Jianhua Qiu

Cortical spreading depression activates and upregulates MMP-9

Cortical spreading depression (CSD) is a propagating wave of neuronal and glial depolarization and has been implicated in disorders of neurovascular regulation such as stroke, head trauma, and migraine. In this study, we found that CSD alters blood-brain barrier (BBB) permeability by activating brain MMPs. Beginning at 3-6 hours, MMP-9 levels increased within cortex ipsilateral to the CSD, reaching a maximum at 24 hours and persisting for at least 48 hours. Gelatinolytic activity was detected earliest within the matrix of cortical blood vessels and later within neurons and pia arachnoid (> or =3 hours), particularly within piriform cortex; this activity was suppressed by injection of the metalloprotease inhibitor GM6001 or in vitro by the addition of a zinc chelator (1,10-phenanthroline). At 3-24 hours, immunoreactive laminin, endothelial barrier antigen, and zona occludens-1 diminished in the ipsilateral cortex, suggesting that CSD altered proteins critical to the integrity of the BBB. At 3 hours after CSD, plasma protein leakage and brain edema developed contemporaneously. Albumin leakage was suppressed by the administration of GM6001. Protein leakage was not detected in MMP-9-null mice, implicating the MMP-9 isoform in barrier disruption. We conclude that intense neuronal and glial depolarization initiates a cascade that disrupts the BBB via an MMP-9-dependent mechanism.

Early release of HMGB-1 from neurons after the onset of brain ischemia

The nuclear protein high-mobility group box 1 (HMGB-1) promotes inflammation in sepsis, but little is known about its role in brain ischemia-induced inflammation. We report that HMGB-1 and its receptors, receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), and TLR4, were expressed in normal brain and in cultured neurons, endothelia, and glial cells. During middle cerebral artery occlusion (MCAO), in mice, HMGB-1 immunostaining rapidly disappeared from all cells within the striatal ischemic core from 1 h after onset of occlusion. High-mobility group box 1 translocation from nucleus to cytoplasm was observed within the cortical periinfarct regions 2 h after ischemic reperfusion (2 h MCAO). High-mobility group box 1 predominantly translocated to the cytoplasm or disappeared in cells that colabeled with the neuronal marker NeuN. Furthermore, RAGE was robustly expressed in the periinfarct region after MCAO. Cellular release of HMGB-1 was detected by immunoblotting of cerebrospinal fluid as early as 2 h after ischemic reperfusion (2 h MCAO). High-mobility group box 1 released from neurons, in vitro, after glutamate excitotoxicity, maintained biologic activity and induced glial expression of tumor necrosis factor α (TNFα). Anti-HMGB-1 antibody suppressed TNFα upregulation in astrocytes exposed to conditioned media from glutamate-treated neurons. Moreover, TNFα and the cytokine intercellular adhesion molecule-1 increased in cultured glia and endothelial cells, respectively, after adding recombinant HMGB-1. In conclusion, HMGB-1 is released early after ischemic injury from neurons and may contribute to the initial stages of the inflammatory response.

BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia

Mitochondria and cytochrome c release play a role in the death of neurons and glia after cerebral ischemia. In the present study, we investigated whether BID, a proapoptotic promoter of cytochrome c release and caspase 8 substrate, was expressed in brain, activated after an ischemic insult in vivo and in vitro, and contributed to ischemic cell death. We detected BID in the cytosol of mouse brain and primary cultured mouse neurons and demonstrated, by using recombinant caspase 8, that neuronal BID also is a caspase 8 substrate. After 2 h of oxygen/glucose deprivation, BID cleavage was detected in neurons concurrent with caspase 8 activation but before caspase 3 cleavage. Bid−/− neurons were resistant to death after oxygen/glucose deprivation, and caspase 3 cleavage was significantly reduced; however, caspase 8 cleavage did not differ from wild type. In vivo, BID was cleaved 4 h after transient middle cerebral artery occlusion. Infarct volumes and cytochrome c release also were less in Bid−/− mice (−67% and −41%, respectively) after mild focal ischemia. These findings suggest that BID and the mitochondrial-amplification pathway promoting caspase activation contributes importantly to neuronal cell death after ischemic insult.

EGF amplifies the replacement of parvalbumin-expressing striatal interneurons after ischemia

EGF promotes proliferation and migration of stem/progenitor cells in the normal adult brain. The effect of epidermal growth factor on neurogenesis in ischemic brain is unknown, however. Here we show that intraventricular administration of EGF and albumin augments 100-fold neuronal replacement in the injured adult mouse striatum after cerebral ischemia. Newly born immature neurons migrate into the ischemic lesion and differentiate into mature parvalbumin-expressing neurons, replacing more than 20% of the interneurons lost by 13 weeks after ischemia and representing 2% of the total BrdU-labeled cells. These data suggest that administration of EGF and albumin could be used to manipulate endogenous neurogenesis in the injured brain and to promote brain self-repair.

Adiponectin Prevents Cerebral Ischemic Injury Through Endothelial Nitric Oxide Synthase–Dependent Mechanisms

Background— Adiponectin is a fat-derived plasma protein that has beneficial actions on cardiovascular disorders. A low level of plasma adiponectin is associated with increased mortality after ischemic stroke; however, the causal role of adiponectin in ischemic stroke is unknown. Methods and Results— To explore the role of adiponectin in the development of acute cerebral injury, we subjected adiponectin-deficient (APN-KO) and wild-type (WT) mice to 1 hour of middle cerebral artery occlusion followed by 23 hours of reperfusion. APN-KO mice exhibited enlarged brain infarction and increased neurological deficits after ischemia-reperfusion compared with WT mice. Conversely, adenovirus-mediated supplementation of adiponectin significantly reduced cerebral infarct size in WT and APN-KO mice. APN-KO mice showed decreased cerebral blood flow during ischemia by laser speckle flowmetry methods. Adiponectin colocalized within the cerebral vascular endothelium under transient ischemic conditions by immunohistochemical analysis. Phosphorylation of endothelial nitric oxide synthase in ischemic brain tissues and the production of nitric oxide metabolites in plasma were attenuated in APN-KO mice compared with WT mice. Adenovirus-mediated administration of adiponectin stimulated endothelial nitric oxide synthase phosphorylation and nitric oxide metabolites during cerebral ischemia in both WT and APN-KO mice. Neuronal nitric oxide synthase expression during ischemia did not differ between WT and APN-KO mice. Adenovirus-mediated delivery of adiponectin did not affect brain infarction in mice deficient in endothelial nitric oxide synthase. Conclusions— These data provide causal evidence that adiponectin exerts a cerebroprotective action through an endothelial nitric oxide synthase–dependent mechanism. Adiponectin could represent a molecular target for the prevention of ischemic stroke.

FGF-2 regulates neurogenesis and degeneration in the dentate gyrus after traumatic brain injury in mice

We studied the role of FGF-2 on regulation of neurogenesis and cell loss in the granule cell layer (GCL) of the hippocampal dentate gyrus after experimental traumatic brain injury (TBI). In both FGF-2(-/-) and FGF-2(+/+) mice subjected to controlled cortical impact, the number of dividing cells labeled with BrdU, injected on posttrauma days 6 through 8, increased at 9 days after TBI, and the number of BrdU-positive cells colabeled with neuron-specific nuclear antigen significantly increased at 35 days. However, in injured FGF-2-/- mice, BrdU-positive cells and BrdU-positive neurons (days 9, 35) were fewer compared with FGF-2(+/+) mice. There was also a decrease in the volume of the GCL and the number of GCL neurons after TBI in both FGF-2(-/-) and FGF-2(+/+) mice, but the decrease in both was greater in FGF-2-/- mice at 35 days. Overexpression of FGF-2 by intracerebral injection of herpes simplex virus-1 amplicon vectors encoding this factor increased numbers of dividing cells (day 9) and BrdU-positive neurons (day 35) significantly in C57BL/6 mice. Furthermore, the decrease in GCL volume was also attenuated. These results suggest that FGF-2 upregulates neurogenesis and protects neurons against degeneration in the adult hippocampus after TBI, and that FGF-2 supplementation via gene transfer can reduce GCL degeneration after TBI.

Antibody against early driver of neurodegeneration cis P-tau blocks brain injury and tauopathy

Traumatic brain injury (TBI), characterized by acute neurological dysfunction, is one of the best known environmental risk factors for chronic traumatic encephalopathy and Alzheimer’s disease, the defining pathologic features of which include tauopathy made of phosphorylated tau protein (P-tau). However, tauopathy has not been detected in the early stages after TBI, and how TBI leads to tauopathy is unknown. Here we find robust cis P-tau pathology after TBI in humans and mice. After TBI in mice and stress in vitro, neurons acutely produce cis P-tau, which disrupts axonal microtubule networks and mitochondrial transport, spreads to other neurons, and leads to apoptosis. This process, which we term ‘cistauosis’, appears long before other tauopathy. Treating TBI mice with cis antibody blocks cistauosis, prevents tauopathy development and spread, and restores many TBI-related structural and functional sequelae. Thus, cis P-tau is a major early driver of disease after TBI and leads to tauopathy in chronic traumatic encephalopathy and Alzheimer’s disease. The cis antibody may be further developed to detect and treat TBI, and prevent progressive neurodegeneration after injury.Traumatic brain injury (TBI), characterized by acute neurological dysfunction, is one of the best known environmental risk factors for chronic traumatic encephalopathy (CTE) and Alzheimers disease (AD), whose defining pathologic features include tauopathy made of phosphorylated tau (p-tau). However, tauopathy has not been detected in early stages after TBI and how TBI leads to tauopathy is unknown. Here we find robust cis p-tau pathology after sport- and military-related TBI in humans and mice. Acutely after TBI in mice and stress in vitro, neurons prominently produce cis p-tau, which disrupts axonal microtubule network and mitochondrial transport, spreads to other neurons, and leads to apoptosis. This process, termed “cistauosis”, appears long before other tauopathy. Treating TBI mice with cis antibody blocks cistauosis, prevents tauopathy development and spread, and restores many TBI-related structural and functional sequelae. Thus, cis p-tau is a major early driver after TBI and leads to tauopathy in CTE and AD, and cis antibody may be further developed to detect and treat TBI, and prevent progressive neurodegeneration after injury.

High-Mobility Group Box 1 Promotes Metalloproteinase-9 Upregulation Through Toll-Like Receptor 4 After Cerebral Ischemia

Background and Purpose— HMGB1 is a nuclear protein and an alarmin that signals cell damage in response to injury. It is believed that after release from injured cells, HMGB1 binds to its receptors to stimulate cross-talk among cells and to drive components of the inflammatory cascade. This study was intended to investigate the role of extracellular HMGB1 in ischemic stroke by examining the response of the zymogen matrix metalloproteinase-9 (MMP-9) to HMGB1 in vivo and in vitro. Methods— Toll-like receptor 2 (TLR2), TLR4, receptor for advanced glycation endproducts (RAGE), and MMP-9 expression was examined using quantitative RT-PCR in primary cultured neurons, astrocytes, and mouse brain after HMGB1 addition. MMP-9 expression/activity was examined using zymography. Middle cerebral artery occlusion was induced for 60 minutes using a filament model. Results— TLR4 is constitutively expressed in neurons, astrocytes, and mouse brain. HMGB1 addition to neuronal and glial cell cultures caused MMP-9 upregulation in a dose- and time-dependent manner. Lack of TLR4 function attenuated MMP-9 expression induced by HMGB1 in vitro. After striatal microinjection of HMGB1, MMP-9 was upregulated, and the response was independent of tumor necrosis factor-&agr;. Interestingly, MMP-9 upregulation was reduced in TLR4 missense mutant mice after ischemia compared with wild-type controls, as was infarct volume. Conclusion— Our results suggest that HMGB1 triggers MMP-9 upregulation in neurons and astrocytes predominantly via TLR4 after cerebral ischemia. Hence, targeting HMGB1/TLRs signaling pathway may reduce the acute inflammatory response and reduce tissue damage in cerebral ischemia.

Protective effects of statins involving both eNOS and tPA in focal cerebral ischemia

Previous studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) protect the brain against ischemic injury by upregulating endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that statins provide additional beneficial effects by also upregulating endogenous tissue plasminogen activator (tPA) and enhancing clot lysis in a mouse model of embolic focal ischemia. Heterologous blood clots (0.2 mm) were injected into the distal internal carotid artery to occlude blood flow in the middle cerebral artery territory after long-term (14 days) simvastatin, atorvastatin or vehicle treatment. Ischemic lesion volume, neurologic deficits, as well as residual blood clots were measured at 22 h. Reverse transcription-polymerase chain reaction assessed mRNA levels of eNOS, tPA, and the endogenous plasminogen activator inhibitor PAI-1. Ischemic lesion volumes and neurologic deficits were significantly reduced in wild-type mice by both simvastatin and atorvastatin. Statins increased eNOS and tPA mRNA levels but did not change mRNA levels of PAI-1. In eNOS knockout mice, atorvastatin reduced the volume of ischemic tissue and improved neurologic outcomes after arterial occlusion by blood clot emboli. In contrast, statins did not have protective effects in tPA knockout mice after embolic focal ischemia, but only in a filament model where focal ischemia was achieved via mechanical occlusion. These results suggest that statins protect against stroke by multiple mechanisms involving both eNOS and tPA. The involvement of each pathway may be revealed depending on the choice of experimental stroke model.

Sonic Hedgehog Regulates Ischemia/Hypoxia-Induced Neural Progenitor Proliferation

Background and Purpose— Sonic hedgehog (Shh) protein is required for the maintenance of neural progenitor cells (NPCs) in the embryonic and adult hippocampus. Brain ischemia causes increased proliferation of hippocampal NPCs. We therefore examined whether Shh regulates the increase in proliferation of NPCs after ischemia/hypoxia. Methods— Male SV129 mice were exposed to a 20-minute middle cerebral artery occlusion; hippocampi were then analyzed for Shh mRNA and protein expression by real-time polymerase chain reaction, immunoblot, and immunohistochemistry. Primary cell cultures of neurons, astrocytes, and NPCs were exposed to 16 hours of hypoxia (1% O2) and analyzed by real-time polymerase chain reaction and immunoblot for Shh expression. Proliferation of NPCs, in vivo and in vitro, was measured by bromodeoxyuridine incorporation. Results— Among the cell types examined in vitro, only NPC and neurons increased Shh mRNA under hypoxic conditions. Furthermore, hypoxia increased proliferation of NPCs and this proliferation was enhanced by the addition of recombinant Shh or blocked by the pathway-specific inhibitor, cyclopamine. Middle cerebral artery occlusion was associated with a transient 2-fold increase in the mRNA encoding both Shh and its transcription factor, Gli1, 0.5 days after ischemia. Within the hippocampus, Shh protein was increased approximately 3-fold 3 and 7 days after ischemia and was observed predominantly within cells in the CA3 and hilar regions. Shh was expressed only in mature neurons. In vivo, cyclopamine suppressed ischemia-induced proliferation of subgranular NPCs. Conclusion— The Shh pathway plays a role in the proliferation of NPCs induced by ischemia/hypoxia and might participate in injury remodeling.