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Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study
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Journal of Microscopy and Ultrastructure xxx (2014) xxx–xxx
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Original Article
Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study Nafisa A. Elbakary, Gihan M. Soliman, Sadika M. Tawfik ∗ , Shaimaa M. Zaher Histology Department, Faculty of Medicine, Tanta University, Egypt
a r t i c l e
i n f o
Article history: Received 29 December 2013 Received in revised form 3 February 2014 Accepted 7 April 2014 Available online xxx
Keywords: Methotrexate Jejumun Vit A Histopathogy Rat
a b s t r a c t Methotrexate (MTX), a folic acid antagonist agent, is mainly used in treatment of malignant tumors and autoimmune diseases. Vitamin A is essential for normal growth and differentiation of epithelial tissues. The aim of this study was to evaluate the effect of methotrexate on the histological structure of jejunal mucosa and to assess the possible protective role of vitamin A. Fifty adult male albino rats divided into three groups; a control, vit A and experimental group subdivided into two groups (B and C), experimental group (B) received 4 mg of methotrexate intraperitoneally as a single dose, and experimental group (C) received methotrexate and 1000 IU of vitamin A given three days before MTX administration and continued until the rats were sacrificed. The specimens were prepared for histological, immunohistochemical and ultrastructure examination. Morphometric study was also done and statistical results were analyzed. Light and electron microscopic studies revealed structural alterations in group (B) in the form of disturbance of villus and crypt architecture. Dilatation of RER cisternae, swollen mitochondria with destroyed cristae vacuolation of the cytoplasm and lipid droplets in some cells were observed with widening of the intercellular space. Corrugation and indentation of the nuclear membrane and exfoliation of epithelial cells. PCNA showed a significant decrease in the number of PCNA-positive nuclei in the crypts of group B. Co-administration of vitamin A with methotrexate (group C) ameliorated these histological changes and there was a increase in the number of PCNA positive nuclei in the crypts. Methotrexate caused mucosal damage improved with vitamin A co-administration. © 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.
1. Introduction Methotrexate (MTX) is a folic acid antagonist. It belongs to the antimetabolites group of cytotoxic drugs. They are S phase-specific agents that are most effective in rapidly
∗ Corresponding author. Tel.: +20 01001085481. E-mail address: sadika [email protected] (S.M. Tawfik).
growing tumors. In addition to their cytotoxic effects on neoplastic cells, the antimetabolites also have immunosuppressant actions. Methotrexate remains the only antifolate agent used for clinical immunosuppression to date. It is a weak dicarboxylic organic acid and is negatively charged at neutral PH, resulting in limited lipid solubility [1–4]. The cytotoxic effect of MTX is not selective for the cancer cells, as it also affects the normal tissues that have a high rate of proliferation, including the haematopoietic cells of
http://dx.doi.org/10.1016/j.jmau.2014.04.002 2213-879X/© 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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Fig. 1. (A) A photomicrograph of jejunum of control group showing mucosa (M), submucosa (S), musculosa (star) and serosa (→) (Hx.&E. Mic. Mag. × 200). (B) Higher magnification of A showing finger-like villi ( ) and simple tubular crypts of Liberkühn opening at the base of the villi (C). The villi are covered with simple columnar cells with oval nuclei and acidophilic cytoplasm (→). Each villus shows a core of lamina propria (L). Notice the presence of goblet cells in between the columnar cells (G) (Hx.&E. Mic. Mag. × 400).
the bone marrow and the actively dividing cells of the gut mucosa. The major toxic effects of MTX is intestinal injury and enterocolitis. Also anorexia, nausea, vomiting, stomatitis, ulceration and mucositis are common [5–7]. Vitamin A is essential for normal growth and differentiation of epithelial tissues. It regulates intestinal epithelial cell proliferation and regeneration. Vitamin A participates in the important metabolic steps that are involved in immune competence and mucosal integrity. It has potential to prevent chemotherapy-induced mucosal lesions. Vitamin A also plays an important role in the regulation of cell division in the small intestine of the rats [8]. 2. Aim of the work The present work aimed to evaluate the possible protective role of vitamin A on the methotrexate induced histological changes on jejunal mucosa.
Fig. 2. A photomicrograph of rat jejunum of control group showing PAS positive brush border (red arrows) and goblet cells (black arrows). (PAS Mic. Mag. × 400).
3. Materials and methods This work was carried out on 50 adult male albino rats. Their weights ranged between 150 and 200 g for each animal. All the animals were housed in suitable clean properly ventilated cages under similar conditions and were fed on a similar commercial laboratory diet and water. They were acclimatized to their environment for one week before starting the experiment. The drug used in this study was methotrexate produced by EIMC United Pharmaceutical (Egypt) in the form of vials. The dose of MTX given was 20 mg/kg [9], intraperitoneally as a single dose. Vitamin A was produced by Kahira Pharm. & Chem. Ind. Co. Cairo (Egypt) in the form of soft gelatin capsules. Each capsule contains 50,000 IU and the dose that was given was 5000 IU/kg. Each capsule was emptied and dissolved in 25 ml of sunflower oil [10], so each 0.5 ml of the prepared solution contained about 1000 IU for each rat given
Fig. 3. A photomicrograph of rat jejunum of control group showing PCNA positive nuclei in the crypt region, the positive nuclei of PCNA attained brown coloration (immunostaining for PCNA and hematoxylin counter stain ×1000).
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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Fig. 4. A photomicrograph of rat jejunum of subgroup (B1) showing disrupted villus and crypt architecture, some of the villi are short and broad, others are fused (bifid arrow). ALSO, crypt loss (star) and formation of crypt cyst (curved arrow) with desquamated epithelial cells inside it (→). Enterocytes appear swollen with rounded nuclei (thick arrow). Notice heavy inflammatory cellular infiltration (I) in the lamina propria (Hx.&E. Mic. Mag. × 400).
orally through a gastric tube every day and continued until rats were sacrificed. The animals were divided into: 1. Control group: included 30 rats that were further subdivided into three subgroups. - Subgroup 1: (included 10 rats) for histological and ultrastructural study of the normal mucosal structure. They were kept without treatment for the same periods as experimental animals (5 rats for 2 days and 5 rats for 6 days). - Subgroup 2: (included 10 rats) that were given 0.5 ml of sunflower oil (the dissolving vehicle for vitamin A) by a gastric tube once daily for the same periods as experimental animals (5 rats for 2 days and 5 rats for 6 days). 2. Group 2 (vitamin A-treated group): included 10 rats that were further subdivided into two subgroups (5 rats each). - Subgroup A1: (5 rats) were given 1000 IU (0.5 ml of the prepared solution) of vitamin A orally by a gastric tube once daily for 5 days. - Subgroup A2: (5 rats) were given 1000 IU (0.5 ml of the prepared solution) of vitamin A orally by a gastric tube once daily for 9 days. 3. Experimental groups: included 20 rats it is subdivided into two experimental groups.
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Fig. 5. A photomicrograph of rat jejunum of subgroup (B1) showing decrease in the number of goblet cells (black arrows) and interrupted stained brush border (red arrow) (PAS Mic. Mag. × 200).
- Experimental group-B (methotrexate-treated group): included 10 rats that were further subdivided into two subgroups (5 rats each). - Subgroup B1: (5 rats) were given 4 mg (0.16 ml) of methotrexate intraperitoneally as a single dose and sacrificed 2 days after MTX injection. - Subgroup B2: (5 rats) were given 4 mg (0.16 ml) of methotrexate intraperitoneally as a single dose and sacrificed 6 days after MTX injection. - Experimental group-C (methotrexate and vitamin A-treated group): included 10 rats that were further subdivided into two subgroups (5 rats each). - Subgroup C1: (5 rats) were administrated 4 mg (0.16 ml) of MTX intraperitoneally as a single dose. Three days before MTX administration, each rat was given 1000 IU (0.5 ml of the prepared solution) of vitamin A orally by a gastric tube once daily and continued until rats were sacrificed 2 days after MTX injection [10]. - Subgroup C2: (5 rats) were administrated 4 mg (0.16 ml) of MTX intraperitoneally as a single dose. Three days before MTX administration, each rat was given 1000 IU (0.5 ml of the prepared solution) of vitamin A orally by a gastric tube once daily and continued until rats were sacrificed 6 days after MTX injection [10]. At the end of each period of the experiment, the jejunum specimens were taken 5 cm far from the end of duodenum and prepared for light and electron microscopic examination.
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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3.2. Electron microscopic study The jejunal specimens were cut in small pieces of 1 mm2 size and fixed in 2.5% glutaraldehyde for 24 h. Specimens were washed in 0.1 M phosphate buffer at 4 c, then post fixed in 1% osmium tetraoxide at room temperature. Specimens were dehydrated in ascending grades of ethyl alcohol, then embedded in Epon resin. Semithin sections (1 m) were stained with toluidine blue in borax and examined with light microscope. Ultrathin sections (50 nm) were cut, mounted on copper grids and stained with uranyl acetate and lead citrate [14,15]. Specimens were examined and photographed with JoeL 100SX transmission electron microscope at EM unit Faculty Of Medicine Tanta University. Fig. 6. A photomicrograph of rat jejunum of subgroup (B1) showing apparent decrease in PCNA positive nuclei in the crypt region (immunostaining for PCNA and hematoxylin counter stain × 400).
The specimens were taken and immediately fixed in 10% formol saline solution for preparation of paraffin blocks. Sections of 5 m in thickness were cut by the microtome for histological study and then stained by the following methods [11,12]:
3.3. Morphometric study and statistical analysis 3.3.1. Morphometric analysis for the mean heights of jejunal villi The heights of jejunal villi (from the tip of the villus to the villus-crypt junction) were measured (in m) in Hx.&E. stained sections. For each specimen 10 randomly different microscopic fields were measured at a magnification power of (×10) objective.
1. Hematoxylin and eosin (Hx.&E.): used for the study of general histological features. 2. Periodic Acid Schiff reagent (PAS): used for detection of neutral mucopolysaccharides.
3.3.2. Morphometric analysis for the mean crypt depths The crypt depth was measured (in m) in Hx.&E. stained sections. For each specimen 10 randomly different microscopic fields were measured at a magnification power of (×10) objective.
3.1. Immunohistochemical study
3.3.3. Morphometric analysis for the mean number of goblet cells The PAS-stained goblet cells were counted under a magnification power of (×20) objective in both villi and crypts. For each specimen a total of 10 randomly microscopic fields of crypts and 10 fields of villi were measured per animals. This was done using Image Analysis System (Leica Q 500) in National Research Center-Pathology Department
Detection of proliferating cell nuclear antigen (PCNA) of the crypt cells in the crypt region was performed using PCNA monoclonal antibody (clone pc 10) from Labvision Inc., Fremont, CA, USA. The avidin–biotin complex technique was used. Negative control staining was done after omission of the primary antibody [13].
Fig. 7. A photomicrograph of rat jejunum of subgroup (B2) showing villus shortening (), fusion (→) and kinking ( lamina propria and crypt cyst formation (curved arrows) (Hx.&E. Mic. Mag. × 200).
), inflammatory infiltration (I) in the
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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Fig. 9. A photomicrograph of rat jejunum of subgroup (B2) showing marked depletion of goblet cells (black arrow) and brush border (red arrows) (PAS Mic. Mag. × 200).
Fig. 8. A photomicrograph of rat jejunum of subgroup (B2) showing desquamation and destruction of surface epithelium ( ). Crypt cyst formation (curved arrow) with desquamated epithelial cells inside it (→) (Hx.&E. Mic. Mag. × 400).
[6,16]. The data were analyzed by ANOVA test (f-test). Differences were regarded as significant if p < 0.05. 4. Results 4.1. Light microscopic studies 4.1.1. Control and vitamin A treated group Examination of sections obtained from rat jejunum of the control and vitamin A treated groups stained with hematoxylin & eosin showed the same histological picture which was identical to the known histological structure of the jejunum. The jejunal mucosa was formed of epithelium, lamina propria and muscularis mucosa. The mucosa appeared as finger-like projections with a core of loose connective tissue containing mononuclear cells (villi) and invaginations in the lamina propria forming simple tubular glands (crypts of Libierkühn). The epithelial cells covering the villi were tall columnar cells with basal oval nuclei and acidophilic cytoplasm. Goblet cells appeared as clear empty spaces in between the columnar cells (Fig. 1). PAS stained sections obtained showed a PAS positive brush border of columnar absorptive cells and numerous goblet cells appeared magenta red (Fig. 2). Immunohistochemical staining for control and vitamin A treated group for PCNA positive cells showed numerous PCNA positive nuclei in the crypts. The positive nuclei of PCNA attained brown coloration (Fig. 3).
4.1.2. Experimental group 4.1.2.1. Group B. Subgroup B1. Examination of Hx.&E. stained sections of subgroup B1 (animals treated with single dose of MTX and sacrificed after 2 days) showed disturbance of normal villus architecture and disrupted crypt areas. Some villi appeared shortened and broad while others appeared fused. Disrupted crypt area with crypt loss and formation of crypt cyst with desquamated epithelial cells in its lumen. Enterocytes appeared swollen with rounded nuclei. Heavy inflammatory cellular infiltration was observed in the lamina propria (Fig. 4). PAS-stained sections obtained from this group revealed decrease in the number of goblet cells and interrupted stained brush border (Fig. 5). Immunohistochemical staining for PCNA showed apparent decrease in the numbers of PCNA positive nuclei in the crypts as compared to the control group (Fig. 6). Subgroup B2. Examination of sections obtained from the jejunum of animals of subgroup B2 (animals treated with single dose of MTX and sacrificed after 6 days) and stained with hematoxylin & eosin showed severe changes
Fig. 10. A photomicrograph of rat jejunum of subgroup (B2) showing marked decrease in the PCNA positive nuclei in the crypt region (immunostaining for PCNA and hematoxylin counter stain 400×).
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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Fig. 11. A photomicrograph of rat jejunum of subgroup (C1) showing abnormal shaped villus (), fusion of the villi (→) and inflammatory cellular infiltration (I) in the lamina propria (Hx.&E. Mic. Mag. × 400).
in the jejunal mucosa in the form of shortening, fusion and kinking of the villi, inflammatory infiltrate in the lamina propria and crypt cyst formation. Desquamation and destruction of surface epithelium and desquamated epithelial cells in the lumen of some crypt cysts could also be noticed (Figs. 7 and 8). PAS-stained sections obtained from this group revealed marked depletion of goblet cells and decrease intensity of PAS reaction at brush border (Fig. 9). Immunohistochemical staining for PCNA showed apparent decrease in the PCNA positive nuclei in the crypts as compared to group B1 (Fig. 10). 4.1.2.2. Group-C. Subgroup C1. Examination of sections obtained from the jejunum of animals of subgroup C1 (animals that were concomitantly treated with both methotrexate and vitamin A and sacrificed after 2 days of MTX injection) and stained with hematoxylin & eosin showed increase in the villus height. While, there were areas showing abnormal shaped villus and fusion of the villi. In addition there were broad and short villi and inflammatory cellular infiltration in the lamina propria (Figs. 11 and 12). PAS-stained sections obtained from this group showed increase in the number of goblet cells and increased intensity of PAS reaction at the brush border (Fig. 13). Immunohistochemical staining for PCNA showed apparent increase in the numbers of PCNA positive nuclei as compared to group B1 (Fig. 14). Subgroup C2. Examination of hematoxylin & eosin sections obtained from the jejunum of animals of subgroup C2 (animals that were concomitantly treated with both methotrexate and vitamin A and sacrificed after 6 days of MTX injection) showed increase in the villus height and improved crypt architecture. While, there were areas showing distortion of epithelial lining of villi, inflammatory cellular infiltration in the lamina propria and shortening of some villi (Fig. 15). PAS-stained sections obtained from this group showed increase in the number of goblet cells both in the villi and
Fig. 12. A photomicrograph of rat jejunum of subgroup (C1) showing broad and short villi (). Notice inflammatory cellular infiltration (I) in the lamina propria. (Hx.&E. Mic. Mag. × 400).
crypts and increased intensity of PAS reaction at the brush border (Fig. 16). Immunohistochemical staining for PCNA showed apparent increase in the numbers of PCNA positive nuclei as compared to group B2 (Fig. 17). 4.2. Electron microscopic study 4.2.1. Control and vitamin A treated groups Ultrathin sections obtained from rat jejunum of control and Vitamin A treated groups showed the same picture which was identical with the normal ultrastructure of the jejunum. The columnar absorptive cells were regularly arranged and contained basal, oval and euchromatic nuclei. They had continuous regular microvillus border, abundant elongated mitochondria and extensive lateral interdigitations. Intercellular junctions were present between adjacent cells and diffuse appearance of the terminal web in addition to the presence of RER cisternae and free ribosomes in the cytoplasm were also detected. Goblet cells were insinuated between enterocytes. The apical wide part of each goblet cell was distended with moderately electrondense mucin secreting granules (Figs. 18 and 19). 4.2.2. Experimental group 4.2.2.1. Group B. Subgroup B1. Examination of ultrathin sections of the jejunum obtained from this subgroup
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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vacuole that caused indentation of an enterocyte nucleus and exfoliation of epithelial cells were detected. Destruction of the luminal surface of goblet cells was also observed (Figs. 23 and 24).
Fig. 13. A photomicrograph of rat jejunum of subgroup (C1) showing increased number of goblet cells (black arrow) and increased intensity of PAS reaction at the brush border (red arrow). (PAS Mic. Mag. × 200).
revealed enterocytes with dilatation of RER cisternae, vacuolization of the cytoplasm and lipid droplets in some focal areas. Widening of the intercellular space and short sparse loosely packed and irregularly arranged microvilli of enterocytes with areas of focal loss of microvillus border were detected. The mitochondria became swollen with destroyed cristae. Moreover, there were corrugation and indentation of the nuclear membrane in some enterocytes (Figs. 20–22). Subgroup B2. Ultrathin sections of the jejunum obtained from this subgroup revealed decreased heights of some enterocytes with short sparse microvilli and focal loss of microvillus border in addition to rarified and vacuolated cytoplasm of some enterocytes. Large cytoplasmic
4.2.2.2. Group-C. Subgroup C1. Examination of ultrathin sections obtained from rat jejunum of subgroup C1 showed some enterocytes regained their normal ultrastructure. They had closely packed regularly arranged microvilli and intact lateral interdigitations, some mitochondria were still swollen with destroyed cristae. The examination revealed a goblet cell, with dilated apical part and containing mucinsecreting granules, insinuated between enterocytes. RER cisternae of enterocytes appeared relatively as the control (Fig. 25). Subgroup C2. Ultrathin sections obtained from rat jejunum of subgroup C2 showed some enterocytes regained their normal heights with regularly arranged but loosely packed microvilli. Some enterocytes lose their microvillus border. Also, the examination revealed elongated mitochondria and intact lateral interdigitations and with relatively normal goblet cells were interspersed between enterocytes (Fig. 26).
4.3. Morphometric results and statistical analysis 4.3.1. Statistical results for the mean height of jejunal villi As assessed by image analyzer, vitamin A treated group revealed non-significant change in the mean heights of the villi when compared with control group. The MTX treated group B (including both subgroup B1 and B2) showed statistically highly significant decrease in the mean height of the villi when compared with control group. Concerning the MTX-vitamin A treated group C (including subgroup C1 and C2) it was found that there was still highly significant decrease in the mean height of the villi when compared with control group. Also, there was significant increase in the mean of the villus height compared to group B.
Villus height
Mean ± SD f-test p-value
Control
B1
B2
CI
C2
346.2 ± 51.6
127 ± 33.4
54 ± 18.1 26.523 0.001
176.5 ± 45.2
154 ± 25.5
Scheffe test Control and BI
Control and B2
Control and C1
Control and C2
0.001*
0.001*
0.001*
0.001*
350
mean value
300 250 200 150 100 50 0 Control
B1
Control
B2
B1
B2
CI
CI
C2
C2
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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4.3.2. Statistical results for the mean crypt depth As assessed by image analyzer, vitamin A treated group revealed non-significant change in the mean crypt depth when compared with control group. The MTX treated group B (including both subgroup B1 and B2) showed statistically highly significant decrease in the mean crypt depth when compared with control group. Concerning the MTX-vitamin A treated group C (including subgroup C1 and C2) it was found that there was still highly significant decrease in the mean crypt depth compared with control group. Also, it was found that statistically highly significant increase the mean crypt depth when compared to group B.
Crypt depth
Mean ± SD f-test p-value
Control
B1
B2
CI
C2
338.8 ± 55.7
150.3 ± 45.8
185.3 ± 64.1 10.362 0.001
183.8 ± 54.5
261.2 ± 68.8
Scheffe test Control and BI
Control and B2
Control and C1
Control and C2
0.001*
0.001*
0.001*
0.001*
350 300
mean value
250 200 150 100 50 0 Control
B1
Control
B2
B1
CI
B2
CI
C2
C2
4.3.3. Statistical results for the mean number of goblet cells As assessed by image analyzer, vitamin A treated group revealed non-significant change in the mean number of
goblet cells when compared with control group. The MTX treated group B (including both subgroup B1 and B2) showed statistically highly significant decrease in the mean number of goblet cells when compared with control group. Concerning the MTX-vitamin A treated group C (including subgroup C1 and C2) it was found that there was still highly significant decrease in the mean number of goblet cells when compared with control group. Also, it was found that statistically highly significant increase the mean number of goblet cells when compared to group B.
Goblet cell number
Mean ± SD f-test p-value
Control
B1
B2
CI
C2
144.2 ± 6.52
53 ± 4.5
25 ± 3.2 9.362 0.001
122 ± 7.5
96 ± 8.8
Scheffe test Control and BI
Control and B2
Control and C1
Control and C2
0.001*
0.001*
0.001*
0.001*
160
mean value
140 120 100 80 60 40 20 0
Control
B1 Control
B2 B1
B2
CI CI
C2
C2
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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Fig. 14. A photomicrograph of rat jejunum of subgroup (C1) showing an increase in PCNA positive nuclei in the crypt region as compared to subgroup B1 (immunostaining for PCNA and hematoxylin counter stain ×400).
4.3.4. Light microscopic results 4.3.5. Electron microscopic results 5. Discussion Methotrexate (MTX) is one of the antimitotic drugs. These drugs act by blocking deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, inducing apoptosis and slowing down or stopping cell proliferation in rapidly proliferating cells such as tumors, bone marrow and gut mucosa cells [17–19] Sufficient vitamin A in the diet maintain the epithelium of gastrointestinal tract which in turn helps the maximal absorption of nutrients, resulting in improved weight gain. In several studies, it was shown that vitamin A may directly regulate intestinal epithelial cell proliferation and regeneration [20–22]. It has been also suggested that vitamin A coadministration prevents MTX-induced decrease in the contents of the small intestinal membrane protein and lipid and preserves partly the activity of thymidine kinase,
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which is a key enzyme in the salvage pathway of DNA synthesis [8]. In this study, the jejunum was chosen as a representative for the small intestine because the epithelial morphology and proliferation are nearly the same along the whole small intestinal axis. Also, the proximal small intestine is the most sensitive region of rat gut. In addition, active absorption of MTX occurs in proximal jejunum [4,23] Normally the crypt cells, which are rapidly generated, migrate to the villus tip and the replacement of the intestinal epithelium is completed in 3 days in humans and in 2 days in rats [24]. Intestinal mucosal damage and barrier function alterations have been described as consequences of different processes: apoptosis, hypoproliferation, inflammatory response, altered absorptive capacity and bacteria proliferation and colonization [25,26] In this research, the experimental group (B) that received MTX as a single dose showed disturbance in villus architecture, crypt loss and crypt cyst formation, epithelial separation with goblet cell depletion and inflammatory cellular infiltration in the lamina propria. These changes were in consistent with [7,27–30] who described similar results in their studies. These changes could be attributed to direct toxic effect of methotrexate on the jejunal mucosa or generation of reactive oxygen species (ROS). As lipid peroxidation results from binding of ROS to cellular macromolecules, particularly membrane lipids and polyunsaturated fatty acids in the endoplasmic reticulum and cellular membranes, nucleotides in DNA and sulfhydryl bonds in proteins [31,32]. This was parallel to previous findings of many investigators who reported that lipid peroxidation is an important cause of destruction and damage to cell membranes and may be a contributing factor in the development of MTX-mediated tissue damage [33–35] The villus damage caused by MTX may be due to reduced antioxidant enzymes capacity, resulting in increased sensitivity of cells to ROS related injury [6,36]. In addition to the direct damaging effects of reactive oxygen species on the tissues, they trigger the accumulation of activated
Fig. 15. A photomicrograph of rat jejunum of subgroup (C2) showing (A) increase in villus height ( ) and improved crypt architecture (C). Distortion of the epithelial lining of some villi (curved arrow) and inflammatory cellular infiltration (I) in the lamina propria (Hx.&E. Mic. Mag. × 200). (B) Some villi are short (arrow head), apparent increase in the number of goblet cells (G) with improvement of the crypts architecture (bifid arrows) (Hx.&E. Mic. Mag. × 400).
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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Fig. 16. A photomicrograph of rat jejunum of subgroup (C2) showing (A) increased number of goblet cells in villi and crypts (→) (PAS Mic. Mag. × 200). (B) Increase intensity of PAS reaction at the brush border (PAS Mic. Mag. × 400).
Fig. 17. A photomicrograph of rat jejunum of subgroup (C2) showing an increase in PCNA positive nuclei in the crypt region as compared to subgroup (B2). (immunostaining for PCNA and hematoxylin counter stain ×400).
leukocytes in the tissues which secrete enzymes such as myeloperoxidase, elastase and proteases and liberate more free radicals. It was reported that myeloperoxidase activity increased in the MTX-treated rats [37–39]. ROS in the intestinal mucosa disrupts microcirculation and plays a key role in desquamation of epithelial cells [40]. Citrulline is a non protein amino acid synthesized by enterocytes and responsible for mucosal integrity and enterocyte mass [41]. It was reported that the concentration of citrulline is reduced in MTX-treated rats. This may explain the reduction of the enterocyte mass [42]. The observed ultrastructural changes in enterocytes in MTX treated group were summarized in the form of dilatation of RER cisternae, vacuolation of the cytoplasm, focal accumulation of lipid droplets, swollen mitochondria with destroyed cristae, short sparse microvilli with focal loss of microvillus border, widening of intercellular space, some nuclei showed variable degrees of indentation and corrugation of their nuclear membrane.
Fig. 18. An electron micrograph of ultrathin section of rat jejunum of control group showing (A) regularly arranged closely packed microvilli () and elongated mitochondria (M). The lateral plasma membranes of adjacent cells interdigitate extensively (bifid arrows). (B) Terminal web ( ) and junctional complexes between enterocytes (→) with the extensive lateral interdigitations (bifid arrow). The cytoplasm contains RER (curved arrows) and free ribosomes ().
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Fig. 19. An electron micrograph of ultrathin section of rat jejunum of control group showing a goblet cell (G) insinuated between absorptive cells. Its apical part is distended with mucin-secreting granules (*).
In the present study, the mitochondrial damage could be attributed to ROS which results in formation of a channel called mitochondrial permeability transition pore. Opening of this channel leads to loss of mitochondrial membrane potential and PH changes, resulting in failure of oxidative phosphorylation which leads to progressive adenosine triphosphate (ATP) depletion. As, the mitochondria contain several proteins, including cytochrome C. These proteins are capable of activating apoptotic pathways. Increased permeability of mitochondrial membrane as a result of its damage may result in leakage of these proteins into the cytosol and death of cell by apoptosis [43,44]. Accumulation of sodium in the cell leads to increase in water content and the cell will swell, a condition called hydropic degeneration. This may explain dilatation of RER and vacuolation of the cytoplasm [45]. Sodium-potassiumATPase activity was significantly diminished in the MTX treated rats and as a result the absorptive function of enterocytes was reduced leading to accumulation of nutrients and fluids in the intercellular spaces and widening of intercellular space [9].
Activation of proteases breaks down membrane and cytoskeletal proteins leading to shortening and focal loss of microvilli [46]. It also leads to destruction of intermediate filaments of nuclear lamins causing indentation and corrugation of nuclear membrane in some enterocytes [47]. In the present work focal accumulation of lipid droplets within enterocytes was attributed to impaired protein synthesis as a result of RER damage. Accordingly this leads to inhibition of lipoprotein manufacture resulting in accumulation of fat inside the cytoplasm of affected cells [48]. The light microscopic examination of group C (concomitantly received a single dose of MTX and a daily dose of vitamin A) revealed some improvement in villus and crypt architecture with increase in the villus height and increase in goblet cell number as compared to group B. But there were areas showing villus shortening and fusion, distortion of epithelial lining of some villi and inflammatory infiltration in the lamina propria. Examination of ultrathin sections obtained from this group revealed some improvements of the ultrastructure
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Fig. 20. An electron micrograph of ultrathin section of rat jejunum of subgroup (B1) (A) showing an enterocyte with dilatation of RER cisternae (curved arrows). Notice a granular goblet cell (star). (B) Showing decrease in goblet cell granules (star). Notice the desquamation of enterocyte in the lumen.
Fig. 21. An electron micrograph of ultrathin section of rat jejunum of subgroup (B1) showing (A) vacuolation of the cytoplasm (→) and lipid droplets ( ) with displaced indented nucleus (N) of an enterocyte. (B) Widening of the intercellular space between enterocytes (bifid arrows) with vacuolation of the cytoplasm (→) and lipid droplets ( ).
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Fig. 22. An electron micrograph of ultrathin section of rat jejunum of subgroup (B1) showing (A) short, sparse, loosely packed and irregularly arranged microvilli of enterocytes with focal loss of microvillus border (). Notice condensation of chromatin, shrinkage and corrugation of the nuclear membrane of an enterocyte nucleus (→). Another cell showing fragmentation of its nucleus ( ). (B) Higher magnification of A showing an enterocyte containing swollen mitochondria with destroyed cristae (bifid arrows) and corrugation of the nuclear membrane (→). A large lipid droplet ( ) could be seen.
Fig. 23. An electron micrograph of ultrathin section of rat jejunum of subgroup (B2) showing decreased heights of enterocytes (curved arrow), focal loss of microvillus border () with rarified and vacuolated cytoplasm (*) and inflammatory cellular infiltration (→) in the lamina propria.
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Fig. 24. An electron micrograph of ultrathin section of rat jejunum of subgroup (B2) showing large cytoplasmic vacuoles (*) of an enterocyte with destroyed luminal surface of a goblet cell (→).
of enterocytes confirmed by morphometric and statistical studies. The protective effect of vitamin A on the small intestine may be explained by stimulation of DNA and RNA biosynthesis. Moreover, it was reported that vitamin A is one of antioxidants that may either block the formation of free radicals or scavenger them once they have formed [10,46]. The protective effect of vitamin A could be also attributed to its role in regulation of differentiation of epithelial cells and goblet cells and maintenance of integrity of mucous membranes [49]. As, the preservation of cell proliferation capacity is a key factor in protecting the epithelium from the consequences of MTX-induced
apoptosis. These authors related this protection to the maintenance of goblet cell function [50]. Immunohistochemical study detected decrease in the number of PCNA positive nuclei in group (B) which indicated a decrease in the cell division in the crypt region. This is in accordance with other studies that MTX induce mitotic arrest of intestinal crypt cells [51]. While, there was increase in the number of PCNA positive nuclei in group (C) in relation to group B indicating that vit A plays a role in stimulating cell division in the intestinal mucosa [22] From the present research, it could be concluded that MTX has a toxic effect on the jejunal mucosa as was proved
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Fig. 25. An electron micrograph of ultrathin section of rat jejunum of subgroup (C1) showing enterocytes containing swollen mitochondria with destroyed cristae (bifid arrow). RER cisternae appear within normal (curved arrows). Notice a goblet cell (G) between enterocytes.
Fig. 26. An electron micrograph of ultrathin section of rat jejunum of subgroup (C2) showing some enterocytes with regularly arranged but loosely packed microvilli () and elongated mitochondria (M). Notice the presence of a goblet cell (G) between the enterocytes.
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histologically and most of the changes were reversible with concomitant administration of vit A. So, it is recommended to give Vit A in combination with MTX as a protective agent against its toxicity.
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Original Article
Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study Nafisa A. Elbakary, Gihan M. Soliman, Sadika M. Tawfik ∗ , Shaimaa M. Zaher Histology Department, Faculty of Medicine, Tanta University, Egypt
a r t i c l e
i n f o
Article history: Received 29 December 2013 Received in revised form 3 February 2014 Accepted 7 April 2014 Available online xxx
Keywords: Methotrexate Jejumun Vit A Histopathogy Rat
a b s t r a c t Methotrexate (MTX), a folic acid antagonist agent, is mainly used in treatment of malignant tumors and autoimmune diseases. Vitamin A is essential for normal growth and differentiation of epithelial tissues. The aim of this study was to evaluate the effect of methotrexate on the histological structure of jejunal mucosa and to assess the possible protective role of vitamin A. Fifty adult male albino rats divided into three groups; a control, vit A and experimental group subdivided into two groups (B and C), experimental group (B) received 4 mg of methotrexate intraperitoneally as a single dose, and experimental group (C) received methotrexate and 1000 IU of vitamin A given three days before MTX administration and continued until the rats were sacrificed. The specimens were prepared for histological, immunohistochemical and ultrastructure examination. Morphometric study was also done and statistical results were analyzed. Light and electron microscopic studies revealed structural alterations in group (B) in the form of disturbance of villus and crypt architecture. Dilatation of RER cisternae, swollen mitochondria with destroyed cristae vacuolation of the cytoplasm and lipid droplets in some cells were observed with widening of the intercellular space. Corrugation and indentation of the nuclear membrane and exfoliation of epithelial cells. PCNA showed a significant decrease in the number of PCNA-positive nuclei in the crypts of group B. Co-administration of vitamin A with methotrexate (group C) ameliorated these histological changes and there was a increase in the number of PCNA positive nuclei in the crypts. Methotrexate caused mucosal damage improved with vitamin A co-administration. © 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.
1. Introduction Methotrexate (MTX) is a folic acid antagonist. It belongs to the antimetabolites group of cytotoxic drugs. They are S phase-specific agents that are most effective in rapidly
∗ Corresponding author. Tel.: +20 01001085481. E-mail address: sadika [email protected] (S.M. Tawfik).
growing tumors. In addition to their cytotoxic effects on neoplastic cells, the antimetabolites also have immunosuppressant actions. Methotrexate remains the only antifolate agent used for clinical immunosuppression to date. It is a weak dicarboxylic organic acid and is negatively charged at neutral PH, resulting in limited lipid solubility [1–4]. The cytotoxic effect of MTX is not selective for the cancer cells, as it also affects the normal tissues that have a high rate of proliferation, including the haematopoietic cells of
http://dx.doi.org/10.1016/j.jmau.2014.04.002 2213-879X/© 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. (A) A photomicrograph of jejunum of control group showing mucosa (M), submucosa (S), musculosa (star) and serosa (→) (Hx.&E. Mic. Mag. × 200). (B) Higher magnification of A showing finger-like villi ( ) and simple tubular crypts of Liberkühn opening at the base of the villi (C). The villi are covered with simple columnar cells with oval nuclei and acidophilic cytoplasm (→). Each villus shows a core of lamina propria (L). Notice the presence of goblet cells in between the columnar cells (G) (Hx.&E. Mic. Mag. × 400).
the bone marrow and the actively dividing cells of the gut mucosa. The major toxic effects of MTX is intestinal injury and enterocolitis. Also anorexia, nausea, vomiting, stomatitis, ulceration and mucositis are common [5–7]. Vitamin A is essential for normal growth and differentiation of epithelial tissues. It regulates intestinal epithelial cell proliferation and regeneration. Vitamin A participates in the important metabolic steps that are involved in immune competence and mucosal integrity. It has potential to prevent chemotherapy-induced mucosal lesions. Vitamin A also plays an important role in the regulation of cell division in the small intestine of the rats [8]. 2. Aim of the work The present work aimed to evaluate the possible protective role of vitamin A on the methotrexate induced histological changes on jejunal mucosa.
Fig. 2. A photomicrograph of rat jejunum of control group showing PAS positive brush border (red arrows) and goblet cells (black arrows). (PAS Mic. Mag. × 400).
3. Materials and methods This work was carried out on 50 adult male albino rats. Their weights ranged between 150 and 200 g for each animal. All the animals were housed in suitable clean properly ventilated cages under similar conditions and were fed on a similar commercial laboratory diet and water. They were acclimatized to their environment for one week before starting the experiment. The drug used in this study was methotrexate produced by EIMC United Pharmaceutical (Egypt) in the form of vials. The dose of MTX given was 20 mg/kg [9], intraperitoneally as a single dose. Vitamin A was produced by Kahira Pharm. & Chem. Ind. Co. Cairo (Egypt) in the form of soft gelatin capsules. Each capsule contains 50,000 IU and the dose that was given was 5000 IU/kg. Each capsule was emptied and dissolved in 25 ml of sunflower oil [10], so each 0.5 ml of the prepared solution contained about 1000 IU for each rat given
Fig. 3. A photomicrograph of rat jejunum of control group showing PCNA positive nuclei in the crypt region, the positive nuclei of PCNA attained brown coloration (immunostaining for PCNA and hematoxylin counter stain ×1000).
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Fig. 4. A photomicrograph of rat jejunum of subgroup (B1) showing disrupted villus and crypt architecture, some of the villi are short and broad, others are fused (bifid arrow). ALSO, crypt loss (star) and formation of crypt cyst (curved arrow) with desquamated epithelial cells inside it (→). Enterocytes appear swollen with rounded nuclei (thick arrow). Notice heavy inflammatory cellular infiltration (I) in the lamina propria (Hx.&E. Mic. Mag. × 400).
orally through a gastric tube every day and continued until rats were sacrificed. The animals were divided into: 1. Control group: included 30 rats that were further subdivided into three subgroups. - Subgroup 1: (included 10 rats) for histological and ultrastructural study of the normal mucosal structure. They were kept without treatment for the same periods as experimental animals (5 rats for 2 days and 5 rats for 6 days). - Subgroup 2: (included 10 rats) that were given 0.5 ml of sunflower oil (the dissolving vehicle for vitamin A) by a gastric tube once daily for the same periods as experimental animals (5 rats for 2 days and 5 rats for 6 days). 2. Group 2 (vitamin A-treated group): included 10 rats that were further subdivided into two subgroups (5 rats each). - Subgroup A1: (5 rats) were given 1000 IU (0.5 ml of the prepared solution) of vitamin A orally by a gastric tube once daily for 5 days. - Subgroup A2: (5 rats) were given 1000 IU (0.5 ml of the prepared solution) of vitamin A orally by a gastric tube once daily for 9 days. 3. Experimental groups: included 20 rats it is subdivided into two experimental groups.
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Fig. 5. A photomicrograph of rat jejunum of subgroup (B1) showing decrease in the number of goblet cells (black arrows) and interrupted stained brush border (red arrow) (PAS Mic. Mag. × 200).
- Experimental group-B (methotrexate-treated group): included 10 rats that were further subdivided into two subgroups (5 rats each). - Subgroup B1: (5 rats) were given 4 mg (0.16 ml) of methotrexate intraperitoneally as a single dose and sacrificed 2 days after MTX injection. - Subgroup B2: (5 rats) were given 4 mg (0.16 ml) of methotrexate intraperitoneally as a single dose and sacrificed 6 days after MTX injection. - Experimental group-C (methotrexate and vitamin A-treated group): included 10 rats that were further subdivided into two subgroups (5 rats each). - Subgroup C1: (5 rats) were administrated 4 mg (0.16 ml) of MTX intraperitoneally as a single dose. Three days before MTX administration, each rat was given 1000 IU (0.5 ml of the prepared solution) of vitamin A orally by a gastric tube once daily and continued until rats were sacrificed 2 days after MTX injection [10]. - Subgroup C2: (5 rats) were administrated 4 mg (0.16 ml) of MTX intraperitoneally as a single dose. Three days before MTX administration, each rat was given 1000 IU (0.5 ml of the prepared solution) of vitamin A orally by a gastric tube once daily and continued until rats were sacrificed 6 days after MTX injection [10]. At the end of each period of the experiment, the jejunum specimens were taken 5 cm far from the end of duodenum and prepared for light and electron microscopic examination.
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3.2. Electron microscopic study The jejunal specimens were cut in small pieces of 1 mm2 size and fixed in 2.5% glutaraldehyde for 24 h. Specimens were washed in 0.1 M phosphate buffer at 4 c, then post fixed in 1% osmium tetraoxide at room temperature. Specimens were dehydrated in ascending grades of ethyl alcohol, then embedded in Epon resin. Semithin sections (1 m) were stained with toluidine blue in borax and examined with light microscope. Ultrathin sections (50 nm) were cut, mounted on copper grids and stained with uranyl acetate and lead citrate [14,15]. Specimens were examined and photographed with JoeL 100SX transmission electron microscope at EM unit Faculty Of Medicine Tanta University. Fig. 6. A photomicrograph of rat jejunum of subgroup (B1) showing apparent decrease in PCNA positive nuclei in the crypt region (immunostaining for PCNA and hematoxylin counter stain × 400).
The specimens were taken and immediately fixed in 10% formol saline solution for preparation of paraffin blocks. Sections of 5 m in thickness were cut by the microtome for histological study and then stained by the following methods [11,12]:
3.3. Morphometric study and statistical analysis 3.3.1. Morphometric analysis for the mean heights of jejunal villi The heights of jejunal villi (from the tip of the villus to the villus-crypt junction) were measured (in m) in Hx.&E. stained sections. For each specimen 10 randomly different microscopic fields were measured at a magnification power of (×10) objective.
1. Hematoxylin and eosin (Hx.&E.): used for the study of general histological features. 2. Periodic Acid Schiff reagent (PAS): used for detection of neutral mucopolysaccharides.
3.3.2. Morphometric analysis for the mean crypt depths The crypt depth was measured (in m) in Hx.&E. stained sections. For each specimen 10 randomly different microscopic fields were measured at a magnification power of (×10) objective.
3.1. Immunohistochemical study
3.3.3. Morphometric analysis for the mean number of goblet cells The PAS-stained goblet cells were counted under a magnification power of (×20) objective in both villi and crypts. For each specimen a total of 10 randomly microscopic fields of crypts and 10 fields of villi were measured per animals. This was done using Image Analysis System (Leica Q 500) in National Research Center-Pathology Department
Detection of proliferating cell nuclear antigen (PCNA) of the crypt cells in the crypt region was performed using PCNA monoclonal antibody (clone pc 10) from Labvision Inc., Fremont, CA, USA. The avidin–biotin complex technique was used. Negative control staining was done after omission of the primary antibody [13].
Fig. 7. A photomicrograph of rat jejunum of subgroup (B2) showing villus shortening (), fusion (→) and kinking ( lamina propria and crypt cyst formation (curved arrows) (Hx.&E. Mic. Mag. × 200).
), inflammatory infiltration (I) in the
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Fig. 9. A photomicrograph of rat jejunum of subgroup (B2) showing marked depletion of goblet cells (black arrow) and brush border (red arrows) (PAS Mic. Mag. × 200).
Fig. 8. A photomicrograph of rat jejunum of subgroup (B2) showing desquamation and destruction of surface epithelium ( ). Crypt cyst formation (curved arrow) with desquamated epithelial cells inside it (→) (Hx.&E. Mic. Mag. × 400).
[6,16]. The data were analyzed by ANOVA test (f-test). Differences were regarded as significant if p < 0.05. 4. Results 4.1. Light microscopic studies 4.1.1. Control and vitamin A treated group Examination of sections obtained from rat jejunum of the control and vitamin A treated groups stained with hematoxylin & eosin showed the same histological picture which was identical to the known histological structure of the jejunum. The jejunal mucosa was formed of epithelium, lamina propria and muscularis mucosa. The mucosa appeared as finger-like projections with a core of loose connective tissue containing mononuclear cells (villi) and invaginations in the lamina propria forming simple tubular glands (crypts of Libierkühn). The epithelial cells covering the villi were tall columnar cells with basal oval nuclei and acidophilic cytoplasm. Goblet cells appeared as clear empty spaces in between the columnar cells (Fig. 1). PAS stained sections obtained showed a PAS positive brush border of columnar absorptive cells and numerous goblet cells appeared magenta red (Fig. 2). Immunohistochemical staining for control and vitamin A treated group for PCNA positive cells showed numerous PCNA positive nuclei in the crypts. The positive nuclei of PCNA attained brown coloration (Fig. 3).
4.1.2. Experimental group 4.1.2.1. Group B. Subgroup B1. Examination of Hx.&E. stained sections of subgroup B1 (animals treated with single dose of MTX and sacrificed after 2 days) showed disturbance of normal villus architecture and disrupted crypt areas. Some villi appeared shortened and broad while others appeared fused. Disrupted crypt area with crypt loss and formation of crypt cyst with desquamated epithelial cells in its lumen. Enterocytes appeared swollen with rounded nuclei. Heavy inflammatory cellular infiltration was observed in the lamina propria (Fig. 4). PAS-stained sections obtained from this group revealed decrease in the number of goblet cells and interrupted stained brush border (Fig. 5). Immunohistochemical staining for PCNA showed apparent decrease in the numbers of PCNA positive nuclei in the crypts as compared to the control group (Fig. 6). Subgroup B2. Examination of sections obtained from the jejunum of animals of subgroup B2 (animals treated with single dose of MTX and sacrificed after 6 days) and stained with hematoxylin & eosin showed severe changes
Fig. 10. A photomicrograph of rat jejunum of subgroup (B2) showing marked decrease in the PCNA positive nuclei in the crypt region (immunostaining for PCNA and hematoxylin counter stain 400×).
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Fig. 11. A photomicrograph of rat jejunum of subgroup (C1) showing abnormal shaped villus (), fusion of the villi (→) and inflammatory cellular infiltration (I) in the lamina propria (Hx.&E. Mic. Mag. × 400).
in the jejunal mucosa in the form of shortening, fusion and kinking of the villi, inflammatory infiltrate in the lamina propria and crypt cyst formation. Desquamation and destruction of surface epithelium and desquamated epithelial cells in the lumen of some crypt cysts could also be noticed (Figs. 7 and 8). PAS-stained sections obtained from this group revealed marked depletion of goblet cells and decrease intensity of PAS reaction at brush border (Fig. 9). Immunohistochemical staining for PCNA showed apparent decrease in the PCNA positive nuclei in the crypts as compared to group B1 (Fig. 10). 4.1.2.2. Group-C. Subgroup C1. Examination of sections obtained from the jejunum of animals of subgroup C1 (animals that were concomitantly treated with both methotrexate and vitamin A and sacrificed after 2 days of MTX injection) and stained with hematoxylin & eosin showed increase in the villus height. While, there were areas showing abnormal shaped villus and fusion of the villi. In addition there were broad and short villi and inflammatory cellular infiltration in the lamina propria (Figs. 11 and 12). PAS-stained sections obtained from this group showed increase in the number of goblet cells and increased intensity of PAS reaction at the brush border (Fig. 13). Immunohistochemical staining for PCNA showed apparent increase in the numbers of PCNA positive nuclei as compared to group B1 (Fig. 14). Subgroup C2. Examination of hematoxylin & eosin sections obtained from the jejunum of animals of subgroup C2 (animals that were concomitantly treated with both methotrexate and vitamin A and sacrificed after 6 days of MTX injection) showed increase in the villus height and improved crypt architecture. While, there were areas showing distortion of epithelial lining of villi, inflammatory cellular infiltration in the lamina propria and shortening of some villi (Fig. 15). PAS-stained sections obtained from this group showed increase in the number of goblet cells both in the villi and
Fig. 12. A photomicrograph of rat jejunum of subgroup (C1) showing broad and short villi (). Notice inflammatory cellular infiltration (I) in the lamina propria. (Hx.&E. Mic. Mag. × 400).
crypts and increased intensity of PAS reaction at the brush border (Fig. 16). Immunohistochemical staining for PCNA showed apparent increase in the numbers of PCNA positive nuclei as compared to group B2 (Fig. 17). 4.2. Electron microscopic study 4.2.1. Control and vitamin A treated groups Ultrathin sections obtained from rat jejunum of control and Vitamin A treated groups showed the same picture which was identical with the normal ultrastructure of the jejunum. The columnar absorptive cells were regularly arranged and contained basal, oval and euchromatic nuclei. They had continuous regular microvillus border, abundant elongated mitochondria and extensive lateral interdigitations. Intercellular junctions were present between adjacent cells and diffuse appearance of the terminal web in addition to the presence of RER cisternae and free ribosomes in the cytoplasm were also detected. Goblet cells were insinuated between enterocytes. The apical wide part of each goblet cell was distended with moderately electrondense mucin secreting granules (Figs. 18 and 19). 4.2.2. Experimental group 4.2.2.1. Group B. Subgroup B1. Examination of ultrathin sections of the jejunum obtained from this subgroup
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vacuole that caused indentation of an enterocyte nucleus and exfoliation of epithelial cells were detected. Destruction of the luminal surface of goblet cells was also observed (Figs. 23 and 24).
Fig. 13. A photomicrograph of rat jejunum of subgroup (C1) showing increased number of goblet cells (black arrow) and increased intensity of PAS reaction at the brush border (red arrow). (PAS Mic. Mag. × 200).
revealed enterocytes with dilatation of RER cisternae, vacuolization of the cytoplasm and lipid droplets in some focal areas. Widening of the intercellular space and short sparse loosely packed and irregularly arranged microvilli of enterocytes with areas of focal loss of microvillus border were detected. The mitochondria became swollen with destroyed cristae. Moreover, there were corrugation and indentation of the nuclear membrane in some enterocytes (Figs. 20–22). Subgroup B2. Ultrathin sections of the jejunum obtained from this subgroup revealed decreased heights of some enterocytes with short sparse microvilli and focal loss of microvillus border in addition to rarified and vacuolated cytoplasm of some enterocytes. Large cytoplasmic
4.2.2.2. Group-C. Subgroup C1. Examination of ultrathin sections obtained from rat jejunum of subgroup C1 showed some enterocytes regained their normal ultrastructure. They had closely packed regularly arranged microvilli and intact lateral interdigitations, some mitochondria were still swollen with destroyed cristae. The examination revealed a goblet cell, with dilated apical part and containing mucinsecreting granules, insinuated between enterocytes. RER cisternae of enterocytes appeared relatively as the control (Fig. 25). Subgroup C2. Ultrathin sections obtained from rat jejunum of subgroup C2 showed some enterocytes regained their normal heights with regularly arranged but loosely packed microvilli. Some enterocytes lose their microvillus border. Also, the examination revealed elongated mitochondria and intact lateral interdigitations and with relatively normal goblet cells were interspersed between enterocytes (Fig. 26).
4.3. Morphometric results and statistical analysis 4.3.1. Statistical results for the mean height of jejunal villi As assessed by image analyzer, vitamin A treated group revealed non-significant change in the mean heights of the villi when compared with control group. The MTX treated group B (including both subgroup B1 and B2) showed statistically highly significant decrease in the mean height of the villi when compared with control group. Concerning the MTX-vitamin A treated group C (including subgroup C1 and C2) it was found that there was still highly significant decrease in the mean height of the villi when compared with control group. Also, there was significant increase in the mean of the villus height compared to group B.
Villus height
Mean ± SD f-test p-value
Control
B1
B2
CI
C2
346.2 ± 51.6
127 ± 33.4
54 ± 18.1 26.523 0.001
176.5 ± 45.2
154 ± 25.5
Scheffe test Control and BI
Control and B2
Control and C1
Control and C2
0.001*
0.001*
0.001*
0.001*
350
mean value
300 250 200 150 100 50 0 Control
B1
Control
B2
B1
B2
CI
CI
C2
C2
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4.3.2. Statistical results for the mean crypt depth As assessed by image analyzer, vitamin A treated group revealed non-significant change in the mean crypt depth when compared with control group. The MTX treated group B (including both subgroup B1 and B2) showed statistically highly significant decrease in the mean crypt depth when compared with control group. Concerning the MTX-vitamin A treated group C (including subgroup C1 and C2) it was found that there was still highly significant decrease in the mean crypt depth compared with control group. Also, it was found that statistically highly significant increase the mean crypt depth when compared to group B.
Crypt depth
Mean ± SD f-test p-value
Control
B1
B2
CI
C2
338.8 ± 55.7
150.3 ± 45.8
185.3 ± 64.1 10.362 0.001
183.8 ± 54.5
261.2 ± 68.8
Scheffe test Control and BI
Control and B2
Control and C1
Control and C2
0.001*
0.001*
0.001*
0.001*
350 300
mean value
250 200 150 100 50 0 Control
B1
Control
B2
B1
CI
B2
CI
C2
C2
4.3.3. Statistical results for the mean number of goblet cells As assessed by image analyzer, vitamin A treated group revealed non-significant change in the mean number of
goblet cells when compared with control group. The MTX treated group B (including both subgroup B1 and B2) showed statistically highly significant decrease in the mean number of goblet cells when compared with control group. Concerning the MTX-vitamin A treated group C (including subgroup C1 and C2) it was found that there was still highly significant decrease in the mean number of goblet cells when compared with control group. Also, it was found that statistically highly significant increase the mean number of goblet cells when compared to group B.
Goblet cell number
Mean ± SD f-test p-value
Control
B1
B2
CI
C2
144.2 ± 6.52
53 ± 4.5
25 ± 3.2 9.362 0.001
122 ± 7.5
96 ± 8.8
Scheffe test Control and BI
Control and B2
Control and C1
Control and C2
0.001*
0.001*
0.001*
0.001*
160
mean value
140 120 100 80 60 40 20 0
Control
B1 Control
B2 B1
B2
CI CI
C2
C2
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Fig. 14. A photomicrograph of rat jejunum of subgroup (C1) showing an increase in PCNA positive nuclei in the crypt region as compared to subgroup B1 (immunostaining for PCNA and hematoxylin counter stain ×400).
4.3.4. Light microscopic results 4.3.5. Electron microscopic results 5. Discussion Methotrexate (MTX) is one of the antimitotic drugs. These drugs act by blocking deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, inducing apoptosis and slowing down or stopping cell proliferation in rapidly proliferating cells such as tumors, bone marrow and gut mucosa cells [17–19] Sufficient vitamin A in the diet maintain the epithelium of gastrointestinal tract which in turn helps the maximal absorption of nutrients, resulting in improved weight gain. In several studies, it was shown that vitamin A may directly regulate intestinal epithelial cell proliferation and regeneration [20–22]. It has been also suggested that vitamin A coadministration prevents MTX-induced decrease in the contents of the small intestinal membrane protein and lipid and preserves partly the activity of thymidine kinase,
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which is a key enzyme in the salvage pathway of DNA synthesis [8]. In this study, the jejunum was chosen as a representative for the small intestine because the epithelial morphology and proliferation are nearly the same along the whole small intestinal axis. Also, the proximal small intestine is the most sensitive region of rat gut. In addition, active absorption of MTX occurs in proximal jejunum [4,23] Normally the crypt cells, which are rapidly generated, migrate to the villus tip and the replacement of the intestinal epithelium is completed in 3 days in humans and in 2 days in rats [24]. Intestinal mucosal damage and barrier function alterations have been described as consequences of different processes: apoptosis, hypoproliferation, inflammatory response, altered absorptive capacity and bacteria proliferation and colonization [25,26] In this research, the experimental group (B) that received MTX as a single dose showed disturbance in villus architecture, crypt loss and crypt cyst formation, epithelial separation with goblet cell depletion and inflammatory cellular infiltration in the lamina propria. These changes were in consistent with [7,27–30] who described similar results in their studies. These changes could be attributed to direct toxic effect of methotrexate on the jejunal mucosa or generation of reactive oxygen species (ROS). As lipid peroxidation results from binding of ROS to cellular macromolecules, particularly membrane lipids and polyunsaturated fatty acids in the endoplasmic reticulum and cellular membranes, nucleotides in DNA and sulfhydryl bonds in proteins [31,32]. This was parallel to previous findings of many investigators who reported that lipid peroxidation is an important cause of destruction and damage to cell membranes and may be a contributing factor in the development of MTX-mediated tissue damage [33–35] The villus damage caused by MTX may be due to reduced antioxidant enzymes capacity, resulting in increased sensitivity of cells to ROS related injury [6,36]. In addition to the direct damaging effects of reactive oxygen species on the tissues, they trigger the accumulation of activated
Fig. 15. A photomicrograph of rat jejunum of subgroup (C2) showing (A) increase in villus height ( ) and improved crypt architecture (C). Distortion of the epithelial lining of some villi (curved arrow) and inflammatory cellular infiltration (I) in the lamina propria (Hx.&E. Mic. Mag. × 200). (B) Some villi are short (arrow head), apparent increase in the number of goblet cells (G) with improvement of the crypts architecture (bifid arrows) (Hx.&E. Mic. Mag. × 400).
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Fig. 16. A photomicrograph of rat jejunum of subgroup (C2) showing (A) increased number of goblet cells in villi and crypts (→) (PAS Mic. Mag. × 200). (B) Increase intensity of PAS reaction at the brush border (PAS Mic. Mag. × 400).
Fig. 17. A photomicrograph of rat jejunum of subgroup (C2) showing an increase in PCNA positive nuclei in the crypt region as compared to subgroup (B2). (immunostaining for PCNA and hematoxylin counter stain ×400).
leukocytes in the tissues which secrete enzymes such as myeloperoxidase, elastase and proteases and liberate more free radicals. It was reported that myeloperoxidase activity increased in the MTX-treated rats [37–39]. ROS in the intestinal mucosa disrupts microcirculation and plays a key role in desquamation of epithelial cells [40]. Citrulline is a non protein amino acid synthesized by enterocytes and responsible for mucosal integrity and enterocyte mass [41]. It was reported that the concentration of citrulline is reduced in MTX-treated rats. This may explain the reduction of the enterocyte mass [42]. The observed ultrastructural changes in enterocytes in MTX treated group were summarized in the form of dilatation of RER cisternae, vacuolation of the cytoplasm, focal accumulation of lipid droplets, swollen mitochondria with destroyed cristae, short sparse microvilli with focal loss of microvillus border, widening of intercellular space, some nuclei showed variable degrees of indentation and corrugation of their nuclear membrane.
Fig. 18. An electron micrograph of ultrathin section of rat jejunum of control group showing (A) regularly arranged closely packed microvilli () and elongated mitochondria (M). The lateral plasma membranes of adjacent cells interdigitate extensively (bifid arrows). (B) Terminal web ( ) and junctional complexes between enterocytes (→) with the extensive lateral interdigitations (bifid arrow). The cytoplasm contains RER (curved arrows) and free ribosomes ().
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Fig. 19. An electron micrograph of ultrathin section of rat jejunum of control group showing a goblet cell (G) insinuated between absorptive cells. Its apical part is distended with mucin-secreting granules (*).
In the present study, the mitochondrial damage could be attributed to ROS which results in formation of a channel called mitochondrial permeability transition pore. Opening of this channel leads to loss of mitochondrial membrane potential and PH changes, resulting in failure of oxidative phosphorylation which leads to progressive adenosine triphosphate (ATP) depletion. As, the mitochondria contain several proteins, including cytochrome C. These proteins are capable of activating apoptotic pathways. Increased permeability of mitochondrial membrane as a result of its damage may result in leakage of these proteins into the cytosol and death of cell by apoptosis [43,44]. Accumulation of sodium in the cell leads to increase in water content and the cell will swell, a condition called hydropic degeneration. This may explain dilatation of RER and vacuolation of the cytoplasm [45]. Sodium-potassiumATPase activity was significantly diminished in the MTX treated rats and as a result the absorptive function of enterocytes was reduced leading to accumulation of nutrients and fluids in the intercellular spaces and widening of intercellular space [9].
Activation of proteases breaks down membrane and cytoskeletal proteins leading to shortening and focal loss of microvilli [46]. It also leads to destruction of intermediate filaments of nuclear lamins causing indentation and corrugation of nuclear membrane in some enterocytes [47]. In the present work focal accumulation of lipid droplets within enterocytes was attributed to impaired protein synthesis as a result of RER damage. Accordingly this leads to inhibition of lipoprotein manufacture resulting in accumulation of fat inside the cytoplasm of affected cells [48]. The light microscopic examination of group C (concomitantly received a single dose of MTX and a daily dose of vitamin A) revealed some improvement in villus and crypt architecture with increase in the villus height and increase in goblet cell number as compared to group B. But there were areas showing villus shortening and fusion, distortion of epithelial lining of some villi and inflammatory infiltration in the lamina propria. Examination of ultrathin sections obtained from this group revealed some improvements of the ultrastructure
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Fig. 20. An electron micrograph of ultrathin section of rat jejunum of subgroup (B1) (A) showing an enterocyte with dilatation of RER cisternae (curved arrows). Notice a granular goblet cell (star). (B) Showing decrease in goblet cell granules (star). Notice the desquamation of enterocyte in the lumen.
Fig. 21. An electron micrograph of ultrathin section of rat jejunum of subgroup (B1) showing (A) vacuolation of the cytoplasm (→) and lipid droplets ( ) with displaced indented nucleus (N) of an enterocyte. (B) Widening of the intercellular space between enterocytes (bifid arrows) with vacuolation of the cytoplasm (→) and lipid droplets ( ).
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Fig. 22. An electron micrograph of ultrathin section of rat jejunum of subgroup (B1) showing (A) short, sparse, loosely packed and irregularly arranged microvilli of enterocytes with focal loss of microvillus border (). Notice condensation of chromatin, shrinkage and corrugation of the nuclear membrane of an enterocyte nucleus (→). Another cell showing fragmentation of its nucleus ( ). (B) Higher magnification of A showing an enterocyte containing swollen mitochondria with destroyed cristae (bifid arrows) and corrugation of the nuclear membrane (→). A large lipid droplet ( ) could be seen.
Fig. 23. An electron micrograph of ultrathin section of rat jejunum of subgroup (B2) showing decreased heights of enterocytes (curved arrow), focal loss of microvillus border () with rarified and vacuolated cytoplasm (*) and inflammatory cellular infiltration (→) in the lamina propria.
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Fig. 24. An electron micrograph of ultrathin section of rat jejunum of subgroup (B2) showing large cytoplasmic vacuoles (*) of an enterocyte with destroyed luminal surface of a goblet cell (→).
of enterocytes confirmed by morphometric and statistical studies. The protective effect of vitamin A on the small intestine may be explained by stimulation of DNA and RNA biosynthesis. Moreover, it was reported that vitamin A is one of antioxidants that may either block the formation of free radicals or scavenger them once they have formed [10,46]. The protective effect of vitamin A could be also attributed to its role in regulation of differentiation of epithelial cells and goblet cells and maintenance of integrity of mucous membranes [49]. As, the preservation of cell proliferation capacity is a key factor in protecting the epithelium from the consequences of MTX-induced
apoptosis. These authors related this protection to the maintenance of goblet cell function [50]. Immunohistochemical study detected decrease in the number of PCNA positive nuclei in group (B) which indicated a decrease in the cell division in the crypt region. This is in accordance with other studies that MTX induce mitotic arrest of intestinal crypt cells [51]. While, there was increase in the number of PCNA positive nuclei in group (C) in relation to group B indicating that vit A plays a role in stimulating cell division in the intestinal mucosa [22] From the present research, it could be concluded that MTX has a toxic effect on the jejunal mucosa as was proved
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Fig. 25. An electron micrograph of ultrathin section of rat jejunum of subgroup (C1) showing enterocytes containing swollen mitochondria with destroyed cristae (bifid arrow). RER cisternae appear within normal (curved arrows). Notice a goblet cell (G) between enterocytes.
Fig. 26. An electron micrograph of ultrathin section of rat jejunum of subgroup (C2) showing some enterocytes with regularly arranged but loosely packed microvilli () and elongated mitochondria (M). Notice the presence of a goblet cell (G) between the enterocytes.
Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002
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histologically and most of the changes were reversible with concomitant administration of vit A. So, it is recommended to give Vit A in combination with MTX as a protective agent against its toxicity.
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Please cite this article in press as: Elbakary NA, et al. Evaluation of the possible protective role of vitamin A on methotrexate-induced changes on the jejunal mucosa of adult male albino rat: Histological and immunohistochemical study. J Microsc Ultrastruct (2014), http://dx.doi.org/10.1016/j.jmau.2014.04.002