A novel approach for locating mice brain regions of Cryptococcus neoformans CNS invasion

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The mechanism of extrapulmonary dissemination to the CNS are not completely understood.The mouse model of experimental hematogenous meningoencephalitis was commonly used as an in vivo mode to investigate the mechanisms of C. neoformans penetration into the brain (Maruvada et al., 2012;Noverr et al., 2003).
Understanding the interactions between C. neoformans and brain cells is fundamental in exploring the mechanisms of the C. neoformans CNS infection.Brain capillaries can be the sites of the interaction of Cryptococcus with brain endothelial cells (Shi et al., 2010).However, brain regions where yeast cells interact with brain cells and invade into brain remain unclear.
Aim of this study was to locate the regions of brain invasion by Cryptococcus.We established a novel approach for locating the brain regions of C. neoformans CNS invasion in the mouse model of experimental hematogenous meningoencephalitis, which will be very useful for further exploring the mechanism of C. neoformans CNS infection.

Yeast strains
C. neoformans strains H99 was purchased from ATCC.GFP-tagged H99 was generated by integration of plasmid containing GFP construct (a generous gift of Prof. Robin May, University of Birmingham, UK) into the genome of the H99 with a BioRad PDS-1000/He biolistic particle delivery system (Voelz et al., 2010).

Intratracheal inoculation
Mice (female; C57BL/6J mice; 10-12 weeks; 20-25 g) model of experimental meningitis was prepared by intratracheal inoculation of C. neoformans strains H99.Mice were anesthetized with intraperitoneal injection of 4% chloral hydrate at 50 mg/kg.A small incision was made in the skin over the trachea.A 30-gauge needle was attached to a tuberculin syringe.The needle was bent and inserted into the trachea, and 30 µL PBS (containing 10 5 CFU Cryptococcus) was delivered.The skin was sutured with a cyanoacrylate adhesive, and the mice recovered with no visible trauma (Noverr et al., 2003).

Immunofluorescence
After 7 days of intratracheal inoculation of C. neoformans, mouse chest was cut open, and mouse was perfused with a mammalian Ringer's solution by transcardiac perfusion through a 23-gauge needle inserted into the left ventricle of the heart under the perfusion pressure of about 100 mmHg for 20 min.The brains were removed and put in liquid nitrogen.Serial cryosections (10 μm) were incubated overnight with a monoclonal rat anti-PECAM-1 primary antibody and/ or a polyclonal rabbit anti-p-cPLA2 primary antibody.Afterward, sections were incubated with Dylight 488 goat Anti-Rat IgG secondary antibody and/or Cy3 goat anti-Rabbit IgG secondary antibody.Slides were imaged with a confocal microscopy (Leica TCS SP5).

Results
In this study, the mouse model of experimental hematogenous meningoencephalitis was prepared by intratracheal inoculation of GFP-tagged C. neoformans H99 (Figure 1A and B).After 7 days of the inoculation, serial cryosections from 3 mice brains were imaged with fluorescence microscopy for locating regions of brain invasion by C. neoformans.GFP-tagged H99 were found in some brain regions such as primary motor cortex-secondary motor cortex (M1-M2), caudate putamen (CPu), stratum lucidum of hippocampus (SLu), field CA1 of hippocampus (CA1), dorsal lateral geniculate nucleus (DLG), lateral posterior thalamic nucleus, laterorostral part (LPLR), lateral posterior thalamic nucleus, mediorostral part (LPMR), retrosplenial agranular cortex (RSA), lateral area of secondary visual cortex (V2L), and lacunosum molecular layer of the hippocampus (LMol) (Figure 1C and D).Furthermore, immunofluorescent imaging for specific regions in Figure 1C  Some evidence from human cases and experimental animal models of C. neoformans meningoencephalitis indicate that cerebral capillaries are the portal of C. neoformans invasion into the brain (Chang et al., 2004 ;Chretien et al., 2002;Lee et al., 1996;Neuville et al., 2002;Shi et al., 2010).In this study, PECAM-1 (platelet/ endothelial cell adhesion molecule-1), a marker of brain vessel endothelial cells, was stained with immunofluorescent antibody to show microvasculature, which enable us to locate both C. neoformans and vasculature.
Our results have showed that C. neoformans invaded into hippocampal regions such as LMol and SLu (Figure 3) after 7 days of intratracheal inoculation.Our previous work showed that host cytosolic phospholipase A2 (cPLA2) contributes to group B Streptococcus penetration of the blood-brain barrier (BBB) (Maruvada et al., 2011).However, the role of cPLA2 in C. neoformans CNS infection remains unclear.cPLA2 had been showed to be involved in Adependent deficits of learning and memory in a mouse model of Alzheimer's disease (Sanchez-Mejia et al., 2008).Therefore, it will be very interesting to examine whether the cPLA2 in hippocampus is involved in C. neoformans invasion and resulting memory impairment of cryptococcal meningoencephalitis.We have found that cPLA2 is extensively active in hippocampus CA1 (Figure 4).The disappearance of p-cPLA2 positive cells in infected areas indicated that C. neoformans invasion into CA1 cause the damage of the tissue in this region (Figure 4).Although more evidence need for clarifying the role of cPLA2 in C. neoformans CNS infection, the preliminary experiment has showed that this approach will be very useful for future studies of C. neoformans CNS infection.

Discussion
Approaches of traditional histology had been used to study the CNS invasion by C. neoformans.Experiments were designed to characterize the anatomical site where C. neoformans crossed the BBB after intravenous injection of Cryptococcus.However, during a natural infection Cryptococcus are gradually released from the lung and enter the blood stream in limited numbers (Shi et al., 2012).Previous study showed that yeasts were observed in or next to the cortical capillaries of the brain and cerebellum and that no yeast cells were observed in the choroid plexus after intravenous inoculation.In this study, mice model of CNS infection was prepared by intratracheal inoculation, which is more close to natural infection as compared with intravenous inoculation.We provided the first evidence that several important brain regions were infected by C. neoformans after 7 days of intratracheal inoculation.
For intravital imaging of C. neoformans CNS invasion, the yeast cells can be labeled with fluorescent dyes (Shi et al., 2010).However, the yeast cell will lose the fluorescent label when it multiplies.Consequently, it will be hard to track the C. neoformans if it begins to replicate.To overcome this disadvantage, we generated GFP-tagged H99 by integration of plasmid containing GFP construct into the genome of the H99.In in vitro models, GFP-tagged Cryptococcus had been used for automated analysis of cryptococcal macrophage parasitism (Voelz et al., 2010) and for the study of interaction between yeast and human brain microvascular endothelial cell (Huang et al., 2011).In our in vivo model of this study, using of GFP-tagged H99 enable us to track and locate the regions of brain invasion by C. neoformans after 7 days of intratracheal inoculation of Cryptococcus.
In addition, this approach also provides some other advantages: Firstly, GFP-tagged Cryptococcus in serial cryosections are readily visualized via fluorescence microscopy.Secondly, brain structures such as vasculature that are morphologically distinguishable from Cryptococcus can both be stained as the same color in fluorescence imaging, which save one light path for fluorescence imaging of other molecular.Thirdly, all cell nucleus DNA can be stained with DAPI as blue color, and any tissue damage (disappearance of blue) is readily detected.
As an example of using this approach in study of CNS infection, we have showed that the molecular such as cPLA2 can be stained with fluorescence dyes and can be visualized by fluorescence microscopy to examine the role of this molecular in CNS infection.In fact, most fluorescence microscopes can have four light path used for fluorescence imaging.In this approach, GFP-tagged H99 and PECAM-1 (green) and DAPI (blue) occupy two light path, and there are two other light path leave   for fluorescence imaging of two other molecular.If gene knockout or transgene mice are used in the model, up to three or four molecular that may be involved in CNS infection can be examined in the same experimental model, which will make this approach a valuable research tool in the field of C. neoformans CNS infection.

Conclusion
We provided the first evidence that several important brain regions were infected by C. neoformans after 7 days of intratracheal inoculation, which will be very useful for further exploring the mechanism of C. neoformans CNS infection.

Figure 3 :
Figure 3: C. neoformans invasion into lacunosum molecular layer of the hippocampus (LMol), dorsal lateral geniculate nucleus (DLG), or stratum lucidum of hippocampus (SLu) Many GFP-tagged Cryptococcus (H99-GFP, green and round) were found in LMol, DLG, or SLu after 7 days of intratracheal inoculation.PECAM-1 of brain vessel endothelial cells was stained as green, microvessels (green but not round) are distinguishable from green and round H99.DAPI stains cell nucleus.A-C, J-L: 10X, bar=250 μm; D-F: Higher magnification (40X) of the area enclosed within the upper right box in A-C, bar = 50 μm.G-I: Higher magnification (40X) of the area enclosed within the left down box in A-C, bar =50 μm.M-O: Higher magnification (40X) of the area enclosed within the box in J-L, bar = 50 μm

Figure 4 :
Figure 4: C. neoformans invasion into field CA1 of hippocampus (CA1)Many GFP-tagged Cryptococcus (H99-GFP, green and round) were found in CA1 after 7 days of intratracheal inoculation.PECAM-1 of brain vessel endothelial cells was stained as green, microvessels (green but not round) are distinguishable from green and round H99.p-cPLA2 , a Ser 505 phosphorylated cPLA2, is the active form of cPLA2.Cells in CA1 highly express p-cPLA2 (red).DAPI stains cell nucleus.Arrow indicates that H99 caused tissues damage in infected regions of CA1.A-D: 10X, bar = 250 μm; E-H: Higher magnification (40X) of the area enclosed within box in A-D, bar = 50 μm.