Method for preparing permanent brain slices and serial slice images for education and MRI correlation

It is important to understand the anatomical structures of the human brain in horizontal planes. Serially sectioned brain slices can easily be made with a meat slicer. The objective of this research was to enhance the educational value of serial brain slices made with a meat slicer through various applications. Two brains were taken out of two cadavers and embedded with gelatin solution to make two brain blocks. The first brain block was serially sectioned at 5 mm thickness using a meat slicer to make 28 horizontal brain slices. Each brain slice was embedded with a synthetic resin mixture to make 28 permanent specimens. The second brain block was magnetic resonance-scanned at 1.4 mm thickness to make 130 horizontal magnetic resonance images, then serially sectioned at the same thickness using the meat slicer to make 130 horizontal brain slices. Each brain slice was scanned into a computer to make a series of slice images. Ten anatomical structures in the slice images were outlined to make segmented images. Corresponding magnetic resonance images, slice images, and segmented images were stacked and volume-reconstructed to make three-dimensional images, which were sectioned and rotated at free angles. We show that the serial brain slices made with a meat slicer can be permanently preserved and used in a variety of educational settings. Anat Rec (Part B: New Anat) 289B:64-71, 2006. (c) 2006 Wiley-Liss, Inc.     

Immunocytochemical visualization of D-glutamate in the rat brain

Using highly specific antisera directed against conjugated d-amino acids, the distribution of d-glutamate-, d-tryptophan-, d-cysteine-, d-tyrosine- and d-methionine-immunoreactive structures in the rat brain was studied. Cell bodies containing d-glutamate, but not d-glutamate-immunoreactive fibers, were found. Perikarya containing this d-amino acid were only found in the mesencephalon and thalamus of the rat CNS. Thus, the highest density of cell bodies containing d-glutamate was observed in the dorsal raphe nucleus, the ventral part of the mesencephalic central gray, the superior colliculus, above the posterior commissure, and in the subparafascicular thalamic nucleus. A moderate density of immunoreactive cell bodies was observed in the dorsal part of the mesencephalic central gray, above the rostral linear nucleus of the raphe, the nucleus of Darkschewitsch, and in the medial habenular nucleus, whereas a low density was found below the medial forebrain bundle and in the posterior thalamic nuclear group. Moreover, no immunoreactive fibers or cell bodies were visualized containing d-tryptophan, d-cysteine, d-tyrosine or d-methionine in the rat brain. The distribution of d-glutamate-immunoreactive cell bodies in the rat brain suggests that this d-amino acid could be involved in several physiological mechanisms. This work reports the first visualization and the morphological characteristics of conjugated d-glutamate-immunoreactive cell bodies in the rat CNS using an indirect immunoperoxidase technique. Our results suggest that the immunoreactive neurons observed have an uptake mechanism for d-glutamate.     

新生鼠脑外伤后神经元变性的电镜观察

目的：从形态学角度探讨新生鼠脑外伤后神经元变性的机理。方法：建立新生7d大鼠顶叶皮质脑挫伤动物模型,在脑外伤后2h、6h、24h对同侧顶叶皮质和海马神经细胞进行电镜观察。结果：神经元有两类改变：(1)神经元树突和胞体呈巨大膨胀。早期内质网池扩大,线粒体致密和浓缩;此后内质网空泡化,线粒体进行性肿胀和空泡化,多聚核糖体从粗面内质网上解离,并散在于胞浆。核的改变出现于胞浆改变明显之后。核染色质由簇状集聚于核膜下呈钟面排列到向中央积聚成轮廓不规则的团块。轴突基本正常。(2)胞浆和胞核均浓缩,胞浆中有大小不等的空泡。结论：内源性兴奋毒对未成熟脑创伤性神经元变性起十分重要的作用。     

High-resolution brain tumor visualization using three-dimensional x-ray phase contrast tomography

We report on significant advances and new results concerning a recently developed method for grating-based hard x-ray phase tomography. We demonstrate how the soft tissue sensitivity of the technique is increased and show in vitro tomographic images of a tumor bearing rat brain sample, without use of contrast agents. In particular, we observe that the brain tumor and the white and gray brain matter structure in a rat's cerebellum are clearly resolved. The results are potentially interesting from a clinical point of view, since a similar approach using three transmission gratings can be implemented with more readily available x-ray sources, such as standard x-ray tubes. Moreover, the results open the way to in vivo experiments in the near future.     

喉组织切片与喉CT图像的对照研究及三维重建

目的对喉部MSCT和组织切片图像及其三维重建的比较研究。方法 30例(21男,9女)结构完整的喉标本,全喉连续大切片,HE染色,专业微距照相系统拍照,专业图像分析;30名(12男,18女)健康志愿者经64排高分辨率薄层MSCT扫描,得到喉部CT图像。在用3D-Doctor软件进行MSCT三维重建。测量组织切片和MSCT三维重建喉甲状软骨、环状软骨和6个切面会厌前间隙和声门旁间隙的面积,进行两组独立样本的t检验。结果 MSCT图像中甲状软骨、会厌软骨和环状软骨能清晰显示,但杓状软骨显示不全。会厌前间隙和声门旁间隙内容结构无法显示,而组织切片清晰显示间隙内容。组织切片和MSCT甲状软骨、环状软骨的测量结果无显著性差异(P0.05)。除了甲状软骨声带附着处至上、下切迹距离等四项数据无性别差异(P0.05)以外,其他数据均有性别差异(P0.05)。MSCT切割平面与组织切片会厌前间隙和声门旁间隙面积的测量结果无显著性差异(P0.05)。结论 MSCT对超过其分辨率的细微结构显示欠佳,其三维重建的细节效果不如组织切片完整清晰。组织切片能对MSCT起到良好的补充作用,使得MSCT及其三维重建作为临床医疗诊断和影像学检查的辅助工具,更适合于临床应用。     

外源性谷氨酸致新生鼠脑兴奋毒性神经变性的形态学研究

目的 :探讨外源性谷氨酸对新生鼠脑细胞的兴奋毒性作用。方法 :新生 7天SD大鼠 ,背部皮下注射单钠谷氨酸 (MSG) 2mg/ g ,在 2h、6～ 8h、2 4～ 2 8h分别光镜和电镜观察脑细胞的形态改变。结果 :光镜下 ,2h未见明显异常 ,此后可见多处脑细胞肿胀 ,进而细胞固缩。电镜着重观察海马神经元 ,变化一为神经元胞体和树突巨大膨胀 ,伴随着内质网膜的空泡化及线粒体由最早期的浓缩到巨大膨胀 ,轴突基本正常 ,核染色质由簇状集聚于核膜下呈钟面排列到向中央积聚成轮廓不规则的团块 ;变化二为胞浆和胞核均浓缩 ,胞奖中有大小不等的空泡。结论 :外源性谷氨酸能引起未成熟脑细胞的死亡 ,为某些食品添加剂对儿童脑细胞的影响的相关研究提供一定的形态学基础。     

Optically based-indentation technique for acute rat brain tissue slices and thin biomaterials

Currently, micro-indentation testing of soft biological materials is limited in its capability to test over long time scales due to accumulated instrumental drift errors. As a result, there is a paucity of measures for mechanical properties such as the equilibrium modulus. In this study, indentation combined with optical coherence tomography (OCT) was used for mechanical testing of thin tissue slices. OCT was used to measure the surface deformation profiles after placing spherical beads onto submerged test samples. Agarose-based hydrogels at low-concentrations (w/v, 0.3-0.6%) and acute rat brain tissue slices were tested using this technique over a 30-min time window. To establish that tissue slices maintained cell viability, allowable testing times were determined by measuring neuronal death or degeneration as a function of incubation time with Fluor-Jade C (FJC) staining. Since large deformations at equilibrium were measured, displacements of surface beads were compared with finite element elastic contact simulations to predict the equilibrium modulus, μ(∞) . Values of μ(∞) for the low-concentration hydrogels ranged from 0.07 to 1.8 kPa, and μ(∞) for acute rat brain tissue slices was 0.13 ± 0.04 kPa for the cortex and 0.09 ± 0.015 kPa for the hippocampus (for Poisson ratio = 0.35). This indentation technique offers a localized, real-time, and high resolution method for long-time scale mechanical testing of very soft materials. This test method may also be adapted for viscoelasticity, for testing of different tissues and biomaterials, and for analyzing changes in internal structures with loading.     

Correlations between the firing of supraoptic neurones in slices of rat brain

Summary These experiments were an attempt to study the possible interactions between cells of the supraoptic nucleus. In isolated brain slices pairs of supraoptic neurones were recorded simultaneously either with a single electrode or with two electrodes and cross-correlograms produced. Correlations were demonstrated in 22 of the 82 pairs studied and were found to be more common between closely neighbouring pairs of cells. Ten of the correlations indicated that the spikes of one cell followed spikes in the other cell. The correlations of another 10 pairs indicated that the cells were coactivated. In only 2 pairs was there a correlation indicative of an inhibitory connexion. That these correlations could result either from synaptic connexions within the nucleus, or from coactivation of cells from an extranuclear site is discussedBeit Memorial Fellow     

Stimulated amino acid imbalance and histidine transport in rat brain slices.

Histidine concentration in the brain decreases rapidly when rats are fed a low protein diet in which an amino acid imbalance is created by addition of an amino acid mixture devoid of histidine. Competition for histidine transport into the brain was suggested as an explanation for this effect. Therefore, animo acid mixtures simulating composition of plasma from rats fed basal or histidine-imbalanced diets were added to media to evaluate their effects on uptake of histidine by brain slices during a 60-min incubation period. At the concentrations actually found in plasma, the unbalanced mixture decreased histidine uptake significantly more than did the basal mixture. Two distinct inhibition patterns were observed with different groups of amino acids: a linear decrease in histidine uptake with a mixture of the small neutral, hydroxyl, basic, and acidic amino acids, and a hyperbolic decrease with a mixture of large neutral amino acids, and a hyperbolic decrease with a mixture of large neutral amino acids. Inhibition of histidine transport by the complete mixtures reflected these two effects. Plasma patterns and concentrations of competitive amino acids as well as the concentration of histidine appear to be factors involved in decreasing histidine transport into the brain.     

Chronic haloperidol treatment attenuates receptor-mediated phosphoinositide turnover in rat brain slices.

The long-term effects of haloperidol on phosphoinositide turnover in rat brain slices were investigated. Continuous treatment with haloperidol decanoate (21 mg/kg I.M. biweekly for 6 weeks) significantly attenuated carbachol- and norepinephrine (NE)-induced inositol phosphate accumulation in rat frontal cortex and hippocampus. In the striatum, the haloperidol treatment also significantly decreased carbachol-stimulated inositol phosphate level but did not significantly affect NE-sensitive phosphoinositide turnover. These effects were not observed in rats treated with a single dose of haloperidol (1.5 mg/kg). Basel levels of inositol phosphate in these 3 brain regions did not change following continuous or single haloperidol doses.     

Characterization of synaptic transmission in the ventrolateral periaqueductal gray of rat brain slices

Synaptic transmission evoked by focal stimulation in the ventrolateral periaqueductal gray was characterized using the whole-cell recording technique in rat brain slices. At resting membrane potential (-62+/-1 mV), focal stimulation (0.05-0.1 ms, 0.03 Hz) usually evoked a 6-cyano-7-nitroquinoxaline-2, 3-dione-sensitive fast excitatory postsynaptic potential and a DL-2-amino-5-phosphonopentanoic acid-sensitive slow excitatory postsynaptic potential with a bicuculline-sensitive inhibitory postsynaptic potential in between. In the presence of kynurenic acid, bicuculline-sensitive inhibitory postsynaptic currents recorded in the voltage-clamp mode displayed a reversal potential of -68+/-3 mV, resembling GABA(A) receptor-mediated inhibitory postsynaptic currents. However, no GABA(B) receptor-mediated inhibitory postsynaptic current was evoked, even at stronger stimulating intensity. 6-Cyano-7-nitroquinoxaline-2,3-dione-sensitive fast excitatory postsynaptic currents were isolated by DL-2-amino-5-phosphonopentanoic acid plus bicuculline and DL-2-amino-5-phosphonopentanoic acid-sensitive slow fast excitatory postsynaptic currents by bicuculline plus 6-cyano-7-nitroquinoxaline-2,3-dione. Both types of excitatory postsynaptic current reversed at potentials near 0 mV. The I-V curve of slow fast excitatory postsynaptic currents or N-methyl-D-aspartate currents displayed a negative slope at potentials more negative than -30 mV in an Mg(2+)-sensitive manner. The control postsynaptic currents reversed at potentials between -50 and -35 mV, inclined to the reversal potential of GABA(A), but not glutamate, receptor channels. It is concluded that, in the ventrolateral periaqueductal gray, focal stimulation elicits both inhibitory and excitatory transmission, while the former is dominant. The inhibitory transmission is mediated by GABA(A) but not GABA(B) receptors. The excitatory transmission is mediated by glutamate acting on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate as well as N-methyl-D-aspartate receptors.     

Granulocyte macrophage-colony stimulating factor activates microglia in rat cortex organotypic brain slices

Cytokines play an important role in the regulation of proliferation and migration in the central nervous system. The aim of this study was to determine if granulocyte macrophage-colony stimulating factor (GM-CSF) activates cells in the cortex of organotypic brain slice cultures. Our data show that murine GM-CSF markedly stimulated the proliferation and migration of small round microglia from a cortex slice. These round cells were strongly positive for integrin CD11b (OX-42), isolectin B4-lectin-binding, the monocytic marker ED1 and partly expressed major histocompatibility complex (MHC) class II antigen (OX-6). Only some differentiated microglia were visible which expressed the integrin CD11c and MHCII. GM-CSF enhanced the proliferation as analyzed by bromodeoxyuridine incorporation. The number of migrated cells decreased during culturing and enhanced terminal dUTP nick-end labelling positive nuclei were found. Taken together, our data conclude that GM-CSF is an important cytokine, which regulates the proliferation and migration of cortical microglia.     

An algorithm for three-dimensional visualization of radiation therapy beams

A computer algorithm to display radiation beams superimposed on three-dimensional (3-D) views of patient anatomy has been developed. It may be implemented as a postprocessing step to existing software for 3-D presentation and display. The algorithm takes as input a shaded 3-D view (reconstructed, for example, from computed tomography scans), together with the associated depth map, and generates as output an enhanced 3-D view highlighting in color the visible points which lie within the projected beam outlines. The algorithm is independent of the method used to generate the 3-D view (surface or volume rendering techniques may be used) and is independent of beam shape (beams may be modified with shielding blocks). It is not restricted to external surfaces and will correctly show radiation beams projected onto cut-away views of internal organs. The method is illustrated by application to a tangential pair for breast malignancy, using 3-D views generated with volume rendering software.     

Number of correlates of membrane metabolism and long-term potentiation in rat brain slices

Long-term potentiation was elicited in living slices of rat olfactory cortex by stimulation of the lateral olfactory tract. A group of interdependent parameters of membrane metabolism was studied, i.e., the kinetics of⁴⁵Ca metabolism, lipid peroxidation, and antioxidant defense; cytochemical measurements were made of Na⁺, K⁺-ATPase activity in neurons and glial cells; the functional (GTPase) activity of G-proteins was also studied. All parameters were compared with the bioelectrical activity of slices at three time points after tetanization, i.e., 3–5, 15, and 30 min. In most cases, regular phasic changes in metabolic parameters occurred, and their functional significance is discussed. Laboratory of Functional Neurochemistry (N. A. Emel'yanov, Director), I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Fiziologicheskii Zhurnal im. I. M. Sechenova, Vol. 81, No. 8, pp. 29–33, August, 1995.     

NMDA induces protein kinase C translocation in hippocampal slices of immature rat brain

N-Methyl-D-aspartate (NMDA)-induced translocation of protein kinase C (PKC) from cytosol to membrane fractions was examined by the methods of [3H]phorbol 12,13-dibutyrate binding and western blotting in rat hippocampal slices. NMDA and L-glutamate induced translocation of PKC from cytosol to membrane fractions in immature rat hippocampal slices, but not in mature ones. The NMDA-induced translocation of PKC was dependent on Ca2+. It was inhibited by the NMDA receptor antagonists, 2-amino-5-phosphonovaleric acid and ketamine, but not by Mg2+ and Zn2+. These results suggest that stimulation of NMDA receptors enhances Ca2+ influx and thereby induces translocation of PKC in immature rat hippocampus.     

Cell-specific spike-timing-dependent plasticity in GABAergic and cholinergic interneurons in corticostriatal rat brain slices

Striatum, the main input nucleus of basal ganglia, is involved in the learning of cognitive and motor sequences in response to environmental stimuli. Striatal output neurons (medium spiny neurons, MSNs) integrate cortical activity and the two main classes of interneurons (GABAergic and cholinergic interneurons) tightly regulate the corticostriatal information transfer. We have explored the transmission between cortex and striatal interneurons and their capability to develop activity-dependent long-term plasticity based on the quasi-coincident cortical and striatal activities (spike-timing-dependent plasticity, STDP). We have observed glutamatergic monosynaptic connections between cortical cells and both striatal interneurons. Excitatory postsynaptic current latencies and rise times revealed that a cortical stimulation activates GABAergic interneurons before cholinergic, and both interneurons before MSNs. In addition, we have observed that striatal interneurons are able to develop bidirectional long-term plasticity and that there is a cell-specificity of STDP among striatal interneurons. Indeed, in GABAergic interneurons, long-term depression (LTD) and long-term potentiation (LTP) are induced by post-pre and pre-post STDP protocols, respectively. Cholinergic interneurons displayed a partially reversed STDP when compared to GABAergic interneurons: post-pre protocols induced LTP as well as LTD (the induction of either LTP or LTD is correlated with rheobase) and pre-post protocols induced LTD. The cell-specificity of STDP also concerned the receptors activated for the induction of LTP and LTD in GABAergic and cholinergic interneurons: in GABAergic interneurons LTP and LTD required NMDA receptor-activation whereas, in cholinergic interneurons, LTP was underlain by NMDA receptor-activation and LTD by metabotropic glutamate receptors.