Exploring Fibrosis Pathophysiology in Lean and Obese Metabolic-Associated Fatty Liver Disease: An In-Depth Comparison.

While obesity-related nonalcoholic fatty liver disease (NAFLD) is linked with metabolic dysfunctions such as insulin resistance and adipose tissue inflammation, lean NAFLD more often progresses to liver fibrosis even in the absence of metabolic syndrome. This review aims to summarize the current knowledge regarding the mechanisms of liver fibrosis in lean NAFLD. The most commonly used lean NAFLD models include a methionine/choline-deficient (MCD) diet, a high-fat diet with carbon tetrachloride (CCl4), and a high-fructose and high-cholesterol diet. The major pro-fibrogenic mechanisms in lean NAFLD models include increased activation of the extracellular signal-regulated kinase (ERK) pathway, elevated expression of α-smooth muscle actin (α-SMA), collagen type I, and TGF-ß, and modulation of fibrogenic markers such as tenascin-X and metalloproteinase inhibitors. Additionally, activation of macrophage signaling pathways promoting hepatic stellate cell (HSC) activation further contributes to fibrosis development. Animal models cannot cover all clinical features that are evident in patients with lean or obese NAFLD, implicating the need for novel models, as well as for deeper comparisons of clinical and experimental studies. Having in mind the prevalence of fibrosis in lean NAFLD patients, by addressing specific pathways, clinical studies can reveal new targeted therapies along with novel biomarkers for early detection and enhancement of clinical management for lean NAFLD patients.

Dose-Dependent Induction of Differential Seizure Phenotypes by Pilocarpine in Rats: Considerations for Translational Potential.

Background and Objectives: Pilocarpine is used in experimental studies for testing antiepileptic drugs, but further characterization of this model is essential for its usage in testing novel drugs. The aim of our study was to study the behavioral and EEG characteristics of acute seizures caused by different doses of pilocarpine in rats. Materials and Methods: Male Wistar rats were treated with a single intraperitoneal dose of 100 mg/kg (P100), 200 mg/kg (P200), or 300 mg/kg (P300) of pilocarpine, and epileptiform behavior and EEG changes followed within 4 h. Results: The intensity and the duration of seizures were significantly higher in P300 vs. the P200 and P100 groups, with status epilepticus dominating in P300 and self-limiting tonic-clonic seizures in the P200 group. The seizure grade was significantly higher in P200 vs. the P100 group only during the first hour after pilocarpine application. The latency of seizures was significantly shorter in P300 and P200 compared with P100 group. Conclusions: Pilocarpine (200 mg/kg) can be used as a suitable model for the initial screening of potential anti-seizure medications, while at a dose of 300 mg/kg, it can be used for study of the mechanisms of epileptogenesis.

The Interconnection between Hepatic Insulin Resistance and Metabolic Dysfunction-Associated Steatotic Liver Disease-The Transition from an Adipocentric to Liver-Centric Approach.

The central mechanism involved in the pathogenesis of MAFLD is insulin resistance with hyperinsulinemia, which stimulates triglyceride synthesis and accumulation in the liver. On the other side, triglyceride and free fatty acid accumulation in hepatocytes promotes insulin resistance via oxidative stress, endoplasmic reticulum stress, lipotoxicity, and the increased secretion of hepatokines. Cytokines and adipokines cause insulin resistance, thus promoting lipolysis in adipose tissue and ectopic fat deposition in the muscles and liver. Free fatty acids along with cytokines and adipokines contribute to insulin resistance in the liver via the activation of numerous signaling pathways. The secretion of hepatokines, hormone-like proteins, primarily by hepatocytes is disturbed and impairs signaling pathways, causing metabolic dysregulation in the liver. ER stress and unfolded protein response play significant roles in insulin resistance aggravation through the activation of apoptosis, inflammatory response, and insulin signaling impairment mediated via IRE1/PERK/ATF6 signaling pathways and the upregulation of SREBP 1c. Circadian rhythm derangement and biological clock desynchronization are related to metabolic disorders, insulin resistance, and NAFLD, suggesting clock genes as a potential target for new therapeutic strategies. This review aims to summarize the mechanisms of hepatic insulin resistance involved in NAFLD development and progression.

Adipose-derived extracellular vesicles - a novel cross-talk mechanism in insulin resistance, non-alcoholic fatty liver disease, and polycystic ovary syndrome.

Obesity is the best described risk factor for the development of non-alcoholic fatty liver disease (NAFLD)/metabolic dysfunction associated steatotic liver disease (MASLD) and polycystic ovary syndrome (PCOS) while the major pathogenic mechanism linking these entities is insulin resistance (IR). IR is primarily caused by increased secretion of proinflammatory cytokines, adipokines, and lipids from visceral adipose tissue. Increased fatty acid mobilization results in ectopic fat deposition in the liver which causes endoplasmic reticulum stress, mitochondrial dysfunction, and oxidative stress resulting in increased cytokine production and subsequent inflammation. Similarly, IR with hyperinsulinemia cause hyperandrogenism, the hallmark of PCOS, and inflammation in the ovaries. Proinflammatory cytokines from both liver and ovaries aggravate IR thus providing a complex interaction between adipose tissue, liver, and ovaries in inducing metabolic abnormalities in obese subjects. Although many pathogenic mechanisms of IR, NAFLD/MASLD, and PCOS are known, there is still no effective therapy for these entities suggesting the need for further evaluation of their pathogenesis. Extracellular vesicles (EVs) represent a novel cross-talk mechanism between organs and include membrane-bound vesicles containing proteins, lipids, and nucleic acids that may change the phenotype and function of target cells. Adipose tissue releases EVs that promote IR, the development of all stages of NAFLD/MASLD and PCOS, while mesenchymal stem cell-derived AVs may alleviate metabolic abnormalities and may represent a novel therapeutic device in NAFLD/MASLD, and PCOS. The purpose of this review is to summarize the current knowledge on the role of adipose tissue-derived EVs in the pathogenesis of IR, NAFLD/MASLD, and PCOS.

Reduced light exposure mitigates streptozotocin-induced vascular changes and gliosis in diabetic retina by an anti-inflammatory effect and increased retinal cholesterol turnover.

Diabetic retinopathy is not cured efficiently and changes of lifestyle measures may delay early retinal injury in diabetes. The aim of our study was to investigate the effects of reduced daily light exposure on retinal vascular changes in streptozotocin (STZ)-induced model of DM with emphasis on inflammation, Aqp4 expression, visual cycle and cholesterol metabolism-related gene expression in rat retina and RPE. Male Wistar rats were divided into the following groups: 1. control; 2. diabetic group (DM) treated with streptozotocin (100 mg/kg); 3. group exposed to light/dark cycle 6/18 h (6/18); 4. diabetic group exposed to light/dark cycle 6/18 h (DM+6/18). Retinal vascular abnormalities were estimated based on lectin staining, while the expression of genes involved in the visual cycle, cholesterol metabolism, and inflammation was determined by qRT-PCR. Reduced light exposure alleviated vasculopathy, gliosis and the expression of IL-1 and TNF-α in the retina with increased perivascular Aqp4 expression. The expression of genes involved in visual cycle and cholesterol metabolism was significantly up-regulated in RPE in DM+6/18 vs. DM group. In the retina only the expression of APOE was significantly higher in DM+6/18 vs. DM group. Reduced light exposure mitigates vascular changes and gliosis in DM via its anti-inflammatory effect, increased retinal cholesterol turnover and perivascular Aqp4 expression.

Neuroactive steroids in the neuroendocrine control of food intake, metabolism, and reproduction.

Neuroactive steroids are a type of steroid hormones produced within the nervous system or in peripheral glands and then transported to the brain to exert their neuromodulatory effects. Neuroactive steroids have pleiotropic effects, that include promoting myelination, neuroplasticity, and brain development. They also regulate important physiological functions, such as metabolism, feeding, reproduction, and stress response. The homoeostatic processes of metabolism and reproduction are closely linked and mutually dependent. Reproductive events, such as pregnancy, bring about significant changes in metabolism, and metabolic status may affect reproductive function in mammals. In females, the regulation of reproduction and energy balance is controlled by the fluctuations of oestradiol and progesterone throughout the menstrual cycle. Neurosteroids play a key role in the neuroendocrine control of reproduction. The synthesis of neuroestradiol and neuroprogesterone within the brain is a crucial process that facilitates the release of GnRH and LH, which in turn, regulate the transition from oestrogen-negative to oestrogen-positive feedback. In addition to their function in the reproductive system, oestrogen has a key role in the regulation of energy homoeostasis by acting at central and peripheral levels. The oestrogenic effects on body weight homoeostasis are primarily mediated by oestrogen receptors-α (ERα), which are abundantly expressed in multiple brain regions that are implicated in the regulation of food intake, basal metabolism, thermogenesis, and brown tissue distribution. The tight interplay between energy balance and reproductive physiology is facilitated by shared regulatory pathways, namely POMC, NPY and kisspeptin neurons, which are targets of oestrogen regulation and likely participate in different aspects of the joint control of energy balance and reproductive function. The aim of this review is to present a summary of the progress made in uncovering shared regulatory pathways that facilitate the tight coupling between energy balance and reproductive physiology, as well as their reciprocal interactions and the modulation induced by neurosteroids.

Shortened Daily Photoperiod Alleviates Anxiety-like Behaviour by Antioxidant Effect and Changes Serum Fatty Acid Profile in Diabetic Rats.

The aim of our study was to investigate the effects of a shortened daily photoperiod on anxiety-like behaviour, brain oxidative stress, lipid status and fatty acid composition of serum lipids in a streptozotocin (STZ)-induced model of diabetes mellitus in rats. Male Wistar rats were divided into the following groups: first group-control group (C12/12); second group-diabetic group (DM12/12; 100 mg/kg STZ); third group-control group exposed to a light/dark cycle 6/18 h (C6/18); fourth group-diabetic group exposed to a light/dark cycle 6/18 h (DM6/18). Anxiety-like behaviour was tested three weeks following STZ injection by elevated plus maze (EPM) and open-field test (OFT). Oxidative stress parameters were measured in the cortex, hippocampus and thalamus, while lipid status and fatty acid methyl esters (FAMEs) were measured in the serum. Both EPM and OFT showed a lower degree of anxiety-like behaviour in the DM6/18 vs. DM12/12 group. Lipid peroxidation in the cortex, hippocampus and thalamus was significantly lower in the DM6/18 vs. DM12/12 group (p < 0.05), associated with an increased level of antioxidant enzymes and protein thiols in the cortex and thalamus. In the DM6/18 group, oleic, vaccenic, dihomo-γ-linolenic and docosahexaenoic acid concentrations were significantly higher in comparison to the DM12/12 group. A shortened daily photoperiod alleviates anxiety-like behaviour in diabetic rats by reduced lipid peroxidation and changes in the serum fatty acids profile.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome Induces Depression-Like Behavior and Learning-Memory Impairment: A Possible Link with Decreased Hippocampal Neurogenesis and Astrocyte Activation.

Pathogenesis of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) remains unclear since it represents an interplay between immunological, endocrine, and neuropsychiatric factors. Patients suffering from CP/CPPS often develop mental health-related disorders such as anxiety, depression, or cognitive impairment. The aim of this study was to investigate depression-like behavior, learning, and memory processes in a rat model of CP/CPPS and to determine the alterations in hippocampal structure and function. Adult male Wistar albino rats (n = 6 in each group) from CP/CPPS (single intraprostatic injection of 3% λ-carrageenan, day 0) and Sham (0.9% NaCl) groups were subjected to pain threshold test (days 2, 3, and 7), depression-like behavior, and learning-memory tests (both on day 7). Decreased pain threshold in the scrotal region and histopathological presence of necrosis and inflammatory infiltrate in prostatic tissue confirmed the development of CP/CPPS. The forced swimming test revealed the depression-like behavior evident through increased floating time, while the modified elevated plus maze test revealed learning and memory impairment through prolonged transfer latency in the CP/CPPS group in comparison with Sham (p < 0.001 and p < 0.001, respectively). Biochemical analysis showed decreased serum levels of testosterone in CP/CPPS group vs. the Sham (p < 0.001). The CP/CPPS induced a significant upregulation of ICAM-1 in rat cortex (p < 0.05) and thalamus (p < 0.01) and increased GFAP expression in the hippocampal astrocytes (p < 0.01) vs. Sham, suggesting subsequent neuroinflammation and astrocytosis. Moreover, a significantly decreased number of DCX+ and Ki67+ neurons in the hippocampus was observed in the CP/CPPS group (p < 0.05) vs. Sham, indicating decreased neurogenesis and neuronal proliferation. Taken together, our data indicates that CP/CPPS induces depression-like behavior and cognitive declines that are at least partly mediated by neuroinflammation and decreased neurogenesis accompanied by astrocyte activation.

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

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.

Experimental chronic sleep fragmentation alters seizure susceptibility and brain levels of interleukins 1ß and 6.

Brain hyperexcitability in sleep apnea is believed to be provoked by hypoxemia, but sleep fragmentation can also play a significant role. Sleep fragmentation can trigger inflammatory mechanisms. The aim of this research was to investigate the effects of chronic sleep fragmentation on seizure susceptibility and brain cytokine profile. Chronic sleep fragmentation in male rats with implanted EEG electrodes was achieved by the treadmill method. Rats were randomized to: treadmill control (TC); activity control (AC) and sleep fragmentation (SF) group. Convulsive behavior was assessed 14 days later by seizure incidence, latency time and seizure severity during 30 min following lindane administration. The number and duration of EEG ictal periods were determined. Levels of IL-1ß and IL-6 were measured in the animals' serum and brain structures (hippocampus, thalamus and cerebral cortex), in separate rat cohort that underwent the same fragmentation protocol except lindane administration. Incidence and severity of seizures were significantly increased, while latency was significantly decreased in SF+L compared with TC+L group. Seizure latency was also significantly decreased in SF+L compared to AC+L group. The number and duration of ictal periods were increased in the SF+L compared to the AC+L group. IL-1ß was significantly increased in the thalamus, cortex and hippocampus in the SF compared to the AC and TC groups. IL-6 was statistically higher only in the cortex of SF animals, while in the thalamic or hippocampal tissue, no difference was observed between the groups. It could be concluded that fourteen-day sleep fragmentation increases seizure susceptibility in rats and modulates brain production of IL-1ß and IL-6.Brain hyperexcitability in sleep apnea is believed to be provoked by hypoxemia, but sleep fragmentation can also play a significant role. Sleep fragmentation can trigger inflammatory mechanisms. The aim of this research was to investigate the effects of chronic sleep fragmentation on seizure susceptibility and brain cytokine profile. Chronic sleep fragmentation in male rats with implanted EEG electrodes was achieved by the treadmill method. Rats were randomized to: treadmill control (TC); activity control (AC) and sleep fragmentation (SF) group. Convulsive behavior was assessed 14 days later by seizure incidence, latency time and seizure severity during 30 min following lindane administration. The number and duration of EEG ictal periods were determined. Levels of IL-1ß and IL-6 were measured in the animals' serum and brain structures (hippocampus, thalamus and cerebral cortex), in separate rat cohort that underwent the same fragmentation protocol except lindane administration. Incidence and severity of seizures were significantly increased, while latency was significantly decreased in SF+L compared with TC+L group. Seizure latency was also significantly decreased in SF+L compared to AC+L group. The number and duration of ictal periods were increased in the SF+L compared to the AC+L group. IL-1ß was significantly increased in the thalamus, cortex and hippocampus in the SF compared to the AC and TC groups. IL-6 was statistically higher only in the cortex of SF animals, while in the thalamic or hippocampal tissue, no difference was observed between the groups. It could be concluded that fourteen-day sleep fragmentation increases seizure susceptibility in rats and modulates brain production of IL-1ß and IL-6.

Anxiogenic Potential of Experimental Sleep Fragmentation Is Duration-Dependent and Mediated via Oxidative Stress State.

Sleep architecture alterations, among which sleep fragmentation is highly prevalent, represent risk factors for a variety of diseases, ranging from cardiovascular to brain disorders, including anxiety. What mediates anxiety occurrence upon sleep fragmentation is still a matter of debate. We hypothesized that the sleep fragmentation effects on anxiety are dependent on its duration and mediated by increased oxidative stress and alterations in the number of parvalbumin (PV+) interneurons in the hippocampus. Sleep was fragmented in rats by the treadmill method during a period of 14 days (SF group). Rats with undisturbed sleep in the treadmill (TC group) and those receiving equal amounts of treadmill belt motion (EC group) served as controls. To assess anxiety, we subjected rats to the open field, elevated plus maze, and light-dark tests on the 0, 7th, and 14th day. Upon the last test, brain structures were sampled for oxidative stress assessment and PV+ interneuron immunohistochemistry. The results of ethological tests of anxiety-linked behavior suggested duration-dependent anxiogenic potential of sleep fragmentation. Rats' anxiety-linked behavior upon sleep fragmentation significantly correlated with oxidative stress. The rats with fragmented sleep (SF) showed significantly higher oxidative stress in the hippocampus, thalamus, and cortex, compared to controls (TC and EC), while the antioxidant enzymes' activity was significantly decreased. No significant differences were observed in hippocampal PV+ interneurons among these groups. Our results showed that duration of sleep fragmentation is a significant determinant of anxiety-linked behavior, and these effects are mediated through oxidative distress in the brain. Herein, it is revealed that the sleep fragmentation-oxidative stress-anxiety axis contributes to our better understanding of pathophysiological processes, occurring due to disrupted sleep patterns.

Short-term sleep fragmentation enhances anxiety-related behavior: The role of hormonal alterations.

INTRODUCTION: The neuroendocrine background of acute sleep fragmentation in obstructive sleep apnea and sleep fragmentation involvement in psychiatric comorbidities, common in these patients, are still largely unknown. The aim of this study was to determine the effects of short-term experimental sleep fragmentation on anxiety -like behavior and hormonal status in rats. METHODS: Male rats were adapted to treadmill (ON and OFF mode with belt speed set on 0.02m/s and 0.00m/s) and randomized to: 1) treadmill control (TC, only OFF mode); 2) motion, activity control (AC, 10min ON and 30min OFF mode) and 3) sleep fragmentation (SF, 30s ON and 90s OFF mode) group. Six hours later, the animals were tested in the open field, elevated plus maze and light/dark test (n = 8/group). Testosterone, estradiol, progesterone and corticosterone were determined in separate animal cohort immediately upon sleep fragmentation (n = 6/group). RESULTS: SF rats showed decreased rearings number, decreased time spent in the central area and increased thigmotaxic index compared to TC and AC rats in the open field test. Similarly, increased anxiety upon sleep fragmentation was observed in the elevated plus maze and the light/dark test. Significantly lower testosterone, estradiol and progesterone levels were determined in SF in comparison to AC and TC groups, while there was no significant difference in the levels of corticosterone. CONCLUSION: Short term sleep fragmentation enhances anxiety-related behavior in rats, which could be partly mediated by the observed hormonal changes presented in the current study in form of testosterone, estradiol and progesterone depletion.

Chronic prostatitis/chronic pelvic pain syndrome increases susceptibility to seizures in rats and alters brain levels of IL-1ß and IL-6.

Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a result of interplay between psychological, immune, neurological and genetic factors, manifested by variety of urological, as well as brain-related symptoms. However, its relation with brain excitability has not been addressed. herefore, our aim was to assess susceptibility to seizures in rats with CP/CPPS. We induced CP/CPPS in adult rats by intraprostatic injection of 3% λ-carrageenan. Sham operated rats served as controls (0.9% NaCl, Sham). On day 7 upon injection, rats were treated with lindane (4 mg/kg) and observed for convulsive behavior (seizure incidence, latency and severity) and EEG manifestations (number and duration of ictal periods). Interleukin levels (IL-1ß and IL-6) were measured in prostate, hippocampus, thalamus and cerebral cortex. Scrotal skin mechanical pain thresholds were determined and prostates were histologically evaluated. Animals with CP/CPPS showed significantly higher incidence, decreased latency time and augmented severity of lindane-induced seizures compared with Sham group. EEG revealed increased number of ictal periods in CP/CPPS rats. Higher levels of IL-1ß and IL-6 were determined in the thalamus and cortex in CP/CPPS animals vs. Sham. IL-1ß level was higher and IL-6 was lower in prostates from CP/CPPS animals comparing to Sham. CP/CPPS development was verified by histological findings of nonbacterial inflammation in the prostates, as well as by significantly decreased scrotal pain threshold in CP/CPPS animals. On the basis of this research, we concluded that CP/CPPS increases susceptibility to lindane-induced seizures in rats associated with increased level of IL-1ß and IL-6 in the cortex and thalamus.

Sleep disruption increases seizure susceptibility: Behavioral and EEG evaluation of an experimental model of sleep apnea.

Sleep disruption accompanies sleep apnea as one of its major symptoms. Obstructive sleep apnea is particularly common in patients with refractory epilepsy, but causing factors underlying this are far from being resolved. Therefore, translational studies regarding this issue are important. Our aim was to investigate the effects of sleep disruption on seizure susceptibility of rats using experimental model of lindane-induced refractory seizures. Sleep disruption in male Wistar rats with implanted EEG electrodes was achieved by treadmill method (belt speed set on 0.02 m/s for working and 0.00 m/s for stop mode, respectively). Animals were assigned to experimental conditions lasting 6h: 1) sleep disruption (sleep interrupted, SI; 30s working and 90 s stop mode every 2 min; 180 cycles in total); 2) activity control (AC, 10 min working and 30 min stop mode, 9 cycles in total); 3) treadmill chamber control (TC, only stop mode). Afterwards, the animals were intraperitoneally treated with lindane (L, 4 mg/kg, SI+L, AC+L and TC+L groups) or dimethylsulfoxide (DMSO, SIc, ACc and TCc groups). Convulsive behavior was assessed by seizure incidence, latency time to first seizure, and its severity during 30 min after drug administration. Number and duration of ictal periods were determined in recorded EEGs. Incidence and severity of lindane-induced seizures were significantly increased, latency time significantly decreased in animals undergoing sleep disruption (SI+L group) compared with the animals from TC+L. Seizure latency was also significantly decreased in SI+L compared to AC+L groups. Number of ictal periods were increased and duration of it presented tendency to increase in SI+L comparing to AC+L. No convulsive signs were observed in TCc, ACc and SIc groups, as well as no ictal periods in EEG. These results indicate sleep disruption facilitates induction of epileptic activity in rodent model of lindane-epilepsy enabling translational research of this phenomenon.