Gfap, S-100 and vimentin proteins in rat after cerebral post-ischemic reperfusion

In the present study astrocytes reactivity during cerebral post-ischemic reperfusion was evaluated immunocytochemically by using antibodies to vimentin, glial fibrillary acidic protein (GFAP) and S-100 protein. At the 7th day of post-ischemic reperfusion few GFAP-positive cells were observed in the hippocampus and cerebellum, the number of GFAP-positive cells increased slightly after 20 days of reperfusion. This poor GFAP-positivity may be due to the inhibition of GFAP polymerization by S-100; in fact, S-100 immuno-reactivity was already evident from the 7th day. Vimentin immuno-staining was evident both at the 7th and 20th day of reperfusion in microglial cells and in oligodendrocytes, suggesting that these cells are involved in the recovery of neurons following brain injury.     

Pre- and post-ischemic administration of dizocilpine (MK-801) reduces cerebral necrosis in the rat

The purpose of this study was to determine the effectiveness of the non-competitive N-methyl-D-aspartate receptor antagonist dizocilpine, or (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801) in mitigating ischemic neuronal necrosis in the rat. Ten minutes of transient forebrain ischemia was induced by a combination of bilateral carotid clamping and hypotension to 50 mm Hg. Control animals received intravenous saline, whereas treated animals received dizocilpine, either 1 mg/kg iv 20 min. pre ischemia, 1 mg/kg iv 20 min. post ischemia, 10 mg/kg iv 20 min. post ischemia, 10 mg/kg ip 2 hrs. post ischemia, 10 mg/kg ip 24 hrs. post ischemia. The groups receiving dizocilpine before or up to 20 min. after ischemia all showed a significant reduction in the number of dead neurons as assessed by quantitative histopathology in hippocampus, caudate nucleus and cerebral cortex after one week of recovery. However, dizocilpine administered either 2 or 24 hrs. after ischemia afforded no protection. These results suggest that the potent non-competitive NMDA antagonist dizocilpine may have some value in protecting the brain from hippocampal and cortical neuronal necrosis after a short insult consisting of dense transient cerebral ischemia. Noteworthy is the fact that pharmacologic intervention in the post-ischemic period was successful in preventing neuronal death, provided that drug administration occurred within dizocilpine's "therapeutic window".     

Comparison of the immunological properties of rat cerebral and aortic endothelium

The immunological properties of cerebral microvascular endothelium were directly compared with those of an extra-cerebral endothelium in vitro. Lymphocyte adhesion to cerebral endothelium is normally low, but is sensitive to induction by interferon-gamma (IFN gamma) and tumour necrosis factor-alpha (TNF alpha). Conversely adhesion to aortic endothelium is normally much higher but it is only marginally sensitive to induction by cytokines. Adhesion to both cell types is Ca2+ and Mg2+ dependent. Mitogen-activated lymphocytes bind more strongly to both endothelia, but adhesion to aortic endothelium is not enhanced further by activation of the endothelium. The observed low binding of lymphocytes to brain endothelium and its rapid induction by cytokines suggest a mechanism to explain why lymphocyte accumulation in brain is normally very low but rapidly increases during immune responses. Both cell types express similar levels of class I major histocompatibility complex (MHC) molecules, and this is enhanced by IFN gamma with similar responsiveness to different levels of IFN gamma. MHC class II molecules are absent from these cells but may be induced: although both endothelia respond to similar levels of cytokines, the surface density induced on brain endothelium is approximately 2- to 3-fold higher at all levels of IFN gamma.     

Effects of norepinephrine depletion in rats during cerebral post-ischemic reperfusion

The present paper reports the effects of norepinephrine depletion in rats, after treatment with N-(2-chloroethyl)-N-ethyl 2-bromobenzylamine (DSP-4) neurotoxin, on partial cerebral ischemia and reperfusion. Histological observations made under experimental conditions of noradrenergic (NA)-depletion demonstrated that neuronal lesions were not exacerbated; in fact, in DSP-4-treated ischemic animals, a minor number of neurons appeared damaged. Our results suggest that neuronal recovery after post-ischemic reperfusion is not affected by NA-depletion. DSP-4 neurotoxin does not induce 5-hydroxy-triptamine (5-HT) depletion.     

Role of cerebral endothelium in brain oedema

Abstract

The view that cerebral endothelial cells represent the cellular basis of the blood-brain barrier has been generally accepted. The regulation of transport processes operating in the cerebral endothelial cells is currently of great interest. Our knowledge of these regulatory mechanisms is briefly reviewed here with special regard to the molecular processes involved in the formation of brain oedema and emphasis on new therapeutic means for its prevention.     

Role of cerebral endothelium in brain oedema

The view that cerebral endothelial cells represent the cellular basis of the blood-brain barrier has been generally accepted. The regulation of transport processes operating in the cerebral endothelial cells is currently of great interest. Our knowledge of these regulatory mechanisms is briefly reviewed here with special regard to the molecular processes involved in the formation of brain oedema and emphasis on new therapeutic means for its prevention.     

Therapeutic effect of post-ischemic hypothermia duration on cerebral ischemic injury

OBJECTIVE: To study the efficacy of post-ischemic mild brain hypothermia lasting for different time intervals on cerebral ischemic reperfusion injury. METHOD: Male Sprague-Dawley rats were divided into a sham-operated group, normothermia (37-38 degrees C) ischemia group and mild hypothermia (31-32 degrees C) group. The last group was subdivided into four groups: 30 minute hypothermia plus 210 minute normothermia, 60 minute hypothermia plus 180 minute nomothermia,120 minute hypothermia plus 120 minute normothermia, and 240 minute hypothermia (n=8). Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model. Brain tissue was collected following a 20 minute cerebral ischemia and 240 minute reperfusion, and was used to measure the levels of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA), dopamine (DA), norepinephrine (NE), serotonin(5-HT) and hydroxyindoleacetic acid (5-HIAA), nitrite (NO(2)), endothelin-1 (ET(1)), tumor necrosis factor alpha(TNFalpha) and interleukin-1beta (IL-1beta). Serum was collected to measure the levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK) and its brain band isoenzyme (CK-BB). RESULTS: Hypothermia lasting for 60-240 minutes delayed the decrease in these amino acids, postponed the decrease in DA, NE and 5-HT and increase in hydroxyindoleacetic acid (5-HIAA), and decreased the levels of IL-1beta, TNFalpha, ET(1) and NO(2) in brain tissue. Hypothermia also decreased the levels of LDH, AST, CK and CK-BB in serum as compared to normothermia group (p<0.05 or p<0.01). Hypothermia lasting for 30 minutes delayed the decreases in these amino acids and 5-HT and increase in 5-HIAA in brain tissue (p<0.05), but failed to influence the levels of IL-1beta, TNFalpha, ET(1) and NO(2) in brain tissue and the amounts of LDH, AST, CK and CK-BB in serum as compared to normothermia ischemia group (p>0.05). CONCLUSIONS: Post-ischemic mild brain hypothermia can significantly suppress the excessive release of amino acids, monoamine neurotransmitters and inflammation response in ischemic tissue. It can also stabilize the function of the cell membrane, which is associated with the mechanism of cerebral protection by mild hypothermia. These results suggest that mild hypothermia should be applied immediately after ischemia and last for more than 60 minutes in order to obtain neuroprotective effects.     

Growth factor- and cytokine-stimulated endothelial progenitor cells in post-ischemic cerebral neovascularization

Endothelial progenitor cells are resident in the bone marrow blood sinusoids and circulate in the peripheral circulation. They mobilize from the bone marrow after vascular injury and home to the site of injury where they differentiate into endothelial cells. Activation and mobilization of endothelial progenitor cells from the bone marrow is induced via the production and release of endothelial progenitor cell-activating factors and includes specific growth factors and cytokines in response to peripheral tissue hypoxia such as after acute ischemic stroke or trauma. Endotheli- al progenitor cells migrate and home to specific sites following ischemic stroke via growth factor/ cytokine gradients. Some growth factors are less stable under acidic conditions of tissue isch- emia, and synthetic analogues that are stable at low pH may provide a more effective therapeutic approach for inducing endothelial progenitor cell mobilization and promoting cerebral neovascularization following ischemic stroke.     

Nitrative stress in cerebral endothelium is mediated by mGluR5 in hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) disrupts nitric oxide (NO) signaling and increases nitrative stress in cerebral microvascular endothelial cells (CMVECs). This is mediated, in part, by protein nitrotyrosinylation (3-nitrotyrosine; 3-NT) though the mechanisms by which extracellular homocysteine (Hcy) generates intracellular 3-NT are unknown. Using a murine model of mild HHcy (cbs(+/-) mouse), we show that 3-NT is significantly elevated in cerebral microvessels with concomitant reductions in serum NO bioavailability as compared with wild-type littermate controls (cbs(+/+)). Directed pharmacology identified a receptor-dependent mechanism for 3-NT formation in CMVECs. Homocysteine increased expression of inducible NO synthase (iNOS) and formation of 3-NT, both of which were blocked by inhibition of metabotropic glutamate receptor-5 (mGluR5) with the specific antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride. Activation of mGluR5 is both sufficient and necessary to drive the nitrative stress because direct activation using the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine also increased iNOS expression and 3-NT formation while knockdown of mGluR5 receptor expression by short hairpin RNA (shRNA) blocked their increase in response to Hcy. Nitric oxide derived from iNOS was required for Hcy-mediated formation of 3-NT because the effect was blocked by 1400W. These results provide the first evidence for a receptor-dependent process that explains how plasma Hcy levels control intracellular nitrative stress in cerebral microvascular endothelium.     

Lipid peroxidation in rat cerebral cortex during post-ischemic reperfusion: effect of exogenous antioxidants and Ca(++)-antagonist drugs.

Although the role of oxidant-antioxidant metabolism in total ischemia and reperfusion in the central nervous system and cardiac myocardium have been well studied, less is known about the consequences of partial ischemic episodes. Here we show that reperfusion contributes to free radical formation as judged by conjugated diene formation. Also, antioxidants and Ca++ antagonists were able to reduce free radical formation. These results would suggest that free radical generation following ischemia and reperfusion may result from more than one injury process in cerebral cortex.     

Osmotic opening of tight junctions in cerebral endothelium

Hyperosmotic solutions of 3 M urea, either infused into one internal carotid artery or applied topically to the pia mater of rabbits, results in the opening of endothelial tight junctions through which horseradish peroxidase passes from blood to extracellular fluid of the brain. The evidence for this opening of the blood-brain barrier to protein is the entry of peroxidase into the extracellular pools between successive tight junctions. In animals not receiving 3 M urea, the interjunctional pools are inaccessible to proteins. Having passed through the endothelial junctions, the peroxidase spreads along the extracellular channels of the perivascular neuropil for approximately 100 μ in 90 seconds. Most of the affected vessels are capillaries, though larger vessels are rendered leaky as well. Calyciform cisterns, that lie beneath shallow notches in the endothelium of untreated rabbits, appear to be enlarged after the administration of 3 m urea. It is undetermined whether these few endothelial cisterns and vesicles are involved in carrying protein from blood to the cerebral extracellular fluid.     

Mechanical reperfusion is associated with post-ischemic hemorrhage in rat brain

A major complication of recanalization therapy after an acute arterial occlusion in brain is hemorrhagic transformation (HT). Although it is known that prolonged ischemia is important in the development of HT, the role of reperfusion in ischemia–reperfusion induced HT is less well studied. To address the effect of reperfusion on HT, we assessed the incidence and severity of hemorrhage in rats after 5 h of middle cerebral artery occlusion (MCAO) followed by 19-hour reperfusion compared to rats with permanent occlusion (PMCAO) at the same 24-hour time point. The incidence and amount of hemorrhage, neurological function, and mortality rates were measured. MCAO (5 h) with 19-hour reperfusion was associated with a significantly higher incidence of cortical hemorrhage compared to PMCAO (81.8% vs 18.2%, p < 0.05). Hemorrhage scores were higher in the 5-hour MCAO/reperfusion group compared to PMCAO rats (17.6 ± 11.5 vs 2.4 ± 5.3 in cortex, 20.4 ± 4.6 vs 9.7 ± 4.5 in striatum, p < 0.01). Neurological function was worse in the ischemia–reperfusion group compared to PMCAO (p < 0.05) and mortality rates were insignificantly higher in the 5-hour MCAO/reperfusion group vs PMCAO group (54.5% vs18.1%; p < 0.08). The results suggest that reperfusion after prolonged ischemia is associated with increased hemorrhagic transformation and neurological deterioration as compared to permanent ischemia. Whether pharmacological treatments prior to reperfusion attenuate post-ischemic HT requires further study.     

Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium

One method of iron transport across the blood brain barrier (BBB) involves the transferrin receptor (TR), which is localized to the specialized brain capillary endothelium [7]. The melanotransferrin (MTf) molecule, also called p97, has been widely described as a melanoma specific molecule, however, its expression in brain tissues has not been addressed. MTf has a high level of sequence homology to transferrin (Tf) and lactoferrin, but is unusual because it predominantly occurs as a membrane bound, glycosylphosphatidylinositol (GPI) anchored molecule, but can also occur as a soluble form. We have recently demonstrated that GPI-anchored MTf provides a novel route for cellular iron uptake which is independent of Tf and its receptor [10]. Here we consider whether MTf may have a role in the transport of iron across the BBB. The distributions of MTf, Tf and the TR were studied immunohistochemically in human brain tissues. The distributions of MTf and TR were remarkably similar, and quite different from that of Tf. In all brain tissues examined, MTf and the TR were highly localized to capillary endothelium, while Tf itself was mainly localized to glial cells. These data suggest that MTf may play a role in iron transport within the human brain.     

Nitric oxide synthase inhibitor augments post-ischemic leukocyte adhesion in the cerebral microcirculation in vivo

Abstract

The objective was to examine the effect of the nitric oxide synthase inhibitor, N^-nitro-L-arginine methyl ester (L-NAME) on leukocyte adhesion in the cerebral microcirculation during reperfusion following partial forebrain ischemia in the rat. intravital fluorescence video-microscopy through a closed cranial window was used to visualize leukocyte-endothelium interaction in small pial veins of 15-100 /im diameter. Forebrain ischemia was produced by the ligation of both common carotid arteries plus elevation of the intracranial pressure to 20 mmHg for 60 min. The number of leukocytes adhering to the endothelium for longer than 3 sec was determined during ischemia (5 min and 60 min) and during reperfusion (5 min and 60 min). Two experimental groups were treated with either L-NAME or its inactive enantiomer D-NAME (20 mg kg~1 i.v.) 30 min prior to reperfusion. In a third group, also treated with D-NAME, post-ischemic hyperemia was prevented by lowering the ICP without removing the occlusion of common carotid arteries (partial reperfusion). The velocity of flow adjacent to the endothelial surface of pial veins was measured by tracking the movement of fluorescently labeled red blood cells as flow markers before and after ischemia. During ischemia, the number of adhering leukocytes increased approximately two-fold at 5 min, and threefold at 60 min. In the D-NAME-treated group with complete reperfusion, leukocyte adhesion returned to the baseline level by 60 min of reperfusion. However, in the L-NAME-treated group, leukocyte adhesion remained elevated at 60 min of reperfusion. Post-ischemic flow velocity was significantly decreased (-66%) from control after L-NAME treatment whereas it was increased (+53%) in the D-NAME-treated group. In the partial reperfusion group, leukocyte adhesion continued to increase after the first hour of ischemia and reached a level 2.7-fold over baseline at 60 min reperfusion. Flow velocity remained below control (-26%) at 60 min reperfusion. Leukocyte adhesion was absent in pial arteries and no plugging by leukocytes was observed in cortical capillaries. The results suggest that leukocyte adhesion in small pial veins increases during I h forebrain ischemia and continues to increase during reperfusion if the velocity of flow or shear rate is low. The increase in leukocyte adhesion is reversible if flow velocity is elevated during reperfusion. L-NAME prevents post-ischemic hyperemia and augments leukocyte adhesion principally via a ,decrease in velocity or shear rate. [Neurol Res 1999; 21: 378-384]     

Ultrastructural localization of lectin receptors on cerebral endothelium

Summary Oligosaccharide residues on the endothelial luminal plasma membrane of rat cerebral cortical vessels were localized using biotinylated lectins. In addition, the effect of pretreatment of brain slices with neuraminidase prior to the binding of cationized ferritin (CF) and certain lectins was studied.Conjugates of biotinylated lectins and avidin-d horseradish peroxidase reaction product were evenly distributed on the endothelium of arterioles, capillaries, and venules. Lectin binding sites were observed on the plasma membrane of pinocytotic vesicles open onto the vascular lumen and at the luminal end of the interendothelial space only. The following sugar residues were localized: -d-mannosyl, -d-glucosyl, -N-acetylglucosaminyl, sialyl,d-galactosyl, -l-fucosyl, and -N-acetyl-d-galactosaminyl.Following pretreatment of brain slices with neuraminidase -d-gal-(1–3)-d-galN-acetyl groups were demonstrated on endothelium. In this respect, cerebral endothelium differs from noncerebral endothelium which is reported to have peanut agglutinin binding sites without neuraminidase pretreatment.Anionic groups on cerebral endothelium were demonstrated at the same locations as the lectin binding sites. Following neuraminidase pretreatment there was reduction, but not absence, of CF binding supporting the observation that surface charge is not wholly due to sialyl groups.The role of monosaccharide residues in states of altered cerebrovascular permeability remains to be determined.Supported by Ontario Heart Foundation grant no. 2-6     

Developmental changes in transferrin and iron uptake by the brain in the rat

The uptake of transferrin and iron by the brain, liver and femurs was investigated in rats using 125I-59Fe-transferrin (Tf), and 131I-albumin in order to measure the plasma content of the organs. Measurements in rats ranging in age from birth to 70 days revealed that the rate of iron uptake by the brain increased rapidly over the first 15 days of life, peaking at 15 days and thereafter declining. A similar pattern occurred in the uptake of 125I-Tf. These changes were accompanied by rapid growth of the brain up to 15 days and a decrease in the concentration of non-haem iron. The turnover of 59Fe and 125I-Tf in the brain was also determined by measuring radioactivity in the brain of 15-day rats at various times after injection from 15 min to 13 days. The amount of 59Fe in the brain increased over the first 4 h and thereafter remained constant. By contrast, the 125I-Tf values increased rapidly during the first 15 min to reach a relatively constant level which was maintained for at least 6 h after which it declined. The patterns of uptake by the brain were different from those found in the liver and femurs, indicating that the changes in the brain were specific for that organ.(ABSTRACT TRUNCATED AT 250 WORDS)