Brain research. Brain research protocols最新文献

The implication of integrated functional sensory relations of the body to space in anxiety disorders is a very important issue which encourages the development of animal models, in particular, for pharmacological perspectives and for the functional assessment of the deficits induced by genetic manipulation in the mouse or the rat. A new experimental device is presented here: It is comprised of a rotating tunnel and a rotating-beam controlled by computer which can be used for multiple visuo-idiothetic and kinesthetic sensory conflict situations during active locomotor behaviour by mice. The system is linked to a digital video system, Video-Track™, designed to track and record the movements of the animals. Anxious BALB/cByJ mice were compared to non-anxious C57BL/6J mice and were seen to display highly disturbed locomotor behaviour in a sensory conflict situation. The model highlights the advantages of video-digital analysis for animal behavioural sciences.

Recent experimental studies have indicated that bone marrow stromal cells (BMSC) improve neurological deficits when transplanted into the animal models of various neurological disorders, although precise mechanism still remains unclear. In this study, we developed a new in vivo fluorescence optical imaging protocol to sequentially track the transplanted into the brain of the living animals subjected to cerebral infarct. Mice BMSC were harvested from transgenic mice expressing green fluorescent protein (BMSC-GFP). They were stereotactically transplanted into the ipsilateral striatum of mice subjected to permanent middle cerebral artery occlusion after 7 days of ischemia (n=12). During 12 weeks after transplantation, the skull was exposed and the green fluorescence emitted from the brain surface was sequentially observed, using in vivo fluorescence optical microscopy. As the results, regional green fluorescence was detected in the ipsilateral parietal region 4–12 weeks after transplantation in all animals and became more apparent over the time. The images obtained through the skull were very similar to those acquired by thinning or removing the skull. Immunohistochemistry evaluation revealed that the transplanted cells migrated towards the ischemic boundary zone and expressed the neuronal or astrocytic marker, supporting the findings on fluorescence optical images. Sequential visualization of the BMSC transplanted into the brain of living animals would be valuable for monitoring the migration, growth and differentiation of the transplanted cells to explore the fate and safety of stem cell transplantation for various neurological disorders.

Recent advances in genetical engineering of the mouse have highlighted the importance of reproducible and less invasive models of cerebral ischemia in mice. In this paper, we developed minimally invasive and reproducible model of distal middle cerebral artery (MCA) occlusion in mice using krypton (Kr) laser-induced photothrombosis.

C57BL/6 or BALB mice (n=8 each) were anesthetized with halothane. The skin was cut, the temporal muscle was retracted, and the right distal MCA was observed through the skull. A Kr laser beam of wavelength 568 nm was focused onto the MCA over the intact skull. Upon laser irradiation, intravenous administration of a rose bengal solution was begun. After 4 min of irradiation, the laser beam was refocused on the MCA just proximal to the first spot, and another 4-min irradiation was performed. Then, the right common carotid artery (CCA) was ligated. Three days later, the brain was removed, and infarct volume was determined.

Infarction confined almost solely to the cortical area was produced in each mouse. Mean infarct volume in C57BL/6 mice was 25.2±13.7 mm³. The BALB mice group showed significantly larger and more reproducible infarction (44.1±5.2 mm³; the coefficient of variation was 12%) than did C57BL/6 mice (P<0.005).

Our photothrombosis model of stroke in mice can be performed without craniectomy, and its reproducibility is satisfactory when using BALB mice.

Recent data suggest that the incidence of focal cerebral ischemia (FCI) and stroke is higher than previously recognized and could account for a large proportion of brain lesions in the preterm and full term neonate. Therefore, it is critically important to develop an appropriate model of FCI in neonatal animals. We describe here a modified model of permanent FCI in rat pups at postnatal day-7 (P7). To produce permanent FCI, a suture embolus with different diameters (180–220 μm) was inserted into the left common carotid artery (CCA) of the pups with different weight (14–19 g). Then the suture embolus was advanced to the middle cerebral artery (MCA) to produce its occlusion. The success of vascular occlusion was evaluated by imaging the ischemic territory on serial brain sections with carbon black staining immediately after permanent FCI. The consistent cerebral infarction was confirmed by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after permanent FCI. Terminal deoxynucleotidyltransferase-mediated 2′-deoxyuridine 5′-triphospate-biotin nick end labeling (TUNEL) staining showed cell death with TUNEL labeling in the ischemic areas, which is one of the features of apoptosis. The present model opens the way for advanced pathophysiological studies of FCI in neonates.

Biotinylated dextran amine (BDA) and biocytin are well transported both retrogradely and anterogradely. Both tracers have stable molecular structure for long-term storage and examination, and their visualizations can be realized by simple histochemical reactions. Therefore, the BDA and biocytin are widely used in neuroanatomical studies as the tract-tracing markers. The results obtained by BDA and biocytin applications to various areas of the nervous system in teleosts were qualitatively identical, and the retrogradely and anterogradely labeled structures could be clearly identified with reference to the counter-staining. Iontophoretic injections or crystal insertions resulted in filling of cell bodies, dendrites and terminals in the core of injection side, revealing morphological details of the local and distant somata, dendritic arborizations and axonal terminals. However, biocytin exhibited superior to BDA in anterograde transport, and could label very thin axons, axonal collaterals and terminal ramifications. In contrast, retrograde transport of BDA was superior to that of biocytin, and resulted in more complete dendritic filling of retrograde labeled neurons including dendritic arborizations and spines.

Characterization of synaptic transmission between the inner ear sensory cells and primary neuron dendrites has been hampered by the limited access to the postsynaptic terminals. Because direct physiological recording of postsynaptic currents are difficult to achieve, no information regarding the synaptic and dendritic events are available. This is due to the small size of the postsynaptic afferent nerve endings that do not allow a clear identification, and thus compromise direct electrophysiological recordings of the buttons. To study the physiology of afferent nerve endings, we have developed a two-photon imaging technique in cochlear and vestibular slice preparations from neonatal rats and turtles. This technique is based on a retrograde labeling of afferent nerve endings with high-affinity calcium-sensitive dyes. Dye filling was achieved by 6 h application of the dextran–amine conjugate of calcium green-1. Calcium changes were measured in afferent nerve endings in line scan and time lap mode. To address recording in a near-physiological situation, iontophoretic application of K⁺ was performed in the area of the stereocilia whereas glutamate was applied at the basal pole of sensory hair cells. Both types of application cause a reversible and sustained increase of Ca²⁺ in the button of afferent nerve fibers. Typical recordings are presented and potential interests for pharmacological studies of inner ear sensory cell synapses are discussed.

Until recently, evidence supporting postnatal neurogenesis was controversial. Much of the debate has centered on the identification of the dividing cells as neurons versus glia. Because neurogenesis has become a well-documented phenomenon, there is a need for reliable protocols to identify recently divided neurons in a wide range of situations. To facilitate the investigation of postnatal neurogenesis of magnocellular neurons in the pig hypothalamus, a sequential immunohistochemical staining technique was developed for use on serial sections of paraffin-embedded tissue. Proliferating neurons were labeled using mouse-derived monoclonal antibodies to detect proliferating cell nuclear antigen (PCNA) and vasopressin (VP). PCNA, a nuclear protein essential for cell division, identifies recently divided cells in the brains of healthy animals. VP is a unique functional marker for a mature neuron. The presence of a cell with VP positive cytoplasm and a PCNA positive nucleus demonstrates the presence of a VP-producing neuron that has recently divided. This protocol allowed us to safely and accurately label recently proliferated neurons in the mature pig hypothalamus and can be used on archived tissue. This data can be used for further morphometric analysis, as serial sectioning allows for three-dimensional reconstruction of hypothalamic nuclei.