Functional Recovery and Serum Angiogenin Changes According to Intensity of Rehabilitation Therapy After Stroke

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 rehabilitation treatments considering starting time, intensity, and frequency, according to the tolerance of the patient. However, there are no homogeneous protocols and the biological effects are under investigation. Objective: To investigate the impact of rehabilitation intensity (hours) after stroke on functional improvement and serum angiogenin (ANG) in a 6-month follow-up study. Methods: A prospective, observational, longitudinal, and multicenter study with three cohorts: strokes in intensive rehabilitation therapy (IRT, minimum 15 h/week) vs. conventional therapy (NO-IRT, <15 h/week), and controls subjects (without known neurological, malignant, or inflammatory diseases). A total of seven centers participated, with functional evaluations and blood sampling during follow-up. The final cohort includes 62 strokes and 43 controls with demographic, clinical, blood samples, and exhaustive functional monitoring. Results: The median (IQR) number of weekly hours of therapy was different: IRT 15 (15–16) vs. NO-IRT 7.5 (5–9), p < 0.01, with progressive and significant improvements in both groups. However, IRT patients showed earlier improvements (within 1 month) on several scales (CAHAI, FMA, and FAC; p < 0.001) and the earliest community ambulation 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.


INTRODUCTION
Stroke is a leading cause of disability, with more than 16.9 million people having a first stroke every year, 5.9 million strokerelated deaths, and a calculated loss of 102 million Disability-Adjusted Life-Years (1,2). In the last decades, there has been a marked decrease in stroke mortality as a result of improved primary care interventions, better neuroimaging diagnosis, and improved stroke management (with specialized stroke units, thrombolytic, and endovascular treatments) (3). Beyond this, the evidence-based approach to achieve functional improvement in daily-life activities and reduce disabilities in stroke survivors has been implementing personalized rehabilitation programs with multidisciplinary teams working under the physiatrist's supervision (4). Recently, an interesting debate has taken place on the need to implement early rehabilitation interventions (first 24 h) which might worsen the outcome at high doses but might be beneficial with high frequency (5,6), highlighting the need for a fine-tuning of the interventions. Additionally, other studies have demonstrated that high-intensity therapies with a larger amount of hours are determinant for a good prognosis (7,8), although the standard time and dose to achieve improvements is planned individually (9,10). This is the case for intensive rehabilitation therapy (IRT), defined as rehabilitation therapy of more than 15 h per week by a physical therapist, an occupational therapist, and/or a speech therapist, with close monitoring of patient progress to adjust the program (11). Other clinical evidence has shown that with highquality, high-dose, high-intensity upper limb neurorehabilitation during a 3-week (90 h) program clinical improvements in upper limb deficits and activity can be achieved in chronic stroke patients starting more than 6 months after the event (12). And in a retrospective analysis comparing this cohort with a conventional low-intensity treatment cohort it is described that, despite responsiveness of both treatments, the high-intensity approach showed a consistent higher impact at all stages poststroke (13).
Neural plasticity and vascular remodeling are assumed as the basis of post-stroke recovery as reported in preclinical models (14,15). In this context, understanding the role of specific biomarkers in pathophysiological brain changes might serve as a bridge between the fundamental science and patients' clinical management, including monitoring rehabilitation goals and the duration of the intervention (16,17). In this regard, we focused on angiogenin (ANG), a member of the ribonuclease superfamily that acts as a potent angiogenic protein triggering cell proliferation, migration, or survival (18, 19), which has been recently identified as a repair-associated factor in post-stroke rehabilitation by our group (15).
In this multicenter study we aimed at studying the influence of the rehabilitation therapy intensity received after stroke on the functional improvements, and for the first time studying the response of a blood biomarker in response to the dose of therapy received during post-stroke recovery.

Study Cohorts
This study comprises a cohort of 62 post-stroke patients recruited in two periods within the prospective, observational, longitudinal, and multicenter SMARRTS study (Studying Markers of Angiogenesis and Repair during Rehabilitation Therapy after Stroke): ischemic stroke patients under IRT (between February 2014 and May 2015) (15) and both ischemic and hemorrhagic stroke patients under IRT or conventional rehabilitation therapy (between February 2017 and June 2018) from seven Spanish hospitals. Inclusion criteria were: first-ever ischemic or hemorrhagic stroke, age ≤ 75 years, modified Rankin scale (mRS) ≤ 2 before stroke and mRS post-stroke from three to five, stable medical condition, and the signature of informed consent. STROBE guidelines for reporting observational studies were followed in this study (20).

Rehabilitation Interventions
All included patients followed a comprehensive rehabilitation program, including physiotherapy, occupational therapy, speech 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 intensive rehabilitation therapies whereas others followed more conventional programs. For the IRT program only subacute post-stroke patients with moderate/severe disabilities in two or more areas (gait, transfers, activities of daily living, swallowing and/or communication), with mRS previous to stroke ≤ 2 who can participate in a minimum of 3 h of comprehensive therapy per day (5 days/week) were included, according to institutional guidelines (21). When the clinical stroke condition was stable patients started mobilizations followed by a comprehensive rehabilitation program defined as IRT (≥15 h per week) or NO-IRT (conventional therapy with <15 h per week). For all centers, a physiatrist designed an initial rehabilitation program according to patients' impairments, and all patients were treated in inpatient rehabilitation units or day-hospital facilities. Rehabilitation continued until completion of a minimum of the proposed objectives or when functional stability was achieved. For further objectives, patients continued an outpatient rehabilitation program.

Study Protocol and Functional Follow-Up
A total of 62 patients were initially included in the study. However, 3 of them voluntarily abandoned the study, and 6 were withdrawn from the study due to the following: decompressive cranial surgery (1), secondary aneurismal hemorrhagic stroke (1), a second ischemic event (1), or moving to other cities (3). This reduced the number of subjects in the follow-up analysis, as represented in the flow diagram in Figure 1. A baseline inclusion visit was conducted before the rehabilitation program started by an experienced physiatrist who collected demographic, clinical, and stroke-related data together with a battery of tests to assess motor and functional status (details in the Supplementary Methods). During the baseline interview, previous physical activity was considered as any cardiovascular exercise routine such as running, swimming, cycling, and obesity was calculated BMI > 29.
We also analyzed changes in the scores during followup visits vs. baseline scores to assess improvements in the neurological function: Rankin improvement was defined as a decrease of ≥1 point. For the FMA, the improvement was defined as an increase of ≥10 points, described previously as the minimal clinical important difference (26). For the Chedoke Arm and Hand Activity Inventory, an improvement was defined as an increase of ≥7 points (27). For the 10-m walk test, the walking velocity was calculated, and improvement was considered if walking velocity increased by >0.3 m/s. The FAC 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.
was categorized into three categories: cannot walk (score 0), dependent walk (scores 1-3), and independent walk (scores 4 and 5), and improvement was defined as a shift to a higher category (28). For the MRC scale, our analysis differentiated classification between normal (score 5) or impaired (scores 0-4) muscle strength. At all visits blood sampling in serum-separating tubes was obtained, centrifuged at 1,500 rpm for 15 min, and serum stored at −80 • C until use.

Angiogenin Measurement
Serum levels of ANG were measured by ELISA (#DAN00; R&D Systems, USA) and analyzed together with previous-obtained results from cohort one using the same test (15). Briefly, 200 µl of diluted serum samples (1:200) were loaded per duplicate, and only values with a coefficient of variation < 20% were accepted for the statistical analysis. To verify low inter-plate variability (7 ELISA plates were analyzed in total) we included a commercial internal control from Sigma-Aldrich (Human serum type AB, male, from clotted, cat#H6914), and a coefficient of variation < 20% was accepted.

Statistical Analysis
The SPSS 20.0 package was used for statistical analyses. The database entries were reviewed by an independent researcher.

Characteristics of the Study Cohorts at Baseline
The baseline characteristics of the stroke cohort and controls are described in

Clinical Characteristics and Functional Outcome
The two rehabilitation groups presented similar clinical characteristics at emergency admission: stroke characteristics, 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) 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 = 0.02. Both groups presented significant improvements over the 6 month follow-up period in functional and motor tests, but the CAHAI score and MAS scale only improved in the IRT group ( Table 5). Regarding the impact of the rehabilitation intensity on motor and functional scores, Figure 2 shows improvements occurring earlier in the IRT group for the CAHAI, FMA, and FAC tests (p < 0.001 at 1 month), but not in the NO-IRT group, which achieved significance later at 3months (p < 0.05). Other tests presented similar profiles regardless of therapy intensity  (Supplementary Figure 1). Importantly, at 3 months the IRT group achieved a full community ambulation level (0.89 m/s), but not the NO-IRT group (0.56 m/s) ( Table 5). However we did not find differences overtime between rehabilitation groups in a mixed model: FAC (p = 0.86), Walking (p = 0.42), mRS (p = 0.82), BI (p = 0.41), FMA (p = 0.10), and CAHAI (p = 0.10). In this regard, the effect of time was independent of the type of therapy (p < 0.01) for all tests and did not change when adjusting for baseline mRS or Barthel.

Angiogenin Temporal Profile Changes With Therapy Intensity
Baseline ANG was significantly higher in strokes than in controls (520 ± 139 vs. 432 ± 155 ng/mL; p < 0.01, see Table 1), with no baseline differences observed between the rehabilitation groups (see Table 4). No correlation was observed between baseline ANG and time-to-start RHB or baseline sampling. Notably, the number of rehabilitation hours were reduced according to the individual patient's achievements as represented in Figure 3A, with a substantial switch from IRT to NO-IRT over time.
For this reason, the ANG level was analyzed only up to the 3rd month. In all strokes the ANG temporal profile showed significant differences (p < 0.001, see Supplementary Figure 2), being elevated at 1 month vs. controls (p = 0.036). Regarding the type of therapy, we found a significant interaction with ANG levels over time (p = 0.030, see Figure 3B). The ANG temporal profile in the NO-IRT and IRT groups showed differences (p < 0.01, see Figure 3C) in opposite directions since ANG increased after 1 month of IRT (p < 0.05 vs. baseline) but it decreased in the NO-IRT group (p < 0.01 vs. 3rd month). Finally, we examined the relationship between outcome scores improvements from admission to 6 months follow-up with ANG, but we could not confirm a predictive value of ANG at any of the tested time points (data not shown).

DISCUSSION
The present study investigates the effects of rehabilitation therapy intensity on both functional outcome and blood levels of ANG, a potential biomarker of recovery. Our results suggest that stroke patients under more intense rehabilitation programs presented better outcomes earlier with a parallel increase of blood ANG. However, we could not confirm a predictive value of ANG.
Rehabilitation treatment is the gold-standard therapy for stroke survivors to recover functional status, and improve quality of life and independence (4) with programs personalized by multi-disciplinary teams, where the dose of the received therapy is a key factor in the recovery process (8,13). In this study, we have followed two cohorts with similar clinical characteristics which primarily differed in the amount of scheduled therapy time at baseline. Our results suggest that patients under IRT receiving the highest therapy dose improved earlier at 1 month of therapy in the CAHAI and FMA scores, which according to the International Classification of Function (ICF-WHO) framework are designed to assess bilateral activity performance for daily life activities and brain structural recovery, respectively (29). Additionally, patients under IRT also achieved full community ambulation earlier during rehabilitation (30) and showed better BI scores earlier than NO-IRT patients, suggesting improved independence for the basic activities of daily living. In this regard and following previously reported dependence categories for the BI scale (31,32), median BI values show that at 1 month IRT lead to a moderate dependence score whereas No-IRT maintained a severe dependence score and later at 3 months IRT lead to a slight independence score whereas No-IRT lead to a moderate dependence score.
Supporting these observations of larger functional gains in the IRT group, Wang and colleagues showed that the daily amount of physical, occupational, and speech and language therapy was also significantly associated with functional improvements (33). Others have previously reported a direct intensityresponse between rehabilitation and functional recovery (8,34). However, the patient's response to rehabilitation is heterogeneous and might depend on the type and amount of therapy received, or on individual endogenous neurorepair responses. In this regard, it is important to elucidate if IRT could further enhance recovery by increasing therapy intensity and/or duration.
A biomarker is an indicator that can be used to measure underlying molecular processes, identify a disease, predict recovery, or monitor treatment responses. In the present study, we show that only patients under IRT presented a significant increase in serum ANG after rehabilitation, which could be linked to the repair process underlying the prompt recovery since this angiogenic factor can trigger a wide range of biological processes (18). This improvement allowed the reduction of the amount of therapy time that paralleled a decrease of the serum ANG levels at the 3rd and 6th months of therapy. Whether extending the IRT therapy would maintain high ANG and result in larger functional/motor improvements remains to be elucidated in future interventional biomarker studies. Other studies have focused on identifying biomarkers for stroke disease (35,36), but very few have focused on long-term outcomes or the influence of rehabilitation therapies. Others have explored the use of molecules related to oxidative stress (37) or changes in neurotransmitter levels during post-stroke rehabilitation, reporting a correlation with motor improvement (38). Serum ANG levels were first reported higher in patients with stroke within 48 h and on days 3 and 7, but decreasing at 14 days compared to control subjects (39), but the rehabilitation interventions were not described in this work. Our recent work also in serum ANG reported that blood ANG was increased in stroke patients after IRT (15), which is being confirmed in the present study with a larger cohort. Additionally, in this previous study, experiments in pre-clinical stroke models also described an ANG increase in the ischemic site when performing task-specific exercise, suggesting an association between rehabilitation and molecular changes in the brain. Moreover, another pre-clinical study from our group has recently described that physical exercise rapidly increased the amount of endogenous ANG in the ipsilateral neurogenic subventricular zone after cerebral ischemia (40). All this evidence points to potential connections between ANG, stroke disease, and rehabilitation, although confirmatory investigations are needed.
To our knowledge this is the first time a blood biomarker is described as differentially modulated by the amount of therapy time received during rehabilitation, suggesting the need to include rehabilitation treatments as a co-variable in stroke biomarker analyses, especially in multicenter studies.
As a limitation, our study presents a different number of patients in both rehabilitation cohorts due to different recruitment achieved by the participant centers, which could impact on group comparisons although baseline clinical and stroke characteristics are very similar. Also, in this observational study, the dose of therapy hours decreased over time in the IRT group based on clinical decisions and individual achievements, which could influence the levels of serum ANG between followup visits. For these reasons new interventional studies to determine the relationship between the dose of therapy and blood biomarkers are crucial to elucidate the true link with the clinical interventions.
In conclusion, our study shows that designing intensive rehabilitation programs results in earlier improvements, and the monitoring of specific blood biomarkers could be a useful part of the multidisciplinary recovery program.

DATA AVAILABILITY STATEMENT
Requests to access the anonymized datasets should be directed to the corresponding author, anna.rosell@vhir.org.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by