Evidence For Morphine-Induced Neuronal Apoptosis And Its Mechanisms In Rats

Opioids are a class of powerful analgesics that are commonly used in acute and chronic pain management. However, the neuronal damage induced by opiate drugs significantly hampers the clinical utility of opioids. As such, the cellular and molecular mechanisms of opiate-induced neuronal damage have been a focus of extensive research interest.Apoptosis, or programmed cell death, is an active process of normal cell death during development and also occurs as a consequence of the cytotoxin effect of various neurotoxins. The induction of apoptosis in neurones has been demonstrated to share the same basic mechanisms with all other cell types. The molecular mechamisms of apoptosis have not been established yet, but various key proteins are involved in the regulation of programmed cell death. Thus, some members of the Bcl-2 family, such as Bcl-2 and Bcl-XL, supresses apoptosis, while the expression of other , such as the homologues Bax and Bak, are pro-apoptotic. Specifically, the Bcl-2 oncoprotein, localized mainly to the mitochondrial membranes, has been shown to play an important role in protecting neurones from apoptotic cell death. Another key element involved in the regulation of apoptosis is the Fas glycoprotein ( also known as CD95 or Apol), a cell surface receptor that belongs to the tumor necrosis factor receptor family (death receptors) and that is expressed abundantly in various tissues.Objective To investigate the effects of chronic morphine on neuronal apoptosis, pro-apoptotic Fas receptor and anti-apoptotic Bcl-2 oncoprotein in rat brain and to explore the mechanism of opiate-induced neuronal damage after long-term exposure. Methods (1)Twenty-four adult male Sprague-Dawley rats weighing 190-210g each were randomly divided into 3 groups of each 8 animals: chronc morphine-dependent group, chronic morphine-abstinent group and control group. Toestablish the model for morphinomania in rats, rats in dependent group and abstinent group were chronically treated with morphine i.p. for 13 days, using an escalating dosage paradigm (5-95mg). Rats in abstinent group were induced withdrawal syndromes with naloxone 5mg/kg i.p. for 30 minutes after chronic morphine. Control rats received 0.9% saline vehicle i.p. at the indicated treatment times. (2)Apoptosis cells were enumerated by immunohistochemistry using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-X nick end labeling (TUNEL) technique. (3)Immunohistochemistry was used to detect the expresion of pro-apoptotic Fas receptor protein and anti-apoptotic Bcl-2 oncoprotein. Results (1)Chronic treatment with morphine, compared with saline solution administration, induced marked neuronal apoptosis in rat brain (12.89 2.81% vs 2.32 1.58%, P<00.1). Similarly, final naloxone injection in morphine- dependent rats significantly increased the number of apoptotic cells in rat brain compared to those injected with saline (12.79 2.98% vs 2.32 1.58%, P< 00.1). (2)Compared with saline solution administration,chronic morphine induced marked increases in the expression of Fas receptor(11.39 2.42% vs 7.07 2.24%, P < 00.1) and decreases in the expresssion of Bcl-2 oncoprotein (7.95 2.81% vs 10.96 2.18%, P < 00.1) in the cerebral cortex. Conclusion The results of these in vivo studies demonstrate that chronic morphine can promote abnormal neuronal apoptosis in rat brain by enhancing the expression of pro-apoptotic Fas receptor protein and damping the expression of anti-apoptotic Bcl-2 oncoprotein, suggesting that morphine-induced neuronal apoptosis might have a relevant role in opiate-induced neuronal damage.