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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.

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The location of extravascular trypanosomes.A. A frame from S6 Video showing GFP trypanosomes in the plane of extracellular collagen. The movement of one trypanosome appeared to constrained by the collagen (arrow). B. Trypanosomes (red, arrow heads) were frequently observed outside small, somewhat irregular, vessels at the level of collagen (green, see S7 Video). C. Trypanosomes were not observed at the level of large pial vessels. Scale bars A,B,C, 20 μm. D. In a 3D reconstruction of a 741s Z-stack acquisition of trypanosomes (green), they are seen to be within about 40 μm under the skull. E. Texas Red infused in the cisterna magna labeled spaces adjacent to pial veins (V), and patches at that level, leaving a space (d) beneath the skull. Blue signal is from skull bone and furamidine-labeled nuclei. The bit of skull giving no SHG signal (c) may correspond to the site where a blood vessel entered the skull. 16 dpi.
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pntd.0003714.g004: The location of extravascular trypanosomes.A. A frame from S6 Video showing GFP trypanosomes in the plane of extracellular collagen. The movement of one trypanosome appeared to constrained by the collagen (arrow). B. Trypanosomes (red, arrow heads) were frequently observed outside small, somewhat irregular, vessels at the level of collagen (green, see S7 Video). C. Trypanosomes were not observed at the level of large pial vessels. Scale bars A,B,C, 20 μm. D. In a 3D reconstruction of a 741s Z-stack acquisition of trypanosomes (green), they are seen to be within about 40 μm under the skull. E. Texas Red infused in the cisterna magna labeled spaces adjacent to pial veins (V), and patches at that level, leaving a space (d) beneath the skull. Blue signal is from skull bone and furamidine-labeled nuclei. The bit of skull giving no SHG signal (c) may correspond to the site where a blood vessel entered the skull. 16 dpi.

Mentions: Recent papers have reported extravascular trypanosomes in the cortical pia mater [43] or the superficial cortical parenchyma [45], while T cells in the spinal meninges have been reported in the leptomeninges (arachnoid plus pia mater [21]). Knowing that the meninges were intact, and benefitting from signals from structural elements, we attempted to define the compartments containing the trypanosomes and T cells. The upper boundary of the compartment was clearly the skull, as trypanosomes were observed immediately below it. Often they were in the same plane as extracellular collagen, the main component of the dura mater (Fig 4A and S6 Video). Also at about this depth were cell nuclei labeled blue by intravenous furamidine (Fig 3D). These cells are likely to be the 'mesothelial lining cells' of the dura that are labeled by intravascular aminoacridines [24]. Defining the lower boundary of the compartment occupied by trypanosomes, like that of the dura itself [46, 47], was not so straightforward. Trypanosomes were observed close to small horizontal vessels, often with an irregular trajectory and close to collagen (Fig 4B and S7 Video), but were not observed at the level of larger horizontal vessels just above the parenchyma (Fig 4C). The latter were clearly in the subarachnoid space; the former were probably branches of the meningeal artery in the dura. In images acquired over many seconds, the trypanosomes traced out apparently isolated volumes within about 40 μm of the skull (Fig 4D). To delineate the extent of the subarachnoid space we infused dye (Texas Red) in the cisterna magna. From this site, dye is known to flow along the perivascular spaces of surface arteries and enter the subarachnoid space over the cortex [26, 29, 48]. As shown in Fig 4E, Texas Red, infused in this way, labeled spaces adjacent to large pial blood vessels and a thin, patchy, space extending across the cortex, in general agreement with histological studies on Murinae [27, 31, 41, 46, 47]. The dye is excluded from a space beneath the skull that contains nuclei labeled by furamidine. It is this "dural space", and not the subarachnoid space, that appears to be occupied by trypanosomes. Qualitative observation through the dissecting microscope, and vertical sections from sample Z-stacks, suggested that the distance from the skull to pial vessels increased in infected mice.


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)

The location of extravascular trypanosomes.A. A frame from S6 Video showing GFP trypanosomes in the plane of extracellular collagen. The movement of one trypanosome appeared to constrained by the collagen (arrow). B. Trypanosomes (red, arrow heads) were frequently observed outside small, somewhat irregular, vessels at the level of collagen (green, see S7 Video). C. Trypanosomes were not observed at the level of large pial vessels. Scale bars A,B,C, 20 μm. D. In a 3D reconstruction of a 741s Z-stack acquisition of trypanosomes (green), they are seen to be within about 40 μm under the skull. E. Texas Red infused in the cisterna magna labeled spaces adjacent to pial veins (V), and patches at that level, leaving a space (d) beneath the skull. Blue signal is from skull bone and furamidine-labeled nuclei. The bit of skull giving no SHG signal (c) may correspond to the site where a blood vessel entered the skull. 16 dpi.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003714.g004: The location of extravascular trypanosomes.A. A frame from S6 Video showing GFP trypanosomes in the plane of extracellular collagen. The movement of one trypanosome appeared to constrained by the collagen (arrow). B. Trypanosomes (red, arrow heads) were frequently observed outside small, somewhat irregular, vessels at the level of collagen (green, see S7 Video). C. Trypanosomes were not observed at the level of large pial vessels. Scale bars A,B,C, 20 μm. D. In a 3D reconstruction of a 741s Z-stack acquisition of trypanosomes (green), they are seen to be within about 40 μm under the skull. E. Texas Red infused in the cisterna magna labeled spaces adjacent to pial veins (V), and patches at that level, leaving a space (d) beneath the skull. Blue signal is from skull bone and furamidine-labeled nuclei. The bit of skull giving no SHG signal (c) may correspond to the site where a blood vessel entered the skull. 16 dpi.
Mentions: Recent papers have reported extravascular trypanosomes in the cortical pia mater [43] or the superficial cortical parenchyma [45], while T cells in the spinal meninges have been reported in the leptomeninges (arachnoid plus pia mater [21]). Knowing that the meninges were intact, and benefitting from signals from structural elements, we attempted to define the compartments containing the trypanosomes and T cells. The upper boundary of the compartment was clearly the skull, as trypanosomes were observed immediately below it. Often they were in the same plane as extracellular collagen, the main component of the dura mater (Fig 4A and S6 Video). Also at about this depth were cell nuclei labeled blue by intravenous furamidine (Fig 3D). These cells are likely to be the 'mesothelial lining cells' of the dura that are labeled by intravascular aminoacridines [24]. Defining the lower boundary of the compartment occupied by trypanosomes, like that of the dura itself [46, 47], was not so straightforward. Trypanosomes were observed close to small horizontal vessels, often with an irregular trajectory and close to collagen (Fig 4B and S7 Video), but were not observed at the level of larger horizontal vessels just above the parenchyma (Fig 4C). The latter were clearly in the subarachnoid space; the former were probably branches of the meningeal artery in the dura. In images acquired over many seconds, the trypanosomes traced out apparently isolated volumes within about 40 μm of the skull (Fig 4D). To delineate the extent of the subarachnoid space we infused dye (Texas Red) in the cisterna magna. From this site, dye is known to flow along the perivascular spaces of surface arteries and enter the subarachnoid space over the cortex [26, 29, 48]. As shown in Fig 4E, Texas Red, infused in this way, labeled spaces adjacent to large pial blood vessels and a thin, patchy, space extending across the cortex, in general agreement with histological studies on Murinae [27, 31, 41, 46, 47]. The dye is excluded from a space beneath the skull that contains nuclei labeled by furamidine. It is this "dural space", and not the subarachnoid space, that appears to be occupied by trypanosomes. Qualitative observation through the dissecting microscope, and vertical sections from sample Z-stacks, suggested that the distance from the skull to pial vessels increased in infected mice.

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