Brain research. Brain research protocols最新文献

The sensitivity of capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) was compared with conventional agarose gel electrophoresis-ethidium bromide-UV method (AE-EUV) for detection and semi-quantitative determination of GFAP mRNA in mouse brain. GFAP expression was induced by the neurotoxin MPTP in C57BL mice. Serially diluted RNA samples (0.0003, 0.003, 0.03, 0.3, and 3 μg total RNA) were subjected to RT-PCR and analyzed by both procedures. The integrated pixel density (AE-EUV) and peak area (CE-LIF) were directly proportional to the amount of RNA. However, the observed high sensitivity of CE-LIF suggests its potential application for detection and semi-quantitative determination of low-abundance mRNA transcripts.

Recombinant adeno-associated virus-2 (rAAV2) under control of the chicken beta actin promoter/truncated CMV enhancer (CBA) was investigated for its ability to transduce primary cultures of rat brain neurons, microglia and astrocytes. This vector was highly effective in all three cell types in heparin-sensitive manners (astrocytes, microglia and neurons transduced by >98%, 75%, and 95%, respectively). However, astrocytes co-cultured with neurons were not transduced. rAAV2/CBA is an important new method for genetic manipulation of brain cells, though this may be modulated by interactions among cell types.

A systematic neuropsychological assessment technique is described for use with severely physically disabled individuals, possibly with combined motor and cognitive disorders. Target neurological conditions may be, e.g., an incomplete locked-in state, a minimally conscious state, or severe combinations of paralysis, agnosia, and apraxia. Neuropsychological assessment in these patients is difficult, because standard neuropsychological tests require fast motor responses, which can be manual, verbal, or both. To assess the cognitive status of patients with residual motor function, tests have been applied that can be answered by a binary (yes/no) signal and whose outcomes were not based on reaction times. Further, a battery of neurophysiological examination procedures based on event-related brain potentials has been developed. These procedures can be performed directly at a patient's bedside (at home or in a hospital) and applied for assessment of cognitive functions even in patients without residual motor function.

Stereology is an important technique for the quantification of neurons in subregions of the central nervous system. A commonly used method of stereology relies upon embedment of tissue in glycol methacrylates to allow production of sections that are resistant to shrinkage in thickness. However, the use of glycol methacrylates for stereology has several disadvantages, including severe constraints on the size of tissue that can be processed and the long duration of time often required for infiltration. We describe a novel method of stereology utilizing tissue sections cut in the frozen state. This new methodology relies upon the staining of sections as free-floating sections and upon the mounting of these sections onto slides with a water-based mounting media. Sections cut in the frozen state and processed by these methods undergo little or no shrinkage in thickness and are ideal for stereological cell counts utilizing either the optical disector or optical fractionator methods of stereology. We demonstrate that frozen sections can be utilized to estimate neuronal number with high degrees of precision and with low coefficients of error. Because large tissue blocks can be cut as frozen sections, this method expands the range of tissues that can be processed efficiently for stereology and readily allows quantification of neurons from multiple brain regions from the same tissue sections. We applied this new methodology to estimate neuronal numbers in the neocortex and hippocampus of 10-day-old mice. The method was useful for estimation of both large, sparsely packed cell populations, such as the neocortex, and small, densely packed cells, such as the dentate gyrus granule cells. Thus, frozen section methodology offers many potential advantages over the use of glycol methacrylate embedment for stereology. These advantages include expansion of the size of tissue blocks that can be processed, reduction in expended time and costs, and ability to quantify mulitiple brain regions from a single set of sections.

Despite its evergrowing use in health-related areas, procedures characterized as meditation have been little or not at all defined operationally, which hinders its use in a standardized manner. In the present study, the authors present a possible operational definition of meditation, which has been used in social and academic projects, developed in Universidade Federal de São Paulo. In this proposal, it is emphasized that, in order to be characterized as meditation, the procedure should encompass the following requirements: (1) the use of a specific technique (clearly defined), (2) muscle relaxation in some moment of the process and (3) “logic relaxation”; (4) it must necessarily be a self-induced state, and (5) use of “self-focus” skill (coined “anchor”).

For pathophysiological studies, it is advantageous to label specific neuronal populations in living animals. This study aimed to establish a method for stable and long-lasting fluorescent labeling of corticospinal neurons in the living animal. The two fluorescent dyes Fluoro-Red and Fluoro-Green were injected in the cervical spinal cord of anesthetized newborn rats. After a recovery period, treated rats were returned to the mother. After 24 h and 14 days, fixed brain sections revealed wide-spread fluorescence in elongated or pyramidal-shaped cell profiles in a discrete internal cortical layer, consistent with layer V pyramidal cells. Labeled neurons displayed spontaneous synaptic activity using the slice patch clamp method. These results suggest that these dyes are effective tools for pathophysiological and slice patch clamp studies focused on specific neuron groups.

Mitochondrial failure to generate ATP can be due to damage to their membranes, which leads to release of solutes, e.g., pyridine nucleotides, from the mitochondrial matrix. We developed a highly sensitive fluorescence assay for detecting a pathologic increase in mitochondrial membrane permeability. The assay is based on coupled enzymatic reactions that produce hydrogen peroxide in the presence of the reduced or oxidized form of nicotinamide adenine dinucleotide (NADH/NAD). The hydrogen peroxide is a substrate for horseradish peroxidase that converts Amplex Red into highly fluorescent Resorufin. The assay is able to detect nanomolar levels of pyridine nucleotides in the medium. Calcium additions to isolated rat brain or liver mitochondria incubated in a potassium-based medium with added enzymes caused osmotic swelling, as detected with light scattering, and production of Resorufin, due to release of NADH/NAD. These events were blocked by cyclosporin A (CsA) or Bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT). These results indicate that the NADH/NAD release assay is a simple, reliable, and sensitive method for detecting mitochondrial damage and for screening of compounds that protect mitochondria from injury.

The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) stain is in wide use for measuring apoptosis in neurons, as well as in other cell types. TUNEL may give false positive results due to variations in labeling technique as well as staining of cells that have undergone non-apoptotic DNA strand breaks. Therefore, in isolation, TUNEL is not a certain indicator of apoptosis. Recently, we have demonstrated the potent apoptotic effect of secreted phospholipase A₂ from group III (sPLA₂-III) on primary cortical neurons from rat. Here we describe a computer-assisted method for quantifying TUNEL-positive neurons after sPLA₂-III induced apoptosis. Extent of TUNEL is normalized to total nuclear content using 4′,6-diamidino-2-phenylindole (DAPI) staining. Furthermore, DAPI counterstaining allows for determination of a nuclear morphology indicator, based on nuclear size and roundness, which we call the nuclear area factor. We found that the nuclear area factor is an early indicator of cell death (significant after 4 h post treatment), while TUNEL staining is significant at later times (26 h). Thus, the independent staining techniques using TUNEL and DAPI complement each other, and with commercially available image analysis software, may be used to indicate early as well as delayed cell injury processes.

In ischemic preconditioning (IPC), brief sublethal ischemia protects neurons from a subsequent lethal ischemia. In vivo models faithfully display preconditioning, yet, these models are technically challenging, time-consuming and expensive. In vitro models of preconditioning have also been developed that are technically easier and less expensive. A drawback of pre-existing in vitro models is that since susceptibility to ischemic injury is age-dependent; neuroprotection is being studied in neurons that have intrinsic resistance to oxygen–glucose deprivation (OGD). This study introduces a new in vitro model of ischemic preconditioning in hippocampal slice cultures isolated from 20–30-day-old rats. Slice cultures show a high susceptibility and sharp thresholds toward ischemia that is comparable to that found in vivo. A 5-min OGD treatment was not neurotoxic to young adult slice cultures, while a 10-min OGD treatment was neurotoxic. In addition, the sublethal 5-min OGD treatment protected against a 10-min OGD treatment that was delivered 24 h later. Neuroprotection was seen in preconditioned slice cultures stained with propidium iodide (PI) or with antisera against the neuron-specific antigen NeuN. Energy failure is hypothesized to trigger ischemic preconditioning and a 5-min OGD treatment induced transient energy failure in young adult slice cultures. This model may assist in the search for new therapeutics for the prevention and/or treatment of stroke.

Since rodent self-grooming behaviours are elicited by both comfort and stressful conditions, traditional measures such as duration, latency of onset and the number of bouts may be not suitable to dissociate between these opposite conditions. The aim of the current study was to improve and optimize ethological measurement of self-grooming in neurobehavioural stress research enabling differentiation between stress and no-stress situations. This protocol assists in the correct interpretation of animal grooming behaviours and detection of stress by measuring alterations in grooming microstructure in different test situations. While a general pattern of self-grooming uninterrupted cephalocaudal progression is normally observed in no-stress (comfort) conditions in mice and other rodents, the percentage of “incorrect” transitions between different stages and the percentage of interrupted grooming bouts may be used as behavioural marker of stress. The protocol can be a useful tool in neurobehavioural stress research including modelling stress-evoked states, pharmacological screening of potential antistress drugs or behavioural phenotyping of genetically modified animals.