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- Therapeutic effects of IkB kinase inhibitor during systemic inflammationPublication . Amaro-Leal, Ângela; Shvachiy, Liana; Pinto, Rui; Geraldes, Vera; Rocha, Isabel; Mota-Filipe, HelderAnimal models of inflammatory diseases support the idea that nuclear factor κB (NF-κB) activation plays a pathophysiological role and is widely implicated in multiple organ dysfunction (MOD). Indeed, the inhibition of the IκB kinase (IKK) complex, involved in the NF-κB pathway, can represent a promising approach to prevent MOD. The present work employed a rat model of systemic inflammation to investigate the preventive effects of Inhibitor of IKK complex (IKK16). In male Wistar rats, systemic inflammation was induced by a tail vein injection of lipopolysaccharides (LPS challenge; 12 mg/kg). Treatment with IKK16 (1 mg/kg body weight) was administered, by tail vein, 15 min post-LPS. Age- and sex-matched healthy rats and LPS rats without treatment were used as controls. At 24 h post-IKK16 treatment, serum enzyme levels indicative of liver, kidney, pancreas and muscle function were evaluated by biochemical analysis, and RT-PCR technique was used to analyze gene expression of pro-inflammatory cytokines. Hemodynamic parameters were also considered to assess the LPS-induced inflammation. IKK16 treatment yielded a strong therapeutic effect in preventing LPS-induced elevation of serological enzyme levels, attenuating hepatic, renal, pancreatic and muscular dysfunction after LPS challenge. Moreover, as expected, LPS promoted a significantly overexpression of TNF-α, IL-6 and IL-1β in the heart, kidney, and liver; which was diminished by IKK16 treatment. The present study provides convincing evidence that selective inhibition of the IκB kinase complex through the action of IKK16, plays a protective role against LPS-induced multiple organ dysfunction by reducing the acute inflammatory response induced by endotoxin exposure.
- Intermittent low-level lead exposure causes anxiety and cardiorespiratory impairmentPublication . Shvachiy, Liana; Geraldes, Vera; Amaro-Leal, Ângela; Rocha, IsabelAim: To characterize behavioural and cardiorespiratory changes in a new, intermittent low-level lead exposure animal model. Introduction: Lead (Pb) is a cumulative toxic metal affecting all body systems that are particularly vulnerable during developmental phase. Permanent lead exposure has been defined as a cause of behavioural changes, cognitive impairment, sympathoexcitation, tachycardia, hypertension and autonomic dysfunction. However, no studies have been performed to describe a new, intermittent low-level lead exposure profile, that has been increased in the past years. Methods: Foetuses were intermittently (PbI) exposed to water containing lead acetate (0.2%, w/v) throughout life until adulthood (28 weeks of age). A control group (without exposure, CTL), matching in age and sex was used. At 26 weeks, behavioural tests were performed for anxiety (Elevated Plus Maze Test) and locomotor activity (Open Field Test) assessment. Blood pressure (BP), electrocardiogram (ECG), heart rate (HR) and respiratory frequency (RF) rates were recorded at 28 weeks of age. Baroreflex gain (BRG) and chemoreflex sensitivity (ChS) were calculated. Student’s T-test was used (significance p < 0.05) for statistical analysis. Results: An intermittent lead exposure causes hypertension (increased diastolic and mean BP), increased RF, decreased baroreflex function and increased ChS, without significant changes in HR, when compared to CTL group. Regarding behavioral changes, the intermittent lead exposure model showed an anxiety-like behaviour without changes in locomotor activity. Conclusion: Intermittent low-level lead exposure induces changes on the cardiorespiratory profile characterized by hypertension, carotid chemosensitivity and baroreflex impairment. According to behavioural tests results, this study also shows that the exposure to lead during developmental phases causes anxiety in adult animals without locomotor activity impairment. In summary, this study brings new insights on the environmental factors that influence nervous and cardiovascular systems during development, which can help creating public policy strategies to prevent and control the adverse effects of Pb toxicity.
- From molecular to functional effects of different environmental lead exposure paradigmsPublication . Shvachiy, Liana; Amaro-Leal, Ângela; Outeiro, Tiago; Rocha, Isabel; Geraldes, VeraLead is a heavy metal whose widespread use has resulted in environmental contamination and significant health problems, particularly if the exposure occurs during developmental stages. It is a cumulative toxicant that affects multiple systems of the body, including the cardiovascular and nervous systems. Chronic lead exposure has been defined as a cause of behavioral changes, inflammation, hypertension, and autonomic dysfunction. However, different environmental lead exposure paradigms can occur, and the different effects of these have not been described in a broad comparative study. In the present study, rats of both sexes were exposed to water containing lead acetate (0.2% w/v), from the fetal period until adulthood. Developmental Pb-exposed (DevPb) pups were exposed to lead until 12 weeks of age (n = 13); intermittent Pb exposure (IntPb) pups drank leaded water until 12 weeks of age, tap water until 20 weeks, and leaded water for a second time from 20 to 28 weeks of age (n = 14); and the permanent (PerPb) exposure group were exposed to lead until 28 weeks of age (n = 14). A control group (without exposure, Ctrl), matched in age and sex was used. After exposure protocols, at 28 weeks of age, behavioral tests were performed for assessment of anxiety (elevated plus maze test), locomotor activity (open-field test), and memory (novel object recognition test). Metabolic parameters were evaluated for 24 h, and the acute experiment was carried out. Blood pressure (BP), electrocardiogram, and heart (HR) and respiratory (RF) rates were recorded. Baroreflex gain, chemoreflex sensitivity, and sympathovagal balance were calculated. Immunohistochemistry protocol for NeuN, Syn, Iba-1, and GFAP staining was performed. All Pb-exposed groups showed hypertension, concomitant with a decrease in baroreflex gain and chemoreceptor hypersensitivity, without significant changes in HR and RF. Long-term memory impairment associated with reactive astrogliosis and microgliosis in the dentate gyrus of the hippocampus, indicating the presence of neuroinflammation, was also observed. However, these alterations seemed to reverse after lead abstinence for a certain period (DevPb) and were enhanced when a second exposure occurred (IntPb), along with a synaptic loss. These results suggest that the duration of Pb exposure is more relevant than the timing of exposure, since the PerPb group presented more pronounced effects and a significant increase in the LF and HF bands and anxiety levels. In summary, this is the first study with the characterization and comparison of physiological, autonomic, behavioral, and molecular changes caused by different low-level environmental lead exposures, from the fetal period to adulthood, where the duration of exposure was the main factor for stronger adverse effects. These kinds of studies are of immense importance, showing the importance of the surrounding environment in health from childhood until adulthood, leading to the creation of new policies for toxicant usage control.
- Lead as an environmental toxicant in models of synucleinopathiesPublication . Shvachiy, Liana; Amaro-Leal, Ângela; Machado, Filipa; Rocha, Isabel; Geraldes, Vera; Outeiro, TiagoLead, a toxic heavy metal, is prevalent in various industrial applications, contributing to environmental contamination and significant health concerns. Lead affects various body systems, especially the brain, causing long-lasting cognitive and behavioral changes. While most studies have focused on continuous lead exposure, intermittent exposure, such as that caused by migration or relocations, has received less attention. Importantly, lead exposure intensifies the severity of Parkinson's disease (PD) and dementia with Lewy bodies, diseases involving the accumulation of alpha-synuclein (aSyn) in the brain and in the gut. Although the precise mechanisms underlying these observations remain unclear, oxidative stress and mitochondrial dysfunction likely play a role. Here, we investigated how two different profiles of lead exposure - continuous and intermittent - affect models of synucleinopathies. We found that lead exposure enhances the formation of aSyn inclusions, resulting in an increase in both their number and size in cell models. In addition, we found that animals injected with aSyn pre-formed fibrils display serine 129-phosphorylated aSyn inclusions and a reduction in astrocytes in the substantia nigra. These animals also display neuronal damage and alterations in locomotor activity, exploration behavior, anxiety, memory impairments and hypertension. Our results suggest a mechanistic link between environmental lead exposure and the onset and progression of diseases associated with aSyn pathology. Understanding the molecular and cellular interactions between lead and aSyn is crucial for shaping public health policies and may provide novel insight into strategies for mitigating the impact of environmental toxins on neurodegenerative processes involved in Parkinson's disease and related synucleinopathies.
- Peripheral multiorgan dysfunction induced by sepsis : the role of NFκB pathway and its impact on brain and heart functionPublication . Amaro-Leal, Ângela; Filipe, Helder Mota; Rosário, LuísSepsis and sepsis-associated multiorgan failure represent a systemic inflammatory state mediated by the innate immune system resulting in an excessive cellular response to severe infection, with high levels of morbidity and mortality. Furthermore, patients who survive sepsis, have long-term cognitive and functional impairment. Animal models are essential to clarify the pathophysiological mechanisms of sepsis. There is mounting evidence that inhibition of nuclear factor NFκB activation can reduce sepsis-associated organ dysfunction and injury. Herein, we deeply characterize an animal model of lipopolysaccharide (LPS)-induced acute inflammation, through an integral functional, behavioural, morphological and molecular evaluation as well as tested different possible therapeutic approaches to determine their efficacy on the modulation of systemic inflammation through the NF-kB pathway. Male and female Wistar rats (12-20 weeks) were injected with LPS (E. coli serotype O127:B8; tail IV) and divided into three groups: LPS 6 (6mg/kg), LPS 12 (12mg/kg) and SHAM (NaCl 0.9%). At 6 h and 24 h after LPS administration, an autonomic evaluation was performed in both conscious animals, with continuous radio-telemetry recording of blood pressure (BP) and heart rate (HR), and anesthetized animals with BP, ECG, HR, tracheal pressure, respiratory frequency (RF) and body temperature continuously monitored. Baro- and chemoreflexes were evaluated with phenylephrine and lobeline, respectively. Behavioural changes were also evaluated through the elevated-plus maze (anxiety), open-field (locomotor/exploratory activity) and Y-maze (short-term spatial working memory) tests. Immunohistochemistry and RT-PCR were executed to determine heart and brain inflammatory state. Serum biomarkers levels for organ disfunction were also measured. For the test of potential therapeutic drugs, animals were exposed with the two LPS doses and treated 15 minutes later with four different drugs: erythropoietin, dexamethasone, IKK 16 and adenosine. The functional, behavioural, and molecular analysis were performed. Overall, the characterization model results showed a rise in BP and HR and elevated RF, indicative of tachycardia and tachypnea. The higher RF was concomitant with a higher chemoreceptor reflex sensitivity. The autonomic data reveals an overexcitation of sympathetic tone with a sympatho-vagal imbalance, concomitant with baroreflex impairment and/or increases in BP, all these were strongly marked in the highest LPS dose. At both time-points, both LPS groups show an anxiety-like behaviour, associated with lower locomotor and exploratory capacity and short-term memory impairment. The molecular studies show reactive astrogliosis and microgliosis, due to inflammatory processes in the hippocampus, as well as, an upregulation of pro-inflammatory factors in the heart and brain. Serum analysis yielded higher levels of biomarkers for renal and liver dysfunction, and pancreatic and neuromuscular injury, mainly in the LPS 12 group. The functional, behavioral, and molecular response between female and male rats was not so different. Both sexes present markedly changes when exposed to higher doses of LPS. Indeed, male animals are more sensitive to endotoxin that females, that can fight inflammation more easily. Of the four therapeutic proposals studied, erythropoietin and dexamethasone were the best to modulate the NF-ΚB pathway in order to reduce the dysfunction of multiple organs as well as attenuate the behavioural changes caused by the exposure to endotoxin. Both IKK 16 and adenosine treatments attenuate the organ dysfunction by reducing the serum renal, hepatic, pancreatic and neuromuscular levels, however these two drugs did not improve the cardiac and behavioral modifications. Further studies are needed in order to better understand the role of these drugs in the early stages of sepsis. Summarizing, higher doses of LPS induces strong alterations in both cardiac and neurological systems, as well as, multiple organ dysfunction, that can be attenuated by the administration of specific NF-κB modulators, such as, erythropoietin and dexamethasone. These findings served to better understand the pathophysiological processes involved in these animal model of acute inflammation as well as showed therapeutic evidence that through NF-κB signalling pathway modulation it is possible to attenuate or even reverse the acute stages before this condition evolves to more aggressive stages, namely septic shock and ultimately dead.