Shihui Xing

Beclin 1 knockdown inhibits autophagic activation and prevents the secondary neurodegenerative damage in the ipsilateral thalamus following focal cerebral infarction.

Cerebral infarction can cause secondary degeneration of thalamus and delay functional recovery. However, the mechanisms underlying secondary degeneration are unclear. The present study aimed to determine the occurrence and contribution of autophagy to the thalamic degeneration after cerebral infarction. Focal cerebral infarction was induced by distal middle cerebral artery occlusion (MCAO). Autophagic activation, Beclin 1 expression and amyloid β (Aβ) deposits were determined by immunofluorescence, immunoblot and electron microscopy. Secondary damage to thalamus was assessed with Nissl staining and immunofluorescence analysis. Apoptosis was determined using TUNEL staining. The contribution of autophagy to the secondary damage was evaluated by shRNA-mediated downregulation of Beclin 1 and the autophagic inhibitor, 3-methyladenine (3-MA). The potential role of Aβ in autophagic activation was determined with N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The results showed that the conversion of LC3-II, the formation of autophagosomes, and the levels of activated cathepsin B and Beclin 1 were significantly increased in the ipsilateral thalamus at 7 and 14 days after MCAO (p < 0.05 or 0.01). Both Beclin 1 knockdown and 3-MA treatment significantly reduced LC3-II conversion and autophagosome formation, which were accompanied by obvious decreases in neuronal loss, gliosis and apoptosis in the ipsilateral thalamus (p < 0.05 or 0.01). Additionally, DAPT treatment markedly reduced Aβ deposits, which coincided with decreases in LC3-II conversion and autophagosome formation (p < 0.01). These results suggest that inhibition of autophagy by Beclin 1 knockdown can attenuate the secondary thalamic damage after focal cerebral infarction. Furthermore, Aβ deposits may be involved in the activation of autophagy.

Secondary Neurodegeneration in Remote Regions After Focal Cerebral Infarction: A New Target for Stroke Management?

Cerebral infarction-induced cessation of function in areas of the brain remote from, but connected to, the primary site of damage was termed “diaschisis” by von Monakow.1 Initially, the concept of diaschisis did not include morphological changes. However, accumulating evidence has shown that histopathologic changes also occur in nonischemic remote brain regions that have synaptic connections with the primary lesion site. For example, after cerebral infarction in the middle cerebral artery (MCA) territory, neuronal death, gliosis, and axonal degeneration have been found in the ipsilateral thalamus, substantia nigra (SN), and distal pyramidal tract, all of which lie outside the MCA territory.2–7 This kind of secondary neurodegeneration occurs selectively in such areas several days or weeks after stroke onset, and this can be detected by neuroimaging techniques.8 For quite a long time, the role of secondary degeneration in stroke recovery has not been well understood. Recently, emerging studies suggest that secondary degeneration is associated with neurological deficits and can predict motor outcome after stroke.9–15 In this review, we aimed to summarize the pathological and neuroimaging evidence of secondary neurodegeneration in the ipsilateral thalamus, SN, and pyramidal tract after MCA infarction and described its potential significance for stroke management. We searched PubMed from 1980 to September 2011, using the terms “cerebral infarction,” “middle cerebral artery,” “Wallerian degeneration,” “anterograde degeneration,” “retrograde degeneration,” and “transneuronal degeneration.” Further articles were identified from the references cited in those articles and through searches of our personal files. The final list of references was selected on the basis of originality and relevance to the topics covered in this review. ### Postmortem Studies Data from postmortem studies provide direct evidence of secondary neurodegeneration after focal cerebral infarction (Figure 1). Histopathologic examination at 4 months after MCA infarction revealed a delayed and selective decrease in the …

Nogo-A is involved in secondary axonal degeneration of thalamus in hypertensive rats with focal cortical infarction

We investigate whether Nogo-A is involved in the secondary axonal degeneration in the thalamus after distal middle cerebral artery occlusion (MCAO) in stroke-prone renovascular hypertensive rats (RHRSP). The expression of Nogo-A in ipsilateral ventroposterior nucleus (VPN) of the thalamus in RHRSP was observed at 1, 2 and 4 weeks after distal MCAO. In addition, intracerebroventricular infusion of NEP1-40, a Nogo-66 receptor (NgR) antagonist peptide, was administered starting 24 h after MCAO and continued for 1, 2 and 4 weeks, respectively. Axonal damage and regeneration were evaluated by analysis of the immunoreactivity (IR) of amyloid betaA4 precursor protein (APP), growth associated protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2) in ipsilateral VPN of the thalamus at 1, 2 and 4 weeks after distal MCAO. Following ischemia, the expression of Nogo-A in oligodendrocytes increased persistently and its localization became redistributed around damaged axons and dendrites. Administration of NEP1-40 downregulated the expression of Nogo-A, reduced axonal injury and enhanced axonal regeneration. Our data suggest that Nogo-A is involved in secondary axonal degeneration and that inhibition of Nogo-A can reduce neuronal damage in the thalamus after distal MCAO.

Reduction of β-Amyloid Deposits by γ-Secretase Inhibitor is Associated with the Attenuation of Secondary Damage in the Ipsilateral Thalamus and Sensory Functional Improvement after Focal Cortical Infarction in Hypertensive Rats

Abnormal β-amyloid (Aβ) deposits in the thalamus have been reported after cerebral cortical infarction. In this study, we investigated the association of Aβ deposits, with the secondary thalamic damage after focal cortical infarction in rats. Thirty-six stroke-prone renovascular hypertensive rats were subjected to distal middle cerebral artery occlusion (MCAO) and then randomly divided into MCAO, vehicle, and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) groups and 12 sham-operated rats as control. The DAPT was administered orally at 72 hours after MCAO. Seven days after MCAO, sensory function, neuron loss, and glial activation and proliferation were evaluated using adhesive removal test, Nissl staining, and immunostaining, respectively. Thalamic Aβ accumulation was evaluated using immunostaining and enzyme-linked immunosorbent assay (ELISA). Compared with vehicle group, the ipsilateral thalamic Aβ, neuronal loss, glial activation and proliferation, and the mean time to remove the stimulus from right forepaw significantly decreased in DAPT group. The mean time to remove the stimulus from the right forepaw and thalamic Aβ burden were both negatively correlated with the number of thalamic neurons. These findings suggest that Aβ deposits are associated with the secondary thalamic damage. Reduction of thalamic Aβ by γ-secretase inhibitor may attenuate the secondary damage and improve sensory function after cerebral cortical infarction.

Autophagosomes accumulation is associated with β-amyloid deposits and secondary damage in the thalamus after focal cortical infarction in hypertensive rats

J. Neurochem. (2012) 120, 564–573.

Neurogenesis and angiogenesis within the ipsilateral thalamus with secondary damage after focal cortical infarction in hypertensive rats

Neurogenesis and angiogenesis in the subventricular zone and peri-infarct region have been confirmed. However, newly formed neuronal cells and blood vessels that appear in the nonischemic ipsilateral ventroposterior nucleus (VPN) of the thalamus with secondary damage after stroke has not been previously studied. Twenty-four stroke-prone renovascular hypertensive rats were subjected to distal right middle cerebral artery occlusion (MCAO) or sham operation. 5′-Bromo-2′-deoxyuridine (BrdU) was used to label cell proliferation. Rats were killed at 7 or 14 days after the operation. Neuronal nuclei (NeuN), OX-42, BrdU, nestin, laminin+, BrdU+/nestin+, BrdU+/NeuN+, nestin+/GFAP+(glial fibrillary acidic protein), and BrdU+/laminin+ immunoreactive cells were detected within the ipsilateral VPN. The primary infarction was confined to the right somatosensory cortex. Within the ipsilateral VPN of the ischemic rats, the number of NeuN+ neurons decreased, the OX-42+ microglia cells were activated, and BrdU+ and nestin+ cells were detected at day 7 after MCAO and increased in number at day 14. Moreover, BrdU+/nestin+ cells and BrdU+/NeuN+ cells were detected at day 14 after MCAO. In addition, the ischemic rats showed a significant increase in vascular density in the ipsilateral VPN compared with the sham-operated rats. These results suggest that secondary damage with neurogenesis and angiogenesis of the ipsilateral VPN of the thalamus occurs after focal cortical infarction.

Longitudinal Cortical Volume Changes Correlate With Motor Recovery in Patients After Acute Local Subcortical Infarction

Background and Purpose— Secondary changes in the volume of motor-related cortical regions and the relationship with functional recovery during the acute stage after cerebral infarction have not been determined. In the present study, we quantified changes in gray matter (GM) volume in motor-related cortical regions and analyzed their correlations to clinical scores in patients with focal cerebral infarct. Methods— Fifteen patients with acute subcortical infarct underwent longitudinal high-resolution structural MRI and clinical assessment 3 times during a 12-week period (weeks 1, 4, and 12). Fourteen age- and sex-matched controls underwent MRI examination. Voxel-based morphometry was used to quantify changes in global GM volume; in addition, relationships between GM volume changes in volumes of interest and clinical scores were analyzed. Results— In patients with cerebral infarction, GM volumes detected by voxel-based morphometry both decreased and increased significantly in diffuse cortical regions during the observation period (P<0.001). GM volumes within volumes of interest decreased significantly in the ipsilateral supplementary motor area and contralateral insula, but they increased in the contralateral supplementary motor area over time (all P<0.017). The changes of GM volumes in the ipsilesional and contralesional supplementary motor area correlated with the changes in the Fugl–Meyer scale scores (ipsilesional, rs=0.52; P=0.048; contralesional, rs=0.74; P=0.002) and Barthel Index (ipsilesional, rs=0.56; P=0.030; contralesional, rs=0.65; P=0.009). Conclusions— These results suggest that secondary GM changes occur in diffuse areas and structural changes in some specific motor-related cortex may inhibit or promote functional recovery after an acute subcortical cerebral infarct.

Early detection of secondary damage in ipsilateral thalamus after acute infarction at unilateral corona radiata by diffusion tensor imaging and magnetic resonance spectroscopy.

BackgroundTraditional magnetic resonance (MR) imaging can identify abnormal changes in ipsilateral thalamus in patients with unilateral middle cerebral artery (MCA) infarcts. However, it is difficult to demonstrate these early changes quantitatively. Diffusion tensor imaging (DTI) and proton magnetic resonance spectroscopy (MRS) are potentially sensitive and quantitative methods of detection in examining changes of tissue microstructure and metabolism. In this study, We used both DTI and MRS to examine possible secondary damage of thalamus in patients with corona radiata infarction.MethodsTwelve patients with unilateral corona radiata infarction underwent MR imaging including DTI and MRS at one week (W1), four weeks (W4), and twelve weeks (W12) after onset of stroke. Twelve age-matched controls were imaged. Mean diffusivity (MD), fractional anisotropy (FA), N-acetylaspartate (NAA), choline(Cho), and creatine(Cr) were measured in thalami.ResultsT1-weighted fluid attenuation inversion recovery (FLAIR), T2-weighted, and T2-FLAIR imaging showed an infarct at unilateral corona radiate but no other lesion in each patient brain. In patients, MD was significantly increased at W12, compared to W1 and W4 (all P< 0.05). NAA was significantly decreased at W4 compared to W1, and at W12 compared to W4 (all P< 0.05) in the ipsilateral thalamus. There was no significant change in FA, Cho, or Cr in the ipsilateral thalamus from W1 to W12. Spearmans rank correlation analysis revealed a significant negative correlation between MD and the peak area of NAA, Cho, and Cr at W1, W4, and W12 and a significant positive correlation of FA with NAA at W1.ConclusionsThese findings indicate that DTI and MRS can detect the early changes indicating secondary damage in the ipsilateral thalamus after unilateral corona radiata infarction. MRS may reveal the progressive course of damage in the ipsilateral thalamus over time.

Cerebrolysin reduces amyloid-β deposits, apoptosis and autophagy in the thalamus and improves functional recovery after cortical infarction

Focal cerebral infarction causes amyloid-β (Aβ) deposits and secondary thalamic neuronal degeneration. The present study aimed to determine the protective effects of Cerebrolysin on Aβ deposits and secondary neuronal damage in thalamus after cerebral infarction. At 24h after distal middle cerebral artery occlusion (MCAO), Cerebrolysin (5 ml/kg) or saline as control was once daily administered for consecutive 13 days by intraperitoneal injection. Sensory function and secondary thalamic damage were assessed with adhesive-removal test, Nissl staining and immunofluorescence at 14 days after MCAO. Aβ deposits, activity of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), apoptosis and autophagy were determined by TUNEL staining, immunofluorescence and immunoblot. The results showed that Cerebrolysin significantly improved sensory deficit compared to controls (p<0.05). Aβ deposits and BACE1 were obviously reduced by Cerebrolysin, which was accompanied by decreases in neuronal loss and astroglial activation compared to controls (all p < 0.05). Coincidently, Cerebrolysin markedly inhibited cleaved caspase-3, conversion of LC3-II, downregulation of Bcl-2 and upregulation of Bax in the ipsilateral thalamus compared to controls (all p<0.05). These findings suggest that Cerebrolysin reduces Aβ deposits, apoptosis and autophagy in the ipsilateral thalamus, which may be associated with amelioration of secondary thalamic damage and functional recovery after cerebral infarction.

Nogo-A is associated with secondary degeneration of substantia nigra in hypertensive rats with focal cortical infarction.

We investigated the association of Nogo-A protein, a myelin-associated inhibitor of axon regeneration, with secondary damage of the ipsilateral substantia nigra pars reticulata (SNr) after distal middle cerebral artery occlusion (dMCAO) in adult stroke-prone, renovascular hypertensive rats. Intracerebroventricular infusion of NEP1-40, a Nogo-66 receptor antagonist peptide, or vehicle was administered starting 24h after dMCAO and continued for 1, 2, or 4 weeks. The expression of Nogo-A in the ipsilateral SNr was assessed by immunohistochemistry. Neuron death and apoptosis were evaluated using Nissl and terminal uridine nick-end labeling (TUNEL) staining. Glial activation was monitored by immunoreactivity of glial fibrillary acidic protein and the oligodendrocyte marker RIP. Axonal damage and regeneration were determined by Bielschowskys silver staining and immunoreactivity of growth associated protein 43 and microtubule associated protein 2. We found progressive damage in the center of the ipsilateral SNr through 4 weeks after dMCAO. The neuronal loss was topographically related to axonal degeneration that occurred indirectly from the infarcted cortex. Nogo-A protein in oligodendrocytes was persistently increased in the damaged SNr. Administration of NEP1-40 inhibited Nogo-A expression, the loss of neurons, apoptosis, and proliferation of oligodendrocytes and astrocytes. It also boosted the regenerative response of injured axons and encouraged compensatory neurite growth in the ipsilateral SNr. Our data suggest that secondary damage in the ipsilateral SNr may be due to trans-synaptic axonal degeneration that followed the cortical infarct. Further, we showed that Nogo-A is involved in axonal degeneration, and NEP1-40 reduces secondary nigral damage after focal cortical ischemia.