Effect of Methotrexate on the Jejunum of Adult Albino Rat and the Possible Protective Role of Vitamin A: Histological and Immunohistochemical Study

Material and Methods: Seventy two adult male albino rats were divided into six equal groups. In all groups the animals were sacrificed 24 hours after the last injection. The jejunal specimens were examined using H& E and Masson’s trichrome stains and immunohistochemical examination to study the alkaline phosphatase activity using light microscopy. Morphometric study measuring villous height, crypt depth, number of goblet cells, percentage area of fibrosis and the optical density of alkaline phosphatase activity was done in all groups.


Introduction
Methotrexate (MTX) is used for the therapy of a wide range of neoplastic disorders including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, breast cancer and testicular tumors [1]. MTX treatment may reduce the number of attacks in patients with recurrent acute anterior uveitis [2]. Recent literature reported that high dose of MTX regimens may be used in induction of abortion as well as liver cholestatic disorders [3]. As methotrexate (MTX) is an anti-metabolite drug, it is widely used in the treatment of many diseases such as rheumatoid arthritis and psoriasis [4].
Concerning the effect of MTX on the small intestine, Miyazono et al. [5] mentioned that the oxidative stress plays an important role in the MTX-induced small intestinal damage. Ciralik et al. [6] mentioned that methotrexate caused damage to the mucosa of small intestine leading to nausea, vomiting, diarrhea, stomatitis, decreased absorption and gastrointestinal (GI) ulceration. The latter authors also reported that biopsies of the small intestinal epithelium of patients treated with MTX exhibited an increase in the oxidative stress and a decrease in antioxidant defenses. Sener et al. [7] postulated that methotrexate exerted its cytotoxic effects on the rapidly dividing cancer cells as well as on the normal tissues with high cell proliferation rate such as the gut mucosa and the hematopoietic cells. Methotrexate also induced mucositis and nitrosative stress in the small intestine of rats [8].
Regarding the concomitant use of vitamin A with methotrexate, Yasuharu et al. [9] reported that vitamin A increased the integrity of small intestine by increasing its protein and lipid content without affecting the absorption of methotrexate. In addition, Swartz -Basile et al. [10] found that vitamin A was essential for normal growth and differentiation of the epithelial tissues. Yuncu et al. [11] assumed that vitamin A might be involved in the regulation of DNA and RNA synthesis in the crypt cells (stem cells) helping their regeneration which further supports the protective effect of vitamin A. Moreover, Ciralik et al. [6] suggested that vitamin A is among the antioxidants which might protect the jejunum from MTX damage.
The aim of the present study is to detect the histological and the immunohistochemical adverse alterations that may occur in the jejunum of the adult albino rats following short and long term methotrexate therapy (two modes of regimen). Determination of the possible protective role of vitamin A against MTX-induced damage will also be done.
washed, dehydrated in ascending grades of alcohol, cleared in xylol and processed for paraffin sections of 5 µm thickness. The specimens were subjected to: 1-Histological examination: using H & E and Masson's trichrome stains.

2-Immunohistochemical examination:
using DAB (diaminobenzidine) for studying the alkaline phosphatase activity using light microscopy.

3-Morphometric study:
sections stained with DAB and Masson's trichrome stain were examined by the image analyzer computer system to measure the optic density of alkaline phosphatase enzyme activity and the percentage area of fibrosis in each group. The data were obtained using Leica Qwin 500 image analyzer computer system (England). The image analyzer consisted of a colored video camera, colored monitor, hard disc of IBM personal computer connected to the microscope, and controlled by Leica Qwin 500 software. The image analyzer was first calibrated automatically to convert the measurement units (pixels) produced by the image analyzer program into actual micrometer units. Using the measuring field menu, the area, optic density and standard measuring frame of a standard area equal to 118476.6um 2 were chosen from the parameters. This step was repeated but the optic density was replaced by area percentage. In each chosen field the jejunal tissue was enclosed inside the standard measuring frame (Figures 1a and 1b) and then the optic density of alkaline phosphatase enzyme activity and the CT area were masked by a blue color to be measured (Figures 1a and  1b). Villus height, crypts depth and goblet cell counting also detected (Figures 1c and 1d).  concentration of 2.5 mg/tablet (a product of EBEWE pharma Austria). Two tablets were dissolved in 10 ml of saline solution to obtain a concentration of 0.5 mg/ml. Methotrexate was then administrated in a dose of 2.5 and 1 mg/kg for three successive days/week for a period of four weeks to detect its short and long term effects [4].
Vitamin A was supplied as vitamin A capsules in a concentration of 50000 IU/capsule (a product of El Kahira pharmaceutical company). Each capsule was dissolved in 10 ml of olive oil to obtain a concentration of 5000 IU/ml. It was given in a dose of 5000 IU/kg for five days (two days before and three days concurrently with methotrexate injection) for one week in short term treated group and for four weeks in long term treated group. All medications were given by intraperitoneal injection.

Animals
The present study was carried on 72 adult male albinos Sprague Dawely rats weighing 180-220 grams each. The animals were housed in separate cages (five rats per cage) under standard laboratory and environmental conditions with free access to food and water. They were obtained from the animal house of the Faculty of Medicine, Cairo University. Only male rats were used in this study to exclude the possible sex differences. The rats were acclimatized in the laboratory for a period of two weeks before carrying out the drug administration. The rats were divided into six equal groups 12 rats each. In all groups the animals were sacrificed 24 hours after the last injection.

Group I (Normal control):
The rats of this group received nothing and according to the time of scarification were subdivided into: -Subgroup Ia: The rats were sacrificed after one week.
-Subgroup Ib: The rats were sacrificed after four weeks.

Group II (Sham control):
The rats of this group were subdivided into 2 subgroups 6 rats each: -Subgroup IIa: The rats received saline solution in a dose of 0.2 ml/Kg body weight for three days [9].
-Subgroup IIb: The rats received saline solution in a dose of 0.2 ml/ Kg bodyweight for three successive days/week for four weeks [9].  (Figures 6c and 6d), ghost nuclei (Figure 6c), These measurements were done using an objective lens of magnification 40, i.e. of total magnification 400. The total area of fibrosis in each specimen was measured and the mean values for each group were obtained. The statistical package for the social science (SPSS version 7.5) was used on data analysis. Data was expressed as mean ± SD. One way analysis of variance (ANOVA) was used.

Results
Histological and Immunohistochemical results for control and sham control groups (groups I and II) Light microscopic examination: The results of control and sham control are the same. The villi appeared as finger like projections thrown into the lumen (Figures 2a and 2b) and lined by a single layer of tall columnar cells with oval basal nuclei (Figure 2c). The lamina propria of villi covered by a single layer of tall columnar cells with oval basal nuclei (Figures 2b and 2d). The goblet cells were scattered

Morphometric measurements
All the measurements of the jejunum regarding the different experimental groups were summarized in Table 1.

The Villus height:
The villus height of short term methotrexate treated group (group III) decreased significantly in comparison  with the corresponding control group. With treatment with vitamin A (group V), there was a significant increase in villus height. With long duration of exposure to methotrexate (group IV), there was a significant decrease in the villus height when compared with the corresponding control group and insignificant increase in the villus height when compared with short term methotrexate treated group (group III). Much improvement was observed in the villus height in long term methotrexate and vitamin A treated group (group VI) when compared with both short term methotrexate treated group (group III) and long term methotrexate treated group (group IV).

The Crypts depth:
The crypts depth of short term methotrexate treated group (group III) increased significantly in comparison with the corresponding control group. Concomitant treatment with vitamin A (group V), there was a significant decrease in crypts depth. With long duration of exposure to methotrexate (group IV), there was a significant increase in the crypts depth when compared with both the corresponding control group and short term methotrexate treated group (group III). Much improvement was observed in the crypts depth in long term methotrexate and vitamin A treated group (group VI) when compared with both short term methotrexate   treated group (group III) and long term methotrexate treated group (group IV).

The number of goblet cells:
The number of goblet cells of short term methotrexate treated group (group III) decreased significantly in comparison with the corresponding control group. With concomitant treatment with vitamin A (group V), there was a significant increase in the number of goblet cells. With long duration of exposure to methotrexate (group IV), there was a significant decrease in the number of goblet cells when compared with the corresponding control group and insignificant decrease in the number of goblet cells when compared with short term methotrexate treated group (group III). There was a significant increase observed in the number of goblet cells in long term methotrexate and vitamin A treated group (group VI) when compared with both short term methotrexate treated group (group III) and long term methotrexate treated group (group IV).

The mean area percentage of fibrosis:
The mean area percentage of fibrosis of short term methotrexate treated group (group III) increased significantly in comparison with the corresponding control group. With treatment with vitamin A (group V), there was a significant decrease in the mean area percentage of fibrosis. With long duration of exposure to methotrexate (group IV), there was a significant increase in the mean area percentage of fibrosis when compared with the corresponding control group and insignificant increase in the mean area percentage of fibrosis when compared with short term methotrexate treated group (group III). Much improvement was observed in the mean area percentage of fibrosis in long term methotrexate and vitamin A treated group (group VI) when compared with both short term methotrexate treated group (group III) and long term methotrexate treated group (group IV).

The optical density of the alkaline phosphatase enzyme activity:
The optical density of the alkaline phosphatase enzyme activity of short term methotrexate treated group (group III) decreased significantly in comparison with the corresponding control group. With concomitant treatment with vitamin A (group V), there was a significant increase in the optical density of the alkaline phosphatase enzyme activity. With long duration of exposure to methotrexate (group IV), there was a significant decrease in the optical density of the alkaline phosphatase enzyme activity when compared with the corresponding control group and insignificant decrease in the optical density of the alkaline phosphatase enzyme activity when compared with short term methotrexate treated group (group III). The optical density of the alkaline phosphatase enzyme activity in long term methotrexate and vitamin A treated group (group VI) was improved when compared with short term methotrexate treated group (group III) and long term methotrexate treated group (group IV) but the improvement was insignificant.

Discussion
Methotrexate (MTX) is one of the most effective antineoplastic drugs inspite of its undesirable side effects on the different organs. Methotrexate was a structurally related to folic acid and acted as an antagonist to that vitamin [12]. Lower doses of MTX had been shown to be very effective for the management of rheumatoid arthritis and psoriasis due to the inhibition of enzymes involved in purine metabolism, leading to accumulation of adenosine or the inhibition of T cell activation and suppression of intercellular adhesion molecule expressed by T cells [13].
In the present study, the short and long term methotrexate treated groups showed the presence of fusion of villi and loss of intervillus spaces. This was in agreement with several authors [4,14] who observed fusion of the villi in rats treated with MTX. With long duration of exposure to the drug the fusion of the villi was seen more obvious with loss of intervillus space with subsequent decrease in the surface area of absorption from the jejunum. This finding might be attributed to the direct irritant, cytotoxic and oxidative effect of the drug as previously reported [5]. One of the most characteristic finding of short term methotrexate treated group (group III) in the present work was the loss of villus architecture. Similar finding was noticed by many reseaerches [15,16] who suggested that the folic acid analogue, MTX, primarily inhibited DNA synthesis by binding to the enzyme dihydrofolate reductase which might lead to an inhibition of proliferation in the crypts of the small intestine.
Garipardic et al. [17] believed that the oxidative stress played an important role in MTX induced mucosal damage and also, fibrosis might cause loss of villus architecture.
In the current work, most of the villi were covered with flattened or cuboidal cells in long term methotrexate treated group (group IV) as previously reported [18]. This might be related to the antimitotic effect of MTX with wide spread apoptosis of the stem cells in the villus tip [16]. Blunting of the apices of the villi was noticed in the current study in short term methotrexate treated group (group III) and progressed to loss of the villi in long term methotrexate treated group (group IV). This was in agreement with previous reports [16,19] suggesting that the injury to the villous tip was due to exposure of the mucosa to the erosive effect of pancreatic and biliary secretions causing aut-odigestion of the intestinal epithelium. Sukhotnik et al. [20] also observed strong inhibitory effects of MTX on enterocyte proliferation using brumodeoxy-uridine (BrdU), a marker for cell proliferation, which was considered as a major mechanism responsible for decreased intestinal cell mass and mucosal hypoplasia with loss of villi.
Complete shedding of the surface epithelium was a characteristic finding in the present study in both short and long term methotrexate treated groups. This was in agreement with Jahovic et al. [21] who found desquamation of surface epithelium in rats treated with MTX. Some authors [5] suggested that this finding was due to the cytotoxic effect of MTX on the intestinal cells while others [18] related it to the inhibition of the cell proliferation in the small intestinal mucosa. The mucosal damage in the present study progressed to ulcer formation in long term methotrexate treated group (group IV). This was also detected by Gao and Horie [22] who noticed neutrophil aggregation in the intestinal mucosa after MTX administration to rats and suggested that neutrophil accumulation in the intestinal mucosa played a key role in the potentiating action of MTX-induced ulceration. Ulceration of the jejunal mucosa noticed in this work might also be due to the decreased number of goblet cells in MTX treated rats causing deficiency in the secretion of mucus by these cells affecting their protective role. In this study, it was found that the alkaline phosphatase enzyme activity of short and long term methotrexate treated groups decreased and the decrease was statistically significant in comparison with the corresponding control group. This was in agreement with many studies [4,23,24] that attributed this finding to the oxidative stress that caused inactivation of the enzyme and the affection of the brush border of the villi. The significant decrease in the activity of alkaline phosphatase enzyme encountered in the present work might be due to the inhibitory effect of MTX on DNA synthesis [15] with subsequent decrease in protein formation resulting in decreased enzyme synthesis. Intravillus hemorrhage was observed in the current study in short term methotrexate treated group (group III). This was also observed by Kremer [25] who attributed that to the increased transendothelial and transepithelial permeability causing inflammation, on the other hand Garipardic et al. [17] discussed that these changes may be due to the antiplatelet action of nitrous oxide or the folic acid deficiency that might cause severe anemia and bleeding.
Distortion of the crypts was also noticed in the present study in short and long term methotrexate treated groups. This was also observed in one study [18] which explained this by inhibition of proliferation of the cells in the crypts of the small intestine.
Cystic dilatation of the crypts in the present work, with flattening of its epithelial lining was observed in short and long term methotrexate treated groups. Similar finding was detected and explained by the antimitotic effect of MTX with wide spread apoptosis of the stem cells in the crypts [11,14].
In the current study, the crypts depth of short and long term methotrexate treated groups increased significantly in comparison with that of the corresponding control group while the villus height decreased significantly in both groups. Similar findings were reported by several authors [16,26] in rats treated with MTX. However other studies [27,28] related this finding to a reduction of intestinal mucosal protein and DNA contents together with starting recovery by proliferation of crypt epithelium. In disagreement with these findings, some studies [4,29] reported significant decrease in the crypts depth due to cytotoxicity of the drug and the resulting inflammation.
In the present study, the short and long term methotrexate treated groups showed dilated blood vessels. Similar findings were found by many previous studies [16,17]. This might be due to inflammation associated with MTX treatment that increased the transendothelial and transepithelial permeability [25]. Marked cellular infiltrate in the lamina propria was observed in the long term methotrexate treated group (group IV) in the present work. This was in agreement with many previous studies [6,11,16] who observed cellular infiltration in the lamina propria after MTX administration. Miyazono et al. [5] explained that this infiltration to the reactive oxygen species (ROS) production. In this study, it was found that the number of goblet cells of short and long term methotrexate treated groups decreased and the decrease was statistically significant in comparison with that of the corresponding control group. This was in agreement with many authors [4,14,16] who found depletion of goblet cells and attributed that to the antimitotic effect of MTX. In contrast, Xian et al. [30] reported that there were repopulation and accumulation of the goblet cells four days after MTX treatment and interpreted that to the commencement of epithelial proliferation.
One of the most characteristic finding of short term methotrexate treated group (group III) in the present work was the presence apoptotic like lesion. Similar finding was reported by different studies [31][32][33].
Cellular vacuolations were also noticed in the current study in the short and long term methotrexate treated groups. This was in agreement with Leitão et al. [34] who noticed the same finding under transmission electron microscope and referred these vacuolations to the occurrence of large residual bodies (secondary lysosomes) containing partially degraded fragments of damaged epithelial cells. Cells showing cytoplasmic and nuclear degeneration with disappearance of their cellular boundaries (ghosts' nuclei) in addition to cellular pyknosis were noticed in this study in the short term methotrexate treated group (group III) and karryorehxis was seen in long term methotrexate treated group (group IV). In agreement with Leitão et al. [34] and Miyazono et al. [5] such finding might be attributed to the oxidative stress.
In the present study, increased amount of collagen fibers in the submucosa and among the villi was observed in the jejunum of rats of short term methotrexate treated group (group III) and this fibrosis was more obvious in the jejunum of rats of the long term methotrexate treated group (group IV). This was in agreement with Dadhania et al. [4] who found that there was a significant increase in the collagen fibers in the short and long term methotrexate treated group when compared with that of the corresponding control group. The possible cause of this fibrosis was either due to the direct toxic effect of the drug or a result of the inflammation [33]. The present work showed that the use of vitamin A in conjunction with methotrexate (groups V and VI) improved partially the histological finding that occurred in the cells of the jejunum as compared to those of the methotrexate treated groups. The improvement was in the form of few shedded cells at the tip of the villi, numerous goblet cells, partial restoration of villus architecture, and significant increase of the villus height, increased mitotic activity and nearly intact brush border. Almost normal crypts lined by columnar epithelium with basal oval nuclei, increased number of crypts, significant decrease of the crypts depth and few cells shedded inside the crypts were also noticed. Similar findings were reported by different authors [11,14,16]. This improvement was explained by Yuncu et al. [11] who suggested that vitamin A might be involved in the regulation of DNA and RNA synthesis in the crypt cells. A significant increase in the number of goblet cells was observed in groups V and VI when compared to that seen in both short and long term methotrexate treated groups. This matched with the finding of Antar et al. [16] who believed in the protective role of vitamin A.
In the present study, much improvement was observed in the mean area percentage of fibrosis seen in groups V and VI when compared with that noticed in the methotrexate treated groups. This finding was confirmed statistically by the significant decrease in the area percentage of fibrosis with the use of vitamin A. Similar finding was reported by previous studies [4,6] which confirmed the beneficial antioxidant effects of vitamin A. In the current study, much improvement was noticed in the alkaline phosphatase enzyme activity in short term methotrexate and vitamin A treated group when compared to that observed in the short term methotrexate group (group III). This finding was confirmed statistically by the significantly increased optical density of the alkaline phosphatase enzyme activity following the use of vitamin A. Yasuharu et al. [9] confirmed the protective role of vitamin A and reported that the lipid and protein contents of the small intestinal cells were significantly higher in mice treated with MTX plus vitamin A than in mice treated with MTX alone without affecting the intestinal absorption of MTX.
On the other hand, in long term methotrexate and vitamin A treated group (group VI) the increase in the optical density of the alkaline phosphatase enzyme activity was insignificant when compared with that seen in the long term methotrexate treated group (group IV). Similar finding was reported by Dadhania et al. [4] who attributed this differential protection to the different antioxidant reserve as well as to the adaptive response of the cells at the target site to the threshold of the chemical insult. Further, the lack significant protection in long term study could be attributed to persistence of low level of oxidative stress over long period of time and the change in the pH of intestine. It can be concluded that vitamin A has a protective role against the adverse alterations and improves partially the toxic changes that may occur in the jejunum of the adult albino rats following short and long term methotrexate therapy and it is advised to be given prior and throughout the methotrexate therapy.