Retinal Microvascular Changes in Subtypes of Ischemic Stroke.

Aims: Retinal microvasculature shares prominent similarities with the brain vasculature. We aimed to assess the association between retinal microvasculature and subtypes of ischemic stroke. Method: We consecutively enrolled ischemic stroke patients within 7 days of onset, who met the criteria of subtype of atherothrombosis (AT), small artery disease (SAD), or cardioembolism (CE) according to a modified version of the Trial of Org 10172 in Acute Stroke Treatment (NEW-TOAST). Digital fundus photographs were taken within 72 h of hospital admission using a digital camera (Topcon TRC-50DX), and fundus photographs were semi-automatically measured by software (Canvus 14 and NeuroLucida) for retinal vasculature parameters. Results: A total of 141 patients were enrolled, including 72 with AT, 54 with SAD, and 15 with CE. AT subtype patients had the widest mean venular diameter within 0.5-1.0 disk diameter (MVD0.5-1.0DD) followed by SAD and CE subtypes (86.37 ± 13.49 vs. 83.55 ± 11.54 vs. 77.90 ± 8.50, respectively, P = 0.047); CE subtype patients had the highest mean arteriovenous ratio within 0.5-1.0 disk diameter (MAVR0.5-1.0DD) followed by the AT and SAD subtype groups (0.97 ± 0.03 vs. 0.89 ± 0.99 vs. 0.89 ± 0.11, respectively, P = 0.010); SAD subtype patients were found with the highest mean venular tortuosity within 0.0-2.0 disk diameter (MVT0.0-2.0DD) followed by the AT and CE subtypes (1.0294 ± 0.0081 vs. 1.0259 ± 0.0084 vs. 1.0243 ± 0.0066, respectively, P = 0.024). After adjusting for clinic characteristics, MVD0.5-1.0DD was significantly different among AT, SAD, and CE subtypes (P = 0.033). By receiver operating characteristic curve analysis, MVD0.5-1.0DD predicted the AT subtype (area 0.690, 95% confidence interval, 0.566-0.815), with a cutoff value of 82.23 µm (sensitivity 61.1%, specificity 73.3%). Conclusion: Retinal MVD0.5-1.0DD (>82.23 µm) might be associated with the AT stroke subtype; however, we need large-scale prospective studies in future to explore the underlying mechanism and causal explanation for this finding.

Changes in synaptic plasticity and expression of glutamate receptor subunits in the CA1 and CA3 areas of the hippocampus after transient global ischemia.

Excess glutamate release from the presynaptic membrane has been thought to be the major cause of ischemic neuronal death. Although both CA1 and CA3 pyramidal neurons receive presynaptic glutamate input, transient cerebral ischemia induces CA1 neurons to die while CA3 neurons remain relatively intact. This suggests that changes in the properties of pyramidal cells may be the main cause related to ischemic neuronal death. Our previous studies have shown that the densities of dendritic spines and asymmetric synapses in the CA1 area are increased at 12h and 24h after ischemia. In the present study, we investigated changes in synaptic structures in the CA3 area and compared the expression of glutamate receptors in the CA1 and CA3 hippocampal regions of rats after ischemia. Our results demonstrated that the NR2B/NR2A ratio became larger after ischemia although the expression of both the NR2B subunit (activation of apoptotic pathway) and NR2A subunit (activation of survival pathway) decreased in the CA1 area from 6h to 48h after reperfusion. Furthermore, expression of the GluR2 subunit (calcium impermeable) of the AMPA receptor class significantly decreased while the GluR1 subunit (calcium permeable) remained unchanged at the same examined reperfusion times, which subsequently caused an increase in the GluR1/GluR2 ratio. Despite these notable differences in subunit expression, there were no obvious changes in the density of synapses or expression of NMDAR and AMPAR subunits in the CA3 area after ischemia. These results suggest that delayed CA1 neuronal death may be related to the dramatic fluctuation in the synaptic structure and relative upregulation of NR2B and GluR1 subunits induced by transient global ischemia.

Effect of Lycium barbarum (Wolfberry) on alleviating axonal degeneration after partial optic nerve transection.

Our previous results showed that the polysaccharides extracted from Lycium barbarum (LBP) could delay secondary degeneration of retinal ganglion cell bodies and improve the function of the retinas after partial optic nerve transection (PONT). Although the common degeneration mechanisms were believed to be shared by both neuronal bodies and axons, recently published data from slow Wallerian degeneration mutant (Wld(s)) mice supported the divergence in the mechanisms of them. Therefore, we want to determine if LBP could also delay the degeneration of axons after PONT. Microglia/macrophages were thought to be a source of reactive oxygen species after central nervous system (CNS) injury. After PONT, however, oxidative stress was believed to occur prior to the activation of microglia/macrophages in the areas vulnerable to secondary degeneration both in the optic nerves (ONs) and the retinas. But the results did not take into account the morphological changes of microglia/macrophages after their activation. So we examined the morphology in addition to the response magnitude of microglia/macrophages to determine their time point of activation. In addition, the effects of LBP on the activation of microglia/macrophages were investigated. The results showed that (1) LBP reduced the loss of axons in the central ONs and preserved the g-ratio (axon diameter/fiber diameter) in the ventral ONs although no significant effect was detected in the dorsal ONs; (2) microglia/macrophages were activated in the ONs by 12 h after PONT; (3) LBP decreased the response magnitude of microglia/macrophages 4 weeks after PONT. In conclusion, our results showed that LBP could delay secondary degeneration of the axons, and LBP could also inhibit the activation of microglia/macrophages. Therefore, LBP could be a promising herbal medicine to delay secondary degeneration in the CNS via modulating the function of microglia/macrophages.

Mechanisms of secondary degeneration after partial optic nerve transection.

Secondary degeneration occurs commonly in the central nervous system after traumatic injuries and following acute and chronic diseases, including glaucoma. A constellation of mechanisms have been shown to be associated with secondary degeneration including apoptosis, necrosis, autophagy, oxidative stress, excitotoxicity, derangements in ionic homeostasis and calcium influx. Glial cells, such as microglia, astrocytes and oligodendrocytes, have also been demonstrated to take part in the process of secondary injury. Partial optic nerve transection is a useful model which was established about 13 years ago. The merit of this model compared with other optic nerve injury models used for glaucoma study, including complete optic nerve transection model and optic nerve crush model, is the possibility to separate primary degeneration from secondary degeneration in location. Therefore, it provides a good tool for the study of secondary degeneration. This review will focus on the research progress of the mechanisms of secondary degeneration using partial optic nerve transection model.

Hyperphosphorylation of tau protein in the ipsilateral thalamus after focal cortical infarction in rats.

Hyperphosphorylation of tau has been considered as an important risk factor for neurodegenerative diseases. It has been found also in the cortex after focal cerebral ischemia. The present study is aimed at investigating changes of tau protein expression in the ipsilateral thalamus remote from the primary ischemic lesion site after distal middle cerebral artery occlusion (MCAO). The number of neurons in the ventroposterior thalamic nucleus (VPN) was evaluated using Nissl staining and neuronal nuclei (NeuN) immunostaining. Total tau and phosphorylated tau at threonine 231 (p-T231-tau) and serine 199 (p-S199-tau) levels, respectively, in the thalamus were measured using immunostaining and immunoblotting. Moreover, apoptosis was detected with terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay. It was found that the numbers of intact neurons and NeuN(+) cells within the ipsilateral VPN were reduced significantly compared with the sham-operated group, but the levels of p-T231-tau and p-S199-tau in the ipsilateral thalamus were increased significantly in rats subjected to ischemia for 3 days, 7 days and 28 days. Furthermore, the number of TUNEL-positive cells was increased in the ipsilateral VPN at 7 days and 28 days after MCAO. Thus, hyperphosphorylated tau protein is observed in ipsilateral thalamus after focal cerebral infarction in this study. Our findings suggest that the expression of hyperphosphorylated tau protein induced by ischemia may be associated with the secondary thalamic damage after focal cortical infarction via an apoptotic pathway.

Changes in retinal morphology, electroretinogram and visual behavior after transient global ischemia in adult rats.

The retina is a light-sensitive tissue of the central nervous system that is vulnerable to ischemia. The pathological mechanism underlying retinal ischemic injury is not fully understood. The purpose of this study was to investigate structural and functional changes of different types of rat retinal neurons and visual behavior following transient global ischemia. Retinal ischemia was induced using a 4-vessel occlusion model. Compared with the normal group, the number of ßIII-tubulin positive retinal ganglion cells and calretinin positive amacrine cells were reduced from 6 h to 48 h following ischemia. The number of recoverin positive cone bipolar cells transiently decreased at 6 h and 12 h after ischemia. However, the fluorescence intensity of rhodopsin positive rod cells and fluorescent peanut agglutinin positive cone cells did not change after reperfusion. An electroretinogram recording showed that the a-wave, b-wave, oscillatory potentials and the photopic negative response were completely lost during ischemia. The amplitudes of the a- and b-waves were partially recovered at 1 h after ischemia, and returned to the control level at 48 h after reperfusion. However, the amplitudes of oscillatory potentials and the photopic negative response were still reduced at 48 h following reperfusion. Visual behavior detection showed there was no significant change in the time spent in the dark chamber between the control and 48 h group, but the distance moved, mean velocity in the black and white chambers and intercompartmental crosses were reduced at 48 h after ischemia. These results indicate that transient global ischemia induces dysfunction of retinal ganglion cells and amacrine cells at molecular and ERG levels. However, transient global ischemia in a 17 minute duration does not appear to affect photoreceptors.

Diversity and fluctuation of spine morphology in CA1 pyramidal neurons after transient global ischemia.

Dendritic spines form postsynaptic components of excitatory synapses in CA1 pyramidal neurons and play a key role in excitatory signal transmission. Transient global ischemia is thought to induce excitotoxicity that triggers delayed neuronal death in the CA1 region. However, the mechanism underlying structural changes of excitatory synapses after ischemia is not completely understood. Here, we demonstrate how dendritic spines change in their density and structure at an acute stage after transient global ischemia. Intracellular staining in vivo showed that the total spine density in basal, proximal, and distal apical dendrites increased at 12 hr and 24 hr after ischemia, but returned to control levels at 48 hr after ischemia. Consistent increase of spine density mainly appeared in non-late depolarizing postsynaptic potential neurons, although late depolarizing postsynaptic potential neurons also showed slight increases in spine density in these dendrites at the same intervals after ischemia. Golgi staining showed increased spine density occurred in less swollen dendrites but decreased spine density appeared in severely swollen dendrites at 12 and 24 hr after ischemia. In addition, the density and percentage of stubby spines reduced at 12 hr and 48 hr, whereas the density of thin spines increased at 12 hr after ischemia. The density and percentage of filopodia increased nearly fivefold at 24 hr after ischemia. Moreover, the density of mushroom spines doubled and its percentage increased by 150% at 48 hr after ischemia. These morphological changes of spines may be related to neuronal injury in CA1 pyramidal neurons after ischemia.

Morphological heterogeneity of CA1 pyramidal neurons in response to ischemia.

We have found, based on the electrophysiological properties, two subtypes of CA1 pyramidal neurons in the CA1 region of the normal hippocampus, late postsynaptic potential (L-PSP) neurons and non-L-PSP neurons. In addition, our previous study has shown that the electrophysiological properties of these two subtypes of pyramidal neurons were differentially modified after ischemia. In the present study, we hypothesized that ischemia might also induce different morphological alterations in these two subtypes of neuron. To test the hypothesis, we compared the changes in the dendritic arborization and soma volume of these two subtypes of neurons in rats subjected to transient global ischemia. We found a significant decrease in the basal dendritic length of L-PSP neurons at 12 hr after reperfusion, resulting mainly from a significant decrease in the dendrite terminal length. The apical dendritic length of L-PSP neurons markedly increased at 24 hr after ischemia, resulting mainly from an increase in the number of branching arbors in the middle part of the apical dendritic trees. The soma size of L-PSP neurons was significantly reduced at 12 hr, but they became slightly larger at 24 hr and 48 hr after reperfusion. In contrast to L-PSP neurons, non-L-PSP neurons showed slight modifications in the dendritic arborization but had persistent swelling of their soma after ischemia. These results indicate that pathological changes in these two subtypes of neurons are different after ischemia.

Apoptosis in the adult striatum after transient forebrain ischemia and the effects of ischemic severity.

The mechanisms of neuronal injury after cerebral ischemia have been under active investigation. The medium-size neurons in the dorsal striatum die within 24 h after transient cerebral ischemia. Using electron microscopy, the present study examined the nature of neuronal death in the striatum of adult rats following transient forebrain ischemia and tested the hypothesis that the ischemic severity might influence the nature of cell death. After severe ischemia (approximately 21 min ischemic depolarization), most neurons in the dorsal striatum died with swollen organelles and small irregular chromatin clumps resembling necrosis. The tissue damage in the dorsomedial striatum was less severe than that in the dorsolateral striatum and approximately 5% of the neurons in this region died with large chromatin clumps and relatively intact organelles resembling apoptosis. Some neurons displayed a mixture of necrotic- and apoptotic-like appearance. In contrast, the neurons with large somata only exhibited mild ultrastructural changes. After moderate ischemia (approximately 15 min ischemic depolarization), the tissue damage was less severe and the process of necrosis was temporally prolonged compared with that after severe ischemia. The apoptotic-like neuronal death was observed not only in the dorsomedial (approximately 6%) but also in the dorsolateral striatum (approximately 7%). The neurons in the striatum showed transient reversible changes after mild ischemia (approximately 10 min ischemic depolarization). The present study demonstrates that both apoptosis and necrosis occur in the adult striatum following transient forebrain ischemia and apoptosis occurs in the regions with less severe ischemia. These results suggest that ischemic severity might be one of the contributing factors to necrosis or apoptosis following transient global ischemia.

Depression of fast excitatory synaptic transmission in large aspiny neurons of the neostriatum after transient forebrain ischemia.

Spiny neurons in the neostriatum die within 24 hr after transient global ischemia, whereas large aspiny (LA) neurons remain intact. To reveal the mechanisms of such selective cell death after ischemia, excitatory neurotransmission was studied in LA neurons before and after ischemia. The intrastriatally evoked fast EPSCs in LA neurons were depressed < or =24 hr after ischemia. The concentration-response curves generated by application of exogenous glutamate in these neurons were approximately the same before and after ischemia. A train of five stimuli (100 Hz) induced progressively smaller EPSCs, but the proportion of decrease in EPSC amplitude at 4 hr after ischemia was significantly smaller compared with control and at 24 hr after ischemia. Parallel depression of NMDA receptor and AMPA receptor-mediated EPSCs was also observed after ischemia, supporting the involvement of presynaptic mechanisms. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the inhibition of evoked EPSCs at 4 hr after ischemia but not at 24 hr after ischemia. Electron microscopic studies demonstrated that the most presynaptic terminals in the striatum had a normal appearance at 4 hr after ischemia but showed degenerating signs at 24 hr after ischemia. These results indicated that the excitatory neurotransmission in LA neurons was depressed after ischemia via presynaptic mechanisms. The depression of EPSCs shortly after ischemia might be attributable to the enhanced adenosine A1 receptor function on synaptic transmission, and the depression at late time points might result from the degeneration of presynaptic terminals.