INTRODUCTION:

Brain development initiates during the uterine period and finish atthe begin of young adult, during this period, brain is highly sensitive to some internal and external factors. During this developmental stage, stress may be associated with neuropsychiatric illness, including anxiety, depression and schizophrenia^1,2. Indeed, many neuropsychiatric disorders often emerge during adolescence.

In rats, the period of adolescence typically starts just after 3^rd postnatal week (weaning stage) and extends until the 12^th postnatal week. This developmental phase is crucial and can influence the onset of various mental health conditions^3,4.

Early chronic stresscan exert lasting and severeimpacts on behavior and physiology of animal. LPS administration in rats was suggested as a model for stress induction in early-stage development. It induces damage to brain cells, resulting in various physiological and behavioral alterations throughout the rodent's life. These effects encompass learning and memory capabilities, as well as increased behavioral, neural, and endocrine responses to stress⁵.

In stress situations, the adrenal gland release cortisol hormone in humans and corticosterone hormone in rodents. These hormones are known to enhance the toxicity of generating oxygen radical, potentially leading to an increase in the basal value of species of reactive oxygen (ROS) in central nervous system tissues like prefrontal cortex and hippocampus. This can contribute to oxidative stress, which has implications for various physiological and cognitive processes⁶.

Reactive oxygen species (ROS) like superoxide and free radicals of hydroxyl, along with peroxide of hydrogen, are naturally synthetized by products of physiological reactions in brain. Notably, dopamine (DA) metabolism is a significant free radical source of the production of free radical of hydroxyl in brain. The breakdown of DA by oxidase monoamineis responsible for production of hydrogen peroxide. Additionally, dopamine can interact with oxygen, forming semiquinones and quinones molecules that contribute to the production of ROS. These processes highlight the intricate relationship between neurotransmitter metabolism and oxidative stress in the brain⁷. Indeed, under the superoxide dismutase (SOD) effect, superoxide species are transformed into hydrogen peroxide, and then the peroxide of hydrogen is further neutralized to molecules of water through the actions of enzymes like glutathione peroxidase (GPx) and catalase. This orchestrated process helps regulate and mitigate the potential damaging effects of reactive oxygen species in the cellular environment⁸. Additionally, catalase activity was reported present in low rate and localized within the peroxisome vesicles in brain cells. Consequently, glutathione peroxidase (GPx) is considered the first implicated enzyme in neutralizing excess hydrogen peroxide excess produced in nervous cells. The specific localization and distribution of these enzymes may be related to maintaining brain redox balance⁸. The hydrogen peroxide can diffuse toward other cells, potentially leading to a high production of hydroxyl radicals. The radical's reactivity is notable for inducing lipid peroxidation leading to damages in membranes and organelles, ultimately leading to cell death. This cascade of reactions underscores the importance of maintaining a delicate balance in oxidative processes within the body⁷. However, the antioxidant defense system is crucial for eliminating reactive oxygen species (ROS), but imbalances, whether in deficiency, may lead to chronic stress. In the induced stress, the released corticosterone may affect the corticolimbic, particularly in laboratory animals. This intricate interplay highlights the importance of understanding the regulatory mechanisms governing both oxidative stress and neurotransmitter systems in response to stress⁹.

Gender is a significant factor in early stress models, with differences in behavioral and neuroendocrine parameters, it appears that male rats are more affected, with high concentrations of corticosterone compared to females. This gender-specific response underscores the consideration of sex dimorphism related to stress research impacting various physiological and behavioral outcomes¹⁰. Glucocorticoids play a moderate role in learning and memory. It is often attributed to the modulation of glucocorticoid receptors in prefrontal cortex and hippocampus, emphasizing the complex interplay between stress hormones and cognitive processes⁸. Previous studies have demonstrated the LPS impact on the physiology and the behavior in adult^4,11. Further research in this area could provide valuable insights into the effects of LPS stress induction in adolescents compared to adults^10,12. Understanding the mechanisms behind cognitive alterations in adolescent rats due to LPS stress is crucial. The involvement of certain brain structures like hippocampus memory and learning functions adds complexity to the potential impacts of LPS administration on spatial learning in these subjects. Investigating specific neural pathways could offer more insights into the observed effects¹³. Spatial memory is not solely governed by the hippocampus. In this study, the focus on the PFC, striatum, and hippocampus aims to unravel the interconnected neural circuits shaping spatial cognition. Notably, prolonged early stress increased significantly glucocorticoid receptors in male-rat brain compared to female-rats, suggesting a gender-specific response to stress¹³. The study aims to address the unresolved question of gender response to early stress. Specifically, it investigates prolonged effects of LPS administration on SOD, GPxand TBARS levels (indicative of lipid peroxidation) in the interconnected neural circuit of striatum, prefrontal cortex, and hippocampus in 2-, 3- and 10-months male and female rats. This research is significant as it explores, for the first time, the lasting impact of LPS on lipid peroxidation and the antioxidant activity in these nervous structures, considering potential sex differences.

MATERIALS AND METHODS:

Materials:

LPS (derived from E.coli) were obtained from our Laboratory and prepared in non-pyrogenic normal saline (PBS). In dark bottle, DL-α-LA was prepared by uing sterile normal saline and NaOH until total solid dissolution. The Thiobarbituric acid solution was prepared by dissolving 1g of TBA in 200mL of deionized water and same volume of glacial acetic acid. Phosphate buffer with EDTA (pH 7.4).

Animals:

Pregnant Wistar rats, housed individually in cages with sawdust-covered floors, maintained controlled conditions including a light period from 07h to 19h, at temperature of 22±2°C, and regular proper cage. Water and food were provided ad libitum, with four to five rats per cage. Ethical standards from Ibn Tofail University and ICLAS were followed. 12 rats were categorized into 4 groups of three rats: (1) LPS-male, (2) Control male, (3) LPS-female, and (4) Control female.

Antioxidant Enzymes Activities:

For evaluating antioxidant enzyme activity in hippocampus, that conserved at temperature of -70°C, a homogenization process performed. This involved homogenizing in 10 vol (w:v) ice-cold 50mM potassium phosphate buffer (pH 7.4) with 1mM EDTA for determining Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx) activities. For Catalase (CAT) activity, samples were homogenized in 10 vol (w:v) ice-cold 10mM potassium phosphate buffer (pH 7.0). After centrifugation at 10000g for 20 min and 4°C. SOD activity was assessed using a kit. Catalase activity, assessing the degradation of hydrogen peroxide (H₂O₂), CAT activity was assessed in terms of H₂O₂micromoles consumed per one minute per one milligram of protein, theused molar extinction coefficient is 43.6 M^-1.cm^-1.

NO assay:

To assess Nitric Oxide (NO) quantity in hippocampus and cortex, NO concentrations derivatives (nitrates and nitrites) were estimated. In a spectrophotometer tank with a 1cm path length, a mixture was created, comprising 100µL Griess reagent, 300µL nitrite sample, and 2.6mL deionized water. The mixture was incubated for 30 minutes at room temperature. Standard range was obtained using NaNO₃ to facilitate accurate measurement of NO concentrations in the samples^10,12.

Lipid peroxidation assay:

Malondialdehyde (MDA), a product of free radical-mediated breakdown of polyunsaturated fatty acids, was assessed by a method involving the formation of a coloured pigment in an acidic and hot environment (100°C). This pigment results from the interaction between MDA and thiobarbiturate (TBA)14.

In the procedure, 1ml of homogenate from both structures was mixed with 1ml of acid trichloroacetic acid (TCA) (20%) and 2ml of Thiobarbituric acid (TBA, 0.67%). After 15 minutes of centrifugation, the optical density was measured on the supernatant using a spectrophotometer (at 530nm). MDA quantity was evaluated using the generated curve with tetraetoxypropane under the same conditions¹⁵.

Statistical analysis:

Statistical analysis was performed using the software GraphPad Prism 7 and SPSS Statistics Version 24. Prior to analysis, each group underwent outlier assessment with Grubbs’ Test, variance homogeneity with Levene’s Test, and evaluation of normality of residuals using the Shapiro-Wilk Test of Normality. Skewness and kurtosis were also assessed through Descriptive Statistics in SPSS.

Result values were presented as mean ± SE and analysed using two-way test ANOVA, with LPS administration and gender as factors. For comparing control, LPS-male, and LPS-female groups, the Kruskal–Wallis test was employed. Significance was confirmed at p≤5%.

RESULT:

Nitric oxide:

Prefrontal cortex:

In 2 and months, the results (Figure 1) show that LPS, after 48hours of injection, increases the NO levels in males and females, with a highly significant interaction between the two sexes. The results demonstrate a significant interaction between LPS and sex (p=0.045), with significant treatment impact (p<0.001) and sex (p=0.05).

In 10 months, NO levels increase in LPS-treated male and LPS-treated female rats comparing to no treated rats. This difference is significant in male rats and not significant in female rats. Statistical analysis shows the absence of significant LPS vs sex interaction (p>0.05), and sex effect (p=0.05), with presence of a significant treatment effect (p<5%) (Figure 1).

Figure 1: Evaluation of no concentration in prefrontal cortex of 2, 3 and 10 months LPS-male and LPS-female rats.

(*, #, $) p <5%, (**, ##, $$) p <1%, and (***, ###, $$$) p <1‰.

Hippocamps:

In 2 and 3 months, the results (Figure 2) show that LPS, after 48 hours of injection, increases the NO levels in males and females, with a highly significant interaction between the males and females. There is no interaction between LPS and gender (p=0.01), with significant treatment effects (p<0.01) and gender (p<0.01).

In 10 months, the results show an increase highly significant in NO levels in LPS-males and LPS-females compared to control rats, with a highly significant interaction between responsemale and female rats related to the LPS. Also, there is a significant interaction LPS vs gender (p<0.05), with significant effects treatment and gender(p<5%) (Figure 2).

Figure 2: No concentration in the hippocampus of 2, 3 and 10 months LPS-male and LPS-female rats.

(*, #, $) p <5%, (**, ##, $$) p <1%, and (***, ###, $$$) p <1‰.

Lipid peroxidation:

Prefrontal cortex:

Figure 3 shows that LPS, after 48 hours of injection, increases the rate of lipid peroxidation in females; on the other hand, there is no difference significant in males, with a highly significant interaction between male and female rats. Significant interaction between the LPS and gender (p<0.001) was demonstrated, with significant treatment effects (p<1%) and gender (p<1‰).

In 10 months, at the level of the prefrontal cortex, the LPO rate increases significantly in females after one month of LPS injection. However, there is no difference significant in males. The interaction between sex and LPS shows a significant difference. The statistical analysis shows a significant LPS vs sex interaction (p<5%), with significant effects of treatment and gender (p<5%) (Figure 3).

Figure 3: TBARS concentration in the prefrontal cortex of 2, 3 and 10 months LPS-male and LPS-female rats.

(*, #, $) p <5%, (**, ##, $$) p <1%, and (***, ###, $$$) p <1‰.

Hippocamps:

In 2 months, the results (Figure 4) show that LPS, after 48hours of injection, increases the rate of lipid peroxidation in males and females, with a highly significant between the two sexes. statistical analysis shows an interaction significantly between LPS and sex (p<1%), with significant effects treatment (p<1‰) and gender (p<5%)

In 10 months, after ten months of LPS injection, the results show at the level of the hippocampus that there is an important increase in LPO level in LPS-male rats when comparing to no treated rats. On the other hand, the results in females do not show a significant difference. statistical analysis shows an interaction significant LPS vs sex (p<5%), and significant treatment effects (p<5%) and gender (p<5%) (Figure 4).

Figure 4: TBARS concentration in the hippocampus 2, 3 and 10 months LPS-male and LPS-female rats.

(*, #, $) p <5%, (**, ##, $$) p <1%, and (***, ###, $$$) p <1‰.

DISCUSSION:

The present study, we assess the oxidative stress prolongedimpact of Lipopolysaccharide on Hippocampus and prefrontal cortex in female and male-rats at different stages of their life, adolescent, young adult and adult.

The lab analyses demonstrated that LPS administration increases the levels of ROS derived from lipid peroxidation which induced by the LPS as neuo-inflammatory, this effect was significantly higher in prefrontal cortex of adolescent male rats. This finding shows the inflammatory effect of LPS in the production of ROS which plays an important role in causing brain injury. Certainly! The brain is susceptible to oxidative damage due to a few key factors. Firstly, the neuronal membranes in the brain are abundant in polyunsaturated fatty acids, which makes them prone to lipid peroxidation reactions and causing production of reactive radicals, leading to oxidative stress. Additionally, the brain has a high rate of oxygen consumption, which generates reactive oxygen species (ROS) as natural byproducts, further increasing the oxidative burden. Furthermore, the brain exhibits high production of reactive species, it contains metal quantities which are implicated in reactive radical production. Like combination of factors puts the brain at a heightened risk of oxidative injury^12,14. Additionally, lipid peroxidation can induce the breakdown and harm of cell membranes, resulting in alterations to membrane fluidity and permeability, along with increased rates of protein degradation¹⁵. The lipid peroxidation can be assessed by itsthiobarbituric acid reactivity. Several animal models have been used in our laboratory to explore the mechanisms of development^17–26 of some diseases including brain dysfunction.

In the face of oxidative stress, tissues usually boost the activity of antioxidant enzymes as a measure of radical damage. This study observed elevated activity of SOD-enzyme in both hippocampus and PFC. Additionally, increase activity of GPx-enzyme in PFC of LPS-males, whereas LPS-females did not exhibit a similar increase. The heightened SOD and GPx activity in the PFC suggests a responsive strategy to counteract rising levels of reactive oxygen species (ROS)²⁷. The demonstrated adaptive response includes an increase in GPx activity along with heightened SOD activity. SOD's role is to convert superoxide radicals into hydrogen peroxide, resulting in elevated hydrogen peroxide levels. Subsequently, GPx takes part in detoxifying hydrogen peroxide, generating hydroxyl radicals in the process. Ultimately, GPx completes the process by converting hydrogen peroxide into water²⁸. The hydroxyl radical's ability to induce lipid peroxidation can result in damage to cell structures like membranes and organelles, and consequently an apoptosis of cell. Insufficient GPx levels may contribute to lipid peroxidation. The observed high TBARS levels in our study might be attributed to an insufficient increase in GPx in the PFC. The rationale behind the increased SOD and GPx activities, along with elevated TBARS levels (indicating lipid peroxidation), aligns with the explanations provided earlier.

According to previous studies^10,12,15, The current study reveals no correspondence between elevatedenzymatic activities and high activity of hippocampus GPx. This suggests that an isolated increase in SOD activity, could potentially be harmful to brain cells. Insufficient GPx may lead to a lack of hydroxyl radical generation after detoxifying hydrogen peroxide, resulting in potentially severe consequences^29,30.

Indeed, various studies have shown that stressful events can elevate dopamine (DA) metabolism in nucleus accumbensand cortex, with extent depending on stress duration. Notably, more factors of stress can increase the metabolism of DA in the brain area³¹. High doses of dopamine (DA) provoke high levels of ROS responsible for oxidative stress. In our study, the observed increase in GPxand SOD activities in PFC indicates a response aimed at scavenging the heightened levels of generated ROS^32-39.

The current work reveals a dimorphism in the response to earl stress by LPS administration on oxidative brain cells. In males, as outlined earlier, there was an increase in SOD and GPx enzyme activities^40,41. Intriguingly, females seem to be shielded from the oxidative stress induced by maternal separation, showing no differences (p>0.05) in GPxand SOD activities or TBARS levels^42-44. Our findings indicate a sex-specific response regarding inflammation, revealing a sexual dimorphism in activation within the hippocampus rather than the prefrontal cortex. Female rats experiencing early stress appear to exhibit distinct patterns compared to males. Additionally, prolonged early stress significantly increases glucocorticoid receptor expression in hippocampus of LPS-male rats, but this effect is not observed in LPS-female rats^45,46. Indeed, corticosterone is known to enhance the oxygen radical toxicity, potentially increasing the ROS levels in the brain structures. The unaffected enzyme activities in LPS-females may be related to insufficient ROS formation, preventing the increase in enzyme activities. Another plausible explanation could be the antioxidant properties of female gonadal hormones, particularly estrogens, which may function as radical scavengers⁷. Certainly, studies have demonstrated that estrogen, specifically estradiol, influences lipid peroxidation particularly at the primary stage, suggesting a potential neuroprotective effect⁷.

In the current study, early-age injection of LPS induces heightened free radical secretion in the hippocampus (HIP) and prefrontal cortex (PFC), with LPS-male rats showing more pronounced effects than LPS-female rats^15,47.

LPS administration appears to have different effects on male rats compared to females, with males exhibiting greater memory impairment in your previous study. The observed higher GPx-enzyme activities, SOD and TBARS levels in response to LPS in male rats compared to females may suggest a sex difference in the activation of DA pathways. These differences require more investigation to underlying^48-51. Additionally, recent studies showed the importance of several medicinal plants also vitamin D supplementation on brain health, particularly in the hippocampus, striatum, and prefrontal cortex (PFC)^52–54. One potential reason could be attributed to the protective effects of female hormones circulating in the body, as estrogens are known for their robust antioxidant and radical-scavenging properties^55,56.

CONCLUSION:

In summary, these findings indicate that diverse stressful experiences during critical brain development may have lasting effects. The early-age injection of LPS triggers heightened free radical secretion in the hippocampus (HIP) and prefrontal cortex (PFC), with male rats being more affected than females, revealing the first evidence of LPS administration have an impact sex-dependent on stress. The mean limitation of this study was the small size of rats focusing in the hippocampus and prefrontal cortex response.

Oxidative stress effect in modulating the stress response appears to be complex and sex-dependent, and requires more researches for a comprehensive understanding of their interplay.

CONFLICT OF INTEREST:

No Conflicts.

ACKNOWLEDGMENTS:

Our thanks are addressed to Professors and PhD students from our laboratory in recognition to their kind support during other lab studies.

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Received on 12.01.2024 Modified on 14.02.2024

Research J. Pharm. and Tech 2024; 17(7):3268-3274.

DOI: 10.52711/0974-360X.2024.00512