Neuroprotective activity of chlorogenic acid from Brazilian Arnica in rodents subjected to status epilepticus induced by lithium-pilocarpine

doi:10.21203/rs.3.rs-3311375/v1

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Neuroprotective activity of chlorogenic acid from Brazilian Arnica in rodents subjected to status epilepticus induced by lithium-pilocarpine

https://doi.org/10.21203/rs.3.rs-3311375/v1

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In this study, we evaluated the neuroprotective properties and antioxidant effects of chlorogenic acid (CGA) in Wistar rats subjected to Status Epilepticus (SE) model using lithium-pilocarpine. After 72 h induction of SE, brains were collected and studied histologically for viable cells in the hippocampus with staining for cresyl-violet (Nissl staining), and for degenerating cells with Fluoro-Jade C (FJC) staining. Additionally, to evaluate oxidative stress, the presence of malondialdehyde (MDA), the final product of lipid peroxidation, and superoxide dismutase (SOD), the enzyme responsible for the conversion of superoxide anion radicals, were quantified using the Indole and the Pyrogallol methods, respectively.

Animals administered with CGA (30 mg/kg) demonstrated a significant decrease of 59% in the number of hippocampal cell loss in the CA3, and of 48% in the hilus layers after SE. A significant reduction of 75% in the cell loss in the CA3, shown by FJC+ staining, was also observed with the administration of CGA (30 mg/Kg). Furthermore, significant decreases of 49% in MDA production and 72% in the activity of SOD were seen, when compared to animals subjected to SE that received vehicle. This study suggests that CGA administration results in an effective inhibition of the proliferation of oxidizing agents that can initiate cellular death, in the lithium-pilocarpine Status Epilepticus (SE) rat epilepsy model.

Neuroprotective

Status Epilepticus

chlorogenic Acid

Antioxidant

Species of the genus Lychnophora are medicinal plants popularly known as “Brazilian arnica” or “arnica da serra” (Keles et al. 2010). They are used in traditional medicine as anti-inflammatory and analgesic agents, their active components are derived from chlorogenic acid (CGA) and C-flavonoids (dos Santos et al. 2010; Naveed et al. 2018). CGA is a phenolic acid with a wide distribution in many plant species and different kinds of food commonly consumed by humans, such as some fruits, olive oil, wine, and coffee. Studies with polyphenols derived from coffee have revealed many health benefits for neurodegenerative diseases (Mikami et al. 2015; Colombo et al. 2020).

Systematic research using in vivo and in vitro models revealed the mechanisms linked with anti-inflammatory and analgesic actions, which seem to be associated with the antioxidant properties of CGA (dos Santos et al. 2005; dos Santos et al. 2006; dos Santos et al. 2010). Previous studies from our group on CGA (Taleb-Contini et al. 2008; Liberato et al. 2023) helped further our understanding of its mechanisms of action and encouraged us to pursue studies in different models of neurological disorders, including epilepsy models.

Status epilepticus (SE) is a severe clinical manifestation of epilepsy that results in structural brain damage (Fujikawa 1996; Madzen et al. 2007; Green et al. 2021). There are physiological and neurological alterations that take place during the seizure activity (Fountain 2000). Notably, oxidative stress can dramatically alter the neuronal and glial function and has been associated with neurochemical changes observed during SE and spontaneous recurrent seizures (SRS) induced by pilocarpine (Miller 1991; Barros et al. 2007).

Pilocarpine is a potent muscarinic cholinergic agonist that exerts its functions by binding to muscarinic M1 receptors (Hamilton et al. 1997; Bymaster et al. 2003; Müller et al. 2009). Administration of pilocarpine induces epileptiform activity in cortical and hippocampal regions, generating a sequence of behavioral changes, which progress to motor seizures and, subsequently, to SE (Turski et al. 1983; Scorza et al. 2009). Seizures induced by pilocarpine produce very large changes related to the production of reactive oxygen species (ROS) (Liu et al. 2010).

The activation of enzymatic compensation systems such as superoxide dismutase (SOD) and some scavengers of ions contributes to the redox balance maintenance in the cell. This balance may be broken by a prolonged increase of ROS or by decreasing the antioxidant cellular systems (i.e., SOD activity) (Pazdernik et al. 2001; Sudha et al. 2001; Liu et al. 2010). The brain is particularly susceptible to ROS due to its high oxygen consumption. Research data carried out in epileptic patients considers oxidative stress parameters such as lipid peroxidation, the activity of SOD, glutathione peroxide, and catalase, i.e., levels of antioxidants (vitamins A, C, and E). It has been reported that patients with epilepsy have higher lipid peroxidation and lower plasma levels of vitamins A, C, and E compared with control subjects (Liao et al. 2004; Deepa et al. 2008). MDA is the final product of lipid peroxidation and is frequently used to define oxidative stress. In this context, several studies focused on MDA levels in neurological disorders (Camkurt et al. 2017).

Current antiepileptic drugs (AEDs) are ineffective, highlighting the need for further studies to identify and develop new and more selective AEDs with fewer unwanted side effects.

CGA and its metabolites are able to cross the blood-brain barrier (Liberato et al. 2023). A recent study revealed that CGA partially decreases neuronal death induced by glutamate via inhibition of Ca²⁺ entry, without altering the production of compounds derived of nitric oxide (NO) (Colombo et al. 2020). The neuroprotective effects of CGA and 4,5-di-O-[E]-caffeoylquinic acid (CQA), isolated from Lychnophora plants, were reported in a rodent glaucoma model(Liberato et al. 2023). Collectively, these studies encouraged us to evaluate the effects of CGA in the lithium-pilocarpine-induced SE model.

2.1. Compounds and Drug Administration

Compounds for animal experiments including lithium chloride, pilocarpine hydrochloride; CGA was purchased from Sigma-Aldrich, USA. Methyl-scopolamine was purchased from Tocris Bioscience, USA; and sodium thiopental from Cristália, Brazil. All compounds were dissolved in the vehicle (0.9% NaCl) and injected via IP, except for methyl-scopolamine that was injected s.c.

Compounds for histological analysis including methanol, ethanol, and Cresyl Violet were purchased from Merck, USA. Chloroform, and pyrogallol, were purchased from Sigma –Aldrich, USA. Additionally, EDTA was purchased from Synth, Brazil, Permount and Fluoro-Jade® C were purchased from Fisher Scientific, USA, Fluoromount was purchased from Millipore, USA, and Tissue-Tek OCT compound was purchased from Jung, Germany.

2.2. Animals

Male Wistar rats (230-250g, n = 162) were maintained in a temperature-controlled room (23 ± 2°C), on a 12 h light/dark cycle (lights on at 7:00 a.m.), with ad libitum access to food and water. All experimental procedures were designed to minimize animal suffering. The experimental protocol was in accordance with and approved by the local Animal Care Committee (protocol number 08.1.834.53.9 CEUA –Campus University of São Paulo, Ribeirão Preto, Brazil). From the group, 12 rats were used in the optimization of pilocarpine doses (30 and 35 mg/Kg), and 22 rats did not reach or maintain SE, these 34 animals were excluded. Therefore, 128 animals were accounted for in the present study.

2.3. Status Epilepticus

Experimental procedures are based on (Turski et al. 1983), with modifications. Briefly, Lithium chloride (127 mg/Kg) was administered to increase the sensitivity of animals to pilocarpine, followed by methyl-scopolamine (1 mg/kg) administration to reduce the peripheral effect of pilocarpine and the administration of 30 mg/kg pilocarpine hydrochloride to elicit SE. One and a half hours later, seizures were stopped with the injection of thiopental (120 mg/kg). Treatments were then given: CGA at 3, 10, 30, or 100 mg/kg, or vehicle. On the second and third days, similar treatments of CGA, or vehicle were given one and a half hours following thiopental administration (120 mg/Kg). Sham animals were subjected to the same interventions, with the omission of pilocarpine and vehicle. Lastly, animals were euthanized by receiving either an overdose of thiopental (200 mg/kg), for histological analysis (80 rats), or by decapitation, for enzymatic analysis (48 rats).

2.5. MDA and SOD Measurements

After the extraction of the brains, hippocampi were individually processed in a Potter-type homogenizer (Thomas Scientific, USA) in two volumes of cold buffer solution (4% PBS). A solution of N-methyl indole was prepared as an MDA standard. In 96 well plates, both the samples and the MDA standard were incubated for one hour at 45ºC, then centrifuged for 15 min at 3000 rpm.min^− 1, the supernatant was collected, and the absorbance was read at 586 nm, using a Shimadzu model 1800 spectrophotometer (Japan), according to the Indole’s method (Foettinger et al. 2007).

The amount of MDA present in the samples was determined with the formula:

MDA = DOA /DOP

Where DOA, is the optical density of the sample and DOP, is the optical density of the MDA standard, and is expressed in µmol/g. The density values optics obtained were divided by the protein values obtained from the Bradford method (1976) for each sample.

To determine SOD activity, 30 µL methanol (99.9%) and 50 µL chloroform (99.9%) were added to each sample of hippocampus homogenized in 4% PBS, shaken and centrifuged for 15 min at 6,000 rpm.min-1, the supernatant was collected and preserved at 4ºC until the time of reaction. A solution of pyrogallol (≥ 98%) and EDTA (98%) was added as a chromophore marker. The blank was obtained in assays containing distilled water instead of a sample. Readings were done at 420 nm using a Shimadzu 1800 spectrophotometer (Japan) (Seclen et al. 2006).

The activity of SOD in the samples was made by calculating the degree of inhibition of the reaction using the formula:

SOD= [(Δ𝐷𝑂𝐴× 100) − 100] × 0,6 / Δ𝐷𝑂𝐵

Where DOA represents the optical density at the time of the sample and DOB. is the optical density of the blank (no sample). The SOD activity is expressed in units of enzymatic activity per milligram of protein (U/mg protein).

2.4. Histology: Nissl and Fluoro-Jade C stain

The rats anesthetized with an overdose of sodium thiopental (200 mg/kg) were perfused with 50 mM PBS for approximately 10 min, through the left ventricle at a pressure of 50 mmHg, using a peristaltic infusion pump (BP-600, Milan Scientific Equipment, Brazil). Brains were harvested, incubated overnight in 30% sucrose solution, placed on a stand, and covered with OCT compound. With the aid of a cryostat (Leica CM 1850; Leica Microsystems, Germany), the frozen brain tissue was transversely sectioned into 20 µm-thick sections, distended on previously gelatinized glass slides. Ten slides were made per animal, with three sections of the hippocampal formation, between the coordinates − 3.3 and − 4.5 mm from the Bregma, according to Paxinos and Watson (2006).

For the Nissl stain, the slides with the brain tissue sections were placed in a glass vat and bathed with ethanol solutions. Then, the slides were bathed with 0.1% Cresyl Violet solution, washed in sequence with distilled water, and immersed in 99.9% final ethanol. Then the slides were bathed in different concentrations of ethanol and xylol. Finally, the slides were covered with a cover slip and Permount was used as a mounting medium.

To analyze neurons in the degenerative process, histochemical staining with the fluorochrome Fluoro-Jade C (FJC) was utilized according to the protocol by Schumed and Hopkins (2000), and the stained cells (FJC+) were counted and divided by the measured area (mm²) of each hippocampal region.

The slides were analyzed using a fluorescence microscope (DM 5000 B, Leica Microsystems, Germany). The integrity of the layers of the CA1 and CA3 regions, as well as the Hilus, was evaluated, in addition to verifying morphological alterations, such as vacuolated cells, gliosis, disorganization of the layers, and pyknotic nuclei.

In the quantitative analysis of neuronal damage, three slides were selected for each animal, from each group, and from each of these chosen three sections of the hippocampal formation. In each of the selected slices, images were captured of the entire extension of the pyramidal cell layer of the CA1, CA3, and Hilus regions. Image capture was performed by a system consisting of a Leica color digital camera (DFC300 FX; Leica Microsystems, Germany) connected to a microscope (DM 5000 B; Leica Microsystems, Germany) and a computer (configuration: Dual Core 2 Duo). The images obtained were visualized with different magnifications. Viable cells were manually counted through a selection made with or aided by the Q-Win software (Leica Microsystems, Germany), as well as the measurement of the analyzed areas. The estimated number of viable cells (n), was determined by averaging the values of three adjacent sections. The actual number of cells is calculated using the Abercrombie correction method:

N (per mm ² ) = n [T/(T + D)] /A

Where N is the actual number of cells, T is the section thickness (20 µm), D is the average diameter of each nucleus in the sample population, which was measured at 1000X magnification; and A is the measured area (in mm²) of each hippocampal region. The values obtained were expressed as the average of the densities (neurons/mm ± SE) (Kwak et al. 2005).

The FJC assays were evaluated using the Student's t-test, whereas One-way ANOVA, followed by Tukey test was performed for comparisons among the remaining tests conducted. The values are expressed as mean ± S.E.M. and n represents the number of animals. p-values < 0.05 were considered statistically significant. All the statistical analyses were performed with the Graph Pad Prism program for Windows version 8.0 (GraphPad, San Diego, USA).

3.2. Effects of CGA on oxidative markers in the hippocampus

The present study aimed to assess the antioxidant effects of CGA on the hippocampal concentrations of malondialdehyde (MDA) and superoxide dismutase (SOD) in rats with pilocarpine-induced status epilepticus (SE). Statistical analysis showed a significant effect between all evaluated groups. Tukey post hoc analysis showed that treatment with 30 and 100 mg/Kg CGA significantly decreases the concentration of MDA in the hippocampus of SE rats by 83% and 81% respectively when compared to SE + Vehicle (F_(6,35) = 52.85; p < 0.0001). On the other hand, no significant reduction in MDA concentration was observed in the hippocampus of rats that received 3 and 10 mg/Kg of CGA, when compared to SE + Vehicle (Fig. 1)

The concentration of hippocampal SOD exhibited significant differences among the studied groups. Subsequent post hoc analysis revealed that the groups treated with 30 and 100 mg/Kg CGA demonstrated a significant reduction in SOD concentration by 57% and 40% respectively, compared to the SE + Vehicle group (F_(6,35) = 95.23; p < 0.001) (Fig. 2). These results highlight the effectiveness of CGA 30 and 100 mg/Kg in decreasing the levels of both MDA and SOD, which serve as important markers of antioxidative activity, in rats with SE.

3.1. Effects of CGA on neuronal death

To investigate the potential neuroprotective effects of CGA against cell death in rats with SE, histological analysis using Nissl and Fluoro-Jade C (FJC) staining was employed. Representative images of coronal sections of hippocampal areas CA3 and hilus stained with Nissl are shown in Fig. 3A-D and Fig. 3F-J, respectively. Vacuolization are indicated by red head arrows and pyknotic nuclei by red arrows.

The quantification of the number of neuronal cells per area is presented in Fig. 3E and 3F. A significant difference in cell densities was observed between animals treated with CGA (30 mg/kg, the most effective dose shown in the graph) and the SE + Vehicle group, as indicated by one-way ANOVA followed by Tukey's post hoc test (F_{(3, 20)} = 18.5; p < 0.0001). Similar results were observed in the hilus regions, where the statistical analysis demonstrated a significant effect among the studied groups (F_{(3, 20)} = 19.5; p < 0.0001). The treatment with CGA a significantly protected the CA3 and hilus regions against neuronal death in approximately rates of 50% and 46%, respectively, compared to the SE + Vehicle group.

In addition, we investigated FCJ + labeling in these regions. Figure 4 shows representative images of coronal sections of CA3 and hilus from hippocampi from animals from group SE + Vehicle (Fig. 4A and Fig. 4D, respectively) and the group treated with 30 mg/Kg CGA (Fig. 4B and Fig. 4E, respectively), whereas Fig. 4C shows the quantification of FJC + neurons in the CA3 region. One Way ANOVA analysis revealed a significant difference between the SE + Vehicle and CGA group in the CA3 (F_(2,15) = 40.90; p < 0.001). The group that received 30 mg/Kg CGA showed a neuroprotective effect reducing the FJC + neurons in CA3 in 48% when compared with the SE + Vehicle (Fig. 4C). Regarding the region of the hippocampal hilus, the group that received 30 mg/Kg CGA showed a reduction of FJC + neurons in hilus (57%), when compared with the SE + Vehicle (F_(2,15) = 9.515; p = 0.0021) (Fig. 4F). There was not significant in the CA1 (F_(2,15) = 0.037; p = 0.963). Taken together, these data show that CGA reduces cell loss in the hippocampal regions CA3 and hilus suggesting a neuroprotective effect in SE rats.

In this study, we demonstrate that CGA has neuroprotective effects after SE in Wistar rats, observed by a significant reduction in cell loss and degeneration in the CA3 and hilus areas of the hippocampus. We suggest that these effects may be due to an antioxidant effect, suggested by the decreased MDA in the hippocampus of rats administered CGA, suggesting a reduction in lipid peroxidation. This is remarkable as it was seen even after one hour and a half of seizures. This period of 1 and a half hours was chosen based on (Curia et al. 2008), and we cogitate that this is a reasonable time frame to administer post-SE treatments as patients convulsing rarely receive treatments immediately, hence our studies are more translational. Future experiments will study the efficacy of CGA to rescue cell death at different time points after SE.

The decrease in SOD activity, however, should be interpreted with caution. There are two large isoform groups of this enzyme, one located in the mitochondrial matrix, with Mn²⁺ in its active site, and the other type with Cu/Zn in its active site, present in the cytoplasm. Both types reduce O₂^- to H₂O_2,^-but one acts to increase and the other to decrease the conversion, independently of the other (Moscone and Mascini 1988). An example of this phenomenon can be observed in a study showing that the peak action of SOD happens during the first 24 hours after the stressful stimuli and this peak can continue up until nine days in constant activity, but with a downward trend and a rapid saturation (Andreyev et al. 2005).

As shown in this study, there was a reduction of MDA intensity after CGA administration. A compound with this profile would be extremely important in the regulation of oxidative stress triggered by pilocarpine in rats of our experiments. We speculate this is true even with a reduction in SOD activity because, as demonstrated, CGA had a neuroprotective effect. This is similar to other studies showing a decrease in SOD activity, as well as a decrease in malondialdehyde thiobarbituric acid reactive substances (MDA-TBARS) under an antioxidant treatment (Chen et al. 2005; Liang et al. 2015). We speculate the possibility that the treatment with CGA induces gene factors responsible for the synthesis of SOD in the neuron, and future studies could unravel this possibility. Therefore, their rapid action could be explained by this type of regulation, which in turn could be verified by the increase in SOD mRNA (Liang et al. 2015). We propose the mechanism of CGA protection in the hippocampus could be similar to those described in a study using a focal cerebral ischemia-reperfusion injury model, increasing the ICAM-1, VCAM-1, EPO, and HIF-1α levels and nerve growth factor in brain tissue. In this article, the authors administered rats with HeYing-Qing-Re Formula, which is rich in CGA (Zhang et al. 2018). Future studies examining levels of these factors might clarify this mechanism.

It is important to point out that there is a wide range of potential health benefits of CGA, including its anti-diabetic, anti-carcinogenic, anti-inflammatory, and anti-obesity properties that may provide a non-pharmacological and noninvasive approach for the treatment of some chronic diseases (Tajik et al. 2017). In addition to demonstrating a beneficial effect in animal models of nervous tissue injury and in human neuropathology therapies, the phenolic acid CGA can also be useful in several animal models of disease systems, such as cardiovascular, immune system, kidneys (Shan et al. 2019). Higher coffee intake, considering that coffee is one source of CGA, was associated with a lower risk of incident acute kidney injury and could present an opportunity for cardiorenal protection through diet. However, further evaluation of the physiological mechanisms underlying the cardiorenal protective effects of coffee consumption is necessary (Tommerdahl et al. 2022). Additionally, coffee consumption seems to be also correlated with a decreased risk of developing some neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, and dementia. Regular coffee intake may also reduce the risk of stroke (Chan et al. 2021). The mechanism underlying these effects is, however, as in our study, still poorly understood (Wasim et al. 2020; Socala et al. 2020). However, toxicity of 6-hydroxydopamine (6-OHDA) in animal and cellular models was decreased by CGA. CGA reduced 6-OHDA-induced ROS production and endoplasmic reticulum (ER) stress in SH-SY5Y cells. CGA might be an effective neuroprotective compound that mitigates oxidative stress and endoplasmic reticulum stress in Parkinson disease (Wasim et al. 2020; Socala et al. 2020).

Another commendable example of neuroprotection involved in glutamate neurotransmission was the study using 3-O-caffeoylquinic acid (3-CQA), an isomer of chlorogenic acid, which has been shown to regulate lipopolysaccharide-induced tumor necrosis factor production in microglia (Socodato et al. 2015). Oxidant (hydrogen peroxide) stimulation dramatically enhances glutamate release from microglia; such release is diminished by the 3-CQA inhibition of c-Src/ROS generation, significantly alleviating cell death in cultures from embryonic neurons (Socodato et al. 2015).

A recently published work by our research group also shows neuroprotective activity of CGA in a rodent retinal lesion model [9]. We noted that CGA protected the retinal layers against matrix disorganization and decreased the number of vacuolized cells and pyknotic nuclei. In addition, CGA reduced neuronal death after ischemic insult. Therefore, this study reinforces the findings in the present work.

CGA was demonstrated to have a significant neuroprotective effect in the SE model, probably by inhibiting a step of the inhibiting lipid peroxidation, thus avoiding the activation of degenerative cascade triggered by pilocarpine. Future studies could evaluate the activation and expression of caspases I and III, important markers of the apoptotic process, and their effects on the entire oxidative cascade.

Considering the antioxidant and neuroprotective effect, future studies may evaluate CGA in a chronic model of epilepsy, as some studies suggest that free radicals and oxidizing systems have a key role in the spread of damage caused by seizures (dos Santos et al. 2011; Mikami et al. 2015).

6-hydroxydopamine, 6-OHDA; analysis of variance, ANOVA; chlorogenic acid, CGA; 4,5-di-O-[E]-caffeoylquinic acid, CQA; Fluoro-Jade C, FJC; Fluoro-Jade C positive, FJC+; endoplasmic reticulum, ER; lithium-pilocarpine, Li-Pilo; malondialdehyde, MDA; nitric oxide, NO; reactive oxygen species, ROS; Status Epilepticus, SE; superoxide dismutase, SOD; spontaneous recurrent seizures, SRS.

Acknowledgment : This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), both from Brazil. We would like to acknowledge Dr. Javier Marín-Prida (Center for Research and Biological Evaluations (CEIEB), Institute of Pharmacy and Food, University of Havana Havana, Cuba) for his help with the oxidative stress protocols.

Ethics approval:

The experimental protocol was in accordance with and approved by the local Animal Care Committee (protocol number 08.1.834.53.9 CEUA –Campus University of São Paulo, Ribeirão Preto, Brazil).

Conflict of interest:

The authors declare that there are no conflicts of interest.

Authors' contributions:

AJC-G, wrote, and edited the first step of the manuscript, also contributed to data analysis tools, these data were part of his master's thesis. JLL, wrote, reviewed, and edited the manuscript, also contributed to data analysis tools. MVC, contributed by doing the experiments and to data analysis tools. NPL, conceived and designed the analysis, wrote the paper and acquired funding. JLCL, conceived and designed the analysis, wrote the manuscript. LGN, collected the data, performed the analysis. ACKF, conceived and designed the analysis, wrote, reviewed and edited the manuscript. WFS, conceived and designed the analysis, wrote, reviewed, and edited the manuscript, and acquired funding. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Data Availability:

The data that supports the fndings of this study are available upon request.

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Neuroprotective activity of chlorogenic acid from Brazilian Arnica in rodents subjected to status epilepticus induced by lithium-pilocarpine

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Abstract

Figures

1. Introduction

2. Material and Methods

2.1. Compounds and Drug Administration

2.2. Animals

2.3. Status Epilepticus

2.5. MDA and SOD Measurements

2.4. Histology: Nissl and Fluoro-Jade C stain

3. Results

3.2. Effects of CGA on oxidative markers in the hippocampus

3.1. Effects of CGA on neuronal death

4. Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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