The Effect of Neuronal Demyelination on Behavioral, Morphometric and Histological Changes of the fornix and Hippocampus of Male Multiple Sclerosis Rats

 Brain morphology using quantitative data is one of the most important research topics in neuroscience. There is little documentation about the anatomical changes in the demyelinated mouse brain. On the other hand, the differences between the changes applied due to demyelination in both genders have not been studied. Therefore, the present study was conducted with the aim of investigating the morphometric aspects of the brain of demyelinated male rats along with evaluating the changes in their behavioral patterns.Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) that affects different areas such as the brain, spinal cord, and optic nerve. The disease is characterized by active plaques containing cytokine-secreting T lymphocytes (1, 2).Nerve demyelination following acute CNS inflammation leads to neurological disability characterized by temporary remission and recurrence or a chronic advanced stage. One of the complications of the disease is cognitive disorders that have been reported in 40-60% of cases and occur in the early or even late stages of the disease (3, 4).MS-related cognitive impairment is often characterized by a relative decrease in neurophysiological function in assessing short-term memory processing speed, attention, spatial visual abilities, and executive function (5, 6).These disorders affect the course of life of individuals and are diagnosed in clinical evaluation to some extent imperceptibly but have not been studied morphometrically. So far, MRI findings have been used to examine the association between structural damage and cognitive impairment. Studies have shown that atrophy of various structures in the gray matter of the brain, such as the cerebral cortex, causes neurological disability (6, 7).Specifically, atrophy of some areas of the cortex, especially the hippocampus, is associated with dysfunction of the cognitive process in the early stages of the disease, while changes in white matter volume did not play a role in the development of these cognitive disorders (8, 9).In one study, imaging findings from patients with Parkinson's showed that the hippocampus and amygdala were atrophied, and this atrophy was concentrated in the cephalic areas of the hippocampus. This dementia has not been observed (10).Also, in patients with Alzheimer's disease, a decrease in the total volume of the fornix and mammals is directly related to cognitive disorders (11).On the other hand, in another study, the findings of people with brain trauma indicated that a reduction in the volume of the hippocampus, fornix and carpus callosum is also seen in the lesion-free group (12).The corpus callosum is the main structure of white matter in the brain that connects the right and left hemispheres. This structure is damaged in MS and its damage causes complex and cognitive disorders. AlonKalron's findings also show that MS reduces the volume of the hippocampus, amygdala and thalamus. Findings of scientists such as Audoin, Wadhwa and Benear also show the role of hippocampus and phoenix in memory and learning (13-15).Given that none of the studies has so far examined the relationship between the histological and anatomical structure of the brain and its function, this study attempts to macroscopically, microscopically and horny areas of the hippocampus and fornix. Study a function in the male rat demilinase model.

 This study was performed on 21 male rats weighing approximately 300-350 g in ShahidChamran Veterinary School of Ahvaz. First, in order to adapt the rats to the environment, they were kept at 23 ± 23 ° C for one week, and the dark-light cycle was maintained for 12 hours without water and food restrictions. All stages of the test were performed according to the instructions of the Committee on Ethics of Working with Laboratory Animals, Faculty of Veterinary Medicine, ShahidChamran University of Ahvaz (EE / 99.3.02.55591 / scu.ac.ir: Code of Ethics). The mice were then randomly divided into three groups of seven consisting of the first group: healthy, the second group: normal saline recipient and the third group: ethidium bromide recipient.For stereotaxic surgery and injection of ethidium bromide using a microinjector, mice were anesthetized with ketamine (80mg / kg) and xylazine (5mg / kg) and the hair on the skull was shaved and then in the midline of the head from the frontal to the oxy. The sagittal shear palate was created to determine the injection site according to the coordinates of the Paxinos atlas (anterior-posterior 0.46, internal-external 1 and dorsal-abdominal 2.2). In the final stage, injection of ethidium bromide 0.02% and normal saline at the rate of 20 μl was performed using a Hamilton syringe in the specified area in mice in the injury group and normal saline, respectively (16). Modified Neurological Severity Scores (MNSS) method was used to check balance. This test consists of several parts that are measured in the balance test of several parameters، Also, in order to investigate the type and manner of animal movement in ethidium bromide, normal saline and healthy rats, footprint test was used. Morris water maze test was used to check the level of spatial learning of the animal،At the end of the twelfth week, the tested groups were anesthetized by intraperitoneal injection of ketamine and xylazine, and after perfusion (with normal saline solution and 4% paraformaldehyde), the animal's head was separated and the brain was removed without damage. After brain extraction, their length and width were measured using a digital caliper and their weight was measured using a digital scale. Then, the weight and volume of the hippocampus region were measured, and the demyelination and nerve cell survival indices in the brain tissue were measured using Siloxal Fast Blue and Cresyl Violet staining in the hippocampus and fornix.

 The results obtained from the balance test showed balance and motor dysfunction in the affected group, which was statistically significant (P <0.05). The results of the Morris water maze test in 5 days (4 days of learning and one day of testing) show that the healthy and saline groups found the platform in less time than the demyelination group. On the other hand, the mean brain indices, The hippocampus and fornix were less in the injury group compared to the healthy and saline groups (P <0.05). Fast blue lux staining also showed severe myelin damage in the ethidium bromide group and also increased the number of damaged nerve cells (dark cell) in curzil violet staining (P <0.05).

 Cerebral demyelination reduces the volume of the hippocampus and fornix in the brain of male rats and also increases the number of damaged cells in direct relation to memory loss and balance. These people can also develop cognitive and behavioral disorders that will negatively affect their daily lives.