Histological, histochemical and immunohistochemical study of the Effect of chronic low dose of methotrexate and folinic acid on long bone of adult male guinea pigs

Introduction: Methotrexate is anti-folate metabolite. It is used in low dose as first line therapy for treatment of rheumatoid arthritis. It is a main treatment for childhood acute lymphoblastic leukaemia. Several studies reported its wide range side effects such as gastrointestinal intolerance and bone defects in the form of fracture and bone growth defect. Aim of the work: the current study examined the structural cellular damages to the bone caused by long term lowdose methotrexate treatment, as well as the potential protective effects of folinic acid supplementary treatment. Material and Methods: Forty adult male guinea pigs were subjected to experiment for nineteen days. The animals were divided into 4 groups, each of 10 guinea pigs as follow: Group I: (control group) were injected subcutaneously (distilled water and normal saline). Group II: received Folinic acid (1 mg/kg b.wt./ day) was injected intraperitoneally. Group III: received methotrexate (0.65 mg/kg b.wt/ day subcutaneously injected) for two separate 5 days courses (5 days on/9 days off/5 days on). Group IV: received Folinic acid (1mg/kg b.wt./ day) intraperitoneally injected 6 hrs after the administration of same dose of methotrexate as group III.On day twenty, the guinea pigs were anaesthetized with ether then sacrificed. Right tibias were removed from guinea pig and dissected free from soft tissues and subjected to histological, histo-chemical and immunohistochemical studies. Results: Histologically and histochemically, tibia of methotrexate treated guinea pig showed that, long term use of low dose methotrexate can cause oesteocytes dysfunction, bone erosion, bone damage, suppress bone formation, and increase bone resorption resulting in overall bone loss, via inducing osteocytes apoptosis. There was marked increase of Masson trichrome staining in resorption cavities of bone with reduction of collagen fibers in thin cortex of bone. Immunohistochemically, in methotrexate treated group showed positive immunoreactivity for caspus 3 in bone osteocytes and positive immunoreactivity for CD 68 in numerous bone osteoclasts which increased in number when compared to control group. On the other hand, histological, histochemical and immunohistochimical examination of the tibia of guinea pig protective group treated with both folinic acid and methotrexate displayed improvement in bone structure, reduce methotrexate damage in the bone formation. It prevents osteoblasts from undergoing apoptosis and suppressing methotrexate-induced osteoclast formation. Conclusion: Toxic effect of methotrexate should be kept in mind during chronic usage. Folinic acid was advised to be administered in concomitant with methotrexate as it could ameliorate its adverse effect on rat testis. [Manar A. Bashandy and Hala elharoun. Histological, histochemical and immunohistochemical study of the Effect of chronic low dose of methotrexate and folinic acid on long bone of adult male guinea pigs. J Am Sci 2014;10(12):47-57]. (ISSN: 1545-1003). http://www.jofamericanscience. org. 5