Nogo-A and its Nogo receptor (NgR) have been shown to inhibit plasticity after central nervous system lesions. Therefore, we hypothesized that Nogo-A and its receptor NgR will be upregulated and will activate RhoA, and thus, they play a role in the damage in the infarction developed. To test this hypothesis, a focal cerebral infarction model was created by coagulation of the right middle cerebral artery (MCA) and ipsilateral common carotid artery (CCA), as well as the simultaneous transient occlusion of the contralateral CCA for 30 min in 60 adult Sprague-Dawley rats. The rat brains were treated at 6 h, 12 h, 24 h, 48 h, 96 h, and 7 d after cerebral infarction. Sham controls were collected to determine histopathologic damage and Nogo-A, NgR, and RhoA expression using hematoxylin-eosin, immunohistochemical staining, Western blot analysis, and fluorimeter-based quantitive reverse transcriptase-polymerase chain reaction. The results indicate that cerebral infarction produced damage and edema on nerve cells in the infarction area, becoming most prominent at 24h after modeling. Meanwhile, a marked increase of Nogo-A, NgR, and RhoA expression was found at 6h in model groups compared with the sham controls, which peaked at 24 h after the operation. Immunohistochemical staining and Western blot analysis also showed upregulated Nogo-A located in the myelin sheath of the infarction area, NgR expressed on the surface of neurons and their processes, and RhoA expressed inside the cytoplasm of neurons in infarction brain. In conclusion, the upregulation of Nogo-A, NgR, and RhoA in the infarction area may be an important feature of cerebral infarction and may play a role in the pathologic progression of this lesion.

Fulminant hepatic failure (FHF) can be dreadful. When coma sets in, brain edema develops taking FHF into a lethal course. Mechanisms of brain extravasation leading to brain edema remain incompletely understood. Matrix metalloproteinase (MMP)-9 is implicated in various brain injuries. We hypothesized that MMP-9 contributes to brain edema in FHF.

MMP-9 and its proform were assayed using SDS-PAGE and in situ gelatin zymographies. Brain extravasation was assessed with Evans blue. Brain water was determined by specific gravity and astrocytic endfoot swelling by electron microscopy. FHF in mice was induced by azoxymethane. MMP inhibitor GM6001 and MMP-9 monoclonal antibody were used.

Active MMP-9 was significantly increased at the onset of coma and brain extravasation in FHF mice. Blocking MMP-9 with either GM6001 or MMP-9 monoclonal antibody significantly attenuated brain extravasation, astrocytic endfoot swelling, and brain edema. Brains of FHF mice did not show MMP-9 activity. In contrast, livers of these animals showed marked up-regulation of MMP-9 activity.

Our findings suggest that MMP-9 contributes to the pathogenesis of brain extravasation and edema in FHF. The necrotic liver is the source of MMP-9 in FHF. Inhibition of MMP-9 may protect against the development of brain edema in FHF.

Ischemic stroke results from a transient or permanent reduction in cerebral blood flow that is restricted to the territory of a major brain artery. The major pathobiological mechanisms of ischemia/reperfusion injury include excitotoxicity, oxidative stress, inflammation, and apoptosis. In the present report, we first investigated the protective effects of anthocyanins against focal cerebral ischemic injury in rats. The pretreatment of anthocyanins (300 mg/kg, p.o.) significantly reduced the brain infarct volume and a number of TUNEL positive cells caused by middle cerebral artery occlusion and reperfusion. In the immunohistochemical observation, anthocyanins remarkably reduced a number of phospho-c-Jun N-terminal kinase (p-JNK) and p53 immunopositive cells in the infarct area. Moreover, Western blotting analysis indicated that anthocyanins suppressed the activation of JNK and up-regulation of p53. Thus, our data suggested that anthocyanins reduced neuronal damage induced by focal cerebral ischemia through blocking the JNK and p53 signaling pathway. These findings suggest that the consumption of anthocyanins may have the possibility of protective effect against neurological disorders such as brain ischemia.

Nix, a hypoxia-sensitive member of the Bcl-2 family, is upregulated at the mRNA level during hypoxia through induction of a hypoxia-inducible factor-1 alpha (HIF-1 alpha) response element in its promoter sequence. However, the mechanism(s) regulating Nix protein activation remain unclear. The present studies examine Nix protein expression and subcellular distribution in response to hypoxic stimuli in vivo and in culture and to two disparate apoptotic stimuli in vitro. Upregulation and translocation of Nix (by day 5) in hypoxic/serum-deprived CHO-K1 cells, was preceded by Bax activation (by day 4) and caspase-3 processing (by day 2), suggesting that initiation of cell death in vitro is a Nix-independent event. In contrast, an early Nix response (upregulation and translocation to the mitochondria) was observed after 6 h of middle cerebral artery occlusion in the rat. Nix translocation was observed in the ipsilateral cortex and striatum before other histological (infarct development, neuronal loss, apoptotic body formation) or biochemical (Bax activation or caspase-3 cleavage) markers of damage were detected. While fundamental differences between hypoxia/ischaemia in culture and in vivo likely explain the different temporal profiles of Nix, Bax, and caspase-3 activation observed, these studies show that like Bax, mitochondrial accumulation is a common event during Nix activation. These are the first studies to show upregulation and translocation of Nix in the ischaemic brain and suggest Nix to be a novel therapeutic target in ischaemic research. Moreover, Nix upregulation in staurosporine-treated SH-SY5Y cells and dexamethasone-treated A1.1 cells supports a more generalized role for Nix in apoptotic cell death.

The authors investigated the expression of p53, p21(WAF-1), Bax protein, and apoptosis to elucidate the cellular response to ischemia-reperfusion of skeletal muscle using the rat lower limb model. The rat left lower limb was dissected in the inguinal region, isolating the bony femoral muscles, and the femoral vessels were clamped to produce an ischemic condition. After 3 or 6 hours, the clamps were removed and the gastrocnemius muscle was resected at various times up to 72 hours after reperfusion. Five specimens of the muscle were obtained at each time point from 5 rats. When any rat died during the study, additional rats were used until 5 specimens could be obtained from 5 rats at each time point. The expression of three proteins was detected by Western blot analysis. The apoptotic cells were detected using terminal deoxytransferase-mediated dUDP (deoxyuridine[-5']diphosphate) nick-end labeling assay. Histopathological study showed severe interstitial edema and leukocyte infiltration at 6 hours of ischemia compared with 3 hours of ischemia. Moreover, at 6 hours of ischemia, muscle fiber fragmentation was observed at 72 hours after reperfusion whereas no fragmentation was found at 3 hours of ischemia. At 3 hours of ischemia, p53 and p21(WAF-1) accumulated after reperfusion, and there was a time lag in the time of onset of elevation and the peak time point between these two proteins. The level of Bax protein did not elevate and the rate of apoptotic cells did not increase. At 6 hours of ischemia, p53 and p21(WAF-1) also accumulated, but the kinetics of p21(WAF-1) were similar to that of p53 in the time of onset of elevation and the peak time point after reperfusion. In addition, the level of Bax protein increased and apoptosis was induced. These results demonstrated that p53 and p21(WAF-1) accumulated after 3 and 6 hours of ischemia of skeletal muscle during reperfusion. Moreover, it was demonstrated that the kinetics of induced p53, p21(WAF-1) and Bax protein differ between 3 hours and 6 hours of ischemia, and it is speculated that this difference plays an important role in determining the consequence of the cell exposed to ischemia.

The authors investigated the expression of p53, p21WAF-1, and Bax proteins, and apoptosis to elucidate the cellular response to ischemia-reperfusion of the skin. The rat left lower limb was dissected at the inguinal region retaining the bone and femoral vessels, and the vessels were clamped to produce an ischemic condition. After 6 hours the clamps were removed, and the plantar skin was resected at various times up to 72 hours after reperfusion. Five skin specimens were obtained at each time point from 5 rats. When a rat died during the study, additional rats were used until five specimens could be obtained from 5 rats at each time point. The expression of the three proteins was detected by Western blot analysis. The apoptotic cells were detected using the terminal deoxytransferase-mediated dUDP nick-end labeling assay. After reperfusion, the levels of p53 and p21WAF-1 were significantly higher in the ischemia-reperfusion rats compared with the sham-operated rats. However, the levels of Bax protein did not show a noticeable increase at any period. The apoptotic cells in both the epidermis and dermis were not evident compared with the sham skin, which were similar to those in the nontreated, normal skin. These results demonstrate that p53 and p21WAF-1 proteins accumulate after 6 hours of ischemia of the skin during reperfusion. Moreover, it is speculated that accumulation of these proteins plays an important role in the survival of the skin by inducing growth arrest of the cells, but not apoptosis.

This review examines the appearance of hallmarks of apoptosis following experimental stroke. The reviewed literature leaves no doubt that ischemic cell death in the brain is active, that is, requires energy; is gene directed, that is, requires new gene expression; and is capase-mediated, that is, uses apoptotic proteolytic machinery. However, sufficient differences to both classical necrosis and apoptosis exist which prevent easy mechanistic classification. It is concluded that ischemic cell death in the brain is neither necrosis nor apoptosis but is a chimera which appears on a continuum that has apoptosis and necrosis at the poles. The position on this continuum could be modulated by the intensity of the ischemic injury, the consequent availability of ATP and new protein synthesis, and both the age and context of the neuron in question. Thus the ischemic neuron may look necrotic but have actively died in an energy dependent manner with new gene expression and destruction via the apoptotic proteolytic machinery.