Virus-induced Lysosomal Enzyme Dissolution of Nasal Turbinate Cartilage Warren W. Schultz, MS, ScD, and Frederick B. Bang, MD
The mechanism of laryngotracheitis virus-induced dissolution of chick nasal turbinate cartilage was studied by lysosomal enzyme histochemistry. Five-day-old chicks were infected by intranasal instillation, and changes in lysosomal enzyme distribution were followed at daily intervals through the tissue regeneration stage, Day 28. In the mucosa the lysosomes were activated beginning on Day 1, and glycerol acid phosphatase and a diffuse form of d-glucuronidase were released concomitant with tissue cell destruction. In the chondrocytes (where glycerol acid phosphatase was absent), beginning on Day 2, particulate (Iysosomal) B-glucuronidase decreased as diffuse ,B-glucuronidase increased and extended out into the matrix. The cartilage lost its metachromatic staining properties and became soft and pliable. The cartilage cells themselves were not affected which indicated preferential enzyme release. Regeneration of the mucosa started on Day 6 and gradual reappearance of metachromatic staining of the cartilage began on Day 8 with considerable recovery of original turbinate structure by Day 12. A lysosomal membrane labilizer, vitamin A, exacerbated the cartilage pathology, whereas a stabilizer, cortisone, retarded it. (Am J Pathol 87:667-684, 1977)
LARYNGOTRACHEITIS VIRUS (LTV) causes a severe, acute desquamation of the nasal turbinate mucosa in young chickens.' This is accompanied by softening of the underlying supportive cartilage which loses its basophilic and metachromatic staining properties. Other studies have shown that chick limb-bone cartilage undergoes similar changes when treated with such agents as papain 2 or vitamin A.3 Further investigations " led to the finding that these agents caused the activation of lvNsosomes, resulting in release of hydrolases which were responsible for the breakdown of the treated cartilage. We examined the possibility that LTV-induced breakdown of chick nasal turbinate cartilage is also the result of lysosomal activation. From the Department of Mlicrobiology, Nasal Medical Research Institute, Bethesda, Marvland, and the Department of Pathobiology, The Johns Hopkins University School of Hygiene and Public Health. Baltimore, Marvland. Supported in part by Naval Medical Research and Development Command, Navy Department, Research Task No. 10095-DN.12.5403 and MR000.01.01.1169 and bv Grant 3R01 Al-12458 from the National Institutes of Health to Johns Hopkins University. The sork w-as submitted by Dr. Schultz in partial fulfilment of the requirements for.the ScD degree at the Johns Hopkins University. The opinions and statements contained herein are the private ones of the authors and are not to be construed as official or relecting the siew-s of the Nays Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratorv Animal Resources, National Research Council. DHE\' Publication NIH 74-23. Accepted for publication February 18, 19747. Address reprint requests to Dr. Warren W. Schultz, Department of Microbiology, Nasal Medical Research Institute. Bethesda, MD 20014. 667
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Materials and Methods White Leghorn chicks were obtained from a local hatchery and used at 5 davs of age. There has been no laryngotracheitis in any of the farm flocks which supply eggs to the hatcherv, and the chicks have always been fully susceptible to the disease. Larvngotracheitis virus was originally obtained from Dr. E. L. Jungheer, University of Connecticut, as a chorioallantoic membrane (CAM) preparation. A stock was maintained by passage on the CAM of 11-day fertilized hen's eggs from the Hopkins White Leghorn AF-flock (free of antibodies to Rous sarcoma and avian leucosis viruses). The supernatant fluids from 10% suspensions of infected or noninfected fresh CAM in phosphate-buffered saline (PBS) were used for inoculating chicks. The chicks were inoculated with two drops of the fluid per nostril delivered through a 23-gauge needle onto the nasal opening, allowing each drop to be inhaled. Several turbinates from inoculated chicks were harvested on Day 3 after inoculation, homogenized in PBS, and inoculated onto CAM to confirm the presence of LTV infection. Infection was demonstrated in everv case, and only 4 birds in more than 200 showed a significant variation in the time of pathologic events. Turbinates were harvested by bisecting the head of the chick in the plane of and slightly to either side of the nasal septum. The exposed turbinate was then excised using a small surgical scalpel. The opposing turbinate was excised with a fresh scalpel blade. Immediatelv after a turbinate was removed, it was either quick-frozen in mounting medium (Tissue-Tek. O.C.T. compound, Ames Co., Elkhart, Ind.), using a dry ice-ethanol bath and kept at -20 C until sectioning, or was fixed first. Fixative for acid phosphatase histochemistrv was 2.5% glutaraldehyde or 4% methanol-free formalin in 0.1 M cacodylate buffer, pH 7.3, with or without 7.5% sucrose. For f-glucuronidase only the formalin fixative was used. The time and temperature of fixation are given in the results section. After fixation the tissues were washed for 2 hours in ice-cold 0.1 M cacodylate buffer, pH 7.3, containing 7.5% sucrose; drained on filter paper; (quick-frozen in mounting medium; and kept at -20 C until sectioned. Tissue blocks were usually cut within 4 hours of their preparation.
Tissue sections were cut from the middle third of the turbinate at 8-,u thickness at -22 C to -30 C on an International cryostat Model CTI (International Equipment Co., Needham, Mass.) and picked up on gelatin-treated microscope slides (0.25% gelatin in 0.5% NaKCrO4). The sections were kept cold until staining for enzyme. The time between sectioning and staining rarely exceeded 1 hour. A modified Gomori technique 7 was employed for localization of glycerol acid phosphatase. Tissue sections were usually incubated for 30 minutes at 37 C in substrate composed of 0.008 M sodium d-glycerol phosphate (Fisher Scientific Co., Fair Lawn, N.J.) in 0.05 M acetate buffer, pH 5.2, with 0.33 M sucrose and 0.014 M lead nitrate, prepared daily from a stock solution. Controls for phosphatase specificity and nonspecific hydrolysis of lead included incubating tissues in medium with the sodium :-glycerol phosphate deleted or complete substrate medium to which 0.01 M sodium fluoride had been added. The procedure used for the localization of naphthol acid phosphatase was based on the work of Burstone 8 and Kaplow.9 Tissue sections were usually incubated for 30 minutes at 37 C in substrate composed of 5 mg of naphthol AS-MX phosphate (Sigma Chemical Co., St. Louis, Mo.) dissolved in 50 ml of 0.1 M acetate buffer, pH 5.2. Just before use, 25 mg of fast blue RR salt (Sigma) was added, mixed for 60 seconds and then filtered. Controls for enzyme specificity included specimens heated at 90 C for 60 seconds or specimens incul)ated in the presence of 0.01 M sodium fluoride. The simultaneous coupling technique 10 was used for the localization of f-glucuronidase. The substrate was the sodium salt of naphthol AS-BI glucuronide (Sigma) and the coupling agent (visualizing agent) was hexazotized pararosanilin prepared as a stock solution from pararosanilin hydrochloride " (Sigma). The working solution was prepared fresh each day. Controls included incubating tissue in medium without the glucuronide or in complete medium but with a specific inhibitor, glucosaccharo-1,4-lactone at a concen-
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tration of 0.025 mM. Coverslips were affixed to all stained preparations using Permount (Fischer), which had a much slower reaction product diffusion rate than other mounting agents. When employing the glycerol acid phosphatase and naphthol acid phosphatase techniques, the deposit of a brown or blue precipitate, respectively, in the form of a 0.5 to 1.0 p spherical body within the cell cytoplasm was construed as being bound lysosomal acid phosphatase. When employing the 3-glucuronidase method, a red deposit was construed as being bound Ivsosomal 0-glucuronidase. If the red stain appeared as an extremely fine granular or diffuse reaction product, it was considered as lysosome-released or free endoplasmic reticulum (-glucuronidase, depending upon the cell type and time of infection. To determine the percent of cartilage cells showing particulate enzyme staining, the entire cross section of the turbinate scroll was examined; a minimum of 300 cells per section were counted, and the percentage expressed to the nearest 5%. A minimum of three sections per slide were examined. Mucosal columnar cells showing particulate or diffuse enzyme stain could not be accurately quantified. Therefore, the epithelium along the entire length of the scroll was examined and roughly assigned percentage values in terms of areas, rather than cells, showing enzyme staining. Methyl green was routinely used as an enzyme counterstain and as an indicator of the condition of the cartilage matrix. A 2.0% (w/v) aqueous solution of crude methyl green (Allied Chemical and Dye Corp., NY) was repeatedly extracted with chloroform until all undesirable blue dyes were removed. A working solution was then prepared by mixing equal volumes of the dye and 0.2 M acetate buffer, pH 4.2. Frozen sections were also routinely stained with hematoxylin and eosin, toluidine blue, and alcian blue. The hematoxylin and eosin stain was a modification of Mayer's hematoxvlin.u For toluidine blue and alcian blue staining, 0.25% (w/v) and 0.1% (w/v) aqueous solutions were used, respectively. In experiments with vitamin A (all-trans-retinoic acid, Eastman Kodak Co., Rochester, N.Y.) and cortisone (cortisone acetate suspension, N.F., 25 mg/ml; Intra Products, Inc., Dayton, Ohio), drug treatments were initiated when the chicks were 4 days old. For each drug, 16 chicks were divided into four groups: Group 1 was the untreated control, Group 2 received LTV, Group 3 received vitamin A or cortisone, and Group 4 received vitamin A or cortisone and LTV. The vitamin (50 mg/ml in Wesson corn oil) and cortisone were given orally at a dosage of 10 mg and 5 mg per 50 g body weight, respectively, daily for 3 days. After a drug-free day the chicks received 5 mg vitamin A or 2.5 mg cortisone acetate daily until termination of the experiment. Laryngotracheitis virus was instilled on Day 5 immediately after the second drug dose. For the vitamin A experiment the turbinates were fixed ovemight at 4 C to elicit maximum particulate staining in the chondrocytes and permit observation of the redistribution of #-glucuronidase from the particulate to diffuse form. For the cortisone experiment, fixation was at 26 C for 2 hours and then overnight at 4 C to minimize particulate and diffuse staining and permit observation of increased staining. This fixation protocol was derived from experiments shown in the results section. Sequential enzvme and pathologic changes were recorded on color film to capture the multicolor enzvme and background stains. The photomicrographs used here were reproduced from color transparencies by a multistep procedure which resulted in some loss of resolution.
Resufts Locaizai of Lyso-l Enmes in the Non_fected MaIay Canch
The structure, function, and general staining properties of the maxillary concha (middle turbinate, Figure IA) have been well described by Bang
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and Bang.1 Briefly, it is composed of an inner noncalcifying cartilage center with an overlying mucociliated epithelium (i.e., ciliated columnar cells, epithelial goblet cells, and acinar gland cells) and a well-vascularized submucosa (Figure 1B). Acid Phosphatases
Nonfixed turbinates contained no particulate or diffuse glycerol acid phosphatase staining in the columnar and mucous cells or the perichondrocytes and chondrocytes (Table 1). Histochemical studies 13,14 have shown that activation of the lysosomal membrane by the action of a fixative (e.g., formalin or glutaraldehyde) is necessary for the Gomori reaction to occur. When turbinates were fixed, particulate staining was observed in the cytoplasm of the columnar cells along the entire length of both the luminal and outer mucosa of the turbinate scroll (Figure 2A). Cytoplasmic particulate glycerol acid phosphatase was also observed in the mucous cells of fixed tissues when incubated in substrate medium containing 0.01 M Mn2+. There was occasional particulate staining in the perichondrial cells, but no staining in the chondrocytes, even when Mn2+ was added (Table 1). Granulocytes within vessels were observed to contain particulate glycerol acid phosphatase. Particulate staining in this tissue was inhibited by 0.01 M NaF. The optimum conditions were shown to be incubation at 37 C for 30 minutes at pH 5.2. Since the enzyme could not be demonstrated in the chondrocytes with glycerol phosphate as a substrate, sodium salts of naphthol AS-MX phosTable 1-Lysosomal Enzymes in the Turbinates of Noninfected Chicks Enzyme staint
BGase BGase GAPase NAPase Fixation* (Particulate) Particulate Diffuse (Particulate) No + i + + Yes + + Mucous No + + Yes +* + + ± Perichondrocytes No + + ± ± Yes + + No Chondrocytes + + + Yes + + + *Glycerol acid phosphatase (GAPase), 1.5 hours at 4 C; naphthol acid phosphatase (NAPase), 1.0 hours at 24 C; f-glucuronidase (BGase), 0.5 hours at 24 C. t After 0.5 hours at 37 C; - = no staining in at least three sections; ± = occasional cytoplasmic particulate staining and number of particles per cell < 10; + = 1 to 100% staining. Diffuse GAPase and NAPese .stairrng- was-not seen. f Only after incubation in substrate containing 0.01 M Mn2 .
Cell type Columnar
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phate and naphthol AS-TR phosphate were tested. With both substrates, particulate staining was observed in the cytoplasm of columnar, mucous, perichondrial and chondral cells of nonfixed and fixed tissues (Table 1). The staining activity of preparations incubated in medium containing 0.01 M NaF or heat-treated (90 C for 60 seconds) was 10% or less of that observed in the test groups. $-Glucuronidase
3-Glucuronidase stain was selected because this enzyme is involved in cartilage catabolism. In nonfixed tissues, particulate ,B-glucuronidase staining was observed in less than 1 % of the cartilage cells along the scroll after 30 to 60 minutes of incubation but was seen in all cells at 90 minutes (Tables 1 and 2). All of the cartilage cells had a finely granular or diffuse, light red staining product throughout the cytoplasm. The latter type of reaction product was also observed in all of the columnar and acinar cells of the mucosa, the intensity slightly increasing when incubation time was extended from 60 to 90 minutes. However, only rare particulate staining was observed in the columnar and perichondrial cells and no particulate staining was observed in mucous or blood cells. In fixed tissues, all of the cartilage cells showed particulate staining in the cytoplasm at the three substrate incubation times (Tables 1 and 2, Figure 3A), but greater numbers of lysosomes per cell were visualized at 60 and 90 minutes than at 30 minutes. Most of the cartilage cells also had the finely granular or diffuse type of light red staining product, but unlike the nonfixed tissues the intensity did not increase with an increased substrate incubation time. In the mucosal cells, there was only rare particulate staining, but all cells displayed diffuse staining which did not increase in intensity with longer incubation. The granulocytes within blood vessels contained discrete, red staining granules. No particulate or diffuse stain was seen in any of the control preparations. Further characterization of the diffuse and particulate types of ,glucuronidase staining was provided by varying the time of formalin fixation as shown in Table 2. In general, as the fixation time, temperature, or both were increased, the amount of enzyme stain decreased. In the mucosa, fixation at 4 C caused little decrease as the time was increased. In the cartilage cells the particulate staining persisted but the diffuse staining was lost by 20 hours of fixation. At 24 C the lability of both particulate and diffuse enzyme to fixation became much more apparent. Fixation of 0.5 hour resulted in some diffuse staining, but the intensity decreased at 1.0 and 1.5 hours, and only faint diffuse stain remained thereafter. The particulate stain in the cartilage cells was not affected until after 1.5 hours
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Table 2-f3-Glucuronidase Staining: Effect of Fixation Time and Temperature and Substrate Incubation Time Cartilage Mucosa Substrate Fixation incubation* time 24 C 4C 24 C 4C time (hrs) (hrs) 100/100 100/100 100/5 100/5 0.5 0.5 100/100 100/100 100/20 100/0 1.0 100/100 100/100 100/10 100/20 1.5 1.0
0.5 1.0 1.5
100/5 100/0 100/20
100/5 100/20 90/10
100/100 100/100 100/100
100/100 100/100 100/100
1.5
0.5 1.0 1.5
100/5 100/0 100/0
90/0 100/0 90/10
100/100 100/100 100/100
40/80 10/100 100/100
2.0
0.5 1.0 1.5
100/0 100/0 100/10
10/0 50/5 50/5
100/100 100/100 100/100
0/40 0/90 10/100
4.0
0.5 1.0 1.5
100/0 100/10 100/20
0/0 0/0 0/0
100/100 100/100 100/100
0/5 0/20 0/20
20.0
0.5 1.0 1.5
90/0 90/5 90/5
0/0 0/0 0/0
0/90 0/100 0/100
0/0 0/0 0/0
*At 37 C. t Percentage cells showing diffuse staining/percentage cells showing particulate staining.
of fixation. At 37 C there was a complete absence of f,-glucuronidase staining in the 2.0 and 4.0 hour specimens (not shown). Control specimens were uniformly negative. From the above data, the fixation time and temperature of 1 hour at 24 C and a substrate incubation time of 30 minutes were selected for further experiments; under these conditions, the optimum pH was 5.2. Pathology of Laryngotracheitis Virus Infection
Chicks which received the LTV inoculum showed an increase in gross viscosity of the mucus in and around the turbinate on Day 2. On Day 3 the mucus was even more viscous and the turbinate appeared blanched. On Day 4, yellow caseous exudate filled the entire nasal cavity. After Day 5 the nasal septum and turbinates were very soft and pliable. A reversal of these conditions began by Day 8, and by Day 12 turbinate rigidity was similar to that seen in the controls. Significant changes were not seen in chicks inoculated with material derived from normal CAM. Microscopically, the first viral cytopathic effects were readily observed
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by 24 hours. Foci of infected cells with Type A intranuclear inclusions were found in the mucosa, particularly the luminal mucosa. The origin of these cells could not be determined. By Day 2 there was increased syncytial formation with some sloughing, and finally, exhaustion of the mucus from the acini. On Day 3 to 4, most of the luminal and outer mucosa showed marked destruction and infiltration by polymorphonuclear leukocytes. There was much vasodilatation of the underlying vessels with engorgement of surrounding tissue. Lymphocyte and plasma cell infiltration was seen on Day 3 to 8. Mucosal cell sloughing was maximal on Day 4 to 5, and the normal basophilia of the cartilage matrix was greatly decreased. Mucosal regeneration commenced about Day 6 with the appearance of a thin layer of epithelial cells. As regeneration continued, the cartilage regained its basophilia. By 3 to 4 weeks most of the mucosa had been repaired. There observations were consistently found, except that in the regeneration stage (Day 8 to 28) individual variation was slightly more pronounced. Methyl green was used as a counterstain since the staining process did not destroy the enzyme stain end-product and provided a striking cartilage background in normal turbinate tissue. It was compared, therefore, to metachromatic staining in infected turbinates as shown in Table 3. The loss in methyl green staining intensity paralleled the loss of toluidine blue metachromasia, indicating the loss of cartilage matrix substance. Since the loss of methyl green staining was observed 1 to 2 days earlier than the loss of toluidine blue, methyl green was considered the more sensitive indicator of cartilage pathology. Table 3-Turbinate Cartilage Staining in Infected Chicks* Twne atter inoculaion (days) 1 2 3 4
Methyl green Nonfixed
Fixed
4+ 4+ 2+ 2+ 0 0 0 1+ 2+ 3+ 4+ 4+
4+ 4+ 4+ 3+ 2+ 0 0 1+ 3+ 4+ 4+ 4+
5 6 7 8 9 10 11 12t * Intensity of staining indicated by 0 to 4+. Each control which stained uniformly 4+. t Further changes not observed through 28 days.
time
Toluidine blue Fixed 4+ 4+ 4+ 2+ 1+ 1+ 1+ 3+ 4+ 4+ 4+ 4+ interval included a noninfected
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Localization of Lysosomal Enzymes in Infected Turbinates Acid Phosphatase
At 2 days postinoculation, there was a large increase in the amount of particulate staining in the columnar cells of nonfixed tissue which was particularly noticeable in the luminal mucosa (Table 4). Large dense focal areas of diffuse glycerol acid phosphatase also appeared in the mucosal cells. Syncytia and individual cells which had sloughed off from both the luminal and outer mucosa did not show any staining. After Day 3, both the particulate and methyl green stain began to decrease and were no longer observed by Day 6. During this period, large numbers of heterophils with glycerol acid phosphatase-positive granules were seen in the mucosa and in material collected in the lumen and outside the turbinate, and macrophage-like cells with large pools of enzyme were observed in the mucosa. On Day 6, particulate staining was again observed in squamous-like cells forming the outer edges of the regenerating mucosa, reaching a maximum on Day 10 and decreasing to control levels as the regenerative cells differentiated into mucociliated epithelium. At 10 to 12 days the stain also appeared in the differentiating cells forming new mucous acini. Paralleling these changes, the cartilage showed a gradual increased uptake of methyl green that reached an intensity comparable to that of the noninfected control at Day 12. In fixed turbinates, at Day 1, particulate staining was similar in control and infected columnar cells. However, the amount of staining progresTable 4-Glycerol Acid Phosphatase Changes in Infected Turbinates Fixed Nonfixed Cartilage Cartilage Time Columnar after Columnar (methyl green (methyl green cells uptake) cells uptake) inoculation* NDf NDt 5 hrs Pt 4+t P PPPP 4+ 21 hrs 4+ 4+ 42-48 hrs PPP,DD PPP,DD 4+ 68-74 hrs 4+ P,D P,D 2+ PO 4-6 days P_0 2+O 3+ 0 01 + PP -PPP PP PPP 0 6-8 days 1 +- 4+ PPPP PPPP 8-10 days 1 +-3+ PPPP PPPP 4+ 10-12 days 2+-4+ PPPP PPPP 0 4+ 12-28 days 4+ Based on three experiments, except values at 5 hours (one experiment) and 21 hours (two experiments). t P = particulate staining, D = diffuse staining; P or D = 1 to 25%, PP or DD = 26 to 50%, etc. Changes within the time period indicated by ; ND = not done. t Intensity of staining indicated by 0 to 4+. *
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sivelv decreased in the infected tissues, and by Day 6 it was absent. Diffuse staining was seen on Days 1 to 3 (Figure 2B). The initial decrease in particulate enzyme, in both fixed and nonfixed tissue, coincided with the increase in diffuse staining and was followed by the loss of methyl green uptake by the cartilage. During the regenerative stage when particulate glycerol acid phosphatase staining was again present, methyl green uptake reappeared. It should be noted that near the attachment end of the turbinate there was often little or no pathology or change in glycerol acid phosphatase staining in the mucosa and no loss of methyl green staining in the cartilage. Naphthol acid phosphatase staining decreased greatly in the mucosa and slightly in the cartilage during the first 5 days of infection. The numbers of granules per cell were also observed to decrease, but the size of the granules was two to three times larger. By Day 5 or 6 the staining in the cartilage had retumed to normal, and by Day 8 the size and number of cartilage cell granules were similar to those of the noninfected controls. On Days 6, 8, and 10, there was greatly increased particulate staining in the undifferentiated replacement cells of the mucosa. By Day 13, in areas of the mucosa where there was complete regeneration, the naphthol acid phosphatase staining was similar to that of the noninfected controls. Comparable results were observed in fixed tissue. $-GIucuronidase
In nonfixed turbinates the mucosal cells showed a loss of diffuse ,glucuronidase staining beginning on Day 2 and progressing and persisting until Day 7 (Table 5). In the chondrocytes there was an increase in particulate stain during the first 2 days, followed by a decrease with concurrent increase in diffuse stain. In the cartilage matrix, as the dglucuronidase stain increased, the methyl green staining decreased and was absent by Day 6. The cartilage cells themselves, however, showed no apparent pathology. In the initial regeneration period, the earlv replacement cells of the surface layer of the mucosa showed intense diffuse ,glucuronidase staining. With advanced regeneration, the staining of the mucosa, the particulate staining in the chondrocytes, and the uptake of methvl green by the cartilage matrix returned to levels like those observed in the controls. The same pattern of changes in enzyme distribution was observed in the fixed tissue (Table 5). At Day 1, formalin treatment of turbinates for 1.5 hours reduced diffuse mucosal ,-glucuronidase staining to 50% of that of comparable nonfixed tissue, and 2-hour treatment eliminated staining completely. At Day 2 to 3, there was an increase of about 100% in total
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Table 5-Changes in ,B-Glucuronidase Staining in Infected Turbinates* Mucosa after fixation Cartilage after fixation Time after 0 hrs inoculation(days) 1.5 hrs 2.0 hrs 1 hrs 1.5 hrs 2.0 hrs 1 DDDD PP DD 0
The mechanism of laryngotracheitis virus-induced dissolution of chick nasal turbinate cartilage was studied by lysosomal enzyme histochemistry. Five-day-old chicks were infected by intranasal instillation, and changes in lysosomal enzyme distribution were followed at daily intervals through the tissue regeneration stage, Day 28. In the mucosa the lysosomes were activated beginning on Day 1, and glycerol acid phosphatase and a diffuse form of d-glucuronidase were released concomitant with tissue cell destruction. In the chondrocytes (where glycerol acid phosphatase was absent), beginning on Day 2, particulate (Iysosomal) B-glucuronidase decreased as diffuse ,B-glucuronidase increased and extended out into the matrix. The cartilage lost its metachromatic staining properties and became soft and pliable. The cartilage cells themselves were not affected which indicated preferential enzyme release. Regeneration of the mucosa started on Day 6 and gradual reappearance of metachromatic staining of the cartilage began on Day 8 with considerable recovery of original turbinate structure by Day 12. A lysosomal membrane labilizer, vitamin A, exacerbated the cartilage pathology, whereas a stabilizer, cortisone, retarded it. (Am J Pathol 87:667-684, 1977)
LARYNGOTRACHEITIS VIRUS (LTV) causes a severe, acute desquamation of the nasal turbinate mucosa in young chickens.' This is accompanied by softening of the underlying supportive cartilage which loses its basophilic and metachromatic staining properties. Other studies have shown that chick limb-bone cartilage undergoes similar changes when treated with such agents as papain 2 or vitamin A.3 Further investigations " led to the finding that these agents caused the activation of lvNsosomes, resulting in release of hydrolases which were responsible for the breakdown of the treated cartilage. We examined the possibility that LTV-induced breakdown of chick nasal turbinate cartilage is also the result of lysosomal activation. From the Department of Mlicrobiology, Nasal Medical Research Institute, Bethesda, Marvland, and the Department of Pathobiology, The Johns Hopkins University School of Hygiene and Public Health. Baltimore, Marvland. Supported in part by Naval Medical Research and Development Command, Navy Department, Research Task No. 10095-DN.12.5403 and MR000.01.01.1169 and bv Grant 3R01 Al-12458 from the National Institutes of Health to Johns Hopkins University. The sork w-as submitted by Dr. Schultz in partial fulfilment of the requirements for.the ScD degree at the Johns Hopkins University. The opinions and statements contained herein are the private ones of the authors and are not to be construed as official or relecting the siew-s of the Nays Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratorv Animal Resources, National Research Council. DHE\' Publication NIH 74-23. Accepted for publication February 18, 19747. Address reprint requests to Dr. Warren W. Schultz, Department of Microbiology, Nasal Medical Research Institute. Bethesda, MD 20014. 667
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Materials and Methods White Leghorn chicks were obtained from a local hatchery and used at 5 davs of age. There has been no laryngotracheitis in any of the farm flocks which supply eggs to the hatcherv, and the chicks have always been fully susceptible to the disease. Larvngotracheitis virus was originally obtained from Dr. E. L. Jungheer, University of Connecticut, as a chorioallantoic membrane (CAM) preparation. A stock was maintained by passage on the CAM of 11-day fertilized hen's eggs from the Hopkins White Leghorn AF-flock (free of antibodies to Rous sarcoma and avian leucosis viruses). The supernatant fluids from 10% suspensions of infected or noninfected fresh CAM in phosphate-buffered saline (PBS) were used for inoculating chicks. The chicks were inoculated with two drops of the fluid per nostril delivered through a 23-gauge needle onto the nasal opening, allowing each drop to be inhaled. Several turbinates from inoculated chicks were harvested on Day 3 after inoculation, homogenized in PBS, and inoculated onto CAM to confirm the presence of LTV infection. Infection was demonstrated in everv case, and only 4 birds in more than 200 showed a significant variation in the time of pathologic events. Turbinates were harvested by bisecting the head of the chick in the plane of and slightly to either side of the nasal septum. The exposed turbinate was then excised using a small surgical scalpel. The opposing turbinate was excised with a fresh scalpel blade. Immediatelv after a turbinate was removed, it was either quick-frozen in mounting medium (Tissue-Tek. O.C.T. compound, Ames Co., Elkhart, Ind.), using a dry ice-ethanol bath and kept at -20 C until sectioning, or was fixed first. Fixative for acid phosphatase histochemistrv was 2.5% glutaraldehyde or 4% methanol-free formalin in 0.1 M cacodylate buffer, pH 7.3, with or without 7.5% sucrose. For f-glucuronidase only the formalin fixative was used. The time and temperature of fixation are given in the results section. After fixation the tissues were washed for 2 hours in ice-cold 0.1 M cacodylate buffer, pH 7.3, containing 7.5% sucrose; drained on filter paper; (quick-frozen in mounting medium; and kept at -20 C until sectioned. Tissue blocks were usually cut within 4 hours of their preparation.
Tissue sections were cut from the middle third of the turbinate at 8-,u thickness at -22 C to -30 C on an International cryostat Model CTI (International Equipment Co., Needham, Mass.) and picked up on gelatin-treated microscope slides (0.25% gelatin in 0.5% NaKCrO4). The sections were kept cold until staining for enzyme. The time between sectioning and staining rarely exceeded 1 hour. A modified Gomori technique 7 was employed for localization of glycerol acid phosphatase. Tissue sections were usually incubated for 30 minutes at 37 C in substrate composed of 0.008 M sodium d-glycerol phosphate (Fisher Scientific Co., Fair Lawn, N.J.) in 0.05 M acetate buffer, pH 5.2, with 0.33 M sucrose and 0.014 M lead nitrate, prepared daily from a stock solution. Controls for phosphatase specificity and nonspecific hydrolysis of lead included incubating tissues in medium with the sodium :-glycerol phosphate deleted or complete substrate medium to which 0.01 M sodium fluoride had been added. The procedure used for the localization of naphthol acid phosphatase was based on the work of Burstone 8 and Kaplow.9 Tissue sections were usually incubated for 30 minutes at 37 C in substrate composed of 5 mg of naphthol AS-MX phosphate (Sigma Chemical Co., St. Louis, Mo.) dissolved in 50 ml of 0.1 M acetate buffer, pH 5.2. Just before use, 25 mg of fast blue RR salt (Sigma) was added, mixed for 60 seconds and then filtered. Controls for enzyme specificity included specimens heated at 90 C for 60 seconds or specimens incul)ated in the presence of 0.01 M sodium fluoride. The simultaneous coupling technique 10 was used for the localization of f-glucuronidase. The substrate was the sodium salt of naphthol AS-BI glucuronide (Sigma) and the coupling agent (visualizing agent) was hexazotized pararosanilin prepared as a stock solution from pararosanilin hydrochloride " (Sigma). The working solution was prepared fresh each day. Controls included incubating tissue in medium without the glucuronide or in complete medium but with a specific inhibitor, glucosaccharo-1,4-lactone at a concen-
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tration of 0.025 mM. Coverslips were affixed to all stained preparations using Permount (Fischer), which had a much slower reaction product diffusion rate than other mounting agents. When employing the glycerol acid phosphatase and naphthol acid phosphatase techniques, the deposit of a brown or blue precipitate, respectively, in the form of a 0.5 to 1.0 p spherical body within the cell cytoplasm was construed as being bound lysosomal acid phosphatase. When employing the 3-glucuronidase method, a red deposit was construed as being bound Ivsosomal 0-glucuronidase. If the red stain appeared as an extremely fine granular or diffuse reaction product, it was considered as lysosome-released or free endoplasmic reticulum (-glucuronidase, depending upon the cell type and time of infection. To determine the percent of cartilage cells showing particulate enzyme staining, the entire cross section of the turbinate scroll was examined; a minimum of 300 cells per section were counted, and the percentage expressed to the nearest 5%. A minimum of three sections per slide were examined. Mucosal columnar cells showing particulate or diffuse enzyme stain could not be accurately quantified. Therefore, the epithelium along the entire length of the scroll was examined and roughly assigned percentage values in terms of areas, rather than cells, showing enzyme staining. Methyl green was routinely used as an enzyme counterstain and as an indicator of the condition of the cartilage matrix. A 2.0% (w/v) aqueous solution of crude methyl green (Allied Chemical and Dye Corp., NY) was repeatedly extracted with chloroform until all undesirable blue dyes were removed. A working solution was then prepared by mixing equal volumes of the dye and 0.2 M acetate buffer, pH 4.2. Frozen sections were also routinely stained with hematoxylin and eosin, toluidine blue, and alcian blue. The hematoxylin and eosin stain was a modification of Mayer's hematoxvlin.u For toluidine blue and alcian blue staining, 0.25% (w/v) and 0.1% (w/v) aqueous solutions were used, respectively. In experiments with vitamin A (all-trans-retinoic acid, Eastman Kodak Co., Rochester, N.Y.) and cortisone (cortisone acetate suspension, N.F., 25 mg/ml; Intra Products, Inc., Dayton, Ohio), drug treatments were initiated when the chicks were 4 days old. For each drug, 16 chicks were divided into four groups: Group 1 was the untreated control, Group 2 received LTV, Group 3 received vitamin A or cortisone, and Group 4 received vitamin A or cortisone and LTV. The vitamin (50 mg/ml in Wesson corn oil) and cortisone were given orally at a dosage of 10 mg and 5 mg per 50 g body weight, respectively, daily for 3 days. After a drug-free day the chicks received 5 mg vitamin A or 2.5 mg cortisone acetate daily until termination of the experiment. Laryngotracheitis virus was instilled on Day 5 immediately after the second drug dose. For the vitamin A experiment the turbinates were fixed ovemight at 4 C to elicit maximum particulate staining in the chondrocytes and permit observation of the redistribution of #-glucuronidase from the particulate to diffuse form. For the cortisone experiment, fixation was at 26 C for 2 hours and then overnight at 4 C to minimize particulate and diffuse staining and permit observation of increased staining. This fixation protocol was derived from experiments shown in the results section. Sequential enzvme and pathologic changes were recorded on color film to capture the multicolor enzvme and background stains. The photomicrographs used here were reproduced from color transparencies by a multistep procedure which resulted in some loss of resolution.
Resufts Locaizai of Lyso-l Enmes in the Non_fected MaIay Canch
The structure, function, and general staining properties of the maxillary concha (middle turbinate, Figure IA) have been well described by Bang
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and Bang.1 Briefly, it is composed of an inner noncalcifying cartilage center with an overlying mucociliated epithelium (i.e., ciliated columnar cells, epithelial goblet cells, and acinar gland cells) and a well-vascularized submucosa (Figure 1B). Acid Phosphatases
Nonfixed turbinates contained no particulate or diffuse glycerol acid phosphatase staining in the columnar and mucous cells or the perichondrocytes and chondrocytes (Table 1). Histochemical studies 13,14 have shown that activation of the lysosomal membrane by the action of a fixative (e.g., formalin or glutaraldehyde) is necessary for the Gomori reaction to occur. When turbinates were fixed, particulate staining was observed in the cytoplasm of the columnar cells along the entire length of both the luminal and outer mucosa of the turbinate scroll (Figure 2A). Cytoplasmic particulate glycerol acid phosphatase was also observed in the mucous cells of fixed tissues when incubated in substrate medium containing 0.01 M Mn2+. There was occasional particulate staining in the perichondrial cells, but no staining in the chondrocytes, even when Mn2+ was added (Table 1). Granulocytes within vessels were observed to contain particulate glycerol acid phosphatase. Particulate staining in this tissue was inhibited by 0.01 M NaF. The optimum conditions were shown to be incubation at 37 C for 30 minutes at pH 5.2. Since the enzyme could not be demonstrated in the chondrocytes with glycerol phosphate as a substrate, sodium salts of naphthol AS-MX phosTable 1-Lysosomal Enzymes in the Turbinates of Noninfected Chicks Enzyme staint
BGase BGase GAPase NAPase Fixation* (Particulate) Particulate Diffuse (Particulate) No + i + + Yes + + Mucous No + + Yes +* + + ± Perichondrocytes No + + ± ± Yes + + No Chondrocytes + + + Yes + + + *Glycerol acid phosphatase (GAPase), 1.5 hours at 4 C; naphthol acid phosphatase (NAPase), 1.0 hours at 24 C; f-glucuronidase (BGase), 0.5 hours at 24 C. t After 0.5 hours at 37 C; - = no staining in at least three sections; ± = occasional cytoplasmic particulate staining and number of particles per cell < 10; + = 1 to 100% staining. Diffuse GAPase and NAPese .stairrng- was-not seen. f Only after incubation in substrate containing 0.01 M Mn2 .
Cell type Columnar
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phate and naphthol AS-TR phosphate were tested. With both substrates, particulate staining was observed in the cytoplasm of columnar, mucous, perichondrial and chondral cells of nonfixed and fixed tissues (Table 1). The staining activity of preparations incubated in medium containing 0.01 M NaF or heat-treated (90 C for 60 seconds) was 10% or less of that observed in the test groups. $-Glucuronidase
3-Glucuronidase stain was selected because this enzyme is involved in cartilage catabolism. In nonfixed tissues, particulate ,B-glucuronidase staining was observed in less than 1 % of the cartilage cells along the scroll after 30 to 60 minutes of incubation but was seen in all cells at 90 minutes (Tables 1 and 2). All of the cartilage cells had a finely granular or diffuse, light red staining product throughout the cytoplasm. The latter type of reaction product was also observed in all of the columnar and acinar cells of the mucosa, the intensity slightly increasing when incubation time was extended from 60 to 90 minutes. However, only rare particulate staining was observed in the columnar and perichondrial cells and no particulate staining was observed in mucous or blood cells. In fixed tissues, all of the cartilage cells showed particulate staining in the cytoplasm at the three substrate incubation times (Tables 1 and 2, Figure 3A), but greater numbers of lysosomes per cell were visualized at 60 and 90 minutes than at 30 minutes. Most of the cartilage cells also had the finely granular or diffuse type of light red staining product, but unlike the nonfixed tissues the intensity did not increase with an increased substrate incubation time. In the mucosal cells, there was only rare particulate staining, but all cells displayed diffuse staining which did not increase in intensity with longer incubation. The granulocytes within blood vessels contained discrete, red staining granules. No particulate or diffuse stain was seen in any of the control preparations. Further characterization of the diffuse and particulate types of ,glucuronidase staining was provided by varying the time of formalin fixation as shown in Table 2. In general, as the fixation time, temperature, or both were increased, the amount of enzyme stain decreased. In the mucosa, fixation at 4 C caused little decrease as the time was increased. In the cartilage cells the particulate staining persisted but the diffuse staining was lost by 20 hours of fixation. At 24 C the lability of both particulate and diffuse enzyme to fixation became much more apparent. Fixation of 0.5 hour resulted in some diffuse staining, but the intensity decreased at 1.0 and 1.5 hours, and only faint diffuse stain remained thereafter. The particulate stain in the cartilage cells was not affected until after 1.5 hours
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Table 2-f3-Glucuronidase Staining: Effect of Fixation Time and Temperature and Substrate Incubation Time Cartilage Mucosa Substrate Fixation incubation* time 24 C 4C 24 C 4C time (hrs) (hrs) 100/100 100/100 100/5 100/5 0.5 0.5 100/100 100/100 100/20 100/0 1.0 100/100 100/100 100/10 100/20 1.5 1.0
0.5 1.0 1.5
100/5 100/0 100/20
100/5 100/20 90/10
100/100 100/100 100/100
100/100 100/100 100/100
1.5
0.5 1.0 1.5
100/5 100/0 100/0
90/0 100/0 90/10
100/100 100/100 100/100
40/80 10/100 100/100
2.0
0.5 1.0 1.5
100/0 100/0 100/10
10/0 50/5 50/5
100/100 100/100 100/100
0/40 0/90 10/100
4.0
0.5 1.0 1.5
100/0 100/10 100/20
0/0 0/0 0/0
100/100 100/100 100/100
0/5 0/20 0/20
20.0
0.5 1.0 1.5
90/0 90/5 90/5
0/0 0/0 0/0
0/90 0/100 0/100
0/0 0/0 0/0
*At 37 C. t Percentage cells showing diffuse staining/percentage cells showing particulate staining.
of fixation. At 37 C there was a complete absence of f,-glucuronidase staining in the 2.0 and 4.0 hour specimens (not shown). Control specimens were uniformly negative. From the above data, the fixation time and temperature of 1 hour at 24 C and a substrate incubation time of 30 minutes were selected for further experiments; under these conditions, the optimum pH was 5.2. Pathology of Laryngotracheitis Virus Infection
Chicks which received the LTV inoculum showed an increase in gross viscosity of the mucus in and around the turbinate on Day 2. On Day 3 the mucus was even more viscous and the turbinate appeared blanched. On Day 4, yellow caseous exudate filled the entire nasal cavity. After Day 5 the nasal septum and turbinates were very soft and pliable. A reversal of these conditions began by Day 8, and by Day 12 turbinate rigidity was similar to that seen in the controls. Significant changes were not seen in chicks inoculated with material derived from normal CAM. Microscopically, the first viral cytopathic effects were readily observed
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by 24 hours. Foci of infected cells with Type A intranuclear inclusions were found in the mucosa, particularly the luminal mucosa. The origin of these cells could not be determined. By Day 2 there was increased syncytial formation with some sloughing, and finally, exhaustion of the mucus from the acini. On Day 3 to 4, most of the luminal and outer mucosa showed marked destruction and infiltration by polymorphonuclear leukocytes. There was much vasodilatation of the underlying vessels with engorgement of surrounding tissue. Lymphocyte and plasma cell infiltration was seen on Day 3 to 8. Mucosal cell sloughing was maximal on Day 4 to 5, and the normal basophilia of the cartilage matrix was greatly decreased. Mucosal regeneration commenced about Day 6 with the appearance of a thin layer of epithelial cells. As regeneration continued, the cartilage regained its basophilia. By 3 to 4 weeks most of the mucosa had been repaired. There observations were consistently found, except that in the regeneration stage (Day 8 to 28) individual variation was slightly more pronounced. Methyl green was used as a counterstain since the staining process did not destroy the enzyme stain end-product and provided a striking cartilage background in normal turbinate tissue. It was compared, therefore, to metachromatic staining in infected turbinates as shown in Table 3. The loss in methyl green staining intensity paralleled the loss of toluidine blue metachromasia, indicating the loss of cartilage matrix substance. Since the loss of methyl green staining was observed 1 to 2 days earlier than the loss of toluidine blue, methyl green was considered the more sensitive indicator of cartilage pathology. Table 3-Turbinate Cartilage Staining in Infected Chicks* Twne atter inoculaion (days) 1 2 3 4
Methyl green Nonfixed
Fixed
4+ 4+ 2+ 2+ 0 0 0 1+ 2+ 3+ 4+ 4+
4+ 4+ 4+ 3+ 2+ 0 0 1+ 3+ 4+ 4+ 4+
5 6 7 8 9 10 11 12t * Intensity of staining indicated by 0 to 4+. Each control which stained uniformly 4+. t Further changes not observed through 28 days.
time
Toluidine blue Fixed 4+ 4+ 4+ 2+ 1+ 1+ 1+ 3+ 4+ 4+ 4+ 4+ interval included a noninfected
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Localization of Lysosomal Enzymes in Infected Turbinates Acid Phosphatase
At 2 days postinoculation, there was a large increase in the amount of particulate staining in the columnar cells of nonfixed tissue which was particularly noticeable in the luminal mucosa (Table 4). Large dense focal areas of diffuse glycerol acid phosphatase also appeared in the mucosal cells. Syncytia and individual cells which had sloughed off from both the luminal and outer mucosa did not show any staining. After Day 3, both the particulate and methyl green stain began to decrease and were no longer observed by Day 6. During this period, large numbers of heterophils with glycerol acid phosphatase-positive granules were seen in the mucosa and in material collected in the lumen and outside the turbinate, and macrophage-like cells with large pools of enzyme were observed in the mucosa. On Day 6, particulate staining was again observed in squamous-like cells forming the outer edges of the regenerating mucosa, reaching a maximum on Day 10 and decreasing to control levels as the regenerative cells differentiated into mucociliated epithelium. At 10 to 12 days the stain also appeared in the differentiating cells forming new mucous acini. Paralleling these changes, the cartilage showed a gradual increased uptake of methyl green that reached an intensity comparable to that of the noninfected control at Day 12. In fixed turbinates, at Day 1, particulate staining was similar in control and infected columnar cells. However, the amount of staining progresTable 4-Glycerol Acid Phosphatase Changes in Infected Turbinates Fixed Nonfixed Cartilage Cartilage Time Columnar after Columnar (methyl green (methyl green cells uptake) cells uptake) inoculation* NDf NDt 5 hrs Pt 4+t P PPPP 4+ 21 hrs 4+ 4+ 42-48 hrs PPP,DD PPP,DD 4+ 68-74 hrs 4+ P,D P,D 2+ PO 4-6 days P_0 2+O 3+ 0 01 + PP -PPP PP PPP 0 6-8 days 1 +- 4+ PPPP PPPP 8-10 days 1 +-3+ PPPP PPPP 4+ 10-12 days 2+-4+ PPPP PPPP 0 4+ 12-28 days 4+ Based on three experiments, except values at 5 hours (one experiment) and 21 hours (two experiments). t P = particulate staining, D = diffuse staining; P or D = 1 to 25%, PP or DD = 26 to 50%, etc. Changes within the time period indicated by ; ND = not done. t Intensity of staining indicated by 0 to 4+. *
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sivelv decreased in the infected tissues, and by Day 6 it was absent. Diffuse staining was seen on Days 1 to 3 (Figure 2B). The initial decrease in particulate enzyme, in both fixed and nonfixed tissue, coincided with the increase in diffuse staining and was followed by the loss of methyl green uptake by the cartilage. During the regenerative stage when particulate glycerol acid phosphatase staining was again present, methyl green uptake reappeared. It should be noted that near the attachment end of the turbinate there was often little or no pathology or change in glycerol acid phosphatase staining in the mucosa and no loss of methyl green staining in the cartilage. Naphthol acid phosphatase staining decreased greatly in the mucosa and slightly in the cartilage during the first 5 days of infection. The numbers of granules per cell were also observed to decrease, but the size of the granules was two to three times larger. By Day 5 or 6 the staining in the cartilage had retumed to normal, and by Day 8 the size and number of cartilage cell granules were similar to those of the noninfected controls. On Days 6, 8, and 10, there was greatly increased particulate staining in the undifferentiated replacement cells of the mucosa. By Day 13, in areas of the mucosa where there was complete regeneration, the naphthol acid phosphatase staining was similar to that of the noninfected controls. Comparable results were observed in fixed tissue. $-GIucuronidase
In nonfixed turbinates the mucosal cells showed a loss of diffuse ,glucuronidase staining beginning on Day 2 and progressing and persisting until Day 7 (Table 5). In the chondrocytes there was an increase in particulate stain during the first 2 days, followed by a decrease with concurrent increase in diffuse stain. In the cartilage matrix, as the dglucuronidase stain increased, the methyl green staining decreased and was absent by Day 6. The cartilage cells themselves, however, showed no apparent pathology. In the initial regeneration period, the earlv replacement cells of the surface layer of the mucosa showed intense diffuse ,glucuronidase staining. With advanced regeneration, the staining of the mucosa, the particulate staining in the chondrocytes, and the uptake of methvl green by the cartilage matrix returned to levels like those observed in the controls. The same pattern of changes in enzyme distribution was observed in the fixed tissue (Table 5). At Day 1, formalin treatment of turbinates for 1.5 hours reduced diffuse mucosal ,-glucuronidase staining to 50% of that of comparable nonfixed tissue, and 2-hour treatment eliminated staining completely. At Day 2 to 3, there was an increase of about 100% in total
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Table 5-Changes in ,B-Glucuronidase Staining in Infected Turbinates* Mucosa after fixation Cartilage after fixation Time after 0 hrs inoculation(days) 1.5 hrs 2.0 hrs 1 hrs 1.5 hrs 2.0 hrs 1 DDDD PP DD 0
