Eun-Cheol Song

Early Intravenous Infusion of Sodium Nitrite Protects Brain Against In Vivo Ischemia-Reperfusion Injury

Background and Purpose— The rate of nitric oxide (NO) generation from nitrite is linearly dependent on reductions in oxygen and pH levels. Recently, nitrite-derived NO has been reported to exert a profound protection against liver and heart ischemia-reperfusion injury. In this study, we hypothesized that nitrite would be reduced to NO in the ischemic brain and exert NO-dependent neuroprotective effects. Methods— Cerebral ischemia-reperfusion injury was induced by intraluminal thread occlusion of middle cerebral artery in the adult male rats. Solutions of sodium nitrite were infused intravenously at the time of reperfusion. Sodium nitrate and carboxy-PTIO (30 minutes before ischemic surgery), a direct NO scavenger, were infused for comparisons. Results— Nitrite reduced infarction volume and enhanced local cerebral blood flow and functional recovery. The effects were observed at concentrations of 48 nmol and 480 nmol, but not at 4800 nmol nitrite and 480 nmol nitrate. The neuroprotective effects of nitrite were inhibited completely by the carboxy-PTIO. The 480 nmol nitrite attenuated dihydroethidium activity, 3-nitrotyrosine formation, and lipid peroxidation in the ischemic brain. Conclusions— Nitrite exerted profound neuroprotective effects with antioxidant properties in the ischemic brains. These results suggest that nitrite, as a biological storage reserve of NO, may be a novel therapeutic agent in the setting of acute stroke.

Systemic transplantation of human adipose stem cells attenuated cerebral inflammation and degeneration in a hemorrhagic stroke model

Adipose-derived stem cells (ASCs) are readily accessible multipotent mesenchymal stem cells and are known to secrete multiple growth factors, and thereby to have cytoprotective effects in various injury models. In the present study, the authors investigated the neuroprotective effect of ASCs in an intracerebral hemorrhage (ICH) model. ICH was induced via the stereotaxic infusion of collagenase, and human ASCs (three million cells per animal) isolated from human fresh fat tissue, were intravenously administered at 24 h post-ICH induction. Acute brain inflammation markers, namely, cell numbers positively stained for terminal transferase dUTP nick end labeling (TUNEL), myeloperoxidase (MPO), or OX-42, and brain water content were checked at 3 days post-ICH. In addition, the authors quantified brain degeneration by measuring hemispheric atrophy and perihematomal glial thickness at 6 weeks post-ICH, and determined modified limb placing behavioral scores weekly over 5 weeks post-ICH. The results showed that brain water content, TUNEL+, and MPO+ cell numbers were significantly reduced in the ASC-transplanted rats. ASC transplantation attenuated neurological deficits from 4 to 5 weeks post-ICH, and reduced both the brain atrophy and the glial proliferation at 6 weeks. Transplanted ASCs were found to densely populate perihematomal areas at 6 weeks, and to express endothelial markers (von Willebrand factor and endothelial barrier antigen), but not neuronal or glial markers. In summary, ASCs transplantation in the ICH model reduced both acute cerebral inflammation and chronic brain degeneration, and promoted long-term functional recovery.

Pharmacological Induction of Ischemic Tolerance by Glutamate Transporter-1 (EAAT2) Upregulation

Background and Purpose— Astrocytic glutamate transporter protein, GLT-1 (EAAT2), recovers extracellular glutamate and ensures that neurons are protected from excess stimulation. Recently, &bgr;-lactam antibiotics, like ceftriaxone (CTX), were reported to induce the upregulation of GLT-1. Here, we investigated ischemic tolerance induction by CTX in an experimental model of focal cerebral ischemia. Methods— CTX (200 mg/kg per day, IP) was administered for 5 consecutive days before transient focal ischemia, which was induced by intraluminal thread occlusion of the middle cerebral artery for 90 minutes or permanently. Results— Repeated CTX injections enhanced GLT-1 mRNA and protein expressions after 3 and 5 days of treatment, respectively. CTX-pretreated animals showed a reduction in infarct volume by 58% (reperfusion) and 39% (permanent), compared with the vehicle-pretreated animals at 24 hours postischemia (P<0.01). Lower doses of CTX (20 mg/kg per day and 100 mg/kg per day) reduced infarct volumes to a lesser degree. The injection of GLT-1 inhibitor (dihydrokainate) at 30 minutes before ischemia ameliorated the effect of CTX pretreatment. However, CTX administration at 30 minutes after ischemia produced no significant reduction in infarct volume. CTX reduced the levels of proinflammatory cytokines (tumor necrosis factor-&agr;, FasL), matrix metalloproteinase (MMP)-9, and activated caspase-9 (P<0.01). In addition, CTX-pretreated animals showed better functional recovery at day 1 to week 5 after ischemia (P<0.05). Conclusions— This study presents evidence that CTX induces ischemic tolerance in focal cerebral ischemia and that this is mediated by GLT-1 upregulation.

Circulating Endothelial Progenitor Cells as a New Marker of Endothelial Dysfunction or Repair in Acute Stroke

Background and Purpose— Understanding on distinct subsets of endothelial progenitor cells may provide insights of endothelial dysfunction or repair in the acute ischemic event. Recent in vitro data have reported the colony-forming unit (CFU) and outgrowth cell population as a subset of endothelial progenitor cells. In this study, we undertook to validate the significance of CFU number and outgrowth cell yield in acute stroke. Methods— Mononuclear cells were isolated from the peripheral blood of 75 patients with acute stroke, 45 patients with chronic stroke, and 40 age-matched healthy volunteers. CFU numbers were counted after culturing them for 7 days, and outgrowth cell appearance was measured during the 2 months of culture. Endothelial progenitor cell function was also evaluated by matrigel plate assays. Independent parameters predicting CFU number and outgrowth cell yield were assessed using logistic regression analysis. Results— The CFU numbers and tube formation abilities in matrigel assays were significantly reduced in patients with acute stroke compared with patients with chronic stroke or healthy control subjects. Moreover, patients with large artery atherosclerosis had much lower CFU numbers and functional activities than ones with cardioembolism. Outgrowth cells were isolated from 10% of healthy control subjects and 22% of patients with chronic stroke during the cultures, but from 71% of patients with stroke. Multivariate analysis identified glycosylated hemoglobin and National Institutes of Health Stroke Scale on admission as significant independent predictors of a low CFU number and a high isolation frequency of outgrowth cells, respectively. Conclusion— CFU number may thus represent an accumulated endothelial progenitor cell dysfunctional status, whereas outgrowth cell appearance may reflect the resilience of the systemic circulation to acute ischemic stress.

Blockade of AT1 Receptor Reduces Apoptosis, Inflammation, and Oxidative Stress in Normotensive Rats with Intracerebral Hemorrhage

Angiotensin II exerts its central nervous system effects primarily via its receptors AT1 and AT2, and it participates in the pathogenesis of ischemia via AT1. The selective AT1 receptor blocker (ARB) is used in the hypertension treatment, and it exerts a variety of pleiotropic effects, including antioxidative, antiapoptotic, and anti-inflammatory effects. In this study, we investigated the therapeutic effect of the ARB telmisartan in experimental intracerebral hemorrhage (ICH) in normotensive rats. ICH was induced via the collagenase infusion or autologous blood injection. Either telmisartan at 30 mg/kg/dose or phosphate-buffered saline was orally administered 2 h after ICH induction. We evaluated hemorrhage volume, brain water content, and functional recovery, and we performed the histological analysis for terminal deoxynucleotidyl transferase dUTP nick-end labeling, leukocyte infiltration, and microglia activation. A variety of intracellular signals, in terms of oxidative stress, apoptotic molecules, and inflammatory mediators, were also measured. Telmisartan reduced hemorrhage volume, brain edema, and inflammatory or apoptotic cells in the perihematomal area. Telmisartan was noted to induce the expression of endothelial nitric-oxide synthase and peroxisome proliferator-activated receptor γ and decrease oxidative stress, apoptotic signal, tumor necrosis factor-α, and cyclooxygenase-2 expression. The telmisartan-treated rats exhibited less pronounced neurological deficits and recovered better. Thus, telmisartan seems to offer neural protection, including antiapoptosis, anti-inflammatory, and antioxidant benefits in the intracerebral hemorrhage rat model.

Pharmacological induction of heat shock protein exerts neuroprotective effects in experimental intracerebral hemorrhage

Heat shock proteins (HSPs) are reported to reduce inflammation and apoptosis in a variety of brain insults. Geranylgeranylacetone (GGA), developed as an antiulcer in Japan, has been known to induce HSP70 and to exert cytoprotective effects. In this study, we investigated whether GGA, as a specific HSP inducer, exerts therapeutic effects in experimentally induced intracerebral hemorrhage (ICH). ICH was induced with male Sprague-Dawley rats via the collagenase infusion. GGA (800 mg/kg) was administered via oral tube according to various schedules of treatment. The treatment with GGA, beginning before the induction of ICH and continuing until day 3, showed the reduction of brain water content and the increased level of HSP70 protein, as compared to the treatment with vehicle, although GGA started after the induction of ICH or administered as a single dose before ICH failed to up-regulate HSP70 and to reduce brain edema. The rats treated with GGA exhibited better functional recovery than those treated with vehicle. In the pre- and post- treatment group, inflammatory cells and cell death in the perihematomal regions were found to have been decreased. The treatment of GGA inhibited the mRNA expression of MMP-9, uPA, IL-6 and MIP-1, with concomitant increment of eNOS and phosphorylated STAT3 and Akt after ICH. We demonstrated that GGA induced a reduction in the brain edema along with marked inhibitory effects on inflammation and cell death after ICH.

Circulating endothelial progenitor cells as a pathogenetic marker of moyamoya disease

Moyamoya disease (MMD) is an unusual form of chronic cerebrovascular occlusive disease that involves the formation of characteristically abnormal vessels. Recent studies have reported that colony-forming unit (CFU) and outgrowth cells represent a subpopulation of endothelial progenitor cells (EPCs). Here, we attempted to determine the significance of CFU number and outgrowth cell yield in MMD. Endothelial progenitor cells were isolated from the blood of 24 adult MMD patients and from 48 age- and risk factor-matched control subjects. After 7 days of culture, CFUs were determined, and yields of outgrowth cells were measured during 2 months of culture. The EPC function was also evaluated using matrigel plate assays. It was found that CFU numbers were significantly lower in MMD patients than in controls. Moreover, during long-term culture, outgrowth cells were isolated from only 10% of control subjects but from 33% of MMD patients, and CFU numbers and tube formation were found to be lower in advanced MMD cases than in those with early stage disease, whereas outgrowth cells were more frequently detected in those with early MMD and moyamoya vessels than in those with advanced disease. These characteristics of circulating EPCs reflect mixed conditions of vascular occlusion and abnormal vasculogenesis during the pathogenesis of MMD.

Region‐specific plasticity in the epileptic rat brain: A hippocampal and extrahippocampal analysis

Purpose:  Recent evidence suggests that aberrant neuro/gliogenesis and/or inflammation play critical roles in epileptogenesis. Although the plastic and inflammatory changes have been described in the postseizure hippocampus, little data is available concerning extrahippocampal regions, notably in the piriform and entorhinal cortices, amygdala, and parts of the thalamus. In this study, we examined histological changes in whole epileptic rat brain, with respect to cell death, cell genesis, and inflammation.

Proteasomal inhibition in intracerebral hemorrhage : Neuroprotective and anti-inflammatory effects of bortezomib

Inflammation is an important pathophysiologic mechanism of injury induced by intracerebral hemorrhage (ICH). The ubiquitin-proteasome system (UPS) regulates the inflammatory responses via the up-regulation of several pro-inflammatory molecules. In this study, we determined that a potent proteasome inhibitor, bortezomib, exerted therapeutic effects in experimental model of ICH. Either bortezomib (0.05, 0.2, 0.5, 1mg/kg) or vehicle was intravenously administered 2h after ICH induction. The high doses of bortezomib caused high mortality rates. Bortezomib at 0.2 mg/kg reduced the early hematoma growth and alleviated hematoma volume and brain edema at 3 days after ICH, compared with the ICH-vehicle group. The numbers of myeloperoxidase(+) neutrophils, Ox42(+) microglia, and TUNEL(+) cells in the perihematomal regions were decreased by bortezomib. Bortezomib induced significant decrements of mRNA expression of TNF-alpha and IL-6. The production of iNOS and COX2 was also reduced significantly by bortezomib. We concluded that the early treatment with bortezomib induced a reduction in the early hematoma growth and mitigated the development of brain edema, coupled with a marked inhibitory effect on inflammation in ICH.

Combined neuroprotective effects of celecoxib and memantine in experimental intracerebral hemorrhage

Memantine, a N-methyl-D-aspartate (NMDA) receptor antagonist, inhibits hematoma expansion and celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, reduces perihematomal inflammation in intracerebral hemorrhage. We examined whether the combination treatment has additive effects in experimental intracerebral hemorrhage (ICH). ICH was induced using stereotaxic infusion of collagenase into brains of adult rats. After the induction of ICH, rats were treated with intraperitoneal injection of memantine (20 mg/kg), celecoxib (20 mg/kg) or both agents. Only vehicles were administrated in rats of the control group. Results showed that the combination treatment of memantine and celecoxib reduced both hematoma volume and brain edema. Combination treatment also induced the better functional recovery with further attenuation of cerebral inflammation and apoptosis compared to the control group. When compared to the single agent groups, the combination treatment showed better effects in neuroprotection and anti-inflammation. These results suggest the feasible combined application of memantine and celecoxib in ICH treatment.