Histological Brain Sections Research Articles

Biodistribution and toxicity evaluation of sesbania mosaic virus nanoparticles in mice.

Sesbania mosaic virus (SeMV), a 30-nm spherical plant sobemovirus, is suitable for developing functionalized nanoparticles for biomedical applications. However, the in vivo behavior of SeMV and the clinical impact following its delivery via the oral or intravenous route are not known. To address this question, we examined the biodistribution, toxicity and histopathological changes in SeMV treated mice. No toxic effects were observed in mice administered high doses (100mg and 200mg per kg body weight orally or 40mg and 80mg per kg body weight intravenously) of SeMV, and they were found to be normal. Analysis of fecal sample showed that SeMV was cleared in 16h when 20mg of the virus per kg body weight was administered orally. RT-PCR analysis of blood samples showed that SeMV was present up to 72h in mice inoculated either intravenously (8mg/kg body weight) or orally (20mg/kg body weight). Further, SeMV was found to be localized up to 72h in spleen and liver tissues of intravenously inoculated mice only. Biochemical and hematological parameters were found to be normal at 6 and 72h after administration of SeMV. Furthermore, no noticeable changes were observed in histological sections of brain, liver, spleen, lungs and kidney tissue samples collected at 6 and 72h from SeMVadministered mice when compared to control mice. Thus, SeMV appears to be a safe and non-toxic platform that can be tailored as a nanocarrier for in vivo biomedical applications.

Oral administration of potassium bromate induces neurobehavioral changes, alters cerebral neurotransmitters level and impairs brain tissue of swiss mice.

BackgroundPotassium bromate (KBrO3) is widely used as a food additive and is a major water disinfection by-product. The present study reports the side effects of KBrO3 administration on the brain functions and behaviour of albino mice.MethodsAnimals were divided into three groups: control, low dose KBrO3 (100 mg/kg/day) and high dose KBrO3 (200 mg/kg/day) groups.ResultsAdministration of KBrO3 led to a significant change in the body weight in the animals of the high dose group in the first, second and the last weeks while water consumption was not significantly changed. Neurobehavioral changes and a reduced Neurotransmitters levels were observed in both KBrO3 groups of mice. Also, the brain level of reduced glutathione (GSH) in KBrO3 receiving animals was decreased. Histological studies favoured these biochemical results showing extensive damage in the histological sections of brain of KBrO3-treated animals.ConclusionsThese results show that KBrO3 has serious damaging effects on the central nervous system and therefore, its use should be avoided.

A Role for P-Glycoprotein in Clearance of Alzheimer Amyloid β -Peptide from the Brain.

Most data indicates that Alzheimer's disease involves an accumulation of amyloid β - peptide (Aβ) in the CNS and that sporadic cases arise from a deficiency in Aβ clearance. Considerable attention has been given to mechanisms by which Aβ might be transported between the brain and blood, and evidence suggests that p-glycoprotein, also known as the multi-drug resistance (MDR) protein (product of the ABCB1 gene), plays a role in Aβ transport across the blood-brain barrier (BBB). We tested this possibility through two approaches: First, wild-type and MDR1A-knockout mice were compared after intravenous injection of [(125)I]-labeled Aβ; after 60 min, homogenates of brain parenchyma were subjected to γ-counting of TCA-precipitable material, and histological sections of brain were subjected to autoradiography. Second, MDR1Aknockout mice were crossed with Tg2576 APP transgenic mice, a line that routinely accumulates Aβ in the brain; SDS and formic acid extracts of brain homogenates were assessed for Aβ levels by ELISA. Each of these approaches yielded data indicating that Aβ accumulates to a greater degree in mice lacking MDR1A. These findings confirm other reports linking p-glycoprotein to Aβ clearance across the BBB and have important implications for Alzheimer's disease genetics, pharmacology, and epidemiology.

Quantitative and Qualitative Analysis of Transient Fetal Compartments during Prenatal Human Brain Development.

The cerebral wall of the human fetal brain is composed of transient cellular compartments, which show characteristic spatiotemporal relationships with intensity of major neurogenic events (cell proliferation, migration, axonal growth, dendritic differentiation, synaptogenesis, cell death, and myelination). The aim of the present study was to obtain new quantitative data describing volume, surface area, and thickness of transient compartments in the human fetal cerebrum. Forty-four postmortem fetal brains aged 13–40 postconceptional weeks (PCW) were included in this study. High-resolution T1 weighted MR images were acquired on 19 fetal brain hemispheres. MR images were processed using in-house software (MNI-ACE toolbox). Delineation of fetal compartments was performed semi-automatically by co-registration of MRI with histological sections of the same brains, or with the age-matched brains from Zagreb Neuroembryological Collection. Growth trajectories of transient fetal compartments were reconstructed. The composition of telencephalic wall was quantitatively assessed. Between 13 and 25 PCW, when the intensity of neuronal proliferation decreases drastically, the relative volume of proliferative (ventricular and subventricular) compartments showed pronounced decline. In contrast, synapse- and extracellular matrix-rich subplate compartment continued to grow during the first two trimesters, occupying up to 45% of telencephalon and reaching its maximum volume and thickness around 30 PCW. This developmental maximum coincides with a period of intensive growth of long cortico-cortical fibers, which enter and wait in subplate before approaching the cortical plate. Although we did not find significant age related changes in mean thickness of the cortical plate, the volume, gyrification index, and surface area of the cortical plate continued to exponentially grow during the last phases of prenatal development. This cortical expansion coincides developmentally with the transformation of embryonic cortical columns, dendritic differentiation, and ingrowth of axons. These results provide a quantitative description of transient human fetal brain compartments observable with MRI. Moreover, they will improve understanding of structural-functional relationships during brain development, will enable correlation between in vitro/in vivo imaging and fine structural histological studies, and will serve as a reference for study of perinatal brain injuries.

Abstract IA27: Functional genomics identifies drivers of medulloblastoma dissemination

Abstract The pediatric brain tumor medulloblastoma shows a characteristic pattern of metastasis, called leptomeningeal dissemination (LMD), in which tumor cells disseminate via the cerebrospinal fluid to the leptomeningeal surfaces of the brain and spinal cord. This pattern distinguishes medulloblastomas from other solid tumors, which metastasize through the bloodstream or lymphatic channels. The invasion-metastasis cascade, a widely accepted paradigm derived from studies of hematogenous spread of carcinomas, is envisioned as a multistep process, governed by three set of genes: initiation, progression, virulence genes. By contrast, our knowledge of the molecular mediators of medulloblastoma LMD is rudimentary. Initial insight into the genetic drivers of LMD came from a transposon insertion mutagenesis study (Wu et al, Nature 482: 529-33, 2012). Mobilizing the Sleeping Beauty transposon in cerebellar neural progenitor cells caused widespread dissemination of typically localized, nonmetastatic medulloblastomas in Patched+/- mice, in which Sonic Hedgehog (Shh) signaling is hyperactive. Candidate LMD genes were identified by sequencing the insertion sites and then mapping these sequences back to the mouse genome. To determine whether genes located at transposon insertion sites directly caused medulloblastomas to disseminate, we overexpressed candidate genes in Nestin+ neural progenitors in the cerebella of mice by retroviral transfer in combination with Shh and examined histological sections of brain and spinal cord during a 4-month observation period. Ectopic expression of the following Sleeping Beauty common insertion site genes shifted the in vivo growth characteristics of Shh-induced medulloblastomas from a localized pattern to a disseminated pattern: Eras (embryonic stem cell-expressed Ras), Lhx1 (LIM homeobox protein), Ccrk (cell cycle-related kinase), Akt (PI3K signaling protein), Arnt (hypoxia-inducible factor-1β), Gdi2 (Rab-GDP dissociation inhibitor), and Smad5 (TGF-β signal transducer). To elucidate the physiological traits conferred by these genes, we overexpressed them in a human medulloblastoma cell line (DAOY) and an immortalized, nontransformed cell line derived from mouse granule neuron precursors (SHH-NPD) and then quantified migration, invasiveness, and anchorage independence, growth characteristics that are closely associated with metastatic competence in carcinomas. Although the effects were quantitatively different in DAOY compared with SHH-NPD, all genes that induced LMD in vivo conferred these traits to cells in culture. We have used the set of genes discovered through Sleeping Beauty mutagenesis to begin constructing an LMD cascade, analogous to the invasion-metastasis cascade. Some LMD driver genes (Eras and Akt) enhanced the growth of the primary tumor, as indicated by shortened animal survival, while others did not. Therefore, we propose a two-category scheme for the LMD cascade, in which genes that increase primary tumor growth (Eras and Akt) are analogous to initiation/progression genes, whereas genes that only promote the growth and survival of disseminating cells (Lhx1, Ccrk, Arnt, Gdi2, and Smad5) are analogous to virulence genes. This simplified scheme will serve as a framework, upon which future research can build more sophisticated versions that better reflect the complexity of LMD. Citation Format: Daniel W. Fults. Functional genomics identifies drivers of medulloblastoma dissemination. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA27.

A Jones matrix formalism for simulating three-dimensional polarized light imaging of brain tissue.

The neuroimaging technique three-dimensional polarized light imaging (3D-PLI) provides a high-resolution reconstruction of nerve fibres in human post-mortem brains. The orientations of the fibres are derived from birefringence measurements of histological brain sections assuming that the nerve fibres—consisting of an axon and a surrounding myelin sheath—are uniaxial birefringent and that the measured optic axis is oriented in the direction of the nerve fibres (macroscopic model). Although experimental studies support this assumption, the molecular structure of the myelin sheath suggests that the birefringence of a nerve fibre can be described more precisely by multiple optic axes oriented radially around the fibre axis (microscopic model). In this paper, we compare the use of the macroscopic and the microscopic model for simulating 3D-PLI by means of the Jones matrix formalism. The simulations show that the macroscopic model ensures a reliable estimation of the fibre orientations as long as the polarimeter does not resolve structures smaller than the diameter of single fibres. In the case of fibre bundles, polarimeters with even higher resolutions can be used without losing reliability. When taking the myelin density into account, the derived fibre orientations are considerably improved.

Overview and assessment of the histochemical methods and reagents for the detection of β-galactosidase activity in transgenic animals.

Bacterial β-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect β-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri- and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing β-galactosidase, and despite its efficiency in staining whole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional β-galactosidase histochemistry. Here, we compare the traditional β-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for β-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri- and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of β-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.

Targeting of cell death and neuroinflammation with peptide-linked iron oxide nanoparticles and Gd-DTPA in a mouse model of Parkinson's disease

Parkinson’s disease (PD) is one of the most common neurodegenerative disease and remains difficult to diagnose by conventional methods of early detection. It is characterized by the apoptotic loss of dopaminergic neurons (DN) and a neuroinflammation mainly located in the ventral midbrain (VM). The aim of this work is to study new vectorized contrast agents for magnetic resonance imaging (MRI) detection of PD injured areas by targeting apoptosis and inflammation. Two peptides selected by phage display were used for the experiments: R826 peptide, selected for its affinity for the phosphatidylserine (PS) exposed at the external surface of apoptotic cells, and 2C peptide, selected for its affinity for TNF-α (tumor necrosis factor alpha), one of the most abundant cytokines secreted during inflammation. These peptides were grafted to pegylated iron oxide nanoparticles (PEG-USPIO) and gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) for in vitro and in vivo studies, respectively. PD was simulated on mice with the MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) neurotoxin and its active metabolite MPP+ (1-methyl-4-phenylpyridinium) for in vitro studies. The results showed that PEG-USPIO-R826 and PEG-USPIO-2C enabled the detection of apoptosis and inflammation, respectively, on MPP+-treated culture cells. PEG-USPIO-2C also allowed detection of inflammation on histological brain sections of MPTP-treated mice. The 2C peptide grafted to Gd-DTPA showed encouraging results in MRI detection of injured brain areas in MPTP-treated mice. These observations suggest that a targeting of damaged cells and injured areas by these new specific contrast agents occurs, offering a new tool for early diagnosis of neurodegenerative disorders like PD by MRI.

High-Throughput Method of Whole-Brain Sectioning, Using the Tape-Transfer Technique.

Cryostat sectioning is a popular but labor-intensive method for preparing histological brain sections. We have developed a modification of the commercially available CryoJane tape collection method that significantly improves the ease of collection and the final quality of the tissue sections. The key modification involves an array of UVLEDs to achieve uniform polymerization of the glass slide and robust adhesion between the section and slide. This report presents system components and detailed procedural steps, and provides examples of end results; that is, 20μm mouse brain sections that have been successfully processed for routine Nissl, myelin staining, DAB histochemistry, and fluorescence. The method is also suitable for larger brains, such as rat and monkey.

Morphology and morphometry of the human embryonic brain: A three-dimensional analysis

The three-dimensional dynamics and morphology of the human embryonic brain have not been previously analyzed using modern imaging techniques. The morphogenesis of the cerebral vesicles and ventricles was analyzed using images derived from human embryo specimens from the Kyoto Collection, which were acquired with a magnetic resonance microscope equipped with a 2.35-T superconducting magnet. A total of 101 embryos between Carnegie stages (CS) 13 and 23, without apparent morphological damage or torsion in the brain ventricles and axes, were studied. To estimate the uneven development of the cerebral vesicles, the volumes of the whole embryo and brain, prosencephalon, mesencephalon, and rhombencephalon with their respective ventricles were measured using image analyzing Amira™ software. The brain volume, excluding the ventricles (brain tissue), was 1.15±0.43mm3 (mean±SD) at CS13 and increased exponentially to 189.10±36.91mm3 at CS23, a 164.4-fold increase, which is consistent with the observed morphological changes. The mean volume of the prosencephalon was 0.26±0.15mm3 at CS13. The volume increased exponentially until CS23, when it reached 110.99±27.58mm3. The mean volumes of the mesencephalon and rhombencephalon were 0.20±0.07mm3 and 0.69±0.23mm3 at CS13, respectively; the volumes reached 21.86±3.30mm3 and 56.45±7.64mm3 at CS23, respectively. The ratio of the cerebellum to the rhombencephalon was approximately 7.2% at CS20, and increased to 12.8% at CS23. The ratio of the volume of the cerebral vesicles to that of the whole embryo remained nearly constant between CS15 and CS23 (11.6–15.5%). The non-uniform thickness of the brain tissue during development, which may indicate the differentiation of the brain, was visualized with surface color mapping by thickness. At CS23, the basal regions of the prosencephalon and rhombencephalon were thicker than the corresponding dorsal regions. The brain was further studied by the serial digital subtraction of layers of tissue from both the external and internal surfaces to visualize the core region (COR) of the thickening brain tissue. The COR, associated with the development of nuclei, became apparent after CS16; this was particularly visible in the prosencephalon. The anatomical positions of the COR were mostly consistent with the formation of the basal ganglia, thalamus, and pyramidal tract. This was confirmed through comparisons with serial histological sections of the human embryonic brain. The approach used in this study may be suitable as a convenient alternative method for estimating the development and differentiation of the neural ganglia and tracts. These findings contribute to a better understanding of brain and cerebral ventricle development.

Understanding fiber mixture by simulation in 3D Polarized Light Imaging

3D Polarized Light Imaging (3D-PLI) is a neuroimaging technique that has opened up new avenues to study the complex architecture of nerve fibers in postmortem brains. The spatial orientations of the fibers are derived from birefringence measurements of unstained histological brain sections that are interpreted by a voxel-based analysis. This, however, implies that a single fiber orientation vector is obtained for each voxel and reflects the net effect of all comprised fibers. The mixture of various fiber orientations within an individual voxel is a priori not accessible by a standard 3D-PLI measurement. In order to better understand the effects of fiber mixture on the measured 3D-PLI signal and to improve the interpretation of real data, we have developed a simulation method referred to as SimPLI. By means of SimPLI, it is possible to reproduce the entire 3D-PLI analysis starting from synthetic fiber models in user-defined arrangements and ending with measurement-like tissue images. For the simulation, each synthetic fiber is considered as an optical retarder, i.e., multiple fibers within one voxel are described by multiple retarder elements. The investigation of different synthetic crossing fiber arrangements generated with SimPLI demonstrated that the derived fiber orientations are strongly influenced by the relative mixture of crossing fibers. In case of perpendicularly crossing fibers, for example, the derived fiber direction corresponds to the predominant fiber direction. The derived fiber inclination turned out to be not only influenced by myelin density but also systematically overestimated due to signal attenuation. Similar observations were made for synthetic models of optic chiasms of a human and a hooded seal which were opposed to experimental 3D-PLI data sets obtained from the chiasms of both species. Our study showed that SimPLI is a powerful method able to test hypotheses on the underlying fiber structure of brain tissue and, therefore, to improve the reliability of the extraction of nerve fiber orientations with 3D-PLI.

B01 Development Of Aptamers For The Specific Detection Of The Amino-terminal Region Of The Huntingtin Protein

Introduction The huntingtin protein (HTT) has 3144 amino acids and undergoes numerous post-translational modifications. Its amino-terminal end is highly conserved and seems to be involved in huntingtin-huntingtin interactions. Several antibodies have been developed for detecting the amino-terminal domain of HTT (including N17, the CAG tract and the polyProline region), but few have been shown to detect endogeneous HTT expression using histological means. Objectives To develop “custom-made” aptamers that can be used to detect HTT in neuritic aggregates in human and rodent histological material. Methods Aptamers are synthetic DNA-based polymers selected in vitro from random-nucleotide combination libraries. For this project, we used single-chain oligonucleotides due to their flexible nature. These oligonucleotides have been selected against a synthetic albumin-bound peptide and with a polypeptide corresponding to the first exon of HTT (67-amino acid-long polypeptide plus a poly-Q stretch of 23 glutamine residues) bound to glutathione-S-transferase (GST) protein. Results The aptamers obtained varied in the recognition of the recombinant and the native protein in Western-Blots and immunocytochemical analysis of histological sections. An aptamer against the first 12 AA of HTT (termed HTT5) was obtained, which is able to recognise not only normal HTT in control brain sections but also mutant HTT in neuritic aggregates and intranuclear inclusions in histological brain sections from Huntington’s disease patients. Conclusions Aptamer HTT5 is a new tool for the detection of normal and mutant HTT that can be used for the specific recognition of this protein in cells and tissues from animal models and HD-affected patients.

Degeneration of neuronal cells: A product of fluoride and aluminium assault to the prefrontal cortex

Studies have raised the possibility that prolonged exposure to fluoride and aluminium in drinking water is capable of causing neurological impairments. Due to the possible chronic exposure to these substances and their ability to readily interact to form a complex which crosses the blood-brain barrier, it is imperative to assess their neurotoxic effects. This study describes the alterations in the nervous system as a result of treatment with 10 mg/kg sodium fluoride and 200 mg/kg aluminium chloride (AlCl3) for 21 days. Histological sections of brain were stained with Hematoxylin and Eosin (H&E), Cresyl fast violet and Periodic-Acid Schiff (PAS) to determine various and distinct changes in the morphology of the cells. Results revealed enlarged cells and membrane degeneration in the treatment group which suggests excitotoxicity and oxidative stress, respectively. The study therefore concludes that fluoride and aluminium have neurotoxic effects by their ability to induce excitotoxicity, oxidative stress and cellular damage. Key words: Fluoride, aluminium, prefrontal cortex, excitotoxicity, oxidative stress.

Neonatal SVZ EGFP-labeled cells produce neurons in the olfactory bulb and astrocytes in the cerebral cortex by in-vivo electroporation

Neural progenitors/stem cells (NSCs) exist in neonatal mouse subventricular zone (SVZ). To explore the differentiation of the NSCs in neonatal mouse SVZ and the distribution of the progeny cells derived from these NSCs in early adulthood, the enhanced green fluorescent protein (EGFP) plasmid was transferred into the NSCs in the lateral ventricle of newborn mice (P0) by in-vivo electroporation to trace these cells and their progeny cells. Thirty days after electroporation, histological sections of mouse brain were prepared for immunofluorescence with cell-specific antibodies to identify the type(s) of cells that were marked by EGFP. The results showed that EGFP-positive cells were distributed mainly in the olfactory bulb (OB), cortex, and SVZ, and double labeled with NeuN (neuron marker) in OB, glial fibrillary acidic protein (GFAP) (astrocyte marker) in the cortex, and Blbp and GFAP (astrocyte marker) in SVZ. However, there was no-EGFP-positive cell in the hippocampus. The present results indicate that the NSCs in SVZ of the neonatal mouse can give rise to neurons in the OB and astrocytes in the cortex in early adulthood, but not generate progeny cells residing in the hippocampus. In addition, there are still neural progenitors in SVZ until early adulthood.

Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents

AimTo explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO).MethodsIsolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed.ResultsNonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity.ConclusionMicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema.

A High-Resolution Enhancer Atlas of the Developing Telencephalon

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

Lipid peptide nanocomplexes for gene delivery and magnetic resonance imaging in the brain

Gadolinium-labelled nanocomplexes offer prospects for the development of real-time, non-invasive imaging strategies to visualise the location of gene delivery by MRI. In this study, targeted nanoparticle formulations were prepared comprising a cationic liposome (L) containing a Gd-chelated lipid at 10, 15 and 20% by weight of total lipid, a receptor-targeted, DNA-binding peptide (P) and plasmid DNA (D), which electrostatically self-assembled into LPD nanocomplexes. The LPD formulation containing the liposome with 15% Gd-chelated lipid displayed optimal peptide-targeted, transfection efficiency. MRI conspicuity peaked at 4h after incubation of the nanocomplexes with cells, suggesting enhancement by cellular uptake and trafficking. This was supported by time course confocal microscopy analysis of transfections with fluorescently-labelled LPD nanocomplexes. Gd-LPD nanocomplexes delivered to rat brains by convection-enhanced delivery were visible by MRI at 6h, 24h and 48h after administration. Histological brain sections analysed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) confirmed that the MRI signal was associated with the distribution of Gd3+ moieties and differentiated MRI signals due to haemorrhage. The transfected brain cells near the injection site appeared to be mostly microglial. This study shows the potential of Gd-LPD nanocomplexes for simultaneous delivery of contrast agents and genes for real-time monitoring of gene therapy in the brain.

Some Hematological and Histological Impact of sub-acute exposure to Mono Sodium Glutamate in Mice

Mono Sodium Glutamate (MSG) is a food additive commonly consumed as a flavor enhancer. However, both animal models and human clinical reports have established its harmful effects. The present study aimed to investigate the effect of MSG consumption on some hematological parameters and histological sections of brain, small intestine, liver and testis tissues of Swiss Albino mice. Albino mice (n=15) of average weight 27.4gm were randomly assigned into three groups A, B and C in each group (n=5). Treatment groups (A & B) were given 3g and 6g per Kg of body weight of MSG (amount of MSG in a single sachet consumed by human is 3g) respectively. The mice were sacrificed on day fifteenth of the experiment. Brain, small intestine, liver and testis were carefully dissected out and immediately fixed in 10% formalin for routine histological procedure. Blood samples were analyzed for hematological parameters. Histological findings of small intestine in the treated groups showed evidence of cellular hypertrophy and increased number of goblet cells. Large nuclei with multi nucleoli were seen in liver sections, while prevascular and preneuronal edema were seen in brain sections and thickening of basement membrane of semineferons tubules. Vacuolation of spermatogonia were seen in testicular tissue sections. Blood analysis showed a significant increase at P>0.01 in lymphocytes count compared to the control which can be considered indication of a compromised immune status and poisoning in the treated animals. Packed Cell Volume (PCV) hemoglobin (Hb) and red blood cells count (RBCs) were all indicative of an anemic condition in the treated animals. Significant increase in body weight at P>0.01 was seen in both treatment groups (A & B). All above findings indicate that MSG has some deleterious effects on some hematological parameters and caused histological changes of examined organs as well as on body weight. It is recommended that further studies aimed at corroborating these findings to be carried out.

Optimal Percoll concentration facilitates flow cytometric analysis for annexin V/propidium iodine‐stained ischemic brain tissues

We sought to determine the optimal Percoll concentration for ischemic rat brain prepared for flow cytometric (FC) measurements. Animals were subjected to the right middle cerebral artery (MCA) occlusion, and were euthanized at 3, 12, 24, and 72 h after reperfusion onset. The brains were processed by different concentrations (unisolated, 20, 25, 30, or 40%) of Percoll and stained with annexin V/propidium iodine (PI). Ischemic brain damage was evaluated by FC analysis and image analysis for histologic sections. The relative susceptibility of different phenotypes of cells to necrotic and apoptotic damage were evaluated by the FC analyses for the immunohistochemistry, PI, and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-processed brain tissues. Our results showed that FC analysis effectively detected the extent and maturation of apoptotic/necrotic brain damage, and the results were consistent with those determined from histologic brain sections. Neuron was more vulnerable to apoptosis than glia, whereas both cellular phenotypes were compatible in susceptibility for necrotic cell death. Percoll at a low concentration (20%) could effectively remove tissue debris without affecting membranous integrity of the injured neurons. Conversely, high percentages of Percoll (30-40%) substantially increased membranous damage for the injured cells. These results supported the application of FC to determine the extent and progression in time, as well as relative phenotypes of apoptotic/necrotic cell deaths following ischemic damage. We highlighted the use of Percoll at low percentages to facilitate the removal of tissue debris and to improve membrane integrity preservation for the injured neurons.

Automated atlas-based segmentation of NISSL-stained mouse brain sections using supervised learning

The problem of segmentation of mouse brain images into anatomical structures is an important stage of practically every analytical procedure for these images. The present study suggests a new approach to automated segmentation of anatomical structures in the images of NISSL-stained histological sections of mouse brain. The segmentation algorithm is based on the method of supervised learning using the existing anatomical labeling of the corresponding sections from a specialized mouse brain atlas. A mouse brain section to be segmented into anatomical structures is preliminarily associated with a section from the mouse brain atlas displaying the maximum similarity. The image of this section is then preprocessed in order to enhance its quality and to make it as close to the corresponding atlas image as possible. An efficient algorithm of luminance equalization, an extension of the well-known Retinex algorithm is proposed. A random forest is trained on pixel feature vectors constructed based on the atlas section images and the corresponding class labels associated with anatomical structures extracted from the atlas anatomical labeling. The trained classifier is then applied to classify pixels of an experimental section into anatomical structures. A new combination of features based on superpixels and location priors is suggested. Accuracy of the obtained result is increased by using Markov random field. Procedures of luminance equalization and subsequent segmentation into anatomical structures have been tested on real experimental sections.