Experimental Chronic Prostatitis/Chronic Pelvic Pain Syndrome Increases Anxiety-Like Behavior: The Role of Brain Oxidative Stress, Serum Corticosterone, and Hippocampal Parvalbumin-Positive Interneurons

Abstract

Mechanisms of the brain-related comorbidities in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) are still largely unknown, although CP/CPPS is one of the major urological problems in middle-aged men, while these neuropsychological incapacities considerably diminish life quality. The objectives of this study were to assess behavioral patterns in rats with CP/CPPS and to determine whether these patterns depend on alterations in the brain oxidative stress, corticosterone, and hippocampal parvalbumin-positive (PV+) interneurons. Adult male Wistar albino rats from CP/CPPS (intraprostatic injection of 3% λ-carrageenan, day 0) and sham (0.9% NaCl) groups were subjected to pain and anxiety-like behavior tests (days 2, 3, and 7). Afterwards, rats were sacrificed and biochemical and immunohistochemical analyses were performed. Scrotal allodynia and prostatitis were proven in CP/CPPS, but not in sham rats. Ethological tests (open field, elevated plus maze, and light/dark tests) revealed significantly increased anxiety-like behavior in rats with CP/CPPS comparing to their sham-operated mates starting from day 3, and there were significant intercorrelations among parameters of these tests. Increased oxidative stress in the hippocampus, thalamus, and cerebral cortex, as well as increased serum corticosterone levels and decreased number of hippocampal PV+ neurons, was shown in CP/CPPS rats, compared to sham rats. Increased anxiety-like behavior in CP/CPPS rats was significantly correlated with these brain biochemical and hippocampal immunohistochemical alterations. Therefore, the potential mechanisms of observed behavioral alterations in CP/CPPS rats could be the result of an interplay between increased brain oxidative stress, elevated serum corticosterone level, and loss of hippocampal PV+ interneurons.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) represents a clinical entity whose dominant symptoms are related to pain or discomfort located in the pelvic region [1]. The prevalence of CP/CPPS is very high with 35–50% of men in all age groups reported to be affected by these symptoms during their lifetime [2].

In addition to chronic pelvic pain, CP/CPPS usually causes urinary problems and sexual dysfunction accompanied by psychosocial disorders [3]. These psychosocial issues could, in turn, worsen the CP/CPPS-related dysfunctions in a positive feedback loop manner. Therefore, the syndrome is rarely discussed, kept in secret, and followed by stigma, although its impact on life quality is significant [4].

CP/CPPS patients display higher stress levels, and 62% of them show anxiety symptoms [5]. Hypothalamic-pituitary-adrenal (HPA) axis dysfunction is suspected to be one of the main mechanisms connecting CP/CPPS with anxiety in these patients [6]. Moreover, pain itself is associated with intense recruitment of the HPA axis and corticosterone release, interfering profoundly in the animal behavior [7]. Clinically relevant anxiety has a higher prevalence among patients with chronic pain. Although clinical and epidemiological studies are convincing regarding the relationship between CP/CPPS and anxiety, there is the limited number of in vivo experimental studies regarding this relationship [8–10], so potential pathophysiological mechanisms underpinning this relationship are still largely unknown. The experimental model of CP/CPPS induced by intraprostatic injection of λ-carrageenan has been shown as one of the valuable models for exploring its etiopathology as well as comorbidities [11].

Nowadays, most experts believe that CP/CPPS is a noninfectious syndrome [12] since no evidence of bacteria in prostate tissue has been found in affected individuals [13]. Tissue injury or stressful events trigger chronic peripheral inflammation or nerve injury [14] resulting in high production of inflammatory mediators and neurotransmitters [15] in the prostate. Consequently, it lowers the threshold of primary nociceptive neurons (peripheral sensitization) and leads to the development of inflammatory/neuropathic pain. Several studies indicated the role of oxidative stress in CP/CPPS patients [16–18]. Namely, prostate tissue inflammation is followed by increased reactive oxygen species (ROS) production [19]. However, there are no studies on the role of ROS in the brain-related comorbidities, including anxiety, in CP/CPPS in experimental settings. Also, it is unknown whether oxidative stress can develop in tissues far from an affected organ, as in the brain [20] and its structures which play important roles in anxiety development: the hippocampus, thalamus, or cortex.

The hippocampus plays an important role in the regulation of the HPA axis and also represents a key point in stress, pain response [21], and emotional processes [22]. Dysfunction of local hippocampal GABAergic inhibitory interneuron networks has been shown in many psychiatric illnesses, including anxiety, depression, and posttraumatic stress disorder [23]. Among these interneurons, parvalbumin-positive (PV+) interneurons are highly represented in the hippocampus [21] and provide inhibitory inputs that regulate the synaptic excitation and control the timing of information flow [23]. Recent studies revealed decreased hippocampal PV+ interneurons in chronic stress [24], chronic pain [21], human psychiatric diseases [23], and oxidative stress-driven neuroinflammation [25]. These findings indicate their potential role in anxiety, as well as their vulnerability to chronic stress-evoked signalling and altered redox status. However, the influence of CP/CPPS on hippocampal PV+ interneurons is still unknown, as well as its contribution to CP/CPPS-related bran comorbidities.

Taking into account current considerations, we hypothesized that CP/CPPS is linked to alterations in anxiety-like behavior concomitantly with imbalances in the brain oxidative stress, serum corticosterone, and hippocampal PV+ interneurons. This relationship has not been previously experimentally investigated to the best of our knowledge. Therefore, the objectives of this study were to assess the influence of experimental CP/CPPS on rat anxiety-like behavior, as well as brain oxidative stress level, serum corticosterone level, and the number of hippocampal PV+ interneurons.

2. Materials and Methods

2.1. Ethical Statement

All experimental procedures were in full compliance with the Directive of the European Parliament and the Council (2010/63/EU) and approved by The Ethical Committee of the University of Belgrade (Permission No. 323-07-01339/2017-05/3).

2.2. Animals and Housing

Adult (three-month-old, weighted 250–350 g, total $n=12$) male Wistar albino rats, obtained from the Military Medical Academy breeding laboratory (Belgrade, Serbia), were used in the experiments. The animals were housed in transparent plexiglass cages ($55\times 35\times 30$ cm) with soft bedding and access to food and water ad libitum during the entire experiment. They were kept under controlled ambient conditions (22-24°C, $50\pm 5\%$ relative humidity, 12/12 h light : dark cycle with the light turned on from 08:00 to 20:00 h). Animals were used only once during the experiment. Acclimatization period to the laboratory ambient lasted for 7 days.

2.3. Experimental Design and Test Protocol

Based on our previous experiments and literature data [11], rats were randomly divided into control, sham-operated (sham, $n=6$), and experimental groups with CP/CPPS (CP/CPPS, $n=6$) on the day of the intraprostatic injection (denoted as day 0 of the experiment). In the experimental group, CP/CPPS was induced by single intraprostatic injection of 3% λ-carrageenan (mucopolysaccharide from the cell walls of the red algae). To assess the development of CP/CPPS, we evaluated mechanical pain thresholds in the scrotal skin using an electronic von Frey aesthesiometer (evF). Three days before intraprostatic injection, rats were adapted to the evF aesthesiometer. Mechanical pain thresholds were determined at different time points, 2 days and 1 day before intraprostatic injection, as well as 2, 3, and 7 days upon intraprostatic injection. The animals from both groups ($n=6$ per group) were subjected to ethological tests to assess the anxiety-related behavior at different time points: 2, 3, and 7 days upon intraprostatic injection.

The standard battery of ethological tests consisted of an open field (OF), elevated plus maze (EPM), and light/dark (L/D) test to which the rats were subjected consecutively (in the following order: OF, EPM, and L/D). The same cohort of CP/CPPS and sham animals that underwent ethological testings ($n=6$ per group) was sacrificed on the 7^th day of the experiment to perform biochemical, histological, and immunohistochemical examinations. It allowed us to correlate experimental output variables from behavioral, biochemical, and immunohistochemical analysis. Serums were collected for corticosterone level determination. Brain structures were isolated for the determination of oxidative stress parameters, as well as the number of hippocampal PV+ interneurons. Prostates were collected for histological examination.

The time course of the experiment is presented schematically in Figure 1.

[figure omitted; refer to PDF]

Further quantitative analysis of anxiety-related output variables 2^nd day postoperatively showed no differences between sham and CP/CPPS groups in any of the analyzed parameters ($p>0.05$, Figures 4(d)–4(f)). On the other hand, differences were revealed between rats who underwent intraprostatic injection of 3% λ-carrageenan (CP/CPPS group) and those who underwent intraprostatic injection of 0.9% saline (sham group) on the 3^rd as well as 7^th day upon the surgery. According to one-way ANOVA with the Tukey-Kramer LSD post hoc test, the number of rearings, an indicator of vertical activity, was significantly decreased in CP/CPPS rats compared to sham rats ($p<0.001$, Figure 4(d)) on the 3^rd and 7^th day upon the surgery. The index of thigmotaxis was significantly higher in CP/CPPS rats in comparison with sham rats on the 3^rd ($p<0.001$, Figure 4(e)) and 7^th day ($p<0.01$, Figure 4(e)) upon the surgery. Also, CP/CPPS rats spent significantly less time in the central area of the open field test compared to the sham rats ($p<0.01$, Figure 4(f)), at the same postoperative days. Besides, there is a highly significant reduction ($p<0.001$) of the time in the central area of the OF test within the CP/CPPS rats on the 3^rd and 7^th postoperative day in comparison to the 2^nd postoperative day values (Figure 4(f)).

3.3.2. Elevated Plus Maze Test (EPM)

Schematic illustration of the EPM is shown in Figure 5(a). No differences were detected between CP/CPPS and sham animals in any of the analyzed parameters derived from EPM ($p>0.05$, Figures 5(b) and 5(c)) on the 2^nd day upon the surgery. However, intraprostatic injection of 3% λ-carrageenan (CP/CPPS group) significantly decreased the time animals spent in the open arms during the test compared to control protocol ($p<0.01$, Figure 5(c)) on the 3^rd as well as 7^th day upon the surgery. Also, there is a highly significant reduction of the time animals spent in the open arms during the test within the CP/CPPS rats on the 3^rd ($p<0.01$, Figure 5(c)) and 7^th day ($p<0.05$, Figure 5(c)) upon the surgery in comparison to values on the 2^nd postoperative day. Further, on the 3^rd as well as 7^th postoperative day, the number of open/closed arm transitions was significantly decreased in the CP/CPPS group compared to the sham group ($p<0.01$, Figure 5(b)). Also, there is a highly significant reduction of the number of open/closed arm transitions within the CP/CPPS rats on the 3^rd ($p<0.01$, Figure 5(b)) and 7^th postoperative day ($p<0.001$, Figure 5(b)) in comparison to basal values.

[figure omitted; refer to PDF]

There were strong negative correlations between the MDA level in the hippocampus and the time animal spent in the center of the OF ($r=-0.95$, $p<0.01$), the time animal spent in the open arms of EPM ($r=-0.87$, $p<0.05$), and the time animal spent in the light compartment of the L/D box ($r=-0.86$, $p<0.05$). There was no statistically significant correlation between the MDA level in the thalamus and any analyzed variable. The MDA level in the cortex significantly positively correlated with the corticosterone serum level ($r=0.94$, $p<0.01$), while it significantly negatively correlated with the time animal spent in the center of the OF ($r=-0.91$, $p<0.05$), the time animal spent in the open arms of EPM ($r=-0.96$, $p<0.01$), and the time animal spent in the light compartment of the L/D box ($r=-0.94$, $p<0.01$). The corticosterone serum level significantly negatively correlated with the time animal spent in the center of the OF ($r=-0.86$, $p<0.05$), the time animal spent in the open arms of EPM ($r=-0.86$, $p<0.05$), and the time animal spent in the light compartment of the L/D box ($r=-0.96$, $p<0.01$). Also, there were statistically significant correlations among parameters of three different anxiety-like behavior tests (Figure 10).

Also, simple regression analysis was performed to assess the link among the number of PV+ interneurons in CA1, CA2/3, and DG regions of the hippocampus and parameters of anxiety-like behavior testing in the CP/CPPS group. The results of these correlation analyses are presented in Figure 11. Simple regression analysis indicated that the number of PV+ interneurons in CA2/3 was positively correlated with time CP/CPPS animals spent in the open arms of the EPM ($r=0.835$, $p<0.05$) and time in the light compartment of the L/D test ($r=0.969$, $p<0.001$). Also, there was a strong positive correlation between the number of PV+ interneurons in DG and time these animals spent in the center of the OF test ($r=0.912$, $p<0.05$), time in the open arms of the EPM test ($r=0.950$, $p<0.01$), and time in the light compartment of the L/D test ($r=0.893$, $p<0.05$). There was no significant correlation for the CA1 region and test parameters of anxiety-like behavior (OF, EPM, and LD tests) in CP/CPPS rats ($r=0.664$, $p>0.05$; $r=0.348$, $p>0.05$; and $r=0.596$, $p>0.05$, respectively). The same hold was for the CA2/3 region and time in the center of the OF ($r=0.794$, $p>0.05$).

[figure omitted; refer to PDF]

4. Discussion

Results of our current study revealed increased anxiety-like behavior in rats with CP/CPPS comparing to corresponding controls, their sham-operated mates. These behavioral changes in CP/CPPS animals were accompanied by increased oxidative stress and alterations in antioxidative capacity in the hippocampus, thalamus, and cerebral cortex, increased serum corticosterone levels, and decreased number of PV+ interneurons in the hippocampus.

We confirmed in our current study the occurrence of CP/CPPS by a functional test, i.e., pain threshold determination, and by histological verification of prostatitis. Namely, NIH-Chronic Prostatitis Symptom Index (NIH-CPSI) showed that chronic pelvic pain and prostatodynia are the most steady and the most important symptoms in patients with CP/CPPS [33]. In our study, CP/CPPS animals showed a statistically significant reduction in mechanical pain threshold in the scrotal skin at 3 and 7 days time points when compared to the sham animals, which is in line with results given in previous studies [26, 27, 34]. Moreover, histological evaluation of prostates from CP/CPPS rats revealed typical pathohistological signs of prostatitis with interstitial leukocyte infiltration and necrosis, cell desquamation, and leukocyte infiltration of tubuloalveolar glands (Figure 3). These outcomes of functional testing and histological image secured face and construct validity of this experimental model, indicating that these time points could be used as a reference point of fully developed CP/CPPS.

4.1. Experimentally Induced CP/CPPS Is Accompanied by Anxiety-Like Behavior

Upon induction of CP/CPPS, we have assessed anxiety-like behavior in rats over one week (time points: 2, 3, and 7 days) using a standard battery of ethological tests, including OF, EPM, and L/D. The results of these behavioral tests revealed the appearance of anxiogenic behavior 3 and 7 days upon intraprostatic λ-carrageenan injection, while behavioral patterns were unaltered in the first 2 days upon prostatitis induction. Rats with CP/CPPS showed an increased anxiety-like pattern of behavior in the OF, manifested as decreased number of rearings (an indicator of vertical activity), higher index of thigmotaxis (showing its tendency to stick to arena’s walls), and lower time spent in the central area, compared to their sham mates (Figure 4). This specific behavioral pattern outlined by these output variables in the OF matches the generally accepted image of the anxiety-like behavior [35]. Also, the remaining two behavioral tests performed herein showed congruent results. Namely, we observed in the EPM decreased time that CP/CPPS animals spent in the open arms and decreased the number of open/closed arm transitions during test 3 and 7 days upon CP/CPPS induction (Figure 5). The EPM is a widely used ethological test for innate anxiety behavior of rodents [36], relying on approach-avoidance behavior easily translational to humans [37]. It is based on rodents’ tendency toward dark, enclosed arms (approach) and an unconditioned fear of heights/open arms (avoidance) [38]. In L/D, shorter time spent in the light compartment and decreased number of L/D compartment transitions were scored in CP/CPPS animals in the same time points (Figure 6). The L/D is a widely applied ethological test of unconditioned anxiety-like behavior in rodents, based on conflict between the drive to explore novel areas and an aversion to brightly lit and open areas [39].

Our results are in line with findings from clinical studies indicating the link between CP/CPPS and anxiety, i.e., a higher prevalence of anxiety in men with CP/CPPS [40, 41]. Hence, we confirmed the hypothesis that experimentally induced CP/CPPS is accompanied by anxiety as one of the neuropsychiatric comorbidities, permitting the exploration of the possible mechanism underpinning this link in experimental conditions. Oxidative stress, as an imbalance between ROS production and antioxidative capacities, is one of these possible mechanisms.

4.2. Brain Oxidative Stress Could Be the Underlying Mediator of CP/CPPS Link with Anxiety-Like Behavior

Oxidative stress is considered to be a significant factor in the inflammatory cascade of chronic prostatitis development [19, 42]. In the process of any chronic pelvic pain-related syndrome, a large amount of ROS was generated and released into the bloodstream [43, 44]. Moreover, extensive inflammation present in chronic prostatitis leads to stromal or epithelial cell damage [45], thus leading to the intense release of ROS, causing changes in protein structure and function, and DNA modifications [46]. Products of lipid peroxidation, such as MDA and unsaturated aldehydes, are capable of inactivating many cellular proteins. Our results showed that animals with CP/CPPS had significantly increased MDA levels, an indicator of lipid peroxidation, in all examined brain structures, i.e., the hippocampus, thalamus, and cerebral cortex (Figure 7). This suggests a higher oxidative burden in the brains of animals with CP/CPPS. The extent of lipid peroxidation in these structures was almost uniform, indicating the strong trigger of oxidative stress. The brain is very susceptible to oxidative stress damage since it generates more free radicals per gram of tissue than does any other organ, as well as it contains high amount of polyunsaturated fatty acids, high oxygen consumption, and a low antioxidant capacity, compared with other organs [47]. The antioxidant enzymes, including SOD and GPx, as well as content of thiol groups, are among the most efficient mechanisms operating in the brain to tackle the threat posed by ROS [48]. On the other hand, in the current study, there was a significant decrease of SOD activity and thiol group content in all three isolated brain structures in CP/CPPS animals, suggesting significant loss of antioxidants which eventually resulted in the development of oxidative stress. However, CP/CPPS animals had increased activity of GPx enzyme in the thalamus and cortex but not in hippocampal structures, compared to sham animals. We can assume adaptive response in these structures, but still insufficient to cope with oxidative stress in the thalamus and cortex, as well as in the hippocampus.

Neurocircuitry models of anxiety disorders are highly complex and encompass different brain regions and circuits including amygdala-cortical interactions. Cortical regions are believed to be specifically involved in some anxiety disorders [49]. The oxidative stress in the brain could be an underpinning mechanism associated with vulnerability to anxiety disorders [50]. Over the past few years, several studies in rodents reported that anxiety disorders may be characterized by increased oxidative damage to proteins, lipids, and nucleic acids, as well as lowered antioxidant defence [51]. Accumulated oxidative stress could damage neurons and alter neuronal function, resulting in the pathogenesis of anxiety [52]. Also, a link between decreased SOD activity and anxiety was reported [53]. Our results showed that increased anxiety-like behavioral pattern correlated with increased lipid peroxidation in the cortex and hippocampus in CP/CPPS rats. Recently, Bouayed and Soulimani [54] provided direct evidence that hydrogen peroxide, a component of oxidative stress, induces high-anxiety-related behavior in rodents. Further evidence about the role of oxidative stress in the genesis of anxiety has been discussed in more detail elsewhere [51, 55]. Therefore, in our research, we support the hypothesis that oxidative stress in the brain could be the underlying mediator of CP/CPPS link with anxiety-like behavior confirmed herein.

4.3. Corticosterone Could Mediate CP/CPPS-Evoked Anxiety-Like Behavior

One of the main features of CP/CPPS, besides prostate inflammation, is chronic pain, and we observed herein marked reduction in pain threshold in CP/CPPS rats. Pain itself is associated with stress and could provoke stress reaction [56]. However, stress, in turn, can be an important causative factor for CP/CPPS and anxiety, independently [9]. Some studies found that high-level stress and lack of social support were associated with a history of prostatitis in men [57].

Results of our study revealed elevated serum corticosterone levels in CP/CPPS rats and its correlation with increased anxiety-like behavior patterns among these rats. This result speaks in favour of increased activity of the HPA axis (CRH-ACTH-corticosterone) and elevated stress level, as well as immune system reactions on stress in CP/CPPS rats. Corticosterone crosses the blood-brain barrier and affects neurons and glial cells producing changes in certain brain regions, such as the prefrontal cortex, amygdala, hippocampus, nucleus accumbens, and hypothalamus [58]. More specifically, corticosterone acts via glucocorticoid and mineralocorticoid receptors in the central nucleus of the amygdala and mediates the HPA and autonomic components of the stress axis [59]. Also, when corticosterone binds at the amygdala, there is an increase in CRH release and the subsequent facilitation of the stress axis since corticosterone might directly affect amygdaloid neurons [60]. Hence, the limbic brain and HPA axis form an interconnected loop as projections from the hippocampus, amygdala, and prefrontal cortex feedback to the hypothalamus and regulate the stress responses and glucocorticoid release [61], together with induction of long-lasting anxiety-like behavior [62]. Recently, Bergamini et al. [63] showed that peripheral pain hypersensitivity increases anxiety by elevating corticosterone serum levels in rats.

Moreover, the decrease in SOD activity in all brain structures described in our study may be due to the release of corticosterone in the response to pain as a stressor. Namely, an earlier study showed that incubation of cortical and hippocampal structures with glucocorticoids resulted in increased ROS accumulation and oxidative stress [64]. We found that elevated serum corticosterone levels positively correlated with lipid peroxidation and oxidative stress in the cortex, thalamus, and hippocampus. It seems that corticosterone, by increasing the availability of glucose and through the Nrf2 signalling pathway, promotes spontaneous ROS generation, increases prooxidant gene transcription, and decreases antioxidant defence mechanisms [65]. Also, excessive ROS production leads to dysregulation of inflammatory response and degradation of NF-κB inhibitors [66]. Therefore, our current results support the hypothesis that activation of the HPA-corticosterone axis could be another underlying mediator of CP/CPPS link with anxiety-like behavior together with oxidative stress.

4.4. Hippocampal PV+ Interneuron Reduction in CPPS-Evoked Anxiety-Like Behavior

Numerous studies suggested an important role of the hippocampus and its alterations in anxiety initiation and development [67–69]. Anxiety is the result of competition between simultaneously available goals or choices that could be associated with conflict or uncertainty [70]. Different hippocampal neural circuits are considered to be the main part of a comparator system to detect it [71]. The hippocampus is organized in the stereotyped anatomical trisynaptic circuit [72]. Cortical inputs from the entorhinal cortex carry spatial and contextual information, synapsing onto dentate gyrus (DG) granule cells and area CA3 pyramidal neurons, which in turn project to area CA2 and CA1 pyramidal neurons that send extrahippocampal projections [73]. Highly interconnected reciprocal circuit, composed of extrahippocampal projections, amygdala, medial prefrontal cortex, hypothalamus, and the bed nucleus of the stria terminals [74] can elicit anxiety-related behavior via direct outputs to brainstem structures like the periaqueductal grey and parabrachial nucleus [75]. Hippocampal PV+ interneurons, predominantly chandelier (or axo-axonic) cells, and a subset of basket-type interneurons [24] are involved in the regulation of cognitive function, circadian rhythms, and behavioral patterns including anxiety, social interaction, and fear extinction [67]. These interneurons provide inhibition in the cortex and hippocampus by control of the memory-related network activity pattern generation which is involved in the obtaining and extinction of fear memories [76]. The main suppressive effect of hippocampal PV+ interneurons on hippocampal CA1, CA 2/3, and DG neuronal activity is mediated mainly by GABAergic neurotransmission [77].

Results of our study showed decreased number of PV+ interneurons in CA1, CA2/3, and DG regions of the hippocampus in CP/CPPS rats. Moreover, this decreased number of hippocampal PV+ interneurons in CA2/3 and DG regions correlated with increased anxiety-like behavioral patterns during OF, EPM, and L/D tests in CP/CPPS rats. The simple and unconditioned ethological laboratory tests of anxiety are based on the conflict between the approach and exploration of the potential danger, but also potential reward (open arm in EPM, the center of the OFT, and light compartment of the L/D test), or avoidance in the safe and enclosed compartments (closed arm in EPM, the periphery of the OFT, and dark compartment of the L/D test) [71]. Taking into account these facts, it could explain why the impaired hippocampal PV+ interneuronal network caused anxiety-like behavior, which even correlated with the degree of PV+ interneuron loss.

There are many speculated mechanisms implicated in alterations of hippocampal plasticity in CP/CPPS rats. Impaired redox status in the brain is observed to be responsible for many immunohistochemical changes in the hippocampus [78]. Namely, redox dysregulation impairs glutamatergic, dopaminergic, immune, and antioxidant signalling in the brain [25], as well as NADPH oxygenase 2 regulation [79], which is causally connected to PV+ interneuron loss. Additionally, corticosterone could generate a considerable reduction in the hippocampal PV+ interneuron expression. Exposure to chronic stress and recruitment of HPA axis with elevated serum level of corticosterone may lead to dendritic retraction without cell death thus compromising hippocampal function [80]. The hippocampus acts as a negative feedback control to the HPA axis by downregulation of CRH production in the hypothalamus [81]. The loss of hippocampal-negative feedback regulation of the HPA axis may be associated with elevated serum corticosterone levels in CP/CPPS rats, which could in turn induce hippocampal changes. Therefore, loss of hippocampal PV+ interneurons is responsible for further HPA axis hyperactivity in the positive-feedback loop manner [82], making CP/CPPS animals more vulnerable to chronic-stress-induced anxiety-like behavioral alterations. Moreover, chronic pain could induce hippocampal maladaptive plastic changes by decreasing hippocampal dendritic complexity and reducing the number of PV+ interneurons [21]. Therefore, our findings indicated the involvement of hippocampal PV+ interneuron reduction in CP/CPPS link with anxiety-like behavior.

4.5. The Interplay between Brain Oxidative Stress, Serum Corticosterone, and Hippocampal PV+ Interneurons in CP/CPPS-Evoked Anxiety-Like Behavior

Anxiety presents primarily a response to the potential danger and could be a protective mechanism to fall into potentially dangerous situations [83], which is used as a base of ethological tests to assess anxiety-like behavior in laboratory animals. Herein, there were strong positive correlations among parameters of three different anxiety-like behavior tests (OF, EPM, and L/D) used in this study, all indicating increased anxiety-like behavior in CP/CPPS rats. On the other hand, we have demonstrated that increased anxiety-like behavior significantly correlated with increased lipid peroxidation in the cortex and hippocampus in CP/CPPS rats measured as the MDA level in these brain structures. Furthermore, elevated serum corticosterone level positively correlated with indicators of anxiety-like behavior but also with lipid peroxidation in the cortex in CP/CPPS rats. We also demonstrated that there was a strong correlation between increased anxiety-like behavior and number of hippocampal PV+ interneurons in CA2/3 and DG regions in CP/CPPS rats. Namely, a lower number of PV+ interneurons in CA2/3 was accompanied by less time CP/CPPS animals spent in the open arms of the EPM and less time in the light compartment of the L/D test. Similar situation existed for PV+ interneurons in DG. Hence, an interplay between brain oxidative stress, hyperactivity of HPA axis seen as elevated serum corticosterone, and loss of hippocampal PV+ interneurons exist in CP/CPPS-evoked anxiety-like behavioral patterns with possible involvement of circulus vitiosus mechanisms.

5. Conclusions

In summary, our current study showed increased anxiety-like behavior in rats with CP/CPPS which strongly correlated with increased oxidative stress in the brain, elevated corticosterone serum level, and hippocampal PV+ interneurons loss. These mechanisms could be potential targets for the amelioration of psychiatric comorbidities in CP/CPPS. Also, we cannot exclude other possible mechanisms of anxiety-like behavior in CP/CPPS, which remains to be elucidated in further studies.

Acknowledgments

This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number 175032.

References

[1] J. M. Potts, "Chronic pelvic pain syndrome: a non-prostatocentric perspective," World Journal of Urology, vol. 21 no. 2, pp. 54-56, DOI: 10.1007/s00345-003-0327-2, 2003.

[2] J. N. Krieger, S. W. H. Lee, J. Jeon, P. Y. Cheah, M. L. Liong, D. E. Riley, "Epidemiology of prostatitis," International Journal of Antimicrobial Agents, vol. 31, pp. S85-S90, DOI: 10.1016/j.ijantimicag.2007.08.028, 2008.

[3] M. B. Passavanti, V. Pota, P. Sansone, C. Aurilio, L. De Nardis, M. C. Pace, "Chronic pelvic pain: assessment, evaluation, and objectivation," Pain Research and Treatment, vol. 2017,DOI: 10.1155/2017/9472925, 2017.

[4] W. Fu, Z. Zhou, S. Liu, Q. Li, J. Yao, W. Li, J. Yan, "The effect of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) on semen parameters in human males: a systematic review and meta-analysis," PLoS One, vol. 9 no. 4, article e94991,DOI: 10.1371/journal.pone.0094991, 2014.

[5] G. X. Zhang, W. J. Bai, T. Xu, X. F. Wang, "A preliminary evaluation of the psychometric profiles in Chinese men with chronic prostatitis/chronic pelvic pain syndrome," Chinese Medical Journal, vol. 124 no. 4, pp. 514-518, 2011.

[6] R. U. Anderson, E. K. Orenberg, A. Morey, N. Chavez, C. A. Chan, "Stress induced hypothalamus-pituitary-adrenal axis responses and disturbances in psychological profiles in men with chronic prostatitis/chronic pelvic pain syndrome," The Journal of Urology, vol. 182 no. 5, pp. 2319-2324, DOI: 10.1016/j.juro.2009.07.042, 2009.

[7] J. L. Rhudy, "The importance of emotional processes in the modulation of pain," Pain, vol. 146 no. 3, pp. 233-234, DOI: 10.1016/j.pain.2009.07.003, 2009.

[8] C. Brünahl, C. Dybowski, R. Albrecht, B. Riegel, J. Höink, M. Fisch, B. Löwe, "Mental disorders in patients with chronic pelvic pain syndrome (CPPS)," Journal of Psychosomatic Research, vol. 98, pp. 19-26, DOI: 10.1016/j.jpsychores.2017.04.011, 2017.

[9] S.-D. Chung, H.-C. Lin, "Association between chronic prostatitis/chronic pelvic pain syndrome and anxiety disorder: a population-based study," PLoS One, vol. 8 no. 5,DOI: 10.1371/journal.pone.0064630, 2013.

[10] C. Hu, H. Yang, Y. Zhao, X. Chen, Y. Dong, L. Li, Y. Dong, J. Cui, T. Zhu, P. Zheng, C.-S. Lin, J. Dai, "The role of inflammatory cytokines and ERK1/2 signaling in chronic prostatitis/chronic pelvic pain syndrome with related mental health disorders," Scientific Reports, vol. 6 no. 1, article 28608,DOI: 10.1038/srep28608, 2016.

[11] N. Šutulović, Ž. Grubač, S. Šuvakov, Đ. Jovanović, N. Puškaš, Đ. Macut, A. R. Marković, T. Simić, O. Stanojlović, D. Hrnčić, "Chronic prostatitis/chronic pelvic pain syndrome increases susceptibility to seizures in rats and alters brain levels of IL-1 β and IL-6," Epilepsy Research, vol. 153, pp. 19-27, DOI: 10.1016/j.eplepsyres.2019.03.014, 2019.

[12] A. C. Strauss, J. D. Dimitrakov, "New treatments for chronic prostatitis/chronic pelvic pain syndrome," Nature Reviews. Urology, vol. 7 no. 3, pp. 127-135, DOI: 10.1038/nrurol.2010.4, 2010.

[13] J. N. Krieger, S. Takahashi, D. E. Riley, "Chronic prostatitis: role of uncommon organisms," European Urology Supplements, vol. 2 no. 2, pp. 19-22, DOI: 10.1016/S1569-9056(02)00195-1, 2003.

[14] M. L. Breser, F. C. Salazar, V. E. Rivero, R. D. Motrich, "Immunological mechanisms underlying chronic pelvic pain and prostate inflammation in chronic pelvic pain syndrome," Frontiers in Immunology, vol. 8,DOI: 10.3389/fimmu.2017.00898, 2017.

[15] R. L. Silva, A. H. Lopes, R. M. Guimarães, T. M. Cunha, "CXCL1/CXCR2 signaling in pathological pain: role in peripheral and central sensitization," Neurobiology of Disease, vol. 105, pp. 109-116, DOI: 10.1016/j.nbd.2017.06.001, 2017.

[16] F. Aoun, A. K. Chemaly, S. Albisinni, M. Zanaty, T. Roumeguere, "In search for a common pathway for health issues in men - the sign of a Holmesian deduction," Asian Pacific Journal of Cancer Prevention, vol. 17 no. 1,DOI: 10.7314/APJCP.2016.17.1.1, 2016.

[17] Y. Hu, X. Niu, G. Wang, J. Huang, M. Liu, B. Peng, "Chronic prostatitis/chronic pelvic pain syndrome impairs erectile function through increased endothelial dysfunction, oxidative stress, apoptosis, and corporal fibrosis in a rat model," Andrology, vol. 4 no. 6, pp. 1209-1216, DOI: 10.1111/andr.12273, 2016.

[18] T. Roumeguère, J. Sfeir, E. El Rassy, S. Albisinni, P. van Antwerpen, K. Zouaoui Boudjeltia, N. Farès, J. Kattan, F. Aoun, "Oxidative stress and prostatic diseases," Molecular and Clinical Oncology, vol. 7 no. 5, pp. 723-728, DOI: 10.3892/mco.2017.1413, 2017.

[19] A. U. Ihsan, F. U. Khan, P. Khongorzul, K. A. Ahmad, M. Naveed, S. Yasmeen, Y. Cao, A. Taleb, R. Maiti, F. Akhter, X. Liao, X. Li, Y. Cheng, H. U. Khan, K. Alam, X. Zhou, "Role of oxidative stress in pathology of chronic prostatitis/chronic pelvic pain syndrome and male infertility and antioxidants function in ameliorating oxidative stress," Biomedicine & Pharmacotherapy, vol. 106, pp. 714-723, DOI: 10.1016/j.biopha.2018.06.139, 2018.

[20] F. F. Pasqualotto, R. K. Sharma, J. M. Potts, D. R. Nelson, A. J. Thomas, A. Agarwal, "Seminal oxidative stress in patients with chronic prostatitis," Urology, vol. 55 no. 6, pp. 881-885, DOI: 10.1016/S0090-4295(99)00613-5, 2000.

[21] M. Tajerian, V. Hung, H. Nguyen, G. Lee, L.-M. Joubert, A. V. Malkovskiy, B. Zou, S. Xie, T.-T. Huang, J. D. Clark, "The hippocampal extracellular matrix regulates pain and memory after injury," Molecular Psychiatry, vol. 23 no. 12, pp. 2302-2313, DOI: 10.1038/s41380-018-0209-z, 2018.

[22] I. Zaletel, D. Filipović, N. Puškaš, "Chronic stress, hippocampus and parvalbumin-positive interneurons: what do we know so far?," Reviews in the Neurosciences, vol. 27 no. 4, pp. 397-409, DOI: 10.1515/revneuro-2015-0042, 2016.

[23] K. A. Pelkey, R. Chittajallu, M. T. Craig, L. Tricoire, J. C. Wester, C. J. McBain, "Hippocampal GABAergic inhibitory interneurons," Physiological Reviews, vol. 97 no. 4, pp. 1619-1747, DOI: 10.1152/physrev.00007.2017, 2017.

[24] B. Czeh, M. Simon, M. G. van der Hart, B. Schmelting, M. B. Hesselink, E. Fuchs, "Chronic stress decreases the number of parvalbumin-immunoreactive interneurons in the hippocampus: prevention by treatment with a substance P receptor (NK1) antagonist," Neuropsychopharmacology, vol. 30 no. 1, pp. 67-79, DOI: 10.1038/sj.npp.1300581, 2005.

[25] P. Steullet, J.-H. Cabungcal, J. Coyle, M. Didriksen, K. Gill, A. A. Grace, T. K. Hensch, A.-S. LaMantia, L. Lindemann, T. M. Maynard, U. Meyer, H. Morishita, P. O’Donnell, M. Puhl, M. Cuenod, K. Q. Do, "Oxidative stress-driven parvalbumin interneuron impairment as a common mechanism in models of schizophrenia," Molecular Psychiatry, vol. 22 no. 7, pp. 936-943, DOI: 10.1038/mp.2017.47, 2017.

[26] R. Radhakrishnan, R. S. Nallu, "Development and characterisation of a novel animal model of prostate inflammation-induced chronic pelvic pain," Inflammopharmacology, vol. 17 no. 1, pp. 23-28, DOI: 10.1007/s10787-008-8051-6, 2009.

[27] F. Zeng, H. Chen, J. Yang, L. Wang, Y. Cui, X. Guan, Z. Wang, J. Niu, X. Zu, L. Qi, X. Zhang, Z. Tang, L. Liu, "Development and validation of an animal model of prostate inflammation-induced chronic pelvic pain: evaluating from inflammation of the prostate to pain behavioral modifications," PLoS One, vol. 9 no. 5, article e96824,DOI: 10.1371/journal.pone.0096824, 2014.

[28] D. Hrncic, J. Mikić, A. Rasic-Markovic, M. Velimirović, T. Stojković, R. Obrenović, B. Rankov-Petrović, V. Šušić, D. Djuric, N. Petronijević, O. Stanojlovic, "Anxiety-related behavior in hyperhomocysteinemia induced by methionine nutritional overload in rats: role of the brain oxidative stress," Canadian Journal of Physiology and Pharmacology, vol. 94 no. 10, pp. 1074-1082, DOI: 10.1139/cjpp-2015-0581, 2016.

[29] J. C. Dousset, M. Trouilh, M. J. Foglietti, "Plasma malonaldehyde levels during myocardial infarction," Clinica Chimica Acta, vol. 129 no. 3, pp. 319-322, DOI: 10.1016/0009-8981(83)90035-9, 1983.

[30] H. Misra, I. Fridovich, "The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase," The Journal of Biological Chemistry, vol. 247 no. 10, pp. 3170-3175, DOI: 10.1016/S0021-9258(19)45228-9, 1972.

[31] W. A. Günzler, H. Vergin, I. Müller, L. Flohé, "Glutathione peroxidase VI: the reaction of glutahione peroxidase with various hydroperoxides," Hoppe-Seyler's Zeitschrift für Physiologische Chemie, vol. 353 no. 6, pp. 1001-1004, 1972.

[32] P. C. Jocelyn, "Spectrophotometric assay of thiols," Methods in Enzymology, vol. 143, pp. 44-67, DOI: 10.1016/0076-6879(87)43013-9, 1987.

[33] J. N. Krieger, L. Nyberg, J. C. Nickel, "NIH consensus definition and classification of prostatitis," Journal of the American Medical Association, vol. 282 no. 3, pp. 236-237, DOI: 10.1001/jama.282.3.236, 1999.

[34] C.-S. Chen, P.-J. Chang, W.-Y. Lin, Y.-C. Huang, D.-R. Ho, "Evidences of the inflammasome pathway in chronic prostatitis and chronic pelvic pain syndrome in an animal model," The Prostate, vol. 73 no. 4, pp. 391-397, DOI: 10.1002/pros.22580, 2013.

[35] L. Prut, C. Belzung, "The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review," European Journal of Pharmacology, vol. 463 no. 1–3,DOI: 10.1016/S0014-2999(03)01272-X, 2003.

[36] S. H. Albani, M. M. Andrawis, R. J. H. Abella, J. T. Fulghum, N. Vafamand, T. C. Dumas, "Behavior in the elevated plus maze is differentially affected by testing conditions in rats under and over three weeks of age," Frontiers in Behavioral Neuroscience, vol. 9,DOI: 10.3389/fnbeh.2015.00031, 2015.

[37] S. V. Biedermann, D. G. Biedermann, F. Wenzlaff, T. Kurjak, S. Nouri, M. K. Auer, K. Wiedemann, P. Briken, J. Haaker, T. B. Lonsdorf, J. Fuss, "An elevated plus-maze in mixed reality for studying human anxiety-related behavior," BMC Biology, vol. 15 no. 1,DOI: 10.1186/s12915-017-0463-6, 2017.

[38] A. A. Walf, C. A. Frye, "The use of the elevated plus maze as an assay of anxiety-related behavior in rodents," Nature Protocols, vol. 2 no. 2, pp. 322-328, DOI: 10.1038/nprot.2007.44, 2007.

[39] A. E. Arrant, N. L. Schramm-Sapyta, C. M. Kuhn, "Use of the light/dark test for anxiety in adult and adolescent male rats," Behavioural Brain Research, vol. 256, pp. 119-127, DOI: 10.1016/j.bbr.2013.05.035, 2013.

[40] J. Q. Clemens, R. T. Meenan, M. C. O’Keeffe Rosetti, T. Kimes, E. A. Calhoun, "Prevalence of and risk factors for prostatitis: population based assessment using physician assigned diagnoses," The Journal of Urology, vol. 178 no. 4, pp. 1333-1337, DOI: 10.1016/j.juro.2007.05.140, 2007.

[41] M. A. Pontari, M. McNaughton-Collins, M. P. O’leary, E. A. Calhoun, T. Jang, J. W. Kusek, J. R. Landis, J. Knauss, M. S. Litwin, CPCRN Study Group, "A case-control study of risk factors in men with chronic pelvic pain syndrome," BJU International, vol. 96 no. 4, pp. 559-565, DOI: 10.1111/j.1464-410X.2005.05684.x, 2005.

[42] A. S. Polackwich, D. A. Shoskes, "Chronic prostatitis/chronic pelvic pain syndrome: a review of evaluation and therapy," Prostate Cancer and Prostatic Diseases, vol. 19 no. 2, pp. 132-138, DOI: 10.1038/pcan.2016.8, 2016.

[43] W. Dröge, "Free radicals in the physiological control of cell function," Physiological Reviews, vol. 82 no. 1, pp. 47-95, DOI: 10.1152/physrev.00018.2001, 2002.

[44] J.-Y. Wang, Y.-L. Wang, S.-L. Zhou, J.-F. Zhou, "May chronic childhood constipation cause oxidative stress and potential free radical damage to children?," Biomedical and Environmental Sciences, vol. 17 no. 3, pp. 266-272, 2004.

[45] A. M. De Marzo, E. A. Platz, S. Sutcliffe, J. Xu, H. Grönberg, C. G. Drake, Y. Nakai, W. B. Isaacs, W. G. Nelson, "Inflammation in prostate carcinogenesis," Nature Reviews Cancer, vol. 7 no. 4, pp. 256-269, DOI: 10.1038/nrc2090, 2007.

[46] R. Olinski, D. Gackowski, M. Foksinski, R. Rozalski, K. Roszkowski, P. Jaruga, "Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome," Free Radical Biology & Medicine, vol. 33 no. 2, pp. 192-200, DOI: 10.1016/S0891-5849(02)00878-X, 2002.

[47] I. Hovatta, J. Juhila, J. Donner, "Oxidative stress in anxiety and comorbid disorders," Neuroscience Research, vol. 68 no. 4, pp. 261-275, DOI: 10.1016/j.neures.2010.08.007, 2010.

[48] R. Kohen, A. Nyska, "Invited review: oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification," Toxicologic Pathology, vol. 30 no. 6, pp. 620-650, DOI: 10.1080/01926230290166724, 2016.

[49] M. R. Milad, S. L. Rauch, "The role of the orbitofrontal cortex in anxiety disorders," Annals of the New York Academy of Sciences, vol. 1121 no. 1, pp. 546-561, DOI: 10.1196/annals.1401.006, 2007.

[50] A. C. Ercan, B. Bahceci, S. Polat, O. C. Cenker, I. Bahceci, A. Koroglu, K. Sahin, C. Hocaoglu, "Oxidative status and prolidase activities in generalized anxiety disorder," Asian Journal of Psychiatry, vol. 25, pp. 118-122, DOI: 10.1016/j.ajp.2016.10.008, 2017.

[51] A. G. Fedoce, F. Ferreira, R. G. Bota, V. Bonet-Costa, P. Y. Sun, K. J. A. Davies, "The role of oxidative stress in anxiety disorder: cause or consequence?," Free Radical Research, vol. 52 no. 7, pp. 737-750, DOI: 10.1080/10715762.2018.1475733, 2018.

[52] I. Smaga, E. Niedzielska, M. Gawlik, A. Moniczewski, J. Krzek, E. Przegaliński, J. Pera, M. Filip, "Oxidative stress as an etiological factor and a potential treatment target of psychiatric disorders. Part 2. Depression, anxiety, schizophrenia and autism," Pharmacological Reports, vol. 67 no. 3, pp. 569-580, DOI: 10.1016/j.pharep.2014.12.015, 2015.

[53] H. Rammal, J. Bouayed, C. Younos, R. Soulimani, "Evidence that oxidative stress is linked to anxiety-related behaviour in mice," Brain, Behavior, and Immunity, vol. 22 no. 8, pp. 1156-1159, DOI: 10.1016/j.bbi.2008.06.005, 2008.

[54] J. Bouayed, R. Soulimani, "Evidence that hydrogen peroxide, a component of oxidative stress, induces high-anxiety-related behaviour in mice," Behavioural Brain Research, vol. 359, pp. 292-297, DOI: 10.1016/j.bbr.2018.11.009, 2019.

[55] W. Hassan, C. Barroso Silva, I. U. Mohammadzai, J. Teixeira da Rocha, J. Landeira-Fernandez, "Association of oxidative stress to the genesis of anxiety: implications for possible therapeutic interventions," Current Neuropharmacology, vol. 12 no. 2, pp. 120-139, DOI: 10.2174/1570159X11666131120232135, 2014.

[56] C. G. Abdallah, P. Geha, "Chronic pain and chronic stress: two sides of the same coin?," Chronic Stress, vol. 1, article 247054701770476,DOI: 10.1177/2470547017704763, 2017.

[57] L. P. Wallner, J. Q. Clemens, A. V. Sarma, "Prevalence of and risk factors for prostatitis in African American men: The Flint Men's Health Study," The Prostate, vol. 69 no. 1, pp. 24-32, DOI: 10.1002/pros.20846, 2009.

[58] B. S. McEwen, "Stress, sex, and neural adaptation to a changing environment: mechanisms of neuronal remodeling," Annals of the New York Academy of Sciences, vol. 1204, pp. E38-E59, DOI: 10.1111/j.1749-6632.2010.05568.x, 2010.

[59] B. Greenwood-Van Meerveld, A. C. Johnson, "Mechanisms of stress-induced visceral pain," Journal of Neurogastroenterology and Motility, vol. 24 no. 1,DOI: 10.5056/jnm17137, 2018.

[60] R. Mitra, R. M. Sapolsky, "Acute corticosterone treatment is sufficient to induce anxiety and amygdaloid dendritic hypertrophy," Proceedings of the National Academy of Sciences of the United States of America, vol. 105 no. 14, pp. 5573-5578, DOI: 10.1073/pnas.0705615105, 2008.

[61] S. J. Lupien, B. S. McEwen, M. R. Gunnar, C. Heim, "Effects of stress throughout the lifespan on the brain, behaviour and cognition," Nature Reviews Neuroscience, vol. 10 no. 6, pp. 434-445, DOI: 10.1038/nrn2639, 2009.

[62] A. Vyas, R. Mitra, B. S. Shankaranarayana Rao, S. Chattarji, "Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons," Journal of Neuroscience, vol. 22 no. 15, pp. 6810-6818, DOI: 10.1523/JNEUROSCI.22-15-06810.2002, 2002.

[63] M. R. Bergamini, M. M. Bernardi, I. B. Sufredini, M. T. Ciaramicoli, R. M. Kodama, F. Kabadayan, C. H. C. Saraceni, "Dentin hypersensitivity induces anxiety and increases corticosterone serum levels in rats," Life Sciences, vol. 98 no. 2, pp. 96-102, DOI: 10.1016/j.lfs.2014.01.004, 2014.

[64] C. Behl, F. Lezoualc’h, T. Trapp, M. Widmann, T. Skutella, F. Holsboer, "Glucocorticoids enhance oxidative stress-induced cell death in hippocampal neurons in vitro," Endocrinology, vol. 138 no. 1, pp. 101-106, DOI: 10.1210/endo.138.1.4835, 1997.

[65] S. H. Ki, I. J. Cho, D. W. Choi, S. G. Kim, "Glucocorticoid receptor (GR)-associated SMRT binding to C/EBP β TAD and Nrf2 Neh4/5: role of SMRT recruited to GR in GSTA2 gene repression," Molecular and Cellular Biology, vol. 25 no. 10, pp. 4150-4165, DOI: 10.1128/MCB.25.10.4150-4165.2005, 2005.

[66] H. Solleiro-Villavicencio, S. Rivas-Arancibia, "Effect of chronic oxidative stress on neuroinflammatory response mediated by CD4+T cells in neurodegenerative diseases," Frontiers in Cellular Neuroscience, vol. 12,DOI: 10.3389/fncel.2018.00114, 2018.

[67] B. Rankov Petrovic, D. Hrncic, D. Mladenovic, T. Simic, S. Suvakov, D. Jovanovic, N. Puskas, I. Zaletel, M. Velimirovic, V. Cirkovic, D. Macut, O. Stanojlovic, A. Rasic-Markovic, "Prenatal androgenization induces anxiety-like behavior in female rats, associated with reduction of inhibitory interneurons and increased BDNF in hippocampus and cortex," BioMed Research International, vol. 2019,DOI: 10.1155/2019/3426092, 2019.

[68] V. Rytova, D. E. Ganella, D. Hawkes, R. A. D. Bathgate, S. Ma, A. L. Gundlach, "Chronic activation of the relaxin-3 receptor on GABA neurons in rat ventral hippocampus promotes anxiety and social avoidance," Hippocampus, vol. 29 no. 10, pp. 905-920, DOI: 10.1002/hipo.23089, 2019.

[69] Y.-L. Wang, Q.-Q. Han, W.-Q. Gong, D.-H. Pan, L.-Z. Wang, W. Hu, M. Yang, B. Li, J. Yu, Q. Liu, "Microglial activation mediates chronic mild stress-induced depressive- and anxiety-like behavior in adult rats," Journal of Neuroinflammation, vol. 15 no. 1,DOI: 10.1186/s12974-018-1054-3, 2018.

[70] C. Barkus, S. B. McHugh, R. Sprengel, P. H. Seeburg, J. N. P. Rawlins, D. M. Bannerman, "Hippocampal NMDA receptors and anxiety: at the interface between cognition and emotion," European Journal of Pharmacology, vol. 626 no. 1, pp. 49-56, DOI: 10.1016/j.ejphar.2009.10.014, 2010.

[71] D. M. Bannerman, R. Sprengel, D. J. Sanderson, S. B. McHugh, J. N. P. Rawlins, H. Monyer, P. H. Seeburg, "Hippocampal synaptic plasticity, spatial memory and anxiety," Nature Reviews Neuroscience, vol. 15 no. 3, pp. 181-192, DOI: 10.1038/nrn3677, 2014.

[72] F. W. Stahnisch, R. Nitsch, "Santiago Ramón y Cajal’s concept of neuronal plasticity: the ambiguity lives on," Trends in Neurosciences, vol. 25 no. 11, pp. 589-591, DOI: 10.1016/S0166-2236(02)02251-8, 2002.

[73] J. C. Jimenez, J. E. Berry, S. C. Lim, S. K. Ong, M. A. Kheirbek, R. Hen, "Contextual fear memory retrieval by correlated ensembles of ventral CA1 neurons," Nature Communications, vol. 11 no. 1, article 3492,DOI: 10.1038/s41467-020-17270-w, 2020.

[74] G. G. Calhoon, K. M. Tye, "Resolving the neural circuits of anxiety," Nature Neuroscience, vol. 18 no. 10, pp. 1394-1404, DOI: 10.1038/nn.4101, 2015.

[75] E. R. Duval, A. Javanbakht, I. Liberzon, "Neural circuits in anxiety and stress disorders: a focused review," Therapeutics and Clinical Risk Management, vol. 11, pp. 115-126, DOI: 10.2147/TCRM.S48528, 2015.

[76] G. Çalışkan, I. Müller, M. Semtner, A. Winkelmann, A. S. Raza, J. O. Hollnagel, A. Rösler, U. Heinemann, O. Stork, J. C. Meier, "Identification of parvalbumin interneurons as cellular substrate of fear memory persistence," Cerebral Cortex, vol. 26 no. 5, pp. 2325-2340, DOI: 10.1093/cercor/bhw001, 2016.

[77] D. Zou, L. Chen, D. Deng, D. Jiang, F. Dong, C. McSweeney, Y. Zhou, L. Liu, G. Chen, Y. Wu, Y. Mao, "DREADD in parvalbumin interneurons of the dentate gyrus modulates anxiety, social interaction and memory extinction," Current Molecular Medicine, vol. 16 no. 1, pp. 91-102, DOI: 10.2174/1566524016666151222150024, 2016.

[78] J. H. Harkness, P. N. Bushana, R. P. Todd, W. C. Clegern, B. A. Sorg, J. P. Wisor, "Sleep disruption elevates oxidative stress in parvalbumin-positive cells of the rat cerebral cortex," Sleep, vol. 42 no. 1,DOI: 10.1093/sleep/zsy201, 2019.

[79] A. C. Rossetti, M. S. Paladini, M. Colombo, P. Gruca, M. Lason-Tyburkiewicz, K. Tota-Glowczyk, M. Papp, M. A. Riva, R. Molteni, "Chronic stress exposure reduces parvalbumin expression in the rat hippocampus through an imbalance of redox mechanisms: restorative effect of the antipsychotic lurasidone," The International Journal of Neuropsychopharmacology, vol. 21 no. 9, pp. 883-893, DOI: 10.1093/ijnp/pyy046, 2018.

[80] C. D. Conrad, "Chronic stress-induced hippocampal vulnerability: the glucocorticoid vulnerability hypothesis," Reviews in the Neurosciences, vol. 19 no. 6, pp. 395-411, DOI: 10.1515/revneuro.2008.19.6.395, 2008.

[81] S. R. Bornstein, W. C. Engeland, M. Ehrhart-Bornstein, J. P. Herman, "Dissociation of ACTH and glucocorticoids," Trends in Endocrinology & Metabolism, vol. 19 no. 5, pp. 175-180, DOI: 10.1016/j.tem.2008.01.009, 2008.

[82] S. K. Godavarthi, P. Dey, M. Maheshwari, N. R. Jana, "Defective glucocorticoid hormone receptor signaling leads to increased stress and anxiety in a mouse model of Angelman syndrome," Human Molecular Genetics, vol. 21 no. 8, pp. 1824-1834, DOI: 10.1093/hmg/ddr614, 2012.

[83] J. A. Gray, N. MacNaughton, The Neuropsychology of Anxiety: An Enquiry into the Functions of the Septo-Hippocampal System,DOI: 10.1093/acprof:oso/9780198522713.001.0001, 2003.