Brain research. Brain research protocols最新文献

We have recently developed an optical spectroscopy technique to monitor light scattering changes of the nervous system in vivo. Near infrared (NIR) spectroscopy emphasizes the detection of light scattering properties, which are prominent within the wavelength range of 700 to 850 nm wavelength. The purpose of this study is to test the hypothesis that demyelination and degeneration of the sciatic nerves after nerve injury will lead to a change in light scattering properties and be detected by the NIR technique. Left spinal nerve ligations (L4, L4 and L5, L5) were performed in rats. The scattering properties of the left (ligated) and right (control) sciatic nerve were measured by the NIR reflectance using a bifurcated needle probe at postoperative days 1, 4, 7, and 14. The results show that there was no significant difference among three types of ligation, and neither did the readings between left and right sciatic nerve at postoperative day 1. Significant decreases in light scattering indexes were found between left and right sciatic nerves at postoperative days 4, 7, and 14. It is concluded that our initial hypothesis is proven, suggesting that the NIR technique may have a potential for clinical application in detecting demyelination and degeneration of the nervous system.

Abide its toxicity, 3,3′-diaminobenzidine-tetrahydrochloride-dihydrate (DAB) was the most potent marker for immunochemistry at the light and electron microscopic level in the last decades. Recently, a sensitive substrate for immunohistochemical staining methods and in-situ hybridization, HistoGreen, was developed for the use with peroxidase. In peroxidase reactions, HistoGreen delivers a green staining product which is suitable for permanent embedding without water. In contrast to DAB, HistoGreen is not toxic. To evaluate its usefulness, we performed comparative immunohistochemistry on angiotensin II (AT₁)-receptors with DAB- and HistoGreen-staining on paraffin embedded slices of the rat brain at the light microscopic level. This also included counterstaining with Mayer's Hemalum and Nuclear Fast Red, respectively. We could demonstrate that HistoGreen delivers a coarsely grained label which is fast detectable in light microscopy. HistoGreen equals DAB in the exact localization of the immunoreaction to a large degree but its reaction product is considerably less stable in alcohol and water than DAB. In combination with Nuclear Fast Red, HistoGreen provides excellent imaging properties for the visualization and documentation of immunoreactive structures paired with an adequate demonstration of cellular details. Its tendency towards rapid over-staining as well as its low stability will restrict the use of HistoGreen in some areas of immunohistochemical research, yet the new chromogen represents an interesting alternative to DAB at the light microscopic level.

The postnatal subventricular zone (SVZ) contains the largest pool of dividing and migrating neural precursors in the adult rodent brain. Neuronal precursors migrate throughout the SVZ and along the rostral migratory stream (RMS) towards the olfactory bulb where they differentiate into interneurons. To facilitate the investigation of cell migration in the SVZ and RMS, an inexpensive migration assay was developed for use in acute brain slices. Acute sagittal slices were kept at 37 °C in 5% O₂/95% CO₂-saturated solution and migrating cells in the SVZ and RMS were visualized using an upright infrared-differential interference contrast microscope. Time-lapse movies were acquired to identify the direction and measure the speed of cell migration. The neurotransmitter GABA and inhibitors of GABA receptors or transporters can be bath applied to determine the function of endogenous GABA on the direction and speed of cell migration. In parallel, the levels of endogenous GABA released from acute SVZ or RMS explants were measured with mass spectrometry. Additional techniques such as electrophysiology and immunohistochemistry confirmed the identity of cells as neuronal precursors and characterized the expression of GABA receptors and transporters. This report describes how modulations in the direction and speed of neuronal precursor migration can be accurately monitored and how changes in local GABA levels can be measured. The described techniques can be used to identify the endogenous factors that regulate cell migration. Identifying such factors is essential for the future therapeutic use of SVZ cells to replace damaged or lost cells.

In the present study, we suggest that long elevated horizontal rod (Suok test, ST) and its light–dark modification (LDST) may be used for behavioral characterization in mice, including simultaneous assessment of their anxiety, activity, and neurological phenotypes. To establish the ST and the LDST as murine models of anxiety, we used several different mouse strains which differ markedly in their anxiety and activity (C57BL/6, 129S1/SvImJ, NMRI, and BALB/c). Here we show that our tests are able to ethologically discriminate between high and low anxiety mouse strains, as assessed by horizontal and directed exploration, stops, and defecation boli. The spatial distribution of the LDST behaviors is also sensitive to these strain-specific anxiety phenotypes, showing clear avoidance of the brightly lit part of the test in stressed (rat exposed) vs. control NMRI mice. In addition, we validated the ST in 129S1/SvImJ and BALB/c mice by assessing the behavioral consequences of acute stress such as rat exposure. Finally, we showed that our test is able to detect high anxiety and poorer motor coordination in 129S1/SvImJ (vs. C57BL/6) mice. The results of our study show that the ST emerges as an experimental tool to analyze anxiety, motor-vestibular anomalies, as well as anxiety-induced motor impairments in mice. Overall, we suggest that the ST can be a useful protocol in neurobehavioral stress research including modeling stress-evoked states, pharmacological screening of potential anti-stress drugs, or behavioral phenotyping of genetically modified animals.

In order to verify the biological aspects of ‘autogeneic’ bone marrow stromal cells (BMSC) transplantation for neurological disorders, we aimed our study towards the assessment of the survival, distribution, and differentiation of autologous BMSC in the central nervous system (CNS). We harvested rat BMSC from femur bones, and the nuclei were then fluorescently labeled by a 24-h co-culture with bis-benzimide. These BMSC were stereotactically injected into the striatum (n=6) or thoracic cord (n=8) of each animal. We evaluated the distribution and differentiation of ‘autogeneic’ BMSC in the brain and spinal cord after 4 weeks, using the immunohistochemistry technique. We found some injected cells in the ipsilateral striatum, hippocampus, neocortex, and bilateral corpus callosum, and approximately 20% and 15% of the engrafted cells expressed neuronal and astrocytic markers, respectively. Other injected cells were distributed in the dorsal funiculus and adjacent gray matter, and about 10% and 15% of these cells expressed neuronal and astrocytic markers, respectively. Although the precise mechanism of BMSC transdifferentiation still remains unclear, the present results show that ‘autogeneic’ BMSC could highly differentiate into their own CNS neural cells, suggesting that they are surrounded by favorable conditions.

Many individuals with acquired brain injury (ABI) report reading problems of oculomotor origin. These may include frequent loss of place, skipping of lines and difficulty shifting to the next line of print. We describe two protocols for the testing and training of reading-related eye movements in adult individuals with ABI (traumatic brain injury [TBI] and stroke with hemianopia), who experience oculomotor-based symptoms when reading. These protocols use objective eye movement recording techniques and computer-based stimulus presentation and analysis. One protocol tests and the other trains basic horizontal and vertical versional eye movements (fixation, saccades and pursuit), as well as reading eye movements using simulated single and multiple line dynamic arrays. In addition, a reading rating-scale questionnaire is administered before and after completion of training to assess subjective reading improvement. In all paradigms, the target consists of a 0.5° luminous square, which is displayed on a computer monitor positioned 40 cm from the subject along the midline. All testing and training are conducted under binocular viewing conditions with optical correction in place. There are two modes of training: normal internal oculomotor visual feedback either alone (4 weeks) or in conjunction with external oculomotor auditory feedback (4 weeks) administered in a counterbalanced manner within each diagnostic group. Training is performed 1 h, twice weekly for the 8 weeks. Oculomotor testing is conducted before, midway and after training. Following training, reading-related eye movements and reading ability improved as assessed both subjectively and objectively. These protocols provide a systematic approach to the quantitative and comprehensive testing and training of reading-related eye movement skills and behaviors in the ABI population manifesting oculomotor-based reading dysfunctions. Furthermore, the training protocol results in the rapid remediation of the eye movement deficits, which appear to transfer to activities of daily living.