Protective Effect of L-Arginine in an Animal Model of Alzheimer's Disease Induced by Intra-Hippocampal Injection of AlCl3
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.344672
Source of Support: None, Conflict of Interest: None
Keywords: AlCl3, Alzheimer's, CA1, βA, L-arginine, L-NAME, memory retrieval, NO, novelty-seeking behavior, rat
Alzheimer's disease (AD) is a progressive neurological disorder characterized by reduced cognitive and daily activities.,, This disease is the most common type of dementia, which is accompanied by symptoms such as loss of information retention and especially short-term memory in old age. It will slowly progress with the loss of time recognition, depression, and lack of speech, isolation, and eventually respiratory distress. Death begins 5 to 10 years after the onset of symptoms. The disease may actually begin about 20 years before the onset of symptoms. The disease leads to the destruction of nerve synapses in some areas of the brain where fibrous protein structures such as neurofibrillary tangles (NFTs) are detected., Loss of cholinergic function is a major cause of AD., The main treatment strategy, thus, is to increase the level of acetylcholine (Ach) in the brains of people with AD using cholinesterase inhibitors such as donepezil and galantamine.
Today, amyloid imaging is used for the early detection of AD as well as the differential diagnosis of early and mild stages of cognitive impairment. There is an emphasis on the diagnosis of the final stage (i.e., dementia). In addition, the diagnosis is mainly based on the use of various imaging techniques such as magnetic resonance imaging, because damage to the hippocampus and cortex is evident in all Alzheimer's patients.,
Despite new criteria that allow early diagnosis of the disease (e.g., measurement of biomarkers derived from cerebrospinal fluid), novelty-seeking study can also inspect the destruction of spatial neurons.,,
Since animal models of AD are often based on the induction of very costly amyloid peptides, the most important thing to know is that the type of these peptides is different in animals than in humans, and that the beta-amyloid accumulated in the brains of animal models can be easily removed from their brains and does not show the same physical and biochemical properties as human amyloid. This raises important issues related to the testing and development of therapeutic agents; therefore, apart from these problems, we investigated the protective effect of L-arginine on Alzheimer's complications using an inexpensive model provided by intra-hippocampal CA1 injection of aluminum chloride (AlCl3).
Materials used in the present study include AlCl3 with 3 H2O (Merck, Germany), L-arginine (Merck, Germany), NG-Nitro-L-arginine methyl ester hydrochloride (Sigma-Aldrich, Germany), and Beta-Amyloid protein (Merck, Germany), and ketamine (10%) and xylazine (2%) (Veterinary Organization, Iran).
The study was conducted on adult male Wistar rats weighing 220 g (purchased from Pasteur Institute of Iran). They were kept in standard PVC cages at 23°C ± 2°C for 12 h light and had free access to adequate water and food ad libitum. Each animal was used only once and was removed after testing according to the ethical principles of working with animals. In this research, all the ethical principles set by Declaration of Helsinki (DoH) for care and use of laboratory animals were considered and the research plan was approved by the local ethics committee (IR.SHAHED.REC.1399.021). Rats were randomly divided into control group (receiving saline in the dorsal hippocampal CA1) and AlCl3-treated group. Saline (1 μL/rat) or AlCl3 (1–200 μg/rat) was microinjected into CA1 under stereotaxic surgery, and a week later, L-arginine (0.05–25 μg/rat) was injected alone or with L-NAME before the test. We also had a reference group (2 μg/rat beta-amyloid protein, intra-CA1).
Novelty seeking behavior
To examine the spatial memory, the two-part conditioned place preference device 30 × 60 × 30 cm was used. After injections into the dorsal hippocampus (under the stereotaxic cannulation surgery), and after recovery, each rat was examined by a novelty environment seeking paradigm. This behavioral test is based on the conditioning method and includes three stages: familiarization, confining (restricting only one side without access to the other side), and test. In the first stage, they were adapted to the box for 10 min (while the guillotine door was located 12 cm up, and the animal freely explored the whole box). All animal behaviors and time spent in each part of the two-compartment CPP box were recorded using the Ethovision system located 120 cm above the box. The records were then reviewed by an observer who was blind to the experiments. Later in the 3-day confining phase, which includes 2 sessions per day and each session lasts 40 min, the rats were placed in only one part and did not have access to the other part (the guillotine door was closed). Finally, on the test day (day five), they were in the same condition as the first day, but before the experiment, they were first given saline (control group) or substance and tested for 10 min and all behaviors and duration of stopping in each part were recorded and compared with the data of the first day.
On the day of the experiment, an hour and a half after the behavioral measurements, the animal was ethically killed by CO2, and after beheading, the animal's brain was immediately removed from the skull. The hippocampus was carefully isolated on ice and kept at −20°C for histological studies.
Before slicing, the brain tissue was immersed in 10% formalin for 48 h–72 h for fixation, and then, the histological preparation process was performed as follows: dehydration by placing them in alcoholic solutions of 50%, 70%, 85%, 95%, 100% and xylol - alcohol and then two stages of xylol for one hour each. The samples were then placed in a paraffin bath (60°C), three times and one hour each time to provide paraffin blocks. Then, sections (3–4 μm) were prepared by microtome (Leica, Italy) and placed on slides coated with poly L-lysine. After 24 h, the slices were dehydrated, clarified, histochemically and immunohistochemically labeled, and affixed with Entellan (Merck, Germany). These protocols are presented in the following sections.
Staining of CA1 area with Congo red
To evaluate the formation of amyloid plaque (in the target area: dorsal hippocampal CA1), a specific staining method was used for the detection of neurofibrillary nodules (NFTs) in the CA1 area of the hippocampus. CA1 sections were first prepared by microtome and the staining process was performed as follows: 3 to 4 μm sections were immersed in saturated salt solution for 20 min and then immersed in 1% Congo red solution (1 g per 100 mL of distilled water) for 30 min, and finally dehydrated, clarified, and glued by Entellan and covered.
Immunohistochemistry of c-fos protein
After sections went through the phase of deparaffinization (xylol 1 and 2 each for 15–20 min), dehydration (descending alcohols 98%, 80%, 70%, 50% each for 5 min), and rinsing with tap water for 1 min, they were exposed to the antigen recovery phase. During this step, the sections were first placed in a tracer buffer (also called antigen recovery solution or TBS: 1.02 g of Tris base and 0.37 g of EDTA dissolved in one liter of distilled water, pH = 9) and then in a 90°C (darkroom) water bath for 40 min. After this step, the sections were washed with distilled water and phosphate buffer solution (PBS, pH = 4.7 for three times, each time for 10 min). The slides were placed twice in a glass containing blocker (1% oxygenated water in methanol) for 15 min and then washed with distilled water and phosphate buffer (3 times each for 10 min). At this stage, the sections were incubated with primary c-fos antibody at a dilution of 1: 100 (in phosphate buffer) in a dark room at 4°C overnight. The sections were then washed with PBS (three times each for 10 min) and exposed to secondary antibodies for 1 h.
SPSS software version 21 was used for data analysis. At first, the homogeneity of variances was checked and if applicable, analysis of variance (ANOVA) was followed to analyze the data. To compare the differences between groups, Tukey's post-hoc test was used. P < 0.05 was considered as the statistically significant value.
AlCl3 dose response in Alzheimer's induction
The time spent in the two parts of CPP box (a factor of spatial memory) on the days of adaptation and test for all groups was calculated and compared with the control group [Figure 1]. The average time spent in the novel part (the side in which the animal was not restricted during the 3 days of confinement) as well as the other side was measured and compared between the days of familiarization and testing, and was shown as the mean ± standard error. As the statistical analysis showed, the answer is significant (P < 0.001), which indicates AD modeling and spatial memory impairment in rats receiving AlCl3.
The effect of L-arginine doses on AlCl3 response in Alzheimer's induction
The time spent in the box parts between the days of familiarization and test for the control group (1 μL/rat) and the different doses of AlCl3 (1–200 μg/rat) were calculated [Figure 2]. Statistical analysis of the mean time spent in the novel (confined) part compared to the opposite side of the CPP device, in comparison between days one to five, was performed in the control and AlCl3-treated groups (P < 0.001), which shows the protective effect of L arginine. The NO precursor restores the spatial memory of L-arginine-receiving rats.
The effect of L-NAME doses alone on Alzheimer's induction
The time spent in the box compartments between the days of adaptation and testing between the control group (1 μL/rat) and the groups receiving different doses of L-NAME (0.05–25 μg/rat) is shown in [Figure 3]. Saline or L-NAME was injected into the rat's CA1 during stereotaxic surgery. Statistical analysis did not show a significant response. This suggests that the antagonist alone is not able to induce the Alzheimer's model.
The effect of L-NAME pretreatment on L-arginine response in AlCl3 induced Alzheimer's model
The time spent in the box sections was measured comparatively on the day of introduction and test for the control group (1 μL/rat) and different doses of AlCl3 (1–200 μg/rat) [Figure 4]. Before the test, first L-NAME (3 or 9 μg/rat) and then L-arginine (3 or 9 μg/rat) were injected into the rat's CA1. The average time spent in the new part compared to the other side between the days of one and five is not significant, which indicates the role of nitric oxide (NO) in information recall.
AlCl3 dose response in the induction of sniffing, rearing, grooming, and compartment entering
Different behavioral components were measured in all groups and compared between experimental and control groups. The results are shown in [Figure 5].
Evaluation of the formation of amyloid plaques and NFTs in the dorsal hippocampus in all study groups using Congo red specific staining
The evidence obtained in this study showed a significant effect: Accumulation of tangles [Figure 6].
Evaluation of c-fos protein expression in the dorsal hippocampus
The expression of protein c-fos is significant, as can be seen in [Figure 7].
In the present study, the hypothesis of induction of memory impairment model and the incidence of AD by intra-hippocampal CA1 injection of AlCl3 in rats was investigated and the protective effect of NO precursor (L-arginine) on its complications was studied.
Memory retention, as indicated by the rats' novelty seeking behavior, failed due to AlCl3 administration, which was consistent with the previous study. However, L-arginine, a NO precursor, showed protection against memory loss as the present finding showed. L-NAME, NO synthase antagonist, although not shown to have a significant effect by intra-CA1 in rats alone, when administered before the precursor, blocked the effects of L-arginine during testing in AlCl3-treated animals. Therefore, the nonprotective effects of L-arginine in the presence of L-NAME indicate the role of NO in in the protective effects of L-arginine. According to the present findings, different seeking behavioral components also appeared differently in animals given AlCl3, β-amyloid protein, L-arginine, and L-NAME.
The dorsal hippocampus (dH) is one of the most important areas of hippocampal formation (consisting of dH, dentate gyrus, and subiculum) with the ability to form short-term memory as well as to consolidate and retrieve memory. This site consists of the Cornu Ammonis (CA) region (CA1 to CA4), next to the DG, where CA1 is exclusively attributed to memory processing. It receives information from the DG and communicates with the entorhinal cortex at the same time. Therefore, this area makes a definite comparison between the information received from the environment at any given time and the previously stored information that already exists in the memory boxes., Damage to the hippocampus, which is involved in the acquisition of spatial memory, reduces memory retention.,
AD, most often occurring in older people, has a wide range of symptoms in which damage to short-term memory (primary disease) is a major symptom. In case of long-term memory impairment, the disease is advanced. Also, as an early symptom of the disease, we may refer to the onset of stress and loss of recall of recent events and intellectual abilities., The authors also add anger to these early signs.
According to previous research, a dose of 100 mg/kg of AlCl3 was given orally for 60 days and induced the animal model AD. The authors have also improved the effects with the help of nanomaterials. In fact, each proposed model is a tool for examining human problems in the laboratory and the results are attributed to humans, but it should not be overlooked that any method may have challenging consequences. For example, the above result is obtained with a high level of substance over a long period of time (2 months), which is questionable in terms of animal rights principles. Others have studied the effect of L-arginine in a model of lipopolysaccharide-induced memory impairment in passive avoidance task and have shown that L-arginine has protective effects against oxidative damage to brain tissue. And other researchers have investigated the effectiveness of L-NAME on the AlCl3-derived oxidative stress parameters in Wistar rats (intra-hippocampal injection) and have reported that L-NAME has antioxidant effects and concluded that NO gives neurotoxicity. Such a conclusion seems a bit hasty because L-NAME is just a competitive inhibitor. However, as a matter of interest in the present study, we may introduce a more appropriate and less invasive model in the laboratory and suggest the possibility of treating AD with the amino acid L-arginine, which can be presented organically. The amino acid L-arginine can be found in many food sources; moreover, it is a precursor to NO that can be metabolically produced by the L-arginine in the presence of NO synthase, molecular oxygen, and the NADPH cofactor. NO is also known as a retrograde neurotransmitter that is involved in memory formation. The protective role of this neurotransmitter in this target area that has been exposed to the neurotoxin colchicine has already been reported in this laboratory. Previous studies have also reported changes in L-arginine metabolism in AD.
The present results showed that pretest L-arginine administration significantly restored the conserved events in Alzheimer's animals. Its effect on improving behavioral symptoms was also evident. However, further studies need to be done to confirm the direct connection between these symptoms and Alzheimer's disease. Therefore, as our suggestion, the exact functional mechanisms of the amino acid of L-arginine should be examined in future studies.
Conclusion: In the present study, L-arginine showed a protective effect against Alzheimer's disease in rats treated with aluminum chloride. There is a possibility of a nitric oxide mediating role that needs to be clarified.
We thank the Neurophysiology Research Center of Shahed University for supporting this research project.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]