Near infrared spectroscopy detection of hemispheric cerebral ischemia following middle cerebral artery occlusion in rats.

Timely and sensitive in vivo estimation of ischemic stroke-induced brain infarction are necessary to guide diagnosis and evaluation of treatments' efficacy. The gold standard for estimation of the cerebral infarction volume is magnetic resonance imaging (MRI), which is expensive and not readily accessible. Measuring regional cerebral blood flow (rCBF) with Laser Doppler flowmetry (LDF) is the status quo for confirming reduced blood flow in experimental ischemic stroke models. However, rCBF reduction following cerebral artery occlusion often does not correlate with subsequent infarct volume. In the present study, we employed the continuous-wave near infrared spectroscopy (NIRS) technique to monitor cerebral oxygenation during 90 min of the intraluminal middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats (n = 8, male). The NIRS device consisted of a controller module and an optical sensor with two LED light sources and two photodiodes making up two parallel channels for monitoring left and right cerebral hemispheres. Optical intensity measurements were converted to deoxyhemoglobin (Hb) and oxyhemoglobin (HbO2) changes relative to a 2-min window prior to MCAO. Area under the curve (auc) for Hb and HbO2 was calculated for the 90-min occlusion period for each hemisphere (ipsilateral and contralateral). To obtain a measure of total ischemia, auc of the contralateral side was subtracted from the ipsilateral side resulting in ΔHb and ΔHbO2 parameters. Infarct volume (IV) was calculated by triphenyl tetrazolium chloride (TTC) staining at 24h reperfusion. Results showed a significant negative correlation (r = -0.81, p = 0.03) between ΔHb and infarct volume. In conclusion, our results show feasibility of using a noninvasive optical imaging instrument, namely NIRS, in monitoring cerebral ischemia in a rodent stroke model. This cost-effective, non-invasive technique may improve the rigor of experimental models of ischemic stroke by enabling in vivo longitudinal assessment of cerebral oxygenation and ischemic injury.

Black Rice (Oryza sativa L., Poaceae) Extract Reduces Hippocampal Neuronal Cell Death Induced by Transient Global Cerebral Ischemia in Mice.

Rice is the most commonly consumed grain in the world. Black rice has been suggested to contain various bioactive compounds including anthocyanin antioxidants. There is currently little information about the nutritional benefits of black rice on brain pathology. Here, we investigated the effects of black rice (Oryza sativa L., Poaceae) extract (BRE) on the hippocampal neuronal damage induced by ischemic insult. BRE (300 mg/kg) was orally administered to adult male C57BL/6 mice once a day for 21 days. Bilateral common carotid artery occlusion (BCCAO) was performed for 23 min on the 8th day of BRE or vehicle administration. Histological analyses conducted on the 22nd day of BRE or vehicle administration revealed that administering BRE profoundly attenuated neuronal cell death, inhibited reactive astrogliosis, and prevented loss of glutathione peroxidase expression in the hippocampus when compared to vehicle treatment. In addition, BRE considerably ameliorated BCCAO-induced memory impairment on the Morris water maze test from the 15th day to the 22nd day of BRE or vehicle administration. These results indicate that chronic administration of BRE is potentially beneficial in cerebral ischemia.

Lin28 overexpression inhibits neurite outgrowth of primary cortical neurons in vitro.

Lin28 has been shown to promote proliferation of progenitors and survival of neurons during cortical neurogenesis. However, the role of Lin28 in the terminal maturation of neurons remains obscured. In this study, we investigated the developmental impact of Lin28 overexpression on neurite outgrowth. Lin28 expression was upregulated by in utero electroporation at E14.5. Two days later, electroporated cortices were dissociated for culturing primary cortical neurons. We found that Lin28 overexpression, which was confirmed immunocytochemically, led to neurite underdevelopment for all time points during culture. Specifically, Lin28-overexpressing cells displayed significantly fewer primary neurites and a decreased dendritic branching index, compared to GFP-expressing controls. Additionally, Lin28 overexpression in primary cortical neurons induced the expression of high mobility group AT-Hook 2 (HMGA2). Taken together, our study demonstrates that constitutive Lin28 expression disrupts cortical neurogenesis resulting in impaired neurite outgrowth with a concomitant induction of HMGA2.

Anthocyanins extracted from black soybean seed coat protect primary cortical neurons against in vitro ischemia.

The present study investigated the neuroprotective effects of anthocyanins extracted from black soybean (cv. Cheongja 3, Glycine max (L.) MERR.) seed coat against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays were employed to assess cell membrane damage and viability of primary neurons, respectively. OGD-induced cell death in 7 d in vitro primary cortical neurons was found to be OGD duration-dependent, and approximately 3.5 h of OGD resulted in ≈60% cell death. Treatment with black soybean anthocyanins dose-dependently prevented membrane damage and increased the viability of primary neurons that were exposed to OGD. Glutamate-induced neuronal cell death was dependent on the glutamate concentration at relatively low concentrations and the number of days the cells remained in culture. Interestingly, black soybean anthocyanins did not protect against glutamate-induced neuronal cell death. They did, however, inhibit the excessive generation of reactive oxygen species (ROS) and preserve mitochondrial membrane potential (MMP) in primary neurons exposed to OGD. In agreement with the neuroprotective effect of crude black soybean anthocyanins, purified cyanidin-3-glucoside (C3G), the major component of anthocyanins, also offered dose-dependent neuroprotection against OGD-induced neuronal cell death. Moreover, black soybean C3G markedly prevented excessive generation of ROS and preserved MMP in primary neurons that were exposed to OGD. Collectively, these results suggest that the neuroprotection of primary rat cortical neurons by anthocyanins that were extracted from black soybean seed coat might be mediated through oxidative stress inhibition and MMP preservation but not through glutamate-induced excitotoxicity attenuation.

Major role of the PI3K/Akt pathway in ischemic tolerance induced by sublethal oxygen-glucose deprivation in cortical neurons in vitro.

Ischemic preconditioning can provide protection to neurons from subsequent lethal ischemia. The molecular mechanisms of neuronal ischemic tolerance, however, are still not well-known. The present study, therefore, examined the role of MAPK and PI3K/Akt pathways in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. Ischemic tolerance was simulated by preconditioning of the neurons with sublethal 1-h OGD imposed 12 h before lethal 3-h OGD. The time-course studies of relative phosphorylation and expression levels of ERK1/2, JNK and p38 MAPK showed lack of their involvement in ischemic tolerance. However, there were significant increases in Akt phosphorylation levels during the reperfusion period following preconditioned lethal OGD. In addition, Bcl-2 associated death promoter (Bad) and GSK-3ß were also found to be inactivated during that reperfusion period. Finally, treatment with an inhibitor of PI3K, wortmannin, applied from 15 min before and during lethal OGD abolished not only the preconditioning-induced neuroprotection but also the Akt activation. Concomitant with blockade of the Akt activation, PI3K inhibition also resulted in activation of Bad and GSK-3ß. The results suggest that ischemic tolerance induced by sublethal OGD preconditioning is primarily mediated through activation of the PI3K/Akt pathway, but not the MAPK pathway, in rat cortical neurons.

The Neuroprotective Potential of Cyanidin-3-glucoside Fraction Extracted from Mulberry Following Oxygen-glucose Deprivation.

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of 50µM glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.

Involvement of ceramide in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation in primary cultured cortical neurons of rats.

The complex molecular cascades of ischemic tolerance in brain cells remain unclear. Recently, sphingolipid-related metabolite ceramide has been implicated as a second messenger in many biological functions, including neuronal survival and death. The present study, therefore, examined the roles of ceramide (Cer) in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation (OGD) using primary cultured cortical neurons of rats. Preconditioning of the neurons with sublethal 1-h OGD produced robust neuroprotection against cell death induced by lethal 3-h OGD imposed 12 h after preconditioning when measured by the MTT assay. Analysis of sphingolipids using LC-MS/MS showed that the ischemic preconditioning resulted in significant increases in the levels of C(16 : 0) Cer, C(18 : 0) Cer, C(20 : 0) Cer, C(24 : 0) Cer, C(24 : 1) Cer and the total ceramide contents compared with the sham-washed control group. However, sphingomyelin contents were not significantly changed by the ischemic preconditioning, suggesting that ceramides were increased through the de novo synthetic pathway. In the case of severe OGD paradigm, levels of ceramide and sphingomyelin in the lethal OGD group were not significantly different from those of the control group or the lethal OGD group with preconditioning at any time points studied. Treatment with an inhibitor of de novo ceramide synthesis, fumonisin B(1), during the ischemic preconditioning period completely blocked preconditioning-induced ischemic tolerance. Moreover, application of a non-cytotoxic concentration of exogenous cell-permeable ceramide produced neuroprotection against lethal OGD. The results suggest that ceramides increased by sublethal OGD preconditioning play an important role in induction of ischemic tolerance.

Mechanisms and prospects of ischemic tolerance induced by cerebral preconditioning.

In the brain, brief episodes of ischemia induce tolerance against a subsequent severe episode of ischemia. This phenomenon of endogenous neuroprotection is known as preconditioning-induced ischemic tolerance. The purpose of this review is to summarize the current state of knowledge about mechanisms and potential applications of cerebral preconditioning and ischemic tolerance. Articles related to the terms ischemic preconditioning and ischemic tolerance were systematically searched via MEDLINE/PubMed, and articles published in English related to the nervous system were selected and analyzed. The past two decades have provided interesting insights into the molecular mechanisms of this neuroprotective phenomenon. Although both rapid and delayed types of tolerance have been documented in experimental settings, the delayed type has been found to be more prominent in the case of neuronal ischemic tolerance. Many intracellular signaling pathways have been implicated regarding ischemic preconditioning. Most of these are associated with membrane receptors, kinase cascades, and transcription factors. Moreover, ischemic tolerance can be induced by exposing animals or cells to diverse types of endogenous and exogenous stimuli that are not necessarily hypoxic or ischemic in nature. These cross-tolerances raise the hope that, in the future, it will be possible to pharmacologically activate or mimic ischemic tolerance in the human brain. Another promising approach is remote preconditioning in which preconditioning of one organ or system leads to the protection of a different (remote) organ that is difficult to target, such as the brain. The preconditioning strategy and related interventions can confer neuroprotection in experimental ischemia, and, thus, have promise for practical applications in cases of vascular neurosurgery and endo-vascular therapy.