Changman Zhou

Neurovascular Protection Reduces Early Brain Injury After Subarachnoid Hemorrhage

Background and Purpose— Cell death, especially apoptosis, occurred in brain tissues after subarachnoid hemorrhage (SAH). We examined the relationships between apoptosis and the disruption of blood–brain barrier (BBB), brain edema, and mortality in an established endovascular perforation model in male Sprague-Dawley rats. Methods— A pan–caspase inhibitor (z-VAD-FMK) was administered intraperitoneally at 1 hour before and 6 hours after SAH. Expression of caspase-3 and positive TUNEL was examined as markers for apoptosis. Results— Apoptosis occurred mostly in cerebral endothelial cells, partially in neurons in the hippocampus, and to a lesser degree in the cerebral cortex. Accordingly, increased BBB permeability and brain water content were observed, accompanied by neurological deficit and a high mortality at 24 hours after SAH. z-VAD-FMK suppressed TUNEL and caspase-3 staining in endothelial cells, decreased caspase-3 activation, reduced BBB permeability, relieved vasospasm, abolished brain edema, and improved neurological outcome. Conclusions— The major effect of z-VAD-FMK on early brain injury after SAH was probably neurovascular protection of cerebral endothelial cells, which results in less damage on BBB.

MMP-9 Deficiency Enhances Collagenase-Induced Intracerebral Hemorrhage and Brain Injury in Mutant Mice

Matrix metalloproteinase-9 (MMP-9) participates in the disregulation of blood–brain barrier during hemorrhagic transformation, and exacerbates brain injury after cerebral ischemia. However, the consequences of long-term inhibition or deficiency of MMP-9 activity (which might affect normal collagen or matrix homeostasis) remains to be determined. The authors investigated how MMP-9 gene deficiency enhances hemorrhage and increases mortality and neurologic deficits in a collagenase-induced intracerebral hemorrhage (ICH) model in MMP-9–knockout mice. MMP-9–knockout and corresponding wild-type mice at 20 to 35 weeks were used to model an aged population (because advanced age is a significant risk factor in human ICH). Collagenase VII-S (0.5 μL, 0.075 U) was injected into the right basal ganglia in mice and mortality, neurologic deficits, brain edema, and hemorrhage size measured. In addition, MMP-9 activity, brain collagen content, blood coagulation, cerebral arterial structure, and expressions of several MMPs were examined. Increased hemorrhage and brain edema that correlated with higher mortality and neurologic deficits were found in MMP-9–knockout mice. No apparent structural changes were observed in cerebral arteries, even though brain collagen content was reduced in MMP-9–knockout mice. MMP-9–knockout mice did exhibit an enhanced expression of MMP-2 and MMP-3 in response to ICH. The results indicate that a deficiency of MMP-9 gene in mutant mice increases collagenase-induced hemorrhage and the resulting brain injury. The intriguing relationship between MMP-9 deficiency and collagenase-induced ICH may reflect the reduction in collagen content and an enhanced expression of MMP-2 and MMP-3.

Inhibition of Apoptosis by Hyperbaric Oxygen in a Rat Focal Cerebral Ischemic Model

The hypothesis was tested that hyperbaric oxygen therapy (HBO) reduced brain infarction by preventing apoptotic death in ischemic cortex in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and subsequently were exposed to HBO (2.5 atmospheres absolute) for 2 h, at 6 h after reperfusion. Rats were killed and brain samples were collected at 24, 48, 72 h, and 7 days after reperfusion. Neurologic deficits, infarction area, and apoptotic changes were evaluated by clinical scores, 2,3,7-triphenyltetrazolium chloride staining, caspase-3 expression, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL)-hematoxylin and eosin (H&E) costaining. In MCAO/R without HBO treatment animals, DNA fragmentation was observed in injured cortex at 24, 48, and 72 h but not in samples at 7 days after reperfusion. Double labeling of brain slides with NeuN and caspase-3 demonstrated neurons in the injured cortex labeled with caspase-3. TUNEL+H&E costaining revealed morphologic apoptotic changes at 24, 48, and 72 h after reperfusion. Hyperbaric oxygen therapy abolished DNA fragmentation and reduced the number of TUNEL-positive cells. Hyperbaric oxygen therapy reduced infarct area and improved neurologic scores at 7 days after reperfusion. One of the molecular mechanisms of HBO-induced brain protection is to prevent apoptosis, and this effect of HBO might preserve more brain tissues and promote neurologic functional recovery.

Caspase Inhibitors Prevent Endothelial Apoptosis and Cerebral Vasospasm in Dog Model of Experimental Subarachnoid Hemorrhage

Apoptosis in the endothelium of major cerebral arteries may play a role in the initiation and maintenance of cerebral vasospasm after subarachnoid hemorrhage (SAH). We tested the therapeutic effect of caspase inhibitors on endothelial apoptosis and on cerebral vasospasm in an established dog double-hemorrhage model. Thirty-one mongrel dogs were divided into five groups: control; SAH; SAH treated with vehicle [DMSO]; SAH treated with Ac-DEVD-CHO [a specific caspase-3 inhibitor]; and SAH treated with Z-VAD-FMK [a broad caspase inhibitor]. The inhibitors (100 μM) were injected into the cisterna magna daily from Day 0 through Day 3. Angiography was performed on Day 0 and Day 7. Histology, TUNEL staining, and immunohistochemistry were conducted on basilar arteries collected on Day 7 after SAH. Positive staining of TUNEL, poly(ADP)-ribose polymerase (PARP), caspase-3, and caspase-8 was observed in the endothelial cells of the spastic arteries. Double fluorescence labeling demonstrated co-localization of TUNEL with caspase-3 and TNFα-receptor-1 (TNFR1). Ac-DEVD-CHO and Z-VAD-FMK prevented endothelial apoptosis and reduced angiographic vasospasm. The mechanism of apoptosis in endothelial cells involves TNFR1 and the caspase-8 and caspase-3 pathways. Caspase inhibitors may have potential in the treatment of cerebral vasospasm.

Mechanisms of Erythropoietin-induced Brain Protection in Neonatal Hypoxia-Ischemia Rat Model:

Erythropoietin, a hemotopoietic growth factor, has brain protective actions. This study investigated the mechanisms of Recombinant Human EPO (rhEPO)-induced brain protection in neonates. An established rat hypoxia-ischemia model was used by ligation of the right common carotid artery of 7-day-old pups, followed by 90 minute of hypoxia (8% 02 and 92% N2) at 37°C. Animals were divided into three groups: control, hypoxia-ischemia, and hypoxia-ischemia plus rhEPO treatment. In rhEPO treated pups, 300 units rhEPO was administered intraperitoneally 24 hours before hypoxia. rhEPO treatment (300 units) was administered daily for an additional 2 days. ELISA and immunohistochemistry examined the expression of EPO and EPOR. Brain weight, morphology, TUNEL assay, and DNA laddering evaluated brain protection. rhEPO abolished mortality (from 19% to 0%) during hypoxia insult, increased brain weight from 52% to 88%, reduced DNA fragmentation, and decreased TUNEL-positive cells. Real-time RT-PCR, Western blot, and immunohistochemistry revealed an enhanced expression of heat shock protein 27 (HSP27) in ischemic brain hemisphere. Double labeling of TUNEL with HSP27 showed most HSP27 positive cells were negative to TUNEL staining. rhEPO reduces brain injury, especially apoptotic cell death after neonatal hypoxia-ischemia, partially mediated by the activation of HSP27.

Early inhibition of HIF-1α with small interfering RNA reduces ischemic–reperfused brain injury in rats

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in cerebral ischemia as a proapoptotic factor. We hypothesized that HIF-1alpha siRNA can protect the brain from ischemic damage by inhibiting HIF-1alpha induced apoptotic pathway at the RNA level in a rat focal ischemic model. Results showed that treatment with HIF-1alpha siRNA reduced the infarct volume, decreased mortality, improved neurological deficits and reduced Evans blue extravasation. The expression of HIF-1alpha mRNA (Real-Time PCR) and protein were significantly silenced and the immunohistochemistry and Western blot revealed the suppression of HIF-1alpha, VEGF, p53 and Caspase-3. Double fluorescence labeling showed HIF-1alpha positive immunoreactive materials were partly colocalized with NeuN, p53 and Caspase-3 in the injured cerebral cortex. This study showed that HIF-1alpha siRNA may protect the ischemic-reperfused neurons in vivo via inhibition of HIF-1alpha, its downstream VEGF and other apoptotic-related proteins such as p53 and Caspase-3 and may have potentials for the early treatment of ischemic cerebral stroke.

Multiple effects of hyperbaric oxygen on the expression of HIF-1α and apoptotic genes in a global ischemia–hypotension rat model

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor specifically activated by hypoxia. Activation of proapoptotic caspase-9 and caspase-3 pathways, by binding with tumor suppressor p53, HIF-1alpha could lead to harmful actions such as apoptosis. We examined whether increasing oxygen levels by hyperbaric oxygen (HBO) offers neuroprotection, at least partially by suppression of HIF-1alpha and apoptotic genes. Male SD rats (n = 78) were randomly divided into 13 groups: 1 sham group, 6 groups of global ischemia-hypotension (GI), and 6 groups of HBO treatment after global ischemia-hypotension (GI + HBO). HBO (3 ATA for 2 h) was applied at 1 h after global ischemia-hypotension. Rats were sacrificed at 6, 12, 24, 48, and 96 h and 7 days. Global ischemia-hypotension (10 min ischemia, 30-35 mm Hg) produced a marked increase of HIF-1alpha expressions in the hippocampus and cortex at 6 h and peaked at 48-96 h. The expressions of p53, caspase-9, and caspase-3 were all increased in a similar time course. These molecular changes were accompanied by massive cell loss in the hippocampal regions and to a lesser degree in the cortex, with features of apoptosis. HBO treatment reduced expressions of HIF-1alpha, p53, caspase-9, and caspase-3 and decreased cell death. The protein levels of proapoptotic caspase-8 and antiapoptotic bcl-2 were increased after global ischemia-hypotension and HBO potentiated the expression of caspase-8 and decreased expression of bcl-2. These results indicate that HBO has multiple actions on apoptotic genes even though the overall effect of HBO was decreased HIF-1alpha expression and reduced apoptosis after global ischemia-hypotension.

Role of p53 and apoptosis in cerebral vasospasm after experimental subarachnoid hemorrhage

Our previous studies indicate that apoptosis in endothelial cells of major cerebral arteries contributes to cerebral vasospasm after subarachnoid hemorrhage (SAH). This study examined the pathologic roles of tumor suppressor p-53 in cerebral vasospasm using an established dog double-hemorrhage model. Twenty mongrel dogs were divided into four groups: (1) control, (2) SAH, (3) SAH+DMSO (vehicle), and (4) SAH+pifithrin-α (PFT) (p53 inhibitor). The p53 inhibitor (200 nmol/L) was injected into the cisterna magna daily from Day 0 through Day 3. Angiogram was performed on Day 0 and Day 7. Western blot, cell proliferation assay, histology, and TUNEL staining were conducted on the basilar arteries collected on Day 7 after SAH. The arterial diameter on Day 7 was 42%±4%, 40%±5%, and 59%±4% for SAH, SAH+DMSO, and SAH+PFT, respectively. In addition, positive staining of TUNEL and increased protein expression of p53, Bax, and PCNA in the basilar artery were observed on Day 7. PFT suppressed apoptosis in endothelial cells and proliferation in smooth muscle cells, and attenuated angiographic vasospasm. In conclusion, p53 may be a key factor in endothelial apoptosis and smooth muscle proliferation after SAH. Inhibition of p53 may potentially reduce or even prevent cerebral vasospasm.

Multiple effects of 2ME2 and D609 on the cortical expression of HIF-1α and apoptotic genes in a middle cerebral artery occlusion-induced focal ischemia rat model

Despite 2‐methoxyestradiol (2ME2) and tricyclodecan‐9‐yl‐xanthogenate (D609) having multiple effects on cancer cells, mechanistically, both of them down‐regulate hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF). We hypothesize HIF‐1α plays an essential role in cerebral ischemia as a pro‐apoptosis regulator; 2ME2 and D609 decrease the levels of HIF‐1α and VEGF, that might contribute to protecting brain from ischemia injury. A total of 102 male Sprague–Dawley rats were split into five groups: sham, middle cerebral artery occlusion (MCAO), MCAO + dimethyl sulfoxide, MCAO + 2ME2, and MCAO + D609. 2ME2 and D609 were injected intraperitoneally 1 h after reperfusion. Rats were killed at 24 h and 7 days. At 24 h, 2ME2 and D609 reduce the levels of HIF‐1α and VEGF (enzyme‐linked immunosorbent assay), depress the expression of HIF‐1α, VEGF, BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and cleaved caspase 3 (western blot and immunohistochemistry) in the brain infarct area. Double fluorescence labeling shows HIF‐1α positive immunoreactive materials are co‐localized with BNIP3 and terminal deoxynucleotidyl transferase biotin‐dUTP nick end labeling inside the nuclei of neurons. At 7 days, 2ME2 and D609 reduce the infarct volume (2,3,7‐triphenyltetrazolium chloride) and blood–brain barrier extravasation, decrease the mortality and improve the neurological deficits. In conclusion, 2ME2 and D609 are powerful agents to protect brain from cerebral ischemic injury by inhibiting HIF‐1α expression, attenuating the superfluous expression of VEGF to avoid blood–brain barrier disruption and suppressing neuronal apoptosis via BNIP3 pathway.

Therapeutic application of gene silencing MMP-9 in a middle cerebral artery occlusion-induced focal ischemia rat model.

RNA interference appears to have a great potential not only as an in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene. We hypothesize that MMP-9 siRNA can be effective as an MMP-9 protein inhibitor in a rat focal ischemia model. Male Sprague-Dawley rats (156) were subjected to 2 h of middle cerebral artery occlusion (by using the suture insertion method) followed by 24 h of reperfusion. In the treatment group, 5 microl MMP-9 siRNA was administrated by intracerebroventricular injection within 60 min after 2 h of focal ischemia. The siRNA transfection was demonstrated by fluorescence conjugated siRNA. Treatment with MMP-9 siRNA produced a significant reduction in the cerebral infarction volume, brain water content, mortality rate and accompanying neurological deficits. The followings were recorded: Evans blue and IgG extravasation were reduced; the expression of MMP-9 mRNA and protein were significantly silenced; and immunohistochemistry and Western blot analysis revealed that the expression of MMP-9 and VEGF were reduced while occludin and collagen-IV were up-regulated in brain tissues. Our findings provide evidence that a liposomal formulation of siRNA might be used in vivo to silence the MMP-9 gene and could potentially serve as an important therapeutic alternative in patients with cerebral ischemia.