Bovine Brain Research Articles

Autophagy and NLRP3 inflammasome crosstalk in neuroinflammation in aged bovine brains

NLRP3 inflammasome is a multiprotein complex that can sense several stimuli such as autophagy dysregulation and increased reactive oxygen species production stimulating inflammation by priming the maturation of proinflammatory cytokines interleukin-1β and interleukin-18 in their active form. In the aging brain, these cytokines can mediate the innate immunity response priming microglial activation. Here, we describe the results of immunohistochemical and molecular analysis carried out on bovine brains. Our results support the hypothesis that the age-related impairment in cellular housekeeping mechanisms and the increased oxidative stress can trigger the inflammatory danger sensor NLRP3. Moreover, according to the recent scientific literature, we demonstrate the presence of an age-related proinflammatory environment in aged brains consisting in an upregulation of interleukin-1β, an increased microglial activation and increased NLRP3 expression. Finally, we suggest that bovine may potentially be a pivotal animal model for brain aging studies.

Evaluation of Kinetic Parameters of Acetylcholinesterase from the Bovine Brain and its Inhibition with Mentha pulegium L. Extract

Evaluation of Kinetic Parameters of Acetylcholinesterase from the Bovine Brain and its Inhibition with Mentha pulegium L. Extract

Regulation of Malate Dehydrogenases and Glutamate Dehydrogenase of Mammalian Brain by Thiamine in vitro and in vivo.

To study the mechanisms of the non-coenzyme action of thiamine and its diphosphate (ThDP) on brain proteins, proteins of acetone extract of bovine brain synaptosomes or the homogenate of rat brain cortex were subjected to affinity chromatography on thiamine-modified Sepharose. In the step-wise eluates by thiamine (at pH 7.4 or 5.6), NaCl, and urea, the occurrence of glutamate dehydrogenase (GDH) and isoenzymes of malate dehydrogenase (MDH) along with the influence of thiamine and/or ThDP on the enzymatic activities were characterized using mass spectrometry and kinetic experiments. Maximal activation of the malate dehydrogenase reaction by thiamine is observed after the protein elution with the acidic thiamine solution, which does not elute the MDH1 isoenzyme. Effects of exogenous thiamine or ThDP on the GDH activity may depend on endogenous enzyme regulators. For example, thiamine and/or ThDP activate the brain GDH in eluates from thiamine-Sepharose but inhibit the enzyme in the crude preparations applied to the sorbent. Inhibition of GDH by ThDP is observed using the ADP-activated enzyme. Compared to the affinity chromatography employing the elution by thiamine at pH 7.4, the procedure at pH 5.6 decreases the activation of GDH by thiamine (but not ThDP) in the eluates with NaCl and urea. Simultaneously, the MDH2 content and total GDH activity are higher after the affinity elution at pH 5.6 than at pH 7.4, suggesting the role of the known interaction of GDH with MDH2 in stabilizing the activity of GDH and in the regulation of GDH by thiamine. The biological potential of thiamine-dependent regulation of the brain GDH is confirmed in vivo by demonstration of changes in regulatory properties of GDH after administration of a high dose of thiamine to rats. Bioinformatics analysis of the thiamine-eluted brain proteins shows a specific enrichment of their annotation terms with "phosphoprotein", "acetylation", and "methylation". The relationship between thiamine and the posttranslational modifications in brain may contribute to the neuroprotective effects of high doses of thiamine, including the regulation of oxidation of the major excitatory neurotransmitter in brain - glutamate.

Absorption of 5G Radiation in Brain Tissue as a Function of Frequency, Power and Time

The rapid release of 5G wireless communications networks has spurred renewed concerns regarding the interactions of higher radiofrequency (RF) radiation with living species. We examine RF exposure and absorption in ex vivo bovine brain tissue and a brain simulating gel at three frequencies: 1.9 GHz, 4 GHz and 39 GHz that are relevant to current (4G), and upcoming (5G) spectra. We introduce a highly sensitive thermal method for the assessment of radiation exposure, and derive experimentally, accurate relations between the temperature rise (ΔT), specific absorption rate (SAR) and the incident power density (F), and tabulate the coefficients, ΔT/ΔF and Δ(SAR)/ΔF, as a function of frequency, depth and time. This new method provides both ΔT and SAR applicable to the frequency range below and above 6 GHz as shown at 1.9, 4 and 39 GHz, and demonstrates the most sensitive experimental assessment of brain tissue exposure to millimeter-wave radiation to date, with a detection limit of 1 mW. We examine the beam penetration, absorption and thermal diffusion at representative 4G and 5G frequencies and show that the RF heating increases rapidly with frequency due to decreasing RF source wavelength and increasing power density with the same incident power and exposure time. We also show the temperature effects of continuous wave, rapid pulse sequences and single pulses with varying pulse duration, and we employ electromagnetic modeling to map the field distributions in the tissue. Finally, using this new methodology, we measure the thermal diffusivity of ex vivo bovine brain tissue experimentally.

Speed of Sound and Attenuation Temperature Dependence of Bovine Brain: Ex Vivo Study.

Brain treatments using focused ultrasound (FUS) offer a new range of noninvasive transcranial therapies. The acoustic energy deposition during these procedures may induce a temperature elevation in the tissue; therefore, noninvasive thermal monitoring is essential. Magnetic resonance imaging is the current adopted monitoring modality, but its high operational costs and limited availability may hinder the accessibility to FUS treatments. Aiming at the development of a thermometric ultrasound (US) method for the brain, the specific objective of this investigation was to study the acoustic thermal response of the speed of sound (SOS) and attenuation coefficient (AC) of different brain tissues: namely white matter (WM) and cortical matter. Sixteen ex vivo bovine brain samples were investigated. These included 7 WM and 9 cortical matter samples. The samples were gradually heated to about 45°C and then repeatedly scanned while cooling using a computerized US system in the through-transmission mode. The temperature was simultaneously registered with thermocouples. From the scans, the normalized SOS and AC for both tissues were calculated. The results demonstrated a characteristic cooldown temporal behavior for the normalized AC and SOS curves, which were related to the temperature. The SOS curves enabled clear differentiation between the tissue types but depicted more scattered trajectories for the WM tissue. As for the AC curves, the WM depicted a linear behavior in relation to the temperature. However, both tissue types had rather similar temperature patterns. These findings may contribute to the development of a US temperature-monitoring method during FUS procedures.

Antifungal and anti-biofilm activity of designed derivatives from kyotorphin

Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous analgesic neuropeptide first isolated from bovine brain in 1979. Previous studies have shown that kyotorphins possess anti-inflammatory and antimicrobial activity. Six kyotorphins—KTP-NH2, KTP–NH2–DL, ibuprofen-conjugated KTP (IbKTP), IbKTP-NH2, N-methyl-D-Tyr-L-Arg, and N-methyl-L-Tyr-D-Arg—were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. This study assessed the antimicrobial and antibiofilm activity of these peptides. The antifungal activity of kyotorphins was determined in representative strains of Candida species, including Candida albicans ATCC 10231, Candida krusei ATCC 6258, and six clinical isolates—Candida dubliniensis 19-S, Candida glabrata 217-S, Candida lusitaniae 14-S, Candida novergensis 51-S, Candida parapsilosis 63, and Candida tropicalis 140-S—obtained from the oral cavity of HIV-positive patients. The peptides were synthesized by standard solution or solid-phase synthesis, purified by RP-HPLC (purity >95 %), and characterized by nuclear magnetic resonance. The results of the broth microdilution assay and scanning electron microscopy showed that IbKTP-NH2 presented significant antifungal activity against Candida strains and antibiofilm activity against the clinical isolates. The absence of toxic activity and survival after infection was assessed after injecting the peptide in larvae of Galleria mellonella as experimental infection model. Furthermore, IbKTP-NH2 had strong antimicrobial activity against multidrug-resistant bacteria and fungi and was not toxic to G. mellonella larvae up to a concentration of 500 mM. These results suggest that IbKTP-NH2, in addition to its known effect on cell membranes, can elicit a cellular immune response and, therefore, is promising for biomedical application.

Melatonin Stimulates Epithelium Migration in Wound Models In Vitro and In Vivo.

We studied the effect of bovine brain gangliosides, individual ganglioside GM1, and melatonin on the rate of wound closure under in vitro conditions and the effect of melatonin on the rate of wound healing under in vivo conditions. It was shown that bovine brain gangliosides and melatonin reliably increased cell migration in the experimental wound model. This effect was detected when the cell cultures were treated with the test preparations after wound infliction and when the cultures of human keratinocytes were pretreated before wounding. Analysis of the effect of melatonin on the rate of wound healing in vivo showed that melatonin accelerated this process, especially at the middle stages corresponding to the proliferation phase (days 3-6 after surgery). Histological analysis revealed intensification of epidermal cell proliferation at the edges of the wound starting from day 4 after surgery.

Interaction of Synaptosomal-Associated Protein 25 with Neutral Sphingomyelinase 2: Functional Impact on the Sphingomyelin Pathway

Neurotransmitter release is mediated by ceramide, which is generated by sphingomyelin hydrolysis. In the present study, we examined whether synaptosomal-associated protein 25 (SNAP-25) is involved in ceramide production and exocytosis. Neutral sphingomyelinase 2 (nSMase2) was partially purified from bovine brain and we found that SNAP-25 was enriched in the nSMase2-containing fractions. In rat synaptosomes and PC12 cells, the immunoprecipitation pellet of anti-SNAP-25 antibody showed higher nSMase activity than the immunoprecipitation pellet of anti-nSMase2 antibody. In PC12 cells, SNAP-25 was colocalized with nSMase2. Transfection of SNAP-25 small interfering RNA (siRNA) significantly inhibited nSMase2 translocation to the plasma membrane. A23187-induced ceramide production was concomitantly reduced in SNAP-25 siRNA-transfected PC12 cells compared with that in scrambled siRNA-transfected cells. Moreover, transfection of SNAP-25 siRNA inhibited dopamine release, whereas addition of C6-ceramide to the siRNA-treated cells moderately reversed this inhibition. Additionally, nSMase2 inhibition reduced dopamine release. Collectively, our results indicate that SNAP-25 interacts with nSMase2 during ceramide production, which mediates exocytosis and neurotransmitter release.

New Simulator for Neuroendoscopy: A Realistic and Attainable Model

To present an attainable and realistic model for neuroendoscopic simulation which replicates exercises of tissue biopsy and coagulation and membrane fenestration. We presented a stepwise method to create a neuroendoscopic simulation model using bovine brain and membrane units made by a soda cup covered by an amniotic membrane inside an expanded polystyrene spherical container. We used face validation for preliminary evaluation. We also rated the students before and after training with the NEVAT global rating scale (GRS) and recorded the time required to complete all 3 procedures (third ventriculostomy, tissue biopsy, and coagulation). The total cost of the model was $5. The experts consider this new model as capable of reproducing real surgical situations with great similarity to the human brain. We tested the model in 20 trainees. The median GRS score before the training was 9 (range, 7-12). After repeated training and performance feedback, the final median GRS score was 41 (range, 37.5-45; P < 0.0001). The time needed to finish the exercises before training was 33 minutes (range, 30.5-42.5 minutes), and after using the model the final median time was 20 minutes (range, 17.5-22 minutes; P<0.0001). Simulators for neuroendoscopy described so far are reliable, but they entail a high cost. Models with live animals, although of lower cost, are questioned from an ethical point of view. In the current work, we describe a high fidelity ventricular neuroendoscopic simulator model that, because of its low cost, can be replicated in any training center that has a neuroendoscope.

Rate-dependent constitutive modeling of brain tissue.

In this paper, the dynamic behavior of bovine brain tissue, measured from a set of in vitro experiments, is investigated and represented through a nonlinear viscoelastic constitutive model. The brain samples were tested by employing unconfined compression tests at three different deformation rates of 10, 100, and 1000mm/s. The tissue exhibited a significant rate-dependent behavior with different compression speeds. Based onthe parallel rheological framework approach, a nonlinear viscoelastic model that captures the key aspects of the rate dependency in large-strain behavior was introduced. The proposed model was numerically calibrated to the tissue test data from three different deformation rates. The determined material parameters provided an excellent constitutive representation of tissue response in comparison with the test results. The obtained material parameters were employed in finite element simulations of tissue under compression loadings and successfully verified by the experimental results, thus demonstrating the computational compatibility of the proposed material model. The results of this paper provide groundwork in developing a characterization framework for large-strain and rate-dependent behavior of brain tissue at moderate to high strain rates which is of thehighest importance in biomechanical analysis of the traumatic brain injury.

Brain lateralization probed by water diffusion at the atomic to micrometric scale

Combined neutron scattering and diffusion nuclear magnetic resonance experiments have been used to reveal significant interregional asymmetries (lateralization) in bovine brain hemispheres in terms of myelin arrangement and water dynamics at micron to atomic scales. Thicker myelin sheaths were found in the left hemisphere using neutron diffraction. 4.7 T dMRI and quasi-elastic neutron experiments highlighted significant differences in the properties of water dynamics in the two hemispheres. The results were interpreted in terms of hemisphere-dependent cellular composition (number of neurons, cell distribution, etc.) as well as specificity of neurological functions (such as preferential networking).

A poro-hyper-viscoelastic rate-dependent constitutive modeling for the analysis of brain tissues

In this paper, the dynamic behavior of bovine brain tissue, measured from in-vitro unconfined compression tests, is examined and represented through a viscoelastic biphasic model. The experiments have been carried out under three compression speeds of 10, 100, and 1000 mm/s. The results exhibited significant rate-dependent behavior. The brain tissue is modeled as a biphasic continuum consisting of a compressible solid matrix, fully saturated with an incompressible interstitial fluid. The governing equations based on conservation of mass and momentum are used to describe the solid-fluid interactions. An inverse scheme is employed in which a finite element model runs iteratively to optimize constitutive constants. The obtained material parameters of the proposed biphasic model show relatively good agreement (R2 ≥ 0.96) with the experimental tissue mechanical responses at different rates. The model can successfully capture the key aspects of the rate-dependency for both solid and fluid phases under large strain deformation. This poro-hyper viscoelastic model can effectively estimate the global and local rate-dependent tissue deformations, the spatial variations in pore spaces, hydrostatic pressure as well as fluid diffusion through the tissue.

Outgrowth, proliferation, viability, angiogenesis and phenotype of primary human endothelial cells in different purchasable endothelial culture media: feed wisely

Function and dysfunction of endothelial cells are regulated by a multitude of factors. Endothelial cell research often requires in vitro cell culture experiments. Hence, various culture media specifically designed to promote endothelial cell growth are available. These strikingly differ in their composition: complex media contain endothelial cell growth supplement (ECGS), an extract produced of bovine brain with undefined amounts of biologically active compounds, whilst defined media contain selected growth factors in defined concentrations. We here compared the effect of seven purchasable endothelial cell culture media on colony outgrowth, proliferation, viability, in vitro angiogenesis and phenotype of mature primary human endothelial cells using feto-placental endothelial cells isolated from chorionic arteries (fpEC). The effect of media on colony outgrowth was additionally tested on umbilical cord blood-derived endothelial progenitor cells (ECFCs). Outgrowth, purity, proliferation and viability differed between media. Outgrowth of fpEC and ECFCs was best in a defined medium containing EGF, FGF2 and VEGF. By contrast, established fpEC isolations proliferated best in complex media containing ECGS, heparin and ascorbic acid. Also viability of cells was higher in complex media. In vitro angiogenesis was most intense in a defined medium containing the highest number of individual growth factors. FACS analysis of surface markers for endothelial cell subtypes revealed that endothelial phenotype of fpEC was unaffected by media composition. Our data demonstrate the fundamental effect of endothelial cell culture media on primary cell isolation success and behaviour. Whether the composition of supplements is suitable also for individual experiments needs to be tested specifically.

Nicotinamide adenine dinucleotides and their precursor NMN have no direct effect on microtubule dynamics in purified brain tubulin.

Microtubules are dynamic cytoskeletal polymers that provide mechanical support for cellular structures, and play important roles in cell division, migration, and intracellular transport. Their intrinsic dynamic instability, primarily controlled by polymerization-dependent GTP hydrolysis, allows for rapid rearrangements of microtubule arrays in response to signaling cues. In neurons, increases in intracellular levels of nicotinamide adenine dinucleotide (NAD+) can protect against microtubule loss and axonal degeneration elicited by axonal transection. The protective effects of NAD+ on microtubule loss have been shown to be indirect in some systems, for example through the sirtuin-3 pathway. However, it is still possible that NAD+ and related metabolites have direct effects on microtubule dynamics to promote assembly or inhibit disassembly. To address this question, we reconstituted microtubule dynamics in an in vitro assay with purified bovine brain tubulin and examined the effects of NAD+, NADH, and NMN. We found that the compounds had only small effects on the dynamics at the plus and minus ends of the microtubules. Furthermore, these effects were not statistically significant. Consequently, our data support earlier findings that NADs and their precursors influence microtubule growth through indirect mechanisms.

Anomalous water dynamics in brain: a combined diffusion magnetic resonance imaging and neutron scattering investigation.

Water diffusion is an optimal tool for investigating the architecture of brain tissue on which modern medical diagnostic imaging techniques rely. However, intrinsic tissue heterogeneity causes systematic deviations from pure free-water diffusion behaviour. To date, numerous theoretical and empirical approaches have been proposed to explain the non-Gaussian profile of this process. The aim of this work is to shed light on the physics piloting water diffusion in brain tissue at the micrometre-to-atomic scale. Combined diffusion magnetic resonance imaging and first pioneering neutron scattering experiments on bovine brain tissue have been performed in order to probe diffusion distances up to macromolecular separation. The coexistence of free-like and confined water populations in brain tissue extracted from a bovine right hemisphere has been revealed at the micrometre and atomic scale. The results are relevant for improving the modelling of the physics driving intra- and extracellular water diffusion in brain, with evident benefit for the diffusion magnetic resonance imaging technique, nowadays widely used to diagnose, at the micrometre scale, brain diseases such as ischemia and tumours.

Optimum recovery time for cyclic compression tests on bovine brain tissue

In conducting mechanical tests on the brain tissue, it is preferred to perform multiple tests on the same sample. In this study we investigated the behavior of the bovine brain tissue in repeated compression tests with six recovery periods (10, 60, 120, 180, 240 and 300 s). Compression tests were performed on cylindrical samples with an average diameter and height of 18.0 mm and 15.0 mm respectively. Two testing protocols were employed: first protocol comprised of experiments with 5, 25 and 125 mm/min loading speed up to 33% strain and the second protocol consisted of tests with 25 and 125 mm/min loading speed up to 17% strain. Each experiment was conducted in two cycles separated by a specific recovery period. Stress-strain data from the first and second cycles were compared using three criteria, namely Normalized root-mean-square error (NRMSE), coefficient of variation (R2) and effective height ratio (EHR). The analysis suggests that the optimum recovery period for the first and second protocols are 120 s and 180 s respectively. Moreover, differences between the first and second cycles of medium and high speed tests were found to be smaller compared to the low-speed experiments.

Biosynthetic Short Neuropeptides: A Rational Theory Based on Experimental Results for the Missing Pain-Relief Opioid Endomorphin Precursor Gene.

Endomorphins are neuropeptides that bind strongly to μ-opioid receptors and are considered to play important roles in pain modulation and other biological functions. Two endomorphins have been identified, to date, endomorphine-1 and -2; both are tetrapeptides and differ by only a single amino acid in the third position. Both peptides were isolated from bovine brains; however, their precursor genes have not been identified. In this study, a nucleotide sequence corresponding to the endomorphin-1 peptide in an expressed sequence tag database has been found and a preproendomorphin-like precursor peptide from human brain complementary DNA (cDNA) has been cloned. The cDNA consists of nucleotide sequences of two already annotated predicted genes, and the putative peptide differs by one amino acid from the isolated endomorphin peptides. It is proposed herein that there is the possibility of unknown short proteins or peptide precursors being missed by automated gene prediction programs based on similarities of known protein sequences. A novel concept of how to produce endomorphins from a similar peptide is described. The oxidatively modified base might provide a clue for understanding discrepancies between nucleotide sequences on the genome and those on cDNAs.

Global DNA Methylation in the Limbic System of Cattle.

To elucidate the extent to which DNA methylation varies across multiple tissues in the brain and between animals, we have quantified global DNA methylation in tissues comprising the limbic system for six Red Angus x Simmental steers. Global DNA methylation was measured in nine regions of the bovine brain: amygdala, the bed nucleus of the stria terminalis, cingulate gyrus, dorsal raphe, hippocampus, hypothalamus, nucleus accumbens, periaqueductal gray and prefrontal cortex. DNA methylation varies among animals for each tissue type and varies among tissue types for each animal. The highest amounts of DNA methylation were found in the amygdala, cingulate gyrus and dorsal raphe, while the bed nucleus of the stria terminalis, nucleus accumbens and periaqueductal gray had the lowest amounts of DNA methylation. A heatmap sorted by k-means clustering was generated to graphically display percent DNA methylation in relation to tissue type and animal number. This is the first study to report measures of DNA methylation in the limbic system of the bovine brain and can be used to inform the cattle genomics community of expected variation in cattle brain methylation.

Procoagulant activity of bovine neutrophils incubated with conditioned media or extracellular vesicles from Histophilus somni stimulated bovine brain endothelial cells

Histophilus somni is a Gram negative coccobacillus that causes respiratory, reproductive and central nervous system disease in cattle. The hallmark of H. somni infection is diffuse vasculitis and intravascular thrombosis that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis (TME). Because neutrophils are major players in the pathophysiology of septic meningitis, we sought to determine their role in H. somni-induced fibrin clot formation in vitro. Bovine brain endothelial cells (TBBE cells) were exposed to H. somni cells at a 1:25 ratio, respectively. Conditioned media (CM) were collected after a 6 h incubation at 37 °C with 5% CO2, and then incubated with bovine peripheral blood polymorphonuclear neutrophils (PMNs). Following incubation, fibrin clot formation and tissue factor activity were assessed by a re-calcified plasma clotting assay. We found greater tissue factor activity in cell lysates and CM from H. somni-stimulated TBBE cells than unstimulated control TBBE cells. In addition, PMNs exposed to CM or extracellular vesicles from H. somni-stimulated TBBE cells expressed von Willenbrand factor, exhibited increased fibrin clot formation, and displayed greater tissue factor activity than PMNs exposed to CM or extracellular vesicles from unstimulated control TBBE cells. These results suggest that bovine PMNs might acquire extracellular vesicles from endothelial cells that leads to thrombus formation in bovine brain microvasculature and contribute to the process that characterizes TME.

Abstract WP547: FTY720 (Fingolimod) Ameliorates Ischemia Reperfusion Injury in Experimental Stroke Model

Background: Fingolimod (FTY720), a widely known sphingosine-1-phosphate (S1P) receptor agonist, was approved as a treatment for multiple sclerosis due to its strong anti-inflammatory effect. Methods: In-vivo study, FTY720 was injected intraperitoneally just before reperfusion. Neurological score and infarct volume were evaluated at Day0, 1, 3, 5, and 7 after MCAO. Evans Blue analysis and immunohistochemistry were conducted at day1 and day7. [18F]DPA-714 PET was performed 2 and 9 days after MCAO. In vitro study, in order to perform OGD, Bovine Brain Microvascular Endothelial Cells (BBMVECs) were incubated in a glucose-free medium under 1% oxygen condition, and then reperfused for 4 hours. In vitro models of ischemia/reperfusion injury were exposed to FTY720, FTY720-P, S1P and SEW (100 nM) immediately after reperfusion. The effects of FTY720 on the blood brain barrier were examined by real-time PCR analysis and immunofluorescence staining. Results: In this study, we observed that FTY720 reduced infarction size and improved neurological score after MCAO. [18F]DPA-714 PET showed a high standardized uptake value (SUV) around the ischemic area 2 days after MCAO. Although SUV was increased further 9 days after MCAO in both treatment groups, the increase was significantly inhibited in 1.5mg/kg group. In addition, FTY720 recovered the integrity of blood brain barrier which has been disrupted by ischemia/reperfusion injury. However, we observed that FTY720 can improve the integrity of blood brain barrier by inducing the translocation of tight junction and adherins junction, but FTY720 cannot ameliorate the expression of tight junction and adherins junction. Conclusions: The present results suggest that FTY720 improves neurological score, reduces infarction size, inflammatory activation and neuronal apoptosis. Especially ameliorates the integrity of Blood-Brain Barrier by inducing the translocation of tight junction and adherins junction after transient MCAO.