Damn it all, write up a fucking protocol so it can be critiqued and refined or improved upon. Because we have no one writing protocols no one knows what else is going on in other areas of the world. As a result stroke survivors are badly served. A great stroke association president would be knocking heads over this lack of professionalism. And yet our fucking failures of stroke associations do nothing to solve this problem. DAMN YOU ALL TO HELL!
Continuous theta-burst stimulation enhances and sustains neurogenesis following ischemic stroke
Abstract
Rationale: Previous work has indicated that continuous theta-burst stimulation (cTBS), a modality of transcranial magnetic stimulation (TMS), may provide neuroprotection and improve neurological function after stroke by preserving the blood-brain barrier, altering glial polarization phenotypes, and supporting peri-infarct angiogenesis. The present study was performed to examine whether cTBS, a noninvasive neurostimulation technique, promotes neurogenesis in a photothrombotic (PT) stroke rat model and contributes to functional recovery.
Methods: Beginning 3 h or 1 week after the induction of PT stroke, once-daily 5-min cTBS treatments were applied to the infarcted hemisphere for 6 days. Samples were collected 6 days, 22 days, and 35 days after PT stroke. Fluorescent labeling, Western blotting, and behavioral tests were performed accordingly.
Results: We found that cTBS therapy significantly expanded the pool of neural progenitor cells (NPCs) and newly generated immature neurons in the cortical peri-infarct region after PT stroke. Likewise, the amount of DCX-positive immature neurons in the peri-infarct area was markedly elevated by cTBS. Application of cTBS strikingly diminished the PT-induced loss of NPCs and newly-formed neurons. In addition, the amount of newly generated mature neurons in the peri-infarct zone was significantly promoted by cTBS. Intriguingly, cTBS reduced reactive gliogenesis significantly while promoting oligodendrogenesis and preserving myelination. Mechanistic studies uncovered that cTBS upregulated brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2). Finally, cTBS-treated animals displayed improved motor functions. To be noted, temozolomide (TMZ), a drug that has been previously used to suppress neurogenesis, could reverse the beneficial effects of cTBS.
Conclusions: Our findings provide new insight into the mechanism by which cTBS promotes functional recovery from stroke. We demonstrated that cTBS effectively enhances and sustains neurogenesis after PT stroke. Both early and delayed cTBS treatment could improve the survival of newly generated neurons and functional recovery, and inhibition of neurogenesis could reverse these therapeutic benefits. Mechanistically, cTBS was effective in upregulating the release of neurotrophic factors, protecting NPC and immature neurons, as well as suppressing excessive gliogenesis.
Introduction
Stroke, the cessation or reduction of blood flow to a part of the brain, is a pressing public health issue in the United States, affecting nearly 800,000 people every year 1. This disease takes a drastic toll on patients; ~150,000 people died from a stroke in 2018 alone, and 50% of survivors developed some form of chronic disability 1, 2. Ischemic stroke is the most common form of stroke, accounting for 87% of cases, and occurs when a cerebral blood vessel is occluded, usually from a clot. Unfortunately, effective treatment options for stroke are currently limited to mechanical thrombectomy and tissue plasminogen activator (tPA) 3. However, both of these treatment strategies must be applied within a short time window to save brain tissue within the core infarct, 4.5 hours for tPA and 6 hours for mechanical thrombectomy 4. As a result, only a few patients will benefit from these treatment interventions, and the functional recovery of survivors is usually limited. Indeed, even if reperfusion means such as intravenous thrombolysis and endovascular therapy are applied, the 90-day good prognosis rate (mRS score 0-2) of patients is only about 50%, with a high recurrence rate of related symptoms. Hence, there is an urgent need to develop new treatment strategies that can effectively reduce disability rates and facilitate functional recovery.
The process of post-stroke neuronal cell death is not immediate, however. While the tissue at the infarct site is lost due to necrotic cell death within four hours of stroke, neurons in the surrounding tissue, called the penumbra or peri-infarct region, slowly die over the next few days through the process of programmed neuronal cell death 5. Thus, if the peri-infarct tissue could be preserved or restored, neurological outcomes for patients could be improved. Therefore, therapies promoting endogenous repair mechanisms are highly sought after and are the goal of extensive biomedical research.
Once thought to be a phenomenon limited to early development, adult neurogenesis has since been well-established as occurring in response to ischemic brain injury 6-8. Shortly after cerebral ischemia, neural progenitor cells (NPCs) from the subventricular zone (SVZ) and the subgranular zone (SGZ) begin proliferating and migrating to the peri-infarct area 9. Once there, they begin to differentiate and attempt to incorporate themselves into the neuronal architecture. Unfortunately, the harsh post-stroke neuronal microenvironment takes a toll on NPCs and newly formed neurons, killing most of them long-term 10, 11. The hostile milieu present after stroke is mediated, in part, by activated astroglial cells 12.
Activated astrocytes release inflammatory factors that can, in the early phase, stimulate a neurogenic response. However, a chronic neuroinflammatory response creates toxic microenvironmental conditions that are harmful to new neurons 13-15. However, astroglia can release anti-inflammatory factors and trophic factors such as brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2) that are critical for supporting new neurons and oligodendrocyte progenitors as they attempt to repair the peri-infarct site 13, 14, 16, 17. Thus, one key to neural repair may be noninvasive treatments like transcranial magnetic stimulation (TMS) that target different components that contribute to the neuronal microenvironment 18.
Continuous theta-burst stimulation (cTBS) is a modality of TMS that has shown many beneficial actions in the context of stroke and other brain injury models 19. In TMS, a strong magnetic field is applied to a specific brain region to induce a current that can either stimulate or suppress local activity 19. This noninvasive therapy is currently used in the clinic for treatment-resistant depression and is praised for its efficacy and minimal adverse effects 20. In our previous work, we applied cTBS to the photothrombotic stroke model in rats, which uses a photochemical reaction to generate a core infarct surrounded by a rim of salvageable peri-infarct tissue 18, 21. We found that cTBS preserved neuronal survival and functional outcomes by ameliorating the hostile post-stroke microenvironment. Furthermore, astroglial and microglial phenotype polarization was shifted from the damaging proinflammatory states to a beneficial anti-inflammatory phenotype in cTBS-treated rats. In addition, cTBS stimulated angiogenesis in the peri-infarct region, which is spatiotemporally coupled with neurogenesis 21, 22. Therefore, we conducted the current study to investigate if cTBS can stimulate and support neurogenesis after experimental stroke and determine whether this is supported by trophic factor release.
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Vincenzo Di Lazzaro
Fioravante Capone
Giovanni Di Pino
Giovanni Pellegrino
Lucia Florio1, 
Davide Simonetti