Vilas et al. The Journal of Headache and Pain https://doi.org/10.1186/s10194-023-01576-3 (2023) 24:41 The Journal of Headache and Pain RESEARCH Open Access Periaqueductal gray matter echogenicity as a marker of migraine chronification: a case control study Dolores Vilas1*, Sara Rubio1, Mireia Gea2, Jose Rios3,4, Lourdes Ispierto1, María Hernández‑Pérez1, Martí Paré1, Mònica Millán1 and Laura Dorado1 Abstract Background Migraine is one of the most prevalent and disabling medical diseases in the world. The periaqueductal gray matter and the red nucleus play an important role in its pathogenesis. Our aim was to evaluate the echogenicity of the periaqueductal gray matter and the red nucleus in patients with migraine, by means of transcranial ultrasound. Methods In this cross-sectional study, a group of patients with migraine (according to the International Classification of Headache Disorders) and a group of control subjects with comparable age-and-sex distribution were prospectively included. We evaluated the area and echogenicity of the periaqueductal gray matter and the red nucleus by means of transcranial ultrasound, both bedside and posteriorly analyzed with the medical image viewer Horos. Results We included 115 subjects: 65 patients with migraine (39 of them with chronic migraine and 26 with episodic migraine), and 50 controls. Median disease duration in patients with chronic migraine was 29 (IQR: 19; 40) years, with a median of 18 (IQR: 14; 27) days of migraine per month. The area of the periaqueductal gray matter was larger in patients with chronic migraine compared to episodic migraine and controls (0.15[95%CI 0.12;0.22]cm2; 0.11[95%CI 0.10;0.14]cm2 and 0.12[95%CI 0.09;0.15]cm2, respectively; p = 0.043). Chronic migraine patients showed an intensity of the periaqueductal gray matter echogenicity lower than controls (90.57[95%CI 70.87;117.26] vs 109.56[95%CI 83.30;122.64]; p = 0.035). The coefficient of variation of periaqueductal gray matter echogenicity was the highest in chronic migraine patients (p = 0.009). No differences were observed regarding the area or intensity of red nucleus echogenicity among groups. Conclusion Patients with chronic migraine showed a larger area of echogenicity of periaqueductal gray matter, a lower intensity of its echogenicity and a higher heterogenicity within this brainstem structure compared to patients with episodic migraine and controls. The echogenicity of the periaqueductal gray matter should be further investi‑ gated as a biomarker of migraine chronification. Keywords Migraine, Chronic migraine, Periaqueductal gray matter, Echogenicity, Transcranial sonography *Correspondence: Dolores Vilas dvilas.germanstrias@gencat.cat Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Vilas et al. The Journal of Headache and Pain (2023) 24:41 Page 2 of 8 Background patients with migraine, as a marker of structural damage, Migraine is one of the most prevalent and disabling and consequent migraine chronification. medical diseases in the world. The World Health Organization (WHO) classifies migraine as the leading Material and methods cause of disability between the ages of 15 and 49, and Study design and patient selection the second leading cause of disability for activities of This is a cross-sectional study conducted at the Head- daily living globally [1]. According to the international ache Unit of the Hospital Universitari Germans Trias classification of headaches (ICHD-3) [2] migraine is i Pujol, from March 2020 to June 2022. A prospective chronic (CM) if patients have headache 15 or more recruitment was carried out among those patients evalu- days per month, of which at least eight meet criteria ated in the unit with a diagnosis of migraine according for migraine, and for minimum during 3 months [2]. to the ICHD-3. Patients with a known neurological dis- Approximately, 3% of patients with episodic migraine ease other than migraine were excluded from the study. (EM) develop CM each year [3], being patients with The coexistence of other primary headaches was not an CM those with a greater disability [4]. exclusion criterion in the patients with migraine group. Although the pathophysiology of migraine is not yet For comparative purposes, a group of control subjects, fully understood, the trigeminovascular system, the without diagnosis of migraine or other neurological hypothalamus, the brainstem nuclei and the cortex are pathologies, and with comparable age and sex distribu- involved in the generation of migraine headache [5]. tion to the cases, was also evaluated. Control subjects Among the brainstem nuclei, the periaqueductal gray were prospectively recruited from non-blood relatives matter (PAG) and the red nucleus (RN) play an important of patients included in the study, as well as from healthy role in the pathogenesis of the migraine. The PAG con- volunteers. A detailed interview, conducted by a head- trols nociceptive responses [6], allows the inhibition of ache-expert neurologist (LD), was performed to ensure the painful stimulus and connects with encephalic struc- that controls had no history of migraine or other primary tures, ascending medullary fibers and pons’ structures headaches. Subjects who were on antidepressant treat- as the raphe nucleus magnus [7]. Magnetic resonance ment or treatment for chronic pain were excluded. imaging studies have demonstrated the presence of iron This study was approved by the Research Ethics Com- deposits in the PAG and RN in patients with migraine mittee of Hospital Germans Trias i Pujol (PI-20–081). All [8, 9]. These iron depositions, and its size have been subjects signed the informed consent form for participa- correlated with the frequency and intensity of migraine tion in the study and use of their clinical data for research attacks, and the time course of the disorder, suggesting purposes. a causal relationship between recurrent attacks and iron accumulation [8, 9]. Clinical variables A precise diagnosis of migraine, especially in CM, may Demographic variables and comorbidities were collected be potentially difficult, since patients might suffer a non- in all the subjects included in the study. In migraine specific pain that can lead to errors in diagnosis [10]. patients, disease duration, characteristics and frequency The identification of migraine biomarkers would help of migraine (headache days per month, migraine days per to increase the accuracy in its diagnosis, to improve the month), and symptomatic treatment were recorded. Dis- knowledge of the underlying pathogenic mechanisms, ability and quality of life associated with migraine were and to predict its progression and response of a thera- assessed using the Migraine Disability Assessment Scale peutic intervention [11]. (MIDAS) [14], the Headache Impact Test (HIT-6) [15] Transcranial ultrasound (TCS) is a noninvasive imaging and the version 2 of the Migraine-Specific Quality-of-Life technique, harmless and easy-to-perform, with a demon- Questionnaire (MSQV2.19) [16]. The Hospital Anxiety strated utility for the visualization of deep brain struc- and Depression Scale (HADS) [17] was completed. tures, such as the substantia nigra (SN), the raphe nuclei or the third ventricle. In particular, TCS has proven its Transcranial sonography usefulness in the diagnostic workout of movement dis- TCS was performed in all participants by a neurologist orders, such as Parkinson’s Disease (PD), where approxi- (DV) experienced in performing and interpreting tran- mately 90% of patients present with hyperechogenicity scranial sonography of deep brain structures [14, 15]. of the substantia nigra, now demonstrated biomarker for This neurologist was not related with the Headache the prodromal phase of Parkinson’s disease (PD) [12, 13]. Unit and was blinded to clinical data. A 2 MHz phased- The aim of the current study was to assess the useful- array transducer was used (Philips Affiniti 70 ultra- ness of TCS for migraine diagnosis. We hypothesize sound machine), with a penetration depth of 14 cm that the echogenicity of the PAG and RN is increased in and a dynamic range of 45–55 db. Image brightness Vilas et al. The Journal of Headache and Pain (2023) 24:41 Page 3 of 8 and time-gain compensation were predetermined and with similar ultrasound systems [18]. The echogenicity were not changed across the study. Tissue harmonic of the brainstem raphe nuclei was graded as hypoecho- imaging settings were applied to increase the tissue genic when this midline structure in the midbrain was contrast. The examination was performed from both interrupted or not visible [19]. The width of the third sides of the head using the transtemporal bone window ventricle (IIIv) was measured by taking the minimum to evaluate the mesencephalic and the thalamic plane. transverse diameter in the thalamic plane and the right Images were analyzed in two different ways. First, dur- and left frontal horn of the lateral ventricles in the same ing the performance of TCS we made a bedside analy- plane. Secondly, the TCS images were de-identified and sis. The PAG was defined, as observed by TCS, as a subsequently analyzed with the medical image viewer structure surrounding the aqueduct of Sylvius, with a Horos by a second blinded explorer (SRG), previously higher echogenicity than the cerebrospinal fluid signal trained. Horos is a free and open source code software observed in the aqueduct. The PAG area was manu- (FOSS) program that is distributed free of charge under ally delimited and measured (Fig. 1). The echogenicity the LGPL license at Horosproject.org and sponsored by of the right and left RN was also evaluated. The RN is Nimble Co LLC d/b/a Purview in Annapolis, MD USA. usually seen as a small white dot near the brainstem All the structures previously evaluated in the bedside midline, posterior to the substantia nigra (SN), which TCS were again assessed by means of Horos, with the is isoechogenic to the basal cisterns. When observed, same methodology. In addition, this program allows to the area of echogenicity of RN was manually delimited quantify the intensity of the echogenicity of a region of and measured. For internal validation purposes, other interest (ROI). We manually outlined the area of echo- deep brain structures were also evaluated. Substan- genicity of the PAG and the RN, and the program gave tia nigra (SN) hyperechogenicity was considered if the us a histogram of each ROI, with the mean, the mini- area of SN echogenic signal area was equal to or greater mum and maximum echogenicity (unnamed units) of than 0.20 cm2, following the cut-off values usually used this region of interest. Fig. 1 Magnetic resonance and transcranial sonography images of midbrain axial sections showing the periacueductal gray matter. The TCS images show images from our sonographic laboratory obtained with an ultrasound system for clinical application (Philips Affiniti 70 ultrasound machine). A MRI of the axial section at the level of the midbrain. The square box shows the area corresponding to the magnified TCS image shown in B. B TCS image of the axial midbrain section showing the area of the periaqueductal gray matter echogenicity Vilas et al. The Journal of Headache and Pain (2023) 24:41 Page 4 of 8 Statistical analysis Results Results are described as median with interquartile We included 115 subjects (65 patients with migraine and range (IQR: 2­ 5th; ­75th percentiles) or with absolute fre- 50 controls). Among patients with migraine, 39 (60%) had quencies and percentages, with inferential statistical CM and 26 (40%) EM. Table 1 shows demographic and analyses between groups by means Fisher’s exact test clinical characteristics of all participants. Neither sta- or Mann–Whitney U test for qualitative or quantita- tistical nor numerical differences were observed among tive variables respectively. Capacity of discrimination groups regarding age and sex. Median disease duration from PAG echogenicity between controls and patients was 29 (IQR: 19; 40) years for CM patients, with a median was assessed by means of the area under the ROC of 18 (IQR: 14; 27) days of migraine per month and 28 curve (AUC) and its 95% confidence interval (95%CI). (IQR: 24; 30) days of headache per month. Regarding EM, To analyze the morphologic changes within the PAG median disease duration was 30 (IQR: 22; 40) years, with and the RN, we performed a texture analysis of both a median of 9 (IQR: 2; 12) days of migraine per month structures, comparing the extracted variables from his- and 9.5 (IQR: 3; 12) days of headache per month. tograms of their echogenicity, obtained from the Horos viewer, from patients with migraine and controls. The Transcranial sonography findings magnitude of the intensity of echogenicity was evalu- Forty-eight (96%) controls and 64 (98.5%) patients with ated with the mean ROI, while the heterogeneity of migraine had an adequate transtemporal bone window to this intensity was estimated with the coefficient of var- assess the echogenicity of the PAG. The echogenicity of iation. Accordingly, not only the intensity of the echo- the right RN was assessed in 65 (56,52%) of participants genicity but also its texture could be evaluated jointly. and echogenicity of the left RN in 63 (54,78%) because of Differences were considered statistically significant for an inadequate bone window. a two-sided type I error of 0.05. SPSS Version 26 (IBM According to the bedside assessment, the median area Corp. Armonk, NY. USA) was used for all statistical of the PAG was higher in patients with CM compared to analyses. EM and controls, with 0.15 (IQR: 0.12;0.22), 0.11 (IQR: 0.10;0.14) and 0.12 (IQR: 0.09;0.15) c­m2, respectively Table 1 Demographic and clinical characteristics of patients with migraine and controls Controls (n = 50) Migraine CM (n = 39) EM (n = 26) p value (C vs M) p value (CM vs EM) patients (n = 65) Age (years) 43.50 45.00 45.00 45.00 0.954 0.981 [28.00;65.00] [38.00;53.00] [38.00;53.00] [38.00;54.00] Sex, female (n,%) 34 (68%) 47 (72.3%) 27 (69.23%) 20 (76.92%) 0.682 0.579 Disease duration (years) - 30.00 29.00 30.00 - 0.384 [20.00;40.00] [19.00;40.00] [22.00;40.00] Duration of CM (months) - - 60.00 - NA [40.00;120.00] Days of migraine per month - 14.00 18.00 9.00 - < 0.001 [10.00;19.00] [14.00;27.00] [2.00;12.00] Days of headache per month - 20.00 28.00 9.50 - < 0.001 [12.00;30.00] [24.00;30.00] [3.00;12.00] NSAIDs overuse (n,%) - - 11 (28.21%) 1 (3.85%) - 0.02 Triptans overuse (n,%) - - 12 (30.77%) 7 (26.92%) - 0.787 MIDAS, total score - 60.00 84.00 21.00 - < 0.001 [20.00;96.00] [56.00;122.00] [3.50;36.50] HIT-6 67.00 68.00 64.00 - 0.004 [62.00;70.00] [65.00;70.00] [55.50;67.00] MSQV2.19 42.85 34.29 63.58 - < 0001 [30.00;65.58] [22.85;51.43] [46.43;88.58] HADS Anxiety, score - 9.00 11.00 7.50 - 0.002 [6.00;14.00] [7.00;16.00] [4.00;10.00] HADS Depression, score 6.00 9.00 4.00 - 0.001 [4.00;10.00] [5.00;12.00] [3.00;8.00] C Controls, M Migraine patients, CM Chronic migraine, EM Episodic migraine, NSAIDs Nonsteroidal anti-inflammatory drugs, MIDAS Migraine Disability Assessment Scale, HIT-6 Headache Impact Test, MSQV2.19 Version 2 of the Migraine-Specific Quality-of-Life Questionnaire, HADS Hospital Anxiety and Depression Scale Vilas et al. The Journal of Headache and Pain (2023) 24:41 Page 5 of 8 (p = 0.043). Also, we found similar results when per- In addition, CM patients had higher coefficient of vari- forming the analysis with the Horos medical viewer ation of the PAG echogenicity (p = 0.009) compared to (Table 2). EM and controls (Table 2). The area of PAG echogenic- PAG intensity of echogenicity was significantly lower in ity and the intensity of the echogenicity of the PAG were CM patients than in controls, 90.57 (IQR: 70.87;117.26) similar among CM or EM patients with and without vs 109.56 (IQR: 83.30;122.64), p = 0.035. EM patients and NSAIDs or triptans overuse (Supplementary Table 1). controls showed similar intensity of the PAG echogenic- No differences were observed regarding the area or ity (p = 0.457). intensity of RN echogenicity among groups, neither in the Table 2 Sonographic variables of patients with migraine and controls Controls Migraine CM (n = 39) EM (n = 26) Controls vs Controls Controls CM vs EM (n = 50) patients (n = 65) migraine patients vs CM vs EM Bed-side analysis PAG area 0.12 0.13 0.15 0.11 0,043* 0,004* 0,895 0.017 [0.09;0.15] [0.11;0.17] [0.12;0.22] [0.10;0.14] Right RN 0.06 0.05 0.05 0.06 0,281 0,336 0,369 0.940 [0.05;0.08] [0.04;0.07] [0.04;0.07] [0.05;0.07] Left RN 0.07 0.07 0.07 0.06 0,868 0,962 0,782 0.763 [0.05;0.09] [0.05;0.08] [0.05;0.09] [0.05;0.08] Right SN area 0.09 0.10 0.11 0.10 0,082 0,013* 0,935 0.127 [0.07;0.12] [0.08;0.13] [0.08;0.14] [0.07;0.12] Left SN area 0.11 0.11 0.11 0.10 0,793 0,478 0,697 0.308 [0.08;0.13] [0.09;0.13] [0.09;0.14] [0.08;0.13] IIIv size 0.33 0.29 0.29 0.30 0,092 0,13 0,205 0.914 [0.27;0.45] [0.21;0.44] [0.21;0.46] [0.25;0.38] Right horn LV 1.40 1.45 1.54 1.39 0,367 0,06 0,535 0.026* [1.26;1.55] [1.32;1.60] [1.40;1.60] [1.25;1.47] Left horn LV 1.50 1.49 1.49 1.54 0,771 0,868 0,462 0.352 [1.32;1.68] [1.35;1.67] [1.31;1.60] [1.35;1.68] Horos analysis PAG area 0.13 0.16 0.16 0.17 0,038* 0,04* 0,191 0.650 [0.12;0.18] [0.13;0.20] [0.13;0.20] [0.12;0.20] PAG echogenicity (intensity) 109.56 93.84 90.57 101.70 0,07 0,035* 0,457 0.221 [83.30;122.64] [71.34;117.26] [70.87;117.26] [76.44;115.17] PAG echogenicity (CV) 25.40 29.26 32.24 25.84 0,12 0,009* 0,688 0.003* [20.08;31.84] [23.69;35.02] [25.74;35.83] [21.78;30.95] Right SN area 0.10 0.12 0.13 0.12 0,006* 0,09* 0,003* 0.260 [0.08;0.14] [0.10;0.15] [0.09;0.14] [0.10;0.15] Rigth SN echogenicity 82.87 79.76 79.76 80.80 0,96 0,769 0,8 0.743 (intensity) [68.57;95.34] [63.52;100.93] [59.48;99.03] [67.74;101.18] Rigth SN echogenicity (CV) 27.08 28.13 29.27 27.48 0,545 0,624 0,608 0,940 [20.92;33.90] [22.45;37.52] [20.37;40.22] [23.30;34.00] Right RN area 0.07 0.08 0.08 0.08 0,954 0,96 0,838 0.612 [0.07;0.08] [0.07;0.09] [0.07;0.09] [0.07;0.10] Rigth RN echogenicity 85.54 69.61 75.59 61.56 0,062 0,077 0,15 1.000 (intensity) [67.46;104.89] [53.52;97.05] [53.52;86.33] [56.12;100.41] Rigth RN echogenicity (CV) 23.85 28,79 28.92 28.30 0,135 0,193 0,193 0.780 [20.04;29.51] [20.59;31.83] [22.10;32.81] [26.43;30.69] Left SN area 0.10 0.11 0.11 0.12 0,01* 0,027* 0,033* 0.632 [0.08;0.12] [0.09;0.16] [0.09;0.16] [0.09;0.16] Left SN echogenicity 82.17 78.06 74.35 80.49 0,972 0,827 0,709 0.500 (intensity) [55.90;102.12] [62.68;95.60] [64.13;94.45] [61.23;106.43] Left SN echogenicity (CV) 26.39 28.43 28.43 27.07 0,163 0,179 0,348 0.848 [20.16;29.87] [21.77;35.20] [23.07;32.66] [21.41;36.30] Left RN area 0.08 0.07 0.07 0.08 0,796 0,837 0,377 0.675 [0.05;0.09] [0.05;0.10] [0.06;0.08] [0.05;0.11] Left RN echogenicity 89.34 8.,45 85.47 76.00 0,235 0,268 0,377 0.884 (intensity) [73.36;108.69] [59.84;96.68] [68.49;96.60] [58.66;106.75] Left RN echogenicity (CV) 25.18 29.86 35.51 23.97 0,383 0,124 0,679 0.051 [22.07;37.25] [22.80;55.92] [24.82;64.38] [21.06;31.34] CM Chronic migraine, EM Episodic migraine, SN Substantia igra, PAG Periaqueductal gray matter, IIIv Third ventricle size, LV Lateral ventricle, CV Coefficient of variation, RN Red nucleus.*p value < 0.05 Vilas et al. The Journal of Headache and Pain (2023) 24:41 Page 6 of 8 bedside analysis or in the analysis with the Horos medical other brainstem structures related to normal antinocic- viewer (Table 2). The area of SN echogenicity was higher eptive function, contributing to migraine chronification in migraine patients than in controls, being the patients [20]. In a recent study, functional biomarkers, including with CM those with a higher SN echogenicity area PAG networks, seems to be the most important MRI fea- (right SN: CM 0.13[0.09;0.14] cm2, EM 0.12[0.10;0.15] tures in classifying migraine patients from controls [21]. cm2, controls 0.12[0.10;0.15]cm2;p = 0.006; left SN: From a sonographic point of view, CM and EM patients CM 0.11[0.09;0.16], EM 0.12[0.09;0.16], controls seems to behave different in our study, suggesting that 0.11[0.09;0.16]; p = 0.01) (see Table 2). the pathophysiology of both entities is different. We did The AUC in the ROC analysis to classify control sub- not find a cutoff point of the PAG with enough sensi- jects and patients with migraine for the area of the PAG tivity and specificity to use it as an aid in the diagnosis and for the intensity of the PAG echogenicity was 0.61 migraine patients. Further studies with larger sample (95%CI 0.51; 0.72) and 0.60 (95%CI 0.50; 0.71), respec- to improve the representativeness of migraine patients tively; and the AUC for control subjects and CM for the might evaluate this aspect. coefficient of variation of the intensity of the PAG echo- We found no differences among patients with migraine genicity was 0.59 (95%CI 0.48; 0.70). and controls regarding the RN echogenicity. This could be explained, at least partly, because we only observed Discussion this brainstem structure in around 55% of participants. The main findings of the current study are that patients It could be also possible that the RN has a lower impli- with CM presented structural changes of the PAG with cation in the pathophysiology of migraine. Functional a larger area, a lower intensity and a higher heterogenic- MRI studies as well as prospective studies with TCS in ity of its echogenicity, compared to patients with EM and patients with migraine could help to clarify this issue. controls. TCS, a noninvasive imaging technique with a demon- Our results suggest that the structure of the PAG is strated utility for the visualization of deep brain struc- altered in patients with CM, compared to controls and, tures, has proven its usefulness in the assessment of interestingly, to EM patients. Also, EM patients and movement disorders, such as PD, where approximately controls are quite similar in terms of PAG and RN echo- 90% of patients present with hyperechogenicity of the SN genicity. To our knowledge, this is the first case–control [17]. TCS has been rarely used in patients with migraine. study that evaluates the PAG and the RN by means of We previously reported that the hypoechogenicty of the TCS. If our results are replicated in further studies, the raphe nuclei is more prevalent in patients with migraine echogenicity of the PAG might emerge as a biomarker of than in controls [22], concordant with results from previ- migraine chronification. ous studies [23–26] and supporting the role of the raphe Although the pathophysiology of migraine is still under nuclei in migraine. In the current study, the first that investigation, the PAG is an important structure that assess the PAG by means of TCS, we also support the role modulates nociceptive transmission. It is thought that of the PAG in the pathophysiology of migraine. the function of brainstem pain modulating circuits con- The significance of the sonographic changes that we tributes to the maintenance of chronic neuropathic pain, observed in the PAG in patients with migraine needs to even predisposing individuals to develop chronic pain [6]. be address. The well-known hyperechogenicity of the SN PAG is activated during migraine attacks, and repeated observed in PD, seems to be due to different structural migraine attacks could lead to free radical damage asso- changes. Different imaging investigations, experimen- ciated with hyperemia and consequent iron deposition tal studies in animal models and postmortem analysis in in this area [9]. Almost 30% of the CM patients included humans, may support the hypothesis that alterations in in our study presented an overuse of anti-inflammatory local iron deposition and changes in cellular composition drugs, which is an important factor for the chronifica- in the SN lead to its hyperechogenicity [27]. In general, tion process. Although, in our cohort, CM patients with the echogenicity of any structure depends on its acous- and without medication overuse showed a similar PAG tic impedance and on the relationship with adjacent area and PAG intensity, further studies should consider structures. An increase in the echogenicity of deep brain this issue. MRI studies in migraine patients showed the structures may result from the composition of the com- presence of iron deposits in the PAG and RN. The size of ponents of neurons, glia, and connecting fibers. On the these deposits correlates with the frequency of migraine other hand, the accumulation of heavy metals, such as attacks, their intensity, and the time course of the disorder iron, copper or manganese, can result in an increase in [8, 9]. It has therefore been hypothesized that iron deposi- echogenicity. The hyperechogenicity of the SN in PD has tion may reflect progressive dysfunction of the PAG and been associated with an increase in the content of heavy Vilas et al. The Journal of Headache and Pain (2023) 24:41 Page 7 of 8 metals such as iron, copper or manganese [28] and oxida- Supplementary Information tive damage [29]. Contrary of what we suspected, inten- The online version contains supplementary material available at https://​doi.​ sity of echogenicity in PAG was lower in CM patients org/​10.​1186/​s10194-​023-​01576-3. than controls and EM. This could reflect that, other Additional file 1: Supplementary Table 1. mechanisms, different to iron deposition, are also pre- sent, leading to a different composition in the tissue of this particular area implicated in pain modulation. Fur- Acknowledgements The authors are grateful to the patients for their participation in this study. ther studies are needed to address this point. Unexpectedly, we also found that the area of SN echo- Authors’ contributions genicity was higher in migraine patients than in controls, DV performed the bedside transcranial sonography, analyzed and interpreted the patient data and was a major contributor in writing the manuscript. SR especially in CM patients. This finding was not previ- analyzed the transcranial sonography images in the medical viewer Horos ously reported and further studies are needed to confirm and analyzed the data. MG, LI, MHP and MM interpreted the results and our results. contributed in writing the manuscript. JR performed the statistical analysis. MP contributed in patient’s recruitment. LD analyzed and interpreted the data Some limitations of our study include the relatively and was a major contributor in writing the manuscript. All authors read and small sample size, the absence of comparison of the echo- approved the final manuscript. genicity of the PAG with previously reported imaging Funding biomarkers such as the MRI iron deposits and, finally, the CERCA Programme, Generalitat de Catalunya. fact that the TCS is an explorer-dependent technique. New tools are needed to reduce the delay in the diagno- Availability of data and materials The datasets used and analysed during the current study are available from sis of migraine, to facilitate the initiation of treatment at the corresponding author on reasonable request. its earlier stages and to detect the predictors of response to treatments. Ideally, these tools should be accessible Declarations to the network of physicians involved in the diagnosis of migraine, cheap and non-invasive. In this direction, if Ethics approval and consent to participate This study was approved by the Research Ethics Committee of Hospital other groups replicate our findings, TCS might be a use- Germans Trias i Pujol (PI-20–081). All subjects signed the informed consent ful tool in the diagnosis of migraine. Longitudinal stud- form for participation in the study and use of their clinical data for research ies in CM patients could also help to elucidate the role of purposes. the TCS in the prediction of response to new treatments, Consent for publication such as anti-calcitonin gene-related peptide monoclonal Not applicable. antibodies, and others recently approved in migraine. Competing interests The authors declare that they have no competing interests. Conclusions Author details The echogenicity of the PAG, evaluated by means of 1 Department of Neurosciences, Neurology Service, University Hospital TCS, seems to be a marker of disease chronification Germans Trias I Pujol, Carretera Canyet s/n, 08916, Badalona, Barcelona, Spain. 2 Institut de Recerca Germans Trias I Pujol (IGTP), Univesitat Autònoma de in migraine. If confirmed by other groups, our findings Barcelona, Badalona, Barcelona, Spain. 3 Department of Clinical Farmacology, suggest that TCS, a cheap, quick and easy-to-apply tech- Hospital Clinic and Medical Statistics Core Facility, Institut d’Investigacions nique, could be useful for the diagnosis and prognosis of Biomèdiques August Pi I Sunyer (IDIBAPS), Barcelona, Spain. 4 Biostatistics Unit, School of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain. patients with migraine. Received: 23 March 2023 Accepted: 10 April 2023 Abbreviations PAG Periaqueductal gray matter RN Red nucleus TCS Transcranial ultrasound References ICHD-3 International Classification of Headache Disorders 1. Global Burden of Disease (2016) Collaborators (2018) Global, regional, CM Chronic migraine and national burden of migraine and tension-type headache, 1990–2016: EM Episodic migraine A systematic analysis for the Global Burden of Disease Study 2016. Lancet MIDAS Migraine Disability Assessment Scale Neurol 17:954–976 HIT-6 Headache Impact Test 2. Headache Classification Committee of the International Headache Soci‑ MSQV2.19 Migraine-Specific Quality-of-Life Questionnaire HADS: Hospital ety (IHS) (2018) The International Classification of Headache Disorders, Anxiety and Depression Scale 3rd edition. Cephalalgia 38(1):1–211 SN Substantia nigra 3. May A, Schulte LH (2016) Chronic migraine: risk factores, mechanisms IIIv Third ventricle and treatment. Nat Rev Neurol 12:455–464 AUC​ Area under curve 4. Lipton RB, Bigal ME, Diamong M, Freitag F, Reed ML, Stewart WF (2007) IQR Interquartilic range Migraine prevalence, disease burden, and the need for preventive ROI Region of interest therapy. Neurology 68:343–349 Vilas et al. The Journal of Headache and Pain (2023) 24:41 Page 8 of 8 5. Ashina M, Hansen JM, Do TP, Melo-Carrillo A, Burstein R, Moskowitwz MA 27. Berg D, Grote C, Rausch M, Wesemann W, Riederer P, Becker G (1999) Iron (2019) Migraine and the trigeminovascular system-40 years and count‑ accumulation in the substantia nigra in rats visualized by ultrasound. ing. Lancet Neurol 18:795–804 Ultrasound Med Biol 25:901–904 6. Mills E, Keay KA, Henderson LA (2021) Brainstem pain-modulation 28. Guaschino E, Ghiotto N, Tassorelli C, Bitetto V, Nappi G, Moglia A, Bosone circuitry and its plasticity in neuropathic pain: insights from human brain D, Sances G (2015) Hyperechogenicity of the periaqueductal gray in imaging investigations. Front Pain Res 2:812209 chronic migraine and episodic migraine as a potential marker of progres‑ 7. Basile GA, Quartu M, Bertino S, Serra MP, Boi M, Bramanti A, Anastasi sive dysfunction: preliminary results with transcranial sonography. J GP, MIlardiCacciola DA (2021) Red nucleus structure and function: from Headache Pain 16:A61 anatomy to clinical neurosciences. Brain Struct Funct 226:69–91 29. Berg D, Roggendorf W, Schr.derKleinTatschner URT, BenzTucha PO, Preier 8. Kruit MC, Overbosch J, van Buchem MA, Ferrari MD (2009) Iron accumula‑ M, LangeReiners KWK, GerlachBecker MG (2002) Echogenicity of the tion in deep brain nuclei in migraine: a population-based magnetic substantia nigra: association with increased iron content and marker for resonance imaging study. Cephalalgia 29:351–359 susceptibility to nigrostriatal injury. Arch Neurol 59:999–1005 9. Dominguez C, Lopez A, Ramos-Cabrer P, Vieites-Prado A, Perez-Mato M, Villalba C, Sobrino T, Rodriguez-Osorio X, Campos F, Castillo J, Leira R (2019) Iron deposition in periaqueductal gray matter as a potential Publisher’s Note biomarker for chronic migraine. Neurology 92:e1076–e1085 Springer Nature remains neutral with regard to jurisdictional claims in pub‑ 10. Dodick DW, Loder EW, Manack Adams A, Buse DC, Fanning KM, Reed ML, lished maps and institutional affiliations. Lipton RB (2016) Assessing barriers to chronic migraine consultation, diagnosis, and treatment: Results from the Chronic Migraine Epidemiol‑ ogy and Outcomes (CaMEO) study. Headache 56:821–834 11. Durham P, Papapetropoulos S (2013) Biomarkers associated with migraine and their potential role in migraine management. Headache 53:1262–1277 12. Vilas D, Iranzo A, Pont-Sunyer C, Serradell M, Gaig C, Santamaria J, Tolosa E (2015) Brainstem raphe and substantia nigra echogenicity in idiopathic REM sleep behavior disorder with comorbid depression. J Neurol 262:1665–1672 13. Vilas D, Ispierto L, Álvarez R, Pont-Sunyer C, Martí MJ, Valldeoriola F, Compta Y, de Fabregues O, Hernández-Vara J, Puente V, Calopa M, Jaumà S, Campdelacreu J, Aguilar M, Quílez P, Casquero P, Lomeña F, Ríos J, Tolosa E (2015) Clinical and imaging markers in premotor LRRK2 G2019S mutation carriers. Parkinsonism Relat Disord 21:1170–1176 14. Stewart WF, Lipton RB, Kolodner K, Liberman J, Sawyer J (1999) Reliability of the migraine disability assessment score in a population-based sample of headache sufferers. Cephalalgia 19:107–114 15. Kosinski M, Bayliss MS, Bjorner JB, Ware JE, Garber WH, Batenhorst A, Cady R, Dahlöf CGH, Dowson A, Tepper S (2003) A six-item short-form survey for measuring headache impact: the HIT-6. Qual Life Res 12:963–974 16. Cole JC, Lin P, Rupnow MFT (2007) Validation of the Migraine-Specific Quality of Life Questionnaire version 2.1 (MSQ v. 2.1) for patients under‑ going prophylactic migraine treatment. Qual Life Res 16:1231–1237 17. Zigmond AS, Snaith RP (1983) The Hospital Anxiety and Depression Scale. Acta Psychiatr Scand 67:361–370 18. Berg D, Godau J, Walter U (2008) Transcranial sonography in movement disorders. Lancet Neurol 7:1044–1055 19. Tao W, Cai X, Shen J, Xue-Gong S, Wang Y (2019) Hypoechogenicity of brainstem raphe correlates with depression in migraine patients. J Head‑ ache Pain 20:53 20. Welch KM et al (2001) Periaqueductal gray matter dysfunction in migraine: cause or the burden of illness? Headache 41:629–637 21. Messina R, Sudre CH, Wei DY, Filippi M, Ourselin S, Goadsby PJ (2022) Bio‑ markers of Migraine and Cluster Headache: Differences and Similarities. Ann Neurol. https://​doi.​org/​10.​1002/​ana.​26583 22. Dorado L, Rubio-Guerra S, Valls-Carbó A, Ispierto L, Hernández-Pérez M, Paré M, Vilas D (2022) Hypoechogenicity of the raphe nuclei as a biomarker of migraine: A case-control study, review, and meta-analysis. J Neuroimaging. https://​doi.​org/​10.​1111/​jon.​13070 Ready to submit your research ? Choose BMC and benefit from: 23. Ayzenberg I, Nastos I, Strassburger-Krogias K, Obermann M, Gold R, Krogias C (2016) Hypoechogenicity of brainstem raphe nuclei is associ‑ • fast, convenient online submission ated with increased attack frequency in episodic migraine. Cephalalgia 36:800–806 • thorough peer review by experienced researchers in your field 24. Hamerla G, Kropp P, Meyer B, Rocco A, JÜrgensWalter U (2017) Midbrain • rapid publication on acceptance raphe hypoechogenicity in migraineurs: an indicator for the use of anal‑ • support for research data, including large and complex data types gesics but not triptans. Cephalalgia 37:1057–1066 25. Tao W, Cai X, Shen J, Shi XG, Wang Y (2019) Hypoechogenicity of brain‑ • gold Open Access which fosters wider collaboration and increased citations stem raphe correlates with depression in migraine patients. J Headache • maximum visibility for your research: over 100M website views per year Pain 20:53 26. Zhang Y, Liu Y, Han R, Liu K, Xing Y (2021) Hypoechogenicity of the At BMC, research is always in progress. midbrain raphe detected by transcranial sonography: an imaging biomarker for depression in migraine patients. Ther Adv Neurol Disord Learn more biomedcentral.com/submissions 14:17562864211007708