Role of Growth Factors & Neurophysiological Markers In Evaluation of Neuroplasticity Using 1Hz rTMS With Physical Therapy In Ischemic Stroke: A Double Blind, Parallel Group, Sham Controlled Randomized Trial

Background: The therapeutic benets of repetitive trans cranial magnetic stimulation along with physical therapy to study the neuroplasticity and neurogenesis in ischemic stroke patients has not been fully elucidated. The aim of this study is to determine the neuroplasticity using serum growth factors as a surrogate marker, using 1Hz rTMS with conventional physiotherapy in patients with sub acute ischemic stroke. Methods: In this study, participants with rst ever ischemic stroke (N = 96), onset within 15 days were randomized after a run-in period of 75 ± 7 days along with standard physical therapy to receive 10 sessions of real 1Hz rTMS (N = 47) on contralesional premotor cortex or to sham stimulation (N = 49) for 2 weeks. Participants, investigators and outcome assessors were blinded. The primary ecacy outcome was change in the level of peripheral serum growth factors VEGF & BDNF at third month. The secondary outcome was measurement of neurophysiological parameters and their correlation with growth factors levels. Results: Modied intention to treat analysis showed signicant up regulation in the mean level of serum VEGF & BDNF from pre to post rTMS in Real rTMS Group. Trend of decrease in Resting Motor Threshold and increase in Motor Evoked Potential in the affected hand was seen. Statistically signicant negative correlation between motor evoked potential and mean VEGF (rho = -1.000, P<0.001) in the affected hand in Real rTMS Group was seen. Conclusion: Total ten sessions of 1Hz rTMS plus physical therapy on contralateral hemisphere resulted in up regulation of serum growth factors possible reecting improved neuroplasticity. in the various recovery at day and at 3 Another showed positive correlation of serum VEGF with long term prognosis in patients with acute ischemia. Another study conducted and team administered electrical stimulation for seven day in adult Wistar rats. Signicant up regulation of both VEGF and BDNF was seen. Ours is the rst study in assessing the role of rTMS using low frequency with conventional physical therapy arm using large sample size of ischemic stroke patients at third month. Compared to healthy controls, higher expression of mean VEGF level was seen at the time of recruitment, signifying good prognosis. Results in our study have shown signicant up regulation of mean VEGF level in Real rTMS Group. Referring to our previous published study, rTMS intervention has shown signicant improvement in the magnitude of functional outcome in the active TMS arm 11 . Although, no signicant correlation of mean VEGF with functional outcome was seen but up regulation of mean VEGF at immediate rTMS could justify the role angiogenesis which might play an essential role in the pathophysiology of stroke recovery mediated by rTMS. polymorphism (val66met) negatively inuences the effect of rTMS on upper limb weakness. The changes in blood levels of BDNF may be due to TMS induced alteration of BDNF-TrkB signaling in the brain. Recent study conducted by Niimi et. al.,2016 27 investigated the molecular effects of rTMS using low frequency on serum levels of BDNF and other growth factors in stroke patients with upper limb hemiparesis. Statistically signicant increase in the serum BDNF level was seen after two weeks combination therapy. A community based cohort study 28 having N = 3440 participants was conducted in understanding the association of serum VEFG and BDNF levels & based on their analysis it was concluded that lower serum BDNF and higher VEGF concentrations were associated with increased risk of incident. Similar results could be seen in our study. Compared to healthy controls, lower levels of serum BDNF (ng/ml) were expressed. Immediate post RTMS showed signicant elevated levels of BDNF in Real rTMS Group. Similar ndings were seen in an observational study conducted by Sobrino et. al., 2020 1 , which reveals that elevated levels of serum endothelial growth factors are associated with good functional outcome at 3 months’ ischemic stroke onset. is the rst randomized controlled study with larger sample size to evaluate the association of both serum VEGF and BDNF levels in correlation to noninvasive approach with routine physical therapy intervention in understating neuroplasticity in ischemic stroke patients. mechanisms seen in other supportive evidences 34,35,36 Our results are consistent with the study done by Du and his team 37 , in which signicant behavior neurophysiological correlation between corticospinal excitability and motor improvement was seen at 3 months that indicates that enhanced motor cortical excitability in the stroke hemisphere is an important precondition for neural plasticity, which may allow the surviving neurons to reorganize in response to therapy. A trend of cortico excitability was seen in the affected arm in the neurophysiological parameters with a decrease in Resting Motor Threshold, increase in the Motor Evoked Potential in Real rTMS Group. and of the study have supported our study hypothesis. Higher levels of serum endothelial growth factors and signicant change in the neurophysiological markers and its signicant correlation with growth factors might play a vital role in understanding the neuroplasticity mechanism in stroke recovery. Follow up randomized controlled studies with large sample size are required to understand the role of TMS intensity and its exhibited ceiling effect in ischemic stroke recovery.


Background
Stroke is the leading cause of adult neurological disability, affecting one in six people worldwide. Although various acute rehabilitative measures have been introduced to improve the outcome, but full recovery is often incomplete. Although reperfusion treatment bene ts 20% of ischemic stroke patients, their effectiveness does not exceed 50-60% 1 . Although various functional enhancing management techniques like physical therapy and conventional tools are available but till date no validated rehabilitative approach has been introduced which could restore the impaired functions by a complete recovery of the damaged tissue 2 As new neuroprotective approaches are limited for clinicians and researchers, a need for new targets for the medium and long term prognosis of ischemic stroke is needed depending upon the size of infarction, speed and e cacy of the acute phase, therapeutic measures and the degree of neuro repair processes mediated by cerebral plasticity mechanism called cellular plasticity and its stimulation using growth factors causing neurogenesis, synaptogenesis mechanism and constitute a more translation therapeutic approach from the lab to the bedside 1 . Few supportive studies have evaluated the motor recovery pattern using non-invasive approach, despite of that more research in understanding the cellular mechanism and neuroprotective and restorative activities using trans cranial magnetic stimulation is needed 3,4 To overcome this problem, neuroplasticity plays a vital role in the functional prognosis of stroke patients, and the same could be modulated by non-invasive neuro rehabilitation tool called trans cranial magnetic stimulation. This interactive mechanism between TMS and neurons produces wide range of electrophysiological, biochemical and molecular, cellular changes and also affects behavior, mood, memory, myelination and neuroplasticity 5 At molecular level, certain neurotrophins like BDNF, VEGF are found to be upregulated in the rst 4 weeks after stroke, creating a " neuroplastic milieu" in which cortical remodeling within the intact brain is optimized 6 Various animal studies have studied the post ischemic molecular events in which growth of new synapses and dendritic branching modi es the intact sensorimotor cortex and subcortical structures contributing to the recovery of cognitive and motor performance, but understanding the neuroplasticity and neurogenesis model in patients with ischemic stroke recovery is yet to be studied 7,8,9 . Few researchers have studied the reduction in the oxidative stress and enhancement in the proliferation and signaling pathways using extremely low magnetic eld with physical rehabilitation with few endothelial biomarkers in acute ischemic stroke patients 10 but understanding the neuro pathophysiology and neurogenesis during Ischemic Stroke recovery stage is still lacking. Therefore, the aim of this study was to assess the serum growth factors in studying the cortical excitability measured through neurophysiological parameters by administrating low frequency rTMS as a non-invasive stimulation approach in combination with conventional physical therapy in sub acute ischemic stroke patients.

Methods
The study methodology protocol was considered and followed from our previous published article 11 , except the growth factor estimation and neurophysiological outcomes which is the secondary objective of our research in this manuscript.

Participants, Setting and Study Design
The study was conducted at the All India Institute of Medical Sciences (AIIMS), New Delhi. This was a single center, randomized, parallel group, double blind, sham controlled trial. AIIMS Ethics committee approved the study protocol. The trial was registered at the Clinical Trial Registry India (CTRI/2016/02/006620) and funded by Indian Council of Medical Research (ICMR), India. All the participants or their caregivers gave written informed consent. We included patients aged 18-75 years, with rst ever-acute ischemic stroke, within last 15 days documented by CT/MRI scan of the head and NIHSS of 4-20. We excluded participants who were medically unstable, pregnant, with any other brain lesion (tumor, infection), comatose or on mechanical ventilation, history of epilepsy or having any surgical implant/ pacemaker.

Procedure
Pre-randomization run-in period A trained licensed physiotherapist provided the standard physical therapy to all recruited participants. All participants received a standard care, which includes passive, active and active assisted exercises. Participants were encouraged to continue motor training at home after discharge and total number of hours of an individual exercise was monitored.

Growth factor estimation
Approximately 5ml non-fasting intravenous blood sample was withdrawn in an EDTA vial for the estimation of serum VEGF (pg/ml) and BDNF (ng/ml) at the time of recruitment, pre-rTMS and post rTMS period. Samples were then immediately centrifuged (1500g/15minutes, and serum was stored at -70 o C until assayed. Both VEGF (pg/ml) and BDNF (ng/ml) levels were measured using standard quantitative sandwich ELISA (Quantikine, USA) kits obtained from R & D Systems. Samples from each individual were analyzed in a triplicate and subsequently used in all further statistical analysis.

Randomization
Participants were randomly assigned in a 1:1 ratio to receive either Real rTMS or Sham rTMS. Randomization was done using a computer-generated randomization sequence. Allocations were concealed using sealed, opaque, numbered envelopes and were opened only during the time of randomization phase. The investigator, physiotherapist and participants were masked to the treatment allocation. Lab technician who was not part of the study was responsible for the random allocation of participants to Sham or Real rTMS Group. The outcome assessor was also blinded to allocation.

Interventions
Low frequency rTMS was performed using Magstim Rapid stimulator, Magstim Ltd, UK equipped with air-cooled gure of eight coil (70 mm) i:e biphasic pulse.
The resting motor evoked potential (MEP) was ascertained using an electromyogram, recording from the Abductor Pollicis Brevis (APB) in keeping with the International Federation of Clinical Neurophysiology (IFCN) recommendations 12 . The coil was placed tangentially to the scalp over the hand area of the primary motor cortex to calculate hot spot. Hot spot was de ned as the location on the scalp where stimulation of a slightly supra threshold intensity elicited the largest motor evoked potential (MEP) in the APB muscle. After the hot spot was identi ed, resting motor threshold was determined using the lowest stimulus intensity to produce motor evoked potential of > 50µV peak-to-peak amplitude in 5 out of 10 subsequent trails. If no MEP was obtained at the time of hot spot calculation in the affected ipsilesion M1, then the hotspot was de ned as the symmetric location to the contra lesion M1.The stimulation parameters were chosen in accordance with the safety guidelines for rTMS 12 Total 750 pulses, 75 trains using low frequency (1Hz) with inter train interval of 45 seconds at calculated intensity of 110% resting motor threshold (RMT) (Fc3/Fc4), was administered to the randomized patient. Localization was done using 10 -20 EEG methods. Sham rTMS pulses were administered using the same stimulation parameters over the contra-lesion premotor cortex area with the gure of eight coil angled at 90 degrees from the scalp. Patient was aroused throughout the rTMS administration.
The rTMS sessions on each day was followed by 45-minute conventional physical therapy regime given by a trained physiotherapist.

Monitoring for complications
All participants were monitored for any adverse events. A checklist of previously reported side effects was used to report any possible adverse events.

Outcomes
The co-primary e cacy outcomes were changes in serum growth factors level VEGF (pg/ml), BDNF (ng/ml) measured at 3 months of stroke onset. The secondary e cacy outcomes were change in the physiological markers Resting Motor Threshold, Motor Evoked Potential and change in the MEP duration during rest at 3 months stroke onset.

Sample size calculation
Sample size was calculated for primary clinical e cacy outcome, mBI with a superiority hypothesis assumption. Calculation was based on the study done by Khedr et. al. 13 who reported 34.6% and 7.7% of good/excellent outcome under rTMS and sham rTMS respectively, which yielded a sample size of 45 in each arm (90% power and a two-sided α level of 5%). Adjusting for 10% attrition we estimated a sample size of 100 (50 in each arm)

Statistical analysis
Statistical analysis was performed using STATA version 14.1 and was based on modi ed intention to treat principle. Normal distribution was tested using Kolmogorov -Smirnov statistic. Study data was not following normal distribution; hence non-parametric tests were applied. For Categorical data, 2x2 table was generated. Chi-square test/ Fisher's exact test was applied to compare the properties in the two groups. Between groups comparisons were carried out using Wilcoxon's rank sum test. Wilcoxon signed rank test was used to assess changes in the scores within the same group. Non-parametric independent test analysis was also done using log algorithm. Nemar test was applied for the categorical variable. For serum estimation, non-parametric independent t-test was applied. For correlation analysis, spearman correlation was applied.

Rationale
The rationale of the study was to conduct the clinical translation research using rTMS along with physiotherapy at sub-acute recovery stage which is considered as the golden period for the initiation of exogenous restorative therapies. Endogenous repair reaches peak levels at this stage followed by the functional reorganization and enhances neural plasticity in the brain 14 .

Results
Between August 2012 and February 2016, 445 participants with acute ischemic stroke were screened and 139 participants ful lling the eligibility criteria were recruited into the pre-rTMS run-in phase. During the run-in period, 35 participants withdrew consent and 4 died after discharge from the hospital.
Randomization was done in 100 participants (Fig. 1). Four participants were excluded after randomization (consent withdrawn after randomization). The baseline characteristics were comparable between Real rTMS (n = 47) and sham rTMS (n = 49). Mean age in Real rTMS arm was 54.85 ± 13.39 vs. 52.89 ± 14.95 years in Sham rTMS arm. There were more of male compared to female in both the groups (Table 1.). Mean serum biomarkers VEGF (pg/ml) and BDNF (ng/ml) were comparable. All study participants received standard of care and at least one session of real or sham rTMS. Seven participants in real rTMS were lost to follow up. One participant had seizure and un-blinding was done for that participant and was managed symptomatically. Modi ed intention to treat analysis was done for 47 participants in real and 49 participants in sham rTMS arm.

Growth Factors Estimation:
Compared to healthy controls, down regulation expression level of mean serum BDNF was seen. On the other hand, mean VEGF was elevated on 5 th day at the time of recruitment. (Table 2 (Table 8. & 9.) One participant in the real TMS arm developed seizure (generalized tonic clonic seizure) 18 hours after the fourth session and about 1 h after waking from sleep in the morning. CT scan of head did not reveal any fresh changes. He was treated with phenytoin and the event was reported to Institute's Ethics Committee. The participant did not receive any more TMS sessions. No other adverse events were reported in the participants.

Discussion
In this Randomized controlled study, we measured the levels of serum growth factors VEGF and BDNF after administrating low frequency rTMS onto the contra lesion hemisphere along with conventional physiotherapy in patients with sub acute ischemic stroke. We assessed the neurophysiological parameters as a secondary outcome. In addition to this, a correlation of growth factors levels with neurophysiological parameters was also done at third month.
Although various studies using human models have been conducted in understanding the motor and clinical recovery but very limited studies in humans have been done in understanding the cellular mechanisms mediating rTMS neuroprotective and restorative activities. We speculated that post stroke noninvasive treatment approach induced neurogenesis might play a future potential target for therapeutic intervention.
Post stroke acute recovery is known to be correlated with endothelial function. With the release of polypeptide growth factors after acute ischemic stroke, they are likely to play a vital role in the both cellular and molecular process underlying wound healing and functional recovery in Stroke patients. Various observational studies reveal that VEGF mRNA up regulates under hypoxic conditions and elevated levels at during 7th day and 3th month are found to be independently associated with good functional outcome 15,1 In our study, we found signi cant change in the mean serum biomarkers VEGF and BDNF in an active arm i:e Real rTMS Group compared with placebo arm i.e. Sham rTMS Group with conventional physical therapy in Stroke patients at third month.
VEGF is the most prominent hypoxia inducible angiogenic factor and a key mediator of angiogenesis, which is an important process leading to reperfusion of ischemic brain tissue after acute stroke, and is also implicated as an essential factor promoting neurogenesis in the adult brain, increasing proliferation and differentiation of endothelial and neuronal progenitor cells 16,17,18 . Increased VEGF might have long term bene cial effects as a result of continued angiogenesis over several months 15 . Various studies have been conducted in understating the VEGF expression various stroke recovery stages. Study conducted by Sobrino et. al. 1 . had shown high serum VEGF level at day 7 and at 3 months which is directly associated with good functional outcome. Another study conducted by Lee and his colleagues 19 showed positive correlation of serum VEGF with long term prognosis in patients with acute ischemia. Another study conducted by Baba and his team 20 administered electrical stimulation for seven day in adult Wistar rats. Signi cant up regulation of both VEGF and BDNF was seen. Ours is the rst study in assessing the role of rTMS using low frequency with conventional physical therapy arm using large sample size of ischemic stroke patients at third month. Compared to healthy controls, higher expression of mean VEGF level was seen at the time of recruitment, signifying good prognosis. Results in our study have shown signi cant up regulation of mean VEGF level in Real rTMS Group. Referring to our previous published study, rTMS intervention has shown signi cant improvement in the magnitude of functional outcome in the active TMS arm 11 . Although, no signi cant correlation of mean VEGF with functional outcome was seen but up regulation of mean VEGF at immediate rTMS could justify the role angiogenesis which might play an essential role in the pathophysiology of stroke recovery mediated by rTMS.
Another neurotropic factor called BDNF is known to be involved in ischemia induced neurogenesis processes, increases recruitment of endogenous progenitors to injured brain regions, mediating repair mechanisms and neuronal plasticity in various brain disorders. It also induces synaptogenesis, morphogenesis, and plasticity of dendritic spines, resulting in synapses with functionality 21,22 and also plays an important role as an effective indicator for rehabilitation interventions 23 . Supportive evidence based studies suggests that rTMS causes enhancement of peripheral BDNF levels in patients suffering from Depression. In healthy human subjects, BDNF has shown activation of BDNF signaling pathways [24][25][26] . There is accumulating evidence on the role of BDNF in therapeutic effects using low frequency rTMS. BDNF polymorphism (val66met) negatively in uences the effect of rTMS on upper limb weakness. The changes in blood levels of BDNF may be due to TMS induced alteration of BDNF-TrkB signaling in the brain. Recent study conducted by Niimi et.
al.,2016 27 investigated the molecular effects of rTMS using low frequency on serum levels of BDNF and other growth factors in stroke patients with upper limb hemiparesis. Statistically signi cant increase in the serum BDNF level was seen after two weeks combination therapy. A community based cohort study 28 having N = 3440 participants was conducted in understanding the association of serum VEFG and BDNF levels & based on their analysis it was concluded that lower serum BDNF and higher VEGF concentrations were associated with increased risk of incident.
Similar results could be seen in our study. Compared to healthy controls, lower levels of serum BDNF (ng/ml) were expressed. Immediate post RTMS showed signi cant elevated levels of BDNF in Real rTMS Group. Similar ndings were seen in an observational study conducted by Sobrino et. al., 2020 1 , which reveals that elevated levels of serum endothelial growth factors are associated with good functional outcome at 3 months' ischemic stroke onset.
Ours is the rst randomized controlled study with larger sample size to evaluate the association of both serum VEGF and BDNF levels in correlation to noninvasive approach with routine physical therapy intervention in understating neuroplasticity in ischemic stroke patients.

Neurophysiological Markers:
The therapeutic effects of non-invasive stimulation i:e Trans cranial magnetic stimulation could only be evaluated by applying either low (inhibitory) on contra lesion or high (excitatory) on ipisilesion hemisphere in stroke patients. rTMS has been known to restore the unbalanced interhemispheric inhibition, which results in increased inhibition in the ischemic hemisphere and subsequent worsening in motor function whereas the neurophysiological effect of rTMS on neuronal activity has not been well explored. TMS is known to be used as an excellent non-invasive tool to map the electrophysiological changes in the cortical hemispheres. In addition, that, they are used to dissect physiological mechanisms underlying motor de cits, spontaneous motor recovery, and the effect of therapeutic interventions in ischemic stroke like motor threshold, motor evoked potential, latency period etc.
Neurophysiological changes occur both in acute and sub acute phase of stroke mostly attributable to pathophysiological changes like reversal of diaschisis, resolution of edema etc. 29 . Neurophysiological parameters like MEP, amplitude, MEP duration could provide an important insight into the motor cortical function. 30 As MEP plays a main dependent variable in understanding the cortical function, recently the role of MEP duration was recently studied in both resting and facilitated state using various stimulus intensities to understand the underlying physiological changes in MEP duration re ecting cortical and nonpropriospinal mechanism involvement in stroke recovery 30 In our study, MEP duration was assessed during resting state at a calculated intensity of 110% RMT and was correlated with the change in serum growth factors level to determine the cortical mechanisms and ceiling effects. MEP duration was measured from MEP onset latency to the time at which the activity returned to baseline. We determined the absolute change in the MEP duration during rest. Compared to control arm, statistically signi cant positive correlation of serum BDNF with delta MEP duration in the unaffected hand was seen in the active arm. Ours is the rst study to correlate serum growth factor with change in the MEP duration in sub acute ischemic stroke patients using TMS as a main dependent factor in understanding the ceiling effect. Marisa and her colleagues measured an increase in duration of MEP during contraction in a group of healthy volunteers and on a group of patients with various neurological disorders. Compared to healthy controls, signi cant difference in the MEP duration at rest was seen 31 An observational study was conducted to understand the physiological process underlying changes in MEP duration using TMS on healthy controls.
Signi cant correlation of cortical inhibition and MEP duration was seen which might contribute as a future biomarker of motor cortical function 30 Recent meta-analysis conducted by Zhang et. al., 2017 32 reported that low frequency rTMS as an add on therapy improves functional recovery in Stroke patients. Trend of decrease in the cortico excitability, re ected in the form of Resting Motor Threshold and increase in the Motor Evoked Potential Amplitude in the affected hemisphere and vice versa has been found to be associated with motor recovery 33 . The pooled effects could also be seen in other supportive evidences 34,35,36 Our results are consistent with the study done by Du and his team 37 , in which signi cant behavior neurophysiological correlation between corticospinal excitability and motor improvement was seen at 3 months that indicates that enhanced motor cortical excitability in the stroke hemisphere is an important precondition for neural plasticity, which may allow the surviving neurons to reorganize in response to therapy. A trend of cortico excitability was seen in the affected arm in the neurophysiological parameters with a decrease in Resting Motor Threshold, increase in the Motor Evoked Potential in Real rTMS Group.

Conclusions
The primary and secondary outcomes of the study have supported our study hypothesis. Higher levels of serum endothelial growth factors and signi cant change in the neurophysiological markers and its signi cant correlation with growth factors might play a vital role in understanding the neuroplasticity mechanism in stroke recovery. Follow up randomized controlled studies with large sample size are required to understand the role of TMS intensity and its exhibited ceiling effect in ischemic stroke recovery.