A Possible Novel Protective Effect of Piceatannol against Isoproterenol (ISO)-Induced Histopathological, Histochemical, and Immunohistochemical Changes in Male Wistar Rats

Abstract

Dry mouth is characterized by lower saliva production and changes in saliva composition. In patients with some salivary gland function remaining, pharmaceutical treatments are not recommended; therefore, new, more effective methods of promoting saliva production are needed. Hence, this study aimed to provide an overview of the histological changes in the salivary gland in the model of isoproterenol (ISO)-induced degenerative changes in male Wistar rats and to evaluate the protective effect of piceatannol. Thirty-two male Wistar rats were randomly divided into four groups: the control group, the ISO group, and the piceatannol (PIC)-1, and -2 groups. After the third day of the experiment, Iso (0.8 mg/100 g) was injected intraperitoneally (IP) twice daily into the animals. PIC was given IP in different daily doses (20 and 40 mg/kg) for three days before ISO and seven days with ISO injection. The salivary glands were rapidly dissected and processed for histological, histochemical, immunohistochemical (Ki-67), and morphometric analysis. Upon seven days of treatment with ISO, marked hypertrophy was observed, along with an increased number of positive Ki-67 cells. Proliferation was increased in some endothelial cells as well as in ducts themselves. Despite the significant decrease in proliferation activity, the control group did not return to the usual activity level after treatment with low-dose PIC. Treatment with a high dose of PIC reduced proliferative activity to the point where it was substantially identical to the results seen in the control group. An ISO-driven xerostomia model showed a novel protective effect of piceatannol. A new era of regenerative medicine is dawning around PIC's promising role.

Keywords: Ki-67; histochemical; histological; isoproterenol; piceatannol; salivary gland.

Figure 1

Photomicrographs of sections of rat major salivary glands from the control group showing salivary glands dissected with adjacent structures intact. Submandibular gland (SM), sublingual gland (SL), parotid gland (P), and mandibular lymph node (LN) (HE; ×5).

Figure 2

A section in the parotid gland stained with HE from the control group (A–C) showing the lobules of the gland, each lobule is formed of serous acini (A) and ducts (D) with wide connective tissue septa filled with a fine network of interlobular connective tissue fibers (CT) serous acini (A) consists of a single layer of pyramidal cells (↑) with basal rounded nuclei surrounding a lumen. Among the acini, intercalated ducts lined by cuboidal cells with rounded nuclei (arrowhead) and striated ducts (D) are observed and lined by a single layer of columnar epithelium (dot arrow) (HE; A × 100, B × 200, C × 400). ISO group (D–F) showing markedly hypertrophic packed secretory serous acini (A). Some of the acini exhibited irregular outline and spaces (s) appear between acini. The cytoplasm and basal lines of acinar cells are blurred, and the vacuolization (V) of acinar cells is obvious. Nuclei of serous acini appear enlarged and irregular in shapes and size with clumped chromatin hyperchromatic (↑) or with pyknotic nuclei (red ↑). Striated duct (D) appears disorderly arranged epithelium lining with loss of basal striation. Execratory ducts (*) appear with loss of pseudo-stratification and stagnated secretion in the lumen. The duct was surrounded by dens fibrous connective tissue (CT) with hyalinization area (h) and congested dilated blood vessels (bifid head). Inset hyper magnification of straited duct (D) (HE; D × 100, E × 200, F × 400; inset × 400). Low dose of PIC (PIC-1 group) (G–I) showing improvement in the histological structure. Some areas of serous acini (A) contain large hyperchromatic nuclei (↑). Apparently large lobules are separated by relatively thin connective tissue septa (s) with few collagens’ fibers deposition. Striated duct (D) with columnar cells and basal striation is detected. Some of the excretory ducts (*) shows loss of pseudo-stratification in some areas. (HE; G × 100, H × 200, I × 400; inset × 400). High dose of PIC (PIC-2 group) (J–L) showing marked improvement in the histological structure. The parotid gland appears nearly as the control group. Each lobule is formed of serous acini (A) and ducts (D) with wide connective tissue septa filled with a fine network of interlobular connective tissue fibers (CT) serous acini (A) consists of a single layer of pyramidal cells (↑) with basal rounded nuclei surrounding a lumen. Among the acini, striated ducts (D) are observed and lined by a single layer of columnar epithelium (dot arrow). (HE; J × 100, K × 200, L × 400; inset × 400).

Figure 3

A section in the sublingual gland stained with HE from the control group (A–C) showing sublingual gland mucous acini and ducts appear with a fine network of interlobular connective tissue (CT). Each mucous acinus (A) consisted of large pyramidal mucous cells with abundant pale blue vacuolated cytoplasm. Acinar mucous cells contain flattened basal nuclei and pale eosinophilic cytoplasm (↑). A serous demilune is seen at the left of the acinus with rounded nuclei, and basal dark linear stain (wavy arrow). The striated duct is lined by columnar cells having eosinophilic cytoplasm and prominent cytoplasmic striations (dot arrow). The excretory ducts (*) have tall columnar epithelium with more apically located nuclei. Myoepithelial cells (yellow arrow) show a narrow cytoplasm and a very flattened nucleus, may be found wrapping ducts and some acini. (HE; A × 100, B × 200, C × 400). ISO group (D–F) showing markedly hypertrophic packed secretory mucous acini (A) with some areas with shrunken (curved arrow) loss of the acini (↑↑). A serous demilune is seen with vacuole-like structures (wavy arrow). Striated duct (D) appears disorderly arranged epithelium lining with loss of basal striation and marked vacuolation (dot arrow). Execratory ducts (*) appear with loss of pseudo-stratification and stagnated secretion in the lumen. Notice numerous mononuclear cellular infiltration (red arrow), hyalinization area (h), and dilated congested blood vessels (bifid arrow) in the connective tissue septa. (HE; D × 100, E × 200, F × 400; inset × 400). Low dose of PIC (PIC-1 group) (G–I) showing almost mucous acini (A) and striated ducts (D) appear nearly as control with a fine network of interlobular connective tissue (CT). A serous demilune at the left of the acinus (wavy arrow) and myoepithelial cells (yellow arrow) are seen. Excretory ducts (*) are possessed normal appearance of cell lining and are filled with a small amount of secretion. Notice few mononuclear cellular infiltrations (red arrow) and mild congested blood vessels (bifid arrow) (HE; G × 100, H × 200, I × 400; inset × 400). High dose of PIC (PIC-2 group) (J–L) showing marked improvement in the histological structure. The sublingual gland appears nearly as the control group. (HE; J × 100, K × 200, L × 400; inset × 400).

Figure 4

A section in the submandibular gland stained with HE from the control group (A–C) showing numerous lobules separated by thin connective tissue septa (CT). The lobules show closely packed mixed secretory acini (A), granular convoluted tubules (D) and execratory ducts (*). The acinar cells are mixtures of serous (↑) and mucous (m) secretory cells. The myoepithelial cells (yellow arrow) appear gasping the acini. The striated ducts are lined by columnar cells having oval nuclei (D). The well-developed granular convoluted duct is lined by columnar cells having eosinophilic cytoplasm (dot arrow). The excretory ducts (*) have tall columnar epithelium with more apically located nuclei and prominent cytoplasmic striations. (HE; A × 100, B × 200, C × 400). ISO group (D–F) showing markedly hypertrophic packed secretory acini (A). The normal architecture of acinar cells is distorted, their nuclei became dark and deeply stained atrophied nuclei (arrowhead). The cytoplasm lost its basophilic character (A). Giant nuclei (↑) are also seen. The cytoplasmic vacuolation in ductal (D) cells were increased with reduced acidophilic content of the cytoplasm of granular duct cells (dot arrow). Execratory ducts (*) appear with loss of pseudo-stratification and stagnated secretion in the lumen. The duct was surrounded by dens fibrous connective tissue (CT) with hyalinization area (h) and congested dilated blood vessels (bifid arrow). (HE; D × 100, E × 200, F × 400). Low dose of PIC (PIC-1 group) (G–I) showing marked improvement in the histological structure of cells of acini (A) as well as cells of ducts lining, and the acini relatively preserve their shape are seen. The numbers of vacuoles decrease, and well-formed striated ducts (D) are also detected. The granular convoluted tubules were lined by simple columnar epithelium with eosinophilic cytoplasm and basal rounded nuclei. (HE; G × 100, H × 200, I × 400). High dose of PIC (PIC-2 group) (J–L) showing marked improvement in the histological structure. The submandibular gland appears nearly as the control group. (HE; J × 100, K × 200, L × 40).

Figure 5

Masson trichrome-stained sections of the three salivary glands (parotid (A–D), sublingual (E–H), and submandibular (I–L) glands) of: control group showing scanty collagen fibers in the connective tissue septa between the lobules surrounding the blood vessels and large excretory ducts (*) are seen. The ISO treated group shows marked deposition of abundant collagen fibers in the connective tissue septa between the lobules that extended to surround the intralobular secretory acini and ducts. The PIC-1 group shows an apparent decrease of collagen fibers deposition in connective tissue septa between the lobules and around the intralobular acini and ducts. The PIC-2 group reveals scanty collagen fibers and appeared similar to the control group. A: acini, D: ducts, CT: connective tissue. (Masson trichrome; ×200).

Figure 6

Sections of the three salivary glands (parotid (A–D), sublingual (E–H), and submandibular (I–L) glands) are stained with Alcian blue for acidic glycoconjugates and counterstained with Nuclear Fast red. Control group strong positive acidic mucin sky-blue reaction in the secretory acini (A) and the lumen of ducts (D) in the sublingual gland with a negative reaction in the serous demilune (↑) and negative reaction in the parotid gland. While submandibular gland showed a negative reaction with faint sky-blue color inside the acinar cells. The ISO treated group shows a faint positive acidic mucin sky-blue reaction in the sublingual secretory acini and the lumen of ducts and a strong positive reaction in the serous demilune. The parotid gland shows a negative reaction with faint sky-blue color inside the acinar cells. The submandibular gland shows a blue positive reaction (↑) in the acinar cell’s cytoplasm. The duct epithelial cells show a strong positive reaction for the secretory granules and the epithelial lining. The PIC-1 group shows strong positive acidic mucin sky-blue reaction except for few acinar cells with faint (*) positive reaction. A negative reaction is shown in most of the acini and ducts of the parotid gland while submandibular gland shows negative reaction in most of the acinar cells. The PIC-2 group appears similar to the control group (Alcian blue; ×200).

Figure 7

Sections of the three salivary glands (parotid (A–D), sublingual (E–H), and submandibular (I–L) glands) are stained with Alcian blue for Periodic Acid-Schiff (PAS) for identification of neutral mucin and counterstained with Hematoxylin. Sections in the parotid and submandibular salivary gland of control groups and in the low dose PIC-1 and high dose PIC-2 treated groups show strong positive PAS reaction (magenta color), appear in both ducts (D) and acini (A) which is observed more at their basement membrane. However, ISO treated group reveal faint positive PAS reaction in both the acini and the ducts. Most of acinar cells of the sublingual gland of control group exhibit faint positive magenta color reaction in the secretory acini (A) and the lumen of ducts (D) and strong positive reaction in the demilunar cells (↑). However, the ISO treated group shows a strong positive reaction in the secretory acini and the lumen of ducts. Demilunar cells show evident faint positive reaction. Interestingly, low dose PIC-1 and high dose PIC-2 appear nearly as control group compared to the previous groups. The duct cells show a slight increase in the intensity of PAS compared to the ISO treated group (PAS; ×200).

Figure 8

Sections of the three salivary glands (parotid (A–D), sublingual (E–H), and submandibular (I–L) glands) are stained. The control group shows few immunopositive staining to Ki-67 in the secretory acini and ducts. The ISO treated group shows marked increased numbers of immunopositive labeled nuclear staining in the secretory acini. The PIC-1 group shows an apparent decrease in the numbers of immunopositively labeled nuclear staining in the secretory acini and ducts as compared to the ISO group. The PIC-2 group reveal few immunopositive staining to Ki-67 in the secretory acini and ducts and appeared similar to the control group (immunohistochemical stain Ki-67; ×200).

Figure 9

Graph of the mean area %age of collagen fibers (%) in the three salivary glands (parotid (A), sublingual (B), and submandibular (C) glands). The mean number of Ki-67 positive nuclei in the three salivary glands (parotid (D), sublingual (E), and submandibular (F) glands). Data are presented as mean ± SD. Differences between groups were identified using one-way ANOVA, followed by Tukey’s multiple comparison post-hock-test, indicated above the bars. (n = 8 rat/group). ** p ≤ 0.01, **** p ≤ 0.00001, ns = non-significant to the corresponding group.