaction in most examined sections, while specimens obtained from subgroup IVb elicited weak positive Ag-Ab reaction in few acinar cells (Figs. 7D, 8D).

Group V: CD-3 stained sections showed moderate positive cytoplasmic reaction in acinar and ductal cells (Fig. 8E).

BAX protein immune-staining

Groups I and II: Light microscopic examination of BAX stained sections obtained from subgroups (Ia and Ib) elicited weak positive immune reaction in few stromal cells around the gland acini and ducts (Figs. 9A, 10A). sections from subgroups (IIa and IIb) revealed weak reaction to BAX antibodies in few stromal and acinar cells (Figs. 9B, 10B).

Fig. 9.

(A) A photomicrograph of normal control group (day 1) eliciting weak reaction to BCL-2 associated protein X (BAX) antibodies in submandibular gland parenchymal cells. (B) A photomicrograph of stress control group (day 1) showing negative BAX antigen-antibody reaction in parenchymal cells cytoplasm (arrow). (C) A photomicrograph of irradiated group (day 1) illustrating strong cytoplasmic and nuclear reactivity in serous acini (arrow) and ducts (arrowheads). (D) A photomicrograph of AgNps – protected group (day 1) indicating weak to moderate reaction in acini-lining cells cytoplasm and nuclei (arrow) BAX, ×200).

Fig. 10.

(A) A photomicrograph of normal control group (day 7) eliciting weak reaction to BCL-2 associated protein X (BAX) antibodies in submandibular gland parenchymal cells. (B) A photomicrograph of stress control group (day 7) showing weak BAX antigen-antibody reaction in parenchymal cells cytoplasm (arrow). (C) A photomicrograph of irradiated group (day 1) illustrating strong cytoplasmic and nuclear reactivity in serous acini (arrows) and ducts (arrow heads). (D) A photomicrograph of AgNps – protected group (day 7) indicating moderate reaction in acini-lining cells cytoplasm (arrow). (E) A photomicrograph from AgNps-treated group (day 7) showing strong BAX reactivity in the cytoplasm (arrow) (BAX, ×200).

Group III: BAX stained sections of group IIIa exhibited strong and extensive positive cytoplasmic and nuclear reaction in acinar and ductal cells, also group IIIb showed cells lining serous acini and ducts with strong reaction to BAX antibodies (Figs. 9C, 10C).

Group IV: Salivary gland tissue obtained from subgroup IVa exhibited mild positive BAX reaction in most examined sections, while specimens obtained from subgroup IVb elicited weak positive reaction in few acinar and ductal cells (Figs. 9D, 10D).

Group V: BAX stained sections showed strong positive cytoplasmic reaction in acinar and ductal cells (Fig. 10E).

Histomorphometric interpretation

Serous acini surface area (mean±SD)

Surface area of the serous acini was measured in µm³ using image-J analyzer software program, and mean values were calculated for each group. Mean surface area of the serous acini in groups Ia and Ib was 53.27±7.5 and 52.61±8.1 respectively. In groups IIa and IIb, mean acinar area was 54.33±9.4 and 57.19±9.78. In irradiated groups (IIIa and IIIb), mean surface area of the acini was decreased to 17.8±2.95 and 25.84±4.28 respectively. While in group IVa, mean surface area of the acini was 29.7±3.12 and in group IVb, it was 27.12±2.08. Mean acinar area in group V was 17.54±4.11 (Fig. 11).

Fig. 11.

A bar chart showing mean acini surface area measured in the different study groups.

Striated duct (SD) diameter (mean±SD)

Striated duct diameter was measured by image J analyzer in µm, and measurements obtained were used to calculate mean±SD values for each group. In groups Ia and Ib, mean±SD diameter were 46.33±6.3 and 44.67±5.71, while in groups IIa and IIb mean values of SD diameter were 42.98±9.11 and 48.10±4.91. In group III, mean values of subgroup IIIa was 65.38±6.8, whereas in group IIIb it was increased to 102.6±8.19. Mean±SD diameter in group IVa was 43.27±4.2 and in group IVb it was 49.01±1.18, while in group V it was 97.2±12.07 (Fig. 12).

Fig. 12.

A bar chart showing mean striated duct (SD) diameter measured in the different study groups.

Optical density (OD) of PAS reaction

Mean OD of PAS staining in groups Ia and Ib were 0.42±0.09 and 0.37±0.11. Similarly, PAS mean OD in groups IIa and IIb were 0.36±0.13 and 0.41±0.09 respectively. In group IIIa, mean OD of PAS staining dropped to 0.1±0.04. Group IIIb mean OD was 0.09±0.008. In AgNps- protected groups (IVa and IVb), mean OD values were 0.277±0.04 and 0.197±0.009 respectively. Group V (sacrificed on day 7) reported OD 0.1±0.09 (Fig. 13).

Fig. 13.

A bar chart showing mean optical density of periodic acid sciff (PAS) reagent in PAS-stained sections among the different study groups.

Mean area % of BAX protein expression

Mean area % of immunohistochemical staining of BAX protein in groups Ia and Ib were 9.93±2.06 and 7.14±1.89 respectively, while in stress control groups (IIa and IIb) mean values of BAX expression were 5.91±2.56 and 8.34±3.30. In irradiated groups, mean area % of BAX reaction were 57.26±12.39 and 59.81±10.23 in groups IIIa and IIIb respectively. While in AgNps-protected groups (IVa and IVb) mean values of BAX protein staining were 35.8±7.71 and 28.91±10.10. Group V elicited mean area % of staining 62.34±8.13 (Table 1).

Table 1.

Mean area % of BAX immune staining in BAX-stained sections and mean positive cell count in CD3 stained sections in the different study groups

Group	Mean area % of BAX immune reaction (%)	Mean cell count of CD3 +ve cells (cell)
Ia	9.93±2.06	5.67±2.08
Ib	7.14±1.89	5.91±1.07
IIa	5.91±2.56	7.33±1.09
IIb	8.34±3.30	6.21±2.01
IIIa	57.26±12.39*	38.67±7.30*
IIIb	59.81±10.23*	46.60±5.80*
IVa	35.8±7.71**	22.35±6.81**
IVb	28.91±10.10**	21.47±9.30**
V	62.34±8.13	29.11±4.71**

BAX, Bcl-2 associated protein X; CD3, cluster of differentiation 3. *Statistically significant compared to groups I and II. **Statistically significant compared to group III.

Cell count of CD3 positive cells (mean ±SD)

CD3 positive cells were counted using Image J analyzer and mean values were calculated through SPSS statistical software program. In groups Ia and Ib, mean CD3 count were 5.67±2.08 and 5.91±1.07, while in groups IIa and IIb they were 7.33±1.09 and 6.21±2.01. Group IIIa elicited mean positive cell count 38.67±7.30, while in group IIIb it was 46.60±5.80. In AgNps-protected groups, mean CD3 cell count were 22.35±6.81 and 21.47±9.30 in groups IVa and IVb respectively. Mean cell count in group V was 29.11±4.71 (Table 1).

Electron microscopy

Control groups (group I and II)

Parenchymal cells of the serous acini showed basal rounded nuclei with regular nuclear membrane, homogenous, well-distributed chromatin, granular and prominent nucleoli. Parallel rough endoplasmic reticulum (rER) appeared basal to the nucleus with few oval mitochondria. Membrane bounded secretory granules of variable size and denisty were elicited surrounding the nucleus. Intercalated duct cells exhibited large centrally located nucleus, few cisternae of rER and secretory granules, normal desmosomal junctions were recognized throughout the boundaries of the cells. Striated duct cells revealed apical microvilli. The cells exhibited large central nuclei with granular appearance bounded by few rER. The lining cells showed parallel plasma membrane infoldings containing abundant rod shaped mitochondria. Granular convoluted tubules (GCTs) cells contained rounded, regular outlined nucleus with prominent nucleolus and abundant mitochondria. Several membrane bounded granules of varying size and density filled the apical two third of the cells. Junctional complexes were also recognized between the basal plasma membrane of GCT cells (Fig. 14).

Fig. 14.

An electron micrograph representing control groups (I and II). (A) Serous acini cell showing eukaryotic nucleus with intact membrane and prominent nucleolus (N), parallel arrays of rough endoplasmic reticulum (rER), oval pale mitochondria (m) and membrane bound secretory vesicles (SV) filling the cytoplasm. (B) Cell lining granular convoluted tubule eliciting oval shaped nucleus with granular chromatin (N), multiple oval shaped mitochondria (m), parallel cisternae of rER, several SV. (C) Parenchymal cell showing oval shaped mitochondria with parallel transverse cristae (arrow), parallel palisades of rER surrounding eukaryotic nucleus (N). (D) Intercalated duct exhibiting epithelial cells with rounded central nuclei (N), spindle shaped myoepithelial cells are detected (arrow), surrounding parenchymal cells are filled with electron dense SV. (E) Striated duct cell lining showing rounded eukaryotic nucleus with homogenous chromatin appearance (N), desmosomal junctions are intact (white arrows), luminal border of the cell shows microvilli (black arrows). (F) Excretory duct cell lining illustrating central homogenous nuclei (N) with basal plasma infoldings (white arrow) and apical microvilli (black arrow).

Irradiated groups

Group IIIa (acute effect)

Acinar cells showed distinct intracellular alterations. Some nuclei showed early apoptotic changes as irregularity of the nuclear membrane and chromatin clumping. Some nuclei appeared pyknotic and shrunken with chromatin condensation. The rER presented dilatation and widening of cisternal spaces, discontinuity and fragmentation. Dilated mitochondria with disrupted cristae were also marked. Secretory granules were reduced with ill-defined outlines. Areas of vacuolar degeneration were also spotted within the acinar cytoplasm. Intercalated duct nuclei appeared dark with chromatin condensation, others were shrunken and pyknotic, rER arrays were dilated. Striated duct nuclei showed signs of degeneration presented by nuclear membrane irregularity and pyknosis. The mitochondria appeared dense and elongated with disruption of their cristae. The rER were sparse, disrupted and abnormally dilated. Moreover, the cytoplasm showed extensive areas of vacuolar degeneration, desmosomal junctions were distorted between the cells. Ultrastructural examination of GCTs showed cells with few electron dense secretory granules surrounded by cytoplasmic vacuolation. Excretory duct lining cells revealed shrunken nuclei, mitochondrial degeneration with dilated, granular rER and few vacuolated areas (Fig. 15).

Fig. 15.

An electron micrograph representing group IIIa. (A) A parenchymal cell showing an irregular shaped heterochromatic nucleus (N) with clumps of chromatin (arrow), electron dense mitochondria (m), disturbed rough endoplasmic reticulum (rER) and ill-defined secretory vesicles (SV). (B) Striated duct showing enfolded lumen, heterochromatic dark nuclei (N), electrolucent cytoplasmic vacuoles (arrows) with few SV. (C) Excretory duct cell lining eliciting irregular shaped nucleus (N) with chromatin clumps (arrow) and large cytoplasmic vacuoles (*). (D) Granular convoluted tubule showing dark heterochromatic nuclei (N), elongated mitochondria (white arrows) and disrupted desmosomal junctions (black arrows). (E) Intercalated duct showing dark irregular shaped nuclei (black arrows), dark shrunken myoepithelial cells (m), some cells show karryolysis (dotted line), others show karryorehxis (arrow). G, electron dense secretory granules.

Group IIIb (recovery group)

Transmission electron microscopy (TEM) examination of irradiated glands that were collected on day 7 elicited indifferent findings from those collected on day 1. Parenchymal cells of the acini elicited nuclei with dense clumps of chromatin, irregular nuclear membrane, few secretory granules appeared in the cytoplasm, along with few dilated cristae of rER. Regarding intralobular ducts its lining cells showed apoptotic changes, shrunken nuclei, clumped chormarin with irregular nuclear membrane. The cytoplasm showed few mitochondria, dilated rER, cytoplasmic vacuoles and loss of apical microvilli (Fig. 16).

Fig. 16.

An electron micrograph representing group IIIb. (A) Parenchymal cells showing heterochromatic nuclei (N) of variable shape and size with clumps of chromatin (arrows), the cytoplasm show secretory vesicles (SV) and electro-lucent cytoplasmic vacuoles. (B) Acini cell exhibiting, heterochromatic dark nuclei (N) with chromatin clumps (arrow) cytoplasm shows few SV. (C) Excretory duct cell eliciting irregular shaped nucleus (N) with chromatin clumps (arrow) and dilated cisternae of rough endoplasmic reticulum (rER). (D) Striated duct showing dark heterochromatic nuclei (N), elongated mitochondria (white arrows) and cytoplasmic vacuoles. (E) Granular convoluted tubule showing clear nuclei of variable shape (N), basal elongated mitochondria (white arrow), desmosomal junctions are disrupted (black arrows).

Group IV a

In this group, nuclei of parenchymal cells appeared normally rounded, granular with well-defined nuclear membrane and homogenously distributed peripheral chromatin with prominent nucleolus. The rER was relatively well organized and arranged in parallel rows with few areas of persistent dilatation. Meanwhile, normally appearing mitochondria were recognized. Intralobular ducts cells showed regain of normal nuclear appearance and uniform arrangement of cisternae of rER was spotted. The cytoplasm showed few well-defined electron dense secretory granules. As well, desmosomal junctions between the cells were clearly distinguishable with no intercellular spaces. Mitochondria were scanty exhibiting transverse cristae (Fig. 17).

Fig. 17.

An electron micrograph representing group IVa. (A) Showing parenchymal cell with eukaryotic nuclei (N) exhibiting homogenous granular chromatin, surrounded with parallel arrays of rough endoplasmic reticulum (rER), oval pale mitochondria (white arrows) with several membrane-bound secretory vesicles (SV). (B) Intercalated duct eliciting central rounded heterochromatic nuclei (N), cytoplasm shows multiple vacuoles (white arrows) with luminal microvilli (black arrow). (C) Granular convoluted tubule showing clear eukaryotic nuclei (N) with electron dense plasma infoldings (white arrows) and elongated mitochondria (m) and multiple apical electron dense granules (G) and apical microvilli are noted (black arrow). (D) Striated duct eliciting heterochromatic nuclei (N) with variable size, basal plasma infoldings (white arrow) and apical electron dense granules (G).

Group IV b

TEM examination of ultra thin sections obtained from this group revealed acini cells with intact desmosomal junctions between them, nuclei appeared normal rounded, with homogenous chromatin and prominent nucleoli. The cytoplasm showed many electron dense secretory granules, oval mitochondria and parallel cisternae of rER. Duct lining cells showed marked ultrastructural improvement; nuclei appeared normal with granular appearance. Cytoplasm was filled with parallel palisades of rER, oval mitochondria, intact cell membrane with well distinguished desmosomal junctions, cells exhibited microvilli at their luminal surface (Fig. 18).

Fig. 18.

An electron micrograph representing group IVb. (A) Showing parenchymal cell with euchromatic nuclei (N) exhibiting granular chromatin, with parallel cisternae of rough endoplasmic reticulum (rER), oval pale mitochondria (white arrows) with electron dense secretory vesicles (SV). (B) Parenchymal cell with eukaryotic nucleus (N), oval mitochondria with transverse cristae (arrow) and secretory vesicle (SV). (C) Granular convoluted tubule showing clear eukaryotic nuclei (N) with multiple apical electron dense granules (G). (D) Striated duct eliciting rounded euchromatic nuclei (N) with variable size, basal plasma infoldings (white arrow) and apical electron dense secretory vesicles (SV). (E) Higher magnification of striated ducts shows clear euchromatic nuclei with prominent nucleoli (N) and elongated electron dense basal striations (white arrows), while (*) indicates electrolucent secretory vesicles.

Group V

Electron microscopic examination of this group showed parenchymal cells with disrupted cell-cell junctions, nuclei appeared shrunken and pyknotic with clumps of chromatin. The cytoplasm showed some electron dense granules, few mitochondria and dilated palisades of rER. Cells lining interlobular ducts showed dense pyknotic nuclei with perinuclear halo. Cytoplasm was filled with cytoplasmic electron lucent vacuoles, cell membrane exhibited disrupted desmosomal junctions, apical microvilli were lost (Fig. 19).

Fig. 19.

An electron micrograph representing group V. (A) Showing parechymal cell with heterochromatic nuclei (N) with chromatin condensation (arrow), desmosomal junctions are disrupted (double headed arrows). (B) Granular convoluted tubule showing heterochromatic irregular shaped nuclei (N), multiple cytoplasmic vacuoles (arrow) and few large secretory vesicles (SV). (C) Intercalated duct eliciting rounded shrunken heterochromatic nuclei (N) with variable size, cytoplasmic vacuoles (*), some cells show complete pyknosis (white arrow). (D) Striated ducts shows eukaryotic nuclei (N) and elongated electron dense basal striations (white arrows), dilated rough endoplasmic reticulum (rER), cytoplasmic vacuoles (*), and SV.

Discussion

Light microscopic examination of H&E-stained sections of the submandibular gland in control groups (I and II) elicited glandular lobules composed of closely arranged, spherical shaped serous acini. The interacinar spaces in between the acini were minimal. The lining cells of the acini were pyramidal in shape exhibiting acidophilic cytoplasm and basophilic nuclei. These results agree with those of Fattah and Omar [23] who identified similar histological features regarding the pure serous composition of the submandibular gland acini. Carvalho-Silva and Reis [24] claimed that the submandibular gland featured both serous and mucinous acini. That could be attributed to the fact that they have performed their study on pre-pubertal rats.

The choice of day 1 and day 7 in the current study design, was based on capturing two biologically distinct phases of radiation-induced tissue response. Day 1 represents the acute phase, characterized by early cellular injury, oxidative stress, and initiation of inflammatory pathways. In contrast, day 7 was selected to reflect the early phase, during which partial tissue regeneration, modulation of inflammation, and restoration of cellular architecture are expected to occur. These time points allow for meaningful comparison with previously published data.

In the present work, H&E-stained sections of group IIIa revealed loss of lobular architecture; acini were atrophied losing their spherical appearance. The acinar cells featured cytoplasmic vocalizations and pyknotic nuclei, Wu and Leung [25] reported similar disrupted architecture of the gland after seven days of irradiation with X-rays. Wang et al. [26] explained that any adopted type ionizing radiations with causes up regulation of the pathways involved in inflammatory process and cellular apoptosis. The latter authors hypothesized that apoptosis causes degeneration of cell organelles leaving cytoplasmic vacuoles and ends with cell lysis. Zhao et al. [27] concluded that direct cellular damage to the DNA is induced by physical interactions, where indirect damage occurs from produced free radicals that chemically interact with the DNA helical structure.

In the present study, group IIIa showed striated ducts and granulated convoluted tubules with variable degrees of ductal dysmorphism; their lining epithelium was shed into their lumen and some cells exhibited cytoplasmic vaculization. Striated duct diameter showed an increase compared to group Ia and IIa. This difference was statistically significant. The forementioned results are almost identical to those reported by Urek et al. [28] and Ahmed et al. [29] who identified similar findings. Both authors hypothesized a close relationship between oxidative stress and radiation-induced tissue damage. The former authors suggested that radiation creates a state of excessive oxidative stress in the cells leading to generation of oxygen free radicles, that affect the cell permeability. The latter authors outlined that cytotoxicity and organ damage occur when the production of ROS exceeds the neutralization capacity of the normal antioxidant mechanisms to scavenge them. Sakat et al. [30] reported similar findings and concluded that oxidative stress and the inflammatory cascades caused by the radiation causes localized tissue edema and blood vessel congestion with appearance of inflammatory exudate and blood infiltration.

In the present study, H&E-stained sections of group IIIb elicited almost identical results to group IIIa. Disrupted acini, ductal metaplasia and congested blood vessels were encountered in the examined sections. Being almost identical to group IIIa, there was a statistically significant difference in the same parameters when group IIIb were compared to groups Ib and IIb. These results are in accordance with Ahmed and Rasmy [31] who elicited similar histopathological findings through examining the effect of gamma rays on submandibular gland after one, seven- and 14-days post-exposure. The latter authors concluded that the intensity of radiation damage to the gland parenchyma increases by time. On the same line, Taha et al. [32] hypothesized that radiation causes unpredictable and irreversible damage to cell DNA that in turn limits the possibility of recovery. In the present work, the whole gland was irradiated with a single non fractionated high dose of gamma rays, and this hindered the ability of the gland to recover. Jensen et al. [8] hypothesized that the degree of severity of glandular damage and their potential for recovery depends on the irradiated gland volume, radiation dose and the ability of the surviving stem cells to repopulate.

Several authors in previous literature stated that ionizing radiation induces salivary gland damage primarily through generation of ROS that overwhelm the ability of endogenous antioxidant enzymes, and that consequently contribute to cellular damage [33, 34]; radiolysis of water leads to formation of highly reactive radicals such as hydroxyl ions, that targets lipids, proteins and nucleic acids. In parallel, oxidative stress activates redox-sensitive signaling pathways, such as NF-kB (nuclear factor protein kappa); enhancing the expression of pro-inflammatory cytokines and perpetuating DNA damage and mitochondrial instability that further trigger gland cellular death [35-37].

In the present study, light microscopic examination of H&E-stained sections of group IVa revealed partial preservation of the gland histology compared to that of group IIIa. The acini were packed side by side with minimal interacinar space. Histomorphmetric study of acinar surface area in group IVa was showed increase compared to group IIIa. This increase was statistically significant. These findings were featured by Abd El-Haleem et al. [20] who proved the potential protective role of AgNPs by examining the antioxidant and anti-inflammatory mechanisms of nano silver in the irradiated submandibular gland. Hamed et al. [38] demonstrated the protective effect of AgNPs on irradiated spleen through their anti-inflammatory mechanism. Krishnan et al. [39] proposed that AgNPs represent a recent agent of free radical scavengers and may be used to scavenge radiation produced ROS, thus they augment the cellular antioxidant mechanism.

Ducts in group IVa was identified with almost regular appearance and intact epithelial linings in H&E-stained sections. The arrangement lining cells was intact. Few others were affected by radiation; the ductal outline was disrupted, and the lining cells exhibited apoptotic changes, Gaillet and Rouanet [40] have tested the toxicity of AgNPs and concluded that according to the dose, duration of exposure and route of administration, toxicological or protective role of AgNPs is determined.

H&E-stained sections in group IVb were similar to those shown in group IVa; the histology of the glandular lobes was preserved; the acini were arranged close to each other with their spherical shape. The duct system was nearly normal, keeping its histological identity. Whereas, striated duct diameter was significantly decreased in group IVb in comparison to group IIIb. Statistical interpretation showed no significant difference between group IVb and Ib and IIb. These results agree with Bagoria et al. [41] who demonstrated similar histological features in gamma -irradiated liver tissue treated with caffeine coated AgNPs after 30 days post irradiation.

AgNPs were used as a radiotherapeutic agent in group V post-irradiation. H&E-stained sections of the submandibular gland in this group showed nearly similar picture to group IIIb. The acini exhibited heterogenous morphology, slightly normal or disrupted appearance. The duct system had damaged histopathological features. Statistical evaluation of the obtained data by the histomorhometric study regarding the mean acinar area and striated duct diameter showed statistically significant difference compared to groups Ib and IIb and statistically insignificant difference compared to group IIIb. This indicates persistent damaging effect of radiation to the gland and poor curative possibility of AgNPs as a radiotherapeutic agent. In line with these findings, Guo et al. [42] hypothesized that AgNPs increase the radioresistant ability of the biological tissues to radiotherapy by relieving the oxidative stress induced by the ionizing radiation, thus increasing the survival rate of the normal cells. This mechanism may be effective with pro-radiotherapy administration of AgNps mostly due to the normal condition of the cellular DNA as absorption of the nanoparticles activates the anti-apoptotic and anti-inflammatory pathways of the undisrupted cellular genome. By reviewing previous literature available on several databases, no workers used nano silver particles after exposure to radiation.

In our work, PAS-stained sections of group IIIa exhibited a weak reaction to the Schiff reagent, indicating depletion of glycoprotein content in both acini and duct epithelial lining. Histomorphmetric study of optical density of group IIIa showed decrease compared to Ia and IIa. This decrease was statistically significant. In line with the obtained results, Redman [43] concluded that radiation damages rER and Golgi apparatus which are the main sites for glycoprotein synthesis. This could be the cause that lead to less PAS reactivity with subsequent decrease in its optical density. In this work, PAS-stained sections in group IIIb exhibited weak staining. There was a decrease in their optical density in histomorphometric study compared to Ib and IIb indicating persistent glycoprotein depletion within the cells. These results agreed with what was outlined by Yoo et al. [44], who investigated the effects of irradiation in an in vitro study on human epithelial parotid cells and elicited a marked decrease in PAS reactivity, due to decreased saliva synthesis and production. PAS staining of group IVb exhibited moderate positive reaction. Histomorphmetric study of the optical density of PAS stain showed an increase compared to IIIb but there was minimal difference compared to Ib and IIb. The increase was statistically significant while the minimal difference was statistically non-significant. PAS reaction in group V showed mild positive reaction. Histomorphometric measures of the optical density were decreased compared to Ib, IIb and IVb. This decrease was statistically significant. However, the minimal difference between group V and group IIIb in histomorphometric study was statistically non-significant. This may be attributed to the failure in restoration of carbohydrate and lipid forming organelles.

In the present work, immunohistochemical stained sections in control groups showed weak expression of CD3 and BAX. Histomorphometric study of CD3 positive cells and mean area % of BAX among the four-control groups showed minimal differences. These differences were statistically non-significant This agrees with Varghese et al. [45] who reported the same findings. Group IIIa showed strong positive CD3 immune reaction. The reaction was widespread in the stroma and the parenchyma. Histomorphomertic study of CD3 positive cells in group IIIa showed evident differences compared to Ia and IIa groups. These differences were statistically significant. These results are with Nair et al. [46] and Konings et al. [47] who reported salivary gland CD3 infiltration, post-irradiation, in animal models. The former authors explained that irradiated gland tissue secretes pro-inflammatory cytokines and up-regulates inflammatory cells adhesion and extravasation that ends in lymphocytic infiltration with increased inflammatory reaction.

In the present work, immunohistochemical study of sections obtained from group IIIa revealed high expression of BAX protein. Histomorphomertic measures of mean area % of BAX in group IIIa showed an increase compared to groups Ia and IIa. This increase was statistically significant. Ren et al. [48] and Ahmed et al. [49] reported similar BAX reactivity findings in mice salivary glands following exposure to radiation. The latter authors concluded that DNA damage caused by irradiation directly activates P53, which directly increases BAX gene transcription, the key regulator of programmed cell death and this increases the rate of cell lysis. Bcl-2 is a key antiapoptotic regulator that plays a critical role in maintaining cellular survival by stabilizing mitochondrial membrane integrity and reducing cytochrome c release; thus evaluation of Bcl-2 provides insights into the balance between pro-survival and pro-apoptotic signaling within the glandular tissue; it particularly enabled us to assess mitochondrial-mediated apoptosis [36].

In the present study, Immunohistochemical results of CD3 stained sections in group IIIb revealed strong, widely expressed reaction. This reveals the continuous influx of inflammatory cells to the gland. Histomorphomertic interpretation of CD3 positive cells showed increased difference compared to groups Ib and IIb. This increase was statistically significant. The fore mentioned results are with Johnson et al. [50] who studied the inflammatory response by estimating many different inflammatory markers. The latter authors explained that radiation causes sustained production of cytokines and decreases the tissue macrophages leading to sustained inflammatory influx of cells with up regulation of the inflammatory markers.

In the present study, BAX-stained sections in group IIIb demonstrated strong positive expressions. Positive BAX immunostaining confirmed continuous apoptosis that occurs to the acini and duct even after only one set of radiation exposure. These results are with Wang et al. [26] who proved the positive inflammatory and BAX immune reaction following irradiation to the submandibular gland which in turn proves the presence of apoptosis. Sun et al. [33] and Liu et al. [36] have confirmed the presence of apoptosis through the positive immune reaction to different apoptotic markers. Sullivan et al. [51] demonstrated radiation-induced cellular apoptosis in human submandibular gland resected during neck dissection following radiotherapy, they elicited increased expression of apoptotic markers in the resected salivary gland. In the present study, statistical analysis of the estimated parameters in group IIIb exhibited statistically significant difference compared to control groups.

Immunohistochemical staining of group IVa showed mild positive immune reactivity to CD3 and BAX. Histomorphmetric study of CD3 and BAX-stained sections in group IVa exhibited decreased expression of the inflammatory and apoptotic markers compared to groups IIIa. This decrease was statistically significant. There was also statistically significant difference in the measures of these parameters in group IVa compared to groups Ia and IIa. This is going with the potential ability of AgNPs in attenuating inflammation and apoptosis induced by irradiation. The down regulation of CD3 expression in AgNPs mechanism was reviewed by Carvalho et al. [52] who hypothesized that during inflammatory process, the released Ag+ions suppress the inflammatory cascade, so they help in attenuating the production of harmful inflammatory cytokines. In addition to that, they activate T-regulatory cells and upregulate growth factors which help in tissue repair. Previous literature highlighted the role of AgNPs in variable inflammatory conditions. Crisan et al. [53] demonstrated the curative outcome of topically applied silver in treating human psoriasis. They reported that AgNPs suppress production of inflammatory cytokines that represent key pathogenic mediators in psoriasis.

Radiation-induced salivary gland injury is a multifactorial process; involving oxidative stress, apoptosis and inflammation. Previous investigations have highlighted the involvement of several molecular markers that further elucidate the underlying pathogenesis. Apoptosis has been demonstrated by up reulation of cleaved caspase 3 and P53 by several authors [6, 54]. Inflammatory pathways also play a central role, as evidenced by elevation of TNF-α, IL-6 and NF-kB [55].

In the present work, Immunohistochemical staining of group IVb showed mild to moderate positive immune reactivity to CD3 and BAX antibodies. Histomorphometric study of positive CD3 cell count and mean area % of BAX showed a decreased differences compared to group IIIb. These differences were statistically significant. Yet, there was a statistically non-significant difference in the same parameters when group IVb were compared to group IVa. In line with the obtained data, Bagoria et al. [41] recently demonstrated the radioprotective role of AgNPs in radiation-induced liver damage.

Immunohistochemical staining of group V showed moderate positive immune reactivity to CD3 and strong positive reaction to BAX antibodies. Histomorphometric study of CD3 positive cell count showed minimal difference compared to group IVb. This difference was statistically non-significant. In the histomorphometric study of BAX-stained sections in group V, there was increased difference compared to group Ib, IIb, and IVb. This increase was statistically significant. On the other hand, a difference that was statistically non-significant when group V was compared to group IIIb was recorded. This may be due to the exhaustion of the normal antioxidant mechanism with further production of ROS. The net result is increased cellular apoptosis and subsequent increase in BAX expression.

In the present study, ultrastructural examination of sections from control groups revealed well recognized cellular outlines and clear intact cellular organelles. The nuclei were rounded with clear intact nuclear membrane, granular chromatin and prominent nucleoli. These results agree with Wu et al. [5] who identified almost identical findings in rodents’ submandibular glands. In our work, control groups elicited rER with palisade cisterna, rod shaped mitochondria and scattered secretory vesicles occupying the cytoplasm. Myoepithelial cells were found surrounding the acini and the intercalated ducts. These results are with Chen et al. [56], who outlined the ultrastructural picture of the gland. Group IIIa revealed degenerative changes in the cells; the nuclei showed enfolded nuclear membrane with different appearances of the chromatin, where clumping and segmentation were detected indicating different stages of apoptosis. rER cisternae were widened losing their palisade arrangement, the mitochondria were swollen with disrupted cristae and minimal secretory vesicles were elicited. These findings detected by TEM validated the rise of pre-apoptotic marker BCL-2 that was detected by IHC study.

These results are in agreement with Wang et al. [57] who demonstrated disrupted cellular structure in the submandibular salivary gland in rat models. The latter author suggested that ionizing radiation disrupts mitochondrial structure and functions, enhances mitochondrial oxidative stress, and increases apoptosis in salivary gland.

Electron microscopic examination of group IIIb revealed disrupted tight junctions between the cells, enfolded nuclear membrane, and chromatin degeneration. The palisades of rER were dilated, the mitochondria were swollen and the secretory vesicles were limited. These results were featured by Wu et al. [58] who used TEM in evaluating the destructive effect of radiation on the submandibular gland. In the present work, ultra structural examination of group IIIb supports the cumulative destructive effect of radiation in the gland. Donnelly et al. [59] and Krishnan Nair et al. [18] clarified the acute radiation syndrome (radiation toxicity or radiation sickness) that occurs when the radiation dose is high enough to penetrate the tissues over a very short period and continues in the following 30 days.

Ultrastructural examination of group IVa revealed eukaryotic, pale nuclei in most acinar and duct cells and well distributed chromatin. The cytoplasm of the acini and epithelial lining of the duct showed preserved cellular organells; rER cisternae and membrane bound secretory granules appeared filling the cytoplasm. The mitochondria were elongated with preserved shape especially in striated ducts. These results are almost identical to Abd El-Haleem et al. [20] who studied the effect of nano silver on the submandibular gland. The latter author concluded that AgNPs could have a protctive role against radiation-induced changes.

Group IVb allocated maintained intact intra cellular homeostasis, the nuclei were eukaryotic, abundant mitochondria and parallel cisternae of rER. Few previous literatures studied the effect of AgNPs on irradiated salivary glands after seven days, however many authors investigated the radioprotective and radio sensitizing potency of AgNPs on other organs. Despite not using the TEM in their study, Zhang et al. [34], elicited the anti-inflammatory role of AgNps in ulcerative colitis and Rheumatoid arthritis in vivo models. Ultrastructural changes elicited in group V showed shrunken nuclei with disrupted chromatin. The tight junctions between the cells were widened. rER lost their palisade arrangement of their cisterna and the mitochondria were swollen. This indicates persistent damage to cellular organelles. By reviewing other literatures available on several data bases, thesis that studied the role of AgNPs as a radiotherapeutic agent after exposure to radiation were lacking.

Conclusion

We concluded that AgNPs has the ability to attenuate the apoptotic changes induced by gamma radiation in the submandibular salivary gland tissue, minimize nuclear alterations, in addition to modulating the lymphocytic infiltration and CD3 expression associated with radiation-induced cell injury.

Limitations

While the study provides important structural and immunohistochemical insights, functional assessment of salivary gland activity was not conducted due to lack of technical inquiries needed for submandibular gland duct cannulation.

Recommendation

We recommend performing further studies to evaluate the long term changes in the salivary glands, associated with exposure to gamma irradiation. Also further studies are required to elicit the role of AgNPs combined with other radioprotective agents in attenuating the radiation-induced alterations.