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 significant 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 beneficial 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 colleagues19 showed positive correlation of serum VEGF with long term prognosis in patients with acute ischemia. Another study conducted by Baba and his team20 administered electrical stimulation for seven day in adult Wistar rats. Significant up regulation of both VEGF and BDNF was seen. Ours is the first 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 significant up regulation of mean VEGF level in Real rTMS Group. Referring to our previous published study, rTMS intervention has shown significant improvement in the magnitude of functional outcome in the active TMS arm 11. Although, no significant 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 24–26. There is accumulating evidence on the role of BDNF in therapeutic effects using low frequency rTMS. BDNF polymorphism (val66met) negatively influences 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.,201627 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 significant increase in the serum BDNF level was seen after two weeks combination therapy. A community based cohort study 28having 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 significant elevated levels of BDNF in Real rTMS Group. Similar findings were seen in an observational study conducted by Sobrino et. al., 20201, 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 first randomized controlled study with larger sample size to evaluate the association of both serum VEGF and BDNF levels in correlation to non- invasive 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 deficits, 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 reflecting cortical and non-propriospinal mechanism involvement in stroke recovery30
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 significant positive correlation of serum BDNF with delta MEP duration in the unaffected hand was seen in the active arm. Ours is the first 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, significant 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. Significant 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., 201732 reported that low frequency rTMS as an add on therapy improves functional recovery in Stroke patients. Trend of decrease in the cortico excitability, reflected 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 team37, in which significant 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.