Rapid Transepithelial Transport of Prions following Inhalation

Anthony E. Kincaid,a,b,c Kathryn F. Hudson,a* Matthew W. Richey,a and Jason C. Bartzc

Department of Physical Therapy,a Department of Biomedical Sciences,b and Department of Medical Microbiology and Immunology,c Creighton University, Omaha,Nebraska, USA

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervoussystem. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across anepithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spa-tial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross thenasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it wasimmediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were pro-cessed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrPd).Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, andwithin lymphatic vessels of the nasal cavity at all time points examined. PrPd was identified within a limited number of M cells 15to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these resultssupport M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the re-spiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately crossthe nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining thenasal cavity.

The prion diseases are a group of fatal neurodegenerative dis-eases that affect animals, including humans. There is consid-

erable evidence to support the hypothesis that the infectious agentis largely, if not entirely, PrPSc, a misfolded conformation of thenormal prion protein, which is designated PrPC (8, 12, 18, 51, 78,85). Infectious prions are thought to be generated when PrPSc

comes into direct contact with PrPC and converts the native pro-tein into a misfolded conformation. The misfolded isomer hasdistinct biological and physical properties, including the ability toself-propagate, aggregate, and cause disease (9, 14, 17, 24, 66).

While human prion diseases are rare, the prevalence of someanimal prion diseases is relatively high and, in some cases, is in-creasing. For example, the prevalence of chronic wasting disease(CWD), a prion disease of deer and elk, can be as high as 15% insome free-ranging populations and 80 to 89% in captive herds (54,55, 57, 87). The distribution of CWD in cervid populations inNorth America has expanded since the disease was first reportedin Colorado and Wyoming in the 1980s, and with the recent iden-tification of infected animals in Michigan, Texas, and Iowa, CWDhas now been detected in 18 states and 2 Canadian provinces (15).One of the characteristic features of prion diseases is that they canbe transmitted horizontally between animals, but neither thesource nor the mode of transmission in free-ranging animal pop-ulations is known (56, 77). There is increasing evidence for theexistence of infectious prions in a number of bodily fluids, includ-ing urine (31, 62, 74, 76), semen (70), saliva (31, 50, 53), nasalsecretions (7), blood (13, 37, 53), milk (43, 47, 48), and feces (45,71, 84). In addition, there is evidence that prion infectivity residesin the placenta (3, 68), decaying carcasses (56), antler velvet (4),and inanimate objects such as fence post and feeding trough sur-faces (49). Infectious prions are particularly stable and have beenshown to retain their infectivity after years in the environment;therefore, the ongoing transmission of disease in free-ranging an-imals is of great concern (10, 27, 40, 72, 75).

The oral route of infection has been established in the spread ofprion diseases caused by the consumption of prion-contaminatedfood sources, including the bovine spongiform encephalopathy(BSE) outbreak in the United Kingdom (16), the spread of kuru inNew Guinea (26), and the rare occurrence of transmissible minkencephalopathy (TME) that affected ranch-raised mink (52).While the oral route of infection may be involved in the transmis-sion of disease in free-ranging animals, other routes of prion entrymay exist (79). Recently the nasal cavity has been demonstratedexperimentally to be an effective route of prion infection in ham-sters, mice, and sheep following intranasal or extranasal (e.n.)inoculation of infected brain homogenate (6, 32, 42, 73). Further-more, aerosolization of prions using a nebulizer, followed by expo-sure of the noses or snouts of mice, also resulted in the developmentof disease in cervidized, wild-type, and immunodeficient mice (19,34). Given that animal populations that are susceptible to prion dis-eases use their highly developed olfactory systems for the detection offood and predators, as well as for reproductive and exploratory pur-poses, the mucosal surface of the nasal cavity may be a site of initialcontact with inhaled materials harboring prions.

Whether the initial site of mucosal contact is in the gut or thenasal cavity, the infectious agent must be transported across amucosal epithelium to gain entry into the body, where it can rep-licate and cause disease. There is disagreement about the location

Received 24 July 2012 Accepted 5 September 2012

Published ahead of print 12 September 2012

Address correspondence to Anthony E. Kincaid, akincaid@creighton.edu.

* Present address: Kathryn F. Hudson, Department of Pharmacology, EmoryUniversity, Atlanta, Georgia, USA.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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and mechanism of the transepithelial transport of prions follow-ing ingestion, with in vitro evidence for M cell transport (35),cotransport of prions with ferritin (58, 80), and laminin-mediatedbinding and endocytosis of prions (60), all across Caco-2 humanepithelial cells. A second in vitro model of prion transport dem-onstrated that a mouse-adapted BSE agent crossed bovine M cellsefficiently (59). In vivo experiments have demonstrated evidencefor both M-cell-mediated and non-M-cell-mediated transepithe-lial transport of prion proteins following oral ingestion in miceand sheep, respectively (2, 20, 25, 36, 38, 46, 64, 83). The temporalparameters and the cell types mediating the transepithelial trans-port of prions within the nasal cavity have not been established.

The experiments reported here were designed to determine thelocation, cell type, and time course of the transepithelial transportof prions within the nasal cavity following inhalation by hamstersin an in vivo model of prion infection. An important componentof these experiments was the ability to test the hypothesis that Mcells located in the follicle-associated epithelium (FAE) that over-lies the nasal mucosa-associated lymphoid tissue (NALT) are re-sponsible for the transport of prions across the epithelium.

MATERIALS AND METHODSAnimal care. Procedures involving animals were preapproved by theCreighton University Institutional Animal Care and Use Committee andwere carried out in accordance with the Guide for the Care and Use ofLaboratory Animals (63). Male adult golden Syrian hamsters from HarlanSprague-Dawley (Indianapolis, IN) were group housed in standard cageswith ad libitum access to food and water.

Animal inoculations. Extranasal inoculations were performed as de-scribed previously (42). Hamsters receiving e.n. inoculations were firstbriefly anesthetized with isoflurane (Webster Veterinary, Kansas City,MO) and then placed in a supine position, and 10 to 100 �l of brainhomogenate was placed just below each nostril (total volume, 20 to 200�l). The brain homogenate was immediately inhaled into the nasal cavity;the animals began to move freely in their cages within 1 to 2 min of thestart of the procedure.

Brain homogenate and control solutions. Dulbecco’s phosphate-buffered saline (Dulbecco’s PBS), India ink (colloidal carbon in water), ora 10% (wt/vol) brain homogenate from either an HY TME-infected ham-ster (containing a 109.3 intracerebral 50% lethal dose [LD50]/g) or anuninfected hamster was used. India ink was used to identify M cells in theFAE of the hamster nasal cavity (69).

Tissue collection. Animals exposed to PBS, India ink, or infected ormock-infected brain homogenate were reanesthetized following a speci-fied survival period and were transcardially perfused with 50 ml of 0.01 MDulbecco’s PBS, followed by 50 to 75 ml of McLean’s paraformaldehyde-lysine-periodate (PLP) fixative. Skulls were removed and were placed inPLP at room temperature overnight; then they were decalcified for 2weeks at room temperature with a solution change at 1 week (decalcifyingsolution; Richard-Allan Scientific, Kalamazoo, MI). By using the hardpalate for orientation, the nasal cavity was excised between the posterioredge of the incisors and the anterior edge of the seventh palatal ridge, alength of about 12 mm. This block of tissue contains the entire nasalcavity, including the NALT and the olfactory and respiratory epithelia, butnot the rostral vestibule, which is lined by a stratified squamous epithe-lium (1). This block was then cut and divided into three 4-mm coronalslices, or sagittally into single right and left halves, which were placed in acassette for paraffin processing and embedding. Serial sections (7 �m)were cut with a rotary microtome and were mounted onto glass slides. Asmall number of animals (2 untreated and 2 PBS-inoculated animals)were not transcardially perfused but were immersion fixed in PLP for 1week prior to decalcification and paraffin processing in order to differen-tiate lymphatic vessels from blood vessels.

Histology. To establish the locations and boundaries of the varioustypes of epithelia found within the nasal cavity, including the location ofM cells, tissue sections from untreated animals or from animals extrana-sally exposed to India ink or PBS were cleared, dehydrated, and stainedwith periodic acid-Schiff stain (PAS) or hematoxylin and eosin (H&E).Pairs of tissue sections not further than 112 �m apart through the rostral-caudal extent of the hamster nasal cavity were examined using an Olym-pus BX 40 light microscope to demonstrate normal nasal mucosal mor-phology and the location of M cells.

IHC. Immunohistochemistry (IHC) was performed to detect PrPd asreported previously, and infected and mock-infected tissues were pro-cessed at the same time using the same reagents (42). In brief, tissuesections were deparaffinized and were subjected to antigen retrieval informic acid (95%) for 10 min at room temperature. All subsequent stepswere carried out at room temperature; incubations were separated by 3rinses with 0.05% (vol/vol) Tween in Tris-buffered saline (TTBS). Endog-enous peroxidase and nonspecific antibody binding were inhibited byincubating the tissue sections in 0.3% H2O2-methanol for 20 min, fol-lowed by incubation in 10% normal horse serum in TTBS for 30 min. Theprion protein was visualized by using the avidin-biotin method. Incuba-tion in an anti-prion protein monoclonal antibody (clone 3F4; dilution,1:600; Chemicon, Temecula, CA) was carried out for 2 h at room temper-ature, followed by incubation in a biotinylated horse anti-mouse antibody(dilution, 1:600; Vector Laboratories, Burlingame, CA) for 30 min. Thesections were placed in ABC solution (dilution, 1:200; Vector Laborato-ries, Burlingame, CA) for 15 to 20 min and were subsequently reacted ina solution containing filtered 0.05% diaminobenzidine tetrachloride(Sigma, St. Louis, MO) with 0.0015% H2O2. The sections were counter-stained with hematoxylin, dehydrated through graded alcohols, andplaced on coverslips with Cytoseal-XYL (Richard-Allan Scientific, Kala-mazoo, MI). Glial fibrillary acidic protein (GFAP) IHC was carried out ina similar manner with the following differences: there was no antigenretrieval step; the blocking step was carried out using normal goat serum;the primary antibody was a rabbit anti-GFAP polyclonal antibody (dilu-tion, 1:800; Dako, Carpinteria, CA); and the secondary antibody was abiotinylated goat anti-rabbit antibody (dilution, 1:800, Vector Labo-ratories, Burlingame, CA). Some sections were processed identicallywith either the primary or secondary antibodies omitted, or with amouse immunoglobulin G isotype control (Abcam, Cambridge, MA)in place of the primary antibody at the same concentration. Pairs oftissue sections not further than 56 �m apart were examined using anOlympus BX 40 light microscope. When additional morphologicaldetail was required, adjacent tissue sections were processed for GFAPIHC or were stained with H&E.

RESULTS

Hamsters are obligate nose breathers, so a small amount of brainhomogenate placed just below their nostrils was immediately in-haled into their nasal cavities. This behavior precluded the needfor placing a pipette inside the nose, where it could injure the nasalmucosa and expose brain homogenate directly to blood vesselsand nerves. Mucosal damage could lead to direct hematogenousspread and/or neuroinvasion, which would complicate the inter-pretation of the transepithelial transport route and mechanism(s).

Following inhalation of PBS (n � 7), India ink (n � 6), andinfected (n � 35) or uninfected (n � 15) brain homogenate, ham-sters were allowed to survive for 1 to 180 min prior to the initiationof transcardial perfusion. Control animals were left untreated(n � 6). Regularly spaced tissue sections were inspected for thepresence of ink, GFAP, or PrPd. The average number of immuno-histochemically processed tissue sections analyzed per animal was366 (range, 150 to 640). Sagittally sectioned nasal cavities pro-duced fewer tissue sections, because the nasal cavity is approxi-mately twice as long as it is wide. GFAP is the principal interme-

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diate filament of astrocytes and therefore is a component of bothinfected and uninfected brain homogenates, while PrPd is foundonly in prion-infected brain homogenate (8, 14, 21). Roughlytwo-thirds of the tissue sections were processed using GFAP IHC,since tissue morphology is better preserved by this procedure thanby PrPd IHC, which requires the use of formic acid for antigenretrieval. The mucosal surface and underlying lamina propria oftissue sections through the rostral-caudal extent of each nasal cav-ity were inspected using a light microscope, and brain homoge-nate was identified with either GFAP or PrPd IHC. Brain homog-enate was identified in the nasal cavity of each experimentalanimal but was not seen in untreated animals or in those whoinhaled PBS. While more brain homogenate was observed withinthe nasal cavities of animals that inhaled larger volumes of brainhomogenate, the volume of brain homogenate was not related tothe occurrence of transport. Therefore, the data for animals withthe same survival time that had inhaled different volumes ofbrain homogenate were combined. Photomicrographs weretaken wherever brain homogenate was identified on the surfaceof, within, or beneath the nasal mucosa. Omission of either theprimary or the secondary antibody or replacement of the primaryantibody with an isotype control resulted in a complete lack ofstaining (data not shown).

Hamster nasal cavity and light microscopic identification ofM cells. The hamster nasal cavity is lined by a variety of morpho-logically distinct epithelia, including the respiratory and olfactoryepithelia and the FAE, which lies adjacent to the NALT (Fig. 1Aand B). M cells are nonciliated with a flattened apical surface anddistinct intraepithelial pockets that contain lymphoid cells (28,44) and were identified in the FAE overlying the NALT in H&E-and PAS-stained tissue sections from untreated animals (Fig. 1C).M cells could also be identified based on their ability to endocytoseinhaled India ink (69). Ink particles were identified within M cellsin all 6 animals that were allowed to survive for 5 to 30 min afterinhalation of 20 to 100 �l of ink (Fig. 1D). Inspection of untreated orPBS-exposed animals did not reveal any visible particulate matter on,or within, the M cells of the FAE of the nasal cavity (Fig. 1C). Theaverage number of tissue sections per animal that were processed forH&E and PAS staining was 178 (range, 136 to 204).

Identification of inhaled prions. Prion-infected or uninfectedbrain homogenate was identified within the nasal cavity of eachanimal following the inhalation of brain homogenate from eitherHY TME-infected or mock-infected animals. By use of GFAP IHC,prion-infected or uninfected brain homogenate was easily identifiedas aggregated or punctate brown particulate matter within thenasal cavity airspace and lining the nasal mucosa (Fig. 2). By use ofPrPd IHC, prion-infected brain homogenate was identified assmall brown punctate particulate matter, while uninfected brainhomogenate was unstained (Fig. 2). PrPd IHC was used on tissuesections adjacent to GFAP-positive sections to confirm the pres-ence of PrPd in brain homogenate in animals that had inhaledinfected brain homogenate. Brain homogenate from HY TME-infected animals was immunopositive for GFAP and PrPd, whilebrain homogenate from uninfected animals was immunopositivefor GFAP but not for PrPd (Fig. 2).

M cell transport of brain homogenate. Based on previous re-ports of M cell involvement in the transport of prions, M cells inthe FAE adjacent to the NALT were examined using a light micro-scope. Infected or mock-infected brain homogenate was identi-fied using GFAP IHC and was seen adhering to the FAE of 44 of 50

animals following extranasal inoculation (Table 1; Fig. 3). Thecriterion used for positive identification of brain homogenate orprions within M cells of the FAE was brown punctate GFAP orPrPd immunoreactivity surrounded by M cell cytoplasm in thesame focal plane. By applying this criterion, brain homogenate,either infected or uninfected, was identified within M cells in 31 ofthe 43 hamsters that survived for 5 min or longer (Table 1; Fig. 3).The number of M cells noted to contain brain homogenate foreach animal at each time point was counted to determine theperiod of maximal M cell transport of brain homogenate. Thegreatest number of M cells containing brain homogenate wasfound in those animals that survived 10 to 60 min prior to theinitiation of perfusion, with a noticeable drop in the number of Mcells containing brain homogenate in those animals that survived180 min (Table 1). Analysis of tissue deep within the M cell layerwas restricted to tissue sections processed for PrPd IHC, sinceGFAP labels thin-diameter nonmyelinating Schwann cells (29,39), which are located deep within M cells, in the NALT, andcoursing through the lamina propria (Fig. 2D and 4E).

Paracellular transport of brain homogenate occurs betweenolfactory epithelium, respiratory epithelium, and FAE cellswithin minutes of extranasal inoculation. Infected and unin-fected brain homogenates were detected penetrating or com-pletely spanning the nasal mucosae in 40 of the 50 animals inoc-ulated extranasally in this experiment (Table 1; Fig. 4A and B).This was determined to be paracellular transport, because brainhomogenate was located between intact epithelial cells, not withincells. This was confirmed by focusing through the depth of thetissue section and by examining the same cells on adjacent tissuesections. Paracellular transport of brain homogenate was identi-fied between cells of the respiratory and olfactory epithelia and theFAE of the nasal cavity (Fig. 4) and could be seen extending intothe underlying lamina propria in some cases (Fig. 4C and D).There were multiple examples of transepithelial transport in allanimals exhibiting transport; in some cases, multiple examples ofparacellular transport were identified on single tissue sections(Fig. 4B, D, and E). While paracellular transport was seen in ani-mals from all survival time points, the number of animals demon-strating paracellular transport decreased by about 50% amongthose that survived 180 min following inoculation (Table 1).There were no examples of brain homogenate crossing the kera-tinized stratified squamous epithelium that lines the vestibule ofthe proximal nasal cavity (33).

Prions quickly enter lymphatic vessels in the lamina propriabut not the NALT. Infected or uninfected brain homogenate wasidentified within the lumens of vessels in the laminae propriae of47 of the 50 inoculated animals in this study (Table 1). Thesevessels were determined to be predominantly lymphatic vesselsand not blood vessels. This distinction was based on the analysis ofvessels from immersion-fixed animals. Immersion fixation doesnot rinse the blood cells from blood vessels, so blood vessels wereeasily distinguished by the presence of red blood cells within theirlumens, and lymphatic vessels by the lack of blood cells. The locationsof lymphatic vessels were found to be relatively constant betweenanimals, especially for medium- and large-diameter lymphatic ves-sels. Infected or uninfected brain homogenate was identified insidethe lumens of lymphatic vessels in the laminae propriae of the nasalcavities of animals at all survival time points (Fig. 5). PrPd was notidentified in the intraepithelial pockets of M cells, adjacent to theNALT, or within the NALT for any animal in this study.

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FIG 1 Hamster nasal cavity epithelial types and the identification of M cells. (A) Low-power view of a PAS-stained portion of a hamster nasal cavity demon-strating the morphology of the olfactory and respiratory epithelia (OE and RE, respectively) (box 1, enlarged in panel B) and the FAE (indicated by asterisks) withM cells overlying the NALT (box 2, enlarged in panel C). (B) The RE can be distinguished from the OE based on the thickness of the epithelial layer and the lengthof cilia. (C) M cells can be identified by their distinct morphology, including intraepithelial pockets (outlined) and the lack of cilia, which distinguish them fromneighboring goblet and respiratory epithelial cells. (D) An H&E-stained tissue section from a hamster extranasally exposed to 20 �l India ink that survived for30 min prior to perfusion. Note the presence of ink particles within a subset of the M cells of the FAE. Asterisks indicate air space. Bars, 200 �m (A) and 20 �m(B, C, and D).

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FIG 2 Uninfected and infected brain homogenates in the hamster nasal cavity after inhalation, identified by GFAP IHC and PrPSc IHC. (A) Low-power view ofa portion of a nasal turbinate in the nasal cavity of a hamster that had been inoculated extranasally with mock-infected brain homogenate and that survived for5 min prior to perfusion, processed using GFAP IHC. The boxed area is enlarged in panel B. (B) Higher-power view of the uninfected brain homogenate identifiedusing GFAP IHC. (C) Higher-power view of an adjacent tissue section processed using PrPSc IHC. Note the lack of punctate immunoreactivity, indicating thatthe brain homogenate contains GFAP but lacks PrPSc. (D) Low-power view of a portion of a nasal turbinate in the nasal cavity of a hamster that had beeninoculated extranasally with HY TME-infected brain homogenate and that survived for 5 min prior to perfusion, processed using GFAP IHC. The boxed area isenlarged in panel E. Note the GFAP-labeled bundle of nonmyelinating Schwann cells in the lamina propria (indicated by an arrow). (E) GFAP-labeled brainhomogenate from a HY TME-infected animal. (F) An adjacent tissue section processed using PrPSc IHC. Note the distinct punctate labeled brain homogenatethat is characteristic of PrPSc, indicating that this section contains GFAP and PrPSc. Contrast the staining in panels C and F to note the distinction betweenPrPSc-negative and PrPSc-positive brain homogenates. Asterisks indicate air space. Bars, 100 �m (A and D) and 20 �m (B, C, E, and F).

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DISCUSSION

The in vivo transport of infected brain homogenate containingPrPd across the nasal mucosa began within minutes of inhalation,and transport could still be detected in some animals as late as 180

min following inhalation. Paracellular transport was detected inanimals that were perfused within 1 to 2 min after inhalation,while M cell transport was first detected in animals that were per-fused beginning 5 min after inhalation. Both types of transport

TABLE 1 Brain homogenate distribution in the nasal cavity following inhalation

Survival timea

(no. of animals) Inoculum (vol [�l])b

No. of animals with brain homogenate at the indicated location/total no.

On FAEInside M cells(no. of M cells)

Crossing betweenepithelial cells

Deep withinepithelium

1–2 min (7) Uninfected bh (50–100) 2/2 0/2 2/2 2/2Infected bh (50–200) 5/5 0/5 4/5 5/5

5 min (16) Uninfected bh (20–100) 6/6 2/6 (5) 5/6 6/6Infected bh (20–200) 10/10 8/10 (15) 9/10 9/10

10–20 min (13) Uninfected bh (100) 2/3 2/3 (7) 1/3 2/3Infected bh (100) 10/10 10/10 (44) 10/10 10/10

60 min (7) Uninfected bh (100) 1/2 1/2 (2) 1/2 2/2Infected bh (50–100) 5/5 5/5 (29) 5/5 5/5

180 min (7) Uninfected bh (100) 1/2 1/2 (3) 1/2 2/2Infected bh (100) 2/5 2/5 (4) 2/5 4/5

a Time to the initiation of perfusion. The time until death included an additional 10 to 12 min.b Total volume of mock-infected or HY TME-infected brain homogenate applied below the nostrils. bh, brain homogenate.

FIG 3 Transcellular transport of prions by M cells in the nasal cavity following inhalation. (A and B) Inhaled mock-infected brain homogenate (A) andprion-infected brain homogenate (B) were identified with GFAP IHC located on the surface of the FAE of hamsters. Note the lack of brain homogenate withinthe M cells of the FAE in both of these sections: one taken from an animal that survived 5 min before perfusion (A) and the other from an animal that survived1 min before perfusion (B). (C and D) Infected brain homogenate identified with GFAP IHC (C) and PrPSc IHC (D) within M cells (indicated by arrows) ofanimals that survived 10 min (C) or 5 min (D) before perfusion. Asterisks indicate air space. Bars, 50 �m (A and B) and 20 �m (C and D).

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were detected in all remaining survival groups, with a noticeabledecrease in the number of animals demonstrating transportamong those that survived 180 min after inhalation. The rapid andprolonged period of transepithelial transport of PrPd in the nasalcavity following inhalation is consistent with the results of a studyperformed in a sheep gut-loop model of prion infection whereprion proteins were detected in the lumens of villous and submu-cosal lymphatics 15 min to 210 min following direct inoculation ofan isolated loop of distal ileum in sheep (38). The time frame, thetype of transport (transcellular or paracellular), and the cells/epi-thelia involved in the in vivo transport of prions following inhala-tion have been identified. Most notably, the paracellular transportof relatively large amounts of brain homogenate between epithe-lial cells in the nasal cavity identified here has not been describedpreviously. While the sequence of events leading to the paracellu-lar transport of prions has not been determined, the mechanism of

transepithelial transport may involve the passage of brain homog-enate through existing gaps in the mucosa created by the naturalshedding of epithelial cells that has been reported in the intestinesof mice and humans (11, 41, 86). Utilization of existing gaps in theepithelia would be consistent with the observed speed and physi-cal dimensions of the transport of brain homogenate reportedhere, where the passage of brain homogenate between cells oftenappeared to be about one epithelial cell in width and occurredwithin minutes of inhalation (Fig. 4).

The persistence and transepithelial transport of brain homog-enate in the nasal cavity for as long as 3 h after inhalation, reportedhere, are surprising, since nasal mucociliary activity results inmeasured mucus flow rates ranging from 0.9 to 11 mm/min indifferent areas of the rat nasal cavity and to an average of 1.3mm/min in the mouse nasal cavity (30, 61). At these rates, inhaledbrain homogenate would be expected to be cleared from the ham-

FIG 4 Paracellular transport of brain homogenate in the nasal cavity following inhalation, identified with GFAP IHC. Shown are inhaled mock-infected (A) andinfected (B) brain homogenates crossing the nasal mucosa between cells of the olfactory epithelium 5 min (A) and 1 min (B) after inhalation. Brain homogenatecould be seen between epithelial cells spanning the complete width of the epithelium and entering the underlying lamina propria on some tissue sections (C).Note that multiple examples of paracellular transport of infected (B, C, and D) and uninfected (E) brain homogenates were observed on some tissue sections.Transport of brain homogenate was noted across the olfactory (C) and respiratory (D) epithelia and the FAE (E). Nonmyelinating Schwann cells deep within theFAE (indicated by arrows) are labeled with GFAP IHC. Note that the width of the paracellular transport appears to be approximately the same as that of a singleepithelial cell in all panels. NS, nasal septum; OE, olfactory epithelium; RE, respiratory epithelium. Bars, 100 �m (A and B) and 25 �m (C, D, and E).

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ster nasal cavity in less than 60 min. This prolonged period oftransport is similar to that reported for the sheep gut-loop model,where prions were detected in submucosal lymphatics for as longas 3.5 h following inoculation (38).

A goal of these experiments was to test the hypothesis thatprion entry following inhalation was mediated via M cells of theFAE in the nasal cavity. Infected or uninfected brain homogenatewas seen within a relatively modest number of M cells (average, 1to 5 per animal) in 31 of 50 animals in this study (Table 1). Giventhat the efficiency of this route of inoculation has been determinedto be 10 to 100 times greater than that of per os inoculation (42),the number of animals demonstrating M cell transport and thenumber of M cells per animal transporting brain homogenate inthis study were lower than might be expected. This is in markedcontrast to the robust paracellular transport, reported here, ofPrPd in brain homogenate that was noted between cells of theolfactory, respiratory, and follicle-associated epithelia that line thenasal cavity (Table 1; Fig. 4). The results of this study are consis-tent with a relatively minor role for M cell transport in the trans-epithelial transport of prions following inhalation, with a greatercontribution coming from the paracellular transport of prions.The limited number of examples of M cell involvement in PrPd

transport following inhalation was not due to a lack of exposure tobrain homogenate, since almost all of the animals examined in thisstudy had multiple examples of brain homogenate resting on FAE(Table 1). There are certain limitations to the interpretations thatcan be made about a dynamic cellular process that occurs across asurface and over a period when one is examining sections taken atspecific time points. First, the transport of brain homogenateacross the M cells could happen very quickly, so that the chance ofidentifying brain homogenate within the cell at any one time pointis reduced, and the numbers reported here would be artificiallylow. However, this scenario would not be expected, based on areport that the uptake and transfer of 600- to 750-nm latex parti-cles across M cells occurred over 90 min after contact with the FAEin a rabbit intestinal loop model (67). Second, the transport ofbrain homogenate through M cells could involve the phagocytosisand movement of very small particulate matter, some of whichcould be beyond the resolution of a light microscope (�0.2 �m)and therefore undetectable (65). The reported diameter of trans-ported microparticles within M cells in other studies is 0.02 to 10

�m (22); therefore, we may have been unable to identify thesmaller range of particulate brain homogenate that was containedwithin the M cells. Regardless of resolution issues, definite identi-fication of transcellular transport of prions within M cells willrequire ultrastructural analysis. While the relative contributionsof the two routes identified in this report to pathogenesis and thedevelopment of disease remain to be determined, the apparentlylesser role of M cell involvement in prion transepithelial transportreported here is consistent with that in with sheep gut-loop modelof prion infection and in sheep orally inoculated with prions (2,38, 46), where the FAE and M cells do not appear to be involved inthe transport of prion proteins across the gut wall.

The results of this study indicate that PrPd can gain access tolymphatic vessels quickly and that transport across the nasal mu-cosa and into the vessels persists for as long as 3 h after inhalation.It is not likely that the persistence of brain homogenate in thelymphatics is simply due to a low rate of lymph flow in the nasalcavity, given that flow rates have been reported to average 3 to 9�m/s in different species (5, 23, 82) and that dye placed in the nosereaches cervical lymph nodes in 14 to 51 min, depending on thespecies (88). The presence of brain homogenate within lymphaticsis consistent with their role of draining fluid and macromoleculesfrom the extracellular matrix of the lamina propria in the nasalcavity. Material located in the lamina propria is propelled towardlymphatic capillaries by interstitial fluid pressure and enters thecapillaries, which are made permeable by the lack of a basementmembrane and the presence of anchoring filaments (for a review,see reference 81). Thus, it appears that the lymphatics, and subse-quently the circulatory system, can serve as a route for the systemicdispersion of prions that have been inhaled into the nasal cavity.Consistent with this pattern of transepithelial transport followinginhalation was the notable lack of brain homogenate or PrPd ad-jacent to, or within, the NALT in all the animals examined in thisstudy. While it is possible that direct neuroinvasion of prions mayoccur via somatic and autonomic nerves located in the laminapropria of the nasal cavity, this has not been reported.

ACKNOWLEDGMENTS

We sincerely thank Jacob Ayers, Albert Lorenzo, Melissa Clouse, ShawnFeilmann, and Theresa Lonmeth for excellent technical assistance withthis work.

FIG 5 Brain homogenate was located within the lumens of lymphatic vessels of the lamina propria of the nasal cavity within minutes of inhalation and was stillvisible for hours after inhalation. Infected (A and D) and uninfected (B and C) brain homogenates (indicated by arrows) were located within lymphatics 5 min(A and B) and 60 min (C and D) after inhalation. Bars, 50 �m.

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This work was supported by the National Institute of NeurologicalDisorders and Stroke (RO1 NS061994 and RO1 NS061994-03S1), HealthFuture Foundation Discretionary Award 200600-713131d, and grantG20RR024001 from the National Center for Research Resources.

The content of this article is solely the responsibility of the authors anddoes not necessarily represent the official views of the National Center forResearch Resources or the National Institutes of Health.

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