Transcranial-Direct-Current-Stimulation Accelerates Motor Recovery After Cortical Infarction in Mice: The Interplay of Structural Cellular Responses and Functional Recovery.

BACKGROUND: Transcranial direct current stimulation (tDCS) promotes recovery after stroke in humans. The underlying mechanisms, however, remain to be elucidated. Animal models suggest tDCS effects on neuroinflammation, stem cell proliferation, neurogenesis, and neural plasticity. OBJECTIVE: In a longitudinal study, we employed tDCS in the subacute and chronic phase after experimental focal cerebral ischemia in mice to explore the relationship between functional recovery and cellular processes. METHODS: Mice received photothrombosis in the right motor cortex, verified by Magnetic Resonance Imaging. A composite neuroscore quantified subsequent functional deficits. Mice received tDCS daily: either 5 sessions from day 5 to 9, or 10 sessions with days 12 to 16 in addition. TDCS with anodal or cathodal polarity was compared to sham stimulation. Further imaging to assess proliferation and neuroinflammation was performed by immunohistochemistry at different time points and Positron Emission Tomography at the end of the observation time of 3 weeks. RESULTS: Cathodal tDCS at 198 kC/m2 (220 A/m2) between days 5 and 9 accelerated functional recovery, increased neurogenesis, decreased microglial activation, and mitigated CD16/32-expression associated with M1-phenotype. Anodal tDCS exerted similar effects on neurogenesis and microglial polarization but not on recovery of function or microglial activation. TDCS on days 12 to 16 after stroke did not induce any further effects, suggesting that the therapeutic time window was closed by then. CONCLUSION: Overall, data suggest that non-invasive neuromodulation by tDCS impacts neurogenesis and microglial activation as critical cellular processes influencing functional recovery during the early phase of regeneration from focal cerebral ischemia.

Transcranial Current Stimulation Alters the Expression of Immune-Mediating Genes.

Despite its extensive use in clinical studies, the molecular mechanisms underlying the effects of transcranial direct current stimulation (tDCS) remain to be elucidated. We previously described subacute effects of tDCS on immune- and stem cells in the rat brain. To investigate the more immediate effects of tDCS regulating those cellular responses, we treated rats with a single session of either anodal or cathodal tDCS, and analyzed the gene expression by microarray; sham-stimulated rats served as control. Anodal tDCS increased expression of several genes coding for the major histocompatibility complex I (MHC I), while cathodal tDCS increased the expression of the immunoregulatory protein osteopontin (OPN). We confirmed the effects of gene upregulation by immunohistochemistry at the protein level. Thus, our data show a novel mechanism for the actions of tDCS on immune- and inflammatory processes, providing a target for future therapeutic studies.

Bioluminescence imaging visualizes osteopontin-induced neurogenesis and neuroblast migration in the mouse brain after stroke.

BACKGROUND: Osteopontin (OPN), an acidic phosphoglycoprotein, is upregulated in the brain after cerebral ischemia. We previously reported that OPN supports migration, survival, and proliferation of neural stem cells (NSC) in primary cell culture, as well as their differentiation into neurons. We here analyzed the effects of OPN on neuroblasts in vivo in the context of cerebral ischemia. METHODS: Transgenic mice expressing luciferase under the control of the neuroblast-specific doublecortin (DCX)-promoter, allowing visualization of neuroblasts in vivo using bioluminescence imaging (BLI), were injected with OPN intracerebroventricularly while control mice were injected with vehicle buffer. To assess the effects of OPN after ischemia, additional mice were subjected to photothrombosis and injected with either OPN or vehicle. RESULTS: OPN enhanced the migration of neuroblasts both in the healthy brain and after ischemia, as quantified by BLI in vivo. Moreover, the number of neural progenitors was increased following OPN treatment, with the maximum effect on the second day after OPN injection into the healthy brain, and 14 days after OPN injection following ischemia. After ischemia, OPN quantitatively promoted the endogenous, ischemia-induced neuroblast expansion, and additionally recruited progenitors from the contralateral hemisphere. CONCLUSIONS: Our results strongly suggest that OPN constitutes a promising substance for the targeted activation of neurogenesis in ischemic stroke.

Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps.

Heartworm disease is a zoonotic vector-borne disease caused by Dirofilaria immitis mainly affecting canids. Infectious third-stage larvae (L3) are transmitted to the definitive hosts via culicid mosquitoes; adult nematodes reside in the pulmonary arteries and in the right heart releasing unsheathed first-stage larvae (microfilariae) into the bloodstream leading to chronic and sometimes fatal disease. So far, early innate immune reactions triggered by these different D. immitis stages in the canine host have scarcely been investigated. Therefore, D. immitis microfilariae and L3 were analyzed for their capacity to induce neutrophil extracellular traps (NETs) in canine polymorphonuclear neutrophils (PMN). Overall, scanning electron microscopy analysis revealed both larval stages as strong inducers of canine NETosis. Co-localization of PMN-derived extracellular DNA with granulocytic histones, neutrophil elastase, or myeloperoxidase in parasite-entrapping structures confirmed the classical characteristics of NETosis. Quantitative analyses showed that both larval stages triggered canine NETs in a time-dependent but dose-independent manner. Moreover, parasite-induced NET formation was not influenced by the parasites viability since heat-inactivated microfilariae and L3 also induced NETs. In addition, parasite/PMN confrontation promoted significant entrapment but not killing of microfilariae and L3. Both, NETosis and larval entrapment was significantly reversed via DNase I treatments while treatments with the NADPH oxidase inhibitor diphenyleneiodonium failed to significantly influence these reactions. Interestingly, different types of NETs were induced by microfilariae and L3 since microfilarial stages merely induced spread and diffuse NETs while the larger L3 additionally triggered aggregated NET formation.

Transcranial Direct Current Stimulation Modulates Neurogenesis and Microglia Activation in the Mouse Brain.

Transcranial direct current stimulation (tDCS) has been suggested as an adjuvant tool to promote recovery of function after stroke, but the mechanisms of its action to date remain poorly understood. Moreover, studies aimed at unraveling those mechanisms have essentially been limited to the rat, where tDCS activates resident microglia as well as endogenous neural stem cells. Here we studied the effects of tDCS on microglia activation and neurogenesis in the mouse brain. Male wild-type mice were subjected to multisession tDCS of either anodal or cathodal polarity; sham-stimulated mice served as control. Activated microglia in the cerebral cortex and neuroblasts generated in the subventricular zone as the major neural stem cell niche were assessed immunohistochemically. Multisession tDCS at a sublesional charge density led to a polarity-dependent downregulation of the constitutive expression of Iba1 by microglia in the mouse cortex. In contrast, both anodal and, to an even greater extent, cathodal tDCS induced neurogenesis from the subventricular zone. Data suggest that tDCS elicits its action through multifacetted mechanisms, including immunomodulation and neurogenesis, and thus support the idea of using tDCS to induce regeneration and to promote recovery of function. Furthermore, data suggest that the effects of tDCS may be animal- and polarity-specific.

Transcranial direct current stimulation in the male mouse to promote recovery after stroke.

Transcranial direct current stimulation (tDCS) constitutes a promising approach for promoting recovery of function after stroke, although the underlying neurobiological mechanisms are unclear. To conduct translational research in animal models, stimulation parameters should not lead to neuronal lesions. Liebetanz et al. recommend charge densities for cathodal stimulation in rats, but parameters for mice are not established. We established tDCS in the wild-type mouse, enabling studies with genetically-engineered mice (GEM). tDCS equipment was adapted to fit the mouse skull. Using different polarities and charge densities, tDCS was safe to apply in the mouse where the charge density was below 198 kC/m(2) for single or repeated stimulations. These findings are crucial for future investigations of the neurobiological mechanisms underlying tDCS using GEM.

Transcranial direct current stimulation promotes the mobility of engrafted NSCs in the rat brain.

Transcranial direct current stimulation (tDCS) is used in numerous clinical studies and considered an effective and versatile add-on therapy in neurorehabilitation. To date, however, the underlying neurobiological mechanisms remain elusive. In a rat model of tDCS, we recently observed a polarity-dependent accumulation of endogenous neural stem cells (NSCs) in the stimulated cortex. Based upon these findings, we hypothesized that tDCS may exert a direct migratory effect on endogenous NSCs towards the stimulated cortex. Using noninvasive imaging, we here investigated whether tDCS may also cause a directed migration of engrafted NSCs. Murine NSCs were labeled with superparamagnetic particles of iron oxide (SPIOs) and implanted into rat striatum and corpus callosum. MRI was performed (i) immediately after implantation and (ii) after 10 tDCS sessions of anodal or cathodal polarity. Sham-stimulated rats served as control. Imaging results were validated ex vivo using immunohistochemistry. Overall migratory activity of NSCs almost doubled after anodal tDCS. However, no directed migration within the electric field (i.e. towards or away from the electrode) could be observed. Rather, an undirected outward migration from the center of the graft was detected. Xenograft transplantation induced a neuroinflammatory response that was significantly enhanced following cathodal tDCS. This inflammatory response did not impact negatively on the survival of implanted NSCs. Data suggest that anodal tDCS increases the undirected migratory activity of implanted NSCs. Since the electric field did not guide implanted NSCs over large distances, previously observed polarity-dependent accumulation of endogenous NSCs in the cortex might have originated from local proliferation. Results enhance our understanding of the neurobiological mechanisms underlying tDCS, and may thereby help to develop a targeted and sustainable application of tDCS in clinical practice.