The Effects of Different Repetitive Transcranial Magnetic Stimulation (rTMS) Protocols on Cortical Gene Expression in a Rat Model of Cerebral Ischemic-Reperfusion Injury
Milos R. Ljubisavljevic | Asma Javid | Joji Oommen | Khatija Parekh | Nico Nagelkerke | Safa Shehab | Thomas E. Adrian
Published: October 2, 2015 | DOI: 10.1371/journal.pone.0139892
Copyright © 2015 PLOS All rights reserved.
Although repetitive Transcranial Magnetic Stimulation (rTMS) in treatment of stroke in humans has been explored over the past decade the data remain controversial in terms of optimal stimulation parameters and the mechanisms of rTMS long-term effects. This study aimed to explore the potential of different rTMS protocols to induce changes in gene expression in rat cortices after acute ischemic-reperfusion brain injury. The stroke was induced by middle cerebral artery occlusion (MCAO) with subsequent reperfusion. Changes in the expression of 96 genes were examined using low-density expression arrays after MCAO alone and after MCAO combined with 1Hz, 5Hz, continuous (cTBS) and intermittent (iTBS) theta-burst rTMS. rTMS over the lesioned hemisphere was given for two weeks (with a 2-day pause) in a single daily session and a total of 2400 pulses. MCAO alone induced significant upregulation in the expression of 44 genes and downregulation in 10. Two weeks of iTBS induced significant increase in the expression of 52 genes. There were no downregulated genes. 1Hz and 5Hz had no significant effects on gene expression, while cTBS effects were negligible. Upregulated genes included those involved in angiogenesis, inflammation, injury response and cellular repair, structural remodeling, neuroprotection, neurotransmission and neuronal plasticity. The results show that long-term rTMS in acute ischemic-reperfusion brain injury induces complex changes in gene expression that span multiple pathways, which generally promote the recovery. They also demonstrate that induced changes primarily depend on the rTMS frequency (1Hz and 5Hz vs. iTBS) and pattern (cTBS vs. iTBS). The results further underlines the premise that one of the benefits of rTMS application in stroke may be to prime the brain, enhancing its potential to cope with the injury and to rewire. This could further augment its potential to favorably respond to rehabilitation, and to restore some of the loss functions.
Transcranial Magnetic Stimulation (TMS) is a well-established, non-invasive technique that allows the assessment and modulation of brain excitability. Repetitive TMS (rTMS), a variant of TMS that involves repeated application of TMS pulses, may facilitate or suppress brain activity with variable behavioral effects. Research generally shows that the functional effects of rTMS on cortical excitability depend on stimulation intensity, frequency and the overall stimulation pattern. It appears that rTMS repeated at fixed high-frequency intervals (> 4 Hz) increase cortical excitability, while stimuli repeated at low-frequency (~ 1Hz) decrease it . rTMS protocols utilizing patterned stimulation like theta-burst patterns (bursts of 3–5 pulses at 50–100 Hz, repeated at 5 Hz, i.e. theta rhythm) appear to enhance cortical excitability if applied continuously (cTBS), whereas if applied intermittently (iTBS) they tend to lower cortical excitability . Furthermore, changes in cortical excitability elicited by rTMS may outlast the duration of the stimulation , a finding that has prompted considerable exploration of the potential of rTMS neurological and psychiatric therapy. Limited but promising data currently exist for the benefit of the rTMS in the treatment of depression, tinnitus, anxiety disorders, neurodegenerative diseases and pain syndromes . In stroke, rTMS was also applied based on a model of interhemispheric competition for sensory and motor processing [5–7], prompting development of two conceptually different stimulation strategies [8,9]: one aiming to increase excitability of the affected hemisphere by excitatory rTMS [10–12] and the other aiming to suppress excitability of the unaffected hemisphere by inhibitory rTMS [13–16]. In patients with chronic post-stroke hemiparesis, for example, stimulation of the affected motor cortex with 5 Hz  or 10 Hz  facilitated practice-dependent plasticity and improved motor learning, whereas inhibition of the contra-lesional hemisphere with 1-Hz rTMS also enhanced motor recovery [13,14,17].
The current understanding of mechanisms aiding functional recovery after stroke suggest complex mechanisms that involve resolution of edema and necrotic tissue, reperfusion of the ischemic penumbra  and a set of neuronal compensatory mechanisms. These mechanisms include the recruitment of new/additional pathways, disinhibition of redundant neuronal connections and formation of new neural networks to take over function of the damaged areas . What remains unclear are the effects of rTMS and how it interacts with such complex cellular and molecular milieu. In animals, rTMS increases the content of ATP and microtubule associated protein-2 expression  while promoting the recovery of the neuronal function , enhancing the long-term potentiation of the hippocampal neurons , preventing ischemic neural damage , enhancing anti-apoptotic mechanisms in the peri-ischemic area  and inducing neuroprotective effects .
However, in human studies, variable effects of rTMS interventions were reported [26,27]. The duration of the effects also seems to vary and depend on several factors, including the timing of the rTMS application (subacute or chronic stroke), the patient’s characteristics and the site of stimulation [28–30].The possibility of varying rTMS parameters (intensity, pattern, duration) makes the potential effects and therapeutic outcomes even more unpredictable. Furthermore, the effectiveness of rTMS may be influenced by the nature of the underlying pathological processes. A common assumption is that therapeutic effects in patients can be predicted based on the modulatory effects of rTMS in healthy subjects. However, there is no real evidence to suggest that this is always the case because the susceptibility to the conditioning effects of rTMS may well depend on the underlying pathology. Finally, it is apparent that the long-term clinical improvements caused by rTMS cannot be entirely explained by immediate electrophysiological processes caused by rTMS. Rather, processes beyond instantaneous electrophysiological modulation of neuronal activity related to adaptive changes in gene expression may be involved in sustaining rTMS effects.
This study primarily aimed to examine whether different rTMS protocols have differential effects on gene expression in lesioned cortices after ischemic-reperfusion brain injury. Thus, we examined the effects of four standard rTMS protocols (1Hz, 5 Hz, cTBS and iTBS) on functional recovery and changes in gene expression in lesioned rat cortices with subacute cerebral ischemic-reperfusion injury induced by middle cerebral artery occlusion (MCAO). We assessed changes in the expression of 98 genes known to be altered by stroke, as well as those potentially involved in promoting recovery after stroke, with real-time RT-PCR after two weeks of rTMS. […]