ORIGINAL RESEARCH published: 25 November 2021 doi: 10.3389/fneur.2021.767484 Functional Recovery and Serum Angiogenin Changes According to Intensity of Rehabilitation Therapy Edited by: After Stroke Adriana Bastos Conforto, Universidade de São Paulo, Brazil Nicolás Garcia-Rodriguez 1,2 , Susana Rodriguez 2 , Pedro Ignacio Tejada 3 , Zuberoa Maite Miranda-Artieda 3 , Natalia Ridao 4 , Xavi Buxó 2 , Reviewed by: Belén Rubio Ballester, María Engracia Pérez-Mesquida 2 , Maria Rosario Beseler 5† , Juan B. Salom 6,7 , Institute for Bioengineering of Laura M. Pérez 8,9 , Marco Inzitari 8,10 , Sergio Otero-Villaverde 11 , Rosa Martin-Mourelle 11 , Catalonia (IBEC), Spain Mercedes Molleda 12 , Manuel Quintana 13 , Marta Olivé-Gadea 14 , Anna Penalba 1 and Pedro M. Pimentel-Coelho, Anna Rosell 1* Federal University of Rio de 1 Janeiro, Brazil Neurovascular Research Laboratory, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Rosalia Mendez-Otero, Spain, 2 Unidad de Rehabilitación Neurológica y Daño Cerebral, Hospital Vall d’Hebron, Barcelona, Spain, 3 Unidad de Daño Federal University of Rio de Cerebral del Hospital de Górliz, Osakidetza, Górliz, Spain, 4 Servei de Medicina Física i Rehabilitació, Parc Taulí Hospital Janeiro, Brazil Universitari, Institut d’Investigació i Innovació Parc Taulí I3PT, Sabadell, Spain, 5 Servicio de Medicina Física y Rehabilitación, Hospital Universitario y Politécnico La Fe, Valencia, Spain, 6 Unidad Mixta de Investigación Cerebrovascular, Instituto de *Correspondence: Investigación Sanitaria La Fe–Universitat de Valencia, Valencia, Spain, 7 Departamento de Fisiología, Universidad de Valencia, Anna Rosell Valencia, Spain, 8 RE-FiT Barcelona Research Group, Vall d’Hebron Institute of Research, Parc Sanitari Pere Virgili, anna.rosell@vhir.org Barcelona, Spain, 9 Parc Sanitari Pere Virgili, Area of Intermediate Care, Barcelona, Spain, 10 Universitat Oberta de Catalunya, † Present address: Barcelona, Spain, 11 Hospital Marítimo de Oza, A Coruña, Spain, 12 Hospital Universitari Germans Tries i Pujol, Badalona, Spain, 13 Epilepsy Research Group and Epilepsy Unit, Vall d’Hebron Research Institute and Vall d’Hebron Hospital, Maria Rosario Beseler, Barcelona, Spain, 14 Stroke Group, Vall d’Hebron Research Institute, Barcelona, Spain Servicio de Medicina Física y Rehabilitación, Hospital Universitario Dr. Peset, Valencia, Spain Background: Rehabilitation is still the only treatment available to improve functional status after the acute phase of stroke. Most clinical guidelines highlight the need to design Specialty section: This article was submitted to rehabilitation treatments considering starting time, intensity, and frequency, according to Stroke, the tolerance of the patient. However, there are no homogeneous protocols and the a section of the journal Frontiers in Neurology biological effects are under investigation. Received: 30 August 2021 Objective: To investigate the impact of rehabilitation intensity (hours) after stroke on Accepted: 22 October 2021 functional improvement and serum angiogenin (ANG) in a 6-month follow-up study. Published: 25 November 2021 Citation: Methods: A prospective, observational, longitudinal, and multicenter study with Garcia-Rodriguez N, Rodriguez S, three cohorts: strokes in intensive rehabilitation therapy (IRT, minimum 15 h/week) vs. Tejada PI, Miranda-Artieda ZM, conventional therapy (NO-IRT, <15 h/week), and controls subjects (without known Ridao N, Buxó X, Pérez-Mesquida ME, Beseler MR, neurological, malignant, or inflammatory diseases). A total of seven centers participated, Salom JB, Pérez LM, Inzitari M, with functional evaluations and blood sampling during follow-up. The final cohort includes Otero-Villaverde S, Martin-Mourelle R, Molleda M, Quintana M, 62 strokes and 43 controls with demographic, clinical, blood samples, and exhaustive Olivé-Gadea M, Penalba A and functional monitoring. Rosell A (2021) Functional Recovery and Serum Angiogenin Changes Results: The median (IQR) number of weekly hours of therapy was different: IRT 15 According to Intensity of Rehabilitation (15–16) vs. NO-IRT 7.5 (5–9), p < 0.01, with progressive and significant improvements Therapy After Stroke. Front. Neurol. 12:767484. in both groups. However, IRT patients showed earlier improvements (within 1 month) on doi: 10.3389/fneur.2021.767484 several scales (CAHAI, FMA, and FAC; p < 0.001) and the earliest community ambulation Frontiers in Neurology | www.frontiersin.org 1 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery achievements (0.89 m/s at 3 months). There was a significant difference in ANG temporal profile between the IRT and NO-IRT groups (p < 0.01). Additionally, ANG was elevated at 1 month only in the IRT group (p < 0.05) whereas it decreased in the NO-IRT group (p < 0.05). Conclusions: Our results suggest an association of rehabilitation intensity with early functional improvements, and connect the rehabilitation process with blood biomarkers. Keywords: angiogenin, intensive therapy, rehabilitation, biomarker, recovery INTRODUCTION superfamily that acts as a potent angiogenic protein triggering cell proliferation, migration, or survival (18, 19), which has been Stroke is a leading cause of disability, with more than 16.9 recently identified as a repair-associated factor in post-stroke million people having a first stroke every year, 5.9 million stroke- rehabilitation by our group (15). related deaths, and a calculated loss of 102 million Disability- In this multicenter study we aimed at studying the influence Adjusted Life-Years (1, 2). In the last decades, there has been of the rehabilitation therapy intensity received after stroke on a marked decrease in stroke mortality as a result of improved the functional improvements, and for the first time studying the primary care interventions, better neuroimaging diagnosis, and response of a blood biomarker in response to the dose of therapy improved stroke management (with specialized stroke units, received during post-stroke recovery. thrombolytic, and endovascular treatments) (3). Beyond this, the evidence-based approach to achieve functional improvement MATERIALS AND METHODS in daily-life activities and reduce disabilities in stroke survivors has been implementing personalized rehabilitation programs Study Cohorts with multidisciplinary teams working under the physiatrist’s This study comprises a cohort of 62 post-stroke patients supervision (4). Recently, an interesting debate has taken place recruited in two periods within the prospective, observational, on the need to implement early rehabilitation interventions longitudinal, and multicenter SMARRTS study (Studying (first 24 h) which might worsen the outcome at high doses but Markers of Angiogenesis and Repair during Rehabilitation might be beneficial with high frequency (5, 6), highlighting Therapy after Stroke): ischemic stroke patients under IRT the need for a fine-tuning of the interventions. Additionally, (between February 2014 and May 2015) (15) and both ischemic other studies have demonstrated that high-intensity therapies and hemorrhagic stroke patients under IRT or conventional with a larger amount of hours are determinant for a good rehabilitation therapy (between February 2017 and June 2018) prognosis (7, 8), although the standard time and dose to from seven Spanish hospitals. Inclusion criteria were: first-ever achieve improvements is planned individually (9, 10). This ischemic or hemorrhagic stroke, age ≤ 75 years, modified Rankin is the case for intensive rehabilitation therapy (IRT), defined scale (mRS) ≤ 2 before stroke and mRS post-stroke from three as rehabilitation therapy of more than 15 h per week by a to five, stable medical condition, and the signature of informed physical therapist, an occupational therapist, and/or a speech consent. The exclusion criteria were: previous stroke or transient therapist, with close monitoring of patient progress to adjust the ischemic attacks, malignant infarct, global aphasia, hemorrhage program (11). Other clinical evidence has shown that with high- from arteriovenous malformations or cerebral aneurysms, quality, high-dose, high-intensity upper limb neurorehabilitation previous cognitive decline, or recent infectious, inflammatory or during a 3-week (90 h) program clinical improvements in upper malignant disease. limb deficits and activity can be achieved in chronic stroke The study was approved by all the clinical research ethics patients starting more than 6 months after the event (12). committee sites [HUVH PR(IR)317/2013-PR(IR)346/2016, And in a retrospective analysis comparing this cohort with a PI16/00981/CEI:PI-17-056, Comité de Ética de Investigación conventional low-intensity treatment cohort it is described that, de A Coruña-Ferrol 2017-125, Corporació Sanitària Parc Taulí despite responsiveness of both treatments, the high-intensity de Sabadell 2017521, Hospital Universitario Politécnico La Fe approach showed a consistent higher impact at all stages post- 2016/0727, Euskadi PI2016168]. Healthy subjects (43) without stroke (13). known neurological, malignant, infectious, or inflammatory Neural plasticity and vascular remodeling are assumed as diseases were enrolled as the control cohort. All subjects signed the basis of post-stroke recovery as reported in preclinical informed consent following the Declaration of Helsinki. models (14, 15). In this context, understanding the role of STROBE guidelines for reporting observational studies were specific biomarkers in pathophysiological brain changes might followed in this study (20). serve as a bridge between the fundamental science and patients’ clinical management, including monitoring rehabilitation goals Rehabilitation Interventions and the duration of the intervention (16, 17). In this regard, we All included patients followed a comprehensive rehabilitation focused on angiogenin (ANG), a member of the ribonuclease program, including physiotherapy, occupational therapy, speech Frontiers in Neurology | www.frontiersin.org 2 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery FIGURE 1 | Study design. Scheme showing the studied cohorts and follow-up visits when a battery of tests to assess motor and functional status was conducted together with blood samples extraction. Controls were recruited in a unique inclusion visit when blood samples were obtained. therapy, and/or neuropsychology, however, some centers offered program started by an experienced physiatrist who collected intensive rehabilitation therapies whereas others followed more demographic, clinical, and stroke-related data together with a conventional programs. For the IRT program only subacute battery of tests to assess motor and functional status (details post-stroke patients with moderate/severe disabilities in two or in the Supplementary Methods). During the baseline interview, more areas (gait, transfers, activities of daily living, swallowing previous physical activity was considered as any cardiovascular and/or communication), with mRS previous to stroke ≤ 2 who exercise routine such as running, swimming, cycling, and obesity can participate in a minimum of 3 h of comprehensive therapy was calculated BMI > 29. per day (5 days/week) were included, according to institutional Follow-up visits were conducted by the same physiatrist at guidelines (21). When the clinical stroke condition was stable 1, 3, and 6 months after the start of rehabilitation including a patients started mobilizations followed by a comprehensive battery of tests to assess neuro-functional status: the mRS (scores rehabilitation program defined as IRT (≥15 h per week) or 0–6), the Granger modified Barthel Index (BI, scores 0–100) (22), NO-IRT (conventional therapy with <15 h per week). For all the Fugl-Meyer Assessment score for the upper extremity (FMA, centers, a physiatrist designed an initial rehabilitation program scores 0–66) (23), the Functional Ambulation Categories (FAC, according to patients’ impairments, and all patients were scores 0–5), the Chedoke Arm and Hand Activity Inventory treated in inpatient rehabilitation units or day-hospital facilities. (CAHAI, scores 13–91) (24), the 10-m walk test (velocity is Rehabilitation continued until completion of a minimum registered), the Medical Research Council scale (MRC, scores 0– of the proposed objectives or when functional stability was 5) of the upper and lower extremities at the proximal/distal level, achieved. For further objectives, patients continued an outpatient and the Modified Asworth Scale (MAS, scores 0–4, including rehabilitation program. 1+) (25). We also analyzed changes in the scores during follow- Study Protocol and Functional Follow-Up up visits vs. baseline scores to assess improvements in the A total of 62 patients were initially included in the study. neurological function: Rankin improvement was defined as a However, 3 of them voluntarily abandoned the study, and 6 were decrease of ≥1 point. For the FMA, the improvement was withdrawn from the study due to the following: decompressive defined as an increase of ≥10 points, described previously as cranial surgery (1), secondary aneurismal hemorrhagic stroke the minimal clinical important difference (26). For the Chedoke (1), a second ischemic event (1), or moving to other cities Arm and Hand Activity Inventory, an improvement was defined (3). This reduced the number of subjects in the follow-up as an increase of ≥7 points (27). For the 10-m walk test, analysis, as represented in the flow diagram in Figure 1. A the walking velocity was calculated, and improvement was baseline inclusion visit was conducted before the rehabilitation considered if walking velocity increased by >0.3 m/s. The FAC Frontiers in Neurology | www.frontiersin.org 3 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery TABLE 1 | Baseline characteristics of the control and stroke cohorts. TABLE 2 | Baseline characteristics of the IRT vs. NO-IRT cohorts. Stroke cohort Control cohort p-value IRT NO-IRT p-value n = 62 n = 43 n = 45 n = 17 Age (years) 57.59 ± 9.7 60.8 ± 10.8 0.11 Age 56.4 ± 9.1 60.6 ± 10.8 0.13 Sex, males 79.7 (51) 20.3 (13) <0.01 Sex, males 82.2 (37) 82.4 (14) 0.72 Risk factors and comorbidities Risk factors and comorbidities Alcohol 27.4 (17) 23.8 (10) 0.65 Alcohol 28.9 (13) 23.5 (4) 0.72 Tobacco 37.1 (23) 16.7 (7) 0.02 Tobacco 40.0 (18) 29.5 (5) 0.53 Atrial fibrillation 9.7 (6) 0 (0) 0.07 Atrial fibrilation 8.9 (4) 11.8 (2) 0.64 Hypertension 61.3 (38) 46.5 (20) 0.16 Hypertension 66.7(30) 47.1 (8) 0.23 Dyslipidemia 40.3 (25) 46.5 (20) 0.52 Dyslipidemia 46.7 (21) 23.5 (4) 0.09 Diabetes mellitus 19.7 (12) 18.6 (8) 0.89 Diabetes mellitus 25.0 (11) 5.9 (1) 0.15 Obesity 29.5 (18) 34.9 (15) 0.56 Obesity 29.5 (13) 29.4 (5) 1 Previous exercise 39.0 (23) 76.7 (33) <0.001 Previous exercise 38.1 (16) 41.2 (7) 0.8 Cardiopathy 6.5 (4) 0 (0.0) 0.14 Cardiopathy 11.8 (2) 4.4 (2) 0.3 Osteoarticular 12.9 (8) 32 (14) 0.01 Osteoarticular 8.9 (4) 23.5 (4) 0.18 Psychiatric 16.1 (10) 9.3 (4) 0.31 Psychiatric 20 (9) 5.9 (1) 0.26 Previous medication Previous medication Anti-platelets 21 (13) 14.3 (6) 0.38 Anti-platelets 22.2 (10) 17.6 (3) 1 Anti-coagulants 4.8 (3) 0 (0.0) 0.27 Anticoagulants 4.4 (2) 5.9 (1) 1 Statins 38.7 (24) 28.6 (12) 0.28 Statins 46.7 (21) 17.6 (3) 0.03 Anti-hypertensives 53.2 (33) 40.5 (17) 0.2 Anti- hypertensives 55.6 (25) 47.1 (8) 0.58 Anti-diabetic 17.7 (11) 14.3 (6) 0.64 Anti-diabetic 22.2(10) 5.9 (1) 0.26 Angiogenin Variables are expressed as a percentage (number of cases) or mean ± SD. Differences Baseline levels (ng/mL) 520.8 ± 139.2 432.8 ± 155.7 <0.01 were assessed with a t-test, chi-square test, or Fisher exact test. Statistically significant differences are highlighted in bold. Variables are expressed as a percentage (number of cases, n) or mean ± SD. Differences were assessed with a t-test, chi-square test, or Fisher exact test. Statistically significant differences are highlighted in bold. Descriptive statistics: categorical variables were reported as frequencies (percentages) and continuous variables as mean was categorized into three categories: cannot walk (score 0), ± standard deviation (SD) or median [interquartile range dependent walk (scores 1–3), and independent walk (scores 4 (IQR)], as appropriate. Missing data were considered at random and 5), and improvement was defined as a shift to a higher and no imputations were used. The normality assumption of category (28). For the MRC scale, our analysis differentiated quantitative variables was checked with the use of quantile- classification between normal (score 5) or impaired (scores 0–4) quantile (Q-Q) plots. Statistical significance was assessed by muscle strength. Pearson’s chi-square or Fisher’s exact test for categorical variables, At all visits blood sampling in serum-separating tubes was Student’s t-test for continuous variables, and the Mann-Whitney obtained, centrifuged at 1,500 rpm for 15 min, and serum stored U test for functional scores and numerical variables without at −80◦ C until use. normal distribution. Temporal profile changes in normally distributed variables were analyzed with repeated measures Angiogenin Measurement ANOVA (Bonferroni post-hoc test) or the Friedman followed Serum levels of ANG were measured by ELISA (#DAN00; R&D by Wilcoxon tests for non-normal distributions. Box plots Systems, USA) and analyzed together with previous-obtained were used to represent the temporal profile of non-normal results from cohort one using the same test (15). Briefly, 200 µl distributed variables (functional scores) and bar-graphs showing of diluted serum samples (1:200) were loaded per duplicate, and mean with 95% confidence interval to represent normally only values with a coefficient of variation < 20% were accepted distributed variables (temporal profile of ANG). Pearson (normal for the statistical analysis. To verify low inter-plate variability (7 distribution) or Spearman (non-normal distribution) tests ELISA plates were analyzed in total) we included a commercial were used. Repeated measures in general linear models were internal control from Sigma-Aldrich (Human serum type AB, conducted to assess changes in the ANG temporal profile between male, from clotted, cat#H6914), and a coefficient of variation < the two rehabilitation groups, controlling for other baseline 20% was accepted. characteristics (CAHAI). The temporal profile changes in the functional scales were analyzed with Mixed Models Analysis Statistical Analysis generated by the SAS System (Version W32_7 PRO) adjusting The SPSS 20.0 package was used for statistical analyses. The for the baseline scores. A p-value < 0.05 was considered database entries were reviewed by an independent researcher. statistically significant. Frontiers in Neurology | www.frontiersin.org 4 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery TABLE 3 | Clinical characteristics of IRT and NO IRT group on admission. TABLE 4 | Post-stroke rehabilitation characteristics at baseline visit. IRT NO-IRT p-value IRT NO-IRT p-value n = 45 n = 17 n = 45 n = 17 Rankin 4.5 (4–5) 5 (4–5) 0.46 Hospitalization regimen NIHSS 12 (7.5–17) 11 (6–14) 0.32 Inpatient rehabilitation 95.6 (43) 100 (17) 1 NIHSS (motor) 7 (4–9) 6.5 (5–11) 0.65 Day-hospital rehabilitation 4.4 (2) 0 (0) 1 Stroke laterality, left 48.9 (22) 41.2 (7) 0.58 Time stroke- RHB program in days 14 (9–19) 11 (8–14) 0.09 Stroke type, ischemic 73.3 (33) 70.6 (11) 0.82 Time stroke-sample in days 14 (8.25–19) 10 (6–13.5) 0.08 Vascular territory 0.7 RHB hour/week at baseline 15 (15–16) 7.5 (5–9) <0.01 Carotid 78.8 (25) 83.3 (10) Functional scores Vertebrobasilar 24.2 (8) 16.7 (2) Rankin 4 (3–5) 5 (3.2–5) 0.2 Ischemic etiology 0.45 Barthel 35 (20–68) 23 (20–36.5) 0.12 Cardioembolic 27.3 (9) 8.3 (1) NIHSS 9 (5–14) 9 (4–11) 0.38 Atherothrombotic 21.2 (7) 25 (3) CAHAI 13 (13–16) 18.5 (13–82) 0.02 Lacunar 18.2 (6) 41.7 (5) FMA 8.5 (4–40.7) 9 (0–59) 0.94 Others 12.1 (4) 8.3 (1) FAC 0 (0–2) 0 (0–2.7) 0.78 Undetermined 21.2(7) 16.7 (2) MRC proximal upper limbs 2 (0–4) 2.5 (0–3) 0.99 OCSP classification 0.35 MRC distal upper limbs 1 (0–4) 0.5 (0–3.5) 0.95 TACI 46.9 (15) 27.3 (3) MRC proximal lower limbs 2 (1–4) 3 (2–4) 0.98 PACI 15.6 (5) 9.1 (1) MRC distal lower limbs 1 (0–4) 1 (0–3) 0.34 LACI 25 (8) 54.5 (6) MAS proximal upper limbs 0 (0–1) 0 (0–0) 0.3 POCI 12.5 (4) 9.1 (1) MAS distal upper limbs 0 (0–0.5) 0.5 (0–3.5) 0.73 Acute treatment MAS proximal lower limbs 0 (0–1) 0 (0–1) 0.82 Thrombolytic therapy 15.4 (4) 11.8 (2) 0.48 MAS distal lower limbs 0 (0–0.5) 0 (0–1) 0.36 Endovascular treatment 6.7 (3) 17.6 (3) 0.33 Angiogenin Hemorrhagic transformation 4 (1) 0 (0) 1 Baseline levels (ng/mL) 502.7 ± 133.8 570.9 ± 145.8 0.09 Stroke type, hemorrhagic 26.7 (12) 29.4 (5) 0.53 Variables are expressed as a percentage (number of cases), mean ± SD, or median Location 0.51 (IQR). mRS, modified Rankin scale; BI, Granger modified Barthel Index; FMA, Fugl-Meyer Deep 91.7 (11) 80 (4) Assessment; FAC, the Functional Ambulation Categories; CAHAI, Chedoke Arm and Hand Lobar 8.3 (1) 20 (1) Activity Inventory; MAS, Modified Ashworth Scale. Differences were assessed with a t-test, Mann-Whitney-U, chi-square test, or Fisher exact test. Hemorrhagic etiology 0.33 Statistically significant differences are highlighted in bold. Hypertensive 83.3 (10) 100 (5) Undetermined 16.7(2) 0 (0) Clinical Characteristics and Functional Variables are expressed as a percentage (number of cases) or median (IQR). OCSP, Oxfordshire Community Stroke Project; TACI, total anterior cerebral infarct; LACI, lacunar Outcome cerebral infarct; PACI, partial anterior cerebral infarct; POCI, posterior cerebral infarct. The two rehabilitation groups presented similar clinical Differences were assessed with a t-test, Mann-Whitney-U, chi-square test, or Fisher characteristics at emergency admission: stroke characteristics, exact test. acute treatment, hospitalization regimen, or baseline functional scores (see Tables 3, 4). Significant differences were only found in the number of rehabilitation hours per week: IRT 15 (15–16) RESULTS vs. NO-IRT 7.5 (5–9), p < 0.01, and in the baseline CAHAI score which was lower in the IRT group: 13 (13–16) vs. 18.5 (13–82), p Characteristics of the Study Cohorts at = 0.02. Baseline Both groups presented significant improvements over the 6 The baseline characteristics of the stroke cohort and controls month follow-up period in functional and motor tests, but the are described in Table 1. Of note, our stroke cohort presented CAHAI score and MAS scale only improved in the IRT group more men (79.7 vs. 20.3%, p < 0.01), more tobacco users (37.1 (Table 5). Regarding the impact of the rehabilitation intensity vs. 16.7%, p = 0.02), and less previous exercise (39 vs. 76.7%, p on motor and functional scores, Figure 2 shows improvements < 0.001) than the control. Importantly, our two rehabilitation occurring earlier in the IRT group for the CAHAI, FMA, and groups presented similar baseline characteristics except for the FAC tests (p < 0.001 at 1 month), but not in the NO-IRT group, previous statins medication which was more frequent in the IRT which achieved significance later at 3months (p < 0.05). Other group (46.7 vs. 17.6%, p = 0.03) as shown in Table 2. tests presented similar profiles regardless of therapy intensity Frontiers in Neurology | www.frontiersin.org 5 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery TABLE 5 | Measures of functional and motor outcome. IRT Group Baseline 1st month 3rd month 6th month BI (0–100)** 35 (20–68) 77 (49–94) 93 (84–100) 100 (93–100) mRS** 4 (3–5) 3 (2–4) 3 (1–3) 2 (1–2) FMA (0–66)* 8.5 (4–40.7) 44 (9–56.5) 47.5 (12.5–61.3) 50.5 (17.8–64.5) FAC (0–5)** 0 (0–2) 2 (1–5) 4 (3–5) 5 (4–5) CAHAI (13–91)** 13 (13–15.7) 43 (13–76) 73 (13–87) 74 (13–90) 10-m walk test (m/s)** NA 0.50 (0–1) 0.89 (0.25–1.22) 0.90 (0.54–1.30) MRC scale superior-proximal (0–5)** 2 (0–1) 4 (2–4) 4 (2.5–5) 4 (2–5) MRC scale superior-distal (0–5)** 1 (0–4) 2.5 (0–4) 4 (0.5–5) 4 (1–5) MRC scale inferior-proximal (0–5)** 1 (0–4) 5 (4–5) 5 (4–5) 5 (4–5) MRC scale inferior-distal (0–5)** 1 (0–4) 4 (1–5) 4 (2–5) 4 (2–5) MAS scale superior-proximal (0–4)** 0 (0–1) 1 (0–1) 1 (0–2) 1 (1–2) MAS scale superior-distal (0–4)** 0 (0–0.5) 1 (0–1.5) 1 (0–2) 1 (1–2) MAS scale inferior-proximal (0–4)** 0 (0–1) 0 (0–1) 0 (0–1) 0 (0–1) MAS scale inferior-distal (0–4)** 0 (0–0.5) 0 (0–1) 1 (0–2) 1 (0–2.25) NO-IRT Group BI (0−100)* 23 (20–36.2) 49.5 (40–93) 88.5 (59–99) 90 (75–100) mRS** 5 (3.2–5) 4 (1.2–4) 2.5 (1–3) 1 (1–3) FMA (0–66)* 9 (0.5–55.2) 31 (7.2–64.7) 40.5 (9–63.5) 50 (18–66) FAC (0–5)* 0 (0–2.7) 2 (1–4.75) 4.5 (3–5) 5 (4–5) CAHAI (13–91) 18.5 (13–82.2) 23 (13–90) 59.5 (13–90) 76 (13–91) 10-m walk test (m/s)** NA 0 (0–0.70) 0.56 (0.28–0.99) 0.95 (0.62–1.10) MRC scale superior-proximal (0–5)* 2.5 (0–3) 3 (0–4) 3 (1.5–5) 4 (2–4) MRC scale superior-distal (0–5)* 0.5 (0–3) 2.5 (0–4) 3.5 (1.5–4) 3.5 (1.5–5) MRC scale inferior-proximal (0–5)* 1 (0–3) 4 (3–4) 4 (3.5–5) 4 (3.5–5) MRC scale inferior-distal (0–5)* 1 (0–3) 2.5 (0–4) 4 (1–5) 4 (2–5) MAS scale superior-proximal (0–4) 0 (0–0) 0.5 (0–1) 0 (0–1) 0 (0–1) MAS scale superior-distal (0–4) 0 (0–0) 0.5 (0–1) 0.5 (0–1.75) 0 (0–2) MAS scale inferior-proximal (0–4) 0 (0–0.75) 0 (0–1) 0.5 (0–1) 0 (0–1) MAS scale inferior-distal (0–4) 0 (0–1) 0.5 (0–1) 1 (0–1.75) 0 (0–2) Data are shown as median (IQR). mRS, modified Rankin scale; BI, Granger modified Barthel Index; FMA, Fugl-Meyer Assessment; FAC, the Functional Ambulation Categories; CAHAI, Chedoke Arm and Hand Activity Inventory; MAS, Modified Ashworth Scale; NA, not applicable. Every scale shows the Median and IRQ Differences were assessed with a Friedman test: *p < 0.05, **p < 0.01. Statistically significant differences are highlighted in bold. (Supplementary Figure 1). Importantly, at 3 months the IRT number of rehabilitation hours were reduced according to the group achieved a full community ambulation level (0.89 m/s), individual patient’s achievements as represented in Figure 3A, but not the NO-IRT group (0.56 m/s) (Table 5). However we did with a substantial switch from IRT to NO-IRT over time. not find differences overtime between rehabilitation groups in a For this reason, the ANG level was analyzed only up to the mixed model: FAC (p = 0.86), Walking (p = 0.42), mRS (p = 3rd month. In all strokes the ANG temporal profile showed 0.82), BI (p = 0.41), FMA (p = 0.10), and CAHAI (p = 0.10). significant differences (p < 0.001, see Supplementary Figure 2), In this regard, the effect of time was independent of the type of being elevated at 1 month vs. controls (p = 0.036). Regarding therapy (p < 0.01) for all tests and did not change when adjusting the type of therapy, we found a significant interaction with ANG for baseline mRS or Barthel. levels over time (p = 0.030, see Figure 3B). The ANG temporal profile in the NO-IRT and IRT groups showed differences (p < Angiogenin Temporal Profile Changes With 0.01, see Figure 3C) in opposite directions since ANG increased Therapy Intensity after 1 month of IRT (p < 0.05 vs. baseline) but it decreased in Baseline ANG was significantly higher in strokes than in controls the NO-IRT group (p < 0.01 vs. 3rd month). (520 ± 139 vs. 432 ± 155 ng/mL; p < 0.01, see Table 1), with no Finally, we examined the relationship between outcome scores baseline differences observed between the rehabilitation groups improvements from admission to 6 months follow–up with (see Table 4). No correlation was observed between baseline ANG, but we could not confirm a predictive value of ANG at any ANG and time-to-start RHB or baseline sampling. Notably, the of the tested time points (data not shown). Frontiers in Neurology | www.frontiersin.org 6 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery FIGURE 2 | Functional outcome. Temporal profile of the tested scales in IRT and NO-IRT cohorts. Note that in the 1st month significant improvements were only achieved in the IRT group. Differences were assessed with the Wilcoxon tests and the Man-Withney U for the transversal analysis. *p < 0.05; **p < 0.01, and ***p < 0.001. Median and IQR are represented in box plots. DISCUSSION is a key factor in the recovery process (8, 13). In this study, we have followed two cohorts with similar clinical characteristics The present study investigates the effects of rehabilitation therapy which primarily differed in the amount of scheduled therapy intensity on both functional outcome and blood levels of ANG, a time at baseline. Our results suggest that patients under IRT potential biomarker of recovery. Our results suggest that stroke receiving the highest therapy dose improved earlier at 1 month of patients under more intense rehabilitation programs presented therapy in the CAHAI and FMA scores, which according to the better outcomes earlier with a parallel increase of blood ANG. International Classification of Function (ICF-WHO) framework However, we could not confirm a predictive value of ANG. are designed to assess bilateral activity performance for daily Rehabilitation treatment is the gold-standard therapy for life activities and brain structural recovery, respectively (29). stroke survivors to recover functional status, and improve quality Additionally, patients under IRT also achieved full community of life and independence (4) with programs personalized by ambulation earlier during rehabilitation (30) and showed better multi-disciplinary teams, where the dose of the received therapy BI scores earlier than NO-IRT patients, suggesting improved Frontiers in Neurology | www.frontiersin.org 7 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery FIGURE 3 | Angiogenin (ANG) blood levels during rehabilitation. (A) Bar graph showing the number of patients under IRT and NO-IRT over time, changing according to early improvements in the IRT group. (B) Graph comparing how time and type of therapy influenced the serum levels of ANG, assessed with ANOVA for repeated measures. (C) Jitter plots showing the temporal profile of ANG levels in NO-IRT and IRT groups; Differences were assessed with Wilcoxon tests, *p < 0.05, **p < 0.01. Mean with 95% CI are represented in graphs. independence for the basic activities of daily living. In this regard 34). However, the patient’s response to rehabilitation is and following previously reported dependence categories for the heterogeneous and might depend on the type and amount BI scale (31, 32), median BI values show that at 1 month IRT lead of therapy received, or on individual endogenous neurorepair to a moderate dependence score whereas No-IRT maintained a responses. In this regard, it is important to elucidate if IRT severe dependence score and later at 3 months IRT lead to a could further enhance recovery by increasing therapy intensity slight independence score whereas No-IRT lead to a moderate and/or duration. dependence score. A biomarker is an indicator that can be used to measure Supporting these observations of larger functional gains in the underlying molecular processes, identify a disease, predict IRT group, Wang and colleagues showed that the daily amount recovery, or monitor treatment responses. In the present study, of physical, occupational, and speech and language therapy we show that only patients under IRT presented a significant was also significantly associated with functional improvements increase in serum ANG after rehabilitation, which could be (33). Others have previously reported a direct intensity- linked to the repair process underlying the prompt recovery response between rehabilitation and functional recovery (8, since this angiogenic factor can trigger a wide range of biological Frontiers in Neurology | www.frontiersin.org 8 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery processes (18). This improvement allowed the reduction of DATA AVAILABILITY STATEMENT the amount of therapy time that paralleled a decrease of the serum ANG levels at the 3rd and 6th months of therapy. Requests to access the anonymized datasets should be directed to Whether extending the IRT therapy would maintain high ANG the corresponding author, anna.rosell@vhir.org. and result in larger functional/motor improvements remains to be elucidated in future interventional biomarker studies. ETHICS STATEMENT Other studies have focused on identifying biomarkers for stroke disease (35, 36), but very few have focused on long-term The studies involving human participants were reviewed outcomes or the influence of rehabilitation therapies. Others and approved by Research Ethics Committee sites [HUVH have explored the use of molecules related to oxidative stress PR(IR)317/2013-PR(IR)346/2016, PI16/00981/CEI:PI-17-056, (37) or changes in neurotransmitter levels during post-stroke Comité de Ética de Investigación de A Coruña-Ferrol 2017-125, rehabilitation, reporting a correlation with motor improvement Corporació Sanitària Parc Taulí de Sabadell 2017521, Hospital (38). Serum ANG levels were first reported higher in patients Universitario Politécnico La Fe 2016/0727, Euskadi PI2016168]. with stroke within 48 h and on days 3 and 7, but decreasing at The patients/participants provided their written informed 14 days compared to control subjects (39), but the rehabilitation consent to participate in this study. interventions were not described in this work. Our recent work also in serum ANG reported that blood ANG was increased in AUTHOR CONTRIBUTIONS stroke patients after IRT (15), which is being confirmed in the present study with a larger cohort. Additionally, in this previous NG-R, SR, XB, and AR participated in the design of the study. study, experiments in pre-clinical stroke models also described an NG-R, SR, PT, ZM-A, XB, NR, MP-M, MB, JS, LP, MI, SO-V, ANG increase in the ischemic site when performing task-specific RM-M, MM, MO-G, and AP participated in patients’ recruitment exercise, suggesting an association between rehabilitation and and data collection. NG-R, XB, MQ, AP, and AR participated in molecular changes in the brain. Moreover, another pre-clinical data analysis. NG-R, SR, PT, ZM-A, NR, XB, MP-M, MB, JS, LP, study from our group has recently described that physical MI, SO-V, RM-M, MM, MQ, MO-G, AP, and AR participated in exercise rapidly increased the amount of endogenous ANG in the the manuscript draft and/or review. All authors contributed to ipsilateral neurogenic subventricular zone after cerebral ischemia the article and approved the submitted version. (40). All this evidence points to potential connections between ANG, stroke disease, and rehabilitation, although confirmatory FUNDING investigations are needed. To our knowledge this is the first time a blood biomarker NG-R holds a VHIR fellowship and MO-G a Joan Margarit VHIR is described as differentially modulated by the amount of fellowship. Research grants: from the Instituto de Salud Carlos therapy time received during rehabilitation, suggesting the need III and European Regional Development Funds (PI16/00981, to include rehabilitation treatments as a co-variable in stroke PI19/00186, RD16/0019/0021, and RD16/0019/0008), 2017-SGR- biomarker analyses, especially in multicenter studies. 1427 program from the Generalitat de Catalunya-AGAUR, and As a limitation, our study presents a different number Clinical Translational Program for Regenerative Medicine in of patients in both rehabilitation cohorts due to different Catalonia (P-CMR [C]). recruitment achieved by the participant centers, which could impact on group comparisons although baseline clinical and ACKNOWLEDGMENTS stroke characteristics are very similar. Also, in this observational study, the dose of therapy hours decreased over time in the IRT NG-R is doing a doctorate at the Departament de Medicina de group based on clinical decisions and individual achievements, la Universitat Autònoma de Barcelona. We are grateful to all which could influence the levels of serum ANG between follow- clinical staff helping in blood sampling and follow-up visits and up visits. For these reasons new interventional studies to to Xavier Vidal from the Clinical Pharmacology Service at HUVH determine the relationship between the dose of therapy and for statistical advice. blood biomarkers are crucial to elucidate the true link with the clinical interventions. SUPPLEMENTARY MATERIAL In conclusion, our study shows that designing intensive rehabilitation programs results in earlier improvements, and the The Supplementary Material for this article can be found monitoring of specific blood biomarkers could be a useful part of online at: https://www.frontiersin.org/articles/10.3389/fneur. the multidisciplinary recovery program. 2021.767484/full#supplementary-material REFERENCES 2. Feigin VL, Forouzanfar MH, Krishnamurthi R, Mensah GA, Connor M, Bennett DA. Global and regional burden of stroke during 1990–2010: findings 1. Giroud M, Jacquin A, Béjot Y. The worldwide landscape of stroke in the 21st from the Global Burden of Disease Study 2010. Lancet. (2014) 383:245– century. Lancet. (2014) 383:195–7. doi: 10.1016/S0140-6736(13)62077-2 54. doi: 10.1016/S0140-6736(13)61953-4 Frontiers in Neurology | www.frontiersin.org 9 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery 3. Licher S, Darweesh SKL, Wolters FJ, Fani L, Heshmatollah A, 22. Mahoney FI, Barthel DW. Functional evaluation. The Barthel Index. Mutlu U. Lifetime risk of common neurological diseases in Maryland State Med J. (1965) 14:61–5. doi: 10.1037/t02366-000 the elderly population. J Neurol Neurosurg Psychiatry. (2019) 23. Hernández ED, Galeano CP, Barbosa NE, Forero SM, Nordin Å, 90:148–56. doi: 10.1136/jnnp-2018-318650 Sunnerhagen KS. Intra- and inter-rater reliability of Fugl-Meyer 4. Langhorne P, Bernhardt J, Kwakkel G. Stroke rehabilitation. Lancet. (2011) Assessment of Upper Extremity in stroke. J Rehabil Med. (2019) 377:1693–702. doi: 10.1016/S0140-6736(11)60325-5 51:652–9. doi: 10.2340/16501977-2590 5. AVERT Trial Collaboration Group. Efficacy and safety of very early 24. Barreca S, Gowland CK, Stratford P, Huijbregts M, Griffiths J, Torresin mobilisation within 24 h of stroke onset (AVERT): a randomised W, et al. Development of the Chedoke Arm and hand activity inventory: controlled trial. Lancet. (2015) 386:46–55. doi: 10.1016/S0140-6736(15) theoretical constructs, item generation, and selection. Top Stroke Rehabil. 60690-0 (2004) 11:31–42. doi: 10.1310/JU8P-UVK6-68VW-CF3W 6. Bernhardt J, Churilov L, Ellery F, Collier J, Chamberlain J, 25. Blackburn M, van Vliet P, Mockett SP. Reliability of measurements obtained Langhorne P. Prespecified dose-response analysis for a very with the modified Ashworth scale in the lower extremities of people with early rehabilitation trial (AVERT). Neurology. (2016) 86:2138– stroke. Phys Ther. (2002) 82:25–34. doi: 10.1093/ptj/82.1.25 45. doi: 10.1212/WNL.0000000000002459 26. Arya KN, Verma R, Garg RK. Estimating the Minimal clinically important 7. Nakazora T, Iwamoto K, Kiyozuka T, Arimoto H, Shirotani T, difference of an upper extremity recovery measure in subacute stroke Domene K. Effectiveness of 7-day versus weekday-only rehabilitation patients. Top Stroke Rehabil. (2011) 18:599–610. doi: 10.1310/tsr18 for stroke patients in an acute-care hospital: a retrospective cohort s01-599 study. Disabil Rehabil. (2018) 40:3050–3. doi: 10.1080/09638288.2017. 27. Barreca SR, Stratford PW, Lambert CL, Masters LM, Streiner DL. Test 1367964 Retest reliability, validity, and sensitivity of the chedoke arm and hand 8. Lang C, Lohse K, Birkenmeier R. Dose and timing in neurorehabilitation: activity inventory: a new measure of upper-limb function for survivors of prescribing motor therapy after stroke. Curr Opin Neurol. (2015) 28:549– stroke. Arch Phys Med Rehabil. (2005) 86:1616–22. doi: 10.1016/j.apmr.2005. 55. doi: 10.1097/WCO.0000000000000256 03.017 9. Imura T, Nagasawa Y, Fukuyama H, Imada N, Oki S, Araki O. Effect 28. Mehrholz J, Wagner K, Rutte K, Meißner D, Pohl M. Predictive of early and intensive rehabilitation in acute stroke patients: retrospective validity and responsiveness of the functional ambulation category in pre-/post-comparison in Japanese hospital. Disabil Rehabil. (2018) 40:1452– hemiparetic patients after stroke. Arch Phys Med Rehabil. (2007) 88:1314– 5. doi: 10.1080/09638288.2017.1300337 9. doi: 10.1016/j.apmr.2007.06.764 10. Lohse KR, Lang CE, Boyd LA. Is more better? Using metadata to explore 29. Yaruss JS, Quesal RW. Stuttering and the international classification of dose-response relationships in stroke rehabilitation. Stroke. (2014) 45:2053– functioning, disability, and health: an update. J Commun Disord. (2004) 8. doi: 10.1161/STROKEAHA.114.004695 37:35–52. doi: 10.1016/S0021-9924(03)00052-2 11. Kamo T, Momosaki R, Suzuki K, Asahi R, Azami M, Ogihara 30. Perry J, Garrett M, Gronley JK, Mulroy SJ. Classification of H, et al. Effectiveness of intensive rehabilitation therapy on walking handicap in the stroke population. Stroke. (1995) functional outcomes after stroke: a propensity score analysis based 26:982–9. doi: 10.1161/01.STR.26.6.982 on japan rehabilitation database. J Stroke Cerebrovasc Dis. (2019) 31. Ploughman M, Beaulieu S, Harris C, Hogan S, Manning OJ, Alderdice 28:2537–42. doi: 10.1016/j.jstrokecerebrovasdis.2019.06.007 PW. The Canadian survey of health, lifestyle and ageing with 12. Ward NS, Brander F, Kelly K. Intensive upper limb neurorehabilitation multiple sclerosis: methodology and initial results. BMJ Open. (2014) in chronic stroke: outcomes from the Queen Square programme. J 4:e005718. doi: 10.1136/bmjopen-2014-005718 Neurol Neurosurg Psychiatry. (2019) 90:498–506. doi: 10.1136/jnnp-2018-31 32. Patricia P. Measures of adult general functional status: the Barthel index, 9954 Katz index of activities of daily living, health assessment questionnaire 13. Ballester BR, Ward NS, Brander F, Maier M, Kelly K, Verschure (HAQ), MACTAR patient preference disability questionnaire, and modified PFMJ. Relationship between intensity and recovery in post-stroke health assessment questionnaire (MHAQ). Arthritis Rheum. (2003) 49:S15– rehabilitation: a retrospective analysis. J Neurol Neurosurg Psychiatry. 27. doi: 10.1002/art.11415 (2021). doi: 10.1136/jnnp-2021-326948. [Epub ahead of print]. 33. Wang H, Camicia M, Terdiman J, Mannava MK, Sidney S, Sandel M. Daily 14. Krakauer JW, Carmichael ST, Corbett D, Wittenberg GF. treatment time and functional gains of stroke patients during inpatient Getting neurorehabilitation right: what can be learned from rehabilitation. PM R. (2013) 5:122–8. doi: 10.1016/j.pmrj.2012.08.013 animal models? Neurorehabil Neural Repair. (2012) 26:923– 34. Jette DU, Warren RL, Wirtalla C. The relation between therapy intensity and 31. doi: 10.1177/1545968312440745 outcomes of rehabilitation in skilled nursing facilities. Arch Phys Med Rehabil. 15. Gabriel-Salazar M, Morancho A, Rodriguez S, Buxó X, García-Rodríguez N, (2005) 86:373–9. doi: 10.1016/j.apmr.2004.10.018 Colell G. Importance of angiogenin and endothelial progenitor cells after 35. Montaner J, Ramiro L, Simats A, Tiedt S, Makris K, Jickling GC. Multilevel rehabilitation both in ischemic stroke patients and in a mouse model of omics for the discovery of biomarkers and therapeutic targets for stroke. Nat cerebral ischemia. Front Neurol. (2018) 9:508. doi: 10.3389/fneur.2018.00508 Rev Neurol. (2020) 16:247–64. doi: 10.1038/s41582-020-0350-6 16. Korivi M, Wu CY, Lin KC. Potential predictive values of inflammatory 36. Ramiro L, Simats A, García-Berrocoso T, Montaner J. Inflammatory biomarkers for stroke rehabilitation outcomes. J Formos Med Assoc. (2013) molecules might become both biomarkers and therapeutic 112:735–7. doi: 10.1016/j.jfma.2013.10.017 targets for stroke management. Ther Adv Neurol Disord. (2018) 17. Whiteley W, Tian Y, Jickling GC. Blood biomarkers in stroke: 11:1756286418789340. doi: 10.1177/1756286418789340 research and clinical practice. Int J Stroke. (2012) 7:435– 37. Hsieh YW, Lin KC, Korivi M, Lee TH, Wu CY, Wu KY. The reliability 9. doi: 10.1111/j.1747-4949.2012.00784.x and predictive ability of a biomarker of oxidative DNA damage on 18. Goncalves KA, Silberstein L, Li S, Severe N, Hu MG, Yang H, et functional outcomes after stroke rehabilitation. Int J Mol Sci. (2014) 15:6504– al. Angiogenin promotes hematopoietic regeneration by dichotomously 16. doi: 10.3390/ijms15046504 regulating quiescence of stem and progenitor cells. Cell. (2016) 166:894– 38. Blicher JU, Near J, Næss-Schmidt E, Stagg CJ, Johansen-Berg H, Nielsen 906. doi: 10.1016/j.cell.2016.06.042 JF. GABA levels are decreased after stroke and GABA changes during 19. Sebastià J, Kieran D, Breen B, King MA, Netteland DF, Joyce D. Angiogenin rehabilitation correlate with motor improvement. Neurorehabil Neural Repair. protects motoneurons against hypoxic injury. Cell Death Differ. (2009) (2015) 29:278–86. doi: 10.1177/1545968314543652 16:1238–47. doi: 10.1038/cdd.2009.52 39. Huang L, Guo H, Cheng M, Zhao Y, Jin X. The kinetic change of the serum 20. Cuschieri S. The STROBE guidelines. Saudi J Anaesth. (2019) 13:S31– angiogenin level in patients with acute cerebral infarction. Eur Neurol. (2007) 4. doi: 10.4103/sja.SJA_543_18 58:224–7. doi: 10.1159/000107944 21. Ictus: guia de pràctica clínica: actualització gener del 2007. 2a ed. Barcelona: 40. Gabriel-Salazar M, Lei T, Grayston A, Costa C, Medina-Gutiérrez E, Agència d’Avaluació de Tecnologia i Recerca Mèdiques (2007). Available Comabella M. Angiogenin in the neurogenic subventricular zone after stroke. online at: https://scientiasalut.gencat.cat/handle/11351/1853 Front Neurol. (2021) 12:662235. doi: 10.3389/fneur.2021.662235 Frontiers in Neurology | www.frontiersin.org 10 November 2021 | Volume 12 | Article 767484 Garcia-Rodriguez et al. Stroke Rehabilitation Intensity, Biomarkers, and Recovery Conflict of Interest: LP and MI have received honoraria from Nestlé for activities be made by its manufacturer, is not guaranteed or endorsed by the unrelated to the present work. publisher. The remaining authors declare that the research was conducted in the absence of Copyright © 2021 Garcia-Rodriguez, Rodriguez, Tejada, Miranda-Artieda, Ridao, any commercial or financial relationships that could be construed as a potential Buxó, Pérez-Mesquida, Beseler, Salom, Pérez, Inzitari, Otero-Villaverde, Martin- conflict of interest. Mourelle, Molleda, Quintana, Olivé-Gadea, Penalba and Rosell. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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