3.1. Toxicity and Viability Analyses of TNK and rtPA
In both normoxic and oxygen glucose deprivation/re-oxygenation (OGD/R) conditions, rtPA (10 and 100 µg/mL) and TNK (2.5 and 25 µg/mL), did not show evidence of toxicity or cell death during 24 h of treatment exposure on endothelial cells (Figure S3) and astrocytes (Figure S4), compared with control (treated with vehicle) or untreated cells. Similar results were observed when both thrombolytic therapies were tested in an independent group of astrocytes subjected to NMDA-induced excitotoxicity (Figure S4B and D). Drug doses employed corresponds to the concentration in the blood of mice treated with 1 and 10 mg/kg of rtPA and 0.25 and 2.5 mg/kg of TNK.
Analysis of toxicity and viability were repeated in primary neuronal culture using the same doses of rtPA (10 and 100 µg/mL) and TNK (2.5 and 25 µg/mL). Flow cytometry and immunofluorescence analyses confirmed a purity of approximately 87% cortical neuron primary cultures (Figure S5). The higher dose of rtPA (100 µg/mL) induced a significant higher toxicity and cell death in both normoxic and OGD/R conditions (Fig. 1A and C), compared with control or untreated cells. Similar effects were observed in combination with NMDA treatment (Fig. 1B and D). In contrast, TNK 2.5 and 25 µg/mL did not show cell toxicity or death in any of the tested conditions.
3.2. Pharmacokinetic Analysis of rtPA and TNK in Healthy Animals
The enzymatic activity in the plasma of healthy animals was evaluated 60 min after treatment, as indicated in Fig. 2A. Vehicle treatment, control group, did not affect fibrinolytic plasma activity (Fig. 2B and C). The plasma activity raised 2- fold in groups of animals treated with 1 mg/kg rtPA and 9-fold in the 10 mg/kg rtPA group, with respect to the baseline value; this increase was maintained over the entire infusion period (Fig. 2B). The pharmacokinetic profile of a single bolus of TNK (Fig. 2C) 0.25 and 2.5 mg/kg lead to a rise of plasma fibrinolytic activity over basal levels of 5 and 13-folds, respectively, and for a follow-up period of at least 60 min. It is worth noting that a single bolus of 2.5 mg/kg TNK was able to induce a higher activity than 10 mg/kg rtPA administered as a continuous infusion, which reflects the longer blood half-life of TNK compared to rtPA.
3.3. Therapeutic Analysis of rtPA and TNK in a Thromboembolic Model of Stroke
In Center 1, to study the therapeutic effects of rtPA and TNK on stroke, infarct volumes and haemorrhages were studied 24 h and 5 days after treatment in a thromboembolic model of stroke induced by the local injection of thrombin into the middle cerebral artery[17, 18] (Fig. 3A).
Infarct volumes determined by MRI showed that control animals (vehicle group), had an average infarct volume of 31.0 ± 2.6 mm3 at 24 h and 10.8 ± 2.2 mm3 at 5 days after stroke. In those animals treated with rtPA, a significant infarct volume reduction was observed only with the higher dose (10 mg/kg) at 24 h and 5 days (15.4 ± 2.5 mm3 and 3.9 ± 1.2 mm3, respectively, P < 0.01 compared with the vehicle group). Similar to rtPA, only the higher dose of TNK (2.5 mg/kg) showed a significant reduction of infarct volume at 24 h compared with the control group (14.4 ± 3.2 mm3, P < 0.01). Five days after stroke onset, both doses of TNK showed a beneficial effect (3.3 ± 1.2 mm3, P < 0.01 in 0.25 mg/kg-TNK and 3.1 ± 1.1 mm3, P < 0.01 in 2.5 mg/kg-TNK) (Fig. 3B).
T2*-weighted images were used to evaluate HT observed as hyposignals due to blood leakage and iron accumulation in the brain tissue[19, 20]. Relevant hyposignals were not observed 24 h after ischemia (Fig. 3C), however, on day 5, an increase in T2* signal was detected in all groups, but there were no significant differences between the treated and control groups.
The most effective doses, 10 mg/kg rtPA and 2.5 mg/kg TNK, were also administered 4.5 h after thrombin injection, to investigate a potential enlargement of the therapeutic window of TNK compared to rtPA. Figure 3D shows a trend on the infarct volume reduction at 24 h (vehicle group: 29.3 ± 3.0 mm3; rtPA group: 19.3 ± 3.8 mm3; TNK group: 18.1 ± 3.1 mm3) and 5 days after ischemia (vehicle group: 9.0 ± 1.3 mm3; rtPA group: 5.7 ± 1.6 mm3; TNK group: 4.3 ± 1.4 mm3), although these differences were not significant. HT evaluated using T2*-weighted images did not show significant differences between the experimental groups (Fig. 3E).
3.4 Analysis of the Risk Haemorrhagic Transformations in Ischemic Animals Exposed to Acute Hyperglycaemia Treated with rtPA and TNK
Chronic hyperglycemia increases endothelial damage, which is closely associated with HT in patients with acute ischemic stroke treated with thrombolysis[21, 22]. For this reason we tested the effective doses of rtPA (10 mg/kg) and TNK (2.5 mg/kg) in animals with thromboembolic stroke and STZ-induced hyperglycemia. Administration of STZ for five consecutive days produces a sustained increase in glucose levels after 10 days (basal glucose levels: 134 ± 5 mg/dL; glucose levels at day 15: 431 ± 5 mg/dL; Figure S6). Ischemia was induced two weeks after the initiation of STZ treatment (Fig. 4A). Infarct volume assessment at 24 h and 5 days revealed that chronic hyperglycemia cancels the therapeutic effect of both thrombolytic agents even when administered early (30 min) after stroke, as no significant differences between control and treated groups were observed 24 h (vehicle group: 26.6 ± 4.2 mm3; rtPA group: 30.8 ± 4.2 mm3; TNK group: 33.2 ± 2.9 mm3) and 5 days after ischemia (vehicle group: 8.5 ± 1.2 mm3; rtPA group: 10.7 ± 1.5 mm3; TNK group: 11.6 ± 1.6 mm3) (Fig. 4B).
HT analyzed using T2*-weighted sequences showed higher hyposignals on day 5 in the groups treated with thrombolytic agents, although no statistical differences were found between the control and treated groups (Fig. 4C). Magnified images of the hyposignals detected in the brain samples are shown in Figures S7-9.
In an independent group of animals, the treatments were administered 4.5 h after surgery. Similar to the early administration in hyperglycemic mice, T2-weighted sequences (Fig. 4D) showed no differences in lesion volumes at 24 h and 5 days. T2*-weighted sequences, performed to study HT associated with late administration, revealed that in the group treated with 2.5 mg/kg TNK, there was a slight increase in the haemorrhagic volume (Fig. 4E). Magnifications of the hyposignals detected in the brain samples are shown in Figures S10-12.
Hemorrhagic lesions observed in this model were classified into four groups: no haemorrhages, petechial events, hemorrhage that represents less than 50% of the lesion, and hemorrhage that represents more than 50% of the lesion (Fig. 5A). Early administration of 10 mg/kg rtPA seemed to increase the risk of hemorrhage compared to TNK (Fig. 5B). However, the risk of hemorrhage was higher with TNK when administered late (Fig. 5C). To verify whether the hyposignals detected using MRI corresponded to haemorrhagic lesions, diamenibenzidine staining of the brain samples was performed (Figure S13).
3.5. Second independent therapeutic analysis of rtPA and TNK in the same thromboembolic model of stroke
A second independent therapeutic analysis was developed in Center 2 using the same thromboembolic model (Fig. 6A). In line with the results obtained in Center 1, the early administration (20 min after stroke) of rtPA (10 mg/kg) and TNK (2.5 mg/kg) significantly reduced the lesion size (51.9% and 41.2% reduction, respectively, P < 0.05 vs vehicle) 24 h after treatment. Similar to results from Center 1, no significant differences were observed with the late administration (4 h after stroke) of the treatments (Fig. 6B). Infarct volume was correlated with the angiographic score, measured at 24h, (Fig. 6C). Under the experimental conditions used in the Center 2, HT was clearly detected in this stroke model without inducing hyperglycemia with STZ pretreatment, as required for in Center 1. The early treatment with TNK showed a reduction of HT rates compared to rtPA (Fig. 6D). This effect was abolished when administered late after stroke onset.