Combustion-Derived Pollutants Linked with Kidney Disease in Low-Lying Flood-Affected Areas in the Balkans.

Tens of thousands of people in southern Europe suffer from Balkan endemic nephropathy (BEN), and four times as many are at risk. Incidental ingestion of aristolochic acids (AAs), stemming from the ubiquitousAristolochia clematitis(birthwort) weed in the region, leads to DNA adduct-induced toxicity in kidney cells, the primary cause of BEN. Numerous cofactors, including toxic organics and metals, have been investigated, but all have shown small contributions to the overall BEN relative to non-BEN village distribution gradients. Here, we reveal that combustion-derived pollutants from wood and coal burning in Serbia also contaminate arable soil and test as plausible causative factors of BEN. Using a GC-MS screening method, biomass-burning-derived furfural and coal-burning-derived medium-chain alkanes were detected in soil samples from BEN endemic areas levels at up to 63-times and 14-times higher, respectively, than in nonendemic areas. Significantly higher amounts were also detected in colocated wheat grains. Coexposure studies with cultured kidney cells showed that these pollutants enhance DNA adduct formation by AA, - the cause of AA nephrotoxicity and carcinogenicity. With the coincidence of birthwort-derived AAs and the widespread practice of biomass and coal burning for household cooking and heating purposes and agricultural burning in rural low-lying flood-affected areas in the Balkans, these results implicate combustion-derived pollutants in promoting the development of BEN.

Complex Hippocampal Response to Thermal Skin Injury and Protocols with Hyperbaric Oxygen Therapy and Filipendula ulmaria Extract in Rats.

The aim of this study was to evaluate the alterations of the hippocampal function that may be related to anxiogenic response to thermal skin injury, including the morpho-functional alterations, and the effects of hyperbaric oxygen (HBO) and Filipendula ulmaria (FU) extract in the treatment of anxiety-like behavior that coincides with thermal skin injury. A rat thermal skin injury experimental model was performed on 2-month-old male Wistar albino rats. The evaluated therapeutic protocols included HBO and/or antioxidant supplementation. HBO was applied for 7 days in the hyperbaric chamber (100% O2, 2.5 ATA, 60 min). Oral administration of FU extract (final concentration of 100 mg/kg b.w.) to achieve antioxidant supplementation was also applied for 7 days. Anxiety level was estimated in the open field and elevated plus-maze test, which was followed by anesthesia, sacrifice, and collection of hippocampal tissue samples. HBO treatment and FU supplementation significantly abolished anxiogenic response to thermal skin injury. This beneficial effect was accompanied by the reduction in hippocampal pro-inflammatory and pro-apoptotic indicators, and enhanced BDNF and GABA-ARα2S gene expression, previously observed in untreated burns. The hippocampal relative gene expression of melatonin receptors and NPY positively responded to the applied protocols, in the same manner as µ and δ opioid receptors, while the opposite response was observed for κ receptors. The results of this study provide some confirmations that adjuvant strategies, such as HBO and antioxidant supplementation, may be simultaneously applied in the treatment of the anxiety-like behavior that coincides with thermal skin injury.

Its all about IFN-λ4: Protective role of IFNL4 polymorphism against COVID-19-related pneumonia in females.

Despite the pivotal role of IFN-λs in the innate immune response, the data on its genetic polymorphism in relation to COVID-19 severity are scarce and contradictory. In the present study, we aimed to determine if the presence of the most frequent functional single nucleotide polymorphisms (SNPs) of the two most important IFN-λs coding genes, namely IFNL3 and IFNL4, alters the likelihood of SARS-CoV-2-infected patients to develop more severe form of the disease. This observational cohort study involved 178 COVID-19 patients hospitalized at the University Clinical Centre Kragujevac, Serbia. Patients' demographics, clinical characteristics, and laboratory parameters were collected at admission. COVID-19 signs and symptoms were assessed during the hospital stay, with the worst condition determining the disease severity. Genotyping for IFNL3 (rs12980275 and rs8099917) and IFNL4 (rs12979860 and rs368234815) SNPs was conducted using TaqMan assays. Our study revealed carriers of IFNL3 and IFNL4 minor alleles to be less likely to progress from mild to moderate COVID-19, that is, to develop COVID-19-related pneumonia. After adjustment for other factors of influence, such as age, sex, and comorbidities, the likelihood of pneumonia development remained significantly associated with IFNL4 polymorphism (odds ratios [ORs] [95% confidence interval (95% CI)]: 0.233 [0.071; 0.761]). When the patients were stratified according to sex, the protective role of IFNL4 minor alleles, controlled for the effect of comorbidities, remained significant only in females (OR [95% CI]: 0.035 [0.003; 0.408]). Our results strongly suggest that IFNL4 rs12979860 and rs368234815 polymorphisms independently predict the risk of COVID-19-related pneumonia development in females.

Simultaneous Administration of Hyperbaric Oxygen Therapy and Antioxidant Supplementation with Filipendula ulmaria Extract in the Treatment of Thermal Skin Injuries Alters Nociceptive Signalling and Wound Healing.

Background and Objectives: Thermal skin injuries are a prevalent cause of skin damage, potentially leading to severe morbidity and significant mortality. In this study, we intended to estimate the effects of HBO (hyperbaric oxygen treatment) and antioxidant supplementation with Filipendula ulmaria extract, individually and simultaneously, in the treatment of thermal skin injuries. Materials and Methods: As a thermal skin injury experimental model, we used two-month-old male Wistar albino rats. Thermal injuries were made with a solid aluminium bar at a constant temperature of 75 °C for 15 s. Hyperbaric oxygen treatment was performed in a specially constructed hyperbaric chamber for rats (HYB-C 300) for seven consecutive days (100% O2 at 2.5 ATA for 60 min). Antioxidant supplementation was performed with oral administration of Filipendula ulmaria extract dissolved in tap water to reach a final concentration of 100 mg/kg b.w. for seven consecutive days. Results: Simultaneous administration of hyperbaric oxygen therapy and antioxidant supplementation with Filipendula ulmaria extract significantly ameliorated the macroscopic and histopathological characteristics of the wound area and healing. Also, this therapeutic approach decreased the local expression of genes for proinflammatory mediators and increased the expression of the µ-opioid receptor and the MT1 and MT2 receptors in the wound area and spinal cord, with a consequent increase in reaction times in behavioural testing. Conclusions: In conclusion, the presented results of our study allow evidence for the advantages of the simultaneous employment of HBO and antioxidant supplementation in the treatment of thermal skin injuries, with special reference to the attenuation of painful sensations accompanied by this type of trauma.

Oral Health and Oral Health-Related Quality of Life in Patients with Primary Sjögren's Syndrome.

Background and Objectives: Oral disorders, frequently observed in patients with primary Sjögren's syndrome, can profoundly affect patients' daily lives and well-being, as oral health represents a fundamental part of general health. Saliva plays an essential part in maintaining and protecting oral health, so the decrease in its quantity and quality leads to chronic oral discomfort alongside a broad spectrum of problems. The objective of the present study was to evaluate the oral health of patients with primary Sjögren's syndrome and establish its effect on the different domains of their oral health-related quality of life (OHRQoL). Materials and Methods: The research was designed as an observational case-control study with prospective data collection. Eighty patients, divided into two groups based on their oral status, participated in the study. All subjects underwent a complete oral examination. The OHRQoL was assessed using the Oral Health Impact Profile-14 (OHIP-14). Results: The most prevalent oral manifestation was exfoliative cheilitis, while 30% of subjects complained of chewing and swallowing difficulties. The OHIP-14 summary score was significantly higher in the patients with oral lesions (26.0 (5.0) vs. 17.0 (4.0), respectively; p < 0.001). Oral manifestations, systemic involvement, medication, and periodontal indexes were significantly associated with OHIP-14 scores. Conclusions: Patients with oral alterations had a substantially decreased OHRQoL. These findings emphasize the importance of oral diseases for patients' well-being. Therefore, it is essential for dentists to be included in the multidisciplinary teams managing primary Sjögren's syndrome, as improving patients' oral status would lead to better oral health and enhanced OHRQoL.

IFNL3/4 polymorphisms as a two-edged sword: An association with COVID-19 outcome.

Coronavirus Disease 2019 (COVID-19) has been ranked among the most fatal infectious diseases worldwide, with host's immune response significantly affecting the prognosis. With an aim to timely predict the most likely outcome of SARS-CoV-2 infection, we investigated the association of IFNL3 and IFNL4 polymorphisms, as well as other potentially relevant factors, with the COVID-19 mortality. This prospective observational case-control study involved 178 COVID-19 patients, hospitalized at Corona Center or Clinic for Infectious Diseases of University Clinical Centre Kragujevac, Serbia, followed up until hospital discharge or in-hospital death. Demographic and clinical data on all participants were retrieved from the electronic medical records, and TaqMan assays were employed in genotyping for IFNL3 and IFNL4 single nucleotide polymorphisms (SNPs), namely rs12980275, rs8099917, rs12979860, and rs368234815. 21.9% and 65.0% of hospitalized and critically ill COVID-19 patients, respectively, died in-hospital. Multivariable logistic regression analysis revealed increased Charlson Comorbidity Index (CCI), N/L, and lactate dehydrogenase (LDH) level to be associated with an increased likelihood of a lethal outcome. Similarly, females and the carriers of at least one variant allele of IFNL3 rs8099917 were almost 36-fold more likely not to survive SARS-CoV-2 infection. On the other hand, the presence of at least one ancestral allele of IFNL4 rs368234815 decreased more than 15-fold the likelihood of mortality from COVID-19. Our results suggest that, in addition to LDH level, N/L ratio, and CCI, IFNL4 rs368234815 and IFNL3 rs8099917 polymorphisms, but also patients' gender, significantly affect the outcome of COVID-19.

Criteria for the Classification of the Interradicular Septum Shape in Maxillary Molars with Clinical Importance for Prosthetic-Driven Immediate Implant Placement.

The aim of this study was to use cone-beam computed tomography (CBCT) to evaluate the morphometric properties of the interradicular septum (IRS) in the maxillary molar region that may be indicative for prosthetic-driven implant placement. Following the repetitive algorithm based on the visual identification of IRS shapes, we described the following IRS shapes: arrow, boat, drop, and palatal and buccal convergence. The incidence of IRS shapes showed significant differences for the first and second maxillary molars (the highest frequency for the arrow shape, and the lowest for the drop shape) with no significant difference between the molars. The most prominent width indicative for implant placement was observed in the palatal convergence shape, whereas the height criteria were the most satisfying in the buccal convergence-shaped IRS for both molars. Apart from the parameters in the coronal view, the image analysis in the axial view revealed that IRS surface area, required for the implant placement, was the most prominent in the palatal convergence shape for the first, and boat shape for the second molars. Our results showed the benefits of CBCT diagnostics in posterior maxilla morphometric analysis. IRS shape classification may be helpful in achieving more rapid and accurate planning for interventions in this region.

Aristolochic acid I as an emerging biogenic contaminant involved in chronic kidney diseases: A comprehensive review on exposure pathways, environmental health issues and future challenges.

Described in the 1950s, Balkan Endemic Nephropathy (BEN) has been recognized as a chronic kidney disease (CKD) with clinical peculiarities and multiple etiological factors. Environmental contaminants - aromatic compounds, mycotoxins and phytotoxins like aristolochic acids (AAs) - polluting food and drinking water sources, were incriminated in BEN, due to their nephrotoxic and carcinogenic properties. The implication of AAs in BEN etiology is currently a highly debated topic due to the fact that they are found within the Aristolochiaceae plants family, used around the globe as traditional medicine and they were also incriminated in Aristolochic Acid Nephropathy (AAN). Exposure pathways have been investigated, but it is unclear to what extent AAs are acting alone or in synergy with other cofactors (environmental, genetics) in triggering kidney damage. Experimental studies strengthen the hypothesis that AAI, the most studied compound in the AAs class, is a significant environmental contaminant and a most important causative factor of BEN. The aim of this review is to compile information about the natural exposure pathways to AAI, via traditional medicinal plants, soil, crop plants, water, food, air. Data that either supports or contradicts the AAI theory concerning BEN etiology was consolidated and available solutions to reduce human exposure were discussed. Because AAI is a phytotoxin with physicochemical properties that allow its transportation in environmental matrices from different types of areas (endemic, nonendemic), and induce CKDs (BEN, AAN) and urinary cancers through bioaccumulation, this review aims to shed a new light on this compound as a biogenic emerging pollutant.

Epidemiological Predictive Modeling of COVID-19 Infection: Development, Testing, and Implementation on the Population of the Benelux Union.

Since the outbreak of coronavirus disease-2019 (COVID-19), the whole world has taken interest in the mechanisms of its spread and development. Mathematical models have been valuable instruments for the study of the spread and control of infectious diseases. For that purpose, we propose a two-way approach in modeling COVID-19 spread: a susceptible, exposed, infected, recovered, deceased (SEIRD) model based on differential equations and a long short-term memory (LSTM) deep learning model. The SEIRD model is a compartmental epidemiological model with included components: susceptible, exposed, infected, recovered, deceased. In the case of the SEIRD model, official statistical data available online for countries of Belgium, Netherlands, and Luxembourg (Benelux) in the period of March 15 2020 to March 15 2021 were used. Based on them, we have calculated key parameters and forward them to the epidemiological model, which will predict the number of infected, deceased, and recovered people. Results show that the SEIRD model is able to accurately predict several peaks for all the three countries of interest, with very small root mean square error (RMSE), except for the mild cases (maximum RMSE was 240.79 ± 90.556), which can be explained by the fact that no official data were available for mild cases, but this number was derived from other statistics. On the other hand, LSTM represents a special kind of recurrent neural network structure that can comparatively learn long-term temporal dependencies. Results show that LSTM is capable of predicting several peaks based on the position of previous peaks with low values of RMSE. Higher values of RMSE are observed in the number of infected cases in Belgium (RMSE was 535.93) and Netherlands (RMSE was 434.28), and are expected because of thousands of people getting infected per day in those countries. In future studies, we will extend the models to include mobility information, variants of concern, as well as a medical intervention, etc. A prognostic model could help us predict epidemic peaks. In that way, we could react in a timely manner by introducing new or tightening existing measures before the health system is overloaded.

Artificial intelligence approach towards assessment of condition of COVID-19 patients - Identification of predictive biomarkers associated with severity of clinical condition and disease progression.

BACKGROUND AND OBJECTIVES: Although ML has been studied for different epidemiological and clinical issues as well as for survival prediction of COVID-19, there is a noticeable shortage of literature dealing with ML usage in prediction of disease severity changes through the course of the disease. In that way, predicting disease progression from mild towards moderate, severe and critical condition, would help not only to respond in a timely manner to prevent lethal results, but also to minimize the number of patients in hospitals where this is not necessary. METHODS: We present a methodology for the classification of patients into 4 distinct categories of the clinical condition of COVID-19 disease. Classification of patients is based on the values of blood biomarkers that were assessed by Gradient boosting regressor and which were selected as biomarkers that have the greatest influence in the classification of patients with COVID-19. RESULTS: The results show that among several tested algorithms, XGBoost classifier achieved best results with an average accuracy of 94% and an average F1-score of 94.3%. We have also extracted 10 best features from blood analysis that are strongly associated with patient condition and based on those features we can predict the severity of the clinical condition. CONCLUSIONS: The main advantage of our system is that it is a decision tree-based algorithm which is easier to interpret, instead of the use of black box models, which are not appealing in medical practice.

Morphological and Morphometric Characteristics of Anterior Maxilla Accessory Canals and Relationship with Nasopalatine Canal Type-A CBCT Study.

This study aimed to evaluate principal morphological and morphometric characteristics of accessory canals (ACs) of the anterior maxilla, as well as to analyze the relationship with nasopalatine canal (NPC) type. The results of our study showed that ACs were observed in almost 50% of participants. They were mostly presented bilaterally and in a curved shape, with a palatal foramen position. The morphometric characteristics of ACs were significantly influenced by NPC type. NPC type had the strongest impact on the distance between the NPC and AC, as well as on the distance between the AC and the facial aspect of buccal bone wall, in inferior parts of the alveolar ridge. On the other hand, the distance between the AC and central incisors was not significantly influenced by NPC shape in the lower region of the anterior maxilla. However, the participants with the banana-type of the NPC expressed the reduction in distance from the AC to the central incisor at the upper part in comparison with the subjects with the cylindrical-type of the NPC. On the basis of the results of this study, the simultaneous estimation of ACs and the NPC seems reasonable, as this approach may be useful in the prevention of complications which could occur during implant surgery interventions.

Morphological Characteristics of the Nasopalatine Canal and the Relationship with the Anterior Maxillary Bone-A Cone Beam Computed Tomography Study.

The aim of this study was to evaluate the interconnection between the type of nasopalatine canal (NPC) and morphometric characteristics of the anterior maxilla. The investigation involved 113 subjects, and the morphometric parameters were obtained using cone beam computed tomography (CBCT). NPC shapes were classified into four types: banana-, hourglass-, cylindrical-, and funnel-shaped (distribution of approximately 9, 25, 31, and 35%, respectively). The analysis revealed that the NPC shape was significantly connected with the morphometric properties of anterior maxilla horizontal dimensions. In general, a banana-shaped NPC was accompanied with an overall reduction in anterior maxilla diameters when compared to other NPC shapes, with no significant difference between the other three NPC shapes. Furthermore, the morphometric characteristics that depend on NPC shape at the sagittal cross-section were significantly correlated with diameters of the incisive foramen, nasal foramen, and NPC length. According to the results of our study, it seems that the presented analyses of morphometric data may allow useful insight into the algorithms of various interconnections between the measures obtained in the region of the anterior maxilla, which could be of interest for a time rationale approach when planning some surgical procedures, such as immediate dental implant placement planning.

Analysis of Polycyclic Aromatic Hydrocarbons and Phthalate Esters in Soil and Food Grains from the Balkan Peninsula: Implication on DNA Adduct Formation by Aristolochic Acid I and Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy affecting residents of rural farming areas in many Balkan countries. Although it is generally believed that BEN is an environmental disease caused by multiple geochemical factors with much attention on aristolochic acids (AAs), its etiology remains controversial. In this study, we tested the hypothesis that environmental contamination and subsequent food contamination by polycyclic aromatic hydrocarbons (PAHs) and phthalate esters are AA toxicity factors and important to BEN development. We identified significantly higher concentrations of phenanthrene, anthracene, diethyl phthalate (DEP), dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP) in both maize and wheat grain samples collected from endemic villages than from nonendemic villages. Other PAHs and phthalate esters were also detected at higher concentrations in the soil samples from endemic villages. Subsequent genotoxicity testing of cultured human kidney cells showed an alarming phenomenon that phenanthrene, DEP, BBP, and DBP can interact synergistically with AAs to form elevated levels of AA-DNA adducts, which are associated with both the nephrotoxicity and carcinogenicity of AAs, further increasing their disease risks. This study provides direct evidence that prolonged coexposure to these environmental contaminants via dietary intake may lead to greater toxicity and accelerated development of BEN.

SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets.

Clinical manifestations of SARS-CoV-2 infection range from mild to critically severe. The aim of the study was to highlight the immunological events associated with the severity of SARS-CoV-2 infection, with an emphasis on cells of innate immunity. Thirty COVID-19 patients with mild/moderate symptoms and 27 patients with severe/critically severe symptoms were recruited from the Clinical Center of Kragujevac during April 2020. Flow cytometric analysis was performed to reveal phenotypic and functional alterations of peripheral blood cells and to correlate them with the severity of the disease. In severe cases, the number of T and B lymphocytes, dendritic cells, NK cells, and HLA-DR-expressing cells was drastically decreased. In the monocyte population proportion between certain subsets was disturbed and cells coexpressing markers of M1 and M2 monocytes were found in intermediate and non-classical subsets. In mild cases decline in lymphocyte number was less pronounced and innate immunity was preserved as indicated by an increased number of myeloid and activated dendritic cells, NK cells that expressed activation marker at the same level as in control and by low expression of M2 marker in monocyte population. In patients with severe disease, both innate and adoptive immunity are devastated, while in patients with mild symptoms decline in lymphocyte number is lesser, and the innate immunity is preserved.

Cigarette smoking and heavy coffee consumption affecting response to olanzapine: The role of genetic polymorphism.

Objectives: To evaluate the effect of cigarette smoking and heavy coffee consumption on efficacy and safety of olanzapine treatment in schizophrenia patients, in relation to genetic polymorphism.Methods: The study involved 120 patients with schizophrenia, treated with olanzapine for 30 days. Therapy efficacy was determined using three different psychiatric scales, and safety by assessing metabolic adverse effects and extrapyramidal symptoms. Genotyping included CYP1A2*1C, CYP1A2*1F and CYP1A1/1A2 intergenic polymorphism, as well as CYP2D6*3, CYP2D6*4 and CYP2D6*6.Results: Cigarette smoking and heavy coffee consumption decreased the efficacy and increased the safety of olanzapine treatment (P < 0.001). Although the effect was detected only in carriers of CYP1A2*1F allele, covariate analysis revealed that it is independent of CYP1A2 genotype. Olanzapine dose was inversely correlated with the drug efficacy (P ≤ 0.002) and LDL level (P = 0.004). Women and older subjects responded better to therapy (P < 0.026), but had more certain adverse effects (P ≤ 0.049). When controlling for other relevant factors, CYP2D6 metabolizer status affects olanzapine efficacy (P = 0.032).Conclusions: We confirm the effect of cigarette smoking and heavy coffee consumption on olanzapine efficacy and safety. The relevance of CYP1A2 genotype for the described effect needs further investigation. Olanzapine treatment outcome is also affected by dose, sex, age and CYP2D6 metabolizer status.

Contemporary surgical management of drug-resistant focal epilepsy.

Introduction: Seizures, which could not be controlled by drug therapy, have profound negative influence on the quality of life of the affected person. If with clear locus of origin and accompanied by loss of consciousness, drug-resistant epilepsy could be treated by surgery.Areas covered: The aim of this article was to review current status of epilepsy surgery through description of the most important operative methods and narrative comparison of their benefits and harms. In total 1154 articles were retrieved from MEDLINE, SCOPUS, EBSCO, and SCINDEKS databases, and 78 included in the review. The review included systematic reviews, meta-analyses, clinical trials, observational studies on humans, case series, and case reports.Expert opinion: Sophisticated diagnostic methods nowadays offer much more precise localization of epileptogenic focus and detailed planning of a surgical procedure which will make minimal damage of neural pathways and structures essential for movements, speech, cognition, and emotions. Advent of perioperative care, and improved diagnostics and surgical techniques resulted with significant drop in rates of postoperative complications, long-term neurological deficit, and mortality in the last decade, while seizure freedom rate and quality of life increased.

Optimal threshold of the prostate health index in predicting aggressive prostate cancer using predefined cost-benefit ratios and prevalence.

PURPOSES: The aim of the study was to determine optimal threshold of the Prostate Health Index (Phi) for predicting aggressive prostate cancer (PCa), taking into account misclassification costs, prevalence, and plausible risk factors. METHODS: This prospective cohort study analyzed patients undergoing prostate biopsy and Phi testing. The primary endpoint was aggressive PCa, defined as biopsy Gleason score ≥ 7. The data about age, total prostate-specific antigen (PSA), percentage of free PSA (%fPSA), and digital rectal examination (DRE) were extracted from the patient files. We divided the patients to the low- and high-risk group. The clinical usefulness of the Phi was assessed by the decision curve analysis. The predictive performance was assessed using the area under the receiver operating characteristic curve (AUC), per-class metrics, and the potential reduction in unnecessary biopsies. The uncertain interval of Phi values was also determined. RESULTS: There were 200 men included in the study, 35 (17.5%) of them having aggressive PCa. Important predictors of aggressive PCa were %fPSA, DRE, Phi, and belonging to the high-risk group. With optimal threshold of 30.7, about 32% unnecessary biopsies would be avoided. The optimal threshold of Phi was lower in the high-risk group than in the low-risk group. The AUC for detection of aggressive PCa was 0.791. Per-class metrics showed that the Phi has insufficient diagnostic accuracy. The lower and upper limits of the uncertain interval were 41.8 and 51.4, respectively. CONCLUSION: Different thresholds of the Phi could be optimal, depending on prevalence, patient characteristics, and misclassification costs. Further studies with a larger patient sample are necessary to confirm our conclusions.

Exercise Attenuates Anabolic Steroids-Induced Anxiety via Hippocampal NPY and MC4 Receptor in Rats.

The aim of our study was to evaluate the effects of chronic administration of nandrolone-decanoate (ND) or testosterone-enanthate (TE) in supraphysiological doses and a prolonged swimming protocol, alone and in combination with ND or TE, on anxiety-like behavior in rats. We investigated the immunohistochemical alterations of the hippocampal neuropeptide Y (NPY) and melanocortin 4 receptor (MC4R) neurons, as a possible underlying mechanism in a modulation of anxiety-like behavior in rats. Both applied anabolic androgenic steroids (AASs) induced anxiogenic effect accompanied with decreased serum and hippocampal NPY. The exercise-induced anxiolytic effect was associated with increased hippocampal NPY expression. ND and TE increased the number of MC4R, while the swimming protocol was followed by the reduction of MC4R in the CA1 region of the hippocampus. However, NPY/MC4R ratio in hippocampus was lowered by AASs and elevated by exercise in all hippocampal regions. An augmentation of this ratio strongly and positively correlated to increased time in open arms of elevated plus maze, in the context that indicates anxiolytic effect. Our findings support the conclusion that alterations in both hippocampal NPY and MC4R expression are involved in anxiety level changes in rats, while their quantitative relationship (NPY/MC4R ratio) is even more valuable in the estimation of anxiety regulation than individual alterations for both NPY and MC4R expression in the hippocampus.