Efficient high-resolution TMS mapping of the human motor cortex by nonlinear regression

Transcranial magnetic stimulation (TMS) is a powerful tool to investigate causal structure-function relationships in the human brain. However, a precise delineation of the effectively stimulated neuronal populations is notoriously impeded by the widespread and complex distribution of the induced electric field. Here, we propose a method that allows rapid and feasible cortical localization at the individual subject level. The functional relationship between electric field and behavioral effect is quantified by combining experimental data with numerically modeled fields to identify the cortical origin of the modulated effect. Motor evoked potentials (MEPs) from three finger muscles were recorded for a set of random stimulations around the primary motor area. All induced electric fields were nonlinearly regressed against the elicited MEPs to identify their cortical origin. We could distinguish cortical muscle representation with high spatial resolution and localized them primarily on the crowns and rims of the precentral gyrus. A post-hoc analysis revealed exponential convergence of the method with the number of stimulations, yielding a minimum of about 180 random stimulations to obtain stable results. Establishing a functional link between the modulated effect and the underlying mode of action, the induced electric field, is a fundamental step to fully exploit the potential of TMS. In contrast to previous approaches, the presented protocol is particularly easy to implement, fast to apply, and very robust due to the random coil positioning and therefore is suitable for practical and clinical applications

Concise review : increasing the validity of cerebrovascular disease models and experimental methods for translational stem cell research

Interspecies differences, anatomical and physiological aspects, as wells as simplified study designs contribute to an overestimation of treatment effects and limit the transferability of experimental results into clinical applications. Confounders of cell therapies for cerebrovascular disorders (CVD) include common CVD comorbidities, frequent medications potentially affecting endogenous and transplanted stem cells, as well as age‐ and immune‐system–related effects. All those can contribute to a substantial modeling bias, ultimately limiting the prospective quality of preclinical research programs regarding the clinical value of a particular cell therapy. In this review, we discuss the nature and impact of most relevant confounders. We provide suggestions on how they can be considered to enhance the validity of CVD models in stem cell research. Acknowledging substantial and sometimes surprising effects of housing conditions, chronobiology, and intersex differences will further augment the translational value of animal models. We finally discuss options for the implementation of high‐quality functional and imaging readout protocols. Altogether, this might help to gain a more holistic picture about the therapeutic impact of a particular cell therapy for CVD, but also on potential side and off‐site effects of the intervention

In Vivo Imaging of Stepwise Vessel Occlusion in Cerebral Photothrombosis of Mice by 19F MRI

Background $^{19}$F magnetic resonance imaging (MRI) was recently introduced as a promising technique for in vivo cell tracking. In the present study we compared $^{19}$F MRI with iron-enhanced MRI in mice with photothrombosis (PT) at 7 Tesla. PT represents a model of focal cerebral ischemia exhibiting acute vessel occlusion and delayed neuroinflammation. Methods/Principal Findings Perfluorocarbons (PFC) or superparamagnetic iron oxide particles (SPIO) were injected intravenously at different time points after photothrombotic infarction. While administration of PFC directly after PT induction led to a strong $^{19}$F signal throughout the entire lesion, two hours delayed application resulted in a rim-like $^{19}$F signal at the outer edge of the lesion. These findings closely resembled the distribution of signal loss on T2-weighted MRI seen after SPIO injection reflecting intravascular accumulation of iron particles trapped in vessel thrombi as confirmed histologically. By sequential administration of two chemically shifted PFC compounds 0 and 2 hours after illumination the different spatial distribution of the $^{19}$F markers (infarct core/rim) could be visualized in the same animal. When PFC were applied at day 6 the fluorine marker was only detected after long acquisition times ex vivo. SPIO-enhanced MRI showed slight signal loss in vivo which was much more prominent ex vivo indicative for neuroinflammation at this late lesion stage. Conclusion Our study shows that vessel occlusion can be followed in vivo by $^{19}$F and SPIO-enhanced high-field MRI while in vivo imaging of neuroinflammation remains challenging. The timing of contrast agent application was the major determinant of the underlying processes depicted by both imaging techniques. Importantly, sequential application of different PFC compounds allowed depiction of ongoing vessel occlusion from the core to the margin of the ischemic lesions in a single MRI measurement

Temporal Pattern of ICAM-I Mediated Regulatory T Cell Recruitment to Sites of Inflammation in Adoptive Transfer Model of Multiple Sclerosis

Migration of immune cells to the target organ plays a key role in autoimmune disorders like multiple sclerosis (MS). However, the exact underlying mechanisms of this active process during autoimmune lesion pathogenesis remain elusive. To test if pro-inflammatory and regulatory T cells migrate via a similar molecular mechanism, we analyzed the expression of different adhesion molecules, as well as the composition of infiltrating T cells in an in vivo model of MS, adoptive transfer experimental autoimmune encephalomyelitis in rats. We found that the upregulation of ICAM-I and VCAM-I parallels the development of clinical disease onset, but persists on elevated levels also in the phase of clinical remission. However, the composition of infiltrating T cells found in the developing versus resolving lesion phase changed over time, containing increased numbers of regulatory T cells (FoxP3) only in the phase of clinical remission. In order to test the relevance of the expression of cell adhesion molecules, animals were treated with purified antibodies to ICAM-I and VCAM-I either in the phase of active disease or in early remission. Treatment with a blocking ICAM-I antibody in the phase of disease progression led to a milder disease course. However, administration during early clinical remission aggravates clinical symptoms. Treatment with anti-VCAM-I at different timepoints had no significant effect on the disease course. In summary, our results indicate that adhesion molecules are not only important for capture and migration of pro-inflammatory T cells into the central nervous system, but also permit access of anti-inflammatory cells, such as regulatory T cells. Therefore it is likely to assume that intervention at the blood brain barrier is time dependent and could result in different therapeutic outcomes depending on the phase of CNS lesion development

Magnetic resonance imaging of blood brain/nerve barrier dysfunction and leukocyte infiltration: closely related or discordant?

Unlike other organs the nervous system is secluded from the rest of the organism by the blood brain barrier (BBB) or blood nerve barrier (BNB) preventing passive influx of fluids from the circulation. Similarly, leukocyte entry to the nervous system is tightly controlled. Breakdown of these barriers and cellular inflammation are hallmarks of inflammatory as well as ischemic neurological diseases and thus represent potential therapeutic targets. The spatiotemporal relationship between BBB/BNB disruption and leukocyte infiltration has been a matter of debate. We here review contrast-enhanced magnetic resonance imaging (MRI) as a non-invasive tool to depict barrier dysfunction and its relation to macrophage infiltration in the central and peripheral nervous system under pathological conditions. Novel experimental contrast agents like Gadofluorine M (Gf) allow more sensitive assessment of BBB dysfunction than conventional Gadolinium (Gd)-DTPA enhanced MRI. In addition, Gf facilitates visualization of functional and transient alterations of the BBB remote from lesions. Cellular contrast agents such as superparamagnetic iron oxide particles (SPIO) and perfluorocarbons enable assessment of leukocyte (mainly macrophage) infiltration by MR technology. Combined use of these MR contrast agents disclosed that leukocytes can enter the nervous system independent from a disturbance of the BBB, and vice versa, a dysfunctional BBB/BNB by itself is not sufficient to attract inflammatory cells from the circulation. We will illustrate these basic imaging findings in animal models of multiple sclerosis, cerebral ischemia, and traumatic nerve injury and review corresponding findings in patients

Isolation and flow cytometric analysis of immune cells from the ischemic mouse brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed

Flow cytometric characterization of brain dendritic cell subsets after murine stroke

Background Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects. It is believed that this determination is initiated relatively early after stroke onset by clearing macrophages and migratory dendritic cells (DC). However, the phenotypic differentiation of macrophages and DC is intricate particularly in the disease context. Here, we utilized a set of surface markers used in mucosal immunity research to investigate the involvement of macrophages and DC subpopulations in post-stroke inflammation in mice. Findings Photothrombotic stroke induced a significant increase of lineage (CD3, B220, Ly6G and CD49b) negative CD11b+ cells in the brain primarily consisting of F4/80+ macrophages and, to a lesser extent, F4/80-/CD11c-/CD11b+ monocytes and F4/80-/CD11c+ DC. The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found. Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere. Conclusions The surface marker combination used in this study allowed a phenotypic differentiation of macrophages and DC subpopulations after stroke, thus providing an important prerequisite to study post-stroke immunity and tolerance

Object-based analysis of astroglial reaction and astrocyte subtype morphology after ischemic brain injury

The astrocytic response to ischemic brain injury is characterized by specific alterations of glial cell morphology and function. Various studies described both beneficial and detrimental aspects of activated astrocytes, suggesting the existence of different subtypes. We investigated this issue using a novel object-based approach to study characteristics of astrogliosis after stroke. Spontaneously hypertensive rats received permanent middle cerebral artery occlusion. After 96 h, brain specimens were removed, fixed and stained for GFAP, glutamine synthetase (GS), S100Beta and Musashi1 (Msh1). Three regions of interest were defined (contralateral hemisphere, ipsilateral remote zone and infarct border zone), and confocal stacks were acquired (n=5 biological with each n=4 technical replicates). The stacks were background-corrected and colocalization between the selected markers and GFAP was determined using an automated thresholding algorithm. The fluorescence and colocalization channels were then converted into 3D-objects using both intensity and volume as filters to ultimately determine the final volumes of marker expression and colocalization, as well as the morphological changes of astrocyte process arborisation. We found that both S100Beta and Msh1 determined the same GFAP-positive astroglial cell population albeit the cellular compartments differed. GFAP stained most of the astrocyte processes and is hence suitable for the analysis of qualitative characteristics of astrogliosis. Due to its peri-nuclear localization, Msh1 is appropriate to estimate the total number of astrocytes even in regions with severe reactive astrogliosis. GS expression in GFAP-positive astrocytes was high in the remote zone and low at the infarct border, indicating the existence of astrocyte subclasses

High-dosage granulocyte colony stimulating factor treatment alters monocyte trafficking to the brain after experimental stroke

Ischemic stroke elicits a prompt inflammatory response that is characterized by a well-timed recruitment of peripheral immune cells to the brain. Among these, monocytes play a particularly important, but multifaceted role and have been increasingly recognized to affect stroke outcome. Granulocyte colony stimulating factor (GCSF) is known for its immunosuppressive actions on mononuclear cells, but previous studies in the stroke field were mainly confined to its neuroprotective actions. Herein, we investigated whether GCSF affects post-stroke inflammation in a mouse model of focal brain ischemia by modulating monocyte responses. Treatment with GCSF was controlled by vehicle injection, sham surgery and naive animals. Despite a significant monocytosis, high-dosage GCSF reduced the number of brain-infiltrating monocytes/macrophages four days after stroke. Lower numbers of mononuclear phagocytes in the brain were associated with smaller cerebral edema and improved motor outcome after stroke. GCSF treatment over 72 h, but not 24 h diminished integrin expression on circulating Ly6C+ inflammatory monocytes. In vitro experiments further revealed that GCSF strongly promotes interleukin (IL)-10 secretion by activated mononuclear cells. Blockade of the IL-10 receptor partly reversed GCSF-induced downregulation of integrin surface expression. Overall, our results suggest that high-dosage GCSF mitigates monocyte infiltration after stroke, likely by attenuating integrin-mediated adhesion to the brain endothelium in an IL-10-dependent manner. Lower amounts of mononuclear cells in the brain translate to less severe brain edema and functional impairment and thus support a harmful role of Ly6C+ inflammatory monocytes in the acute stage of stroke