Nitroxidergic modulation of behavioral and immunological responses in Morphine treated rats

S Surcheva1, A Tsakova1, K Surchev1, K Simeonova1, I Altankova2, Ts Marinova3, M Vlaskovska1. 1Medical University Sofia, Department of Pharmacology, Medical Faculty, 1431 Sofia, Bulgaria, 2University Hospital St. Ivan Rilski, 1431 Sofia, Bulgaria, 3St. Kliment Ohridsky University, Department of Biology, Medical Genetics and Microbiology, Medical Faculty, 1000 Sofia, Bulgaria

Opioids are widely used to treat severe pain. A major adverse effect upon long-term treatment with opioids is the development of tolerance/dependence. Despite recent advances in understanding the modulation of behavioural and immune responses through opioid tolerance/dependence the role of nitroxidergic mechanisms in this modulation is far from being fully understood. The aim of the present investigation was to study the effects of NOS inhibition: (i) in vivo on the development of tolerance to analgesic and cardiovascular effects of Morphine (ii) in vitro on the leukocyte proliferation, DNA fragmentation and thymocyte apoptosis in opioid tolerant/dependent state. Animals: Male rats (Wistar, 220-240g, Animal House of the Medical University Sofia) were used. Experiments have been approved by the Ethics Committee of Medical University, Sofia. Drugs: Morphine hydrochloride, L-NAME, Concanavaline A (CON A) were purchased from Sigma.

Methodology: The analgesic tolerance to Morphine (Mo; 5 mg/kg s.c.) and Mo plus L-NAME (15 mg/kg i.p.) was determined by ED50 using tail-flick test (group of 8-10 rats). The effects of repeated i.v. Mo or Mo plus L-NAME administration on blood pressure and heart rate was studied in conscious chronic-instrumented rats. CON A-induced lymphocyte proliferation was studied in splenic tissue by 3H-Tymidine labeling. DNA fragmentation was revealed by agarose electrophoresis and thymocyte apoptosis was studied by TUNEL reaction. Statistical comparisons were made by an analysis of variance ANOVA. Values are expressed as the means S.E.M.

Results: It was found that NOS inhibition suppressed development of tolerance to the analgesic effect of Mo in tail flick test. Results showed that ED50 increased from 4.9 ± 0.6 mg/kg (naive) to 17.0 ± 1.6 mg/kg (tolerant rats). Co-administration of Mo with L-NAME decreased ED50 to 7.5 ± 0.6. Morphine (0.5 mg/kg) evoked transient hypotension and bradycardia which were abolished after the second Mo-application but did persist in the presence of L-NAME (6 mg/kg). After repeated administration of morphine on conscious rats tolerance to its cardiovascular effects developed which was attenuated by the NOS-inhibitor.

Chronic administration of Mo activated spinal astrocytes and microglia.

Morphine suppressed and L-NAME stimulated splenocyte proliferation (see below).

L-NAME alleviated Morphine-induced thymocyte DNA fragmentation and apoptosis.

Conclusion: It is plausible to suggest that opioid behavioural and immune responses can be alleviated by nitroxidergic inhibition.

Acknowledgements: The research was partially supported by the Council of Medical Sciences of Medical University Sofia (Grant 29 / 2011) and DNTS/BG-SI/01/9/2011, Ministry of Higher Education, Youth and Science, Bulgaria.