Masaaki Hokari

Incidence and Clinical Features of Disease Progression in Adult Moyamoya Disease

Background and Purpose— The progression of occlusive lesions in the major intracranial arteries was believed to be very rare in adult patients with moyamoya disease. The present study aims to clarify the incidence and clinical features of disease progression in adult moyamoya disease. Methods— For the past 15 years, 120 adult Japanese patients were diagnosed with moyamoya disease. Of these, 63 patients were enrolled in this historical prospective cohort study on a total of 86 nonoperated hemispheres. All were followed up with a mean period of 73.6 months. MRI and magnetic resonance angiography were repeated every 6 to 12 months, and cerebral angiography was performed when disease progression was suspected on MRI and magnetic resonance angiography. Results— Disease progression occurred in 15 of 86 nonoperated hemispheres (17.4% per hemisphere) or in 15 of 63 patients (23.8% per patient) during the follow-up period. Occlusive arterial lesions progressed in both anterior and posterior circulations, in both symptomatic and asymptomatic patients, and in both bilateral and unilateral types. Eight of 15 patients developed ischemic or hemorrhagic events in relation to disease progression. Multivariate analysis revealed that the odds ratio conferred by a male patient was 0.20 (95% CI, 0.04 to 0.97). Conclusions— The incidence of disease progression in adult moyamoya disease is much higher than recognized before, and female patients may be at higher risk for it than male patients. Careful follow-up would be essential to prevent additional stroke occurrence in medically treated adult patients with moyamoya disease, even if they are asymptomatic or are diagnosed as having unilateral moyamoya disease.

Bone marrow stromal cells protect and repair damaged neurons through multiple mechanisms

A surprising shortage of information surrounds the mechanism by which bone marrow stromal cells (BMSC) restore lost neurologic functions when transplanted into the damaged central nervous system. To clarify the issue, the BMSC were cocultured with the neurons using two paradigms: the cell‐mixing coculture technique and three‐dimensional coculture technique. The green fluorescent protein (GFP)‐expressing BMSC were cocultured with the PKH‐26‐labelled neurons, using cell mixing coculture technique. GFP‐positive, PKH‐26‐negative cells morphologically simulated the neurons and significantly increased the expression of MAP‐2, Tuj‐1, nestin, and GFAP. GFP/nestin‐positive, PKH‐26‐negative cells increased from 13.6% ± 6.7% to 32.1% ± 15.5% over 7 days of coculture. They further enhanced Tuj‐1 expression when cocultured with neurons exposed to 100 μM of glutamate for 10 min. About 20–30% of GFP‐positive cells became positive for PKH‐26 through coculture with the neurons, but the doubly positive cells did not increase when cocultured with glutamate‐exposed neurons. Alternatively, the BMSC significantly ameliorated glutamate‐induced neuronal damage when cocultured with the three‐dimensional coculture technique. The protective effect was more prominent when coculture was started prior to glutamate exposure than when coculture was started just after glutamate exposure. ELISA analysis revealed that the BMSC physiologically produce NGF, BDNF, SDF‐1α, HGF, TGFβ‐1, and IGF‐1 and significantly enhanced the production of NGF and BDNF when cocultured with glutamate‐exposed neurons. These findings strongly suggest that the BMSC may protect and repair the damaged neurons through multiple mechanisms, including transdifferentiation, cell fusion, and production of growth factors.

Microglial P2Y12 Deficiency/Inhibition Protects against Brain Ischemia

Objective Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury. Methods Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12−/− or P2Y12+/−), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed. Results The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/−) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB). Interpretation P2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.

TRANSPLANTED BONE MARROW STROMAL CELLS PROMOTE AXONAL REGENERATION AND IMPROVE MOTOR FUNCTION IN A RAT SPINAL CORD INJURY MODEL

OBJECTIVERecent studies have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurological function when transplanted into animal models of spinal cord injury (SCI). However, it is still unclear how the transplanted BMSCs promote functional recovery after SCI. In this study, therefore, we evaluated how the transplanted BMSCs restore the function of the dorsal corticospinal tracts in the injured spinal cord. METHODSThe rats were subjected to incomplete SCI by means of a pneumatic impact device. BMSC or vehicle transplantation into the rostral site of SCI was performed at 7 days after injury. Neurological symptoms were assessed throughout the experiments. Fluoro-Ruby was injected into the dorsal funiculus of the rostral site of SCI at 63 days after injury. The fate of the transplanted BMSCs was examined using immunohistochemistry. RESULTSBMSC transplantation significantly enhanced functional recovery of the hind limbs. The number of Fluoro-Ruby–labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI was significantly higher in the BMSC-transplanted animals than in the vehicle-transplanted animals. Some of the engrafted BMSCs were positive for Fluoro-Ruby, NeuN, and MAP2 in the gray matter, suggesting that they acquired neuronal phenotypes and built synaptic connection with the hosts neural circuits. Others in the white matter morphologically simulated the astrocytes and were also positive for glial fibrillary acidic protein. CONCLUSIONThe findings suggest that the transplanted BMSCs acquire neural cell phenotypes around the injury site and contribute to rebuild the neural circuits, including the corticospinal tract, promoting functional recovery of the hind limbs.

Noninvasive transplantation of bone marrow stromal cells for ischemic stroke: preliminary study with a thermoreversible gelation polymer hydrogel.

OBJECTIVERecent studies have indicated that bone marrow stromal cells (BMSCs) have the potential to improve neurological function when transplanted into animal models of cerebral infarct. However, it is still undetermined how the BMSCs should be transplanted to obtain the most efficient therapeutic benefits safely. The aim of this study was to assess whether a thermoreversible gelation polymer (TGP) hydrogel acts as a noninvasive, valuable scaffold in BMSC transplantation for infarct brain. METHODSThe mice were subjected to permanent middle cerebral artery occlusion. Vehicle, BMSC suspension, or the BMSC-TGP construct was transplanted onto the ipsilateral intact neocortex at 7 days after the insult. Neurological symptoms were assessed throughout the experiments. The fate of the transplanted BMSC was examined 8 weeks after transplantation with immunohistochemistry. RESULTSTGP hydrogel completely disappeared and provoked no inflammation in the host brain. Many transplanted cells were widely engrafted in the ipsilateral cerebrum, including the dorsal neocortex adjacent to the cerebral infarct in the BMSC-TGP construct—treated mice. Their number was significantly larger than in the BMSC-treated mice. The majority were positive for both NeuN and MAP2 and morphologically simulated the neurons. CONCLUSIONThe findings suggest that surgical transplantation of tissue-engineered BMSCs onto the intact neocortex enhances the engraftment of donor cells around the cerebral infarct. These data may be useful in developing a noninvasive but efficient paradigm in neural tissue engineering. TGP hydrogel can be a promising candidate for valuable scaffolds in BMSC transplantation for central nervous system disorders because of its unique biochemical properties.

Overexpression of mitochondrial transcription factor A (TFAM) ameliorates delayed neuronal death due to transient forebrain ischemia in mice

Mitochondrial transcription factor A (TFAM) is an important regulator to maintain mitochondrial DNA copy number. However, no studies have denoted its roles in cerebral ischemia. Therefore, this study was aimed to assess whether the forced overexpression of TFAM ameliorates delayed neuronal death following transient forebrain ischemia. We have established human TFAM‐transgenic (Tg) mice. Wild type (WT) and TFAM‐Tg mice were subjected to 20‐min bilateral common carotid artery occlusion (BCCAO). Immunostaining against cytochrome c was performed to estimate its release from mitochondria at 24 h after 20‐min BCCAO. Histological analysis was performed to evaluate the effect of TFAM overexpression on delayed neuronal death at 72 h after 20‐min BCCAO. The number of cytochrome c‐positive neurons in the hippocampal CA1 sector was significantly smaller in TFAM‐Tg mice than in WT mice (P = 0.005). The percentage of viable neurons in the hippocampal CA1 sector was significantly higher in TFAM‐Tg mice than in WT mice (P < 0.001), and the number of TUNEL‐positive neurons was significantly smaller in TFAM‐Tg mice than in WT mice (P < 0.001). Our data strongly suggest that TFAM overexpression can reduce mitochondrial permeability transition and ameliorate delayed neuronal death in the hippocampus after transient forebrain ischemia.

Impact of oxygen extraction fraction on long-term prognosis in patients with reduced blood flow and vasoreactivity because of occlusive carotid artery disease

BACKGROUND Reduced cerebral blood flow and cerebrovascular reactivity to acetazolamide (type 3 ischemia) is believed as an independent predictor for subsequent ischemic stroke in patients with occlusive carotid artery diseases. However, recent studies have shown that type 3 patients can be divided into 2 pathophysiologically different subgroups as follows: those with elevated OEF and those with normal OEF. This study was aimed to clarify whether there is a difference in the prognosis between patients with type 3 and elevated OEF and those with type 3 but normal OEF. METHODS Twenty type 3 patients were enrolled in this prospective, longitudinal cohort study. Hemodynamic and metabolic parameters were quantitatively determined by (15)O-gas PET. All of them were medically treated. RESULTS Oxygen extraction fraction was elevated in 9 patients but was normal in other 11. During an average follow-up period of 45.6 months, 3 of 9 patients with type 3 and elevated OEF developed ipsilateral ischemic stroke. The annual risk was 10.6%. The location and shape of cerebral infarction strongly suggested a key role of hemodynamic compromise in their recurrence. On the other hand, no subsequent stroke occurred in none of 11 patients with type 3 but normal OEF. There was a statistically significant difference in the incidence of ipsilateral ischemic stroke between 2 groups (P = .0303). CONCLUSION Type 3 patients may be categorized into 2 subgroups as follows: those with elevated OEF and higher stroke risk and those with normal OEF and lower stroke risk, although larger number of subjects should be analyzed.

Synergistic effects of bone marrow stromal cells and a Rho kinase (ROCK) inhibitor, Fasudil on axon regeneration in rat spinal cord injury

Transplanted bone marrow stromal cells (BMSC) promote functional recovery after spinal cord injury (SCI) through multiple mechanisms. A Rho kinase inhibitor, Fasudil also enhances axonal regeneration. This study was aimed to evaluate whether combination therapy of BMSC transplantation and Fasudil further enhances axonal regeneration and functional recovery in rats subjected to SCI. Fasudil or vehicle was injected for 2 weeks. BMSC or vehicle transplantation into the rostral site of SCI was performed at 7 days after injury. Neurological symptoms were assessed throughout the experiments. Fluoro‐Ruby was injected into the dorsal funiculus of the rostral site of SCI at 63 days after injury. The fate of the transplanted BMSC was examined using immunohistochemistry. BMSC transplantation significantly increased the number of Fluoro‐Ruby ‐labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI, enhancing functional recovery of the hind limbs. Some of the engrafted BMSC were positive for Fluoro‐Ruby, neuronal specific nuclear protein and microtubule‐associated protein‐2, suggesting that they acquired neuronal phenotypes and built synaptic connection with the hosts neural circuits. Fasudil treatment also improved axonal continuity, but did not promote functional recovery. Combination therapy dramatically increased the number of Fluoro‐Ruby‐labeled fibers of the dorsal corticospinal tracts at the caudal site of SCI, but did not further boost the therapeutic effects on locomotor function by BMSC transplantation. The findings suggest that BMSC transplantation and Fasudil provide synergistic effects on axon regeneration after SCI, although further studies would be necessary to further enhance functional recovery.

Graded model of diffuse axonal injury for studying head injury‐induced cognitive dysfunction in rats

Diffuse axonal injury (DAI) plays a major role in the development of cognitive dysfunction, emotional difficulties and behavioral disturbances in patients following closed head injury, even when they have no definite abnormalities on conventional MRI. This study aimed to develop a highly controlled and reproducible model for DAI that simulates post‐traumatic cognitive dysfunction in humans. Sprague‐Dawley (SD) rats were subjected to impact acceleration head injury, using a pneumatic impact targeted to a steel disc centered onto their skull. The severity of injury was graded as three levels by adjusting the driving pressure at 60, 70 or 80 pounds per square inch. In vivo MRI was obtained 2 days post‐injury. Cognitive function was evaluated using the Morris water maze at 1 and 2 weeks post‐injury. HE staining and immunohistochemistry were performed to assess neuronal and axonal damages after 2 weeks. MRI demonstrated that this model induced no gross structural modification in the brain. The degree and duration of cognitive dysfunction were dependent on the force of impact. Histological analysis revealed the force‐dependent damage of the neurons and microtubule‐associated protein 2‐positive axons in the neocortex. Hippocampal damage was much less pronounced and was not linked to cognitive dysfunction. This is the first report that precisely evaluates the threshold of impact energy to lead to neocortical damage and cognitive dysfunction in rodents. This model would be suitable for clarifying the complex mechanisms of post‐traumatic brain damage and testing novel therapeutic approaches against post‐traumatic cognitive dysfunction due to diffuse axonal damage.

Transplanted bone marrow stromal cells improves cognitive dysfunction due to diffuse axonal injury in rats

Diffuse axonal injury (DAI) often leads to persistent cognitive dysfunction in spite of the lack of gross lesions on MRI. Therefore, this study was aimed to evaluate whether transplanted bone marrow stromal cells (BMSC) can improve DAI‐induced cognitive dysfunction or not. The rats were subjected to impact acceleration head injury, using a pneumatic high‐velocity impactor. The BMSC were harvested from the mice and were cultured. The BMSC (4.0 × 105 cells) or vehicle were stereotactically transplanted into the right striatum at 10 days post‐injury. Cognitive function analysis was repeated at 1, 2, and 4 weeks post‐injury, using the Morris water maze test. Histological analysis was performed at 2, 8 and 20 weeks post‐injury, using double fluorescence immunohistochemistry. Transplanted BMSC were widely distributed in the injured brain and gradually acquired the phenotypes of neurons and astrocytes over 20 weeks. In addition, they significantly improved DAI‐induced cognitive dysfunction as early as 2 weeks post‐injury, although their processes of neuronal differentiation were not completed at this time point. The findings suggest that the engrafted BMSC may exhibit this early beneficial effect on cognitive function by producing neuroprotective or neurotrophic factors. In conclusion, direct transplantation of BMSC may serve as a novel therapeutic strategy to enhance the recovery from DAI‐induced cognitive impairment.