EPR Study of the Intensity of the Nitric Oxide Production in Rat Brain After Ischemic Stroke

Electron paramagnetic resonance of (DETC)₂–Fe²⁺–NO complexes has been used as a method to detect the formation of nitric oxide (NO) in the brain tissues of Wistar rats. The content of nitric oxide in the center of ischemia (left brain hemisphere), in the non-ischemic part of the left hemisphere, and also in the regions conditionally not affected by ischemia, the cerebral cortex of the right hemisphere and the cerebellum of rats was studied in 5, 9, 24 and 72 h after modeling an ischemic stroke. It is found that in the ischemic part of the left hemisphere in 5 h after modeling the ischemic stroke, the NO content in the spin trap and R-conformer compositions decreases by 5 times and 30%, respectively. This decrease in the NO content is found in 9 and 24 h after the stroke. In the cerebral cortex of the right hemisphere and non-ischemic parts of the cerebral cortex of the left hemisphere, the NO content in the composition of the spin trap in 5, 9 and 24 h after the stroke decreases by 40–50%. In the composition of the R-conformer it does not vary. In 72 h, the partial restoration of the NO content in all regions except for the ischemia zone is observed.     

Effect of neutrophil depletion on gelatinase expression,edema formation and hemorrhagic transformation after focal ischemic stroke

Background  

While gelatinase (MMP-2 and -9) activity is increased after focal ischemia/reperfusion injury in the brain, the relative contribution of neutrophils to the MMP activity and to the development of hemorrhagic transformation remains unknown.     

Enhanced cerebrovascular expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 via the MEK/ERK pathway during cerebral ischemia in the rat

Background  

Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1).     

Real-time monitoring of ischemic and contralateral brain pO2 during stroke by variable length multisite resonators

Purpose

Electron paramagnetic resonance (EPR) oximetry using variable length multi-probe implantable resonator (IR), was used to investigate the temporal changes in the ischemic and contralateral brain pO₂ during stroke in rats.

Material and methods

The EPR signal to noise ratio (S/N) of the IR with four sensor loops at a depth of up to 11 mm were compared with direct implantation of lithium phthalocyanine (LiPc, oximetry probe) deposits in vitro. These IRs were used to follow the temporal changes in pO₂ at two sites in each hemisphere during ischemia induced by left middle cerebral artery occlusion (MCAO) in rats breathing 30% O₂ or 100% O₂.

Results

The S/N ratios of the IRs were significantly greater than the LiPc deposits. A similar pO₂ at two sites in each hemisphere prior to the onset of ischemia was observed in rats breathing 30% O₂. However, a significant decline in the pO₂ of the left cortex and striatum occurred during ischemia, but no change in the pO₂ of the contralateral brain was observed. A significant increase in the pO₂ of only the contralateral non-ischemic brain was observed in the rats breathing 100% O₂. No significant difference in the infarct volume was evident between the animals breathing 30% O₂ or 100% O₂ during ischemia.

Conclusions

EPR oximetry with IRs can repeatedly assess temporal changes in the brain pO₂ at four sites simultaneously during stroke. This oximetry approach can be used to test and develop interventions to rescue ischemic tissue by modulating cerebral pO₂ during stroke.     

Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

Background

Minocycline, a second-generation tetracycline with anti-inflammatory and anti-apoptotic properties, has been shown to promote therapeutic benefits in experimental stroke. However, equally compelling evidence demonstrates that the drug exerts variable and even detrimental effects in many neurological disease models. Assessment of the mechanism underlying minocycline neuroprotection should clarify the drug's clinical value in acute stroke setting.

Results

Here, we demonstrate that minocycline attenuates both in vitro (oxygen glucose deprivation) and in vivo (middle cerebral artery occlusion) experimentally induced ischemic deficits by direct inhibition of apoptotic-like neuronal cell death involving the anti-apoptotic Bcl-2/cytochrome c pathway. Such anti-apoptotic effect of minocycline is seen in neurons, but not apparent in astrocytes. Our data further indicate that the neuroprotection is dose-dependent, in that only low dose minocycline inhibits neuronal cell death cascades at the acute stroke phase, whereas the high dose exacerbates the ischemic injury.

Conclusion

The present study advises our community to proceed with caution to use the minimally invasive intravenous delivery of low dose minocycline in order to afford neuroprotection that is safe for stroke.     

Enhanced expressions of microvascular smooth muscle receptors after focal cerebral ischemia occur via the MAPK MEK/ERK pathway

Background  

MEK1/2 is a serine/threonine protein that phosphorylates extracellular signal-regulated kinase (ERK1/2). Cerebral ischemia results in enhanced expression of cerebrovascular contractile receptors in the middle cerebral artery (MCA) leading to the ischemic region. Here we explored the role of the MEK/ERK pathway in receptor expression following ischemic brain injury using the specific MEK1 inhibitor U0126.     

Delayed neuronal cell death in brainstem after transient brainstem ischemia in gerbils

Background  

Because of the lack of reproducible brainstem ischemia models in rodents, the temporal profile of ischemic lesions in the brainstem after transient brainstem ischemia has not been evaluated intensively. Previously, we produced a reproducible brainstem ischemia model of Mongolian gerbils. Here, we showed the temporal profile of ischemic lesions after transient brainstem ischemia.     

Non-receptor tyrosine kinase Src is required for ischemia-stimulated neuronal cell proliferation via Raf/ERK/CREB activation in the dentate gyrus

Background  

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. However, Molecular mechanisms that regulate neuronal cell proliferation in the dentate gyrus (DG) following ischemic stroke insult are poorly understood. This study was designed to investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated cell proliferation in the adult DG and its underlying mechanism.     

Matrix metalloproteinase-7 facilitates immune access to the CNS in experimental autoimmune encephalomyelitis

Background  

Metalloproteinase inhibitors can protect mice against experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Matrix metalloproteinase-9 (MMP-9) has been implicated, but it is not clear if other MMPs are also involved, including matrilysin/MMP-7 – an enzyme capable of cleaving proteins that are essential for blood brain barrier integrity and immune suppression.     

The neuroblast and angioblast chemotaxic factor SDF-1 (CXCL12) expression is briefly up regulated by reactive astrocytes in brain following neonatal hypoxic-ischemic injury

Background  

Stromal cell-derived factor 1 (SDF-1 or CXCL12) is chemotaxic for CXCR4 expressing bone marrow-derived cells. It functions in brain embryonic development and in response to ischemic injury in helping guide neuroblast migration and vasculogenesis. In experimental adult stroke models SDF-1 is expressed perivascularly in the injured region up to 30 days after the injury, suggesting it could be a therapeutic target for tissue repair strategies. We hypothesized that SDF-1 would be expressed in similar temporal and spatial patterns following hypoxic-ischemic (HI) injury in neonatal brain.     

Different methods for administering 17β-estradiol to ovariectomized rats result in opposite effects on ischemic brain damage

Background  

Numerous stroke studies have controversially shown estrogens to be either neuroprotective or neurodamaging. The discordant results observed in rat brain ischemia models may be a consequence of discrepancies in estrogen administration modes resulting in plasma concentration profiles far from those intended. To test this hypothesis we reproduced in detail and extended an earlier study from our lab using a different mode of 17β-estradiol administration; home-made silastic capsules instead of commercial slow-release 17β-estradiol pellets. Four groups of female rats (n = 12) were ovariectomized and administered 17β-estradiol or placebo via silastic capsules. All animals underwent MCAo fourteen days after ovariectomy and were sacrificed three days later.     

Reduced blood brain barrier breakdown in P-selectin deficient mice following transient ischemic stroke: a future therapeutic target for treatment of stroke

Background  

The link between early blood- brain barrier (BBB) breakdown and endothelial cell activation in acute stroke remain poorly defined. We hypothesized that P-selectin, a mediator of the early phase of leukocyte recruitment in acute ischemia is also a major contributor to early BBB dysfunction following stroke. This was investigated by examining the relationship between BBB alterations following transient ischemic stroke and expression of cellular adhesion molecule P-selectin using a combination of magnetic resonance molecular imaging (MRMI), intravital microscopy and immunohistochemistry. MRMI was performed using the contrast, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated to Sialyl Lewis X (Sle^x) where the latter is known to bind to activated endothelium via E- or P selectins. Middle cerebral artery occlusion was induced in male C57/BL 6 wild-type (WT) mice and P-selectin-knockout (KO) mice. At 24 hours following middle cerebral artery occlusion, T₁ maps were acquired prior to and following contrast injection. In addition to measuring P- and E-selectin expression in brain homogenates, alterations in BBB function were determined immunohistochemically by assessing the extravasation of immunoglobulin G (IgG) or staining for polymorphonuclear (PMN) leukocytes. In vivo assessment of BBB dysfunction was also investigated optically using intravital microscopy of the pial circulation following the injection of Fluorescein Isothiocyanate (FITC)-dextran (MW 2000 kDa).     

Transplanted bone marrow stem cells relocate to infarct penumbra and co-express endogenous proliferative and immature neuronal markers in a mouse model of ischemic cerebral stroke

Background  

Several studies demonstrate that neurogenesis may be induced or activated following vascular insults, which may be important for neuronal regeneration and functional recovery. Understanding the cellular mechanism underlying stroke-associated neurogenesis is of neurobiological as well as neurological/clinical relevance. The present study attempted to explore potential homing and early development of transplanted bone marrow stem cells in mouse forebrain after focal occlusion of the middle cerebral artery, an experimental model of ischemic stroke.     

Estrogen-Astrocyte interactions: Implications for neuroprotection

Background  

Recent work has suggested that the ovarian steroid 17β-estradiol, at physiological concentrations, may exert protective effects in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and acute ischemic stroke. While physiological concentrations of estrogen have consistently been shown to be protective in vivo, direct protection upon purified neurons is controversial, with many investigators unable to show a direct protection in highly purified primary neuronal cultures. These findings suggest that while direct protection may occur in some instances, an alternative or parallel pathway for protection may exist which could involve another cell type in the brain.     

Administration of the GABA<Subscript>A</Subscript> receptor antagonist picrotoxin into rat supramammillary nucleus induces c-Fos in reward-related brain structures. Supramammillary picrotoxin and c-Fos expression

Background  

Picrotoxin blocks GABA_A receptors, whose activation typically inhibits neuronal firing activity. We recently found that rats learn to selectively self-administer picrotoxin or bicuculline, another GABA_A receptor antagonist, into the supramammillary nucleus (SuM), a posterior hypothalamic structure localized anterior to the ventral tegmental area. Other drugs such as nicotine or the excitatory amino acid AMPA are also self-administered into the SuM. The SuM appears to be functionally linked with the mesolimbic dopamine system and is closely connected with other brain structures that are implicated in motivational processes, including the prefrontal cortex, septal area, preoptic area, lateral hypothalamic area and dorsal raphe nucleus. Here, we hypothesized that these brain structures are activated by picrotoxin injections into the SuM.     

Contribution of constitutively proliferating precursor cell subtypes to dentate neurogenesis after cortical infarcts

Background  

It is well known that focal ischemia increases neurogenesis in the adult dentate gyrus of the hippocampal formation but the cellular mechanisms underlying this proliferative response are only poorly understood. We here investigated whether precursor cells which constitutively proliferate before the ischemic infarct contribute to post-ischemic neurogenesis. To this purpose, transgenic mice expressing green fluorescent protein (GFP) under the control of the nestin promoter received repetitive injections of the proliferation marker bromodeoxyuridine (BrdU) prior to induction of cortical infarcts. We then immunocytochemically analyzed the fate of these BrdU-positive precursor cell subtypes from day 4 to day 28 after the lesion.     

Berberine chloride can ameliorate the spatial memory impairment and increase the expression of interleukin-1beta and inducible nitric oxide synthase in the rat model of Alzheimer's disease

Background  

Berberine is the major alkaloidal component of Rhizoma coptidis, and has multiple pharmacological effects including inhibiting acetylcholinesterase, reducing cholesterol and glucose, lowering mortality in patients with chronic congestive heart failure and anti-inflammation etc. Thus berberine is a promising drug for diabetes, hyperlipemia, coronary artery disease and ischemic stroke etc. The present study was carried out to investigate the effect of berberine chloride on the spatial memory, inflammation factors interleukin-1 beta (IL-1beta) and inducible nitric oxide synthase (iNOS) expression in the rat model of Alzheimer's disease (AD) which was established by injecting Abeta (1–40) (5 microgram) into the rats hippocampuses bilaterally.     

Src kinase up-regulates the ERK cascade through inactivation of protein phosphatase 2A following cerebral ischemia

Background  

The regulation of protein phosphorylation requires a balance in the activity of protein kinases and protein phosphatases. Our previous data indicates that Src can increase ERK activity through Raf kinase in response to ischemic stimuli. This study examined the molecular mechanisms by which Src activates ERK cascade through protein phosphatases following cerebral ischemia.     

Calcium signals in the nucleus accumbens: Activation of astrocytes by ATP and succinate

Background

Late cerebral ischemia carries high morbidity and mortality after subarachnoid hemorrhage (SAH) due to reduced cerebral blood flow (CBF) and the subsequent cerebral ischemia which is associated with upregulation of contractile receptors in the vascular smooth muscle cells (SMC) via activation of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide a therapeutic window for treatment of cerebral ischemia after SAH.

Results

Here we demonstrate that SAH increases the phosphorylation level of ERK1/2 in cerebral vessels and reduces the neurology score in rats in additional with the CBF measured by an autoradiographic method. The intracisternal administration of SB-386023-b, a specific inhibitor of raf, given 6 h after SAH, aborts the receptor changes and protects the brain from the development of late cerebral ischemia at 48 h. This is accompanied by reduced phosphorylation of ERK1/2 in cerebrovascular SMC. SAH per se enhances contractile responses to endothelin-1 (ET-1), 5-carboxamidotryptamine (5-CT) and angiotensin II (Ang II), upregulates ET_B, 5-HT_1B and AT₁ receptor mRNA and protein levels. Treatment with SB-386023-b given as late as at 6 h but not at 12 h after the SAH significantly decreased the receptor upregulation, the reduction in CBF and the neurology score.

Conclusion

These results provide evidence for a role of the ERK1/2 pathway in regulation of expression of cerebrovascular SMC receptors. It is suggested that raf inhibition may reduce late cerebral ischemia after SAH and provides a realistic time window for therapy.