Morphine-alcohol treatment impairs cognitive functions and increases neuro-inflammatory responses in the medial prefrontal cortex of juvenile male rats

Abstract

In the developed and developing world, opioid consumption in combination with alcohol has become one of the substances abused. In this experiment, we examined the effects of alcohol, morphine, and morphine+alcohol combination on cognitive functions and neuroinflammatory responses in the medial prefrontal cortex (mPFC) of juvenile male rats. Alcohol (1.0 ml of 15% v/v ethanol twice daily, subcutaneously, 7 hours apart), morphine (0.5 ml/kg of 0.4 mg/kg morphine chlorate twice daily, subcutaneously, 7 hours apart), morphine+alcohol co-treatment (0.5 ml/kg of 0.4 mg/kg morphine chlorate+1.0 ml of 15% v/v ethanol twice daily, subcutaneously, 7 hours apart) were administered for 21 days. Treatment with morphine+alcohol significantly impairs cognition functions in the Morris water maze, passive avoidance, and novel object recognition tests, furthermore, the treatment significantly increased the quantitative count of astrocytic cells and also conferred marked neuronal cell death in the mPFC, which were studied by glial fibrillary acidic protein immunochemistry for astrocytes and Cresyl violet for Nissl's substance distribution in neurons respectively. These results suggest that alcohol, morphine, and morphine+alcohol co-treatment may trigger cognitive deficits and neuroinflammatory responses in the brain.

Keywords: Alcohols; Astrocytes; Cognition; Morphine; Neurodegeneration.

Fig. 1. A schematic representation of the behavioral tests.

Fig. 2. The latency of time of Morris water maze test across the groups (n=5 per group at P<0.001). ^a)Significant difference between vehicle and the other groups. ^b)Significant difference between alcohol, morphine, and morphine+alcohol groups. ^c)Significance difference between morphine and morphine+alcohol group.

Fig. 3. The latency of time of passive avoidance test across the groups (n=5 per group at P<0.001). ^a)Significant difference between vehicle and the other groups. ^b)Significant difference between alcohol, morphine, and morphine+alcohol groups. ^c)Significance difference between morphine and morphine+alcohol group.

Fig. 4. The memory index of novel object recognition test across the groups (n=5 per group at P<0.001). ^a)Significant difference between vehicle and the other groups. ^b)Significant difference between alcohol, morphine, and morphine+alcohol groups. ^c)Significance difference between morphine and morphine+alcohol group.

Fig. 5. (A) Photomicrographs of the medial prefrontal cortex of the representative rats in the vehicle, alcohol, morphine, and morphine+alcohol treated groups, respectively. In the vehicle group, the neurons are with intact cytoplasmic contents devoid of perineuronal vacuolation or cavitation, and the Nissl substances are well preserved; in the alcohol treated group, the neurons are with features of chromatolysis, fragmented cytoplasm and peri-nuclear Nissl deposits, pyknotic neurons, neurons with ruptured membrane; in the morphine treated group, Nissl distribution in the neurons are altered. The neurons are with neurodegenerative features such as; neuronal vacuolation, irregular distribution of Nissl's substances and chromatolysis, and peri-nuclear Nissl deposits, vascular structure; in the morphine+alcohol treated group, the neurons have condensed nuclei with nuclear karyorrhectic with concurrent swelling of neuronal dendrites (Cresyl violet staining, ×40). (B) Effect of treatments on the number of normal neurons in the medial prefrontal cortex obtained from the cresyl fast violet stained section (n=5 per group at P<0.001). ^a)Significant difference between vehicle and the other groups. ^b)Significant difference between alcohol, morphine and morphine+alcohol groups while. ^c)Significance difference between morphine and morphine+alcohol group.

Fig. 6. (A) Anti–glial fibrillary acidic protein (GFAP) antibody labelled sections of the medial prefrontal cortex (mPFC) of the representative rats in the vehicle, alcohol, morphine, and morphine+alcohol treated groups respectively. There was a uniform distribution of GFAP immunopositive reactivity in the mPFC of the rat in the vehicle group showing astrocyte with normal cytological features. In the alcohol treated group, there were more GFAP immunopositive astrocytes with intensively stained cell bodies and elongated astrogliotic processes with scar formation (black circles). In the morphine treated group, few astrocytic densities were observed with prominent astroglial scar (black circle); few of the visible neurons are sparsely stained. In the morphine+alcohol treated group, there were many neurons with vacuolations; each of these vacuolated neurons are surrounded by GFAP immunopositive astrocytes; the section is also characterized by obvious patches of glia scars. Note: vascular structures (GFAP, ×40). (B) The graphical representative of the number of GFAP positive of glial cell in mPFC (n=5 per group at P<0.001). ^a)Significant difference between vehicle and the other groups. ^b)Significant difference between alcohol, morphine and morphine+alcohol groups while. ^c)Significance difference between morphine and morphine+alcohol group.