myelin-basic-protein and Ischemic-Attack--Transient

Oligodendrocytes play a crucial role in creating the myelin sheath that is an important component in neural transmission. In an animal model of transient cerebral ischemia, application of oligodendrocyte progenitor cells (OPCs) has not yet been reported. In this study, the effects of F3.Olig2 transplantation on memory and cognitive dysfunction were investigated in the aged gerbil in which ischemic stroke was induced. To investigate the possible mechanisms underlying repair, changes in the expression of myelin basic protein (MBP), oligodendrocyte-specific protein (OSP), and brain-derived neurotrophic factor (BDNF) were examined. Experimental ischemic stroke was induced by occlusion of bilateral common carotid arteries in aged gerbils. Gerbils (n=31 per group) were randomly divided into three groups: (1) vehicle sham group, (2) vehicle ischemia group, and (3) F3.Olig2 ischemia group. After 1, 3, and 7 days of ischemiareperfusion (I-R), saline or F3.Olig2 cells (1106 cells in 100 l) were injected into the gerbils intravenously. The gerbils were sacrificed 10 days after I-R for identification of grafted F3.Olig2 cells, and 15 and 30 days after I-R for tissue analysis after conducting passive avoidance and novel object recognition test. Injected F3.Olig2 cells and MBP, OSP, and BDNF were detected by specific antibodies using immunohistochemistry and/or Western blots. Memory and cognition were significantly increased in the F3.Olig2 ischemia group compared with the vehicle ischemia group. In the F3.Olig2 ischemia group, the neurons were not protected from ischemic damage; however, MBP, OSP, and BDNF expressions were significantly increased. Our results show that injection of F3.Olig2 cells significantly improved impaired memory and cognition, which might be related to increased MBP expression via increasing OSP and BDNF expression in the aged gerbil hippocampus following transient cerebral ischemia.

Topics: Animals; Brain-Derived Neurotrophic Factor; Calcium-Binding Proteins; Cell Line; Claudins; Gerbillinae; Glial Fibrillary Acidic Protein; Hippocampus; Humans; Immunohistochemistry; Ischemic Attack, Transient; Male; Myelin Basic Protein; Nerve Tissue Proteins; Neural Stem Cells; Reperfusion Injury

White matter injury induced by ischemic stroke elicits sensorimotor impairments, which can be further deteriorated by persistent proinflammatory responses. We previously reported that delayed and repeated treatments with omega-3 polyunsaturated fatty acids (n-3 PUFAs) improve spatial cognitive functions and hippocampal integrity after ischemic stroke. In the present study, we report a post-stroke n-3 PUFA therapeutic regimen that not only confers protection against neuronal loss in the gray matter but also promotes white matter integrity. Beginning 2 h after 60 min of middle cerebral artery occlusion (MCAO), mice were randomly assigned to receive intraperitoneal docosahexaenoic acid (DHA) injections (10 mg/kg, daily for 14 days), alone or in combination with dietary fish oil (FO) supplements starting 5 days after MCAO. Sensorimotor functions, gray and white matter injury, and microglial responses were examined up to 28 days after MCAO. Our results showed that DHA and FO combined treatment-facilitated long-term sensorimotor recovery and demonstrated greater beneficial effect than DHA injections alone. Mechanistically, n-3 PUFAs not only offered direct protection on white matter components, such as oligodendrocytes, but also potentiated microglial M2 polarization, which may be important for white matter repair. Notably, the improved white matter integrity and increased M2 microglia were strongly linked to the mitigation of sensorimotor deficits after stroke upon n-3 PUFA treatments. Together, our results suggest that post-stroke DHA injections in combination with FO dietary supplement benefit white matter restoration and microglial responses, thereby dictating long-term functional improvements.

Topics: Animals; Antigens, CD; Calcium-Binding Proteins; Cell Proliferation; Cerebrovascular Circulation; Corpus Callosum; Disease Models, Animal; Docosahexaenoic Acids; Eicosapentaenoic Acid; Ischemic Attack, Transient; Leukoencephalopathies; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Myelin Basic Protein; Nerve Tissue Proteins; Nervous System Diseases; Stroke; Time Factors

Transient global ischemia causes delayed white matter injury to the brain with oligodendrocyte (OLG) death and myelin breakdown. There is increasing evidence that hypoxia may be involved in several diseases of the white matter, including multiple sclerosis, vascular dementia, and ischemia. Matrix metalloproteinases (MMPs) are increased in rat and mouse models of hypoxic hypoperfusion and have been associated with OLG death. However, whether the MMPs act on myelin or OLGs remains unresolved. We hypothesized that delayed expression of MMPs caused OLG death and myelin breakdown. To test the hypothesis, adult mice underwent hypoxic hypoperfusion with transient bilateral occlusion of the carotid arteries. After 3 days of reperfusion, ischemic white matter had increased reactivity of astrocytes and microglia, MMP-2 localization in astrocytes, and increased protein expression and activity of MMP-2. In addition, there was a significant loss of myelin basic protein (MBP) by Western blot and caspase-3- mediated OLG death. Treatment with the broad-spectrum MMP inhibitor, BB-94, significantly decreased astrocyte reactivity and MMP-2 activity. More importantly, it reduced MBP breakdown. However, MMP inhibition had no effect on OLG loss. Our results implicate MMPs released by reactive astrocytes in delayed myelin degradation, while OLG death occurs by an MMP-independent mechanism. We propose that MMP-mediated myelin loss is important in hypoxic injury to the white matter.

Topics: Animals; Caspase 3; Cell Death; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Ischemic Attack, Transient; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin Sheath; Oligodendroglia; Phenylalanine; Thiophenes; Time Factors

After an ischemic stroke, there is a prolonged inflammatory response and secondary phase of injury that is more amenable to treatment than acute neurotoxicity. Surprisingly, little is known about temporal and spatial relationships between inflammation and white matter injury. Here, we quantified development of white matter damage, inflammation, and a glial limitans at 1, 3, and 7 days after transient ischemia in the rat striatum using immunohistochemistry. Quantitative analysis showed that decreased staining for myelin basic protein and increased staining for damaged myelin basic protein began in the core, coincided with neutrophil infiltration, and progressed outward over time. Axon damage (i.e. accumulation of amyloid precursor protein) began at the edge of the lesion, coinciding with substantial microglia/macrophage activation, and progressed into the core. During the 7 days, activated microglia/macrophages dramatically increased only in the core and edge of the lesion. Detailed spatial analyses revealed that activated microglia/macrophages that surrounded undamaged axon bundles did not express ED1, a marker of phagocytic cells, whereas those inside damaged bundles expressed ED1. These results imply different contributions of neutrophils and microglia/macrophages to white matter injury after ischemic stroke. The distinct localizations of activated microglia/macrophages imply complex signals that regulate their migration toward and infiltration of damaged white matter.

Topics: Amyloid beta-Protein Precursor; Animals; Astrocytes; Brain Infarction; Calcium-Binding Proteins; Corpus Striatum; Disease Models, Animal; Disease Progression; DNA-Binding Proteins; Ectodysplasins; Functional Laterality; Glial Fibrillary Acidic Protein; Inflammation; Ischemic Attack, Transient; Male; Microfilament Proteins; Myelin Basic Protein; Nerve Fibers, Myelinated; Neuroglia; Protease Nexins; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Tetrazolium Salts; Time Factors

The vulnerability of oligodendrocytes to ischemic injury may contribute to functional loss in diseases of central white matter. Immunocytochemical methods to identify oligodendrocyte injury in experimental models rely on epitope availability, and fail to discriminate structural changes in oligodendrocyte morphology. We previously described the use of a lentiviral vector (LV) carrying enhanced green fluorescent protein (eGFP) under the myelin basic protein (MBP) promoter for selective visualization of oligodendrocyte cell bodies and processes. In this study, we used LV-MBP-eGFP to label oligodendrocytes in rat cerebral white matter prior to transient focal cerebral ischemia, and examined oligodendrocyte injury 24 h, 48 h and 1 week post-reperfusion by quantifying cell survival and assaying the integrity of myelin processes. There was progressive loss of GFP+ oligodendrocytes in ischemic white matter at 24 and 48 h. Surviving GFP+ cells had non-pyknotic nuclear morphology and were terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-negative, but there was marked fragmentation of myelin processes as early as 24 h after stroke. One week after stroke, we observed a restoration of GFP+ oligodendrocytes in ischemic white matter, reflected both by cell counts and by structural integrity of myelin processes. Proliferating cells were not the main source of GFP+ oligodendrocytes, as revealed by bromodeoxyuridine (BrdU) incorporation. These observations identify novel transient structural changes in oligodendrocyte cell bodies and myelinating processes, which may have consequences for white matter function after stroke.

Topics: Animals; Cell Proliferation; Green Fluorescent Proteins; HIV; Humans; Ischemic Attack, Transient; Male; Myelin Basic Protein; Myelin Sheath; Neural Stem Cells; Oligodendroglia; Rats; Rats, Sprague-Dawley; Time Factors

Insulin-like growth factor (IGF-1) markedly increases myelination and glial numbers in white matter after ischemia in near-term fetal sheep; however, it is unclear whether this is due to reduced cell loss or increased secondary proliferation. Brain injury was induced in near-term fetal sheep by 30 minutes of bilateral carotid artery occlusion. Ninety minutes after the occlusion, fetuses were given, intracerebroventricularly, either a single dose of IGF-1 (either 3 or 30 micro g), or 3 micro g followed by 3 micro g over 24 hours (3 + 3 micro g). White matter was assessed 4 days after reperfusion. Three micrograms, but not 30 micro g of IGF-1 prevented loss of oligodendrocytes and myelin basic protein density (P < 0.001) compared to the vehicle-treated ischemia controls. No additional effect was observed in the 3 + 3 micro g group. IGF-1 treatment was associated with reduced caspase-3 activation and increased glial proliferation in a similar dose-dependent manner. Caspase-3 was only expressed in oligodendrocytes that showed apoptotic morphology. Proliferating cell nuclear antigen co-localized with both oligodendrocytes and astrocytes and microglia. Thus, increased oligodendrocyte numbers after IGF-1 treatment is partly due to suppression of apoptosis, and partly to increased proliferation. In contrast, the increase in reactive glia was related only to proliferation. Speculatively, reactive glia may partly mediate IGF-1 white matter protection.

Topics: Animals; Apoptosis; Astrocytes; Caspase 3; Caspases; Cell Division; Cytoplasm; Enzyme Activation; Female; Gene Expression; Gestational Age; Glial Fibrillary Acidic Protein; Glycoproteins; Immunohistochemistry; Injections, Intraventricular; Insulin-Like Growth Factor I; Ischemic Attack, Transient; Lectins; Myelin Basic Protein; Oligodendroglia; Pregnancy; Proliferating Cell Nuclear Antigen; Proteolipids; RNA, Messenger; Sheep

Deleterious processes of extracellular proteolysis may contribute to the progression of tissue damage after acute brain injury. We recently showed that matrix metalloproteinase-9 (MMP-9) knock-out mice were protected against ischemic and traumatic brain injury. In this study, we examined the mechanisms involved by focusing on relevant MMP-9 substrates in blood-brain barrier, matrix, and white matter. MMP-9 knock-out and wild-type mice were subjected to transient focal ischemia. MMP-9 levels increased after ischemia in wild-type brain, with expression primarily present in vascular endothelium. Western blots showed that the blood-brain barrier-associated protein and MMP-9 substrate zonae occludens-1 was degraded after ischemia, but this was reduced in knock-out mice. There were no detectable changes in another blood-brain barrier-associated protein, occludin. Correspondingly, blood-brain barrier disruption assessed via Evans Blue leakage was significantly attenuated in MMP-9 knock-out mice compared with wild types. In white matter, ischemic degradation of the MMP-9 substrate myelin basic protein was significantly reduced in knock-out mice compared with wild types, whereas there was no degradation of other myelin proteins that are not MMP substrates (proteolipid protein and DM20). There were no detectable changes in the ubiquitous structural protein actin or the extracellular matrix protein laminin. Finally, 24 hr lesion volumes were significantly reduced in knock-out mice compared with wild types. These data demonstrate that the protective effects of MMP-9 gene knock-out after transient focal ischemia may be mediated by reduced proteolytic degradation of critical blood-brain barrier and white matter components.

Topics: Actins; Animals; Blood-Brain Barrier; Blotting, Western; Brain; Cell Survival; Disease Models, Animal; Extracellular Matrix; Immunohistochemistry; Ischemic Attack, Transient; Laminin; Male; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Knockout; Myelin Basic Protein; Nerve Fibers, Myelinated; Peptide Hydrolases; Phosphoproteins; Zonula Occludens-1 Protein

Phosphorylation of cyclic adenosine monophosphate (AMP) response element binding protein (CREB) was examined immunohistochemically in the corpus callosum of the rat brain at various time points after 90-minute focal cerebral ischemia. Focal ischemia was induced by occlusion of the middle cerebral artery (MCA) using the intraluminal suture method. Sham animals showed that numerous oligodendrocytes (OLGs) constitutively express unphosphorylated CREB. Local cerebral blood flow (lCBF) measured by the 14C-iodoantipyrine method was reduced from 44.2 +/- 15.4 (mL 100 g(-1) min(-1)) to 18.4 +/- 3.8 and from 53.9 +/- 14.4 to 4.8 +/- 4.5 in the medial and the lateral regions of the corpus callosum, respectively, during MCA occlusion (MCAO). After release of the MCAO, lCBF recovered to the control level in each region. The medial region of the corpus callosum showed a marked increase in phosphorylated CREB-positive OLGs at 3.5 hours of recirculation, and it remained increased until 2 weeks of recirculation as it gradually declined. The activation of CREB phosphorylation in the OLGs was accompanied by expression of antiapoptotic protein bcl-2, normal staining with cresyl violet, and negative TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) staining. Myelination detected by immunostaining with anti-myelin basic protein (MBP) antibody and anti-myelin associated glycoprotein (MAG) antibody remained normal in the medial region of the corpus callosum. The lateral region of the corpus callosum showed a significant but only transient increase in phosphorylated CREB-positive OLGs at 3.5 hours of recirculation, which was followed by a rapid decrease during the subsequent recirculation period. Expression of bcl-2 was suppressed in this region, and demyelination became apparent. These findings suggest that signal transduction through CREB phosphorylation may be closely associated with survival of OLGs and maintenance of myelination in the corpus callosum after cerebral ischemia.

Topics: Adenomatous Polyposis Coli Protein; Animals; Antipyrine; Autoradiography; Carbon Radioisotopes; Cerebrovascular Circulation; Corpus Callosum; Cyclic AMP Response Element-Binding Protein; Cytoskeletal Proteins; Functional Laterality; Ischemic Attack, Transient; Myelin Basic Protein; Oligodendroglia; Phosphorylation; Rats

In order to achieve a better understanding of the pathophysiology of ischemic white matter lesions, oligodendrocytic degeneration and subsequent proliferation were examined in the mouse model of middle cerebral artery occlusion. In situ hybridization histochemistry for proteolipid protein messenger RNA was employed as a sensitive and specific marker of oligodendrocytes, and immunohistochemistry for myelin basic protein was used as a compact myelin marker. Immunohistochemistry for microtubule-associated protein 2 and albumin was employed to monitor neuronal degeneration and the breakdown of the blood brain barrier, respectively. In the ischemic core of the caudoputamen, the immunoreactivity for microtubule-associated protein 2 disappeared and massive albumin extravasation occurred several hours after vessel occlusion, while proteolipid protein messenger RNA signals remained relatively strong at this time. The messenger RNA signals began to attenuate 12 h after ischemia and were hardly detectable 24 h after ischemia in the whole ischemic lesion. In situ end-labeling of fragmented DNA showed some cells with proteolipid protein messenger RNAs to have DNA fragmentation at this period. In contrast to proteolipid protein messenger RNA signals, the immunoreactivity for myelin basic protein was detected as long as five days after ischemia. An apparent increase in the cells possessing strong proteolipid protein messenger RNA signals was found five days after ischemia, mainly in the corpus callosum and the cortex bordering the infarcted areas. A double simultaneous procedure with in situ hybridization for proteolipid protein messenger RNA and immunohistochemistry for glial fibrillary acid protein or lectin histochemistry for macrophages/microglia showed proliferating oligodendrocytes to be co-localized with reactive astrocytes and macrophages/microglia. These findings show that oligodendrocytic damage occurred following ischemic neuronal damage and the breakdown of the blood brain barrier, but preceded the breakdown of myelin proteins in the ischemic lesion, that an apoptosis-like process was involved in ischemic oligodendrocytic death, and that surviving oligodendrocytes responded and proliferated in the outer border of the infarcted area.

Topics: Animals; Astrocytes; Blood-Brain Barrier; Brain; Cell Division; Glial Fibrillary Acidic Protein; Immunoenzyme Techniques; Immunohistochemistry; In Situ Hybridization; Ischemic Attack, Transient; Macrophages; Male; Mice; Mice, Inbred C57BL; Microglia; Microtubule-Associated Proteins; Myelin Basic Protein; Myelin Proteolipid Protein; Nerve Degeneration; Nerve Tissue Proteins; Oligodendroglia; Reference Values; Reperfusion; RNA, Messenger; Serum Albumin

No marker that reflects and predicts brain injury due to subarachnoid hemorrhage (SAH) and cerebral vasospasm has been reported. We hypothesized that membrane-bound tissue factor (mTF) and thrombin-antithrombin III complex (TAT) in the cerebrospinal fluid (CSF) of patients with SAH become markers indicating brain injury. To evaluate the hypothesis, we correlated levels of mTF and TAT in the CSF of patients with SAH with clinical severity, the degree of SAH, and outcome.. We assayed CSF mTF, TAT and myelin basic protein (MBP) in patients with SAH at intervals that included days 0 to 4 and days 5 to 9 after ictus. Classification of clinical severity of disease on admission was based on Hunt and Hess grade, degree of SAH on CT on Fisher's grading, and outcome 3 months after SAH on the Glasgow Outcome Scale.. In the interval from days 0 to 4, mTF and TAT correlated with Hunt and Hess and Fisher grades, and occurrence of cerebral infarction due to vasospasm. Only mTF correlated significantly in this period with outcome. TAT, mTF, and MBP all correlated significantly with each other. From days 5 to 9, only mTF correlated with cerebral infarction, infarction volume, MBP levels, and outcome.. Both mTF and TAT reflected brain injury from SAH and predicted vasospasm, though mTF was more sensitive and a better predictor of outcome. Unlike mTF, TAT did not correlate with vasospasm during the interval when it most commonly occurs, which raised doubt about thrombin activation as a cause.

Topics: Adult; Aged; Aged, 80 and over; Antithrombin III; Biomarkers; Cerebral Infarction; Female; Hospitalization; Humans; Ischemic Attack, Transient; Male; Middle Aged; Myelin Basic Protein; Osmolar Concentration; Peptide Hydrolases; Severity of Illness Index; Subarachnoid Hemorrhage; Thromboplastin; Tomography, X-Ray Computed; Treatment Outcome

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor beta1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor beta1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

Topics: Animals; Cerebrovascular Disorders; Corticosterone; Hypersensitivity, Delayed; Immune Tolerance; Immunohistochemistry; Ischemic Attack, Transient; Male; Myelin Basic Protein; Rats; Rats, Inbred Lew; Transforming Growth Factor beta

We have extended the results from our previous investigation of the reactivity of leucocytes from patients with multiple sclerosis and some other neurological diseases to bovine encephalitogenic protein, using the migration in agarose technique. 1. Reactivity (i.e. behaviour in the test deviating from that expected with cells from healthy subjects) is manifested either as an enhancement or an inhibition of leucocyte migration. 2. The incidence of subjects with cells showing reactivity to encephalitogenic protein is higher in the group of patients with multiple sclerosis than in the group of patients with other neurological diseases. 3. Reactivity to encephalitogenic protein is correlated to disease course score; patients with a chronic progression of the disease show the strongest reactivity in the test. 4. The tryptophan-containing region of the encephalitogenic protein appears to be a major determinant of significance for this test. 5. Patients tested during corticotrophin treatment tend to show a higher degree of reactivity in the test than expected-the increased reactivity seems to be primarily directed to the tryptophan-containing region.

Topics: Adrenocorticotropic Hormone; Amyotrophic Lateral Sclerosis; Cell Migration Inhibition; Humans; Ischemic Attack, Transient; Leukocytes; Multiple Sclerosis; Myelin Basic Protein