Therapeutic effects of JLX001 on cerebral ischemia through inhibiting platelet activation and thrombus formation in rats.

(3ß,5α,16α,20S)-4,4,14-trimethyl-3,20-bis(methylamino)-9,19-cyclopregnan-16-ol-dihydrochloride (JLX001), a derivative of cyclovirobuxine D (CVB-D), is a novel compound from synthesis. This study aims to confirm the therapeutic effect of JLX001 on cerebral ischemia and researchits antiplatelet and antithrombosis activities via thromboxane (TXA2)/phospholipase C-ß-3(PLCß3)/protein kinase C (PKC) pathway suppression. The therapeutic effects of JLX001 was evaluated by infarct sizes, brain edema and neurological scores in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Brain TXA2 and prostacyclin (PGI2) were measured by enzyme-linked immunosorbentassay (ELISA). P-PLCß3and activated PKC were detected by immunohistochemical method. Adenosine diphosphate (ADP) or 9, 11-dieoxy-11α, 9α-epoxymethanoeprostaglandin F2α (U46619) was used as platelet agonist in the in vivo and in vitro platelet aggregation experiments. Clotting time and bleeding time were determined. Besides, two whole-animal experiments including arteriovenous shunt thrombosis and pulmonary thromboembolism model were conducted. Results showed that JLX001 treatment markedly alleviated cerebral infarcts, edema, and neurological scores in permanent middle cerebral artery occlusion (pMCAO) rats. Brain TXA2 level, p-PLCß3and activated PKC were decreased, while PGI2level had no significant change. Besides, JLX001 inhibited platelet aggregation induced by ADP or U46619 and exhibited anti-coagulation effects with a minor bleeding risk. In the two whole-animal experiments, JLX001 inhibited thrombus formation. In summary, JLX001 attenuates cerebral ischemia injury and the underlying mechanisms relate to inhibiting platelet activation and thrombus formation via TXA2/PLCß3/PKC pathway suppression.

Emodin from Polygonum multiflorum ameliorates oxidative toxicity in HT22 cells and deficits in photothrombotic ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum multiflorum Thunb. has been used widely in East Asia in treatment of diseases associated with aging. Emodin, an active component from Polygonum multiflorum Thunb., provides benefits for brain disturbances induced by severe cerebral injury. AIM OF THE STUDY: We investigated the neuroprotective effect of emodin from Polygonum multiflorum Thunb. against glutamate-induced oxidative toxicity and cerebral ischemia. MATERIALS AND METHODS: For examination of neuroprotective effects of emodin, cell viability, cytotoxicity, flow cytometry, and Western blot were performed in HT22 cells and infarct volume, behavioral tests and Western blot in a mouse model of photothrombotic ischemic stroke. RESULTS: Pretreatment with emodin resulted in significantly reduced glutamate-induced apoptotic cell death in HT22 cells. However, blocking of phosphatidylinositol-3 kinase (PI3K) activity with LY294002 resulted in significantly inhibited cell survival by emodin. Exposure of glutamate-treated cells to emodin induced an increase in the level of Bcl-2 expression, whereas the expression of Bax and active caspase-3 proteins was significantly reduced. In addition, treatment with emodin resulted in increased phosphorylation of Akt and cAMP response element binding protein (CREB), and expression of mature brain-derived neurotrophic factor (BDNF). This expression by emodin was also significantly inhibited by blocking of PI3K activity. In a photothrombotic ischemic stroke model, treatment with emodin resulted in significantly reduced infarct volume and improved motor function. We confirmed the critical role of the expression levels of Bcl-2/Bax, active caspase-3, phosphorylated (p)Akt, p-CREB, and mature BDNF for potent neuroprotective effects of emodin in cerebral ischemia. CONCLUSIONS: These results suggest that emodin may afford a significant neuroprotective effect against glutamate-induced apoptosis through activation of the PI3K/Akt signaling pathway, and subsequently enhance behavioral function in cerebral ischemia.

Effect of task-specific training on functional recovery and corticospinal tract plasticity after stroke.

PURPOSE: To determine the optimal timing of rehabilitation and its role in corticospinal tract (CST) plasticity after stroke. METHODS: Rats were subjected to photothrombotic infarct. The large stroke (LS) and small stroke (SS) groups were subdivided and task-specific training (TST) was initiated at 1, 5, or 14 days poststroke. Behavioral tests were performed at 2, 7, 14, 21, 28, and 35 days poststroke. The differences of axonal sprouting in the cortex, red nucleus, cerebral peduncle, and pyramid level were compared by immunohistochemistry. RESULTS: SS groups with TST starting at 1 day and 5 days showed significantly better recovery in the behavioral tests. LS group with TST starting at 5 days showed better recovery, while those with TST starting at 1 day showed worse recovery. Contralesional axonal sprouting was increased in both groups with TST starting at 5 days. However, it was decreased in the LS group with TST starting at 1 day. Transcallosal axonal sprouting from the contralesional motor cortex was increased in the LS group with TST starting at 5 days. CONCLUSIONS: Functional recovery after stroke may vary, depending on the lesion size and the timing of rehabilitation. The underlying mechanism may involve contralesional CST plasticity and transcallosal axonal sprouting.

Neuropathological analysis of lacunes and microvascular lesions in late-onset depression.

AIMS: Previous neuropathological studies documented that small vascular and microvascular pathology is associated with cognitive decline. More recently, we showed that thalamic and basal ganglia lacunes are associated with post-stroke depression and may affect emotional regulation. The present study examines whether this is also the case for late-onset depression. METHODS: We performed a detailed analysis of small macrovascular and microvascular pathology in the post mortem brains of 38 patients with late-onset major depression (LOD) and 29 healthy elderly controls. A clinical diagnosis of LOD was established while the subjects were alive using the DSM-IV criteria. Additionally, we retrospectively reviewed all charts for the presence of clinical criteria of vascular depression. Neuropathological evaluation included bilateral semi-quantitative assessment of lacunes, deep white matter and periventricular demyelination, cortical microinfarcts and both focal and diffuse gliosis. The association between vascular burden and LOD was investigated using Fisher's exact test and univariate and multivariate logistic regression models. RESULTS: Neither the existence of lacunes nor the presence of microvascular ischaemic lesions was related to occurrence of LOD. Similarly, there was no relationship between vascular lesion scores and LOD. This was also the case within the subgroup of LOD patients fulfilling the clinical criteria for vascular depression. CONCLUSIONS: Our results challenge the vascular depression hypothesis by showing that neither deep white matter nor periventricular demyelination is associated with LOD. In conjunction with our previous observations in stroke patients, they also imply that the impact of lacunes on mood may be significant solely in the presence of acute brain compromise.

Hyperthermia-induced morphological changes in cerebral tissue of the rat.

The sensitivity of cerebral tissue to hyperthermia, its immediate effect, manifested by histological changes and the role of local blood flow, blood rheological properties, and the possible role of free radicals in development of mentioned changes have been studied. Through the cranial window local area of cerebral surface was irrigated by artificial CSF heated up to 41, 43 or 45 degrees C. Serial brain coronal sections 50 microm thick were analyzed under light microscope. Local Cerebral Blood Flow was measured by thermo-clearance method. Blood rheological properties were changed by injection of Dextran T-500 and free radicals existence was controlled by DMSO injection. High sensitivity of cerebral tissue to hyperthermic exposure even at a temperature of 41 degrees C has been confirmed. We consider cerebrovascular thrombosis as one of the most significant complication of brain hyperthermia. Deteriorated blood rheological properties aggravates hyperthermia-induced cerebral lesion. Administration of free radicals scavengers can partially lessen hyperthermia induced cerebral lesion.

Experimental model of brainstem stroke in rabbits via endovascular occlusion of the basilar artery.

BACKGROUND: Basilar artery thrombosis remains a significant clinical problem, and no reproducible animal model has been established to study the stroke within the vertebrobasilar distribution. We report a study designed to pilot test a novel model of brainstem stroke in rabbits, created by selective endovascular occlusion of the basilar artery. METHODS: Basilar artery occlusion was induced in 8 New Zealand white rabbits by injection of the autologous clot through the microcatheter positioned within the distal vertebral artery. Animals were divided into subgroups (I and II) based on the length of produced ischemia (3 and 6 hours, respectively). Magnetic resonance (MR) imaging of the brain and MR angiography of the intracranial vessels were performed before the procedure, and at 3 hours after induced ischemia for groups I and II, with continued imaging up to 6 hours for group II, with diffusion-weighted images acquired approximately every 30 minutes. Animals were killed at the end of the 3-hour (group I) or 6-hour (group II) ischemia time. RESULTS: Brainstem stroke was successfully induced in all animals, with pathological changes documented in all cases. The earliest changes of ischemia on MR diffusion-weighted images were identified at only 4.5 hours of basilar artery occlusion. CONCLUSION: These results suggest that a reproducible model of brainstem stroke can be induced in rabbits using selective endovascular occlusion of the basilar artery. The availability of such a model, integrated with state-of-the-art imaging techniques, holds promise for preclinical investigations of emergent therapeutic approaches in stroke.

[Combination and transformation of toxin and blood stasis in etiopathogenesis of thrombotic cerebro-cardiovascular diseases].

According to the basic theory of traditional Chinese medicine (TCM), the pathogenetic factors such as platelet activation, adhesion, congregation and thrombosis fall into the category of blood stasis, while the pathological changes such as tissue necrosis, oxidative stress injury and inflammation, etc, are far beyond the etiological category of blood stasis. The toxin or the combination and transformation of toxin and blood stasis of TCM are involved in the pathogenesis of thrombotic cerebro-cardiovascular diseases. It is significant to recognize and stress the combination and transformation of toxin and stasis in pathogenicity so as to enrich TCM etiology and improve TCM clinical efficacy in the treatment of cerebro-cardiovascular and thrombotic diseases.

[Differential diagnosis of bilateral thalamic lesions]. / Differenzialdiagnose bilateraler Thalamusläsionen.

A multitude of different diseases can result in bilateral thalamic lesions. These include vascular pathologies requiring prompt therapeutic intervention, such as basilar thrombosis or thrombosis of the internal cerebral veins, as well as tumors, infectious or demyelinating diseases, and toxic-metabolic lesions. Therefore, detailed knowledge of the typical radiological findings for the various diseases is essential for determining the correct diagnosis. This review provides a synopsis of the radiological findings for the most important bithalamic lesions and an overview of the literature.

[Effects of ginkgolides injection on experimental cerebral ischemia in mice and rats].

OBJECTIVE: To investigate the effects of ginkgolides injection on experimental cerebral ischemia and its related mechanism of action. METHOD: The middle cerebral artery occlusion (MACO) model was induced by the FeCl3-occluding method to explore the protective effects of ginkgolides injection on the score of neurological deficits, the rate of cerebral infarction and the histomorphology of cerbral ischemia in rats. Thrombosis formation in vivo was induced by adrenaline-collagen in mice to explore the antithrombotic effect. Platelet aggregation was induced by ADP and hemorrheological parameters with hyper-viscosity by dextran T-500 were used to explore the effects of antiplatelet aggregation and decreasing viscosity of blood. RESULT: Ginkgolides injection could markedly decrease the infarct size and behavior deficits score, inhibit the thrombus formation in mice, decrease blood viscosity and ameliorate hemorrheological parameters in rat. CONCLUSION: Ginkgolides injection has the protective effects on focal cerebral ischemia, and its mechanism may be relative to its inhibition of platelet-dependent thrombosis and amelioration of hemarheological partments.

Upregulated expression of 14-3-3 proteins in astrocytes from human cerebrovascular ischemic lesions.

BACKGROUND AND PURPOSE: Several types of chaperone proteins, such as heat shock proteins, have been reported to be associated with brain ischemia. The purpose of this study was to investigate whether an abnormal expression of 14-3-3 proteins, a novel type of molecular chaperones, occurs in human gray and white matter ischemic lesions. METHODS: We prepared formalin-fixed, paraffin-embedded sections from 33 autopsied brains, consisting of 7 normal controls, 4 cases with cerebral thrombosis, 5 cases with cerebral embolism, 8 cases with multiple lacunar infarctions, and 9 cases with Binswanger disease. Deparaffinized sections from all cases were immunostained with anti-14-3-3 antibodies using the avidin-biotin-peroxidase complex method, and some sections were also double-immunostained for 14-3-3 and glial markers. RESULTS: In the normal control brains, 14-3-3 immunoreactivity was mainly localized to the neuronal somata and processes. Strongly 14-3-3-immunopositive astrocytes were distributed in the infarct lesions and were particularly abundant in infarcts at the chronic stage. Intensely 14-3-3-immunolabeled astrocytes were also observed in the ischemic white matter lesions, and in the severely affected white matter lesions from patients with Binswanger disease, dense 14-3-3 immunoreactivity was found in clasmatodendritic astroglia as well as in reactive astrocytes. CONCLUSIONS: Our results suggest that 14-3-3 proteins may be induced mainly in astrocytes from human cerebrovascular ischemic lesions, and that the upregulated expression of 14-3-3 proteins in astrocytes may be involved in the formation of astrogliosis.

Cerebral venous thrombosis in a gentleman presenting with fever, convulsion and frontotemporal haemorrhages.

Cerebral venous thrombosis (CVT) is an uncommon but serious type of stroke. Thrombosis may involve the cortical or deep veins or the venous sinuses. The presenting clinical features are non-specific. We report a 48-year-old man with CVT who presented with fever, bitemporal throbbing headache, and generalised convulsion. Computed tomography (CT) of the brain revealed acute haemorrhages over right anterior frontal and posterior temporal regions with surrounding oedema and right anterior temporal subcortical oedema. The initial diagnosis was herpes simplex encephalitis. Absence of venous flow over the right transverse and sigmoid sinuses during the venous phase of digital subtraction angiography (DSA) revealed CVT. He was anti-coagulated for 6 months. An underlying cause of CVT was not detected. A high index of suspicion is required when risk factors of CVT are present. CT brain may be normal or showing non-specific findings. Magnetic resonance imaging plus venography, CT venography, or DSA is diagnostic.

Cerebral sinovenous thrombosis in children: another reason to treat iron deficiency anemia.

Iron deficiency anemia is a rare cause of cerebral sinovenous thrombosis in children. We report three cases of cerebral sinovenous thrombosis and iron deficiency anemia treated at Primary Children's Medical Center in Salt Lake City, Utah, between 1998 and 2001. The children were 9, 19, and 27 months old at the time of admission. Hemoglobin levels ranged from 6.6 to 7.0 g/dL, mean corpuscular volume levels from 45 to 56 fL, and platelet counts from 248,000 to 586,000/microL. Magnetic resonance imaging and magnetic resonance venography revealed thrombosis of the straight sinus and internal cerebral veins in all three children, with the addition of the vein of Galen, left transverse and sigmoid sinuses, and upper left internal jugular vein in one child. Recovery ranged from excellent to poor in 3 months to 3 years of follow-up. Four additional cases, ages 6 to 22 months, were found in the English-language literature. Evaluation for prothrombotic disorders was negative in all children, including the current cases. Treatments have included thrombectomy, corticosteroids, mannitol, heparin, low-molecular-weight heparin, warfarin, aspirin, blood transfusion, and iron supplementation, but there is no consensus regarding therapy, other than to correct the anemia and treat iron deficiency. Iron deficiency anemia, a preventable cause of cerebral sinovenous thrombosis, deserves consideration when cerebral sinovenous thrombosis is detected in young children.

Three-dimensional MRI of cerebral projections in rat brain in vivo after intracortical injection of MnCl2.

In this study we investigated the potential of in vivo MRI detection of axonal Mn2+ transport for tracing neuronal projections originating in the sensorimotor cortex in healthy and lesioned rat brains. Special attention was given to the potential of visualizing neuronal sprouting of central nervous system across the midline. After injecting unchelated MnCl2 into the forelimb area of sensorimotor cortex of 18 healthy and 10 lesioned rats corticofugal projections could be traced through the internal capsule to the cerebral peduncle and the pyramidal decussation. Although the neuronal tract was visible as early as 6 h after MnCl2 injection, best contrast was achieved after 24-48 h. Beside the cortico-spinal tract, the cortico-thalamic fibres were also visualized by anterograde Mn2+ transport. Cortico-striatal fibres were partially masked by the very high signal near the MnCl2 injection site but could be discerned as well. Slight, diffuse signal enhancement of cortical tissue contralateral to the MnCl2 injection site in healthy rat brains suggests interhemispheric connections or passive diffusion of Mn2+. However, enhanced fibre tract contrast connecting both hemispheres was visible 16 weeks after onset of focal photothrombotic cortical injury. In conclusion our study has shown that we were able to visualize reproducibly the main descending corticofugal projections and interhemispheric connections by non-invasive MRI after localized injection of MnCl2. The appearance of interhemispheric Mn2+-enhanced fibres after photothrombotic focal injury indicates that the method may bear potential to follow non-invasively gross plastic changes of connectivity in the brain after injury.

Widespread and long-lasting alterations in GABA(A)-receptor subtypes after focal cortical infarcts in rats: mediation by NMDA-dependent processes.

Impairment of inhibitory neurotransmission has been reported to occur in widespread, structurally intact brain regions after focal ischemic stroke. These long-lasting alterations contribute to the functional deficit and influence long-term recovery. Inhibitory neurotransmission is primarily mediated by gamma-aminobutyric acid (GABA)A receptors assembled of five subunits that allow a variety of adaptive changes. In this study, the regional distribution of five major GABA(A)-receptor subunits (alpha1, alpha2, alpha3, alpha5, and gamma2) was analyzed immunohistochemically 1, 7, and 30 days after photochemically induced cortical infarcts. When compared with sham-operated controls, a general and regionally differential reduction in immunostaining was found within the cortex, hippocampus, and thalamus of both hemispheres for almost all subunits. Within ipsilateral and contralateral neocortical areas, a specific pattern of changes with a differential decrease of subunits alpha1, alpha2, alpha5, and gamma2 and a significant upregulation of subunit alpha3 was observed in the contralateral cortex homotopic to the infarct. This dysregulation was most prominent at day 7 and still present at day 30. Interestingly, a single application of the noncompetitive N-methyl-D-aspartate-receptor antagonist MK-801 during lesion induction completely blocked these bihemispheric alterations. Cortical spreading depressions induced by topical application of KCl do not change GABA(A)-receptor subunit expression. As alterations in subtype distribution crucially influence inhibitory function, ischemia-induced modifications in GABA(A)-receptor subtype expression may be of relevance for functional recovery after stroke.

Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.

In this study, we adapted the original rat photothrombosis model of Watson et al. (Ann Neurol 17 (1985) 497) for use in mice by refining the application route of the dye, illumination and stereotactic parameters. After intraperitoneal injection of the photosensitive dye Rose bengal, subsequent focal illumination of the brain with a cold light source through the intact skull led to focal cortical infarcts of reproducible size, location and geometry. Cresyl violet histology displayed well-demarcated infarcts that matured with time in a predictable manner. Microglial responses, as assessed by immunocytochemistry, against F4/80 and CD11b antigens were rapid and complete at the infarct site, but delayed and incomplete in degenerating fiber tracts and ipsilateral thalamic nuclei. In contrast to the rat, where the expression of CD4 and CD8 antigens discriminate distinct subpopulations of lesion-associated phagocytes, the expression of both markers was low to absent in the mouse model. In both rats and mice, cerebral photothrombosis shares essential inflammatory responses with focal ischemia induced by middle cerebral artery occlusion. It may provide a useful model to study functional aspects of lesion-associated and remote molecular responses in transgenic mice.