Antisense preproendothelin-oligoDNA therapy for vasospasm in a canine model of subarachnoid hemorrhage

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Abstract
Object. The purpose of this study is twofold: 1) to test antisense genetic techniques used in the prevention of cerebral vasospasm in a canine model of subarachnoid hemorrhage (SAH), targeting the endothelin-1 (ET-I) gene; and 2) to determine if fibrinolysis of subarachnoid clot with recombinant tissue plasminogen activator (rtPA) could enhance the effect of antisense treatment. Methods. A total of 39 dogs were studied in this experiment. Placebo (six animals), rtPA (six animals), antisense preproET-1 oligodeoxynucleotide (ASOD; five animals), or rtPA plus ASOD (combined treatment; six animals) was injected into the cisterna magna 30 minutes after a second SAH was induced on the 2nd day of the experiment. The animals were observed until Day 7, when they underwent follow-up angiography and then were killed; their basilar arteries were removed for analysis. Control animals included in this study (two animals in each group) received placebo, rtPA, ASOD, or rtPA plus ASOD without induction of SAH, or rtPA with mismatched (nonsense) preproET-1 oligodeoxynucleotide following SAH. Six additional dogs were analyzed earlier following SAH. Dogs that received placebo developed severe vasospasm (51 +/- 8% of baseline caliber). Administration of ASOD alone resulted in a mild reduction in vasospasm (64 +/- 13% of baseline caliber) and rtPA alone resulted in a moderate reduction in vasospasm (81 +/- 5% of baseline caliber); however, the combined therapy of rtPA plus ASOD almost completely prevented vasospasm (95 +/- 6% of baseline caliber), which was significantly different from all other groups (p < 0.05). Morphological analysis of the basilar arteries yielded results similar to angiography with respect to vasospasm severity. The ASOD treatment combined with rtPA resulted in reduced ET-1 expression, as demonstrated by immunohistochemical staining of the arteries, and reduced preproET-1 levels on Day 4, as measured by reverse transcription-polymerase chain reaction. Nonsense DNA sequences had no effect on the vessels. Conclusions. Antisense preproET-1 oligodeoxynucleotide treatment, when combined with clot lysis caused by rtPA, reduced vasospasm in the canine model of SAH, and this effect appeared to be related to reduced ET-1 synthesis. The results of this experiment support a causative role for ET-1 early in the course of vasospasm development in dogs. The apparent additive therapeutic effects of antisense and fibrinolytic treatments could be due to clot lysis, which allows better delivery of oligodeoxynucleotides to arteries within the subarachnoid space.