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Rapid Transepithelial Transport of Prions Following Inhalation 2

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Running Title: In Vivo Transport of Prions Across Nasal Mucosa 4

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Anthony E. Kincaid1,2,3# Kathryn F. Hudson1*, Matthew W. Richey1 and Jason C. Bartz3 6

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Department of Physical Therapy,1 Department of Biomedical Sciences,2 and 8

Department of Medical Microbiology and Immunology,3 Creighton University, Omaha, 9

Nebraska 68178 10

*Current address: Department of Pharmacology, Emory University, Atlanta, Georgia 11

30322-4218 12

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Abstract word count: 245 15

Text word count: 4,490 16

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Corresponding author E-mail: akincaid@creighton.edu 18

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.01930-12 JVI Accepts, published online ahead of print on 12 September 2012

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Abstract 19

Prion infection and pathogenesis is dependent upon the agent crossing an epithelial 20

barrier to gain access to the recipient nervous system. Several routes of infection have 21

been identified, but the mechanism(s) and timing of in vivo prion transport across an 22

epithelium have not been determined. The hamster model of nasal cavity infection was 23

used to determine the temporal and spatial parameters of prion-infected brain 24

homogenate uptake following inhalation and to test the hypothesis that prions cross the 25

nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was 26

placed below each nostril where it was immediately inhaled into the nasal cavity. 27

Regularly-spaced tissue sections through the entire extent of the nasal cavity were 28

processed immunohistochemically to identify brain homogenate and the disease-29

associated isoform of the prion protein (PrPd). Infected or uninfected brain homogenate 30

was identified adhering to M cells, passing between cells of the nasal mucosa and 31

within lymphatic vessels of the nasal cavity at all time points examined. PrPd was 32

identified within a limited number of M cells 15-180 minutes following inoculation, but 33

not in the adjacent nasal associated lymphoid tissue (NALT). While these results 34

support M cell transport of prions, larger amounts of infected brain homogenate were 35

transported paracellularly across the respiratory, olfactory and follicle associated 36

epithelia of the nasal cavity. These results indicate prions can immediately cross the 37

nasal mucosa via multiple routes and quickly enter lymphatics where they can spread 38

systemically via lymph draining the nasal cavity. 39

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Introduction 40

The prion diseases are a group of fatal neurodegenerative diseases that affect animals, 41

including humans. There is considerable evidence to support the hypothesis that the 42

infectious agent is largely if not entirely PrPSc, a misfolded conformation of the normal 43

prion protein, which is designated as PrPC (8, 12, 18, 51, 78, 85). The generation of 44

infectious prions is thought to occur when PrPSc comes into direct contact with PrPC and 45

converts the native protein into a misfolded conformation. The misfolded isomer has 46

distinct biological and physical properties; including the ability to self-propagate, 47

aggregate and cause disease (9, 14, 17, 24, 66). 48

While human prion diseases are rare, the prevalence of some animal prion diseases is 49

relatively high and in some cases increasing. For example the prevalence of chronic 50

wasting disease (CWD), a prion disease of deer and elk, can be as high as 15% in 51

some free-ranging populations and 80-89% in captive herds [54, 55, 57, 87). The 52

distribution of CWD in cervid populations in North America has expanded since it was 53

first reported in Colorado and Wyoming in the 1980s and with the recent identification of 54

infected animals in Michigan, Texas and Iowa has now been detected in 18 states and 2 55

Canadian provinces [http://www.cdc.gov/ncidod/dvrd/cwd/]. One of the characteristic 56

features of prion diseases is that they can be horizontally transmitted between animals, 57

but neither the source nor the mode of transmission in free ranging populations of 58

animals is known (56, 77). There is increasing evidence for infectious prions existing in 59

a number of bodily fluids including: urine (31, 62, 74, 76), semen (70), saliva (31, 50 60

53), nasal secretions (7), blood (13, 37, 53), milk (43, 47, 48) and feces (45, 71, 84). In 61

addition there is evidence that prion infectivity resides in: placenta (2, 68), decaying 62

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carcasses (56), antler velvet (4) and inanimate objects such as fence post and feeding 63

trough surfaces (49). Infectious prions are particularly stable and have been shown to 64

retain their infectivity after years in the environment, therefore the ongoing transmission 65

of disease in free-ranging animals is of great concern (10, 30, 40, 72,75). 66

The oral route of infection has been established in the spread of prion diseases caused 67

by the consumption of prion-contaminated food sources including the bovine spongiform 68

encephalopathy (BSE) outbreak in the United Kingdom (16), the spread of kuru in New 69

Guinea (26), and the rare occurrence of transmissible mink encephalopathy (TME) that 70

affected ranch-raised mink (52). While the oral route of infection may be involved in the 71

transmission of disease in free-ranging animals, other routes of prion entry may exist 72

(79). Recently the nasal cavity has been demonstrated experimentally to be an 73

effective route of prion infection in hamsters, mice and sheep following intra- or 74

extranasal inoculation of infected brain homogenate (6, 32, 42, 73). Furthermore, 75

aerosolization of prions using a nebulizer followed by exposure to the nose or snout of 76

mice also resulted in the development of disease in cervidized, wild-type and 77

immunodeficient mice (19, 34). Given that animal populations that are susceptible to 78

prion diseases use their highly developed olfactory systems for the detection of food 79

and predators, and for reproductive and exploratory purposes the mucosal surface of 80

the nasal cavity may be a site of initial contact of inhaled materials harboring prions. 81

Whether the initial site of mucosal contact is in the gut or the nasal cavity, the infectious 82

agent must be transported across a mucosal epithelium to gain entry into the body 83

where it can replicate and cause disease. There is disagreement about the location and 84

mechanism of the transepithelial transport of prions following ingestion with in-vitro 85

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evidence for M cell transport [35], co-transport of prions with ferritin [58, 80], and 86

laminin-mediated binding and endocytosis of prions (60), all across Caco-2 human 87

epithelial cells. A second in vitro model of prion transport demonstrated that murine-88

adapted BSE crossed bovine M cells efficiently (59). In vivo experiments have 89

demonstrated evidence for both M cell-mediated, and non M cell-mediated 90

transepithelial transport of prion proteins following oral ingestion in mice and sheep 91

respectively (3, 20, 25, 36, 38, 46, 64, 83). The temporal parameters and the cell types 92

mediating the transepithelial transport of prions within the nasal cavity have not been 93

established. 94

The experiments reported here were designed to determine the location, cell type and 95

time course of the transepithelial transport of prions within the nasal cavity following 96

inhalation by hamsters in an in-vivo model of prion infection. An important component 97

of these experiments was the ability to test the hypothesis that M cells located in the 98

follicle associated epithelium (FAE) that overlies the NALT are responsible for the 99

transport of prions across the epithelium. 100

MATERIALS AND METHODS 101

Animal care. Procedures involving animals were preapproved by the Creighton 102

University Institutional Animal Care and Use Committee and done in accordance with 103

the Guide for the Care and Use of Laboratory Animals (63). Male adult golden Syrian 104

hamsters from Harlan Sprague-Dawley (Indianapolis, IN) were group housed in 105

standard cages with ad libitum access to food and water. 106

107

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Animal Inoculations. Extranasal (e.n.) inoculations were performed as described 108

previously (42). Hamsters receiving e.n. inoculations were briefly anesthetized with 109

isoflurane (Webster Veterinary, Kansas City, MO), placed in a supine position and 10-110

100µl of brain homogenate was placed just inferior to each nostril (20-200µl total 111

volume). The brain homogenate was immediately inhaled into their nasal cavity; the 112

animals began to move freely in their cages within 1-2 minutes of the start of the 113

procedure. 114

Brain homogenate and control solutions. Dulbecco’s phosphate buffered saline 115

(PBS), India ink (colloidal carbon in water), or a 10% (wt/vol) brain homogenate from 116

either an HY TME-infected hamster (containing a 109.3 intracerebral 50% lethal dose 117

[LD50]/g) or uninfected hamster was used. India ink was used to identify M cells in the 118

FAE of the hamster nasal cavity (69). 119

Tissue Collection. Animals exposed to PBS, India ink, infected- or mock-infected brain 120

homogenate were reanesthetized following a specified survival period and transcardially 121

perfused with 50 ml of 0.01 M Dulbecco’s PBS followed by 50-75 ml of McLean’s 122

paraformaldehyde-lysine-periodate (PLP) fixative. Skulls were removed and placed in 123

PLP at room temperature overnight, then decalcified for 2 weeks at room temperature 124

with a solution change at 1 week (Decalcifying Solution, Richard-Allan Scientific, 125

Kalamazoo, MI). Using the hard palate for orientation the nasal cavity was excised 126

between the posterior edge of the incisors and the anterior edge of the seventh palatal 127

ridge, a length of about 12 mm. This block of tissue contains the entire nasal cavity, 128

including the NALT, olfactory and respiratory epithelia, but not the rostral vestibule 129

which is lined by a stratified squamous epithelium (1). This block was then cut and 130

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divided into three 4 mm coronal slices, or sagittaly into single right and left halves, and 131

placed into a cassette for paraffin processing and embedding. Serial sections (7 µm) 132

were cut with a rotary microtome and mounted onto glass slides. A small number of 133

animals (2 untreated and 2 PBS inoculated animals) were immersion fixed in PLP for 1 134

week prior to decalcification and paraffin processing, not transcardially perfused, to 135

differentiate lymphatic vessels from blood vessels. 136

Histology. To establish the location and boundaries of the various types of epithelia 137

found within the nasal cavity, including the location of M cells, tissue sections from 138

untreated animals, or animals extranasally exposed to India ink or PBS were cleared, 139

dehydrated and stained with periodic acid-Schiff (PAS) or hematoxylin and eosin (H&E). 140

Pairs of tissue sections not further than 112 µm apart through the rostral-caudal extent 141

of the hamster nasal cavity were examined using an Olympus BX 40 light microscope to 142

demonstrate normal nasal mucosa morphology and the location of M cells. 143

Immunohistochemistry. Immunohistochemistry was performed to detect PrPd as 144

reported previously, and infected and mock-infected tissues were processed at the 145

same time using the same reagents (42). In brief, tissue sections were deparaffinized 146

and subjected to antigen retrieval in formic acid (95%) for 10 min at room temperature. 147

All of the following steps were carried out at room temperature; incubations were 148

separated by 3 rinses with 0.05% v/v Tween in Tris-buffered saline (TTBS). 149

Endogenous peroxidase and non-specific antibody binding were inhibited by incubating 150

the tissue sections in 0.3% H2O2/methanol for 20 min followed by incubation in 10% 151

normal horse serum in TTBS for 30 min. Visualization of the prion protein was carried 152

out using the avidin-biotin method. Incubation in anti-prion protein monoclonal antibody 153

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(Clone 3F4; 1:600, Chemicon, Temecula, CA) was carried out for 2 hrs at room 154

temperature followed by incubation in biotinylated horse anti-mouse antibody (1:600, 155

Vector Laboratories, Burlingame, CA) for 30 min. The sections were placed in ABC 156

solution (1:200, Vector Laboratories, Burlingame, CA) for 15-20 min and subsequently 157

reacted in a solution containing filtered 0.05% diaminobenzidine tetrachloride (Sigma, 158

St. Louis, MO) with 0.0015% H2O2. The sections were counterstained with hematoxylin, 159

dehydrated through graded alcohols and coverslipped with Cytoseal-XYL (Richard Allan 160

Scientific, Kalamazoo, MI). GFAP IHC was carried out in a similar manner with the 161

following differences: there was no antigen retrieval step, the blocking step was carried 162

out using normal goat serum, the primary antibody was a rabbit anti GFAP polyclonal 163

(1:800, DAKO, Carpinteria, CA), and the secondary antibody was biotinylated goat anti-164

rabbit (1:800, Vector Laboratories, Burlingame, CA). Some sections were processed 165

identically with either the primary or secondary antibodies omitted, or with a mouse 166

immunoglobulin G isotype control (Abcam, Cambridge, MA) in place of the primary 167

antibody at the same concentration. Pairs of tissue sections not further apart than 56 168

µm were examined using an Olympus BX 40 light microscope. When additional 169

morphological detail was required adjacent tissue sections were processed for GFAP 170

IHC or stained with H&E. 171

RESULTS 172

Hamsters are obligate nose breathers, so a small amount of brain homogenate placed 173

just inferior to their nostrils was immediately inhaled into their nasal cavity. This 174

behavior precluded the need for placing a pipette inside the nose where it could injure 175

the nasal mucosa and expose brain homogenate directly to blood vessels and nerves. 176

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Mucosal damage could lead to direct hematogenous spread and/or neuroinvasion, 177

which would complicate the interpretation of the transepithelial transport route and 178

mechanism(s). 179

Following inhalation of PBS (n=7), India ink (n=6), and infected (n= 35) or uninfected 180

(n= 15) brain homogenate hamsters were allowed to survive for 1-180 minutes prior to 181

initiating transcardial perfusion. Control animals were untreated (n=6). Regularly-182

spaced tissue sections were inspected for the presence of ink, GFAP or PrPd. The 183

average number of immunohistochemically-processed tissue sections analyzed per 184

animal was 366 (range: 150-640). Sagittaly sectioned nasal cavities produced fewer 185

tissue sections, as the nasal cavity is approximately twice as long as it is wide. GFAP is 186

the principle intermediate filament of astrocytes and therefore a component of both 187

infected and uninfected brain homogenate, while PrPd is found only in prion-infected 188

brain homogenate (8, 14, 21). Roughly 2/3rds of the tissue sections were processed 189

using GFAP IHC, since tissue morphology is better preserved using this procedure than 190

PrPd IHC, which requires the use of formic acid for antigen retrieval. The mucosal 191

surface and underlying lamina propria of tissue sections through the rostral-caudal 192

extent of each nasal cavity were inspected using a light microscope and brain 193

homogenate was identified with either GFAP or PrPd IHC. Brain homogenate was 194

identified in the nasal cavity of each experimental animal, but was not seen in untreated 195

animals or those who inhaled PBS. While there was more brain homogenate observed 196

within the nasal cavities of animals that inhaled larger volumes of brain homogenate, 197

the volume of brain homogenate was not related to the occurrence of transport. 198

Therefore the data for animals that inhaled different volumes of brain homogenate with 199

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the same survival time was combined. Photomicrographs were taken wherever brain 200

homogenate was identified on the surface of, within, or deep to the nasal mucosa. 201

Omission of either the primary or secondary antibody or replacement of the primary 202

antibody with an isotype control resulted in a complete lack of staining (data not shown). 203

Hamster nasal cavity and light microscopic identification of M cells. The hamster 204

nasal cavity is lined by a variety of morphologically-distinct epithelia, including 205

respiratory, olfactory and FAE that lies adjacent to the NALT (Fig. 1A, 1B). M cells are 206

non-ciliated with a flattened apical surface and distinct intraepithelial pockets that 207

contain lymphoid cells (27, 44) and were identified in the FAE overlying the NALT in 208

H&E and PAS-stained tissue sections from untreated animals (Fig. 1C). M cells could 209

also be identified based on their ability to endocytose inhaled India ink (69). Ink particles 210

were identified within M cells in all 6 animals allowed to survive for from 5 to 30 minutes 211

after inhalation of 20-100µl of ink (Fig. 1D). Inspection of untreated or PBS-exposed 212

animals did not reveal any visible particulate matter on, or within, the M cells of the FAE 213

of the nasal cavity (Fig. 1C). The average number of tissue sections per animal that 214

were processed for H&E and PAS was 178 (range: 136-204). 215

Identification of inhaled prions. Prion-infected or uninfected brain homogenate was 216

identified within the nasal cavity of each animal following inhalation of brain homogenate 217

from either HY TME-infected, or mock-infected animals. Prion-infected or uninfected 218

brain homogenate was easily identified as aggregated or punctate brown particulate 219

matter within the nasal cavity airspace and lining the nasal mucosa using GFAP IHC 220

(Fig. 2). Prion-infected brain homogenate was identified using PrPd IHC as small brown 221

punctate particulate matter, while uninfected brain homogenate was unstained (Fig. 2). 222

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PrPd IHC was used on tissue sections adjacent to GFAP-positive sections to confirm the 223

presence of PrPd in brain homogenate in animals that inhaled infected brain 224

homogenate. Brain homogenate from HY TME-infected animals was immunopositive 225

for GFAP and PrPd, while brain homogenate from uninfected animals was 226

immunopositive for GFAP but not for PrPd (Fig. 2). 227

M cell transport of brain homogenate. Based on previous reports of M cell 228

involvement in the transport of prions, M cells in the FAE adjacent to the NALT were 229

examined using a light microscope. Infected or mock-infected brain homogenate was 230

identified using GFAP IHC and was seen adhering to the FAE of 44 of 50 animals 231

following extranasal inoculation (Table 1; Fig. 3). The criterion used for positive 232

identification of brain homogenate or prions within M cells of the FAE was brown 233

punctate GFAP or PrPd immunoreactivity surrounded by M cell cytoplasm in the same 234

focal plane. Applying this criterion brain homogenate, either infected or uninfected, was 235

identified within M cells in 31 of the 43 hamsters that survived for 5 minutes or longer 236

(Table 1; Fig. 3). The number of M cells noted to contain brain homogenate for each 237

animal at each time point was counted to determine the period of maximal M cell 238

transport of brain homogenate. The greatest number of M cells containing brain 239

homogenate was in those animals that survived 10-60 minutes prior to initiating 240

perfusion, with a noticeable drop in the number of M cells containing brain homogenate 241

in those animals that survived 180 minutes (Table 1). Analysis of tissue deep to the M 242

cell layer was restricted to tissue sections processed for PrPd IHC since GFAP labels 243

thin diameter nonmyelinating Schwann cells (28, 39) which are located deep to M cells, 244

in the NALT, and coursing through the lamina propria (Fig. 2D and 4E). 245

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Paracellular transport of brain homogenate occurs between olfactory, respiratory 246

and FAE cells within minutes of extranasal inoculation. Infected and uninfected 247

brain homogenate was detected penetrating or completely spanning the nasal mucosa 248

in 40 of the 50 animals extranasally inoculated in this experiment (Table 1; Fig. 4A and 249

4B). This was determined to be paracellular transport as brain homogenate was 250

located between intact epithelial cells, not within cells. This was confirmed by focusing 251

through the depth of the tissue section, and by examining the same cells on adjacent 252

tissue sections. Paracellular transport of brain homogenate was identified between 253

cells of the respiratory, olfactory and FAE of the nasal cavity (Fig. 4) and could be seen 254

extending into the underlying lamina propria in some cases (Fig. 4C and 4D). There 255

were multiple examples of transepithelial transport in all animals exhibiting transport; in 256

some cases multiple examples of paracellular transport were identified on single tissue 257

sections (Fig. 4B, 4D, and 4E). While paracellular transport was seen in animals from 258

all survival time points the number of animals that demonstrated paracellular transport 259

decreased in those animals that survived 180 minutes following inoculation by about 260

50% (Table 1). There were no examples of brain homogenate crossing the keratinized 261

stratified squamous epithelium that lines the vestibule of the proximal nasal cavity (33). 262

Prions quickly enter lymphatic vessels in the lamina propria but not the NALT. 263

Infected or uninfected brain homogenate was identified within the lumen of vessels in 264

the lamina propria of 47 of the 50 inoculated animals in this study (Table 1). These 265

vessels were determined to be predominantly lymphatic vessels and not blood vessels. 266

This distinction was based on the analysis of vessels from immersion fixed animals. 267

Immersion fixation does not rinse the blood cells from blood vessels, so blood vessels 268

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were easily distinguished by the presence of red blood cells within their lumen, and 269

lymphatic vessels by the lack of blood cells. The location of lymphatic vessels was 270

found to be relatively constant between animals, especially for medium and large 271

diameter lymphatic vessels. Infected or uninfected brain homogenate was identified 272

inside the lumen of lymphatic vessels in the lamina propria of the nasal cavity of animals 273

at all survival time points (Figure 5). PrPd was not identified in the intraepithelial 274

pockets of M cells, or adjacent to, or within the NALT in any animal in this study. 275

DISCUSSION 276

The in vivo transport of infected brain homogenate containing PrPd across the nasal 277

mucosa began within minutes of inhalation and transport could still be detected in some 278

animals as long as 180 minutes following inhalation. Paracellular transport was 279

detected in animals that were perfused within 1-2 minutes after inhalation, while M cell 280

transport was first detected in animals that were perfused beginning 5 minutes after 281

inhalation. Both types of transport were detected in all remaining survival groups, with a 282

noticeable decrease in the number of animals demonstrating transport in the animals 283

that survived 180 minutes after inhalation. The rapid and prolonged period of 284

transepithelial transport of PrPd in the nasal cavity following inhalation is consistent with 285

the results of a study performed in a sheep gut-loop model of prion infection where prion 286

proteins were detected in the lumen of villous and submucosal lymphatics 15 minutes to 287

210 minutes following direct inoculation of an isolated loop of distal ileum in sheep (38). 288

The time frame, the type of transport (transcellular or paracellular) and the 289

cells/epithelia involved in the in vivo transport of prions following inhalation have been 290

identified. Most notably the paracellular transport of relatively large amounts of brain 291

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homogenate between epithelial cells in the nasal cavity identified here has not been 292

described previously. While the sequence of events leading to the paracellular 293

transport of prions has not been determined, the mechanism of transepithelial transport 294

may involve passage of brain homogenate through existing gaps in the mucosa created 295

by the natural shedding of epithelial cells that has been reported in the intestine of mice 296

and humans (11, 41, 86). Utilizing existing gaps in the epithelia would be consistent 297

with the observed speed and physical dimensions of the transport of brain homogenate 298

reported here, where the passage of brain homogenate between cells often appeared to 299

be about one epithelial cell in width and occurred within minutes of inhalation (Figure 4). 300

The persistence and transepithelial transport of brain homogenate in the nasal cavity for 301

up to 3 hours after inhalation reported here is surprising as nasal mucociliary activity 302

results in measured mucus flow rates ranging from 0.9 to 11 mm/minute in different 303

areas of the rat nasal cavity and to average 1.3 mm/minute in mouse nasal cavity (29, 304

61). At these rates inhaled brain homogenate would be expected to be cleared from the 305

hamster nasal cavity in less than 60 minutes. This prolonged period of transport is 306

similar to what has been reported in the sheep gut-loop model where prions were 307

detected in submucosal lymphatics for up to 3.5 hours following inoculation (38). 308

A goal of these experiments was to test the hypothesis that prion entry following 309

inhalation was mediated via M cells of the FAE in the nasal cavity. Infected or 310

uninfected brain homogenate was seen within a relatively modest number of M cells 311

(average 1-5 per animal) in 31 of 50 animals in this study (Table 1). Given that the 312

efficiency of this route of inoculation has been determined to be 10-100 times greater 313

than per os inoculation (42), the number of animals demonstrating M cell transport, and 314

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the number of M cells per animal transporting brain homogenate in this study were 315

lower than might be expected. This is in marked contrast to the robust paracellular 316

transport of PrPd in brain homogenate that was noted between cells of the olfactory, 317

respiratory and follicle associated epithelia that line the nasal cavity that has been 318

reported here (Table 1; Figure 4). The results of this study are consistent with a 319

relatively minor role for M cell transport in the transepithelial transport of prions following 320

inhalation, with a greater contribution coming from the paracellular transport of prions. 321

The limited number of examples of M cell involvement in PrPd transport following 322

inhalation was not due to a lack of exposure to brain homogenate, as almost all of the 323

animals examined in this study had multiple examples of brain homogenate resting on 324

FAE (Table 1). There are certain limitations to the interpretations that can be made 325

about a dynamic cellular process that occurs across a surface and over a period of time 326

when one is examining sections taken at specific time points. First, the transport of 327

brain homogenate across the M cells could happen very quickly, so that the chance of 328

identifying brain homogenate within the cell at any one time point is reduced, and the 329

numbers reported here would be artificially low. However, this scenario would not be 330

expected based on a report that the uptake and transfer of 600-750 nm latex particles 331

across M cells occurred over 90 minutes after contact with the FAE in a rabbit intestinal 332

loop model (67). Second, the transport of brain homogenate through M cells could 333

involve the phagocytosis and movement of very small particulate matter, some of which 334

could be beyond the resolution of a light microscope (~0.2 µm) and therefore not 335

detectable (65). The reported diameter of transported microparticles within M cells in 336

other studies is 0.02 to10 µm (22); therefore we may have been unable to identify the 337

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smaller range of particulate brain homogenate that was contained within the M cells. 338

Regardless of resolution issues, definite identification of transcellular transport of prions 339

within M cells will require ultrastructural analysis. While the relative contribution to the 340

pathogenesis and the development of disease from the two routes identified in this 341

report remains to be determined, the apparently lesser role of M cell involvement in 342

prion transepithelial transport reported here is consistent with the sheep gut-loop model 343

of prion infection and in sheep orally inoculated with prions (3, 38, 46), where the FAE 344

and M cells do not appear to be involved in the transport of prion proteins across the gut 345

wall. 346

The results of this study indicate that PrPd can gain access to lymphatic vessels quickly 347

and that transport across the nasal mucosa and into the vessels persists for up to 3 348

hours after inhalation. It is not likely that the persistence of brain homogenate in the 349

lymphatics is simply due to a slow rate of lymph flow in the nasal cavity given that flow 350

rates have been reported to average 3-9 µm/second in different species (5, 23, 82) and 351

that dye placed in the nose reaches cervical lymph nodes in anywhere from 14-51 352

minutes, depending on the species (88). The presence of brain homogenate within 353

lymphatics is consistent with their role of draining fluid and macromolecules from the 354

extracellular matrix of the lamina propria in the nasal cavity. Material located in the 355

lamina propria is propelled towards lymphatic capillaries by interstitial fluid pressure and 356

enters the capillaries, which are made permeable by lack of a basement membrane and 357

the presence of anchoring filaments (for review see 81). Thus it appears that the 358

lymphatics, and subsequently the circulatory system, can serve as a route for the 359

systemic dispersion of prions that have been inhaled into the nasal cavity. Consistent 360

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with this pattern of transepithelial transport following inhalation was the notable lack of 361

brain homogenate or PrPd adjacent to, or within, the NALT in any of the animals 362

examined in this study. While it is possible that direct neuroinvasion of prions may 363

occur via somatic and autonomic nerves located in the lamina propria of the nasal 364

cavity, this has not been reported. 365

366

Acknowledgments: The authors would like to sincerely thank Dr. Jacob Ayers, Albert 367

Lorenzo, Melissa Clouse, Shawn Feilmann and Theresa Lonmeth for their excellent 368

technical assistance with this work. 369

This work was supported by the National Institutes for Neurological Disorders and 370

Stroke (RO1 NS061994 and RO1 NS061994-03S1), Health Future Foundation 371

Discretionary Award #200600-713131d and by Grant G20RR024001 from the National 372

Center for Research Resources. The content is solely the responsibility of the authors 373

and does not necessarily represent the official views of the National Center for 374

Research Resources or the National Institutes of Health. 375

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Table 1 Brain homogenate distribution in nasal cavity following inhalation 376

377

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a Survival time till initiation of perfusion. Time till death included additional 10-12 minutes 378 b Total volume of mock-infected or HY-TME infected brain homogenate applied inferior to nostrils 379

FAE, follicle associated epithelium; bh, brain homogenate 380

Survival timea

Inoculumb

Location of brain homogenate

On FAE

Inside M cells

(# M cells)

Crossing between

epithelial cells

Deep to epithelium

1-2 min

(n=7)

Uninfected bh

(50-100 µl)

Infected bh

(50-200 µl)

2/2 0/2 2/2 2/2

5/5 0/5 4/5 5/5

5 min

(n=16)

Uninfected bh

(20-100 µl)

Infected bh

(20-200 µl)

6/6 2/6 (5) 5/6 6/6

10/10 8/10 (15) 9/10 9/10

10-20 min

(n=13)

Uninfected bh

(100 µl)

Infected bh

(100 µl)

2/3 2/3 (7) 1/3 2/3

10/10 10/10 (44) 10/10 10/10

60 min

(n=7)

Uninfected bh

(100 µl)

Infected bh

(50-100 µl)

1/2 1/2 (2) 1/2 2/2

5/5 5/5 (29) 5/5 5/5

180 min

(n=7)

Uninfected bh

(100 µl)

Infected bh

(100 µl)

1/2 1/2 (3) 1/2 2/2

2/5 2/5 (4) 2/5 4/5

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Figure Legends 381

Figure 1. Hamster nasal cavity epithelial types and the identification of M cells. (A) Low 382

power view of a PAS-stained portion of hamster nasal cavity demonstrating morphology 383

of olfactory and respiratory epithelia (Box 1, enlarged in panel B) and FAE with M cells 384

overlying the NALT (Box 2, enlarged in panel C; FAE indicated by asterisks). (B) RE 385

can be distinguished from OE based on the thickness of the epithelial layer and the 386

length of cilia. (C) M cells can be identified by their distinct morphology, including 387

intraepithelial pockets (outlined) and lack of cilia which distinguish them from 388

neighboring goblet and respiratory epithelial cells. (D) An H&E stained tissue section 389

from a hamster extranasally exposed to 20 µl India ink that survived for 30 minutes prior 390

to perfusion; note the presence of ink particles within a subset of M cells of the FAE. 391

Asterisks indicate air space. OE, olfactory epithelium; RE, respiratory epithelium; FAE, 392

follicle associated epithelium; NALT, nasal associated lymphoid tissue. Bars: A, 200 µm; 393

B,C,D 20 µm. 394

Figure 2. Uninfected and infected brain homogenate in the hamster nasal cavity after 395

inhalation identified with GFAP and PrPSc IHC. (A) A low power view of a portion of a 396

nasal turbinate in the nasal cavity of a hamster extranasally inoculated with mock-397

infected brain homogenate that survived for 5 minutes prior to perfusion, processed 398

using GFAP IHC. The area outlined in the box is enlarged in panel B. (B) Higher power 399

view of the uninfected brain homogenate identified using GFAP IHC and an adjacent 400

tissue section (C) processed using PrPSc IHC. Note the lack of punctate 401

immunoreactivity in C, indicating the brain homogenate contains GFAP, but lacks PrPSc. 402

(D) A low power view of a portion of a nasal turbinate in the nasal cavity of a hamster 403

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extranasally inoculated with HY TME-infected brain homogenate that survived for 5 404

minutes prior to perfusion, processed using GFAP IHC. The area outlined in the box is 405

enlarged in panel E. Note the GFAP labeled bundle of nonmyelinating Schwann cells in 406

the lamina propria indicated by an arrow. (E) GFAP labeled brain homogenate from HY 407

TME-infected animal and an adjacent tissue section (F) processed using PrPSc IHC. 408

Note the distinct punctate labeled brain homogenate that is characteristic of PrPSc, 409

indicating this section contains GFAP and PrPSc. Contrast the staining in panels C and 410

F to note the distinction between PrPSc-negative and PrPSc-positive brain homogenate. 411

Asterisks indicate air space. Bars, A,D 100 µm; B,C,E,F 20 µm. 412

Figure 3. Transcellular transport of prions by M cells in the nasal cavity following 413

inhalation. Inhaled mock-infected brain homogenate (A) and prion-infected brain 414

homogenate (B) identified with GFAP IHC located on the surface of the FAE of 415

hamsters. Note the lack of brain homogenate within the M cells of the FAE in either of 416

these sections; (A) taken from an animal that survived 5 minutes till perfusion and (B) 417

from an animal that survived 1 minute till perfusion. Infected brain homogenate 418

identified with GFAP IHC (C) and PrPSc IHC (D) within M cells (indicated by arrows) of 419

animals that survived 10 minutes (C) and 5 minutes (D) till perfusion. Asterisks indicate 420

air space. Bars, A,B 50 µm; C,D 20 µm. 421

Figure 4. Paracellular transport of brain homogenate in the nasal cavity following 422

inhalation identified with GFAP IHC. Inhaled mock (A) and infected (B) brain 423

homogenate crossing the nasal mucosa between cells of the olfactory epithelium 5 424

minutes (A) and 1 minute (B) after inhalation. Brain homogenate could be seen 425

between epithelial cells spanning the complete width of the epithelium and entering the 426

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underlying lamina propria on some tissue sections (C). Note that multiple examples of 427

paracellular transport of infected (B, C, D) and uninfected (E) brain homogenate were 428

observed on some tissue sections. Transport of brain homogenate was noted across 429

olfactory (C), respiratory (D) and FAE (E). Nonmyelinating Schwann cells deep to the 430

FAE are labeled with GFAP IHC and indicated by arrows. Note the width of the 431

paracellular transport appears to be approximately the same width as that of a single 432

epithelial cell in all the panels. NS, nasal septum. Bars, A,B 100 µm; C,D,E 25 µm. 433

Figure 5. Brain homogenate was located within the lumen of lymphatic vessels of the 434

lamina propria of the nasal cavity within minutes of inhalation, and was still visible for 435

hours after inhalation. Infected (A,D) and uninfected (B,C) brain homogenate was 436

located within lymphatics 5 min (A,B) and 60 min (C,D) after inhalation. Bars, A,B,C,D 437

50 µm. 438

439

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