|Year : 2019 | Volume
| Issue : 3 | Page : 276-284
Falcaria vulgaris attenuates morphine toxicity in prefrontal cortex in rats
Shiva Roshankhah1, Cyrus Jalili2, Mohammad Reza Salahshoor1
1 Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Anatomical Sciences, Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
|Date of Submission||24-Mar-2019|
|Date of Acceptance||30-May-2019|
|Date of Web Publication||26-Sep-2019|
Mohammad Reza Salahshoor
Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah, 6714673159
Source of Support: None, Conflict of Interest: None
Background Morphine is a major risk factor in the development of functional disorder of several organs. Falcaria vulgaris is a vegetable that contains antioxidant ingredients.
Objective This study was designed to evaluate the effects of F. vulgaris against morphine-induced damage to the prefrontal cortex of rats.
Materials and methods In this study, 64 Wistar male rats were randomly assigned to eight groups: sham group, morphine group (20 mg/kg once daily in the first 5 days and double per day in the following 5 days; on the 11th to 20th day, 30 mg/kg, doubles each day), F. vulgaris groups (50, 100, and 150 mg/kg), and morphine+F. vulgaris groups (50, 100, and 150 mg/kg). Treatments were administered intraperitoneally daily for 20 days. Ferric reducing/antioxidant power method was applied to determine the total antioxidant capacity. The number of dendritic spines was investigated by Golgi staining. Cresyl violet staining method was used to determine the number of neurons in prefrontal region. Moreover, Griess technique was used to determine serum nitrite oxide level.
Results Morphine administration increased significantly nitrite oxide level and total antioxidant capacity and decreased the number of neuronal dendritic spines and neurons compared with the sham group (P<0.05). In the F. vulgaris and morphine+F. vulgaris groups, in all dosages, the number of neurons and neuronal dendritic spines increased significantly whereas nitrite oxide level and total antioxidant capacity decreased compared with the morphine group (P<0.05).
Conclusion It seems that F. vulgaris administration improves brain’s prefrontal region injury in rats due to morphine.
Keywords: Falcaria vulgaris, morphine, prefrontal cortex
|How to cite this article:|
Roshankhah S, Jalili C, Salahshoor MR. Falcaria vulgaris attenuates morphine toxicity in prefrontal cortex in rats. Egypt Pharmaceut J 2019;18:276-84
|How to cite this URL:|
Roshankhah S, Jalili C, Salahshoor MR. Falcaria vulgaris attenuates morphine toxicity in prefrontal cortex in rats. Egypt Pharmaceut J [serial online] 2019 [cited 2020 Feb 22];18:276-84. Available from: http://www.epj.eg.net/text.asp?2019/18/3/276/264089
| Introduction|| |
Opioids produce free radicals and cause apoptosis in some cells. Morphine is an opioid analgesic drug, and the main psychoactive chemical in opium . Morphine is addictive and causes physiological dependence . Morphine spreads rapidly in brain tissue within 10–20 s and attaches to the nicotinic acetylcholine receptors (nAChRs) . Morphine rapidly passes through the blood–brain barrier and stimulates the mesolimbic dopamine system. This substance can regulate brain neurotransmitters, including catecholamine and serotonin A . Dopaminergic structure shows a vigorous role in controlling memory and mainly rewards behaviors . Morphine acetylcholine receptors are found in pathways, for instance, accumbens nucleus and ventral tegmental. Stimulation of these receptors increases dopamine release in accumbens nucleus and prefrontal cortex and induces feeling of joyfulness in the user . However, morphine can induce oxidative stress in some organs including the brain . Pathologic changes associated with neuronal apoptosis have been reported owing to the use of morphine . Moreover, morphine can induce increased oxidative stress and neuronal apoptosis, destroy DNA, and produce reactive oxygen species . This compound seems to activate the areas of the brain that play an important role in drug addiction and learning process. Among the brain areas greatly affected by morphine are mesocorticolimbic and brain’s prefrontal regions . The prefrontal cortex of brain shows a key role in character and state of mind . The purpose for studying the prefrontal region is for the reason that of the function this cortex has in regulatory performance, judgment, and behavior . The Falcaria vulgaris is a vegetable belonging to the Apiaceae (Umbelliferae) family that grows in the west and southwest of Iran as an annual wild plant . F. vulgaris has antioxidant properties . This plant has a stem and its height is 30 cm on average. In the west of Iran, this plant has been traditionally used to treat skin ulcers, gastric disorders, including gastric ulcer, liver disease, kidney stones, and bladder . Phytochemical studies on this medicinal plant have shown the presence of tannins and saponin . This plant contains vitamin C, phytosterol, protein, and starchy materials and is a rich source of tannins and ascorbic acid. Similar to antibiotics, it can be used for the treatment of skin ulcers . Considering the effects of morphine toxicity on the brain and the therapeutic properties of F. vulgaris, the present study was designed and conducted to investigate the effects of F. vulgaris on morphine-induced toxicity in the brain’s prefrontal region of male rats.
| Materials and methods|| |
In this experimental study, 64 male Wistar rats) weighing 220–250 g (were purchased from the Pasteur Institute and transferred to the animal house in the medical school. During the study, the animals were kept under standard conditions (i.e. 12 h light/12 h dark and 22±2°C), humidity of 50–60%, in special cages and on a straw bed. Water and food were freely available to the animals. Standard food and treated municipal water were used to feed the animals. All investigations conformed to the ethical and human principles of research and were approved by the Ethics Committee (ethics certificate no. 97564) .
F. vulgaris plant was obtained from a local store (time to pick and buy this plant in the spring in the western of Iran), and its impurities were removed. After endorsement by a botanist, the plant was cleaned. The leaves and stems were desiccated in shadow for 5 days and ground using a grinder. Next, 100 g of the powder was added to 70% ethanol. The acquired solution was reserved in a warm water bath (36°C) under dark condition. Thereafter, the solution was progressively poured on Buchner funnel filter paper and cleaned by a vacuum pump. It was then transferred to a rotary device to obtain the extra solvent. The isolation process continued until the concentrated extract was obtained. The extract was dissolved in distilled water and administered intraperitoneally per a kilogram of animal’s weight. It was sterilized after double filtration through a 0.2-μm filter .
Study groups and treatment of animals
A total of 64 male rats were randomly divided into eight groups, and eight rats were placed in each group. The first group was the sham group, which received normal saline through intraperitoneal injection equivalent to the amount of experimental groups. In the second morphine group, morphine was administered via an intraperitoneal injection (20 mg/kg once daily in the first 5 days and double per day in the following 5 days; on the 11th to 20th day, a dose of up to 30 mg/kg doubles each day). The third to fifth groups included the F. vulgaris groups, in which each animal correspondingly received 50, 100, and 150 mg/kg of F. vulgaris once daily, intraperitoneally, on days 1–20. The sixth to eighth groups include morphine+F. vulgaris groups, in which each animal on days 1–20 received 50, 100, and 150 mg/kg of F. vulgaris once daily plus morphine intraperitoneally ,.
The transcardiac method was used for fixation. In this process, 24 h after the last injection of the drug, animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). The chest was opened in the midline, and the apex of the left ventricle was pierced after the completion of thoracotomy. Next, a glass cannula of 1 mm diameter was inserted and fixed in the ascending aorta. The pericardium and the right ventricle were cut. The left ventricle pathway was cut and the ascending aorta was connected to a plastic tube by the glass cannula, and descending aorta was clamped right above the diaphragm. The cannula linked to the normal saline solution was implanted into the aorta through making an incision in the left ventricle. The descending aorta was fastened, and after washing the brain, the solution was removed through the incision made in the right atrium. Formalin 5% and buffer phosphate 7% were inoculated into the brain by the cannula, and the brain was fixed for 15 min. After perfusion, the brains were separated from the skull and stored in the same perfusion solution for 3 days .
Golgi staining method
The Golgi staining method was used to observe neuron dendrites in the brain prefrontal cortex. This method was applied using potassium dichromate followed by silver nitrate. After brain fixation, tissue blocks were put inside 3% potassium dichromate solution for 48 h in a dark environment. The blocks were washed in 0.75% silver nitrate solution and were put inside the solution for 72 h. The tissues were washed in 1% silver nitrate solution. Then, tissue processing, counting dehydration, clearing, and embedding were performed. Microscopic sections (5 μm) were prepared and examined morphologically .
Cresyl violet method
The cresyl violet staining method was used to determine the number of live cells in the prefrontal cortex. In brief, the slips were stable again (10 min) in 4% paraformaldehyde solution. Slides were immersed in 70 and 100% ethanol and in xylene for 20 min. They were then immersed back through the ethanol descent concentrations. They were stained for 5 min in filtered cresyl violet solution. The slides were then dehydrated again in ethanol. They were located in xylene for another 10 min and then cover slipped. After preparing the photograph, the number of cells was counted in 1 mm2). In the slides stained by means of cresyl violet technique, the round cells without peak nose were considered as live cells .
The dendritic thorn count was made via microscopic examination with an optical microscope and Motic software and Image tool IT (version 3) software. In the slides stained through Golgi staining technique, neurons entirely stained with cell bodies in the central part of the tissue slices distant from the surrounding stained neurons were included. The dendritic tree of pyramidal neurons was demonstrated through camera lucida at ×750 magnification and the dendritic exclusion order from the cell body was used for counting the dendritic pieces .
Nitrite oxide measurement was done by Griess assay using microplate technique. Through this process, zinc sulfate powder (6 mg) was mixed with serum samples (400 µl), and vortexed for 1 min. The samples were centrifuged at 4°C for 10 min at 12 000 rpm, and the supernatant was used to measure the nitrite oxide. In brief, 50 µl of sample was added to 100 µl of Griess reagent (Sigma, St. louis, MO, USA), and the reaction mixture was incubated for ∼30 min at room temperature. The optical density (OD) of the sample was measured by an ELISA reader (Hyperion, Miami, FL, USA) at a wavelength of 540 nm according to the manufacturer’s protocol .
Ferric reducing/antioxidant power method
Ferric reducing/antioxidant power method was used to the measure total antioxidant capacity of the serum (Abcam, ab234626, Calif, Ml, USA). Before transcardiac method, venipuncture from the animals’ hearts (right ventricle) was done using a 5 ml syringe. The blood sample was incubated for 15 min at 37°C to clot. Then the clotted blood was then centrifuged for 15 min at 3000 rpm until the serum was separated. The separated serum was stored at the temperature of −70°C until antioxidant capacity level was measured. In this technique, the ability of the plasma to reinstate ferric ions was measured. This process required a great quantity of FeIII. A blue stain was formed when the compound of FeIII-TPTZ in acidic pH returned to FeII and absorption at the maximum wavelength was measured at 532 nm on spectrometer (Spectro, Germany). The only factor defining the speed of the FeII-TPTZ and the blue color was the vitalizing power of the sample. Total antioxidant capacity values were strategized by means of the standard curve with diverse concentrations of iron sulfate .
The Kruskal–Wallis test was used to examine data normality and the homogeneity of variance at a significance level of 0.05. The one-way analysis of variance was used for statistical analysis, and Tukey post-hoc test was used to determine the difference between the groups. SPSS 16 (IBM, SPSS version 16.0, New York) was used for data analysis. The obtained results were expressed as mean±SE and P value less than 0.05 was considered statistically significant.
| Results|| |
The results regarding the number of neurons in the brain prefrontal cortex showed a significant decrease in morphine group compared with the sham group (P<0.05). The mean number of neurons was not significant in all F. vulgaris groups compared with the sham group (P>0.05). Moreover, the mean number of pyramidal neurons increases significantly in F. vulgaris and morphine+F. vulgaris groups in all doses compared with the morphine group (P<0.05) ([Figure 1] and [Figure 2]).
|Figure 1 Effect of morphine and Falcaria vulgaris administration on the number of neurons in the brain prefrontal region. *Significant decrease in the mean number of neurons in the morphine group compared with the sham group (P<0.05). †Significant increase in all F. vulgaris groups compared with the morphine groups (P<0.05). ¶Significant increase in all morphine+F. vulgaris groups compared with the morphine group (P<0.05).|
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|Figure 2 Microscopic images of brain prefrontal cortex in male rats in different groups (5 µm thick sections, cresyl violet staining, at ×100 magnification). Micrograph of the prefrontal cortex section in the sham group (a), showing normal number of neurons in the prefrontal cortex. Micrograph of the prefrontal cortex section in morphine group (b), showing decreased neurons cells. Micrograph of the prefrontal region section in Falcaria vulgaris (150 mg/kg) group (c), showing normal number of neurons. Micrograph of brain prefrontal cortex section in morphine+F. vulgaris (150 mg/kg) (d), showing normal number of neurons.|
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The mean number of neuronal dendritic spines in experimental groups showed a significant decrease between the sham group and morphine group (P<0.05). Moreover, the mean number of neuronal dendritic spines was not significant in all F. vulgaris groups compared with the sham group (P>0.05). Furthermore, at the F. vulgaris and morphine+F. vulgaris groups, the mean number of neuronal dendritic spines increases significantly in all treated groups compared with the morphine group (P<0.05) ([Figure 3] and [Figure 4]).
|Figure 3 Comparison of morphine, Falcaria vulgaris, and morphine+F. vulgaris groups regarding the number of dendritic spines in brain prefrontal cortex. *Significant decrease in the morphine group compared with the sham group (P<0.05). †Significant increase in all F. vulgaris groups compared with the morphine group (P<0.05). ¶Significant increase for all morphine+F. vulgaris groups compared with the morphine group (P<0.05).|
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|Figure 4 Microscopic images of neuronal dendritic and dendritic spines in brain prefrontal cortex in male rats in different groups (5 μm thick sections, Golgi staining, magnification ×400). Micrograph of the brain prefrontal section in the sham group (a, neuronal dendritic, e, dendritic spines), showing normal structure. Micrograph of the brain prefrontal cortex section in morphine group (b, neuronal dendritic, f, dendritic spines) showing decreased number of dendritic spines owing to the oxidative stress caused by morphine. Micrograph of the prefrontal cortex section in the Falcaria vulgaris (150 mg/kg) group (c, neuronal dendritic, g, dendritic spines) showing normal structure. Micrograph of prefrontal section in morphine+F. vulgaris (150 mg/kg) (d, neuronal dendritic, h, dendritic spines) groups, showing normal structure.|
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The results of blood serum nitrite oxide measurement showed a significant increase in morphine group compared with the sham group (P<0.05). The mean nitrite oxide in the blood serum was not significant in all F. vulgaris groups compared with the sham group (P>0.05). Moreover, the mean level of nitrite oxide in blood serum declined significantly in F. vulgaris and morphine+F. vulgaris groups in all doses compared with the morphine group (P<0.05) ([Figure 5]).
|Figure 5 Effects of Falcaria vulgaris, morphine, and morphine+F. vulgaris on the mean of nitrite oxide level. *Significant increase of nitrite oxide in the morphine group compared with the sham group (P<0.05). †Significant decrease in all F. vulgaris groups compared with the morphine group (P<0.05). ¶Significant decrease in all morphine+F. vulgaris groups compared with the morphine group (P<0.05).|
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Total antioxidant capacity
The results displayed that the total antioxidant capacity serum level reduced significantly in the morphine group compared with the sham group (P<0.05). The total antioxidant capacity level enhanced significantly in all F. vulgaris and morphine+F. vulgaris groups compared with the morphine group (P<0.05) ([Figure 6]).
|Figure 6 Comparison of total antioxidant capacity in morphine, Falcaria vulgaris, and morphine+F. vulgaris groups. *Significant decrease in the morphine group compared with the sham group (P<0.05). †Significant increase in all F. vulgaris groups compared with the morphine group (P<0.05). ‡Significant increase in all morphine+F. vulgaris groups compared with the morphine group (P<0.05).|
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| Discussion|| |
The prefrontal cortex of the brain displays an important role in personality . Morphine produces many implications and adverse effects by influencing the central nervous system . The patients experiencing shocks in the prefrontal region mislay community consciousness, skill to conversation, decision, and initiative, so that they would hardly be able to live . The present study was aimed to investigate the effects of F. vulgaris on morphine-induced disorders in the prefrontal cortex. Based on the results of this study, it can be generally argued that morphine has destructive effects on the prefrontal cortex. The results of the current study showed that the number of neurons and dendritic thorns decreased significantly in morphine group in comparison with the sham group. In all F. vulgaris and morphine+F. vulgaris groups, there was a significant increase in the number of dendritic thorns compared with the morphine group. The results may indicate the control of apoptosis and neurodegeneration by administering different doses of F. vulgaris . The results of Montel et al.  were consistent with those of the present study that showed morphine could damage the cells in the brain cortex by increased protein accumulation in the membrane and reduced cell size. It seems that morphine induces oxidative stress which can cause cell damage . Generated free radicals following oxidative stress may have the potential to damage cellular compositions, including proteins, lipids, and DNA . The lipid in the membrane of the nerve cells has a high content of oxidized unsaturated fatty acids. Therefore, it seems that morphine can produce reactive oxygen species via P-450 enzyme and cause the destruction of the nucleus in neurons . Dendritic thorns play a major role in synaptic transmission . Morphine can reduce the length and the number of dendritic thorns in nucleus accumbens by affecting the neurotrophic factors in the striatum . A study by Robinson and Kolb  showed that morphine injections could reduce the length and the number of dendritic thorns, which is consistent with the results of our study. It seems that morphine can destroy dendritic thorns by β2-nAChRs deactivation at postsynaptic cells in prefrontal cortex . Moreover, morphine can reduce the number of thorns by deactivating α4β2-nAChRs in the presynaptic membrane and by disrupting the release of glutamate neurotransmitters . F. vulgaris is a purifier of reactive oxygen species and has the potential to destroy oxidative stress . The results of the study by Rafiey et al.  confirmed the results of the present study that F. vulgaris could prevent cell death and development of oxidative stress owing to STZ administration. F. vulgaris seems to inhibit oxidative stress of quinolinic acid . F. vulgaris extract can increase the effects of antioxidant enzymes such as catalase and superoxide dismutase and reduce ROS production . The results of this study showed that there was a significant increase in serum nitrite oxide and serum total antioxidant levels in the morphine group compared with the sham group. In all F. vulgaris and morphine+F. vulgaris groups, there was a significant decrease in serum nitrite oxide and serum total antioxidant levels in comparison with the morphine group. Nitrite oxide is a free radical and can regulate angiogenesis, apoptosis, cell cycle, and metastasis in cells . Morphine can stimulate nitrite oxide receptors in the brain and increase glutamate release and NMDA activation. The activation of NMDA may increase the formation of nitrite oxide in the prefrontal cortex . The results of a study by Keser et al.  showed that exposure to morphine increases the activity of nitrite oxide in the frontal cortex in the mouse brain, which is consistent with the results of the present study. Jalili et al.  showed that nicotine decreases neuron cell count and dendritic spines compared with the control group, which is consistent with the results of the current study. The reduction in total antioxidant capacity level in this study shows the effects of oxidative stress from morphine on the prefrontal neuron. Morphine induces oxidative stress in neural tissue, which is demonstrated as a growth in the levels of ROS and a reduction in the action of antioxidant enzymes like total antioxidant capacity . In the present study, improved levels of total antioxidant capacity in rats treated with F. vulgaris highlight the antioxidant effects of F. vulgaris . A total antioxidant level increase owing to the administration of morphine indicates the positive effect of F. vulgaris on magnified antioxidant effects. Further, it is assumed that saponins in the extract of F. vulgaris inhibit the synthesis of nitrite oxide induction enzyme . The results of the study by Salahshoor et al.  are consistent with the results of the present study, which indicated that F. vulgaris administration could inhibit nitrite oxide production. The results of the present study showed that F. vulgaris administration in all doses studied might have a positive effect on morphine-induced toxicity, as an oxidative stress, in the neurons of the prefrontal cortex, and these effects were not associated with an increase in the dose of Falcaria extract.
| Conclusion|| |
It appears that F. vulgaris provides protection against oxidative stress resulting from morphine in the prefrontal cortex. Such an ability of the F. vulgaris might be owing to its strong potential antioxidant attributes. F. vulgaris administration moderates the antioxidant agents in the extract. As a result, it leads to prefrontal cortex recovery and prevention of morphine adverse effects on total antioxidant capacity, nitrite oxide, number of neurons, and dendritic spines as evidenced in the male rats.
The authors acknowledge the Research Council of Kermanshah University of Medical Sciences (No: 97564) for the financial support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]