Two-photon Laser Scanning Microscopy Research Articles

Complete Visualization of T Follicular Helper Cells in Germinal Centers by Light Sheet Fluorescence Microscopy.

Long-lasting immunity depends on generation of antibody forming cells in germinal centers (GCs). Conventional methods such as immunohistology and intravital live imaging have been used extensively to investigate the location of cellular assemblies within tissues as well as their dynamic motility and cellular interactions. Two photon laser scanning microscopy (TPLSM) intravital imaging allows scanning of large areas within tissues and reveals multiple immune cell niches. Nonetheless, this type of imaging is limited by the depth of penetration and cannot capture effectively all of the GC niches within lymphoid organs. Here we describe a method to visualize antigen-specific T and B cells in multiple microanatomical locations and niches at the level of a whole organ. This large-scale imaging approach can greatly increase our understanding of the spatial distribution of immune cells and help obtain detailed 3D maps of their locations and quantities.

Establishment of a Pre-vascularized 3D Lung Cancer Model in Fibrin Gel-Influence of Hypoxia and Cancer-Specific Therapeutics.

Lung cancer is the most frequently diagnosed cancer worldwide and the one that causes the highest mortality. In order to understand the disease and to develop new treatments, in vitro human lung cancer model systems which imitate the physiological conditions is of high significance. In this study, a human 3D lung cancer model was established that features the organization of a tumor with focus on tumor angiogenesis. Vascular networks were formed by co-culture of human umbilical vein endothelial cells and adipose tissue-derived mesenchymal stem cells (ASC) for 14 days in fibrin. A part of the pre-vascularized fibrin gel was replaced by fibrin gel containing lung cancer cells (A549) to form tri-cultures. This 3D cancer model system was cultured under different culture conditions and its behaviour after treatment with different concentrations of tumor-specific therapeutics was evaluated. The evaluation was performed by measurement of metabolic activity, viability, quantification of two-photon laser scanning microscopy and measurement of the proangiogenic factor vascular endothelial growth factor in the supernatant. Hypoxic conditions promoted vascularization compared to normoxic cultured controls in co- and tri-cultures as shown by significantly increased vascular structures, longer structures with a higher area and volume, and secretion of vascular endothelial growth factor. Cancer cells also promoted vascularization. Treatment with 50 µM gefitinib or 50 nM paclitaxel decreased the vascularization significantly. VEGF secretion was only reduced after treatment with gefitinib, while in contrast secretion remained constant during medication with paclitaxel. The findings suggest that the herein described 3D lung cancer model provides a novel platform to investigate the angiogenic potential of cancer cells and its responses to therapeutics. Thus, it can serve as a promising approach for the development and patient-specific pre-selection of anticancer treatment.

Calcium dysregulation and compensation in cortical pyramidal neurons of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal, hereditary neurodegenerative disorder that predominantly affects striatal medium-sized spiny neurons and cortical pyramidal neurons (CPNs). It has been proposed that perturbations in Ca2+ homeostasis could play a role in CPN alterations. To test this hypothesis, we used the R6/2 mouse model of juvenile HD at different stages of disease progression; presymptomatic, early symptomatic, and late symptomatic. We combined whole-cell patch-clamp recordings of layer 2/3 CPNs with two-photon laser scanning microscopy to image somatic and dendritic Ca2+ transients associated with evoked action potentials (APs). We found that the amplitude of AP-induced Ca2+ transients recorded at the somata of CPNs was significantly reduced in presymptomatic and late symptomatic R6/2 mice compared with wild-type (WT) littermates. However, reduced amplitudes were compensated by increases in decay times, so that Ca2+ transient areas were similar between genotypes. AP-induced Ca2+ transients in CPN proximal dendrites were variable and differences did not reach statistical significance, except for reduced areas in the late symptomatic group. In late symptomatic mice, a specific store-operated Ca2+ channel antagonist, EVP4593, reduced somatic Ca2+ transient amplitude similarly in WT and R6/2 CPNs. In contrast, dantrolene, a ryanodine receptor (RyR) antagonist, and nifedipine, an L-type Ca2+ channel blocker, significantly reduced both somatic Ca2+ transient amplitude and area in R6/2 but not WT CPNs. These findings demonstrate that perturbations of Ca2+ homeostasis and compensation occur in CPNs before and after the onset of overt symptoms, and suggest RyRs and L-type Ca2+ channels as potential targets for therapeutic intervention.NEW & NOTEWORTHY We used two-photon microscopy to examine calcium influx induced by action potentials in cortical pyramidal neurons from a mouse model of Huntington's disease (HD), the R6/2. The amplitude of somatic calcium transients was reduced in R6/2 mice compared with controls. This reduction was compensated by increased decay times, which could lead to reduced calcium buffering capacity. L-type calcium channel and ryanodine receptor blockers reduced calcium transient area in HD neurons, suggesting new therapeutic avenues.

Prostaglandin E2 Dilates Intracerebral Arterioles When Applied to Capillaries: Implications for Small Vessel Diseases.

Prostaglandin E2 (PGE2) has been widely proposed to mediate neurovascular coupling by dilating brain parenchymal arterioles through activation of prostanoid EP4 receptors. However, our previous report that direct application of PGE2 induces an EP1-mediated constriction strongly argues against its direct action on arterioles during neurovascular coupling, the mechanisms sustaining functional hyperemia. Recent advances have highlighted the role of capillaries in sensing neuronal activity and propagating vasodilatory signals to the upstream penetrating parenchymal arteriole. Here, we examined the effect of capillary stimulation with PGE2 on upstream arteriolar diameter using an ex vivo capillary-parenchymal arteriole preparation and in vivo cerebral blood flow measurements with two-photon laser-scanning microscopy. We found that PGE2 caused upstream arteriolar dilation when applied onto capillaries with an EC50 of 70 nM. The response was inhibited by EP1 receptor antagonist and was greatly reduced, but not abolished, by blocking the strong inward-rectifier K+ channel. We further observed a blunted dilatory response to capillary stimulation with PGE2 in a genetic mouse model of cerebral small vessel disease with impaired functional hyperemia. This evidence casts previous findings in a different light, indicating that capillaries are the locus of PGE2 action to induce upstream arteriolar dilation in the control of brain blood flow, thereby providing a paradigm-shifting view that nonetheless remains coherent with the broad contours of a substantial body of existing literature.

BSCI-08. In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer

Abstract Background Brain metastases represent a common complication of lung cancer and dramatically limit prognosis in affected patients. The influence of tumor-associated macrophages and microglia (TAM/M) and their receptor CX3CR1 on different steps of brain metastasis formation from lung cancer is poorly characterized, but might be of therapeutic relevance. Methods We established an orthotopic cerebral metastasis model using CX3CR1-proficient (CX3CR1GFP/wt) and -deficient (CX3CR1GFP/GFP) mice with green-fluorescent TAM/M. A cranial window was prepared, and intracarotid injection of red-fluorescent Lewis Lung Carcinoma-cells (tdtLLC) was performed two weeks later. Formation of brain metastases was followed by repetitive two-photon laser scanning microscopy. Results After intracarotid injection, intravascular tumor cells extravasated into the cerebral parenchyma and eventually formed micrometastases (≤50 cells) and mature macrometastases (>50 cells). We observed phagocytosis of extravasated tumor cells by TAM/M during early steps of metastatic growth. Notably, these anti-tumor effects of TAM/M diminished during later steps of metastasis formation and were accompanied by TAM/M accumulation and activation. CX3CR1-deficiency resulted in a lower number of extravasated tumor cells, and only a small number of TAM/M were visualized during early steps of metastasis formation (extravasation, formation of micrometastases) in such mice. In contrast, progression of extravasated tumor cells into micrometastases was more frequently found in CX3CR1-deficient mice. Overall, these mechanisms resulted in a comparable number of mature macrometastases between CX3CR1-deficient and -proficient mice. Conclusion Our findings indicate that unspecific inhibition of CX3CR1 might not be a suitable therapeutic approach to prevent cerebral dissemination of lung cancer cells. Given the close interaction between TAM/M and tumour cells during metastasis formation, other therapeutic approaches targeting TAM/M function warrant evaluation. Such concepts might be evaluated in vivo using the herein established orthotopic mouse model.

Methods for calculating the stereotaxic coordinates of rat brain structures by pixel coordinates of the image obtained by confocal and two-photon laser scanning microscopy

BackgroundThere is a challenge to determine stereotaxic coordinates of a target structure with the accuracy, comparable to their size, when imaging the rat brain through cranial windows using confocal (multiphoton) microscopy in vivo. Some methods based on the estimation of the linear displacement from the intersections of the cerebral vessels are most often used for these purposes, but their accuracy can be improved. New methodA new method for converting pixel coordinates of points of interest on an image obtained in two-photon microscopy into stereotaxic ones using quadratic approximation with L2 regularization has been developed. A comparative analysis of several methods for converting pixel coordinates into stereotaxic ones was carried out. The current study is aimed to select a method which minimizes the error of coordinate conversion within the a priori specified threshold value. ResultsA method for determining the stereotaxic coordinates of each pixel in an image obtained by laser scanning in two-photon and / or confocal modes with an accuracy of several tens of microns is proposed. Comparison with existing method(s)It is shown that the error probability of the most common method based on calculating the points of interest coordinates as displacements relative to the selected vessels intersections can be reduced by using the quadratic approximation with L2 regularization. Our proposed method allows us to improve the accuracy of determining the coordinates of points of interest on 10–30 µm. ConclusionsThe proposed approach will be useful in research where precise positioning of microelectrodes, sensors, etc. for implantation in specified brain structures or groups of neurons determined by functional mapping is required.

A multielectrode array-based hypoxia model for the analysis of electrical activity in murine retinae.

Several eye diseases, for example, retinal artery occlusion, diabetic retinopathy, and glaucoma, are associated with retinal hypoxia. The lack of oxygen in the retina, especially in retinal ganglion cells (RGCs), causes cell damage up to cell degeneration and leads to blindness. Using multielectrode array recordings, an ex vivo hypoxia acute model was established to analyze the electrical activity of murine wild-type retinae under hypoxic stress conditions. Hypoxia was induced by exchanging the perfusion with oxygen-saturated medium by nitrogen-saturated medium. Hypoxic periods of 0min (control) up to 60min were tested on the retinae of adult female C57BL/6J mice. The electrical RGC activity vanished during hypoxia, but conditionally returned after the reestablishment of conventional test conditions. With increasing duration of hypoxia, the returning RGC activity decreased. After a hypoxic period of 30min and a subsequent recovery time of 30min, 59.43±11.35% of the initially active channels showed a restored RGC activity. The survival rate of retinal cells after hypoxic stress was analyzed by a live/dead staining assay using two-photon laser scanning microscopy. For detailed information about molecular changes caused by hypoxia, a microarray gene expression analysis was performed. Furthermore, the effect of 2-aminoethanesulfonic acid (taurine, 1mM) on retinae under hypoxic stress was tested. Treatment with taurine resulted in an increase in the RGC response rate after hypoxia and also increased the survival rate of retinal cells under hypoxic stress, confirming its potential as promising candidate for neuroprotective therapies of eye diseases.

Hematopoietic stem cell stretches and moves in its bone marrow niche.

Hematopoietic stem cells are the most illustrious inhabitants of the bone marrow. Direct visualization of endogenous hematopoietic stem cells in this niche is essential to study their functions. Until recently this was not possible in live animals. Recent studies, using state-of-the-art technologies, including sophisticated in vivo inducible genetic approaches in combination with two-photon laser scanning microscopy, allow the follow-up of endogenous hematopoietic stem cells' behavior in their habitat. Strikingly, the new findings reveal that quiescent hematopoietic stem cells are more mobile than previously thought, and link their retained steady state within the niche to a mobile behavior. The arising knowledge from this research will be critical for the therapy of several hematological diseases. Here, we review recent progress in our understanding of hematopoietic stem cell biology in their niches.

Vascular permeability of skeletal muscle microvessels in rat arterial ligation model: in vivo analysis using two-photon laser scanning microscopy.

Peripheral artery disease (PAD) in the lower limb compromises oxygen supply due to arterial occlusion. Ischemic skeletal muscle is accompanied by capillary structural deformation. Therefore, using novel microscopy techniques, we tested the hypothesis that endothelial cell swelling temporally and quantitatively corresponds to enhanced microvascular permeability. Hindlimb ischemia was created in male Wistar rat's by iliac artery ligation (AL). The tibialis anterior (TA) muscle microcirculation was imaged using intravenously infused rhodamine B isothiocyanate dextran fluorescent dye via two-photon laser scanning microscopy (TPLSM) and dye extravasation at 3 and 7 days post-AL quantified to assess microvascular permeability. The TA microvascular endothelial ultrastructure was analyzed by transmission electron microscopy (TEM). Compared with control (0.40 ± 0.15 μm3 × 106), using TPLSM, the volumetrically determined interstitial leakage of fluorescent dye measured at 3 (3.0 ± 0.40 μm3 × 106) and 7 (2.5 ± 0.8 μm3 × 106) days was increased (both P < 0.05). Capillary wall thickness was also elevated at 3 (0.21 ± 0.06 μm) and 7 (0.21 ± 0.08 μm) days versus control (0.11 ± 0.03 μm, both P < 0.05). Capillary endothelial cell swelling was temporally and quantitatively associated with elevated vascular permeability in the AL model of PAD but these changes occurred in the absence of elevations in protein levels of vascular endothelial growth factor (VEGF) its receptor (VEGFR2 which decreased by AL-7 day) or matrix metalloproteinase. The temporal coherence of endothelial cell swelling and increased vascular permeability supports a common upstream mediator. TPLSM, in combination with TEM, provides a sensitive and spatially discrete technique to assess the mechanistic bases for, and efficacy of, therapeutic countermeasures to the pernicious sequelae of compromised peripheral arterial function.

Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope.

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.

ER71/ETV2 Promotes Hair Regeneration from Chemotherapeutic Drug-Induced Hair Loss by Enhancing Angiogenesis.

Chemotherapy-induced alopecia and hair loss can be stressful in patients with cancer. The hair grows back, but sometimes the hair tends to stay thin. Therefore, understanding mechanisms regulating hair regeneration may improve the management of chemotherapy-induced alopecia. Previous studies have revealed that chemotherapeutic agents induce a hair follicle vascular injury. As hair growth is associated with micro-vessel regeneration, we postulated that the stimulation of angiogenesis might enhance hair regeneration. In particular, mice treated with 5-fluorouracil (5-FU) showed delayed anagen initiation and reduced capillary density when compared with untreated controls, suggesting that the retardation of anagen initiation by 5-FU treatment may be attributed to the loss of perifollicular micro-vessels. We investigated whether the ETS transcription factor ETV2 (aka ER71), critical for vascular development and regeneration, can promote angiogenesis and hair regrowth in a 5-FU-induced alopecia mouse model. Tie2-Cre; Etv2 conditional knockout (CKO) mice, which lack Etv2 in endothelial cells, presented similar hair regrowth rates as the control mice after depilation. Following 5-FU treatment, Tie2-Cre; Etv2 CKO mice revealed a significant reduction in capillary density, anagen induction, and hair restoration when compared with controls. Mice receiving lentiviral Etv2 injection after 5-FU treatment showed significantly improved anagen induction and hair regrowth. Two-photon laser scanning microscopy revealed that enforced Etv2 expression restored normal vessel morphology after 5-FU mediated vessel injury. Our data suggest that vessel regeneration strategies may improve hair regrowth after chemotherapeutic treatment.

IoT-Based Research Equipment Sharing System for Remotely Controlled Two-Photon Laser Scanning Microscopy.

In this study, two-photon laser scanning microscopy (TPLSM) based on the internet of things (IoT) is proposed as a remote research equipment sharing system, which enables the remote sharing economy. IoT modules, where data are transmitted to and received from the remote users in the web service via IoT, instead of a data acquisition (DAQ) system embedded in the conventional TPLSM, are installed in the IoT-based TPLSM (IoT-TPLSM). The performance for each IoT module is evaluated independently, and it is confirmed that it works well even in a personal computer-free environment. In addition, a message queuing telemetry transport (MQTT) protocol is applied to the DAQ interface in the web service, and a graphic user interface for enabling the remote users to operate IoT-TPLSM remotely is also designed and implemented. For the image acquisition demonstration, the stained cellular images and the autofluorescent tissue images are obtained in IoT-TPLSM. Lastly, it is confirmed that the comparable performance is provided with the conventional TPLSM by evaluating the imaging conditions and qualities of the three-dimensional image stacks processed in IoT-TPLSM.

Improved Cerebral Perfusion Pressure and Microcirculation byDrag Reducing Polymer-Enforced Resuscitation Fluid After Traumatic Brain Injury and Hemorrhagic Shock.

Hemorrhagic shock (HS) after traumatic brain injury (TBI) reduces cerebral perfusion pressure (CPP) and cerebral blood flow (CBF), increasing hypoxia and doubling mortality. Volume expansion with resuscitation fluids (RFs) for HS does not improve CBF and tissue oxygen, while hypervolemia exacerbates brain edema and elevates intracranial pressure (ICP). We tested whether drag-reducing polymers (DRPs), added to isotonic Hetastarch (HES), would improve CBF but prevent ICP increase. TBI was induced in rats by fluid percussion, followed by controlled hemorrhage to mean arterial pressure (MAP)=40mmHg. HES-DRP or HES was infused to MAP=60mmHg for 1h, followed by blood reinfusion to MAP=70mmHg. Temperature, MAP, ICP, cortical Doppler flux, blood gases, and electrolytes were monitored. Microvascular CBF, tissue hypoxia, and neuronal necrosis were monitored by two-photon laser scanning microscopy 5h after TBI/HS. TBI/HS reduced CPP and CBF, causing tissue hypoxia. HES-DRP (1.9±0.8mL) more than HES (4.5±1.8mL) improved CBF and tissue oxygenation (p<0.05). In the HES group, ICP increased to 23±4mmHg (p<0.05) but in HES-DRP to 12±2mmHg. The number of dead neurons, microthrombosis, and the contusion volume in HES-DRP were significantly less than in the HES group (p<0.05). HES-DRP required a smaller volume, which reduced ICP and brain edema.

Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy

This protocol describes a novel technique to investigate the microcirculation dynamics underlying the pathology in the small intestine of neonatal mice using two-photon laser-scanning microscopy (TPLSM). Recent technological advances in multi-photon microscopy allow intravital analysis of different organs such as the liver, brain and intestine. Despite these advances, live visualization and analysis of the small intestine in neonatal rodents remain technically challenging. We herein provide a detailed description of a novel method to capture high resolution and stable images of the small intestine in neonatal mice as early as postnatal day 0. This imaging technique allows a comprehensive understanding of the development and blood flow dynamics in small intestine microcirculation.

Changes in Synaptic Terminal Structure in Adolescent Rat during Pregnancy; the Action Potential Propagation and Synaptic Transmission

Synaptic plasticity is a biological system of specific pattern of synaptic activity result in changes in synaptic strength. This influence puberty, pregnancy hormones, sensory experiences, and brain disorders. Long-term synaptic plasticity is accompanied by protein synthesis and trafficking, leading to structural changes of the synapse. Increasing evidence connects the terminal synaptic changes with potential propagation in adolescent and pregnancy. We investigate on the synaptic structural plasticity, which has mainly been studied with in vivo two-photon laser scanning microscopy. We also discuss how a different type of synapses, the multi-contact synapses associated with pregnancy

Optimization aspects of electrodeposition of photoluminescent conductive polymer layer onto neural microelectrode arrays

In neuroscience the use of a microelectrode array allows the detection of neuroelectric signals with high temporal resolution in a confined space within the tissue, while two-photon laser scanning microscopy provides high spatial resolution in a wide region of interest. The combination of these two techniques promises better understanding of the operation of neural pathways. To facilitate this connection, we studied the direct electrochemical deposition of the conductive polymer poly-2,3-ethylenedioxy-thiophene onto different Pt and Pt/Ir electrode surfaces from non-aqueous solvents, such as ionic liquid and propylene carbonate. We show the effects of electrochemical deposition technique (pulsed or continuous), monomer concentration range and solvent electrolyte type on the formation of photoluminescent - conductive films. For these variables we determined the optimal deposition parameters given as 0.025–0.050 M EDOT monomer concentration in BMIMBF4 ionic liquid and the use of pulsed deposition process to form an adherent, uniform functional electrode coating.

Sural Nerve Perfusion in Mice.

Peripheral nerve function is metabolically demanding and nerve energy failure has been implicated in the onset and development of diabetic peripheral neuropathy and neuropathic pain conditions. Distal peripheral nerve oxygen supply relies on the distribution of red blood cells (RBCs) in just a few, nearby capillary-sized vessels and is therefore technically challenging to characterize. We developed an approach to characterize distal sural nerve hemodynamics in anesthetized, adult male mice using in vivo two-photon laser scanning microscopy. Our results show that RBC velocities in mouse sural nerve vessels are higher than those previously measured in mouse brain, and are sensitive to hindlimb temperatures. Nerve blood flow, measured as RBC flux, however, was similar to that of mouse brain and unaffected by local temperature. Power spectral density analysis of fluctuations in RBC velocities over short time intervals suggest that the technique is sufficiently sensitive and robust to detect subtle flow oscillations over time scales from 0.1 to tens of seconds. We conclude that in vivo two-photon laser scanning microscopy provides a suitable approach to study peripheral nerve hemodynamics in mice, and that local temperature control is important during such measurements.

Changes in Synaptic Terminal Structure in Adolescent Rat During Pregnancy; The Action Potential Propagation and Synaptic Transmission

Synaptic plasticity is a biological system of specific pattern of synaptic activity result in changes in synaptic strength. This influence puberty, pregnancy hormones, sensory experiences, and brain disorders. Long-term synaptic plasticity is accompanied by protein synthesis and trafficking, leading to structural changes of the synapse. Increasing evidence connects the terminal synaptic changes with potential propagation in adolescent and pregnancy. We investigate on the synaptic structural plasticity, which has mainly been studied with in vivo two-photon laser scanning microscopy. We also discuss how a different type of synapses, the multi-contact synapses associated with pregnancy.

High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

Understanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

Functional characteristics of Th1, Th17, and ex-Th17 cells in EAE revealed by intravital two-photon microscopy

BackgroundT helper (Th) 17 cells are a highly plastic subset of T cells, which in the context of neuroinflammation, are able to acquire pathogenic features originally attributed to Th1 cells (resulting in so called ex-Th17 cells). Thus, a strict separation between the two T cell subsets in the context of experimental autoimmune encephalomyelitis (EAE) is difficult. High variability in culture and EAE induction protocols contributed to previous conflicting results concerning the differential contribution of Th1 and Th17 cells in EAE. Here, we systematically evaluate the role of different T cell differentiation and transfer protocols for EAE disease development and investigate the functional dynamics of encephalitogenic T cells directly within the inflamed central nervous system (CNS) tissue.MethodsWe compiled the currently used EAE induction protocols reported in literature and investigated the influence of the different Th1 and Th17 differentiation protocols as well as EAE induction protocols on the EAE disease course. Moreover, we assessed the cytokine profile and functional dynamics of both encephalitogenic Th1 and Th17 cells in the inflamed CNS using flow cytometry and intravital two-photon laser scanning microscopy. Lastly, we used astrocyte culture and adoptive transfer EAE to evaluate the impact of Th1 and Th17 cells on astrocyte adhesion molecule expression in vitro and in vivo.ResultsWe show that EAE courses are highly dependent on in vitro differentiation and transfer protocols. Moreover, using genetically encoded reporter mice (B6.IL17A-EGFP.acRFP x 2d2/2d2.RFP), we show that the motility of interferon (IFN)γ-producing ex-Th17 cells more closely resembles Th1 cells than Th17 cells in transfer EAE. Mechanistically, IFNγ-producing Th1 cells selectively induce the expression of cellular adhesion molecules I-CAM1 while Th1 as well as ex-Th17 induce V-CAM1 on astrocytes.ConclusionsThe behavior of ex-Th17 cells in EAE lesions in vivo resembles Th1 rather than Th17 cells, underlining that their change in cytokine production is associated with functional phenotype alterations of these cells.