Tehran University Medical Journal

Background: Ultrasound (US) has been used in neuroprotection after cerebral ischemia, however the mechanism of action remains unclearly. We have previously shown the protective effect of ultrasound on infarction volume and brain edema in ischemic brain injured at normothermic condition. Ultrasound may also amplify the effect of fibrinolytic medications in thrombolysis process .We have also shown that hyperthermia can exacerbate cerebral ischemic injury and that the efficacy of tissue plasminogen activator (tPA) is reduced in the presence of hyperthermia. In this study, the effects of US alone or in combination with tPA on brain ischemic injury were evaluated.

Methods: Focal ischemic brain injury was induced by emblazing a pre-formed clot into the middle cerebral artery in rats. Principally, we examined whether US can reduce the perfusion deficits and, the damage of blood- brain barrier (BBB) in the ischemic injured brain. There are two series of experiments at this study .in the first series, animals were randomly assigned to four groups (n=7 per group) as follows: 1-control (saline), 2-US (1W/cm², 10 duty cycle ), 3- US+high- tPA (1W/cm², 10 duty cycle +20 mg/kg) and 4- high -tPA (20 mg/kg). We also examined the effects of US and tPA on BBB integrity after ischemic injury. The animals were assigned into four groups (n=7 per group), treatment is the same as above. BBB permeability was assessed by the Evans blue (EB) extravasations method at 8 h after MCA occlusion. BBB permeability was evaluated by fluorescent detection of extravagated Evans blue dye and Perfusion deficits were analyzed using an Evans blue staining procedure. The perfused microvessels in the brain were visualized using fluorescent microscopy. Areas of perfusion deficits in the brain were traced, calculated and expressed in mm².

Results: The results showed that US improved neurological deficits significantly (p<0.05). The administration of US significantly decreased perfusion deficits and BBB permeability. In the control set, for the US+high tPA, high tPA only and US only groups, the mean perfusion deficits (±SD) were 14.32±3.15, 7.03±4.08, 5.92±1.90 and 9.14±3.37 mm², respectively, 8 h after MCA occlusion (P<0.05).

Conclusions: These studies suggest that US is protective in a rat embolic model of stroke due to decreased perfusion deficits.

Background: Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agentsused in patients with vascular occlusions such as acute ischemic stroke or myocardial infarction. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae (L. tarentolae), a non-pathogenic trypanosomatid protozoa, has come under consideration because of its safety and immunogenicity as a vaccine vector and special attributes in the expression of complex proteins. This study was done to improve rt-PA expression in this protozoon and create the opportunity for the replacement of rt-PA gene with other genes for the production of other complex proteins.
Methods: Two expression cassettes were used for the integration of two copies of t-PA cDNA, one copy in each cassette, into the parasite genome by electroporation. The transformed clones were selected by antibiotic resistancy. The expression of active secreted rt-PA was confirmed by Western blot analysis and Chromolize assay.
Results: Appearance of a 64 kD band in nitrocellulose membrane in the Western blot analysis confirmed the presence of full-length rt-PA in the culture media. Chromolize assay showed the expression levels of active recombinant t-PA in single and double transfected L. tarentolae clones- 375 IU/ml and 480 IU/ml of the culture media,respectively.
Conclusion: The produced rt-PA in the culture media containing the transfected cells was at least seven times higher than what has been reported in previous studies on L. tarentolae or on some other eukaryotic systems.