Ultrasound localization microscopy and functional ultrasound imaging reveal atypical features of the trigeminal ganglion vasculature

ARTICLE https://doi.org/10.1038/s42003-022-03273-4 OPEN Ultrasound localization microscopy and functional ultrasound imaging reveal atypical features of the trigeminal ganglion vasculature 1234567890():,; Annabelle Réaux-Le-Goazigo1, Benoit Beliard2, Lauriane Delay 2, Line Rahal2, Julien Claron2, Noémi Renaudin2, Isabelle Rivals 3, Miguel Thibaut2, Mohamed Nouhoum2,4, Thomas Deffieux2, Mickael Tanter 2 & Sophie Pezet 2 ✉ The functional imaging within the trigeminal ganglion (TG) is highly challenging due to its small size and deep localization. This study combined a methodological framework able to dive into the rat trigeminal nociceptive system by jointly providing 1) imaging of the TG blood vasculature at microscopic resolution, and 2) the measurement of hemodynamic responses evoked by orofacial stimulations in anesthetized rats. Despite the small number of sensory neurons within the TG, functional ultrasound imaging was able to image and quantify a strong and highly localized hemodynamic response in the ipsilateral TG, evoked not only by mechanical or chemical stimulations of corneal nociceptive fibers, but also by cutaneous mechanical stimulations of the ophthalmic and maxillary orofacial regions using a von Frey hair. The in vivo quantitative imaging of the TG’s vasculature using ultrasound localization microscopy combined with in toto labelling reveals particular features of the vascularization of the area containing the sensory neurons, that are likely the origin of this strong vasotrigeminal response. This innovative imaging approach opens the path for future studies on the mechanisms underlying changes in trigeminal local blood flow and evoked hemodynamic responses, key mechanisms for the understanding and treatment of debilitating trigeminal pain conditions. 1 Sorbonne Université, INSERM, CNRS, Institut de la vision, 17 rue Moreau, 75012 Paris, France. 2 Physics for Medicine Paris, Inserm, ESPCI Paris, CNRS, PSL Research University, 17 rue Moreau, 75012 Paris, France. 3 Equipe de Statistique Appliquée, ESPCI Paris, PSL Research University, UMRS 1158, 10 rue Vauquelin, 75005 Paris, France. 4 Iconeus, 27 Rue du Faubourg Saint-Jacques, 75014 Paris, France. ✉email: COMMUNICATIONS BIOLOGY | (2022)5:330 | https://doi.org/10.1038/s42003-022-03273-4 | www.nature.com/commsbio 1 ARTICLE T COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-03273-4 he trigeminal ganglion (TG) contains the cell body of the primary sensory neurons from the ophthalmic (V1), the maxillary (V2), and the mandibular (V3) nerves. These sensory neurons are highly specialized, as they detect and respond to a variety of chemical, mechanical, and thermal stimuli applied on these regions. Because the TG is relatively small and localized in Meckel’s trigeminal cave in both human and rodents, only a limited number of studies were able to perform functional neuroimaging studies. While Bererra’s and Borsook’s teams published seminal works on the existence of a vascular response in the human TG1–4, only one preclinical contrast MRI study imaged macrophage infiltration in the mouse TG using ultrasmall superparamagnetic iron oxide nanoparticle contrast in a model of alkali burn cornea5. But, to the best of our knowledge, dynamic functional imaging of the TG was never performed in rodents due to the difficulties to access the TG. Preclinical studies investigating the physiological activity within the TG of rodents are classically based on electrophysiological recordings of single and/or clusters of neurons6,7, as well as immunohistochemical staining using indirect markers of neuronal activation (see ref. 8 for review). Despite the cellular resolution of these surrogates, this approach lacks the ability to follow the dynamics of these neuronal changes. Recently, the visualization of trigeminal sensory neuron activities in response to orofacial stimuli was reported ex vivo using either voltage-sensitive dye approach in decerebrated animals9 or calcium imaging in GCaMP6 mouse line10,11. However, these highly invasive experimental paradigms require the decerebration of the animal, and therefore the disconnection between TG and the CNS. Functional ultrasound (fUS) imaging is a relatively new versatile neuroimaging modality that allows imaging and measurement of cerebral blood volume in both human12,13, non-human primates14 and rodents15–19 with excellent spatial (100–300 µm) and temporal resolutions (down to 20 ms). One of its biggest advantages is its high sensitivity compared to fMRI20–22. Indeed, during a task, the locally increased neuronal activity due to the neurovascular coupling leads to a hemodynamic response23. The direct link between fUS signal and neuronal activity was recently described, as well as the hemodynamic response function21,24. In the past, fUS imaging has proven sensitive enough to measure the cortical hemodynamic changes induced by optogenetic stimulations22,25, sensory18,25, olfactory26, and visual27,28 stimuli in anesthetized animals, as well as auditory stimuli29 and motor tasks14–16 in awake animals. Interestingly, fUS can be coupled on the same device with another emerging modality, Ultrasound Localization Microscopy (ULM), enabling the observation of the brain vascular anatomy and blood flow up to microscopic resolution both in rodents30 and humans31. The corneal trigeminal system is particularly interesting as the cornea is the most densely innervated tissue in the body8 whose nerve terminals are directly accessible for stimulation. Moreover, the cornea is exclusively innervated by unmyelinated C- and thinly myelinated A delta fibers, including mechano-nociceptors that are triggered by noxious mechanical stimulation, polymodal nociceptors that are excited by mechanical, chemical, and thermal stimuli, and cold thermoreceptors that are activated by cooling8,32,33. Taking advantage of the high sensitivity of fUS imaging, this study had several main objectives: first to localize the TG in anesthetized rats, second to measure the velocity of blood flow in the TG using ULM, and third to detect and measure the functional activation in the TG induced by peripheral stimulations of various orofacial trigeminal divisions (ophthalmic V1 and maxillary V2). We provide the first proof of concept of imaging the rat’s TG, with a detection of local blood flow at a microscopic 2 scale, and of the measurement of the hemodynamic responses evoked by the activation of trigeminal nociceptors in anesthetized animals. Our results bring forward an innovative approach to study the TG’s evoked hemodynamic responses, a key element for deciphering the mechanisms of trigeminal sensitization and concomitant pain characteristic of trigeminal pathologies. Results Localization/imaging of the rat trigeminal ganglia using ultrafast Doppler imaging. Taking into account that TG is a deep structure, we imaged much deeper under the brain as compared to previous studies in anaesthetized rodents18,19. Despite t (...truncated)