Immunohistopathological Studies on Rats Injected with CCl4 and Treated with Propolis and Honey Bee

The first group of 10 rats serves as control. The second were maintained as CCl4, then third and fourth maintained as honey and propolis respectively for period of experiment (6 weeks). Pieces of liver and kidney were subjected to histopathological and immunohistochemistry procdures and the obtained sections (5 μm thick) were stained with H&E also with silver nitrate stain and anti laminin antibody for immunohistochemistry.


Introduction
Carbon tetrachloride is widely used as hepatotoxic compound for screening the anti-hepatotoxic/hepatoprotective activity of drugs in experimental model systems, because CCl 4 -induced hepatotoxicity is regarded as an analogue of liver injury caused by a variety of hepatotoxicity in man. It has been generally reported and accepted that CCl 4 -induced hepatotoxicity due to its hepatotoxic metabolites and trichloromethyl free radicals (•CCl 3 ) induced lipid peroxidation [1,2]. Therefore, one of the therapeutic strategies against liver injury is to find antioxidant compounds that are able to block liver injury through scavenging of trichloromethyl free radicals generated by CCl 4 [2].
Propolis is a resinous hive product collected by honey bees from exudates and buds of plants and mixed with wax and bee enzymes [3,4] It also contained flavonoids, sugar, and aliphatic acids. Flavonoids are thought to be responsible for many of its biological and pharmacological activities including anticancer [5], anti-inflammatory [6] and antioxidant effects [7]. Propolis has several biological and pharmacological properties, as antimicrobial [8], anti-inflammatory [9], antioxidant [10,11] antiparasitic [12], immune modularity and immune stimulant effects and it increased the percentage of protected animals suggesting its use in vaccines as an adjuvant [13,14].
Honey is one of honey bees' products which are used in medicine in many cultures since ancients' times. Honey is the main source of concentrated sweetness in the diet of many people and contained about 80% carbohydrate, 20% water and traces of protein and ash [15]. Honey is known to exhibit a broad spectrum of activities including antiviral, antibacterial and immunostimulant [16]. It was found to have antioxidant activity due to its high content of flavonoids [17,18]. The aim of the present study is to determine the pathological findings in liver and kidney tissues due to CCl 4 toxicity and the therapy of honey and propolis. Also, Immuno-staining of laminin in kidney and liver tissues before and after therapy.
(Muri Lab Prep) (diluted 1:50 in blocking buffer) were applied to slides overnight at 4°C in humified chamber, then sections washed in PBS 3 times. 100 µl of biotinylated swine anti-rabbit antibody diluted 1:500 in blocking buffer were applied for 2 hours in humified chamber. After washing in PBS , 100 µl of Avidin -Biotin complex was added for two hours in humified chamer. 100 µl of freshly prepared diaminobezidine-HCl (Sigma) were added to devlope colour for 12 minutes. Sections were washed in running water to stop colour development . Sections were dehydrated, counterstained and coverslip in Permount [32]. Assessment of HBeAg and its antibodies may be an important predictor of which chronic carriers of HBV will develop chronic active liver disease [33].

Liver histopathology
H/E stain: The hepatocytes showed acidophilic cytoplasm with single central rounded vesicular nuclei and some of the cells were binucleated ( Figure 1). In group II (CCL 4 alone group), most of the hepatocytes contained multiple, large cytoplasmic vacuoles and some hepatocytes had deeply acidophilic cytoplasm and deeply stained nuclei ( Figure 2). After P and H administration, remarkable improvement in the hepatocytes was noticed as they appeared nearly similar to that of the control rats. Only few hepatocytes appeared with slight vacuolated cytoplasm ( Figure 3). On the other hand, in the CCL 4 /recovery group the liver was still markedly affected. Hepatocytes appeared with highly vacuolated cytoplasm and deeply stained nuclei ( Figure 4).

Masson's trichrome stain:
In Masson's trichrome stained sections 0.5 mg/kg of b. wt. of CCl 4 twice weekly for six weeks. Honey group: injected ip with 0.5 mg/kg of b. wt. of CCl 4 twice weekly for six weeks and treated with 10% honey bee in drinking water daily. Popolis group: injected ip with 0.5 mg/kg of b. wt. of CCl 4 twice weekly for six weeks and treated with propolis orally, (40 µl/rat/day) day after day for six weeks.

Histological study
For light microscopic examination: Liver and kidney samples were collected and fixed in 10% buffered formalin for 24 hours. Serial 5 μm paraffin sections were prepared for pathological study, with hematoxylin and eosin (H&E), Masson Trichrome (MT), silver nitrate and immunohistochemical examination with anti laminin antibody.

H/E stain
Liver tissue specimens were fixed in 10% formalin and embedded in paraffin wax and 5 micrometer thickness were cut. Sections were stained with H/E stain according to Drury and Wallington [21].

Masson trichrome stain
The method was done according to Carson [22]. Briefly, liver specimens were fixed in Zinker solution for 24 hours, washed in 50% alcohol and treated with diluted iodine solution, then washed in 3% sodium thiosulphate. Sections were stained firstly with haematoxylen, then with Masson A solution for 5 minutes. They were washed in distilled water and stained with Masson B solution for 5 minutes and differentiated in 1% phosphomolybdic acid for 5 minutes, stained with 2.5% fast green solution for 2 minutes, then differentiated in 1% glacial acetic acid. Sections were dehydrated, cleared in xylene and mounted with DPX.

Silver stain
Many methods of silver staining have been developed. Two general classifications are :-1) silver amine or alkaline method, and 2) silver nitrate or acidic method [23]. Detection levels of proteins using the various staining methods are determined by how quickly the back-ground develops. The silver amine methods usually have lower background than the silver nitrate methods and, therefore, have typically been more sensitive. A drawback to the use of the silver diammine method is the use of glutaraldehyde for fixing and sensitization [24,25]. Glutaraldehyde is not compatible with matrixassisted laser desorption/ion-ization mass spectrometry (MALDI-MS) [26][27][28] because it causes protein-protein cross-linkages. The use of various types of sensitizers with the silver nitrate method has been shown to decrease the background and increase detection sensitivity. The ProteoSilver™ Kits are silver nitrate-based with a unique sensitizer solution and optimized protocols which produces better sensitivity (0.1 ng/mm 2 ) than typically claimed using a silver amine method, but is MALDI-MS compatible [29,30]. The ProteoSilver Plus Kit also contains destaining solutions for removal of silver ions from excised bands for enhanced MALDI-MS sensitivity [31].

Immunohistochemistry
Hepatic tissues were processed to paraffin blocks. Sections were dewaxed in xylene and rehydrated in descending grades of alcohol. Microwave antigen retrieval in 10 mM sodium citrate at pH 6 was carried out for 20 minutes. Sections were subsequently covered with 3% hydrogen peroxide for 10 minutes. Secttions were hydrated in descending series of alcohol. Sections were covered with 100 µl of PBS containing 10% normal swine serum for 1 hour in humified chamber to reduce unspecific background staining. 100 µl of rabbit anti-laminin  the parenchyma of the liver in control groups appeared to be supported with a stroma of very delicate meshwork of collagenous fibers. Few collagenous fibers surrounding the central veins, in portal area and in capsules were seen ( Figure 5). In subgroupIIA (CCL 4 alone group), the stroma was well defined. There was thick connective tissue capsule and an apparent increase in the collagen fibers around the central veins, in between hepatocyte cords and in portal areas ( Figure 6). In CCL 4 /P and H groups, few collagen fibers were detected (Figure 7) while in the CCL 4 /recovery group the collagen fibers were very numerous ( Figure  8).

Silver stain:
In silver nitrate stained sections, Histology of liver sections of normal control animals (group 1) showed normal liver architecture with central vein, and cytoplasm and prominent nucleus and nucleolus were preserved (Figure 9). The liver sections of CCL 4 treated animals (group 2) showed degeneration in sinusoids and become as vacuoles ( Figure 10). Honey bee and propolis treatment (group 3,4) appeared to significantly prevent the CCL 4 toxicity as revealed by the hepatic cells which were preserved cytoplasm. This also caused a marked decrease in inflammatory cells (Figures 11 and 12).

Kidney histopathology
H/E satin: In Heamatoxilin and eosin stained sections, CCl 4 administration alone caused prominent histopathological damage in the kidney compared with the control rats ( Figure 13). The kidney sections from the control-and honey-treated groups showed normal glomeruli, and tubulointerstitial cells (Figure 14). In contrast, the      kidneys of CCl 4 -treated rats showed marked deleterious histological changes. The kidney sections showed significant glomerular and tubular degenerations varying from, glomerular basement thickening, interstitial inflammation, tubular cell swelling, pycnotic nuclei, medullary vascular congestion and moderate to severe necrosis ( Figure  15). propolis-treated group of rats preserved normal morphology of the kidney and shows normal architecture of the kidney (Figure 16).

Masson trichrome stain:
In masson trichrome stained sections, histology of Kidney sections of normal control animals (group 1) showed normal kidney architecture ( Figure 17). The kidney sections of CCL 4 treated animals (group 2) showed clear vacuolation of cells and dilation in space of bowmans capsule (Figure 18). Honey bee and propolis treatment (group 3,4) appeared to significantly prevent the CCL 4 toxicity with regeneration in glomeruli and interstitial spaces nearly as seen in normal control (Figures 19 and 20).

Sliver nitrate:
In silver nitrate stained sections Histopathological changes in Kidney sections of normal control animals (group 1) showed normal structure of glomerulus and bowman capsule ( Figure  21).The kidney sections of CCL 4 treated animals (group 2) showed clear vacuolation of cells and dilation in space of bowman's capsule ( Figure 22). Honey bee and propolis treatment (group 3,4) appeared to significantly prevent the ccl4 toxicity with regeneration in glomeruli and interstitial spaces nearly as seen in normal control (Figures 23 and  24).      of the control liver tissues stained with anti-laminin showed normal structure of the liver (Figure 25 a). In CCl 4 injected rats, hepatic tissues stained with anti-laminin showed extensive hepatocellular necrosis (Figure 25 b).

Immunohistochemical examination: Immunohistchemistry
The pathology of hepatic tissues stained with anti-laminin in CCl 4 injected rats and treated with honey showed improvement of general hepatic architecture and slight hepatocellular fibrosis (Figure 25 c).
The pathology of hepatic tissues stained with anti-laminin in CCl 4 injected rats and treated with propolis showed normal liver lobular architecture with slight hepatocellular fibrosis (Figure 25 d).

Discussion
The present study showed that, CCl 4 administration for sex weeks, resulted in loss of the usual hepatic architecture, many vacuoles with dark stained nuclei in most of the liver cells. This structural damage was due to edema of the organelles [33].
Liver fibrosis was clearly evidenced, in the present work, by a significant increase in area percentage of the collagen fibers in Masson trichrome stained section. It was explained that hepatic fibrosis is usually initiated by hepatocytes damage leading to activation of Kupffer cells and subsequent release of cytokines and growth factors. These factors activate HSCs which proliferate and transform into myofibroblasts-like cells that deposit large amounts of connective tissue components [34]. Moreover, CCL4 causes oxidative stress that activates HSCs [35].
One of the structural glycoproteins is a high molecular weight molecule called laminin [31]. It is well known from previous immunohistochemical studies that laminin and type IV collagen are permanent constituents of the basement membrane of late and early chorionic villi and of placental blood vessels [36][37][38]. It has also been reported that basement membrane components such as type IV collagen and laminin can be synthesized by trophoblastic cells [39]. The basement membranes may control selective permeability for macromolecules as well as providing structural tissue support. Laminin, which binds to itself, to type IV collagen, to heparin, and to cell surface receptors, is able to promote the adhesion and growth of various epithelial cells and tumour cells [40,41], as well as the outgrowth of neurites [42]. The interactions of cells with various tissue matrices may result in quite different cellular responses, and modification of the extra cellular matrix structure is likely to play an important role in the regulation of cell behaviour during developmental processes laminin might be an essential constituent of placental basement membranes and might play an important role in the function and structure of the villi. In addition, significantly enhanced laminin expression in the basement membrane beneath the trophoblastic epithelium might modulate the characteristics of the trophoblastic cells in hydatidiform moles in an autocrine and/or paracrine fashion. Serum laminin P-1 concentrations are reported to be high in patients with fibrosis of the liver, e.g. chronic hepatitis, liver cirrhosis, alcoholic liver disease [43][44][45], various malignant tumours [46][47][48][49], and pregnancy. In the case of liver fibrosis, increased deposition of basement membrane components, such as type IV collagen and laminin, in the peri -sinusoidal walls of the liver has been reported [50][51][52][53]. In the case of malignancy, [50] reported that serum laminin P-1 values were increased in ~50% of cancer patients. This increase may be due to the destruction of the basement membrane by tumour invasion and increased synthesis of fibrous components surrounding the tumour.