Cresyl Violet Research Articles

Preclinical Assessment of EPO-Derived Peptide JM4 in Experimental Autoimmune Neuritis (P10-5.008)

<h3>Objective:</h3> Determine clinical efficacy of JM4, a synthetic erythropoietin-derived 19-mer peptide in experimental autoimmune neuritis (EAN). <h3>Background:</h3> Guillain-Barré syndrome, an acute inflammatory demyelinating polyneuropathy (AIDP), is an autoimmune disease causing injuries to myelinated nerves. Mild AIDP cases result in temporary motor and sensory deficits, but severe AIDP cases can also cause dysautonomia and respiratory failure leading to disability or death. Treatments include intravenous immune globulin or plasma exchange, but only 30–40% of patients experience clinical improvement. More effective interventions are needed to improve AIDP clinical outcomes. JM4 is a 19-mer peptide derived from human erythropoietin, which does not have an unwanted side effect of erythropoiesis. We demonstrated JM4 therapy had prolonged therapeutic benefits in an experimental autoimmune encephalomyelitis (EAE) model. Since inflammatory mechanisms underlying EAE are shared with EAN, an animal model for AIDP, we reasoned JM4 would also have clinical benefit in EAN. <h3>Design/Methods:</h3> EAN was induced in C57BL/6 mouse and in Lewis rat using murine peripheral nerve myelin P0 antigen and bovine myelin P2 peptide, respectively. EAN clinical scoring paradigm was used to assess neurological deficits. At deficit onset, EAN mice were administered JM4 intravenously (IV) for 10 days and EAN rats were administered JM4 IV for 3 days followed by intraperitoneal injection for 7 days. Sciatic nerves from EAN rodents were stained with Luxol fast blue to assess demyelination and with cresyl violet to assess mononuclear cell infiltration. <h3>Results:</h3> JM4 treatment reduced peak EAN clinical scores compared to sham treatment. Based on staining analysis, EAN induction caused mononuclear cell infiltration and demyelination in the sciatic nerve. <h3>Conclusions:</h3> Preliminary clinical scoring data from rodent models suggest JM4 may have therapeutic benefits in EAN models. Further studies are needed to correlate improvements in clinical scoring with histological analysis of demyelination, mononuclear cell infiltration and neuroinflammatory markers in EAN models. <b>Disclosure:</b> Dr. Arismendi has nothing to disclose. The institution of Dr. Choi has received research support from Department of Veterans Affairs. Dr. Husar has nothing to disclose. Peter C. Dowling, MD has nothing to disclose. Dr. Lu has nothing to disclose.

Enriched environment ameliorates postsurgery sleep deprivation-induced cognitive impairments through the AMPA receptor GluA1 subunit.

As a common postsurgery complication, sleep deprivation (SD) can severely deteriorate the cognitive function of patients. Enriched environment (EE) exposure can increase children's cognitive ability, and whether EE exposure could be utilized to alleviate postsurgery SD-induced cognitive impairments is investigated in this study. Open inguinal hernia repair surgery without skin/muscle retraction was performed on Sprague-Dawley male rats (9-week-old), which were further exposed to EE or standard environment (SE). Elevated plus maze (EPM), novel object recognition (NOR), object location memory (OLM), and Morris Water Maze assays were utilized to monitor cognitive functions. Cresyl violet acetate staining in the Cornusammonis 3 (CA3) region of rat hippocampus was used to detect neuron loss. The relative expression of brain-derived neurotrophic factor (BDNF) and synaptic glutamate receptor 1 (GluA1) subunits in the hippocampus were detected with quantitative reverse transcription polymerase chain reaction (RT-qPCR), Western blots, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence. EE restored normal levels of time spent in the center, time in distal open arms, open/total arms ratio, and total distance traveled in the EPM test; EE restored normal levels of recognition index in the NOR and OLM test; EE restored normal levels of time in the target quadrant, escape latencies, and platform site crossings in the Morris Water Maze test. EE exposure decreased neuron loss in the CA3 region of the hippocampus with increased BDNF and phosphorylated (p)-GluA1 (ser845) expression. EE ameliorates postsurgery SD-induced cognitive impairments, which may be mediated by the axis of BDNF/GluA1. EE exposure could be considered as an aid in promoting cognitive function in postsurgery SD.

Effects of maternal vitamin deficiency on the microstructure of the maternal hippocampus and behavior in offspring

ObjectivesNutrition plays a critical role in the brain's function and development. Vitamin B6 in the form of pyridoxal phosphate (PLP) is required for the biosynthesis of several neurotransmitters. As vitamin B6 is not endogenously synthesized, the availability of dietary sources becomes imperative. Due to its contribution to neurological functions, severe vitamin B6 deficiency leads to an increased risk of psychiatric disorders, dementia, and neurodevelopmental disorders. This study aimed to establish a vitamin B6-deficient model in experimental animals and assess the neurodevelopmental effects in their offspring. MethodsTwo- to three-month-old female C57BL/6J mice were used in the study. They were randomly divided into control and vitamin B6-deficient groups. The control group was fed a regular diet containing 6 mg vitamin B6/kg and the vitamin B6-deficient group was fed a customized diet containing 0 mg vitamin B6/kg, for 5 weeks (n = 6). After 5 weeks, plasma PLP was assessed. The animals were bred to generate offspring. The dams were killed following weaning, and the hippocampal neurons were quantified using cresyl violet staining. The offspring were assigned the respective diet post-weaning up to 2 months of age. Learning and memory were assessed using the Morris water maze test. ResultsThe plasma PLP levels confirmed the deficiency in the deficient group compared to the control group. The viable pyramidal neurons in the cornu ammonis 3 (CA3) region of the hippocampus showed a significant difference between the control and deficient groups. Offspring born to deficient dams showed a substantial increase in latency to reach the target quadrant during the probe trial compared to the controls. ConclusionVitamin B6 deficiency reduces memory in dams and their offspring, suggesting the importance of vitamin B6 for both brain function and development.

Hiperbarik oksijen tedavisinin MCF-7 meme kanseri hücrelerinin morfolojik özellikleri ve sağkalımı üzerindeki etkileri

Aim: This study aims to determine the effects of hyperbaric oxygen therapy at different pressure values on cell morphology and cell survival in the MCF-7 breast cancer cell line. Materials and Methods: The experimental groups were formed by applying 100% oxygen to MCF-7 breast cancer cells at 1.5, 2, and 2.5 atmospheres for 2 hours. The control group did not receive treatment. At the end of the experiment, cell survival was investigated by CCK-8 analysis, cell shapes were determined by cresyl violet staining, and cell surface morphologies were determined by scanning electron microscope. Results: Cell viability was significantly reduced at atmospheric pressure of 1.5, 2, and 2.5 compared to the control group (p &amp;lt; 0.005). As pressure increased, the surface area of the cell decreased, nuclear condensation increased, and the cell borders became irregular. Cell membrane bleb and cell membrane porosity increased at 2 and 2.5 atmospheres. Conclusion: Hyperbaric oxygen therapy severely reduces the viability of MCF-7 breast cancer cells under increased pressure. It can induce apoptosis and change the shape and surface morphology of MCF-7 breast cancer cells. Although further studies are needed, our study supports the potential use of hyperbaric oxygen therapy in the treatment of breast cancer.

Electromagnetic Field Effect on Bone Marrow Stem Cells Differentiation and Nucleoli AgNOR

Purpose: Numerous studies have described the effect of Electromagnetic Fields (EMFs) in the promotion of Bone Marrow Stem Cell (BMSC) differentiation. We aimed to investigate the influence of frequency (10 and 100 Hz) and different pulse shapes (sine, rectangular, and triangular) of EMF on rats' BMSCs.&#x0D; Materials and Methods: The BMSCs in 6 groups were exposed to EMF for 1 h/ 7 days. The BMSCs viability was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The cresyl violet labeled the Nissl bodies, and the silver nitrate staining was done to evaluate the BMSCs nucleoli AgNORs.&#x0D; Results: The MTT test verified that EMF and pulse shape did not affect cell viability. In Nissl bodies staining most of the large neurons were related to the rectangular 10 Hz EMF group. The majority of the differentiated BMSCs were astrocytes, microglia, and oligodendrocyte in the triangular 100 Hz EMF group. Although the silver nitrate staining confirmed the effect of 10 Hz EMF, pulse shape alteration did not affect AgNOR parameters. In conclusion, we presented a low-magnetic flux density EMF (400 µT) to assess the responses of BMSCs nuclei.&#x0D; Conclusion: The findings showed that BMSCs differentiation was frequency-dependent. Further investigations are recommended for recognizing the function of EMF on BMSCs.

Vitamin D3 promotes oligodendrogenesis and modulates synucleinopathy in lead-induced nigral pars compacta neurotoxicity in rats.

Lead-induced neurotoxicity was marked with locomotor and Parkinsonian-like changes. Oligodendrocytes and synucleinopathy were signed to in the pathophysiology of some neurodegenerative diseases. Vitamin D3's (D3) role in substantia nigra pars compacta (SNpc) disorders is debated between neuroscientists. The aim of the study was to investigate lead-induced SNpc neurotoxic changes and explore the possible neuroprotective role of D3 and the possible involvement of oligodendrocytes and α-synuclein. This study included 40 adult Wistar rats assigned into four equal groups: control, lead (Pb) (in drinking water, 1,000 mg/L), Pb + D3 (D3 injection, 1,000 IU/kg IM; 3 days/week), and D3. After 8 weeks, the rats were sacrificed, and their midbrain underwent biochemical and immunoblotting analysis. Midbrain paraffin blocks were stained for histological and immunohistochemical assessment. Lead (Pb) had increased significantly (p < 0.05) nigral α-synuclein and caspase-11 by immunoblotting analysis. Histologically, it induced neurodegeneration in SNpc and significantly decreased neuronal cell density by cresyl violet staining. Pb also significantly reduced SNpc tyrosine hydroxylase immunoreaction, significantly elevated glial fibrillatory acid protein (GFAP) and α-synuclein immunoreaction associated with a mild but significant increase in caspase-3. In the Pb + D3 group, all the previous deleterious changes were significantly alleviated in addition to significant upregulation of anti-oligodendrocytes immunoexpression. Lead (Pb) may induce SNpc neurotoxicity presumably via activation of caspase-11 and α-synuclein. D3 may modulate this neurotoxicity probably through an oligodendrogenic effect.

Alleviation of cognitive deficits in a rat model of glutamate-induced excitotoxicity, using an N-type voltage-gated calcium channel ligand, extracted from Agelena labyrinthica crude venom.

Excitotoxicity is a common pathological process in Alzheimer's disease (AD) which is caused by the over-activity of N-Methyl-D-Aspartate receptors (NMDARs). The release of neurotransmitters depends on the activity of voltage-gated calcium channels (VGCCs). Hyper-stimulation of NMDARs can enhance the releasement of neurotransmitters through the VGCCs. This malfunction of channels can be blocked by selective and potent N-type VGCCs ligand. Under excitotoxicity condition, glutamate has negative effects on the pyramidal cells of the hippocampus, which ends in synaptic loss and elimination of these cells. These events leads to learning and memory elimination through the hippocampus circuit's dysfunction. A suitable ligand has a high affinity to receptor or channel and is selective for its target. The bioactive small proteins of venom have these characteristics. Therefore, peptides and small proteins of animal venom are precious sources for pharmacological applications. The omega-agatoxin-Aa2a was purified, and identified from Agelena labyrinthica specimens, as an N-type VGCCs ligand for this study. The effect of the omega-agatoxin-Aa2a on the glutamate-induced excitotoxicity in rats was evaluated through behavioral tests including Morris Water Maze, and Passive avoidance. The syntaxin1A (SY1A), synaptotagmin1 (SYT1), and synaptophysin (SYN) genes expression were measured via Real-Time PCR. The local expression of synaptosomal-associated protein, 25 k Da (SNAP-25) was visualized using an immunofluorescence assay for synaptic quantification. Electrophysiological amplitude of field excitatory postsynaptic potentials (fEPSPs) in the input-output and LTP curves of mossy fiber were recorded. The cresyl violet staining of hippocampus sections was performed for the groups. Our results demonstrated that the omega-agatoxin-Aa2a treatment could recover the learning, and memory impairment caused by NMDA-induced excitotoxicity in rat hippocampus.

Relating the Induced Free Charge Density Gradient in a Room-Temperature Ionic Liquid to Molecular-Scale Organization.

We report on dilution-dependent changes in the local environments of chromophores incorporated into room-temperature ionic liquid (RTIL)-molecular solvent binary systems where the ionic liquid cation and molecular solvent possess the same alkyl chain length. We have used the RTIL 1-decyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide (DMPyrr+TFSI-) and the molecular solvent 1-decanol. Perylene was used as a non-polar probe, and cresyl violet (CV+) was used as a polar probe chromophore. We observe that in both regions there is a change in the chromophore local environments with increasing 1-decanol content. The changes in the nonpolar regions of the binary RTIL-molecular solvent system occur at a lower 1-decanol concentration than changes in the polar regions. Both chromophores reorient as oblate rotors in this binary system, allowing detailed information on the relative values of the Cartesian components of the rotational diffusion constants to be extracted from the experimental data. The induced free charge density gradient, ρf, known to exist in RTILs, persists to high 1-decanol content (1-decanol mole fraction of 0.75), with the structural details of the gradient being reflected in depth-dependent changes in the Cartesian components of the rotational diffusion constants of CV+. This is the first time that changes in molecular organization have been correlated with ρf.

Therapeutic Treatment with Pycnogenol® Attenuates Ischemic Brain Injury in Gerbils Focusing on Cognitive Impairment, Neuronal Death, BBB Leakage and Neuroinflammation in the Hippocampus

A gerbil model of ischemia and reperfusion (IR) injury in the forebrain has been developed for studies on mechanisms, prevention and therapeutic strategies of IR injury in the forebrain. Pycnogenol® (PYC), a standardized extract of French maritime pine tree (Pinus pinaster Aiton) has been exploited as an additive for dietary supplement. In the present study, we investigated the neuroprotective effects of post-treatment with PYC and its therapeutic mechanisms in gerbils. The gerbils were given sham and IR operation and intraperitoneally injected with vehicle and Pycnogenol® (25, 50 and 100 mg/kg, respectively) immediately, at 24 hours and 48 hours after sham and IR operation. Through 8-arm radial maze test and passive avoidance test, each spatial memory and short-term memory function was assessed. To examine the neuroprotection of Pycnogenol®, we conducted cresyl violet staining, immunohistochemistry for neuronal nuclei, and Fluoro-Jade B histofluorescence. Moreover, we carried out immunohistochemistry for immunoglobulin G (IgG) to investigate blood-brain barrier (BBB) leakage and interleukin-1β (IL-1β) to examine change in pro-inflammatory cytokine. We found that IR-induced memory deficits were significantly ameliorated when 100 mg/kg Pycnogenol® was treated. In addition, treatment with 100 mg/kg Pycnogenol®, not 25 mg/kg nor 50 mg/kg, conferred neuroprotective effect against IR injury. For its mechanisms, we found that 100 mg/kg Pycnogenol® significantly reduced BBB leakage and inhibited the expression of IL-1β. Therapeutic treatment (post-treatment) with Pycnogenol® after IR effectively attenuated ischemic brain injury in gerbils. Based on these results, we suggest that PYC can be employed as an important material for ischemic drugs.

Lithography-free fabrication of scalable 3D nanopillars as ultrasensitive SERS substrates

Surface-enhanced Raman spectroscopy (SERS) detection of analyte molecules at ultra-low concentrations requires highly-efficient plasmonic nanostructures enabling a high hot-spot density. However, a facile and cost-effective strategy toward large-area fabrication of efficient nanostructures with significant electromagnetic field enhancement remains a great challenge. Further, SERS faces reliability issues with the molecular fingerprint at ultra-low concentrations. This work shows a one-step rapid fabrication technique utilizing glancing angle deposition for growing 3D nanopillars of Ag or Au, which is facile, scalable and cost-effective. The 3D nanopillar substrates can reliably detect analyte molecules with concentrations as low as 10-18 M with a high signal-to-noise ratio molecular fingerprint proven for Cresyl violet, p-aminothiophenol and Rhodamine 6G. The ultra-high enhancement is realized in conjunction with the formation of a high hot-spot density due to localized surface plasmons and surface plasmons at metal/air interface. A portable handheld Raman spectrometer is used to evaluate the potential application of the nanopillars for on-site diagnostics. It avoids the need for sophisticated tabletop instruments yet provides high-precision molecular specificity outside specialized laboratories. The 3D nanopillar substrates show excellent molecular detection limits at 10−15 M concentrations when tested with a handheld Raman spectrometer. The uniqueness of the 3D nanopillar features with the formation of a high density of hot-spots and one-step nanofabrication methods provide a platform to unravel on-site diagnostics with cost-effective approaches.

Vanadium improves memory and spatial learning and protects the pyramidal cells of the hippocampus in juvenile hydrocephalic mice.

Hydrocephalus is a neurological condition known to cause learning and memory disabilities due to its damaging effect on the hippocampal neurons, especially pyramidal neurons. Vanadium at low doses has been observed to improve learning and memory abilities in neurological disorders but it is uncertain whether such protection will be provided in hydrocephalus. We investigated the morphology of hippocampal pyramidal neurons and neurobehavior in vanadium-treated and control juvenile hydrocephalic mice. Hydrocephalus was induced by intra-cisternal injection of sterile-kaolin into juvenile mice which were then allocated into 4 groups of 10 pups each, with one group serving as an untreated hydrocephalic control while others were treated with 0.15, 0.3 and 3 mg/kg i.p of vanadium compound respectively, starting 7 days post-induction for 28 days. Non-hydrocephalic sham controls (n = 10) were sham operated without any treatment. Mice were weighed before dosing and sacrifice. Y-maze, Morris Water Maze and Novel Object Recognition tests were carried out before the sacrifice, the brains harvested, and processed for Cresyl Violet and immunohistochemistry for neurons (NeuN) and astrocytes (GFAP). The pyramidal neurons of the CA1 and CA3 regions of the hippocampus were assessed qualitatively and quantitatively. Data were analyzed using GraphPad prism 8. Escape latencies of vanadium-treated groups were significantly shorter (45.30 ± 26.30 s, 46.50 ± 26.35 s, 42.99 ± 18.44 s) than untreated group (62.06 ± 24.02 s) suggesting improvements in learning abilities. Time spent in the correct quadrant was significantly shorter in the untreated group (21.19 ± 4.15 s) compared to control (34.15 ± 9.44 s) and 3 mg/kg vanadium-treated group (34.35 ± 9.74 s). Recognition index and mean % alternation were lowest in untreated group (p = 0.0431, p=0.0158) suggesting memory impairments, with insignificant improvements in vanadium-treated groups. NeuN immuno-stained CA1 revealed loss of apical dendrites of the pyramidal cells in untreated hydrocephalus group relative to control and a gradual reversal attempt in the vanadium-treated groups. Astrocytic activation (GFAP stain) in the untreated hydrocephalus group were attenuated in the vanadium-treated groups under the GFAP stain. Pyknotic index in CA1 pyramidal layer of untreated (18.82 ± 2.59) and 0.15mg/kg vanadium-treated groups (18.14 ± 5.92) were significantly higher than control (11.11 ± 0.93; p = 0.0205, p = 0.0373) while there was no significant difference in CA3 pyknotic index across all groups. Our results suggest that vanadium has a dose-dependent protective effect on the pyramidal cells of the hippocampus and on memory and spatial learning functions in juvenile hydrocephalic mice.

Abstract TP193: The Protective Effects Of Exercise On Cerebral Ischemia Injury In Diabetes

Ischemic stroke (IS) causes neurological dysfunction due to a loss of cerebral blood flow with exaggerated cerebral damage in diabetes. Exercise has shown beneficial effects on the vascular system and diabetes by mediating inter-organ communications. However, whether and how exercise contributes preventatively to the brain after IS in type 2 diabetes (T2D) is undefined. Here, we aimed to determine the effects of exercise on the metabolism, cerebral injury, neurological function, and protein expression in the brain after IS. T2D diabetic mice (db/db, 7-8 wks), and age/sex-matched controls (db/c) were subjected to exercise (10 m/min, 5 days/wk for 8-wks) or sedentary. Body weight and blood glucose were recorded once a week. One day after exercise, middle cerebral artery occlusion surgery was performed to induce IS. Sensorimotor deficits were assessed by the adhesive removal and corner tests two days after surgery. Afterward, the brain samples were collected for measuring the infarct size by cresyl violet staining. The proteins from the ipsilateral and contralateral brain were used for Western Blot to measure the levels of endothelial nitric oxide synthase (eNOS), neuronal NOS, NADPH oxidase (Nox2), Nox4, nuclear factor-kappa B (NF-κB), NF-κ light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα). We found: 1) exercise could stabilize the blood glucose in male db/db mice and have effects on preventing blood glucose increase at the early age of female db/db mice; 2) exercise could improve sensorimotor deficits by reducing tape contact and removal time and balancing the corner turning times (p&lt; 0.05); 3) the infarct size is decreased in exercised group in both db/c (18.5 ± 2.2% and 22.2 ± 2.5%), and db/db mice (25.6 ± 3.1% and 35.6 ± 3.8%, exercise vs. sedentary, p&lt; 0.05); 4) proteins related to oxidative stress and inflammation were downregulated in the brain of exercised diabetic IS mice. In conclusion, exercise could provide protective effects on stabilizing metabolism, improving neurological function, and reducing brain injury by downregulating the protein expression related to inflammation and oxidative stress in the brain.

Abstract TMP119: Increased Stroke Severity In A Model Of Preeclampsia Is Associated With Poor Collateral Flow, Oxidative Stress, And Elevated Thrombin

Background: Pregnancy is associated with an increased risk of stroke largely driven by the comorbidity of preeclampsia (PE), a hypertensive disorder that complicates 4-8% of all pregnancies. Although stroke treatment has improved in recent years, pregnant women have been excluded from stroke clinical trials and thus little is known about treatment and stroke outcome in this population. We investigated stroke outcome (infarct and hemorrhagic transformation, HT) in normal pregnancy (Preg; n=13) and in a model of preeclampsia (PE; n=6) induced by feeding pregnant Sprague Dawley rats a high cholesterol diet on gestational days 7-21. Nonpregnant (NP; n=7) rats were a control for pregnancy. Methods: Middle cerebral artery occlusion (MCAO) was used to induce focal ischemia for 3 hours with 1 hour of reperfusion. Perfusion deficit in the core MCA and collateral territories were measured using multi-site laser Doppler. Infarction and oxidative stress were determined by cresyl violet and 3-nitrotyrosine staining of fixed brain tissue, respectively. Endothelial oxidative stress and hemostatic factors were measured by ELISA for 8-isoprostane and thrombin. Results: Perfusion deficit in core MCA was similar between groups; however, collateral flow was significantly impaired in ePE vs. NP and LP: -51±8% vs. -21±15% and -25±13% (p&lt;0.05). Infarct (%) was increased in ePE vs. NP and LP: 33±5% vs. 22±6% and 14±3% (p&lt;0.05). Importantly, the majority of NP animals had HT (4/7), but none of the Preg or ePE suggesting pregnancy was protective of hemorrhage. The increased incidence of HT in NP was associated with increased 3-NT in brain tissue. However, 8-iso was increased in serum of ePE rats vs. NP and LP: 778±16 vs. 208±25 and 274±54 pg/mL (p&lt;0.01), as was plasma thrombin: 6.6±1.2 vs. 0.49±0.01 and 0.56±0.01 pg/mL (p&lt;0.01). Conclusions: Increased stroke severity in ePE animals was associated with poor collateral flow, oxidative stress and elevated thrombin, all of which could contribute to poor ischemic tolerance and increased infarction. Pregnancy-induced protection of HT may be due to high sex steroids that was present in both LP and ePE groups. Understanding contributors to stroke outcome in this population could help manage these high risk patients.

Abstract WP216: Intraperitoneal Delivery Of Capsinoids (Non-pungent TRPV1 Agonists) Promotes Mild Hypothermia And Improved Long-term Outcome Following Stroke

Intro: Mild hypothermia has been shown to improve outcomes in cardiac arrest and experimental stroke models. Unfortunately, hypothermia protocols in awake subjects are hampered by inconsistent temperature management due to incomplete suppression of cold defense mechanisms (e.g. shiver response). To address a need for better strategies for cooling conscious subjects, we sought to promote mild hypothermia by pharmacological activation of heat-sensing nerve fibers in the thermoregulatory system. We hypothesize that capsinoids - non-pungent agonists of the transient receptor potential vanilloid 1 (TRPV1) channel - activate vagal afferents, triggering thermoregulatory regions of the brain to induce mechanisms of whole-body cooling, protecting the brain and preventing injury progression after a stroke. Methods: C57BL6 male mice of 11wks underwent permanent distal middle cerebral artery occlusion (pdMCAO) or sham. Mice recovered for 2hrs in warming cages to maintain normal body temperatures during post-stroke recovery before being administered capsinoids or vehicle (4 IP injections, 90 min interval) to produce 6 hours of mild hypothermia (or normothermic control). After 1 month, mice underwent functional tests and brains were collected for histological analysis. Results: Capsinoids provided a significant and consistent hypothermia whereas vehicle maintained a baseline temperature (two-way ANOVA, n=7, p=0.0036). Capsinoid-treated mice showed ~50% infarct volume reduction 1 month after stroke, measured by cresyl-violet staining (0.7% ± 0.1% SEM vs 1.5% ± 0.2% SEM, n=7, two-tailed unpaired t-test: p=0.0046). Additionally, a stroke-induced breathing dysfunction (decrease in tidal volume by plethysmography) was significantly restored toward normal in the capsinoid-treated group (7.7e-3 ± 5.6e-4 SEM vs 5.8e-3 ± 2.4e-4 SEM, n=8, one-way ANOVA: p=0.033). Conclusion: Capsinoids delivered to TRPV1-containing visceral afferents promotes reversible mild hypothermia in freely moving conscious mice. Application of this form of cooling in the pdMCAO model provides significant reduction of stroke injury volume and long-term improvement in functional outcome.

Exploring the Epigenetic Regulated Modulation of Fibroblast Growth Factor 21 Involvement in High-Fat Diet Associated Parkinson's Disease in Rats.

Imbalance in brain glucose metabolism and epigenetic modulation during the disease course of insulin resistance (IR) associated with Parkinson's disease (PD) risk remains a prime concern. Fibroblast growth factor 21 (FGF21), the metabolic hormone, improves insulin sensitivity and elicits anti-diabetic properties. Chronic stress during brain IR may modulate the FGF21 expression and its dynamic release via epigenetic modifications. Metformin regulates and increases the expression of FGF21 which can be modulating in obesity, diabetes, and IR. Hence, this study was designed to investigate the FGF21 expression modulation via an epigenetic mechanism in PD and whether metformin (MF), an autophagy activator, and sodium butyrate (NaB), a pan histone deacetylase inhibitor, alone and in combination, exert any therapeutic benefit in PD pathology exacerbated by high-fat diet (HFD). Our results portray that the combination treatment with MF and NaB potentially attenuated the abnormal lipid profile and increased motor performance for the rats fed with HFD for 8 weeks followed by intrastriatal 6-hydroxy dopamine administration. The enzyme-linked immunosorbent assay (ELISA) estimations of C-reactive protein, tumor necrosis factor-α, interleukin-1 beta and 6, and FGF21 exhibited extensive downregulation after treatment with the combination. Lastly, mRNA, western blot, histological, and cresyl violet staining depicted that the combination treatment can restore degenerated neuronal density and increase the protein level compared to the disease group. The findings from the study effectively conclude that the epigenetic mechanism involved in FGF21 mediated functional abnormalities in IR-linked PD pathology. Hence, combined treatment with MF and NaB may prove to be a novel combination in ameliorating IR-associated PD in rats, probably via the upregulation of FGF21 expression.

Neuregulin-1/PI3K signaling effects on oligodendrocyte proliferation, remyelination and behaviors deficit in a male mouse model of ischemic stroke

In this study, we investigated the effect of neuregulin-1 (NRG1) on demyelination and neurological function in an ischemic stroke model, and further explored its neuroprotective mechanisms. Adult male ICR mice underwent photothrombotic ischemia surgery and were injected with NRG1 beginning 30 min after ischemia. Cylinder and grid walking tests were performed to evaluate the forepaw function. In addition, the effect of NRG1 on neuronal damage/death (Cresyl violet, CV), neuronal nuclei (NeuN), nestin, doublecortin (DCX), myelin basic protein (MBP), non-phosphorylated neurofilaments (SMI-32), adenomatous polyposis coli (APC), erythroblastic leukemia viral oncogene homolog (ErbB) 2, 4 and serine-threonine protein kinase (Akt) in cortex were evaluated using immunohistochemistry, immunofluorescence and western blot. The cylinder and grid walking tests exposed that treatment of NRG1 observably regained the forepaw function. NRG1 treatment reduced cerebral infarction, restored forepaw function, promoted proliferation and differentiation of neuron and increased oligodendrogliogenesis. The neuroprotective effect of NRG1 is involved in its activation of PI3K/Akt signaling pathway via ErbB2, as shown by the suppression of the effect of NRG1 by the PI3K inhibitor LY294002. Our results demonstrate that NRG1 is effective in ameliorating the both acute phase neuroprotection and long-term neurological functions via resumption of neuronal proliferation and differentiation and oligodendrogliogenesis in a male mouse model of ischemic stroke.

Bone marrow mesenchymal stem cell-derived exosomal lncRNA KLF3-AS1 stabilizes Sirt1 protein to improve cerebral ischemia/reperfusion injury via miR-206/USP22 axis

BackgroundCerebral ischemia/reperfusion (I/R) is a pathological process that occurs in ischemic stroke. Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have been verified to relieve cerebral I/R-induced inflammatory injury. Hence, we intended to clarify the function of BMSC-Exos-delivered lncRNA KLF3-AS1 (BMSC-Exos KLF3-AS1) in neuroprotection and investigated its potential mechanism.MethodsTo mimic cerebral I/R injury in vivo and in vitro, middle cerebral artery occlusion (MCAO) mice model and oxygen–glucose deprivation (OGD) BV-2 cell model were established. BMSC-Exos KLF3-AS1 were administered in MCAO mice or OGD-exposed cells. The modified neurological severity score (mNSS), shuttle box test, and cresyl violet staining were performed to measure the neuroprotective functions, while cell injury was evaluated with MTT, TUNEL and reactive oxygen species (ROS) assays. Targeted genes and proteins were detected using western blot, qRT-PCR, and immunohistochemistry. The molecular interactions were assessed using RNA immunoprecipitation, co-immunoprecipitation and luciferase assays.ResultsBMSC-Exos KLF3-AS1 reduced cerebral infarction and improved neurological function in MCAO mice. Similarly, it also promoted cell viability, suppressed apoptosis, inflammatory injury and ROS production in cells exposed to OGD. BMSC-Exos KLF3-AS1 upregulated the decreased Sirt1 induced by cerebral I/R. Mechanistically, KLF3-AS1 inhibited the ubiquitination of Sirt1 protein through inducing USP22. Additionally, KLF3-AS1 sponged miR-206 to upregulate USP22 expression. Overexpression of miR-206 or silencing of Sirt1 abolished KLF3-AS1-mediated protective effects.ConclusionBMSC-Exos KLF3-AS1 promoted the Sirt1 deubiquitinating to ameliorate cerebral I/R-induced inflammatory injury via KLF3-AS1/miR-206/USP22 network.

Enhanced yield of cholinergic neurons from induced pluripotent stem cells (iPSC): A two-step induction protocol.

Cholinergic neurons, a type of neurons found in central nervous system, play a vital role in muscle movement and activities. Cholinergic neurons degeneration is the main pathological symptom of neurodegenerative diseases. Among a variety of stem cells, iPSCs have emerged as a promising candidate for transplantation to improve the repair of neuronal lesion sites. However, the establishment of an appropriate induction method to yield large numbers of cholinergic neurons has yet to be determined. Here, we studied the differentiation potential of iPSCs to generate cholinergic neurons by developing a new optimized differentiation protocol. The iPSCs were harvested on 6-well matrigel-coated plate and incubated with serum‑free DMEM/F12 with 2 % B27 supplement, 20 ng/ml the basic fibroblast growth factor and 20 ng/ml epidermal growth factor for 48 hours. Then, the pre-induced cells were treated in neuronal induction medium supplemented with all-trans retinoic acid, sonic hedgehog, 100 ng/ml glial-derived neurotrophic factor and 200 ng/ml brain-derived neurotrophic factor for 7 days. Cell viability during induction stages was tested by MTT assay. Differentiated cells were evaluated with crystal violet staining, immunocytochemistry and real‑time PCR. Our results showed that the survival rate of iPSCs leveled out and was similar to that in the control group following the differentiation process. Immunochemistry results revealed that the expression of ChAT was observed in cells in both pre‑induction and induction stages with a significantly higher expression level at the induction stage as compared to the pre-induction stage. However, none of these markers was expressed in the iPSCs. Cresyl violet staining confirmed the neuronal phenotype of differentiated cells. The induction group significantly expressed the higher levels of Islet1, Olig2 and HB9, whereas pluripotency markers including those of Oct4 and Nestin plunged. Our investigation represents a highly efficient protocol for iPSCs differentiation toward cholinergic neurons which could be used for further preclinical transplantation studies (Tab. 1, Fig. 5, Ref. 35). Text in PDF www.elis.sk Keywords: induced pluripotent stem cells, cholinergic neurons, neurotrophic factors, induction protocol, preclinical transplantation.

Study of FRET between Zn-Cu-In-S/ZnS core/shell quantum dots-Cresyl violet dye system

This study reports the fluorescence resonance energy transfer between Zn-Cu-In-S/ZnS core–shell quantum dots and Cresyl Violet dye. The quenching of Zn-Cu-In-S/ZnS core–shell QD (donor) emission and lifetime and simultaneous enhancement in Cresyl Violet (acceptor) emission validates and confirms the non-radiative resonance energy transfer from Zn-Cu-In-S/ZnS QDs to Cresyl violet. The values of spectral overlap, Förster distance, the distance between donor and acceptor pair and FRET efficiency were determined using the steady state as well as time-resolved fluorescence spectroscopic techniques. This study demonstrates that this systemexhibits efficient FRET and can act as a very sensitive chemical sensor. Since Zn-Cu-In-S/ZnS QDs are nontoxic compared to popular cadmium-based quantum dots, the application of FRET in biological systems will be advantageous. It can find vast applications in photonics and biomedical applications.

Effect of induced diabetes on morphometric indexes of the cerebellar cortex and gene expression in C57BL mice.

Diabetes is a metabolic disorder that affects the development of the central nervous system and plays an important role in learning and memory. Diabetes increases the reactive oxygen species (ROS) level in cells and changes the expression of several genes, including SYP, BDNF, PAX7, and SYNCAM1, through the FOXO transcription factor. This study was done to assess the effect of diabetes on morphometric indexes of the cerebellar cortex and gene expression in mice. Diabetes was induced in twelve adult, male C57BL mice using an injection of streptozotocin. After two months, the mice were dissected, and the cerebellum was stored for further analysis. For the morphometric analysis, tissue sections were stained with cresyl violet and examined with a light microscope. For gene expression analysis, the RNA was extracted, and cDNA was synthesized. The mRNA levels of SYP, BDNF, PAX7, and SYNCAM1 genes were measured by the real-time PCR method. The thickness of the molecular layer and Purkinje layer, and the number of Purkinje and granular cells in the diabetic group were significantly reduced compared to controls P<0.0 1). The area, perimeter, and diameter of Purkinje cells in the diabetic group were significantly reduced compared to controls P<0.0 1). The expression of PAX7, SYP, and BDNF genes of the diabetic group was significantly reduced. However, SYNCAM1 expression in the cerebellum of the diabetic group was significantly increased compared to controls (P<0.05). Induced diabetes in mice can decrease the expression of memory-related genes in the cerebellum. Also, these genes affect the morphology and thickness of the cerebellum.