Brain Homogenates Research Articles

Rescue of CUMS-induced HPA axis hyperfunction and hypothalamic synaptic deficits by Citrus aurantium L. cv. Daidai essential oil via the cAMP/PKA/Grin2b pathway.

Traditional Chinese medicine has historically used Citrus aurantium L. cv. Daidai to alleviate anxiety and improve mood. Essential oils are the primary active component of Citrus aurantium L. cv. Daidai, but little research has been conducted on the active substances and mechanisms of the antidepressant effect of Citrus aurantium L. cv. Daidai essential oil (CEO). This research used network pharmacology, molecular docking, transcriptomics, and in vitro and in vivo experimental validation to assess the effectiveness and therapeutic mechanism of CEO. We used gas chromatography‒mass spectrometry(GC-MS) to identify and quantify CEO components and brain-penetrating chemicals. CEO was given to chronic and unpredictable mild stimulation (CUMS) mice for 4 weeks. Depression was assessed using sucrose preference, forced swimming, tail suspension, elevated plus-maze, and open field tests. Analyzing brain homogenates and serum biochemistry data simultaneously revealed neurotransmitter and hormone alterations. Tissue samples were collected for histological and protein analysis. Furthermore, transcriptome and network pharmacology were used to investigate the mechanism by which CEO alleviates depression, and in vitro glutamate(Glu)-induced PC12 cell injury assays were conducted to validate this new mechanism. CEO inhalation altered neurotransmitter and hormone expression and improved CUMS-induced weight and depression-like behavior in mice. Compared with CUMS mice, CEO mice presented less pathological brain damage, as demonstrated by HE staining, immunohistochemistry, Golgi staining, transmission electron microscopy, and immunofluorescence staining. We discovered that 13 of the active chemicals in CEO act on 522 targets, 96 of which are linked to depression. PRKACA was identified as the core target by a modular analysis of the PPI network. Network pharmacology and transcriptomics revealed that CEO influences depression via the cAMP/PKA/Grin2b pathway and Glu synaptic modulation. In vivo studies indicated that CEO administration reduced PKA and Grin2b phosphorylation in CUMS mice, inhibited the cAMP/PKA/Grin2b pathway, protected against synaptic deficits, and restored HPA axis function. In vitro investigations revealed that the survival rate of PC12 cells treated with CEO increased, the apoptotic rate decreased, and the expression of LDH, Ca2+, and MDA decreased. Western blot and immunofluorescence staining indicated that CEO inhibits the cAMP/PKA/Grin2b pathway to regulate Glu-induced PC12 cells and that 8-Bromo-cAMP pretreatment reduces the protective effect of CEO. The active chemicals in CEO can inhibit the cAMP/PKA/Grin2b pathway, reduce anxiety and depression, alleviate excitotoxicity caused by Glu synaptic overactivation, protect against hypothalamic synaptic deficits, and restore HPA axis function. This research could serve as a reference for the field of illness prevention and complementary therapies for depression.

Detection of chronic wasting disease prions in the farm soil of the Republic of Korea.

Chronic wasting disease (CWD) is a highly contagious prion disease occurring in free-ranging and farmed cervids. CWD continues to spread uncontrolled across North America, and cases continue to be detected almost every year in the Republic of Korea. CWD-infected animals contaminate the soil by releasing infectious prions through their excreta, and shed prions accumulate and remain infectious in the soil for years. Given that the upper soil levels can become contaminated with prions and serve as infectivity reservoirs facilitating horizontal transmission of CWD, the ability to detect prions in the soil is needed for monitoring and managing CWD spread. Using the protein misfolding cyclic amplification (PMCA) technique, we investigated whether prions could be amplified and detected in farm soil experimentally exposed to CWD-infected brain homogenate as well as in the soil of CWD-affected farms. From each soil sample, we performed 10 serial extractions and used these 10 extracts as PMCA templates. Here, we show that prion seeding activity was detected in extracts from farm soil following 4 years of incubation with CWD-infected brain homogenate. More importantly, 13 of 38 soil samples collected from six CWD-affected farms displayed prion seeding activity, with at least one soil sample in each farm being PMCA positive. Mouse bioassays confirmed the presence of prion infectivity in the soil extracts in which PMCA seeding activity was detected. This is the first report describing the successful detection of prions in soil collected from CWD-affected farms, suggesting that PMCA conducted on serial soil extracts is a sensitive means for prion detection in CWD-contaminated soil.IMPORTANCEChronic wasting disease (CWD) is a highly contagious prion disease affecting free-ranging and farmed cervids. CWD continues to spread uncontrollably across North America, and multiple cases are detected annually in the Republic of Korea. Prions shed from CWD-infected animals remain infectious in the soil for years, serving as infectivity reservoirs that facilitate horizontal transmission of the disease. Therefore, the ability to detect CWD prions in soil is crucial for monitoring and managing the spread of the disease. In this study, we have demonstrated for the first time that prions in the soil of CWD-affected farms can be reliably detected using a combination of serial soil extraction and a prion amplification technique. Our data, in which at least one soil sample tested positive for CWD in each of the six CWD-affected farms analyzed, suggest that the approach employed in this study is a sensitive method for prion detection in CWD-contaminated soil.

Cocaine and aortic dissection: the need for collaboration to overcome the underreporting bias.

The dissection of the aorta is a serious and potentially fatal consequence of cocaine use. Nonetheless, the underlying mechanisms and characteristics of this phenomenon remain to be deeply studied. The autopsy case of a 46-year-old white male found irresponsive and unconscious in his house and had a history of abusing cocaine is presented. Autopsy findings showed aortic arch and thoracic aorta of regular calibre, with evidence of a mid-adventitial dissecting aneurysm of the ascending intrapericardial portion of the aorta. Forensic toxicological analyses evidenced a positivity to cocaine and its main metabolite, benzoylecgonine, in all fluids (peripheral blood, bile and urine), liver and brain homogenates. Data also evidenced a positivity to alcohol, confirming a past history of intake. The presented case confirms the connection between cocaine addiction and the risk of aortic dissection, emphasizing the need for increased knowledge about the risks connected to this drug. It is emphasized the need of prompt examination and effective treatment of patients exhibiting cardiac symptoms linked to cocaine use, as well as the need of an accurate anamnesis to evidence use/abuse of other cardiotoxic substances.

Preparation and pharmacokinetic evaluation of a sertraline-methylpropyphenazone prodrug: a comparative metabolic study on the plasma and brain tissues of rats using LC-MS/MS analysis.

A mutual prodrug of sertraline-methylpropyphenazone (SER-MP) was prepared and characterized using a spectral method. The yield of the prepared SER-MP was 90%, and its purity reached 98.8%. The metabolic fate of the prepared SER-MP versus sertraline (SER) was investigated in the plasma and brain tissues of rats. A solid-phase extraction procedure was developed and validated for the optimal recovery of SER, 3-hydroxymethylpropyphenazone (3-OHMP), and SER-MP from plasma and brain tissues. The extraction efficiency for the targeted analytes was improved from 93.5% to 98.0% using Chromabond® C8-100 mg solid-phase extraction columns. A high-performance liquid chromatography-triple-quad-mass spectrometric method was developed and validated to quantify the SER, SER-MP, and potential metabolites. The pharmacokinetic parameters showed that the time to reach the maximum plasma concentration (t max) for both SER and SER-MP was 6 hours, and the maximum plasma concentration of SER-MP reached 192 ng mL-1 with an elimination half-life time of 50 hours. The plasma level of SER, which was released as a metabolite of orally administered SER-MP, was increased by 2.4 times compared to SER HCl administered at equimolar doses. The concentration of SER-MP in the rat brains remained approximately stable at 100 ng g-1 for 0.5 to 192 hours. The serotonin level in the rat brain homogenate was 50 to 90 ng g-1 for both the group receiving SER and that receiving an equal molar dose of SER-MP. This observation was consistent during a time range of 1 to 192 h from oral administration. Thus, this approach could lead to the development of more efficient antidepressant therapies with reduced side effects. The findings indicate that SER-MP could minimize serotonin syndrome risks by maintaining steady serotonin levels in the brain, thus improving patient safety and compliance.

Inclusion Complex of Nitrate γ-Cyclodextrine with Conjugate of Tetrahydrocarbasol and Aminoadamantane

The possibility of obtaining the inclusion complex of γ-cyclodextrin nitrate with tetrahydrocarbazole and aminoadamantane conjugate, a promising drug for the treatment of neurodegenerative diseases, was investigated and proved using XRD, DSC, HPLC and NMR. It was found that the obtained complex, unlike the active substance itself, is soluble in water, and its cellular toxicity does not exceed that of the active substance, which contributes to the increase of its bioavailability and provides the possibility of conducting the necessary biological studies. It was found that the formed inclusion complex preserves the medicinal properties of the active substance, exhibiting mitoprotective and microtubule-stabilizing activities, and also possesses a pronounced antioxidant activity, effectively inhibiting spontaneous lipid peroxidation in rat brain homogenate.

Improvement of antioxidant protection and energy production in neurodegenerative processes in the brain caused by long-term alcoholism under the influence of Lophantus chinensis Benth extract

Long-term alcohol abuse causes psycho-emotional and cognitive impairment, including severe dementia. Oxidative stress is considered one of the main mechanisms in the cognitive disorders pathophysiology caused by long-term alcoholism. In this regard, the search for substances capable of correcting mitochondrial dysfunction and oxidative stress that arise as a result of prolonged ethanol consumption is relevant. Of particular interest in the complex treatment of alcoholic encephalopathy is Chinese hyssop (Lophanthus chinensis), used in Tibetan and Mongolian traditional medicine for liver diseases, as well as a means of improving the functional state of the body and metabolism, slowing down the aging process. Material and methods. Alcohol intoxication was modeled in Wistar rats by per os administration of a 40 % ethanol solution in a volume of 10 ml/kg for six weeks. The L. chinensis dry extract at a dose of 100 mg/kg was administered to animals per os starting from the third week, an hour after ethanol. On day 45, the content of malondialdehyde (MDA), reduced glutathione (GSH), activity of catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase, NADH dehydrogenase and succinate dehydrogenase complex, concentration of ATP werei determined spectrophotometrically in the animal brain homogenate. Results. It was found that L. chinensis extract increases the activity of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase (by 10 %, р = 0.016, by 31 %, р = 0.001, by 30 %, р = 0.041, by 29 %, р = 0.009, respectively) and GSH content (by 24 %, р = 0.019), and also reduces the concentration of MDA (by 20 %, р = 0.014) in brain tissue. A decrease in the intensity of oxidative stress enhances the functioning of enzymatic complexes I (by 23 %, р = 0.017) and II of the mitochondrial respiratory chain (by 72 %, р = 0.001) and increases the ATP content (by 23 %, р = 0.029) in the rat’s brain. Conclusions. L. chinensis extract exhibits antioxidant effects and stimulates energy processes in neurodegenerative processes in the brain caused by long-term alcoholism.

Bupropion Showed Neuroprotective Impacts Against Cerebral Ischemia/Reperfusion Injury by Reducing Oxidative Stress and Inflammation

Background: Cerebral ischemia/reperfusion (I/R) injury is the most prevalent form of brain stroke, affecting many patients worldwide. It is believed that oxidative stress and inflammation play major roles in the damage that occurs after the initiation of the disease. Objectives: Therefore, for the first time, the current study aimed to investigate the neuroprotective effects of bupropion against cerebral I/R damage in a rat model. Methods: Forty male rats were divided into four groups: Control, cerebral I/R, and two diseased groups that received 60 and 100 mg/kg of bupropion. One day after induction of the disease, behavioral tests, including grid walking, novel object recognition, and modified neurological severity score (mNSS), were performed on the rats. The levels of inflammatory cytokines, including IL-1β, TNF-α, IL-6, and IL-10, were measured in the rats' brain homogenates. Additionally, the levels of MDA, catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), and NO2- were measured. Results: Bupropion administration was associated with improved performance in the novel object recognition and grid walking behavioral tests, as well as in the neurological disorder scores, in cerebral I/R rats. Moreover, BCAAO-induced inflammation was reduced by the administration of this drug, evidenced by reduced levels of cytokines IL-1β, TNF-α, and IL-6 and upregulation of IL-10. Additionally, membrane lipid peroxidation was reduced in the cerebral I/R rats receiving 100 mg/kg bupropion, and the level of SOD activity was improved in these animals. Finally, the administration of bupropion prevented the increase in NO2- levels induced by BCAAO. Conclusions: In conclusion, bupropion has neuroprotective effects against cerebral I/R damage by reducing inflammation and oxidative stress in the brain.

Subacute symptoms of depression and anxiety in stress-exposed mice: Role of Schinus terebinthifolia Raddi leaf lectin (SteLL).

Anxiety and depression are leading causes of disability worldwide, often exacerbated by chronic stress. Schinus terebinthifolia Raddi. has been used in traditional medicine for several purposes. Among these, the use of bark-and-leaf tea and leaf decoction to treat depression has been reported. Previous studies showed that the S. terebinthifolia leaf lectin (SteLL) can ameliorate anxiety and depression symptoms in mice. To investigate SteLL as a compound from S. terebinthifolia leaf able to alleviate symptoms of depression and anxiety in an unpredictable chronic mild stress (UCMS) animal model. Mice were subjected to four-week UCMS and then treated with SteLL at 2 and 4mg/kg (i.p.) or with fluoxetine at 10mg/kg i.p. (positive control) for 21 days. Behavioral assessments were conducted using the open field test, elevated plus maze, tail suspension test, and sucrose preference test. Serum corticosterone and inflammatory markers (cytokines) levels were determined. The levels of cytokine, oxidative stress indicators and monoamines in brain homogenates were also measured to understand the biochemical changes induced by SteLL treatment. SteLL treatment at both doses significantly (p<0.05) alleviated the stress-induced behavior in mice, reducing the anxiety and depression signals in all tests. SteLL administration increased the brain levels of monoamines noradrenaline and serotonin in comparison with UCMS control mice that received only vehicle. SteLL reduced superoxide production, lipid peroxidation and improved reduced glutathione (GSH) levels in the brain. The lectin also increased serum and brain levels of anti-inflammatory cytokine IL-4, while reducing levels of pro-inflammatory cytokines. Serum corticosterone levels were not decreased by lectin treatment. Our findings highlight SteLL as a neuromodulatory agent from S. terebinthifolia leaves effective in subacute and stress-induced anxiety and depression through modulation of monoaminergic, oxidative stress, and inflammatory pathways. The data shows the potential of this lectin as a therapeutic agent for stress-related neuropsychological disorders.

Study of edaravone impact on oxidative stress biomarkers in serum and brain of rats with traumatic brain injury

It is known that traumatic brain injury (TBI) is one of the main causes of death and disability among people of all ages. Recently, the risk of TBI has increased dramatically in Ukraine, not only for the military, but also for civilians. After traumatic brain injury a cascade of multidirectional metabolic changes begins in the brain, including protein carbonylation, increased lipid peroxidation, free radicals formation, impaired neurotransmitters release and energy imbalance, which are associated with the development of various functional disorders. The search for effective drugs to eliminate or prevent these complications is very important. Nowadays, although edaravone has a limited range of indications (alleviation of neurological symptoms and functional disorders associated with acute ischemic stroke, as well as slowing the progression of functional disorders in amyotrophic lateral sclerosis), the known mechanisms of its pharmacological action may be useful in other conditions of the central nervous system, where oxidative stress plays a leading role in pathogenesis. The aim of this study was to examine the effect of edaravone on biomarkers of carbonyl oxidative stress in rats with TBI. Modeling of TBI was carried out by applying a directed mechanical injury to the area of the scalped skull with a metal bar (450 g) falling from a height of 170 cm. Advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS), protein carbonyls (PC370, PC430), the content of lactate and ischemia modified albumin (IMA) in serum and S1 fractions of the cerebral cortex and hippocampus of experimental animals were studied using spectrophotometry. It was established, that the modeling of TBI was associated with an elevation of PC430, AOPP and lactate in serum. Simultaneously, the levels of TBARS, IMA and lactate also were increased in the brain homogenates in addition to the above-mentioned changes. In our study edaravone demonstrated efficacy in reducing the levels of some specific biomarkers of oxidative stress. In particular, the administration of the drug was accompanied by a decrease in elevated contents of PC430, AOPP and lactate in serum, indicating its ability to systemically attenuate oxidative damage. It should be noted that a characteristic feature of the drug in case of TBI was its effect on the course of oxidative stress in the hippocampus. In addition, the ability of edaravone to a moderate increase in lactate production can be explained not by the phenomena of lactic acidosis, but by the activation of glia to meet the energy needs of the neurons in the injured brain. The results obtained provide valuable insight into the potential therapeutic effects of edaravone in TBI, but require further studies to elucidate more detailed mechanisms of therapeutic action of this drug.

Sulfamethizole Attenuates Pentylenetetrazole-Induced Seizures in Mice via mTOR Inhibition.

Epilepsy affects at least 1% of the global population of all socioeconomic backgrounds. Data obtained from previous studies suggest the role of mTOR signaling in epileptogenesis. The present study aimed to investigate the hypothesis that mTOR inhibitor sulfamethizole might produce antiepileptic effects in pentylenetetrazole (PTZ)-induced kindling seizures in mice. For induction of kindling, mice were administered 40 mg/kg PTZ on alternate days for 13 days. The severity of kindling was analyzed using a seizure intensity score. Rotarod performance, actophotometer, and chimney tests were performed to check muscle coordination and locomotor functions. mTOR and IL-6 levels were measured in the brain homogenate. Histological analyses were done using hematoxylin-eosin and cresyl violet stains. Sulfamethizole was administered daily at 10 and 50 mg/kg doses. PTZ administration resulted in kindling seizures in the PTZ-veh group. In addition, mice from the PTZ-veh group showed decreased fall time in rotarod performance, reduced locomotor activity, and failed chimney tests. mTOR and IL-6 levels were also increased in the brain, along with neuronal degeneration and a decreased layer of neuronal cells in the hippocampus. Treatment with sulfamethizole at 50 mg/kg significantly ameliorated seizure intensity score, seizure latency and duration, muscle coordination, and locomotor functions compared to the PTZ-veh group. It also downregulated brain mTOR and IL-6 expression significantly. In conclusion, sulfamethizole showed antiepileptic activity against PTZ-induced kindling seizure in mice via mTOR inhibition.

Antioxidant Effects of Moringa oleifera Against Abamectin-Induced Oxidative Stress in the Brain and Erythrocytes of Rats.

The current study was conducted to explore the phytochemical composition and in vitro antioxidant activity of Moringa oleifera leaves aqueous extract (MOLE), as well as its in vivo modulatory effects on abamectin (ABM)-induced oxidative stress in rat erythrocytes and brain tissue. Following extraction, the total phenolic, flavonoid, condensed tannin and ortho-diphenolic contents of MOLE were determined. High-performance liquid chromatography (HPLC) analysis allowed the identification and the quantification of 12 bioactive compounds: gallic acid, chlorogenic acid, caffeic acid, vanillic acid, quercetin, ferulic acid, ascorbic acid, alizarin, hesperidin, neohesperidin, resveratrol, and naringin. In vitro study: the assessment of the antioxidant activity of MOLE on the 2,2-diphenyl-1-picrylhydrazyl radical DPPH and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS), its ferric reducing power and its antioxidant effect on the β-carotene bleaching indicated that MOLE exhibited potent antioxidant activity, with an IC50 of 0.125mg/mL against DPPH radical, and an IC50 of 0.06mg/mL against ABTS radical. It also demonstrated notable ferric-reducing ability, with an EC50 of 1.4mg/mL and a strong inhibition of β-carotene bleaching with an IC50 of 1.36mg/mL. In vivo study: Twenty rats were equally divided into four groups. The first group served as a control and received distilled water by gavage. The second group (negative control) received ABM in drinking water at a dose of 1mg/kg body weight. The third group received MOLE at a dose of 200mg/kg of body weight by gavage. The fourth group received a combination of ABM and MOLE in the same manner and doses as described, for 3weeks. Body weight, brain relative and absolute weights, and nitric oxide levels were not affected by ABM. However, ABM significantly inhibited acetylcholinesterase (AChE) activity (p<0.001), decreased the activities of antioxidant enzymes, specifically superoxide dismutase (SOD) and glutathione S-transferase (GST) in cerebral tissue, and catalase (CAT) in erythrocytes (p<0.001). ABM also decreased reduced glutathione (GSH) levels in both the brain (p<0.001) and erythrocytes (p<0.05). In addition, malondialdehyde (MDA) levels significantly increased in the brains of ABM-intoxicated rats (p<0.01) compared to the control group. These results were accompanied by histopathological changes, notably the remarkable vacuolization of neuropil in brain tissue. Supplementation with MOLE in ABM-treated rats significantly ameliorated brain AChE (p<0.05) and GST activities, decreased MDA content, and improved GSH levels in both brain and erythrocyte homogenates (p<0.01). MOLE also restored the histopathological alterations observed in the ABM group. Computational modeling revealed that some of the tested molecules, including some present in the studied extract, bound human peroxiredoxin 5, CAT, and glutathione peroxidase with acceptable affinities, which, together with the established molecular interactions and tight embedding satisfactory support the in vivo results. Thus, it may be concluded that ABM impairs brain and erythrocyte function through oxidative damage, and these effects could be prevented by MOLE, likely due to its antioxidant activity.

Increased GABBR2 Expression on Cell Membranes Causes Increased Ca2 + Inward Flow, Associated with Cognitive Impairment in Early Alzheimer's Disease.

Alzheimer's disease (AD) and mild cognitive impairment (MCI) are a serious global public health problem. The aim of this study was to analyze the key molecular pathological mechanisms that occur in early AD progression as well as MCI. Expression profiling data from brain homogenates of 8 normal volunteers, and 6 patients with prodromal AD who had developed MCI were analyzed, and the data were obtained from GSE12685. Further, overexpression of GABBR2 was achieved in human neuroblastoma cell lines SH-SY5Y and BE(2)-M17 using expression plasmid transfection. GABBR2 was significantly overexpressed in brain tissues of patients with prodromal AD who had developed MCI, as compared to normal brains. Moreover, GABBR2 overexpressing cells showed a significant increase in intracellular Ca2+ concentration, a large amount of reactive oxygen species production, a large opening of the mitochondrial permeability transition pore and a significant increase in apoptosis compared with control cells. GABBR2 overexpression was significantly involved in early AD progression and MCI by causing cellular events such as intracellular Ca2+ imbalance, oxidative stress, and mitochondrial dysfunction.

Ultrasensitive Protein Aggregate Quantification Assays for Neurodegenerative Diseases on the Simoa Platform.

Nanoscale aggregates play a key role in the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. However, quantifying these aggregates in complex biological samples, such as biofluids and postmortem brain tissue, has been challenging due to their low concentration and small size, necessitating the development of methods with high sensitivity and specificity. Here, we have developed ultrasensitive assays utilizing the Quanterix Simoa platform to detect α-synuclein, β-amyloid and tau aggregates, including those with common posttranslational modifications such as truncation of α-synuclein and AT8 phosphorylation of tau aggregates. All assays had a detection limit in the low pM range. As a part of this work, we developed silica-nanoparticle calibrators, allowing for the quantification of all aggregates. These assays were validated for aggregate and target specificity through denaturation and cross-reactivity experiments. We then applied these assays to brain homogenate samples from Alzheimer's disease and control samples, demonstrating their applicability to postmortem tissue. Lastly, we explored the potential of these assays for blood-based diagnostics by detecting aggregates in serum samples from early Alzheimer's disease patients.

A Novel Galantamine–Curcumin Hybrid Inhibits Butyrylcholinesterase: A Molecular Dynamics Study

Cholinesterases are enzymes that break down the neurotransmitter acetylcholine in the nervous system. The two main types are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). AChE inhibitors are used to treat Alzheimer’s disease by increasing acetylcholine levels. BChE activity increases in later stages of Alzheimer’s, suggesting it might contribute to the disease. In previous experiments, it was found that a newly designed hybrid of galantamine (GAL) and curcumin (CCN) (compound 4b) decreases the activity of BChE in murine brain homogenates. Here, we explore this observation using molecular dynamics simulations. GAL and CCN were also studied for comparison. The structures of the complexes between the BChE and the ligands were predicted by molecular docking. Then, molecular dynamics simulations were performed to evaluate the stability of the complexes and the interactions between the ligands and the enzyme over a simulated time of 1 μs. All three ligands formed stable complexes with BChE. Compound 4b formed more hydrogen bonds and other interactions with BChE compared to GAL and CCN, suggesting a stronger binding affinity. The stronger binding of 4b to BChE might explain its superior anti-BChE activity observed in previous experiments.

Edaravone reduces the markers of oxidative stress and neuroinflammation in neocortex of rats with acute intracerebral hemorrhage and type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is associated with a higher incidence of hemorrhagic stroke in a severe form. The aim of this study was to estimate the markers of oxidative stress and neuroinflammation in the brain of rats with acute intracerebral hemorrhage (ICH) and T2DM after treatment with edaravone. T2DM was induced by a single intraperitoneal injection of nicotinamide/streptozotocin, ICH – by stereotactic microinjection of bacterial collagenase. Rats were randomized into four groups: 1 – intact control; 2 – T2DM; 3 – T2DM+ICH; 4 – T2DM+ICH+edaravone 6 mg/kg/day. Edaravone (a drug to treat neural injury after acute cerebral ischemic stroke) was administered intraperitoneally for 10 days starting from the 60th day after diabetes mellitus induction and 30 min after ICH induction. Brain homogenates were assessed for the content of advanced glycation end products (AGEs) and advanced oxidation protein products (AOPPs). The levels of TNF-α and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured with ELISA. The increased content of 8-OHdG and TNF-α in brain homogenates of animals of T2DM group compared to the control was shown. It was revealed that in brain homogenates of animals of T2DM+ICH group the content of these markers­ significantly exceeds that for T2DM group, and in addition, an elevated AOPPs level was observed. Our results demonstrated that edaravone prevented the elevation of TNF-α level, reduced oxidative DNA damage by decreasing 8-OHdG content, and attenuated the formation of AGEs and AOPPs in the brains of experimental animals. These findings suggest that edaravone may have therapeutic potential in diabetic patients with acute ICH. Keywords: edaravone, intracerebral hemorrhage, neuroinflammation, oxidative stress, TNF-α, type 2 diabetes mellitus

Neuroprotective Effects of Curcuma longa and Rosmarinus officinalis Extracts on Oxidative Stress and Inflammation in the Brains of Alcohol-treated Rats

Background: Neurological disorders remain a global health challenge. Chronic alcohol consumption induces damage to the brain that can cause various forms of dementia. An abundance of acetaldehyde is produced by excessive alcohol consumption and accumulates in the body to induce oxidative stress, apoptosis, and inflammation in neuronal cells, which results in brain damage leading to memory loss. This study is aimed at investigating the effect of mixed ethanol extract of Curcuma longa rhizome and Rosmarinus officinalis leaf on oxidative stress and proinflammatory markers in the brain of alcohol- treated male Wistar rats. Methods: Twenty-five male Wistar rats were divided into five groups: control, mixed extract only (600 mg/kg), alcohol only (15 ml/kg of 40% ethanol), and mixed extract + alcohol groups at 300 mg/kg and 600 mg/kg doses. Treatments were administered orally for 21 days. Brain homogenates were analyzed for superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), reduced glutathione (GSH), malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and AChE activity. Histopathological evaluations assessed neuroprotective effects. Results: Alcohol administration significantly decreased SOD (16.2 ± 1.3 U/mg protein), catalase (12.8 ± 0.9 U/mg protein), GPx (10.7 ± 0.8 U/mg protein), and GSH (9.2 ± 0.6 mg/g protein) compared to the control group (32.8 ± 2.2 U/mg, 23.5 ± 1.7 U/mg, 18.9 ± 1.2 U/mg, and 18.3 ± 1.1 mg/g, respectively; p&lt;0.05). MDA levels increased to 7.6 ± 0.3 nmol/mg protein compared to 2.8 ± 0.2 nmol/mg in the control group. Treatment with 600 mg/kg of mixed extracts significantly restored SOD (31.5 ± 2.4 U/mg), catalase (22.6 ± 1.4 U/mg), GPx (17.8 ± 1.0 U/mg), GSH (17.4 ± 1.2 mg/g), and reduced MDA (3.2 ± 0.3 nmol/mg; p&lt;0.05). TNF-α and IL-1β levels were elevated in alcohol-only rats (123.5 ± 4.3 pg/mL and 85.6 ± 3.2 pg/mL) compared to controls (68.3 ± 2.8 pg/mL and 43.2 ± 1.7 pg/mL, p&lt;0.05) but were significantly reduced with mixed extract treatment. Histopathological analyses confirmed reduced neurodegeneration and restoration of normal brain architecture in treated groups. Conclusion: The mixed extract demonstrated significant neuroprotective effects by mitigating oxidative stress, inflammation, and neuronal damage in alcohol-treated rats. These findings suggest its potential as a therapeutic candidate for preventing alcohol-induced dementia.

Abstract A020: Liposomal topotecan with low-intensity focused ultrasound utilization to improve drug penetration with brain parenchyma in pediatric brain tumors

Abstract The blood-brain barrier (BBB) remains a significant obstacle in delivering effective chemotherapeutics to treat pediatric brain tumors, limiting drug penetration and reducing therapeutic outcomes. To overcome this challenge, we investigated the use of liposomal topotecan (TPT), encapsulated in a sonosensitive formulation, designed for controlled release triggered by low-intensity focused ultrasound (LIFU). In this study, we evaluated the in vitro efficacy of a sonosensitive liposomal topotecan formulation. The liposomal topotecan was created using remote loading with an ammonium sulfate gradient. TPT was loaded at a drug-to-lipid ratio of 1:10. The liposome was composed of hydrogenated soy PC, cholesterol, and DSPE-PEG2000. IC50 assay comparing free topotecan to the liposomal topotecan indicated that the cytoxicity of free TPT is lower than liposomal TPT indicating a sustained release of drug which would decrease long term toxicity. In addition, pharmcokinetic fluorescent quantification of liposomal topotecan showed linear increase in free drug (ug/uL) as liposomal TPT concentration is increased in brain homogenates. We hypothesize that tumor growth inhibition and overall survival would be significantly improved in mice receiving LIFU-mediated liposomal topotecan delivery. Our future work includes additional pharmokinetics with survival analysis in vivo with and without low intensity focused ultrasound. These results highlight the potential of combining liposomal drug formulations with focused ultrasound to overcome the challenges of BBB permeability, offering a promising therapeutic strategy for improving drug accumulation and survival outcomes in pediatric brain tumor models. Citation Format: Avani Mangoli, Sarah Kim, Kathryn Blethen, Angela Everhart, Sasha Nikiforov, Gerry Grant, Luke Avigliano. Liposomal topotecan with low-intensity focused ultrasound utilization to improve drug penetration with brain parenchyma in pediatric brain tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A020.

Human brain microglia responses to amyloid beta and tau pathology in high pathology controls and across disease stages

AbstractBackgroundMicroglia responses to Aβ and tau pathology and the dysregulation of the microglial role in synaptic function may determine the onset and course of Alzheimer’s disease (AD). While significant work has been performed in mouse models, we still lack a complete understanding of physiological and pathological microglial states and functions in human AD brain.MethodFor immunoblotting of brain homogenates against multiple microglial markers, and flow cytometry (FC) analysis of synaptosomal fractions (SNAP25/CD47/Aβ(10G4)/phospho‐tau(AT8)), 49 cryopreserved human parietal cortex samples were categorized into four groups: low pathology control (LPC), high Aβ control (HAC), high pathology control (HPC), and AD. Selected microglia markers were assessed in the snRNAseq dataset (Rexach et al., 2023, bioRxiv) and validated by immunohistochemistry (IHC) analysis.ResultIn the LPC group, only 22.5% of Aβ‐positive (+)/p‐tau‐negative (‐) synaptosomes expressed CD47 on their surface, with a significant progressive increase in the percentage (%) of CD47+ events in the Aβ+/p‐tau‐ group across disease stages, reaching 53.4% in the AD group. Surprisingly, Aβ+/tau+ synaptosomes expressed the highest CD47 levels, suggesting protection from elimination. Higher levels of P2ry12 and CD206 were found in the LPC group compared to all other groups in brain homogenates. Interestingly, the levels of P2ry12, CD206, and Axl1 were significantly higher, and the level of Clec7a was significantly lower in the HPC group compared to the AD group. Linear regression modeling revealed a significant negative association between P2ry12 and synaptic tau pathology, as well as CD47 expression in p‐tau‐positive synaptosomes. Interestingly, in primary tauopathy microglia (but not AD microglia), IKFZ1‐regulated genes, including P2ry12, were upregulated according to snRNAseq and around p‐tau‐bearing neurons by IHC.ConclusionOur data suggest that in normal aging, synaptic Aβ accumulation leads to a diminishing of synaptic CD47 (‘don’t eat me’) signal, accelerating the clearance of malfunctioning Aβ but not p‐tau positive synapses. Upregulation of P2ry12 and CD206, accompanied by downregulation of Clec7a, may represent an early and potentially protective response to pre‐tangle tau pathology. Later in disease progression, P2ry12 reduction may drive a more reactive, phagocytic phenotype in response to tau pathology, leading to an increase in synaptic CD47 levels and synaptic pathology accumulation.

Establishing validated RT-qPCR workflow for the analysis of oligodendrocyte gene expression in the developing murine brain.

Myelination is essential for the proper conduction of impulses across neuronal networks. Mature, myelinating glia differentiate from progenitor cells through distinct stages that correspond to oligodendrocyte-specific gene expression markers. Reverse transcription quantiatative PCR (RT-qPCR) is a common technique used to quantify gene expression across cell development; however, a lack of standardization and transparency in methodology may lead to irreproducible data. Here, we have designed and validated RT-qPCR assays for oligodendrocyte genes and reference genes in the developing C57BL6/J mouse brain that align with the MIQE guidelines, including quality controls for primer specificity, temperature dependence, and efficiency. A panel of eight commonly used reference genes was ranked using a series of reference gene stability methods that consistently identified Gapdh, Sdha, Hmbs, Hprt1, and Pgk1 as the top candidates for normalization across brain regions. In the cerebrum, myelin genes peaked in expression at postnatal day 21, which corresponds to the peak of developmental myelination. The gene expression patterns from the brain homogenate were in agreement with previously reported RNA-seq and microarray profiles from oligodendrocyte lineage cells. The validated RT-qPCR assays begin to build a framework for future investigation into the molecular mechanisms that regulate myelination in mouse models of brain development, aging, and disease.

Characterization and Evaluation of Alpha‐Synuclein Aggregates: Unveiling Their Potential as a Biomarker for Parkinson's Disease (PD)

AbstractBackgroundParkinson's disease (PD) is a debilitating condition that affects millions of people worldwide, yet there are currently no reliable biomarkers for its diagnosis. Alpha‐synuclein aggregation is a well‐known hallmark of PD pathology, but the behavior and kinetics of these aggregates are poorly understood. To address this gap in knowledge, this study utilized several approaches to evaluate the potential of alpha‐synuclein aggregates as potential biomarker for PD.MethodFirstly, the aggregation behavior of alpha‐synuclein (aSyn) oligomers and fibrils was evaluated using a Real‐time quaking induced conversion (RT‐QuIC) assay. This assay differentiated PD from healthy samples in human skin, brain homogenates, and cerebrospinal fluid (CSF) samples. Secondly, a symmetric ECL assay was used to quantify alpha‐synuclein oligomers and fibrils. Finally, direct stochastic optical reconstruction microscopy (dSTORM) was utilized to visualize the size and shape of recombinant alpha‐synuclein fibrils and RT‐QuIC end products (fibrils) at high resolution.ResultThe results demonstrated that fast‐aggregating samples had higher concentrations of aSyn oligomers in skin and other samples compared to slow‐aggregating samples. Interestingly, human skin as a biological matrix showed a better and significant differentiation of healthy and PD samples, compared to brain or CSF indicating skin as a promising specimen for detecting alpha‐synuclein aggregation. Preliminary analysis of the average length and width of alpha‐synuclein aggregates showed promise, and further experiments with a larger sample size are required to prove whether different species of alpha‐synuclein fibrils/oligomers exist in healthy and disease samples.ConclusionThese results provide a foundation for the continued investigation of alpha‐synuclein aggregation as a potential biomarker for PD.