Changes in early endosomes in rat hippocampal CA1 neurons after transient global cerebral ischaemia.

INTRODUCTION: Transient global ischaemia in rodents causes selective loss of hippocampal CA1 neurons, but the potential involvement of endocytic pathways has not been fully explored. The aim of this study was to investigate the changes in early endosomes in the CA1 subfield after ischaemia and reperfusion. MATERIALS AND METHODS: A four-vessel occlusion (4-VO) model was established in Wistar rats to induce 13 minutes of global cerebral ischaemia. Neuronal death was detected by Fluoro-Jade B (FJ-B) staining at various intervals after reperfusion, and intracellular membrane changes in ischaemic neurons were revealed using DiOC6(3), a lipophilic fluorescent probe. Ras-related protein Rab5 (Rab5) immunostaining was performed to detect changes in early endosomes in ischaemic neurons. Western blot analysis was used to confirm the morphological observations on Rab5 in the CA1 hippocampal subfield. RESULTS: FJ-B staining confirmed progressive neuronal death in the CA1 subfield in ischaemic rats after reperfusion. DiOC6(3) staining revealed abnormally increased membranous components in ischaemic CA1 neurons. Specifically, early endosomes, as labelled by Rab5 immunostaining, significantly increased in number and size in CA1 neurons at 1.5 and 2 days post-reperfusion, followed by rupture at day 3 and a decrease in staining intensity at day 7 post-reperfusion. Western blot analysis confirmed a significant upregulation of Rab5 protein levels at day 2, which returned to near control levels by day 7. CONCLUSIONS: Our study revealed significant changes in the dynamics of early endosomes in CA1 neurons after ischaemia-reperfusion injury. The initial increase in the area fraction of early endosomes in CA1 neurons may reflect an upregulation of endocytic activity, whereas the fragmentation and reduction of early endosomes at the later stage may indicate a failure of adaptive mechanisms of ischaemic neurons against ischaemia-induced death. Understanding the temporal dynamics of early endosomes provides critical insights into the cellular mechanisms that govern fate of CA1 hippocampal neuronsl after ischaemia/reperfusion.

The detection methods currently available for protein aggregation in neurological diseases.

Protein aggregation is a pathological feature in various neurodegenerative diseases and is thought to play a crucial role in the onset and progression of neurological disorders. This pathological phenomenon has attracted increasing attention from researchers, but the underlying mechanism has not been fully elucidated yet. Researchers are increasingly interested in identifying chemicals or methods that can effectively detect protein aggregation or maintain protein stability to prevent aggregation formation. To date, several methods are available for detecting protein aggregates, including fluorescence correlation spectroscopy, electron microscopy, and molecular detection methods. Unfortunately, there is still a lack of methods to observe protein aggregation in situ under a microscope. This article reviews the two main aspects of protein aggregation: the mechanisms and detection methods of protein aggregation. The aim is to provide clues for the development of new methods to study this pathological phenomenon.

A novel detection method for neuronal death indicates abnormalities in intracellular membranous components in neuronal cells that underwent delayed death.

Acute neuronal degeneration is always preceded under the light and electron microscopes by a stage called microvacuolation, which is characterized by a finely vacuolar alteration in the cytoplasm of the neurons destined to death. In this study, we reported a method for detecting neuronal death using two membrane-bound dyes, rhodamine R6 and DiOC6(3), which may be associated with the so-called microvacuolation. This new method produced a spatiotemporally similar staining pattern to Fluoro-Jade B in kainic acid-damaged brains in mice. Further experiments showed that increased staining of rhodamine R6 and DiOC6(3) was observed only in degenerated neurons, but not in glia, erythrocytes, or meninges. Different from Fluoro-Jade-related dyes, rhodamine R6 and DiOC6(3) staining is highly sensitive to solvent extraction and detergent exposure. Staining with Nile red for phospholipids and filipin III for non-esterified cholesterol supports that the increased staining of rhodamine R6 and DiOC6(3) might be associated with increased levels of phospholipids and free cholesterol in the perinuclear cytoplasm of damaged neurons. In addition to kainic acid-injected neuronal death, rhodamine R6 and DiOC6(3) were similarly useful for detecting neuronal death in ischemic models either in vivo or in vitro. As far as we know, the staining with rhodamine R6 or DiOC6(3) is one of a few histochemical methods for detecting neuronal death whose target molecules have been well defined and therefore may be useful for explaining experimental results as well as exploring the mechanisms of neuronal death.

The temporal and spatial changes of actin cytoskeleton in the hippocampal CA1 neurons following transient global ischemia.

Transient global ischemia usually results in delayed neuronal death in selective brain regions, prior to which a rapid loss of dendritic spines has been widely reported in these regions. Dendritic spines are characterized by a highly branched meshwork of actin cytoskeleton (F-actin), which is extremely vulnerable to the ATP-depleted conditions such as hypoxia/ischemia. However, the ischemia-induced changes of F-actin are still not clarified in the vulnerable brain areas. This study was designed to examine the temporal and spatial alterations of F-actin in the CA1 subfield of rat hippocampus following reperfusion after global cerebral ischemia. Phalloidin staining and confocal microscopic examination showed that F-actin disappeared from the dentritic spines in the CA1 stratum radiatum, but aggregated into thread- or fiber-like structures on days 1.5-2 after ischemia. This was followed by a nearly complete loss of F-actin in the CA1 subfield on days 3-7 after ischemia. Colocalization analysis demonstrated that the F-actin threads or fibers were located mainly within the dentritic trunks. As revealed by Nissl and Fluoro-Jade B staining, the decrease of F-actin proceeded concurrently with the evolution of ischemic damage. Consistently, western blots detected a significant decrease of F-/G-actin ratio in the dissected CA1 subfield after ischemia. To our knowledge, this is the first report on the change of F-actin in the ischemic brain. Although the underlying mechanisms remain to be elucidated, our findings may provide an important structural clue for the neuronal dysfunction induced by ischemia.

The effects of epothilone D on microtubule degradation and delayed neuronal death in the hippocampus following transient global ischemia.

Disruption of microtubule cytoskeleton plays an important role during the evolution of brain damage after transient cerebral ischemia. However, it is still unclear whether microtubule-stabilizing drugs such as epothilone D (EpoD) have a neuroprotective action against the ischemia-induced brain injury. This study examined the effects of pre- and postischemic treatment with different doses of EpoD on the microtubule damage and the delayed neuronal death in the hippocampal CA1 subfield on day 2 following reperfusion after 13-min global cerebral ischemia. Our results showed that systemic treatment with 0.5 mg/kg EpoD only slightly alleviated the microtubule disruption and the CA1 neuronal death, while treatment with 3.0 mg/kg EpoD was not only ineffective against the CA1 neuronal death, but also produced additional damage in the dentate gyrus in some ischemic rats. Since the pyramidal cells in the CA1 subfield and the granule neurons in the dentate gyrus are known to be equipped with dynamically different microtubule systems, this finding indicates that the effects of microtubule-disrupting drugs may be unpredictably complicated in the central nervous system.

The effects of calcineurin inhibitor FK506 on actin cytoskeleton, neuronal survival and glial reactions after pilocarpine-induced status epilepticus in mice.

After status epilepticus (SE), actin cytoskeleton (F-actin) becomes progressively deconstructed in the hippocampus, which is consistent with the delayed pyramidal cell death in both time course and spatial distribution. A variety of experiments show that calcineurin inhibitors such as FK506 are able to inhibit the SE-induced actin depolymerization. However, it is still unclear what changes happen to the F-actin in the epileptic brain after FK506 treatment. A pilocarpine model of SE in mice was used to examine the effects of FK506 on the F-actin in the hippocampal neurons. The post SE (PSE) mice with or without FK506 treatment were monitored consecutively for 14 days to examine the frequency and duration of spontaneous seizures. The effects of FK506 on the activity of cofilin and actin dynamics were assessed at 7 and 14 d PSE by western blots. The organization of F-actin, neuronal cell death, and glial reactions were investigated by phalloidin staining, histological and immunocytochemical staining, respectively. As compared to the PSE + vehicle mice, FK506 treatment significantly decreased the frequency and duration of spontaneous seizures. Relative to the PSE + vehicle mice, western blots detected a partial restoration of phosphorylated cofilin and a significant increase of F/G ratio in the hippocampus after FK506 treatment. In the PSE + vehicle mice, almost no F-actin puncta were left in the CA1 and CA3 subfields at 7 and 14 d PSE. FK506-treated PSE mice showed a similar decrease of F-actin, but the extent of damage was significantly ameliorated. Consistently, the surviving neurons became significantly increased in number after FK506 treatment, relative to the PSE + vehicle groups. After FK506 treatment, microglial reaction was partially inhibited, but the expression of GFAP was not significantly changed, compared to the PSE + vehicle mice. The results suggest that post-epileptic treatment with FK506 ameliorated, but could not stop the deconstruction of F-actin or the delayed neuronal loss in the PSE mice.

[The Application of the L-Curve Method in the Retrieval of Temperature Profiles Using Ground-Based Hyper-Spectral Infrared Radiance].

The thermodynamic profiles of Planetary Boundary Layer could be retrieved by using ground-based hyper-spectral infrared radiance. The AERIoe algorithm has a better performance at the dependency of initial profiles than the "onion peeling" method which was originally applied in the Atmospheric Emitted Radiance Interferometer. The regularization parameter is the key to the AERIoe algorithm, and the strategy for choosing the regularization parameter in the retrieval algorithm is based on the empirical method, which requires too much time for computation while the empirical method needs many iteration steps. A L-curve criterion is proposed to calculate the regularization parameter in AERIoe algorithm. The L-curve criterion is based on a log-log plot of corresponding values of the residual and solution norms, and the optimal regularization parameter corresponds to a point on the curve near the "corner" of the L-shaped region. Therefore, the L-curve criterion has better theoretical basis than the traditional empirical method. The result of retrieval experiment using the observed data collected at the SGP site of the year 2011 shows that, the L-curve method has a good performance in terms of stability, convergence and accuracy of the retrieval. Compared with empirical method, L-curve algorithm converges more quickly which saves much computation time when retrieving the temperature profiles. When considering the retrieval accuracy, the L-curve method has a better behavior at the middle and top of the boundary layer, with an improvement of 0.2 K of RMSE at the altitude of 1～3 km than the empirical method. Therefore, the L-curve algorithm has a better performance compared with the empirical method when choosing the regularization parameter in the retrieval of temperature profiles using the ground-based hyper-spectral infrared radiance.

[Research on the Noise Reduction with Hyper-Resolution Infrared Spectrum Based on Improved PCV Method].

The noise reduction with observed high resolution infrared radiance is crucial to improve the accuracy and stability of the retrieval of thermodynamic profiles. When applying the principal component analysis noise filter algorithm to the observed radiance, the optimal number k of principal components that used in the algorithm was mostly calculated with the statistical and empirical method. The percent cumulative variance method is one of the statistical methods that have been commonly used to calculate k, however, the threshold of the percent cumulative variance was determined subjectively and arbitrarily, which limits the application of this method. While the empirical method need the real-time Noise-Equivalent Spectral Radiance (NESR) to normalize non uniform noise in the observed data, but the real-time NESR needs the raw data of complex spectrum which is not easy to obtain in most cases. Aiming at the solving the problems above, a PCA noise filter based on the Improved PCV algorithm is proposed, of which the threshold is determined by iteratively calculating the difference between the simulated and reconstructed spectrum using different principal components, whereby k is determined such that the PCV is larger than the threshold. The new method solves the problem of arbitrary of the determination of k, and at the same time it doesn't need the real-time NESR to normalize the observed radiance. First, the impact of normalization on the noise reduction is analyzed using physical retrieval of temperature profiles; the result shows that the impact is very small, which less than the impact of calculation error of k is caused by normalization on the retrieval of temperature profiles. Then, the noise reduction of the representative radiance data which covers four quarters of 2011 shows that, the RMSE of the retrieved temperature profile using the Improved PCV method is improved by 0.1 K compared to the factor indicator function method when the real-time NESR is not available, and it is almost the same with the latter when the normalization is done. Under the condition that the NESR is not available, the method proposed in this article could objectively and reasonably reduce the noise level of the ground-based high resolution infrared radiance.

Pilocarpine-induced epilepsy is associated with actin cytoskeleton reorganization in the mossy fiber-CA3 synapses.

Dramatic structural changes have been demonstrated in the mossy fiber-CA3 synapses in the post status epilepticus (SE) animals, suggesting a potential reorganization of filamentous actin (F-actin) network occurring in the hippocampus. However, until now the long-term effects of SE on the synaptic F-actin have still not been reported. In this study, phalloidin labeling combined with confocal microscopy and protein analyses were adopted to investigate the effects of pilocarpine treatment on the F-actin in the C57BL/6 mice. As compared to the controls, there was ∼ 43% reduction in F-actin density in the post SE mice. Quantitative analysis showed that the labeling density and the puncta number were significantly decreased after pilocarpine treatment (p<0.01, n=5 mice per group, Student's t-test). The puncta of F-actin in the post SE group tended to be highly clustered, while those in the controls were generally distributed evenly. The mean puncta size of F-actin puncta was 0.73±0.19µm(2) (n=1102 puncta from 5 SE mice) in the experimental group, significantly larger than that in the controls (0.51±0.10µm(2), n=1983 puncta from 5 aged-matched control mice, p<0.01, Student's t-test). These observations were well consistent with the alterations of postsynaptic densities in the same region, revealed by immunostaining of PSD95, suggesting the reorganization of F-actin occurred mainly postsynaptically. Our results are indicative of important cytoskeletal changes in the mossy fiber-CA3 synapses after pilocarpine treatment, which may contribute to the excessive excitatory output in the hippocampal trisynaptic circuit.

The rearrangement of filamentous actin in mossy fiber synapses in pentylenetetrazol-kindled C57BL/6 mice.

Chemical kindling, as an experimental model of epileptogenesis, is induced by repetitive administration of subconvulsive amount of excitatory drugs. Kindled mice do not typically display spontaneous recurrent seizures, but are instead characterized by enhanced seizure susceptibility to convulsive stimulations. In order to provide insights into the aberrant synaptic plasticity during kindling, this study investigated the effect of pentylenetetrazol (PTZ) kindling on filamentous actin (F-actin) in mossy fiber synapses in C57BL/6 mice. Phalloidin labeling of F-actin showed that F-actin puncta were increased in number in the stratum lucidum of CA3 region in the hippocampus after kindling. The rearrangement of F-actin seemed to occur presynaptically, since synapsin I, a specific marker for mossy fiber terminals, was also up-regulated. Such subtle structural modifications occurring in the synapses are thought to contribute to the long-lasting increased sensitivity in the PTZ-kindled C57BL/6 mice.

Human umbilical cord mesenchymal stem cells derived from Wharton's jelly differentiate into cholinergic-like neurons in vitro.

In Alzheimer's disease patients, dysfunction of the cholinergic neurons is one of the causes of cognitive disorders. Although there is still no effective cure for theses diseases and conditions, some promising strategies are currently available to replace these damaged cells. Wharton's jelly mesenchymal stem cells (WJ-MSCs) derived from umbilical cord appeared as a promising cell source for cell replacement therapy. However, the capacity of WJ-MSCs to differentiate into cholinergic-like neurons remains undetermined. In this study, we examined whether WJ-MSCs could differentiate into cholinergic-like neurons in vitro. After induction, the spindle-shaped or fibroblast-like WJ-MSCs changed into bulbous cells. The induced cells positively expressed cholinergic neuron's markers, and an acetylcholine secretion of the induced WJ-MSCs was significantly elevated. These results demonstrated that WJ-MSCs had capability to differentiate into cholinergic-like neurons.