Pieces Of Brain Tissue Research Articles

Using neurogenic explants to assess the role of the stem cell niche structure fractones in adult neurogenesis and pathology

Neurogenesis occurs throughout adulthood in restricted zones of the adult brain. Growth factors influence neural stem cell proliferation and differentiation. However, the mechanisms orchestrating growth factors to regulate neurogenesis are not well known. In the adult brain, neural stem cells and their immediate progeny reside in a nurturing stem cell niche environment. These niches are located in the ventricular/sub ventricular zone (VZ/SVZ) of the lateral ventricles. In the SVZ, stem cells are directly contacted by extracellular matrix (ECM) structures that we have named fractones. Using in vivo experiments, we have previously shown that fractones contain heparan sulfate proteoglycans (HSPG) that bind, concentrate and dispatch growth factors to the target stem cells to ultimately regulate stem cell fate. Here, an ex vivo model was devised to expand our ability to study fractones, stem cells, and growth factor interactions in culture in a near‐physiological context. This model consists of brain tissue pieces of the neurogenic zone, termed neurogenic explant. Neurogenic explants survive for months in a simple culture medium. They contain neural stem cells, their immediate progeny, ependymocytes, fractones, all forming the VZ/SVZ, plus the adjacent superficial part of the caudate nucleus. Using mini‐incubator and fluorescence microscopy, we demonstrate that we can visualize fractones, cell proliferation and growth factor binding on the living neurogenic explants. The ease in altering the ECM of explants and assessing the effects on neurogenic stem cells will allow us to understand the role of fractones in neurogenesis. By using explants originating from animal models, we will also be able to understand the relationship between disrupted adult neurogenesis and neural pathology. We anticipate that we can determine from the neurogenic explant model which HSPGs are responsible for the binding of individual growth factors. This will provide insight into the possibility of manipulating HSPG expression in explants to ultimately alter the cell‐fractone interactions responsible for the progression of disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Brain tissue banking for stem cells for our future

In our lab we study neurogenesis and the development of brain tumors. We work towards treatment strategies for glioblastoma and towards using autologous neural stem cells for tissue regeneration strategies for brain damage and neurodegenerative disorders. It has been our policy to try to establish living cell cultures from all human biopsy material that we obtain. We hypothesized that small pieces of brain tissue could be cryopreserved and that live neural stem cells could be recovered at a later time. DMSO has been shown to possess a remarkable ability to diffuse through cell membranes and pass into cell interiors. Its chemical properties prevent the formation of damaging ice crystals thus allowing cell storage at or below −180 C. We report here a protocol for successful freezing of small pieces of tissue derived from human brain and human brain tumours. Virtually all specimens could be successfully revived. Assays of phenotype and behaviour show that the cell cultures derived were equivalent to those cultures previously derived from fresh tissue.

Interactive 3D visualization of structural changes in the brain of a person with corticobasal syndrome.

The visualization of the progression of brain tissue loss in neurodegenerative diseases like corticobasal syndrome (CBS) can provide not only information about the localization and distribution of the volume loss, but also helps to understand the course and the causes of this neurodegenerative disorder. The visualization of such medical imaging data is often based on 2D sections, because they show both internal and external structures in one image. Spatial information, however, is lost. 3D visualization of imaging data is capable to solve this problem, but it faces the difficulty that more internally located structures may be occluded by structures near the surface. Here, we present an application with two designs for the 3D visualization of the human brain to address these challenges. In the first design, brain anatomy is displayed semi-transparently; it is supplemented by an anatomical section and cortical areas for spatial orientation, and the volumetric data of volume loss. The second design is guided by the principle of importance-driven volume rendering: A direct line-of-sight to the relevant structures in the deeper parts of the brain is provided by cutting out a frustum-like piece of brain tissue. The application was developed to run in both, standard desktop environments and in immersive virtual reality environments with stereoscopic viewing for improving the depth perception. We conclude, that the presented application facilitates the perception of the extent of brain degeneration with respect to its localization and affected regions.

Is neurology ready to see the light?

Is neurology ready to see the light?

Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue

This protocol uses rat tail-derived type I collagen hydrogels to analyze key processes in developmental neurobiology, such as chemorepulsion and chemoattraction. The method is based on culturing small pieces of brain tissue from embryonic or early perinatal mice inside a 3D hydrogel formed by rat tail-derived type I collagen or, alternatively, by commercial Matrigel. The neural tissue is placed in the hydrogel with other brain tissue pieces or cell aggregates genetically modified to secrete a particular molecule that can generate a gradient inside the hydrogel. The present method is uncomplicated and generally reproducible, and only a few specific details need to be considered during its preparation. Moreover, the degree and behavior of axonal growth or neural migration can be observed directly using phase-contrast, fluorescence microscopy or immunocytochemical methods. This protocol can be carried out in 4 weeks.

My ‘brush’ with kuru research

In November 1966, I received, to my surprise, formalin-fixed pieces of brain tissue from a young kuru patient (Tabaso, female, 12 years) from Dr Michael Alpers at the NIH with the request for electron microscopy (EM). At that time, I was engaged in EM studies of infectious diseases of the central

Quantitative imaging of zinc, copper and lead in three distinct regions of the human brain by laser ablation inductively coupled plasma mass spectrometry

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to determine the distribution of the trace elements zinc, copper and lead in insular, central and hippocampal areas of thin tissue sections (thickness 20 μm) through an entire human brain hemisphere. For the investigation of the tissue samples, a commercial laser ablation system was coupled to a double-focusing sector field ICP-MS. The regions of interest of healthy brain tissue (thickness 20 μm) were scanned (raster area ∼200 mm 2) with a focused laser beam (wavelength 266 nm, diameter of laser crater 200 μm and laser power density 3 × 10 9 W cm −2). The ion intensities of 64Zn +, 63Cu + and 208Pb + were measured by LA-ICP-MS within the ablated area. For quantification purposes, matrix-matched laboratory standards were prepared by means of dosing of each analyte to the pieces of brain tissue. The mass spectrometric analysis yielded inhomogeneous and largely reciprocal distributions of Zn and Cu in the selected areas of investigated brain samples. The highest concentrations of Zn and Cu with the most distinct distribution pattern were found in the hippocampus (up to 15 μg g −1). In contrast to zinc and copper, for lead, a more homogeneous distribution throughout all regions examined was found at a low concentration (in the ng g −1 range) level within the analytical range of LA-ICP-MS.

Paeonol reduced cerebral infarction involving the superoxide anion and microglia activation in ischemia-reperfusion injured rats

Both Moutan cortex of Paeonia suffruticosa Andrews (MC) and the root of Paeonia lactiflora Pall (PL) are important Traditional Chinese herbs used commonly to treat inflammatory and pyretic disorders. Paeonol, a common component of MC causes anti-platelet aggregation and scavenges free radicals. Therefore, the aim of the present study is to investigate the effects of Paeonol on cerebral infarct. A total of 60 male Sprague–Dawley (SD) rats were studied. An animal model of cerebral infarct was established by occluding both common carotid arteries and the right middle cerebral artery for 90 min, followed by a 24 h period of reperfusion. The percentage of cerebral infarction area to total brain area in each piece of brain tissue, and neuro-deficit score were measured. Superoxide anion was determined by the number of lucigenin-chemiluminescence (CL) counts. ED1 (mouse anti rat CD68) and interleukin-1β (IL-1β) immunostaining in the cerebral infarction region were also investigated for activation of microglia. The results indicated that Paeonol 15 and 20 mg/kg pretreatment and 20 mg posttreatment reduced the cerebral infarction area; Paeonol 15 and 20 mg/kg pretreatment reduced the neuro-deficit score. In addition, Paeonol 20 mg/kg pretreatment reduced the lucigenin-CL counts at 2 h period of reperfusion. The number of ED1 and IL-1β immunoreactive cells also reduced in the cerebral infarction region; there were no significant changes in blood sugar levels. The results show that Paeonol reduced cerebral infarct and neuro-deficit in rat, suggesting Paeonol might play a similar role in reducing cerebral infarction in humans. Paeonol suppresses and scavenges superoxide anion, and inhibit microglia activation and IL-1β in ischemia-reperfusion injured rats.

Independent accumulations of tau and amyloid β-protein in the human entorhinal cortex

Previous studies have repeatedly described that neurofibrillary tangles arise earlier than senile plaques (SPs) in the entorhinal cortex, but one study suggested that SPs, if present, enhance the former lesions. All of these studies were performed at the histologic or immunocytochemical level, which may not accurately reflect the actual levels of amyloid beta-protein (Abeta) and tau. To determine whether there is significant interaction between Abeta and tau in the human entorhinal cortex with regard to the Braak stage. Biochemical studies were conducted on 50 brains from elderly people, who were mainly at Braak stages I to III. All the cases were examined neuropathologically and staged according to Braak and Braak. A small piece of brain tissue for each case was dissected from the anterior portion of the right entorhinal cortex. The amounts of tau and Abeta in the insoluble fraction of the tissue were quantified using western blotting. The levels of tau and possibly Abeta42 in the entorhinal cortex appeared to rise steeply at approximately age 75. The levels of insoluble tau increased as the Braak stage increased from I to II; however, it had a tendency to remain between stages II and III. The levels of Abeta42 showed a small increase, whereas those of Abeta40 increased continuously as the Braak stage advanced. In contrast, the extent of Abeta42 accumulation increased with increasing Braak stage for SPs. There was no significant correlation between the levels of insoluble tau and Abeta42 in the entorhinal cortex. Even if Abeta did not accumulate to significant extents, substantial accumulation of insoluble tau occurred. Accumulations of tau and amyloid beta-protein occur independently in the human entorhinal cortex.

Feeding active neurons: (re)emergence of a nursing role for astrocytes

Despite unquestionable evidence that glucose is the major energy substrate for the brain, data collected over several decades with different approaches suggest that lactate may represent a supplementary metabolic substrate for neurons. Starting with the pioneering work of McIlwain in the early 1950s which showed that lactate can sustain the respiratory rate of small brain tissue pieces, this idea receives confirmation with more recent studies using nuclear magnetic resonance spectroscopy undoubtedly demonstrating that lactate is efficiently oxidized by neurons, both in vitro and in vivo. Not only is lactate able to maintain ATP levels and promote neuronal survival but it was also found to support neuronal activity, at least if low levels of glucose are present. Despite the early suggestion for a role of astrocytes in metabolic supply to neurons, it is only recently however that they have been considered as a potential source of lactate for neurons. Moreover, it has been proposed that astrocytes might provide lactate to neurons in response to enhanced synaptic activity by a well-characterized mechanism involving glutamate uptake. The description of specific transporters for lactate on both astrocytes and neurons further suggest that there exist a coordinated mechanism of lactate exchange between the two cell types. Thus it is proposed that astrocytes play a nursing role toward neurons by providing lactate as an additional energy substrate especially during periods of enhanced synaptic activity. The importance of this metabolic cooperation within the central nervous system, although not unique if compared to other organs, still remains to be explored.

前頚部より刺入し頭蓋内に達した鉄筋棒による穿通性脳損傷の１例

A 15-year-old boy with a neck injury transferred to our emergency room was alert and cooperative. During surgical closure of the wound, he vomited and became somnolent. A piece of brain tissue was found in vomit. Computed tomography (CT) showed pneumocephalus and a small hemorrhage in the left frontal lobe. Surgery undertaken found an irregular bone defect in the plamun sphenoidale and a dural laceration in the frontal base. The dura of the left frontal convexity was also lacerated. These findings suggested that an unknown foreign body penetrated the right lateral neck and the skull base through the nasopharynx, then reached up to the frontal bone. Postoperative course was uneventful. He witnessed later that he hit his neck on to an iron rod protruding from concrete blocks when he fell. He had known that this rod had penetrated his neck. Orbitocranial stab wounds may associate penetrating brain injuries, but it seems quite rare that a cervical stab wound complicated a penetrating brain injury. It should be stressed that physicians and paramedics must glean information about the modality of penetrating injuries.

Transplantation of ventral forebrain cholinergic neurons to the hippocampus ameliorates impairment of radial-arm maze learning in rats with AF64A treatment

Two types of cholinergic neurons were transplanted into the hippoccampus of adult rats chemically damaged by lateral ventricular administration of AF64A, a cholinergic neurotoxin, and the effects were compared with respect to their ability to reinnervate the hippocampus and to repair behavioral deficit. Pieces of brain tissue containing the nucleus basalis magnocellularis (NBM) or the striatum were taken for grafting from 17-day rat fetuses. About 3 months after transplantation, the rats with bilateral NBM grafts showed significant amelioration in radial-arm maze performance and habituation to a novel environment in an open field box, although they had not recovered to the control level. In rats with NBM grafts that showed a good performance, there were surviving grafts and many ingrowths of AChE-positive fibers in the hippocampus. By contrast, rats with striatal grafts showed hardly any significant improvement in these behavioral measures. The AChE staining revealed poor outgrowth of the striatal grafts into the hippocampus. These results indicate that grafting of NBM cholinergic neurons, which are anatomically similar to septal neurons, into the hippocampus produces a partial restorative effect on the cognitive impairment associated with hypofunction of the septohippocampal system.

Transplantation of ventral forebrain cholinergic neurons to the hippocampus ameliorates impairment of radial-arm maze learning in rats with AF64A treatment

Abstract Two types of cholinergic neurons were transplanted into the hippocampus of adult rats chemically damaged by lateral ventricular administration of AF64A, a cholinergic neurotoxin, and the effects were compared with respect to their ability to reinnervate the hippocampus and to repair behavioral deficit. Pieces of brain tissue containing the nucleus basalis magnocellularis (NBM) or the striatum were taken for grafting from 17-day rat fetuses. About 3 months after transplantation, the rats with bilateral NBM grafts showed significant amelioration in radial-arm maze performance and habituation to a novel environment in an open field box, although they had not recovered to the control level. In rats with NBM grafts that showed a good performance, there were surviving grafts and many ingrowths of AChE-positive fibers in the hippocampus. By contrast, rats with striatal grafts showed hardly any significant improvement in these behavioral measures. The AChE staining revealed poor outgrowth of the striatal grafts into the hippocampus. These results indicate that grafting of NBM cholinergic neurons, which are anatomically similar to septal neurons, into the hippocampus produces a partial restorative effect on the cognitive impairment associated with hypofunction of the septohippocampal system.

Cytoprotective effect of iloprost on isolated cortical brain tissue grafts in rats

The cytoprotective effect of iloprost was studied on isolated embryonic cortical brain tissue grafts of rats, using light and transmission electronmicroscopy. The brain tissue pieces were stored either in saline or 50 ng/ml iloprost solution for 30 minutes, 3, 6, 24 hours at +4 °C. It was demonstrated that iloprost significantly protected the neuronal integration of the tissue pieces compared with saline preserved pieces. Tissues preserved in iloprost showed only minimal dissolution of the tissue with minimal extracellular edema only in the later stages of preservation. The mechanism of action of the cytoprotective effect of iloprost is discussed.

Multiple ring enhancement in a case of acute reversible demyelinating disease in childhood suggestive of acute multiple sclerosis

A 5-year-old Japanese boy was admitted to our hospital because of progressive visual disturbance and quadriplegia. Computed tomography (CT) revealed multiple low density areas in the cerebral white matter, and intravenous injection of contrast medium resulted in ring enhancement around these areas. Exploratory aspiration of one of the low density areas gave a xanthochromic, viscous fluid. Pathologic examination of the drained fluid demonstrated small pieces of brain tissue with necrotizing lesions surrounded by demyelinating areas. The patient has been followed up for 3 years with corticosteroid treatment, and marked improvement has been achieved both in neurological examination and CT findings. From these clinicopathological findings an acute form of multiple sclerosis (MS) in childhood was suspected.

A manually-operated brain tissue slicer suitable for neurotransmitter release studies

A simple, inexpensive device has been designed to prepare slices or prisms of brain tissue for metabolic and release experiments by means of a hand-operated method. It is particularly suitable for very rapid slicing of small, irregular pieces of brain tissue dissected from animals or specimens available at biopsy. This simple apparatus was produced as an alternative to the more widely used motor-driven tissue choppers. A comparison of the morphological and metabolic integrity of tissue produced by the new slicer and a conventional tissue chopper is presented. The hand tissue slicer has been used to measure the release of monoamine neurotransmitters and specific neuropeptides in vitro.

Metastatic tumor cell invasion of brain organ tissue cultured on cellulose polyacetate strips.

Brain organ cells were cultured on cellulose polyacetate and other substrates in order to investigate metastatic tumor cell invasion in vitro. The cultures consisted of small pieces (approximately 1 X 3 mm) of neonatal mouse cerebrum or cerebellum tissue. Brain tissue pieces were allowed to attach to the substrates and were then cultured for 14-15 days in roller tubes. Cellulose polyacetate was found to be the best substrate for the attachment of brain tissue, and it eliminated some of the undesirable tissue movements that occurred using other substrates. Also, the invasion assays were the most reproducible using this tissue support. In culture, both cerebrum and cerebellum tissue achieved stable structures by 14 days, but the neurons and astrocytes in these tissues continued to exhibit structural changes such as extension of cellular processes. Murine B16 melanoma cells selected in vivo 15 times for brain colonization bound rapidly to and invaded brain tissue, infiltrating deep into the tissue within 4 hours and displacing the entire tissue by 5 days. Many of the B16 tumor cells extended pseudopodia and filopodia during invasion, suggesting that their tissue infiltration was an active invasive process. However, some B16 cells remained spherical in shape with numerous surface microvilli, but these same tumor cells also moved into the brain tissue. Brain tissue attached to cellulose polyacetate appeared to be the most useful system for elucidating the invasive interactions of malignant cells with brain tissues in vitro.

Actin‐ and myosin‐like filaments in rat brain pericytes

Heavy meromyosin (HMM) labeling was used to identify the nature of the filaments which form bundles in the cytoplasm of the pericytes in brain tissue. Rat brain tissue pieces were incubated in glycerol solutions at 4 degrees and then transferred into buffer (pH 7.0), (1) without HMM, (2) with HMM, (3) with HMM + 5 mM ATP, and (4) with HMM + 2.5 mM Na+ pyrophosphate. In pericytes from untreated tissue, smooth-surfaced microfilaments, averaging 6 nm in diameter, appear to branch and anastomose and to anchor on the plasma membrane. After exposure to HMM, the number and the density of the microfilaments are strikingly increased. These tightly-packed microfilaments are now heavily coated with exogeneous HMM thus increasing in width to 18-20 mm. They intertwine in closely-woven networks. After incubation in HMM solutions containing ATP or Na+ phosphate, they are no longer coated with thick sidearms. It can thus be concluded that these microfilaments are of actin-like nature. In addition, after incubation in ATP, they are intermingled with, and converge onto the surfaces of, thick, tapered filaments, which we have tentatively identified as of myosin-like nature. Thus, it appears that certain of the major elements necessary for contraction are present in brain pericytes.

Actin-like filaments in the endothelial cells of adult rat brain capillaries

While studying glycerinated adult rat brain tissue by means of heavy meromyosin (HMM) labeling, the presence of actin-like filaments within the cytoplasm of capillary and postcapillary venule endothelial cells was observed. After incubation in cold glycerol solutions, rat brain tissue pieces were transferred into buffer, pH 7.0: (i) with HMM, (ii) without HMM, (iii) with HMM + 5 m m ATP, and (iv) with HMM + 2.5 m m Na + pyrophosphate. In the endothelial cells from untreated nervous tissue, we observed smooth-surfaced microfilaments, averaging 6 nm in diameter, branched and anastomosed, intertwining in loosely meshed networks, and anchored to the plasma membrane. After exposure to HMM, the microfilaments reacted with the latter molecules. They increased in width to 18 to 20 nm and were tightly packed in portions of the endothelial cells. When the HMM solutions contained adenosine 5′-triphosphate (ATP) or Na + pyrophosphate, the microfilaments were no longer coated with thick sidearms. Thus, we demonstrated that the microfilaments present within the endothelial cells of capillary and postcapillary venules in adult rat brain tissue are of actin-like nature. In addition, after incubation with ATP, the microfilaments were intermingled with, and converged onto the surfaces of, thick, tapered filaments, which we tentatively identified as of myosin-like nature. However, the number of contractile filaments within the endothelial cells was small compared to that observed in the pericyte of the same capillary and postcapillary venule. Several possible functions of the contractile filaments in the endothelial cells are presented.

Counterstaining Golgi-Cox impregnations with luxol fast blue as a myelin stain.

Immerse pieces of brain tissue 4 wk in solutions A and B, mixed just before use: A. K2Cr2O7, 1 gm; HgCl2, 1 gm; boiling distilled water, 85 ml. Boil A for 15 min, cool to 2 C and add: B. K2CrO4, 0.8 gm; Na2WO4, 0.5 gm; distilled water, 20 ml. Rinse in water and immerse 24 hr in LiOH, 0.5 gm; KNO3, 15 gm; distilled water, 100 ml. Wash 24 hr in several changes of 0.2% acetic acid and then for 2 hr in tap water. Dehydrate and embed in celloidin. Process a 60 μ section through 70 and 95% ethanol, a 3:1 mixture of absolute ethanol and chloroform, and toluene. Immerse it for 5 min in a solution containing methyl benzoate, 25 ml; benzyl alcohol, 100 ml; chloroform, 75 ml. Orient the section on a chemically clean slide and let air-dry 5–10 min. Process through toluene, 3:1 ethanol-chloroform and 95% ethanol. Place the section for 5–60 min at 60 C in a solution made up of: Luxol fast blue G (Matheson, Coleman and Bell), 1 gm; 95% ethanol, 1000 ml; 10% acetic acid, 5 ml. Hydrate to water and immerse in 0.05% Li2CO3...