Mammalian Target Of Rapamycin (mTOR) Inhibition Reduces Cerebral Vasospasm Following A Subarachnoid Hemorrhage Injury In Canines

Background:Subarachnoid hemorrhage (SAH) is a subtype of devastating stroke that can lead to a variety of consequences including early brain injury and cerebral vasospasm. Despite promising therapeutic approaches, successful treatment following SAH remains inadequate. This is partly attributed to the poor strategic approach when dealing with cerebral ischemia as a result of cerebral vasospasm (CVS), one of the major consequences seen following an SAH. Although much has been discovered with regards to the mechanistic understanding of CVS, it continues to puzzle most scientists.Mammalian target of rapamycin (mTOR) pathway is a serine/threonine protein kinase that plays a vital role in regulating growth, proliferation, survival, and protein synthesis among cells. As a member of the PI3K family (phosphatidylinositol3-kinase-related kinase), mTOR has been shown to orchestrate the phosphorylation of key downstream proteins including P70S6K1(proteins p70ribosomal S6kinase) and4E-BP1(eukaryotic initiation factor4E binding protein1).As a protein kinase, mTOR forms two distinct multiprotein complexes called mTORC1and mTORC2. The mTORC1is inhibited by rapamycin. On the other hand, AZD8055is a potent and selective mTOR kinase inhibitor that acts on both the C1and C2prototypes, along with other downstream substrates. However, mTOR inhibitors role in cerebral vasospasm has not been investigated. Subject:In the present study, we investigated the role of the mTOR protein kinase following SAH brain injury in canines, specifically investigating its position as a key orchestrator of cerebral vasospasm. We used an mTOR C1inhibitor (rapamycin) and an mTOR C1/mTOR C2simultaneous inhibitor (AZD8055) to explore potential mechanistic theories while measuring direct anti-proliferation activity through P70S6K1,4E-BP1and PCNA (proliferating cell nuclear antigen) expression.Materials and Methods:Thirty male/female beagle dogs weighing15to20kg were housed in a12-hour light/dark cycle at a controlled temperature and humidity with free access to food and water. All animals were randomly assigned to one of five groups-Sham (n=6), SAH (n=6), SAH+DMSO (Dimethyl sulfoxide; n=6), SAH+RAPA (Rapamycin,2mg/kg, mTORC1inhibitor, n=6), and SAH+AZD8055(2mg/kg, a mTORC1/mTORC2inhibitor, n=6). Animals were anesthetized with a cepromazine, atropine, and xylazine cocktail. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on days0and2. Angiography was performed at days0and7. Iodixanol was injected to acquire an image of the basilar artery. After angiography,0.5mL/kg of blood taken from the femoral artery was injected into the cisterna magna at day0and then repeated at day2. Three behavioral examinations were modified from a previous study and performed daily after SAH to record appetite, activity, and neurological deficits. In groups, histology, immunohistochemistry, and Western blot of mTOR, P70S6K1,4E-BP1and PCNA (proliferating cell nuclear antigen) in the basilar arteries were examined.Results:Severe SAH was particularly pronounced around the Circle of Willis and along the ventral brainstem following injury. The animals in the SAH and SAH+DMSO groups developed severe vasospasm (p<0.05vs Sham) as shown by angiography on day7. The mean values of the residual diameter of the basilar artery on day7, as a percentage of that on day0, was34.3±19.84%in SAH,38.4±10.26in SAH+DMSO, and93.9±5.01%in Sham respectively. In the SAH+RAPA and SAH+AZD8055groups, a moderate vasospasm,62.3±15.92%and65.2±10.34%was observed (p <0.05versus SAH and SAH+DMSO).The behavior scores are shown that the appetite score in both Rapamycin and AZD8055treatment groups were better than in the SAH group from days2to4, even though statistical significance was achieved at day5to7(p<0.05, ANOVA). No statistical difference was found between SAH+Rapamycin and SAH+AZD8055groups (p>0.05, ANOVA). The activity scores in Rapamycin and AZD8055treatment groups were significantly better than in the SAH and SAH+DMSO groups (p<0.05, ANOVA) at day6and7. Most animals did not show any signs of serious neurological deficits which was evident by the lack of statistical significance among the observed groups.Morphological vasospasm was observed in animals assigned to the SAH group. This was characterized by corrugation of the internal elastic lamina, contraction of smooth muscle cells, and increased thickness of the vessel wall, which was a sign of severe vasospasm. Treatment with vehicle of DMSO did not get any improvement. Moderate vasospasm was observed in the SAH+RAPA and SAH+AZD8055animal groups. Sham animals were the only group that did not show any signs of vasospasm.In the SAH and SAH+DMSO groups, notable immunohistochemical staining of mTOR, P70S6K1,4E-BP1, and PCNA were observed across all layers of the basilar artery, especially among smooth muscle cells compared to Sham animals. Animals treated with both Rapamycin and AZD8055where characterized by a notable reduction in staining of mTOR, P70S6K1,4E-BP1, and PCNA. Double and triple fluorescence immunohistochemistry staining revealed a marked elevation in staining with mTOR, P70S6K1and PCNA across all layers of the basilar artery, especially in smooth muscle layer; merging double and triple of these images indicated that mTOR co-localized with P70S6K1and PCNA. Using a high magnification zoom, the stain demonstrated that mTOR, P70S6K1and PCNA co-localized in the smooth muscle cells.Western blotting revealed that the expressions of mTOR, P70S6K1,4E-BP1, and PCNA in the basilar artery samples in Sham, SAH, SAH+DMSO, SAH+RAPA and SAH+AZD8055groups. When the values in the sham basilar arteries were regarded as100%, there was a significant enhancement of expression of mTOR, P70S6K1,4E-BP1, and PCNA in the spastic basilar arteries from the SAH and SAH+DMSO animals sacrificed at day7(p<0.05vs. Sham). Treatment with Rapamycin and AZD8055significantly suppressed the expression of mTOR, P70S6K1,4E-BP1, and PCNA (p<0.05vs. Sham). No significant differences were noted between the two inhibitor treatment groups and between the SAH and SAH+DMSO groups.Conclusion:Our study suggests that vascular smooth muscle cell proliferation mediated by the mTOR pathway may in fact play a significant role in cerebral vasospasm following SAH injury. The expressions of mTOR, P70S6K1and4E-BP1were accompanied with PCNA in the basilar arteries. By blocking the activation of the mTOR pathway, the attenuation of angiographic vasospasm was attributed to the anti-proliferation that ensues following cerebral vasospasm.The mTOR molecular signaling pathway plays a significant role in cerebral vasospasm following SAH, and the inhibition of the mTOR pathway has the potential to become an attractive strategy to treat vasospasm following SAH. However, More studies evaluating the exact mechanistic target of the mTOR pathway within vasospasm are warranted.