A Novel Bioabsorbable Sheet That Delivers NF-κB Decoy Oligonucleotide Restrains Abdominal Aortic Aneurysm Development in Rats.

For the suppression of inflammation in the aneurysm development, we focused on inhibition of an important transcription factor, nuclear factor-kappa B (NF-κB), using a decoy strategy. We newly developed a novel bioabsorbable sheet that delivers NF-κB decoy oligodeoxynucleotide (ODN).We treated 5-week-old SD rats that were induced with abdominal aortic aneurysm (AAA) using 0.5 M CaCl2 with an NF-κB decoy sheet. Four weeks after AAA induction, aortic tissue was excised for further examinations. We showed that this bioabsorbable sheet could deliver the decoy ODN into the target tissues and dissolve within a week. Treatment with the NF-κB decoy sheet reduced the aneurysm size compared with the controls. It also suppressed inflammation due to the effect of NF-κB decoy ODN. Immunohistochemistry revealed that the expression of CD31, CD4, and CD11b in the NF-κB decoy sheet group was significantly lower than in the control sheet group. The NF-κB decoy sheet was absorbed on the target tissue.We have revealed that the bioabsorbable sheet mediated decoy ODN is effective for transfection into target organs. We have also indicated that NF-κB decoy ODN transfection using this sheet has the potential to suppress the dilatation of aneurysm. The bioabsorbable sheet mediated transfection of the decoy ODN can be beneficial for the clinical treatment of AAA and other NF-κB-related cardiovascular diseases.

Angiotensin II Peptide Vaccine Protects Ischemic Brain Through Reducing Oxidative Stress.

BACKGROUND AND PURPOSE: Medication nonadherence is one of major risk factors for the poor outcome in ischemic stroke. Vaccination is expected to solve such a problem because of its long-lasting effects, but its effect on ischemic brain damage is still unknown. Here, we focused on vaccination for renin-angiotensin system and examined the effects of angiotensin II (Ang II) peptide vaccine in permanent middle cerebral artery occlusion model in rats. METHODS: Male Wistar rats were exposed to permanent middle cerebral artery occlusion after 3× injections of Ang II peptide vaccine, and the serum or brain level of anti-Ang II antibody was examined. The effects of the vaccine were evaluated by differences in infarction volume, brain renin-angiotensin system components, and markers for neurodegeneration and oxidative stress. RESULTS: Ang II vaccination successfully produced anti-Ang II antibodies in serum without concomitant change in blood pressure. Sufficient production of serum anti-Ang II antibody led to reduction of infarct volume and induced the penetration of anti-Ang II antibody in ischemic hemisphere, with suppressed expression of Ang II type 1 receptor mRNA. Vaccinated rats with sufficient antibody production showed the reduction of Fluoro-Jade B-positive cells, spectrin fragmentation, 4-hydroxynonenal-positive cells, and Nox 2 mRNA expression. CONCLUSIONS: Our findings indicate that Ang II vaccination exerts neuroprotective and antioxidative effects in cerebral ischemia, with renin-angiotensin system blockade by penetration of anti-Ang II antibodies into ischemic brain lesion. Ang II peptide vaccination could be a promising approach to treat ischemic stroke.

OPG/RANKL/RANK axis is a critical inflammatory signaling system in ischemic brain in mice.

Osteoprotegerin (OPG) is a soluble secreted protein and a decoy receptor, which inhibits a receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)/the receptor activator of NF-κB (RANK) signaling. Recent clinical studies have shown that a high-serum-OPG level is associated with unfavorable outcome in ischemic stroke, but it is unclear whether OPG is a culprit or an innocent bystander. Here we demonstrate that enhanced RANKL/RANK signaling in OPG(-/-) mice or recombinant RANKL-treated mice contributed to the reduction of infarct volume and brain edema via reduced postischemic inflammation. On the contrary, infarct volume was increased by reduced RANKL/RANK signaling in OPG(-/-) mice and WT mice treated with anti-RANKL neutralizing antibody. OPG, RANKL, and RANK mRNA were increased in the acute stage and were expressed in activated microglia and macrophages. Although enhanced RANKL/RANK signaling had no effects in glutamate, CoCl2, or H2O2-stimulated neuronal culture, enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture, suggesting that RANKL/RANK signaling can attenuate inflammation through a Toll-like receptor signaling pathway in microglia. Our findings propose that increased OPG could be a causal factor of reducing RANKL/RANK signaling and increasing postischemic inflammation. Thus, the OPG/RANKL/RANK axis plays critical roles in controlling inflammation in ischemic brains.

Porphyromonas gingivalis, a periodontal pathogen, enhances myocardial vulnerability, thereby promoting post-infarct cardiac rupture.

There is a strong association between periodontal disease (PD) and atherosclerosis. However, it remains unknown whether PD is also involved in myocardial damage. We hypothesized that infection with periodontal pathogens could cause an adverse outcome after myocardial infarction (MI). C57BL/6J mice were inoculated with Porphyromonas gingivalis (P.g.), a major periodontal pathogen, or injected with phosphate-buffered saline (PBS) into a subcutaneously-implanted steelcoil chamber before and after coronary artery ligation. A significant increase in mortality, due to cardiac rupture, was observed in the P.g.-inoculated MI mice. Ultrastructural examinations revealed that P.g. invaded the ischemic myocardium of the P.g.-inoculated MI mice. The expression of p18 Bax, an active form of pro-apoptotic Bax protein, markedly increased in the P.g.-inoculated MI hearts. In vitro experiments demonstrated that gingipain, a protease uniquely secreted from P.g., cleaved wild type Bax at Arg34, as evidenced by the observation that the cleavage of Bax by gingipain was completely abolished by the Arg34Ala mutation in Bax. Treatment with immunoglobulin Y against gingipain significantly decreased the mortality of the P.g.-inoculated MI mice caused by cardiac rupture. Furthermore, inoculation of P.g. also resulted in an increase of MMP-9 activity in the post-MI myocardium by enhancing oxidative stress, possibly through impairing the selective autophagy-mediated clearance of damaged mitochondria. In conclusion, infection with P.g. during MI plays a detrimental role in the healing process of the infarcted myocardium by invasion of P.g. into the myocardium, thereby promoting apoptosis and the MMP-9 activity of the myocardium, which, in turn, causes cardiac rupture.

Periodontitis May Deteriorate Sinus of Valsalva Dilatation in Marfan Syndrome Patients.

Marfan syndrome (MFS) is a systemic connective tissue disorder that is caused by mutations of fibrillin-1. While MFS patients are at a high risk of periodontitis and aortic diseases, little causal information has been provided to date. To clarify the relationship, their oral condition and sinus of Valsalva (SoV) were evaluated.The subjects were patients with MFS (n = 33) who attended the University of Tokyo Hospital. We divided them into two groups; MFS patients with highly dilated (the diameters were equal to or more than 39 mm) SoV (high group, n = 18) and MFS patients with mildly dilated (less than 39 mm) SoV (mild group, n = 15). Blood examinations, echocardiograms, and full-mouth clinical measurements, including number of teeth, probing pocket depth (PPD), bleeding on probing (BOP), and community periodontal index (CPI) were performed.We found that the high group patients had greater rates of BOP compared to that of the mild group. Furthermore, the high group tended to have higher serum levels of C-reactive protein, matrix metalloproteinase-9, and transforming growth factor-ß compared to the mild group.Periodontitis may deteriorate SoV dilatation in MFS patients.

Incidence of periodontitis in Japanese patients with cardiovascular diseases: a comparison between abdominal aortic aneurysm and arrhythmia.

Although there is a link between periodontitis and cardiovascular disease (CVD), the influence of periodontitis on CVD is unclear. The aim of this study was to assess the relationship between periodontal bacterial burden and CVD. We studied 142 patients with tachyarrhythmia (TA) and 25 patients with abdominal aortic aneurysm (AAA). We examined periodontitis and the presence of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Prevotella intermedia in the patients' saliva and subgingival plaque using PCR. We also measured serum antibody titers against the pathogens using ELISA. We found that the patients with AAA had fewer remaining teeth (14.6 ± 2.0 vs. 20.9 ± 0.7, P < 0.05) and deeper pocket depth (3.01 ± 0.26 vs. 2.52 ± 0.05 mm, P < 0.05) compared to the TA patients. The existence of each periodontal bacterium in their saliva or subgingival plaque and serum antibody titers was comparable between the two groups. Periodontitis may have a larger affect on aneurysm progression compared to arrhythmia.

High incidence of periodontitis in Japanese patients with abdominal aortic aneurysm.

Periodontitis is known to be a risk factor for abdominal aortic aneurysm (AAA). However, the influence of periodontitis on AAA in Japanese patients has not yet been elucidated. The aim of this clinical investigation was to assess the relationship between periodontal bacterial burden in AAA patients.We studied 12 AAA patients and 24 age- and sex-matched non-AAA cardiovascular patients. We examined periodontitis and the presence of the periodontal pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia in oral samples using polymerase chain reaction assays.We found that the AAA patients had deeper pocket depth compared to the non-AAA patients (3.53 ± 0.38 mm versus 2.67 ± 0.17 mm, P < 0.05). However, the populations of periodontal bacteria were comparable between the two groups. Periodontitis may have a greater effect on aneurysm progression compared to other cardiovascular diseases.

PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum.

Pigment epithelium-derived factor (PEDF) is a serine protease inhibitor (serpin) protein with well established neuroprotective and anti-angiogenic properties. Recent studies have also shown that PEDF enhances renewal of adult subventricular zone (SVZ) neural precursors. In neurosphere cultures prepared from the SVZ of adult mice, we found that addition of recombinant PEDF to the medium enhanced expressions of oligodendroglial lineage markers (NG2 and PDGFrα) and transcription factors (Olig1, Olig2, and Sox10). Similarly, continuous PEDF administration into the lateral ventricles of adult glial fibrillary acidic protein:green fluorescent protein (GFAP:GFP) transgenic mice increased the proportions of GFAP:GFP+ and GFAP:GFP- SVZ neural precursors coexpressing oligodendroglial lineage markers and transcription factors. Notably, PEDF infusion also resulted in an induction of doublecortin- and Sox10 double-positive cells in the adult SVZ. Immunoreactive PEDF receptor was detectable in multiple cell types in both adult SVZ and corpus callosum. Furthermore, PEDF intracerebral infusion enhanced survival and maturation of newly born oligodendroglial progenitor cells in the normal corpus callosum, and accelerated oligodendroglial regeneration in lysolecithin-induced corpus callosum demyelinative lesions. Western blot analysis showed a robust upregulation of endogenous PEDF in the corpus callosum upon lysolecithin-induced demyelination. Our results document previously unrecognized oligodendrotrophic effects of recombinant PEDF on the adult SVZ and corpus callosum, demonstrate induction of endogenous CNS PEDF production following demyelination, and make PEDF a strong candidate for pharmacological intervention in demyelinative diseases.

Periodontal bacteria aggravate experimental autoimmune myocarditis in mice.

Periodontitis is one of the most common infections in humans. Recently, published reports assert that periodontitis is associated with cardiovascular disease. Although it is said that viral, bacterial infections and autoimmune diseases may be the cause of myocarditis, the pathogenesis of it remains unclear. The aim of this study was to investigate the influence of a periodontal pathogen on experimental autoimmune myocarditis (EAM). Porphyromonas gingivalis (P.g.), PBS as a control, were injected into the mice. Histopathological and immunohistochemical analyses were performed. We examined heart mRNA levels using quantitative RT-PCR. The anti-P.g. IgG antibody level in plasma samples of the P.g.-injected group significantly increased compared with the PBS-injected group. Histopathological analysis detected that the myocarditis-affected areas and the fibrotic area in the P.g.-injected EAM group significantly increased compared with the PBS-injected EAM group (P < 0.05). Immunohistochemical analysis detected that more CD11b-positive cells were shown in the heart of the P.g.-injected EAM group compared with the PBS EAM-injected group (P < 0.05). Hearts from the P.g.-injected EAM group showed significantly increased expression of monocyte chemoattractant protein-1, IFN-γ, and matrix metalloproteinase-9 (MMP-9) mRNA compared with the hearts from the PBS-injected EAM group (P < 0.05). On day 7, serum levels of IL-6 were significantly enhanced in the P.g.-injected EAM group compared with the PBS-injected EAM group (P < 0.05). These results showed that P.g. injection could deteriorate EAM in mice through CD11b-positive cells, cytokines, and MMP-9 expression.

High incidence of Aggregatibacter actinomycetemcomitans infection in patients with cerebral infarction and diabetic renal failure: a cross-sectional study.

BACKGROUND: Recent epidemiological studies suggest that periodontitis is a major risk factor for renal failure and cerebral infarction. The aim of this study was to evaluate the association among periodontitis, renal failure, and cerebral infarction, focusing on microbiological and immunological features. METHODS: Twenty-one patients treated with hemodialysis (HD) were enrolled in this study. They were 8 with diabetic nephropathy and 13 with non-diabetic nephropathy. Blood examination, periodontal examination, brain magnetic resonance image (MRI), and dental radiography were performed on all patients. Subgingival plaque, saliva, and blood samples were analyzed for the periodontal pathogens, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Porphyromonas gingivalis (P. gingivalis), and Prevotella intermedia (P. intermedia) using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that the patients with diabetic nephropathy had more A. actinomycetemcomitans compared with non-diabetic nephropathy (P = 0.038) in dental plaque. Furthermore, the patients with diabetic nephropathy showed a significantly higher incidence of cerebral infarction compared with those with non-diabetic nephropathy (P = 0.029). Clinical oral and radiographic scores tended to be higher among patients in the diabetic nephropathy group than in the non-diabetic nephropathy group. CONCLUSIONS: Periodontal pathogens, particularly A. actinomycetemcomitans, may play a role, at least a part, in the development of cerebral infarction in Japanese HD patients with diabetic nephropathy.

ZPK/DLK, a mitogen-activated protein kinase kinase kinase, is a critical mediator of programmed cell death of motoneurons.

Activation of mitogen-activated protein kinase pathways is critically involved in naturally occurring programmed cell death of motoneurons during development, but the upstream mediators remain undetermined. We found that mice deficient in ZPK, also called DLK (ZPK/DLK), an upstream kinase in these pathways, have twice as many spinal motoneurons as do their wild-type littermates. Nuclear HB9/MNX1-positive motoneuron pools were generated similarly in the spinal cord of both ZPK/DLK-deficient and wild-type embryos. Thereafter, however, significantly less apoptotic motoneurons were found in ZPK/DLK-deficient embryos compared with wild-type embryos, resulting in retention of excess numbers of motoneurons after birth. Notably, these excess motoneurons remained viable without atrophic changes in the ZPK/DLK-deficient mice surviving into adulthood. Analysis of the diaphragm and the phrenic nerve revealed that clustering and innervation of neuromuscular junctions were indistinguishable between ZPK/DLK-deficient and wild-type mice, whereas the proximal portion of the phrenic nerve of ZPK/DLK-deficient mice contained significantly more axons than the distal portion. This result supports the hypothesis that some excess ZPK/DLK-deficient motoneurons survived without atrophy despite failure to establish axonal contact with their targets. This study provides compelling evidence for a critical role for ZPK/DLK in naturally occurring programmed cell death of motoneurons and suggests that ZPK/DLK could become a strategic therapeutic target in motor neuron diseases in which aberrant activation of the apoptogenic cascade is involved.

Role of central nervous system periostin in cerebral ischemia.

BACKGROUND AND PURPOSE: Although periostin, an extracellular matrix glycoprotein, plays pivotal roles in survival, migration, and regeneration in various cells, its expression and function in the brain are still unknown. Here, we investigated the expression and role of periostin in the ischemic brain. METHODS: Expression of full-length periostin (periostin 1 [Pn1]) and its splicing variant lacking exon 17 (periostin 2 [Pn2]) was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining in male C57BL6/J mice. The actions of periostin were examined in adult primary neuronal culture and in a transient middle cerebral artery occlusion (tMCAo) model. RESULTS: Expression of Pn2, but not of Pn1, mRNA was markedly changed after tMCAo. Pn2 mRNA was decreased in the ischemic core at 3 hours after ischemia. At 24 hours, Pn2 mRNA was significantly increased in both the peri-ischemic and ischemic regions. Periostin was mainly observed in neurons in normal brain. However, neuronal expression of periostin was decreased temporarily in the ischemic region, but increased in astrocytes and around endothelial cells at 24 hours after tMCAo. Of importance, intracerebroventricular injection of Pn2 resulted in a significant reduction in infarct volume at 24 hours after tMCAo associated with phosphorylation of Akt. Also, the Pn2-treated mice survived longer until 1 week after tMCAo. Pn2 significantly inhibited neuronal death under hypoxia and stimulated neurite outgrowth. CONCLUSIONS: Here, we demonstrated that periostin was expressed in the brain, and exogenous Pn2 exhibited neuroprotective effects and accelerated neurite outgrowth. Additional studies on periostin may provide new insights into the treatment of ischemic stroke.

Interferon regulatory factor 8/interferon consensus sequence binding protein is a critical transcription factor for the physiological phenotype of microglia.

BACKGROUND: Recent fate-mapping studies establish that microglia, the resident mononuclear phagocytes of the CNS, are distinct in origin from the bone marrow-derived myeloid lineage. Interferon regulatory factor 8 (IRF8, also known as interferon consensus sequence binding protein) plays essential roles in development and function of the bone marrow-derived myeloid lineage. However, little is known about its roles in microglia. METHODS: The CNS tissues of IRF8-deficient mice were immunohistochemically analyzed. Pure microglia isolated from wild-type and IRF8-deficient mice were studied in vitro by proliferation, immunocytochemical and phagocytosis assays. Microglial response in vivo was compared between wild-type and IRF8-deficient mice in the cuprizon-induced demyelination model. RESULTS: Our analysis of IRF8-deficient mice revealed that, in contrast to compromised development of IRF8-deficient bone marrow myeloid lineage cells, development and colonization of microglia are not obviously affected by loss of IRF8. However, IRF8-deficient microglia demonstrate several defective phenotypes. In vivo, IRF8-deficient microglia have fewer elaborated processes with reduced expression of IBA1/AIF1 compared with wild-type microglia, suggesting a defective phenotype. IRF8-deficient microglia are significantly less proliferative in mixed glial cultures than wild-type microglia. Unlike IRF8-deficient bone marrow myeloid progenitors, exogenous macrophage colony stimulating factor (colony stimulating factor 1) (M-CSF (CSF1)) restores their proliferation in mixed glial cultures. In addition, IRF8-deficient microglia exhibit an exaggerated growth response to exogenous granulocyte-macrophage colony stimulating factor (colony stimulating factor 2) (GM-CSF (CSF2)) in the presence of other glial cells. IRF8-deficient microglia also demonstrate altered cytokine expressions in response to interferon-gamma and lipopolysaccharide in vitro. Moreover, the maximum phagocytic capacity of IRF8-deficient microglia is reduced, although their engulfment of zymosan particles is not overtly impaired. Defective scavenging activity of IRF8-deficient microglia was further confirmed in vivo in the cuprizone-induced demyelination model in mice. CONCLUSIONS: This study is the first to demonstrate the essential contribution of IRF8-mediated transcription to a broad range of microglial phenotype. Microglia are distinct from the bone marrow myeloid lineage with respect to their dependence on IRF8-mediated transcription.

Prevention of vascular dementia via immunotherapeutic blockade of renin-angiotensin system in a rat model.

INTRODUCTION: As several clinical trials have revealed that angiotensin-converting enzyme inhibitors and angiotensin II (Ang II) receptor blockers may be efficient in treating vascular dementia (VaD), the long-acting blockade of the renin-angiotensin system (RAS) would be useful considering the poor adherence of antihypertensive drugs. Accordingly, we continuously blocked RAS via vaccination and examined the effectiveness of the VaD model in rats. METHODS: Male Wistar rats were exposed to two-vessel occlusions (2VO) after three injections of Ang II peptide vaccine. The effects of the vaccine were evaluated in the novel object recognition test, brain RAS components, and markers for oligodendrocytes. RESULTS: In the vaccinated rats, anti-Ang II antibody titer level was increased in serum until Day 168, but not in cerebral parenchyma. Vaccinated rats showed better object recognition memory with inhibited demyelination in the corpus callosum and activation of astrocytes and microglia. Also, levels of BrdU/GSTπ-positive cells and the phosphorylation of cAMP response element binding protein was increased in vaccinated rats, indicating that the differentiation of oligodendrocyte progenitor cells to mature oligodendrocytes was accelerated. Vaccinated rats showed increased expression of fibroblast growth factor-2 (FGF2), which was observed in endothelial cells. Angiotensinogen mRNA was decreased at 7 days after 2VO but increased at 14 and 28 days. CONCLUSION: Ang II vaccine might have promoted oligodendrocyte differentiation and inhibited astrocytic and microglial activation by stimulating FGF2 signaling in the endothelial cells-oligodendrocyte/astrocyte/microglia coupling. These data indicate the feasibility of Ang II vaccine for preventing progression of vascular dementia.

Maintenance of the relative proportion of oligodendrocytes to axons even in the absence of BAX and BAK.

Highly purified oligodendroglial lineage cells from mice lacking functional bax and bak genes were resistant to apoptosis after in-vitro differentiation, indicating an essential role of the intrinsic apoptotic pathway in apoptosis of oligodendrocytes in the absence of neurons (axons) and other glial cells. These mice therefore provide a valuable tool with which to evaluate the significance of the intrinsic apoptotic pathway in regulating the population sizes of oligodendrocytes and oligodendroglial progenitor cells. Quantitative analysis of the optic nerves and the dorsal columns of the spinal cord revealed that the absolute numbers of mature oligodendrocytes immunolabeled for aspartoacylase and adult glial progenitor cells expressing NG2 chondroitin sulfate proteoglycan were increased in both white matter tracts of adult bax/bak-deficient mice and, to a lesser extent, bax-deficient mice, except that there was no increase in NG2-positive progenitor cells in the dorsal columns of these strains of mutant mice. These increases in mature oligodendrocytes and progenitor cells in bax/bak-deficient mice were unexpectedly proportional to increases in numbers of axons in these white matter tracts, thus retaining the oligodendroglial lineage to axon ratios of at most 1.3-fold of the physiological numbers. This is in contrast to the prominent expansion in numbers of neural precursor cells in the subventricular zones of these adult mutant mice. Our study indicates that homeostatic control of cell number is different for progenitors of the oligodendroglial and neuronal lineages. Furthermore, regulatory mechanism(s) operating in addition to apoptotic elimination through the intrinsic pathway, appear to prevent the overproduction of highly mitotic oligodendroglial progenitor cells.

NF-kappaB is a key mediator of cerebral aneurysm formation.

BACKGROUND: Subarachnoid hemorrhage caused by the rupture of cerebral aneurysm (CA) remains a life-threatening disease despite recent diagnostic and therapeutic advancements. Recent studies strongly suggest the active participation of macrophage-mediated chronic inflammatory response in the pathogenesis of CA. We examined the role of nuclear factor-kappaB (NF-kappaB) in the pathogenesis of CA formation in this study. METHODS AND RESULTS: In experimentally induced CAs in rats, NF-kappaB was activated in cerebral arterial walls in the early stage of aneurysm formation with upregulated expression of downstream genes. NF-kappaB p50 subunit-deficient mice showed a decreased incidence of CA formation with less macrophage infiltration into the arterial wall. NF-kappaB decoy oligodeoxynucleotide also prevented CA formation when it was administered at the early stage of aneurysm formation in rats. Macrophage infiltration and expression of downstream genes were dramatically inhibited by NF-kappaB decoy oligodeoxynucleotide. In human CA walls, NF-kappaB also was activated, especially in the intima. CONCLUSIONS: Our data indicate that NF-kappaB plays a crucial role as a key regulator in the initiation of CA development by inducing some inflammatory genes related to macrophage recruitment and activation. NF-kappaB may represent a therapeutic target of a novel medical treatment for CA.

A model of cerebrovascular injury in rats.

Although the pathophysiology of post-angioplasty restenosis has been extensively studied in extracranial arteries using transluminal vascular injury model in rodents, it is still not well known in the intracranial arteries, which have quite different structures from extracranial arteries. Here, we examined whether 1-min placement of modified intraluminal suture could induce an injury in the internal carotid artery (ICA) in rats and observed temporal profile of histological change after the injury. HE staining showed that the injured intracranial ICA was dilated, while the media was markedly thinned at 1 day after injury. The internal elastic lamina was not observed, and the media contained few cells. At 1 week after injury, a thin layer of neointimal hyperplasia was observed on the luminal side of the internal elastic lamina. Neointimal hyperplasia developed until at least 4 weeks after injury. Morphometric analysis demonstrated that the healing process of the injury was related to arterial remodeling. Immunohistochemical staining for alpha-smooth muscle actin and electron microscopic analysis showed that the neointima was composed of smooth muscle cells. Re-endothelialization was observed from 1 to 4 weeks after injury by immunohistochemical staining for von Willebrand's factor and electron microscopic analysis. Vascular endothelial growth factor was expressed in neointima on days 7 and 14. Interestingly, superoxide anion was not increased in injured arteries on day 3, when the infiltration of macrophages was intensive, but increased on day 7, when infiltrating macrophages almost disappeared. These findings might shed new light on pathophysiology of post-angioplasty restenosis in intracranial arteries.

Therapeutic Effects of Systemic Administration of the Novel RANKL-Modified Peptide, MHP1, for Ischemic Stroke in Mice.

Microglial healing peptide 1, "MHP1", is a newly developed synthetic peptide composed of the DE and a part of the EF loop of the receptor activator of nuclear factor-кB (NFκB) ligand (RANKL). Our previous report demonstrated that MHP1 significantly inhibits Toll-like receptor (TLR) 2- and 4-induced inflammation in microglia/macrophages through RANK signaling without osteoclast activation. However, its inhibitory effects on ischemic stroke when administered intravenously have not been clarified. First, we examined whether MHP1 could penetrate the brain parenchyma. Intravenous injection of FITC-conjugated MHP1 demonstrated that MHP1 could cross the blood-brain-barrier in peri-infarct regions, but not in intact regions. Because MHP1 in the parenchyma was reduced at 60 minutes after injection, we speculated that continuous injection was necessary to achieve the therapeutic effects. To check the possible deactivation of MHP1 by continuous injection, the anti-inflammatory effects were checked in MG6 cells after incubation in 37°C for 24 hours. Although the inhibitory effects for IL6 and TNFα were reduced compared to nonincubated MHP1, its anti-inflammatory efficacy remained, indicating that continuous administration with pump was possible. The single and successive continuous administration of MHP1 starting from 4 or 6 hours after cerebral ischemia successfully reduced infarct volume and prevented the exacerbation of neurological deficits with reduced activation of microglia/macrophages and inflammatory cytokines. Different from recombinant RANKL, MHP1 did not activate osteoclasts in the paralytic arm. Although further modification of MHP1 is necessary for stabilization, the MHP1 could be a novel agent for the treatment ischemic stroke.

Delayed postischemic treatment with fluvastatin improved cognitive impairment after stroke in rats.

BACKGROUND AND PURPOSE: Recent clinical evidences indicate that statins may have beneficial effects on the functional recovery after ischemic stroke. However, the effect of delayed postischemic treatment with statins is still unclear. In the present study, we evaluated the effects of fluvastatin in the chronic stage of cerebral infarction in a rat model. METHODS: Rats exposed to permanent middle cerebral artery occlusion were treated for 3 months with fluvastatin beginning from 7 days after stroke. MRI, behavioral analysis, and immunohistochemistry were performed. RESULTS: Two months of treatment with fluvastatin showed the significant recovery in spatial learning without the decrease in serum total cholesterol level and worsening of infarction. Microangiography showed a significant increase in capillary density in the peri-infarct region in fluvastatin-treated rats after 3 months of treatment. Consistently, BrdU/CD31-positive cells were significantly increased in fluvastatin-treated rats after 7 days of treatment. MAP1B-positive neurites were also increased in the peri-infarct region in fluvastatin-treated rats. In addition, rats treated with fluvastatin showed the reduction of superoxide anion after 7 days of treatment and the reduction of A beta deposits in the thalamic nuclei after 3 months of treatment. CONCLUSIONS: Thus, delayed postischemic administration of fluvastatin had beneficial effects on the recovery of cognitive function without affecting the infarction size after ischemic stroke. Pleiotropic effects of fluvastatin, such as angiogenesis, neuritogenesis, and inhibition of superoxide production and A beta deposition, might be associated with a favorable outcome.

Expression of hepatocyte growth factor and c-Met after spinal cord injury in rats.

Since hepatocyte growth factor (HGF) plays a pivotal role in the development of the central nervous system and pathological conditions, we examined the long-term changes in the mRNA and protein expression of HGF and its receptor c-Met after spinal cord injury (SCI) in rats. HGF mRNA was significantly increased from 7 days after SCI in the injured segment, and the peak was at 7 days after SCI as assessed by real-time RT-PCR. Importantly, c-met mRNA expression was up-regulated from 1 day after SCI, and reached a peak at 14 days after SCI. Although up-regulation of HGF and c-met mRNA expression in the injured segment gradually decreased, the increased expression level persisted until 56 days after SCI. Consistent with HGF mRNA expression, HGF protein level was significantly increased mainly in the injured region, which persisted until 56 days after SCI. Immunohistochemistry showed that most of GFAP-positive reactive astrocytes expressed HGF and c-Met both on 14 days and 56 days after SCI. Staining with the mitotic indicator, bromodeoxyuridine (BrdU), revealed that a small number of BrdU-incorporated cells were co-localized with HGF/GFAP-positive or c-Met/GFAP-positive cells both on 14 and 56 days. These data suggest that HGF and c-Met were up-regulated mainly in the reactive astrocytes around the injured region in the subacute to chronic stage of spinal cord injury. Since HGF plays a critical role in neurotrophic activity, activation of the HGF/c-Met signaling system might be involved in the process of post-traumatic regeneration.