Takuma Wakai

Interleukin 6-preconditioned neural stem cells reduce ischaemic injury in stroke mice

Transplantation of neural stem cells provides a promising therapy for stroke. Its efficacy, however, might be limited because of massive grafted-cell death after transplantation, and its insufficient capability for tissue repair. Interleukin 6 is a pro-inflammatory cytokine involved in the pathogenesis of various neurological disorders. Paradoxically, interleukin 6 promotes a pro-survival signalling pathway through activation of signal transducer and activator of transcription 3. In this study, we investigated whether cellular reprogramming of neural stem cells with interleukin 6 facilitates the effectiveness of cell transplantation therapy in ischaemic stroke. Neural stem cells harvested from the subventricular zone of foetal mice were preconditioned with interleukin 6 in vitro and transplanted into mouse brains 6 h or 7 days after transient middle cerebral artery occlusion. Interleukin 6 preconditioning protected the grafted neural stem cells from ischaemic reperfusion injury through signal transducer and activator of transcription 3-mediated upregulation of manganese superoxide dismutase, a primary mitochondrial antioxidant enzyme. In addition, interleukin 6 preconditioning induced secretion of vascular endothelial growth factor from the neural stem cells through activation of signal transducer and activator of transcription 3, resulting in promotion of angiogenesis in the ischaemic brain. Furthermore, transplantation of interleukin 6-preconditioned neural stem cells significantly attenuated infarct size and improved neurological performance compared with non-preconditioned neural stem cells. This interleukin 6-induced amelioration of ischaemic insults was abolished by transfecting the neural stem cells with signal transducer and activator of transcription 3 small interfering RNA before transplantation. These results indicate that preconditioning with interleukin 6, which reprograms neural stem cells to tolerate oxidative stress after ischaemic reperfusion injury and to induce angiogenesis through activation of signal transducer and activator of transcription 3, is a simple and beneficial approach for enhancing the effectiveness of cell transplantation therapy in ischaemic stroke.

Neural Stem Cells Genetically Modified to Overexpress Cu/Zn-Superoxide Dismutase Enhance Amelioration of Ischemic Stroke in Mice

Background and Purpose— The harsh host brain microenvironment caused by production of reactive oxygen species after ischemic reperfusion injury offers a significant challenge to survival of transplanted neural stem cells (NSCs) after ischemic stroke. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We have investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted stem cells and accelerates amelioration of ischemic stroke. Methods— NSCs genetically modified to overexpress or downexpress SOD1 were administered intracerebrally 2 days after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from Days 0 to 28 after stroke. Results— Overexpression of SOD1 suppressed production of superoxide anions after ischemic reperfusion injury and reduced NSC death after transplantation. In contrast, downexpression of SOD1 promoted superoxide generation and increased oxidative stress-mediated NSC death. Transplantation of SOD1-overexpressing NSCs enhanced angiogenesis in the ischemic border zone through upregulation of vascular endothelial growth factor. Moreover, grafted SOD1-overexpressing NSCs reduced infarct size and improved behavioral performance compared with NSCs that were not genetically modified. Conclusions— Our findings reveal a strong involvement of SOD1 expression in NSC survival after ischemic reperfusion injury. We propose that conferring antioxidant properties on NSCs by genetic manipulation of SOD1 is a potential approach for enhancing the effectiveness of cell transplantation therapy in ischemic stroke.

Indocyanine green videoangiography to detect aneurysm and related vascular structures buried in subarachnoid clots

This 44-year-old woman presented with a ruptured anterior communicating artery aneurysm. Intraoperative indocyanine green (ICG) videoangiography demonstrated the aneurysm neck and dome, which were buried in subarachnoid clots. Dissection and aspiration of the clots around the neck were safely performed without touching the ruptured points. The aneurysm was successfully clipped. The patients postoperative course was excellent. This case illustrates the use of intraoperative ICG videoangiography to provide information about the anatomical location of the aneurysm neck and dome despite their being completely obscured by subarachnoid clots. Intraoperative ICG videoangiography allowed safer dissection of the ruptured aneurysm from the blood clots.

Transplantation of neural stem cells that overexpress SOD1 enhances amelioration of intracerebral hemorrhage in mice.

Previous studies have shown that intraparenchymal transplantation of neural stem cells (NSCs) ameliorates neurologic deficits in animals with intracerebral hemorrhage (ICH). However, massive grafted cell death after transplantation, possibly caused by a hostile host brain environment, lessens the effectiveness of this approach. We focused on the effect of oxidative stress against grafted NSCs and hypothesized that conferring antioxidant properties to transplanted NSCs may overcome their death and enhance neuroprotection after ICH. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted NSCs and accelerates amelioration of ICH. Neural stem cells that overexpress SOD1 were administered intracerebrally 3 days after ICH in a mouse model. Histologic and behavioral tests were examined after ICH. Copper/zinc-superoxide dismutase overexpression protected the grafted NSCs via a decrease in production of reactive oxygen species. This resulted in an increase in paracrine factors released by the NSCs, and an increase in surviving neurons in the striatum and a reduction in striatal atrophy. In addition, SOD1 overexpression showed progressive improvement in behavioral recovery. Our results suggest that enhanced antioxidative activity in NSCs improves efficacy of stem cell therapy for ICH.

The Role of Parl and HtrA2 in Striatal Neuronal Injury After Transient Global Cerebral Ischemia

The presenilin-associated rhomboid-like (PARL) protein and high temperature requirement factor A2 (HtrA2) are key regulators of mitochondrial integrity and play pivotal roles in apoptosis. However, their roles after cerebral ischemia have not been thoroughly elucidated. To clarify these roles, mice were subjected to transient global cerebral ischemia, and striatal neuronal injury was assessed. Western blot and coimmunoprecipitation analyses revealed that PARL and processed HtrA2 localized to mitochondria, and that PARL was bound to HtrA2 in sham animals. Expression of PARL and processed HtrA2 in mitochondria significantly decreased 6 to 72 hours after ischemia, and the binding of PARL to HtrA2 disappeared after ischemia. In contrast, expression of processed HtrA2 increased 24 hours after ischemia in the cytosol, where HtrA2 was bound to X chromosome-linked inhibitor-of-apoptosis protein (XIAP). Administration of PARL small interfering RNA inhibited HtrA2 processing and worsened ischemic neuronal injury. Our results show that downregulation of PARL after ischemia is a key step in ischemic neuronal injury, and that it decreases HtrA2 processing and increases neuronal vulnerability. In addition, processed HtrA2 released into the cytosol after ischemia contributes to neuronal injury via inhibition of XIAP.

Hypoxic preconditioning enhances neural stem cell transplantation therapy after intracerebral hemorrhage in mice.

Previous studies have shown that intraparenchymal transplantation of neural stem cells ameliorates neurological deficits in animals with intracerebral hemorrhage. However, hemoglobin in the host brain environment causes massive grafted cell death and reduces the effectiveness of this approach. Several studies have shown that preconditioning induced by sublethal hypoxia can markedly improve the tolerance of treated subjects to more severe insults. Therefore, we investigated whether hypoxic preconditioning enhances neural stem cell resilience to the hemorrhagic stroke environment and improves therapeutic effects in mice. To assess whether hypoxic preconditioning enhances neural stem cell survival when exposed to hemoglobin, neural stem cells were exposed to 5% hypoxia for 24 hours before exposure to hemoglobin. To study the effectiveness of hypoxic preconditioning on grafted-neural stem cell recovery, neural stem cells subjected to hypoxic preconditioning were grafted into the parenchyma 3 days after intracerebral hemorrhage. Hypoxic preconditioning significantly enhanced viability of the neural stem cells exposed to hemoglobin and increased grafted-cell survival in the intracerebral hemorrhage brain. Hypoxic preconditioning also increased neural stem cell secretion of vascular endothelial growth factor. Finally, transplanted neural stem cells with hypoxic preconditioning exhibited enhanced tissue-protective capability that accelerated behavioral recovery. Our results suggest that hypoxic preconditioning in neural stem cells improves efficacy of stem cell therapy for intracerebral hemorrhage.

Effects of valsartan on neuroprotection and neurogenesis after ischemia.

Experimental and clinical studies have revealed that angiotensin II type 1 receptor blocker has protective effects against ischemic brain injury, but the mechanism is still obscure. Angiotensin II type 1 receptor blocker may also have effects on neurogenesis through the activation of unblocked angiotensin II type 2 receptors. In this study, we showed that valsartan significantly suppressed superoxide production and cytochrome C release into the cytosol after transient forebrain ischemia and consequently attenuated ischemic neuronal damage without affecting the blood pressure in rats. However, valsartan has none of the expected effects on neurogenesis after ischemia. These results suggest that valsartan has neuroprotective effects on ischemic injury through the suppression of oxidative stress and mitochondrial injury.

Cerebral hemodynamic disturbance in dural arteriovenous fistula with retrograde leptomeningeal venous drainage: a prospective study using 123I-iodoamphetamine single photon emission computed tomography

OBJECT The severity of cerebral hemodynamic disturbance caused by retrograde leptomeningeal venous drainage (RLVD) of a dural arteriovenous fistula (dAVF) is related to neurological morbidity and unfavorable outcome. However, the cerebral hemodynamics of this disorder have not been elucidated well. The aim of this study was to assess the relationship between the cerebral venous congestive encephalopathy represented as a high-intensity area (HIA) on T2-weighted MR images and the cerebral hemodynamics examined by (123)I-iodoamphetamine (IMP) single photon emission computed tomography (SPECT), as well as the predictive value of (123)I-IMP SPECT for the development and reversibility of venous congestion encephalopathy. METHODS Based on the pre- and posttreatment T2 HIAs associated with venous congestion encephalopathy, patients were divided into 3 groups: a normal group, an edema group, and an infarction group. The regional cerebral blood flow (rCBF) at the region with RLVD was analyzed by (123)I-IMP SPECT, and the results were compared among the groups. RESULTS There were 11, 6, and 3 patients in the normal, edema, and infarction groups, respectively. No patients in the normal group showed any symptoms related to venous congestion. In contrast, all patients in the edema and infarction groups developed neurological symptoms. The rCBF in the edema group was significantly lower than that in the normal group, and significantly higher than that in the infarction group. The cerebral vascular reactivity (CVR) of the infarction group was significantly lower than that of the normal and edema groups. After treatment, the neurological signs disappeared in the edema group, but only partial improvement was seen in the infarction group. The rCBF also significantly increased in the normal and edema groups, but not in the infarction group. CONCLUSIONS Quantitative rCBF measurement is useful for evaluating hemodynamic disturbance in dAVF with RLVD. The reduction of rCBF was strongly correlated with the severity of venous congestive encephalopathy, and loss of CVR is a reliable indicator of irreversible venous infarction caused by RLVD.

Efficacy of Endoscopic Fluorescein Video Angiography in Aneurysm Surgery—Novel and Innovative Assessment of Vascular Blood Flow in the Dead Angles of the Microscope

BACKGROUND In aneurysm surgery, assessment of the blood flow around the aneurysm is crucial. Recently, intraoperative fluorescence video angiography has been widely adopted for this purpose. However, the observation field of this procedure is limited to the microscopic view, and it is difficult to visualize blood flow obscured by the skull base anatomy, parent arteries, and aneurysm. OBJECTIVE To demonstrate the efficacy of a new small-caliber endoscopic fluorescence video angiography system employing sodium fluorescein in aneurysm surgery for the first time. METHODS Eighteen patients with 18 cerebral aneurysms were enrolled in this study. Both microscopic fluorescence angiography and endoscopic fluorescein video angiography were performed before and after clip placement. RESULTS Endoscopic fluorescein video angiography provided bright fluorescence imaging even with a 2.7-mm-diameter endoscope and clearly revealed blood flow within the vessels in the dead angle areas of the microscope in all 18 aneurysms. Consequently, it revealed information about aneurysmal occlusion and perforator patency in 15 aneurysms (83.3%) that was not obtainable with microscopic fluorescence video angiography. Furthermore, only endoscopic video angiography detected the incomplete clipping in 2 aneurysms and the occlusion of the perforating branches in 3 aneurysms, which led to the reapplication of clips in 2 aneurysms. CONCLUSION The innovative endoscopic fluorescein video angiography system we developed features a small-caliber endoscope and bright fluorescence images. Because it reveals blood flow in the dead angle areas of the microscope, this novel system could contribute to the safety and long-term effectiveness of aneurysm surgery even in a narrow operative field.

Oxidative Stress in White Matter Injury

White matter is the region of the brain underlying gray matter and comprises over half the human brain. Its elements, axons, oligodendrocytes (myelin-producing cells), and oligodendroglia progenitor cells, are exceedingly vulnerable to oxidative stress, since axons contain abundant mitochondria (organelles that are a main source of reactive oxygen species), and the myelin sheath contains numerous lipids, which can be peroxidized after oxidative stress. In addition, low levels of reduced glutathione and high levels of iron content in oligodendrocytes and oligodendrocyte progenitors contribute to this vulnerability. White matter is at risk for oxidative ischemic injury throughout life, from periventricular white matter injury in neonates to stroke and vascular dementia in later life. Prevention of oxidative stress could be a clinical strategy for ischemic white matter injury.