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

Extravascular trypanosomes appear in the meninges.A. GFP trypanosomes in the meninges at 32 dpi. Blue: collagen. Faint magenta: blood marker (Qdots). B. At the level of vertical penetrating vessels and parenchymal capillaries, intravascular trypanosomes were detected (arrow), but extravascular trypanosomes were not. 16 dpi. C. A trypanosome labeled by intravenous injection of furamidine. In this case, the nucleus gave red fluorescence (arrow) and the cytoplasm gave blue fluorescence. D. Trypanosomes (identifiable by their rapid movement as in S5 Video) giving red fluorescence after labeling with i.v. furamidine. Furamidine also produced blue fluorescence from endogenous nuclei. Single plane, 25 dpi, CD-1 mouse. E. Numbers of trypanosomes in the meninges as a function of dpi (green triangles). Their appearance tends to be later than the increase in T cells (red lozenges). In 8 mice, T cell number had increased but no trypanosomes were observed in the meninges. F. The number of trypanosomes in the dura shows no dependence on paristemia. G. Numbers of trypanosomes in the dura 20–40 dpi in C57BL/6 mice and CD1 mice (from [20]).
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003714.g003: Extravascular trypanosomes appear in the meninges.A. GFP trypanosomes in the meninges at 32 dpi. Blue: collagen. Faint magenta: blood marker (Qdots). B. At the level of vertical penetrating vessels and parenchymal capillaries, intravascular trypanosomes were detected (arrow), but extravascular trypanosomes were not. 16 dpi. C. A trypanosome labeled by intravenous injection of furamidine. In this case, the nucleus gave red fluorescence (arrow) and the cytoplasm gave blue fluorescence. D. Trypanosomes (identifiable by their rapid movement as in S5 Video) giving red fluorescence after labeling with i.v. furamidine. Furamidine also produced blue fluorescence from endogenous nuclei. Single plane, 25 dpi, CD-1 mouse. E. Numbers of trypanosomes in the meninges as a function of dpi (green triangles). Their appearance tends to be later than the increase in T cells (red lozenges). In 8 mice, T cell number had increased but no trypanosomes were observed in the meninges. F. The number of trypanosomes in the dura shows no dependence on paristemia. G. Numbers of trypanosomes in the dura 20–40 dpi in C57BL/6 mice and CD1 mice (from [20]).

Mentions: In mice infected with T.b.brucei GVR35 expressing a fluorescent protein (FP), such as EGFP or tdTomato, extravascular (as well as intravascular) trypanosomes were observed in the meninges, usually from about 11 dpi (Fig 3A and S2 Video). In contrast, in the superficial parenchyma only intravascular ones were observed (Fig 3B), in agreement with other reports that extravascular trypanosomes are rare in the sub-pial cortex of Murinae [17, 20, 42, 43]. Rare extravascular trypanosomes were observed in ventral areas of brain slices (S3 Video). Since the extravascular trypanosomes moved rapidly, they could not be counted without imaging rapidly, so we scanned in one nominal XY plane at the maximum rate of 8 f.p.s. (S4 Video). However, in any one image field, trypanosomes were almost all confined within a depth extending about 5 μm thick above and below the plane of maximum density. Given the finite depth of focus, and the up-and-down motion of the trypanosomes (so they crossed this plane), we could make an approximate count by examining videos of 100 or more frames.


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)

Extravascular trypanosomes appear in the meninges.A. GFP trypanosomes in the meninges at 32 dpi. Blue: collagen. Faint magenta: blood marker (Qdots). B. At the level of vertical penetrating vessels and parenchymal capillaries, intravascular trypanosomes were detected (arrow), but extravascular trypanosomes were not. 16 dpi. C. A trypanosome labeled by intravenous injection of furamidine. In this case, the nucleus gave red fluorescence (arrow) and the cytoplasm gave blue fluorescence. D. Trypanosomes (identifiable by their rapid movement as in S5 Video) giving red fluorescence after labeling with i.v. furamidine. Furamidine also produced blue fluorescence from endogenous nuclei. Single plane, 25 dpi, CD-1 mouse. E. Numbers of trypanosomes in the meninges as a function of dpi (green triangles). Their appearance tends to be later than the increase in T cells (red lozenges). In 8 mice, T cell number had increased but no trypanosomes were observed in the meninges. F. The number of trypanosomes in the dura shows no dependence on paristemia. G. Numbers of trypanosomes in the dura 20–40 dpi in C57BL/6 mice and CD1 mice (from [20]).
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003714.g003: Extravascular trypanosomes appear in the meninges.A. GFP trypanosomes in the meninges at 32 dpi. Blue: collagen. Faint magenta: blood marker (Qdots). B. At the level of vertical penetrating vessels and parenchymal capillaries, intravascular trypanosomes were detected (arrow), but extravascular trypanosomes were not. 16 dpi. C. A trypanosome labeled by intravenous injection of furamidine. In this case, the nucleus gave red fluorescence (arrow) and the cytoplasm gave blue fluorescence. D. Trypanosomes (identifiable by their rapid movement as in S5 Video) giving red fluorescence after labeling with i.v. furamidine. Furamidine also produced blue fluorescence from endogenous nuclei. Single plane, 25 dpi, CD-1 mouse. E. Numbers of trypanosomes in the meninges as a function of dpi (green triangles). Their appearance tends to be later than the increase in T cells (red lozenges). In 8 mice, T cell number had increased but no trypanosomes were observed in the meninges. F. The number of trypanosomes in the dura shows no dependence on paristemia. G. Numbers of trypanosomes in the dura 20–40 dpi in C57BL/6 mice and CD1 mice (from [20]).
Mentions: In mice infected with T.b.brucei GVR35 expressing a fluorescent protein (FP), such as EGFP or tdTomato, extravascular (as well as intravascular) trypanosomes were observed in the meninges, usually from about 11 dpi (Fig 3A and S2 Video). In contrast, in the superficial parenchyma only intravascular ones were observed (Fig 3B), in agreement with other reports that extravascular trypanosomes are rare in the sub-pial cortex of Murinae [17, 20, 42, 43]. Rare extravascular trypanosomes were observed in ventral areas of brain slices (S3 Video). Since the extravascular trypanosomes moved rapidly, they could not be counted without imaging rapidly, so we scanned in one nominal XY plane at the maximum rate of 8 f.p.s. (S4 Video). However, in any one image field, trypanosomes were almost all confined within a depth extending about 5 μm thick above and below the plane of maximum density. Given the finite depth of focus, and the up-and-down motion of the trypanosomes (so they crossed this plane), we could make an approximate count by examining videos of 100 or more frames.

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