The Role of Heat Shock Protein 90 in Mitigating Facial Allodynia and Improving Blood Brain Barrier Integrity in a Migraine Model Utilizing Cortical Spreading Depression

Abstract

The Blood-brain barrier and related pharmacokinetics are subjects of intense research in neurological pathologies such as traumatic brain injury, ischemic stroke, epilepsy, and encephalopathies. Aside from direct physical damage, the barrier also undergoes stress from the surrounding microenvironment. There is evidence which suggests chronic pain can also compromise blood-brain barrier integrity through various mechanisms, including inflammation. In recent years migraine with aura, a neurological disease afflicting billion world-wide, has been a subject of debate as to whether the disease affects blood-brain barrier integrity. Preclinical research has shown that cortical spreading depression (CSD), the underlying mechanism of an aura, can at least cause transient paracellular leaks. Clinical studies utilizing imaging technology to observe the vasculature of the brain suggests that if these changes are occurring, they are not significant. However, clinical reports have shown that migraine with aura is strongly correlated with increased likelihood of stroke. Additionally, studying the effects of CSD on the microenvironment of the neurovascular unit is crucial in relation to pharmacokinetics and drug delivery. There is evidence which suggest dysregulation of pH in the brain during and after a CSD event. In this study we observed the effects of inhibiting an acid-sensing channel (ASIC) sodium-hydrogen exchanger 1 (NHE1) during CSD conditions on blood brain barrier modulation, periorbital allodynia, and drug delivery. We first explored how NHE1 inhibition, with specific NHE1 inhibitor zoniporide, would affect the drug delivery across a blood-endothelial barrier in vitro. Results showed increased sumatriptan transport across the BEB in the presence of zoniporide and high extracellular KCl. We observed that NHE1 inhibition with zoniporide alone caused periorbital allodynia and did not ameliorate the effects of KCl induced CSD in female rats. However, increased the efficacy of sumatriptan in CSD induced female rats. Tissue western blot results revealed a decrease in NHE1 expression during post CSD event correlating with pH dysregulation. This suggests pH changes may affect transporters of the BBB and modulate drug delivery to the brain. To better understand the increased efficacy of sumatriptan in rats and seek a mechanism for increased sumatriptan transport in vitro we studied Oatp1a4 in the context of CSD. Oatp1a4 had previously been suggested to have a role in sumatriptan transport. Our studies revealed no change in expression or localization of Oatp1a4 in high extracellular KCl in vitro in bEnd.3 mouse cells. Secondly, in an in vitro evaluation of sumatriptan transport E3S, an Oatp inhibitor, was used to treat the BEB monolayer decreasing the amount of sumatriptan transport in the presence of high extracellular KCl. In vivo in situ brain perfusions with3H-sumatriptan were performed utilizing E3S pretreatment before CSD induction we observed no significant difference in sumatriptan transport post CSD but we did observe a time dependent effect on transport of sumatriptan across ass groups. This study indicates Oatp1a4 is not a major transporter of sumatriptan and depending on the timing from CSD induction there is less sumatriptan uptake in the brain. Molecular mechanisms for BBB dysregulation and pain behavior and modulation are poorly understood in migraine with aura. In this study we researched the effect of HSP90 inhibition in our models of CSD in relation to migraine with aura. HSP90 inhibition has previously been shown to be neuroprotective of the BBB in a ischemic stroke model. In this study we explored how it would affect the BBB in conditions related to spreading depression. In vitro HSP90 inhibition reduced the drop in TEER of a BEB monolayer of bEnd.3 mouse cells caused by the presence of high extracellular KCl. Also in vitro pretreated BEB monolayers were shown to have reduced paracellular leak in the presence of high extracellular KCl. In vivo insitu brain using 14C-sucrose assessed BBB integrity in female rats, 17-AAG pretreated animals showed a significant reduction in sucrose uptake in the cortex of the brain suggesting reduced paracellular leak. Tissue western blots revealed animals pretreated with 17-AAG had increased protein detection levels of tight junction protein claudin 5 in the cortex and PAG regions of the brain showing HSP90 inhibition and upregulation of claudin 5 to be neuroprotective of the BBB in CSD conditions. Regarding pain associated with migraine with aura we utilized our in vivo CSD model in female rats and assessed periorbital allodynia with pretreatment of 17-AAG. We observed HSP90 inhibition showed ameliorating effects on CSD induced periorbital allodynia with an intermediate dose of 0.5 nmol over the dura mater. The theory of clinical endocannabinoid deficiency in patients with headache has shown to correlate with data collected in our lab, HSP90 has co-chaperones which affect the endocannabinoid system. In this study we tested the relationship of HSP90 inhibition and the CB1R system in vivo. We repeated behavioral experiments with 17-AAG pretreatment of female rats, 30 minutes before CSD induction in 17-AAG pretreated rats we gave CB1R antagonist/invers agonist rimonabant. We observed the ameliorative effects of 17-AAG pretreatment to be ablated suggesting CB1R involvement in pain modulation associated with HSP90 inhibition during CSD. For molecular experiments ex vivo we focused on the PAG of the brain as we previously observed significant effects from CSD on HSP90 protein expression as well as previous research suggest endocannabinoid dysregulation in this region. We found that 17-AAG pretreatment significantly increased anandamide (AEA) in the PAG 90 minutes post CSD induction using LC/MS. We also report that 17-AAG pretreatment caused a significant decrease in expression of AEA hydrolysis enzyme FAAH 90 minutes post CSD induction. We also observed no change in expression of the CB1R using western blots and a right ward shift in CB1R activity using 35S-GTPγ functional binding in PAG tissue pretreated with 17-AAG and harvested 90 minutes post CSD induction in female rats. These results suggest HSP90 inhibition positively modulates the endocannabinoid system to cause pain relieving effects through descending pain modulation post CSD to prevent headache associated periorbital allodynia in female rats. To further explore possible therapeutics for migraine with aura we assessed the efficacy of ABHD6 inhibition to increase 2-AG levels in our in vivo model of CSD in female rats. Previous work has shown decreased 2-AG levels in the brain could lead to headache pain. In this study we observed ABHD6 inhibition to be effective in both the prevention and reversal of periorbital allodynia post CSD induction. Also, we utilized both CB1 and CB2 receptor inhibitors in the prevention and reversal paradigm to understand which receptor may play a role in headache pain modulation. We report the CB1R to have a major role in ABHD6 inhibition in the reversal of headache pain but not prevention leaving further studies to be warranted. We also investigated the role of 2-AG in BBB maintenance in vitro and in vivo. We report in this study that with use of LEI106, a DAGLα inhibitor, as 2-AG depletion mechanism there was a decrease in TEER using a bEnd3 BEB monolayer. Using in situ brain perfusions with 14C-sucrose we assessed BBB integrity 120m minutes post LEI106 I.P. injections into female rats. We observed a significant increase of sucrose in the PAG region of the brain and a near significant increase in the cortex suggesting 2-AG is important for BBB maintenance in key regions of the brain associated with headache and migraine pathology. Finally, in this study we assessed BBB integrity after acute and chronic morphine treatment like what is observed in medication induced headache. Previous results showed a decrease in 2-AG in the PAG of female rats with morphine induced medication over-use headache (MOH). As 2-AG depletion has shown to be important for BBB maintenance in the PAG specifically we investigated how morphine induced MOH affected the BEB in vitro using bEnd3 mouse cells. We report no significant change in TEER between acute and chronic morphine. To further investigate the effects of chronic morphine on paracellular leak we used 14C-sucrose to assess the integrity of the BEB in vitro, morphine alone had no effect on paracellular leak however morphine in the presence of high extracellular KCl seemed to reduce the transient leak caused by KCl. In vivo assessment of female rats continuously administered morphine for seven days with MOH induced periorbital behavior were assessed using in situ brain perfusions of 14C-sucrose for 15 minutes to determine BBB integrity. We show significant uptake of sucrose in the PAG region of the brain, however at this time more control animals are necessary for confirmation. The results presented in this dissertation demonstrate that CSD affects ASIC channels which can alter the microenvironment of the BBB which can affect drug delivery. These changes can be led to new strategies for treatment of migraine pain. Also, HSP90 inhibition with 17-AAG has proven to be useful in ameliorating BBB leak and headache pain associated with migraine with aura. 17-AAG has also provided insight into molecular mechanisms associated with the positive effect of HSP90 inhibition and provided new avenues for research topics in the field of headache and migraine. Topics related to claudin 5 regarding its neuroprotective effects of the BBB and further knowledge into how clinical endocannabinoid deficiency can be regulated to provide pain relief for headache pain. Evidence is provided for ABHD6 inhibition as a therapeutic target for headache. Its mechanism of action is reliant on CB1R activity. Investigation into the PAG region for confirmation that the therapy is due to action in descending modulation is further needed. Low 2-AG levels also affect BBB integrity further evidenced by the effect of morphine induced MOH on the BBB in the PAG. Based on previous results with HSP90 inhibition, future studies involving HSP90 in morphine induced MOH models may be of interest.