Low dose tPA plus annexin A2 combination attenuates tPA delayed treatment- associated hemorrhage and improves recovery in rat embolic focal stroke

Abstract

We previously have shown that tissue type plasminogen activator (tPA) in combination with its receptor annexin A2 (rA2) protein significantly improved tPA thrombolytic efficacy. In this study we aimed to examine the therapeutic effects of the combination when treated at delayed 4-hourwindow after stroke compared to standard conventional tPA alone in an embolic focal stroke rat model. We compared effects of intravenous tPA alone (10mg/kg) versus a combination of low-dose tPA (5mg/kg) plus 10 mg/kg rA2. Totally 152 rats were used. Our results showed that: (1) at 24 hours after stroke, the combination slightly reduced brain infarction compared to saline (9.2% reduction), and tPA (7.4% reduction), although the reductions did not reach statistical significance; while the combination significantly reduced (22.2% reduction) the conventional tPA-elevated intracerebral hemorrhagic (ICH) transformation; (2)at 7 days after stroke, the combination significantly attenuated conventional tPA alone-elevated iron deposition at peri-lesion area (68.2% reduction); (3) at 28 days after stroke, the combination significantly improved performance of adhesive tape-removal test, which was accompanied by asignificantly higher microvessel density at peri-infarct areas compared to conventional tPA alone group. In conclusion, compared to conventional tPA alone, when treated at delayed 4-hour after stroke, the combination of low-dose tPA plus rA2 therapy provides a safer profile by lowering risk of ICH transformation and improves neurological function recovery after stroke.

Animal Models of Focal Cerebral Ischaemia and Haemorrhagic Transformation: Considerations in Experimental Stroke Study Design

“…The thromboembolic model more closely mimics the situation in clinical stroke and can be used to study clot-related mechanisms of injury, including the influence of different clot preparation [62] and composition [63][64][65], and thrombolytic interventions, such as tPA [66][67][68]. Several variations of this model have been performed using different methods of clot preparation; either injection of spontaneously formed or thrombin-induced clots into either the MCA or ICA via the ECA [62,69], or direct injection of thrombin into the vasculature [70][71][72][73] or brain tissue [74,75].…”

“…These models are based on adaptations to common animal models of ischaemic stroke, the most commonly used models being the intraluminal thread or thromboembolic models. In the thromboembolic model, the most common adaptation for induction of haemorrhagic transformation is delayed recanalisation of the occluded artery by delayed administration of tPA [43,66,124]. Delayed recanalisation resulted in the development of macroscopic haemorrhage in 50-100% of animals [43,106,124,125].…”

“…Intracerebral haemorrhages can also be identified by iron stains such as Perl's Prussian blue stain which will stain ferric iron, formed by the breakdown of haemoglobin during red blood cell lysis. Iron staining has only been used in a small number of studies assessing haemorrhagic transformation [66,121,129]. It has been suggested that the effectiveness of Prussian blue staining might be dependent on the age of the bleed being less sensitive in detecting fresh haemorrhage [144], although Prussian blue has been used successfully to identify intracerebral haemorrhages at 24 h after reperfusion [129].…”

“…These tests are relatively simple to perform and allow easy comparison between animals but the information they provide can be limited as the score does not describe the specific deficit observed [106,112,124,140,142,[156][157][158]. Other tests have been used in these studies to provide more detailed information, notably beam walking [113,132,134,159], grip strength [113,160], foot fault [66,114] and bilateral asymmetry tests [66,114]. While many studies into haemorrhagic transformation include tests of neurological function most studies to-date have involved a short recovery period (24-72h) post-stroke [114,121,145,156,161,162], this will limit the extent of functional testing which can be performed.…”

“…Interpretation of these findings may be complicated by the different techniques used to measure haemorrhagic transformation (incidence [112,161,162], volume [161,162], haemorrhage type [115,130,131,162,163], haemoglobin content [66,142,143,159]).…”