Acth4-10pro8-Gly9-Pro10 Administration Attenuate Ketamine-Induced Neurotoxicity in Post Natal Day-7 (Pnd-7) Rats: Effect on Caspase-3, Bcl-2, and BDNF Activity

Received: 03-Dec-2021;Accepted Date: Dec 17, 2021; Published: 24-Jan-2022

Introduction

Anesthesia in children has developed rapidly over the last 20 years. There are many children around the world who have undergone surgical procedures under general anesthesia. Based on an epidemiological study by the National Survey of Ambulatory Surgery (NSAS) in the United States in 2006, there were 202,412 children under 18 years old and 1,177,327 children from 0 to 5 years old who received anesthesia [1]. In another study, more than 450,000 children under18 years old also underwent surgery under general anesthesia each year and 115,000 children of them were under 3 years old. So in general, anesthesia is considered a safe intervention in children [2].

An anesthetic drug that is commonly used for anesthesia in children is ketamine. The use of ketamine has been extensively documented in studies with more than 11,000 children undergoing anesthesia and has demonstrated excellent safety and efficacy. Ketamine has a strong analgesic effect and can maintain spontaneous respiration and hemodynamic stability. The anesthetic effect of ketamine is rapid and does not depress the heart. Ranging from small doses for sedation to induction of general anesthesia, ketamine can be used in uncooperative and frightened children [3]. Ketamine is also combined with opioids for the management of postoperative pain in children [4]. Ketamine is also the main choice for pediatric patients who require light sedation and invasive procedures performed in the Emergency Room (ER), Intensive Care Unit (ICU), and radiology room. A large number of clinical studies have shown that the combination of ketamine with midazolam, ketamine with dexmedetomidine, or ketamine with propofol is very useful and safe for sedation and pain relief in pediatric patients admitted to the emergency department or UPI, especially in patients with prolonged ventilator use at UPI [5-8]

A number of animal studies have provided strong evidence that repeated exposure to ketamine results in impaired brain function that is associated with neuroapoptotic injury in the immature brain. This finding has made practitioners review the safety of using ketamine in anesthesia in children [9]. In fact, the United States Food and Drug Administration issued a warning about the safety of anesthetic drugs given repeatedly or more than 3 hours in surgery or procedures on children under 3 years of age, or in 3rd trimester pregnant women which will affect the child’s brain development [10].

Although the mechanism underlying the effects of ketamine on brain neurodevelopment is not fully understood, it is thought to arise during the synaptogenesis process in the growth spurt period, and it is found that brain neurons are very sensitive to disturbances in the synaptic system, and in general there are two mechanisms that are considered to play an important role. Ketamine neurotoxicity [11,12].

Neuropeptides play an important role in the incidence of neurotoxicity. In several studies, the administration of neuropeptides can improve levels of apoptotic biomarkers. One of the neuropeptide compounds that has been clinically proven to reduce neurotoxic effects is the peptide compound ACTH4-10Pro8-Gly9-Pro10 [13,14]. The compound ACTH4-10Pro8-Gly9-Pro10 is a heptapeptide with the arrangement Met(hionine)v-Glu(tamine)-His(tidine) Phe(nylalanine)-Pro(line)-Gly(cine) Pro (line), with nomenclature single letter MEHFPGP [15].

The dose of ACTH4-10Pro8-Gly9-Pro10 given in animal studies varies and depends on the desired effect, including antioxidant, antihypoxic, angioprotective, and ischemic effects. Based on the description above, the researcher was interested in analyze the effect of giving ACTH4-10Pro8- Gly9-Pro10 on ketamine neurotoxicity in Rattus novergicus Sprague Dawley strain Post Natal Day-7 (PND7) by assessing the expression of caspase 3, Bcl-2, and BDNF.

Methods

This study was true experimental research with a post-test only control group design. Randomization of treatment was carried out using a simple randomization technique with double blinds. All samples were labeled with numbers 1 to 33 by the analyst at random. Furthermore, the group was divided randomly into 33 rats with the help of the Microsoft Excel computer program. In practice, this study applied a double-blind, where this randomization process and the provision of drugs were carried out by the analyst without being known by the researcher as the treatment provider and also without being known by the histopathological examiner. The sample was divided into three negative control group, given intranasal placebo (0.9% NaCl) and subcutaneous placebo injection (0.9% NaCl), positive control group, given intranasal placebo (0.9%NaCl) and ketamine 40 mg/kgBW subcutaneously, and treatment group, given ACTH4-10Pro8-Gly9-Pro10 at a dose of 50 mcg/kgBW intranasally and ketamine at a dose of 40 mg/kgBW subcutaneously. The research approval was obtained from the health research ethics commission, Faculty of Medicine, University of North Sumatra (381/KEP/USU/2020)

The care of experimental animals and examination of the expression of caspase 3, Bcl-2, and BDNF were carried out at the Laboratory of Experimental Animal Care Unit Faculty of Medicine, Brawijaya Malang. The research was conducted from January 2021.

The object of research were Rattus norvegicus rats, male, 7 days old, weighing 15-20 grams, Spraque-Dawley strain, obtained from the Experimental Animal Care Unit. The subjects were divided into three groups, each with 11 rats. This study’s inclusion criteria were male rat. The purpose of choosing male sex was to avoid hormonal influences on the final results of the study, healthy and active. It was characterized by active movement and based on morphological appearance (no wounds and body defects), 7 days old, and weight 15-20 grams. Exclusion criteria were animals that behave aggressively in observation by attacking other groups. Drop out criteria was rats that did not survive to the end of the study.

The research was carried out using animal treatment procedures in terms of the 3R principles (Reduction, replacement, refinement) and the 5 F principles (Freedom from hunger and thirst, Freedom from discomfort, Freedom from pain, injury or disease, Freedom express, normal behavior, Freedom from fear and distress) and the dropout criteria are applied if the research subject experiences illness or death so that he cannot fulfill the research procedure [16].

Rattus novergicus, male, PND-7, Spraque-Dawley strain, obtained from the animal care unit as many as 33 rats. Before treatment, the rats were weighed; rectal temperature measured, and kept in cages measuring (40 x 20 x 20) cm3 each containing 11 rats. The temperature in the cage was set at room temperature. The rats used in vivo experiments were all Sprague-Dawley with 7 days postnatal. PND7 rats were randomly assigned to 3 treatment groups. Test rats were grouped randomly into a negative control group, a positive control group, and a treatment group. The negative control group was given an intranasal placebo of 0.05 ml of 0.09% NaCl and a placebo injection of 0.4 ml of 0.9% NaCl subcutaneously. The positive control group was given an intranasal placebo (0.4 ml NaCl 0.9%) and ketamine 40 mg/kg (0.4 ml) subcutaneously. The treatment group was given Semax^® at a dose of 50 mcg/kgBW intranasally and ketamine at a dose of 40 mg/kgBW subcutaneously. The rats were decapitated 6 hours after administration of ketamine. Then the rat brain was separated for paraffin block. Brain tissue collection for examination of BDNF Bcl-2 and caspase-3 expression was performed on all groups of rats. The brain was taken especially in the hippocampus area, on the basis of the theory that BDNF is most abundant and active in cornuamnosis 1 (CA1) in the hippocampus, while caspase-3 and Bcl-2 can be found in all brain areas, including the hippocampus. The ACTH4-10Pro8-Gly9-Pro10 therapy adminis-tered was under the patent name Semax^® produced by The Institute of Molecular Genetics Russian Academy of Sciences. The ketamine administered had a patent name Ketamine-Hameln^®, manufactured by PT. Combiphar Indonesia.

The examination of caspase-3, Bcl-2, and BDNF was by staining IHC preparations. Immunohis-tochemistry (IHC) is a process of identifying specific proteins in tissues or cells using antibodies. The binding site of the antibody to a specific protein is identified by the marker attached to the antibody and can be visualized directly or by the reaction to identify the marker. IHC assay is a very important method that specifically visualizes the distribution and number of certain molecules in tissues using specific antigen-antibody reactions. The IHC examination is superior to many other laboratory tests because the examination is carried out without destroying the histological architecture, so that it can be carried out an assessment of molecular expression patterns in the context of the microenvironment [17]. The results of the measurement of BDNF, Bcl-2, and caspase-3 brain tissue of control and treatment rats were statistically analyzed using the SPSS 25.0 program with a significance level of 5% and a 95% confidence level (α=0.05).

Results

The samples in this study were 11 rat’s rattus norvegicus each group, male, 7 days old, and weigh 15 grams-20 grams, Spraque-Dawley strain, obtained from the Experimental Animal Care Unit. However, during the research process, 2 rats died in the placebo group and 1 rat died in the treatment group. Thus, 9 rats were obtained in the negative control group, 11 rats in the positive treatment group, and 10 rats in the treatment group.

Caspase-3 expression in the brain of Rattus norvegicus Sprague Dawley strain PND 7 receiving placebo and ketamine are shown in Table 1. It was found that the expression of caspase-3 in the group given ketamine 40 mg/ kgBW experienced an increase in the distribution of the percentage of cells expressing caspase-3 about 1.2 times that of the negative control group (placebo) with a distribution percentage of 34.1  4.8. Percent and statistically significant (p=0.046) (Figure 1).

Table 1: Expression of caspase-3 in positive control and treatment groups.

Figure 1: Distribution of Caspase-3 staining in the brain of Rattus norvegicus Sprague Dawley strain PND 7 receiving ketamine and ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine. The observation of the preparations used a microscope with a magnification of 400x and image analysis using ImageJ 1.53c software.

Expression of Bcl-2 in the brain of Rattus norvegicus Sprague Dawley strain PND 7 receiving ketamine and ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine are shown in Table 2. it was found that the expression of Bcl-2 in the group receiving ACTH4-10Pro8- Gly9-Pro10 before the administration of ketamine and the positive control group (subcutaneous ketamine) almost had the same number of distributions of the percentage of cells expressing Bcl-2 (p=0.99) (Figure 2). BDNF expression in the brain of Rattus norvegicus Sprague Dawley strain PND 7 receiving ketamine and ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine are shown in Tabel 3. It was found that the expression of BDNF in the group that received ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine experienced an increase in the distribution of the percentage of cells expressing BDNF about 1.55 times that of the positive control group (subcutaneous ketamine) with a distribution percentage of 31.0  7.9 percent and statistically significant (p=0.001) (Figure 3).

Table 2: Expression of Bcl-2 in the positive control and treatment groups.

Figure 2: Distribution of Bcl-2 staining in the brain of Rattus norvegicus Sprague Dawley strain PND 7 receiving ketamine and ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine. The observation of the preparations used a microscope with a magnification of 400x and image analysis using ImageJ 1.53c software.

Figure 3: Distribution of BDNF staining in the brain of Rattus norvegicus Sprague Dawley strain PND 7 receiving ketamine and ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine. The observation of the preparations used a microscope with a magnification of 400x and image analysis using ImageJ 1.53c software.

Comparison of the mean distribution of caspase-3, Bcl-2, and BDNF expression staining values in the brain of Rattus norvegicus Sprague Dawley strain PND-7 in the placebo group (0.9% NaCl), the subcutaneous ketamine group, and the group receiving ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine is presented in tabular (Tables 3 and 4). It was found that there was no difference in the distribution of the percentage of cells expressing caspase-3 and Bcl-2 in the three groups with p-values of 0.145 and 0.943, respectively. However, in the distribution of the percentage of cells expressing BDNF there was a significant difference between the three groups with p<0.001.

Table 3: Expression of BDNF in positive control and treatment groups.

Table 4: Expression of caspase-3, Bcl-2, and BDNF in the three groups.

Discussion

This study was conducted to prove that the administration of ACTH4-10Pro8-Gly9 Pro10 can reduce the incidence of neurotoxicity due to the administration of ketamine in Rattus novergicus Sprague Dawley strain PND 7 by assessing the expression of caspase 3, BDNF and Bcl-2 in the brain. The rat samples tested were 33 rats, in which the samples were divided into three, i.e. the negative control group (P0) with rats given an intranasal placebo (0.9% NaCl) and placebo injection (0.9% NaCl) subcutaneously, the positive control group (P1) with rats given placebo intranasal (NaCl 0.9%) and ketamine 40 mg/kgBW subcutaneously, and the treatment group (P2) with rats given Semax® at a dose of 50 mcg/kgBW intranasal and ketamine at a dose of 40 mg/ kgBW subcutaneously.

Ketamine induces neuronal death through compensatory upregulation of the NMDA receptor subunit and overstimulation of the glutamate neurotransmitter system that follows. It has been shown that the expression of the NR1 receptor (NMDA receptor subunit 1) is significantly increased in the brains of ketamine-treated rats compared to controls, and it is hypothesized that this NMDA receptor upregulation plays a role in inducing neurotoxicity because this receptor causes toxic intracellular calcium accumulation. Ketamine at certain doses triggers massive and widespread neuronal death through apoptotic mechanisms in immature rat brain. Repeated ketamine injection also caused marked apoptosis in the hippocampus and dentate gyrus areas of neonates undergoing growth spurts [18].

In this study, it was found that the expression of caspase-3 in the group that received ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine decreased the distribution of the percentage of cells expressing caspase-3 about 0.88 times that of the positive control group (subcutaneous ketamine) with the percentage distribution 30.3  7.3 percent, but not statistically significant (p=0.178). There were no previous studies that have conducted research on differences in caspase-3 activity between administration of saline and administration of ACTH4-10Pro8-Gly9-Pro10 followed by ketamine.

There are two apoptotic pathways, i.e. the intrinsic pathway involving caspase activation due to cytochrome c released by mitochondria and the extrinsic pathway involving Death Receptors (DR), Fas protein and TNF-α which undergo sensitization followed by the apoptotic pathway consisting of a number of enzymes such as F Associated death domain (FAAD), tumor necrosis factor receptor (TNFR)-associated death domain (TRADD), caspase-8 and 10 as effector activators of apoptosis [19,20]. These two apoptotic pathways meet in caspase [21]. In various recent studies, it was proven that ACTH4-10Pro8-Gly9-Pro10 can prevent apoptosis by inducing activation of protein kinase A, inhibiting nuclear factor activated T-cells (NFAT) and NF-kB, reducing levels of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, including caspase-3 [22].

In this study it was found that the expression of Bcl-2 in the group receiving ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine and the positive control group (subcutaneous ketamine) almost had the same number of distributions of the percentage of cells expressing Bcl-2 (p=0.99). Ketamine causes apoptosis mainly through the mitochondrial pathway (intrinsic pathway) and Bcl-2 protein is anti-apoptotic and has been shown to prevent ketamine- induced apoptosis by inhibiting proapoptotic proteins in this intrinsic pathway [23].

A literature study of Bcl-2 implicated as a tissue biomarker of cerebral cortex, human neurons in head injury cases to explain apoptosis and cellular responses to such injury showed that Bcl-2 protein had increased expression in injured but surviving neurons, while Bcl-2 was not detected in cells that have undergone apoptosis [24]. This may explain the findings in this study, where there was no difference between the positive control group and the treatment group. Existing studies on the neuroprotective effects of ACTH4-10Pro8-Gly9-Pro10 were conducted at different time intervals and there was no definitive timeframe.

Further research is needed to ensure that most neurons are protected before being subjected to neuro-damaging treatments such as ketamine administration.

In this study, it was found that the expression of BDNF in the group that received ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine experienced an increase in the distribution of the percentage of cells expressing BDNF about 1.55 times that of the positive control group (subcutaneous ketamine) with a distribution percentage of 31.0  7.9 percent and was statistically significant (p=0.001). The experimental animal was a 2-month-old male Wistar rat which was made into an incomplete global ischemic model by bilateral carotid artery irreversible occlusion procedure injected with physiological saline and a max of 10 g/100 g body weight. In animal test treated with ischemia, ACTH4-10Pro8-Gly9-Pro10 administration showed increased BDNF at 12 hours after occlusion, when gene expression was optimally inhibited in the hippocampus of ischemic control animals. In a study that examined glial cells that were cultured and subjected to oxidative stress, an increase in BDNF expression was found with ACTH4- 10Pro8-Gly9-Pro10 administration. In an in vivo test with rats, an increase in BDNF protein levels was also found in the hippocampus of healthy rats [25]. This is also in line with studies in rats given ketamine which was an NMDA receptor inhibitor and found an increase in neurodegeneration, BDNF levels, and TrkB cDNA protein products, where the increase in BDNF expression was thought to be a protective response to neurotoxicity caused by ketamine [26].

In this study, it was found that there was no difference in the distribution of the percentage of cells expressing caspase-3 and Bcl-2 in the three groups with p values of 0.145 and 0.943, respectively. However, in the distribution of the percentage of cells expressing BDNF there was a significant difference between the three groups with p<0.001. In terms of caspase-3 expression, there was a difference in the mean that was consistent and in accordance with existing research [22,27,28], where in Soriano’s study there was a 5-fold increase in caspase-3 expression in the group treated with ketamine compared to the saline group and in Lyu’s study caspase-3 expression increased significantly after the administration of ketamine at 3 and 6 hours compared to 0 and 1 hours (F=46.28, P=0.002, 3 hours vs. 0 hours; F=18.32, P=0.014, 3 hours vs. 1 hour; F=46.67, P=0.002, 6 hours vs. 0 hours; and F=15.94, P=0.016, 6 hours vs. 1 hour). In this study, there was also an increase in the mean of the negative control group and the positive control group (28.4  7.0 vs. 34.1  4.8). Decreased caspase-3 expression, as in the study of ACTH4-10Pro8-Gly9-Pro10, could reduce caspase-3 levels measured at 3 and 6 hours (10.67 vs 13.67; p ^¼ 0.000) [29].

In this study, the treatment group that received Semax before giving ketamine compared to the positive control group experienced a decrease in caspase-3 expression from 34.1  4.8 to 30.3  7.3. However, this difference was not statistically significant, it can occur due to several things, including the lack of a large number of research samples, less varied doses, or different research treatment factors from previous studies.

Significant differences were found in the last biomarker in this study, BDNF. BDNF is the most abundant neurotrophin in the brain and is essential for neuronal survival during neuronal development and integration in the adult brain. BDNF is also involved in synaptic plasticity, neuron differentiation, and neuronal survival [30]. Administration of ACTH4-10Pro8-Gly9-Pro10 causes the most pronounced changes in the expression of neurotrophin genes and their receptors in the hippocampus, where ACTH4-10Pro8- Gly9-Pro10 will activate gene expression in most cases [25]. ACTH4-10Pro8-Gly9-Pro10 has a positive effect on the expression level of the gene encoding growth factor involved in trophic levels, BDNF [22]. The neuroprotective effect of ACTH4-10Pro8-Gly9-Pro10 is thought to be mediated by an increase in the content of neurotrophic factors, such as BDNF, which have clear neuroprotective properties and can prevent dopaminergic neuronal death in both in vitro and in vivo experiments. In experiments with glial cells cultured 30 minutes and 20 hours after ACTH4-10Pro8- Gly9-Pro10 treatment, significant 5 and 8-fold increases in nerve growth factor and BDNF mRNA expression were found, respectively. An increase in the content of BDNF in the hippocampus and striatum of rats was also found in in vivo experiments 3 and 24 hours after a single intranasal administration of ACTH4-10Pro8-Gly9-Pro10 at a dose of 50 or 250 g/kg [14]. In a study, given ACTH4-10Pro8- Gly9-Pro10 in experimental rats, observations in the rat cortex found an upregulation of BDNF mRNA expression at 24 hours. Administration of ACTH4-10Pro8-Gly9-Pro10 to rats inhibited the transcription of neurotrophin receptors (TrkB, TrkA) in the cortex after 3 hours [31].

Those findings are in line with the results in this study, where the expression of BDNF in the group receiving ACTH4-10Pro8-Gly9-Pro10 before the administration of ketamine compared to the group receiving placebo or ketamine alone experienced a significant increase in the distribution of the percentage of cells expressing BDNF (15.1  5.4 vs 19.9  4.9 vs 31.0  7.9). With this study, it is proven that the administration of ACTH4-10Pro8-Gly9-Pro10 can reduce the neurotoxic effect of ketamine caused by apoptotic mechanisms through various pathways including the intrinsic pathway of caspase-3 activation and many other signaling pathways such as PI3k/Akt that affect Bcl-2, and direct inhibition of the receptor tyrosine kinase TrkB which is directly related to NMDA receptor and BDNF expression.

Conclusion

In conclusion, ACTH4-10Pro8-Gly9-Pro10 administration can attenuate ketamine-induced neurotoxicity in post natal day-7 (pnd-7) rats and reduce distribution of the percentage of cells expressing BDNF significantly, but there was no significant difference in the distribution of the percentage of cells expressing caspase-3 and Bcl-2.

Acknowledgement

We would like to thank Institut Biosains UB. Universitas Brawijaya and Laboratory of Experimental Animal Care Unit Faculty of Medicine, Brawijaya Malan.

Conflict of Interest

The authors declare that there is no conflict of interest

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