Jouji Kokuzawa

Gene Transfer of Hepatocyte Growth Factor to Subarachnoid Space in Cerebral Hypoperfusion Model

Although cerebral hypoperfusion caused by cerebral occlusive disease leads to cerebral ischemic events, an effective treatment has not yet been established. Recently, a novel therapeutic strategy for ischemic disease using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. Therapeutic angiogenesis might be useful for the treatment of cerebral occlusive disease. Hepatocyte growth factor (HGF) is a potent angiogenic factor, in addition to vascular endothelial growth factor (VEGF), whereas in the nervous system HGF also acts as neurotrophic factor. Therefore, we hypothesized that gene transfer of these angiogenic growth factors could induce angiogenesis, thus providing an effective therapy for cerebral hypoperfusion or stroke. In this study, we employed a highly efficient gene transfer method, the viral envelop (Hemagglutinating Virus of Japan [HVJ]-liposome) method, because we previously documented that &bgr;-galactosidase gene could be transfected into the brain by the HVJ-liposome method. Indeed, we confirmed wide distribution of transgene expression using &bgr;-galactosidase via injection into the subarachnoid space. Of importance, transfection of HGF or VEGF gene into the subarachnoid space 7 days before occlusion induced angiogenesis on the brain surface as assessed by alkaline phosphatase staining (P <0.01). In addition, significant improvement of cerebral blood flow (CBF) was observed by laser Doppler imaging (LDI) 7 days after occlusion (P <0.01). Unexpectedly, transfection of HGF or VEGF gene into the subarachnoid space immediately after occlusion of the bilateral carotid arteries also induced angiogenesis on the brain surface and had a significant protective effect on the impairment of CBF by carotid occlusion (P <0.01). Interestingly, coinjection of recombinant HGF with HGF gene transfer revealed a further increase in CBF (P <0.01). Here, we demonstrated successful therapeutic angiogenesis using HGF or VEGF gene transfer into the subarachnoid space to improve cerebral hypoperfusion, thus providing a new therapeutic strategy for cerebral ischemic disease.

Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos

Hepatocyte growth factor (HGF), originally cloned as a hepatocyte mitogen, has recently been reported to exhibit neurotrophic activity in addition to being expressed in different parts of the nervous system. At present, the effects of HGF on neural stem cells (NSCs) are not known. In this study, we first report the promoting effect of HGF on the proliferation of neurospheres and neuronal differentiation of NSCs. Medium containing only HGF was capable of inducing neurosphere formation. Addition of HGF to medium containing fibroblast growth factor 2 or epidermal growth factor increased both the size and number of newly formed neurospheres. More neurons were also obtained when HGF was added in differentiation medium. In contrast, neurosphere numbers were reduced after repeated subculture by mechanical dissociation, suggesting that HGF-formed neurospheres comprised predominantly progenitor cells committed to neuronal or glial lines. Together, these results suggest that HGF promotes proliferation of neurospheres and neuronal differentiation of NSCs derived from mouse embyos.

Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells

Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP‐expressing D3‐ES cells. The spheres contained nestin‐positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self‐renewal ability. The cells differentiated into microtubule‐associated protein‐2 (MAP2)‐positive neuronal cells and glial fibrillary acidic protein (GFAP)‐positive glial cells. γ‐Aminobutyric acid‐positive cells and tyrosine hydroxylase‐positive cells were also observed in the spheres. The percentages of the MAP2‐ or GFAP‐positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue.

Neuronal protein NP25 interacts with F-actin

Neuronal protein NP25 is a neuron-specific protein present in highly differentiated neural cells, but its functional properties have not been well characterized. NP25 shows high amino acid sequence homology with the smooth muscle cell cytoskeleton-associated proteins, SM22, mp20, and calponin. To gain an insight into the biological functions of NP25, we first examined its subcellular localization in the human neuroblastoma cell line, SK-N-SH. NP25 diffusely distributed in the cytoplasm and fiber-like staining was also observed. It showed that NP25 co-localized with F-actin on stress fibers. A co-sedimentation assay demonstrated that NP25 bound to filamentous actin. Further investigations using fluorescence resonance energy transfer (FRET) technique revealed intracellular binding of NP25 and actin. The significance of the interaction between NP25 and F-actin is discussed.

Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells.

We previously reported that hepatocyte growth factor (HGF) promoted proliferation of neurospheres and neuronal differentiation of neural stem cells (NSCs) derived from mouse embryonic brain. In this study, spheres from mouse embryonic stem (ES) cells were generated by floating culture following co-culture on PA6 stromal cells. In contrast to the behavior of the neurospheres derived from embryonic brain, addition of HGF to the growth medium of the floating cultures decreased the number of spheres derived from ES cells. When spheres were stained using a MAP-2 antibody, more MAP-2-positive cells were observed in spheres cultured with HGF. When HGF was added to the growth and/or differentiation medium, more MAP-2-positive cells were also obtained. These results suggest that HGF promotes neuronal differentiation of NSCs derived from ES cells.

Superficial temporal artery-middle cerebral artery bypass using local anesthesia and a sedative without endotracheal general anesthesia.

OBJECT Superficial temporal artery (STA)-middle cerebral artery (MCA) bypasses have continually evolved, and new strategies have been advocated for reducing anesthetic or surgical morbidity and mortality. Further simplifying, and decreasing the invasiveness of, STA-MCA bypass by performing this operation without endotracheal general anesthesia was believed to be feasible in certain subsets of patients. METHODS The authors performed STA-MCA bypass using local anesthesia with a sedative in 10 patients with hemodynamically compromised occlusive cerebrovascular disease, as well as multiple comorbidities, between February 2010 and September 2011. The technique is based on the preoperative identification of the point at which the donor and recipient vessels are in closest proximity. Preoperative use of CT angiography allowed the authors to identify the target point precisely and use a minimally invasive procedure. All patients received dexmedetomidine as the sole sedative agent, together with scalp-blocking local anesthesia, with an unsecured airway. RESULTS Successful STA-MCA bypass surgeries were achieved via a preselected minimally invasive approach in all cases. There was good hemodynamic stability throughout surgery. No airway or ventilation complications occurred, and no patients were converted to general anesthesia. Subjectively, patients tolerated the technique well with a high rate of satisfaction. There were no perioperative morbidities or deaths. Postoperative MR angiography confirmed a patent bypass in all patients. All patients remained symptom free and returned to normal daily life following the operation. CONCLUSIONS This initial experience confirms the feasibility of performing STA-MCA bypass without endotracheal general anesthesia. This novel technique produced a high degree of patient satisfaction.

Diabetes mellitus and carotid artery plaques exhibiting high-intensity signals on MR angiography are related to increased platelet reactivity after carotid artery stenting

Background Increased platelet reactivity after carotid artery stenting (CAS) may cause thromboembolic complications. Objective This study aimed to investigate the incidence of increased platelet reactivity after CAS and to determine the factors related to it. Methods Patients who underwent CAS were recruited prospectively. They received pre-procedural antiplatelet therapy comprising some combination of aspirin (100 mg/day), clopidogrel (75 mg/day), and/or cilostazol (200 mg/day) for a minimum of 7 days. ADP- and collagen-induced platelet aggregation were measured before and 4 days after CAS. Changes in platelet reactivity were reported as changes in the categorized platelet reactivity grade based on the effective dose 50%. Clinical characteristics of patients with and without increased platelet reactivity were compared. Results Among 38 consecutive patients who underwent CAS, 18 (47%) exhibited increased platelet reactivity. Diabetes mellitus (OR 15.0; 95% CI 2.1 to 106.5; p=0.007) and carotid artery plaques exhibiting high-intensity signals (HIS) on time-of-flight MR angiography (TOF-MRA) (OR 25.2; 95% CI 2.0 to 316.2; p=0.013) were independently associated with increased platelet reactivity in a multivariate analysis. Conclusions Increased platelet reactivity occurred in nearly half of the studied patients subjected to CAS and was independently associated with diabetes mellitus and carotid artery plaques exhibiting HIS on TOF-MRA.

STA-MCA Bypass Under Local Anesthesia

BACKGROUND AND AIMS The superficial temporal artery to middle cerebral artery (STA-MCA) bypass procedure has continually evolved and new strategies have been advocated to reduce anesthetic or surgical mortality and morbidity. Further simplifying and decreasing the invasiveness of STA-MCA bypass by performing this operation without endotracheal general anesthesia was deemed feasible in certain subsets of patients. METHODS We performed STA-MCA bypass using local anesthesia using a sedative in 45 patients with hemodynamically compromised cerebrovascular occlusive disease as well as multiple comorbidities in the period between February 2010 and April 2016. The technique is based on preoperative identification of the point at which the donor and recipient vessels are in closest proximity. The preoperative use of computed tomography angiography allowed us to identify the target point precisely and use a minimally invasive procedure. All patients received dexmedetomidine as the sole sedative agent, together with scalp block local anesthesia with an unsecured airway. RESULTS Successful STA-MCA bypass surgeries were achieved via a preselected minimally invasive approach in all cases. There was good hemodynamic stability throughout surgery. No airway/ventilation complications occurred and no patient was converted to general anesthesia. The patients subjectively tolerated this technique well with a high rate of satisfaction. Postoperative magnetic resonance angiography confirmed patent bypass in 44 of 45 patients (patency rate of 97.8%). There were two postoperative hyper-perfusion syndromes and one cerebral ischemia with transient neurological symptoms (postoperative complication rate of 6.3%). No recurrence of ipsilateral cerebral ischemia was observed during the follow-up periods. There was one contralateral cardiogenic cerebral embolism during the follow-up period. The overall stroke rate was calculated as 1%/patient/year. CONCLUSIONS Our initial experience confirms the feasibility of performing STA-MCA bypass under local anesthesia without endotracheal general anesthesia.