Cerebrolysin Attenuates Heat Shock Protein (HSP 72 KD) Expression in the Rat Spinal Cord Following Morphine Dependence and Withdrawal: Possible New Therapy for Pain Management

Abstract

The possibility that pain perception and processing in the CNS results in cellular stress and may influence heat shock protein (HSP) expression was examined in a rat model of morphine dependence and withdrawal. Since activation of pain pathways result in exhaustion of growth factors, we examined the influence of cerebrolysin, a mixture of potent growth factors (BDNF, GDNF, NGF, CNTF etc,) on morphine induced HSP expression. Rats were administered morphine (10 mg/kg, s.c. /day) for 12 days and the spontaneous withdrawal symptoms were developed by cessation of the drug administration on day 13(th) that were prominent on day 14(th) and continued up to day 15(th) (24 to 72 h periods). In a separate group of rats, cerebrolysin was infused intravenously (5 ml/kg) once daily from day one until day 15(th). In these animals, morphine dependence and withdrawal along with HSP immunoreactivity was examined using standard protocol. In untreated group mild HSP immunoreaction was observed during morphine tolerance, whereas massive upregulation of HSP was seen in CNS during withdrawal phase that correlated well with the withdrawal symptoms and neuronal damage. Pretreatment with cerebrolysin did not affect morphine tolerance but reduced the HSP expression during this phase. Furthermore, cerebrolysin reduced the withdrawal symptoms on day 14(th) to 15(th). Taken together these observations suggest that cellular stress plays an important role in morphine induced pain pathology and exogenous supplement of growth factors, i.e. cerebrolysin attenuates HSP expression in the CNS and induce neuroprotection. This indicates a new therapeutic role of cerebrolysin in the pathophysiology of drugs of abuse, not reported earlier.

Keywords: Morphine; analgesia.; cerebrolysin; growth factors; heat shock proteins (HSP 72 kD); morphine tolerance; pain perception; stress reaction; withdrawal symptoms.

Fig. (1)

Heat shock protein (HSP) 72 kD, a marker of cellular stress immunoreactivity in the spinal cord of morphine dependent rats on day 12 (MD12) and following 2nd day of morphine withdrawal (MWD2). Expression of HSP 72 kD is seen in the ventral (a) and lateral (b) horns in C-5 segment (arrows) on the day 12 of morphine dependent rats. The magnitude and intensity of HSP expression is further increased on day 2 following morphine withdrawal in (MWD2, c,d, arrows). HSP expression is mainly seen in the cell cytoplasm. However, some nerve cells showed staining of cell nucleus as well. Bar = a.b = 40 µm, c,d = 30 µm [Reproduced with permission [12].

Fig. (2)

Semiquantitative analysis of HSP 72 kD expression in the CNS and its modification with cerebrolysin. The number of HSP positive cells significantly increased in the cerebral cortex, hippocampus and in spinal cord following morphine dependence by day 12 (MD 12) as well as after morphine withdrawal day 1 (MW1) and day 2 (MW2) as compared to control group. Cerebrolysin (CBL) given daily in control animals did not induce HSP expression (CBL+ Cont) but is able to significantly reduce the HSP expression in both morphine dependent (MD) and morphine withdrawal (MW) rats on day 1 and 2. ** = P < 0.01 from control group, Chi Square test.

Fig. (3)

Light micrograph showing HSP expression in the cortex and thalamus is reduced by cerebrolysin treatment in morphine dependent rats on day 10 and 12 (MD 10, MD 12) and after morphine withdrawal day 1 and 2 (MWD1, MWD2). Only few scattered labeled neurons and dendrites particularly in the cell cytoplasm could be seen expressing weak HSP activity in cerebrolysin treated morphine dependent rats that was most prominent on day 10 (MD 10) as compared to day 12 (MD 12). On the other hand, cerebrolysin was able to prevent HSP expression in cortex and thalamus after morphine withdrawals day 1 and 2. Bar = 40 µm.

Fig. (4)

Ultrastructural changes in the nerve cell, myelin and neuropil in morphine dependent rats (day 12, MD 12) and following 2nd day of withdrawal (MWD2). A. Vacuolation (*), vesiculation of myelin (arrow) and degenerative changes in the neuropil are common in morphine dependent rat on day 12 (a). The magnitude and intensity of these structural changes, e.g., myelin vesiculation (arrows), membrane damage and vacuolation (') are much more frequent in animals following 2nd day of morphine withdrawal (A.b). One nerve cell showing dark and condensed cell cytoplasm in the ventral medial thalamic nucleus (c) is clearly seen on the 2nd day of morphine withdrawal in the rat. The surrounding neuropil showed many degenerative changes (arrows). B. Vacuolation and degenerative changes in the piriform cortex in one morphine dependent rat on day 12 (MD 12, B.a). These degenerative changes are much more prominent in animals on the 2nd day of morphine withdrawal in the cortex (B.b) and in cerebellum (B.c). In the cerebellum, one Purkinje cell showed condensed cytoplasm with marked degenerative changes in the surroundings. Vacuolation (*) and degenerative changes are clearly seen around the nerve cell and the granule cell or astrocyte (MWD2 c). Bar: A.a = 1 µm, b,c = 0.8 µm; B.a,b = 1.5 µm, c = 0.8 µm [Reproduced with permission [12].

Fig. (5)

Representative examples of lanthanum extravasation across the blood-brain (a) and blood-spinal cord (b) barriers following 48 h after spontaneous morphine withdrawal in dependent rat. Infiltration of lanthanum is seen (arrowheads) across the endothelial cell of one microvessel from the cerebral cortex (a) and from the cervical spinal cord (b). Occurrences of many microvesicular profiles are clearly evident in the spinal cord endothelial cells (b). The tight junctions appear to be closed for lanthanum (arrows) in the cerebral (c) and spinal cord (d) microvessels. Edematous swelling of perivascular astrocytes is clearly visible (a-d). Bar = 500 nm (a); 600 nm (b), 300 nm (c); 400 nm (d).

Fig. (6)

Effect of Cerebrolysin on lanthanum extravasation across the cerebral microvessels in morphine dependent animals on day 12 (MD12) and following 2^nd day of morphine withdrawal (MWD2). At the ultrastructural level, cerebrolysin was able to thwart lanthanum extravasation from lumen to the endothelial cell cytoplasm or in neuropil in MWD2 rat although; mild perivascular edema is also seen around this microvessel (*). Whereas, extravasation of lanthanum across the neuropil was largely abolished except in some very minor areas of the endothelial cell membrane (arrow head) in morphine dependent animal on the day 12(MD12). Signs of perivascular edema and structural changes are considerably reduced by cerebrolysin treatment.