Thursday, 11 May 2017

Imaging T cells in real time is Grizzly

Part of my job is to discuss animal work as it relates to MS.

Some of you aren't interested in the science and it is often years before animal work reaches the clinic.

Imaging cells in real time has provided many insights into biology and is something that just can't be done in humans.

The advent of two photon microscopy has allowed to to be done.

Finsh your Breakfast lunch or Tea(Dinner) before reading this.

Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a depth of about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite fluorescent dyes. However for each excitation, two photons of the infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. However, the resolution remains diffraction-limited. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection and reduced phototoxicity. The concept of two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. An excitation results in the subsequent emission of a fluorescence photon, typically at a higher energy than either of the two the two excitatory photons.



Haghayegh Jahromi N, Tardent H, Enzmann G, Deutsch U, Kawakami N, Bittner S, Vestweber D, Zipp F, Stein JV, Engelhardt B. A Novel Cervical Spinal Cord Window Preparation Allows for Two-Photon Imaging of T-Cell Interactions with the Cervical Spinal Cord Microvasculature during Experimental Autoimmune Encephalomyelitis. Front Immunol. 2017 Apr 11;8:406. doi: 10.3389/fimmu.2017.00406. eCollection 2017.

T-cell migration across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Two-photon intravital microscopy (2P-IVM) has been established as a powerful tool to study cell-cell interactions in inflammatory EAE lesions in living animals. In EAE, central nervous system inflammation is strongly pronounced in the spinal cord, an organ in which 2P-IVM imaging is technically very challenging and has been limited to the lumbar spinal cord. Here, we describe a novel spinal cord window preparation allowing to use 2P-IVM to image immune cell interactions with the cervical spinal cord microvascular endothelium during EAE. We describe differences in the angioarchitecture of the cervical spinal cord versus the lumbar spinal cord, which will entail different hemodynamic parameters in these different vascular beds. Using T cells as an example, we demonstrate the suitability of this novel methodology in imaging the post-arrest multistep T-cell extravasation across the cervical spinal cord microvessels. The novel methodology includes an outlook to the analysis of the cellular pathway of T-cell diapedesis across the BBB by establishing visualization of endothelial junctions in this vascular bed.


The images are publically available and shows you what is done.
Gross isn't it.

This grizzy set-up is the stuff of anti-vivisectionist, but the animal is anaesthetised and will never wake-up



This allows you to monitor cells entering the CNS is the living animal and so can tell you how it is done...and how to stop it.


6 comments:

  1. The figures above prove that trespassing cells in EAE prefer tiny vessels to make their entrance into CNS: In these vessels blood flow is slower making it easier for the cells to attach themselves onto the vessel wall.

    Each one of these immune cells creates a pigment of demyelination in the proximity of the entry point. These pigments put together side by side shape the macroscopically visible EAE lesion.

    I guess you all agree with this, right? So, here is the question:
    Do EAE lesions exhibit ring enhancement as MS lesions do? Please provide a link if possible.


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  2. Hi VV, hope you are well, long time no (critism :-)
    To answer your question. Do EAE lesions expand like MS?
    Not to my knowledge but the lesions last a few days not a few weeks as occurs in MS

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  3. Hi, never been better. Best wishes to you and your readers.

    To the point, i did some search and found no report of ring enhancement in EAE lesions. Since your experience confirms this, it seems that we might take it for granted.

    All histological studies of MS lesions conclude that they are singular entities, they are not unions of smaller demyelinating spots, and this reflects in the ring enhancement they exhibit: A singular lesion has borders, whereas a set of unified demyelinating spots has not. To support this view, MS lesions have a distinct, macroscopically visible vein inside them.

    Therefore, if you hypothesize that tissue damage in MS comes from immune cells that trespass the BBB, you should accept that all of them emerge from a certain point of that central vein and then expand to all directions leading to the typical ovoid MS lesions. You should also accept that the progress uniformly, exert their damage with the same speed, and then all of them stop at the same time, at the lesion borders.

    And then you must explain why they chose that specific entrance point of that central vein, where the blood flows much faster than inside tiny vessels, making it extremely difficult for the immune cells to adhere the BBB.

    So, it is rather clear that nothing in that pictures happens in MS.

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    Replies
    1. Ah, I knew which way this was going, I was hoping (forlornly as it turns out) you'd be trialling some new material VV. Still, good to have you back ;-)

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    2. Can't teach old dog new tricks, it applies to both (all) of us.

      Still, despite your workload, I would appreciate some comments to refute my arguments. I think your readers might find them convincing enough.

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    3. Perhaps we might differ as for us, when the evidence changes, we change our minds. There have been a number of studies now that have shown that the CCSVI theory is disproven.
      We have never said that EAE is MS but certain models (particularly ours of course) can mimic certain aspects of MS but it isn't MS. I think we have been clear about that. It is certainly useful for areas such as neurodegeneration, where the processes do seem to mimic what is seen in MS and which is our chief focus these days, though MD has got back into some serious immunology of MS recently.

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