Limits...
Intravital imaging of a massive lymphocyte response in the cortical dura of mice after peripheral infection by trypanosomes.

Coles JA, Myburgh E, Ritchie R, Hamilton A, Rodgers J, Mottram JC, Barrett MP, Brewer JM - PLoS Negl Trop Dis (2015)

Bottom Line: Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells.Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007).The population and motility of the trypanosomes tended to decline after about 30 dpi.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom; Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sickness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice infected with T.b.brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells. Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007). The T cells occasionally made contact lasting tens of minutes with dendritic cells, indicative of antigen presentation. The population and motility of the trypanosomes tended to decline after about 30 dpi. We suggest that the lymphocyte infiltration of the meninges may later contribute to encephalitis, but have no evidence that the dural trypanosomes invade the parenchyma.

Show MeSH

Related in: MedlinePlus

Peripheral infection increases the number of T cells in the meninges.A. The thinned skull, showing parts of the metal plate surrounding the 5 mm hole. Generally, arteries arrive from the left, veins leave to the right. Inset: the approximate position of the area imaged. B. 3D reconstruction from a Z-stack showing the skull (green) and larger blood vessels, horizontal in the meninges and vertical in the brain parenchyma. C. Z-projection 30 μm thick in an uninfected mouse showing sparse CD2+ cells (DsRed) close to the skull. The skull is slightly inclined to the XY plane and is seen in green to the lower left. Cell nuclei labeled by i.v. injection of furamidine are blue. Blood vessels (cyan) are labeled with quantum dots. D. At deeper levels, surface vessels turn downwards and vertical vessels in the parenchyma are visible (arrows). Three T cells are at about the level of the horizontal vessel. This is another 20 μm Z-projection of the same XY field as (C). E,F. Deep Z-projections (143 μm and 188 μm respectively) at 7 dpi (E) and 39 dpi (F), showing increased numbers of T cells. The blue in (E) is from the skull. Blood vessels in (F) are green. G. Numbers per mm2 of meningeal T cells plotted on a log scale against dpi. Each symbol corresponds to one mouse. The dashed line is a fitted sigmoid.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400075&req=5

pntd.0003714.g001: Peripheral infection increases the number of T cells in the meninges.A. The thinned skull, showing parts of the metal plate surrounding the 5 mm hole. Generally, arteries arrive from the left, veins leave to the right. Inset: the approximate position of the area imaged. B. 3D reconstruction from a Z-stack showing the skull (green) and larger blood vessels, horizontal in the meninges and vertical in the brain parenchyma. C. Z-projection 30 μm thick in an uninfected mouse showing sparse CD2+ cells (DsRed) close to the skull. The skull is slightly inclined to the XY plane and is seen in green to the lower left. Cell nuclei labeled by i.v. injection of furamidine are blue. Blood vessels (cyan) are labeled with quantum dots. D. At deeper levels, surface vessels turn downwards and vertical vessels in the parenchyma are visible (arrows). Three T cells are at about the level of the horizontal vessel. This is another 20 μm Z-projection of the same XY field as (C). E,F. Deep Z-projections (143 μm and 188 μm respectively) at 7 dpi (E) and 39 dpi (F), showing increased numbers of T cells. The blue in (E) is from the skull. Blood vessels in (F) are green. G. Numbers per mm2 of meningeal T cells plotted on a log scale against dpi. Each symbol corresponds to one mouse. The dashed line is a fitted sigmoid.

Mentions: Optical access to the parietal cortex was gained by thinning the skull (Fig 1A) and imaging was started within about 30 min after the beginning of skull surgery. Skull bone was visualized by its second harmonic signal (SHG, Fig 1B and S3 Fig) and blood vessels by a blood marker (red in Fig 1B). With averaging of scans, it was possible to resolve large blood vessels to a depth of 400 μm (Fig 1B); objects, that were smaller and moving, such as T cells, could be detected down to about 150 μm below the skull. All quantitative data are from C67BL/6 mice infected with T.b.brucei GVR35, with some supporting illustrations from other strains of mice or T.b.brucei.


Intravital imaging of a massive lymphocyte response in the cortical dura of mice after peripheral infection by trypanosomes.

Coles JA, Myburgh E, Ritchie R, Hamilton A, Rodgers J, Mottram JC, Barrett MP, Brewer JM - PLoS Negl Trop Dis (2015)

Peripheral infection increases the number of T cells in the meninges.A. The thinned skull, showing parts of the metal plate surrounding the 5 mm hole. Generally, arteries arrive from the left, veins leave to the right. Inset: the approximate position of the area imaged. B. 3D reconstruction from a Z-stack showing the skull (green) and larger blood vessels, horizontal in the meninges and vertical in the brain parenchyma. C. Z-projection 30 μm thick in an uninfected mouse showing sparse CD2+ cells (DsRed) close to the skull. The skull is slightly inclined to the XY plane and is seen in green to the lower left. Cell nuclei labeled by i.v. injection of furamidine are blue. Blood vessels (cyan) are labeled with quantum dots. D. At deeper levels, surface vessels turn downwards and vertical vessels in the parenchyma are visible (arrows). Three T cells are at about the level of the horizontal vessel. This is another 20 μm Z-projection of the same XY field as (C). E,F. Deep Z-projections (143 μm and 188 μm respectively) at 7 dpi (E) and 39 dpi (F), showing increased numbers of T cells. The blue in (E) is from the skull. Blood vessels in (F) are green. G. Numbers per mm2 of meningeal T cells plotted on a log scale against dpi. Each symbol corresponds to one mouse. The dashed line is a fitted sigmoid.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400075&req=5

pntd.0003714.g001: Peripheral infection increases the number of T cells in the meninges.A. The thinned skull, showing parts of the metal plate surrounding the 5 mm hole. Generally, arteries arrive from the left, veins leave to the right. Inset: the approximate position of the area imaged. B. 3D reconstruction from a Z-stack showing the skull (green) and larger blood vessels, horizontal in the meninges and vertical in the brain parenchyma. C. Z-projection 30 μm thick in an uninfected mouse showing sparse CD2+ cells (DsRed) close to the skull. The skull is slightly inclined to the XY plane and is seen in green to the lower left. Cell nuclei labeled by i.v. injection of furamidine are blue. Blood vessels (cyan) are labeled with quantum dots. D. At deeper levels, surface vessels turn downwards and vertical vessels in the parenchyma are visible (arrows). Three T cells are at about the level of the horizontal vessel. This is another 20 μm Z-projection of the same XY field as (C). E,F. Deep Z-projections (143 μm and 188 μm respectively) at 7 dpi (E) and 39 dpi (F), showing increased numbers of T cells. The blue in (E) is from the skull. Blood vessels in (F) are green. G. Numbers per mm2 of meningeal T cells plotted on a log scale against dpi. Each symbol corresponds to one mouse. The dashed line is a fitted sigmoid.
Mentions: Optical access to the parietal cortex was gained by thinning the skull (Fig 1A) and imaging was started within about 30 min after the beginning of skull surgery. Skull bone was visualized by its second harmonic signal (SHG, Fig 1B and S3 Fig) and blood vessels by a blood marker (red in Fig 1B). With averaging of scans, it was possible to resolve large blood vessels to a depth of 400 μm (Fig 1B); objects, that were smaller and moving, such as T cells, could be detected down to about 150 μm below the skull. All quantitative data are from C67BL/6 mice infected with T.b.brucei GVR35, with some supporting illustrations from other strains of mice or T.b.brucei.

Bottom Line: Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells.Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007).The population and motility of the trypanosomes tended to decline after about 30 dpi.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom; Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sickness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice infected with T.b.brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells. Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007). The T cells occasionally made contact lasting tens of minutes with dendritic cells, indicative of antigen presentation. The population and motility of the trypanosomes tended to decline after about 30 dpi. We suggest that the lymphocyte infiltration of the meninges may later contribute to encephalitis, but have no evidence that the dural trypanosomes invade the parenchyma.

Show MeSH
Related in: MedlinePlus