# Brain Hsp90 Inhibition Mitigates Facial Allodynia in a Rat Model of CSD Headache and Upregulates Endocannabinoid Signaling in the PAG

## TL;DR

Inhibiting Hsp90 in the brain reduces facial pain in a rat model of headache and boosts endocannabinoid signaling.

## Contribution

This study is the first to show that Hsp90 inhibition mitigates facial allodynia and modulates endocannabinoid signaling in the PAG during cortical spreading depression.

## Key findings

- Hsp90 inhibition with 17-AAG prevents facial allodynia in a rat model of CSD headache.
- Hsp90 inhibition increases anandamide levels in the PAG by upregulating NAPE-PLD and downregulating FAAH.
- The analgesic effect of Hsp90 inhibition is blocked by a CB1 receptor antagonist, indicating endocannabinoid system involvement.

## Abstract

Background/Objectives: The role of the molecular chaperone heat shock protein 90 (Hsp90) in pain and analgesia has been recognized; however, no study to date has investigated its role in facial allodynia during headache. In the current study, we examined the role of Hsp90 and its possible connection to the endocannabinoid system utilizing a rodent model of cortical spreading depression (CSD). Methods: CSD, a physiological phenomenon associated with headache disorders, was induced by cortical injection of KCl in female Sprague Dawley rats. To selectively inhibit Hsp90, 17-AAG was applied on the dura mater 24 h before CSD induction. Periorbital allodynia was assessed by von Frey filaments, while tissue samples were subjected to LC-MS, qPCR, Western immunoblotting, and the GTPγS coupling assay. Results: Increased expression of Hsp90 was selectively observed in the periaqueductal gray (PAG) harvested 90 min after cortical KCl injection, suggesting increased cellular stress from CSD induction. Application of 17-AAG (0.5 nmol) on dura mater 24 h before CSD induction significantly prevented facial allodynia as measured by von Frey filaments. This effect was blocked by injection of the CB1R antagonist rimonabant (1 mg/kg, ip). The pretreatment with 17-AAG significantly increased the level of anandamide (AEA) in PAG 90 min after cortical insult, as measured by LC-MS. This effect was accompanied by reduced expression of FAAH and increased expression of NAPE-PLD in the same nuclei. Conclusions: These results suggest that Hsp90 inhibition positively modulates the endocannabinoid system, causing pain relief through descending pain modulation in PAG post-CSD.