Exploring the effects of extracranial injections of botulinum toxin type A on activation and sensitization of central trigeminovascular neurons by cortical spreading depression in male and female rats.

Background: OnabotulinumtoxinA (onabotA), is assumed to achieve its therapeutic effect in migraine through blocking activation of unmyelinated meningeal nociceptors and their downstream communications with central dura-sensitive trigeminovascular neurons in the spinal trigeminal nucleus (SPV). The present study investigated the mechanism of action of onabotA by assessing its effect on activation and sensitization of dura-sensitive neurons in the SPV by cortical spreading depression (CSD). It is a follow up to our recent study on onabotA effects on activation and sensitization of peripheral trigeminovascular neurons.
Methods: In anesthetized male and female rats, single-unit recordings were used to assess effects of extracranial injections of onabotA (five injections, one unit each, diluted in 5 μl of saline were made along the lambdoid (two injection sites) and sagittal (two injection sites) suture) vs. vehicle on CSD-induced activation and sensitization of high-threshold (HT) and wide-dynamic range (WDR) dura-sensitive neurons in the SPV.
Results: Single cell analysis of onabotA pretreatment effects on CSD-induced activation and sensitization of central trigeminovascular neurons in the SPV revealed the ability of this neurotoxin to prevent activation and sensitization of WDR neurons (13/20 (65%) vs. 4/16 (25%) activated neurons in the control vs. treated groups, p  = 0.022, Fisher's exact). By contrast, onabotA pretreatment effects on CSD-induced activation and sensitization of HT neurons had no effect on their activation (12/18 (67%) vs. 4/7 (36%) activated neurons in the control vs. treated groups, p  = 0.14, Fisher's exact). Regarding sensitization, we found that onabotA pretreatment prevented the enhanced responses to mechanical stimulation of the skin (i.e. responses reflecting central sensitization) in both WDR and HT neurons. In control but not treated WDR neurons, responses to brush ( p =  0.004 vs. p =  0.007), pressure ( p  = 0.002 vs. p  = 0.79) and pinch ( p  = 0.007 vs. 0.79) increased significantly two hours after CSD. Similarly, in control but not treated HT neurons, responses to brush ( p =  0.002 vs. p =  0.79), pressure ( p  = 0.002 vs. p  = 0.72) and pinch ( p  = 0.0006 vs. p  = 0.28) increased significantly two hours after CSD. Unexpectedly, onabotA pretreatment prevented the enhanced responses of both WDR and HT neurons to mechanical stimulation of the dura (commonly reflecting peripheral sensitization). In control vs. treated WDR and HT neurons, responses to dural stimulation were enhanced in 70 vs. 25% ( p  = 0.017) and 78 vs. 27% ( p  = 0.017), respectively.
Conclusions: The ability of onabotA to prevent activation and sensitization of WDR neurons is attributed to its preferential inhibitory effects on unmyelinated C-fibers. The inability of onabotA to prevent activation of HT neurons is attributed to its less extensive inhibitory effects on the thinly myelinated Aδ-fibers. These findings provide further pre-clinical evidence about differences and potentially complementary mechanisms of action of onabotA and calcitonin gene-related peptide-signaling neutralizing drugs.
Competing Interests: Declaration of conflicting interestsThe authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: RB is the John Hedley-Whyte Professor of Anesthesia and Neuroscience at the Beth Israel Deaconess Medical Center and Harvard Medical School. He has received research support from the NIH: R01 NS094198-01A1, R37 NS079678, R01NS095655, R01 NS104296, R21 NS106345, Allergan, Teva, Dr Ready, Eli Lilly, Trigemina and the Migraine Research Foundation. He is a reviewer for NINDS, holds stock options in AllayLamp and Percept; serves as consultant, advisory board member, or has received honoraria from: Alder, Allergan, Biohaven, Dr Reddy's Laboratory, Eli Lilly, GlaxoSmithKline, Merck, Teva and Trigemina. CME fees from Healthlogix, Medlogix, WebMD/Medscape, and Patents 9061025, 11732265.1, 10806890, US2021-0015908, WO21007165, US2021-0128724, WO210054RBr, BD, AMA and MFB are employees of Allergan, an AbbVie Company.The other authors declare that they have no conflicts of interest.