Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 43, Num. 5, 2011, pp. 512-515

Indian Journal of Pharmacology, Vol. 43, No. 5, September-October, 2011, pp. 512-515

Research Article

Evaluation of the role of erythropoietin and methotrexate in multiple sclerosis

Sandipan Dasgupta¹, Bhaskar Mazumder¹, Y Roja Ramani², Subhra Prakash Bhattacharyya³, Mukesh K Das⁴

¹ Department of Pharmaceutical Sciences, Dibrugarh University, Assam, India
² M.K.C.G. Medical College and Hospital, Department of Pharmacology, Berhampur, Orissa, India
³ Department of Pharmaceutics, Dr. B.C. Roy College of Pharmacy and AHS, Durgapur, West Bengal, India
⁴ Raghu College of Pharmacy, Dakamarri, Bheemunipatnam Mandal, Visakhapatnam, Andhra Pradesh, India
Correspondence Address: Sandipan Dasgupta, Department of Pharmaceutical Sciences, Dibrugarh University, Assam, India, s_dg2006@yahoo.co.in

Date of Submission: 24-May-2010
Date of Decision: 08-Feb-2011
Date of Acceptance: 01-Jul-2011

Code Number: ph11139

DOI: 10.4103/0253-7613.84955

Abstract

Keywords: Demyelination, encephalomyelitis, erythropoietin, methotrexate

Introduction

Experimental autoimmune encephalomyelitis (EAE) is a principal model of multiple sclerosis (MS). ^[1] In this model, the animals are injected with the whole or part of various proteins that make up myelin, the insulating sheath that surrounds the neurons. These proteins induce an autoimmune response against the myelin. ^[2],[3] The animals develop a disease process that closely resembles multiple sclerosis in humans. Erythropoietin (Epo) is the main regulator of erythropoiesis in mammals. ^[4] It has been shown to reduce apoptosis of erythroid progenitors by interacting with a high-affinity receptor on these cells. It is becoming increasingly clear that erythropoietin has additional neurotrophic and neuroprotective effects in different in vivo models of brain injury. ^{[5],[6],[7],[8]} Furthermore, erythropoietin has been demonstrated to delay the onset and to reduce the severity of clinical symptoms in an EAE model induced by myelin basic protein (MBP) in rodents. ^[7] In a recent study, it has been shown that these effects were caused by an Epo-induced modulation of cytokine expression. ^[8] More effective therapies are needed to treat multiple sclerosis. One of the drugs used for this disease is high dose methotrexate (Mtx) at 1-4 weeks interval which nevertheless appears to produce modest clinical benefit associated with life threatening adverse effects. There is a good scientific rationale for the use of combination therapy in multiple sclerosis and a need for its evaluation. In the present study, we tested the role of 'early' and 'late' Epo in combination with Mtx in EAE, a commonly used animal model of MS and other demyelinating human diseases.

Materials and Methods

Prior approval from the Institutional Animal Ethical Committee (IAEC) was obtained and the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines were followed throughout the study period.

Study Design

Thirty six female C57BL/J6 mice (18-20g), were divided into six groups after one week of acclimatization (n0 = 6 in each group). Subsequently they were immunized with 200 mg of MBP (whole protein) emulsified in complete Freund's adjuvant (CFA) (Sigma Aldrich, USA) supplemented with 1 mg/ml killed mycobacterium tuberculosis (MBP: CFA in 1:1 ratio). A volume of 0.1 ml of the mixture was injected on the either side of the back subcutaneously (both flanks) for each mouse. Pertussis toxin, 500 ng (Serum Institute, Kasauli, Himachal Pradesh) was injected intraperitonially (i.p) immediately and repeated 48 hours later to enhance the immunological reaction.

Group-I received normal saline 0.1 ml/mice. Group-II received the standard drug Mtx (Dabur Pharma, Baddi, Himachal Pradesh) from the day of immunization for 8 days. Epo (Shanta Biotech, Hyderabad) was administered to group-III to VI 5000 U/kg i.p. Among them, group-III received Epo from the day of immunization for 8 days (preventive schedule). Group-IV received the Epo from the day of onset of clinical symptoms for 8 days (therapeutic schedule). Group-V received a combination of Epo (5000 U/kg) and Mtx (1 mg/kg) from the day of immunization for 8 days (preventive schedule). Group-VI received a combination of Epo (5000 U/kg) and Mtx (1 mg/kg) from the day of onset of disease till 8 days (therapeutic schedule).

Evaluation of Experimental Allergic Encephalomyelitis (EAE) ^[9],[10]

Clinical assessment was performed using a scoring system. Scoring was done by a blinded observer on a 0-5 scale as follows: 1 - limp tail or waddling gait with tail tonicity; 2- waddling gait with limp tail (ataxia); 2.5 - ataxia with partial limb paralysis; 3 - full paralysis of one limb; 3.5 - full paralysis of one limb with partial paralysis of second limb; 4 - full paralysis of two limbs; 4.5 - moribund; 5 - death. Survival was also monitored from the day of immunization until the end of the study.

Histopathology

On day 45, post-immunization mice were extensively perfused and spinal cords were harvested. Mice were perfused with 4% paraformaledehyde (PFA), the spinal vertebrae were taken and post fixed in Bouin's fixative for 2-3 days to be decalcified. Using a fine forceps, vertebra was removed and spinal cord was soaked in ethanol 70% until the day of embedding. For paraffin embedding, tissues were first dehydrated in 90, 96, and 100% each for 1 hour and then cleared by incubation in ethanol/toluene (2:1), ethanol/toluene (1:1), toluene 100% (2 changes) each for 30-45 minutes. Finally the embedding was started by toluene/paraffin (1:1) for half an hour and continued for four changes of paraffin baths, each for 15 minutes. Blocks were allowed to be cooled and sectioned by using a rotary microtome at 7 mm. Five-micrometer sections were stained with HandE and Luxol fast blue (myelin stain) for inflammation and demyelination, respectively. Slides were assessed in a blinded fashion for inflammation and demyelination.

For inflammation, the following scale was used: 0, none; 1, a few inflammatory cells; 2, organization of perivascular infiltrates; 3, increasing severity of perivascular cuffing with extension into the adjacent tissue. For demyelination, the following scale was used: 0, none; 1, rare foci; 2, a few areas of demyelination; 3, large (confluent) areas of demyelination.

Data on clinical score were compared using nonparametric Kruskal-Wallis test. Values were expressed as mean ± SEM. 'P' value ≤ 0.05 was regarded as significant.

Results

Onset of Disease

Disease manifestation was significantly (' P' < 0.05) delayed in all the test groups with respect to control. Vehicle-treated animals developed symptoms on day 15.25 ± 0.25 after immunization. Animals treated with Mtx as a monotherapy from day 1 to day 8 of MBP-EAE (1 mg/kg) showed the first neurological symptoms on day 23 ± 1.73 after immunization. In the early Epo treatment group, (daily application of Epo from the day of immunization until day 8), the first neurological symptoms occurred on day 20.25 ± 0.25 comparable to that of the Mtx treated group. Disease onset was at day 17 in the late Epo group (Epo given from disease manifestation onwards until day 8 of EAE). The early Epo and Mtx group (1 mg/kg Mtx given from day 1 to day 8 of EAE in addition to the early Epo treatment) developed first signs of EAE on day 19. In the late Epo and Mtx group (1 mg/kg Mtx. given from day 1 to day 8 of EAE in addition to the late Epo treatment), disease onset occurred 17 days after immunization.

Disease Score

The clinical scores were compared on the day of maximum disease manifestation in the control group. The clinical score remained highest on day 43 onwards of the disease in vehicle-treated animals (4.75 ± 0.25). There was no significant change in the disease score in the group treated with late Epo (4.5 ± 0.3), as well as in late Epo-Mtx treated animals (4.25 ± 0.48). Significant ( P < 0.05) improvement was observed in Mtx treated (2.25 ± 0.95), early Epo treated (2.0 ± 1.0) and in those treated with an early combination therapy (2.5 ± 0.87). Therefore, animals that received Epo from the day of immunization showed a tendency to a better clinical outcome which was statistically significant (P < 0.05). Treated animals displayed a discernible improvement in alertness, spontaneous mobility, tone and motor function.

Histopathological Evaluation

The effect of each treatment on central nervous system (CNS) inflammation was determined by scoring leukocyte infiltration into spinal cords on day 45 (end of study period). Early Epo group had a significant reduction in inflammation score compared to the normal saline(NS) treated controls. The combination of early Epo and Mtx was most effective at reducing CNS inflammation with a very low total inflammation score of 0.25 ± 0.25 compared to 2.75 ± 0.25 for NS-treated controls. The late Epo (2.25 ± 0.48) and late Epo combined with Mtx (2.25 ± 0.25) did not have any significant effect on the inflammatory infiltrates [Table - 1].

Early Epo (1 ± 0.41) also had a significant effect on reducing the demyelination and mice regained body weight. The combination treatment (preventive schedule) led to reduced inflammation and demyelination but was not as effective as Epo alone. Mtx (2.25 ± 0.25) alone did not have a significant effect on demyelination as also its combination with Epo as therapeutic schedule (2.5 ± 0.29).

Percent survival of mice was monitored from the day of immunization. Only one animal survived till 45 days in the control group which was the reason for fixing that particular day as end of study period. Survival in the Mtx, Epo (preventive schedule), and the early combination group was 75% whereas the survival rate in the therapeutic schedule groups was 25 (Epo alone) and 50% (combination), respectively.

Discussion

In MS, the most frequent inflammatory demyelinating disease of the CNS in humans, repair capacities exist but seem to be insufficient. It is a challenging disease to treat. Three pharmacological treatments are emphasized: Steroids, immunomodulation, and immunosuppressants. Traditional immunosuppressants such as cyclophosphamide and azathioprine have been used in MS for some time showing a variable degree of benefit. ^[11],[12] According to one theory, both inflammatory and degenerative disease processes are present from the beginning and proceed independently with inflammation being most prominent in the early phases of the disease whereas neurodegeneration dominates during the later stages. ^[13] Approaches which allow CNS repair by inhibiting inflammation and/or simultaneously protecting neurons and oligodendrocytes from damage could thus be effective therapies for multiple sclerosis. In the present study, disease severity in C57BL/J6 mice correlated with the degree of CNS inflammation and demyelination as evidenced from the histopathology reports. Epo treated C57BL/J6 mice developed less severe MBP-induced EAE which was characterized by late onset, less severe paralysis, decreased mortality, and lesser degree of demyelination and inflammatory infiltration compared with that of the saline administered control. Epo reduced neuronal damage and improved functional outcome in tested animal model of MS. Epo-mediated neuroprotection may have a variety of mechanisms with involvement of neuronal, glial, and endothelial cell functions like that in experimental brain injury models. Neurorepair, including remyelination, is an important physiological process and a therapeutic strategy. Therefore, novel therapies that promote repair as well as regeneration in CNS should be the target to control the dysregulated CNS inflammation and neurodegeneration seen in MS. Epo has been reported to promote angiogenesis, neurogenesis as well as differentiation of oligodendrocytes. ^[14] This could be an important effect of Epo in the model of EAE used in our work. In fact, a recent report has indicated that Epo increases oligodendrocytes progenitor cell proliferation in a different model of EAE, induced in SJL/J mice by immunization with myelin proteolipid protein peptide 139-151 and augments brain-derive neurotrophic factor (BDNF) expression in the CNS in this and other experimental conditions. ^[15] Finally, it is important to consider Epo in the context of the pathogenesis of MS. Blood brain barrier disruption takes place in EAE and Epo has been shown to diminish its leakage in EAE. ^[16] Since leakage contributes to the inflammatory process and vice versa, it may be a consequence of inflammation ^[17] and this mechanism could be part of the overall anti-neuroinflammatory action of Epo in EAE.

To conclude, the present study reveals that Mtx alone significantly reduces the disease progression which is in agreement with the available literature. Apart from that, when combined with Epo in two different schedules, it produces variable effects on inflammation and demyelination. Epo (early) and Mtx when compared to Epo alone improve the inflammatory component whereas they do not have any effect on demyelination. The early combination when compared to Mtx alone is superior for both inflammation as well as demyelination. Also this combination is significantly more effective than the Epo (late) and Mtx. By comparing different treatment protocols, we showed that early application of a neuroprotective agent such as Epo together with Mtx during the acute stage of the disease is an effective strategy to ameliorate disease progression in inflammatory autoimmune CNS disease. However, the clinical significance of the above results needs to be established.

References

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