Tsuyoshi Uesugi

Administration of hematopoietic cytokines increases the expression of anti-inflammatory cytokine (IL-10) mRNA in the subacute phase after stroke

We investigated the effect of the subcutaneous administration of hematopoietic cytokines, granulocyte colony-stimulating factor (G-CSF)+stem cell factor (SCF), on mRNA expression of tissue cytokines in the acute or subacute phase after focal ischemia in male C57 BL/6J mice. The expression of IL-10 mRNA was elevated at 4-14 days after occlusion when cytokines were given in the acute phase (days 1-10). The expression of IL-10 mRNA was markedly elevated at 14 days after occlusion, then remained high until 28 days when cytokines were given in the subacute phase (days 11-20). However, there were no significant changes in IL-6, TGF-beta1, TNF, G-CSF, SCF and iNOS expression following either acute- or subacute-phase treatment. Further, hematopoietic cytokine treatment in the subacute phase, but not in the acute phase, reduced ED1-positive microglia/macrophages in the infarcted brain. Our recent study showed that the subacute-phase treatment is effective for functional recovery, enhancing generation of neuronal cells from both bone-marrow-derived and neural stem/progenitor cells. Taken together, these results suggest that cytokine treatment in the subacute phase may provide a favorable microenvironment for neurogenesis after ischemic stroke through the up-regulation of IL-10.

Phase I Study of Intravenous Low-dose Granulocyte Colony-stimulating Factor in Acute and Subacute Ischemic Stroke

BACKGROUND Granulocyte colony-stimulating factor (G-CSF; filgrastim) may be useful for the treatment of acute ischemic stroke because of its neuroprotective and neurogenesis-promoting properties, but an excessive increase of neutrophils may lead to brain injury. We examined the safety and tolerability of low-dose G-CSF and investigated the effectiveness of G-CSF given intravenously in the acute phase (at 24 hours) or subacute phase (at 7 days) of ischemic stroke. METHODS Three intravenous dose regimens (150, 300, or 450 μg/body/day, divided into 2 doses for 5 days) of G-CSF were examined in 18 patients with magnetic resonance imaging (MRI)-confirmed infarct in the territory of the middle cerebral artery. Nine patients received the first dose at 24 hours poststroke (acute group) and 9 patients received the first dose on day 7 poststroke (subacute group; n = 3 at each dose in each group). A scheduled administration of G-CSF was skipped if the patients leukocyte count exceeded 40,000/μL. Patients received neurologic and MRI examinations. RESULTS We found neither serious adverse event, drug-related platelet reduction nor splenomegaly. Leukocyte levels remained below 40,000/μL at 150 and 300 μg G-CSF/body/day, but rose above 40,000/μL at 450 μg G-CSF/body/day. Neurologic function improvement between baseline and day 90 was more marked after treatment in the acute phase versus the subacute phase (Barthel index 49.4 ± 28.1 v 15.0 ± 22.0; P < .01). CONCLUSIONS Low-dose G-CSF (150 and 300 μg/body/day) was safe and well tolerated in ischemic stroke patients, and leukocyte levels remained below 40,000/μL.

A sartan derivative with a very low angiotensin II receptor affinity ameliorates ischemic cerebral damage

Angiotensin II receptor blockers (ARBs) have a potent ability to inhibit oxidative stress and advanced glycation, in addition to their protective effects originated from blood pressure lowering and angiotensin II type 1 receptor (AT1)-blockade. To obtain a pharmacological tool to dissect the mechanisms of ARBs’ protective benefits in experimental stroke, we synthesized a novel ARB-derivative, R-147176, which is 6,700 times less potent than olmesartan in AT1-binding inhibition and therefore has a minimal antihypertensive effect, but retains marked inhibitory effects on oxidative stress and advanced glycation. We evaluated the effect of R-147176 (10–30 mg/kg per day), administered orally or intravenously, on brain infarct volume in transient thread occlusion and photothrombotic models in rats. The antioxidative and antiinflammatory properties were also investigated. R-147176 significantly reduced infarct volume, without influence on blood pressure, in both models. R-147176 significantly reduced the numbers of ED-1-positive cells and of TUNEL-positive cells, and protein carbonyl formation in the damaged brain. This ARB derivative, despite its significantly lower AT1 affinity and virtually no antihypertensive effect, ameliorated ischemic cerebral damage through antioxidative and antiinflammatory properties. These findings suggest potential usefulness of R-147176 as a pharmacological tool to investigate the ARBs’ protective effect in experimental stroke and open new therapeutic avenues.

A novel prolyl hydroxylase inhibitor protects against cell death after hypoxia.

Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.

Cerebral Microbleeds on T2*-Weighted Images and Hemorrhagic Transformation after Antithrombotic Therapies for Ischemic Stroke

To assess the predictive value of cerebral microbleeds (CMBs) on gradient-echo T2*-weighted magnetic resonance imaging for hemorrhagic transformation (HT) after antithrombotic therapy for an acute ischemic stroke, we prospectively examined the relationship between CMBs on T2*-weighted images before the start of therapy and the appearance of HT in a series of patients treated with antithrombotic therapies. The subjects were consecutive acute ischemic stroke patients admitted to Tokai University Hospital (187 subjects, mean age±SD: 74±11 years). The prevalence of CMBs was not significantly different between the subjects with and without HT on computed tomography (CT) (19% versus 36%, P=.081). In both the subgroup of patients treated with anticoagulants and the subgroup treated with antiplatelets, the prevalence of HT was not significantly different between the subjects with and without CMBs (anticoagulants, 9% versus 21%, P=.161; antiplatelets, 0% versus 9%, P=.542). The odds ratios (ORs) of increasing the National Institutes of Health Stroke Scale score (1.14, 95% confidence interval [CI]: 1.04-1.26, P=.005) and decreasing the Alberta Stroke Program Early CT Score on diffusion-weighted images (ASPECTS-DWI) (1.32, 95% CI: 1.10-1.59, P=.003) were significantly increased for the appearance of HT, but the OR of CMBs (.35, 95% CI: .09-1.41, P=.140) was not significantly increased for the appearance of HT. In conclusion, the severity of neurological deficits and the ASPECTS-DWI are closely correlated to the development of HT related to anticoagulants/antiplatelets but not to CMBs on T2*-weighted images.

Trigeminal Neuropathy Accompanied by a Pontine Lesion on MRI

A 63-year-old man presented with the loss of the sensations of pain and temperature sensation in the right facial region innervated by the trigeminal nerve (V1 to 3). He showed abnormal lesions in the pons and the trigeminal nerve on magnetic resonance imaging (MRI). He had recurrent herpes in the nasal cavity, and a history of left facial palsy. We herein present the unique MRI findings and suggest that herpes simplex infection may cause trigeminal neuropathy. This is the first reported case of dissociated trigeminal neuropathy with herpes simplex infection which was accompanied by a pontine lesion on MRI.

[Hemichorea improved by carotid artery stenting in a 73-year-old man with hypoperfusion of the basal ganglia].

A 73-year-old man presented with continuous hemichoreic movement of right arm and leg and with dyskinesia in his tongue. Magnetic resonance image (MRI) showed no ischemic lesion within the basal ganglia, but magnetic resonance angiography (MRA) and carotid duplex ultrasonography showed the left internal carotid occlusion and 80% stenosis in the right common carotid artery. Tc-99m-ECD-SPECT showed hypoperfusion of the frontal lobe, temporal lobe, parietal lobe, basal ganglia and thalamus. A trial of haloperidol had no effect; therefore, the right carotid artery stenting was performed. Hypoperfusion in the left internal carotid artery area was improved by cross flow from the right side, and his hemichorea gradually improved. This result supports the notion that hypoperfusion-related hemichorea may occur, even in the absence of cerebral ischemia.

Effect of atorvastatin co‐treatment on inhibition of platelet activation by clopidogrel in patients with ischemic stroke

Clopidogrel is used to achieve sustained platelet inhibition in ischemic stroke patients (1). 3-Hydroxy-3methylglutaryl-CoA reductase inhibitors also reduce the stroke events (2). We hypothesized that an interaction between clopidogrel and 3-Hydroxy-3methylglutaryl-CoA reductase inhibitors would decrease P2Y12 receptor and inhibit platelet activation by acting on lipid rafts (3,4). To test this idea, we evaluated the effect of coadministration of atorvastatin on the inhibition of platelet activation in ischemic stroke patients receiving clopidogrel. Twenty ischemic stroke patients (13 atherothrombotic, 7 lacunar; mean age 68 ± 12, 65% male) receiving clopidogrel with dyslipidemia were recruited. Written informed consent was obtained from all patients, and the study was approved by Tokai University Ethics Committee. Blood samples were taken before, and at twoweeks and two-months after the administration of 10-mg/day atorvastatin. PAC1, CD62P, and WBC-platelet complex were quantified by means of three-color flow cytometry, according to our previous reports (5–8). Coadministration of atorvastatin for two-months significantly reduced LDLcholesterol from 143 ± 26 to 92 ± 20 mg/dl (P < 0·01). No significant difference in PAC-1-positive platelets beyond the cutoff value was observed (Fig. 1a). However, the percentage of CD62Ppositive platelets beyond the cutoff value was significantly reduced by coadministration of atorvastatin for two-months, compared with clopidogrel alone (Fig. 1b, P < 0·05). The percentage of WBCplatelet complex beyond the cutoff value was also significantly reduced by coadministration of atorvastatin for twoweeks and two-months, compared with clopidogrel alone (Fig. 1c, P < 0·05). The suppression of CD62P may suggest that the translocation of P2Y12 receptors away from lipid rafts, due to reduction of lipid rafts by atorvastatin (4), inhibits platelet activation by suppressing adenylate cyclase activation via Gi (9). Further, WBC-platelet complex is associated with expression of P-selectin (CD62P), which mediates adhesion of platelets to leukocytes (10). Our results indicate that coadministration of atorvastatin in ischemic stroke patients receiving clopidogrel further enhances the inhibition of platelet activation.