Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis

Abstract

Central nervous system (CNS) infections in ruminant livestock, such as listeriosis, are of major concern for veterinary and public health. To date, no host-specific in vitro models for ruminant CNS infections are available. Here, we established and evaluated the suitability of organotypic brain-slices of ruminant origin as in vitro model to study mechanisms of Listeria monocytogenes CNS infection. Ruminants are frequently affected by fatal listeric rhombencephalitis that closely resembles the same condition occurring in humans. Better insight into host-pathogen interactions in ruminants is therefore of interest, not only from a veterinary but also from a public health perspective. Brains were obtained at the slaughterhouse, and hippocampal and cerebellar brain-slices were cultured up to 49 days. Viability as well as the composition of cell populations was assessed weekly. Viable neurons, astrocytes, microglia and oligodendrocytes were observed up to 49 days in vitro. Slice cultures were infected with L. monocytogenes, and infection kinetics were monitored. Infected brain cells were identified by double immunofluorescence, and results were compared to natural cases of listeric rhombencephalitis. Similar to the natural infection, infected brain-slices showed focal replication of L. monocytogenes and bacteria were predominantly observed in microglia, but also in astrocytes, and associated with axons. These results demonstrate that organotypic brain-slice cultures of bovine origin survive for extended periods and can be infected easily with L. monocytogenes. Therefore, they are a suitable model to study aspects of host-pathogen interaction in listeric encephalitis and potentially in other neuroinfectious diseases.

Figure 1

(a) First, the head was cut off from the animal directly after slaughter and cranial nerves were cut with fine sterile scissors through the foramen magnum. (b) In a multistep sampling procedure with several insertions of the spoon tool (inset) the caudal brainstem was removed. (c) Then, the cerebellum was removed. Once the whole cerebellum was extracted from the scull, access to the telencephalon including the hippocampus was possible. (d) Final samples of hippocampus (right) and cerebellum (left) are shown.

Figure 2

HE-stained cryosections from hippocampal and cerebellar brain-slices are compared to fluorescent images from the viability-staining. In HE-stained sections, hippocampal slices show normal architecture of the dentate gyrus (20×, from top right to bottom left within each image) and granule neurons with normal nuclear morphology (insets). Cerebellar slices show normal architecture of the granule cell layer throughout the experiment, although at day 49 a marked decrease in cell density and nuclear fragmentation can be observed. At days 7 and 49 necrotic Purkinje cells are present. Fluorescent images (20×) were taken on whole slices and represent the same region as the corresponding HE stains. Total number of nuclei is stained in red (propidium iodide after permeabilization) and nuclei of dead cells are shown in blue (eBioscience fixable-viability-stain 660). At days 7 and 49, a significant number of viable cells are present in the dentate gyrus. In the cerebellar granule cell layer, cell density is decreased at day 49 and the proportion of dead cells is higher as in the dentate gyrus. Fluorescent images were digitally enhanced and gamma settings were adjusted using the Fluoview software (Olympus FV10-ASW Version 01.07.01.00). d, day.

Figure 3

(a) The average proportion of viable cells as estimated from dead cell and total cell counts are indicated at different time points for hippocampal and cerebellar tissue-slice cultures from six different calves (error bars represent standard deviations). (b) Total cell numbers in 0.6 mm² of the same hippocampal and cerebellar tissue-slices as in (a) at different time points.

Figure 4

(a) Double-immunofluorescence with antibodies specific for neurons (NeuN, green) on cryosections of hippocampal and cerebellar brain-slices of the same experiment as in Figure 3 at days 0, 28 and 49 in vitro. Nuclei were stained with TOTO-3 (blue). Top row: 20× magnification of the dentate gyrus of the hippocampus. Middle row: 100× magnification of the dentate gyrus of the hippocampus. Bottom row: 100× magnification of the granule cell layer in the cerebellar cortex. (b) Cryosections of hippocampal slices from the same experiment as in Figure 3 were stained (green) with antibodies specific for astrocytes (GFAP), microglia (Iba-1) or oligodendrocytes (CNP). Nuclei were stained with TOTO-3 (blue). Images are taken from the dentate gyrus. Magnification: 100×. (c) Cryosections of cerebellar brain-slice cultures from the same experiment as in Figure 3 were stained with the same antibodies as the hippocampal slices (days 0, 28 and 49 in vitro, 100× magnification). Images were digitally enhanced and gamma settings were adjusted using the FluoView software (Olympus FV10-ASW Version 01.07.01.00).

Figure 5

(a) Quantification of colony forming units (CFU) in seven hippocampal slices and six cerebellar slices per time-point. Hippocampal and cerebellar brain-slices from five different animals were infected with Listeria monocytogenes on days 7–13 in vitro (animal 1: day 12; animal 2: day 7, animal 3; day 13, animal 4: day 12; animal 5: day 7). Brain-slices were lysed at different time-points (6, 24, 48 h) after infection. (b) CFU’s were determined by plating lysates and medium on blood agar plates. Photographs of the agar plates are shown: lysate (left) and culture medium (middle) of hippocampal brain-slices infected with L. monocytogenes. Lysate from a Listeri innocua-infected brain-slice culture served as control (right).

Figure 6

Comparison of infected brain-slices with natural infections. In all images, Listeria monocytogenes are stained in red (LM). Microglia (CD68), neuronal processes (NF), astrocytes (GFAP) and oligodendrocytes (CNP) are stained in green. Nuclei (TOTO-3) are blue. Images were digitally enhanced and gamma settings were adjusted using the FluoView software (Olympus FV10-ASW Version 01.07.01.00). (a) Overview: focal growth of L. monocytogenes (red) in brain-slices and natural cases of listeriosis. On the left microglia are stained in green (10× magnification). On the right astrocytes are stained in green (20× magnification). (b) Immunofluorescence on cryosections of infected brain-slices. CD68: L. monocytogenes are present in microglial cells (arrowhead, 300× magnification). Neurofilament (NF): L. monocytogenes are closely associated with axons (arrowheads, 200× magnification). GFAP: L. monocytogenes are closely associated with astrocytic processes (arrowhead, 200× magnification). CNP: in brain-slices, L. monocytogenes are only rarely found inside oligodendrocytes (arrowhead, 600× magnification). (c) Immunofluorescence on paraffin-sections of a natural case of listeric encephalitis. CD68: L. monocytogenes are present in microglial cells (arrowhead, 300× magnification). Neurofilament (NF): L. monocytogenes are closely associated with axons (arrowhead, 300× magnification). GFAP: L. monocytogenes are closely associated with astrocytic processes (arrowhead, 400× magnification). CNP: in natural infections no L. monocytogenes were found within oligodendrocytes (400× magnification).