Hyung Soo Han

Neuroprotective mechanisms of hypothermia in brain ischaemia

Cooling can reduce primary injury and prevent secondary injury to the brain after insults in certain clinical settings and in animal models of brain insult. The mechanisms that underlie the protective effects of cooling — also known as therapeutic hypothermia — are slowly beginning to be understood. Hypothermia influences multiple aspects of brain physiology in the acute, subacute and chronic stages of ischaemia. It affects pathways leading to excitotoxicity, apoptosis, inflammation and free radical production, as well as blood flow, metabolism and blood–brain barrier integrity. Hypothermia may also influence neurogenesis, gliogenesis and angiogenesis after injury. It is likely that no single factor can explain the neuroprotection provided by hypothermia, but understanding its myriad effects may shed light on important neuroprotective mechanisms.

Mild Hypothermia Inhibits Inflammation After Experimental Stroke and Brain Inflammation

Background and Purpose— We previously showed that mild hypothermia protects against experimental stroke, even when cooling was delayed by 2 hours. Protection may be due in part to inhibiting inflammation. To clarify, we examined leukocyte infiltration, microglial activation, and adhesion molecule expression in models of stroke and pure brain inflammation. Methods— Rats underwent 2-hour middle cerebral artery occlusion (MCAO; n=36) or intravenous injection with 5 mg/kg lipopolysaccharide (LPS; n=22). Temperature was lowered to 33°C for 2 hours or kept at 37°C. In MCAO, cooling was applied intraischemically or on reperfusion (delayed). In the LPS model, cooling began after injection. One and 3 days later, brains were assessed for neutrophils, monocytes/microglia, major histocompatibility complex class II antigen, and intercellular adhesion molecule-1 (ICAM-1). Results— One day after MCAO, both intraischemic and delayed hypothermia decreased ICAM-1 (51% and 60%, respectively, versus normothermia; P <0.001), monocytes (63% and 57%; P <0.01), and microglia (55% and 53%; P <0.001). Similar decreases were seen at 3 days for ICAM-1 (91% and 93%; P <0.001), monocytes (62% and 54%; P <0.01), and microglia (55% and 53%; P <0.001). In the LPS model, ED-1–positive cells were not observed in the brain, but hypothermia decreased ICAM-1 (26%; P <0.05), OX6 (56%; P <0.01), and microglia (47%; P <0.01) at 1 day. Conclusions— Mild hypothermia decreases inflammatory responses in both brain inflammation and stroke, implicating a direct anti-inflammatory effect of cooling. This suggests that hypothermia can attenuate factors contributing to delayed ischemic injury.

Bone Marrow‐Derived Mesenchymal Stem Cells Promote Neuronal Networks with Functional Synaptic Transmission After Transplantation into Mice with Neurodegeneration

Recent studies have shown that bone marrow‐derived MSCs (BM‐MSCs) improve neurological deficits when transplanted into animal models of neurological disorders. However, the precise mechanism by which this occurs remains unknown. Herein we demonstrate that BM‐MSCs are able to promote neuronal networks with functional synaptic transmission after transplantation into Niemann‐Pick disease type C (NP‐C) mouse cerebellum. To address the mechanism by which this occurs, we used gene microarray, whole‐cell patch‐clamp recordings, and immunohistochemistry to evaluate expression of neurotransmitter receptors on Purkinje neurons in the NP‐C cerebellum. Gene microarray analysis revealed upregulation of genes involved in both excitatory and inhibitory neurotransmission encoding subunits of the ionotropic glutamate receptors (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, AMPA) GluR4 and GABAA receptor β2. We also demonstrated that BM‐MSCs, when originated by fusion‐like events with existing Purkinje neurons, develop into electrically active Purkinje neurons with functional synaptic formation. This study provides the first in vivo evidence that upregulation of neurotransmitter receptors may contribute to synapse formation via cell fusion‐like processes after BM‐MSC transplantation into mice with neurodegenerative disease.

Mild Hypothermia Inhibits Nuclear Factor-κB Translocation in Experimental Stroke

Nuclear factor-κB (NFκB) is a transcription factor that is activated after cerebral ischemia. NFκB activation leads to the expression of many inflammatory genes involved in the pathogenesis of stroke. The authors previously showed that mild hypothermia is protective even when cooling begins 2 h after stroke onset. In the present study, they examined the influence of hypothermia on NFκB activation. Rats underwent 2 h of transient middle cerebral artery occlusion. Brains were cooled to 33°C immediately after or 2 h after occlusion, and maintained for 2 h. After normothermic ischemia (brain temperature at 38°C), NFκB cytoplasmic expression, nuclear translocation, and binding activity were observed as early as 2 h in the ischemic hemisphere and persisted at 24 h. Hypothermia decreased NFκB translocation and binding activity but did not alter overall expression. Hypothermia also affected the levels of NFκB regulatory proteins by suppressing phosphorylation of NFκBs inhibitory protein (IκB-α) and IκB kinase (IKK-γ) and decreasing IKK activity, but did not alter overall IKK levels. Hypothermia suppressed the expression of two NFκB target genes: inducible nitric oxide synthase and TNF-α. These data suggest that the protective effect of hypothermia on cerebral injury is, in part, related to NFκB inhibition due to decreased activity of IKK.

Influence of hypothermia on post-ischemic inflammation: Role of nuclear factor kappa B (NFκB)

Mild hypothermia is one of the most robust neuroprotectant studied in the laboratory to date. The reasons for this protective effect are likely multifactorial, but work from our laboratory and others have shown that this protection is associated with remarkable suppression of the inflammatory response that accompanies brain ischemia. Consistently, laboratories have shown that small decreases in brain temperature to 30-34 degrees C result in reduced inflammatory cell infiltrate, less microglial activation, and reduction of a variety of inflammatory mediators such as nitric oxide, inflammatory cytokines and superoxide. Nuclear factor-kappaB (NFkappaB) is a transcription factor that is activated after cerebral ischemia. NFkappaB activation leads to the expression of many inflammatory genes involved in the pathogenesis of stroke. Our laboratory has shown that hypothermia decreases NFkappaB translocation and binding activity, by affecting NFkappaB regulatory proteins. Mild hypothermia appears to suppress phosphorylation of NFkappaBs inhibitory protein (IkappaB-alpha) by decreasing expression and activity of IkappaB kinase-gamma (IKK). As a consequence, hypothermia suppressed gene expression of two NFkappaB target genes, inducible nitric oxide synthase and TNF-alpha. These data suggest that the protective effect of hypothermia on cerebral injury is, in part, related to NFkappaB inhibition due to decreased activity of IKK.

Obovatol attenuates microglia‐mediated neuroinflammation by modulating redox regulation

Background and purpose:  Obovatol isolated from the medicinal herb Magnolia obovata exhibits a variety of biological activities. Here, the effect of obovatol and its mechanism of action on microglial activation, neuroinflammation and neurodegeneration were investigated.

The Function and Integrity of the Neurovascular Unit Rests Upon the Integration of the Vascular and Inflammatory Cell Systems

The neurovascular unit is composed of a microvascular endothelium, neuron, and glial cell elements that are in physical proximity to the endothelium. The vascular system provides oxygen, glucose, and hormones for brain cells and guides the cells to appropriately respond to the local environment. Conversely, the brain cells, especially glial cells, can regulate the function of blood vessels in response to local requirements. The disruption of the neurovascular coordination was observed in a variety of inflammation-related diseases in brain, such as infectious diseases, stroke, vascular dementia, and multiple sclerosis. Inflammatory responses resulting from infections or injury of the brain activate the endothelium and glial cells to various degrees depending on the type, titer, or strength and duration of exposure to the agents or insults. The activation of endothelial and microglial cells may be modulated by the action of cytokines or other substances secreted from these cells. In an effort to understand the pathogenesis and find rational treatments against inflammatory disorders in brain, studies have been separately carried out using either endothelial cells or microglia. Increasing evidence, however, indicates that a crosstalk between these two cell types is important for the brain inflammation. Here, we review recent advances that provide insights into the coordinated interaction between the vascular and microglial systems, including the role of the specialized endothelium in regulating the immune response that occurs within CNS, the influence of microglial cells on the properties of endothelial cells, and the effects of endothelium on the state of microglial activation.

Lipocalin-2 deficiency attenuates neuroinflammation and brain injury after transient middle cerebral artery occlusion in mice

Lipocalin-2 (LCN2) is a secreted protein of the lipocalin family, but little is known about the expression or the role of LCN2 in the central nervous system. Here, we investigated the role of LCN2 in ischemic stroke using a rodent model of transient cerebral ischemia. Lipocalin-2 expression was highly induced in the ischemic brain and peaked at 24 hours after reperfusion. After transient middle cerebral artery occlusion, LCN2 was predominantly expressed in astrocytes and endothelial cells, whereas its receptor (24p3R) was mainly detected in neurons, astrocytes, and endothelial cells. Brain infarct volumes, neurologic scores, blood–brain barrier (BBB) permeabilities, glial activation, and inflammatory mediator expression were significantly lower in LCN2-defkient mice than in wild-type animals. Lipocalin-2 deficiency also attenuated glial neurotoxicity in astrocyte/neuron cocultures after oxygen-glucose deprivation. Our results indicate LCN2 has a critical role in brain injury after ischemia/reperfusion, and that LCN2 may contribute to neuronal cell death in the ischemic brain by promoting neurotoxic glial activation, neuroinflammation, and BBB disruption.

Tissue-Specific Upregulation of Angiotensin-Converting Enzyme 1 in Spontaneously Hypertensive Rats Through Histone Code Modifications

The renin-angiotensin system has been implicated in the development of hypertension and damages several organs. The expressions of the components of a local renin-angiotensin system (RAS) in the hypertensive rats differ from those of the normotensive rats. We hypothesized that local tissue-specific upregulation of angiotensin-converting enzyme 1 (ACE1) in hypertension is caused by epigenetic changes. Adrenal gland, aorta, heart, kidney, liver, and lung tissues were excised from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Ace1 mRNA and protein expressions were measured by real-time PCR and Western blot, respectively. Promoter methylation was revealed by bisulfite sequencing. Histone modifications, such as histone 3 acetylation (H3Ac), fourth lysine trimethylation (H3K4me3), and ninth lysine dimethylation (H3K9me2), were quantified by chromatin immunoprecipitation (ChIP), followed by real-time PCR. The expressions and associations of chromatin remodeling genes were analyzed by real-time PCR and ChIP, respectively. Local tissues from SHRs showed higher expressions of Ace1 mRNA and protein than those from the WKY rats. Ace1 promoter was mostly unmethylated in all of the tissues from both strains. The Ace1 promoter regions of SHR tissues were more enriched with H3Ac and H3K4me3, except in the lungs. The adrenal glands, hearts, and kidneys of SHRs showed less enrichment with H3K9me2. Valsartan treatment in SHRs decreased local Ace1 mRNA and protein expressions, which were accompanied by higher H3K9me2, as well as less H3Ac and H3K4me3. In conclusion, ACE1 is upregulated in local tissues of SHRs via histone code modifications.

Expression of Na+-K+-2Cl− Cotransporter 1 Is Epigenetically Regulated During Postnatal Development of Hypertension

BACKGROUND The expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is upregulated in spontaneously hypertensive rat (SHR). We investigated whether expression of NKCC1 is epigenetically regulated during postnatal development of hypertension. METHODS The mesenteric arteries from 5-, 10-, and 18-week-old Wistar-Kyoto rats (WKY) and SHRs were subjected to vascular contraction. We determined expression levels of Nkcc1 mRNA and protein, methylation status, and histone modification of Nkcc1 promoter, and DNA methyltransferase (DNMT) activity. RESULTS The inhibition of dose-response curves by bumetanide, an inhibitor of NKCC1, as well as the expression of Nkcc1 mRNA and protein was comparable between 5-week-old SHR and age-matched WKY, but greater in 18-week-old SHR than in age-matched WKY. Nkcc1 promoter in WKY was getting methylated with age whereas that in SHR mostly remained hypomethylated after development of hypertension. DNMT3B was highly associated with the promoter of WKY, whereas the CXXC finger protein 1 (Cfp1) was highly bound to the promoter of SHR. At the age of 18 weeks, the DNMT activity in aorta of WKY was about threefold higher than that of SHR. The transcription-activating histone code acetyl H3 was higher in SHR than in WKY, whereas suppressive histone code dimethyl H3K9 was greater in WKY than in SHR. CONCLUSION It is concluded that expression of NKCC1 is epigenetically upregulated during postnatal development of hypertension. Our data indicate that maintenance of hypomethylation in Nkcc1 promoter of SHR resulting from low DNMT activity plays an important role in the upregulation of NKCC1 during development of spontaneous hypertension.