Quantitative Detection of Thrombin Activity in an Ischemic Stroke Model

Bushi, Doron; Chapman, Joab; Katzav, Aviva; Shavit‐Stein, Efrat; Molshatzki, Noa; Maggio, Nicola; Tanné, David

doi:10.1007/s12031-013-0072-y

Cited by 49 publications

(67 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thrombin activity was measured by use of a fluorometric assay as described previously with minor modifications42. Briefly, animals were reanesthetized and perfused with saline.…”

Section: Methodsmentioning

confidence: 99%

Nafamostat mesilate attenuates neuronal damage in a rat model of transient focal cerebral ischemia through thrombin inhibition

Chen

Wang

et al. 2014

Sci Rep

View full text Add to dashboard Cite

Evidence suggests that thrombin, a blood coagulation serine protease, mediates neuronal injury in experimental cerebral ischemia. Here, we test the hypothesis that nafamostat mesilate, a serine protease inhibitor, may ameliorate ischemia-induced neuronal damage through thrombin inhibition after ischemic stroke. Focal ischemia was induced in adult Sprague-Dawley rats by occlusion of the middle cerebral artery for 2 hours followed by 22 hours of reperfusion. The administration of nafamostat mesilate during ischemia and reperfusion reduced the brain infarct volume, edema volume and neurological deficit. Thrombin expression and activity in the ipsilateral striatum were increased after ischemia, whereas the administration of nafamostat mesilate significantly inhibited thrombin expression and activity. Immunostaining showed that the majority of thrombin was expressed in neurons. TUNEL staining showed that nafamostat mesilate reduced the number of dying cells during ischemia. A rat behavioral test showed that nafamostat mesilate treatment significantly improved the learning ability of ischemic rats. These results suggest that nafamostat mesilate may have a potential therapeutic role for neuroprotection against focal cerebral ischemia through thrombin inhibition.

show abstract

“…Thrombin activity was measured by use of a fluorometric assay as described previously with minor modifications42. Briefly, animals were reanesthetized and perfused with saline.…”

Section: Methodsmentioning

confidence: 99%

Nafamostat mesilate attenuates neuronal damage in a rat model of transient focal cerebral ischemia through thrombin inhibition

Chen

Wang

et al. 2014

Sci Rep

View full text Add to dashboard Cite

show abstract

“…The detrimental effect of TF during brain I/R injury is most likely mediated via thrombin-dependent mechanisms. High levels of prothrombin mRNA expression and thrombin activity have been detected in brain tissue after acute ischemic stroke (36, 37). In addition, thrombin has been shown to cause synaptic dysfunction, and thrombin activity was associated with neuronal damage (38, 39).…”

Section: Discussionmentioning

confidence: 99%

Protective and detrimental effects of neuroectodermal cell–derived tissue factor in mouse models of stroke

et al. 2016

View full text Add to dashboard Cite

Within the CNS, a dysregulated hemostatic response contributes to both hemorrhagic and ischemic strokes. Tissue factor (TF), the primary initiator of the extrinsic coagulation cascade, plays an essential role in hemostasis and also contributes to thrombosis. Using both genetic and pharmacologic approaches, we characterized the contribution of neuroectodermal (NE) cell TF to the pathophysiology of stroke. We used mice with various levels of TF expression and found that astrocyte TF activity reduced to ~5% of WT levels was still sufficient to maintain hemostasis after hemorrhagic stroke but was also low enough to attenuate inflammation, reduce damage to the blood-brain barrier, and improve outcomes following ischemic stroke. Pharmacologic inhibition of TF during the reperfusion phase of ischemic stroke attenuated neuronal damage, improved behavioral deficit, and prevented mortality of mice. Our data demonstrate that NE cell TF limits bleeding complications associated with the transition from ischemic to hemorrhagic stroke and also contributes to the reperfusion injury after ischemic stroke. The high level of TF expression in the CNS is likely the result of selective pressure to limit intracerebral hemorrhage (ICH) after traumatic brain injury but, in the modern era, poses the additional risk of increased ischemia-reperfusion injury after ischemic stroke.

show abstract

“…This type of anesthesia is common for this stroke model. PMCAo was performed using the common filament model based on our previously reported technique (12), using silicone-coated filament (Doccol Corp, Redlands, CA, USA). Mice were sacrificed using 100 µl pental by i.p.…”

Section: Methodsmentioning

confidence: 99%

“…Another group of healthy mice served as control ( n = 5). Sample sizes were chosen based on the magnitude of thrombin changes in our previous experiences (12, 38). Exclusion criteria include massive bleeding during surgery and/or intracerebral hemorrhage.…”

Section: Methodsmentioning

confidence: 99%

“…Activation of this receptor by low concentrations of thrombin may have neuroprotective effects, while at higher concentrations thrombin has deleterious effects (7–11). In the setting of an acute ischemic stroke, high thrombin levels have been detected in the infarct area, possibly as a consequence of blood–brain barrier opening (12, 13) or as a result of its local synthesis in the brain (14, 15) or both. Functionally, thrombin has been shown to cause synaptic dysfunction (15–19) and later, on vascular disruption, inflammatory response and neuronal damage (20–22), through the activation of PAR1 (13, 20).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Linear Temporal Increase in Thrombin Activity and Loss of Its Receptor in Mouse Brain following Ischemic Stroke

et al. 2017

Self Cite

View full text Add to dashboard Cite

BackgroundBrain thrombin activity is increased following acute ischemic stroke and may play a pathogenic role through the protease-activated receptor 1 (PAR1). In order to better assess these factors, we obtained a novel detailed temporal and spatial profile of thrombin activity in a mouse model of permanent middle cerebral artery occlusion (pMCAo).MethodsThrombin activity was measured by fluorescence spectroscopy on coronal slices taken from the ipsilateral and contralateral hemispheres 2, 5, and 24 h following pMCAo (n = 5, 6, 5 mice, respectively). Its spatial distribution was determined by punch samples taken from the ischemic core and penumbra and further confirmed using an enzyme histochemistry technique (n = 4). Levels of PAR1 were determined using western blot.ResultsTwo hours following pMCAo, thrombin activity in the stroke core was already significantly higher than the contralateral area (11 ± 5 vs. 2 ± 1 mU/ml). At 5 and 24 h, thrombin activity continued to rise linearly (r = 0.998, p = 0.001) and to expand in the ischemic hemisphere beyond the ischemic core reaching deleterious levels of 271 ± 117 and 123 ± 14 mU/ml (mean ± SEM) in the basal ganglia and ischemic cortex, respectively. The peak elevation of thrombin activity in the ischemic core that was confirmed by fluorescence histochemistry was in good correlation with the infarcts areas. PAR1 levels in the ischemic core decreased as stroke progressed and thrombin activity increased.ConclusionIn conclusion, there is a time- and space-related increase in brain thrombin activity in acute ischemic stroke that is closely related to the progression of brain damage. These results may be useful in the development of therapeutic strategies for ischemic stroke that involve the thrombin-PAR1 pathway in order to prevent secondary thrombin related brain damage.

show abstract

Quantitative Detection of Thrombin Activity in an Ischemic Stroke Model

Cited by 49 publications

References 28 publications

Nafamostat mesilate attenuates neuronal damage in a rat model of transient focal cerebral ischemia through thrombin inhibition

Nafamostat mesilate attenuates neuronal damage in a rat model of transient focal cerebral ischemia through thrombin inhibition

Protective and detrimental effects of neuroectodermal cell–derived tissue factor in mouse models of stroke

A Linear Temporal Increase in Thrombin Activity and Loss of Its Receptor in Mouse Brain following Ischemic Stroke

Contact Info

Product

Resources

About