Research Article

Degeneration of Pyramidal cells of CA1, CA2, and CA3 area of the Hippocampus in male rats under the influence of Simvastatin

Mohammad Saleh Ranaiy*,Farah Farokhi , Farrin Babaei-Balderlou , 

Department of Biology,, Faculty of Sciences, Urmia University, Urmia, Iran

*Corresponding author: Dr. Farah Farokhi, Department of Biology, Urmia University, Urmia County, West Azerbaijan, Iran. 533000. Tel: 989107421580+; E-mail: st_ms.ranaiy@urmia.ac.ir

Abstract

Background: Simvastatin is a lipophilic statin and can cross the blood-brain barrier. This study aimed to evaluate the Effect of Simvastatin on pyramidal cells in CA1, CA2, and CA3 regions of the hippocampus in healthy male rats. Method: For the experiment, 36 male Wistar rats with an average weight of 300-250 g were divided into six groups (each group comprised six heads). Groups: 1): Control group, 2): Vitamin D dose 5 µg/kg, 3): Simvastatin group dose 1 mg/kg, 4): Simvastatin group dose 10 mg/kg, 5): Simvastatin group dose 1 mg/kg + vitamin D dose 5 µg/kg, 6): Simvastatin group dose 10 mg/kg + vitamin D dose 5 µg/ kg. The duration of drug use was 28 days. At the end of the drug administration period, the animals were slaughtered, then blood samples were taken from the animals (to check TC and LDL-C), and finally, the brain tissue of the animals was extracted to prepare microscopic sections. Results: Statistical results of total cholesterol and LDL-C in rat serum showed that the groups receiving simvastatin 10 mg/kg and simvastatin 10 mg/kg with vitamin D supplement 5 µg/kg were significantly different compared to the control group (P <0.05).

Statistical results showed a significant increase (P <0.05) in degenerated cells in the groups receiving simvastatin 10 mg/kg and simvastatin 10 mg/kg with vitamin D supplementation compared with the control group in CA1 and CA2 regions. The statistical results of the study of the percentage of The statistical results of the study of the rate of degenerative cells in CA3 area CA3 of the hippocampus showed that all groups except the vitamin D group had a significant difference (P <0.50) in the percentage of degenerative cells in this region compared to the control group.

Conclusion: : The results of the present study showed that Simvastatin, due to its lipophilic nature, easily crosses the blood-brain barrier and reduces cholesterol in the brain, thus causing the degeneration of pyramidal cells in the hippocampus.

Keywords: Cell degeneration; Cholesterol; Hippocampus; Simvastatin; Vitamin D.

Background

The first Statins were discovered and isolated from microorganisms in 1972 by Dr. Akira Ando’s research team [1]. Statins, the inhibitor of 3-hydroxy-3-methylglutaryl A (HMG-CoA), are known as plasma cholesterol-lowering drugs and are widely used in patients with blood cholesterol disorders. The primary function of Statins is to lower cholesterol by competitively and reversibly inhibiting the enzyme HMG-COA reductase in the rate-limiting stage of cholesterol production. HMG-CoA combines Acetyl-CoA and Acetoacetyl-CoA, converted to mevalonate by HMG-CoA reductase and NADPH cofactors [2].

The liver mainly absorbs Statins, and in the liver, inhibiting the enzyme (HMGCR), the primary enzyme involved in cholesterol synthesis, lowers serum cholesterol levels [3]. Statins inhibit HMG-CoA reductase in the liver by regulating hepatocyte LDL receptors, increasing circulating LDL-C clearance, and decreasing plasma LDL-cholesterol levels¬ [4]. Simvastatin is a member of the lipophilic family of Statins [5]. Lipophilic Statins can cross the blood-brain barrier and, as a result, can affect most high-cholesterol organs such as the brain [6]. Following chronic use of Statins, a decrease in 24S-hydroxycholesterol levels in plasma and cerebrospinal fluid (CSF) has been observed, which is parallel to the reduction in cholesterol in the brain of long- term treatment with Statins. Statins simultaneously affect the concentration of cholesterol in the brain [7,3].

Statins, like other drugs, have side effects. One of the main barriers to statin treatment is statin-induced muscle myopathy [8]. Some studies show that Simvastatin has protective effects on the nervous system [9,10]. Still, there are other reports of the adverse effects of Simvastatin on dose- dependent and time-dependent nerve system cells [11,12] The hippocampus is part of the limbic system, which is in the middle temporal lobe of the brain and is involved in memory and learning [13]. The predominantly lipophilic Statins include Simvastatin, fluvastatin, lovastatin, pitavastatin, and atorvastatin [14]. Lipophilic drugs such as Simvastatin can cross the blood-brain barrier and affect brain cholesterol [15]; as a result, they can cause changes in the hippocampal tissue. Tens of millions of patients worldwide are currently receiving Statins to treat hypercholesterolemia [16]. In a 2005 study by Lindberg et al., Examining the effects of Statins on microglia, they concluded that Statins, destructive impact on brain cells was highly dose-dependent. They figured that Simvastatin could be toxic to neurons and glia [17]. Because of the importance of Statins, in the present study, the Effect of Simvastatin at 1 mg/kg and 10 mg/kg for 28 days on rat hippocampal tissue Healthy and without brain damage were evaluated. It should be noted that the use of vitamin D supplements in some groups was done because, in some cases, vitamin D was used as a supplement with Statins [18].

Materials and Methods

Thirty-six male Westar rats weighing 250-300g were purchased from the animal house of Urmia University. All procedures were followed according to the National Institute of Health guide for the care and use of Laboratory Animals (NIH Publications No. 8023, revised 1978), and local guidelines for compassionate use of animals in research; Rats were kept in cages with open access to standard tap water and compact chow. The animals were held in the same laboratory conditions (18°C to 23°C room temperature and controlled humidity), with alternating 12-h light and dark cycles. The Ethics Committee has approved the proposal of this study of Urmia University (Ethics Code: IR-UU- AEC-3/1033 / DA).

Animal grouping:

.1. Healthy control group who received normal water and food and were not injected (C). 2. Vitamin D group who received only vitamin D at a dose of 5 µg/kg (200 IU) [19], by intraperitoneal injection (VD). 3. Simvastatin group with a dose of 1 mg/kg [20], received a low dose of Simvastatin orally (SimL). 4. Simvastatin group with a dose of 10 mg/kg [21], received high doses of Simvastatin orally daily (SimH). 5. Simvastatin group with a low dose of 1 mg/kg with vitamin D supplement 5 µg/kg (SimL + VD). 6. Simvastatin group with a high dose of 10 mg/kg with vitamin D supplement VD 5 µg/kg (SimH + VD).

The drugs were administered once daily for 28 days. The drug simvastatin used in the present study was produced by Timova Pharmaceutical Company (Denmark).

Blood sampling

At the end of 28 days of treatment, the animals were anesthetized with ether and sacrificed after the blood collection phase. After one hour, all blood samples were centrifuged at 1500 rpm for 10 minutes to isolate their serum. The obtained serum was carefully separated by a sampler and stored in Ependrov microtubes in a freezer at -70 ° C until biochemical tests were performed. Serum levels of Cholesterol Total and LDL-C were measured by spectrophotometry (enzymatic colorimetry), and using special measuring kits (Pars Azmoun Company).

Tissue study method

The brains of mice were transferred to 10% formalin for fixation and then subjected to dehydration, clarification, paraffin, and molding, then six micron-thick sections were prepared by microtome. After staining with hematoxylin and eosin, they were examined and photographed with a light microscope. The hippocampus was explicitly studied.

For cell counting under a light microscope with x400 magnification and with the help of an Eyepiece micrometer grid, it was counted in a square with an area of 100 micrometers (Nikon, DXM 120 USA). Degeneration of neurons (degeneration: abnormal changes in cell appearance including cell shrinkage, membrane uniformity, cytoplasm density), were examined by light microscopy [22].

Cell counts in the hippocampus were performed separately in three regions: CA1, CA2, CA3.

Statistical analysis

Data were analyzed using SPSS19 software using one- way analysis of variance and Tukey test. The results were presented as mean ± standard error. P <0.05 was considered as a significant level.

Results

Results for serum LDL-C

Statistical results of serum LDL-C showed that rats SimH and SimH + VD had a significant difference in reducing LDL-C compared to the control group (P <0.05), but other groups did not show any significant differences compared to the control group (P> 0.05), Diagram 1.

Results for serum TC .

Statistical results of total serum cholesterol showed that the SimH and SimH + VD groups had a significant decrease in total cholesterol compared to the control group (P <0.05), but other groups showed no significant difference they did not deliver to the control group (P> 0.05), Diagram 2.


      Results for serum LDL-C

                                                              

Diagram 1: Comparison of the effect of treatments on serum LDL-C levels of Meane ± SEM. 1): Control group (C), 2): VD (vitamin D dose 5 µg /kg), 3): SimL group (Simvastatin dose 1 mg /kg), 4): SimH group (Simvastatin dose 10 mg/kg), 5): SimL + VD group (Simvastatin dose 1 mg/kg+ vitamin D dose 5 µg/kg), 6): SimH + VD group (Simvastatin dose 10 mg /kg + vitamin D dose 5 µg /kg).

In all diagrams, the asterisk (*) indicates a significant difference (P <0.05), compared to the control group.

    TC results

                                                              

Diagram 2: Comparison of the effect of treatments on serum TC levels of Meane ± SEM. 1): Control group (C), 2): VD (vitamin D dose 5 µg/kg), 3): SimL group (Simvastatin dose 1 mg/kg), 4): SimH group (Simvastatin dose 10 mg/kg), 5): SimL + VD group (Simvastatin dose 1 mg / kg + vitamin D dose 5 µg/kg), 6): SimH

+ VD group (Simvastatin dose 10 mg/kg + vitamin D dose 5 µg/kg).

Results on the percentage of degenerative cells in the CA1 region of the hippocampus

The study’s statistical results of the percentage of degenerative cells in the CA1 region (pyramidal neurons), of the rat hippocampus showed that SimH and the SimH + VD group significantly increased the percentage of degenerative cells in this region compared to the control group (P <0.05), but other groups did not differ significantly from the control group (P> 0.05), Diagram 3.

Results on the percentage of degenerative cells in the CA2 region of the hippocampus

The study’s statistical results of the percentage of degenerative cells in the CA2 region (pyramidal neurons), of the rat hippocampus showed that SimH and the SimH + VD group significantly increased the percentage of degenerative cells in this region compared to the control group (P <0.05), but other groups did not differ significantly from the control group (P> 0.05), Diagram 4.

Results on the percentage of degenerative cells in the CA3 region of the hippocampus

The study’s statistical results of the percentage of degenerative cells in the CA3 region (pyramidal neurons), of the rat hippocampus showed that, except for the VD group, other groups had a significant difference in the percentage of degenerative cells in this region compared to the control group (P <0.05), Diagram 5.

   Results on the percentage of degenerative cells in the CA1 region of the hippocampus

                                                              

Diagram 3: Comparison of the effect of treatments on the percentage of degenerative cells in region CA1 of the serum hippocampus Meane ± SEM. 1): Control group (C), 2): VD (vitamin D dose 5 µg/kg), 3): SimL group (Simvastatin dose 1 mg/kg), 4): SimH group (Simvastatin dose 10 mg/kg), 5): SimL + VD group (Simvastatin dose 1 mg/kg+ vitamin D dose 5 µg/kg), 6): SimH + VD group (Simvastatin dose 10 mg/kg + vitamin D dose 5 µg/kg).

Discussion

Statins are valuable drugs in the treatment of atherosclerotic cardiovascular disease. This family of drugs reduces mortality from these diseases in primary (Patients with no clinical evidence of coronary heart disease), and secondary prevention (Patients with specific coronary heart disease), even if serum cholesterol levels are normal. Independent studies in patients with heart failure that have been associated with LDL-C levels have reported beneficial effects of statin administration [23,24]. Statins have an impact on serum cholesterol concentration [25]. Controlled trials have shown that drug treatments with Statins reduce LDL-C levels and reduce the risk of cardiovascular disease [26-29].

All Statins competitively bind to the catalytic domain of HMG-COA reductase. This family of drugs prevents HMG- CoA from reaching the active site of the LDL-C receptor. As a result, it enhances the reabsorption of LDL-C and LDL-C precursors from the systemic circulation. Proteins that bind to the sterol regulatory unit (SREBP), can sense changes in cholesterol levels. Then subsequently increase the expression of LDL-C receptors to reabsorb LDL-C from serum to compensate for the decrease in cellular cholesterol; as a result, a significant proportion of cholesterol-lowering Statins indirectly clear LDL-C from plasma [30]. Simvastatin treatment reduced serum cholesterol levels [31].

Simvastatin is inactive as a prodrug then is converted to active beta-hydroxy derivatives in the gastrointestinal

  Results on the percentage of degenerative cells in the CA3 region of the hippocampus

                                                              

Diagram 5: Comparison of the effect of treatments on the percentage of degenerative cells in region CA3 of the serum hippocampus Meane ± SEM. 1): Control group (C), 2): VD (vitamin D dose 5 µg / kg), 3): SimL group (Simvastatin dose 1 mg/kg), 4): SimH group (Simvastatin dose 10 mg/kg), 5): SimL + VD group (Simvastatin dose 1 mg/kg+ vitamin D dose 5 µg/kg), 6): SimH + VD group (Simvastatin dose 10 mg /kg + vitamin D dose 5 µg/kg).

tract. The most significant impact of this drug is on the liver. This preferential impact can be attributed to differences in different tissues in removing the drug from the blood. Plasma triglycerides decrease, and HDL-C levels increase during treatment with this drug. The HMG-COA inhibitor is effective in patients with elevated plasma LDL levels [32- 36]. The present study results, performed on male rats for 28 days, showed a significant decrease in serum LDL-C levels in the SimH and SimH + VD groups. In male rats, significant serum TC levels were significantly reduced in SimH and SimH + VD groups (Diagrams 1 and 2). These results indicate the positive impact of Simvastatin on cholesterol reduction [37]. However, other groups did not significantly impact these two factors compared to the healthy control group, and this lack of effect is due to the low dose of Sim 1 mg/kg, which is a low dose.

Cholesterol is an essential biological molecule in all cells and cell membranes and in the blood that includes many functions, including myelin sheath formation, expression of neurotransmitter receptors, neuronal synapses, production of steroid hormones (e.g., testosterone, estrogen, etc.). Cortisol and vitamin D) are involved in signaling the central and peripheral nervous systems, as well as in healthy cell function and the transport of antioxidants such as vitamin E, carotenoids, and coenzyme Q 10 Important for energy production, cellular function, and defense of the body against free radicals [38]. Lipophilic Statins such as Simvastatin can cross the blood-brain barrier and, as a result, can affect most high-cholesterol organs such as the brain [6]. Studies have shown that high doses of Simvastatin (100 mg/kg) affect brain cholesterol synthesis, most likely due to the drug passing directly through the blood- brain barrier, lipophilic, and locally inhibiting cholesterol synthesis in the brain. It seems that the ability of different Statins to inhibit cholesterol synthesis in the CNS depends on their lipophilicity and distribution in the brain. The use of lipophilic Statins such as Simvastatin in high doses may affect the cholesterol synthesis of the brain in humans [15].

The study results of Ajaib S. Paintlia and Manjeet K. Paintlia et al. showed no side effects of different Statins on the expression of proteins directly involved in cholesterol synthesis. However, cholesterol levels in the cerebral cortex decreased slightly during the experiment [39]. Given the vital role of cholesterol in the CNS, altering its metabolism can have devastating consequences in humans [40]. In addition, research has shown that HMGR activity is low in the brainstem, while higher activity is observed in the hippocampus, cortex, and cerebellum [41]. In the present study, the effects of Simvastatin at doses of 1 mg/kg and 10 mg/kg orally over 28 years on healthy adult male rats were evaluated for the Effect of Simvastatin on hippocampal tissue. The study results of serum profiles showed that Simvastatin at a dose of 10 mg/kg causes a significant reduction in LDL-C and total serum cholesterol Diagrams 1 and 2. In this regard, in the present study, the results of the examination of hippocampal tissue in male rats that received the drugs for 28 days showed that the pyramidal cells of CA1 regions in the SimH group Figure 4- B and SimH + VD group Figure 6-B have degenerated. The staining of these cells during passage Tissue enhanced Diagram 3.

Interestingly, vitamin D supplementation in the CA1 group in the SimH + VD group reduces the percentage of degenerate cells compared to the SimH group, which indicates the positive effect of vitamin D supplementation in reducing the negative impact Simvastatin. However, this significant positive effect was not observed in CA2 and CA3 regions Figure 4, 6-C, D. The statistical results of the study of pyramidal cells in the CA2 area showed a significant percentage of degenerate cells in the SimH Figure 4-C and SimH + VD groups Figure 6-C compared to the control group and other groups Diagrams 4. However, in the groups receiving SimL, SimL + VD, and VD, no significant effect

                       

Figure: 1 Longitudinal microscopic images of hippocampal tissue of control group. A (X100). B, C, D (X400). The blue arrow with the H tag indicates a healthy cell in all images, and the black arrow with the D tag indicates a degenerate cell.

 

 

                       

            Figure: 2 Longitudinal microscopic images of hippocampal tissue of Vitamin D group. A (X100). B, C, D (X400).

 

 

                       

            Figure: 3 Longitudinal microscopic images of hippocampal tissue of SimL group. A (X100). B, C, D (X400).

 

 

                       

           Figure: 4 Longitudinal microscopic images of hippocampal tissue of SimH group. A (X100). B, C, D (X400).

 

                       

           Figure: 5 Longitudinal microscopic images of hippocampal tissue of SimL + VD group. A (X100). B, C, D (X400).

 

                       

           Figure: 6 Longitudinal microscopic images of hippocampal tissue of SimH + VD group. A (X100). B, C, D (X400).

was observed on the degenerate of cells in the CA2 region compared to the control group. The statistical results of the study of pyramidal cells in the CA3 area show a significant percentage of degenerate cells in the groups receiving SimH Figure 4-D, SimH + VD Figure 6-D, SimL Figure 3-D and, SimL + VD Figure 5-D compared to the control group Diagrams 5

The results of degenerative pyramid cell studies in CA1, CA2, and CA3 regions may seem very strange. Perhaps this difference in outcomes in these hippocampal regions is due to the physiological and biochemical differences of cells in the different areas of the hippocampus. Despite the significant Effect of Simvastatin on cell destruction in all three hippocampal regions at a dose of 10 mg/kg, Simvastatin at a dose of 1 mg/kg showed only a significant adverse effect on cells in the CA3 region. Measures that directly study cerebral structure, cerebral blood flow, cholesterol circulation, and neuronal activity can be used to understand how Statins affect the CNS. Still, there are few studies in this area, and their results are different. Decreased hippocampal volume along with memory impairment has been studied in some age-related studies. Few studies have been performed on the impact of Statins on the hippocampus. There are many differences in the results of these studies [42,43]. Previous and current research on the effects of statin drugs has shown a significant effect of Simvastatin on the hippocampus [41].

Simvastatin-induced toxicity has been reported on different cell types [11]. Simvastatin interferes with myelin repair in myelinated mice [44]. The research results by Kamminskey et al. Showed that the treatment of astrocytes with Simvastatin induced apoptosis in cells depending on dose and time [11]. Statins lower cholesterol [45], and because cholesterol is an integral part of animal cell membranes, any drug that affects cholesterol levels can affect animal cell membrane cholesterol. Because Statins are cholesterol-lowering drugs, they can affect cell membranes. Cholesterol does not cross the blood-brain barrier, and brain cholesterol is produced by its production in this organ [46]. Any interference with the concentration of cholesterol in the brain can affect the cell wall. As a result of the effect of Statins on cholesterol and increase in cell membrane permeability, the cholesterol balance of the cell wall is disturbed. In preparing tissue sections for microscopic studies, the dye can enter the cell and affect the results of the studies. Cells in different areas of the hippocampus are no exception. Each region of the hippocampus has cells with different functions from other areas of the hippocampus, and the cells in each region have different cellular characteristics and even various afferents and efferents from other regions [47-49].

Some studies of the adverse effects of Statins include cessation of DNA synthesis [50] and proliferation cell death in primary neuronal cell culture [12, 51], end of neuroblasts [52], loss of synapses [53], and negative impact on cognitive function in clinical trials [54]. The study results by Beitan et al., Showed that the use of Simvastatin for four weeks at a dose of 10 mg/kg per day causes memory impairment in the Barnes maze test. However, this effect was not observed in mice taking Simvastatin at a dose of 30 mg/kg/day [21].

The results of the present study, which was performed to evaluate the effect of oral administration of Simvastatin for four weeks on healthy mice, show a significant negative effect of Simvastatin on pyramidal cells of CA1, CA2 and CA3 regions of the hippocampus at a dose of 10 mg/kg simvastatin. This result obtained in the present study confirms the negative effect of 10 mg/kg simvastatin in the research of Beitan et al., [21]. It should be noted that the hippocampus is one of the main components in the memory process [13]. In this regard, the results of research by Zongmin and Steven et al., Showed the harmful effects of Simvastatin on the myelination of rat cerebellar neurons [55]. Simvastatin may affect oligodendrocyte cell survival signaling by blocking isoprenylation [52]. Simvastatin interferes with myelin repair by inhibiting lipid-associated signaling and cholesterol-dependent processes [56].

The findings of Sierra et al. To evaluate Statins as neuroprotectants showed the preventive properties of Statins as an effective strategy in protecting the brain against stroke.

After careful study of some parameters, the researchers stated that Simvastatin is the best Statins to prevent neurological diseases [57]. The results of some studies suggest that Simvastatin was effective in inhibiting hippocampal cell apoptosis and inflammation in mice with Alzheimer’s disease and that Simvastatin had protective effects on the nervous system [9,58].

Research shows that treatment with Simvastatin has potent inhibitory effects on hippocampal nerve apoptosis, thus helping to improve memory [59,60]. Sun, J., Et al., In a study to investigate Simvastatin in the hippocampus, reported that treatment with Simvastatin significantly reduced nerve damage and reduced apoptosis in the hippocampus [61]. However, the present study results showed that the Effect of Simvastatin on the hippocampal tissue of healthy male rats was inconsistent with the positive effects of Simvastatin on the hippocampal tissue mentioned above.

Declarations

Statins are at the forefront of the fight against atherosclerosis, as they effectively lower LDL-C, so their pleiotropic effects show the valuable role of Statins in preventing cardiovascular disease [62]. Due to Simvastatin adverse Effects on Simvastatin in the present study and other harmful effects on the nervous system and other organs of the body [11], it is necessary to conduct more detailed research on this drug in the future physicians should prescribe Simvastatin with caution.

Conclusion

The present study results showed that Simvastatin, due to its lipophilic nature, readily crosses the blood-brain barrier and reduces cholesterol in the brain, thus causing the degeneration of pyramidal cells in the hippocampus.

Abbreviations

TC: Total cholesterol LDL-C: Low-Density Lipoproteins HDL-C: High-Density Lipoproteins HMGCR: Hydroxy Methyl Glutaryl CoA-reductase VD: Vitamin D Sim: Simvastatin SimL: Low dose of Simvastatin (1 mg/Kg) SimH: High doses of Simvastatin (10 mg/Kg) µg: Micrograms mg: Milligram kg: Kilogram

Authors’ Contributions

All authors read and approved the final manuscript.

Ethical Approval and Consent to participate

This article is extracted from the dissertation of Mohammad Saleh Ranaiy, and all authors are satisfied with their participation. The Ethics Committee has approved the proposal of this study of Urmia University (Ethics Code: IR- UU-AEC-3/1033 / DA).

Consent for publication

All authors are delighted with the publication of this article.

Availability of supporting data

There is data to support this article.

Competing interests

There is no authors’ conflict of interest.

Funding

Urmia University was the sponsor of this article.

Authors’ contributions

All authors have contributed to this article.

Acknowledgements

This article is taken from the dissertation of Mohammad Saleh Ranaiy. We thank the Department of Development and Physiology of Urmia University for providing the basics of this research.

Authors’ information

.

Mohammad Saleh Ranaiy (MSc), E-Mail: st_ms.ranaiy@ urmia.ac.ir, Department of Biology, Faculty of Sciences, Urmia University, Urmia, Iran

Farah Farokhi1 (Ph.D.), E-Mail: f.farokhi@urmia.ac.ir, Department of Biology, Faculty of Sciences, Urmia University, Urmia, Iran

Farrin Babaei-Balderlou (Ph.D.), E-Mail: f.babaei@urmia. ac.ir, Department of Biology, Faculty of Sciences, Urmia University, Urmia, Iran

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