|Year : 2019 | Volume
| Issue : 2 | Page : 124-128
Impacts of low-protein diet on the hippocampal CA1 neurons and learning deficits in rats
Shiva Roshankhah1, Ehsan Sadeghi2, Cyrus Jalili3, Mohammad Reza Salahshoor1
1 Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Nutritional Science, Public Health School, Kermanshah University of Medical Sciences, Kermanshah, Iran
3 Department of Anatomical Sciences, Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
|Date of Web Publication||8-May-2019|
Mohammad Reza Salahshoor
Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah
Source of Support: None, Conflict of Interest: None
Introduction: Proteins are the essential part of all organism cells. Nutrition plays the most important role in the structure and function of the brain. CA1 region belongs to hippocampus and plays a vital role in converting short-term to long-term memory. This study was designed to assess the effects of low-protein diet on hippocampal region CA1 and learning deficit in rats. Materials and Methods: In this study, 30 male rats were randomly assigned to two groups: control group and low-protein diet group (8% protein). Animals in a low-protein diet group have eaten food with low protein daily for 10 months. Body weight was measured. Transcardiac perfusion method was applied to tissue fixation. Passive avoidance learning of animals was examined by the shuttle-box apparatus technique. The number of dendritic spines was investigated by the Golgi staining technique. Furthermore, Cresyl violet staining method was used to determine the number of neurons in the hippocampal region CA1. Results: The passive avoidance learning of the low-protein diet rats was reduced significantly compared to the control ones (P < 0.001). Low-protein diet decreased the body weight, number of neuronal dendritic spines and neurons compared to the control group (P < 0.001). Conclusion: It seems that administration of low-protein diet had harmful effects of structure and function of hippocampal region CA1 in rats.
Keywords: CA1, hippocampus, learning, low protein
|How to cite this article:|
Roshankhah S, Sadeghi E, Jalili C, Salahshoor MR. Impacts of low-protein diet on the hippocampal CA1 neurons and learning deficits in rats. Adv Hum Biol 2019;9:124-8
|How to cite this URL:|
Roshankhah S, Sadeghi E, Jalili C, Salahshoor MR. Impacts of low-protein diet on the hippocampal CA1 neurons and learning deficits in rats. Adv Hum Biol [serial online] 2019 [cited 2020 Jul 7];9:124-8. Available from: http://www.aihbonline.com/text.asp?2019/9/2/124/257813
| Introduction|| |
This is a fact that the brain has a life cycle for physiological, biochemical and anatomical evolution. It is not surprising that nutrition factors are considered to be effective in the function and structure of the brain. Nearly two-third of metabolic items of the body are comprised proteins. The need for protein for per kilogram of weight in childhood is daily 1.2 g/kg, and it seems that 10%–16% of total daily calorie of the body is obtained from proteins. Protein deficiency may affect chemical structure of the brain neurons and even behaviour. There are a lot of unanswered questions regarding how diets can affect neurological and psychological disorders. The relationship between a good diet and appropriate evolution of the brain, specifically in children, has always been discussed. Natural evolution of different brain region includes numerous brain cells, migration and organisation of neurons which starts from neonatal period up to some times after birth. Development of neurite in neuron cells includes dendrites and axons depending on the nature of the stimulus received by the brain and the quality of an appropriate diet. Myelin structures, glial cells and neurons are full of protein; therefore, an appropriate diet, specifically a diet full of proteins, is required for the evolution of different brain regions. Biosynthesis of brain proteins depends on the efficient and consistent receipt of amino acids. Furthermore, it seems that proteins are necessary for providing the required energy of metabolisms and synthesis of neurotransmitters in the brain. Hippocampus has structures that play an important role in the formation of long-term memory, and their function is associated with stimulation reward system. Hippocampus is a part of the limbic system and seems to be essential in the formation of different types of learning and memory. CA1 area belongs to the hippocampus and plays a vigorous role in converting short-term to long-term memory. The reported results of Kinzig et al. indicated that, in rats which were fed with low-protein diet, hypothalamus nucleus had malformation. With regard to the importance of protein in brain evolution, and since the impacts of low-protein diet on amount of learning and changes in neurons of the CA1 region is a research gap, the present study aims to investigate the impacts of impacts of low-protein diet on changes in neurons of the CA1 region of hippocampus and the amount of learning in rats.
| Materials and Methods|| |
In this experimental study, 30 male Wistar rats (weighing 220–250 g) were purchased from the Pasteur Institute and transferred to the animal house in medical school. The average age of the animals was 2 months (8 weeks). During the study, the animals were kept under standard conditions (i.e., 12 h light/12 h dark and 22°C ± 2°C) in special cages and on a straw bed. Water and food were freely available to the animals. Standard food was including plate and treated municipal water was 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. 97497).
Grouping and diet
A total of 30 male rats were randomly divided into two groups, and 15 rats were placed in each group. The first group, called the control group, used a normal diet including 17.5% protein. In low-protein diet group, a low-protein diet was used; hence that, this diet merely included 8% protein. Base energy in both diets was similar. In the first group with normal diet, base energy was 2531 kcal, and in the second group with low-protein diet, base energy was 2534 kcal. Energy-to-protein ratio in the first group was 145 and 317 in the second group. The required food for both groups was provided from Pars Animal Food Company in Tehran. These foods were given to the rats after their transmission to the specified plates during 10 months. The required food of rats was 5–6 g for per 100 g of their weight in each day.
The passive-avoidance learning ability of rats was assessed using shuttle-box device. The device contained two isolated compartments (20 cm × 30 cm × 20 cm) that were divided through a guillotine entrance from which. When it was open, the rats could cross it. The bottom and walls of one of the compartments were white and for another one was black. The base of both rooms had parallel metal bars through which electrical stimulation with favourite time and voltage could be carried to animals' feet through the stimulator involved to them. Passive-avoidance learning was assessed in 3 phase's base of the usual technique.
Weight of animals
The body weight was measured using a microbalance sensitive up to 0.001 mg (Precisa 125A; Switzerland) and the average weights of the kidneys were calculated and recorded.
The transcardiac method was used for fixation. In this process, animals were intraperitoneally anaesthetised with ketamine (70 mg/kg) and diazepam (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 the 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 in 15 min. After perfusion, the brains were separated from the skull and stored in the same perfusion solution for 3 days.
The Golgi method was used to observe neuron dendrites in the hippocampus CA1 region. 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 hippocampus CA1 region. For this purpose, six rat heads from each group and five slides from each rat were taken to be stained. Subsequently, after producing 5 μm cuts by microtome and performing tissue processing, the left hemispheres were stained using Cresyl violet staining technique. After preparing the photo, 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 through microscopic examination with an optical microscope and Motic software and Image tool IT (version 3, Moticam 2000, Spain) 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 ×400 and the dendritic exclusion order from the cell body was used for counting the dendritic pieces.
After extracting the information, Kolmogorov–Smirnov test was first conducted to confirm the data compliance of the normal distribution. The t-test analysis was used for statistical analysis and difference between the groups. SPSS 16 (SPSS Inc, Chicago, Illinois, USA) was used for data analysis. The obtained results were expressed as mean ± standard error and P < 0.05 was considered statistically significant.
| Results|| |
Low-protein diet was shown to decrease the mean of animals' weight compared to the control group (P < 0.001) [Figure 1].
|Figure 1: Effect of low-protein diet on weight of animals. *Significant decrease in low-protein diet group compared to the control group (P < 0.001).|
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Passive-avoidance learning ability
The outcomes of the shuttle-box apparatus shown that passive-avoidance learning ability (retention latencies) of low-protein diet animals group was significantly reduced compared to the control group (P < 0.001) [Figure 2].
|Figure 2: Effect of low-protein diet on the step-through latency (learning ability). *Significant decrease in low-protein diet group compared to the control group (P < 0.001).|
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The results of a number of neurons in the hippocampal region CA1 showed a statistically significant decrease in the low-protein diet group compared to the control group (P > 0.001) [Figure 3] and [Figure 4].
|Figure 3: Effect of low-protein diet on the number of neurons in the CA1 region. *Significant decrease in the mean number of neurons in the low-protein diet group compared to the control group (P < 0.001).|
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|Figure 4: Microscopic images of CA1 region in male rats in different groups (5-μm thick sections, Cresyl violet staining, at ×100). Micrograph of the CA1 section in the control group (a), the normal number of neurons in the CA1 region; Micrograph of the CA1 section in low-protein diet group (b), reducing the number of neurons cells can be seen.|
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The mean number of neuronal dendritic spines showed a statistically significant decrease between the control group and the low-protein diet group (P < 0.001) [Figure 5] and [Figure 6].
|Figure 5: Comparison of low-protein diet and normal protein diet groups at the number of dendritic spines in hippocampal region CA1. *Significant decrease in the low-protein diet group compared to the control group (P < 0.001).|
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|Figure 6: Microscopic images of neuronal dendritic spines in hippocampal region CA1 in male rats in different groups (5-μ thick sections, Golgi staining, ×100). Micrograph of the CA1 section in the control group (a), normal structure. Micrograph of the hippocampal region CA1 section in the low-protein diet group (b), decreased number of dendritic spines due to the low-protein diet.|
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| Discussion|| |
The body needs nutrition to preserve its health and growth. One of the most important nutritions, which should be provided daily is specific doses, is protein. Proteins are the necessary cell components required for maintaining the balance between destruction and regeneration of cell components. Malnutrition caused by protein deficiency is one of the most common types of malnutrition in the world. It seems that this kind of malnutrition affects the neurons in some brain regions such as hippocampus. The present study investigates the impacts of low-protein diet on changes in brain neurons of hippocampus region and amount of learning in rats. The results of this study indicated that low-protein diet reduces the weight of the rats significantly. Furthermore, in the low-protein group, livelihood was reduced in rats and their hair started to fall after 2 months. Since some parts of energy and calorie of the body are provided by proteins, reduced amount of received calories is considered to be one of the reasons for weight loss of rats. When cells are filled up to their maximum protein content, each extra amino acid decomposes in body fluids and converts to energy or is mostly saved as fats. It is believed that the role of reduced amount of proteins in malformation of different sections of the body and cell and tissue repair is of great importance. When the body receives low amount of protein, a specific percentage of proteins decompose to amino acids and then oxidise leading to weight loss of rats in this study. The reported results of Snoeck et al. are consistent with the results of this study and indicated that low-protein diet in pregnancy causes severe weight loss of newly born rats. The results of the current study showed that the number of neurons and dendritic horns decreased significantly in low-protein diet group in comparison to the control group. The results may indicate the increase of apoptosis and neurodegeneration by administering low-protein diet. The results of Mattson et al. were consistent with those of the present study that showed low-protein diet could damage the cells in the brain by increased protein accumulation in the membrane and reduced cell size. It seems that low-protein diet in the long term induces oxidative stress and consequently, the production of free radicals such as superoxide and hydroxyl radicals, 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 reported results of Andrade et al. confirm the results of this study indicating that low-protein diet in rats causes reduced volume and number of neurons in the subiculum region of the brain. Dendritic thorns play a major role in synaptic transmission. In this regard, many brain diseases are associated with changes in the morphology and density of dendritic spines. Low-protein diet can reduce the length and the number of dendritic spines in nucleus accumbens by affecting the neurotrophic factors in the striatum. A study by West and Kemper showed that low-protein diet could reduce the length and density of synaptic thorns; that is consistent with the results of this study. It seems that reduce of protein can destroy dendritic thorns by β2 nicotinic acetylcholine receptors (β2-nAChRs) deactivation at postsynaptic cells in the hippocampus region. Moreover, protein deficiency can reduce the number of thorns by deactivating α4 β2-nAChRs in the pre-synaptic membrane and by disrupting the release of glutamate neurotransmitters. It appears that protein deficiency induced cellular destruction in CA1, related to amplified expression of some markers involved in apoptosis and cell cycle such as Cyclin B1 which can interfere with neuronal death. The results of the current study showed that the low-protein diet seizure has deleterious consequences on the learning ability as indicated by passive-avoidance test. A reduction in the learning ability has been reported in clinical studies in children with protein deficiency. Problems of reduced amount of learning in the rats of this study can be caused by reduced number of neurons and their dendrites, and consequently, reduced density of synapses in CA1 regions of hippocampus due to longtime protein deficiency. Protein is one of the main factors in building neurotransmitters. Hence, its deficiency can cause serious disorder in the functioning of synapses in hippocampus. Reyes-Castro et al. reported that increase the amount of protein in the daily diet, enhanced spatial memory, which is in line with the findings of the present study. The results of the present study showed that low-protein diet administration might have a negative effect on the learning ability and neurons of the hippocampus CA1 region.
| Conclusion|| |
It appears that low-protein diet had harmful effects on the CA1 region. Low-protein diet may decrease the learning ability and degenerative effects in animals. As a result, low-protein diet leads to reduction in the learning ability through CA1 tissue loss and adverse effects on number of neurons and dendritic spines in male rats.
Financial support and sponsorship
We gratefully acknowledge the Research Council of Kermanshah University of Medical Sciences (No: 97497) for the financial support.
Conflicts of interest
There are no conflicts of interest.
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