Regionally specific neuroprotective effect of progesterone in rat cerebral hypoperfusion model

Pro-oksidativne i pro-apoptotske promene biomolekula, uključujući lipide, proteine i DNK, koje nastaju usled smanjenja protoka krvi kroz mozak (moždana hipoperfuzija, MH) za posledicu mogu imati narušavanje strukture i funkcije ćelija prečeone moždane kore i hipokampusa. Do sada je testiran efekat brojnih jedinjenja kako bi se ublažile posledice MH, ali adekvatna terapija još uvek ne postoji. Steroidni hormon progesteron (P4) se nametnuo kao tretman u brojnim modelima neuroloških bolesti, jer smanjuje otok mozga, povećava motorne i kognitivne sposobnosti i drugo, koje nastaju usled oksidativnog/nitrozativnog stresa (OS/NS), prevazilaženja kapaciteta antioksidativne (AO) zaštite i apoptoze. Za potrebe proučavanja efekata P4 u stanju eksperimentalno izazvane trajne MH, odrasli mužjaci pacova Wistar soja podeljeni su u tri grupe: lažno operisane jedinke injecirane lanenim uljem (1 mg/kg/7 dana, kontrole), jedinke sa podvezanim karotidnim arterijama injecirane lanenim uljem (1 mg/kg/7 dana) ili P4 (1.7 mg/kg/7 dana). Potencijalne promene su praćene pomoću odgovarajućih testova ponašanja, biohemijskih i histoloških metoda. Analiza ispitivanih parametara ukazuje na to da je P4 sposoban da ublaži MH-izazvane poremećaje ponašanja, smanji obim ćelijske smrti i održi „normalnu“ morfologiju ćelija uočenu kod kontrola, kao i da regionalno-specifično moduliše indikatore OS/NS, pospeši AO i anti-apoptotsku odbranu delovanjem na enzimsku aktivnost, gensku i proteinsku ekspresiju. Predstavljeni rezultati mogu pružiti bazu za dalja ispitivanja koja bi upotpunila saznanja o efektima P4 u MH i sličnim povredama mozga.Pro-oxidative and pro-apoptotic modifications of biomolecules, including lipids, proteins and DNA, due to cerebral blood flow restriction (cerebral hypoperfusion, MH) may induce structural and functional alterations of prefrontal cortex and hippocampus cells. To date, the effect of a numerous compounds has been tested to mitigate the outcomes of MH, but adequate therapy does not yet exist. Steroid hormone progesterone (P4) has been used as a treatment in many neurological models since it reduces brain swelling, increases motor and cognitive abilities, etc, arised due to oxidative/nitrosative stress (OS/NS), deteriorated anti-oxidative (AO) capacity and apoptosis. To study the effects of P4 in experimentally induced permanent MH, adult male Wistar rats were divided into three groups: sham operated animals injected with flaxseed oil (1 mg/kg/7 days, controls), animals with permanently occluded carotid arteries treated either with flaxseed oil (1 mg/kg/7 days) or P4 (1.7 mg/kg/7 days). Potential changes were monitored using appropriate behavioural tests, biochemical and histological methods. The obtained results indicate that P4 is capable to ameliorate MH-induced behavioural disorders, reduce the extent of cell death and maintain "normal" cell morphology as observed in controls, as well as to modulate the action of tested OS/NS indicators, enhance AO and anti-apoptotic defence by acting on enzymatic activity, gene and protein expression in regional specific manner. Herein presented findings could provide a basis for further studies that would fulfill the knowledge of P4 outcomes in MH and similar brain insults

Neuroprotective progesterone potential

Poremećena moždana cirkulacija, koja je karakteristična za starenje i mnoga cerebrovaskularna i/ili neurodegenerativna oboljenja, povezuje se sa prooksidativnim i proapoptotskim promenama mnogobrojnih biomolekula, uključujući lipide, proteine i DNK, i narušavanjem strukture i funkcije ćelija prečeone moždane kore (PFC) i hipokampusa (HIPP). Do sada je testiran efekat brojnih jedinjenja da bi se ublažile posledice narušenog protoka krvi kroz moždano tkivo, ali adekvatna terapija još uvek nije dostupna. Ipak, kao jedan od mogućih terapeutika, izdvaja se steroidni hormon, progesteron (pregn-4-en-3,20-dion, P4), za kojeg je pokazano da ispoljava zaštitne efekte u mnogim životinjskim eksperimentalnim modelima kojima se oponašaju pojedini aspekti poremećaja strukture i funkcije moždanih ćelija primećeni kod starijih ljudi i obolelih od cerebrovaskularnih i/ili neurodegenerativnih bolesti. Stoga, fokus ovog rada je pružanje mogućnosti boljeg razumevanja molekulskih mehanizama delovanja P4 u mozgu u fiziološkom stanju, kao i terapijskog potencijala koji iskazuje u animalnim modelima cerebrovaskularnih i neurodegenerativnih bolesti, sa posebnim osvrtom na trajnu moždanu hipoperfuziju (MH) gde pokazuje regionalno-specifično dejstvo. Iako se P4, za sada, u humanoj populaciji pokazao kao neadekvatan terapeutik u lečenju patološkog stanja povezanog sa poremećenom moždanom cirkulacijom, dodatne kliničke studije bi obezbedile saznanja o efektu ovog potentnog neurosteroida kod obolelih od drugih cerebrovaskularnih i/ili neurodegenerativnih bolesti, dok bi u slučaju ohrabrujućih rezultata, P4 terapija mogla znatno olakšati kvalitet života obolelih.Disturbed cerebral circulation, characteristic for aging and many cerebrovascular and/or neurodegenerative diseases, is associated with prooxidative and proapoptotic changes of various biomolecules, including lipids, proteins and DNA, and with compromised cell structure and function in prefrontal cortex (PFC) and hippocampus (HIPP). Numerous compounds, until today, have been tested to alleviate the outcomes of impaired blood flow through brain tissue, but adequate therapy is not yet available. However, one of the possible therapeutics is singled out, the steroid hormone, progesterone (pregn-4-en-3,20-dione, P4), which has been shown to exert protective effects in many animal experimental models that mimic certain aspects of brain structural and functional changes observed in the elderly and those suffering from cerebrovascular and/or neurodegenerative diseases. Therefore, this paper is focused on providing a better understanding of physiological P4-mediated brain molecular mechanisms, as well as its therapeutic potential in animal models of cerebrovascular and neurodegenerative diseases with the emphasis on permanent cerebral hypoperfusion (CH) where the regional-specific response is observed. Although P4 is shown as inadequate for treating a pathological condition associated with disturbed cerebral circulation, additional clinical studies would provide insights into the potential therapeutic capacity of this potent neurosteroid in other cerebrovascular and/or neurodegenerative diseases and conditions. In the case of encouraging results, P4 therapy could significantly improve the quality of patients’ life

Acute Toxicity Assessment of Orally Administered Microplastic Particles in Adult Male Wistar Rats

While the effects of chronic exposure to microplastic particles (MPs) are extensively studied, the outcomes of a single treatment have received relatively less attention. To investigate MPs’ potential acute toxicity, including their impact on general health status (victual consumption, sensorimotor deficits, and clinical toxicity signs) and serum biochemical parameters (markers of organ/tissue function and oxidative stress indicators), we administered thoroughly characterized MPs (1.4, 35, or 125 mg/kg), generated from polyethylene terephthalate (PET) bottles, to adult male Wistar rats via oral gavage. The MPs’ short-term effects were assessed with well-established tests and methods. The results point to the absence of sensorimotor deficits and clinical toxicity signs, while levels of markers of liver, heart, and kidney function were altered in all MP groups. Decreased victual consumption and increased levels of oxidative stress indicators were evident following treatment with the two higher MP doses. Presented data indicate that examined MPs are able to initiate the development of local changes in tissues and organs within a short time frame, potentially leading to their damage and dysfunction. This study may increase the awareness of the detrimental effects of plastic contamination, as even a single exposure to MPs may provoke adverse health outcomes

Ecto-5'-nucleotidase marks amoeboid microglial cells in the rat model of neurodegeneration

Adenosine 5'-triphosphate (ATP) and adenosine are versatile signaling molecules involved in many pathophysiological processes in the nervous system. They can be released from all types of brain cells in the extracellular space and activates purinergic receptors. Signaling via extracellular ATP is regulated by cell-surface located ectonucleotidases. Extracellular AMP resulting from the hydrolysis of ATP and ADP can in turn be hydrolyzed into adenosine by ecto-5'-nucleotidase (eN). We examined the involvement of purinergic signaling components in the rat model of trimethyltin (TMT)-induced hippocampal neurodegeneration (8mg/kg, single ip), which results in behavioral and neurological dysfunction similar as in Alzheimer's disease models. Enzyme histochemistry and immunohistochemistry (ir) showed that products of AMPase activity and eN-ir were accumulated in the neuronal strata, infiltrating within neuronal cell layers, depicting individual round-shaped elements that covered neuronal layers with pronounced cell death mostly at the late stage of TMT-induced neurodegeneration. Co-localization with Iba1+ specifically marked eN at amoeboid microglial cells. Neither of the tested pro-inflammatory cytokines (IL-1β, TNF-α, IL10) and C3 nor polarization marker iNOS was found in association with those Iba1/eN+ -cells. Iba1-ir cells co-localized with Arg1-ir and phagocytic marker CD68- ir. Marked induction of P2Y12R-, P2Y6R-, and P2X4-mRNA at the early stage of TMT-induced neurodegeneration might reflect the migration, and chemotaxis of microglia, while induction of P2X7R at amoeboid cells probably modulates their phagocytic role. These findings may contribute to a better understanding of the involvement of purinergic signaling components in the progression of neurodegenerative disorders that could be target molecules for development of novel therapies.Poster Session: Neuroimmunoendocrine Interaction

Time-Related Sex Diffrences in Cerebral Hypoperfusion-Induced Brain Injury

Although the model of cerebral hypoperfusion in rats has been a matter of many investigations over the years, the exact intracellular and biochemical mechanisms that lead to neuron loss and memory decline have not been clearly identified. In the current study, we examined whether cerebral hypoperfusion causes changes in hippocampal protein expression of apoptotic markers in the synaptosomal fraction and neurodegeneration in a time-dependent and sex-specific manner. Adult male and female Wistar rats were divided into two main groups, controls that underwent sham operation, and animals subjected to permanent bilateral occlusion of common carotid arteries. Both male and female rats were killed 3, 7 or 90 days following the insult. The obtained results indicate that the peak of processes that lead to apoptosis occured on postoperative day 7 and that they were more prominent in males, indicating that neuroprotective effects of certain substances (planned for future experiments), should be tested at this time point

Long-term alprazolam treatment may cause tolerance development by modulating components of glutamatergic neurotransmission in the hippocampus of male Wistar rats

The benzodiazepine alprazolam (ALP) is commonly prescribed to treat anxiety, panic, and sleep disorders. However, ALP is often abused for prolonged periods of time, leading to severe side effects such as tolerance, dependence, and withdrawal syndrome. Previous literature data suggest that neuroadaptive changes at synaptic receptors, such as gammaaminobutyric acid receptor type A (GABAAR) and glutamatergic receptors, may be responsible for the occurrence and development of the aforementioned side effects. Therefore, the present study investigated the potential effects of prolonged ALP treatment (2 mg/kg, ip.) on the α1-subunit containing GABAAR and components of glutamatergic neurotransmission in the hippocampus of adult male Wistar rats. The study revealed behavioral changes consistent with a possible onset of tolerance and associated changes in the GABAergic and glutamatergic systems. The primary target of ALP, the α1-subunit containing GABAAR, was decreased indicating its potential downregulation by prolonged agonist (ALP) action. Considering studied glutamatergic components, an increase in NMDAR subunits, a decrease in vGlut1, and differential modulation of excitatory amino acid transporters 1 and 2 (EAAT1/2, in vivo and in vitro) were observed. These changes may all together indicate a compensatory mechanism due to the sustained suppression of glutamatergic neurons by enhanced inhibitory impulses from GABAergic neurons. The data presented provide valuable and, to our knowledge, the first information on components of glutamatergic neurotransmission after prolonged ALP treatment and their potential impact on the development of side effects. However, further research is needed to examine the observed changes in detail.Poster Session: Bran Stimulation & Signalling, Phase Separation and Open Dat

Dietary supplementation with flaxseed oil ameliorates trimethyltin (TMT)-induced neurodegeneration and gliosis in female Wistar rats

It is increasingly apparent that the prevention/treatment of neurodegenerative disorders is not only achieved through pharmacological therapy but also through the consumption of natural products. Flaxseed oil (or linseed oil, FSO) derived from the seeds of the flax (Linum usitatissimum L.) gained worldwide awareness as a neuroprotective agent due to its high content of omega-3 polyunsaturated fatty acids (n-3 PUFAs). Thus, the aim of this study was to examine the preventive effects of dietary FSO in trimethyltin (TMT) - induced hippocampal neurodegeneration and gliosis in female Wistar rats. Animals were continuously treated with FSO (1 ml/kg, orally) for two weeks, then received a single dose of TMT (8 mg/kg, i.p.), and application of FSO continued for twenty-one days. Data have convincingly shown that FSO continuous treatment ameliorated TMT-induced neuronal loss in the CA3 hippocampal region and ameliorated astrogliosis and microgliosis. FSO treatment elevated all tested n-3 fatty acids in the hippocampus: α-linolenic acid (ALA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), and consequently increased total amount of n-3 PUFA. However, no changes in n-6 fatty acids due to FSO treatment were observed. Consequently, FSO lowered n-6/n-3 ratio compared to TMT, having a protective effect on fatty acid profile in hippocampus. These findings support beneficial neuroprotective properties of FSO against TMT-induced model of neurodegeneration and hint at a promising preventive use of FSO in hippocampal degeneration and dysfunctionPoster Session: Brain Metabolism & Dietary Intervention

Prolonged zaleplon treatment enhance GABAergic and glutamatergic signaling in the hippocampus of male Wistar rats

Zaleplon, a member of Z-drugs, is a pyrazolopyrimidine hypnotic with sedative, anxiolytic, anticonvulsant and muscle relaxant properties. Zaleplon is approved for the short-term management of insomnia since acting as positive γ-aminobutyric acid (GABA) receptor allosteric modulator increases efficacy of inhibition on brain excitability. Importantly, for the proper functioning of the brain a balance between inhibitory (i.e., GABAergic) and excitatory (i.e., glutamatergic) system must be accomplished. This may be fulfilled by control of presynaptic elements (synthesis or degradation of glutamate and GABA neurotransmitters, their compartmentation, releasing and recycling) and regulation of expression and function of glutamate and GABA receptors. Hence, we aimed to investigate effects of prolonged zaleplon treatment on the expression of proteins involved in the gabaergic and glutamatergic signalization in the hippocampus of adult male Wistar rats. Five-day intraperitoneal administration increased level of components of GABAergic signalization (glutamate decarboxylase 67-GAD67, vesicular GABA transporter-VGAT and α1 subunit of GABA receptor-GABAAα1). This was accompanied by increased level of glutamatergic components (vesicular glutamate transporter 1-vGlut1 and subunits of glutamate N-Methyl-d-aspartate receptor-NMDAR, namely NR1, NR2A, NR2B), which clearly indicate maintenance of balance between main inhibitory and excitatory neurotransmitters. Given the importance of equilibrium of these systems for neuronal excitability, synaptic plasticity and cognitive functions, as well as its involvement in the mood, feeding behavior, reproductive functions, pain sensitivity, aging, etc., the current and prospective pharmaceuticals increasingly rely on GABA/glutamate balancePoster Session: Bran Stimulation & Signalling, Phase Separation and Open Dat

Prolonged zaleplon treatment enhance GABAergic and glutamatergic signaling in the hippocampus of male Wistar rats

Zaleplon, a member of Z-drugs, is a pyrazolopyrimidine hypnotic with sedative, anxiolytic, anticonvulsant and muscle relaxant properties. Zaleplon is approved for the short-term management of insomnia since acting as positive γ-aminobutyric acid (GABA) receptor allosteric modulator increases efficacy of inhibition on brain excitability. Importantly, for the proper functioning of the brain a balance between inhibitory (i.e., GABAergic) and excitatory (i.e., glutamatergic) system must be accomplished. This may be fulfilled by control of presynaptic elements (synthesis or degradation of glutamate and GABA neurotransmitters, their compartmentation, releasing and recycling) and regulation of expression and function of glutamate and GABA receptors. Hence, we aimed to investigate effects of prolonged zaleplon treatment on the expression of proteins involved in the gabaergic and glutamatergic signalization in the hippocampus of adult male Wistar rats. Five-day intraperitoneal administration increased level of components of GABAergic signalization (glutamate decarboxylase 67-GAD67, vesicular GABA transporter-VGAT and α1 subunit of GABA receptor-GABAAα1). This was accompanied by increased level of glutamatergic components (vesicular glutamate transporter 1-vGlut1 and subunits of glutamate N-Methyl-d-aspartate receptor-NMDAR, namely NR1, NR2A, NR2B), which clearly indicate maintenance of balance between main inhibitory and excitatory neurotransmitters. Given the importance of equilibrium of these systems for neuronal excitability, synaptic plasticity and cognitive functions, as well as its involvement in the mood, feeding behavior, reproductive functions, pain sensitivity, aging, etc., the current and prospective pharmaceuticals increasingly rely on GABA/glutamate balancePoster Session: Bran Stimulation & Signalling, Phase Separation and Open Dat