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Glutathione Reductase Activity assay

A study of some of the effects of aqueous extract of leaves of Cannabis sativa in the visual cortex of adult Wistar rats

TEXT

Introduction

Hallucination in the broadest sense, is the subjective perception of an object or event no stimulus or situation is such present1, 2. In a strict sense, is defined as a perception in a conscious and awake state in the absence external stimuli, which have properties perception, real estate abroad space2 goal. It can occur in any modality Sensory: visual, auditory, olfactory, gustatory, tactile, proprioceptive, equilibrioceptive, nociceptive and chronoceptive3 thermoceptive. A mild form of the disorder is called, and can occur in both directions. Hallucination may be benevolent (it is, tell someone good about himself or malicious (eg, cursing someone) and occurs in both healthy and diseased individuals4. The most common form of hallucination including the visual phenomenon of seeing things which are not present. Different causes of visual hallucinations were classified as psychobiochemical (alteration of neurotransmitters) psychophysiological (A modification of the structure of the brain) and psychological / psychodynamic (eg, significant experience in consciousness5 interference. Manford and Andermann (1998) 6 summarizes the three pathophysiologic mechanisms of visual hallucination, so:

1. i. irritation of the visual cortex, both the (areas Brodmann 18 and 19) primary (Brodmann area 17) and association
2. ii. deafferentation of the visual system may lead to the release phenomenon cortical
3. iii. effects on the reticular activating system

An injury of any kind and from any source in the visual system, particularly involving the visual cortex will result in visual hallucinations. Studies have shown that extracts of medicinal plants and have a partnership with visual hallucinations. One of the major plants that have been attributed to cannabis sativa visual hallucination 7.

Cannabis sativa, known as marijuana or cannabis is an annual plant of the family Cannabaceae and an herb that has been used through the history of man as a source of fiber, for its oil, food, like a drug and medicine purposes8 and spiritual character. In its action on nerve and higher centers may lead intoxication with stimulants hallucination7. His property was a widely used psychoactive drug Nigeria, despite the legal consequences in their possession and use. The World Drug Report 2006 (WDR 2006) published by the United Nations Office cons Drugs and crime 9 showed that the annual prevalence of cannabis in Nigeria is about 13.8% as in 20,002 years. It should be noted that the prevalence Annual consumption of cannabis is the percentage of the population of young adults (15-64 years) who have used drugs once in the last survey year. Nigeria, with this value, has the highest prevalence of cannabis in the world9 9.

Methodology

600 g of dried leaves Cannabis sativa, obtained from the control of the Kwara State NDLEA was crushed in a mortar and pestle local powder. Power 100 is soaked in 100 g ml of distilled water for 72 hours and filtered after 72 hours without a Whatman filter paper for filtering 800ml. The filtrate is dried in an oven at a temperature of 600C for 7 days to form a dark brown paste of 10 grams was dissolved in 50 ml of phosphate buffer saline 200mg/ml an aqueous solution of cannabis sativa.

Sixteen (16) adult Wistar rats with an average weight of 200g weight range (170g to 240g is) were purchased from the pet department of zoology University of Ilorin. They were of both sexes of rats with 8 male and 8 female rats. standard diet of rat food was purchased Animal Bendel, Taiwo Road, Ilorin.

The animals were bred in the Animal Farm of the Faculty of Basic Sciences, University Medical Ilorin. Were made to acclimatize for two (2) weeks before the start of administration of plant extracts. Have were fed a normal diet was purchased from Bendel feed at a time to avoid a change in the composition of the diet and also given ad libitum tap water. They were given free assess to food and water. They were kept in wooden cages standard laboratory in groups of four (4) with males and females reared apart. They were treated in general, under conditions standardized, well-lit laboratory at moderate temperature and adequate ventilation and a clean environment. They were weighed daily Saltul systematically weighing balance.

The animals were divided into two (2) experimental and control groups A and B, each group of animals were four male and four female rats were kept in separate apartments to avoid mating and pregnancy in females. The animals in group A were treated with 300 mg / kg bw / d (0.3 ml) for 21 days Cannabis sativa while group B animals were treated with an equal volume of saline phosphate buffer of each dose of the extract (0.3 ml) per day for 21 days. The administration of the extract and phosphate buffer saline was orally with the help of the orogastric tube at 07.00 hours each day.

The animals were killed by cervical dislocation 24 hours after 21 days of administration. Brain tissue was carefully removed from the skull and occipital cortex quickly removed for preparation of homogenates tissue while the whole brain tissue was fixed in formalin 10% calcium for 72 hours, after which they were removed right occipital cortex separately for histological further. The left occipital cortex removed was placed in the mortar (Lao Style) after being weighed with the delicate balance weight. 1 ml of 0.25 M (6.625%) was added a solution of sucrose and the fine homogenate. tissue homogenate were collected in the vial with 5 ml of normal saline for enzyme assay.

Data were analyzed using software version SPSS Statistics 17. Mean and standard error of the mean (SEM) values of each group was determined. The average values of experimental groups were compared by Student t test, confidence interval 95% to determine the level of statistical significance between the mean values

Results

The animals were observed closely throughout the research period. During acclimation, all animals appeared normal, hair supposedly without problems since the back and pink eyes. They eat very long. On the first day of the extract of the plant, animal group Experimental seemed agitated for several hours after administration. They had hair on the back foot, bright eyes and pink. Appeared dull and inactive as the day went and took out food and water. Probably appeared normal during the last 12 hours a day and eat more moderately. As the end of administration, the animals in the experimental groups particular seems to be more docile and less active. The animals in group B (control animals) appeared to retain the status of compliance before the administration throughout this investigation.

animal weights were monitored at 07.00 am each day and recorded. Before the plant extract, all animals in both experimental and control groups were gaining weight. From the third day of treatment, animals in the experimental group was observed they begin to lose weight. Her weight remained stable over the first and second days of the administration (no increase or decrease). The animals in the control group, but still gain weight constant. Statistical analysis weight changes indicate no statistically significant differences in changes in weight of animals in the experimental group, compared the control group, p> 0.05. The average weight on certain days, the average standard deviation of the mean (SEM) and the importance level of around 95% shown in Tables 1, 2 and 3.

There was an increased level of LDH in homogenates of experimental animals, compared to control animals, with a statistically significant difference in the average level of LDH (P <0.05). The results of the LDH level means with standard error of the mean (SEM) and significance level are shown in Tables 4 and 5. There was also an increase in the level of G6PDH homogenized with a statistically significantly the average level of G6PDH (P <0.05) in animals in the experimental group compared with the control group. The results of G6PD levels mean error error of the mean (SEM) and significance level are shown in Tables 6 and 7.

Visual sections stained with hematoxylin and eosin bark showed vacuolation of neurons, glial cells and pyramidal cells in the experimental groups. cell population, using stereological network, appeared more numerous in group B sctions to control sections for the experimental group. Nissl bodies are widely affectted cresyl fast sections stained purple experimental group of animals and this was evidenced by the perinuclear space and many smaller the proportion of neurons using the network of stereological sections. The sections of DNA Feulgen DNA showed black-black animals against the pink-red proposed by Rossenbeck Feulgen and observed in the control group.

Discussion

The plant extract appears to have a first psycho-stimulant effects of animals, making them more agitated and aggressive few hours immediately after administration. Several hours after administration extracts of plants, animals entered into inactivity, but not to sleep. One could say that the sedative effects of the extract began psychological after effects stimulants have fallen, but the plant extract they seem to have hypnotic effects.

The plant extract also appears be effective in weight loss. The mean weight on days 1, 8, 15 and 22 in the result (Table 1) showed a reduction in days of laboratory animals and an increase for the control animals, although the average weight loss in comparison, is not statistically significant. The plant extract also appears increase the activity of G6PDH and LDH in neurons of the visual cortex, respectively.

The increase in G6PDH levels suggests the formation of oxidants stress after the administration of plant extracts. G6PD is a cytoplasmic enzyme that plays a protective role during oxidative stress in animal eukaryotic because they provide co-enzymes and substrates, the main antioxidant enzymes10. By virtue of its ability to produce NADPH with glutathione reductase, G6PDH is traditionally considered a supporter of the System11 antioxidant. Antioxidant enzymes regulate free radical reactions by compacting, repairing, development and the chain breaking reactions12, 13.

Oxidative stress occurs when free radical generation or the capacity increase free radicals and / or repair both14 oxidation decreases modification of macromolecules. This imbalance leads to the accumulation of oxidized molecules modified mainly final products of superoxide (O2) and hydroxyl ions (OH-) 15,16. reactive O2 species (ROS) has been shown to mediate cell damage. ROS protection requires the maintenance of endogenous thiol groups, more importantly, Reduced Glutathione (GSH) by NADPH17. Affect G6PDH producing the reduced form cytosolic adenosine dinucleotide phosphate nicotinamide coenzyme (NADPH), control the passage of glucose-6-phosphate 6-phosphogluconate in the pentose (What is the direct oxidation of glucose) 18. Salvemini et al (1996) 19 showed that G6PDH expression is enhanced by oxidative stress induced by agents that either increase the intracellular concentration of oxygen or decrease the pool of glutathione.

the high oxygen consumption headwaters and intracellular metabolic activity increased leakage of electrons from the mitochondrial transport system and causes increased oxidative stress and production of free radicals with vulnerability to cell damage increasesd 20.

Following this agreement, the plant extract, used as stressors induced oxidase has been shown to mediate cellular damage, either by increasing the intracellular concentration of oxygen and thus produce more ROS or reduced glutathione pool, where the levels of G6PDH (follower of anti-oxidant) increased. G6PDH levels in homogenates the visual cortex of animals treated with cannabis is the average value of 1.298mmol / L, which is statistically significant at the average values of G6PDH 0.724mmol / L in control animals.

LDH is present in almost all tissues of the body and is used to detect changes in tissue body21. Is responsible for the conversion of pyruvate to lactate and enzyme Key in the anaerobic decomposition of glucose to pyruvate during glycolysis. Due its wide dissemination throughout the body, cellular damage causes an increase in serum total and HDL tissues, so that when an injury to tissues, cell LDH increased and, consequently, release into the blood circulation, which is identified in more than values22 normal.

oxygen consumption elevated during increased metabolic activity with increased leakage of electrons transport system and oxidative stress and mitochondrial increased production of free radicals that damage cellular components increased levels of LDH homogenized tissue of animals in the experimental group in this study. The average level of LDH in the treated group of cannabis 16100u / L is statistically significant compared with the control group (mean LDH 5940u / L).

In this study, higher levels of G6PDH and LDH in the experimental groups suggest that a certain amount of damage Cell was held in the visual cortex of animals in the group. The increase in enzyme levels also suggest that neurons in the visual cortex metabolize carbohydrate carbon through both pentose phosphate pathway, which is an aerobic and glycolytic (anaerobic) pathway. The rate of metabolism is by aerobic pentose phosphate pathway. G6PDH activity increased in animals treated with the extract of the plant suggest that the production of ribose – 5-phosphate in the pentose phosphate pathway, which is used in the synthesis of nucleotides and nucleic acids.

visual cortex sections were mounted on the light microscope and examined critically. Articles in laboratory animals have shown vacoulations in neurons and glial cells pyramidal cells in H & E stained slides, showing no cell death in neurons of animals. Slides of these animals were slightly stained to show that there were fewer neurons to take the stain (H and E). Perinucleus large space and reduces the proportion of neurons in the slides stained CFV laboratory animals have been very concerned about the Nissl substance in animals. The abnormal DNA dark-black on the slats of the animals in group experimental compared to normal colouration23 pink-red when viewed from behind the scenes of animals in the control group indicated that the extract affects the nuclear substance, even if she is responsible for this could be explained later.

Conclusion

This study showed that the aqueous leaf extract of cannabis has detrimental effects on the visual cortex of adult Wistar rats. Biochemical leaf extract cause cell damage through the mediation of oxidative stress and release of reactive oxygen species (ROS) with greater effects in the treatment group cannabis. Histologically, sections of the visual cortex showed vacuolation and perinuclear spaces, Nissl substance widely affected as evidenced reduced proportion of abnormal cells and DNA staining in groups of animals treated with cannabis sativa. Therefore, the use of this plant for its hallucinogenic effects and other reasons why a man should be made with great caution, because there is a greater vulnerability to toxicity.

About the Author

Authors: *A. A Tijani and **A. M Adekilekun

* Department of Anatomy, Faculty of Basic Medical Sciences

College of Health Sciences, Osun State University, Osogbo.

**School of Basic Midwifery, University of Ilorin Teaching Hospital

Ilorin, Kwara State.

Name of Department and Institution in which the work was done:

Department of Anatomy, Faculty of Basic Medical Sciences

College of Health Science, University of Ilorin.

Name and Address of Corresponding Author:

A.A Tijani, jidekilekun3@yahoo.com, 08038063582

Department of Anatomy, Faculty of Basic Medical Sciences

College of Health Sciences, Osun State University, Osogbo.